Proceedings of the Fourth International Conference

ISSN 1313-7735

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES

Publisher

Bulgarian National Multidisciplinary Scientific Network of the Professional Society for Research work

Volume 2

LOZENEC, BULGARIA 12– 16 JUNE, 2013

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES

Fourth Conference

Publisher

Bulgarian National Multidisciplinary Scientific Network of the Professional Society for Research Work

with the support of

Bulgarian National Society of Agricultural Engineers “Engineering and Research for Agriculture” “Angel Kunchev” University of Rousse, Bulgaria National Institute of Research-Development for Machines and Installations Designed to Agriculture and Food Industry, Bucharest, Romania University of Agricultural Sciences and Veterinary Medicine of Banat Timişoara, Faculty of Zootechny and Biotechnology, Romania Society “ECOLOGICA”, Belgrade, Serbia

Volume 2

Social and human sciences. Economic sciences. Ecology and Environment protection, Land & Water Use and Environment, Physics, Chemistry, Biology, Computer Systems and Technologies

LOZENEC, BULGARIA 12– 16 JUNE, 2013

Organizing Committee

Atanasov Atanas, Bulgaria - Editor in Chief Mihailov Nikolay, Bulgaria Vezirov Chavdar, Bulgaria Kehayov Dimitar, Bulgaria Bikic Sinisa, Serbia Vlăduţ Valentin, Romania Dochev Veselin, Bulgaria Bungescu Sorin, Romania Atanasova Polina, Bulgaria Kneževiš Dario, Croatia Biriş Sorin-Ştefan, Romania Sandric Maria, Romania

International Scientific Committee

Vezirov Chavdar, Bulgaria Banaj Đuro, Croatia Paradjikovic Nada, Croatia Vulkov Lubin, Bulgaria Inoue Keiichi, Japan Vlăduţ Valentin, Romania Martinov Milan, Serbia Popescu Simion, Romania Bilalis Dimitrios, Greece Fröba Norbert, Germany Kehayov Dimitar, Bulgaria Perkoviš Anica, Croatia Zheliazkova Irina, Bulgaria Bungescu Sorin, Romania Kaya Yalcin, Turkey Biriş Sorin-Ştefan, Romania Jovanoviš Larisa, Serbia

ISSN 1313-7735

Printed in Bulgaria Printing House “Angel Kunchev” University of Rousse 8, Studentska Street, 7017 Rousse, Bulgaria

PREFACE

Dear Colleagues! The International Scientific Conference “Research People and Actual Tasks on Multidisciplinary Sciences” is fourth International Conference organized in Bulgaria with basic purpose to create the framework for the presentation, debate and publication of the valuable scientific results obtained by both the young members. United by the idea of Multidisciplinary Sciences, the researchers and faculty will report the results of their research. Thus, the scientists will contribute is to promote exchange of research results, scientific ideas and their practical implementation and development work in the various disciplines. We hope this meeting will initiate new joint research projects, new friendships. We owe special thanks to all participants, and especially to the supporting organizations.

Chief Editor Assoc. Prof. Dr. Atanas Atanasov

CONTENTS VOLUME 2

Social and human sciences. Economic sciences. Ecology and Environment protection, Land & Water Use and Environment, Physics, Chemistry, Biology, Computer Systems and Technologies

1. STABILIZATION OF L-ASCORBIC ACID WATER SOLUTIONS DURING ITS DETERMINATION BY HORSERADISH PEROXIDASE Vladan Djuriš, Nebojša Deletiš, Slaviša Stojkoviš, Nenad Draškoviš, Suzana Stankoviš…………………………………………………………………………1

2. IRON CONTENT IN PSEUDOGLEYOF WESTERN SERBIA AS DETERMINED BY DIFFERENT EXTRACTION METHODS M. Jelic, J. Milivojevic, G. Dugalic, O. Nikolic, S. Stamenkovic………………………6

3. THE ASSESSMENT OF ANTIOXIDANT CAPACITY OF MEDICINAL HERBS IN LOW PH CONDITIONS D.S. Stef, I. Gergen, T.I. Trasca, A. Rivis, L. Stef, R. Heghedus-Mindru………..….11

4. THE EVALUATION OF FOOD QUALITY BY USING DIFFERENT METHODS D.S. Stef, R. Heghedus-Mindru, L. Stef, G. Heghedus-Mindru, M. Cazacu, G.Bujanca, F. Berbentea………………………………………………………………..16

5. ANGIOTENSIN I CONVERTING ENZYME INHIBITORY ACTIVITY OF PUMPKIN OIL CAKE 12S GLOBULIN HYDROLYSATES Ž. Vaštag, Lj. Popoviš, S. Popoviš, D. Peričin, and V. Krimer-Maleševiš...... 21

6. THE PLANTLETS ROOTING OF CHILLI PEPPER OBTAINED IN VITRO CULTURE CONDITIONS A. Lazăr, C. Petolescu, G. Velicevici, I. Creţescu, S. Popescu, O.M. Boldura, G.Poşta and V. Berar………………………………………………………………….…26

7. THE SUCROSE INFLUENCE ON CHILLI PEPPERS PLANTLETS GROWING IN THE IN VITRO CULTURE CONDITION A. Lazăr, C. Petolescu, G. Velicevici, I. Creţescu, S. Popescu, O.M. Boldura, G.Poşta and V. Berar………………………………………………………………….…31

8. THE DETERMINATION OF THE OPTIMAL CHLORINE DOSE FOR THE DISINFECTION OF A MICROBIAL CONTAMINATED WASTEWATER V.V. Safta, Viviana-Carmen Ciucă...... 35

9. STUDY ON MEAT QUALITY DEPENDING ON OXIDATIVE STRESS OF THE ANIMALS BEFORE SLAUGHTER Iuliana Manea, Laur Constantin Manea………………………………………………40

10. THE DYNAMIC OF PHENOLS ACCUMULATION AND THE INFLUENCE ON THE ANTIOXIDANT ACTIVITY OF VITIS VINIFERA L. CALLUS CULTURE EXTRACTS R. Mihai, F. Helepciuc, and M. Mitoi……………………………………………………44

11. DETERMINATION OF SOME ANIONS CONCENTRATION IN MINERAL WATERS FROM NORTH REGION OF ROMANIA, BY HIGH PERFORMANCE ION CHROMATOGRAPHY (HPIC) R. C. Heghedűş-Mîndru, G. Heghedűş-Mîndru, P. Negrea, , R.L. Şumălan, T.I. Traşcă, A. Riviş, D.S. Ştef and L.M. Rusnac……………………………………….…50

12. DETERMINATION OF SOME ANIONS CONCENTRATION IN MINERAL WATERS FROM WEST REGION OF ROMANIA, BY HIGH PERFORMANCE ION CHROMATOGRAPHY (HPIC) G. Heghedűş-Mîndru, R.C. Heghedűş-Mîndru, P. Negrea, , R.L. Şumălan, T.I. Traşcă, A. Riviş, D.S. Ştef and L.M. Rusnac…………………………………………..56

13. DETERMINATION OF SOME ANIONS CONCENTRATION IN MINERAL WATERS FROM CENTRE REGION OF ROMANIA, BY HIGH PERFORMANCE ION CHROMATOGRAPHY (HPIC) G. Heghedűş-Mîndru, R.C. Heghedűş-Mîndru, P. Negrea, , R.L. Şumălan, T.I. Traşcă, A. Riviş, D.S. Ştef and L.M. Rusnac………………………………………….62

14. MICROARCHITECTURE OF QUATERNIZED POLYSULFONE/POLY (VINYLIDENE FLUORIDE) BLEND FOR SCAFFOLDS IN TISSUE ENGINEERING L. I. Buruiana, E. Avram, A. Popa, and S. Ioan...... 67

15. ANTIOXIDANT CAPACITY OF SALVIA OFFICINALIS EXTRACT REGARDING THE SUNFLOWER OIL USED IN FOOD THERMAL APPLICATIONS Nadina Buta, N. Popa, Nicoleta Bordea, Mariana Poiană and T.I. Traşcă...... 72

16. IMPROVE PERFORMANCE OF THE POLYSULFONE MEMBRANES INDUCED BY PRESENCE OF POLYVINYL ALCOHOL A. Filimon1, E. Avram1, and S. Ioan1…………………………………………………...77

17. DEVELOPMENT AND ANALYSIS OF A TOPICAL PHARMACEUTICAL PRODUCT CONTAINING RIBES NIGRUM LEAVES EXTRACT L.E. Dutu, .T.D.Balaci, C.E.Gird, L.M. Popescu, I. Dutu……………………………..82

18. PRELIMINARY RESEARCH REGARDING A DERMATO-COSMETIC PRODUCT WITH VEGETAL EXTRACTS USEFUL FOR THE TREATMENT OF HYPERPIGMENTATION C. E. Gîrd, T. D. Balaci, L. E. Duţu, I. Nencu, E. A. Ciocan...... 88

19. ISOTROPIC AND ANISOTROPIC PHASES IN CELLULOSE LIQUID CRYSTALLINE SOLUTIONS M. Dobos1, M. D. Onofrei1, and S. Ioan1...... 95

20. DIELECTRIC PROPERTIES OF NEW POLYSULFONES WITH CHELATING GROUPS R.M. Albu1, E. Avram1, V.E. Musteata1, and S. Ioan1………………………………100

21. EFFECT OF AMMONIUM VANADATE ON VIABILITY AND PROLIFERATION OF CULTURED ANIMAL CELLS Abdulkadir Abudalleh, Pavel Mitrenga, Tanya Zhivkova, Lora Dyakova, Stoyan Shishkov, Radostina Alexandrova……………………………………………..……105

22. THE FORMULATION, PREPARATION AND CHARACTERIZATION OF A COMPRESSED MULTIPARTICULATE SYSTEM Mircea Hîrjău, Dumitru Lupuliasa, Mirela Mitu, Anca Nicoară, Victoria Hîrjău…..111

23. STUDIES CONCERNING THE INCORPORATION OF ORIGANUM L. SYRIACUM ESSENTIAL OIL IN LIQUID AND SOLID FORMULATIONS FOR ORAL CAVITY USE Zeinab Lakis, Teodora Balaci, Cerasela Gird, Ancuţa Fiţa, Andreea Stanescu, Denisa Mihele…………………………………………………………………………..118

24. THE MICROBIOLOGICAL MONITORING OF SOME BATCHES OF MILK ORIGINATED FROM PRIVATE HOUSEHOLDS IN THE WEST PLAIN OF ROMANIA Mişcă Corina Dana, Mişcă C. B., Rinovetz A., Bujancă G., David I., Rădoi P. B., Rinovetz Adina...... 124

25. THE MONITORING OF ENZYME ACTIVITY OF GLUCOAMYLASE AND ENDOXYLANASE ON THE BREAD DOUGH David I., Misca Corina, Bujanca G., Rinovetz A., Berbentea F., Georgescu L., Danci M...... 129

26. VARIATION OF HEAT TRANSFER AND TEMPERATURE OF TOMATO PASTE AT STERILIZATION AND COOLING PROCESSES G. Bujancă. C. Jianu, A. Rinovetz, F. Crista, I. David, Corina Dana Mişcă, D. Ştef, Antoanela Cozma, Mihaela Cazacu, Zorica Adina Riniovetz……………………..136

27. AMENDING FACTORS OF ANTHOCYANIN DYES IN FRUIT JUICES G. Bujancă, C. Jianu, A. Rinovetz, Corina Dana Mişcă, I. David, B. Rădoi, D. Ştef, Ariana Velciov, Mihaela Cazacu, Adina, Zorica Adina Rinovetz...... 142

28. IMPROVEMENT OF PUMPKIN OIL CAKE PROTEIN GELATION PROPERTIES THROUGH ENZYMATIC POLYMERYZATION WITH TRANSGLUTAMINASE Lj. Popoviš, Ž. Vaštag, S. Popoviš, D. Peričin, V. Krimer-Maleševiš...... 148

29. VIABILITY ASSESSMENT OF Ruscus aculeatus L. GERMPLASM EXPOSED TO EXTREME LOW TEMPERATURES Banciu Cristian, Anca Aiftimie-Păunescu…………………………………………...153

30. STUDIES CONCERNING THE VARIABILITY OF SOME BLACK PINE (PINUS NIGRA) POPULATIONS FROM ROMANIA AND GREECE I.Sotiropoulos., E.Madoşă, Oana Danci, and M.Danci……………………………...158

31. AN ASSESSMENT ON THE TROPHIC STATE OF LAKE ŠELIJE, SERBIA S. Širiš, B. Miloševiš, J. Kneževiš and Z. Spasiš...... 163

32. DISCOVER TRADITIONAL GASTRONOMY ORIGINATING FROM THE VRANCEA COUNTY C. C. Vişan and C. I. Băra...... 168

33. ENVIRONMENTAL DAMAGES, WHICH MUST BE COMPENSATED DURING THE PROCESS COMPULSORY PURCHASE OF LAND IN UKRAINE

Alina Lizunova, Mariia Mykhalova…………………………………………………….173 34. SYSTEMATIC AND ECOLOGICAL ANALYSIS ON STINK BUGS (PENTATOMIDAE, HEMIPTERA) IN THE ECOSYSTEMS OF DURRESI REGION, ALBANIA Eltjon Halimi1,Hesat Aliu2 , Anila Paparisto1, Dritan Topi1…………………………..177

35. INTERDISCIPLINARY STUDY ON INFLUENCE OF VARIOUS TYPES OF WATER ON HERBS AND AROMATIC PLANTS IN CULTURE Gabriela Vlăsceanu1, Ştefan Manea1, Georgeta Negru1, Marosy Zoltán2, Alexandru Cristian Ion3...... 184

36. NEW PERSPECTIVES FOR SPIRULINA PLATENSIS G. Iordachescu, G. Vlasceanu, St. Manea...... 190

37. DNA IDENTIFICATION OF HUMAN REMAINS EXHUMED IN KOSOVO AND METOHIA DURING THE PERIOD 2001-2011 Milanka Miletiš, Nebojša Deletiš, Zvezdan Milanoviš, Suzana Matejiš, Sonja Smiljiš……………………………………………………………………………..195

38. ANALYSIS OF THE MOTIVATING FACTORS AND THE SATISFACTION OF THE EMPLOYEES OF A COMPANY FROM TIMISOARA (ROMANIA) C. C. Ruset, G. Buzamat, G. Popescu, K.I. Lato, and I. Cretescu…………………200

39. THE ANALYSIS OF THE FINANCIAL POSITION IN AN AGRICULTURAL COMPANY FROM ROMANIA Genoveva Buzamat, Corina Ruset, Elena Pet, Iuliana Cretescu...... 205

40. THE PROBLEM OF SYNTACTIC STRUCTURES IN CONTEMPORARY LINGUISTICS L.Ramazanova...... 210

41. ENGLISH FOR SPECIFIC PURPOSES IN CAREER OPPORTUNITIES Rodica Silvia Stan………………………………………………………………………215

42. Opportunities and prospects of improvement of differentiation of lands in Ukraine A.N. Lychogrud…………………………………………………………………………..220

43. Usage of renewable energy resources in Serbia Larisa Jovanoviš…………………………………………………………………………224

44. Application of the multi-criteria decision analysis for assessing the student’s programming task performance I. Zheliazkova and O. Kir…………………………………………………………….....229

45. Environmental applications of zeolite-based nanomaterials. hospital wastewater treatment investigation C.I.Covaliu, L.Toma, G. Paraschiv, S.Şt. Biriş……………………………………….237

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

STABILIZATION OF L-ASCORBIC ACID WATER SOLUTIONS DURING ITS DETERMINATION BY HORSERADISH PEROXIDASE

Vladan Djuriš, Nebojša Deletiš, Slaviša Stojkoviš, Nenad Draškoviš, Suzana Stankoviš

Abstract: Metaphosphoric acid has been shown to be the best medium for homogenization of vitamin C, and it stabilized the vitamin even in the presence of oxygen. Metaphosphoric acid is often used for extraction of ascorbate from natural products, in order to precipitate proteins from sample and to inhibit endogenous ascorbidase. During the study, several different stabilizing substances have been tested and compared with the protection effect of metaphosphoric acid. Mixture of EDTA and acetic acid showed the greatest protective effect and expressed synergism in regard to simple components. Key words: Ascorbic Acid, Horseradish, Peroxidase, Stability, Determination.

INTRODUCTION Globalization of modern society sets new standards in every area of human activity, so a special attention is paid to production and processing of food and water, as precondition of living quality. Nutritionists and pharmacists additionally advocate correction of food nutritive value using supplementation and fortification by various vitamins and minerals, with the aim to protect and improve nutritive properties of food products. This concept necessarily includes systems for safety control in every stage of food production and distribution, together with inventing new methods and modifying existing ones for chemical analysis of versatile sample types. The all vitamins are essential nutrients that ought to be present in diet, and their deficiency cause a row of structural and functional disorders. For that reason their presence in dietary supplements is almost inevitable. Vitamin C is L enantiomer of ascorbic acid and represents a derivative of glucose soluble in water. Although it is necessary cofactor for several enzymes involved in various metabolic processes, the highest attention in last few decades is attracted by its antioxidant activity (Padayatty et al., 2003). Thus, vitamin C is one of the most important nonenzymatic antioxidants, capable to neutralize most of reactive oxygen species (by donating hydrogen), above all thanks to its low redox potential (0.18 and 0.08 V at pH 4.5 and 6.4, respectively) (Shekhovtsova et al., 2006). L-ascorbic acid is highly reactive compound, sensitive to action of air and light. Stability of its water solutions depends on pH, temperature, pressure and presence of metal ions and organic matter. During oxidation, ascorbic acid is converted to dehydroascorbic acid (Welch et al., 1995). For that reason, water solutions and extracts containing vitamin C ought to be stabilized immediately after sampling by adding suitable substances, in order to get more relevant data. L-ascorbic acid is stabler at lower pH values, so that solutions of acids (metaphosphoric, phosphoric, hydrochloric, etc.) are usually used as mediums. Metaphosphoric acid ((HPO3)3) has been shown to be the best medium for homogenization of vitamin C, and it stabilized the vitamin even in the presence of oxygen. (HPO3)3 is often used for extraction of ascorbate from natural products, in order to precipitate proteins from sample and to inhibit endogenous ascorbate oxidase (Davey et al., 2000). Although there are plenty of methods for determination of vitamin C published in literature, as regarding food products as biological fluids, a constant effort is directed to find better and more accurate technics. Within a wider study aimed to standardize method by which the total antioxidant capacity of certain sample would be expressed as equivalent of corresponding amount of 1 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA vitamin C, for determination of vitamin itself was used the enzymatic kinetic method with horseradish peroxidase (Arnao et al., 1996). It is highly sensitive method with detection limit below 0.1 μM. As the most of kinetic methods, this method is also limited by numerous interferences with complex chemical system composed of many reactants and having relatively low frequency of carrying out. Involving new components, such as stabilizers of vitamin C, gives an additional load to this system, especially because metaphosphoric acid, like other mineral acids, shows strong inhibitory effect to peroxidase enzyme, which affects final results of measurement (Đurić et al., 2012). For that reason several alternative stabilizing substances have been tested, such as acetic, oxalic and ethylenediaminetetraacetic acid (EDTA), with the aim to obtain consistent results during enough long period of time, needed for carrying out the experiment.

MATERIAL AND METHODS The trial was set so that different concentrations of L-ascorbic acid in the presence of various stabilizing agents were exposed to laboratory atmosphere in open glasses at room temperature. Concentration of ascorbic acid was determined by the standard volumetric method with 2,6-dichlorophenolindophenol (DCPIP) and by spectrophotometry, based on horseradish peroxidase activity. Coefficient of direction of the obtained curves was used as a measure of vitamin C oxidation speed. During this study, the following solutions and equipment were used: Solutions of L-ascorbic acid (Galenika, Serbia) used in this trial were within range of concentration between 0.1 and 10 mM. Buffer systems: acetate buffer pH 4 (made in the laboratory), citrate buffer pH 5 (Farmitalia Carlo Erba, Italy), citrate buffer pH 6 (Farmitalia Carlo Erba, Italy), phosphate buffer pH 7 (Fisher Scientific, UK) and borate buffer pH 8 (Farmitalia Carlo Erba, Italy). The following stabilizing substances were used: metaphosphoric acid, ortophosphoric acid, hydrochloric acid, acetic acid, oxalic acid and EDTA. Preparation of horseradish peroxidase of purity level 200 kU/g was made by Merck (Germany). Enzyme solutions were prepared immediately before trials. Relative activity of horseradish peroxidase was determined by spectrophotometry, measuring dependence of absorbance of o-tolidin oxidized form (Centrohem, Serbia), at wavelength of 630 nm, from the moment of H2O2 injection. For that purpose spectrophotometer Beckman DU-65 (UK) was used. Solutions of hydrogen peroxide (Merck, Germany) were checked by standard permanganometric method. As the dilutant for preparing basic solutions we used deionized water, with 0.5 μS/cm2 of conductivity, while for preparing series of ascorbic acid water solutions we used distilled water, with 15 μS/cm2 of conductivity.

RESULTS AND DISCUSSION Stability of L-ascorbic acid water solutions is time-dependent and decreases by rise of dilution (figure 1a). Speed of vitamin C degradation is a reaction of first order (Vieira et al., 2000; Van der Broeck et al., 1998) and, as can be seen, dependent on concentration. Logarithm of ascorbic acid concentration is linearly dependent on time (Imer et al., 2003). Solutions of higher concentration showed a lower degree of degradation, which pointed to certain degree of autoprotection of L-ascorbic acid molecules.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

a) b)

1E-3

0.01

2 pH 4

AscH pH 5 AscH2 10mM pH 6

1E-3

log C AscH

1E-4

log C C log 1E-4 AscH2 1mM pH 7

1E-5

AscH2 0.1mM pH 8

0 24 48 72 96 120 0 24 48 72 96 120 Time (h) Time (h)

Figure 1 a) Speed of L-ascorbic acid transformation to dehydroascorbic acid in water solutions 10; 1 i 0.1 mM of concentration during 96 hours. Coefficients of line direction were: -0.0126; -0.0144; and -0.0169, respectively. b) Dependence of L-ascorbic acid transformation speed to dehydroascorbic acid of medium pH, without use of stabilizing substances.

The speed of transformation AscH2 into DHA, in aerobic conditions, is faster at higher pH values, in other words, L-ascorbic acid is stabler in acid environment, as presented on figure 1b. Content of vitamin C in acetate buffer pH 4; citrate buffers pH 5 and pH 6; phosphate buffer pH 7 and borate buffer pH 8, after 96 hours amounted: 59%, 40.7%, 30.05%, 6.6% and 1.53% of the initial value, respectively. Application of standard stabilizing substances significantly decelerated degradation of ascorbic acid in water solution (figure 2a).

a) b)

1E-3 (HPO ) 2% 1E-3 3 3 CH3COOH 0.1M + EDTA 1mM

(HPO ) 1%

2 3 3

CH3COOH 0.1M + EDTA 0.5mM 2 AscH H2C2O4 0.1M

H PO 3% AscH 3 4 CH3COOH 0.1M

log C C log HCl 2% EDTA 1mM CH COOH 0.05M log C C log 3

8E-4

5E-4 EDTA 0.5mM HCl 1%

0 24 48 72 96 120 0 24 48 72 96 120 Time (h) Time (h)

Figure 2 a) Speed of ascorbic acid degradation when standard (inorganic) stabilizing substances were used. b) Speed of ascorbic acid degradation when organic stabilizing substances were used.

As expected, the most efficient stabilizing agent was metaphosphoric acid and, when it was present, concentration of vitamin C during 24 hours, stayed almost unchanged. After 96 hours, content of ascorbic acid determined by the standard DCPIP method, in 2% and 1% solutions of metaphosphoric acid, 3% solution of ortophosphoric acid and 2% and 1% solutions of hydrochloric acid, amounted 98.0%, 95.5%, 90.5%, 88.2% and 72.4% of the initial value, respectively. Results of the same samples, obtained using kinetic spectrophotometric method, differed significantly in regard to the expected values, and showed unrealistically higher content of vitamin C (tab. 1). Application of organic acids as stabilizing agents in the prescribed doses increased content of ascorbic acid after 96 hours by more than 10 times in regard to the reference 3 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA value without using stabilizers (figure 2b). As organic stabilizers, CH3COOH, H2C2O4 and EDTA, as well as their mixtures, were applied. Different concentrations of these substances showed different protective effect on content of ascorbic acid in solutions. A satisfactory stabilizing effect, during first 48 hours, for determination by the standard DCPIP method and enzymatic method using horseradish peroxidase, was shown by mixture of CH3COOH and EDTA (tab. 1).

Table 1 Stabilizing effect of some substances on vitamin C in water solutions during 48 hours (presented as percent in regard to 1 mM of vitamin C).

CH3COOH 0.1M No (HPO3)3 H2C2O4 CH3COOH EDTA 1 EDTA Reaction type stabilizers 2% HCl 2% 0.1M 0.1M mM 1mM DCPIP 0h 98.6 100 100.2 101.0 100.6 99.5 100.1 DCPIP 24h 61.4 100.1 91.2 94.1 94.5 85.6 99.3 DCPIP 48h 19.2 99.5 80.5 89.2 85.5 79.7 98.5 Peroxidase 0 h 103.1 137.4 129.3 107.0 100.8 99.7 101.4 Peroxidase 24 h 66.0 135.8 121.6 103.3 95.4 87.8 99.8 Peroxidase 48 h 27.3 135.0 120.2 96.6 88.1 82.3 97.8

Mixture of CH3COOH and EDTA showed a synergistic effect in regard to simple components and did not change activity of horseradish peroxidase enzyme.

CONCLUSIONS AND FUTURE WORK On the basis of the above mentioned, the following conclusions can be drawn out:  L-ascorbic acid, physiologically active form of vitamin C, can easily undergo to oxidation process of transformation to inactive dehydroascorbic acid, which can significantly affect content of this vitamin in regard to the declared values for various food products.  Lower concentrations of L-ascorbic acid undergo faster to oxidation than solutions of higher concentration, which suggests possible autoreduction mechanism.  Process of L-ascorbic acid oxidation accelerates significantly by increasing pH value of medium.  Among the applied classical stabilizers ((HPO3)3, H3PO4 and HCl), the highest protective effect was shown by metaphosphoric acid. Mixture of CH3COOH and EDTA (ratio 100:1) showed almost the same protective effect as metaphosphoric acid.  Mixture of CH3COOH and EDTA did not affect peroxidase activity at any observable level which could disturb measuring L-ascorbic acid using the mentioned enzymatic system, while inorganic stabilizers inhibit peroxidase activity, and so they are useless for application in this enzymatic method.  Price of (HPO3)3 is several times bigger than price of CH3COOH and EDTA.

REFERENCES [1]. Arnao, M.B., Cano, A., Hernandez-Ruiz, J., Garcia-Canovas, F. and M. Acosta. 1996. Inhibition by L-ascorbic acid and other antioxidants of the 2,2‘-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid) oxidation catalyzed by peroxidase: a new approach for determining total antioxidant status of foods. Anal. Biochem., 236, 255-261.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

[2]. Davey, M.W., Montagu, M.V., Inzé, D., Sanmartin, M., Kanellis, A., Smirnoff, N., Benzie, I.J., Strain, J.J., Favell, D. and J. Fletcher. 2000. Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. J. Sci. Food Agric., 80, 825–860. [3]. Djuriš, V.R., Deletiš, N.R., Stankov-Jovanoviš, V.P. and R.M. Simonoviš. 2012. Inhibitory effect of retinol acetate on horseradish peroxidase. Hemijska industrija, On Line- First (00), 95-95. [4]. Imer, F., Sonmezoglu, I.C. and M. Kozcaz. 2003. The role of buffers on thr kinetics of l-ascorbic acid oxidation catalyzed by copper (II). Italian Journal of Food Science, 15, 521- 529. [5]. Padayatty, S.J., Katz, A., Wang, Y., Eck, P., Kwon, O., Lee, J., Chen, S., Corpe, C. and A. Dutta. 2003. Vitamin C as an antioxidant: evaluation of its role in disease prevention. Journal of the American College of Nutrition, 22(1), 18-35. [6]. Shekhovtsova, T.N., Muginova, S.V., Luchinina, J.A. and A.Z. Galimova. 2006. Enzymatic metods in food analysis: determination of ascorbic acid. Anal. Chim. Acta, 573- 574, 125-132. [7]. Van der Broeck, I., Ludikhuyz,e L., Weemaes, C., Van Loey, A. and M. Hendricks. 1998. Kinetics of isobaric-isothermal degradation of L-ascorbic acid. Process optimisation and minimal processing of foods. 4, 12-17. [8]. Vieira, M.C., Teixeira, A.A. and C.L.M Silva. 2000. Mathematical modeling of the thermal degradation kinetics of vitamin C in cupuaçu (Theobroma grandiflorum) nectar. Journal of Food Engineering, 3(1), 1-7. [9]. Welch, R.W., Wang, Y., Crossman, A., Park, J.B., Kirk, K.L. and M. Levine. 1995. Accumulation of vitamin C (Ascorbate) and Its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J. Biol. Chem., 270(21), 12584-12592.

ABOUT THE AUTHORS V. Djuriš, MSc, Teaching Assistant, University of Priština, Faculty of Agriculture Lešak, Kopaonička Street bb 38219 Lešak, Serbia, E-mail: [email protected] N. Deletiš, PhD, Associate Professor, University of Priština, Faculty of Agriculture Lešak, Kopaonička Street bb 38219 Lešak, Serbia, E-mail: [email protected] S. Stojkoviš, PhD, Associate Professor, University of Priština, Faculty of Agriculture Lešak, Kopaonička Street bb 38219 Lešak, Serbia, E-mail: [email protected] N. Draškoviš, PhD, Associate Professor, University of Priština, Faculty of Agriculture Lešak, Kopaonička Street bb 38219 Lešak, Serbia, E-mail: [email protected] S. Stankoviš, PhD, Senior Research Associate, Mining and Metallurgy Institute, 35 Zeleni Bulevar 19210 Bor, Serbia, E-mail: [email protected]

5 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

IRON CONTENT IN PSEUDOGLEYOF WESTERN SERBIA AS DETERMINED BY DIFFERENT EXTRACTION METHODS

M. Jelic, J. Milivojevic, G. Dugalic, O. Nikolic, S. Stamenkovic

Abstract: This study was conducted on pseudogley soil having different physico-chemical characteristics. Soil samples were collected from tilled fields and meadows at six different locations in Western Serbia. A extraction methods was applied with the extraction of total iron (HNO3-HclO4-HF) and available forms (0.1 M HCl- and 0.005 M DTPA-extractable). The physico-chemical characteristics of the test pseudogleys had a significant effect on the content and forms of iron in different locations in Western Serbia. Soil pH, CEC and the content of finer fractions (silt and clay) had the highest effect on the distribution of different forms of iron The extraction methods showed similar forms of iron in tilled field and meadow pseudogleys. However, the content of DTPA-extractable iron did not correspond to the total content, which led to the common incidence of iron deficiency in Western Serbian pseudogleys regardless of the high levels of total iron in the soil.

Key words: Content, Extraction method, Iron, Location, Pseudogley, Soil.

INTRODUCTION Pseudogleys cover significant areas of Serbia, accounting for about 285,000 ha or 78.73% of the total land area in Western Serbia (Tanasijevic et al., 1966). Pseudogleys are rather poor in alkalis, being medium to strongly acid in reaction. They have a highly unfavorable structure, and a low content of organic matter. The acid reaction of pseudogley, its low humus content, and a low supply with available phosphorus and potassium are limiting factors for higher crop yields (Dugalic et al., 2005). Iron is one of the most common elements in the Earth´s crust, ranking fourth in abundance after oxygen, silicon and aluminium. In the soil, iron forms a variety of minerals such as hematite, goethite, limonite, notronite, and pyrite. Iron is also present in the form of grain-coatings in oxidized material, and in many secondary minerals (Kostic et al., 2002). Iron hydroxides play a key role in the biogeochemical cycle, in the bioavailability of iron itself and in the bioavailability of other elements (Hesterberg, 1998). The bioavailability, mobility and chemical reactivity of heavy metals in soils are often associated with their distribution among certain soil fractions and the dynamic equilibrium among them (Kreamer, 2004; Sharma et al., 2008). It is well known that main factors affecting the mobility of heavy metals in soil include: pH, content and quality of soil organic matter, content and quality of clay fraction, iron and manganese oxides. Apart from soil pH, which is a key parameter, the content and mainly the quality of soil organic matter can influence the availability of heavy metals in soil (Makovnikova, 2000). Iron is an important micronutrient whose availability could be significantly affected by soil properties. Generally, the total iron increases with increasing cation exchange capacity (CEC) and the clay and silt content (Sharma et al., 2008). Complexation of iron by soil organic matter may result in increased plant availability, and microbial exudates can supply additional iron to plant routes (Mackowiak and Grossl, 2001). Likewise, exchangeable iron absorbed onto inorganic sites and DTPA extractable iron increase with increasing soil organic matter but decrease with higher soil pH and calcium carbonate content (Sharma et al., 2008; Jakovljevic et al., 2005).

MATERIAL AND METHODS The investigations were conducted on pseudogley soil taken from the Ah horizon at six different localities in Serbia: Kraljevo, Koceljevo, Lajkovac, Gorobilje, Mionica and

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Vladimirci. The sub-samples were taken from field and meadow ecosystems at a depth of 0 to 20 cm, after which they were air-dried, crushed in a porcelain mortar and sieved through stainless steel screens. Particles 2 mm in size were used for soil characterization and Fe-fraction analyses Soil pH was determined in a suspension with water and 1M KCl mixture, with the ratio of soil/solution 1:2.5 after a 0.5 hour equilibration period; the organic content was determined using the humus method by Kotzmann (1966), available P2O5 and K2O - using the Al method by Egner-Riehm (Egner et al., 1960). CEC - using 1M NH4OAc, pH 7, and particle size distribution - by a pipette method. The total iron content was determined by atomic adsorption spectrophotometry (AAS, Model Carl Zeis Jena AAS 1N) after soil digestion in acid (HF, HNO3 and HClO4) mixtures. A cold extraction method was used for the determination of the total and pseudo-total iron in which 0.5±0.001 g of sample was transferred into a centrifuge tube and then 10 ml of 0.5 M HCl was added. The solution was shaken, subjected to vortex for a short time and placed on a shaking-table for agitation for 1 h. After agitation, the solution was centrifuged at 3000 rpm (1.900xg) for 15 min, and them filtered through a 0.45-µm syringe filter to remove particulates (Ure, 1995). The results obtained for different contents of iron (total iron, accessible iron and different chemical fractions) in pseudogley soil were subjected to statistical analysis using Student and Pearson correlation coefficients (Mead et al., 1996).

RESULTS AND DISCUSSION Table 1. gives main characteristics of the test pseudogley samples. The test soils had very poor physical and chemical characteristics. Particle size analysis showed that, apart from an average content of clay particles (< 2 μm) of 48.3% (tilled fields) and 45.4% (meadows), the soils had a high percentage of silt particles (2-50 μm), averaging 39.3% (tilled field) and 42.9% (meadow). The total percentage of silt and clay particles in the soil was very high, i.e. above 86%, whereas the sand fraction content (0.05-2.0 mm) was very low – about 12%. According to the above properties, these soils were classified as very silt loam soils.

Table 1. The physico-chemical characteristics of pseudogleys in Western Serbia Soil characteristics Tilled field Meadow Mean Range Standard Mean Range Standard deviation deviation pH(H2O) 5.4 4.9-6.1 0.4 5.5 5.1-3.2 0.4 pH(KCl) 4.3 3.9-5.6 0.4 4.4 4.0-4.9 0.4 Humus content,% 2.9 2.2-3.7 0.5 4.2 2.9-6.6 1.2 -1 P2O5 mg 100 g 7.6 3.4-10.1 2.2 7.6 4.4-10.2 1.9 -1 K2O 100 g 10.8 8.8-13.5 1.6 9.7 8.2-11.6 1.3 CEC m.e 100 g-1 19.0 14.1-24.8 4.0 18.2 13.3-25.7 4.3 Sand, % 12.4 5.5-21.8 5.7 11.7 4.8-20.6 5.8 Silt, % 39.3 30.2-48.2 6.9 42.9 31.0-55.2 8.3 Clay, % 48.3 44.0-53.9 3.0 45.4 37.9-50.2 3.7 Silt + Clay, % 86.6 78.6-92.4 4.9 88.3 80.3-95.4 5.8

Soil pH ranged from 3.9 to 5.6, averaging 4.3, which makes this soil extremely acid. The cation adsorption capacity (CEC) was relatively high, ranging from 13.3 to 25.7 m.e 100 g-1 soil, with an average of about 19.0 m.e 100 g-1. The average humus content of tilled field was low (2.9%), whereas that of meadow soil was considerably higher (4.2%).

7 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The content of readily available phosphorus in the test locations was low, ranging from 3.4-10.1 mg 100 g-1 in samples taken from tilled fields and 4.4-10.2 mg 100 g-1 in meadow soil samples. The pseudogleys in the test locations had a higher supply of K2O than of P2O5. The average content of total iron (HNO3-HClO4-HF extraction) in the test pseudogley samples was 2.76% (tilled field) and 2.73% (meadow), the values being within the range of 1.88 to 3.61% (Table 2). The values were typical of the test soil type. The variations between samples were the result of differences between the test soil samples in both physical and chemical characteristics across locations. Soil metals having a higher clay level and higher CEC values are more strongly bound to soil components and they are not susceptible to leaching into deeper soil layers. Hence, the total iron content in these soils is higher than in soils having a lower content of clay fraction and lower CEC values (Adriano, 1986). The available forms of 0.1 M HCl- and 0.005 M DTPA-extractable iron were extremely low as compared to their total amount in the test soil (Table 2). The content of 0.1 M HCl- extractable iron was twofold higher than that of 0.005 M DTPA- extractable iron. The average content of 0.1 M HCl-extractable iron was 177.6 mg kg-1 (tilled field) and 159.2 mg kg-1 (meadow), whereas that of available 0.005 M DTPA-extractable Fe was 85.3 mg kg-1 (tilled field) and 82.3 mg kg-1 (meadow). The relative values of HNO3-extractable iron (pseudo-total content) show considerable variations in both groups of test pseudogleys. The average content of pseudo-total forms of iron was about 2.4% in both pseudogley varieties. The percentage of HNO3-extractable forms as compared to the total iron content (HF) in the test locations was 88.5% on average in tilled field pseudogley and 88.1% in meadow pseudogleys. The data suggest that 11 to 12% of the total iron in the soil is bound to the structure of primary and secondary minerals. The results are in agreement with those obtained by Han et al. (2007) who studied the content of 4 M HNO3 extractable iron in vertisol in the Mississippi River Delta.

Table 2. Iron content in the tested pseudogley of West Serbia as determined using different extraction methods (X±sd and interval, mg kg-1) Location HF HNO3 0.1 M HCl 0.005 DTPA Field (n=6) 27600±5946 24300±4694 177.6±20.5 85.3±15.9 18800-35000 17500-31000 150.0-220.0 60.0-120.0 Meadow 27300±5192 24000±4076 159.2±58.5 82.3.±14.9 (n=6) 20500-36100 18500-29800 70.0-240.0 60.0-110.0 t-test 0.615 0.650 1.227 0.581 There is no statistical significance at p ˂ 0.05

The percentage of water-soluble iron in relation to the total iron content is given in Table 3. In the tilled field pseudogley, the average percentage of water-soluble 0.1 M HCl- extractable iron relative to the total iron was very low (0.63%), whereas that in the meadow pseudogley was lower (0.58%), regardless of similarity in their total and pseudo-total contents. The content of HCl-extractable iron in certain samples showed significant variations, ranging from 0.31 to 0.88, and was found to be independent of the total iron content. Therefore, in most locations, the relative content of 0.1 M HCl-extractable iron decreased with increasing concentration of total iron. However, similar differences were observed between certain samples in the distribution of 0.1 M HCl-extractable iron, resulting from differences in pH between the test samples. Particularly important were the samples with the lowest pH values which had the highest content of HCl-extractable Fe.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

During 0.005 M DTPA extraction, the average percentage of iron was identical in both pseudogleys (0.31%). As compared to 0.1 M HCl-extractable Fe, the obtained values for DTPA-extractable Fe decreased by over 50% on average in both soils. The distribution of DTPA-extractable iron in both pseudogley soils exhibited marked heterogeneity. The observed distribution dynamics across locations was consistent with the distribution of 0.1 M HCl-extractable iron, but inconsistent with the total and pseudo-total iron contents of the test soil. The values of DTPA-extractable Fe in the soil samples confirm the common incidence of iron deficiency regardless of the high amount of total iron in the soils (Sharma et al., 2008).

Table 3. The relative content of iron in different extraction agents (in % of HF- total) Location/Extraction HF HNO3 0.1 M HCl 0.005 M DTPA method Tilled field Kraljevo 33000 93.9 0.63 0.26 Koceljevo 18800 93.6 0.87 0.36 Lajkovac 35000 80.3 0.44 0.28 Gorobilje 30200 82.1 0.55 0.30 Mionica 26500 91.3 0.67 0.26 Vladimirci 22000 90.0 0.88 0.39 X 27600 88.5 0.67 0.31 Meadow Kraljevo 30600 92.4 0.76 0.21 Koceljevo 20600 89.3 0.87 0.47 Lajkovac 36000 82.7 0.61 0.28 Gorobilje 28400 85.4 0.49 0.26 Mionica 24500 90.1 0.45 0.37 Vladimirci 23800 88.6 0.31 0.27 X 27300 88.1 0.58 0.31

The results by Jeliš et al. (2011) show that the test vertisols in Serbia had a similar distribution and content of total and pseudo-total forms of Fe, and an extremely low i.e. critical content of DTPA- extractable Fe.

CONCLUSIONS AND FUTURE WORK The physico-chemical characteristics of the test pseudogleys had a significant effect on the content and forms of iron in different locations in Western Serbia. Soil pH, CEC and the content of finer fractions (silt and clay) had the highest effect on the distribution of different forms of iron. The extraction methods showed similar forms of iron in tilled field and meadow pseudogleys. However, the content of DTPA-extractable iron did not correspond to the total content, which led to the common incidence of iron deficiency in Western Serbian pseudogleys regardless of the high levels of total iron in the soil.

ACKNOWLEDGEMENTS The authors would like to thank the Serbian Ministry of Science and Environmental protection for supporting this work through project No. TR 31054.

9 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

REFERENCES [1]. Adriano, D. C., 1986. Trace elements in the Terrestrial Environmen, Springer–Verlag, Inc, New York. [2]. Dugalic, G., et al. 2005. Agrochemical characteristics of pseudogley in the Kraljevo basen. Proceedings. XL. Croatian Symposium on Agriculture, 15-18. February, Croatia, pp 415-416. [3]. Egner, H., et al. 1960. Laboratory Manual of Soil and Water Chemistry. Berlin, 26, (In Germany). [4]. Han, X. F., et al., 2007. Effects of land uses on solid-phase distribution of micronutrients in selected vertisols of the Mississippi River Delta. Geoderma, 142, 96-103. [5]. Hesterberg, D., 1998. Biogeochemical cycles processes leading to changes in mobility of chemical in soils. Agric. Ecos. Emviron. 67, 121-133. [6]. Kostic, M. N., et al., 2002. Geochemistry of iron in soils of Vojvodina, Sumadija and Northern Pomoravlje. Proceedings for Natural Sciences, Matica Srpska, Novi Sad, 103, 17-28. [7]. Kreamer, M. S. 2004. Iron oxide dissolution and solubility in the presence of siderophores. Aquat. Sci. 66, 1, 130-138. [8]. Jakovljevic, M., et al. 2005. Some negative chemical properties of acid soils. J. Serb. Chem. Soc., 70 (5) 765-774. [9]. Jelic, Z. M., et al., 2011. Distribution and forms of iron in the vertisols of Serbia. J. Serb. Chem. Soc. 76, 781-794. [10]. Mackowiak, C. L. and R. P. Grossl, 2001. Beneficial effect of humic acid on micronutrient availability to wheat. Soil Sci. Soc. Ame. J. 65, 1744-1750. [11]. Makovnikova, J., 2000. Relationship between selected soil parameters and available content of cadmium, lead, copper and zinc. Rostl. Vyr., 46, 289-296. [12]. Manual for Soil chemical Analysis. Yugoslav Society of Soil Science. Belgrade, 1966. [13]. Mead, R. R., et al. 1996. Statistical methods in agriculturae and experimental biology. Chapman & Hall, London, p. 410. [14]. Sharma, B. D., et al. 2008. Forms of iron and their association with soil arid properties in four soil tahonomic orders of arid and semi soils of Punjab, India. Communications in Soil Science and Plant Analysis 39, 2550-2567. [15]. Tanasijevic, Đ., et al., 1966. Pedologic composition of the western and northwestern Serbia. Belgrade, Belgrade. [16]. Ure, M. A., 1995., in Heavy metals in soils, 2nd ed., B. J. Allovay, Blackie Academic & Professional, Glasgow, UK, p.58.

ABOUT THE AUTHORS M. Jelic, University of Pristina-Kosovska Mitrovica, Faculty of Agriculture, Kopaonicka bb, 38219 Lesak, Serbia, E-mail: [email protected] J. Milivojevic, Small Grains Research Center Kragujevac, S. Kovacevic 31, 34000 Kragujevac, Serbia, E-mail: [email protected] G. Dugalic, University of Kragujevac, Faculty of Agronomy Cacak, cara Dusana 34, 32000 Cacak, Serbia, E-mail: [email protected] O. Nikolic, University of EDUCONS, Faculty of Ecological Agriculture,Svetog Save 60, 35210 Svilajnac, Serbia, E-mail: [email protected] S. Stamenkovic, University of Pristina-Kosovska Mitrovica, Faculty of Agriculture, Kopaonicka bb, 38219 Lesak, Serbia, E-mail: [email protected]

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

THE ASSESSMENT OF ANTIOXIDANT CAPACITY OF MEDICINAL HERBS IN LOW PH CONDITIONS

D.S. Stef, I. Gergen, T.I. Trasca, A. Rivis, L. Stef, R. Heghedus-Mindru

Abstract: The aim of this paper was to evaluate the antioxidant activity of medicinal herbs in extraction solution with lower pH, to simulate the acidity of the stomach. Eighteen medicinal plants were analyzed both for total phenols content and for total antioxidant capacity. The results were compared with experimental variants which used alcoholic extracts. The highest TACFRAP value was obtained for Rosa canina (362 μM Fe2+ ·g-1), followed by Crataegus monogyna (192 μmoli Trolox/g), Hippophae rhamnoides (169 μM Fe2+ ·g-1) and Echinaceae herba (134 μM Fe2+ ·g-1). The smallest value was noticed for Pinus (31 μM Fe2+ ·g-1). The highest total phenols values were identified for Rosa Canina (730 μmoli Trolox/g), followed by Arnica montana (490 μmoli Trolox/g) and Rhamnus frangula (317 μmoli Trolox/g). The medium values were noticed for: Thy mi herba (227 μmoli Trolox/g), Echinaceae herba (250 μmoli Trolox/g) and Crataegus monogyna (266 μmoli Trolox/g). Cynara scolymus (64 μmoli Trolox/g) and Pinus (56 μmoli Trolox/g) shown the lower values. Keywords: antioxidant activity, medicinal plants, alcoholic extracts, low pH

INTRODUCTION Today it is widely accepted that synthetic antioxidants (butylated hydroxyltoluene and butylated hydroxylanisole) must be replaced with natural antioxidants. There are a lot of reasons for this replacement. The natural antioxidants are involved in the prevention or treatment of diseases and in the ageing process; they have antimicrobial, cardioprotective, antimutagenic and anticarginogenic properties [2] [9] [1] [4]. Also, many people believe that the natural antioxidants are safer than synthetic ones [3]. Polyphenols are secondary plant metabolites. They contain one or more hydroxil groups and can be used in human diet. The main classes of polyphenols are: phenolic acids, flavonoids, stilbenes [6]; and lignans [8]. The highest antioxidant activity was measured for ortho-diphenols [5]. The most assessments of the antioxidant activity are made on alcoholic extracts. In human or animals bodies there are lower values for pH. It can affects the antioxidant activity of extracts which are used. Therefore the aim of this paper was to evaluate the antioxidant activity of medicinal herbs in lower pH conditions.

MATERIAL AND METHODS 1. Reagents and equipment: All chemicals and reagents were analytical grade or purest quality purchased from Sigma, Merck, Aldrich and Fluka. Deionized water was used. Absorption determination for FRAP and DPPH methods was made using SPECORD 205 spectrophotometer by Analytik Jena. 2. Plant material and sampling: In the present study, 18 medicinal plants were analyzed both for total phenols content and for total antioxidant capacity. These medicinal herbs have been purchased on the local market. The results were compared with experimental variants which used alcoholic extracts [7]. For antioxidant compounds extraction were prepared ethanolic (50%) extracts in ratio 10/20. After 30 minutes all the extracts were filtered and diluted 1/10 with deionized water. 3. Evaluation of total antioxidant capacity (TAC) by FRAP method FRAP method depend upon the reduction of ferric tripyridyltriazine complex to the ferrous tripyridyltriazine by a reductant at low pH. This ferrous tripyridyltriazine complex has an intensive blue color and can be monitored at 593 nm (Benzie and Strain, 1996). Reagents: acetate buffer, 300mM/L, pH 3.6 (3.1g sodium acetate 3H2O and 16 mL conc. Acetic acid per 1L of buffer solution); 10 mM/L TPTZ (2,4,6-tripyridyl-s-triazine) in 40 11 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA mM/L HCl; 20 mM/L FeCl36H2O in distilled water. FRAP working solution: 25 mL acetate buffer, 2.5 mL TPTZ solution and 2.5 mL FeCl3 solution. The working solution must be always freshly prepared. Aqueous solution of known Fe (II) concentration was used for calibration, in a range of 0.1-0.8 mM/L. For the preparation of calibration curve 0.5 mL aliquot of 0.1, 0.2, 0.4, 0.6, 0.8 M/mL aqueous Fe(II) as Mohr salts solution (1mM) were mixed with 2.5 mL FRAP working solution; FRAP reagent was used as blank. The absorption was read after 10 min. at 25°C and 593 nm. 4. The amount of polyphenolic compounds: It was used the following reagents: 2.0 M Folin-Ciocalteu phenol reagent, Trolox and anhydrous carbonate. The content of total polyphenolic compounds in medicinal herbs and microelements mixture ethanol extracts diluted 1/10 was determined by Folin-Ciocalteu method (1927). For the preparation of calibration curve 0.5 mL aliquot of 0.2, 0.3, 0.4, 0.8 and 1.2 M/mL aqueous Trolox solution were mixed with 2.5 mL Folin-Ciocalteu reagent (diluted ten-fold) and 2.0 mL sodium carbonate solution (7.5%). The absorption was read after 2 h at 20°C, at 750 nm. All determinations were performed in triplicate. Total content of polyphenols in samples in Trolox equivalents was calculated. Squared correlation coefficient (r2) for calibration curve was 0.9706.

RESULTS AND DISCUSSION The results for total antioxidant capacity (TAC) by FRAP method and total phenols for the studied medicinal herbs are presented in Table 1 and more suggestive in Figures 1 and 2. Table 1 Total antioxidant capacity (TAC) by FRAP method and total phenols of eighteen medicinal herbs Total antioxidant Total phenols No. Specification capacity – FRAP [μmoli Trolox/g] [μM Fe2+ ·g-1] 1 Arnica montana 490 115 2 Cynara scolymus 64 45 3 Hippophae rhamnoides 115 169 4 Thy mi herba 227 86 5 Equisetum arvense 144 86 6 Achillea millefolium 109 125 7 Rhamnus frangula 317 131 8 Echinaceae herba 250 134 9 Phoeniculus 118 42 10 Calendula officinalis 173 58 11 Rosa canina 730 362 12 Mentha piperita 157 99 13 Matricaria chamomilla 128 58 14 Malva silvestris 112 61 15 Crataegus monogyna 266 192 16 Taraxacum officinale 115 83 17 Artemisia absinthium 83 38 18 Pinus 58 31

TACFRAP is a measure of the presence in medicinal herbs of the compounds with reducing power.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Arnica montana 400 Cynara scolymus 362 Hippophae rhamnoides 350 Thy mi herba Equisetum arvense 300 Achillea millefolium Rhamnus frangula 250 Echinaceae herba Phoeniculus 200 192 169 Calendula officinalis

150 Rosa canina 134

131 Mentha piperita 115 125 99 Matricaria chamomilla 100 8686 83 58 58 61 Malva silvestris 45 42 Crataegus monogyna 50 38 31 Taraxacum officinale 0 Artemisia absinthium 1 Pinus Figure 1 Total antioxidant capacity (TAC) by FRAP method for medicinal herbs

Among all medicinal herbs the highest TACFRAP value was obtained for Rosa canina (362 μM Fe2+ ·g-1), followed by Crataegus monogyna (192 μmoli Trolox/g), Hippophae rhamnoides (169 μM Fe2+ ·g-1) and Echinaceae herba (134 μM Fe2+ ·g-1). The smallest value was noticed for Pinus (31 μM Fe2+ ·g-1).

800 Arnica montana Cynara scolymus 700 Hippophae rhamnoides Thy mi herba 600 Equisetum arvense Achillea millefolium 500 Rhamnus frangula Echinaceae herba 400 Phoeniculus Calendula officinalis 300 Rosa canina Mentha piperita 200 Matricaria chamomilla Malva silvestris 100 Crataegus monogyna Taraxacum officinale 0 Artemisia absinthium 1 Pinus

Figure 2 Total phenols for medicinal herbs

Total phenols determined by Folin-Ciocalteu methods for analyzed samples are presented in Table1 and Figure 2. The highest total phenols values were identified for Rosa Canina (730 μmoli Trolox/g), followed by Arnica montana (490 μmoli Trolox/g) and Rhamnus frangula (317 μmoli Trolox/g). The medium values were noticed for: Thy mi herba (227 μmoli Trolox/g), Echinaceae herba (250 μmoli Trolox/g) and Crataegus monogyna (266 μmoli Trolox/g). Cynara scolymus (64 μmoli Trolox/g) and Pinus (56 μmoli Trolox/g) shown the lower values. To highlight the differences that occur between alcoholic extracts and alcoholic extracts with low pH we plotted graphically the results. The differences for total phenols content of alcoholic extracts with different pH are shown in Figure 3. Almost all alcoholic extracts with low pH had a lower content of polyphenols comparatively with the other extracts. The differences range from -20.44% at Equisetum arvense to -72.77% at Pinus. Only two herbs from studied group behave differently.

13 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Total phenols Extract alcoholic EA Total phenols EA and small pH

900 800 700 600 500 400 300 200 100

Pinus

Rosa c Rosa

Malva s Malva

Thy mi h mi Thy

Arnica m Arnica

Cynara s Cynara p Mentha

Achillea m Achillea

Rhamnus f Rhamnus

Artemisia a Artemisia

Matricaria c Matricaria

Calendula o Calendula

Hippophae r Hippophae

Phoeniculus

Equisetum a Equisetum

Crataegus m Crataegus Taraxacum o Taraxacum Echinaceae h Echinaceae Figure 3 The differences for total phenols content (alcoholic extracts with different pH)

Total phenols for Malva silvestris have increased with 12% and total phenols for Rosa canina have increased with 98,37%. In next chart are presented the differences for total antioxidant capacity (TAC) by FRAP method.

Extract alcoholic EA EA and small pH

400 362 350 300

250

202 202

199

192

186 185 184

200 180

169

163

147

134 131

150 130

125 125

115

109

101

86 86

100 83

58 58 58

38 33

50 31 26

Pinus

Rosac

Malva s Malva

Thy mi h mi Thy

Arnicam s Cynara

Mentha p Mentha

Achilleam

Rhamnus f Rhamnus

Artemisia a Artemisia

Matricaria c Matricaria

Calendulao

Phoeniculus

Hippophaer

Equisetum a Equisetum

Crataegus m Crataegus o Taraxacum Echinaceaeh

Figure 4 The differences for total antioxidant capacity (TAC) by FRAP method (alcoholic extracts with different pH)

According with presented dates it can say that most medicinal herbs have shown lower total antioxidant capacities for extracts with low pH. Calendula officinalis had the same total antioxidant capacity (58) for both types of alcoholic extracts. Cynara scolymus, Hippophae rhamnoides, Rhamnus frangula and Echinaceae herba were 4 medicinal herbs with small differences of total antioxidant capacities. In contrast, total antioxidant capacity of Rosa canina almost doubled (+96.74%). Also, total antioxidant capacity for Malva silvestris has grown very much.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

CONCLUSIONS AND FUTURE WORK The antioxidant potential of medicinal plants is significantly influenced by the pH value. Both total phenolics and total antioxidant capacity by FRAP method of analyzed medicinal herbs have shown lower values for low pH. For both situations Rosa canina behaves differently. It has shown the highest increasings for total phenols (98.37%) and for total antioxidant capacity by FRAP method (96.74%).

REFERENCES [1]. Caccetta, R. A. A., Croft, K. D., Beilin, L. J. and Puddey, I. B. 2000. Ingestion of red wine significantly increases plasma phenolic acid concentrations but does not acutely affect ex vivo lipoprotein oxidizability. American Journal of Clinical Nutrition, 71, 67-7 4. [2]. Friedman, M. and Jurgens, H. S. 2000. Effect of pH on the stability of plant phenolic compounds. Journal of Agricultural and Food Chemistry, 48, 2101-2110. [3]. Jayasinhe, C., Gotoh, N., Aoki, T., & Wada, S. 2003. Phenolic composition and antioxidant activity of sweet basil (Ocimum basilicum L.). Journal of Agricultural and Food [4]. Kampa, M., Alexaki, V. I., Notas, G., Nifli, A. P., Nistikaki, A., Hatzoglou, A., et al. 2004. Antiproliferative and apoptotic effects of selective phenolic acids on T47D human breast cancer cells: potential mechanisms of action. Breast Cancer Research, 6, R63-R74. [5]. Rice-Evans, C. A., Miller, N. J. and Paganga, G. 1996. Structure- antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20, 933-956. [6]. Scalbert, A. and Williamson, G. 2000. Dietary intake and bioavailability of polyphenols. Journal of Nutrition, 130, 2073S-2085S. [7]. Stef D.S.,Gergen I., Trasca T.I., Harmanescu M., Stef L., Druga M., Biron R., Heghedus-Mindru G. 2010. Screening of 33 Medicinal Plants for he Microelements Content. Scientific Papers: Animal Science and Biotechnologies, 43 (1), 127-132 [8]. Tapiero, H., Tew, K. D., Ba, G. N. and Mathe, G. 2002. Polyphenols: do they play a role in the prevention of human pathologies? Biomedicine & Pharmacotherapy, 56, 200- 207. [9]. Wen, A. M., Delaquis, P., Stanich, K. and Toivonen, P. 2003. Antilisterial activity of selected phenolic acids. Food Microbiology, 20, 305-311.

ABOUT THE AUTHORS D.S. Stef, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] I. Gergen, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] T.I. Trasca, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] A. Rivis, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] L. Stef, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] R. Heghedus-Mindru, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

15 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

THE EVALUATION OF FOOD QUALITY BY USING DIFFERENT METHODS

D.S. Stef, R. Heghedus-Mindru, L. Stef, G. Heghedus-Mindru, M. Cazacu, G.Bujanca, F. Berbentea

Abstract: The goal of paper was to assess the ice cream quality using both sensorial and non- sensorial properties. Also, it presents an easy sensory evaluation method accessible to consumers without great experience. The study was carried for vanilla ice cream and it used the total quality index. Total quality indicator method takes into account both the sensory properties and physico-chemical properties. It allows you to use the non sensory characteristics making them measurable characteristics. Thus, it is more easily to make a hierarchy to different food kinds based on their quality. The best sample of ice-cream with vanilla was number 2, which was with 19 percent better than control sample and with 37 percent better than standard. The lowest total indicator quality was noticed for the experimental sample 3 which was with 5 percent lower than the experimental sample 1 and with 22 percent lower than the experimental sample 2. Keywords: sensorial, non sensorial, food quality, ice cream, total quality indicator

INTRODUCTION In the last time, it became very important listening the voice of the consumer. There are many reasons for this: It is necessary in the development or improvement of products; [1] [3-5] [7]; to solve marketing problems; in academic researches. The most popular tools of marketing research are preference mapping techniques [2] [9]. Intensity scaling is frequently used in evaluation of sensory properties. Many experts in sensory evaluation use nine-point scale [6]. Although this method can be easily used by researchers and experts in the field it presents some disadvantages. On the hand this kind of evaluation can be difficult to apply to the final consumer. On the other hand it does not consider the role of non-sensory factors. Therefore the aim of this paper was to assess the food quality using both sensorial and non-sensorial properties, and to presents an easy sensory evaluation method accessible to consumers without great experience. The study was carried out for vanilla ice cream.

MATERIAL AND METHODS Order to accomplish this work it was used a control sample that was made in the laboratory and three samples from local market. 3 egg yolks, 100 g sugar, 500 ml milk, 250 ml cream and 25 g vanilla were used for making of the control sample. The evaluation and measurement of quality were done in three stages. The first stage was the evaluation of the sensory characteristics. At the same time the dry matter, the fat and the acidity of samples were measured. Finally, based on sensory evaluation and the laboratory determinations, we calculated the total quality indicator. The methodology for calculating this indicator is presented below. The simple indices are calculated for each characteristic with one of following relations:

X a X b a  or a  [1] X b X a where: Ia – the analytical indicator of quality; Xa – the estimated value of characteristic; Xb - the standard value (reference) of the characteristic.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The simple indices are calculated both for sensorial and chemical characteristics. Further analytical indicators weighted (Iap) are calculated:

I ap  I a  p [2] By summing the analytical indicators weighted (indices x weighting factor) results the total index of product quality. In our case the weighting coefficients were established as it follows: odour - 0.20; taste – 0.20; colour - 0.15; structure and consistency – 0.15; dry matter - 0.10; fats - 0.10 and acidity - 0.10 [8].

RESULTS AND DISCUSSION The results for tasting of ice cream with vanilla are shown in Table 1. Each sample was assessed by eleven consumers (non experts in the field). The following grades were given: very good, good, satisfactory and unsatisfactory.

Table 1. The results for tasting of ice cream with vanilla Specification Control Sample 1 Sample 2 Sample 3 Structure and 3 VG + 7 G + 1 S 11 VG 8 VG + 3 G 11 VG consistency Color 8 VG + 3 G 7 VG + 4 G 7 VG + 4 G 7 VG + 4 G Odor 7 VG + 5 G 8 VG + 3 G 8 VG + 3 G 8 VG + 2 G + 1S 6 VG + 4 G + 1 Taste 9 VG + 2 G 8 VG + 2 G + 1 S 5 VG + 6G S VG: Very Good; .G: Good; S: satisfactory.

Then for each grade (VG, G and S) was given a score using the scoring scale 0 to 1. The next values were used: 1.0 for VG, 0.67 for G, 0.33 for S and 0.1 for US (unsatisfactory). It was calculated the average score for each sensory characteristic (table 2). Table 2. The average scores of sensory characteristics Specification Control* Sample 1 Sample 2 Sample 3

Structure and consistency 0.69 0.94 0.91 0.94 Color 0.91 0.88 0.88 0.88 Odor 0.85 0.91 0.91 0.87 Taste 0.94 0.87 0.82 0.81 * (3 x 1.0 + 7 x 0.67 + 1 x 0.33) / 11

Considering these data, it is not clear which was the best sample. The control sample was the best for odor but the worst for structure and consistency. The samples 1 and 4 had the same result (0.94) for structure and consistency. Also, samples 2 and 3 have shown the same result (0.91) for odor. Also, the mean scores of the analyzed samples are presented in figure number 1. Neither the tabular results nor the graphical representation clarify the hierarchy of studied samples. This is the main reason to use another method that takes into account both the sensory and the chemical determinations.

17 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

1 0.94 0.94 0.94 0.91 0.91 0.91 0.91 0.88 0.88 0.88 0.9 0.85 0.87 0.87 0.82 0.81 0.8 0.69 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 Control Sample 1 Sample 2 Sample 3

Structure and consistency Colour Odor Taste

Figure 1. The average score of sensory characteristics

The results of chemical measurements are presented in table 3 and in Figure 2.

Table 3. The results for chemical determinations Samples Specification STAS Control 1 2 3 Dry matter, [%] min. 33 32.02 34.08 36.92 35.37 Acidity, [°T] max. 24 15.50 16.20 19.80 18.70 Fat, [%] 5 7.6 10.2 11.5 10.8

40 36.92 35.37 35 34.08 33 32.02 30

25 24 19.8 20 18.7 15.5 16.2 15 11.5 10.2 10.8 10 7.6 5 5

0 STAS Control 1 2 3

Dry matter, [%] Acidity, [°T] Fat, [%]

Figure 2. The results for chemical determinations

According with these data, it can say that the dry matter ranged between 32.02% and 36.92%. The lowest value (32.02%) was registered by the control sample, which did not respect the minimal requirement for this characteristic. The other experimental samples have exceeded the minimum limit with values between 1.08 and 3.92 percentage points.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The best for acidity was control sample which registered 15.5°T. This shows that in the process of getting ice cream were used fresh ingredients. It can be explained by the use of fresh milk as raw materials. The second place was occupied by sample 1 (16.2°T), followed by sample 3 (18.7°T). The fat percentage varied between 5 and 10%. The average value of this parameter was 7.20% and it had with 2.20% percentage points over the limit. The values of the sensorial characteristics and the chemical composition do not show a clear hierarchy individually of the experimental samples. The total quality index might be a good solution for this problem. The estimation methodology for total index quality is presented in tables 4 and 5.

Table 4. The calculation of the total indicator of quality Standard p Control sample Sample 1 Specification (Xb) Xa Ia Iap Xa Ia Iap Structure and 0.21 1.40 0.94 0.15 1.02 0.69 0.15 ٭0.67 consistency Colour 0.67 0.15 0.91 1.35 0.20 0.88 1.31 0.19 Odor 0.67 0.20 0.85 1.26 0.25 0.91 1.35 0.27 Taste 0.67 0.20 0.94 1.40 0.28 0.87 1.29 0.25 Dry matter, min. 33 0.10 32.02 0.97 0.09 34.08 1.03 0.10 [%] Acidity, [°T] max. 24 0.10 15.50 1.54 0.15 16.20 1.48 0.14 Fat, [%] 5 0.10 7.6 0.65 0.06 10.2 0.49 0.04 Total - 1.00 - - 1.18 - - 1.20 *the standard refers to a good sample in sensorial characteristics

Table 5. The calculation of the total indicator of quality Standard p Sample 2 Sample 3 Specification (Xb) Xa Ia Iap Xa Ia Iap Structure and 0.67 0.15 0.91 1.35 0.21 0.94 1.40 0.21 consistency Colour 0.67 0.15 0.88 1.31 0.19 0.88 1.31 0.19 Odor 0.67 0.20 0.91 1.35 0.27 0.87 1.29 0.25 Taste 0.67 0.20 0.82 1.22 0.24 0.81 1.20 0.24 Dry matter, min. 33 0.10 36.92 1.11 0.11 35.37 1.07 0.10 [%] Acidity, [°T] max. 24 0.10 19.80 1.21 0.12 18.70 1.28 0.12 Fat, [%] 5 0.10 11.5 2.3 0.23 10.8 0.46 0.04 Total - 1.00 - - 1.37 - - 1.15

According with tabular data it can say that the best sample of ice cream was number 2, which was with 19 percent better than control sample and with 37 percent better than standard. On second place stood the experimental sample 1 which was 2 percent better than the control sample and 20 percent better than standard. The lowest total indicator quality was noticed for the experimental sample 3 which was with 5 percent lower than the experimental sample 1 and with 22 percent lower than the experimental sample 2.

19 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

CONCLUSIONS AND FUTURE WORK This method (total quality indicator) takes into account both the sensory properties and physico-chemical properties. It allows you to use any non sensory characteristics making them measurable characteristics. Also, it is a lot more easily to make a hierarchy for many kinds of food products rely on their quality. The best sample of ice cream was number 2, which was with 19 percent better than control sample and with 37 percent better than standard. The lowest total indicator quality was noticed for the experimental sample 3.

REFERENCES [1]. Arditti, S. 1997: Preference mapping; A case study. Food Quality and Preference, 8(5/6), 323–327. [2]. Cooper, L. G. 1983: A review of multidimensional scaling in marketing research. Applied Psychological Measurement, 7(4), 427–450. [3]. Garber, L. L., Hyatt, E. M. and Starr, R. G. 2003: Measuring consumer response to food products. Food Quality and Preference, 14(1), 3–15. [4]. Jaeger, S. R., Rossiter, K. L., Wismer, W. V. and Harker, F. R. 2003: Consumer-driven product development in the kiwi-fruit industry. Food Quality and Preference, 14(3), 187– 198. [5]. Kaul, A. and Rao, V. R. 1995: Research for product positioning and design decisions: An integrative review. International Journal of Research in Marketing, 12, 293–320. [6]. Park, J.Y., O‘Mahony, M. and Kwang-Ok Kim. 2007: ‗Different-stimulus‘ scaling errors; effects of scale length. Food Quality and Preference 18, 362–368. [7]. Slater, S. F. and Narver, J. C. 2000: Intelligence generation and superior customer value. Journal of the Academy of Marketing Science, 28(1), 120–127. [8]. Stef D.S. 2007: Food products commodities. Part I, Second Edition, Mirton Publishing house, 218 pp. [9]. Urban, G. L. and Hauser, J. R. 1993: Design and marketing of new products (2nd ed.). New Jersey: Prentice Hall.

ABOUT THE AUTHORS D.S. Stef, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] R. Heghedus-Mindru, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] L. Stef, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] G. Heghedus-Mindru, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] M. Cazacu, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] G.Bujanca, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected] F. Berbentea, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Aradului Street 300365 Timisoara, Romania, E-mail: [email protected]

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

ANGIOTENSIN I CONVERTING ENZYME INHIBITORY ACTIVITY OF PUMPKIN OIL CAKE 12S GLOBULIN HYDROLYSATES

Ţ. Vaštag, Lj. Popoviš, S. Popoviš, D. Peričin, and V. Krimer-Maleševiš

Abstract: Angiotensin I converting enzyme (ACE) is the key enzyme in the rennin angiotensin aldosterone system regulating blood pressure and its inhibition is considered the first line of therapy for hypertension. Synthetic ACE inhibitors have already used in human therapy, but there has been a new trend towards developing safer, natural ACE inhibitors, mostly peptides from food protein enzymatic hydrolysates. After cold pressing of pumpkin oil, defatted hull-less pumpkin oil cake (PUOC) 12S globulin (cucurbitin) was utilised as raw material for preparation of protein hydrolysates, containing bioactive peptides. In this study, cucurbitin was hydrolysed with alcalase, flavorzyme and pepsin at optimum reaction conditions (temperature, pH) for each protease, during 60 min and the in vitro angiotensin I converting enzyme (ACE) inhibitory activity of hydrolysates was tested. All hydrolysates regardless of protease used, exhibited ACE inhibition. The apparent IC50 values were 0.0244 mg (0.163 mg/ml), 0.0899 mg (0.599 mg/ml) and 0.0445 mg (0.3 mg/ml) for alcalase, flavorzyme and pepsin hydrolysate, respectively. Based on these results, cucurbitin showed potential as starting material for production of innovative antihypertensive agents. Key words: Cucurbitin, alcalase, flavorzyme, pepsin, protein hydrolysates, ACE inhibitors. .

INTRODUCTION Hypertension is one major risk factor for cardiovascular diseases (atherosclerosis, stroke, and so forth). Angiotensin I converting enzyme, ACE (EC 3.4.15.1) is the key enzyme in the rennin angiotensin aldosterone system (RAAS) regulating blood pressure. ACE converts angiotensin I into angiotensin II and inactivates bradykinin [1]. Inhibition of ACE is considered the first line of therapy for hypertension and several synthetic ACE inhibitors (captopril, enalapril, etc.) have already been used in human therapy. However, as they can cause adverse side effects, new trend has been towards developing safer, natural ACE inhibitors [2]. The main recent interest has been directed toward food derived ACE inhibitory peptides and lot of animal and plant proteins have been revealed as sources of ACE inhibitory peptides, including from milk, meat, fish, soybean, sunflower, corn, etc. [3]. Several products containing peptides with ACE inhibitory properties are currently on the market or in development [4]. ACE inhibitory activity of peptides depends on their amino acid composition and sequence, which is related to the specificity of the protease and hydrolysis conditions used for their production. Using optimised procedures ACE inhibitory peptides may be released. Thus, thorough studies on these bioprocesses are fundamental regarding the development of innovative ACE inhibitors. Besides the most freuqently cultivated oil crops (soybean, rapeseed, sunflower), pumpkin (Cucurbita sp.) seed, especially the hull-less (naked) varieties, has been used for edible oil production in Austria, Hungary, Slovenia and Serbia (province of Vojvodina) for a long time. Pumpkin oil cake (PuOC), the main byproduct of the oil processing, has been mostly used as animal feed despite its components that could be attractive as ingredients in food or pharmaceutical industry [5, 6]. Defatted PUOC has about 65% of proteins, majorly composed of salt-soluble 12S globulin (cucurbitin) and albumins. In order to valorize PUOC, diverse enzymatic processes for modification of PUOC protein isolate and cucurbitin have been developed in our laboratory. The bioprocesses have been aimed at production of functional proteins, either using proteases or enzymes for cross-linking [6, 7]. The improved functionality of hydrolysed PUOC proteins can involve improved techno- functional properties (solubility, emulsifying or foaming activity), as well as bioactivity (antioxidant, ACE inhibitory activity) [7, 8, 9, 10]. Depending on the protease and reaction conditions various bio-functional hydrolysates may be produced.

21 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

In this study the ACE inhibitory activity of cucurbitin's hydrolysates are demonstrated. The hydrolysates were produced under the optimal reaction conditions (temperature, pH) for three selected food-grade proteases, namely alcalase, flavorzyme and pepsin, at the same enzyme/substrate ratio, during 60 min. In this order, the effect of the type of protease on the properties of hydrolysates can be considered.

MATERIAL AND METHODS Isolation and enzymatic hydrolysis of cucurbitin Cucurbitin was prepared by the procedure described by Peričin et al. [5]. The enzymatic hydrolysis of cucurbitin was carried out under following controlled reaction conditions; for alcalase and flavourzyme at pH 8.00, temperature of 50°C, and NaCl concentration of 40 mg/ml, and for pepsin at pH 3.00 and temperature of 37°C. The enzyme to substrate (E/S) ratio was 0.02 g /1g for each enzyme. After 60 min, the reaction mixture was heated (100°C, 5 min), centrifuged and the supernatants were used for further analysis. The degree of hydrolysis (DH) was determined by method described by Peričin et al. [6]. The protein content of each analysed sample was determined by method of Lowry et al. [11], using bovine serum albumin as the standard protein. In vitro assay for ACE inhibitory activity The ACE inhibitory test was performed according to Yoshie-Stark et al. [12]. The samples were dissolved in 0.2 mol/L potassium phosphate buffer (pH 8.30) was incubated at 37 ºC for 80 min with hippuryl-His-Leu (HHL) in 0.2 mol/L potassium phosphate buffer containing 300 mmol/L NaCl (pH 8.30) and the ACE solution. The final concentrations of the HHL and ACE were 10 mmol/l and 25 mU/ml, respectively. The reaction was stopped by adding 110 μl of 1 mol/L HCl. The hippuric acid (HA) liberated from HHL by catalytic activity of ACE was quantified with reverse-phase high performance liquid chromatography (RP-HPLC). 20 μl of the each sample was injected onto Zorbax Eclipse XDB-C18 column (4.6 Id x150 mm, 5μm, 80Å). The column was eluted with 50% methanol and 0.1% trifluoroacetic acid (in water), with flow rate of 1 ml/min, at 22°C and the absorbance was measured at 228 nm. From the chromatograms, the peak area (correspodening to HA) was integrated, and the amount of HA in the samles was quantified. The ACE inhibition activity calculated as follows (Eqn. 1):

(1) where A is the amount of HA in reaction without an inhibitor, B is the amount of HA in reaction with potent inhibitor, while A0 and B0 are the respected blanks (where HCl was added in the test tube before the enzyme solution). Statistical analysis Data were expressed as mean ± standard deviation for triplicate determinations and a least significant difference test with a confidence interval of 95% was used to compare the means.

RESULTS AND DISCUSSION The hydrolysis of a protein is usually measured in terms of degree of hydrolysis (DH). Under the reaction conditions used in this study, the final DH in alcalase (AH), flavorzyme (FH) and pepsin hydrolysate (PH) were 26.944±1.1%, 8.33±0.5%, and 18.7037±1.2%, respectively (Figure 1). Different DH values achieved at the same E/S ratio and reaction

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

time indicates that these proteases hydrolysed the same protein at different peptide bonds dictated by their specificity, resulting in different peptide composition of hydrolysates.

Pepsin 18,7037

Flavorzyme 8,33

Alcalase 26,944

0 5 10 15 20 25 30

Degree of hydrolysis, DH [%]

Figure 1. Final DH values of cucurbitin hydrolysates with alcalase, flavorzyme and pepsin.

ACE inhibitory activity of cucurbitin's hydrolysates The in vitro activity of hydrolysates reflects potential in vivo antihypertensive effect as studies have demonstrated that inhibition of ACE could result in decreased blood pressure in animal models [3]. In the ACE inhibitory assay, HHL acts as the substrate for ACE which catalyses its conversion to hippuric acid (HA) and the dipeptide, histidyl-leucine (HL). All tested hydrolysates exhibited ACE inhibiting activity, which was dependent on their concentration (Figure 2).

100

90 80

60 50 Log. (Alcalase) 40 y = 17,443ln(x) + 114,76, R² = 0,9205

30 Log. (Pepsin) y = 24,185ln(x) + 125,28, R² = 0,9718 20

10 Log. (Flavourzyme) [%] activity inhibitory ACE y = 16,6ln(x) + 89,994, R² = 0,9894 0 0 0,05 0,1 0,15 0,2 0,25 0,3 Final protein concentration [mg] Figure 2. The ACE inhibitory activity of cucurbitin hydrolysates vs. protein concentration, for ♦Alcalase hydrolysate (AH); ▲Flavorzyme hydrolysate and ■Pepsin hydrolysate (PH).

23 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The activities were examined for sample amount range from 0.0024 to 0.37 mg, i.e. from 0.016 up to 2.5 mg/ml (in the test tube, based on protein content). Figure 2 shows that logarithmic curves could be fit adequately to represent the ACE inhibitory activity in function of sample amount. The apparent IC50 value of hydrolysates represents the concentration of proteins in the test tube, required to inhibitin 50% of ACE under the described reaction conditions. The IC50 values were determined through established equations (Figure 2), being 0.0244 mg (0.163 mg/ml), 0.0899 mg (0.599 mg/ml) and 0.0445 mg (0.3 mg/ml) (final protein concentration in the test tube), for tested AH, FH and PH respectively. Based on these results AH had the highest activity, followed by PH and FH. These results are similar to inhibitory activity reported for other similar plant protein hydrolysates [10], indicating that cucurbitin hydrolysates can be valuable sources of natural ACE inhibitory peptides.

CONCLUSIONS AND FUTURE WORK In this work the in vitro ACE inhibitory activity of cucurbitin hydrolysates were investigated. The results show that cucurbitin, the major storage protein of pumpkin oil cake is a potential starting material for enzyme mediated production of protein hydrolysates containing ACE inhibitory peptides that can be used as ingredients in functional foods or nutraceuticals. However, further studies are necessary to purify and characterise the individual ACE inhibitory peptides within these hydrolysates.

REFERENCES [1]. Atlas, S. A. 2007. The Renin-Angiotensin Aldosterone System: Pathophysiological Role and Pharmacologic Inhibition. Journal of Managed Care Pharmacy 18, pp S9– S20. [2]. Acharya, K. R., et al. 2003. Ace revisited: A new target for structure-based drug design. Nature Reviews Drug Discovery 2, pp 891-902. [3]. Hong, F., et al. 2008. The anti-hypertensive effect of peptides: A novel alternative to drugs? Peptides 29, pp 1062-1071. [4]. Korhonen, H. 2009. Milk-derived bioactive peptides: From science to application. Journal of Functional Foods 1, pp 177–187. [5]. Peričin, D., et al. 2008. Evaluation of solubility of pumpkin seed globulins by response surface method. Journal of Food Engineering 84, pp. 591-594. [6]. Peričin, D., et al. 2009. Enzymatic hydrolysis of protein isolate from hull-less pumpkin oil cake: Application of response surface methodology. Food Chemistry 115, pp 753- 757. [7]. Popoviš, Lj., et al. 2013. Optimization of transglutaminase cross-linking of pumpkin oil cake globulin; Improvement of the solubility and gelation properties. Food and Bioprocess Technology 6, pp 1105-1111. [8]. Popoviš, Lj., et al. 2011. Optimization of enzymatic hydrolysis of cucurbitin using Response Surface Methodology: Improvement of the functional properties. International Journal of Food Engineering 7, Article 18. [9]. Vaštag, Ţ., et al. 2010. Hydrolysis of pumpkin oil cake protein isolate and free radical scavenging activity of hydrolysates; Influence of temperature, enzyme/substrate ratio and time. Food Bioproducts Processing 88, pp. 277-282. [10]. Vaštag, Ţ., et al. 2011. Production of enzymatic hydrolysates with antioxidant and angiotensin-I converting enzyme inhibitory activity from pumpkin oil cake protein isolate. Food Chemistry 124, 1316-1321.

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[11]. Lowry, O., et al. 1951. Protein measurement with the Folin–phenol reagent. Journal of Biological Chemistry 193, pp. 265-275. [12]. Yoshie-Stark, Y., et al. 2004. Functional properties, lipoxygenase activity, and health aspects of Lupinus albus protein isolates. Journal of Agriculture and Food Chemistry 52, pp 7681-7689.

ABOUT THE AUTHORS Ţuţana Vaštag, Ph.D., Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia, E-mail: [email protected], Ljiljana Popoviš, Ph. D., Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia, E-mail: [email protected], Senka Popoviš, Ph. D., Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia, E-mail: [email protected] , Prof dr Draginja Peričin, Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia, E-mail: [email protected] Vera Krimer-Maleševiš, Public Health Institute, Zmaj Jovina 30, 24000 Subotica, Serbia, E-mail: [email protected]

Acknowledgements This work was supported by grant number III 46010 from Ministry of Education and Science of Republic of Serbia.

25 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

THE PLANTLETS ROOTING OF CHILLI PEPPER OBTAINED IN VITRO CULTURE CONDITIONS

A. Lazăr, C. Petolescu, G. Velicevici, I. Creţescu, S. Popescu, O.M. Boldura, G.Poşta and V. Berar

Abstract: The study proposed to establish the existent interrelations and phytohormones influence and its concentration in the culture medium on plantlets rooting of chilli pepper. The plantlets of chilli pepper, local population Aldesti were obtained in in vitro conditions through the culture of axillar shoots. It were added in the base culture medium (Murashige-Skoog) three classes of phytohormones (auxins, citokinins and gibbereline) in five concentrations (0.1; 1.0; 1.5; 2.0; 2.5 mg·L-1). The plantlets rooting was strongly stimulated by the auxins, thus, indifferently the acid indole-3 acetic and acid 1- naphthaleneacetic concentrations were, in all the treated variants with auxins the plantlets proportion which the roots had been emitted was superior, comparative with the one registered in the variants in which the citokinins and gibberelic acid were applied.

Key words: chilli pepper, phytohormones, axillar shoots culture, Capsicum annuum L..

INTRODUCTION The genus Capsicum has a wide genetic diversity, consist of about 27 species, being five domesticated and 22 semi-domesticated and wild ones [1]. Of the domesticated species, Capsicum annuum L. is the most economically important and includes both mild and pungent fruit types. Chillies contain numerous chemicals including steam-volatile oil, fatty oils, capsaicinoids, carotenoids, vitamins, protein, fibre and mineral elements [2] and are variously used for different purposes because of their nutritional value, flavour, aroma, texture, pungency and color in a wide assortment of foods, drugs, and cosmetics, while some are cultivated ornamentally, especially for their brightly glossy fruits with a wide range color, shape and size [3].

MATERIAL AND METHODS The biologic material used in experiment, was represented by local population of chilli pepper of Aldesti. The plantlets chilli pepper, were obtained in vitro conditions through axillar bud culture. For rooting induction of chilli pepper plantlets, in environment base of Murashige- Skoog [4] were added five phytohormones from three classes of phytohormones, such as: auxins: - 1-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA); cytokinins:- 2- izopentenil-adenin (2IP), kinetin (KIN) and gibberellin: - gibberellic acid (GA3). An experimental factor analyzed in our experiences was the phytohormones concentration, using for every phytohormones the following concentrations: 0,5; 1,0; 1,5; 2,0; 2,5 mg ·L-1.

RESULTS AND DISCUSSION The phytohormones interaction and its concentrations to in vitro multiplication of Capsicum annuum L. determined considerable differences regarding the plantlets rooting from the 25th variant, the results obtained being presented in table 1. The effect combined between phytohormones and concentrations manifested through obtaining a huge variability for the proportion of rooted plantlets, with limits of variability between 13,0% (KIN- 2,5 mg·L-1) and 94,7% (NAA- 2,5 mg·L-1).

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The experience average for the percentage of rooted plantlets, in the 25th variants, was of 54,7%. Comparative with that average, considered testifier, it was obtained differences statistically assured to 20 variants. Through those, ten variants presented superior deviations, and those ten inferior towards the average, with different levels of statistically assurance.

Table 1 The effect combined of phytohormones and its concentrations on production of chilli pepper plantlets rooted (%).

Rooted Relative value Difference Experimental Concentration Difference No. -1 plantlets face to the from the variants (mg·L ) signification (%) average (%) average (±) 1. 0,5 80,7 147,5 26,0 ** 2. 1,0 83,7 152,9 29,0 *** 3. IAA 1,5 84,7 154,8 30,0 *** 4. 2,0 89,3 163,3 34,6 *** 5. 2,5 93,7 171,2 39,0 *** 6. 0,5 91,0 166,3 36,3 *** 7. 1,0 90,3 165,1 35,6 *** 8. NAA 1,5 92,7 169,4 38,8 *** 9. 2,0 92,7 169,4 38,8 *** 10. 2,5 94,7 173,0 40,0 *** 11. 0,5 46,0 84,1 -8,7 - 12. 1,0 42,7 78,0 -12,0 - סס KIN 1,5 29,3 53,6 -25,4 .13 ססס -38,4 29,9 16,3 2,0 .14 ססס -41,7 23,8 13,0 2,5 .15 16. 0,5 43,0 78,6 -11,7 - 17. 1,0 42,7 78,0 -12,0 - 18. 2IP 1,5 47,3 86,5 -7,4 - סס -26,4 51,8 28,3 2,0 .19 ססס -34,7 36,6 20,0 2,5 .20 ס -22,0 59,7 32,7 0,5 .21 סס -25,4 53,6 29,3 1,0 .22 סס GA3 1,5 25,3 46,3 -29,4 .23 סס -23,7 56,7 31,0 2,0 .24 סס -27,4 50,0 27,3 2,5 .25 Average (Blank) 54,7 100,0 - - LSD 5% = 17,7 LSD 1% = 23,6 LSD 0,1% = 30,8

Proportion the biggest of rooted plantlets registered in the variants where the auxins participated, to which, indifferently the concentrations being, the differences from the experience average were nearly very superior significant. Cytokinins and gibberellic acid had an effect lower on plantlets rooting of chilli pepper, comparative with indole-3-acetic acid and 1-naphthaleneacetic acid. The lowest rooting of plantlets registered through kinetin application in concentrations of 2,0 and 2,5 mg·L-1 (16,3 and 13,0%) and cytokinin 2-izopentenil-adenin in concentration of 2,5 mg·L-1. The three variants presented a rooting percentage inferior very significant to experience average. The differences significantly distinct inferior were obtained also in variants KIN- 1,5 mg·L-1 and 2IP- 2,0 mg·L-1. The reduced values registered through gibberellic acid usage, indifferently the experimental concentration will be. In all the five variants in which the acid gibberellic effect had studied, the percentage of plantlets rooted was inferior to the one obtained on the entire experience, with differences statistically assured. 27 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The results obtained for the plantlets percentage rooted to the species Capsicum annuum L. illustrated the effect that the combined action of hormones and concentrations had on the plantlets rooted obtained from sample explants of the same species. In case it followed the plantlets rooted obtaining, it could recommended, on base of obtained data, that in vitro multiplication to Capsicum annuum L. species took place through phytohormones and concentrations usage which in experience determined the best percentage of plantlets rooted. The results concerning phytohormones on plantlets rooting to the species Capsicum annuum L., indifferently the concentration level was, it were presented in table 2.

Table 2 The effect of phytohormone type used in the culture medium on the percentage of rooted plantlets (%), to the chilli pepper

Rooted Relative value face Difference from Difference No. Phytohormone type plantlets (%) to the average (%) the average (±) signification 1. IAA 86,4 157,9 31,7 *** 2. NAA 92,3 168,7 37,6 *** ססס KIN 29,5 53,9 -25,2 3 ססס 2IP 36,3 66,3 -18,4 .4 ססס GA3 29,1 53,3 -25,6 .5 Average (Blank) 54,7 100,0 - - LSD 5 %=7,9 LSD 1 %=10,6 LSD 0,1%=13,8

From presented data it could observe that the chilli pepper plantlets rooting were significantly influenced by the phytohormone type used. The differences resulted for plantlets rooted proportion in case of all phytohormones used in experience had presented deviations statistically assured, comparative with the experience average. The best rooting of plantlets in vitro the auxins assured, as it followed: 1- naphthaleneacetic acid (92,3%) and indole-3-acetic acid (86,4%), with differences significant superior to the experience average (figure 1).

Figure 1. The influence of phytohormone type used on the rooted plantlets number

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The bad results for the analyzed characteristics obtained through cytokinins and gibberellic acid, to which the rooted plantlets proportion was between 29,1% and 36,3%, all with very significant differences inferior on experience average. From the analysis of data obtained, resulted that the increase regulators had a different influence on plantlets rooting of chilli pepper. Surely, the best rooting it obtained through auxins application in the culture medium. The influence of growth regulators concentration on the plantlets rooted proportion to the species Capsicum annuum L. was presented in table 3.

Table 3 The effect of phytohormones concentration used on rooted plantlets proportion

Phytohormones Rooted Relative value face Difference from Difference No. -1 concentration (mg·L ) plantlets (%) to the average (%) the average (±) signification 1. 0,5 58,7 107,2 4,0 - 2. 1,0 57,7 105,5 3,0 - 3. 1,5 55,9 102,1 1,2 - 4. 2,0 51,5 94,2 -3,2 - 5. 2,5 49,7 90,9 -5,0 - Average (Blank) 54,7 100,0 - - LSD 5 %=7,9 LSD 1 %=10,6 LSD 0,1% = 13,8

The percentage of rooted plantlets in function of the concentration level oscillated between 49,7% (2,5 mg·L-1) and 58,7% (0,5 mg·L-1) (figure 2).

Figure 2. The influence of phytohormones concentration used on plantlets rooting of chilli pepper

It observed that to Capsicum annuum L., the unilateral effect of phytohormones concentrations didn‘t determine the obtaining of some rooted plantlets percentages which differed face to the experience average. It resulted that the concentrations influence on plantlets rooting wasn‘t significantly, so the differences through the plantlets percentage that emitted roots in vitro conditions, to different levels of concentrations, hadn‘t real causes, so it wasn‘t owed to doses in which were the regulators applied. If as opposed to concentrations, the regulators type influenced in significantly way the plantlets rooting, it would mentioned that, thus, to the chilli pepper it observed a 29 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA decreasing of rooting percentage, in measure of its concentration increase. Apparently the percentage of plantlets rooting was inversely proportional with the concentration level of phytohormones administrated in the culture medium

CONCLUSIONS AND FUTURE WORK The percentage of rooted plantlets in the experimental variants resulted through the hormones combination with the concentrations were extremely diversified. The plantlets rooting was strongly stimulated by auxins, thus, indifferently the concentrations of indole-3-acetic acid and 1-naphthaleneacetic acid were, in all the variants treated with auxins the plantlets proportion which emitted roots was superior, comparative with the on registered in variants where cytokinins and gibberellic acid were applied. Through statistically calculations it determined that auxins formed a common group, distinct through its effect on plantlets rooting face to cytokinins and gibberellic acid. The last regulators (KIN, 2IP and GA3) formed a common group, its stimulated influence on rooting capacity of plantlets being relatively uniform, without significant differentiations. The concentration didn‘t influenced the rooting, on experience ensemble, probably thanks to doses effects in which the hormones were it had cancelled, in great part, by phytohormones type.

Acknowledgement This work was published during the project ―POSTDOCTORAL SCHOOL OF AGRICULTURE AND VETERINARY MEDICINE", POSDRU/89/1.5/S/62371, co-financed by the European Social Fund through the Sectorial Operational Programme for the Human Resources Development 2007-2013.

REFERENCES [1]. Reifschneider, J.B., 2000. Capsicum - Pigmentas e pimentos no Brasil. EMBRAPA, Brasilia, pp.113. [2]. Bosland, P.W. and Votava, E.J., 2000. Peppers: Vegetables and Spice Capsicums. Crop Production Science in Horticulture 12. CAB International Publishing, Wallingford, England, UK., pp. 204. [3]. Sanatombi K. and Sharma, G.J., 2007. Micropropagation of Capsicum annuum L., Not. Bot. Hort. Agrobot. 35(1), pp. 57, Cluj. [4]. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures, Physiol. Plant., 15, pp. 473-497.

ABOUT THE AUTHORS A. Lazăr, C. Petolescu, G. Velicevici, I. Creţescu, S. Popescu, O.M. Boldura, G.Poşta, V. Berar, Banat‘s University of Agricultural Sciences and Veterinary Medicine, 119 Calea Aradului, Timişoara, Romania, E-mail: [email protected]

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

THE SUCROSE INFLUENCE ON CHILLI PEPPERS PLANTLETS GROWING IN THE IN VITRO CULTURE CONDITION

A. Lazăr, C. Petolescu, G. Velicevici, I. Creţescu, S. Popescu, O.M. Boldura, G.Poşta and V. Berar

Abstract: The paper had the purpose to emphasize the sucrose role in chilli peppers plantlets evolution in its in vitro culture conditions. It was followed the influence of carbon source on the plantlets altitude and on internodes number. In the Murashige-Skoog culture medium were administrated four sucrose concentrations, such as: 5, 10, 20 and 30 g·L-1. Regarding the plantlets altitude and internodes number, the highest values registered in case of some concentrations usage of 30 g·L-1 sucrose, the others experimental variants presenting an evolution much under its average value.

Key words: chilli pepper, phytohormones, axillar shoots culture, Capsicum annuum L.

INTRODUCTION In vitro culture of plants multiplied through micropropagation, the culture medium represented the success key to all species with that kind of multiplication. It was well known the role the carbohydrates took place in the culture medium. The meristem evolution and all the tissue cultures were dependent by the existence of carbohydrates source. Thanks to the culture medium used in vitro cultures photosynthesis developed at a very low level, for the compensation of that effect it needed a heterotroph source of carbon. In consecrated culture medium, the best carbohydrates concentration had that purpose the conditions assurance for increasing and developing every type of culture one by one, but also indication of organogenic tissue evolution. The abroad literature emphasized the quantitative and qualitative importance of energetic support [1]. In the same context, it supposed the existence of some indirect influences on regulator substances metabolism of endogen increasing with major implications in organogenesis [2]. Thus it was clearly that it couldn‘t conceive a culture medium without carbohydrates source, it was important to be known the best limit so that its effect wasn‘t inhibitor of photosynthesis functions. The carbohydrates shape the usually met in plant was the sucrose which had been spread from the leaves tissue in phloem and ulterior towards the meristematic tissue [1].

MATERIAL AND METHODS The biologic material used was represented by local population of chilli pepper of Aldesti. The chilli pepper plantlets were obtained in vitro conditions through axillar buds culture [3]. In culture medium Murashige- Skoog [4] had been administrated four concentrations of sucrose, such as: 5, 10, 20 and 30 g·L-1. The experimental variant with a concentration of 30 g·L-1 sucrose was considered testifier variant. The experiment followed the influence of carbon source on plantlets altitude and on internodes number/plantlet.

RESULTS AND DISCUSSION Regarding the plant altitude, the highest values were registered in case of some -1 -1 concentrations usage of 30 g·L (VM) and the lowest in case of 5 g·L (V5). Comparative

31 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA with the testifier (VM) all the others variants presented an evolution more inferior to its average value (table 1). The variability coefficient presented the highest values in case of variant V10, respectively 75,46% (figure 1).

Table 1 The average values, percentage and variability coefficient for height plantlets of chilli pepper

No. Experimental variant x  sx % Variability coefficient

1. V5 19.97 ± 2.38 43.66 51.98

2. V10 26.76 ± 5.04 58.51 75.46

3. V20 25.01 ± 3.52 54.69 64.62

4. VM 45.73 ± 6.75 100 62.63

Figure 1. Schedule representation of average values of height plantlets of chilli pepper

It observed very significant differences between variant V5 and testifier, distinct significantly between V20 and testifier and significant differences for V10 variant as it followed in table 2.

Table 2 The differences significance between the average values of chilli pepper plantlets height in function of sucrose concentration applied

Experimental No. Difference GL t Signification variant

1. V20 - VM -20,73 37 -2,83 **

2. V10 - VM -18,97 32 -2,20 *

3. V5 - VM -25,76 35 -3,67 ***

4. V20 - V10 -1,75 35 -0,29 -

5. V20 - V5 5,04 38 1,15 -

6. V10 - V5 6,79 33 1,28 -

Regarding the internodes number, it observed the same evolution of parameters studied (table 3). Thus, the biggest number of internodes registered to variant VM (4,25).

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Table 3 The average values, percentage and variability coefficient for the chilli pepper internodes number

No. Experimental variant x  sx % Variability coefficient

1. V5 1.26 ± 0.12 29.65 44.49

2. V10 1.68 ± 0.19 39.53 47.00

3. V20 1.83 ± 0.23 43.06 59.31

4. VM 4.25 ± 0.59 100 59.61

Figure 2. Schedule representation of average values of chilli pepper internodes number

The biggest number of internodes was a productive character to chilli pepper and that high value obtained to VM variant could be taken in view (figure 2). In case of that variant it obtained the highest variability coefficient of 59,61%, the most reduced coefficient registering in case of V5 variant (44,49%)

Table 4 The differences significance among the average value of internodes number in function of sucrose concentration applied

Experimental No. Difference GL t Signification variant

1. V20 - VM -2,41 37 -3,87 ***

2. V10 - VM -2,56 32 -3,87 ***

3. V5 - VM -2,99 35 -5,01 ***

4. V20 - V10 0,15 35 0,46 -

5. V20 - V5 0,57 38 2,05 *

6. V10 - V5 0,42 33 1,84 -

Difference between average values of internodes number were very significantly for all the five variants V5, V10, V20, comparative with the variant VM (table 4). Also, it could observe significant differences between the experimental variants V20 and V5.

33 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

CONCLUSIONS AND FUTURE WORK Regarding the plantlets altitude and internodes number, the highest values registered in case of some concentrations usage of 30 g·L-1 sucrose, the others experimental variants presenting an evolution much under its average value.

REFERENCES [1]. Cachiţa, C.D.,1984. Tehnica cultivării in vitro a celulelor şi ţesuturilor vegetale, Culturi de celule şi ţesuturi vegetale – aplicaţii în agricultură, Ed. Ceres, Bucureşti, , pp.7-94. [2]. Margara, J., 1982. Bases de la multiplication vegetative. Les meristemes et l‘organogenese. Bot.Kozl., Budapest, 60, pp. 265-270. [3]. Sanatombi K. and Sharma, G.J., 2007. Micropropagation of Capsicum annuum L., Not. Bot. Hort. Agrobot. 35(1), pp. 57, Cluj. [4]. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures, Physiol. Plant., 15, pp. 473-497.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

THE DETERMINATION OF THE OPTIMAL CHLORINE DOSE FOR THE DISINFECTION OF A MICROBIAL CONTAMINATED WASTEWATER

V.V. Safta, Viviana-Carmen Ciucă

Abstract: This paper reports an experimental study for the determination of the optimal chlorine dose required for the disinfection of the wastewater resulted from the manufacturing process of biological products (vaccines, sera, diagnostic reagents) in the National Society “INSTITUTUL PASTEUR” S.A. of Bucharest, which is a microbial contaminated wastewater. For the disinfection of the wastewater, the local treatment plant, who discharges the effluent into the municipal sewerage system, is equipped with a chlorination installation (JESCO type – Germany) with chlorine gas. In order to evaluate the optimal chlorine dosage we have chlorinated the water stream with different chlorine doses ranging from 50 to 200g/h. Different chlorine doses were introduced in the water flow submitted to the chlorination treatment (90 m3/h), progressive for a period of 8 hours. For each chlorine dose we have added, we have determined the microbial count of the influent and of the effluent (aerobic mesophilic bacteria at 37°C (NTG 37°C), total coliforms, faecal coliforms, faecal streptococci, sulphite reducing clostridia and clostridium perfringens, pseudomonas aeruginosa and salmonella) and the residual chlorine content in the effluent (determined by two methods: a titrimetric method, using methyl orange, and a spectrophotometric method, using o-tolidine). The experimental study indicates the optimal chlorine dose for the disinfection which provides an effluent whose parameters regarding the microbial count and the residual chlorine content required by the official Romanian regulation concerning the quality of the effluent discharged into municipal sewerage (ORD.MS.208/07.03.2012 and HGR 352/2005).

Key words: Microbial Contaminated Wastewater, Disinfection, Chlorine Optimal Dose.

INTRODUCTION The National Society ―INSTITUTUL PASTEUR‖ S.A. of Bucharest is an institution wherein are held research and development activities and manufacturing of a wide range of veterinary pharmaceutical products, preponderantly biologically active products (vaccines, sera, diagnostic reagents). From these activities it results microbial contaminated wastewater, which must de evacuated in the central sewerage system of Bucharest city. In order to respect the Romanian legislation concerning the quality of the effluent discharged into municipal sewarages (ORD.MS.208/07.03.2012 and HGR 352/2005), the National Society ―INSTITUTUL PASTEUR‖ S.A. of Bucharest purchased a local wastewater treatment plant in which there are treated the wastewater resulted from the work unit (it must be mentioned that in the local wastewater treatment plant reach both the wastewater resulted from the technological processes and the rainwater colected by the local sewerage system). The local wastewater treatment plant of the National Society ―INSTITUTUL PASTEUR‖ S.A. of Bucharest has as preponderant activity the disinfection of the wastewater so that the quality of the evacuated effluent fit in the legal requirements. The structure of the local wastewater treatment plant is composed of a mechanical unit (consisting of a screening plant and smoothing-retention basins) wherein the wastewater is treated by physico-mechanical methods (screening, settling) and of a chemical unit (consisting of a chlorine dosing and injection installation and reaction tanks) wherein the wastewater is disinfected by chlorination with chlorine gas, after which the resulted effluent is discharged to municipal sewerage. It is mentioned that the circulation of the wastewater submitted to the treatment is achieved by a pumping station equipped with centrifugal pumps with the effective flow of 90 m3/h, that takes the wastewater from the smoothing-

35 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA retention basins and transmit it through a pipeline (in which is performed the chlorine injection) to the reaction tanks. The chlorine dosing and injection installation is Lutz-Jesco type (Germany) [3] and its structure is presented in figure 1.

Fig. 1 Chlorination installation with chlorine gas type Lutz-Jesco (Germany) [3] (1-chlorine cylinder, 2-full-vacuum chlorinator C2211, 3-vacuum manifold, 4-safety valve (chlorine), 5-activated carbon cartridge, 6-chlorine changeover equipment C7522, 7-back/safety shutoff valve, 8-measuring glasses/distribution block, 9-ejector non-return valve (chlorine), 10-ejector, 11-vacuum breaker, 12-control valve C7700, 13-booster pump DE-1-4, 14-ditr trap, 15-safety valve (water), 16-pressure reducing valve with pressure gauge, 17-solenoid valve, 18-ball non-return valve, 19-chlorine solution injector, 20-chlorine gas warning device GW404, 21-sensor for gas warning device)

It is mentioned that the disinfection (chlorination treatment) of the wastewater is continuously and permanently performed for 8 hours per day (during the shift work).

MATERIAL AND METHODS For the determination of the optimal chlorine dose for the disinfection of the microbial contaminated wastewater an experimental study was conducted as follows:

- it was performed a permanent chlorination of the wastewater in period of 8 hours (during a shift work) with progressive chlorine doses between 50 – 200 g Cl2/h (during a chlorination period the chlorine dose being maintained constant); - the flow of wastewater submitted to the chlorination treatment (90 m3/h) was maintained constant during all the duration of the experimental study; - during the study, to every 2 hours, samples were taken from both the influent and

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

effluent (namely at the point of discharge of the effluent in the municipal sewerage) of the disinfection installation for the determination of the microbial count and of the residual free chlorine; - laboratory analyses were performed for the determination of the microbial count and of the residual free chlorine of the taken samples, using the specification presented in table 1 (it is mentioned that there were used both classical methods [1, 2] stipulated by the Romanian standards and methods currently used in Europe and worldwide);

Table 1

The analysed parameter Maximum allowable Analysis Specification [Unit] values

Total number of mesophilic bacteria that Multiple dilution method grow at 37°C SR EN ISO 6222/2004 <300 NTG 37°C [UFC/ml] SR EN ISO 8199/2008 Multiple dilution method Probable number of total coliforms ISO 9308-2/1990 5000 bacteria [UFC/100ml] SR EN ISO 8199/2008 Multiple dilution method Probable number of faecal coliforms ISO 9308-2/1990 2000 bacteria [UFC/100ml] SR EN ISO 8199/2008 Multiple dilution method Probable number of faecal Streptococci ISO 7899-2/2002 1000 bacteria [UFC/100ml] SR EN ISO 8199/2008 Multiple dilution method Probable number of Escherichia coli ISO 9308-2/1990 0 bacteria [UFC/100ml] SR EN ISO 8199/2008 STAS 3001-91 Bacteria of the genus Clostridium Method L458/2002 0 Perfringens [UFC/100ml] (consolidated form in 2011) Bacteria of the genus Pseudomonas STAS 3001-91 0 Aeruginosa [UFC/100ml] pr EN ISO 6222 Pre-enrichment, enrichment, dispersion and isolation on selective media and Bacteria of the genus Salmonella biochemical and serological 0 [UFC/100ml] confirmation ISO 19250/2010 Free residual choline [mg/l] SR EN ISO 7393/2/2002 0,50

- In addition to the routine analyses required by the Romanian and European standards, namely: the total number of mesophilic bacteria that grows at 37° C (NTG 37° C), the probable number of total coliforms, the probable number of faecal coliforms, the probable number of faecal Streptococci, in the experimental study, were added the following additional complementary analysis: the probable number of Escherichia coli bacteria, Clostridium perfringens bacteria, Pseudomonas aeruginosa bacteria and Salmonella bacteria; - for the determination of the residual chlorine content in the samples there were used two methods: a titrimetric method, using methyl orange, and a spectrophotometric method, using o-tolidine, and similar results were obtained; we have to mention that the spectrophotometric method using o-tolidine has been validated and is being used in the Quality Control Laboratory of National Society ―INSTITUTUL PASTEUR‖ S.A. (figure 2 shows the calibration curve: the optical density D.O. is plotted against the free residual chlorine content [mg/l];

37 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Fig. 2 The calibration curve for the spectrophotometric method using o-tolidine: the optical density D.O. is plotted against the free residual chlorine content [mg/l]

RESULTS AND DISCUSSION In table 2 are presented the values of microbiological parameters and free residual chlorine ranges in the influent samples, resulted from the experimental study.

Table 2

The analysed parameter Value ranges [Unit]

NTG 37°C [UFC/ml] 3950 - 3200

Total coliforms [UFC/100ml] 8210 - 7980

Faecal coliforms [UFC/100ml] 3800 - 3500

Faecal Streptococi [UFC/100ml] 2700 - 2500

Escherichia coli [UFC/100ml] 0

Clostridium Perfringens [UFC/100ml] 0

Pseudomonas Aeruginosa [UFC/100ml] 0

Salmonella [UFC/100ml] 0

Free residual choline [mg/l] 0,01 - 0,03

In table 3 there are presented the values of microbiological parameters and free residual chlorine ranges in the effluent samples, at the point of discharge of the effluent in the urban sewerage, resulted from the experimental study. If we analyze the data provided in the table 3 it can be observed that to a chlorine dose of 50 gCl2/h some of the microbiological parameters exceed the maximum allowable limit, while in the case of chlorine doses of 150 and 200 gCl2/h, the free residual chlorine exceeds the permissible limit. The optimal dose of chlorine is 100 gCl2/h, where both the microbiological parameters and the free residual chlorine reside freely within the permitted limits.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Table 3

The chlorine doze [g/h] The analyzed 50 100 150 200 parameter Chlorination period Chlorination period Chlorination period Chlorination period [Unit] [h] [h] [h] [h] 2 4 6 8 2 4 6 8 2 4 6 8 2 4 6 8

NTG 37°C 2250 2200 2000 1100 6,5 7,1 7,5 8,5 2,5 2,8 3,1 3,4 <0,5 <0,5 <0,5 0,5 [UFC/ml]

Total coliforms 2210 2180 1980 1040 0 0 0 0 0 0 0 0 0 0 0 0 [UFC/100ml] Faecal coliforms 330 310 283 148 0 0 0 0 0 0 0 0 0 0 0 0 [UFC/100ml] Faecal Streptococi 1700 1600 1500 1180 0 0 0 0 0 0 0 0 0 0 0 0 [UFC/100ml] Escherichia coli 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 [UFC/100ml] Clostridium Perfringens 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 [UFC/100ml] Pseudomonas Aeruginosa 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 [UFC/100ml]

Salmonella 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 [UFC/100ml]

Free residual 0,2 0,4 choline 0,04 0,05 0,06 0,08 0,20 0,25 0,28 0,40 0,41 0,52 0,63 0,62 0,64 0,68 3 3 [mg/l]

CONCLUSIONS AND FUTURE WORK The paper presents an experimental study to determine the optimal dose of chlorine for the disinfection of a microbiologically contaminated wastewater disposed in municipal sewerage. For this purpose, throughout the study, there were periodically determined the microbiological parameters and the residual chlorine both of the raw influent and of the effluent discharged to the sewerage, the optimal dose of chlorine corresponding to the situation in which all the analyzed parameters are within acceptable limits. In the future it is expected to carry out experimental studies to determine the interdependence of the free residual chlorine of the treated water and its pH or ammonia compounds content.

REFERENCES [1]. Mănescu S., Cucu M. and Diaconescu Mona-Ligia. 1994. Chimia sanitară a mediului. Editura Medicală, Editura Medicală, Bucureşti, 1994, ISBN 973-0249-7, pp. 155-168. [2]. Munteanu, C., Dumitraşcu Mioara and Iliuţă R.A. 2011. Ecologie şi protecţia calităţii mediului. Editura Balneară, Bucureşti, 2011, ISBN 978-606-92826-9-4, pp. 50-60 [3]. *** – Chlorination Installation. Operating Manual – Lutz-Jesco GmbH, Germany

ABOUT THE AUTHORS V.V. Safta, ―POLITEHNICA‖ University of Bucharest, 313 Splaiul Independenţei, Sector 6, 060042, Bucharest, Romania, E-mail: [email protected] Viviana-Carmen Ciucă , N.S. ―INSTITUTUL PASTEUR‖ S.A., 333 Calea Giuleşti, Sector 6, 060269, Bucharest, Romania, E-mail: [email protected]

39 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

STUDY ON MEAT QUALITY DEPENDING ON OXIDATIVE STRESS OF THE ANIMALS BEFORE SLAUGHTER

Iuliana Manea, Laur Constantin Manea

Abstract: The work was designed to assess the state of stress before slaughter cows, followed by determination of quality parameters of meat obtained. Preslaughter stress negatively affect meat obtained. The determination of the pH was performed at 2 hours after slaughter. Meat has less glycogen and maturation and conservation are reduced. This situation is a disadvantage of technological point of view for meat obtained, because meat has less glycogen and maturation and conservation are low. Monitoring stress level by blood lactatdehidrogenase and proteins measurement, especially albumins may help to improve meat quality, can take measures to avoid or reduce to a minimum stress before slaughter. Key words: Preslaughter stress, lactatdehydrogenase, total protein content.

INTRODUCTION Physiological state of the animals at slaughter influences the quality and wholesomeness of meat slaughter animals in the slaughterhouse. [6, 13] Immediately after cutting the animal have severe disruptions of the thermoregulation system, resulting in an increase in the temperature of the carcasses. [1,9] After blood flow it causes a stop to the intake of nutrients and oxygen to the tissue, switching to anaerobic metabolism, followed by the production of lactic acid. The lactic acid product is stored in muscles which results in a significant decrease in pH, phenomenon known as rigor mortis. Acidity is necessary in maintaining postmortem wholesomeness carcasses, because it prevented further development of bacteria that can contaminate carcasses accidentally. Decrease in muscle acidity is manifested by the phenomenon of contracture of all the muscles.[3, 5,12] Various factors may affect muscle rigidity. Thus, high temperature environment hastens the installation and disappearance of rigidity state, low temperature prolongs state of rigidity. Febrile animals, malnourished, those with septicemic condition before cutting, those with muscular dystrophic processes in those states agony are poorly expressed and transient rigor mortis. With the degradation of glycogen, lactic acid is formed, phosphocreatine content decreases and form hydrophobic complex actomyozin, which is a disadvantage in terms of technology for meat produced because water retention capacity is low. [2, 7, 14,15] Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate with coenzyme NADH to NAD+. In muscle tissue, LDH is crucial to maintaining levels of NADH and NAD+ especially under anaerobic conditions. [8,10] When LDH decreases, preslaughter stress is high. Therefore, the aim of this study was to establish a link between beef quality and various physiological variables and to select the variables that show potential to serve as monitoring tools for controlling preslaughter stress levels.

MATERIAL AND METHODS In this study it was examined blood from cows which were then slaughtered and meat production by their slaughtering. The blood taken from the veins of the cows aged between 2 and 8 years. Immediately after prelevation, blood collection tubes were stored in a cooler at + 4 ºC. In the maximum 20 min the blood samples were centrifuged (5 min at 4 000 rpm). Serum obtained was put into cryotubes of 2 ml and stored at + 4 ºC pending analysis.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The obtained meat was preservation at + 4 ºC. To assess pre-slaughter stress were dosed calcium, lactatdehydrogenase and total proteins. These parameters were correlated with indicators of meat quality: pH and total protein content. Calcium, lactatdehydrogenase and serum proteins were dosed of blood serum with an automatic analyzer type ARKRAY.The pH values were measured with a HACH pH-meter. The determination of the pH was performed at 2 hours after slaughter.

RESULTS AND DISCUSSION Serum LDH activity, the calcium and the proteins levels were presented in the figure 1.

Fig. 1 LDH activity, the calcium and the proteins levels in the blood serum

In samples 1-3, the amount of LDH was less than 2 IU / ml, the serum protein content was higher from 9.2 to 9.3 g / dl, with an exception to the sample 1, which was 6.6 g /dl, probably due to a deficiency or failure to absorb food. Also, in the same samples 1-3, calcium content was higher than 11mg/dl. In the samples 4-8, the average of LDH was 2,3698 IU/ml and the average of serum protein content was 8,44g/dl, indicating a precarious metabolic state of the animals. The calcium level was relatively constant in all samples, approximately 11mg/dl, being highest in sample 1 (12,3 mg/dl) and minimum in sample 8 (10,3 mg/dl). At 2 hours after slaughter these animals, the pH in meat obtained are represented in figure 2. In the samples 1-3, pH was close to 7 (6.9-7,01) and the samples 4-8 were slightly acidic, which leads to rapid muscle rigidity, decreased ability to hydration and water binding. Meat has less glycogen and maturation and conservation are low. This situation is a disadvantage of technological point of view for meat obtained, because meat has less glycogen and maturation and conservation are low. Monitoring stress level by blood lactatdehidrogenase and proteins measurement, especially albumins may help to improve meat quality, can take measures to avoid or reduce to a minimum stress before slaughter. LDH and in specially isoenzymes LDH 4 and LDH 5 with found in skeletal muscle and liver shows increases in the case of intense muscular effort of cattle. The normal values of serum LDH is less than 1445 IU /L. [4]

41 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Fig. 2 The pH value of meat obtained immediately after slaughter

CONCLUSIONS AND FUTURE WORK Stress immediately before slaughter cattle has negative consequences on meat quality, because it is hard processed. Serum indicators for identifying any stress before slaughter cows are LDH, the protein content, the calcium levels. PH values in meat produced reflect the state of preslaughter of the animal. Increase LDH indicates a muscular effort before slaughter. The low protein content show the presence of metabolic disorders or poor eating cows.

REFERENCES [1]. Bee, G., Lonergan, S. M., & Huff-Lonergan, E. (2004). Early postmortem pH influences proteolysis of cytoskeletal proteins during aging in porcine longissimus muscle. Journal of Animal Science, 82(Suppl. 1), 13. [2]. Bertram, H. C., Purslow, P. P., & Andersen, H. J. (2002). Relationship between meat structure, water mobility, and distribution: a lowfield nuclear magnetic resonance study. Journal of Agricultural and Food Chemistry, 50, 824–829. [3]. Bowker, B. C., Grant, A. L., Forrest, J. C. and Gerrard, D. E., (2000), Muscle metabolism and PSE pork, Proceedings of the American Society of Animal Science. [4]. Ghergariu, S., (1994). Bazele patologiei medicale a animalelor, Ed. ALL, Bucuresti [5]. Harris, S. E., Huff-Lonergan, E., Lonergan, S. M., Jones, W. R., & Rankins, D. (2001). Antioxidant status affects color stability and tenderness of calcium chloride-injected beef. Journal of Animal Science, 79, 666–677. [6]. Hambrecht, E., Eissen, J. J., Nooijen, R. I. J., Ducro, B. J., Smits, C. H. M., den Hartog, L. A., Verstegen, M. W. A. (2004). Preslaughter stress and muscle energy largely determine pork quality at two commercial processing plants, J ANIM SCI 2004, 82:1401- 1409. [7]. Huff-Lonergan, E., Lonergan, S.M. (2005). Mechanisms of water-holding capacity of meat: The role of postmortem biochemical and structural changes, Meat Science 71, 194–204. [8]. Holbrook, J.J., A. Liljas, S.J. Steindel & M.G. Rossmann (1975) Lactate dehydrogenase. The Enaymes 11: 191-292

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

[9]. Karolyi, D., Salajpal K., Sinjeri Ţ., Kovačiš D., Juriš I., ĐIKIŠ M., Meat quality, blood stress indicators and trimmed cut yield comparison of black slavonian pig with modern pigs in the production of slavonian kulen, Acta agriculturae slovenica, suplement 1(avgust 2004), 67–72. [10]. Kim S., Experiencing Biochemistry, Isolation and Characterization of an Enzyme LDH [11]. Melody, J. L., Lonergan, S. M., Rowe, L. J., Huiatt, T. W., Mayes, M. S., & Huff- Lonergan, E. (2004). Early postmortem biochemical factors influence tenderness and water-holding capacity of three porcine muscles. Journal of Animal Science, 82, 1195– 1205. [12]. Rathgeber, B. M., Boles, J. A. and Shand P. J., (1999), Rapid Postmortem pH Decline and Delayed Chilling Reduce Quality of Turkey Breast Meat, Poultry Science 78:477–484 [13]. Šimek, J./ Grolichová, M./ Steinhauserová, I./ Steinhauser, L. Carcass and meat quality of selected final hybrids of pigs in the Czech Republic. Meat Science, 66(2004), 383–386. [14]. Virgili, R./ Schivazappa, C. Muscle traits for long matured dried meats. Meat Science, 62(2002), 331–343. [15]. Warner, R. D., R. G. Kauffman, and M. L. Greaser. 1997. Muscle protein changes postmortem in relation to pork quality traits. Meat Sci. 45:339-352.

ABOUT THE AUTHORS Iuliana Manea, Valahia University of Targoviste, Faculty of Environmental Engineering and Biotechnology, Department of Food Engineering, 18-24 Unirii Bd., Targoviste, Romania, E-mail: [email protected] Laur Constantin Manea, Valahia University of Targoviste, Faculty of Environmental Engineering and Biotechnology, Department of Environmental Engineering, 18-24 Unirii Bd., Targoviste, Romania, E-mail: [email protected]

43 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

THE DYNAMIC OF PHENOLS ACCUMULATION AND THE INFLUENCE ON THE ANTIOXIDANT ACTIVITY OF VITIS VINIFERA L. CALLUS CULTURE EXTRACTS

R. Mihai, F. Helepciuc, and M. Mitoi

Abstract: Nowadays a considerably increasing interest consists in finding naturally occurring antioxidants from plant sources used in food industry and in preventive medicine in order to replace synthetic antioxidants restricted due to their possible carcinogenic effect. Polyphenolic compounds play an important role in adsorbing and neutralizing free radicals, quenching singlet oxygen or decomposing peroxides. Their synthesis and accumulation is generally stimulated in response to biotic/abiotic stresses. In the present study, total phenolic content and the evaluation of antioxidant potential were comparatively analyzed due to the influence of some biotic and abiotic elicitors in the long- term callus extracts of Vitis vinifera cvs Isabelle. The highest antioxidant activity recorded in case of using as a two-stage culture system the elicitors combination of mannitol (0.1 mM) and jasmonic acid (40 µM), suggests that this culture system could be used as a new method for improving natural antioxidants biosynthesis. The result was supported by a high concentration of total phenols and resveratrol, known in literature as strong antioxidants, in the extracts of the callus grown under the influence of this culture system. Important results were also obtained using biotic elicitors such as Fusarium oxysporum extract, but not comparable with the positive results obtained by using the abiotic culture system previously presented.

Key words: Resveratrol, Elicitors, Polyphenols, Antioxidants, Vitis vinifera L..

INTRODUCTION It has been established that oxidative stress is among the major causative factors in induction chronic and degenerative diseases such as: artherosclerosis, diabetes mellitus, cancer, Parkinson`s disease and immune dysfunction [1]. Antioxidants can be effective for the suppression of such disorders and in prevention of the free radical formation by scavenging or promotion of their composition. In recent years much attention has been devoted to the natural antioxidants and their association with health benefits [2], plants being an important potential source of its. Plant phenolics have been reported to have multiple biological effects, including antioxidant activity, mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers [3]. It has been shown that phenolic compounds (catechins, stilbenes, anthocyanins) extracted from red wine inhibit the oxidation of human low-density lipoproteins significantly more than α-tocopherol, and the some of them prevent in vitro platelet aggregation [4]. Resveratrol is a phenolic compound found in many families of plants. It was first reported in the peel of grape berries for disease resistance and, later, in wines to benefit health. Resveratrol has been reported to have strong antioxidant activity in vitro [5] and antiinflammatory activities [6], to exhibit cancer chemopreventive activity [7] and to modulate low-density lipoprotein oxidation [8]. Plant cell cultures have been shown as feasible systems for the production of these secondary metabolites, being the elicitation with biotic or abiotic stimuli the most efficient strategy for increasing the production of those metabolites. The aim of this study was to evaluate the elicitor effect of some biotic and abiotic factors on Vitis vinifera L. long-term callus culture regarding the antioxidant potential. This study was achieved by carrying out a quantitative analysis of the role of elicitors on resveratrol and total phenols production, compounds in general responsible for the antioxidant activities, in Vitis vinifera L callus culture. A DPPH test for the antioxidant activity was also achieved.

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MATERIAL AND METHODS Plant Material and Extraction Procedures

The study was conducted using a long-term Vitis vinifera L. cv. Isabelle pericarp, maintained through periodic subcultivations on a variant of basal Gamborg-B5 (1968) medium, supplemented with 0.1 mg/l NAA (α-naphtalene acetic acid), 0.2 mg/l kinetine, 2 g/l casein hydrolisate, 30g/l sucrose, 8g/l agar (Difco), which has been referred in the experiment as the control variant. To investigate the influence of different types and concentrations of elicitors on the callus culture, a two-stage experiment was performed. Eight variants of the experimental medium were analyzed (Table 1) for 37 days and each treatment was repeated 4 times. For the first stage of the experiment the long- term callus was grown 30 days on the medium variants supplemented with mannitol in concentration 2mM to ensure the callus proliferation. Further subcultivation of the callus for 7 days during the second stage of the experiment ensured the biosynthetic capacity of the callus. The fungi extract was obtained by centrifugation of Fusarium oxysporum sonicated pellets culture, the fungi medium being represented by the supernatant filtrate.

Table 1. Types of elicitors and the concentration used in the two-stage culture system.

Sample preparation and extraction of polyphenols Extraction procedure was carried out using established methods as a reference with minor modifications. Three grams of callus from all medium variants were ruptured with a pistil in a porcelain mortar aided by sea sand. The extraction was performed with 3 ml of solvent consisting of 0.1% (w/v) HCl in mixture of methanol, supported by the pistil friction until the liquid was saturated with red color and then left to stand for 24 hours in o refrigerator, at 4 C in a dark environment. The saturated extract was decanted after centrifugation (4000 rpm, 15 min). The extracts were filtered through 0.45 lm syringe filter and transferred to an auto sampler vial. Determination of polyphenols and resveratrol was performed immediately after the extraction.

Total Phenolic Assay Determination of total phenolic content, measured as GA equivalents, was made using Folin – Ciocalteu phenol reagent and expressed as gallic acid equivalent GAE/ fresh weight (mg/100g) according to the method of Singleton and Rossi (1965) [9] with slight modification.

45 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

1 ml Vitis vinifera callus extract, diluted 5-10 times with methanol (to obtain absorbance within the range of the prepared calibration curve) was mixed with 5 ml of Folin-Ciocalteu reagent (Sigma) previously diluted with distilled water. A volume of 4 ml 7.5% sodium carbonate solution was added to the mixture let it to stand for 30 min and the blue color formed was measured at 765 nm with a spectrophotometer (Hellios Gamma Thermo-Scientific). Gallic acid was used as a standard for the calibration curve.

Calibration and Determination of trans-resveratrol concentration using the HPLC analysis

Resveratrol was dissolved in methanol at a concentration of 1 mgl-1 and stored away from direct light at 4 0C until used. The standard methanolic solution of resveratrol was diluted and reference solutions in the ‗‗working‘‘ range from 10 µgl-1 to 10 mgl-1 were obtained. These solutions were analyzed and the corresponding peak areas were compared the concentration of resveratrol injected. Twenty microlitre of each solution was injected into the HPLC. Trans-resveratrol was determined by the HPLC method using the JASCO Model AS- 2051 liquid chromatograph equipped with Jasco MD-2015 Plusmultiwavelenght Detector and the Jasco FP-2020 Plus Inteligent Fluorescent Detector coupled to the PROGRAM Jasco-Chrompass Chromatography Data System. The sample was injected into the column filled with the stationary 100 C18 5 UM 25X 0,4 reversephase Nucleosil (TEKNOKROMA). The separation was carried out at room temperature. The isocratic elution at a flow rate of 1.0 ml/min used the mobile phase A of 1% acetic acid in demineralized water, and the mobile phase B of 100% methanol. For the detection of trans- resveratrol, the optimal wavelength of 312 ± 2 nm was used.

Antioxidant activity by DPPH assay:

The hydrogen atom or electron donation ability of the corresponding extracts was measured from the bleaching of purple colored methanol solution of DPPH. This spectrophotometric assay uses stable radical diphenypicrylhydrazyl (DPPH) as a reagent (Buritis and Bucar, 2000). Fifty microliters of various extracts in methanol and standard ascorbic acid solution (0.1ml) were added to 5 ml of a 0.004% methanol solution of DPPH. An equal amount of methanol and DPPH served as control. After 30 min incubation period at room temperature the absorbance was read against a blank at 517 nm. Inhibition free radical DPPH in percent (I%) was calculated as follows: I% = (Ablank-Asample/Ablank) x 100

Where Ablank is the absorbance of the control reaction (containing all reagents except the test compound) and Asample is the absorbance of the test compound. Tests were carried out in triplicate and ascorbic acid was used as positive control. The antioxidant activity of the extract was expressed as IC50. The IC50 value was defined as the concentration (in µg/ml) of extracts that inhibits the formation of DPPH radicals by 50%.

RESULTS AND DISCUSSION 1. Total phenolic content of the extracts The two-stage culture system containing the combination of elicitors mannitol (2mM) and JA (40µM) proved to be the most efficient regarding the total phenol accumulation at the level of Vitis vinifera L. cvs. Isabelle callus extract followed by the combination of the elicitors mannitol (2mM) and ethephon (0.1mM).

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The results are sustained by postulated data which indicate that sugars, such as sorbitol and mannitol, which cannot act as substrates for plant metabolism, play an important role as osmotic stress signals in triggering induction or promotion of phenolic compounds biosynthesis in plant cells [10]. Ethephon is an ethylene precursor. Ethylene treatment increases flavonoid, anthocyanin and stilbenoid production (phenolic compounds) via up-regulation their biosynthetic genes in grape (Vitis vinifera L.) cell cultures [11]. Exogenous application of JA to the plant cell culture or intact plant stimulates biosynthesis of a wide variety of plant secondary metabolites including flavonoids, phenylpropanoids plus many other type of natural products, demonstrating the obtained results of the experiment (Fig.1).

1,6

1,4

1,2

1 GAE 0,8

0,6

0,4

0,2

C ontrol B lank Man- E th Man- J A 40 Man- ABA 20Man-Man 20 Man- F ungi medium Man- F ungi extract Figure 1. Total phenolic content of the calli extracts in response to the elicitors treatment in a two-stage culture system.

2. Determination of trans-resveratrol content in callus extracts

Trans-resveratrol (3, 5, 4`-trihydroxystilbene) is produced by plants in response to fungus infections or to diverse abiotic factors, such as the presence of metallic ions, hydric stress, or exposure to UV light, acting like phytoalexins [12]. The presence of this compound in response to fungus infection is also demonstrated by the enhancement content of resveratrol in the callus under the influence of Fusarium oxysporum fungal extract.(Fig.2) The two-stage culture system containing mannitol (2mM) and ethephon (0.1mM) had a positively influence on the resveratrol accumulation but the most significantly effect was achieved by the elicitors treatment such as mannitol (2mM) in combination with jasmonic acid (40 µM).(Fig. 2) The JA positively influence on resveratrol biosynthesis can be explained by JA pathway which is generally regarded as an integral signal or elicitor signal transducer in the induction of phytoalexins [13].

47 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

140 120 100 80 60

40 (µg/ml)

20 Resveratrol Resveratrol

concentrations concentrations 0

Blank Control Man- Eth Man- JA 40 Man- ABA 20Man- Man 20

Man- Fungi extract Man- Fungi medium Medium variants

Figure 2. The trans-resveratrol content of the Vitis vinifera L. cvs. Isabelle callus extract in response to different elicitors treatment.

3. DPPH radical-scavenging activity DPPH is a stable radical at room temperature and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The significant decrease in the concentration of the DPPH radical is due to the scavenging ability of the extract from the callus grown under the influence of the combined elicitors mannitol (0.1mM) and jasmonic acid (40µM) followed by the combined elicitors Mannitol (2mM) and ethephon (0.1mM) (Fig 3).

2500 2000 1500

IC50 1000 500 0

Blank Control Man- Eth Man- JA 40 Man- ABA 20 Man-Man 20 Ascorbic acid

Man- Fungi extract Man- Fungi medium

Medium variants

Figure 3. Free radical scavenging activity of Vitis vinifera L. cvs. Isabelle callus extract in response to elicitors treatment.

The data were sustained by the previously results of the experiment which demonstrates that this combination of elicitors enhance the accumulation of phenolic compounds that have been reported to have multiple biological effects, including antioxidant activity. The antioxidant activity of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quencers [14]. The total phenolic content of the callus in response to the elicitor treatment with elicitors significantly inhibited the activity of DPPH radicals, having a higher scavenging activity than the ascorbic acid.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

CONCLUSIONS AND FUTURE WORK On the basis of this study results, the Vitis vinifera L. cvs. Isabelle callus extracts grown in the two-stage system with the elicitors mannitol (2mM) and jasmonic acid (40µM) have significant antioxidant activity compared to others. The antioxidant activity may be attributed to the high content of phenolic compounds and resveratrol in the callus extract subject to this combined elicitors influenced. Moreover, this two-stage culture system for the Vitis vinifera L. cvs Isabelle callus extract can be used as an easily accessible and rich source of natural antioxidants.

REFERENCES [1]. Souri E., et al., 2008. Screening of thirteen medicinal plant extracts for antioxidant activity. Iran. J. Pharm. Res., 7, pp. 149- 154. [2]. Arnous A. et al., 2001. Effect of principal polyphenolic components in relation to antioxidant characteristics of aged red wines. J. Agric. Food. Chem. 49, pp. 5736-5742. [3]. Huda-Faujan, N. et al., 2009, Antioxidant activity of plants methanolic extracts containing phenolic compounds, African Journal of Biotechnology 8 (3), pp. 484-489. [4]. Varache-Lembe`ge, M. et al., 2000. Structure-activity relationships of polyhydroxystilbene derivatives extracted from Vitis vinifera cell cultures as inhibitors of human platelet aggregation. Med. Chem. Res. 10, pp. 253–267. [5]. Fauconneau, B., et al. 1997. Comparative study of radical scavenger and antioxidant properties of phenolic compounds from Vitis vinifera cell cultures using in vitro tests. Life Sci. 61(21), pp. 2103-2110. [6]. Kawada, N., et al. 1998. Effect of antioxidants, resveratrol, quercetin, and N- acetylcysteine, on the functions of cultured rat hepatic stellate cells and Kupffer cells. Hepatology 27(5), pp. 1265-1274. [7]. Jang, M.,et al.1997. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Sci. 275, pp. 218-220. [8]. Frankel, E. N., et al. 1993. Inhibition of human LDL oxidation by resveratrol. Lancet. 341, pp. 1103-1104. [9]. Singleton, V.L. and Rossi, J.A. 1965. Colorimetry of total phenolic substances. US: American Chemical Society Symposium series.26, pp. 47-70. [11]. El-Kereamy, A. et al., 2003: Exogenous ethylene stimulates the long-term expression of genes related to anthocyanin biosynthesis in grape berries. Physiol. Plant. 119, pp. 175- 182. [12]. Roggero, J.P. and Garcia Parilla, M.C. 1995. Effects of ultraviolet irradiation on resveratrol and changes in resveratrol and various of its derivatives in the skins of ripening grapes.Sci. Aliments 15, pp. 411-422. [13]. Brader, G., et al. 2001. Jasmonate–dependent induction of indole glucosinolates in Arabidopsis by culture filtrates of the nonspecific pathogen Erwinia carotovora. Plant physiology 126, pp. 849-860. [14]. Rice Evans, C.A., et al. 1995. The relative antioxidant activities of plant derived polyphenolic flavonoids. Free. Radical. Res. 23, pp. 375-383.

ABOUT THE AUTHORS R. Mihai, 296 Splaiul independentei, 060031 Bucharest, Romania, E-mail: [email protected] F. Helepciuc, 296 Splaiul independentei, 060031 Bucharest, Romania, E-mail: [email protected] M. Mitoi, 296 Splaiul independentei, 060031 Bucharest, Romania, E-mail: [email protected]. 49 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

DETERMINATION OF SOME ANIONS CONCENTRATION IN MINERAL WATERS FROM NORTH REGION OF ROMANIA, BY HIGH PERFORMANCE ION CHROMATOGRAPHY (HPIC)

R. C. Heghedűş-Mîndru, G. Heghedűş-Mîndru, P. Negrea, , R.L. Şumălan, T.I. Traşcă, A. Riviş, D.S. Ştef and L.M. Rusnac

Abstract: This paper describe the some anions concentration in mineral waters from north regions of Romania by high performance ionic chromatography (HPIC). In sample A3 three anions were identified (fluorides, chlorides, and sulfates), in sample B3 were identified chlorides, nitrates and sulfates.The sample C3 presented four anions identified (fluorides, chlorides, nitrates and sulfates). The maximum anions content in the north region of Romania, was recorded in sample B3. In the samples of mineral natural waters analyzed, nitrites and phosphates are not identified. Key words: Mineral Water, North Regions of Romania, Anions, High Performance Ionic Chromatography (HPIC),

INTRODUCTION Natural mineral water is a water clearly distinguishable from ordinary drinking water because, it is characterized by its content of certain mineral salts and their relative proportions and the presence of trace elements or of other constituents; it is obtained directly from natural or drilled sources from underground water bearing strata for which all possible precautions should be taken within the protected perimeters to avoid any pollution of, or external influence on, the chemical and physical qualities of natural mineral water; of the constancy of its composition and the stability of its discharge and its temperature, due account being taken of the cycles of minor natural fluctuations; it is collected under conditions which guarantee the original microbiological purity and chemical composition of essential components; it is packaged close to the point of emergence of the source with particular hygienic precautions; it is not subjected to any treatment other than those permitted by this standard. [5] The use of ionic chromatography has appeared due to the necessity of analyzing the mixtures of anions, cations, or polar compounds – a difficult process or impossible to achieve by other variants of liquid chromatography. This variant of HPIC (high performance ionic chromatography) is based on using the ions exchange columns, using the materials that resist to acids, bases or salts aggressively, substances which their aqueous solutions serve as elluents. [1-4, 7-8] One of the most important application is an organic species analysis, like cations and anions analysis from waters, solutions or biological fluids but also for the analysis of same + polluants (like NH4 ion from natural waters) [9-13] HPIC is suited to the analysis of either anions or cations (depending on whether an anion-exchange or cation-exchange resin is used) and both ionorganic and organic ions can be separated using this technique. In this experiment an anion-exchange column will be used. In HPIC, the separation of analyte ions involves the electrostatic interaction between solute anions and the positively charges sites on the stationary phase (the greater the degree of electrostatic attraction, the stronger the anion is retained on the column). [14] This paper describe the some anions concentration in mineral waters from north regions of Romania by high performance ionic chromatography (HPIC)

MATERIALS AND METHODS Materials  DIONEX - ICS 3000 apparatus;

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

 DP type Pump – 35 MPa;  Column: AF12;  Mobile phase, elluent: Na carbonate 3,5mM + sodium bicarbonate 5mM;  Elluent flaw: 1,2 mL/min;  Conductometric detector – CD;  Operating program (HPIC) - Chromeleon Dionex – 6.80 SP1Version [6] HPIC conditions The calibration curves obtained relating to anions determinations and the corresponding retention times, are shown in table 1.

Table1. Retention time, calibration curves and calibration points to stock solution No. Anions Calibration Calibration Retention crt. curves points time 1. Fluoride Linear 5 2,96 2. Chloride Linear 5 4,16 3. Nitrite Linear 5 4,98 4. Nitrate Linear 5 7,24 5. Phosphate Linear 5 9,74 6 Sulfate Linear 5 12,07

Determination of the anions from the north region of Romania The chromatogram obtained by analyzing anions for the mineral water sample A3 is shown in figure 1. As it can be seen from figure 1 along with data from table 1 corresponding to stack solution, it was found out that in mineral water sample A3 the following anions were - - 2- present F , Cl and SO4 . The quantitative analysis, based on the standard curve method has lead to the following results shown in table 2.

Figure 1. Anions chromatogram for sample A3

51 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Table 2. Retention times corresponding to existent anions in the analyzed mineral water sample A3 No. Retention Anions Peak Area Area Concentration crt. time length µS×min % ppm µS 1 2,91 Fluoride 0,020 0,002 1,65 0,556 2 3,60 Unidentified 0,068 0,013 10,98 Unidentified 3 4,03 Chloride 0,177 0,025 20,81 5,175 4 11,20 Sulfate 0,212 0,079 66,56 44,468 Total 0,477 0,119 100 50,196

The chromatogram obtained by analyzing anions for the mineral water sample B3 is shown in figure 2. As it can be seen from figure 2 along with data from table 1 corresponding to stack solution, it was found out that in mineral water sample B3 the following anions were - - 2 present Cl , NO3 and SO4 -. The quantitative analysis, based on the standard curve method has lead to the following results shown in table.

Figure 2. Anions chromatogram for sample B3

Table 3. Retention times corresponding to existent anions in the analyzed mineral water sample B3 No. Retention Anions Peak Area Area Concentration crt. time length µS×min % ppm µS 1 3,60 Unidentified 0,099 0,018 5,20 Unidentified 2 4,04 Chloride 2,134 0,263 75,99 104,943 3 6,92 Nitrate 0,029 0,007 1,98 5,167 4 11,22 Sulfate 0,148 0,058 16,82 32,700 Total 2,410 0,345 100 142,811

The chromatogram obtained by analyzing anions for the mineral water sample C3 is shown in figure 3.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

As it can be seen from figure 3 along with data from table 1 corresponding to stack solution, it was found out that in mineral water sample C3 the following anions were - -, - 2- present F , Cl NO3 , and SO4 . The quantitative analysis, based on the standard curve method has lead to the following results shown in table 4.

Figure 3. Anions chromatogram for sample C3

Table 4. Retention times corresponding to existent anions in the analyzed mineral water sample C3 No. Retention Anions Peak Area Area Concentration crt. time length µS×min % ppm µS 1 2,91 Fluoride 0,033 0,003 2,42 0,377 2 3,63 Unidentified 0,133 0,025 20,03 Unidentified 3 4,04 Chloride 0,248 0,036 29,10 4,397 4 6,93 Nitrate 0,024 0,005 3,93 1,629 5 11,22 Sulfate 0,152 0,055 44,51 13,754 Total 0,589 0,124 100 20,156

RESULTS AND DISCUSSIONS The HPIC analysis of the mineral waters allowed identification of the anions in the north region of Romania as follows:  in sample A3 three anions were identified (fluorides, chlorides, and sulfates), at retention time 2,91 minutes there was obtained 1,65% fluorides, at retention time 4,03% minutes there was obtained 20,81% chlorides, and at retention time 11,20 minutes there was obtained 66,56% sulfates, (figure 1);  in sample B3 three anions were identified (chlorides, nitrates and sulfates), at retention time 4,04 minutes there was obtained 75,99% chlorides, at retention time 6,92 minutes there was obtained 1,98 % nitrates, and at retention time 11,22 minutes there was obtained 16,82% sulfates, (figure 2);  in sample C3 four anions were identified (fluorides, chlorides, nitrates and sulfates), at retention time 2,91 minutes there was obtained 2,42% fluorides, at retention time 4,04 minutes there was obtained 29,10% chlorides, at retention time 6,93 minutes there was obtained 3,93 % nitrates, and at retention time 11,22 minutes there was obtained 44,51% sulfates, (figure 3). 53 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

CONCLUSION AND FUTURE WORK For the north region, the maximum quantity for fluorides was recorded in sample A3 (0,556 mg/L), the minimum quantity being recorded in sample C3 (0,377 mg/L), and in sample B3 no fluorides were identified. The maximum quantity of chloride was recorded in sample B3 (104,943 mg/L), sample A3 (5,175 mg/L), and minimum recorded was in sample C3 (4,397 mg/L). For north region the maximum content of nitrates was recorded for sample B3 (5,167 mg/L), the minimum being of 1,629 mg/L for sample C3. In sample A3 no nitrates were identified. The maximum value for sulfates for north zone was obtained in sample A3 (44,468 mg/L), sample B3 value was 32,7 mg/L, and the minimum value appears for sample C3 (13,754 mg/L). In the samples of mineral natural waters analyzed, from the north region of Romania, nitrites and phosphates are not identified. The maximum anions content in the north region of Romania, was recorded in sample B3.

ACKNOWLEDGEMENTS This work was carried out during the project ―Post-doctoral School of Agriculture and Veterinary Medicine Posdru/89/1.5/S/62371‖ co-financed by the European Social Fund the Sectorial Operational Programme for the Human Resources Development 2007-2013.

REFERENCES [1]. ***ASRO, Standard Român, SR EN ISO 7899-2, Septembrie 2002; [2]. ***ASRO, Standard Român, SR EN ISO 12780, Martie 2003; [3]. ***ASRO, Standard Român, SR EN ISO 6222, Ianuarie 2004; [4]. ***ASRO, Standard Român, SR EN ISO 9308-1, Februarie 2004; [5]. ***Codex Standard for natural mineral waters, Codex Standard 108-1981, Adopted 1981. Amendment 2001, 2011. Revisions 1997, 2008. [6]. ***Dionex ICS-3000, 2006, Ion Chromatography System Operator's, Manual Document No. 065031 Revision 03 September; [7]. Heghedűş-Mîndru G., 2008, Contribuţii la studiul posibilităţilor de îmbunătăţire a indicatorilor de calitate a apelor minerale prin utilizarea tehnicilor moderne de simulare, modelare şi conducere automată, Seria 4: Inginerie chimică, nr. 16, Editura POLITEHNICA, ISSN: 1842-8223, ISBN: 978-973-625-705-6, 226 pp; [8]. Heghedűş-Mîndru G., Biron R. C., Perju D. M., Rusnac L. M., Riviş A., & Ştef D. S., 2008, Cations content in the natural mineral waters of Romania with the HPIC method, Journal of Food, Agriculture & Environment – JFAE, Vol 6. (3 & 4), , Print ISSN: 1459- 0255, Online ISSN: 1459-0263, pag 506-509; [9]. *** IRS Standard Român, 1997, Ape minerale naturale, SR 4450, Indice de clasificare N 85, Decembrie. [10]. Kostic, Vesna., & Nikodinoski, M., 2006, The usage of ion chromatography (IC) in water analysis of inorganic anions and cations, Ovidius University Annals of Chemistry, Volume 17, No.1, Constanţa-România; [11]. Naşcu, H.I., & Jäntschi, L., 2006, Chimie Analitică şi Instrumentală, Editura Academic Press & AcademicDirect; [12]. Vernescu, M., 1988, Apele minerale, Editura Tehnică, Bucureşti;

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[13]. Vernescu, M., 1984, Un nou sistem codificat de caracterizare şi clasificare a apelor minerale şi termale. In: Studii tehnice şi economice, seria E - Hidrogeologie, nr. 14, Institutul de Geologie şi Geofizică, Bucureşti; [14].http://www.chemistry.adelaide.edu.au/external/soc-rel/content/expts/hpicexpt.htm)

ABOUT THE AUTORS Ramona Cristina Heghedűş-Mîndru - Banat‘s University of Agricultural Sciences and Veterinary Medicine Timisoara, Faculty of Food Processing Technologies, 119, Calea Aradului, 300645, Timisoara, Romania, Email: [email protected]

55 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

DETERMINATION OF SOME ANIONS CONCENTRATION IN MINERAL WATERS FROM WEST REGION OF ROMANIA, BY HIGH PERFORMANCE ION CHROMATOGRAPHY (HPIC)

G. Heghedűş-Mîndru, R.C. Heghedűş-Mîndru, P. Negrea, , R.L. Şumălan, T.I. Traşcă, A. Riviş, D.S. Ştef and L.M. Rusnac

Abstract: The present paper deals with the identification and determination of anion concentration in the natural mineral water of the west region from Romania. The anions determination was done with high- performance ion chromatography (HPIC).The anions analyzed and identified are fluoride, chloride, nitrate and sulfate. The chloride (126,659 mg/L) and sulfate (44,468 mg/L) anions have the highest concentration. The present paper aims at revealing the complex chemical composition of mineral waters, underlying the differences between the compositions of mineral waters of various sources, as well as signaling the chemical pollution of water quality. Key words: Mineral Water, High Performance Ionic Chromatography (HPIC), Fluorides, Chlorides, Nitrites, Nitrates, Phosphates, Sulfates

INTRODUCTION Natural mineral water is a water that lies in an aquiferous soil or underground deposit, protected from any pollution risk, comes from an exploit source by one or many natural emerges or drillings and is bottled near the source, with special hygiene precautions [1, 3, 4] There are different categories of waters intended for human consumption and natural mineral waters are just one of them. Natural mineral waters are characterized by their purity at source and their constant level of minerals. The Directive 2009/54/EC, defines the provisions applicable to the marketing and exploitation of natural mineral waters. Commission Directive 2003/40/EC of 16 May 2003 establishing the list, concentration limits and labeling requirements for the constituents of natural mineral waters and the conditions for using ozone-enriched air for the treatment of natural mineral waters and spring waters. [6] This paper describe the some anions concentration in mineral waters from west regions of Romania by high performance ionic chromatography (HPIC) Ionic chromatography (ionic exchange), is an analysis method that depends on a porous stationary solid phase formed from specific materials – ions exchangers – substances with a solid loosened network, organic or anorganic, on which are found grafted, by obtaining process, some ionic exchange centers. [2] This paper describe the some anions concentration in mineral waters from west regions of Romania by high performance ionic chromatography (HPIC)

MATERIALS AND METHODS Materials: DIONEX - ICS 3000 apparatus; DP type Pump – 35 MPa; Column: AF12; Mobile phase, elluent: Na carbonate 3,5mM + sodium bicarbonate 5mM; Elluent flaw: 1,2 mL/min; Conductometric detector – CD; Operating program (HPIC) - Chromeleon Dionex – 6.80 SP1Version. [6] The water samples were taken from west regions of Romania as follows: mineral water source1 (A1), source2 (B1), and source 3 (C1). HPIC conditions: The calibration curves obtained relating to anions determinations and the corresponding retention times, are shown in figure 1, table 1, respectively. [6]

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Fluoride Chloride

Nitrite Nitrate

Phosphate Sulfates

Figure 1. Calibration curves for anions

Determination of the anions from the west region of Romania The chromatogram obtained by analyzing anions for the mineral water sample A1 in shown is figure 2. As it can be seen from figure 2 along with data from table 1 and figure 1 corresponding to stack solution, it was found out that in mineral water sample A1 the - 2-. following anions were present Cl and SO4

57 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The quantitative analysis, based on the standard curve method has lead to the following results shown in table 2.

Figure 2. Anions chromatogram for sample A1

Table 2. Retention times corresponding to existent anions in the analyzed mineral water sample A1 No. Retention Anions Peak Area Area Concentration crt. time length µS×min % ppm µS 1 3,70 Unidentified 0,073 0,011 1,10 Unidentified 2 4,18 Chlorides 8,039 0,959 96,14 126,659 3 12,06 Sulfates 0,070 0,028 2,76 22,823 Total 8,182 0,998 100 149,481

The chromatogram obtained by analyzing anions for the mineral water sample B1 in shown is figure 3. As it can be seen from figure 3 along with data from table 1 and figure 1 corresponding to stack solution, it was found out that in mineral water sample B1 the - - 2-. following anions were present F , Cl and SO4 The quantitative analysis, based on the standard curve method has lead to the following results shown in table 3.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

. Figure 3. Anions chromatogram for sample B1

Table 3. Retention times corresponding to existent anions in the analyzed mineral water sample B1 No. Retention Anions Peak Area Area Concentration crt. time length µS×min % ppm µS 1 2,91 Fluoride 0,009 0,001 0,20 0,099 2 3,61 Unidentified 0,107 0,019 4,92 Unidentified 3 4,04 Chloride 1,591 0,197 50,24 34,454 4 11,21 Sulfate 0,471 0,175 44,63 43,803 Total 2,178 0,392 100 78,356

The chromatogram obtained by analyzing anions for the mineral water sample C1 is shown in figure 4. As it can be seen from figure 4 along with data from table 1 corresponding to stack solution, it was found out that in mineral water sample C1 the - 2- - following anions were present Cl , SO4 and NO3 . The quantitative analysis, based on the standard curve method has lead to the following results shown in table 4.

Table 4. Retention times corresponding to existent anions in the analyzed mineral water sample C1 No. Retention Anions Peak Area Area Concentration crt. time length µS×min % ppm µS 1 3,61 Unidentified 0,100 0,022 15,04 Unidentified 2 4,03 Chloride 0,095 0,015 10,33 0,450 3 6,91 Nitrate 0,048 0,010 7,32 3,511 4 11,21 Sulfate 0,263 0,096 67,32 24,111 Total 0,505 0,143 100 28,072

59 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Figure 4. Anions chromatogram for sample C1

RESULTS AND DISCUSSIONS The HPIC analysis of the mineral waters allowed identification of the anions in the west region of Romania as follows:  in sample A1 two anions were identified (chlorides and sulfates), at retention time 4,18 min there was obtained 96,14% chlorides and at retention time 12,06 min there was obtained 2,76% sulfates, respectively (figure 1);  in sample B1, three anions were identified (fluorides, chlorides and sulfates), at retention time 2,91 minutes there was obtained 0,20% fluorides, at retention time 4,04 minutes there was obtained 50,24 % chlorides, and at retention time 11,21 minutes there was obtained 44,63 % sulfates, respectively (figure 2);  in sample C1, three anions were identified (chlorides, nitrates and sulfates), at retention time 4,03 minutes there was obtained 10,33% chlorides, at retention time 6,91% minutes there was obtained 7,32% nitrates, and at retention time 11,21 minutes there was obtained 67,32 % sulfates, respectively (figure 3).

CONCLUSION AND FUTURE WORK From the HPIC analysis of the mineral waters for identification of the anions from the west region of Romania were shown the following conclusions:  in the west region fluorides were identified only in sample B1 (0,099 mg/L), in the other A1 and C1 samples no fluorides were identified;  for chloride anion the values recorded in ranged between 0,450 mg/L and 126,659 mg/L, with a maximum in sample A1 (126,659 mg/L), sample B1 (34,454 mg/L) and minimum recorded was in sample C1 (0,450 mg/L);  the only samples from west area center region, where nitrates identified are C1 (3,511 mg/L) and C2 (1,328 mg/L) respectively;  sulfates were identified in west region with maximum values for sample B1 (43,803 mg/L), sample C1 with a value of 24,111 mg/L, the lowest value being recorded in sample A1 (22,823 mg/L);  in mineral waters analyzed no nitrites or phosphates anions were identified. The mineral waters analyzed do not presented risks on health from the point of view of mineral elements (anions) contents and ranges between the legal limits foreseen in standard. [3]

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The sample with the highest quantity of anions recorded, was in A1(149,481 mg/L).,

REFERENCES [1]. Asada, K., 2000, The water-water cycle as alternative photon and electron sinks, Phill. Trans. R. Soc. Lond. B., 355:1419-1431; [2]. Binghui, Z., Zhixiong, Z., Jing, Y., 2006 - Ion chromatographic determination of trace iodate, chlorite, chlorate, bromide, bromate and nitrite in drinking water using suppressed conductivity detection and visible detection, Journal of Chromatography A Volume 1118, Issue 1, Pag. 106-110; [3]. Bouwer, H., 2003, Integrated water management for 21st century: Problems and solutions, J. Food, Agric. & Environ, 1:118-127; [4]. Brănescu, S., Andru, C., David, E., Popescu, A., & Barbu, C., 2007, Determinarea concentraţiei unor metale grele din apele uzate evacuate de pe platforma chimică Rm. Vâlcea în scopul determinării gradului de poluare, Revista de chimie, 58 (11), pp. 1142- 1144; [6]. ***Dionex ICS-3000, 2006, Ion Chromatography System Operator's, Manual Document No. 065031 Revision 03 September; [7]. http://ec.europa.eu/food/food/labellingnutrition/water/

ABOUT THE AUTORS Gabriel Heghedűş-Mîndru - Banat‘s University of Agricultural Sciences and Veterinary Medicine Timisoara, Faculty of Food Processing Technologies, 119, Calea Aradului, 300645, Timisoara, Romania, Email: [email protected]

61 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

DETERMINATION OF SOME ANIONS CONCENTRATION IN MINERAL WATERS FROM CENTRE REGION OF ROMANIA, BY HIGH PERFORMANCE ION CHROMATOGRAPHY (HPIC)

G. Heghedűş-Mîndru, R.C. Heghedűş-Mîndru, P. Negrea, , R.L. Şumălan, T.I. Traşcă, A. Riviş, D.S. Ştef and L.M. Rusnac

Abstract: The aim of this study was to determine the concentration of anions in three mineral waters samples, collected from centre region of Romania. For anions determination, was used the ion chromatography high-performance (HPIC)- Dionex – ICS 3000 type, particularized for anions. The highest fluoride concentration , proved to be was in the sample A2 (2,14 mg/L). The only sample has been identified nitrates, was the C2 (1,328 mg/L). The maximum values for sulfates content, was identified in sample A2 (25,435 mg/L), and the minimum content in sample B2 (16,280 mg/L).

Key words: Mineral Water, Anions, High Performance Ionic Chromatography (HPIC), Centre Regions of Romania

INTRODUCTION The natural mineral water is clearly different from the spring water, due it‘s specific natural content in dissolved mineral salts, characteristic relative proportions, oligoelements presence or other constituents and native microbiological purity, which could give some health favorable properties. [7] Besides macro elements, proteic, glucidic and lipidic nutrients, vitamins, the human body needs some metallic elements also, which are found in low quantity, the so-called microelements or trace elements. These are generally transitional metals, components of numerous enzymatic systems. Beside their healthy effect, even indispensable to life, these trace elements in high quantities can damage or be highly toxic for living organisms. [2, 5] Several chemicals from water can be adsorbed in soil and in aquifers. Sorption is the change in concentration of a chemical in a solid matter as a result of mass transfer amongst solution and solute. Sorption processes can be subdivided into adsorption, absorption and ion exchange. Adsorption is a surface phenomenon while absorption and ion exchange are chemical and electro chemical phenomenon respectively. The relevance of the process is subject to solvent and solute property, apart from other factors such as temperature and pH. [6] The most widely spread are the ions exchange resins – organic – that have an organic skeleton support – a porous polymer. On this center, takes place a ionic reaction or an interaction between the substance from the solution (electrolyte or not electrolyte) and the function chemically bounded on the solid support called ionic exchange reaction. [3, 4] HPIC has made significant practical impact on the areas of pharmaceutical, clinical, forensic, environmental and industrial research and development analyses, and is an ideal complementary technique to HPLC. Preparative ion-chromatography has also found an important use in the isolation and purification of various compounds. [8] In this paper was evaluated the some anions concentration in mineral waters from centre region of Romania by high performance ion chromatography (HPIC)

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MATERIALS AND METHODS The apparatus used was: DIONEX - ICS 3000, DP type Pump – 35 MPa; Column: AF12; Mobile phase, elluent: Na carbonate 3,5mM + sodium bicarbonate 5mM; Elluent flaw: 1,2 mL/min; Conductometric detector – CD; Operating program (HPIC) - Chromeleon Dionex – 6.80 SP1Version. [1] The water samples were taken from center regions of Romania as follows: mineral water source1 (A2), source2 (B2), and source 3 (C2). HPIC conditions: The calibration curves obtained relating to anions determinations and the corresponding retention times, are shown table 1. [1]

Determination of the anions from the center region of Romania The chromatogram obtained by analyzing anions for the mineral water sample A2 is shown in figure 1. As it can be seen from figure 1 along with data from table 1 corresponding to stack solution, it was found out that in mineral water sample A2 the following anions were - - 2- present F , Cl and SO4 . The quantitative analysis, based on the standard curve method has lead to the following results shown in table 2.

Figure 1. Anions chromatogram for sample A2

63 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Table 2. Retention times corresponding to existent anions in the analyzed mineral water sample A2 No. Retention Anions Peak Area Area Concentration crt. time length µS×min % ppm µS 1 2,99 Fluoride 0,026 0,002 1,37 2,140 2 3,76 Unidentified 0,120 0,021 11,78 Unidentified 3 4,24 Chloride 0,823 0,111 63,74 13,953 4 12,59 Sulfate 0,097 0,040 23,11 25,435 Total 1,066 0,175 100 41,528

The chromatogram obtained by analyzing anions for the mineral water sample B2 is shown in figure 2. As it can be seen from figure 2 along with data from table 1 corresponding to stack solution, it was found out that in mineral water sample B2 the following anions were - - 2- present F , Cl and SO4 . The quantitative analysis, based on the standard curve method has lead to the following results shown in table 3.

Figure 2. Anions chromatogram for sample B2

Table 3. Retention times corresponding to existent anions in the analyzed mineral water sample B2 No. Retention Anions Peak Area Area Concentration crt. time length µS×min % ppm µS 1 2,99 Fluoride 0,028 0,005 1,29 0,490 2 3,86 Unidentified 0,371 0,084 19,82 Unidentified 3 4,29 Chloride 0,149 0,017 3,97 0,272 4 12,56 Sulfate 0,762 0,316 74,92 16,280 Total 1,310 0,422 100 17,042

The chromatogram obtained by analyzing anions for the mineral water sample C2 is shown in figure 3.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

As it can be seen from figure 3 along with data from table 1 corresponding to stack solution, it was found out that in mineral water sample C2 the following anions were present F-, Cl- and NO3- The quantitative analysis, based on the standard curve method has lead to the following results shown in table 4.

Figure 3. Anions chromatogram for sample C2

Table 4. Retention times corresponding to existent anions in the analyzed mineral water sample C2 No. Retention Anions Peak Area Area Concentration crt. time length µS×min % ppm µS 1 2,94 Fluoride 0,004 0,000 0,71 0,126 2 3,60 Unidentified 0,073 0,014 21,75 Unidentified 3 4,03 Chloride 0,362 0,047 74,73 14,439 4 6,96 Nitrate 0,010 0,002 2,81 1,328 Total 0,450 0,063 100 15,893

RESULTS AND DISCUSSIONS The HPIC analysis of the mineral waters allowed identification of anion in the centre regions of Romania as follows:  in sample A2 three anions were identified (fluorides, chlorides, and sulfates), at retention time 2,99 minutes there was obtained 1,37% fluorides, at retention time 4,24% minutes there was obtained 63,74% chlorides, and at retention time 12,59 minutes there was obtained 23,11 % sulfates, (figure 1);  in sample B2 three anions were identified (fluorides, chlorides, and sulfates), at retention time 2,99 minutes there was obtained 1,29% fluorides, at retention time 4,29% minutes there was obtained 3,97% chlorides, and at retention time 12,56 minutes there was obtained 74,92 % sulfates, (figure 2);  in sample C2 three anions were identified (fluorides, chlorides, and nitrates), at retention time 2,94 minutes there was obtained 0,71% fluorides, at retention time 4,03% minutes there was obtained 74,73% chlorides, and at retention time 6,96 minutes there was obtained 2,81% nitrates, (figure 3).

65 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

CONCLUSION AND FUTURE WORK The highest content of fluorides was recorded in center region in sample A2 (2,14 mg/L), the other B2 and C2 samples having a fluorides content that ranged between 0,49 and 0,126 mg/L. The highest quantity of chloride identified in the center region was recorded in sample C2 (14,439 mg/L), sample A2 (13,953 mg/L), and minimum recorded was in sample B2 (0,272 mg/L). The only samples from center region, where nitrates identified are C2 (1,328 mg/L). For the center region, the maximum sulfates content was identified in sample A2 (25,435 mg/L), and the minimum content in sample B2 (16,280 mg/L). In sample C2, no sulfates were identified. The sample with the lowest quantity of anions is recorded for samples C2 (15,893 mg/L)

REFERENCES [1]. ***Dionex ICS-3000, 2006, Ion Chromatography System Operator's, Manual Document No. 065031 Revision 03 September; [2]. ***Drinking-Water Quality, 2004, Guidelines for Third Edition Volume 1, World Health Organization, Geneva. [3]. Heghedűş-Mîndru G., 2008, Contribuţii la studiul posibilităţilor de îmbunătăţire a indicatorilor de calitate a apelor minerale prin utilizarea tehnicilor moderne de simulare, modelare şi conducere automată, Seria 4: Inginerie chimică, nr. 16, Editura POLITEHNICA, ISSN: 1842-8223, ISBN: 978-973-625-705-6, 226 pp; [4]. Heghedűş-Mîndru G., Biron R. C., Perju D. M., Rusnac L. M., Riviş A., & Ştef D. S., 2008, Cations content in the natural mineral waters of Romania with the HPIC method, Journal of Food, Agriculture & Environment – JFAE, Vol 6. (3 & 4), , Print ISSN: 1459- 0255, Online ISSN: 1459-0263, pag 506-509; [5]. ***IRS Standard Român, 1997, Ape minerale naturale, SR 4450, Indice de clasificare N 85, Decembrie. [6]. Jalgaonkar A., 2008 - Microanalysis of groundwater elements with respect to time and depth in the hortobágy region in Hungary, Carpth. Journal of Earth and Environmental Sciences, Vol. 3, No. 1, p. 39 – 47; [7]. Vernescu, M., 1984, Un nou sistem codificat de caracterizare şi clasificare a apelor minerale şi termale. In: Studii tehnice şi economice, seria E - Hidrogeologie, nr. 14, Institutul de Geologie şi Geofizică, Bucureşti; [8]. http://www.chemistry.adelaide.edu.au/external/soc-rel/content/expts/hpicexpt.htm)

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MICROARCHITECTURE OF QUATERNIZED POLYSULFONE/POLY(VINYLIDENE FLUORIDE) BLEND FOR SCAFFOLDS IN TISSUE ENGINEERING

L. I. Buruiana, E. Avram, A. Popa, and S. Ioan

Abstract: Blends of quaternized polysulfones with triphenylphosphonium pendant groups/poly(vinylidene fluoride) were prepared and then analyzed by atomic force microscopy and scanning electron microscopy, in order to establish their specific surface morphology and to correlate it with prospective applications in biomedicine. The history of film preparation by a dry-cast process reveals a hydrophobic network-like structure with cluster formations and interconnected pores, more pronounced once the quaternized polysulfone is added in the system. Also, the micrographs point out that quaternized polysulfone/poly(vinylidene fluoride) blend accomplish the required properties for specific application, making them good tissue-engineered candidates. Key words: Polysulfone Blend, Microarchitecture, Network Structure.

INTRODUCTION Polymer blending is designed to generate materials with optimized structural, mechanical, morphological and biological properties [1,2]. Polysulfones, having superior properties, such as chemical, mechanical, and thermal resistance, represent attractive materials for the manufacture of commercial membranes and biomedical applications, such as hemodialysis, nano and ultrafiltration, cell culture, biological processes or blood purification [3-5]. However, due to the emerging problem of fouling, either blending with an additional polymer or other modification of polysulfones membrane has been found to be necessary [6,7]. By using polymer-grafted phosphonium salts, a number of advantages are achieved, like preventing substrate contamination, solving toxicity problems, and improving antibacterial activity [8]. Consequently, polysulfones may be modified by electrophilic substitution reactions, which include chloromethylation, followed by phosphonation. In the last years, the interest for phosphorus-based polymers used in biomedical fields has been accelerating, as they are biodegradable (by enzymatic digestion of phosphate linkages under physiological conditions), blood-compatible and, also, as they present structural similarities to the naturally occurring nucleic and teichoic acids, reduce protein adsorption and lead to strong interactions with dentin, enamel or bones. Also, phosphorus-functionalized polymers play a major role as carriers for biologically-active substances [9]. On the other hand, among fluoropolymers, polyvinylidene fluoride (PVDF) has a wide applications range - from electronics to medicine – having high chemical resistance to many acids and alkalis and good blood compatibility. PVDF films have been used as attractive membrane materials in ultrafiltration, microfiltration, and pervaporation, especially in protein filtration, because their hydrophobicity can reduce surface fouling. In medical technology, PVDF has been reported as a scaffold for cell culture, because its piezoelectric properties can enhance cellular adherence and proliferation [10,11]. Blending of PVDF with some biodegradable polymers, such as poly(lactic acid) or poly(glycolic acid), reduces of protein adsorption and platelet adhesion, thus becoming a good thrombus-resisting material. Based on these properties, relevant biological applications have been developed to highlight the considerable contributions of PVDF to the medical domain [12,13]. Therefore, it is of interest to find out whether polysulfones modified by quaternization can be designed for biomedical applications, in blends with poly(vinylidene fluoride), and to establish their impact on different properties.

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The aim of the present paper was to study the correlation between the chemical structures of novel quaternized polysulfone, poly(vinylidene fluoride), and those of the corresponding blends on their surface properties. Solvent‘s influence on the morphology of PSFP/PVDF blend films, as well as its ability to improve the film-forming characteristics, necessary for a better understanding and control of their prospective applications in plasma separation or biomedicine, were studied.

MATERIAL AND METHODS

UDEL-1700 polysulfone (Union Carbide) (PSF) (Mn=39000 g/mol; Mw/Mn=1.625), a commercial product, was purified by repeated reprecipitations from chloroform and dried for 24h in vacuum, at 400C, before being used in the synthesis of chloromethylated polysulfone. A mixture of commercial paraformaldehyde with an equimolar amount of chlorotrimethylsilane (Me3SiCl) as a chloromethylation agent, and stannic tetrachloride 0 (SnCl4) as a catalyst, was used for the chloromethylation reaction of polysulfone, at 50 C. The reaction time necessary to obtain chloromethylated polysulfones (CMPSF) was 72h. Finally, the samples were dried under vacuum, at 400C. Polysulfone with triphenylphosphonium pendant groups, PSFP, was synthesized by reacting chloromethylated polysulfone (6.29% chlorine content) with triphenylphosphine, in the presence of dioxane [14]. The mixture was maintained under stirring in nitrogen atmosphere, for 15h at 900C. The thus obtained viscous product was filtered, washed with dioxane and ethyl ether, and finally dried. The contents of ionic chlorine, Cli, and total chlorine, Clt, were determined by potentiometric titration (Titrator TTT1C Copenhagen), with 0.02 N AgNO3 aqueous solutions. The ratios between the ionic chlorine and total chlorine contents show the quaternization reaction of CMPSF at a transformation degree around 46%. Poly(vinylidene fluoride) (-CH2-CF2-)n (Mw = 534000) and N,N-dimethylformamide (DMF) were purchased from Aldrich Chemical Company Inc. and were used as received, without further purification. Chemical structure of PSFP was illustrated in scheme 1:

- Cl C H + 6 5 CH P C6H5 CH 2 C H 3 6 5 O C O SO [ 2 ] n CH (1) 3

Scheme 1. Chemical structure of quaternized polysulfone with triphenilphosphonium pendant group.

PSFP/PVDF blend solutions (PSFP and PVDF, 8g/dL concentration) for film preparation were performed in DMF. The solutions were mixed and stirred for 8h at room temperature, subsequently degassed and poured on glass plates. The composition of the two polymers in the blends was varied from 0/100, 20/80, 30/70, 40/60, and 100/0 v/v PSFP/PVDF, respectively. The films prepared from solutions-cast blends were initially solidified by slow drying, in a saturated atmosphere of the used solvent, for 72h and finally under vacuum, at 30°C for 24h. Films morphology was studied with a Quanta200 type environmental scanning electron microscope (EDX-SEM), operating at 30 kV, with secondary electrons in low

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vacuum. Also, atomic force microscopy (AFM) images were obtained on a SPM SOLVER Pro-M instrument that operates in semi-contact mode, at 3030 m2 scan area. A cantilever - NSG10/Au silicon type - with a 10 nm curvature radius and 286 kHz oscillation mean frequency was used to investigate the solvent influence on the surface modification of the polymers.

RESULTS AND DISCUSSION The AFM images of the pure components prepared by casting solution in DMF exhibit 2 different structures. For the PVDF film, the scanning area of 3030 m reveals a hydrophobic network-like aspect with cluster formations and interconnected pores; also, for the PSFP film, at the same scanning area, the obtained images show a porous structure (Figure 1).

Figure 1. 2D and 3D AFM images for (a, a‘) PSFP and (b, b‘) PVDF films.

SEM images (Figure 2) of 20/80, 30/70, and 40/60 PSFP/PVDF films, prepared from DMF solutions, emphasize that presence of PSFP maintains cluster formations (with diameter values which decrease from around 4 m through 1 m at higher contents), and enhances surface roughness. The interconnected pores become more pronounced and, at the same time, addition of modified polysulfone may arrest their morphological evolution [15]. Therefore, the average dimensions of pores are around 2 m . This type of membranes - with surface topography and roughness as important factors in determining the response of cells to a foreign material - represents an excellent scaffold for applications in cellular and tissue engineering. Mention should be made over the fact that fibroblasts and chondrocyte cells were shown to grow well in a 3D porous membrane, underlining their superior properties (specific molecular microarchitecture and controlled porosity) for tissue regeneration applications [16].

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Figure 2. SEM micrographs of (a) 20/80, (b) 30/70, and (c) 40/60 v/v PSFP/PVDF films.

The results show that, for tissue engineering, obtaining of some porous and interconnected 3D polymer networks is recommended. Thereby, PSFP/PVDF blends can be used to accomplish the required properties for specific application, making them good tissue-engineered candidates. As a result, considering the traditional processes and the recently developed techniques, the improved ability to control the porosity and molecular microarchitecture of the PSFP/PVDF hydrophobic membranes will drive the research closer to proposed goals.

CONCLUSIONS AND FUTURE WORK New quaternized polysulfone (prepared by quaternization of chloromethylated polysulfone with triphenylphosphine)/poly(vinylidene fluoride) blends was investigated for getting information on their specific porous microarchitecture. According to the history of blends film preparation by a dry-cast process, the obtained results pointed out a hydrophobic network-like structure with interconnected pores, more pronounced once the PSFP was added into the system. This specific microarchitecture, with desired surface roughness and controlled porosity, represents an excellent scaffold for applications as porous membranes in tissue engineering. As a future work it is possible not only to specifically control individual and group pore architecture, but also to take the next step to create microvascular features to improve integration within host tissues. Yet, the improvement in the structure and increased pore interconnectivity of the porous scaffolds is claimed for the development of artificial blood vessels or peripheral nerve growth.

Acknowledgment: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS-UEFISCDI, project number PN-II-RU-TE-2012-3- 0143 (2013 stage).

REFERENCES [1]. Meredith, J. C., et al. 2003. Combinatorial Characterization of Cell Interactions with Polymer Surfaces. Journal of Biomedical Materials Research 66A, 483-491. [2]. Averous, L. 2000. Blends of Thermoplastic Starch and Polyesteramide: Processing and Properties. Journal of Applied Polymer Science 76, 1117-1128. [3]. Zornoza, B., et al. 2009. Mesoporous Silica Sphere-Polysulfone Mixed Matrix Membranes for Gas Separation. Langmuir 25, 5903–5909.

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[4]. Silva, A. I. and Mateus, M. 2009. Development of a Polysulfone Hollow Fiber Vascular Bio-artificial Pancreas Device for In vitro Studies. Journal of Biotechnology 139, 236-249. [5]. Yang, Y.S., et al. 2004. Synthesis of Sulfonated Polysulfone-block-PVDF Copolymers: Enhancement of Proton Conductivity in Low Ion Exchange Capacity Membranes. Macromolecules 37, 1678–1681. [6]. Malaisamy, R., et al. 2002. Cellulose Acetate and Sulfonated Polysulfone Blend Ultrafiltration Membranes. II. Pore Statistics, Molecular Weight Cutoff and Morphological Studies. Journal of Applied Polymer Science 84, 430-444. [7]. Velu, S. et al. 2011. Performance Enhancement of Polysulfone Ultrafiltration Membrane by Blending with Polyurethane Hydrophilic Polymer. International Journal of Chemical and Analytical Science 2, 87-92. [8]. Popa, A., et al. 2003. Study of Quaternary ‗Onium‘ Salts Grafted on Polymers: Antibacterial Activity of Quaternary Phosphonium Salts Grafted on ‗Gel-Type‘ Styrene- Divinylbenzene Copolymers. Reactive and Functional Polymers 55, 151-158. [9]. Dahiyat B. I., et al. 1995. Controlled Release from Poly(phosphoester) Matrices. Journal of Controlled Release 33, 13–21. [10]. Chen, M. H., et al. 2005. Interactions of Acinar Cells on Biomaterials with Various Surface Properties. Journal of Biomedical Materials Research 74, 254-262. [11]. Rodrigues, M. T., et al. 2008. β - PVDF Membranes Induce Cellular Proliferation and Differentiation in Static and Dynamic Conditions. Materials Science Forum 587, 72-76. [12]. Liu T. Y., et al. 2005. Surface Characteristics and Hemocompatibility of PAN/PVDF Blend Membranes. Polymers for Advanced Technologies 16, 413-419. [13]. Chen H. C., et al. 2011. Mechanical Properties and Biocompatibility of Electrospun Polylactide/Poly(vinylidene fluoride) Mats. Journal of Polymer Research 18, 319-327. [14]. Buruiana L. I., et al. 2012. Electrical Conductivity and Optical Properties of a New Quaternized Polysulfone. Polymer Bulletin 68, 1641–1661. [15]. Park J. Y., et al. 2006. Polysulfone-Graft-Poly(ethylene glycol) Graft Copolymers for Surface Modification of Polysulfone Membranes. Biomaterials 27, 856-865. [16]. Annabi N., et al. 2010. Controlling the Porosity and Microarchitecture of Hydrogels for Tissue Engineering. Tissue Engineering Part B 16, 371-383.

ABOUT THE AUTHORS L. I. Buruiana, E. Avram, S. Ioan ―Petru Poni‖ Institute of Macromolecular Chemistry, 700487 Iasi, Romania, E-mail: [email protected] A. Popa, Institute of Chemistry Timisoara of Romanian Academy, 300223 Timisoara, Romania, E-mail: [email protected]

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ANTIOXIDANT CAPACITY OF SALVIA OFFICINALIS EXTRACT REGARDING THE SUNFLOWER OIL USED IN FOOD THERMAL APPLICATIONS

Nadina Buta, N. Popa, Nicoleta Bordea, Mariana Poiană and T.I. Traşcă

This work was published during the program: ―DOCTORAL STUDIES FOR TRENING IN RESEARCH (FOR-CE)‖, POSDRU/CPP107/DMI1.5/S/80127 PROJECT

Abstract: To investigate the retarding lipid oxidation of sunflower oil using sage extract compared with butylated hydroxytoluene (BHT) refined sunflower oil free of additives and supplemented by three concentration levels of sage extract (200, 600 and 1000 ppm) and one level of butylated hydroxytoluene (200 ppm) were subjected to convective heating for 1, 4, 8, 12, and 16 hours. The analyses which were made in order to watch the progress of lipid oxidation are the peroxid value (PV), p-anisidine value (p-AV) and TOTOX value. FRAP value and Total phenolics value were made in order to establish the antioxidant characteristics of sage extract. According to the survey result that sage extract shows a significantly inhibitory effect on lipid oxidation during heat treatment.

Key words: sunflower oil, convective heating, sage extract, antioxidant, oxidative stability

INTRODUCTION Food industries and scientists have turned the interest to natural phenolic antioxidants in order to minimize the unwanted effects of the synthetic food preservatives in human health. Because of their scavenging activity against free radicals that promote lipid oxidation in food, natural phenolic antioxidants gained interest. Frying is an important process and widely used for the preparation of different fried foods providing desirable flavour, colour and texture but can also negatively affect oils deteriorating them. Sunflower oil is a vegetable trygliceride oil produced from sunflower seeds and contains 60-75% linoleic acid. Different types of sunflower oil were developed such as high oleic, mid oleic and high linoleic [1]. Sunflower oil is commonly used in food as a frying oil and is the fourth largest oil source in the world, after soybean, palm and canola oil. In some countries is the most popular vegetable oil and it is used as a source of essential linoleic acid. The main deterioration process that occurs during thermal processing is lipid oxidation and the decomposition of oxidation products which result in decreased nutritional value and sensory quality [2, 3]. To protect against oxidation synthetic antioxidants are added for improving oxidative stability of vegetable oils. This addition of synthetic antioxidants is not recommended because of their toxicity and carcinogenicity instead finding natural antioxidants sources is encouraged and the interest for these increased [3]. Salvia officinalis (also known as sage) belongs to the family of Lamiaceae and has a long history of medicinal and culinary use. The essential oil of sage contains borneol, cineole and thujone. Sage leaf contains oleic acid, ursonic acid, ursolic acid, tannic acid, cornsolic acid, chlorogenic acid, caffeic acid, fumaric acid, niacin, flavones, and estrogenic substances [4].

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MATERIAL AND METHODS Materials. The sun flower oil without addition of any antioxidants used was ―Tip‖ oil. Sage extract (SE) was obtained from dried leaves originated from Romania (Fares). All other chemicals and solvents used were purchased from Merck (Darmstadt, Germany) and were of analytical grade. Rotary evaporator-Heidolph Laborata 4000 Convection oven-Esmach, Italy, 1200W Spectrophotometer-Analytic Jena Specord 205

Antioxidant Activity (FRAP Assay) The antioxidant activity of sage extract was measured using the ferric reducing antioxidant power (FRAP) assay [5]. In order to evaluate antioxidant activity, 0,1 g SE, respectively BHT were mixed with 20 mL ethanol/water (70:30, v/v) for 10 min, then the solution was filtered and used for analysis. FRAP values were obtained reading the absorbance changes at 595 nm using a UV-VIS spectrophotometer. Results were expressed as mM Fe 3+ equivalents/g sage extract, respectively BHT.

Total Phenols Assay Total phenolic content of sage was determined using the Folin-Ciocalteu colorimetric method [6]. A calibration curve using gallic acid was prepared and the absorbance of the standards and samples were measured at 750 nm. Results were expressed as mM gallic acid equivalents/g.

Peroxid Value (PV) Weigh 2.00 g oil sample into a 250 mL Erlenmeyer flask and then were added 10 mL of chloroform and 15 mL of glacial acetic acid. The flask was swirled until the sample was dissolved and then was added 1 mL of saturated potassium iodine (KI) solution. The flask was closed and let for 1 min in dark and after 5 more minutes to stand. 75 mL of distilled water was added and then in presence of starch was slowly titrate with 0.01 N sodium thiosulfate (Na2S2O3) [7].

The p-anisidine value (p-AV) The p-anisidine value is a measurement of carbonyl content in the oils and was detrmined by the standard method according to AOCS [8]. It is based on the reactiveness of the aldehyde carbonyl bond on the p-anisidine amine group, leading to the formation of a Schiff base that absorbs at 350 nm. 2 g of the sunflower oil samples were dissolved in 25 mL isooctane and absorbance (A1) of this fat solution was measured at 350 nm against a blank of isooctane. An aliquot (5 mL) of this solution, respectively 5 mL of isooctane was transferred to each of two test tubes of 10 mL and 1 mL of anisidine solution (0.25% g/v glacial acetic acid) was added to each. After 10 min the absorbance (A2) was measyred at 350 nm against isooctane containing p-anisidine. p-AV was calculated according to the formula: p-AV = 25 x 1.2 x A2 - A1 / w

TOTOX value The total oxidation value (TOTOX) was used to estimate the oxidative deterioration of lipids. Totox value is defined as the sum of both values (PV and p-AV) to total oxidation and was calculated according to the formula: TOTOX value = 2 x PV + p-AV

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RESULTS AND DISCUSSION Antioxidant characteristics of sage extract (SE) and butylated hydroxytoluene (BHT) are presented in table 1.

Table 1. Antioxidant characteristics of SE and BHT Sample FRAP value (mM Fe Total phenolics (mM 3+ /g) GAE/g) Sage extract (SE) 6.56 101.2 BHT 1.36 -

Peroxid value was used as indicator for the primary oxidation of sunflower oil. The primary products of lipid oxidation are hydroperoxides and determination of peroxides can be used as oxidation index for the early stages of lipid oxidation. The results of PV are presented in table 2.

Table 2. Effect of SE and BHT on peroxid value during sunflower heating in convection oven Time Control BHT SE SE SE (hours) 200 ppm 200 ppm 600 ppm 1000 ppm 1 4.87 4.10 3.83 3.28 2.94 4 8.52 8.28 7.95 6.73 5.59 8 13.13 12.92 12.87 11.56 10.55 12 18.65 17.52 17.49 16.75 16.11 16 21.02 20.83 19.61 18.57 17.36

According to the results we can state that SE in a dose of 200 ppm behave similary to BHT. In a dose of 600 ppm and 1000 ppm sage extract shows better results regarding the peroxid value of sunflower oil.

p-AV value is a mesurement of the secondary oxidation products (alcohols, ketones, aliphatic aldehydes and acids). Results regarding p-AV value are presented in table 3.

Table 3. Effect of SE and BHT on p-AV during sunflower oil heating in convection oven Time Control BHT SE SE SE (hours) 200 ppm 200 ppm 600 ppm 1000 ppm 1 17.27 16.02 15.85 14.74 13.52 4 28.00 26.48 25.94 24.66 23.71 8 45.44 43.72 42.80 40.49 39.54 12 54.12 52.46 52.37 50.05 48.21 16 68.16 67.44 67.33 65.11 63.62

The results obtained following the p-AV determination confirm that sage extract in doses of 200 and 600 and 1000 ppm reduced the lipid oxidation in a more efficient manner than BHT.

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CONCLUSIONS AND FUTURE WORK The present research was carried out in refined sunflower oil free of additives, supplemented by three concentration levels of sage extract (200, 600, 1000 ppm) and one level of BHT (200 ppm). The samples were thermal treated in convective oven for 1, 4, 8, 12 and 16 hours. According to the results it can be observed that because of thermal treatments oxidation of sunflower oil increased. Supplementation with sage extract (SE) and BHT reduced the lipid oxidation resulting significant differences. The inhibitory effect of SE against primary oxidation of lipids was concentration-dependent. Significant differences were also observed at p-AV results and it can be seen that phenolic compounds from sage had a strong inhibitory effect on the secondary lipid oxidation. The efficiency of sage extract regarding oxidative stability of sunflower oil during thermal applications increased with increasing concentration of the natural extract and prove that sage extract is a very efficient inhibitor of lipid oxidation.

REFERENCES [1]. Frank D. Gunstone, The chemistry of oils and fats source, composition,properties and uses, Australia, 2004, Blackwell Publishing Ltd [2]. Poiana, M.A., Enhancing Oxidative Stability of Sunflower Oil during Convective and Microwave Heating Using Grape Seed Extract; International Journal of Molecular Sciences; 2012, 13:9240-9259. [3]. Zhang, Y., Yang, L., Zu, Y., Chen, X., Wang, F., Liu, F., Oxidative stability of sunflower oil by carnosic acid compared with synthetic antioxidants during accelerated storage; Food Chem; 2010, 118:656-662. [4]. http://en.wikipedia.org/wiki/Salvia_officinalis [5]. Benzie, I.F.F., Strain, L., Ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: The FRAP assay; Anal. Biochem., 1996 [6]. Singleton, V.L., Orthofer, R., Lamuela-Raventos, R.M., Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Meth. Enzymol, 1999 [7]. Hohmann, J., Zupko, I., Redei, D., Csanyi, M., Falkay, G., Mathe, I., Protective effects of the aerial parts of Salvia officinalis, Melissa Officinalis and Lavandula angustifolia and their constituents against enzyme- dependent and enzyme-independent lipid peroxidation, Planta Medica, 1999, 65:576-8. [8]. Association of Coaching Supervisors. Official and Recommended Practices of the American Oil Chemists‘ Society, Official Methods and Recommended Practices, 5th ed.; Firestone, D., Ed.; AOAC Press: Champaign, IL, USA, 1998.

ABOUT THE AUTHORS Nadina Buta, Faculty of Food Processing Technology, Banat‘s University of Agricultural Sciences and Veterinary Medicine from Timisoara, Calea Aradului 119, RO 300645, Timisoara Romania; E-mail: [email protected] N. Popa, Faculty of Food Processing Technology, Banat‘s University of Agricultural Sciences and Veterinary Medicine from Timisoara, Calea Aradului 119, RO 300645, Timisoara Romania; E-mail: [email protected] Nicoleta Bordea, Faculty of Food Processing Technology, Banat‘s University of Agricultural Sciences and Veterinary Medicine from Timisoara, Calea Aradului 119, RO 300645, Timisoara Romania; E-mail: @yahoo.com

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Mariana Poiană, Faculty of Food Processing Technology, Banat‘s University of Agricultural Sciences and Veterinary Medicine from Timisoara, Calea Aradului 119, RO 300645, Timisoara Romania; E-mail: [email protected] T.I. Traşcă, Faculty of Food Processing Technology, Banat‘s University of Agricultural Sciences and Veterinary Medicine from Timisoara, Calea Aradului 119, RO 300645, Timisoara Romania; E-mail: [email protected]

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IMPROVE PERFORMANCE OF THE POLYSULFONE MEMBRANES INDUCED BY PRESENCE OF POLYVINYL ALCOHOL

A. Filimon1, E. Avram1, and S. Ioan1

Abstract: Understanding of mechanisms which involve the specific interactions developed in polysulfone/polyvinyl alcohol blends in N-methyl-2-pyrrolidone, and the manner in which these interactions affect the rheological properties were studied. These results were discussed according to the hydrophobic (i.e., polysulfones)/hydrophilic (i.e., polyvinyl alcohol) characteristics and to their different properties, such as flexibility and surface morphology. Consequently, knowledge on the rheological properties of these blends is important for establishment of the macroscopic properties specific to ternary systems, allowing anticipation of the microstructural properties of polysulfone membranes achieved in presence of the polyvinyl alcohol. Key words: Polysulfone, Polyvinyl Alcohol, Polymer Blends, Specific Interactions.

INTRODUCTION Polymeric materials are used for a wide variety of applications, namely, in separation technology, food processing, biological processes, medical devices and blood purification or hemodialysis [1]. In this context, polysulfone (PSF) is one of the materials most important and widely used; thus, PSF possessing superior properties, namely, good optical properties, high thermal and chemical stabilities, and most importantly, excellent biocompatibility and film-forming properties [2,3], and therefore, represent the ideal candidate for the manufacture of commercial membranes and biomedical applications [4,5]. However, appear some disadvantages in its applicability, due to the hydrophobic nature of main chain, so as blending with an additional polymer or other modification becoming necessary [6]. Therefore, polysulfone can be modified to improve its performance [7], by chloromethylation, a reaction of considerable interest from both theoretical and practical points of view, such as obtaining of precursors for functional membranes, coatings, ion exchange resins, ion exchange fibers and selectively permeable membranes [8]. Furthermore, the chemical modification of chloromethylated polysulfones by the quaternization of ammonium groups is an efficient method for increasing the hydrophilicity. In recent decades, an alternative in order to obtain of new polymeric materials is represented by the blending of the already existing polymers. Consequently, polymer blends are extensively investigated because of their growing interest for practical applications in high-performance areas like biotechnology, medicine or pharmacy [9,10]. In this context, in the present study, polyvinyl alcohol (PVA) was selected due to the excellent its physical properties and also for its capacity to improve the performance of PSF membranes. Based on these versatile properties, PVA has been widely used, especially in fiber coating, adhesives, films for packing and farming, and also as coating agent for tablets and capsules [11]. Previous studies have been carried out on the quaternization reaction of chloromethylated polysulfones for obtaining water soluble polymers with various amounts of ionic chlorine and have been investigated some properties related to morphological and structural-conformational-rheological aspects [12]. The purpose of the present work is to investigate the flow behavior of the blends obtained from PSF and PVA in N-methyl-2- pyrrolidone (NMP), and to obtain information concerning competition between the interactions exhibited in ternary system, at different compositions of PVA. In addition, the rheological properties were correlated with the surface properties which derive from the characteristics of the polymeric chains. These

77 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA results are useful in future investigations concerning utilization of these blends as semipermeable membranes in biomedical field.

MATERIAL AND METHODS Commercial polysulfone powder form (PSF) (UDEL-1700) was obtained from Union Carbide Company (Texas City; number-average molecular weight, Mn  39,000 g/mol). Polyvinyl alcohol (PVA) (Celvol) was purchased from Celanese Corporation (Texas). According to product specifications, Celvol PVA has a hydrolysis degree ranging between 98.0-98.8% and average weight molecular weight, Mn  23,000 g/mol. Homogeneous solution of 15 g/dL PSF was prepared by dissolution in N-methyl-2-pyrrolidone (NMP) for 24 h at room temperature. In the same time, solution of 10 g/dL PVA was obtained by dissolution in NMP heated at 80°C, and then the dissolution continued in a water bath with a constant temparature of 80°C by continuous stirring for 7 h, and subsequently degassed. The PSF/PVA blends were prepared by mixing the two homogeneous solutions in order to obtain different mixing ratios. Rheological measurements for PSF/PVA blend in NMP were carried out with a Bohlin CS50 rheometer manufactured by Malvern Instruments. The measurements were performed using a measuring system cone–plate geometry, with a cone angle of 4 and a diameter of 40 mm. Shear viscosities were registered over the 0.5–1500 1/s shear rate domain, at temperature of 25 °C. During oscillatory shear tests, frequency was varied between 0.1 and 60 Hz. Rheological tests were obtained with an accuracy of ±5 %. Atomic force microscopy (AFM) images were obtained on a SPM SOLVER Pro-M instrument. A NSG10/Au Silicon tip with a 35 nm curvature radius and 255 kHz oscillation mean frequency was used to investigate film surface morphology. The apparatus was operated in semi-contact mode, over 2 x 2 μm2 scan area, 512 x 512 scan point size images being thus obtained.

RESULTS AND DISCUSSION Synergistic effects of polymer blends on dynamic viscosity and viscoelasticity Studies performed in solutions of polymer blends represent a particular importance, due to their complex behavior, influenced by the hydrophilic/hydrophobic characteristics of each polymer in the system. Additionally, the polymer structures, their mixing ratios and used solvent influence the specific interactions from the system and therefore, the rheological behavior. In agreement with these remarks, the flow behavior of PSF/PVA blend in NMP, in different mixing ratios, was observed from the dynamic viscosity - shear rate dependence (Figure 1).

Figure 1. Logarithmic plot of dynamic viscosity as Figure 2. Activation energy versus a function of shear rate for PSF/PVA blend in weight fraction of PVA at 25°C different mixing ratios at 25°C

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According to Figure 1, a Newtonian behavior for pure polymer components and different blends with a minimum content of PVA appears, while for blends with higher content of PVA the thinning behavior is predominantly. Thus, as one can see, introduction of PVA over the 0.5 weight fraction, resulted in an increase of the blends dynamic viscosity at low shear rates, and determines decrease of the blends dynamic viscosity at higher shear rates. In this context, one can conclude that these blends present properties of the pseudoplastic materials, characterized by a reduced entanglement density, and an enhancing number of oriented segments, as a result of the increasing shear rates. Similar to dynamic viscosity, activation energy (Ea , evaluated from Arrhenius law [13]) gets modified, being affected by the nature groups from the PSF and PVA, and also by PVA composition and solvent. Thus, examination of Figure 2 reveals that, the Ea value for 100/0 PSF/PVA blend is higher than for 0/100 PSF/PVA blend, being influence by the PSF high rigidity comparatively with PVA. In case of other blends (25/75, 50/50 and 75/25 wt/wt) the values are influenced by the rigidity/flexibility of PSF or PVA chains, and also by competition between homogeneous and heterogeneous interactions. Therefore, addition of PVA to the solution of PSF favors polymer (1)-polymer (2) interactions, while the PVA chains dissolved in NMP exhibits the ability to form aggregates through hydrogen- bonding and association phenomena. The effect of hydrophobic/hydrophilic characteristics of polymers and blends composition on the mobility of the segments from the shear field, is reflected in the storage, G' , and loss, G" , moduli. Thus, Figure 3 illustrates the variation of storage and loss moduli versus frequency for PSF, PVA and PSF/PVA at different mixing ratios.

Figure 3. Log-log plots of storage and loss moduli as a function of frequency for different compositions of PSF/PVA blends, at 25°C

As one can see, for all blends that, over the low frequency regime, viscoelastic properties are characteristic for a viscoelastic fluid, where the is always higher than the 2 1 . Generally, in this range G'~ f and G"~ f , respectively; this dependence is observed 1.4÷2.0 0.8÷1.0 for all blends, where and are proportional to f and f , respectively. Also, one can remark that, the frequencies corresponding to the crossover point, which delimits the viscous flow from the elastic one, and for which G'= G", exhibit lower values for PSF in NMP, and become higher with increasing the PVA content in polymer blends.

Morphological aspects Investigation of the rheological properties of PSF/PVA blend is essential for better understanding the morphology developed by their films. These combined studies contribute to a better knowledge on the nature of the interactions in the system, and on the relation between shear deformation and texture. AFM investigation of the PSF/PVA films can reflect the influence of these interactions on the morphological properties. Thus, the 79 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

AFM images (Figure 4) reveal the morphology of films changes, because of the hydrophobic/hydrophilic characteristics and compositions of blend polymers. Two- dimensional (2D) AFM images show that all investigated blends films are not smooth, there are ordered domains, in which pores and nodules with different size and intensity are distributed. This is also highlighted by the higher values of the root mean square roughness parameter, Sq , (more than 1 nm); thus, Sq =1.21 nm for PSF membrane comparatively with PVA membrane ( Sq =12.41 nm). Moreover, different morphologies suggested that the structure of the modified membranes had been altered due to the addition of the flexible and hydrophilic groups of PVA in the casting polysulfone solutions. When the membrane was modified with PVA in different ratios, the surface presence more nodules compared with membrane unmodified. Additionally, the hydrogen-bonding and homogeneous and heterogeneous interactions exhibited in PSF/PVA/NMP ternary system should be among the factors affecting the morphology of their films surface.

Figure 4. 2D AFM images for PSF/PVA blend films in different mixing ratios: (a) 100/0, (b) 0/100, (c) 25/75, (d) 50/50, (e) 75/25

CONCLUSIONS AND FUTURE WORK Influence of the structural characteristics of polymers and composition of mixed polymers is reflected on the rheological properties. The rheological behavior of PSF/PVA blends is affected by specific interactions, including hydrogen-bonding and association phenomena, generated by polyvinyl alcohol presence in the system. A Newtonian flow was observed over the entire studied shear rate range for the PSF, PVA and blends with small content of PVA (75/25 wt/wt PSF/PVA), while with increasing composition of PVA (50/50 and 25/75 wt/wt PSF/PVA blends) the solutions exhibits a shear thinning behavior. The flow activation energy shows that PVA which possesses a higher flexibility, comparatively with PSF, takes lower values, while the more rigid structure of the PSF increase the activation energy. On the other hand, the presence of PVA favors polymer (1)-polymer (2) interactions, and exhibit the ability to form aggregates through hydrogen- bonding and association phenomena, leading to the increases the activation energy. The storage and loss moduli exhibit the power law dependence on frequency, where the exponents are characteristic to viscoelastic fluids. Over the studied frequency range, these properties are influenced by the chemical structures and composition of PVA. Additionally, the surface morphology derives from the characteristics of the polymeric chains and, also is influenced by the history of the membranes realized from their

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solutions. Specific interactions, association and structured phenomena, generated by hydrogen-bonding, should be among the factors affecting the flow behavior and surface properties of the PSF/PVA blends. The results of this study have demonstrated that PSF/PVA blends may provide some important advantages concerning to their specific applications. Knowledge on their rheological properties in correlation with the morphological aspects, lays at the basis their future applications, such as obtaining semipermeable membranes used in biomedical field.

Acknowledgment: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number PN-II-RU-TE-2012- 3-0143 (2013 stage).

REFERENCES [1]. Barzin, J., et al. 2004. Effect of Polyvinylpyrrolidone on Morphology and Performance of Hemodialysis Membranes Prepared from Polyether sulfone. Journal of Applied Polymer Science 92, 3804-3813. [2]. Wang, T., et al. 2006. Antifouling Ultrafiltration Membrane Composed of Polyethersulfone and Sulfobetaine Copolymer. Journal of Membrane Science 280(1/2), 343-350. [3]. Ran, F., et al. 2011. Biocompatibility of Modified Polyethersulfone Membranes by Blending an Amphiphilic Triblock Co-polymer of Poly(vinylpyrrolidone)-b- Poly(methylmethacrylate)-b-Poly(vinyl pyrrolidone). Acta Biomaterialia 7, 3370-3381. [4]. Zornoza, B., et al. 2009. Mesoporous Silica Sphere−Polysulfone Mixed Matrix Membranes for Gas Separation. Langmuir 25, 5903-5909. [5]. Velu, S. and Muruganandam, L. 2011. Development of PES Membranes for Separation of Metal Ions: Effect of Polymer Composition. International Journal of Chemistry Sciences 9(2), 757-768. [6]. Guiver, D., et al. 2000. Functionalized Polysulfones: Methods for Chemical Modification and Membrane Applications in Membrane Formation and Modification, Eds. Pinnau, I., Freeman, B.D., American Chemical Society, Chap. 10, pp. 137. [7]. Tomaszewska, M., et al. 2002. Physical and Chemical Characteristics of Polymer Coatings in CRF Formulation. Desalination 146(1), 319-323. [8]. Huang, Y. and Xiao, C. 2007. Miscibility and Mechanical Properties of Quaternized Polysulfone/Benzoyl Guar Gum Blends. Polymer 48, 371–381. [9]. Peng, Y., et al. 2012. Characterization and Performance of PVA/PSF Composite Hollow Fiber UF Membrane Prepared with Interfacial Polymerization. Polymer Engineering Science 52, 557-565. [10]. Akbari, A., et al. 2010. Synthesis and Characterization of Composite Polysulfone Membranes for Desalination in Nanofiltration Technique. Water Science Technology 62, 2655-2663. [11]. Kawai, F. and Hu, X. 2009. Biochemistry of Microbial Polyvinyl Alcohol Degradation. Applied Microbiology and Biotechnology 84, 227-237. [12]. Ioan, S. and Filimon, A. 2012. Biocompatibility and Antimicrobial Activity of Some Quaternized Polysulfones, in Antibacterial Agents/book 2. Ed. Varaprasad, B., InTech Publisher, Rijeka, Chap. 13, pp. 249-274. [13]. de Vasconcelos, C.L., et al. 2002. Rheology of Polyurethane Solutions with Different Solvents. Polymer International 51, 69–74.

ABOUT THE AUTHORS 1 A Filimon*, E. Avram, S. Ioan, ―Petru Poni‖ Institute of Macromolecular Chemistry, Grigore Ghica Voda Alley 41A, Iasi, Romania, E-mail: [email protected] 81 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

DEVELOPMENT AND ANALYSIS OF A TOPICAL PHARMACEUTICAL PRODUCT CONTAINING RIBES NIGRUM LEAVES EXTRACT

L.E. Dutu, .T.D.Balaci, C.E.Gird, L.M. Popescu, I. Dutu

Abstract: This study proposes a comparative pharmacognostic examination of black currant leaves (Ribes nigrum) and red currant leaves (Ribes rubrum), obtaining a selective dry extract which will be finally including in a topic anti-rheumatic preparation. Using specific reaction and spectrophotometric methods it was established that Ribes nigrum leaves are more rich in bioactive principles. Using Ribes nigrum leaves as vegetal raw material, a selective dry extract was obtained and physico-chemical characterized. Finally, the extract was included in an ointment carbopol base. Thus, the manufactured gel was described from a physical and rheological point of view. Key words: Ribes nigrum leaves, extract, ointment

INTRODUCTION The Ribes genus consists of about 50 species, most of them being woody species. There are nine species which are known in Romania flora. These are: Ribes nigrum, R. rubrum, R. grossularia, R. heteromrphum, R. petraeum, R. alpinum, R. aureum, R. sanguineum and R. spicatum. The most frequently cited in the scientific literature are Ribes nigrum L. and Ribes rubrum L. [ 6,8]. Ribes nigrum (black currant) is a species native to northern Asia and northern Europe. The fruit (berry-type) has a dark purple to black skin (Fig. 1). According to phytochemical data, black currant fruit have a hight content of anthocyanins (delphynidine- glycosides and cyanidine-glycosides), tannins, vitamin C, minerals (potassium, iron, zinc, phosphorus). Due to these compounds the fruitsare well-known in therapy as capillary- protective agents, vitamins and anti-oxidants. Ribes rubrum (red currant) is a species native to western Europe. The fruit is a red translucent berry, usually about 1 cm diameter (Fig. 2). According to scientific data, the content of organic compounds and minerals are less, so red currant has especially culinary uses [ 6, 8. 13].

Fig. 1. Ribes nigrum (black currant) Fig. 2. Ribes rubrum (red currant

A comparative chemical study of fresh fruit of Ribes nigrum and Ribes rubrum L. was already reported and published by us [2]. The leaves harvested from these two species are less known. The infusion and the ointment is cited in folk medicine. The infusion is used for the treatment of arteriosclerosis,

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

hypertension, rheumatism, nephrolithiasis and gout. The ointment, prepared by mixing powdered leaves with hot butter, is used in rheumatism. This study proposes a comparative pharmacognostic examination of the leaves harvested from black and red currant leaves, in view to predict if therapeutic activities differences exist. After that, a selective extract will be obtained, and it be will be finally included in a topic anti-rheumatic preparation.

MATERIAL AND METHODS The raw materials are leaves of Ribes rubrum (red currant) and Ribes nigrum L. (black currant), harvested from Ialomitadistrict (Romania), at their full maturity, in June 2011. The leaves were naturally dried in the shadow, and conserved in laboratory conditions. 1. Pharmacognostic characterization of raw materials The identity was verified through a macroscopic exam and a microscopic exam (powder preparations, clarified with a chloral hydrate 800 g/L; Zeiss Imager D1 microscope, ob. 10x and 40x). For the qualitative analysis the raw material was successively extracted with different solvents (ethyl ether, alcohol, purified water). Half of the above alcoholic and aqueous solutions were hydrolyzed. Specific reactions were carried out in initial and hydrolyzed solutions, with the purpose to identify the active principles [1,3,4]. 2. Obtaining and chemical characterization of selective extract Preliminary, four extractive solutions were obtained, using black currant and red currant leaves as vegetal raw materials, and purified water and alcohol 70 per cent v/v as solvents. Flavones and polyphenolcarboxylic acids were selected as analytical markers. Spectrophotometric methods were used for to evaluate the content of flavones (reaction with aluminium chloride, etalone curve of rutin, detection at 427 nm) and polyphenolcarboxylic acids (formation of oxymes in the presence of sodium nitrite / hydrochloric acid and sodium hydroxide, etalone curve of caffeic acid, detection at 510 nm). For the spectrophotometric assay a UV-VIS Cecil Series 2000 spectrophotometer was used. Preliminary, parameter ,,loss on drying‖ was carried out [3,4,11,12]. The alcoholic black currant extract has the highest content of flavones and polyphenolcarboxylic acids, so we selected this extract for the following researches. For to obtain a selective extract, the vegetal drug was heat under a reflux condenser, using alcohol 70 per cent v/v as solvent, the extractive solution was concentrate in a rotary evaporator (Rotavapor R-215, Buchi) and then was evaporated to dryness in a lyophilizer(Christ Alpha 1-2, B. Braun Biotech International). The dry extract was characterized, by the following parameters: loss on drying, the content of flavones and the content of polyphenolcarboxylic acids. Foregoing methods were applied. 3. Formulation, preparation and quality control of the ointment: Ointment base with hydrophyllic polymer (polyacrylic acid) was prepared, in concentration of 1%.The ointment base was selected according to some of the known principles from pharmaceutical technology such as: stability, plastic and pseudoplastic flowing, slow release of drug, optimum viscosity and penetrability.The preparation method depends on the nature of the macromolecule [5,7,10]. Dry vegetal extract was incorporated in this ointment base, in concentration of 10 % (Table I)

83 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Table I. Hydrogel with vegetal extract Components Quantity (g) Ribes Nigri Extract 10.00 Polyacrilic acid (Carbopol 940) 1.00 Glycerin 15.00 Ethylic alcohol 5.00 Tween 80 0.60 Triethanolamine 0.65 Preservative solution to 100.00

The organoleptic properties, the pH (by potentiometry) and some rheologic properties of the gel, such as spreadability (by the extensiometric method Ojeda - Arbussa) and viscosity (using a Rheotec RC1 apparatus) were determined. The Rheotec RC 1 apparatus is a rotational rheometer cone / plate with an electronic unit allowing the digital control of the rotation speed and the direct recording of the measured values. [9,11,12]. The determinations were made after preparation and after certain periods of time.

RESULTS AND DISCUSSION 1. Black-currant leaves and red-currant leaves are simple, palmately lobed (with 3 - 5 lobes), with double dentate margin, slightly pubescent especially on the abaxial surface. The aspects of our leaf‘ samples correspond to those are mentioned in scientific literature for the leaves of Ribes nigrum and Ribes rubrum, so we considered that the identity of the two raw materials was verified [8,13]. The following anatomic characteristics were observed in both leaf‘microscopic preparations: epidermal cells with anomocytic stomata, spirally and annularly thichened vessels, long covering trichomes with striated cuticle. Carotenoids, saponins, catechic tannins, reducing compounds, flavones and polyphenolcarboxilic acids were identified in both leaves‘samples, by specific chemical reactions. 2. The foregoing active principles, except carotenoids, were identified in all four extractive soolutions. The intensity of the reactions was different, due to the vegetal raw material and the solvent. The results for quantitative analysis of extractive solutions are enclosed in table II.

Table II. Qualitative analysis of extractive solutions Vegetal raw Solvent Flavones (mg Polyphenolcarboxylic material rutoside %) acids (mg caffeic acid %) Ribes Purified water 215.02 693.68 nigrum Alcohol 70 %, v/v 330.88 1630.10 leaves Ribes Purified water 187.28 632.26 rubrum Alcohol 70 %, v/v 258.59 1522.07 leaves

The results suggest that Ribes nigrum leaves has a higher content of active principles than Ribes rubrum leaves, and alcohol 70% is the proper solvent. So, we decided to select the alcoholic black currant extract in our following studies.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Using 20 g vegetal drug, 1. 6 g dry extract was obtained. The selective extract is a green-brownish, odourless powder (fig.3). The results for quantitative analysis are enclosed in table III.

Fig. 3. Ribes nigrum dry extract Fig. 4. Ribes nigrum gel

Table III: Black currant extract - quantitative analysis Parameter Method Result Loss on drying (%) Eu. Ph, general method (105 0C) 4.87 Flavones spectrophotometric method 892.64 (mg %, expressed as rutoside) Polyphenolcarboxilic acids spectrophotometric method 6255.13 (mg %, expressed as caffeic acid)

The experimental gel presented a homogeneous appearance, a brown-red colour and a pleasant odour, characteristic to the vegetal products (fig. 4). These properties remain constant during preservation. The value of the gel‘s pH is within the limits approved by the Romanian Pharmacopoeia X-th edition, within the interval 6,5 – 6,6. These values are compatible with the mucosa and the skin, and do not lead to irritation after cutaneous application. The appearance of the extensiometric curves shows the gel‖s spreadability. The spreadability of the gel increases with the power of the application. The formulation has a good plasticity (table IV, figure 5).

Table IV. The plasticity of the gel Weight (g) Area (mm2) After preparation After 30 days After 90 days 145 2139.00 2073.94 1962.5 245 2677.29 2640.74 2444.21 345 3095.91 2978.73 2658.98 645 4924.02 4800.46 4534.16

85 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

6000

5000

4000 La preparare 3000 Dupa 30 zile

Dupa 90 zile Aria (mm2) Aria 2000

1000

0 145 245 345 645 Greutate (g)

Figure 5. The plasticity of the gel upon preparation and after 30 and 90 days

The rheogram shows that this gel presents a pseudoplastic flow, (typycal for such systems). By incorporating the vegetal extract in the polyacrylic acid gel, their rheologic properties are slightly modified, compared to the Carbopol gel (Figure 6).

Figure 6. The viscosity of the ointment base and hidrogel with Ribes nigri extract

CONCLUSIONS AND FUTURE WORK There are not different morphologic and anatomic characteristics between Ribes nigrum leaves and Ribes rubrum leaves. The qualitative and quantitative results suggest Ribes nigrum leaves has a higher content of active principles (flavones, polyphenolcarboxilic acids). A dry selective extract (obtained by using the leaves fron Ribes nigrum as vegetal raw material and alcohol 70 per cent as solvent) was obtained, was physico.chemical characterized and finally was included in an ointment formulation. Preliminary research results regarding the formulation and the preparation of laboratory-scale ointment carbopol base, combined with the results of preclinical and

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

clinical experiments, can be the basis for obtaining a new type of topic preparation with anti-rheumatic activity.

REFERENCES [1] L.E.Duţu. Pharmacognostic Methods for Analysis of Herbal Drugs, According to European Pharmacopoeia. Purusotam Basnet (Ed), Promising Pharmaceuticals: InTech, 2012 [2] L.E.Dutu, C.E. Gird. Ribes rubrum L. – a Potential Source of Bioactive Principles. Proceeding of international conference ,,Research People and Actual Tasks on Multidisciplinary Sciences‖, Lozenec, Bulgaria, 2009 [3] C.E. Gird, L.E. Dutu, L.M. Popescu, M. Pavel, A.T. Iordache, I. Tudor. Bazele teoretice si practice ale analizei farmacognostice, 2th edition, volume I , Bucharest: Curtea Veche, 2010 [4] C.E. Gird, L.E. Dutu, L.M. Popescu, M. Pavel, A.T. Iordache, I. Tudor. Bazele teoretice si practice ale analizei farmacognostice, volume II , Bucharest: Curtea Veche, 2009 [5] Hîrjău V., Lupuleasa D., Dumitrescu A.M., Dermo-cosmetologie, Bucharest: Polirom, 1998 [6] V. Istudor. Fitoterapia bolilor metabolice, Bucharest: Tehnoplast Company, 2008 [7] S. Leucuţa. Tehnologie farmaceutică industriala, Cluj-Napoca: Dacia, 2001. [8] C-tin Parvu. Universul plantelor, Bucharest: ASAB, 2006 [9] A. Popovici. Reologia formelor farmaceutice, Bucharest: Medicală,1985 [10] Popovici I., Lupuleasa D., Tehnologie farmaceutica, vol II, Iasi: Polirom, 2008; [11] XXX – European Pharmacopoeia, 7th edition, Strasbourg: Council of Europe, 2011. [12] XXX - Farmacopeea Romana, 10th edition, Bucharest: Medicala, 1993. [13] XXX - PDR for herbal medicines, 3th edition. Montvale: Thomson, 2005, pp. 102

ABOUT THE AUTHORS L.E. Dutu, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmacognosy Phytochemistry Phytotherapy, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] T.D. Balaci, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmaceutical Technology, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] C.E. Gird, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmacognosy Phytochemistry Phytotherapy, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] L.M. Popescu, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmacognosy Phytochemistry Phytotherapy, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected]

87 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

PRELIMINARY RESEARCH REGARDING A DERMATO-COSMETIC PRODUCT WITH VEGETAL EXTRACTS USEFUL FOR THE TREATMENT OF HYPERPIGMENTATION

C. E. Gîrd, T. D. Balaci, L. E. Duţu, I. Nencu, E. A. Ciocan

Abstract: Starting from the premise that people are more concerned with pleasing appearance of the skin, this paper presents the research conducted in order to obtain a topical phytopreparation with whitening effect. Based on the scientific literature, the following herbal drugs were selected: Althaeae folium, Calendulae flos, Gei rhizoma, Polygoni avicularis herba, Uvae-ursi folium. The quality of the raw materials was established using pharmacognostical analysis. Subsequentlly, selective dry extracts were obtained and physico-chemical characterized. Finally, in order to obtain a semi-solid dermato-cosmetic product, the optimum formula was selected, by testing some ointment fatty-bases. Physical and rhelogical parameters of the product were determined, and its stability was monitorized. Key words: vegetal extracts, dermato-cosmetic products.

INTRODUCTION The need to maintain a pleasant aspect of the skin has urged scientists to research and study the skin structure, functions and its imperfections. In their attempt the researchers try to develop new and diverse products to treat skin problems, trying to obtain products that are closer to the patient needs. An example of skin problems are hyperpigmentation spots. This dark spots appears on the face, decolletage and hands, beeing more frequent at elder people, pregnant women or after some medication. The mechanism involved in the appearance of the dark spots relies on the increase of melanin synthesis in the melanocytes cells. Unfortunaly, the hyperpigmentation spots are hard to treat, taking into account that only a third of the patients with skin problems, can respond to products that contain hydroquinone, azelaic acid, tretinoin, glycolic acid, lactic acid and others alpha hydroxy acids in combination with sun protection creams. Taking into consideration that the phyto-preparations (with different types of extracts) are well-tolerated by the skin (compared to synthetic substances), this paper presents a preliminary study regarding the obtaining of products with topical use, that may be used in the treatment of hyperpigmentation spots. The selection of the raw materials was done according to scientific data. The vegetal parts were processed into dry extracts, that subsequently were embedded as dermato- cosmetic product. The selected raw materials were: Althaeae folium, for the presence of mucilages with emollient effect; Calendulae flos, for the content of flavonoid, saponins (oleanolic acid), mucilages and essential oil, compounds responsable for the vascular protection-, antiinflammatory-, healing-, emollient and antiseptic effects of the raw material; Gei rhizoma, for the presence of tannins and eugenol, compounds with antiseptic and astringent activities; Polygoni avicularis herba, for the content of flavonoids (rutin), with capillary-protective activity; Uvae-ursi folium, that contains hydroquinone and its derivatives, with bleaching effect through the inhibition of melanin in the epithelial cells [2].

MATERIAL AND METHODS The natural products were bought from the Romanian pharmaceutical market: Polygoni avicularis herba, Gei rhizoma, Althaeae folium, Uvae-ursi folium and Calendulae flores and the corresponding liophylized dry extracts.

RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

The pharmacognostic analysis was used [1]. The study was conducted in several stages: establishing the quality of the raw materials; obtaining the dry vegetal extracts standardised in flavonoids; processing the extracts as pharmaceutical products with topical use (ointment / cream). Phase I. The quality of the raw materials was established using a macroscopic exam (to verify the morphologic characters); microscopic exam (using clarified powder – preparations using 80% chloral hydrate as a clarified agent); qualitative chemical exam (to identify the main active principles using commune or specific chemical reactions); quantitative chemical exam (the assay of flavonoids by chelating reaction with aluminium chloride solution; the assay of polyphenolcarboxylic acids by forming of oxime derivatives with Arnow reagent). Phase II. The obtaining of the dry vegetal extracts using a lyophilizer, after the extraction of the raw materials with different concentrations of alcohol. Polygoni avicularis herba, Calendulae flores and Althaeae folium were extracted with alcohol 200, using reflux apparatus for 30 minutes. Gei rhizoma was treated with ethanol 500, using the same procedure of extraction for 60 minutes; Uvae ursi folium was extracted using absolute ethanol (ethanol 960) (for the hydroquinone extraction) and a reflux condenser for 45 minutes. After extraction and cooling (at room temperature), the mixture of raw material and solvent was kept at room temperature for 60 minutes, for the complete extraction of mucilages that are present in high amount in Polygoni avicularis herba, Calendulae flores şi Althaeae folium. After 60 minute, the mixtures of raw materials and solvents were separated by filtration. 50 g of raw materials was used. The solid-to-solvent ratio used was 1: 10 for the first extraction, 1:6 for the second extraction and 1:2 for the third extraction. The resulted filtrates were combined and subjected to a rotary evaporator. The obtained dry residues were lyophilised. The extracts were standardised in flavonoids. Liophylized extracts were included in ointment bases which contain: wool fat, beeswax, cetaceum, sunflower oil, cocoa butter, cholesterol, cetyl alcohol, soft paraffin. Other components were added , too. Etapa III. Prelucrarea extractelor liofilizate sub forma de creme: Finally, in order to obtain a semi-solid dermato-cosmetic product, the optimum formula was selected, by testing some ointment fatty-bases. The following formulas was selected (table I). Description for the obtaining process: Fatty phase is melt at about 60 -70 0C. Separately, aqous phase is heat to a highter temperature, then is added in the fatty phase and vigurosly shake for 15 minutes. The emulsion is cooled at 30 0C. Separately, vegetal extracts, Benzoin tincture and vitamin A are dispersed in distilated water, and this mixture is added in the previous emulsion. In view to obtain a stable and homogenic finish product, agitation is a very important parameter [3,6].

Table I. Dermato-cosmetic products - qualitative and quantitative formulas

Component Formula I (g) Formula II (g) Uvae-ursi extract 3 3 Calendulae extract 1.5 1.5 Polygoni avicularis extract 1 1 Althaeae extract 0.5 0.5 Gei extract 0.5 0.5 Yellow beeswax - 5 Wool fat - 20 Sunflower oil - 30 89 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Soft white paraffin 10 - Cetaceum 10 5 Cocoa butter 6.5 5 Cholesterol 0.5 0.2 Cetyl alcohol 0.5 5 Sodium laurilsulfate 0.5 - Sodium tetraborate - 1 Vitamin A 300000 IU 300000 IU Methylparaben 0.2 0.2 Benzoin tincture - 3 Distilad water, up to 100 100

The organoleptic properties, the pH (by potentiometry) and some rheologic properties of the products, such as spreadability (by the extensiometric method Ojeda – Arbussa ) and the viscosity (using a Rheotec RC1 apparatus) were determined. The Rheotec RC 1 apparatus is a rotational rheometer cone / plate with an electronic unit allowing the digital control of the rotation speed and the direct recording of the measured values [4,5,7,8]. The determinations were made after preparation and after certain intervals in time. Testing the stability of the creams. Because the composition of the creams is complex, and most of the organic components are unsaturated, the finished products may be degradated by atmospheric oxygen. So, modifications of the structure may appear. These are materialized in changeing of colour, forming of oxygenate grups and free radicals, and so the degradation process will continue. This is the reason for the actual accelerated thermo- and photodegradation studies. The samples were plased in closed recipients, at 50 0C, for 1 hour.

RESULTS AND DISCUSSION The macroscopic and microscopic exam confirmed the identity of the raw materials. The characters, observed on our raw materials, are the same with the ones that are mentioned in scientific data for all vegetal drugs [1,2]..

Polygoni avicularis herba: fragments of stem with leaves; greenish-white or red flowers; astringent taste; epidermis with anisocytic stomata (fig. nr. 1), clorophylian parenchyma, annularly and spirally thickened vessels

Fig. 1. Epidermis cells with stoma Gei rhizoma: fragments of rhizome slightly striated on the surface, outer surface brown and inner surface brownish-red, astringent taste, bitter, faint, aromatic; resembles clove flowers; fragments of reticulate or scalariform thickening vessels (fig.2), parenchyma cortical cells, suber

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Fig. 2. Reticulate thickening vessels Althaeae folium: leaves fragments densely pubescent and velutinous on both sides, more so beneath (velvety leaves); greyish- green in colour and mucilaginous taste; numerous covering trichomes united to form stellate structures (fig. 3), cluster crystals of calcium oxalate, isolated or in the parenchyma of the mesophyll; annular vessels Fig. 3. United covering trichomes

Uvae-ursi folium: coriaceous leaves with waxy and shiny upper surface; green to brownish-green, odourless, astringent to bitter taste, unicellular thick-walled trichomes (fig.4), slightly sinuous cells of the lower epiderminis,

Fig. 4. Thick-walled trichomes Calendulae flores: numerouse ligulate florets with yellowish- orange ligule; brown tubular florets; bitter and mucilaginous taste; characteristic, odour; spherical pollen grains, with exine finely granular (fig.5), endothecium, papillae, covering trichomes biseriate, glandular trichomes biseriate Fig. 5. Pollen grains with exine finely granular The qualitative exam showed the presence of compounds with capillary- protective activity (flavonoids), antiinflammatory effect (saponins, flavonoids), astringent and antiseptic activities (tannins), emollient effect (mucilages) and with bleaching effect (hydroquinone, fig. 6). It is important to mentioned that the intensity of the chemical reactions varies with the type of vegetal product. Fig. 6. Isolated cristals of hydroquinone

The chemical quantitative exam showed that the active principles are found in higher amounts in some raw materials than in the others. Calendulae flos has a higher content of flavonoids than Polygoni avicularis herba. Althaeae folium has a higher content of mucilages than Uvae-ursi folium (table II). 91 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Tabel II. Results of the quantitative exam Test Raw material Flavonoids, g% Mucilages, g% Polygoni 0.134-0.144 0.12-0.21 avicularis herba Gei rhizoma 0.189-1.232 x 10-3 0.009-0.01 Althaeae 0.104-0.123 1.13-1.87 folium Uvae-ursi 0.345-0.412 x 10-3 0.01-0.03 folium Calendulae 0.234-0.314 0.85-0.98 flos

The dry extracts have the same characteristics as the corresponding raw materials. The chemical exams (both qualitative and quantitative) showed no degradation of the active principles during the concentration and liophylisation procedures (table III).

Tabel III. Dry extracts characterization Dry extract Characters Flavonoids, g% Polygoni avicularis Powder, greenish- 1.32-1.84 extractum brown, faint astringent taste Gei extractum Powder, brownish-red, 0.123-0.234 astringent taste Althaeae extractum Powder, green, 1.24-1.59 mucilaginous taste Uvae-ursi extractum Powder, greenish- 0.109-0.112 brown, astringent taste Calendulae Powder, brownish- 1.67-1.89 extractum yellow, bitter and mucilaginous taste

Cream characteristics are showed in table IV and figure 7. They are not modified during storage. Table IV. Organoleptic and physical characteristics of the creams Characteristics Results Aspect Homogenous cream Colour Brownish-yellow Odour Pleasant PH 6.6 (form I); 6.7 (form II) Thermostability Separation of the phases is not observed

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Fig. 7. Crems (formula I; formula II)

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 10 20 60 100 150 200 250 300

Fig. 8. Viscosity variation depending on shear strength - formula I

0.8

0.6

0.4

0.2

0 10 20 60 100 150 200 250 300

Fig. 9. Viscosity variation depending on shear strength - formula II

The rheograms are presented in figures 8 and 9. The viscosity decreases with the increase of the shear strength. The creams have pseudoplastic flow, and so, their viscosity depend on temperature and the forces exerted on them. When the forces stope the viscosity returns to baseline. We consider that sensorial properties of the two creams are very good (s.a. easy to apply, easy absorbtion to the skin and without fatty stain, their pleasant aspect, odour and colour). We apply the creams to some volunteer patients and they said that the creams do not dry the skin, give a feeling of softness of the skin, improve skin appearance, and make it smooth.

CONCLUSIONS Two dermato-cosmetic products containing vegetal extracts was obtained. These creams meet the quality standards and give a pleasing appearance to the application.

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REFERENCES [1]. Gird C.E., et al. 2010. Bazele teoretice si practice ale analizei farmacognostice, 2th edition, volume I , Bucharest: Curtea Veche, 81, 93, 139, 145, 238-241, 270, 342, 371, 477, 521-524 [2]. Istudor V., 1998. Farmacognozie, Fitochimie, Fitoterapie, volume I, Bucharest: Editura Medicală, 84, 106, 164, 200, 314 [3]. Hîrjău Victoria., Lupuleasa D., Dumitrescu A.M., 1998. Dermo – cosmetologie, Editura Polirom [4]. Leucuta S., 2001. Tehnologie Farmaceutica Industriala, Ed. Dacia, Cluj [5]. Popovici Adriana, 1985. Reologia formelor farmaceutice, Editura Medicală, Bucureşti [6]. Popovici Iuliana, Lupuleasa D., 2008. Tehnologie farmaceutica, vol II, Ed. Polirom, Iasi [7]. xxx – Farmacopeea Română ed aXa, 1993. Ed. Medicala, Bucuresti [8]. xxx –European Pharmacopoeia 7thEdition, Strasbourg, 2011

ABOUT THE AUTHORS C.E. Gird, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmacognosy Phytochemistry Phytotherapy, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] T.D. Balaci, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmaceutical Technology, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] L.E. Dutu, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmacognosy Phytochemistry Phytotherapy, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] I.Nencu, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmacognosy Phytochemistry Phytotherapy, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected]

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ISOTROPIC AND ANISOTROPIC PHASES IN CELLULOSE LIQUID CRYSTALLINE SOLUTIONS

A. M. Dobos1, M. D. Onofrei1, and S. Ioan1 1) ―Petru Poni‖ Institute of Macromolecular Chemistry, Grigore Ghica Voda Alley, 41A, Iasi, Romania, E-mail: [email protected]

Abstract: The present work investigates a series of blends obtained from cellulose acetate phthalate (CAP)/ethyl cellulose (EC) in N,N-dimethylacetamide (DMAc). The viscosity-shear rate dependence for CAP exhibits a Newtonian plateau, while the shear experiments performed on lyotropic EC solutions in DMAc presents regions typically for liquid crystalline solution. In the case of viscoelastic properties, from dependence of moduli on frequency, one can observe for both polymers a characteristic behaviour for viscoelastic fluid. These studies contribute to diversification of cellulose derivatives applications in electronic domain. Key words: Cellulose derivatives, Rheology, Liquid crystalline phases.

INTRODUCTION With their broad range of properties, polymer blends are widely used in adhesion, colloidal stability, the design of composite and biocompatible materials, and other areas. As the science and technology of polymer blends advances, an increasing number of polymer blend systems and applications continue to be developed. The understanding of the mechanisms involved in polymer‘s mixing, starting from their fundamental interactions, and manner in which they affect their final properties was possible due to the improvement of characterization techniques. It is well-known that the properties of polymer blends depend on the rheology of their solutions. When a component becomes an anisotropic fluid, it is expected that the anisotropic properties would greatly affect blend‘s properties [1]. Composite material based on liquid crystal (LC) matrix give rise to system relevant for electro-optical applications, exhibiting electro-optical properties similar to those observed in standard polymer dispersed liquid crystal (PDLC) systems [2]. The self-aligning nature of LC is used for obtaining organic polymer thin-film transistors (OP-TFTs) in liquid crystal display devices [3]. In this context, cellulose acetate phthalate and ethyl cellulose were selected for the present work. CAP is a mixed ester of cellulose obtained through phthaloylation of cellulose acetate. It is utilized in different domains as a pharmaceutical excipient, due to the pH dependent solubility of the aqueous medium. Enteric coatings of CAP are resistant to the gastric acid and easily soluble in the slightly alkaline environment of the intestine. Recently, the potential of this polymer of inhibiting the infections caused by several types of herpes virus, such as Herpes Simplex type 1, and other sexually-transmitted diseases was analysed, in vitro [4-7]. As a derivative of cellulose, ethyl cellulose is a type of semirigid and thermoplastic liquid crystalline material, which has interesting physical and chemical properties, being quite attractive for many applications. The flexible ethyl groups confer lyotropic and thermotropic properties on EC [8]. Flow behaviour is the most thoroughly studied rheological property. Onogi and Asada [9] hypothesized the universal existence of three shear flow regimes to describe the viscosity of polymer liquid crystals: a shear thinning regime at low shear rates (Region I), a Newtonian plateau at intermediate shear rates (Region II), and another shear thinning regime at high shear rates (Region III). Literature [9] shows also that cellulose derivatives do not always cover the entire domain from Region I to Region III, because not every regime lies within the accessible shear rate range. In the present work, a series of blends obtained from CAP and EC, in DMAc, at different mixing ratios and concentrations are rheological investigated, to identify the 95 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA specific interactions from the system which modify the liquid crystalline properties of cellulose derivatives. Results will be used in future research for applications in electronic domains. MATERIAL AND METHODS CAP (degree of substitution for acetyl and phthalyl groups are 1.07 and 0.77, respectively) with molecular weight 2534.12 was purchased from Sigma-Aldrich, USA. EC was purchased from Sigma-Aldrich, USA. According to product specifications, EC has a viscosity of 22 cP in toluene/ethanol 80:20, extent of labeling: 48% ethoxyl. Viscometric and oscillatory measurements for CAP/EC in DMAc were obtained with a Bohlin Instrument, by the cone and plate measuring system; the cone had an angle of 4° and a diameter of 40 mm. Shear viscosities were registered over the 0.07–1000 s−1 shear rate domain, at several temperatures (25–55 °C). During the oscillatory shear tests, the frequency was varied between 0.1–150 Hz, and a shear stress,  , of 3 Pa was applied.

RESULTS AND DISCUSSION Dynamic viscosity of CAP, EC and CAP/EC blend Polymer-solvent interactions of the lyotropic mesophases were derived from rheological studies of CAP, EC and their blends in DMAc, where anisotropic comportment appear in specific conditions of temperature, solvent, concentration and/or blend composition. These variables influence the interactions from systems and thus ordered domains in rheological behaviour.

Figure 1 Logarithmic plot of dynamic viscosity as a function of shear rate for 0/100, 50/50, 0/100 CAP/EC wt/wt in DMAc at different concentrations at 25 oC.

In this context, Figure 1 plots the modification of dynamic viscosity,  , versus shear rate,  , for 0/100, 50/50, 0/100 CAP/EC wt/wt in DMAc at c = 25, 30 and 35 g/dL concentrations and 25 °C. As can see, CAP at lower concentrations present a Newtonian behaviour, while at higher concentrations a thinning behaviour appears. The shear experiments performed on lyotropic EC solutions in DMAc reveal the occurrence of three flow regions specific to liquid crystals polymers, from the viscosity-shear rate dependence

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at higher concentrations. As concentration increases in pure sample of EC, the Newtonian plateau becomes smaller, being shifted to lower shear rates. Appearance of the three regions with different expanses in function of concentration and EC content (Figure 1c) is also observed for 50/50 wt/wt CAP/EC blends. The interaction between chain segments is directly related to the positive disengagement ( Edis ) and to the negative association ( Eass ) contributions on the flow activation energy, Ea , (Eqs. (1) and (2)) [10]. These contributions can be influenced by the polymer entanglements and hydrogen bonding of the system:

Ea  Edis  Eass ( 1) E ( ln   ln   a 0 RT 2) where 0 is zero-shear-rate viscosity, R is the universal gas constant and T is the absolute temperature. Figure 2 shows that the flow activation energies for CAP and EC in DMAc, determined in the region of shear rate with Newtonian behaviour, increase with concentration and are higher for EC. In addition, for CAP/EC blends, the values of activation energy are higher than those corresponding to pure samples because, besides the polymer/solvent interactions, polymer/polymer/solvent interactions, implying the hydrogen bonding, also appear. On the other hand, the positive values of flow activation energy obtained for all polymer blends involve a good dissolution when Edis  Eass , according to Eq. 1.

Figure 2 Arrhenius plots for 0/100, 50/50, 0/100 CAP/EC wt/wt in DMAc at (■) 25 g/dL, (▲) 30 g/dL, and (▼) 35 g/dL concentrations. Viscoelastic properties of CAP, EC and CAP/EC blends

Viscoelastic measurements significantly contribute to the knowledge and differentiation of polymer systems, completing the rheological studies developed in shear regime. The ordering tendency in casting solutions of CAP, EC and their blends is observed from dependence of G' and G" moduli on frequency. 97 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Figure 3 Log-log plots of storage (●) and Figure 4 Log-log plots of storage (●) and loss (■) moduli as a function of frequency loss (■) moduli as a function of frequency for different compositions of CAP/EC blends for different compositions of CAP/EC blends at a concentration of 25 g/dL at a concentration of 35 g/dL

The storage, G', and loss moduli, G", are proportional to frequency (within low frequencies domain of 0.2-0.9 Hz) - where the exponents are in the ranges 1.6-0.9 and 1.0-0.7, respectively, versus CAP content in polymeric mixtures and concentrations. In these conditions, the viscoelastic fluid characteristic (where G' G" [11-13]) is maintained. The exponents decrease with increasing polymer concentrations and for higher EC compositions in the polymer blend when the lyotropic phase becomes predominant. In addition, the frequencies corresponding to the crossover point, which delimits the viscous flow from the elastic one, and for which G' G", exhibit lower values for EC in DMAc, and become higher with increasing the CAP content in polymer blends (i.e Figure 3 and 4).

CONCLUSIONS New data on the rheological properties of cellulose acetate phthalate, ethyl cellulose solution and their corresponding blends are reported, showing the existence of lyotropic phases, depending on the concentration and composition of CAP/EC mixtures. Thus, a Newtonian and thinning behaviour of dynamic viscosity at lower and respectively, higher shear rate, was observed for CAP and EC solutions in N,N-dimethylacetamide, depending on solution concentrations. The Newtonian plateau is smaller for EC comparatively with CAP, and it is shifted to lower shear rates as concentration increases. Similar data were obtained for the CAP/EC blend at highest concentrations, where increasing of the EC content reduces the Newtonian regime and leads to apparition of the three regions specific for liquid crystalline polymers. The flow activation energy takes lower values for CAP, which possesses a higher flexibility, and higher values for EC with more rigid structures. On the other hand, the presence of hydrogen bonding, which enhances rigidity in the case of CAP/EC systems, increases the activation energy. In addition, the viscoelastic fluid properties were observed for all samples. The storage and loss moduli for CAP exhibit the power law dependence on frequency, where

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the exponents are characteristic to viscoelastic fluids. For blends with a higher concentration and content of EC, the exponents for G' and G" decreases, behaviour accompanied by the occurrence of lyotropic liquid crystal phases. Knowledge on the rheological properties of these polymers lies at the basis of future exploration and exploitation of their performances in electrical devices.

REFERENCES [1]. Dutta, D., et al. 1990. Polymer Blends Containing Liquid Crystals: A Review. Polymer Engeneering Science 30, 1005–1018. [2]. Godinho, M.H., et al. 1999. Liquid Crystal and Cellulose Derivatives Composites Used in Electro-Optical Applications. Molecular Crystals and Liquid Crystals 331, 173–179. [3]. Chae, B., et al. 2003. Sequence of the Rubbing-Induced Reorientations of Polymer Chain Segments in Nanofilms of a Well-Defined Brush Polyimide With a Fully Rodlike Backbone as Determined by Polarized FTIR Spectroscopy and Two-Dimensional Correlation Analysis. Langmuir 19, 9459–9465. [4]. Stone, A. 2002. Microbicides: a New Approach to Preventing HIV and Other Sexually Transmitted Infections. Nature Reviews Drug Discovery 1, 977-985. [5]. Manson, K.H., et al. 2000. Effect of a Cellulose Acetate Phthalate Topical Cream on Vaginal Transmission of Simian Immunodeficiency Virus in Rhesus Monkeys. Antimicrobial Agents and Chemotherapy 44, 3199-3202. [6]. Neurath, A.R., et al. 2001. Cellulose Acetate Phthalate, a Common Pharmaceutical Excipient, Inactivates HIV-1 and Blocks the Coreceptor Binding Site on the Virus Envelope Glycoprotein gp120. BMC Infectious Diseases 1, 17-28. [7]. Neurath, A.R., et al. 2002. Anti-HIV-1 Activity of Anionic Polymers: A Comparative Study of Candidate Microbicides. BMC Infectious Diseases 2, 27-37. [8]. Zhihao, Z., et al. 2006. Thermotropic Liquid Crystallinity, Thermal Decomposition Behavior, and Aggregated Structure of Poly(propylene carbonate)/Ethyl Cellulose Blends. Journal of Applied Polymer Science 100, 584-592. [9]. Onogi, S., and Asada, T. 1980. Rheology. Plenum. In: Astarita G, Marucci G, Nicolais L (eds), New York, vol I. [10]. de Vasconcelos, C.L., et al. 2001. Rheology of Polyurethane Solutions With Different Solvents. Polymer International 51, 69-74. [11]. Ferry, J,D. 1980. Viscoelasticity Properties of Polymers. Willey Interscience, New York, 3rd Edition. [12]. Tirtaatmadja, V., et al. 1997. Superposition of Oscillations on Steady Shear Flow as a Technique for Investigating the Structure of Associative Polymers. Macromolecules 30, 1426-1433. [13]. Cassagnau, Ph., and Melis, F. 2003. Non-Linear Viscoelastic Behaviour and Modulus Recovery in Silica Filled Polymers. Polymer 44, 6607-6615.

Acknowledgment: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number PN-II-RU-TE-2012-3-0143 (2013 stage).

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DIELECTRIC PROPERTIES OF NEW POLYSULFONES WITH CHELATING GROUPS

R.M. Albu1, E. Avram1, V.E. Musteata1, and S. Ioan1 1 ―Petru Poni‖ Institute of Macromolecular Chemistry, Grigore Ghica Voda Alley, 41A Iasi, Romania, E-mail: [email protected]

Abstract: New quaternized polysulfones with N,N-dimethyloctylamine and methyl 3-pyridyl ketone groups (KQPSF) and chelate quaternized polysulfone (KQPSFCu) were investigated by dielectric spectroscopy at different temperatures (-1500C’1500C) and frequencies (100÷106 Hz). The transient metal ions in polysulfonic films affects different properties in two distinct ways: primarily, complex formation between metal cations and electron donor groups of the polymer chains leads to crosslinking formations, decreasing chain mobility and increasing the glass transition temperature; on the other hand, the predominant effect of metal ions generates distributed complexes in polymer matrices which disrupts the uniformity of polymer chains and decreases the glass transition temperature. In this context, dielectric spectra reveal that a higher interval of dielectric constant,  ' , (from 28.5 to 4.3) appears in the frequencies range of 100 ÷ 106 Hz for KPSF, and a decreasing of this interval is observed for KQPSF (from 17.4 to 3.4), where the reactive ketone alternate with ammonium quaternary side groups), and, especially for KQPSFCu (from 8.2 to 4.7), with complex structures. The rapid increase of dielectric constant at higher temperature was attributed to the increase in chaotic thermal oscillations of the molecules and to the diminishing order degree of dipoles orientation. From technological point of view, these studies are interesting especially in environmental applications.

Key words: Polysulfones, Dielectric properties, Metal cations

INTRODUCTION Derivative polysulfones - possesing excellent dielectric properties - have been employed in a wide variety of applications. Their chain rigidity is derived from the relatively inflexible and immobile phenyl and SO2 groups, while toughness is derived from the connecting ether oxygen. The presence of these groups enhances electrical and thermal stability, oxidation resistance and rigidity even at elevated temperatures. In this context, the chemical modification of polysulfones, especially the chloromethylation and quaternization reactions [1-4], is a subject of considerable scientific interest from both theoretical and practical points of view, including their area of applications. These polysulfones have higher hydrophilicity (of special interest for biomedical applications) [5], allowing permeability and water separation [6, 7], influence the antimicrobial activity [8, 9] and solubility characteristics [2, 10]. The functional groups are an intrinsic requirement for affinity, ion exchange and other special membranes [11]. In the same context, these has been previously stated that the electrical properties, such as dielectric spectrum, relaxation process or electrical conduction, in different complex forms of polysulfones, are very important for the investigation of molecular motion and induced polarization in correlation with different structures [12]. In addition, introduction of chelating units onto the modified polysulfone structure, such as methyl 3-pyridyl ketone, led to new potential applications [13], such as surface coatings on metals and glasses, adhesives, high-temperature lubricants, electrical insulators, semiconductors, reduction of heavy metal pollution in ecosystems, etc. The presence of transient metal ions in polysulfonic films affects different properties in two distinct ways [13, 14]: on one hand, complex structure between metal cations and electron donor groups of the polymer chains leads to crosslinking formations, decreasing chain mobility and increasing the glass transition temperature; on the other hand, the predominant effect is generated by the complexes distributed in polymer matrices, which disrupts the uniformity of polymer chains and decreases the glass transition temperature. From a technological point of view, the polysulfone metal chelates

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considered in this work are interesting especially for environmental applications [15, 16]. In this context, the present study analyzes the relationships between structure and molecular mobility of chelated polysulfones containing methyl 3-pyridyl ketone, and also the complex formation between metal cations, Cu2+, and electron donor groups of the polymer chains, by dielectric spectroscopy.

MATERIAL AND METHODS Chelate-modified polysulfones (KQPSFCu) were obtained, by a method discussed in a previous paper [13]. First, quaternized polysulfone containing chelating groups (KQPSF) was prepared from chloromethylated polysulfone (CMPSF, Scheme 1a).

CH Cl O CH 3 2 C [ O C O - SO2 ]n Cl + CH3 N CH3 CH2 CH 3 [ O C O SO2 ]x

CH3 (a) CMPSF (b) KPSF O

C Cl- - Cl CH 3 + CH3 + N N CH 3 ( ) CH CH CH 2 7 3 CH 2 CH2 3 CH 3 [ O C O SO ] [ O C O 2 x SO2 ]y CH 3 CH3 (c) KQPSF - Cl CH 3 + N CH 3 (CH 2)7 CH3 CH CH2 3 CH 3

[ O C O SO ] [ O C O SO ] 2 x 2 y CH 3 CH2 CH3 + Cl- N CH3 C O 2+ Cu 2

O - - Cl CH 3 Cl C + + N CH 3 N CH3 ( ) CH CH CH 2 7 3 CH 2 CH2 3 CH 3

[ O C O SO ] [ O C O SO ] 2 x 2 y CH 3 CH3 (d) KQPSFCu

Scheme 1 General structure of chloromethylated polysulfone (a, CMPSF), chelating polysulfone containing ketone units (b, KPSF), chelating polysulfone containing quaternary and reactive ketone units (c, KQPSF), and chelate-modified polysulfones containing quaternary and reactive ketone units (d, KQPSFCu)

Thus, CMPSF was dissolved in N,N-dimethylformamide (DMF) vacuum distilled over P2O5, after which methyl 3-pyridyl ketone and potassium carbonate catalyst (K2CO3) were added for attaining a molar degree of the partially quaternized polysulfone (KPSF, Scheme 1b) of 0.4. The reaction occurred at 62 °C, for 17 h. Secondly, N,N-dimethyloctylamine (DMOA) was added to react with the remaining chloromethylene groups, at the same 101 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA temperature, for 24 h. The obtained product, KQPSF (Scheme 1c), contains 60 % quaternary and 40 % reactive ketone units. The ability of forming KQPSFCu complexes is presented in Scheme 1d [13]. Dielectric Spectroscopy (DS) measurements in the 1 Hz to 1 MHz frequency range were carried out using a Novocontrol Concept 40 broadband dielectric spectrometer [14]. Temperature was controlled with a 0.1 ºC device by the Novocontrol Quatro Cryosystem, in dry nitrogen atmosphere. The samples were sandwiched between two gold-coated brass electrodes and then tested. Samples were around 2 mm thick, with slightly larger diameters than those of the upper electrode (20 mm). Dielectric constant,  ' , of KPSF, KQPSF, and KQPSFCu were determined by sweeping the frequency between 1 Hz and 1 MHz at fixed temperatures, at 4 ºC intervals, for a temperature domain between -150 and + 150 ºC, at an increasing temperature rate of 2 ºC/min. This temperature range does not exceed the thermal decomposition temperatures. Dielectric spectra measurements were performed in nitrogen atmosphere, thus avoiding water adsorption.

RESULTS AND DISCUSSION AC-dielectric Measurements at Different Frequencies and Temperatures The dielectric properties of KPSF, KQPSF, and KQPSFCu films, such as dielectrical constant,  ' , were measured over a wide temperature and frequency range. Thus, the amount of dipole alignment, as well as different polarization processes (atomic, electronic, or interfacial) are reflected in the dielectric constant, whereas the energy required to align dipoles or move ions is given by the dielectric loss. According to Figs. 1 and 2, the dielectric constants increase slightly with temperature for all samples, due to the increase of total polarization arising from dipoles orientation and trapped charge carriers, and decrease with increasing frequency, due to dielectric dispersion, as a result of the lag of molecules behind the alternation of the electric field at higher frequency [19]. In this context, one can mention that the dielectric constants depend on the chemical structures of the modified polysulfones. Thus, Fig. 2 shows that, at 25 °C, dielectric constants have values in the 28.5 – 4.3, 17.4 – 3.4 and 8.2 – 4.5 ranges, over the 1 – 1.106 Hz frequency interval, for KPSF, KQPSF and KQPSFCu, respectively. In addition, previous paper provide dielectric constants values of 2817 – 5.99 in the same frequency range, for quaternized polysulfone with N,N-dimethyloctylamine (PSF-DMOA) pendant groups – outside the reactive ketone units [14]. Therefore, the presence of N,N- dimethyloctylamine pendant groups, with higher molecular flexibility and mobility, determines an increase of electronic conjugation from the quaternized polysulfone main chain, and a higher value of  ' , comparatively with the more bulky reactive ketone pendant groups from KPSF. On the other hand, in KQPSF, the cumulative effect of both pendant groups reduces even more the dielectric constant. A higher interval of the dielectric constants appears in the 100 – 106 Hz range for KPSF, and a narrowing of this interval is observed for KQPSF (where the reactive ketone side groups alternate with the ammonium quaternary side groups), and especially for KQPSFCu, where the retention of copper ions led to the formation of complex structures.

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Fig. 1 Frequency and temperature dependencies of the dielectric constants for KPSF, KQPSF, and KQPSFCu films

Fig. 2 Modification of dielectric constans versus temperature at different frequencies for KPSF, KQPSF, and KQPSFCu films

The rapid increase of  ' at higher temperatures for all studied samples was attributed to the increase in chaotic thermal oscillations of the molecules and to the diminishing order degree of dipoles orientation, near the glass transition temperature.

CONCLUSIONS AND FUTURE WORK The relationships between structure and molecular mobility in partially quaternized polysulfones, quaternized polysulfones containing chelating groups and chelated quaternized polysulfones with Cu2+ were investigated by dielectric spectroscopy. For all samples, the dielectric constant decreases with increasing frequency and increases with temperature. Moreover, the dielectric constants are influenced by the total polarization arising from dipoles orientation, and by trapped charge carriers. The interval of variation with frequency at different temperatures becomes narrower for chelated polysulfones with metal ions, whose the complex structure induces some cumulative effects.

REFERENCES [1]. Filimon, A., et al. 2010. Effect of Alkyl Side Chain on the Conformational Properties of Polysulfones with Quaternary Groups. Journal of Macromolecular Science Part B Physics 49, 207-217.

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[2]. Ioan, S., et al. 2006. Influence of the Degree of Substitution on the Solution Properties of Chloromethylated Polysulfone. Journal of Applied Polymer Science 101, 524- 531. [3]. Ioan, S., et al. 2006. Conformational and Visometric Behavior of Quaternized Polysulfone in Dilute Solution. Polymer Engineering and Science 46, 827-836. [4]. Filimon, A., et al. 2013. Impact of Association Phenomena on the Thermodynamic Properties of Modified Polysulfones in Solutions. Journal of Macromolecular Science Part B Physics 52,1-16. [5]. Guan, R., et al. 2005. Polyethersulfone Sulfonated by Chlorosulfonic Acid and its Membrane Characteristics. European Polymer Journal 41, 1554-1560. [6]. Idrisa, A., et al. 2007. Synthesis, Characterization and Performance of Asymmetric Polyethersulfone (PES) Ultrafiltration Membranes with Polyethylene Glycol of Different Molecular Weights as Additives. Desalination 207, 324-339. [7]. Kochkodan, V., et al. 2008. Adhesion of Microorganisms to Polymer Membranes: a Photobactericidal Effect of Surface Treatment with TiO2. Desalination 220, 380-385. [8]. Yu, H., et al. 2007. Preparation and Characterization of a Quaternary Ammonium Derivative of Konjac Glucomannan. Carbohydrate Polymers 69, 29-40. [9]. Filimon, A., et al. 2009. Surface Properties and Antibacterial Activity of Quaternized Polysulfones. Journal of Applied Polymer Science 112, 1808-1816. [10]. Filimon, A., et al. 2007. Influence of Mixed Solvents and Temperature on the Solution Properties of Quaternized Polysulfones. Journal of Macromolecular Science Part B Physics 46, 503-520. [11]. Guiver, M. D., et al. 1993. Functionalized Polysulfone Membranes by Heterogeneous Lithiation. Journal of Applied Polymer Science 48, 1597-1606. [12]. Albu, R. M., et al. 2011. Opto-electrical Properties of some Quaternized Polysulfones. High Performance Polymers 23, 85-96. [13]. Albu, R. M., et al. 2012. Polysulfones with Chelating Groups for Heavy Metals Retention. Polymer Composites 33, 573-581. [14]. Esmaeili, M., et al. 2010. The Dependence of Morphology of Solid Polymer Electrolyte Membranes on Transient Salt Type: Effect of Cation Type. Polymer International 59, 1006-1013. [15]. Li, W., et al. 2002. Synthesis and Characterisation of a Polyacrylamide–polyacrylic Acid Copolymer Hydrogel for Environmental Analysis of Cu and Cd. Reactive and Functional Polymers 52, 31-41. [16]. Pizarro, G. C., et al. 2007. Preparation, Characterization, and Thermal Properties of Hydrophilic Polymers: p-Chlorophenylmaleimides with Hydroxyethyl Methacrylate and β- methylitaconate. Polymer International 56, 93-103.

Acknowledgments: This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS-UEFISCDI, project nr. PN-II-RU-TE-2012-3-0143, 2013 stage.

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EFFECT OF AMMONIUM VANADATE ON VIABILITY AND PROLIFERATION OF CULTURED ANIMAL CELLS

Abdulkadir Abudalleh, Pavel Mitrenga, Tanya Zhivkova, Lora Dyakova, Stoyan Shishkov, Radostina Alexandrova

Abstract: The aim of the present study was to evaluate the effect of ammonium vanadate (NH4VO3) on viability and proliferation of cultured animal cells. Permanent cell lines MDBK (bovine kidney) and BALB/c 3T3 (mouse embryonal fibroblasts) were used as model systems in our investigations.The experiments were performed by thiazolyl blue tetrazolium bromide (MTT) test, neutral red uptake cytotoxicity assay, double staining with acridine orange and propidium iodide staining.The results obtained revealed that applied at concentrations of 0.1–20µg/ml for 24 h, 48h and 72h, NH4VO3 decreased (in a time and concentration dependent manner) the viability and proliferation of the treated murine fibroblasts whereas bovine kidney cells seem to be more resistant.

Key words: Ammonium vanadate,cytotoxicity assays, bovine kidney cells, mouse embryonal fibroblasts.

INTRODUCTION Vanadium, one of the essential micronutrients that are required in small amounts for normal metabolism of animals and humans, has attracted the attention of scientists during the recent years, because of at least three reasons: i) its widely distribution in nature; ii) the daily exposure of living organisms to vanadium compounds due to their application in current industry; iii) the promising antitumor and antidiabetisc properties of vanadium compounds. [Alexandrova, 1999; Mukherjee et al., 2004; Kostova, 2009].It has been found in our previous investigations that ammonium vanadate (NH4VO3) decreases significantly viability and proliferation of the treated chicken , rat and human tumor cells [Abudalleh et al., 2012, 2013 a,b].In order to increase our knowledge about cytotoxicity of vanadium and its compounds the aim of the present study was to evaluate the effect of NH4VO3 on viability and proliferation of cultured non-tumor animal cells.

MATERIAL AND METHODS 1. Chemicals and other materials Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-Invitrogen (UK). Dimethyl sulfoxide (DMSO), neutral red and trypsin were obtained from AppliChem (Germany); thiazolyl blue tetrazolium bromide (MTT) is from Sigma-Aldrich Chemie GmbH (Germany). All other chemicals of the highest purity commercially available were purchased from local agents and distributors. All sterile plastic and syringe filters were from Orange Scientific (Belgium). 2. Compound Ammonium vanadate (Valerus - Bulgaria) was dissolved initially in bidistilled water and sterilized by filtration (diameter of pores 0.2µm) and then diluted in culture medium. The concentration of the compound in stock solution was 1 mg/ml. 3. Cell cultures and cultivation Permanent cell lines BALB/c 3T3 (mouse fibroblasts) and MDBK (bovine kidney) were obtained from Cell Culture Collection of IEMPAM-BAS. The cytotoxic effect of NH4VO3 on MDBK cells was studied in our previous investigations [Abudalleh et al., 2012] and this cell line was included in the present study for comparative purposes. The cells were grown as monolayer cultures in DMEM medium, supplemented with 5- 10% FBS, 100 U/mL penicillin and 100 g/mL streptomycin. The cultures were maintained at 37 ºC in a humidified CO2 incubator (Thermo scientific, Hepa class 100). For routine

105 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA passages adherent cells were detached using a mixture of 0.05% trypsin and 0.02% EDTA. The experiments were performed during the exponential phase of cell growth. 4. Cytotoxicity assays The cells were seeded in 96-well flat-bottomed microplates at a concentration of 1×104 cells/well. After the cells were grown for 24 h to a subconfluent state (~ 60-70%), the culture medium was removed and changed with media modified with different concentrations (0.1, 0.5, 1, 5, 10 and 20 µg/ml) of NH4VO3. Each solution was applied into 4 to 6 wells. Samples of cells grown in non-modified medium served as controls. After 24 h, 48 h and 72 h of incubation, the effect of the compound on cell viability and proliferation was examined by MTT (thiazolyl blue tetrazolium bromide) test [Mossman, 1983] and neutral red uptake cytotoxicity assay (NR) [Borenfreund and Puerner, 1985]. Optical density was measured at 540 nm using an automatic microplate reader (TECAN, SunriseTM, Austria). Relative cell viability, expressed as a percentage of the untreated control (100% viability), was calculated for each concentration. ―Concentration – response‖ curves were prepared and the effective concentrations of the compound - CC50 (causing a 50% reduction of cell viability) and/or CC90 (causing a 90% reduction of cell viability) were estimated (where possible) from these curves using Origin 6.1. All data points represent an average of three independent assays. 5. Double staining with acridine orange (AO) and propidium iodide (PI) The cells were grown on sterile cover slips in 6-well plates in the presence of the compounds tested. Non-treated cells served as controls. After 24, 48 and 72h of incubation, the coverslips were removed and the ability of the compounds to induce cytopathological changes was assessed using double staining method with acridine orange (AO) and propidium iodide (PI) according to the standard procedures [Wahab et al., 2009]. The coverslips were washed with PBS for 2 min. Equal volumes of fluorescent dyes containing AO (10 µg/mL) and PI (10 µg/mL) were added to the cells. Fresh stained cells were placed on a glass slide and examined under fluorescence microscope (Leika DM 500B, Wetzlar, Germany) within 30 mun before the fluorescent color started to fade. 6. Statistical analysis between control and treated groups were assessed using one-way analysis of variance (ANOVA) followed by Dunnett post-hoc test and Origin 6.1TM .

RESULTS AND DISCUSSION RESULTS The influence of NH4VO3 on viability and proliferation of cultured murine fibroblasts (BALB/c 3T3) and bovine kidney (MDBK) cells was evaluated by thiazolyl blue tetrazolium bromide (MTT) test, neutral red uptake cytotoxicity assay (NR) and double staining with acridine orange and propidium iodide (AO/PI). The data obtained are summarized in Figures 1-3 and Table 1. They revealed that: 1) Applied at a concentration range of 0.1- 20 µg/ml NH4VO3 reduced significantly the viability and proliferation of murine fibroblasts (BALB/c 3T3) whereas bovine kidney MDBK cells seemed to be more resistant. For BALB/c 3T3 cells cytotoxic concentrations 50 (CC50) and 90 (CC90) were calculated from concentration-response curves and summarized in Table 1. CC50 of ammonium vanadate for MDBK cells was not determined because at all concentrations tested the cell viability was > 50%. 2) The combined staining with acridine orange and propidium iodide (AO/PI) revealed significant cell losses in BALB/c 3T3 cells treated with NH4VO3 whereas the

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available cells are swollen with picnotic nuclei, chromatin condensation and blebbing (Fig. 3). 3) A positive correlations between the data coming from MTT (which reflects damage to mitochondria) and NR (indicates damage to lysosomes and Golgi apparatus) methods were observed. Discussion In this study we present data about the ability of ammonium vanadate to decrease in a time- and concentration-dependent manner the viability and proliferation of BALB/c 3T3 cells. In contrast to murine embryo fibroblasts bovine kidney MDBK cells demonstrate lower sensitivity to cytotoxic/cytostatic effects of NH4VO3 that is in accordance with our previous investigations [Abudallech et al., 2012].The differences in sencitivity/resistance of these cell lines (BALB/c 3T3 and MDBK) to ammonium vanadate could be at least partially explained by cell specific response. The data about influence of vanadium compounds on BALB/c 3T3 cells have have also been reported by other research groups. Thus, it has been found that while vanadium(IV) at concentrations of 3 x 10(-6) M and 10(-5) M was not transforming in the cells, vanadium(V) showed neoplastic transforming activity. The intensity of vanadium(V)- induced cytotoxicity and morphological transformation in BALB/3T3 cells has been suggested to be dependent on glutathione-mediated bioreduction to vanadium(IV) [Sabbioni et al., 1993].It has been shown by Wozniak and Blasiak [2004] that vanadyl sulfate can be genotoxic for human normal lymphocytes. On one hand vanadium and its compounds were proved to express biologically important activity such as anticancer, immunostimulating and antidiabetis [Alexandrova, 1999; Alexandrova, Alexandrov, Nikolova, 2002; Mukherjee et al., 2004; Alexandrova, Alexandrov, 2004; Kostova, 2009]. At the same time there are data about their transforming ability and genotoxicity [Sabbioni et al., 1991, 1993; Wozniak and Blasiak, 2004].

CONCLUSIONS AND FUTURE WORK In conculsion the results obtained revealed that applied at concentrations of 0.1– 20µg/ml for 24h, 48h and 72h, NH4VO3 decreased (in a time and concentration dependent manner) the viability and proliferation of the treated murine fibroblasts whereas bovine kidney cells seem to be more resistant. In future we plan expriments to clarify the mechanism(s) of action and cellular targets of vanadium, to reveal the potential risks to human and animal health and the possibilities of its application in medicine.

Acknowledgement This study was supported by European Social Fund and Republic of Bulgaria, Operational programme "Development of Human Resources" 2007-2013, Grant No BG051PO001-3.3.06-0048.

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A B

200 150 Control Control 175 MTT 125 MTT 150 NR 100 NR 125

100 75 Control Control 75 50 50

25 Cell viability, % of the of % viability, Cell Cell viability, % of the of % viability, Cell 25 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Concentration g/ml Concentration g/ml

150 Control 125 MTT

100 NR

75 Control 50

25 Cell viability, % of the of % viability, Cell

0 0 5 10 15 20 25 Concentration g/ml

Fig.1. Effect of NH4VO3 on viability and proliferation of murine BALB/c 3T3 fibroblasts. The compound was applied at concentrations of 0.1, 0.5, 1, 5, 10 and 20µg/ml for 24h(A), 48h(B) and 72h(C). The investigations were carried out by MTT test and neutral red uptake cytotoxicity assay (NR) for 24h, 48h and 72h.

Table 1. Cytotoxicity (CC50 and CC90, μg/ml) of ammonium vanadate (NH4VO3) against murine BALB/c 3T3 fibroblasts Cytotoxicity Assay MTT NR Treatment Period 24h 48h 72h 24h 48h 72h

CC50 n.d. 11.7 3.7 2.6 3.2 3.1

CC90 n.d. n.d. 19.1 7.8 8.9 6.4

MTT = thiazolyl blue tetrazolium bromide (MTT) test, NR = neutral red uptake cytotoxicity assay; CC50 and CC90 – cytotoxic concentrations that reduce the percent of viable cells by 50% and 90%, respectively; n.d. – not determined 150 Control 125 MTT

100 NR CV

75 Control 50

Cell viability, %of the %of viability, Cell 25

0 Control MTT NR CV Cytotoxicity assay Fig. 2. Effect of NH4VO3 on viability and proliferation of bovine kidney MDBK cells. The compound was applied at a concentration of 0.1 µg/ml for 72 h. The investigations were performed by thiazolyl blue tetrazolium bromide (MTT) test, neutral red uptake cytotoxicity assay (NR) and crystal violet staining (CVS).

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Fig.3. Cultured murine BALB/c 3T3 fibroblasts:control (A) and treated for 48h withNH4VO3 at a concentration of 5 µg/ml (B) and 10 µg/ml (C). AO/PI staining, Bar = 20 μm.

REFERENCES [1]. Abudalleh A., et al. 2013b. Ammonium Vanadate decreases viability and proliferationof cultured Retrovirus-Transformed chicken hepatoma cells. Bulgarian Journal of Agricultural Science19 (2) (in press). [2]. Abdel Wahab S. I., et al. 2009. J. Biomed. Biotechnol, 46–55. [3]. Abudalleh A., et al. 2012. Effect of ammonium vanadate on viability and proliferation of human and animal tumor and nontumor cells.Journal of BioScience and Biotechnology, Special edition, pp. 25-29. [4]. Abudalleh A., et al. 2013a .Ammonium Vanadate Decreases Viability and Proliferation Activity of Cultured Virustransformed Rat Sarcoma Cells. Compt. Rend. Acad. Bulg. Sci., , 66(1), 61-66. [5]. Alexandrova R., et al. 2002. Effect of orally administered ammonium vanadate on the immune response of experimental animals. Compt. Rend. Bulg. Acad. Sci.55: 69 –72. [6]. Alexandrova R,and Alexandrov I. 2004. Effect of orally administered ammonium vanadate on the immune response of tumour-bearing mice. Compt. rend. Acad. Bulg. Sci.57:77-80. [7]. Alexandrova R. 1999. The secrets of vanadium. Exp. Pathol. Parasitol 2: 39-44. [8]. Borenfreund E and Puerner JA. 1985. Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol Lett. 24(2-3):119-24. [9]. Kostova I. 2009.Titanium and vanadium complexes as anticancer agents. Anticancer Agents Med. Chem.9(8):827-42. [10]. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth.65(1-2):55-63. [11]. Mukherjee B., et al. 2004. Vanadium—an element ofatypical biological significance. Toxicol Lett.150:135–143. [12]. Sabbioni E., et al. 1993. The intensity of vanadium(V)-induced cytotoxicity and morphological transformation in BALB/3T3 cells is dependent on glutathione-mediated bioreduction to vanadium(IV). Carcinogenesis 14(12):2565-8. [13]. Sabbioni E., et al. 1991.Cellular retention, cytotoxicity and morphological transformation by vanadium(IV) and vanadium(V) in BALB/3T3 cell lines. Carcinogenesis12(1):47-52. [14]. Wozniak K.and Blasiak J. 2003. Vanadyl sulfate can differentially damage DNA in human lymphocytes and HeLa cells. Arch. Toxicol. 78(1):7-15.

ABOUT THE AUTHORS A. Abudalleh, Ph.Dstudent, Faculty of Biology,Sofia University ―St. Kliment Ohridski‖ , 8 Dragan Tsankov Blvd. Sofia 1164, Bulgaria and Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences. Acad. Georgi Bonchev Str., Block 25, Sofia 1113, Bulgaria, E-mail:[email protected]. P.Mitrenga,Master of Science, Institute of Experimental Morphology, Pathology and 109 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Anthropology with Museum, Bulgarian Academy of Sciences. Acad. Georgi Bonchev Str., Block 25, Sofia 1113, Bulgaria, E-mail: [email protected] . T.Zhivkova, Ph.Dstudent, Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences. Acad. Georgi Bonchev Str., Block 25, Sofia 1113, Bulgaria, E-mail: [email protected]. L.Dyakova, Ph.Dstudent, Institute of Neurobiology, Bulgarian Academy of Sciences. Acad. Georgi Bonchev Str.,Block 23, Sofia 1113, Bulgaria, E-mail: [email protected]. S.Shishkov, Assoc. Prof., Faculty of Biology,Sofia University ―St. Kliment Ohridski‖ , 8 Dragan Tsankov Blvd. Sofia 1164, Bulgaria, E-mail: [email protected]. Radostina Alexandrova, Assoc. Prof., Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences. Acad. Georgi Bonchev Str., Block 25, Sofia 1113, Bulgaria, E-mail:[email protected].

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THE FORMULATION, PREPARATION AND CHARACTERIZATION OF A COMPRESSED MULTIPARTICULATE SYSTEM

Mircea Hîrjău, Dumitru Lupuliasa, Mirela Mitu, Anca Nicoară, Victoria Hîrjău ―Carol Davila‖ University of Medicine and Pharmacy, Faculty of Pharmacy, Bucharest, Romania

Abstract: The objective of this study was to formulate and obtain by compression a multiparticulate pellet system. Such systems provide the advantages of both pellets (a modified release of the drug by further film-coating and decreasing the risks of adverse reactions) and single-unit dosage forms (applying a well- known manufacturing process, relatively low costs of production). In this case, the major challenge is to produce a tablet with adequate hardness and friability, without damaging the integrity of the pellets or their coating during compression. Two formulations of pellet cores were prepared by extrusion-spheronization, using a blend of microcrystalline cellulose and lactose. The pellet cores could be used as a substrate for subsequent drug- layering and film-coating. The pellets were compressed into tablets, employing directly compressible excipients (Tablettose and Avicel PH102). The pellets were characterized regarding size, shape, properties essential for compression into tablets. Tablet hardness, friability, mass uniformity and microscopic fracture analysis of the resulting tablets were also determined. A blend of Tablettose and Avicel was revealed as a suitable cushioning agent for the pellets during compression.

Key words: Pellets, Extrusion-Spheronization, Compressed multiparticulate system.

INTRODUCTION In the last decades, the interest research and development of multiparticulate drug delivery systems has known an increase, more than 25% of the modified release drugs being authorized on the USA and European market beings such systems, in the form of spheres, pellets and microtablets [1 – 6]. Pellets are spherical, small (0.2 – 2 mm in diameter), dense and free-flowing granules, used in multiparticulate drug-delivery systems with per oral administration. Pellets can be obtained through a variety of techniques, the most often employed being compaction (by extrusion and subsequent spheronization). Different drugs can be contained within the pellets (matrix systems) or on their surface, by drug-layering inert pellet cores with a solution or suspension of the drug [7 – 13]. By film-coating the resulting pellets with specific polymers, a sustained, delayed, targeted or pulsatile release of the drug can be achieved, along with a better chemical stability of the drug, a decrease of the risk of adverse reactions or accidents (reduced risks of local irritation and toxicity and of dose-dumping), compared to monolithic dosage forms. Such pellets can be filled in hard gelatin capsules or compressed into pelletized tablets. A compressed multiparticulate system offers several advantages over capsules: high production rates, at a lower cost. If a high dose strength is required, the large size of the capsules can result in a low patient compliance, especially in patients with swallowing difficulties. Thus, the compression of pellets into pelletized tablets is an atractive alternative, because it presents the advantages of both pellets and tablets. The main challenge in compressing pellets into tablets is to maintain the integrity of the pellets and of the coating film, if any, at the high forces developed during compression, resulting in an altered drug release profile. Considering these facts, an important role in protecting the integrity of the pellets is played by the excipients in the tablet formulation, which are desired to have cushioning properties, and by the compression forces applied to the pellets [14].

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The aim of this study was to obtain two formulations of pellet cores by extrusion- spheronization and to compress the pellets into tablets with adequate hardness and friability and to study the impact of the excipients used in the pellet formulation on some properties relevant for the production of the tablets (pellet size, shape and flowing behavior) and of the excipients associated to the pellets in the tablet formulation on some characteristics of the compressed multiparticulate system (tablet hardness, friability, mass uniformity and microscopic fracture analysis).

MATERIAL AND METHODS The formulation of the pellet cores included the following substances of pharmaceutical grade: microcrystalline cellulose (Vivapur 102, S&D Chemicals Ltd.), lactose monohydrate (Mastermind International Ltd., China), polyvinylpyrrolidone K 30 (BASF AG, Germany). In the formulation of the pelletized tablets, the following excipients were associated to the pellet cores: directly compressible (DC) lactose (Tablettose, Meglle Pharma, Germany), Avicel PH102 (FMC Biopolymer, USA), talc (Luzenac Val Chisone, Italy), magnesia stearate (Union Derivan SA / Arnaud Romania). Two formulations of pellet cores were prepared by extrusion-spheronization technique, while the pelletized tablets resulted by applying a direct compression process to the corresponding mix of pellet cores and tableting excipients for each of the three tablet formulations. The pharmaco-technical evaluation of the pellet cores was performed for the following parameters: pellet size and shape. The tableting blends were characterized for flow behavior. The control on the multiparticulate tablets was conducted using methods described in the Romanian Pharmacopoeia for the following parameters: uniformity of mass, hardness, friability.

1. The formulation of the pellet cores The objectives of the formulation of the pellet cores were the selection of the excipients and correlated with the processing parameters (spheronization speed and duration), in order to result in pellets with an adequate size (0.8 – 1.1 mm), shape (as spherical as possible) and flowing behavior. Two formulations of pellet cores were taken into study. Table I The formulations of pellet cores Quantity (g% w/w in composition of Pellet components Role in formulation wet mass) Formula 1 Formula 2 Microcrystalline cellulose Filler / Spheronizing agent 40 25 -Lactose monohydrate Filler 25 40 Polyvinylpyrrolidone K Binder 30, 2% (w/w) aqueous 35 35 solution Total 100 100

2. The preparation of pellet cores The pelletization of the materials was done by an extrusion-spheronization technique, following these stages: weighing the solid components (microcrystalline cellulose and lactose), dry mixing (10 minutes), addition of the binder aqueous solution, wet mixing (10 minutes), extrusion of the wet mass (Caleva model 25 Extruder, equipped with a sieve opening of 1 mm in diameter) and spheronization of the extrudates (Caleva model 120

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Spheronizer, equipped with a friction plate with waffle-pattern grooves and operating at 1000 rpm; the spheronization duration was different – 2 minutes for the first formulation and 3 minutes for the second), drying of the pellet cores (UE/BE 200 – 800 (Memmert GmbH + Co. KG, Germany) oven at 37°C, for 30 minutes).

3. The formulation of the multiparticulate tablets The selection of filler excipients with cushioning properties was a critical aspect in the formulation of the pelletized tablets, in order to obtain tablets with satisfactory hardness and friability, while maintaining the integrity of the pellets. Two such cushioning excipients were selected and three pelletized formulations were developed. The formulations of tablets are shown in the following table. Table II The formulations of pelletized tablets Quantity (% w/w in composition of Pellet components Role in formulation tableting blend) Formula 1 Formula 2 Formula 3 Pellet cores Multiparticulates 40 40 40 DC lactose (Tablettose) Filler / cushioning 40 56 – excipient Microcrystalline cellulose Filler / cushioning 16 – 56 (Avicel PH 102) excipient Talc 3 3 3 Magnesia stearate 1 1 1 Total 100 100 100

4. The preparation of compressed multiparticulate system The direct compression applied for the formulas 1 – 3 comprises of the following stages: weighing the components, dry mixing, lubricating the mix and compression (Triowin machine with a set of 10 mm punches and die). Tablets with an average mass of 0.280 g were obtained.

5. Pharmaco-technical evaluation of the pellets and multiparticulate tablets The pellets were evaluated for: size distribution, by placing a weighed sample of pellets in a CISA Sieve Shaker (CISA Cedaceria Industrial, Spain), equipped with a set of standardized sieves and weighing the fraction of pellets on each sieve; shape – by taking photographs of 100 individual pellets for each pellet core formulation, using a Visioscan camera (Courage+Khazaka Electronics GmbH, Germany) with a magnification degree of 400X, and analysing the images using the ImageJ software (National Institute for Health, USA), which calculates a circularity coefficient for the pellets in each image. Also, the tableting blends corresponding to the three multiparticulate tablet formulations were analysed for flow behavior (using an automatic operating system PTG- S3 Pharmatest, Germany; the apparatus measures the sample‘s ability to flow in specified, compendial conditions, using an optical sensor). The test is relevant for ensuring a constant fill of the tablet punch and the mass and content uniformity of the tablets. The following parameters were evaluated for the compressed multiparticulate formulations, using compendial methods (Romanian Pharmacopoeia, Xth ed.): appearance (surface appearance, color), tablet thickness and hardness (VanKel VK 200 Tablet Hardness Tester), friability (VanKel Friability Tester), mass uniformity (Mettler Toledo pharmaceutical scale) of the tablets. Also, using the Visioscan camera, photographs of the tablet fracture after hardness testing were taken, in order to study the distribution of the pellets in the tablet and their integrity.

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RESULTS AND DISCUSSION Size distribution of the pellet cores The results are shown in the following figure.

Figure 1. Size distribution of the pellet cores

Both formulations have lead to a high proportion of pellets with size within the 0.8 – 1.18 mm interval (89.43% for formulation 1 and 89.17 for formulation 2). In the case of Formulation 2, the percentage of pellets larger than 1.18 mm in diameter (5.79%, compared to 0.1% for Formulation 1) can be explained by the higher content in lactose, while the pellets obtained with the formula 1 have shown a higher proportion on the 0.6 mm sieve (5.23% for formula 1 and 4.32% for formula 2). A higher content in microcrystalline cellulose has translated into slightly more brittle pellets, in the conditions in which the spheronization process was performed.

Pellet core shape The following figure shows representative images for the shape of the pellet cores obtained for the two formulations.

(a) (b) Figure 2. Pellet core shape for Formulation 1 (a) and Formulation 2 (b) By analysing the images with the ImageJ software, the values obtained for the circularity index were of 0.67937 for Formulation 1, and of 0.63556, respectively, for Formulation 2.

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Both formulations have resulted in relatively spherical pellet cores. The rounder shape of the pellets obtained for Formulation 1 can be explained by a higher proportion of microcrystalline cellulose proportion in the formula, an excipient which promotes spheronization.

Flowing behavior of tableting blend All three formulations have shown a good flowability, expressed by a short flow time in the compendial conditions (the average values recorded for three determinations were of 17 seconds for Formula 1, 20 seconds for Formula 3 and 28 seconds for Formula 2). It can be observed that the flow time decreases with the increase of the Avicel PH 102 proportion in the tableting blend formula. Thus, all tableting blend formulations (pellets and cushioning agents) have a good potential to be used in a tableting process.

Appearance of pelletized tablets For all four formulas, we have obtained disc-shaped tablets, white, convex, spotless, with no cracks and with intact margins. All parameters meet the before-mentioned requirements.

Tablet hardness The average values recorded for tablet hardness are shown in the following table. Table III Average tablet hardness values for the three pelletized tablet formulations Tablet hardness Formulation (kPonds) Formula 1 2.11 Formula 2 7.1 Formula 3 3.2

The results show that the tablet hardness increases with the proportion of Avicel PH 102 in the tablet formulation. This suggests that Avicel acts as a binder better than DC lactose. By studying the images obtained with the Visioscan, all formulations have shown that the integrity of the pellets is well maintained. Formulation 3 has presented the best cushioning properties.

(a) (b) (c) Figure 2. Images of the pelletized tablet fracture for Formulation 1 (a), Formulation 2 (b) and Formulation 3 (c)

Friability of multiparticulate tablets

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All formulations have shown a satisfactory values for friability (under 1%). The results allow a ranking of the three samples, the formulation with the lowest friability was represented by Formula 2, which contains only Avicel PH 102 as a cushioning agent.

Mass uniformity of multiparticulate tablets The average mass calculated for the three formulations are shown in table IV. Table IV Mass uniformity of multiparticulate tablets Formulation Average mass Deviation from declared mass Formula 1 0.2845 + 1.60% Formula 2 0.2737 - 2.25% Formula 3 0.2806 + 0.21%

The compression process applied for all three formulas has lead to homogenous batches of multiparticulate tablets regarding mass uniformity. The largest deviation from the intended tablet mass (0.280 g) was recorded for formula 2, which can be explained due to the slightly poorer flowability of the corresponding tableting blend.

CONCLUSIONS AND FUTURE WORK Two formulations of pellet cores were developed and prepared, based on microcrystalline cellulose (filler and spheronizing agent), lactose (filler) and polyvinylpyrrolidone (binder). The study of certain parameters, such as size and shape, which we have considered relevant for further processing of the pellet cores into multiparticulate tablets, has revealed the formulation containing a higher proportion of microcrystalline cellulose as being more suitable for tableting. The goal of the tableting process was to conduct to tablets with a sufficient hardness and a low friability, uniform in mass, while still maintaining the pellet core integrity during the production of the tablets. Two excipients with cushioning properties were selected (DC lactose and microcrystalline cellulose) and the influence of their proportion in the tableting blend on the characteristics of the tablets was studied. The study has shown that multiparticulate tablets with an adequate hardness and friability can be obtained using a high proportion (60% of the tableting blend) of a mix of the two cushioning excipients. The data collected in this study can be used in production of drug-loaded pellets, based on the formula for the pellet cores. These can be subsequently film-coated and compressed into multiparticulate tablets. We intend to study further the integrity of film- coated drug-loaded pellets after compression.

REFERENCES [1]. Tomuţa I., Leucuţa S.E., Validation of the laboratory-scale technological process preparation of colonic release pharmaceutical system with pH and time-control, Farmacia, 2009, Vol LIII, 4, 272-281, 57; [2]. Chetty D.J., Dangor C.M., The development of an oral controlled release pellets formulation of diethylpropion hydrochloride, Drug Dev. Ind. Pharm., 20 (6),993-1005, (1994); [3]. Gopal Venktesh Shavi et al., Multiparticulate drug delivery system of Aceclofenac:development and in vitro study, Drug Dev. Ind. Pharm., 35: 252-258, 2009;

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[4]. Neau S.H. et. al., Formulation and process consideration for beads containing Carbopol 974P,NF resin made by extrusion-spheronization, International Journal of Pharmaceutics, 199, (2000), 129-140 ; [5]. Tho I., Sande S.A., Kleinebudde P., Pectinic acid, a novel excipient for production of pellets by extrusion/spheronisation: preliminary studies, Eur. J. Pharm. Biopharm., 54 (2002) 95–99.; [6]. Keroe Janez, Challenges and opportunities in multiparticulate drug delivery, Strokovno Glasilo Slovenske Farmacije (Pharmaceutical Journal of Slovenia), vol. 59, september 2008, Published by Slovenian Pharmaceutical Society, ISSN 0014-8229 (SLO ISSN), 7th Central European Symposium on Pharmaceutical Technology and Biodelivery Systems, september 18-20, 2008, Ljubljana, Slovenia, pp. 25; [7]. Matei I.E., Pelete, în Iuliana Popovici, Dumitru Lupuleasa, Tehnologie Farmaceutică, Vol.3, Editura Polirom, Iaşi, 2009, 355-381; [8]. Ghebre-Sellassie I.,Ed., Pharmaceutical Pelletization Technology, (Marcel Dekker), New York, 1989, 20-120; [9]. Leucuţa S. E. , Ed. Dacia, ; [10]. Ghebre-Sellasie I., Knoch A.: Pelletization techniques, in: Swarbrick J., Boylan J.C.: Encyclopedia of Pharmaceutical Technology, Ed. 2002, by Marcel Dekker, New York, 2002, vol 3, 2057 – 2080; [11]. Gajdos, B., 1983. Rotorgranulatoren: Verfahrenstechnische Bewertung der Pelletherstellung mit Hilfe der faktoriellen Design, Pharm. Ind., 45, 1-7.; [12]. Gajdos, B., 1984. Rotary granulators - Evaluation of process technology for pellet production using factorial design, Drugs Made Ger., 27, 30-36.; [13]. Dukiš-Ott A., Thommes M., Remon J.P., Kleinebudde P., Vervaet C., Production of pellets via extrusion–spheronisation without the incorporation of microcrystalline cellulose: A critical review, European Journal of Pharmaceutics and Biopharmaceutics, 71 (2009) 38–46; [14]. Dias V. D., Fegely K., Gothoskar A. V., Rajabi-Siahboomi A. R., Application of an aqueous ethycellulose dispersion in multiple-unit pellet system, poster reprint Controlled Release Society, July 2007.

ABOUT THE AUTHORS M. Hîrjău, University of Medicine and Pharmacy ―Carol Davila‖, Faculty of Pharmacy, department of Pharmaceutical Technology, Str. Traian Vuia, no. 6, Sector 2, Bucharest, Romania, e-mail: [email protected]

AKNOWLEDGEMENTS The study was supported by a grant of the Romanian Autority for Scientific Research, CNDI – UEFISCDI, project number PN-II-RU-TE-2011-3-0228.

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STUDIES CONCERNING THE INCORPORATION OF ORIGANUM L. SYRIACUM ESSENTIAL OIL IN LIQUID AND SOLID FORMULATIONS FOR ORAL CAVITY USE

Zeinab Lakis, Teodora Balaci, Cerasela Gird, Ancuţa Fiţa, Andreea Stanescu, Denisa Mihele

Abstract: The current study aims to prove that adding the Origanum Syriacum essential oil in products designed for oral administration (mouth washes and lozenges) is truly beneficial. The essential oil was obtained from Origanum syriacum by steam distillation. The fact that the previous studies related to the chimical composition showed that essential oil”s has antimicrobial effect (due to the hight content of thymol and carvacrol) encouraged us to create new antiseptical oral products. The strong antibacterial effect was proved on standard strains and wild-type strains of Staphylococcus aureus and Streptococcus β-hemolytic. The antifungal activity was tested on Candida albicans standard and wild-type strins collected from the oral cavity. The essential oil was dispersed into the mouthwash solutions by micellar solubilisation mechanism. In addition, the lozenges, which are solid single dose preparations meant to provide a local effect in the mouth and throat area, have incorporated the essential oil into a flavored and sweetened base. Key words: Origanum Syriacum essential oil, Mouthwash, Lozenges.

INTRODUCTION The essential oils extracted from different species of Lamiaceae family are very popular herbal medicine products. Thymus vulgaris and Origanum syriacum’s essential oils are indicated as adjuvant therapy for many diseases. In traditional medicine, these essential oils are indicated for oral use (due to their upper respiratory tract catarrh, bronchitis, cough and antihelmintic action); cutaneous use especially for pruritus, inhalations, antiseptic gargle, bath additive etc. We previous determined by GC/MS, the chemical composition of Thymus vulgaris (Romania) and Origanum syriacum (Lebanon) essential oils and the results revealed that these oils are rich in phenolic compounds like thymol, carvacrol, terpinen, linalol, p-cymen, β-myrcene. The Origanum syriacum essential oil has an increased concentration of carvacrol (66.64%) while Thymus vulgaris essential oil is rich in thymol (29.74%) and p- cymen (30.26%). The thymol and the carvacrol are the main phenolic compounds mentioned by scientific literature for their many therapeutic properties. Our previous studies proved that both Thymus vulgaris essential oil (29.74% thymol) and Origanum syriacum essential oil (66.64% carvacrol) have a strong antimicrobial and antifungal effect on standard and wild type strains of Staphylococcus aureus, Streptococcus pneumoniae and Candida albicans. Due to our previous results, we aimed in this study to formulate a new mouthwash and lozenges with antimicrobial and antifungal properties with Origanum syriacum essential oil.

MATERIAL AND METHODS The essential oils of both Thymus vulgaris and Origanum syriacum were obtained from the aerial part of the plants by water extraction using Neo-Clavenger equipment. The chemical composition of the essential oils was determined by GC/MS. We used a gas chromatograph Fisons Instruments GC 8000, coupled with mass spectrometer with impact ionization quadrupole analyzer, model MD 800. Ionization energy was 70eV. The column used was a capillary fused silica column with 5% phenyl poly-(-dimetilsiloxan).

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The operating conditions were:

 Injector split spliless (injection mode - split flow dividing ratio 1/30) at 280 C, ion source temperature at 200C and at 280C to interface;  The initial temperature of the column was 40C is scheduled as follows: 3 min at 40 C, 4  C / min up to 250 C, isothermal system (250 C) for 10 minutes;  The flow rate of the carrier gas (helium) was 2 ml / minute;  Sample volume injected was 2 ml; Data acquisition was performed with software MassLab, with a mass in the range of 30-600 u, with a scanning speed of 1 scan / s. Identification of compounds detected was based on Kovats retention indices and comparison of their mass spectra with those in databases (NIST, WILEY and its own database, which contains about 600 spectra). Kovats indices were determined using two sets of C8-C20 n-alkanes and C21-C40. The experimental values obtained for Kovats indices were compared with those reported in the literature. The strong antibacterial effect was proved on standard strains and wild-type strains of Staphylococcus aureus and Streptococcus β-hemolytic. The antifungal activity was tested on Candida albicans standard and wild-type strins collected from the oral cavity. Further were formulated and prepared with essential oil researched the mouthwashes, based on the formula of pharmaceutical products on the market, using micellar solubilization method in order to obtain preparations the most stable over time. Four solutions were prepared for mouthwashes with the following active ingredients: menthol, thyme essential oil, benzoic acid. The solubilizers used were Cremophor RH 40 and Lutrol F 127. In the first formula is used, with the role of co-solubilizers, a mixture of Lutrol and glycerol. In the second formula to maintain, the composition of the first mouthwash formulations, but it decreased to half the amount of alcohol. The third preparation containing polyethylene glycols with the role of co-solubilizer, glycerol, and Lutrol and in the fourth preparation have reduced the amount of alcohol in half to the third formula. In all formulations studied were taken into account recommendations of expert guides, on (about) the concentration of each substance used. The composition of mouthwashes who are the subject of this study is shown in Table I.

Table I. The formulation of mouthwashes Components Formula 1 Formula 2 Formula 3 Formula 4

Essential oil 1 g 1 g 1 g 1 g Menthol 0.05 g 0.05 g 0.05 g 0.05 g Benzoic acid 0.10 g 0.10 g 0.10 g 0.10 g Saccharin sodium 0.25 g 0.25 g 0.25 g 0.25 g Sodium citrate 0.20 g 0.20 g 0.20 g 0.20 g Citric acid 0.50 g 0.50 g 0.50 g 0.50 g Cremophor RH 40 4.00 g 4.50 g 4.00 g 4.50 g Lutrol F 127 5.00 g 6.00 g 5.00 g 6.00 g Propylene glycol - - 7.50 g 7.50 g Glycerol 17.50 g 17.50 g 17.50 g 17.50 g Ethanol 96% 21.50 g 10.75 g 21.50 g 10.75 g Distilled water to 100 g 100 g 100 g 100 g

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Then, were formulated and prepared lozenges (soft and hard pills) with essential oil at a concentration of 1%, with the composition presented in Table II. Soft Pills Composition Amount (g) Menthol 0,5 Active substances Origanum syriacum essential oil 1 Ethanol 96% 2 Gelatine 20 Auxiliary Glycerol 37 substances Methylparaben-Propylparaben 3:1 (0,05) Distilled water 40 Lozenges Composition Amount (g) Active substances Origanum syriacum essential oil 1 Menthol 0,5 Ethanol 96% 2 Glucose 16 Auxiliary Starch 20 substances Sugar 48 Methylparaben-propylparaben 3:1 (0,05) Distilled water to 100

Table II. The formulation of lozenges

After preparation, solutions and lozenges were subjected to quality control, in which we determined: For mouthwashes: - Organoleptic characteristics; - pH (by potenttiometric method); - Viscosity with rheometer RC 1 (RheoTec GmbH Messtechnik GmbH, Radenburg, Germany), a rheometru rotational cone / plate provided with an electronic unit that allows digital control of the speed of rotation and direct recording of measured values. For measurements were determined under the test conditions as follows: shear rate as a function of the number of revolutions / minute, during the determination of the number of readings taken for determination. For lozenges: - Appearance ( margins of lozenges must be intact, mass of pill should be transparent, opaque or specific colored but do not show air bubbles in); - Smell, taste and color; - Moisture content; - pH; - Uniformity of mass; - Volatile oil content.

RESULTS AND DISCUSSION Chemical composition of the essential oil is shown in Table III and is illustrated in Figure 1. Figure 2 shows the chromatogram of volatile oil of Thymus vulgaris species

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(Romania). In the volatile oil obtained from vegetable product harvested from Lebanon dominates carvacrol (66.64%), which explains the different flavors like Romanian product; In the volatile oil obtained from the Romanian product dominate p-cymene (30.26%) and thymol (29.74%).

Table III. The results of chemical analysis by gas chromatography for volatile oil of Origani syriacae herba (Lebanon)

Compound RT, min KI Area % α-thujene 6.44 927 1.00 α-pinene 6.62 934 0.40 Camphene 7.12 950 0.03 Sabinene 7.91 973 0.08 β-pinene 8.01 976 0.08 β-myrcene 8.54 991 0.92 α-phellandrene 8.99 1003 0.16 3-δ-carene 9.07 1006 0.04 α-terpinene 9.35 1016 2.06 p-cymene 9.60 1024 2.94 Limonene 9.77 1030 0.26 β-ocimene 10.41 1050 0.04 γ-terpinene 10.76 1061 23.43 Linalool 12.15 1099 0.05 1-terpinen-4-ol 14.60 1180 0.05 Thymol 17.98 1292 0.11 Carvacrol 18.28 1302 66.64 Trans-caryophyllene 21.40 1416 0.11 bicyclogermacrene 23.41 1492 0.05 α-farnesene 23.76 1506 1.40 α-bisabolene 24.58 1540 0.10 α-muurolol 28.05 1684 0.03

9.602 10.759 18.281 100 9.345

23.762

% 6.436 8.537

6.621

4.032 17.976 7.906 21.400 24.580

0 tR 6.000 8.000 10.000 12.000 14.000 16.000 18.000 20.000 22.000 24.000 26.000 28.000 (min)

Figure 1. The chromatogram of volatile oil - Origanum syriacum (Lebanon)

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9.613 10.713 18.012 100 18.192

12.111 21.388

9.331 6.601

8.509 6.408 7.087 14.284 16.107

14.567 8.276 16.372 13.470 24.011 25.546 17.719 23.850 4.032 22.880 20.227 27.019 0 tR 6.000 8.000 10.000 12.000 14.000 16.000 18.000 20.000 22.000 24.000 26.000 (min)

Figure 2. Chromatogram of volatile oil - Thymus vulgaris (Romania) The results of mouthwashes quality control are shown in Table IV. Table IV. Mouthwashes quality control Parameter Formula 1 Formula 2 Formula 3 Formula 4 Analyzed Aspect Easly opalescent Clear Easly opalescent Opalescent Color Light yellow Transparent Light yellow White Mint and Mint and Mint and Mint and Odor/Taste volatil oil volatil oil volatil oil volatil oil pH 6,5 6,5 6,6 6,7 Viscosity 8,5 cP 7,5 cP 8,3 cP 7,4 cP

The experimental lozenges shown conical shape, entire margins, without air bubbles, transparent, white-yellow colored, have the characteristic smell of volatile oil, pH = 6.8, average weight at 2.5 g, and the volatile oil content of 1%.

CONCLUSIONS The present study aimed to capitalizing volatile oil obtained by distillation from the leaves of Origanum L. Syriacum to obtain pharmaceutical preparations, liquid and solid: mouthwashes and lozenges with antiseptic action. Mouthwashes containing essential oil with other active ingredients and uses micellar solubilization to obtain stable formulations as time. The lozenges contain active ingredients incorporated into a flavored and sweetened base. Substances in the formulation of lozenges must present slow dissolution characteristics to ensure maintenance of the product as long as possible in the buccal cavity, to obtain a local actions as long.

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REFERENCES [1]. Gird C.E., et al., Bazele teoretice si practice ale analizei farmacognostice, 2th edition, volume I, Bucharest, Curtea Veche, 2010; [2]. Hîrjău Victoria., Lupuleasa D., Dumitrescu A.M., Dermo – cosmetologie, Editura Polirom, 1998; [3]. Lakis Z., Gird C., Radulescu V., Ilies D.C., Adrelean S., Mihele D., ―Contributions at phytochemical of thymus vulgaris L., species from Lebanon.‖, 2012, Vasile Goldis University Press, Arad; [4]. Lakis Z., Mihele D., Nicorescu I., Vulturescu V., Udeanu D.I., ―The antimicrobial activity of Thymus vulgaris and Origanum syriacum essential oils on Staphylococcus aureus, Streptococcus pneumoniae and Candida albicans‖, Farmacia, Bucuresti, 2012; [5]. Leucuta S., Tehnologie Farmaceutica Industriala, Ed. Dacia, Cluj 2001; [6]. Pavel M., Radulescu V., Ilies D.C., GC-MS analysis of essential oil obtained from the species Thymus Comosus Heuff. Lamiaceae. Farmacia, 2009; [7]. Popovici Iuliana, Lupuleasa D., Tehnologie farmaceutica, vol I,II,III, Ed. Polirom, Iasi; [8]. xxx – Farmacopeea Română ed aXa, Ed. Medicala, Bucuresti, 1993; [9]. xxx – European Pharmacopoeia 7thEdition, Strasbourg, 2011.

ABOUT THE AUTHORS Z. Lakis, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] T.D. Balaci, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmaceutical Technology, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] C.E. Gird, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmacognosy Phytochemistry Phytotherapy, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] A.C. Fita, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmaceutical Technology, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] A.A. Stanescu, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Pharmaceutical Technology, 6 Traian Vuia Street, 2nd District, Bucharest, Romania, E-mail: [email protected] D. Mihele, University of Medicine and Pharmacy ,,Carol Davila―, Faculty of Pharmacy, Departament of Clinical Laborator, 6 Traian Vuia Street, 2nd District, Bucharest, Romania.

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THE MICROBIOLOGICAL MONITORING OF SOME BATCHES OF MILK ORIGINATED FROM PRIVATE HOUSEHOLDS IN THE WEST PLAIN OF ROMANIA

Mişcă Corina Dana, Mişcă C. B., Rinovetz A., Bujancă G., David I., Rădoi P. B., Rinovetz Adina

Abstract. The paper proposes the detection of the microbiological load and the identification of species with a pathological potential from raw milk collected from samples from rural areas - West Plain area of Romania. The total microbiological load of the milk was determined and there were indentified contamination microorganisms formed by species of coliforms. According to the value of the microbiological load identified measures of decontamination through pasteurization, of restricting consumption for some batches of analyzed milk were recommended, while others did not present any type of danger, fulfilling all the microbiological rigors of the standards in effect, which can be consumed without any restrictions.

Keywords: milk, UFC, coliforms, E.coli

INTRODUCTION The microbiological analysis of milk permits the identification and the removal from the technical and economic route of the raw material which doesn‘t meet the needs and demands imposed by legislation. Ensuring the hygienic conditionsin each step of the technological process determines the aparition of wholesome and quality products. Microbiological hazards are the main risk of milk quality, wholesomeness and safety. Either the microbiological analysis of the raw material is required or the analysis of drinking milk in order to detect the microbiological pollution [2]. According to law, the targeted microbiological parameters are: CFU(colony forming units), coliforms, E.coli and Salmonella. There is a tendency to replace the traditional techniques of microbiological analysis, which are long-drawn, with alternative microbiological techniques (dry rehydratable film and direct epifluorescent filter technique), however these techniques also require a period of 18-24 hours to obtain the results but with bigger costs in comparison with the clasic techniques [3]. The most accesible method for quick highlighting of the aproximate number of CFU, from milk is the method of reduction with Methylene blue or the method with resazurin, which requires a time period of 1-6 hours. According to these results, the milk can be consumer oriented, after thermal processing – pasteurization or UHT, or toward processing obtaining dairy products [1]. The legal requirements for safety conditions, of quality and production, are currently implemented in the states of the European Union through Directives 89/362/CEE and 92/46/CEEC, specifying the minimum requirements that the raw milk should be provided by healthy animals, and should not contain chemical substances that endanger the health of the consumer.

MATERIAL AND METHODS This paper proposes highlighting the microbiological load and spectrum of raw milk collected from the traditionaly bred domestic specimens from different areas (A, B, C), of the West Plain of Romania. After the collection there were established 10 homogeneous samples: 4 samples from the A complex, noted 1- 4, 3 from the B complex, noted 5 -7 and 3 from the C complex, noted 8 - 10. The samples were microbiologically analyzed through

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the imposition and the compliance with standardized specifications for raw milk in the legislative regulations of the Ministry of Health, for which the following parameters were checked for microbiological quality: 1) The total number of mesophilic aerobic germs (CFU) through rapid method: Methylene blue reduction test and the test of resazurin and also the Koch method, more accurate, but requires a longer period of time; 2) The total number of coliform bacteria and E.coli, using ADCL modified by Levine – as selective cultural medium, and for confirmation of the species of coliform bacteria, there were applied identification biochemical tests – TSI, MIU, FAD, Simmons citrate.

RESULTS AND DISCUSSION a) For a fast determination of the number of aerobic mesophilic germs the reduction test with Methylen blue and resazurin was used. The readings for resazurin were realised at different time periods ( =0; 25, 45, 60 min.), and for the samples with Methylen blue the readings were made at 6 hours.Evaluating by the colorimetric aspect, at the four moments – certain observations can be drawn regarding the microbiological quality of the analysed samples, so: - samples 9 and 10 at =60 minutes, good microbiological quality; - samples 1, 2, 3, 5, 7, at =60 minutes, satisfying microbiological quality; - samples 4, 6, 8, at =60 minutes, unsatisfying microbiological quality. In parallel with the sample of resazurin for the 10 samples of milk taken into analysis, the reduction tests with Methylen blue was performed aswell to compare the reliability of results. Evaluating the aspect of the samples at =120 min after treating with Methylen blue, we can notice a visible paleness of the samples 4,6 and 8 which indicates an unsatisfying microbiological quality. The observations made at =300 min indicates the discoloration of the samples 1, 2, 3, 5 and 7, which means a satisfying microbiological quality. At the end of the 6 hours of exposure, =360 min, it is noticed that samples 9 and 10 don‘t present any type of color modification which indicates a good microbiological quality. For a fair determination of the CFU for the 10 samples of milk, the Koch method was used. The dillution of 10-4 was inoculated in Petri plates with simple agar, the incubation period was 24 hours at 37oC, the evolution of these being presented in figure 1. Each determination was realised in triplicate. A maximum is revealed in the case of sample 6 and a minimum in the case of sample 10. Analyzing the results obtained for the three methods previously made, which targets the same microbiological parameter(CFU), we can state that the methods are in full compliance, relevance and practicaly complementary, being established among themselves a interdependence. Reporting the obtained results to the law, for raw milk, we can specify that from the 10 samples of analyzed milk, only 5 respect the required conditions, and only 5 samples could be given for consumption. From the data analyzed allowed for consumption are samples 1, 3, 5, 9 şi 10, the others (2, 4, 6, 7, 8), being considered to represent a high risk and should be disposed of. b) The determination of the number of coliform bacteria. The determination of the number of coliform germs from the 10 analyzed samples using the dilution of 10-2, is made using the cultural medium modified ADCL, and the results are presented in figure 2. Each determination was made in triplicate. The grafic presents the average values obtained from the efectuated repetitions.

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Figure 1. The microbiologycal Figure 2. The average of coliform load of the 10 samples, originating germs identified in the 10 milk samples from the three different areas of the originated from three different zones of the West Plain of Romania. West Plain of Romania.

The graphic interpretation from figure 2 for the 10 samples taken for analysis, reveals a maximum in the case of sample 6 (zone B) and a minimum is recorded in the case of sample 10 (zone C). The biochemical confirmation targets the identification of the coliform species presented. The aproximate number of germs belonging to the Escherichia coli genus – sanitation indicator which allows or not admission in consumer product. According to the european legislation, the total number of coliform germs from raw, unprocessed milk, must not exceed 103/ml sample, and the number of Escherichia coli germs must not exceed the value of 102/ml sample. Analyzing the graphic for the 10 samples, there can be observed a maximum in the case of sample 6 (areal B) and a minimum for sample 10 (areal C). The tests of confirmation, for all the milk samples, have identified 6 species with an ununiforme distribution in the analyzed samples presented in table no.1. Some identified species present a high risk of pathogenity, which leads to the removal from consumption of the milk polluated with these species.

Figure 3. The average value of Escherichia coli germs identified in the 10 samples of milk originated from the three different areas of the West Plain of Romania.

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Table 1. The distribution of coliform species in the 10 analyzed samples No. crt. Areal Sample Coliforms 1 1 Escherichia coli, Proteus mirabilis, Enterobacter aerogenes Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, 2 2 Citrobacter A Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, 3 3 Citrobacter Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, 4 4 Citrobacter, Shigella flexneri 5 5 Escherichia coli, Proteus mirabilis, Citrobacter Escherichia coli, Proteus mirabilis, Enterobacter aerogenes, 6 B 6 Citrobacter, Shigella flexneri, Salmonella typhi 7 7 Escherichia coli, Proteus mirabilis, Shigella flexneri, Citrobacter 8 8 Escherichia coli, Proteus mirabilis, Shigella flexneri, Citrobacter 9 C 9 Escherichia coli, Proteus mirabilis, Enterobacter aerogenes 10 10 Escherichia coli, Proteus mirabilis

As a result of these observations, the application of thermical treatments to the milk is mandatory imposed before it is given to consumption and it is highly recommended to avoid the consumption of raw milk. Although the consumption of a heat-treated milk that was very contaminated with microorganisms before the thermical process isn‘t recommended, because even if the coliform germs were destroyed, being asporulate, they have the capacity to synthesize certain toxines with a high thermoresistance – which can cause food poisoning.

CONCLUSIONS AND FUTURE WORK As a result to the analysis performed it has been found that from the 10 samples of milk tested, three samples presented a totally unsatisfying microbiological quality, five samples – a satisfying microbiological quality and two samples – a good microbiological quality. The number of CFU from the three unsatisfying samples were between 4.700.000 - 7.900.000 germs/ml; for the samples of milk with a satisfying microbiological quality the range of CFU was between 740.000 - 2.300.000 germs/ml, and for the two samples which present a good microbiological quality CFU was 490.000 respective 470.000 germs/ml. The microbiological spectrum contained the following germs: - E. coli, Proteus, Salmonella, Shigella. Milk producers must be verified in terms of respecting the hygene regulations, because some of the microorganisms found in the milk are by human origin and not by animal, for example Proteus mirabilis.

REFERENCES [1] Mişcă Corina Dana, 2001, Îndrumător pentru lucrări practice – Microbiologie agroalimentară, Ed. Solness, Timişoara, ISBN 973-8145-43-0. [2] Purnedu, C. V., 1993, rapid Method and Automation in Dairy Microbiology, J. Dairy Science, vol. 76(10), 3101-3113. [3] Rosmini, M.R., Signorini, M.L., Schneider, R., Bonazza, J.C., 2004, Evaluation of two alternative techniques for counting mesophilic, Food Control, vol. 15(1), 39–44.

ABOUT THE AUTHORS Mişcă Corina Dana, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119,

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Romania, phone numbers 0256/277327, fax numbers 0256/277261. E-mail: [email protected] Mişcă C. B., C. M. I. Dr. Misca phone numbers 0040 0744 554 463 Rinovetz A., Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Bujancă G., Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 David I., Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Rădoi P. B., Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Rinovetz Adina, Theoretical High School ―J. L. Calderon‖, 300115 Timişoara, Pestalozzi 14, Romania, phone numbers 0256/203406

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THE MONITORING OF ENZYME ACTIVITY OF GLUCOAMYLASE AND ENDOXYLANASE ON THE BREAD DOUGH

David I., Misca Corina, Bujanca G., Rinovetz A., Berbentea F., Georgescu L., Danci M.

Abstract. This study presents the action of two enzyme preparation: based on glucoamylase and endoxylanase, in the bread dough. The determination of the rheological characteristics of the dough is obtained by alveographic method and baking test. The addition of glucoamylase and endoxylanase enzymes allows adjustment of the rheological characteristics of dough according to the needs of the technological process. The specific effect of glucoamylase and endoxylanase on the physical properties of dough show a positive influence on the volume and porosity of the bread, also on reducing the kneading process of the dough and the energy consumption for the technological process.

Key words: bread, glucoamylase, endoxylanase, alveograph method, baking test.

INTRODUCTION Enzymes applications have grown to be a common practice in the baking industry with advantage of being considered as natural additives. The enzymes preparation are being used in the baking industry to improve dough-handling properties. The synthetically additives can be replaced with natural additives, as enzymes. Bread is the most comun traditional food product in the entire world. It has a high nutritive value due to the content of easily retainable sugars, lipids and proteins. γ-amylase (glucoamylase or α-1 ,4-D-glucan-glucohidrolaza) is an exoamylase used in food industry, that catalyzes the hydrolysis of α-1 ,4-linkages of glycosidic starch / glycogen (from the end of the unreduced polisugar) to form glucose. Compared with other amylases (α or β) γ-amylase is active at acidic pH (pH optimum is 3). Most glucoamylases can split and bond α-1 ,6-glycosidic, but at a rate lower than the connection α-1, 4. A single species of fungs, Aspergillus oryzae is used on an industrial scale preparation of α- amylase and glucoamylase. Glucoamilase from A. Niger contains 3 regions: (i) the region containing the catalytic (amino acids 1-470), which is the portion glycosylated region 441- 470, (ii) a region binder (amino acids 471-508) in which serine or threonine residues are O-manosylate and region (iii) attaching insoluble starch chain (amino acids 509-616). Glucoamylase acts against aging bread, but also helps prevent steakiness of core. Also works during Maillard reactions between low molecular weight dextrins and protein materials, and can lead to a darker bread crust. Endoxilanaza is most effective hemicellulases in the manufacture of bread. It hydrolyses araboxylans both water-soluble and water-insoluble, in relatively large oligomers. Degradation into smaller fragments occurs strongly with these types of enzymes. Adding endoxylanase of Aspergillus niger for bread leads to increased disolving araboxylans up to 10 to 30%. Without addition of enzyme, 15 to 20% of non-extractable pentosans are solubilized by endogenous xylanase present in wheat flour. Adding endoxylanase double this amount, leading to an increase of 40-65% for viscosity of dough. The breaking of pentosans chains and their disolving helps to improve dough tolerance and a higher volume of the bread. Endoxylanase contribute to greater stability through effects against aging, increased warranty period, improved freshness and improve the texture of bread with a softer core, fine and uniform pore structure, stability of frozen dough, increased crispiness and decreased wettability. Are also used to increase the volume of bread products with high

129 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA content of fiber, to improve the quality of bread flour. The color and flavor are also improved due to the browning effect of the crust and whitening effect of the core.

MATERIAL AND METHODS Samples preparation. The samples are obtained by mixing 250g flour with a solution of salt, yeast and enzyme preparation in the mixer for 15 minutes to form dough. The amount of water is adjusted according to the absorption capacity of flour. Enzyme preparations used are: - ALPHAMALT GA 5071 - based on glucoamylase enzyme preparation; Features: improves browning features, increase the power of slow fermentation; Dosage: 1- 10g/100kg flour - ALPHAMALT HCJ – based on endoxylanase enzyme preparation; Features: improved extensibility characteristics of dough, increase volume; Dosage: 5-15g/100kg flour The standard sample (Proba 0) has only 250g flour composition with a solution of salt, yeast and water. Does not contain enzyme preparations.. Each dough sample is divided in five circular consecutive dough patties witch are rested 20 min in the alveograph in a temperature-regulated compartment at 25 °C. Each dough patty is tested individually and the result is the average of the five dough patties. Methods of analysis. 1.The alveographic method relies on measuring the resistance to biaxial stretch under air pressure of a dough sample prepared in standard conditions and is based on three-dimensional expansion of the pieces of dough (made from flour, salt, water), which under the action of a current of air pressure forms a bubble. Equipment used to determine the alveogrames is the Alveo-Consistograph NG. Alveo-Consistograf NG determines rheological characteristics of flour and is known throughout the world, and the method used is recognized by ISO, ICC, AACC. From the alveogram the following indicators were obtained: • P Value is the force required to blow the bubble of dough. It is indicated by the maximum height of the curve and is expressed in millimeters (mm). It is also known as the viscosity or the value of maximum pressure that is in relationship to the resistance of the deforming dough (mm H2O). • L Value is the extensibility of the dough before the bubble breaks. It is indicated by the length of the curve that begins from the origin until the perpendicular point that corresponds to decreasing pressure due to rupture of air bubble and is expressed in millimeters (mm). • G Value is the expansion index G being the average of the expansion index on the graphic of cellules and corresponds to breaking the abscise L, G =2.226L, where L – air volume (cm3) used to stretch the dough under bubble form. • P/L Ratio is the balance between dough strength and extensibility. It is the rapport of configuration of the curve. • W Value is the area under the curve. It is a combination of dough strength (P value) and extensibility (L value) and is expressed in joules. It represents the action of deformation of the dough, based on a gram of dough, evaluated at 10 E – 4 joule, calculated as follows: W= 1.32 x (V/L) x S, where V- air volume in mm3; L- the average abscise at breaking point in mm; S- surface of the curve, cm2. • Ie – elasticity index, represents the raport between the measured pressures, 3 expressed in mm H2O to form bubbles after the insufflations of 200 cm of air in dough form, that correspond to a length L of 40 mm or an index of expansion G from 14,1 and the maximum of the curve P: Ie%= P200/Pmax.

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2. To determine the characteristics of flours and baking influence of various additives on the quality of their bread, the most reliable method is the baking test. Flour quality assessment and its behavior in the presence of additives is based on the quality of bread produced. The baking test allows tracking the rheological properties of dough during the technological process. Baking tests were performed using both equipment and laboratory facilities, specific, and industrial equipment. Baking process requires a baking oven and a fermenting room. Samples after kneading are put in fermenting room for 45-50 min at a temperature of 33-35 ° C. After that samples are placed in the baking oven at a temperature of 245 ° C for 50min. After baking, the breads are left to cool for 20 min. After cooling, the breads are cut in the middle and measure with a ruler and determined the section height.

RESULTS AND DISCUSSION a) The dough samples alevograms are represented in Fig. 1, Fig. 2, Fig. 3. Each dough sample alveorgame show the five dough patties tested (marked with different colors) and the parameters registered at the testing moment. The results of the samples are represented by the average value obtained from the values of the dough patties tests for each dough sample. In Fig. 1 the alveograme of the standard sample (Proba 0) - no added enzyme preparation are shown the dough‘s rheological characteristics of a sample that contains no enzyme. Compared to optimal values (P = [65 – 75mm], L = [130 – 150mm], G = [20 – 30], P/L = [0,5 – 0,6] and W > 180 10– 4J.) are found: - Dough resistance to deformation (P0) show lower 10mmH2O; - Dough extensibility (L0) has a value less than the normal 75mm; - Also lower values with 8.1 for the extensibility index (G0); - - Total energy absorbed by the dough to stretch (W0) is presented below with 63x10 4J / g; - And curve configuration ratio (P/L0) is 0.85, which is higher with 0.20 than the optimal values for bread dough. The dough sample shows a low extensibility and low tensile strength, suggesting that it is not resistant and bread obtained is undeveloped, flat shape with cracks in the upper crust.

Figure 1. Alveograme sample Proba 0 - no added enzyme preparation

131 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

In the alveograme of sample Proba 1 - with the addition of glucoamylase we can notice an improvement of rheological properties of dough due to the addition of this enzyme. It can be seen that: - The amount of dough resistance to deformation (P1) increased with 12mmH2O in comparison sample Proba 0 - no added enzyme and fits within normal parameters for bread dough. - Dough extensibility (L1) has a value greater than sample Proba 0 with 44mm, but is still much lower than normal bread dough with 21mm. - A significant increase is observed in the value of the extensibility (G1) compared to G0, but compared with normal values is less than 1.8. -4 - Total energy absorbed by the dough to stretch (W1) shows an increase of 133x10 J / g compared to W0 and curve configuration ratio (P/L1) has a lower value with 24 than the standard sample and compared to normal conditions these parameters are within normal parameters for bread dough. Thereby demonstrating that the enzyme preparation based on glucoalylase improved dough quality compared with standard sample. When dough elasticity and extensibility is high enough, it results in loose bread, with developed volume and a core containing pores with thin walls. This sample Proba 1contains the optimal conditions used in bread dough.

Figure 2. Aleograme sample Proba 1 – with addition of enzyme preparation based on glucoamylase

In Fig. 3 the alveograme of sample Proba 2 - with added endoxylanase shows a small improvement in all indicators compared to sample Proba 0, but not achieving the parameters of Proba 1 or the normal conditions for bread dough. The results show: - Dough resistance to deformation (P2) shows a small increase of 3mmH2O in comparison to the standard sample, but in comparison to sample Proba 1 is less with only 9 mm H2O. - also there is a small increase in the properties of dough extensibility (L2 and G2) compared to standard sample,this means that L2 is only 18mm higher and G2 only 2.4 higher, but unlike sample Proba 1 these parameters are much more smaller. - Elasticity index (Ie2) decreased by 5.1% compared to Ie0 and with 12.7% compared to sample Proba 1. - The total amount of energy absorbed by the dough to stretch (W2) increased with - 4 - 4 22x10 J / g compared to W0, but compared to W1 is presented below with 111x10 J / g

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Compared with standard sample parameters were improved, but compared to sample Proba 1 or optimal values for bread dough, these parameters do not qualify for bread dough. Bread made from this dough has a small volume, pale, and with a dense core.

Figure 3. Aleograme sample Proba 2 – with addition of enzyme preparation based on endoxylanase

Table 1. Alveograph results of the dough samples: Proba 0 - no added enzyme preparation; Proba 1 – with addition of enzyme preparation based on glucoamylase; Proba 2 – with addition of enzyme preparation based on endoxylanase Sample Proba 0 - no Proba 1 – with Proba 2 – with added addition of enzyme addition of enzyme enzyme preparation based on preparation based on preparation glucoamylase endoxylanase

P(mmH2O) 55 67 58 L(mm) 65 109 83 G 17,9 23,2 20,3 W(10 – 4J/g) 137 270 159 P/L 0,85 0,61 0,70 Ie(%) 57,8% 65,4 52,7 In Tabel 1. there are presented the characteristics of dough samples obtained by alveographic method. The dough sample Proba 1- with addition of enzyme preparation based on glucoamylase has the highest value for the following indicators, the dough extensibility characteristics (L and G), the total quantity of absorbed energy during the dough deformation (W) compared to the dough samples Proba 2- with addition of enzyme preparation based on endoxylanase and Proba 0 - no added enzyme preparation. The dough sample Proba 2- with addition of enzyme preparation based on endoxylanase presents improvments of the dough extensibility characteristics compared to the standard sample. The corresponding enzyme preparation enhances flour quality and provides an appropriate characteristics for bread dough by creating a dough which is more easly handled during the mechanical process, which becomes more dry, elastic, resulting in loose bread, developed volume and a core containing pores with thin walls. b) regarding the baking test the results are as follows (Table 2):

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The sample Proba 1 - with the addition of glucoamylase has the best volume and Proba 2 - with addition of endoxylanase is at 1.4 cm difference. Unlike Proba 0 – with no added enzyme preparation which has a very low volume and cracks in the crust, the other two samples show a significant increase in volume and pleasant look.

Table 2. The baking test results of the dough samples: Proba 0 - no added enzyme preparation; Proba 1 – with addition of enzyme preparation based on glucoamylase; Proba 2 – with addition of enzyme preparation based on endoxylanase Probe Proba 0 - no Proba 1 – Proba 2 – cu with added with the addition of enzyme addition of endoxylanase Parametrii glucoamylase Volumul (h) cm 5,9 7,9 6,5

In the figures below we can notice the significant difference of the breads made from this samples:

Figure 4. Proba 0 - no added enzyme preparation

   Figure 5. Proba 1 – with addition of  Figure 6. Proba 2 – with addition of glucoamylase endoxylanase

CONCLUSIONS AND FUTURE WORK The additive actions of complex enzymes as ameliorator on flour have positive effects on the rheological characteristics of dough. The technological characteristics of the flour and the nutritive value of the bread are characterized by the following variables: initial volume, fermentation time, flexibility, the dough condition to fermentation, water retention, maximum resistance, extensibility, final rise to baking, final volume of the bread, nutritive value, and energy value In order to improve these variables, different additives and substances are used in the bread manufacture, some of these being native components of the flour. The alveograph test provides results that are common specifications used by flour millers and processors to ensure a more consistent process and product. The alveograph is well suited for measuring the dough characteristics of weak gluten wheat. Weak gluten flour with low P value (strength of gluten) and long L value (extensibility), is preferred for cakes and other confectionery products. Strong gluten flour will have high P values amylase and is preferred for breads.

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The two enzyme preparations used are designed to improve the quality of dough used in the preparation of bakery products. They do not change the manufacturing process and do not have a high cost. Choosing suitable enzyme preparation is based on dough rheological characteristics and the correct amount to be added to the dough will be one that will bring maximum value for these features. Glucoamylase acts against aging bread, but also helps prevent steakiness of core. Also works during Maillard reactions between low molecular weight dextrins and protein materials, and can lead to a darker bread crust. Endoxylanase contribute to greater stability through effects against aging, increased warranty period, improved freshness and improve the texture of bread with a softer core, fine and uniform pore structure, stability of frozen dough, increased crispiness and decreased wettability. Are also used to increase the volume of bread products with high content of fiber, to improve the quality of bread flour. The color and flavor are also improved due to the browning effect of the crust and whitening effect of the core. Enzyme preparations are used to obtain quality breads and more natural. These products are of most interest to all consumers..

REFERENCES [1] Bordei. D., 2005, ―Tehnologia moderna a panificatiei‖, AGIR Editure, Bucharest [2] Diaconescu. D., Ivan. E., 2004,―Amelioratori enzimatici folositi in panificatie‖, Informative bulletine of ,miiling and bread making, vol. 15, no. 3 [3] Mencinicopschi Gh., David I., Brăgărea Şt., Zarnea G. 2008, ―Biotehnologii alimentare, Vol II‖, Editura Mirton, Timişoara. [4] Mencinicopschi Gh., David I., 2008 ―Calitatea alimentului. Dozarea activităţii enzimatice. Metode de analiză a activităţii enzimelor utilizate în fabricarea alimentelor‖, Editura Mirton, Timişoara,. [5] Roumanian Standards ISO 5530-4:2005. Făină de grâu. Caracteristici fizice ale aluatului. Partea 4: Determinarea proprietăţilor reologice folosind alveograful.

ABOUT THE AUTHORS David I, U.S.A.M.V.B., Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technologies, Calea Aradului 199, Timisoara, Romania, +40-744 586 970; E-mail: [email protected] Misca Corina, U.S.A.M.V.B., Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technologies, Calea Aradului 199, Timisoara, Romania, +40-744 586 970 Bujanca G., U.S.A.M.V.B., Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technologies, Calea Aradului 199, Timisoara, Romania, +40-744 586 970 Rinovetz A., U.S.A.M.V.B., Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technologies, Calea Aradului 199, Timisoara, Romania, +40-744 586 970 Berbentea F., U.S.A.M.V.B., Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technologies, Calea Aradului 199, Timisoara, Romania, +40-744 586 970 Georgescu L., U.S.A.M.V.B., Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technologies, Calea Aradului 199, Timisoara, Romania, +40-744 586 970 Danci M., U.S.A.M.V.B., Banat University of Agricultural Sciences and Veterinary Medicine, Faculty of Horticulture, Calea Aradului 199, Timisoara, Romania, +40 256 277021, E-mail: [email protected] 135 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

VARIATION OF HEAT TRANSFER AND TEMPERATURE OF TOMATO PASTE AT STERILIZATION AND COOLING PROCESSES

G. Bujancă. C. Jianu, A. Rinovetz, F. Crista, I. David, Corina Dana Mişcă, D. Ştef, Antoanela Cozma, Mihaela Cazacu, Zorica Adina Riniovetz

Abstract: An experimental work was performed for the election arrangements to enhance cooling water poured tomato paste in large packs. By heating or cooling the product to obtain the most complete heat transfer and faster. In this case the product temperature near the wall and in the center has no appreciable difference, all mass particles are subjected to the same heat treatment, this being a very important factor for maintaining product quality.

Key words: tomato paste, heat treatment.

INTRODUCTION When canned products are heated or cooled, the heat transfer from the outside to the inside of the can or vice versa takes place through heat conductivity, convection or both. The consistency of the sterilized product is the one that determines the way through which heat transfer takes place, and its reaction to cooling. [1] In regard to its consistency, tomato paste is a viscous product. Therefore, convection is mush slowed down, and heat is transferred especially through heat conductivity. This explains also the fact that when tomato paste is sterilized in small packages, heat is transmitted very slowly to the middle of the box. [2] Tomato paste canned in small recipients, in tins no.14 and in glass jars type 83-3, at a temperature of 85ºC, is sterilized in autoclave or in KSJ sterilizers. After sterilization, the boxes and jars are cooled with water. [3] During heating (10 minutes) and sterilization (30 minutes), the temperature of tomato paste in cans type 83-1 rises from 83ºC to 93ºC, in cans no. 9 and 58-1 after 20 minutes of sterilization - from 80ºC to 93ºC or 95ºC. The product takes a long time to cool down. It takes 33 minutes to cool the paste in cans type 58-1 to 45ºC, while for paste in cans no. 9 it takes 43 minutes, and for the paste in cans type 83-1 it takes 58 minutes. When the paste canned in cans type 14 is sterilized in an autoclave, after 30 minutes of sterilization the temperature in the center rises by 1ºC, while near the walls of the recipient, the temperature rises by 8ºC. During the cooling process (30 minutes), the temperature in the center of the tin drops by 2.5ºC, and near the walls it drops to -5ºC. After the cans are exposed to the air, the temperature becomes more uniform, and after 2.5 to 3 hours it drops to 45 - 50ºC. In order for the temperature of the paste in the center of can no. 14 to become 95ºC, it must be sterilized for 2 hours. It takes the same amount of time for the average temperature to cool down to 50ºC, in water.

MATERIAL AND METHODS Many factories use KSJ sterilizers; these are devices that function continuously, where tomato paste canned in cans no. 14 is sterilized at a temperature of 98 to 100 ºC, and is after that water cooled to 25 - 30ºC. During the sterilization process, after 33 minutes, the temperature in the can centre rises by 1ºC (from 85ºC to 86ºC), and near the walls by 6ºC. The cans are slow to cool, after 14 minutes the temperature drops to 80ºC in the center and to 62ºC near the walls, and that is why the stacked cans need air cooling for 3 additional hours. In order for the

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temperature in cans no. 14 to be lowered to 45 - 50ºC, the cans need additional water cooling for 40 more minutes. Thus, the sterilization regime of the tomato paste in cans no. 14, which is currently used in factories, keeps a temperature of 85 - 86ºC in the centre of the can for 45 minutes. The outer layers of tomato paste are heated to 90 ºC, and are cooled faster. A large part of the paste is canned in factories at the temperature of 92ºC to 95ºC, in large containers (83-3, 83-4, no.15), which are then air-cooled. Experiments were made on water-cooling the tomato paste. After put into 83-3 glass jars, the paste was air-cooled after 300 minutes, and then in running water, in fixed position, after 145 minutes. If tomato paste is poured with high temperature in 83-4 type jars, it takes a longer time for it to cool. Observations made in factories revealed that the temperature of paste temperature in air-cooled jars decreases to 45ºC after 640 minutes (10 hours and 40 minutes). If water-cooled, then the temperature drops faster, but even in this case, for the average temperature to drop to 45ºC, it needs 380 minutes (6 hours and 20 minutes). Table 1 presents the average data for the cooling duration of tomato paste canned in large containers, poured when hot.

Table 1. The average values for the cooling time for tomato paste cooled in large containers Paste Paste temperature after cooling Type of temperature at Time [ºC] Cooling procedure recipient canning [min] in the centre near the walls [ºC] 83-3 85 air 310 45 43.5 83-3 94 air 335 46 43 83-3 87 water 145 46 41 83-4 95 air 640 47 42 83-4 95 water 380 45.5 36 no. 15 85 air 600 47 44 no.15 87 water 240 48 42

Observations show that the paste canned at 85ºC to 95ºC in large containers (3 l to 10 l) takes a long time to cool. When the paste is canned in 83-3 jars, the paste temperature is 70ºC to 95ºC for two hours, and this is harmful for the quality of the product. The same temperature is maintained for 3h to 3.5 hours in the paste canned in 83-5 jars and in tins no.15. If we take into consideration that before being poured in cans the paste is for a long time warmed to 95ºC, it becomes clear that the tomato paste packing and sterilization regimes that factories use nowadays damage the product considerably. It is not common practice for factories to cool tomato paste with water, especially if sterilization has not been performed. The experiments revealed that in the case of jars and cans between 3 and 10 l, in a fixed position, water cooling is slow and large amounts of water are required for cooling the paste to the necessary temperature, therefore it implies high water consumption. An experiment was made for choosing the regimes which would intensify the water cooling process of tomato paste in large containers. The most complete and fastest heat transfer is obtained in heating or cooling the tomato paste in agitation retorts. In this case, there is no significant difference between the temperature in the centre and near the walls; all particles are subjected to an identical heat treatment, which is very important for the quality of the product. Paste agitation can be made by rotating the cans around their own axis, which highly accelerates the cooling process. Research has been made on the part played by the

137 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA package rotation speed, the container filling degree and the paste dry content in the cooling speed of the tomato paste.

RESULTS AND DISCUSSION The influence of the degree to which the can is filled. If the package is not entirely filled with product, a space is left in it that is filled with air, which helps in agitating the paste during rotation. The layers of cooled paste near the walls move towards the centre, and hot pasta is projected towards the walls. When agitation takes place, the difference between paste temperature in the centre of the container and the temperature in the outer layers is insignificant. On the other hand, without agitation, the temperature in the center can be much higher than the temperature near the walls. During the experiment, the rotation of the cans was maintained constant and equal to 41 rotations per minute; the cans were cooled with water and the only difference appeared in filling the cans with paste. For the glass jars, the temperature of the cooling water was gradually lowered from 60ºC to 20ºC. For the cans, the temperature of the cooling water was 20ºC. Can cooling ended when the temperature of the product reached 45ºC. At filling, paste temperature was 85 ºC. Figure 1 shows the cooling speed variation of the paste in 83-3 jars in relation to the weight of the packed product, while Figure 2 shows the same variation for cans no. 14. Cooling time for 83-3 jars with 3200g and 3300g net weight is almost the same. Nevertheless, increasing the weight to 3400 g doubles the cooling time. For can no.14, the optimal net weight was 2900 g, for which the time required for cooling was 18 minutes. By comparison, cooling the can with 3000 g net weight took 31 minutes, and if the can was filled with 3100 g net weight, the cooling time was significantly increased: it became 82 minutes.

r e

, y a l

l ] a r C t o n [

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speed rotion [rot/min] speed rotion [rot/min] Figure 1. Variation of the cooling speed in Figure 2. Variation of the cooling speed in 83-3 jars in relation to the weight of the cans no. 14, in relation to the weight of the product: 1 - 3.200g; 2 - 3.300g; 3 - 3400g product: 1- 2900g; 2 - 3000g; 3 - 3100g

Figure 3 presents the cooling time variation of the paste preserved in 83-4 jars and cans no. 15, in relation to the weight of the product. Although the capacity of can no.15 is three times bigger than can no.14, there is little difference between the cooling time of paste in such a container and cans and jars that can hold 3 liters. In jars 83-4, tomato paste cools down considerably slower. 70 minutes are required in order to obtain a temperature of 45 ºC of paste in the centre of the 10800g net weight jars. After 30 minutes of cooling, the paste temperature drops to 67 ºC on average.

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] C o [

e r u t a r e p m e t

speed rotion [rot/min] Figure 3. Variation of the cooling speed in cans no. 15 and 83-4 jars in relation to the weight of the product: 1 - 8800g (cans no 15); 2 - 9000g (cans no 15); 3 - 9100g (cans no 15); 4 - 10800g (83-4); 5 - 10900g (83-4)

The data presented reveal that the lower the filling degree of the container, the shorter the cooling time of the product. Nevertheless, from an economic point of view, it is more profitable for the container to be filled as much as possible. The price list for tomato paste preserved in large containers is in relation with the net weight, effectively obtained at the factory. 3.1 kg is the maximum possible filling for can no. 14, and 3.1 kg is the maximum possible filling for 83-3 jars, but when paste is sterilized in 83-3 jars, the usual net weight is 3.2 kg. For cans no. 15, the adopted net weight is 9 or 9.1 kg, and for 83-4 jars the adopted net weight is 10.9 kg. The optimal net weight for the above-mentioned containers was chosen taking into consideration the maximum possible use of the container, as well as the cooling time. When the product is cooled by rotating the cans, the following degree of filling with tomato paste is recommended.

Table 2. The recommended values for filling the containers with tomato paste Type of container Net weight in kilograms no. 14 3.0 no. 15 9.0 SKO 83-3 3.3 SKO 83-4 10.8

The influence of the rotation speed of the container. For every product there is an optimal rotation speed that renders the best agitation of the product and hence its fastest cooling. If the speed is slower than the optimal one, the product does not agitate sufficiently, and thus the entire cooling process is slowed down. If, on the other hand, the speed is higher than the optimal one, the centrifugal forces will decrease the mobility of the content, which also damages product agitation. Figures 4 and 5 illustrate the variation of the tomato paste cooling speed in relation to the rotation speed, as resulted from the research. The graphs show that the optimal rotation speed for cans no. 14 and 15 is 40 rotations a minute. For slower rotation speed (22 rotations per minute), as well as for higher rotation speed (67 rotations per minute), the cooling time is prolonged on the average to twice that time. In order to determine the temperature to which tomato paste must be cooled in the water cooler, the mixed cooling process was tried out: the paste was water-cooled to 60ºC, and then air-cooled to 45ºC. The results of the experiment showed that 140 minutes are needed to lower the temperature in the centre of can no. 15 from 60ºC to 45ºC, while water cooling only requires 15 minutes. Taking into consideration that the effect of a temperature of 50ºC to 60ºC for a few hours has little influence over the 139 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA quality of tomato paste, water cooling can be finished when the temperature in the centre of the can has reached 60ºC.

] ] C C o o [ [

e e r r u u t t a a r r e e p p m m e t e t

speed rotion [rot/min] speed rotion [rot/min] Figure 4. Figure 5. Figures 4 and 5. Variation of the tomato paste cooling speed in relation to the rotation speed, as resulted from the research

CONCLUSIONS AND FUTURE WORK The influence of the dry matter content in tomato paste. Apart from the experiments for cooling tomato paste containing 30% dry matter, research was performed for water- cooling tomato paste containing 40% dry matter. The optimal cooling conditions established for tomato paste with 30% dry matter content were met; the net weight was recalculated in relation with the density of the paste with 40% dry matter content. The empty space in cans was the same as in the case of the paste with 30% dry matter content. The observations revealed that the cooling time for the tomato paste with 40% dry matter content is 3 and even 4 times longer than the cooling time for the tomato paste with 30% dry matter content. This can be explained primarily by the fact that the viscosity of the tomato paste with 40% dry matter content is bigger than that in the tomato paste with 30% dry matter content. Figure 6 presents the variation curves of the cooling time in relation to the concentration of the tomato paste.

85 80 75 30% dry matter ]

C 70 o 40% dry matter [

e 65 r u

t 60 a r

e 55 p

m 50 e t 45 5

speed rotion [rot/min] Figure 6. The variation of cooling speed in relation to the concentration of the tomato paste and to the type of container: 1. 83-3 jars; 2. cans no. 15; 3. cans no. 14

Thus, the research made in order to determine the optimal conditions for cooling tomato paste revealed that water cooling is efficient for small containers in fixed position. For large containers, such cooling is not useful; rotating the cans during water cooling significantly accelerates the process of reducing the temperature of the product.

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REFERENCES [1] Guţulescu L., E. Dima, Tehnologia prelucrării legumelor. Bucureşti: Ed. Didactică şi pedagogică, 1997 [2] Opriş S., Îndrumător pentru industria conservelor vegetale. Bucureşti: Ed. Tehnică, 1993 [3] Segal B., Tehnologia conservării legumelor. Bucureşti: Ed. Didactică şi pedagogică, 1994

ABOUT THE AUTHORS G. Bujancă, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261. E-mail: [email protected] C. Jianu, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 A. Rinovetz, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 F. Crista, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Agriculture, 300645-Timişoara, Calea Aradului 119, Romania, phone and fax numbers 0256/277007 I. David, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Corina Dana Mişcă, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 D. Ştef, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Antoanela Cozma, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Mihaela Cazacu, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Zorica Adina Rinovetz, Theoretical High School ―J. L. Calderon‖, 300115 Timişoara, Pestalozzi 14, Romania, phone numbers 0256/203406

141 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

AMENDING FACTORS OF ANTHOCYANIN DYES IN FRUIT JUICES

G. Bujancă, C. Jianu, A. Rinovetz, Corina Dana Mişcă, I. David, B. Rădoi, D. Ştef, Ariana Velciov, Mihaela Cazacu, Adina, Zorica Adina Rinovetz

Abstract: In canning industry, fruit syrups are produced that contain added sugar or fruits that are covered with sugar syrup. Diluting juice with water or syrup may result in discoloration, modifications in the acidity and viscosity. The color in these conditions can vary not only because of dilution, but also because of the changes in medium pH. This paper presents the research on color variation in relation to the pH of the medium. The study was made on mixtures of anthocyanins obtained from fresh fruit juice: wild strawberries, sour cherries and rose petals. Key words: : fruit juice, anthocyanin.

INTRODUCTION Fruit syrups with added sugar are often used in canning industry. There is another procedure commonly used in the same industry: the fruits are covered with sugar syrup. In these cases, one must take into consideration the characteristics of the anthocyanin pigments in fruits. Dilution of juices with water or syrup may bring about changes in color, acidity and viscosity of the medium. Under these conditions, color may vary not only because of the dilution, but also because of the change in the pH of the medium. [2, 4] In most cases, the pH value in fruit juices falls within the limits of 2.5 to 5. Under these conditions, it would seem that anthocyanin pigments would only have shades of red. Still, in reality, even in an acid medium, the structure of the anthocyanins may vary, which leads to colorless variants. [1,3] Anthocyanins are found in fruits in a complex mixture of organic substances, and for this reason the data published in specialty literature referring to crystalline anthocyanins do not allow of accurate assessment of their variation in natural juices during technological processing [2, 4].

MATERIAL AND METHODS The aim of the present paper was to establish the optimal conditions of fabrication by mixture with sugar syrup or by mixture of various juices and compote. For this, the researchers studied the variation of color intensity in natural mixtures of anthocyanins depending on the variation in juice pH, and, related to this, of their buffering capacities in water dilution and dilution with sugar syrup. The study of color variation in relation to the pH of the medium was performed on anthocyanin mixtures obtained from fresh fruit juices: wild strawberries, sour cherries and rose petals. The anthocyanins were isolated in the following manner: methanol tissue extracts were precipitated with lead acetate. The precipitate was washed in methanol and ethanol, then decomposed with concentrated hydrochloric acid and precipitated three times in methyl alcohol with sulphur ether. Using buffer substances of citrate-phosphate, different values of medium pH were obtained. Color intensity was measured with the help of a SF-2M spectrophotometer, and the pH value was measured with glass and calomel electrodes.The buffer capacity was determined for juices of grapes, apples, mirabelles, sour cherries, wild strawberries, pomegranate and viburnum berries. The spectrograms of the solutions of crude anthocyanins in wild strawberries for different pH values (Figures 1 and 2) show not only that the intensity of the anthocyanin color depends on its concentration in the solution, but also that it is inversely proportional to the pH of the medium. At the same time, contrary to the data in the specialized literature, the research did not highlight the pH limits within which the color intensity would remain invariable. The study proved that for pH from 4.8 to 5.3, the orange shades in the solutions characteristic for wild strawberries practically

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disappear. It is probably within these pH limits that the entire anthocyanin chloride transforms into a colorless compound or pseudo base. In a neutral or alkaline medium, an increase could be noticed of the intensity of violet blue, characteristic for the quinoid bases and the salts of alkali metals with anthocyanins. On the spectrogram, this is expressed by the displacement of the main absorption peak into the short wavelength spectrum.

RESULTS AND DISCUSSION One particularity of the anthocyanins in wild strawberries is that in a very acid medium parallel with anthocyanin chlorides, colored in red and orange which render the main absorption peak in acid solution at 500 µm, there are other anthocyanin forms that determine the peak at 420 µm. Jarde's data show the same thing. The balance between these forms can be assessed by the ratio between the optical density for a wavelength of 420 µm (D1), which do not depend on the concentration and are constant for the given value of the pH. (Table 1). The variation curve of the ratio D/D1 in relation to the pH of the solutions is shown in Figure 3.

Table 1. Optical density in relation to pH values pH values of the wild Ratio between the optical density at strawberry anthocyanin solutions 500 µm and optical density at 420 µm 0.1 1.42 0.5 1.41 1.0 1.39 2.0 1.38 2.5 1.36 3.0 1.10 3.5 1.05 4.0 1.04 4.5 0.68 5.0 0.61 5.3 0.60 6.0 0.72 9.2 0.90

2,0

y 1,5 t i y s t i e

1,5 s d e

l d

a l 1,0 c a i t c

1,0 i p t p o o 0,5 0,5

400 500 600 400 500 600 wavelenght [m] wavelenght [m] Figure 1. The optical density variation of the Figure 2. The optical density variation of the solutions of wild strawberries anthocyanins with solutions of wild strawberries anthocyanins with the the concentration of 108 mg/ml for different pH concentration of 29 mg/ml for different pH values: 1 values: 1 - 0.1; 2 - 1.0; 3 - 1.6; 4 - 2.5; 5 - 3.3; 6 - - 0.5; 2 - 1.0; 3 - 2.0; 4 - 3.4; 5 - 4.8; 6 - 5.3; 7 - 6.0; 7.0; 7 - 12.0 8 - 12.0

143 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

1,5 1 D / D

i t y

s 1,0 n e d

l a c i t

p 0,5 o

o i t a r

1 3 6 9 12 pH values Figure 3. The variation curve of the ratio between the optical density at a wavelength of 500 µm and optical density of 420 µm in relation to the pH of the solutions

Analysis of the intensity curves of the color of anthocyanins in wild strawberries for different concentrations (Table 2) also proves that the decrease in color intensity does not depend on the concentration of anthocyanin in the solution, but that it only depends on the pH. Within pH limits 0.1 and 4.8 - 5.3, the appearance of the visible red color for every pH value is almost constant. Spectrograms of solutions of crude anthocyanin from roses (Figure 4) and sour cherries (Figure 5) for different pH values are almost analogous. Irrespective of the pH value in acid medium, there is a distinct peak with the maximum absorption point within wavelength limits 515 - 520 µm for rose and 530 - 535 µm for sour cherries. Similarly with wild strawberries, there is an obvious gradual decrease of anthocyanin color intensity from the maximum value at pH 0.1 to the minimum value at pH value 4.8. In the pH interval from 2 to 3 , solution discoloration appears, and for pH 4.8, the red color disappears completely. On the spectrogram, the peak disappears at wavelength 515 - 525 µm. The first moment after alkalinization, compounds appear which render the peak at wavelength 580 - 590 mµ (Figure 4 and Figure 6). After keeping the alkali solutions for a longer period, the maximum absorption point for rose anthocyanins (Figure 6) and sour cherry anthocyanins, just as in the case of wild berries, move towards shorter wavelengths (to 42 µm).

Table 2. Intensity curves of the color of anthocyanins in wild strawberries, sour cherries and rose petals for different concentrations Color intensity of sour cherry Color intensity of wild strawberry anthocyanins or rose petal anthocyanins, pH value anthocyanins, % of the maximum value [%] of the maximum value of the for a concentration of for a concentration of solution 108 29 Average 180 73 Average [mg/mL] [mg/mL] [mg/mL] [mg/mL] 0.1 100.0 100.0 100.0 100.0 100.0 100.0 0.5 96.0 94.7 95.3 96.0 96.3 96.1 1.0 85.4 86.7 86.0 90.5 92.5 91.5 1.5 81.4 78.7 80.2 86.5 87.2 86.8 2.0 72.1 66.9 69.5 70.2 69.3 69.7 2.5 65.5 55.9 61.2 52.0 50.0 51.0 3.0 37.0 34.5 35.8 19.3 17.0 18.0 3.5 20.0 22.1 21.0 8.4 10.3 9.4 4.0 12.0 14.1 13.1 3.4 3.7 3.5 4.5 6.6 6.1 6.3 1.3 1.0 1.1 5.0 0.0 0.0 0.0 1.6 2.6 2.1 5.3 0.0 0.0 0.0 0.0 0.0 0.0

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i t

y 1,0 s n e d

0,75 l a c i t 0,5 p o

0,25

400 520 640 wavelenght [m] Figure 4. Optical density variation in the Figure 5. Optical density variation in the solutions of rose anthocyanins immediately after solutions of sour cherry anthocyanins preparation, for different pH values: 1 - 2.3, 2 - immediately after preparation, for different pH 2.7; 3 - 3.1; 4 - 4.6;5 - 5.8; 6 - 8.0 values: 1 - 0.1, 2 - 1.0; 3 - 1.9; 4 - 3.1;5 - 4.8; 6 - 6.8; 7 - 8.0

i t y s n e

d 0,5

l a c i t p

o 0,25

400 520 640 wavelenght [m] Figure 6. Optical density variation in the solutions of rose anthocyanins after being exposed to low temperatures for two weeks, for different pH values: 1 - 2.3, 2 - 2.7; 3 - 3.1; 4 - 4.6;5 - 5.8; 6 - 8.0

Such a difference between behaviors in alkali media of pelargonidin (anthocyanin in wild strawberries) and cyanidin (anthocyanin in rose petals and sour cherries) is explained by the structural particularities of the molecules of these anthocyanins. Probably pelargonidin - derivatives with one hydroxyl group in the side ring can give a single type of compound with an alkali metal. Cyanidin - derivatives with two OH groups in the side ring, is capable of giving 2 or even 3 types, where the balance in favor of the main form with the maximum absorption point at wavelength of 420 µm is established after prolonged exposure of solutions. In acid medium, the color intensity variation of anthocyanins in sour cherries and rose petals does not depend on their concentration in the given solution. Instead, just as in the case of wild strawberries, it depends on the pH value (see Table 2). Therefore, the color intensity of anthocyanins in fruit juices varies in relation to the pH value (between 0.1 and 5.3) within limits from 100.0 to 0.0. Total disappearance of red and orange in the case of the anthocyanins in wild strawberries takes place at pH 5.3, total disappearance of red in the anthocyanins in sour cherries and rose petals takes place at pH 4.8, hence movement of pH values towards alkalinity is not desirable. In natural fruit juices with pH between 2.7 and 4.7, the color intensity corresponds to 60% - 10% of the maximum value in the content of anthocyanins in juices.

145 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Table 2. Continuation Water or pH value of the juices of sugar wild sour viburnum added to apple grape mirabelle plum pomegranate the juice strawberries cherries berries [%] with water dilution 0.0 3.38 4.63 3.39 3.72 4.15 4.55 2.77 4.10 4.7 3.39 4.63 3.40 3.72 4.15 4.55 2.79 4.10 13.0 3.39 4.63 3.41 3.72 4.15 4.55 2.80 4.10 20.0 3.39 4.63 3.43 3.72 4.15 4.55 2.81 4.10 33.3 3.42 4.64 3.47 3.71 4.15 4.55 2.84 4.18 41.1 3.43 4.64 3.49 3.72 4.16 4.55 2.85 4.18 50.0 3.42 4.63 3.50 3.72 4.19 4.58 2.86 4.19 60.0 3.44 4.65 3.50 3.73 4.23 4.62 2.88 4.21 with syrup sugar dilution 0.0 3.38 4.63 3.39 3.72 4.15 4.55 2.77 4.10 4.7 3.39 4.63 3.43 3.73 4.15 4.58 2.77 4.10 13.0 3.40 4.64 3.46 3.73 4.15 4.58 2.77 4.10 20.0 3.40 4.65 3.46 3.73 4.15 4.58 2.78 4.09 33.3 3.39 4.63 3.49 3.72 4.14 4.57 2.80 4.10 41.1 3.41 4.61 3.49 3.72 4.15 4.57 2.80 4.16 50.0 3.41 4.63 3.49 3.72 4.15 4.57 2.79 4.17 60.0 3.42 4.64 3.48 3.72 4.15 4.56 2.82 4.19

CONCLUSIONS AND FUTURE WORK The study on the buffering capacity of juices (Table 3) shows that, when the juices are diluted with water or sugar syrup, pH moves towards alkalization, by 0.01 to 0.13. Maximum movement can be observed when adding water, and the minimum movement when adding sugar syrup. Juices with pH below 3.5 (pomegranate, wild strawberries, apples) have a less stable buffering system than juices with higher pH values. In the case of wild strawberry juice, even with a dilution of only 5%, pH varies by 0.04, and then it increases to 3.49 (with 33% dilution). The latter pH value stays constant up to a dilution of 66%. The pH value in pomegranate juice still varies for 9% dilution, and then it increases.It is worth noting that pH movement by 0.1 in wild strawberry juice and in pomegranate juice leads to a 5% decrease in color intensity. Consequently, such juices must not be diluted. Preparing syrups with natural juices will help avoid unwanted and unjustified deterioration of the natural color of the products. With water dilution, and especially with sugar syrup dilution, in a proportion of 20 to 40% (which are the limits commonly used in food industry), the buffer system is the most stable in juices where the pH is over 3.5 (mirabelles, plums, grapes, sour cherries). Of all juices under research, the maximum buffering capacity was found in sour cherry juices and mirabelle juices. In the case of sour cherry juice, movement of the pH value only starts when the dilution is 1.5 times the juice. Mirabelle juice forms very stable buffering system in the case of sugar syrup dilution. When water is added, pH movement starts after 40% dilution. Therefore, the most stable buffering system was found in the sour cherry juice. When 50% water or 60% sugar syrup is added, the pH value of the juice remains constant. Pomegranate and wild strawberry juices have the least stable buffering system. Only syrup prepared with natural juices can be added to them. Thus, regulating the pH value of the medium, color intensity of the product can be greatly improved.

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REFERENCES [1] Bujancă G., S. Bungescu and D. Ştef, "The axerophtol and pro-vitamins in the vegetables and fruits from Beregsău Mare, Timiş County, Romania", ISSN 1313-7735, pp. 10 - 12, Lozenec, Bulgaria: June 2009 [2] Corlăţeanu, S., Valorificarea fructelor de pădure. Bucureşti: Ed. Agrosilvică, 1995 [3] Riviş A., Mariana Nistor, Ramona Biron, G. Bujancă, A. Rinovetz, C. Cobrac, "Influenţa unor factori fizico-chimici în analiza şi controlul calităţii sucurilor", Cercetări ştiinţifice, Procese şi Tehnologii Agroalimentare, Seria 7, ISSN 1453 - 1399, pp. 71 - 74, 2001 [4] Segal B., Tehnologia conservării fructelor. Bucureşti: Ed. Didactică şi pedagogică, 1994

ABOUT THE AUTHORS G. Bujancă, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261. E-mail: [email protected] C. Jianu, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 A. Rinovetz, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Corina Dana Mişcă, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 I. David, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 B. Rădoi, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 D. Ştef, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Ariana Velciov, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Mihaela Cazacu, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Processing Technology, 300645-Timişoara, Calea Aradului 119, Romania, phone numbers 0256/277327, fax numbers 0256/277261 Zorica Adina Rinovetz, Theoretical High School ―J. L. Calderon‖, 300115 Timişoara, Pestalozzi 14, Romania, phone numbers 0256/203406

147 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

IMPROVEMENT OF PUMPKIN OIL CAKE PROTEIN GELATION PROPERTIES THROUGH ENZYMATIC POLYMERYZATION WITH TRANSGLUTAMINASE

Lj. Popoviš, Ţ. Vaštag, S. Popoviš, D. Peričin, V. Krimer-Maleševiš

Abstract: The use of plant proteins in the formulation of new food products or as replacement for more expensive animal proteins in conventional foods has been the focus of various researches. Moreover, different protein rich by-products of food industry are proved to be good as protein resources. Oil extraction from pumpkin seeds yields a large amount of defatted pumpkin oil cake (PuOC), which, among other nutritional components, contains a significant amount of protein, but it is usually used only as animal feed. Valorization of this by-product is of great importance from economical as well as environmental reasons. Enzymatic modification with transglutaminase (TG) was used to enhance the gelation of pumpkin oil cake 12S globulin, named cucurbitin and thus improve its potential as a food ingredient. The effects of deegree of polymerization (DP) on cucurbitin gelation properties were evaluated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to show that cross-linking of subunits occurred through TG treatment. The gelation properties of polymerized proteins were improved over the pH range 5.0- 8.0. The greatest decrease of least gelation concentration has been made at pH 9.0 (for 50%).

Key words: Cucurbitin, transglutaminase, gelation properties, oil seed proteins, functional properties.

INTRODUCTION Cultivation of pumpkin (Cucurbita pepo) in the middle-south Europe region (Austria, Hungary, Slovenia, Croatia and Serbia) is wide-spread, primarily for oil production from seed. Defatted pumpkin cake, which is a by-product of oil extraction, is usually used only as animal feed. However, pumpkin oil cake contains about 60% protein which could be useful for human nutrition. The protein exploitation from pumpkin oil cake has been described as a way to increase the value of this agricultural by-product [1-4]. Cucurbitin is the major storage protein in pumpkin seed. It is a 12S, hexameric globulin, with molecular size of about 350 kDa. Although, globulins from various plant seeds play important roles in many foodstuffs, both because of their nutritional and functional properties, there is no much information about cucurbitins applications in this purpose. Hence, investigation of methods for improving some functional properties of cucurbitin would be of great importance in order to make better use of cucurbitin as food ingredient. It has been shown that an attractive mean for improving protein functional properties is enzymatic cross-linking by microbial TG (EC 2.3.2.13) ref. The objective of this research was to improve the gelation properties of cucurbitin through enzymatic crosslinking with transglutaminase (TG). Such improvements could offer new markets for the by-product pumpkin oil cake. TG is widely used in the food industry for the purpose of cross-linking proteins [1-4]. It has the potential to improve gelation through the formation of covalent intra- and intermolecular bonds. These isodipeptide bonds form between glutamine and lysine residues in proteins resulting in e- (g-Glu)-Lys cross-links. TG are reported to: improve solubility, gelation properties, as well as, water- and fat binding capacity of oat globulin, also improve solubility of gluten, and soy protein isolate, improve foaming and emulsifying properties of legumes protein isolate. The degree of cross linking on one hand and effect of cross linking on the other hand is not well investigated. This study was focused on utility of cucurbitin as substrate for TG cross-linking, which could be suitable way for its potential application in food formulations. Enzyme/substrate (E/S) ratio and time of reaction were investigated to determine their

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influence on the DP. Also, the polymerization process of cucurbitin was accompanied and confirmed through the electrophoresis profile. Gelation properties of cross linked protein with different DP values were determined and compared with those of cucurbitin.

MATERIAL AND METHODS Materials The hull-less pumpkin (Cucurbita pepo. c. v. Olinka) oil cake (PuOC) was collected after seed pressing from a local Oil Industry ‖Pan Union‖, Novi Sad, Serbia. It was stored at the temperature of 4°C and ground in a coffee-grinder before use. The microbial transglutaminase, (ACTIVA WM) was a gift from Ajinomoto Food Europe (100 Ug-1). All the other chemicals were at least of analytical grade and were from Sigma-Aldrich (USA).

Preparation of cucurbitin The protein was extracted by the procedure of Blagrove and Lilley [6]. Briefly, 10 g of the defatted ground PuOC meal was extracted sequentially with 200 ml of water and then with 200 ml of 10% NaCl solution for 3 h at room temperature. Cucurbitin was precipitated by the gradual addition of 800 ml of water. The precipitate was dissolved in 100 ml standard buffer (1 mol/l KCl in 0.1 mol/l sodium phosphate buffer, pH 7.0), after centrifugation the cucurbitin was precipitated by the addition of 200 ml of water. The final protein residue was collected and air dried at room temperature.

Protein cross-linking Protein was mixed with phosphate buffer pH 7.0 (0.1 mol/l) to a final concentration of 10 mg/ml. The protein suspension was incubated at the 50°C, and reaction was started by the addition of the enzyme. Aliquots (1.5 ml) were taken out after certain time intervals and immediately centrifuged at 4 500 rpm (Eppendorf Mini spin plus) for 5 min and the supernatant was used for further analysis.

Determination of DP The DP was determined according to the modified method by Church, Swaisgood & Catignani [7]. The method is based on quantification of free amino groups. Decreases in free amino groups were determined spectrophotometrically using the o – phthaldehyde (OPA). OPA (40 mg) was dissolved in 1 ml of ethanol. In separate solution 1.905g of di- sodium-tetraborate decahydrate and 50 mg of sodium dodecylsulfate were dissolved in 40 ml of water. The two solutions were mixed and volume brought to 50 ml with water. 0.1 ml of 2-mercaptoethanol was mixed with 50 ml of OPA reagent just before the use in the assay. To 50 µl aliquot of the supernatant, obtained after the enzymatic reaction, 2.5 ml of OPA reagent was added and absorbance read at 340 nm. The DP value was calculated according the equation (1): Ac  As DP(%)  *100 Ac (1) where Ac is absorbance at 340 nm of cucurbitin without enzyme and As is absorbance at 340 nm of sample obtained after the enzymatic reaction.

SDS-PAGE SDS-PAGE of protein samples was carried out by the method of Laemmli [8]. Electrophoresis (Pharmacia LKB – Multi Drive XL) was carried out at 100 mA until the tracker dye reached the bottom of each gel. After electrophoresis, the gels were stained

149 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA with 0.2/100ml Coomassie Brilliant Blue R-250 (in 10ml/100ml acetic acid: 50ml/100ml methanol) and distained with 10ml/100ml acetic acid containing 40ml/100ml methanol.

Least gelation concentration (LGC) Appropriate sample suspensions of 2, 4, 6, 8, 10, 12, 14, 16 and 18 % (w/v) were prepared in 1 ml 0.01 mol/l sodium phosphate buffer pH 7.0. The test tubes were heated for 1 hour in boiling water followed by rapid cooling using chilled water at 4oC for 2 hours. The LGC was taken as the concentration when the sample from the inverted test-tube did not fall down or slip.

RESULTS AND DISCUSSION The progress of cucurbitin polymerization was monitored as the change in DP during reaction time up to 60 min (Figure 1). Polymerization with higher TG concentration proceeded at a rapid rate during the initial 30 min and then slowed, whereas the rate of the lowest TG concentration gradually increased during the all 60 min. The data suggest that the polymers with various DP values can be obtained by selecting the enzyme concentration and the time of reaction.

Figure 1. Cross-linking of cucurbitin by TG with different E/S ratio.

The cross-linking of cucurbitin through the action of TG was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This is considered an acceptable method to monitor the cross-linking of proteins by TG, because it shows intermolecular cross-linking through the formation of new high-molecular-weight bands. The electrophoresis profile (Figure 2) showed that the enzyme treatment lead to appearance of new high-molecular-weight protein bands with molecular weights of about 80 kDa. These was accompanied by a loss of low molecular weight bands of 33 and 14 kDa, indicating that they formed the high molecular weight polypeptides via cross-linking. TG treatment on cucurbitin resulted in the formation of two new bands with molecular weights of about 80 kDa (lines 3, 4 and 5), which intensity is dependant of DP values.

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Figure 2. SDS PAGE electrophoretograms of molecular weight markers (line 1); control-cucurbitin (line 2); polymers of cucurbitin obtained by activity of TG: DP= 30% (line 3);DP= 40%(line 4),DP= 50%(line 5).

Gelation properties The LGC was taken as a measure of the gelation capacity. LGC value as a function of the pH is presented in Figure 3 for TG cross-linked cucurbitin with DP=50% and control.

Figure 3. Effect of pH on the gelation properties of cucurbitin and TG polymer

LGC value of cucurbitin is lowest (6%) at pH 5.0 which is the pH of the lowest solubility of cucurbitin. TG treatment reduced LGC value in the whole studied pH range which is in agreement with many authors who showed that cross-linking is an important way to improve gel formation [9]. The best LGC (4%) of cross-linked cucurbitin is achieved also at pH 5.0. The highest decrease of LGC has been made at pH 9.0 (for 50%).

CONCLUSIONS AND FUTURE WORK The results demonstrate that modified proteins produced in the present study have improved gelation properties indicating their possible use in different food systems. The use of TG for production of highly functional proteins from oil cakes can be a feasible 151 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA technology to make these by-products a utilized resource and use them as a food ingredient for direct human consumption.

REFERENCES [1]. Vaštag, Ţ. et al. 2011. Production of Enzymatic Hydrolysates with Antioxidant and Angiotensin-I Converting Enzyme Inhibitory Activity from Pumpkin Oil Cake Protein Isolate. Food Chemistry, 124, 1316-1321. [2]. Peričin, D. et al.2008. Evaluation of solubility of pumpkin seed globulins by response surface method. Journal of Food Engineering, 84, 591-594. [3]. Popoviš, Lj., et al. 2013. Optimization of transglutaminase cross-linking of pumpkin oil cake globulin; Improvement of the solubility and gelation properties. Food and Bioprocess Technology 6, pp 1105-1111. [4]. Popoviš, Lj., et al. 2011. Optimization of enzymatic hydrolysis of cucurbitin using Response Surface Methodology: Improvement of the functional properties. International Journal of Food Engineering 7, Article 18. [5]. Aguilera, J. M., et al. 2004. Protein gels. In R. Y. Yada (Ed.), Proteins in food processing (pp. 468–482). Boca Raton: CRCPress LLC. [6]. Blagrove R, Lilley G 1980. Characterisation of cucurbitin from various species of the Cucurbitaceae. Europ J Biochem 103: 577-584. [7]. Church, F.C et al. 1983. Spectrophotometric assay using o-phthaldehyde for determination of proteolysis in milk and isolated milk proteins. Journal of Dairy Science, 66, 1219-1227. [8]. Laemmli UK, Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227: 680-685 (1970). [9]. Sun, X.D., & Arntfield, S.D. 2011. Gelation properties of salt-extracted pea protein isolate catalyzed by microbial transglutaminase cross-linking. Food Hydrocolloids, 25, 25-31.

ABOUT THE AUTHORS Ljiljana Popoviš, Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia, E-mail: [email protected], Ţuţana Vaštag, Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia, E-mail: [email protected], Senka Popoviš, Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia, E-mail: [email protected] , Draginja Peričin, Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, Novi Sad, Serbia, E-mail: [email protected] Vera Krimer-Maleševiš, Public Health Institute, Zmaj Jovina 30, 24000 Subotica, Serbia, E-mail: [email protected]

Acknowledgements This work was supported by grant number 46010 from Ministry of Education and Science of Republic of Serbia.

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VIABILITY ASSESSMENT OF Ruscus aculeatus L. GERMPLASM EXPOSED TO EXTREME LOW TEMPERATURES

Banciu Cristian, Anca Aiftimie-Păunescu

Abstract: The present study concerns the viability assessment after revitalization of regenerants of Ruscus aculeatus L. species from Comana Natural Park, Romania. After a pre-treatment with two cryoprotectants in different concentrations, the explants have been slow cooled and immersed in liquid nitrogen. Fluorescence microscopy images done on viability coloured sections revealed plantlet survival. Key words: Ruscus aculeatus, cryopreservation, fluorescence, viability.

INTRODUCTION Ruscus aculeatus L., perennial species by its horizontal rhizomes, is presented as a pyramid-shaped evergreen shrub ovata 20-70 cm high, sometimes exceeding 1 meter. Aerial stems are metamorphosed into phylloclades - rigid formations, sclerified, with the role of assimilation, attenuated into a short spin. The plant is sexuate propagated by seeds and vegetative by rhizome extension [5]. It is a protected species included on the Red List of vascular plants in Romania [11] with the status of rare species, the Dihoru‘s Red List [6] has the status of vulnerable species and in the Red List published by the team from Cluj [4] has the status of endangered species. Worldwide are numerous studies of medicinal properties of this plant, due to secondary metabolic compounds production including saponins (ruscogenin, neoruscogenin). Also there are studies on the effect of extracts of Ruscus sp. on hamsters and dogs, showing vasoconstrictive properties [12]. All these studies were based on freshly harvested plant material in botanical gardens or natural habitat. In Romania pharmacological properties of plant were studied by scientists in Cluj- Napoca [1]. Studies on cell cultures to obtain extracting bioactive compounds from Ruscus aculeatus are very few. We mention a recent study aimed obtaining callus from immature embryos for extraction of saponins [9]. Recent studies on in vitro multiplication of Ruscus aculeatus have also been performed by bulgarian colleagues from Sofia with successful results for the efforts on conservations of local populations [7]. Cryopreservation is a time saving method to maintain the original variability of the germplasm for long and very long time (from days to years and even hundreds of years) in independent conditions. The results are a model for preserving other plant species with ornamental, pharmacological or conservative importance.

MATERIAL AND METHODS The analyzed material is represented by fragments of Ruscus aculeatus from in vitro culture (Fig. 2) obtained according to previous publications [ 2, 3], maintained for one week at cold (10oC). The cryoprotective treatment consisted in a solution of liquid half mineral concentration MS medium [10] supplemented with 60 g/L sucrose, and two versions of cryoprotectants: 5% Dimethyl sulfoxide (DMSO) + 5% glycerol respectively 10% Dimethyl sulfoxide (DMSO) + 10% glycerol, applied each for 30 minutes to 10 samples of plant material [15]. In the following step the cryotubes containing 0.5 mL of solution and a 5 mm fragment of plant material were loaded to the controlled rate unit (from CryoLogic) using a computerized program (CryoGenesys) as in the following graphic (Fig.1). The steps of the program consisted in cooling with 2oC/min. to 0oC, then with 1oC/min. to -6oC, waiting for nucleation process 7 minutes and cooling with 0.3oC/min. to -32oC and then with 0.5oC/min. to -42oC. After the treatment, the explants have been immersed in (LN) liquid 153 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA nitrogen (at -196oC) for 24 hours. The thawing process took place in a water bath at 37oC followed by recultivation on MS medium supplemented as described above, without cryoprotectants. The viability test was performed by treating the explants for 5 minutes with fluorescein diacetate (FDA) in concentration of 0.1% (w/v) at room temperature [14] and observing the sample on optical microscope Imager M1 from Leica. During the reaction, the FDA is hydrolyzed to fluorescein (colored in UV light) and malic acid [13].

Figure 1. Slow freezing temperature graphic (CryoGenesys 5). Y-temperature (oC), X-time (minutes); below line-protocol parameters, above line-achieved parameters

3 cm 1cm a b

Figure 2. Regenerants of Ruscus aculeatus from in vitro culture (original).

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RESULTS AND DISCUSSION The protocol used for plant cryopreservation consisted in two treatments: a chemical one with two cryoprotective solutions (DMSO and glycerol) and a thermal treatment by gradually cooling the specimens. The program chosen allows the most important phenomena (the ice nucleation) to take part in the intercellular space avoiding ice crystals to grow in the cells and break the cell membrane. In this way the cooling program permit the extracellular water to ice, reducing the concentration of liquid phase and attracting the intracellular water outside the cell by osmosis which is replaced by cryoprotectants as DMSO and glycerol. The plant explants appeared green after thawing and were visualized on fluorescence microscopy using 3 fluorochromes: DAPI (4‘, 6-diamidino-2-phenylindole), GFP (green fluorescence protein) and ROD (Rhodamine). The fluorescence reflected by the tissues confirms the viability of the cells. The structure of the fragment as seen in the figure 3 presents a vascular tissue well developed and surrounding parenchymal tissue. In UV light, using GFP filter vascular bundles are visible with the evidentiation of surrounding structure; concluding that these tissues with intense transport activity have survived and the esterase from intracellular space hydrolyzed FDA (Fig. 4). In the figure 5 the parenchymal tissue displayed in UV light using ROD as filter was intense red colored, revealing that internal tissues have survived with the help of cryoprotective agents. The picture colored in blue visualized by UV light using DAPI filter give a general image on the viable tissue with the intensity on central vascular bundles and parenchimatic tissue (Fig. 6). Viability test of Ruscus aculeatus specimens exposed to extreme cold conditions in LN at -196oC confirmed the capacity of plants to adapt to cold in experimental conditions. Avoiding the intracellular ice crystal formation is the key factor in cells survival in cryopreservation processes. The thawing procedure is also important due to the risk of repeating the same phenomena during ice melting and water re-entering in the cells. If the thawing process in too slow the water has enough time to form ice crystals in the vacuom and the cell membranes are broken. Other authors used encapsulation method for cryopreservation of plant species [8] starting with spores and seeds. In that conditions exposure to LN had even a stimulating role for the immature seeds. We have used the slow freezing procedure due to low concentration of cryoprotectants (avoiding in this way cyto-toxicity of the cytoplasm content) and less shocking treatment of the cells that avoids osmotic stress. These results are a good start for long term conservation for a wide category of plant species threatened by natural and antropic factors, but also for special germplasm collections with economical importance.

155 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Figure 3. Phylloclade tissue of regenerant Figure 4. Phylloclade tissue in UV light visualized in visible light (original). by GFP filter (original).

Figure 5. Phylloclade tissue in UV light Figure 6. Phylloclade tissue in UV light visualized by Rhodamine filter (original). visualized by DAPI filter (original).

CONCLUSIONS AND FUTURE WORK The protocol established for extreme cold treatment for Ruscus aculeatus L. species was successful, after thawing the explants maintaining the viability. It allows further experiments on other endangered plant species. The next step is to confirm the viability of the cryopreserved plant material by in vitro multiplication and regeneration of the plantlets.

ACKNOWLEDGEMENTS This work was cofinanced from the European Social Fund through Sectorial Operational Programme Human Resources Development 2007-2013, project number POSDRU/89/1.5/S/63258 ‖Postdoctoral school for zootechnical biodiversity and food biotechnology based on the eco-economy and the bio-economy required by eco-san- genesys‖.This project was also conducted with the support of the project financed by the Romanian Academy no. RO1567-IBB06/2012.

REFERENCES [1]. Balica, G., Deliu, C., Tămaş, M., (2005): Biotehnologii aplicate la specia Ruscus aculeatus L. (Liliaceae), Hameiul şi plantele medicinale, 25(1-2): 163-166.

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[2]. Banciu C., Aiftimie-Păunescu A., 2012, In vitro propagation of rare species Ruscus aculeatus L. and histological peculiarities of the regenerants, Analele Universităţii din Oradea - Fascicula Biologie, Tom. XIX, Issue: 1, pp. 67-73. [3]. Banciu, C., Brezeanu A., (2008): Potenţialul regenerativ in vitro al explantelor de ţesut somatic, de diferite origini la Ruscus aculeatus L., în: Cachiţă-Cosma D (Ed.) Biotehnologii vegetale pentru secolul XXI, Ed. Risoprint, Cluj-Napoca, pp. 62-68. [4]. Boscaiu N., Coldea G., Horeanu C., 1994, Lista rosie a plantelor vasculare disparute, periclitate, vulnerabile si rare din Flora Romaniei, Ocrotirea Naturii si Mediului Inconjurator, 38(1), 45-56. [5]. Ciocârlan, V., (2000): Flora ilustrată a României, Pterydofita et Spermatophyta, Ed. Ceres, București. [6]. Dihoru, G., Dihoru, A., (1994): Plante rare, periclitate și endemice în Flora României –lista Roșie. Acta. Bot. Hort. Bucurestiensis: 173-197. [7]. Ivanova T., Gussev C., Bosseva Y., Stoeva T., 2011, In vitro conservation of micro- propagated Ruscus aculeatus L. (Liliaceae) plants, Botanica Serbica, 35 (1): 61-66 . [8]. Mikuła A.., Jata K., Rybczyński J. J. 2009. Cryopreservation strategies for Cyathea australis (R. Br.)Domin.CryoLetters 30(6): 429-439. [9]. Moyano, E., Montero, M., Bonfill, M., Cusido, R. M., Palazon, J., Pinol M., T., (2006): In vitro micropropagation of Ruscus aculeatus. Biologia Plantarum, 50(3): 441-443 [10]. Murashige, T., Skoog, F., (1962): A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol.Plant. 15: 473-497. [11]. Oltean, M., Negrean, G., Popescu, A., Dihoru, G., Sanda, V., Mihăilescu, S., (1994): Lista Roșie a plantelor superioare din România, Studii, sinteze, documentații de ecologie, Acad. Rom., I. Biol., 1: 1-52. [12]. Palazón,J., Moyano,E., Bonfill,M., Osuna, L., Cusidó, R.M., Piñol, M.T., (2006): Effect of organogenesis on steroidal saponin biosynthesis in calli cultures of Ruscus aculeatus. Fitoterapia, 77(3): 216-220. [13]. Păunescu, A., (2008): Biotehnologii de conservare a plantelor, Ed. Princeps, Iași. [14]. Reinert J. & Bajaj Y. P. S. 1977. Applied and Fundamental Aspects of Plant Cell, Tissue, and Organ Culture. Springer-Verlag Berlin-Heidelberg. 803 pp. [15]. Withers, L. A. & Williams, J. T., 1985, Research on long term storage and exchange of in vitro plant germplasm. In: Biotechnology in International Agricultural Research, Manilla:11-24.

ABOUT THE AUTHORS Cristian Banciu, Institute of Biology of Romanian Academy, 296 Splaiul Independentei 060031 Bucharest, Romania, Phone: 0040212239072/302; E-mail: [email protected] ; www.ibiol.ro Anca Aiftimie-Păunescu, Institute of Biology of Romanian Academy, 296 Splaiul Independentei 060031 Bucharest, Romania, Phone: 0040212239072/302; E-mail: [email protected] ; www.ibiol.ro

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STUDIES CONCERNING THE VARIABILITY OF SOME BLACK PINE (PINUS NIGRA) POPULATIONS FROM ROMANIA AND GREECE

I.Sotiropoulos., E.Madoşă, Oana Danci, and M.Danci

Abstract: The study was designed to assess the variability of morphological characters and establish the degree of similarity between populations of black pine from south western Romania and central Greece. Banatica variety is extended in southwestern Romania, but some believe that this is not a distinct variety of the Pinus nigra specie. The studied biological material came from two Romanian locations (Herculane and Sviniţa) and two Greek locations (Ano Polidrossos and Kerasia). By biometric measurements the trunk diameter, leaf size and dimensions of cones, were evaluated. It was found that the populations of Romania have a greater variability for all traits. Dendrogram study showed that the highest similarity for analyzed traits has been between Herculane and Ano Polidrossos populations (87.99%) that make up a subset of the first cluster. Svinita population shows a diversity of about 26% compared to the two anterior presented populations. Kerasia population represents a separate cluster showing a similarity of approximately 53% compared to Herculane and Ano Polidrossos populations. Svinita population presents high values of trunk diameter and cones diameter associated with a number of cone scales superior to experience average. In Herculane and Ano Polidrossos populations the longest cones correlated with a lower number of scales/cone and medium values of trunk diameter. Kerasia population presented a large length and width of leaves.

Key words: Black Pine, Inter Population Variability, Biometric Features

INTRODUCTION Pinus nigra belongs to one of the largest coniferous genera, the genus Pinus that contains around 100 species of pines, spread around the world [12]. The genus Pinus belongs to the phylum Coniferophyta, the cone-bearing plants (conifers), that are all considered gymnosperms, because they bear their seeds naked, rather than within an ovary as in the angiosperms (flowering plants) [7]. Pinus nigra is a tall tree growing 40 m, with a pyramidal crown, especially to young trees and swivel roots with vigorous branches. Young branches are bright green, pine buds are ovate-oblong, 6 -12 mm long, sharp, not resinous. Leaves of all pines are needle-like and are joined at the base by two, 4-6 cm long, rigid, pointed, blue-green or green-gray. Cones are oval-conical, 2.5-7 cm long, brown-gray and at maturity they open to release the seed [3, 12, 9, 10]. It is spread from the mountain level to the hills level all over the world. In Europe it is spread especially in 13 countries, occupying an area of 2,300,000 ha on 13 degrees latitude (from 13o to 48o North latitude) and 36 degrees longitude (from 6o in Western Spain to 42o in Eastern of Turkey [10]. In Romania black pine, is spread mostly in the mountain regions. The endemic species found in the South-Western part of Romania the subspecies (Pinus nigra ssp. banatica) is a tertiary relict found on rocks, especially limestone [8]. In Greece the black pine occupies a large area stretching from the northern border with Albania and Macedonia to Parnon and Taygetos, and the islands belonging to Greece [1] . The aim of this study is to evaluate morphological features variability of four black pine populations in order to establish the degree of genetic and phenotypic similarity between Greek and Romanian populations of black pine.

MATERIAL AND METHODS The biological material constituted of populations of black pine collected from national parks from in both countries: two from South Western Romania, namely National Park Domogled – Cerna Valley and National Park Iron Gates and two from Central Greece, one from National Park of Parnassos and the other one from Evia Island. Experimental data

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were collected using biometric measurement on trunk diameter, leaves sizes and elements characteristic of cones. Processing of the experimental data was carried out by methods appropriate to evaluation of characters variability in a population [2]. and determining the degree of relatedness between populations using dendrograms [5].

RESULTS AND DISCUSSION Study of variability of main morphological characters show differences between the populations studied. Trunk diameter presents the higher variability especially to Herculane population and the lowest variability was shown by the population from Ano Polidrossos. Trees with the highest trunk diameters were found in the population of Sviniţa that are significant statistically different than those of Herculane. Leaves dimensions showed a moderate variability. Leaves from Romanian populations presented no statistically differences than those of Ano Polidrossos, only individuals from Kerasia presented wider needles. Population of Kerasia presented also longer leaves comparing with the other populations. Leaves difference recorded to the population of Kerasia might be due to the fact that its individuals were collected from Island Evia that is around 80 km far from the mainland Greece [6] (table 1). Table 1 The significance of differences between black pine populations in terms of trunk diameter and leaf dimensions No. Population Trunk diameter (cm) Leaf length (cm) Leaf width (cm) x  s s% s% s% x 1 Herculane bc 30.02+2.51 40.69 b 8.51+0.22 12.47 b 1.04+0.05 25.01 2 Sviniţa a 44.48+2.65 30.56 c 7.81+0.18 11.33 b 1.06+0.04 20.03 3 Kerasia - Evia c 25.33+1.58 29.87 a 11.76+0.40 16.24 a 1.31+0.02 8.61 4 Ano Polidrossos b 33.38+1.48 22.29 bc 8.02+0.17 10.51 b 1.02+0.04 17.79 5 Exper. mean 33.30+1.17 18.20 9.03+0.16 8.60 1.11+0.02 9.72 LSD 5%5.50 cm LSD 5%0.66 cm LSD 5%0.10 cm LSD 1%7.28 cm LSD 1%0.88 cm LSD 1%0.14 cm LSD 0.1 %9.38 cm LSD 0.1 %1.12 cm LSD 0.1 %0.18 cm

The characteristic elements of the cone have a moderate variability in all populations. Length cones in Greek populations differ from each other and to those from Romania, but Romanian populations are similar. Shorter cones were present in the population of Ano Polidrossos. The smallest diameter was obtained in average to the cones of Kerasia whilst the highest diameter was shown by the cones of Sviniţa. Populations of Kerasia and Sviniţa presented the greatest differences from the others. For this character populations of Herculane and Ano Polidrossos were similar. Shape index highlights the population of Sviniţa, that differ statistically significant than the others. The number of scales per cone was significantly lower to the population of Herculane that presented cones with larger scales than of other populations (table 2). The study of characters variability it is found that there are differences between the populations studied, but also similarities. On cannot say that there is a population that differs significantly from each other for all the characters studied. For some characters there are similarities between the populations of Greece and Romania, but for other features differences exist even between the Romanian populations. These results may be due to the forests age. Very interesting is the similarity between Greek populations and population of Sviniţa regarding the number of scales per cone. This feature can offer much information on propagation potential of black pine. Population of Herculane is different than the others, presenting a smaller number of scales/cone and a high variation in trunk diameter because the conditions of that area are more unfavorable and the rocks are 159 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA much steeper [8]. Also, population of Kerasia differs from other populations for cone (shorter and with lower diameter cones) and leaves dimensions (longer but wider needles) due to the fact that this population comes from an island where even the climatic conditions are different than those from the continent. These elements are reflected in the following dendrogram (figure 1). Table 2 The significance of differences between black pine populations in terms of cones characteristics No. Population Cones length (cm) Cones diameter (cm) Shape index Scales number/cone x  s s% s% s% s% x 1 Herculane b 6.32+0.13 10.15 b 3.81+0.21 27.26 a 1.79+0.12 31.30 b 14.32 84.43+2.49 2 Sviniţa b 6.22+0.16 12.95 a 4.67+0.13 13.91 b 1.35+0.04 15.77 a 12.39 94.60+2.43 3 Kerasia - c 5.74+0.14 11.31 c 2.97+0.06 9.39 a 1.94+0.04 9.10 a 11.02 Evia 96.83+2.23 4 Ano a 6.97+0.13 9.26 b 3.96+0.15 17.86 a 1.80+0.07 18.15 a 12.59 Polidrossos 93.22+2.31 5 Exper. mean 6.31+0.06 4.46 3.85+0.07 9.25 1.65+0.04 10.94 92.27+1.13 5.94 LSD 5%0.35 cm LSD 5%0.37 cm LSD 5%0.18 LSD 5%5.95 LSD 1%0.46 cm LSD 1%0.49 cm LSD 1%0.24 LSD 1%7.87 LSD 0.1 %0.60 cm LSD 0.1 %0.63 cm LSD 0.1 %0.31 LSD 0.1 %10.14

Herculane

Ano Polidrosos

Svinita

Kerasia - Evia

0.4 0.5 0.6 0.7 0.8 0.9 1.0 Similarity coefficient Figure 1. UPGMA clustering of black pine populations

Basing on the phenotypic similarity between the four black pine populations described by the seventh features analyzed dendrogram was realized using clusters method. This dendrogram shows the existence of two principal phenotypic groups among that a medium phenotypic diversity of 57% manifests. The highest similarity to the characters analyzed was recorded between populations Herculane and Ano Polidrossos (87.99 %) that form a subgroup in the first cluster. Population of Sviniţa shows a diversity of approx. 26% of the two populations described above. Population of

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Kerasia Evia respresents a separated cluster manifesting a similarity of approximately 53% of the populations Herculane and Ano Polidrossos (figure 1). This situation may have some explanations. Population of Kerasia-Evia comes from an island, where some conditions differ from the mainland. Populations of Herculane and Ano Polidrossos are natural endemic populations formed in similar relief conditions and population of Sviniţa is different from the others because it might be a planted population and not a natural one as that of Herculane that can be a tertiary relict [8]. Romanian populations of black pine are not morphologically very similar. Curiously population of Herculane is much more similar to that of Ano Polidrossos that with that of Sviniţa. This situation motivates further study by more accurate methods (molecular) to bring new evidence to support the existence of the subspecies banatica. 3 Sn

2 Td S 1 K - E Nl Cd

A- P PC2 PC2 (29.98%) -1 Nw H Cl -2 Si

-3

-3 -2 -1 0 1 2 3 PC1 (68.22%) H-Herculane; S-Svinita; K-E - Kerasia Evia; A-P - Ano Polidrossos Td-trunk diameter; Nl-leaf length; Nw-leaf width; Cl-cones length; Cd-cones diameter; Si-shape index of cones; Sn-scales number/cone. Figura 2 Biplot for the studied black pine populations and traits

Bidimensional diagram based on the first two principal components expresses 99.21% of the variability of the seven characters in the study. Based on population projections on vectors of different characters can be observed that population of Sviniţa shows higher values for trunk and cones diameter related with a number of scales on the cone above the experience average, reduced leaf size and a flattened cone shape. Populations of Herculane and Ano Polidrossos present the longest cones correlated with a small number of scales/cone and medium values for trunk and cones diameter. Population of Kerasia-Evia shows longer and wider needles, cones with smaller dimensions, of more elongated shape and a greater number of scales/cone but lower values for trunk diameter (figure 2). In view of the vectors of different characters, on notice that populations studied shows the highest differentiation for trunk diameter and leaves length while in terms of length of cones and number of scales/cone differences between populations are considerably lower.

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CONCLUSIONS AND FUTURE WORK 1. Studied populations show a high variability to trunk diameter and leaf size vriabilitate moderate and cones. 2. Population with the most vigorous trees is Sviniţa population. 3. Populations differ from each other in terms of trunk diameter and length of the leaves and are similar in terms of length of cones and the number of cone scales. 4. Considering all the characters populations of Herculane and Ano Polidrossos presents a similarity of 87.99%, but population of Sviniţa differs from them in a proportion of 26%. 5. Kerasia population has a unique behavior, being different, and the degree of similarity to Herculane population is of only 53%.

REFERENCES [1] Athanasiadis N. 1986. Forest botany, Part II. Thessaloniki: Giahoudis-Giapoulis Publications (in Greek). [2] Ciulca, S. 2006. Metodologii de experimentare in agricultura si biologie, Ed.Agroprint, Timisoara [3] Cronquist, A., Takhtajan, A., Zimmermann, W. 1966. On the higher taxa of Embryobionta Taxon, 15(15): 129-168. [4] Cronquist, A., Takhtajan, A., Zimmermann, W. 1996. Pinophyta in Phytologia 79(2): 70. [5] Fielding A.H. 2007. Cluster and Classification techniques for the biosciences, Cambridge Univ,Press. [6]. Matziris, D.L. 1984. Genetic variation in morphological and anatomical needle characteristics in the black pine of Peloponnesos. Silv. Genet 33(4-5): 164-169. [7] Mirov, N.T. 1967. The Genus Pinus. Ronald Press, New York. [8] Patroescu, M., Chincea, I., Rozylowicz L., Sorescu, C. 2007. Banat black pine forests. NATURA 2000 site, Web page: www.pinusnigrabanatica.ro [9] Reveal, J.L. 1996. Newly required suprageneric names in Pinophyta. Phytologia 79: 68–76. [10] Richardson, D. M. (ed.). 1998. Ecology and Biogeography of Pinus. Cambridge University Press. [11] Şofletea, N. 2001. Dendrologie, volumele I şi II, Editura „Pentru Viaţă‖, Braşov. [12] Vidakovic, M. 1991. Conifers. Morphology and variation. Zagreb.

ABOUT THE AUTHORS I.Sotiropoulos, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului 119, Timişoara, Romania, e-mail: [email protected] E.Madoşă, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului 119, Timişoara, Romania, e-mail: [email protected]

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AN ASSESSMENT ON THE TROPHIC STATE OF LAKE ĆELIJE, SERBIA

S. Širiš, B. Miloševiš, J. Kneţeviš and Z. Spasiš

Abstract: The aim of this study was to determine the trophic level of Lake Ćelije, in hydrological and thermal unfavorable year of 2011.The sampling of lake water was carried out at representative locations, at different depths. The concentrations of orthophosphates and total phosphorus, as well as chlorophyll "a" concentration, were determined. Carlson's indexes for the total phosphorus were about 65. Indexes for the chlorophyll "a", which were considerably lower than the indexes for phosphorus, indicating incompletely realized trophic potential in 2011, which was largely the result of violations of phytoplankton in March. Key words: Lake, Trophic state, Total phosphorus, Chlorophyll "a".

INTRODUCTION Lake Šelije is located between 43°23' and 43°26' north latitude and between 21°9' and 21°12' eastern longitude, 30 km from the town of Kruševac in central Serbia. It lies in the north-south direction. The lake was formed shortly after the beginning of the middle course of the river of Rasina where the dam was constructed in 1979. The shapes of the former riverbed and surrounding terrain have caused the complex lake morphometrics (Fig. 1), which, going downstream, consists of three basins: Zlatari, Vasiši and Vodozahvat. Basins successively have increasing depth, and thus the volume, and the last basin Vodozahvat contains more than half of the lake water. By Carlson's trophic indexes, the lake had a trophic level of about 50, twenty years ago [7]. In the absence of any form of protection, anthropogenic influences have accelerated the eutrophication and led to the establishment of toxic blue-green algae in the lake in 2003. The first bloom affected the lake of Šelije in the summer 1988. In the previous winter 1987/88, the lake level has been lowered by 4 m, while the volume decreased by approximately 9x106 m3, which is more than one-fifth of the entire volume of the lake. The consequence of such large fluctuations in the level of lake water was rapid multiplication of blue-green algae Microcystis aeruginosa, which was soon suppressed by nitrogen-fixating species Aphanizomenon flos-aquae. This led to the creation of 50 cm thick scum on the water surface [5]. During the war 1999, the lowering of the level of the lake for 7 m led to the reducing of the lake volume for 17x106 m3, ie. for 41%. However, due to heavy rainfall and the runoff which was above average, the next bloom occured to a lasser extent in 2001. In 2003, more complex bloom occured. It was caused by three species, A. flos- aquae, M. aeruginosa and Anabaena circinalis, of which the last two produced microcystins. Bloom was extended to winter, and as a permanent phenomenon in the lake, continued in the period 2004-2010. During the peak bloom 2003, the trophic level of the lake Šelije by Carlson's indexes of total phosphorus and chlorophyll a ranged from 70 to 75. The aim of this examination was to determine the trophic level of the lake after entering the relatively large amounts of phosphorus and almost complete destruction of planktonic settlements in hydrological and thermal unfavorable situation for the blue-green algae in 2011.

MATERIAL AND METHODS Samples of lake water were taken using Van Dorn's 3 l bottle at five stations (Fig. 1), at depths of 0.5, 3, 6, 12, 22, 30 and 35 m. Zlatari basin was sampled at two stations, Vasiši at one station and Vodozahvat at two stations (Fig. 1). Water temperature was measured immediately after sampling with a mercury thermometer with 0.2°C precision.

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Then, the samples were transported in coolers to the laboratory of the Drinking Water Plant in Majdevo near Kruševac, and processed. Orthophosphates and total phosphorus were determined by american standard methods with amoniumheptomolibdate; total phosphorus was determined after digestion of the samples wirh nitric and sulfuric acids [4]. Chlorophyll a is also determined by the U.S. standard spectrophotometrically. The concentration of chlorophyll a in the presence of pheophytin was calculated by the equation: (mg/l) (1) where: A664 = absorbance at 664 nm; A750 = absorbance at 750 nm; A665 = absorbance at 665 nm after acidification; A750ac = absorbance at 750 nm after acidification; V = volume of the sample (l); Ve = volume of extract (l); L = way of the light, the lenght of cuvette (cm).

Figure 1. Lake Šelije with its three basins and sampling points 1-5

For Zlatari and Vodozahvat with two sampling stations each, concentrations of all parameters, including the density of taxa in a single layer of water Ci, were calculated by the approximation that the changes from one to the other depth is linear: (2)

where: C1H1, C1H2 = values of the parameter at two depths of the first station; C2H1, C2H2 = values of the parameter at two depths of the second station.

For Vasiši which has one station, a simple arithmetic mean is applied: (3)

where CH1 and CH2 = concentrations at two depths.

The parameters values in the basins CB were obtained as follows:

(4) where Vi = volumes of water layers in the basin. Similarly, the parameters values in the epilimnion and hypolimnion Ce and Ch, were obtained as follows:

, (5, 6)

where Ve and Vh = volumes of epilimnion and hypolimnion.

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In the same way the values of the parameters for the whole lake were calculated, but all of three basins were included simultaneously. As Carlson's indexes tracking make sense only on long time scales, their annual mean values is calculated. Annual values of Carlson's indexes for phosphorus and chlorophyll a can not be obtained by averaging the values obtained in individual samples, but on the basis of average annual concentrations of phosphorus and chlorophyll a. These concentrations are also not obtained by simple averaging but on the basis of equation 4, as the ratio of 1) the sum of product of the concentrations and individual water layers volumes from all samplings, and 2) the sum of individual layers volumes [1, 2].

RESULTS AND DISCUSSION The values of the temperature of the lake water during the year are shown in fig. 2. According to its thermal regime, the lake should belong to the category of temperate climate lakes with two seasonal stagnation, summer and winter, and two seasonal circulation, autumn and spring, as is the case with so far studied lakes in this region [3, 6]. However, the results show a different picture: a long stagnation with a gradual drop in temperature from the surface to the bottom during the spring, summer and autumn, and full circulation during the winter. This situation is typical for the subtropical lakes in areas with long, hot and dry summers, and mild winters, what otherwise characterized the climate of investigated region in 2011.

23.0 20.5 18.0

15.5 C)

13.0° (

10.5 8.0

5.5 Watertemperature 3.0 I III V VI VII VIII IX X XI XII Month epilimnion hypolimnion benthos

Figure 2. Mean values of temperature of lake Šelije water by month

The results show that the total phosphorus in the basins, except Zlatari basin which is directly exposed to the inflow, has a relatively uniform concentration (Fig. 3). During the summer, the concentration of phosphorus in the epilimnion decreased due to intense utilization of orthophosphates by phytoplankton, whereas in the hypolimnion increased partly due to the decomposition of detritus that fell to the bottom, and partly because of the mobilization of the orthophosphate ion from hydroxyapatite and apatite of bottom sediments in anoxic condition. In March, the obvious enormous intake of phosphorus in the lake was caused by the large amount of rainfall. The concentrations of phosphorus in March, 60% higher than usual, are striking evidence of the influence of inflow to the lake ecosystem (Fig. 3). In this month, very low concentrations of chlorophyll a show that the plankton was practically removed from the lake (Fig. 4). After the high water level in March, low temperatures in April and late spring moved the seasonal fluctuations of lake biocenosis. Usually, the first maximum of phytoplankton is in the second half of May till the first half of June, and the second maximum is in the second half of August till the first half of September. In the lake Šelije, provided that the bloom is not occured, the first maximum of phytoplankton is typically larger than the other. However, because of the disturbance of phytoplankton populations in March 2011, the second maximum was greater than the first in all three basins (Fig. 4). According to earlier studies of lake Šelije, completely utilization of orthophosphates in the epilimnion during the 165 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA first maximum of phytoplankton and their minor restitution after that, limit primary production. However, the trophic potential which is provided by the presence of phosphorus, is realized to a greater extent during the bloom. Then the blue-green algae, which were capable to storage the intracellular phosphorus due to residual inorganic nitrogen in the epilimnion, and a favorable relation of inorganic nitrogen and inorganic phosphorus, supressed the other algae from their niches and became the dominant primary producers [5]. Average annual values of the examined parameters showed the highest values of total phosphorus and chlorophyll a in Zlatari basin, because this basin is directly influenced by inflow (Fig. 5). 0.070 0.060

0.120

0.100 0.050

(mg/l) 0.040

0.080

3 -

0.060 4 0.030

PO (mg/l) 0.040 0.020

0.020 0.010 Total phosphorus Total 0.000 0.000 I III V VI VII VIII IX X XI XII I III V VI VII VIII IX X XI XII Month Month epilimnion hypolimnion benthos epilimnion hypolimnion benthos

Figure 3. Mean concentrations of total phosphorus and orthophosphates in lake water

0.014 0.077 0.070 0.012

a 0.063

0.010 0.056 0.008 0.049

0.042

(mg/l) mg/l 0.006 0.035 0.028 Chlorophyll Chlorophyll 0.004 0.021 0.002 0.014 0.000 0.007 I III V VI VII VIII IX X XI XII 0.000 Zlatari Vasići Vodozahvat Ćelije Month epilimnion hypolimnion benthos Orthophosphates Total phosphorus Chlorophyll a Figure 4. Mean values of chlorophyl a Figure 5. Mean annual values of parameters in lake concentration in lake water Although Carlson's trophic indexes are calculated annually, here are calculated individually for each sample in order to assess fluctuations in their values over the year. Calculations at the annual level (Table 1) showed that the indexes of phosphorus were relatively uniform for the lake and basins, while the indexes of chlorophyll a decreased from Zlatari toward Vodozahvat, and did not correspond with the indexes of phosphorus because they were much lower. Table 1. Carlson's indexes of lake Šelije Zlatari Vasići Vodozahvat Ćelije Month IP IChla IP IChla IP IChla IP IChla I - - - - 62.0 19.5 64.0 20.7 III 72.1 2.2 62.7 18.6 69.5 1.3 71.0 3.4 V 63.4 49.1 71.4 6.3 62.1 33.5 63.1 39.3 VI 62.1 50.7 61.6 36.2 63.6 42.7 63.5 44.8 VII 66.8 56.2 63.5 44.0 61.9 41.4 63.1 46.0 VIII 62.6 54.3 62.5 42.9 63.5 45.0 64.0 47.2 IX 61.4 57.5 64.6 45.4 64.3 37.0 64.1 45.8 X 61.9 49.4 64.1 44.8 64.1 35.7 64.0 40.8 XI 65.5 44.2 64.2 40.9 63.5 41.5 63.3 42.3 XII 65.9 49.4 61.1 42.5 63.6 33.1 63.4 38.3 Ʃ 66.5 51.0 64.1 39.9 64.1 37.5 64.4 41.5

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CONCLUSIONS AND FUTURE WORK In the lake Šelije, total phosphorus concentrations were relatively uniform throughout the year. Uniformity of Carlson's trophic indexes of phosphorus between lake basins and entire lake showed that in relation to phosphorus which is Liebig's macroelement in minimum, basins had almost the same trophic potential. Carlson's indexes of chlorophyll a which were much lower than the indexes of phosphorus induced not fully realized trophic potential in 2011. This was in a large part a consequence of disturbing of phytoplankton populations in March. Carlson's indexes of chlorophyll a in the basins were successively reduced. Based on this, it follows that the Vodozahvat has the lowest trophic level which is consistent with the best quality of water in it.

REFERENCES [1]. Carlson, R. E. (1977): A trophic state index for lakes, Limnology and Oceanography, 22: 361-369. [2]. Carlson, R. E. (1980): More complications in the chlorophyll-Secchi disk relationship, Limnology and Oceanography, 25: 378-382. [3]. Cibuliš, V. (1998): Oxigen regime of reservoir Barje and its importance, "Water protection", Proceedings, Kotor: 55-60. [4]. Clesceri, L. S.; Greenberg, A. E.; Trussell, R. R.; Franson, M. A. H. (1997): Standard methods for the examination of water and wastewater, 19th edition, American Public Health Association, American Water Works Association, Water Pollution Control Federation, Washington, D. C. [5]. Grašiš, S.; Vasiljeviš, B.; Markoviš, B.; Nikoliš, G.; Tadiš, S.; Jovanoviš, B. (2004): Cyanobacterial bloom of lake Šelije, Proceedings of the 33rd conference on actual problems of water use and protection "WATER 2004", Lake Bor: 207-212. [6]. Nikoliš, N. (2002): Assessment of the quality of water of the reservoir Grlište to assess further use for water supply, Thesis, University of Novi Sad, Faculty of Science, Novi Sad, Serbia. [7]. Seleši, Đ. (1989): Investigations on the determination of "zero state" of quality of lake Šelije water, University of Novi Sad, Faculty of Civil Engineering, Subotica, Serbia.

ACKNOWLEDGEMENTS This study is a part of the project TR 31001 "An environmental approach and implementation of modern biotechnologies as a basis for the improvement of ruminant breeding technology" financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia.

ABOUT THE AUTHORS Slavica Širiš, University of Priština, Faculty of Agriculture, 38219 Lešak, Serbia, E- mail: [email protected] Boţidar Miloševiš, University of Priština, Faculty of Agriculture, 38219 Lešak, Serbia, E-mail: [email protected] Jasmina Kneţeviš, University of Priština, Faculty of Agriculture, 38219 Lešak, Serbia, E-mail: [email protected] Zvonko Spasiš, University of Priština, Faculty of Agriculture, 38219 Lešak, Serbia, E- mail: [email protected]

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DISCOVER TRADITIONAL GASTRONOMY ORIGINATING FROM THE VRANCEA COUNTY

C. C. Vişan and C. I. Băra

Abstract: The traditional culinary products originating from the Vrancea county are obtained from environmentally-sound, organic raw materials, specific to the region, by following traditional recipes, inherited along centuries, by using both traditional and modern utensils. The products obtained comply with the national and international directives on consumer protection and environment protection, hence being used not only for private consumption, but also being part of the touristic chain, thus leading to the development of agrotourism in the region. We invite you to the Vrancea county to enjoy the flavour of traditional culinary customs, which so well showcase this region, located at the confluence of oriental and western influences. Key words: traditional gastronomy, environmentally-sound, organic raw materials, agrotourism, sustainability, consumer protection.

INTRODUCTION Romania has a true tourist potential, providing the visitor with a wealth of amenities and beautiful landscapes, ―a heritage of traditions, customs, lived history‖, whole-hearted and hospitable inhabitants, welcoming one with abundant meals. The interference of oriental and western influences confers Romanian gastronomy uniqueness in the world, with sometimes stunning, but undoubtedly original dishes. This confluence results in a varied cuisine, in which both simple dishes, as those present on the farmer´s table, as well as haute cuisine, particularly attractive and tasty, co-exist. A trip to Vrancea is not only an opportunity to contemplate landscapes, spectacular in their beauty and diversity, a tap into a genuine spiritual universe, but also a meeting with a surprisingly varied traditional kitchen, rich in flavours and colours, meeting most demanding tastes and preferences. Both gourmets and the less initiates are invited to tap into it to get to know it better, to sample foods and enjoy unparalleled wines, originating from vineyards like Odobeşti, Panciu and Coteşti, featured in Europe, America and Asia due to their quality. We look forward to meeting you in the Vrancea county to experience its ecological and cultural agrotourism and, why not, to harness the potential of the area as future investors.

MATERIAL AND METHODS The purpose of the work consists in deepening the role of gastronomy in the development of agro-tourism and in promoting traditional gastronomy in the Vrancea county, under the conditions of meeting consumer requirements. At confluence of culinary customs, the gastronomy of the Vrancea county has become more and more refined, but has preserved the specific features and traditions inherited. Traditional holidays and harvest celebrations are events of joy, dance and good mood, but also of gastronomy festivals, with tables decorated with a lot of good taste, with delicious foods. But how can we find out the truth about a region, how can we appreciate its customs and dishes better than tasting a good meal ? Glutton or not, here's where you can eat excellently ! Being good Christians, the people of Vrancea keep all religious fasting periods of the year. According to the season, they cook bean soup prepared with sour mirabelles (Prunus cerasifera), called in the region, ―muşmuşe‖ [mu´mu::e], which make the pottage

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taste sourer, thus opening the appetite, cabbage rolls for fasting, stuffed with rice and spices, steamed mushrooms with garlic, ―iahnie‖ [jah´ni:e], – pottage of beans and pickles. They know how to feast and have fun. Not only roast lamb, ―drob‖ [drob], (seasoned lamb entrails cooked in oven), pork sausages, ―tochitura‖ [toki´tu:rə], (pan-fried, fatty pork chops), ―caltaboş‖ [kalta´b::] (pork sausage with rice and spices) take pride of place, but also desserts like cakes, flat pancakes with plenty of cheese, and cakes with syrup and plenty of nuts. Shall one choose to visit Vrancea, they will meet hard-working people, honest and hospitable, who will accommodate you in their big house and will serve you their delicious dishes: meat soups with sour ―bors‖ [´bɔ r] (wheat / rye bran broth), cabbage juice, unriped grapes or mirabelles and seasoned with sour cream and eggs, different sorts of cheese with warm polenta, savory dishes made of vegetables and meat, seasoned with dill, parsley, lovage, thyme, chilli pepper (―chipăruş‖) [kipə´ru], garlic, which lure one with their flavour, gently sprinkled with home-made, natural, organic wines. And you can't rise from the table without tasting the doughnuts powdered with a lot of sugar, the dumplings filled with plums or pancakes with cheese.

RESULTS AND DISCUSSION The traditional gastronomy of the Vrancea county includes delicacies the locals boast about: smoked green cheese and ―cheese puppets‖, the cheese of Soveja, smoked trout by using fir tree branches, last, but not least, the wine obtained from the Vrancea vineyards.

The green cheese and the cheese puppets of Negrileşti The Negrileşti locality in the Vrancea Mountains is renowned in the whole region, because here, its inhabitants have been producing for centuries smoked green cheese, and doll-shaped cheese chops, by following traditional preparation methods. (Picture 1) The moulds in which the green cheese is pressed into are round, with solar shapes on the surface, as found in the European folk art; the rectangular cheese puppets originating from Vrancea have figures and faces, making them unique in time and space. Small works of art, the Vrancea Picture 1. Puppet moulds of Negrileşti moulds are the ―most touching illustration of humans in the entire Romanian folk art‖. Proud of their wood carving skills for producing moulds and for preparing cheese puppets, the local handicraftsmen try to preserve and transmit the inherited tradition from generation to generation.

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The Cheese originating from Soveja The carved cheese of Soveja is obtained by following a traditional recipe, inherited over the centuries and known only to the natives. The green cheese, obtained from cow milk, is packed into puppet-like moulds, made of sycamore wood, bearing symbols whose meaning dates back to paleochristian times: the ram horn symbolizes Fertility, the braided rope suggests Continuity, the sun is God, the wolf teeth bordering the cheese remind of the Durability of fir tree leaves. During the curing process, when the cheese ―is resting‖ in the puppet-like moulds, the symbols are taking shape ―by being sculpted‖ into the cheese wheels. The cheese puppets can be smoked with cold sawdust and evergreen of fir tree that confers cheese an unmistakable, superior flavour. (Picture 2) Fruit of Romanian folk art, carried forward from generation to generation, the moulds are sometimes hung on the beam of the house, as Picture 2. The Cheese decorative elements. originating from Soveja

Smoked trout in evergreens of fir tree Archaic methods for the preparation of smoked trout by using evergreens of fir tree are still kept alive in the mountain area of the Vrancea county, more exactly in the Lepşa village. The primary processing of fresh fish consists of the following steps: the scales are taken off, the guts are removed, the rest is rinsed and put into salt for about 12 hours (it is salted depending on the customer´s preferences). Then the smoking process in an oven follows, carried out by using beechwood, for approximately 24 hours; the method is known as hot smoking (curing with warm smoke). (Picture 3)

Picture 3. Smoked trout Picture 4. Smoked trout in ―hârzob‖

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After being smoked, the fish is packed into a basked made of fir tree evergreen, known by the name of ―hârzob‖ [hər´zob], which will confer the fish an evergreen taste. (Picture 4) The fish is consumed seasoned with lemon and wine from the Vrancea vineyards; ―hârzobul‖ can be kept for a few days in a cold, well ventilated room, to ensure a proper fish quality. The people of the Vrancea county are unique, the only ones curing trout with different wood essences: fir, beech, mulberry, plum tree. Beechwood curing gives fish a sweetish taste, while cherrywood or even fir tree curing, used in other areas, confers fish a bitter aftertaste.

Sheep cheese The inhabitants of the Vizantea-Livezi village invite you to taste from their traditional sheep cheese (Picture 5). The abundant and less polluted grasslands in the area impart a great taste to the sheep´s milk, and the hardworking hands of the housewives make this cheese be highly valued.

Picture 5. Sheep cheese with warm polenta

CONCLUSIONS The disappearance of peasantry causes us to gradually break with the origins of Romanian traditional gastronomy. Nowadays, when the world is changing at the speed of light, only traditional culinary customs keep us linked to the original rhythm of nature. To which it is good to remain linked! Products specific to the region:  the sparkling wine of Panciu;  the wines of Odobeşti, Panciu and Coteşti;  trout in fir-tree ―hârzob‖;  cured, smoked cheese in sculpted wooden moulds (the ―dolls‖ made of cheese) - constitute a brand for the Vrancea county, making it known nationally, and may become a brand also in the European Union. Shall you arrive in this part of the world, please do not hesitate to discover the specific flavour of cooked dishes, sprinkled generously with wonderful, natural, white or red wines, in a pleasant ambience, under the watchful eyes of your host, urging you to go on serving. And you can't resist this invitation because the harmony of tastes and flavours you try to make you eat much more than you wish to.

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BIBLIOGRAPHY [1]. Ghidiceanu, M., ş.a., 2003, Ghidul gastronomic al României, Colecţia Gastronomica, Editura House of Guides, Bucureşti. [2]. Glăvan, N., 2000, Turismul în România, Editura Economică, Bucureşti. [3]. Deşliu, A., 2000, Vrancea, ghid turistic, Editura Terra, Focşani. [4]. ***, Dumuri de vacanţă, 2005-2006, ghid turistic naţional, numărul 3.

ABOUT THE AUTHORS Vişan Carmen Corina, ―Mihail Kogălniceanu‖ Economic College of Focşani, Bd. Gării nr. 25, Focsani, 620083, Vrancea, Romania, e-mail: [email protected] Băra Csilla Iuliana, ―Al. I. Cuza‖ University of Iassy, B-dul Carol I, 20A, Iassy, Romania, e-mail: [email protected]

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ENVIRONMENTAL DAMAGES, WHICH MUST BE COMPENSATED DURING THE PROCESS COMPULSORY PURCHASE OF LAND IN UKRAINE

Alina Lizunova, Mariia Mykhalova

Abstract: This article is devoted to the problems of calculation of compensation for land owmers. Identified environmental factors that affect on the level of the compensation. Key words: compulsory purchase of land, public needs, compensation, environmental damages.

INTRODUCTION The European Convention on Human Rights and Fundamental Freedoms of one of the fundamental legal preconditions is that land alienation may be caused only to the public interest. Every individual or entity is entitled to the peaceful enjoyment of his possessions. No one shall be deprived of his possessions except in the public interest and subject to the conditions provided for by law and by the general principles of international law. Subject to paragraph 5 of Article 41 of the Constitution of Ukraine [1], no one may be deprived of property rights. The right of private property is inviolable. The compulsory expropriation of private property may be applied only in exceptional cases for reasons of public necessity, on the grounds and in the manner prescribed by law and subject to advance and complete compensation of their value. There is need of compulsory purchase of property right for land and objects of immovable property owned by individual or entity for public needs and due with public necessity in Ukraine as well as in other countries.

MATERIAL AND METHODS The compulsory purchase of private property shall be applied only as an exception for the common good on the basis of and according to the rules and procedure established by the Law of Ukraine "On purchase and other immovable property which they are located, which are in private property for public needs or for reasons of public necessity", and on condition of prior and full compensation of its value. The land is valued by legal entities that have suitable license. Negotiations may follow where questions of compensation, time and provisions of land purchase are dealing. The agency that decided to purchase land for public needs, shall compensate individuals and legal entities - owners and users of adjacent land losses that were caused by their purchase of land for public needs. [3] Procedure for identification and compensation to land owners and land users identified by resolution of the Cabinet of Ministers of Ukraine ―On the order of and compensation to land owners and land users‖ [5]. This resolution provides that landowners and land users are paying compensation for damages caused by withdrawal (redemption) and temporary occupation of land plots, restrictions on their use, degradation of soil and other useful properties of land or bringing them unfit for use and loss of income due to the temporarily used land. Subjects of compensation are: the cost of houses, industrial and other buildings and structures, including construction in progress; cost of perennial plants; the cost of forest; cost of water sources (wells, ponds, reservoirs, wells, etc.), irrigation and drainage systems; costs incurred to improve the quality of land for the period of use of land, taking

173 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA into account economic indicators, progress on agriculture, the intelligence and design; other losses of landowners and land users, including tenants. The amount of losses determined in full accordance with the actual value of the property at the time of causing damage, costs spent on improving the quality of land (including market or replacement value). But there are a number of factors which are not included in compensation to land owners. This primarily environmental factors that directly affect on the value of property unit at the moment or can change value in the future, if at that time they were not found. There are two types of environmental factors: positive and negative. We review only negative factors. The negative environmental factors include the consequences and manifestations of negative impact on the environment. Environmental pollution, in addition to serious negative consequences for human health, leading to considerable losses, resulting in a decrease in property values, you have additional costs associated with the aftermath of damages, direct losses of revenue from degradation and reduced profitability different categories of land. The list of such environmental factors may be quite broad. In each case the valuator must determine which factor will affect on the value of a particular property unit. The action of one and the same environmental factors may have a different affect on different types of real estate. Those factors that reduce the cost of land for housing, may not affect the value of office and industrial buildings, etc.. In Ukraine the construction, repair, reconstruction and maintenance of line objects and engineering and transport infrastructure is a public need for which is often used compulsory purchase of land. Namely the construction and further operation of linear objects and engineering and transport infrastructure is a major source of environmental pollution in cities, causing the need for compensation of environmental damages. Results of interaction the road with the environment depending on traffic, vehicle‘s characteristics, location and size of road,it‘s transport-operational, and operating systems. Highway in the environmental aspect is considered not only as an engineering structure, as well as extended in a line enterprise that carries out transportation work and interact with the environment. The main factors that collectively determine position and environmental problems in cities are: • Air pollution; • High levels of radiation; • High level of noise regime; • Pollution surface water and groundwater. As noted above, environmental damage can change the value of property unit in the future. Therefore, we note that value of property unit may change (increase or decrease) in that cases when property units are not compulsory purchased for public needs. Owners of such property unit shall also receive redemption value. The degree of air pollution in a particular area is determined by comparing the quantity and composition of emissions and their ability to cleanse itself. Size of damages for excess emission of one ton of pollutant in the ambient air is calculated. It based on the minimum wage, established at the time of detection multiplied by a factor of 1.1, subject to adjustment coefficients and the relative hazard rate of each pollutant substance.

The amount of damages is calculated by the formula

З = m х 1,1П х A х К1 х К2 , where

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З - the amount of damages, UAN; m - mass of the pollutants released into the air excess, t; 1.1 П - the minimum wage (П) at the time detect violations per ton of pollutant multiplied by a factor of (1.1), UAH / t; A - dimensionless parameter relative danger of the pollutant; K1 - coefficient taking into account the territorial socio-ecological features; K2 - coefficient depending on the level of air pollution of settlement with pollutants. (6) The total amount of compensation is calculated as the sum of size of losses on excess air emissions of each pollutant. One source of environmental degradation in the settlements is a source of electromagnetic radiation, which includes the transmission network, which consists of high-voltage overhead transmission lines and electrical substations. One of the negative effects of car traffic is noise that is not less harmful than pollution of air or water. This problem occurs especially on roads that cross the residential development, near hospitals, sanatoria, rest homes, resort, park roads and high-speed roads of major cities. Traffic noise degrades the quality of the environment around the road area. The problem of increasing the noise has a great impact on the cost of land, especially for settlements. According to the legislative and regulatory framework of land evaluation location of the land in the buffer zone, in an area exceeding the allowable noise level reduces their consumer quality through reduction factors influence health and hygiene factors. If the widty of the protective strip of the road is increased on average by 50%, thus increasing the area of the territory, which will apply reduction factors for monetary evaluation of property unit If the compulsory purchase for public purposes or for reasons of public necessity is necessary for the construction and reconstruction of public roads, market value of property unit will be compensated for property owners.

CONCLUSIONS Today in calculating the monetary value land by buying out during road construction shall not be considered negative factors that affect the portion of land that has remained the owner or user. Value is calculated only on that part of the property unit, which according to the schedule of compulsory purchase falls into the easement road, but the negative impact is experiencing much larger area. It this article established a number of adverse environmental factors affecting on the state of environment, which in turn affects the reduction of the market value of property unit. For a comprehensive account of the negative impact of engineering and transport infrastructure on the environment need to work towards a system objective scale of values, which include all aspects of areas.

REFERENCES 1. Constitution of Ukraine (28.06.1996) – Information of Verkhovna Rada of Ukraine from 23.07.1996 - 1996, № 30, Article 141 – http://zakon1.rada.gov.ua/cgi-bin/laws/ 2. Land Code of Ukraine (25.10.2001 № 2768-III) – Suffrage of Ukraine 20.11.2001 - № 217 - http://zakon1.rada.gov.ua/cgi-bin/laws/ 3. Law of Ukraine ―On Alienation of Land Plots and Other Objects of Immovable Property Located On Them in Private Ownership for the Social Needs and on the Grounds of

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Social Necessity‖ (17.11.2009 № 1559-VI)– Information of Verkhovna Rada of Ukraine from 08.01.2010 – 2010, № 1, page 3, Article 2 – http://zakon.rada.gov.ua/cgi-bin/laws/ 4. Civil Code of Ukraine (16.01.2003 № 435-IV) – Information of Verkhovna Rada of Ukraine from 03.10.2003 – 2003, № 40, Article 356 – http://zakon.rada.gov.ua/cgi- bin/laws/ 5. Resolution of Cabinet of Ministers of Ukraine ―On the order of and compensation to land owners and land users‖ http://zakon1.rada.gov.ua/cgi- bin/laws/main.cgi?nreg=284-93-%EF 6. Decree of the Ministry of environment of Ukraine «Methods for determining the amount of damages that are caused to the state as a result of excessive emissions pollutants into the air‖ (21.01.2009 №639) http://zakon4.rada.gov.ua/laws/show/z0048- 09

ABOUT THE AUTHORS Alina Lizunova graduates Kyiv National University of Construction and Architecture (KNUCA), Kyiv Ukraine in 2001. From 2002 till 2003 she passed Master program in Kungliga Tekniska Hogskolan, Sweden and receive diploma of Master of Science in Land Management. She holds a Ph.D. in Land Management in 2007 in topic of Property registration in KNUCA. In 2008 she receives scientific degree of docent in land management. Currently she is doctoral student in Kyiv National University of Construction and Architecture, and has been researching in the direction of compulsory purchase of land for public needs. She is also work as an Associate Professor on the Land Management and Cadastre Department in KNUCA. She is the author of more than thirty scientific articles in field of Land Management and Cadastre. E-mail: [email protected] Mariia Mykhalova graduates Kyiv National University of Construction and Architecture (KNUCA), Kyiv Ukraine in 2011. Currently She is Ph.d student in the Department of Land Management and Cadastre, Kyiv National University of Construction and Architecture, and has been researching in the direction of compulsory purchase of land for public needs. She is the author of five scientific articles in field of Land Management and Cadastre. E-mail: [email protected]

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SYSTEMATIC AND ECOLOGICAL ANALYSIS ON STINK BUGS (PENTATOMIDAE, HEMIPTERA) IN THE ECOSYSTEMS OF DURRESI REGION, ALBANIA

Phd. Eltjon Halimi1, MSc. Hesat Aliu2 PhD. Anila Paparisto1, PhD. Dritan Topi 1 1 Department of Biology, University of Tirana, Faculty of Natural Sciences, Tirana, Albania 2 Department of Biology, State University of Tetovo, Faculty of Natural Sciences, Macedonia

Abstract: This study aims to present a systematic and ecological analysis on the Pentatomidae family, stink bugs (Hemiptera), from the different ecosystems of Durresi region, Albania. The collection of biological material is performed during the period 2011-2012. In the paper are presented results of 89 individuals, which represented 21 genus and 33 species. The collected biological material revealed the Aelia genera are more represented, with 4 species and a frequency of 12.12%. Habitats of Shkozet station were represented by more species compared to other stations, 20 species and frequency of 60.61%. While with less represented species was Lalzi Bay station with 9 species and a frequency of 27.27%. Based on the “Jaccard index”, Shkozet and Rrashbull stations, present higher coefficient of similarity compared to other stations, 41.66% and 10 common species, which mean a similarity between these habitats. Zoogeographic Paleartcic regions represent most of the species, with 11 species and frequency 33.33%. Keywords: Stink Bugs, Pentatomidae, Ecosystem, Zoogeographic regions, Aelia

INTRODUCTION Stink Bugs (Pentatomidae Family Leach, are insects with antennas that are constituted by 5 segments. Their body is of oval shape and covered by a solid mantel. Their scutellum is big and of triangle shape as ‗mantel‘ [1]. They present green, yellow and metallic bright colors. These families include individuals of middle to big dimensions and are predominantly small. Tarsus is constituted by 2 or 3 segments [2]. They are classified as phytophagy species. Their negative impact in agriculture is mainly encountered in crops, rice, fruit trees, etc. They stand grouped and sack the liquid of the hosted tree and present considerable resistance toward pesticides [3], [4], [5]. Also, in this group are included predators that are feed with other insects [6], [7]. Our study considers species of this family in the ecosystems of coastal areas or hilly habitats of the Durresi Region, attempting to present a general panorama of this family in this habitat. Conclusions are drawn up through the analysis.

MATERIALS AND METHODS Collection of the biological samples was conducted for the period 2011-2012, in different habitats of the Durresi Region, sampling stations: Sukth, Shkozet, Hamallaj, Rrashbull and Lalzi Bay. The collection of individuals was achieved through random procedures during the warm seasons of year May-September, for each station, and the day time 0900-1500. Instruments used for collection were mainly entomological nets of diameter 80 cm. Shaving of the insects was conducted in diagonal equal surfaces of 100 m2 (10m x 10m), passing 5 times across each rectangle diagonals according to Colas [8]. In some cases of the field expedites were employed also air nets. After the field collection, individuals were placed in plastic bottles, and labeled by giving information on place and date, respectively. Regarding to the tinny samples they were placed in plastic flacons of 150-200cc. The biological material, in scientific laboratory,

177 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA was kept in bottles with Ethanol solution (95%), acetic acid, distilled water in ratio 80:5:20 (v/v/v) and some ether drops added consequently [8], [9]. Scientific determination of the biological material was conducted through investigation with stereomicroscope ZEISS (Carl Zeiss, Stemi 2000-C, Series 455044-9901, Optics: W- Pi 10x/23). The individuals were determined by using the determination keys for each family, collections and previous scientific publications [10], [11], [12], [13], [14], [15].

RESULTS AND DISCUSSIONS Determination of species that belong to the Pentatomidae family (Table 1) includes species encountered in the lower altitude and hilly-mountainous ecosystems of Durresi. For every species we have presented information on the number on each station: Sukth, Shkozet, Hamallaj, Rrashbull and Lalzi Bay, accompanied with information regarding to the zoogeographical region.

Table 1: List of species for family Pentatomidae

N Zoogeographical

Scientific name

o. Region

No.exemplar Sukth Shkozet Hamallaj Rrashbull Gj.Lalzit Genus1 Aelia Aelia1 acuminata Linnaeus, 1758 2 + + Palearctic Euro- Aelia2 rostrata Boheman, 1852 1 + Mediterranean Aelia3 virgata Herrich-Schaeffer, 1841 3 + + + European Aelia4 klugi Hahn, 1833 4 + + + Euro-Siberian Genus2 Ancyrosoma Ancyrosoma leucogrammes Gmelin, 17905 2 + + Mediterranean Genus3 Bagrada Bagrada6 abeillei Puton, 1881 4 + + + Mediterranean Genus4 Carpocoris Carpocoris7 fuscispinus Boheman, 1853 5 + + + Euro-Siberian Carpocoris purpureipennis De Geer, 17738 3 + + Euro-Siberian Genus5 Codophila Codophila9 varia Fabricius, 1787 2 + + Palearctic Genus6 Dolycoris 1 0 Dolycoris baccarum Linnaeus, 1758 3 + + + Palearctic Genus7 Eurydema 1 1 Eurydema ornate Linnaeus, 1758 3 + + + Palearctic 1 2 Euryderma oleracea Linnaeus, 1758 3 + + + Palearctic Genus8 Eysarcoris Eysarcoris1 inconspicous Herrich – 3 Schâffer, 1844 4 + + + Palearctic 1 4 Eysarcoris fabricii Kirkaldy, 1904 2 + Palearctic Genus9 Graphosoma 1 5 Graphosoma lineatum Linnaeus, 1758 3 + + + Palearctic Graphosoma1 semipunctatum Fabricius, 3 + + + Mediterranean

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6 1775 1 0 Genus Holcostethus 1 7 Holcostethus fissiceps Horvàth, 1906 2 + + Holarctic Holcostethus1 sphacelatus Fabricius, 8 1794 1 + Euro-Africans 1 9 Holcostethus vernalis Wolff, 1804 1 + Holarctic 1 1 Genus Mustha 2 0 Mustha spinosula Lefebvre, 1831 5 + + + + Mediterranean 1 2 Genus Neottiglossa Neottiglossa2 leporina Herrich – Schâffer, 1 1930 2 + + Mediterranean Neottiglossa2 lineolata (Mulsant & Rey, 2 1852) 2 + + Mediterranean 1 3 Genus Pentatoma 2 3 Pentatoma rufipes Linnaeus, 1758 3 + + + Palearctic 1 4 Genus Picromerus Picromerus2 conformis Herrich – 4 Schâffer, 1894 2 + Palearctic 1 5 Genus Piezodorus 2 Central 5 Piezodorus lituratus Fabricius, 1794 2 + + European-Asiatic 1 6 Genus Pitedia 2 6 Pitedia pinicola Mulsant & Rey, 1852 2 + European 1 7 Genus Rhaphigaster 2 7 Rhaphigaster nebulosa Poda, 1761 5 + + Palearctic 1 8 Genus Sciocori 2 8 Sciocori sculatus Fieber, 1851 3 + + Euro-Siberian 1 9 Genus Stagonomus 2 9 Stagonomus amoenus Brullé, 1832 2 + + Mediterranean 3 0 Stagonomus bipunctatus Linnaeus, 1758 4 + + + Mediterranean Stagonomus3 pusillus Herrich – Schâffer, 1 1830 2 + + Mediterranean 2 0 Genus Thalagmus 3 2 Thalagmus flavolineatus Fabricius, 1798 2 + Mediterranean 2 1 Genus Zicrona 3 3 Zicrona caerulea Linnaeus, 1758 2 + + Holarctic

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From our investigation of the biological samples were encountered 89 individuals of Pentatomida family, which represented 21 genera and 33 species (Table 2). In that aspect was determined also the frequency per every species according to equation:

Where: n- number of species for each family; N- number of species in total encountered

Table 2: Number of species according to genera

Species Species frequency Nr Scientific name number (%) 1 Aelia 4 12.12 2 Ancyrosoma 1 3.03 3 Bagrada 1 3.03 4 Carpocoris 2 6.06 5 Codophila 1 3.03 6 Dolycoris 1 3.03 7 Eurydema 2 6.06 8 Eysarcoris 2 6.06 9 Graphosoma 2 6.06 10 Holcostethus 3 9.09 11 Mustha 1 3.03 12 Neottiglossa 2 6.06 13 Pentatoma 1 3.03 14 Picromerus 1 3.03 15 Piezodorus 1 3.03 16 Pitedia 1 3.03 17 Rhafigaster 1 3.03 18 Sciocori 1 3.03 19 Stagonomus 3 9.09 20 Thalagmus 1 3.03 21 Zicrona 1 3.03 Total 33 100

Analysis and interpretation of the data results in our study, indicates that according to species‘ diversity, Aelia genera is represented with more species, 4 respectively, and frequency 12.12%, Holcostethus and Stagonomus genera are represented by three species and frequency 9.09%, Carpocoris, Eurydema, Eysarcoris, Graphosoma, Neottiglosa, genera are represented by two species and frequency 6.06%, while other genera were represented by only one species and frequency 3.70%. According to the species variety, more represented resulted Shkozet station, respectively with 20 species, or frequency 60.61%, followed by Hamallaj station with 18 species or frequency 54.55%, Rrashbull station with 14 species and frequency 42.42%, Sukth station with 12 species or 36.36% and last resulted Lalzi Bay station with 9 species or 27.27% frequency (Table 3).

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Table 3: Number of species according to station

Station Number of Species frequency species Sukth 12 36.36 Shkozet 20 60.61 Hamallaj 18 54.55 Rrashbull 14 42.42 Lalzi Bay 9 27.27

Calculation of the coefficient of similarity Jaccard gave an indication on the species similarity among the stations [16]. In the table are presented the numbers of common species (C), according to the stations, and the coefficient of similarity (CJ) for each station (Table 4). Stations present according to their geographical distribution a diversification of the ecological conditions and ecosystems. Hence is studied the correlation between the species to highlight the influence of the conditions in the species distribution, by taking in consideration the species itself, own their ecological valence.

Table 4: Number of common species and similarity coefficient according to each station

Sukth Shkozet Hamallaj Rrashbull Lalzy Bay Sukth C = 5 C = 7 C = 5 C = 3 CJ = 19.23% CJ = 31.81% CJ =25 % CJ = 18.75% Shkozet C = 9 C = 10 C = 4 CJ = CJ =41.66% CJ =16.66% 31.04% Hamallaj C = 5 C = 4 CJ =18.51 CJ =18.18 % % Rrashbull C = 3 CJ =15.78 %

From analysis, we concluded that the higher values of the coefficient of species‘ similarity stands among Shkozet and Rrashbull stations by 41.66% and 10 common species, followed from similarity among stations Sukth and Hamallaj by 31.81% with 5 common species, Shkozet and Hamallaj by 31.04% and 9 common species. The lowest value stands among Sukth and Rrashbull, by 25.00% and 5 species, Sukth and Shkozet by 19.23% and 5 common species, Sukth and Lalzi Bay with coefficient 18.75% with 3 common species, Hamallaj and Rrashbull with 18.51% and 5 species, Hamallaj and Lalzi Bay station by 18.18% and 4 common species, Hamallaj and Lalzi Bay by 16.66% and 4 species, finally between Rrashbull and Lalzi Bay by 15.78% and 3 common species. Analysis of similarity of species structure, give indication on the affinity regarding to species structure for these stations, as well as impact of ecological factors in general, and particularly the anthropogenic factor impact. From the study of the zoogeographical groups (Figure 1), the nucleus of the Pentatomidae family is the Palearctic Zoogeographic group with 11 species and frequency 33.33%, and consecutively Mediterranean Zoogeographic group with 10 species and frequency 30.30%, and in very small number of species Euro-Siberian group with 4 species or 12.12%, Holarctic with 3 species or 9.09%, European by 2 species or 6.06%, Euro-Africans, Euro-Mediterranean and Central European-Asian by 1 species or 3.70%.

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Figure 1: Distribution of No. species according to zoogeographical regions

In conclusion, this study presents a systematic and ecological analysis for 67 exemplars of Pentatomidae family, in ecosystems of Durresi region. These exemplars represented by 21 genera and 33 species. Results give indication that Aelia genera is presented with the maximum values of diversity, by 4 species and frequency 12.12%. Shkozet station dominates regarding to the species diversity, by 20 species or 60.61%, and last resulted Lalzi Bay station with 8 species or 24.24% frequency. Maximum value of coefficient of species‘ similarity stands between Shkozet and Rrashbull stations, by 41.66% and 10 common species, the minimum value of coefficient stands among Rrashbull and Lalzi Bay by 15.78% and 3 common species. More represented with species is Palearctic zoogeographic region by 11 species or 33.33%, Euro-Africans, Euro-Mediterranean and Central European-Asian by 1 species or 3.70%.

REFERENCES [1] Servadei A. Fauna d‘Italia. Rhynchota: Heteroptera, Homoptera, Auchenorrhyncha. Edizione Calderini, Bologna, Italy, 1967. [2] Tremblay E. Entomologia applicata. Volume II Parte I. 1 ed. Liguori Editore. Napoli, Italy, 1981 [3] Gennaro V. Lotta biologica e integrata. Liguori Editore. Napoli, Italy, 1977. [4] Miller NCE. The Biology of the Heteroptera 2nd Ed. Hill, London, UK, 1971. [5] Pollini A. Manuale di entomologia applicata. Edagricole. Bologna. Italy, 2002. [6] Silvestri F. Compendio di Entomologia Aplicata. Parte Specialie. Portici Tipografia Bellavista. Vol I, Rome, Italy, 1939. [7] Servadei A, Zangheri S, Masutti L. Entomologia generale ed applicata. CEDAM. Padova. Italy, 1972. [8] Colas G. Guide de L‘Entomologist. Edition N. Boubee & C-ie Paris, France, 1969. [9] Chapman RF. The Insects, Structure and Function. 4th Ed. University Press. Cambridge. United Kingdom, 1998. [10] Aukema B, Rieger C. Catalogue of the Heteroptera of the Palearctic Biology and Diversity. Oxford University Press. 2nd ed. London, United Kingdom, 1999. [11] Dolling WR. The Hemiptera. Oxford University Press. London, UK, 1911.

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[12] Halimi E, Paparisto A, Misja K. Some Systematics and Ecological data for True Bugs (Hemiptera) in Some Habitats in Albania. Journal Science and Popular Scencei-Natura Montenegrina. Pogdorica- Montenegro. Vol. 9 (3). 469-479, 2012. [13] Schuh R, Slater JA. True Bugs of the World (Hemiptera: Heteroptera). Classification and Natural History. Ed. Cornell University Press, Ithaca, USA, 1995. [14] Ribes J, Pagola C, Zabalegui I. One Some Palaeartic Carpocorini (Hemiptera: Pentatomidae: Pentatominae). In: Heteropterus Revista de Entomología. Barcelona. Spain, 2008. [15] Tremblay E. Entomologia Aplicata; Volume Generalità e mezzi di controllo; Collembolli-Riconti; Liguori Editore. Napoli, Italy, 1990. [16] Jaccard P (1901). Étude comparative de la distribution florale dans une portion des Alpes et des Jura. Bulletin del la Société Vaudoise des Sciences Naturelles. 37, 547-579.

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INTERDISCIPLINARY STUDY ON INFLUENCE OF VARIOUS TYPES OF WATER ON HERBS AND AROMATIC PLANTS IN CULTURE

Gabriela Vlăsceanu1, Ştefan Manea1, 1 2 3 Georgeta Negru , Marosy Zoltán , Alexandru Cristian Ion

1 Hofigal Export Import SA, 2 Intr. Serelor, district 4, code 042124, Bucharest, Romania, [email protected] 2 Ecological University of Bucharest, Bucharest, Bd. V. Milea 1G, district 6, [email protected] 3 Association of Terotechnic şi Terotechnology from România, 16 N. Bălcescu, district 1, Bucharest, [email protected]

Abstract: Water is the basis of life on Earth and all beings develops due to its specific biological properties. Current studies are developing the concept of structured water as stimulus for germination and plant growth. Different scientific research supporting the hypothesis that the water can be structured with minerals, rare metals or the magnetic fields. Use of minerals to stimulate plant germination is an old method but slightly used at the present time. Also structured water using various variable magnetic fields brings a significant contribution to the plant grow after germination. Use of water with colloidal silver is necessary in order to stimulate certain bio-chemical cellular functions. This method, it becomes more accessible through the development of low-cost technology for the acquisition of colloidal silver, it opens new perspectives in the stimulating of the plants growth and to protect these agains various pathogens, fits in specific requests of the ecological agriculture. Using a protocol for interdisciplinary work in the development trials have been done more research, studying is the main effects which appear in plants by the use of different dilutions and concentrations from the structured water. Results were correlated with the environmental factors, the soil qualities and the frequency does get wet, with the biologically rate of the plants and have been carried out statistics for the impact calculation.

Key words: germination, water, magnetism, minerals, colloidal silver

INTRODUCTION The chemically composition of water is made up of two hydrogen atoms covalently bound to one oxygen atom. H2O molecule has a spatial structure, with the two chemical bonds of H-O with approximately equal in length, 0.96 Å and valence angles of 104,50. In the intimacy of water molecule oxygen atom ionization occurs by absorption of an electron by a covalent bond with a hydrogen atom, the second electron being transferred to another hydrogen atom, forming an electric dipole form HO- - H+. This structure provides a spatial arrangement of atoms in the molecule of water as a tetrahedrical shape. Specific structure of the molecule of water brings an increased capacity to structure in the presence of magnetic fields, minerals and rare earth metals. At the appearance of external magnetic field the water molecule will have a diamagnetic response by the appearance of a precession movement of electrons from the electron cloud of oxygen ion. At a certain value of magnetic induction, without leading to the destruction of chemical bonds, the movement of precession would be large enough in order to cause a rotation of the whole molecule around the direction of magnetic flux passing through the nucleus of oxygen ion. This phenomenon was later called Giromagnetical Molecular Movement - MGM (Fig.1) [2].

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Figure 1. The electronic precession movement create an induction of a currently magnetic moment of water molecule dependent on the size of exterior magnetic induction. Studying the way in which water treated like this, into an experimental device influence the biological environment was observed, the act, that there is a direct dependence between the magnetic field action on water and plant growth. On the other hand, due to the high water-soluble capacity have been carried out a series of experiments to demonstrate plants development becawith the mineral called Sunnite (shungit – rom.). Sunnite was discovered in Karelia – north of Russia, but it also can be found in India, Africa and Canada too. There are also distributors in all over the Europe [1]. This mineral is used apparently starting with the 20th century, but it is not new at all. The estimative age of the mineral is almost 2 billion years. It looks like coal but it has better properties, and obviously different, compare to coal. The origin of the Sunnite is controversial because it was formed before any living organisms on the Earth. The researchers said that the origin of the mineral is actually unknown, so they suppose that the mineral has an extraterrestrial origin, or is volcanic, or is mineral, but nothing exactly. An inedyte study has shown the ability of colloidal silver to streamline the germination capacity and development of shoots. The beneficial effect of silver water has been known for ages. It has been used with success in Greece, Rome, Macedonia and ancient Fenicia for preventing and dealing various infections. However, few studies have been carried out to observe the reaction of planta to the silver colloidal presence in water. From a theoretical point of view silver helps a rapid exchange of water and minerals through the cellular membrane, disinfected water and present an advantage against pests which may inhibit its germination and then the plants development [3].. Repeated studies in the framework of organic Hofigal greenhousea, Bucharest, Romania, have shown beneficial influence on the ability of germination and medicinal plants growth due to use of the different types of water - structured by: magnetism, using mineral sunnite or with different dilutions of coloidal silver. Each type of water creates a certain specific effect and can be used at different times of development of the aromatic and medicinal herbs in culture [4,5].

MATERIALS AND METHODS First study material is structured water magnetic obtained by processing a quantity of 500 ml of ubcarbonated beverage water, for 3 days, in a device with variable magnetic fields in which induction coils are arranged three-dimensional layout. Water structured so it was delivered put into the glass container and ferrite beads of light or intense electromagnetic radiations, accompanied by a written material for advice on how to use and storage the water. For the survey have been used successive dilutions of 1/500 to 1/2000 depending on the plants and soil characteristics. For simplification will continue to use for structured water in three-dimensional magnetic device acronym M3D. For the second type of studied water, have been kept in contact a quantity of 100 g. of shungit to 1000 ml of water, for a period of 12 days. During this time water extracted mineral requirement and has been enriched with a high concentration of fulerenic carbon. Due the special properties of sunnite the water tends to structure in a dodecaedric form, 185 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA which supports the coloid state of the minerals from its composition. The structured solution and with significant uptake of minerals used for wetting aromatic and medicinal herbs ofrom greenhouse. For simplification will continue to use for the water with sunnite acronym SHU. The third study materials is aqueous solution of colloidal silver. The literature did not find a similar study and it took a theoretical thorough documentation. From the studies carried out it was found that the colloidal silver maintain the membrane elasticity and it supports cellular hydration. From this point of view it was considered that germination could be far more effective than if used water is simple. Due to the lack of oxygen in the water and an increased acidity, the addition of silver atoms might balance and neutralize the water pH. On the other hand, anti-bacterial properties are another reason for which it was considered that the water with colloidal silver could protect the seeds against bacterias and viruses that can attack during the first few days of germination. For simplification will continue to use for water with colloidal silver acronym ARG. Protocol for study suppose tests on 2 medicinal plants: momordica (Momordica charantia) and thistle (Silybum marianum L.) on growing period, till maturity. Studies have begun in the first phase in laboratory, through seed sampling into Petri dishes and recorded the germination, then the experiment assumed siphoning in pots of 30-40 plants and monitoring development to maturity. Both stages of the study was carried out in Hofigal Bucharest laboratories and greenhouses. Recordings were carried out in several stages: measuring the number of germinates seeds, the length of the shoots, leaves sizes, plant growth and development; further growth and development of flowers and seeds.

RESULTS AND DISCUSSION The results represent effects of a combination of factors that have acted synergies at the level of each stage of natural and biological rhythm of the plants. They have not been induced no acceleration or radical change in the rate of natural plant development.

Germination As a result of studies carried out it was found that the magnetic structured water (M3D) tends to brake in the first stage seeds germination. Observations made during the first 3 days of the study on water M3D water are already known from previous experiences [4]. It was noted a clear difference between plants which have germinate with simple water or with treated water M3D. By repeat has been highlighted that M3D water is not indicated in the first stage of plant development. As regards water with sunnite (SHU = shungit) there are differences depending on the type of plant. As general observation, it has to be noted that sunnite has a tendency to retain water, and after plant wetting the phenomenon can induce mildew of seeds due provided with a very large quantitie of water. Studies on water with colloidal silver (ARG) have shown that plant germination is quick and reduces the danger of seeds and young plants illing wetting whit this kind of water. Basis of numerous experiments and observations made in different locations, too, from the outside of Hofigal laboratory, it was prove that the effects are similar to the plants in culture also, which supports the initial hypothesis of study.

Plant growth after Germination Researches with M3D water are the most advanced (these have been initiated since 2010 in Hofigal). It has noted a rapid increase and development of plants wetting with M3D water. In this case the difficulty of the laboratory studies was due to insufficient light; the color of plants has varied with the amount of outside daylight. Due the accelerated

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grown the plant needs an increased intake in light and nutrients. After the transplant of young plants in separate pots, moved in greenhouses, and after a significant nutritional intake stheir color has changed, noticing a bright green and the appearance of fleshy leaves.

SHU water has great significance at this stage of plant development. Studies performed both in the lab and greenhouse demonstrates that the plants are moisturize very gently with this kind of water, and are more resistant to small periods of drought or in variations in the quantity of water. Even if at the level of growing aren‘t noted different dimensions versus simple water, can be observe a visible thickening of stem and a particular branching to the roots level. This has been noted also in other studies, outside Hofigal, which demonstrate the ability of the water with shungit at this stage of plants development. The ARG water (with colloidal silver) does not particularly note at this stage of plant growth. The colloids role is to keep safety the seed of bacteria and viruses, especially during the period of germination and development.

Plants growth The experiments carried out in the Hofigal greenhouses have highlighted the particular capacity of M3D water to support the development of shoots, especially at Momordica charantia. Depends on needed, the plants may be wetting with M3D in the different stages of growth and development, in order to obtain a specific desired effect. This issue that appears at this time is the soil specificity. A lack in nutrients in soil can create a specific "bloom" in shoots. It is a phenomenon encountered in several studies, primarily as lack on nitrogen. For the plants as thistle (Silybum marianum L.) it has noted that the SHU water (with shungit) is most indicated. Plant development is characterised by increasing of leaves sizes and producing of more stems in order to flowering stage. Studies have shown that combination of the two types of water (M3D and SHU) can create multiple beneficial effects, directing the plant development toward a desired result (to sprouting or/and development of already existing leaves) (Fig 3).

b) thistle wet with a mix of M3D şi SHU water a) thistle wet with simple water

Figure 3 Properties joint of the magnetic structured water with its of water with shungit has created an opportunity of harmonious development of plants

Because of the existence of many diseases and pests that cann‘t efficiently handle by bio methods, ARG water shows again its quality and efficiency. Knowing that ionic or

187 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA nuclear silver has increased action against pests, the currently studies are concentrated on highlighting the necessary dilutions and stages in which should be sprayed plant leaves. At this stage the efficiency in production silver atoms in colloidal form is the main issue and is concerned externalised of Hofigal company, together with a team of physicists.

Period of maturity Studies performed in greenhouses and field have revealed that the M3D water supports the development of new shoots; it is therefore obvious the possibility of developing more flowers. Wasn‘t observed any change in the natural and biological rhythm of plants by wetting with M3D and no change in the ability of flowering. SHU water supports the flowering and increases the addition of water in specific areas. It is noted the increased number of flowers and a great capacity of resistance to temperature fluctuations. The joint properties of M3D with SHU water increases considerably plant, favoring especially development of flowers. Even if there are not studies in greenhouse and field for this purpose, it is known that colloidal silver can support favorable the development of flowers. A sufficient dilution sufficient of colloidal silver may increase the number of flowers and, in so doing, may increase the number of seeds, in the case where it is proposed. At this stage the join of the three types of water creates direction for the future growth of the plant, providing improving the crops efficiency. In the period 2010-2013 has been designed a protocol for highlighting the efficiency use of different types of water in obtaining and growing crops of medicinal plants and aromatic, with stimulating effect on certain required characters of plants (Table 1).

Table. 1 Usage stages of different types of water in development of medicinal & aromatic herbs in crop. Plant development Plant Period of periods Germination Plant growth development maturity after germination

Type of water M3D Increase efficiency Efficiency on Not Maximum Magnetic in the development of new plants which are recommended efficiency for sprouting structurated water shoots, stems, flowers developing fleshy fruit SHU Maximum Effective for Recommend Maximum efficiency Water with efficiency in roots crops which need ed with moderation for leaves grown sunnite increase water ARG Efficiency on Maximum High efficiency Indicated for usage Water with plants from being efficiency for the sprouting period against pests colloidal silver harvested flowers

CONCLUSIONS 1. This article presents synthetic the studies of an interdisciplinary teams, who has carried out researches, since 2010, makes in the greenhouses and on ecological fields of company Hofigal Import Export Bucharest, Romania. 2. Reffering to the M3D water there were studies carried out in several institutions of higher education in Romania, so that the experiments at Hofigal have started with an advanced theoretical and practical base. On the way, by raising of problems regarding the plant growth and their harmony development the onservations have been completed using water with sunnite and more recently, water with colloidal Ag. 3. The conclusion of our interdisciplinary research team is that the synergic action of several types of water can support the grown of ecological crops of aromatic and

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medicinal herbs. 4. Correlating knowledges from different branches of science there is a specific development of a new branche of knowledge, defined by efficiency, reduced prices, ease-of-implementation and ot ultimately - a positive impact on the environment.

REFERENCES [1]. Bîra A., (2012), Economical Advantages Of Shungite Mineral Using In Organic Farming, Review ‖The Danube River‖, No. 12 / 2012, Bucharest, ISSN 1583-7513, pag. 10-14; [2]. Marosy Z.I., Kacsó L.,(2010)-Water treatment in tri-dimensional magnetic field, Environmental Engineering and Management Journal, Vol.9, No. 11/2010, Iaşi, Romania, ISSN:1582-9596,p.1503-1508; [3]. Marosy Z. I., Ion A. C. (2012), New Studies Regarding The Low-Cost Industrial Production Of Colloidal Silver For Water Decontamination, Journal of the International environmental conference,―New technologies for wastewater treatment‖,EXPOAPA 2012, Bucharest, ISBN 978-606-92682-7-8, p.147-158; [4]. Vlăsceanu G., Manea S., Negru G, Marosy Z.I. (2011), Influence of structured water in three-dimensional magnetic device on germination and plant growth, Proceedings of BENA 2011, Alba-Iulia, Romania; [5]. Vlăsceanu G., Manea S., Popescu M., Marosy Z. I., Bîra A. (2012), Cultivation Of Willow Like Eco-Energy Plant And The Valorisation Of It’s Wastes, Proceedings of the International environmental conference, ‖Sustainable Landscape Planning Safe Environment‖, Istanbul Technical University, BENA Istanbul 2012, p. 429-435.

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NEW PERSPECTIVES FOR SPIRULINA PLATENSIS

G. Iordachescu, G. Vlasceanu, St. Manea

Abstract: The algae world is very complex and supplies a lot of possibilities for food researchers and producers. Spirulina platensis contains the same type of amino acids as humans do. This property as well as the colour, and being at the in the top of sea weed production, the safety, and being a well known marine product recommends this algae for its use as additive in some selected food products. The study wants to identify which products are appropriate for the use of this additive, and the influence of this on their sensorial aspect. Being a nonconventional source of proteins (up 70 % protein content reported to dry wheight), with a protein quality among the best in green sources. Spirulina platensis can be used with good nutritional and sensorial effects in bread, pasta, ice cream, yogurt, frozen yogurt, concentrated apple juice and sauces. The QDA method was used. The results showed that the colour in almost all cases was not an impediment, the nutritional value was really improved and the green message sent by the product had a positive impact on the consumer.

Key words: Spirulina platensis, frozen yogurt, pasta.

INTRODUCTION Being in the food world, everyone can find a lot of chalanges in terms of safety, nutritive value, appearence, colour, aroma, and convenience. However the sensorial aspect is a major factor in decision making when selection foods. This is the advantage in the producers‘ hand to determine the by color, texture, taste and aroma to buy a safe and nutritive product. The algae can be used as food additive to improve the sensorial properties, but mainly contribute to the nutritive value due to their rich and high quality protein content. The algae are photosynthetic organisms which vary from small single celled forms to complex multicelular forms. The organism appears to be capable of adaptation to very different habitats [1]. On the other hand a daily consumption of 3-6 g helps the human body to fight against diabetes, hypercholesterolemia, ulcer, leaver diseases. The use of this organism to improve the nutritional and sensorial quality of some products is recommended. The possible products are concentrated apple juice, bread, pasta, frozen yogurt, apple juice. The products selected for the research were pasta and frozen yogurt. Pasta is the second most consumed product after the bread and eaten everywhere around the world [2]. Pasta is natural, versatile, available in a variety of shapes and easy stored for long periods and serve for both decoration and acts as a carrier for the different types of sauce. [3], [4]. Initially prepared from durum wheat was after diversified with different types of flours, chick pea flour, soy flour, defatted soy flour for example, adapted to new intolerances and colored with different colors red from beetroot, spinach, chlorophyll, black with squid ink. Spirulina platensis can be an alternative which improve also the nutritional value of the product. Frozen yogurt is a frozen dessert that combines the flavors and textures of ice cream and sherbet [5]. This one may be prepared with lactic acid bacteria cultures or using probiotic bacteria. Probiotics are defined as live microorganisms that, when administrated in adequate amount, confer a health benefit to the host [5]. Consumers have shown a great interest in frozen yogurt because it‘s potential as a low fat replacement for ice cream and the perceived probiotic effects of the lactic acid bacteria used in its manufacture [6]. The presence of frozen yogurt in the market is increasing because of the positive health

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effect of probiotic bacteria and for the opportunity of substituting the regular high fat content of ice cream [7]. Different aroma (banana [8], peach, strawberry, and apple) may impose one color on this product, and Spirulina platensis’s color may be easy associated with the mint refreshing taste and also with the image of bio green products. The aim of this work is to identify the influence of the use of Spirulina platensis in pasta and frozen probiotic yogurt on the protein and fat content in both cases and on the acidity in the frozen yogurt case. Another aim is to evaluate the sensory impact on these products.

MATERIAL AND METHODS The products

The pasta was obtained using a traditional home made recipe using wheat flour (100 g), 1 egg. Three samples were prepared: one with 0,5 % Spirulina platensis , one with 1% and third one blank. The dough was kneaded to an elastic consistency, left to rest 20 minutes at room temperature, stretched and cut into thin strips. Afterwards, these were dried for 24 hour at room temperature. Finally they were boiled for the sensory analysis and to estimate the Optimum Cooking Time (OCT). The frozen yogurt was obtained using Probio-Tec Freeze dried cultures obtained from Christian Hansen and milk UHT from Romanian market with 3,5 % fat. The milk was inoculated and fermented under continuous stirring at 370C for 8 hours. Three samples were prepared: 0,5 %, 1 % Spirulina platensis and one blank. The freezing process was conducted at – 180C. The acidity was determined using AOAC method. Protein content was evaluated by Lowry method – proteins forms complex combinations colored in purple blue with Cu2+ alkaline medium. The complex protein Cu2+ reduces Folin – Ciocalteu reagent. The optical density is evaluated and on etalon curve the amount of proteins is calculated. 10 g product was dispersed in 100 ml boiling water. Every minute one piece was pressed between two glass plates and compressed. Optimum Cooking Time after the continuous green line visible at the center of the pasta strands disappears.

Sensory analysis was made using 20 a trained panelists. To evaluate the pasta and frozen was used Quantitative Descriptive Analysis (QDA). The attributes proposed by Meligaard, Civille and Carr (2007) were adapted. These involve the case of pasta color, texture, aroma, taste and overall acceptability, in the case of frozen yogurt appearance, color, texture, sourness, sweetness, creaminess, aroma, body, aftertaste, overall impression. The training was made in three sessions. In the first one were defined the attributes, in the second one the panelists were trained with standards and in the third they were familiarized with the use of the scale. For recording the sensory evaluation was used a 12,5 cm structured scale. The sensory analysis was conducted on a Lazy Susan table.

The room temperature was 200C and relative humidity 45 %. The samples were coded with three digit random numbers. They rinsed the mouth with water between samples.

RESULTS AND DISCUSSION The protein content and the acidity of the pasta with 0,5 % or 1 % are presented in figure 1. 191 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Figure 1. Protein content (mg/100g product) for the pasta samples with Spirulina platensis

The OCT was 10 minutes for the blank and 12 minutes for the both samples. The pasta with Spirulina platensis presents slowly bigger protein content this rate being constant in the case of protein loss in water. The acidity of frozen yogurt is presented in figure 2.

Figure 2. The acidity (% lactic acid) for frozen yogurt Figure 3. The protein content (mg/100 g prod) for Frozen yogurt

The acidity presents small differences between the blank and the samples with 0,5 respectively 1 % Spirulina platensis. This aspect can influence the sensorial characteristic but the protein content which presents also variations influence the nutritive value of the probiotic product. The sensory evaluation showed (figure 4 for pasta, respectively figure 5 for frozen yogurt):

Figure 4. Sensorial properties of pasta with 0,5 % respectively 1 % Spirulina platensis

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All the sensorial attributes present almost the same intensity but the color presented a clear difference this attribute influencing negative the product color in the case of 1,5 % added Spirulina.

Fig.5. The sensory analysis of frozen yogurt

The same characteristics presented the frozen yogurt, no big differences in all the sensorial attributes excepting the color which influenced negatively the product. The overall impression was more than a half of the scale in the both cases.

CONCLUSIONS AND FUTURE WORK This is the first step in the research on Spirulina platensis use in this products. More elaborated studies are imposed in the future with the increase of the number of physic- chemical characteristics and the number of sensorial attributes. The influence of algae content is beneficial on the nutritive value, this bringing a good quality value on protein content. The sensorial analysis showed that the algae has a positive influence on sensorial attributes, the color being the only one which needs a very strictly control in terms of added quantities.

REFERENCES [1]. Ciferi, O., 1983, Spirulina the edible microorganism. Microbiological reviews 47, 551-578. [2]. Torres, A. et al, 2007, Chemical, biological and sensory evaluation of pasta products supplemented with -galactoside-free lupine flours, Journal of the Science of Food and Agriculture, 87:74-81 [3]. D‘Edigio, M.G, 2012, How to cook the perfect pasta -special report, ISO Focus, November-December, 10-12 [4]. Bashir, K. et al, 2012. Physico-chemical and sensory characteristics of pasta fortified with chick pea flour and defatted soy flour, IOSR Journal of environmental science, I: 34-39. [5]. Tungrugsasut, Wet al, 2012, Probiotic frozen yogurt, Journal of Pharmaceutical Sciences, 39(3-4):24-31. [6]. Ordonez, G.A., et al. 2000, Manufacture of frozen yogurt with ultra filtered milk and probiotic lactic acid bacteria, Journal of Food Processing Preservation, 24:163-176

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[7]. Miguel, D.P. et al, 2004, Sensory a chemical aspects of frozen soy yogurt fermented with Enterococcus faecium and Lactobaccilus jugurti, Alim. Nutr. Araraquara, 15(3):197-201. [8]. Ahmed, S.Kh. et al. 2010. Banana frozen yogurt from camel milk, Pakistan Journal of Nutrition, 9 (10): 955-956 [9]. Meligaard, M., Civille, G.V., Carr, B.T., 2007, Sensory evaluation techniques. CRC press.

ABOUT THE AUTHORS G Iordachescu, Dunarea de Jos University, 47 Domneasca Str. 800008, Galati, Romania, E-mail: [email protected] G.A. Vlasceanu, Hofigal Export Import SA, Intrarea Serelor nr.2, cod 042124, sector 4, Bucuresti, Romania, E-mail: [email protected] St. Manea, Hofigal Export Import SA, Intrarea Serelor nr.2, cod 0412124, sector 4, Bucuresti, Romania, E-mail: [email protected]

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DNA IDENTIFICATION OF HUMAN REMAINS EXHUMED IN KOSOVO AND METOHIA DURING THE PERIOD 2001-2011

Milanka Miletiš, Nebojša Deletiš, Zvezdan Milanoviš, Suzana Matejiš, Sonja Smiljiš

Abstract: This paper gives a detailed description of exhumed remains identification process. One of the study objectives has been a comparison between DNA results and traditional forensic identification methods. This paper deals with the identification of human remains that were exhumed in Kosovo and Metohia in the period 2001-2011. The experience of this process and the experience of other countries show that there is a need to organize appropriate services for identification of human remains in Serbia, capable to react effectively in case of mass disaster. Key words: Identification, Exhumation, Human Remains, Kosovo, Metohia.

INTRODUCTION In the last twenty years or so, many unexpected challenges have appeared in the identification of unknown human remains, which led to significant advances in this field. The most famous mass identification campaigns, capable of giving us some lessons to be learned, were those after the attack on World Trade Center (WTC) on 9/11 2001 (USNIJ, 2006), Indian Ocean tsunami in 2004 (Deng et al., 2005), as well as experiences of human remains identification after the armed conflicts in Croatia and Bosnia and Herzegovina (Huffine et al., 2001; Anđelinović et al., 2005), and in Kosovo and Metohia (Dobričanin et al., 2001; Matejić, 2005). During armed conflict, and especially after the establishment of UN administration in Kosovo, in the presence of international military and police forces, disappeared 1,441 people of Serbian, Montenegrin and other ethnicities, including certain number of Albanians loyal to Serbia. Available data indicate that most of these people were killed, and their remains were buried in different ways – in hidden and unmarked individual graves or in mass graves. The fact that Kosovo and Metohia are not under the jurisdiction of Serbia for more than dozen years limited participation of Serbian institutions in the process of recovering and identifying human remains. Therefore, this paper deals with the identification of human remains that were exhumed in Kosovo and Metohia in the period 2001-2011, belonging to Serbs and other non-Albanian ethnic communities (Montenegrins, Bosniaks, Roma, Gorani, and others), as well as much lower number of Albanians who were also killed during the war and post-war period. This paper has been aimed to consider certain aspects of the exhumed human remains identification, which are consequences of local specificity and a lot of peculiar events. One of the study objectives has been a comparison between DNA results and traditional forensic identification methods.

MATERIAL AND METHODS Happenings connected with the armed conflict in Kosovo and Metohia resulted in lots of people missing: in the period 1998/99 and during the NATO air campaign in 1999 more than 4,000 people belonging the all ethnic groups were missing, while after establishment of UN administration additional thousand people went away, mainly those of non-Albanian origin. Associations of missing persons‘ families in Serbia claim about 1,400 people, about two-thirds of whom disappeared after the war. The process of human remains exhumation and identification practically began during the mere armed conflict, but its full extent was reached after establishment of UN administration in Kosovo and Metohia. In the beginning, during 1999 and 2000, the highest number of identified victims belonged to the Albanian ethnic group, and after that, in fact 195 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA since spring 2002, identification of victims who belonged to other ethnic groups, primarily Serbs, was initiated. To the date, joint teams featured by our experts have exhumed remains of 418 persons, which were then examined along with remains of 51 persons exhumed without attendance of our experts. The main task of this process was to reconstruct the facts surrounding grave sites and collect the all relevant information that could be useful for determining cause and manner of death and victim identification (Kirschner and Hannibal, 1994; Matejić, 2005). Examination of the exhumed human remains was carried out in the Office of Missing Persons and Forensics. This institution was established by UN Mission in Kosovo (UNMIK) in June 2002, and it ran within that mission under the name UNMIK OMPF up to 12/9 2008 (UNMIK, 2008), when it fell under jurisdiction of the European Union Rule of Law Mission (EULEX), under the changed name EULEX OMPF. OMPF headquarters and mortuary facility were established in Orahovac, where most of the autopsies were performed, although greater part of this institution was moved to Priština, so that later autopsies were carried out there. During postmortem examination of human remains, they were analyzed by specialists in forensic medicine and anthropology. In addition, clothing and personal effects found with human remains were examined, packed and labeled by the same label used to mark the corpse on which, or next to which, they were found. Skeletal remains were also examined in an anthropological manner. The entire procedure was documented by photographs and video recordings. When necessary, radiographic methods were used. Samples for DNA analysis were collected mostly from long bones, teeth, cranial bones or ribs, and were processed in the laboratories of the International Commission on Missing Persons (ICMP). Ante mortem data (AM data) were collected from families of persons missing in connection with the armed conflict in Kosovo and Metohia, in cooperation with the International Committee of the Red Cross and other organizations. These are data regarding physical characteristics of missing persons, birthmarks, dental status, health status, etc., that may be of importance in the process of human remains identifying. Along with the collection of AM data, blood samples for reference DNA analysis were taken from relatives of missing persons. The whole procedure of analyzing DNA sampled from unidentified human remains, DNA analysis of reference samples of missing persons‘ relatives and expert interpretation of the obtained results, was organized by ICMP, and that institution was entirely responsible for the process.

RESULTS AND DISCUSSION Since June 1999, exhumations were performed by ad hoc teams of international experts in forensic medicine, who worked for the International Criminal Tribunal for the Former Yugoslavia (ICTY). Between June and November 1999 the 2,108 bodies were exhumed at 195 locations. In the second phase, conducted between April and November 2000, ICTY exhumed 1,557 bodies and 285 incomplete remains from 325 locations. On July 17th 2001, the tribunal reported that the total of 4,392 bodies had been exhumed from 876 graves in Kosovo. Forensic expert teams who worked for ICTY performed postmortem examination and other forms of forensic expertise (anthropology, forensic odontology, etc.) in 3,620 of those human remains, and then successfully identified 2,099 victims, whose remains were returned to their families for burial. Afterwards, approximately 1,500 unidentified bodies were buried in two UNMIK cemeteries and other municipal cemeteries for "later" identification (Amnesty Int., 2009). When exhumations discussed in this paper are considered, in 2001 three bodies exhumed earlier have been identified and delivered to their families, so that year is

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included in this paper. The number of bodies exhumed by year is shown in table 1 and graph 1.

Table 1. Number of exhumed and postmortem examined human remains by years Postmortem examined Year Exhumed human remains human remains 2001 0 0 2002 110 110 2003 161 161 2004 22 22 2005 70 70 2006 4 50 2007 16 18 2008 0 3 2009 11 11 2010 18 18 2011 6 6 Total 418 469

Graph 1. Percent of exhumed human remains by years

As it can be seen, most of exhumations were performed in 2002 and 2003 (total 271 or 64.83%). Reasons for such an initial "success" are numerous. There was a great political pressure, sufficient amount of (mainly international) money was coming, and information regarding potential group and individual grave locations were more easily available. Since 2004, a significant reduction of international forces occurred. Also, process of jurisdiction transfer to temporary administrative bodies progressed from year to year, and culminated in 2007 with unilateral declaration of Kosovo independence, which slowed down the process of identifying primarily non-Albanian victims. Number of postmortem examined human remains (table 1 and graph 1) followed a similar tendency, and 57.78% were autopsied in 2002 and 2003. Increase in the number of exhumed and postmortem examined bodies observed in 2005 and 2006 is the result of discovery of several mass graves, especially those in locations Volujak near Klina and Široko near Suva Reka.

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Graph 2. Share of identified persons in the total number of exhumed human remains

The total number of postmortem examined human remains was greater than the number of the exhumed ones, because 51 bodies were exhumed without our experts present at exhumation, but involved in other stages of the process. Of the total number of postmortem examined human remains (469), 333 of them were identified during the studied period, while 136 bodies remained unidentified, which was nearly one-third of the total body count (graph 2). Share of identified persons in the total number of missing persons was much lower, and there are numerous reasons for that.

Graph 3. Share of the applied identification methods

At the very beginning (the first half of 2003), DNA samples were taken and analyzed as an addition to conventional methods when they were not able to assure positive identification, then as the only method when conventional identification was not possible, or at family request for confirmation of identity. Later, the DNA identification was required. Hence, 32 people were identified solely by classical methods, and in 43 persons DNA identification was performed as an addition to classical methods. Afterwards, 13 families requested DNA analysis to confirm identity, so that at the end 19 people were identified only by classical identification, 56 persons by classical and DNA identification combined, while for other 234 identified persons DNA analysis was the method of choice (graph 3). In 14 cases, when results of DNA analysis indicated match, but were inconclusive (probability of matching 96-99.9%), confirmation of identity was accomplished by classical methods.

CONCLUSIONS AND FUTURE WORK Less than a third of the total number of missing persons was identified (according to some sources less than a quarter). The process of DNA identification was carried out in laboratories of the International Commission on Missing Persons (ICMP). The whole process was fully controlled by this institution. Results of DNA analyses were delivered in encrypted form, in a completely non-transparent way. Based on our experience and the

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experience of other countries it is clear that there is a need for establishing appropriate agency for identifying human remains of unknown identity in Serbia, in order to ensure an effective reaction in case of mass disaster.

REFERENCES [1]. Amnesty International. 2009. Burying the past – 10 years of impunity for enforced disappearances and abductions in Kosovo. Amnesty International, Special Report EUR 70/07/2009 (available at: www.amnesty.org). [2]. Andjelinoviš, S., Sutloviš, D., Erceg-Ivkošiš, I., Skaro, V., Ivkošiš, A., Paiš, F., Reziš, B., Definis-Gojanoviš, M. and D. Primorac. 2005. Twelve-year experience in identification of skeletal remains from mass graves. Croat. Med. J., 46(4), 530-539. [3]. Deng, Y.J., Li, Y.Z., Yu, X.G., Li, L., Wu, D.Y., Zhou, J., Man, T.Y., Yang, G., Yan, J.W., Cai, D.Q., Wang, J., Yang, H.M., Li, S.B. and J.Yu. 2005. Preliminary DNA identification for the tsunami victims in Thailand. Genomics, Proteomics & Bioinformatics / Beijing Genomics Institute, 3(3), 143-157. [4]. Dobričanin, S., Matejiš, S., Miloševiš, M. and V. Jakšiš. 2001. Sudsko-medicinska ekspertiza u slučaju Klečka. Praxis medica, 31(1-2 Suppl.), 11-14. [5]. Huffine, E., Crews, J., Kennedy, B., Bomberger, K. and A. Zinbo. 2001. Mass identification of persons missing from the break-up of the former Yugoslavia: Structure, function, and role of the international commission on missing persons. Croat. Med. J., 42(3), 271-275. [6]. Kirschner, R.H. and K.E. Hannibal. 1994. The application of forensic science to human right investigations. Med. Law, 13, 451-460. [7]. Matejiš, S. 2005. Sudskomedicinska identifikacija leševa klasičnim metodama i metodom DNK – uporedna analiza. Doktorska disertacija. Univerzitet u Prištini, Medicinski fakultet, Kosovska Mitrovica. [8]. UNMIK. 2008. UNMIK fact sheet February 2008. (available at: http://unmikonline.org). [9]. US National Institute of Justice Special Report. 2006. Lessons learned from 9/11: DNA identification in mass fatality incidents. U.S. Department of Justice, Office of Justice Programs, Washington DC, USA.

ABOUT THE AUTHORS M. Miletiš, MSc, Research Associate, University of Priština, Faculty of Medicine Kosovska Mitrovica, Anri Dinana Street bb 38220 Kosovska Mitrovica, Serbia, E-mail: [email protected] N. Deletiš, PhD, Associate Professor, University of Priština, Faculty of Agriculture Lešak, Kopaonička Street bb 38219 Lešak, Serbia, E-mail: [email protected] Z. Milanoviš, PhD, Professor, University of Priština, Faculty of Medicine Kosovska Mitrovica, Anri Dinana Street bb 38220 Kosovska Mitrovica, Serbia, E-mail: [email protected] S. Matejiš, PhD, Associate Professor, University of Priština, Faculty of Medicine Kosovska Mitrovica, Anri Dinana Street bb 38220 Kosovska Mitrovica, Serbia, E-mail: [email protected] S. Smiljiš, PhD, Associate Professor, University of Priština, Faculty of Medicine Kosovska Mitrovica, Anri Dinana Street bb 38220 Kosovska Mitrovica, Serbia, E-mail: [email protected]

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ANALYSIS OF THE MOTIVATING FACTORS AND THE SATISFACTION OF THE EMPLOYEES OF A COMPANY FROM TIMISOARA (ROMANIA)

C. C. Ruset, G. Buzamat, G. Popescu, K.I. Lato, and I. Cretescu

Abstract: This study presents is very important because where there is motivation there will be also productivity and performance and both the employees and the managers will be satisfied. The main objective of this study is to highlight the relation between the motivating factors and the employee satisfaction. The study proposes an analysis of the importance of the employees’ needs and their satisfaction, motivation and remuneration satisfaction, the work conditions offered by the employers, the quality of the collaboration with the top management, safety at the work place, the compensation of the performances and other motivating factors. The research was realized in a company from Timisoara (Romania) based on survey sent to several employees from different departments, with different incomes, different ages and sexes. Analyzing the results of the survey we noticed that the main motivating factor is the salary, followed by the work conditions and safety at the work place.

Key words: Human Resources, Motivating, Manager, Satisfaction,

INTRODUCTION The human resource is very important in any business; the other resources (land, buildings, cars, money, etc) have a secondary role. The motivation represents the correlation of the satisfaction of the needs of some individuals with the realization of the objectives and tasks established by the managers. At the basis of the human behavior stands most of the time the motivation which can be intrinsic and extrinsic. [2]. Whatsoever the form it has the motivation process must fulfill simultaneously three characteristics: to be complex (to use different stimuli), to be different (to take into account the specificities of each individual) and to be gradual (to satisfy successively the members‘ needs depending on how they fulfill their tasks [1].

MATERIAL AND METHODS This study has an investigation purpose, observing the configuration way of the motivational factors and the employees‘ satisfaction in a company which offers transportation services in Timisoara city (Romania). The study was realized on an availability sample, and as investigation method we used a questionnaire sent to 38 employees. The questionnaire had questions with answers to be chosen, the subjects were insured for their anonymity but were asked to provide information about their sex, age and seniority in the company.

RESULTS AND DISCUSSION The study proposes an analysis of the importance of the employees‘ needs and their satisfaction, motivation and remuneration satisfaction, the work conditions offered by the employers, the quality of the collaboration with the top management, safety at the work place, the compensation of the performances and other motivating factors. For the question: How satisfied you are by the company and the present job? 73% of the questioned employees are satisfied by their job, but 21% are not satisfied because they are overloaded and they want to change the company and only 6% were very satisfied by their job. (fig.1)

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6% 21%

73%

Fig.1. The satisfaction rate related to the job

For the question: Are you satisfied with the relation with your line manager? From figure 2 we can say that 81% of the company‘s employees are satisfied with the relation with their line manager and 19% are not satisfied because they consider that the communication with the line manager is not very good. The dissatisfaction is an indicator of the motivation and when the employees are unsatisfied the company becomes inefficient and the clients which benefit from the company‘s services will notice this and will contact other companies.

19%

81%

Fig.2. The relation between line manager and subordinates (%)

The subjects had to choose elements which stimulate them in their activity. From figure 3 we can notice that 55% of the subjects consider as main motivating element the salary, followed by work conditions (20%) and work safety (19%) and 6% have chosen other benefits (phone, company car, etc).

201 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

6% 19%

55% 20%

Fig.3. Main elements which stimulate the employees

Another question was referring to the employees‘ satisfaction related to the salary. In figure 4 we can notice that 64% of the subjects are satisfied with their salary. The 16% of the unsatisfied employees and the 20% of quite satisfied employees should be taken into account by the company managers for an action plan because the main motivating factor of this study is the salary.

16%

20% 64%

Fig.4. The satisfaction rate related to the salary For the question: How much should increase the salary in order to consider it motivating? 59% of the subjects answered that the salary should increase with 20-50% in order to motivate them, 37% of the subjects answered that it should increase with more than 50% and 4% answered that it should increase with 100%.

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37%

59%

Figure 5 The Percentage of the salary increase

For the question: Do you believe that the managers try to create motivating conditions? 68% of the subjects answered ―yes‖, and 32% consider that the managers don‘t try to create motivating conditions.

50 32

40 % 30

0 yes no

Figure 6 Creation of motivating work conditions (%)

CONCLUSIONS AND FUTURE WORK In conclusion we can state based on the analysis of the answers that 81% of the employees of the company are satisfied with the relation with their line manager and only 19% are unsatisfied because they consider that the communication between them is poor. From this study we noticed that 55% of the subjects consider that the main motivating element is the salary, followed by the work conditions (20%) and work safety (19%) and 6% answered other benefits. The 16% of the unsatisfied employees and the 20% quite satisfied employees should be taken into account by the company managers. The managers should identify the reasons which lead to dissatisfaction and take corrective measures so that the employees should be fully satisfied. In order to increase 203 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA the satisfaction degree of the employees we consider that the managers should take into account also the employees‘ desires and recommendations.

REFERENCES [1]. Novac E. 1995, Factorul uman in conditiile managementului modern, Ed. Mirton Timisoara, [2]. Olaru S. 2005. Managementul intreprinderii, Editura ASE Bucuresti, pp.150-151. [3]. Petrescu I., 1995, Management, Editura Tipocart. [4]. Ruset C. 2006 Managementul resurselor umane, Editura Solness Timisoara. [5]. Ruset C. 2008 Management, Editura Solness Timisoara.

ABOUT THE AUTHORS C. Ruset, Banat‘s University of Agricultural Sciences and Veterinary Medicine from Timisoara, Calea Aradului street 119, Timişoara, Romania, E-mail: [email protected] G. Buzamat, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului street 119, Timişoara, Romania, E-mail: [email protected] G. Popescu, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului street 119, Timişoara, Romania, E-mail: [email protected]. K.I. Lato Banat‘s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului street 119, Timişoara, Romania, E-mail: [email protected] I Cretescu, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului street 119, Timişoara, Romania, E-mail: [email protected]

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THE ANALYSIS OF THE FINANCIAL POSITION IN AN AGRICULTURAL COMPANY FROM ROMANIA

Genoveva Buzamat, Corina Ruset, Elena Pet, Iuliana Cretescu

Abstract: In this study we present the analysis of the financial situation during the period 2009 – 2011 of an agricultural company having as main activity cereal growing. The study presents first of all the main theoretic concepts related to the financial analysis, and later on we present information related to the studied company: turnover evolution, main clients, suppliers, competition position. The purpose of the study is to identify the main problems the company is facing and to find solutions to solve them because the financial analysis of the companies from the market represents the direction followed by the research from the microeconomic domain. Highlighting the factors which contributed to the evolution of the economic phenomena and processes represents a central objective of the management of the companies with direct implications in the business portfolio development of the company. The main aspects analyzed the structure of the balance, the indicators for reliability and performance, the indicators of the financial balance.

Key words: financial position, performance, reliability, financial balance.

INTRODUCTION The evaluation of the financial and economic situation of the companies from the market represents a direction where the research from the microeconomic domain is heading. This means and imposes the usage of some very strict investigation methods in order to highlight the financial and economic stability of the companies. Only in this way it is possible to elaborate a series of long term strategies which direct a certain behavior of the company.

MATERIAL AND METHODS The study was realized in an agricultural company with main activity growing of cereals and for which we calculated the debt ratio, the solvency ratio and profitability ratio, the instruments used for the calculation of these indicators are the financial and accounting balances.

RESULTS AND DISCUSSION After calculating and analyzing the above mentioned indicators we can notice the following aspects: The global debt ratio overpassed the maximum level from 2010 (66.71), because the long and medium term debts increased. In 2011 this ratio reaches 59.04%, this is an favorable situation because part of the long and medium term credits were reimbursed but it does not increase the safety of the company because it did not reach the level of 50%.

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Figure nr 1 The global debt ratio

The term debt rate did not reach the maximum level considered in the analyzed period; this is not a favorable situation for the company because it is not at insolvency risk.

Figure nr 2 The term debt rate

The analysis of liquidity and solvency rates In table 6 we present the liquidity and solvency rates calculated based on the financial balance. Table nr 1 liquidity and solvency rates

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Table nr 1 liquidity and solvency rates Specification 2009 2010 2011 Current liquidity rate 1.12 0.98 0.76 Fast liquidity rate 0.21 0.49 0.32 Immediate liquidity rate 0.02 0.48 0.02 General solvency rate 228.33 149.90 169.37 Patrimony solvency 0.50% 0.03% 0.08%

Current liquidity rate is decreasing during the entire analyzed period. In 2009 the level of the current liquidity rate was 1.1 in the limits of the reference period (1;2); in the period 2010 – 2011 the level of the current liquidity is the reference interval. The company is at risk to accumulate pending payments which generate supplementary costs and reduce the financial results. The fast liquidity rate is below the reference interval during the entire analyzed period (0.6;1) and reflects a slow coverage of the production process, specific for the agricultural companies. The immediate liquidity rate was in the reference interval (0.2; 0.6). In 2009 and 2011 this was far below the level of the reference interval, registering significant decreases compared to 2011 due to the income and payment flow. The general solvency rate was above the reference level (150%) during the entire analyzed period, reflecting the fact that the total debts are covered by the total actives in case of bankruptcy. The patrimony solvency rate was below the reference level during the entire analyzed period (0.4; 0.6) due to the fact that the company maintained its social capital at the minimum level mentioned by the law. The analysis of the profitability rates The economic profitability calculated based on the gross operating surplus and on the invested capitals and presented in the below figure reached a level of 30.27% in 2009, which is considered a good percentage, the company had a good capacity to renew this capital. In the following two years this decreased to 2.68% in 2010 due to the decrease of the gross operating surplus and in 2011 reaches a level of 14.09%.

Figure nr 3 economic profitability

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The financial profitability presented in figure 4, calculated based on the net profit and the invested capital, reached the level of 44.91% in 2009 and decreases to 6.4% due to the decrease of the net profit in 2010 and increases to 30.94% in 2011.

Figure nr 4 financial profitability

Table nr 2 SWOT analysis Strengths Weaknesses Liquidity below the reference During the entire analyzed level period the company had profit Patrimony solvency below the reference level Global debt rate below 50%, does not provide increased safety Opportunities Threats Possibilities to access structural Natural factors funds Juridical factors related to grant Possibilities of downstream subventions for agriculture integration Strong competition from foreign Possibilities to refocus on bio companies products

CONCLUSIONS AND FUTURE WORK The agricultural company registered profit during the entire analyzed period. The global debt rate exceeded the maximum level considered in 2010 (66.71%) because of the long and medium term debts increase. In 2011 reaches the level of 59.04%, representing a favorable situation due to the fact that part of the long and medium term credits were reimbursed but does not provide an increased safety for the company because it did not reach 50%. The economic profitability calculated based on the gross operating surplus and on the invested capitals and presented in the below figure reached a level of 30.27% in 2009, which is considered a good percentage, the company had a good capacity to renew this

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capital. In the following two years this decreased to 2.68% in 2010 due to the decrease of the gross operating surplus and in 2011 reaches a level of 14.09%.

REFERENCES [1]. Alexander, D.,Nobes,1999 C Fianacial Acountig an International Introduction Pearson Limited pp. 50-52 [2]. Herbei, M., H., Mazaru Luminița 2001 Gestiunea financiară întreprinderilor Ed. Economică București, pp 122-123 [3]. Niculescu Martia 2005 diagnostic financiar Ed Economică București, pp. 186-187 [4]. Popa L.,I., Miculeac Melania Elena 2009 Analiză economicco financiară Ed.Mirton timișoara pp. 274-275

ABOUT THE AUTHORS G. Buzamat, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului street 119, Timişoara, Romania,E-mail: [email protected] C. Ruset, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului street 119, Timişoara, Romania,E-mail: [email protected] E. Pet Banat‘s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului street 119, Timişoara, Romania,E-mail [email protected] Cretescu, Banat‘s University of Agricultural Sciences and Veterinary Medicine, Calea Aradului street 119, Timişoara, Romania,E-mail: [email protected]

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THE PROBLEM OF SYNTACTIC STRUCTURES IN CONTEMPORARY LINGUISTICS

L.Ramazanova

Abstract: Though linguists have already described the traditional grammar of most languages, structural linguistics, with its descriptive inclination, still brings up an issue concerning syntactic structures of a language. This research is an attempt to deal with syntactic structures of Indo-European languages of various branches (the English and Russian languages) as opposed to semantics. The paper examines the concept of syntactic complexity and semantics on the basis of the works of some eminent linguists (Noam Chomsky, Teun A. van Dijk, Wallace Chafe, etc.) and tries to clarify whether semantics or syntactic structures should be considered the main component of a language. Key words: Semantics, Syntactic Structures, Word Order.

INTRODUCTION Linguists have gathered a lot of empiric material of facts and observations about a language so far. Despite this, traditional grammar and structural linguistics of many languages still remain on the level of mere classifications. In his work ‗Syntactic Structures‘, Noam Chomsky tried to change the situation suggesting that the main problem of linguistics is not in the lack of data and facts but quite opposite. Linguists cannot cope with that great number of facts and simply continue to describe a language instead of explaining and arranging the accumulated information. According to Noam Chomsky, generative grammar neither analyses nor describes expressions but, like logic- mathematical calculi, generates all possible grammatical sentences of a language. The main attention in the linguist‘s work was paid to the formal correctness of an expression rather than its notion and validity. Noam Chomsky tried to prove that language behaviour is innate, not learned. He strongly believed that all languages share the same underlying grammatical base. However, Wallace Chafe, an American linguist, casts doubts on Chomskian generative grammar. Supporting a cognitive approach to a language, he considers semantics to be its main component. Since then, over the past 25 years, a lot of linguists have displayed a growing interest for syntactic structures and the idea that they carry meanings independent of the meanings of specific words. The object of this work is to generalize the points of view of some linguists working on the language structure and semantics and to try to clarify whether these two conceptions coexist. The expressions of the English and Russian languages are the basis of the work. Before going over to the factual research into syntactic structures and the meaning they carry, it is necessary to give a preliminary typological description of the languages abovementioned: 1) The English language is mainly analytic, and, unlike most other Indo-European languages, has developed features such as modal verbs and word order as resources for conveying meaning. Auxiliary verbs mark constructions such as questions, negative polarity, the passive voice and progressive aspect. 2) The Russian language as a synthetic language has preserved an Indo-European synthetic-inflectional structure, although considerable changes have taken place. Morphology in the Russian language is more important than syntax.

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MATERIAL AND METHODS Both of the languages abovementioned belong to the same family (Indo-European languages) though to different branches/groups (the English language- the West Germanic branch, the Russian language – the East Slavonic branch). So, syntactic structures and semantics of the two languages are expected to share similar features (if not the same) with slight divergences. However, in should be noted that by ‗syntactic structures‘ large units of syntax are considered, mostly word order in a sentence. The word order in a sentence suggests a basic order in which a sentence mainly consists of the subject, the verb, and objects. In ‗Syntactic Structures‘, Noam Chomsky analyzed parts of the sentence and showed how complex and compound sentences can be formed from simple ones. After introducing the conception of ‗deep structure‘ and ‗surface structure‘, the linguist noted that they also change syntactic relations in a sentence in accordance with the rules of a language. As the current research was conducted with the use of comparative, contrastive, and descriptive methods of linguistics, I would like to demonstrate how the basic word order can be presented in the English language and the Russian language. As is generally known languages can be classified into six types according to the order of words in a sentence: SVO , SOV, VSO , VOS, OSV, OVS. We know that both English and Russian are referred to the SVO type when it concerns a simple declarative sentence. However, see the differences:

Russian English SVO — Subject Verb Object Саша читает книгу Sasha reads a book. SOV — Subject Object Verb Саша книгу читает ----- VSO — Verb Subject Object Читает Саша книгу ----- VOS — Verb Object Subject Читает книгу Саша ----- OSV — Object Subject Verb Книгу Саша читает ----- OVS — Object Verb Subject Книгу читает Саша -----

As we can observe it, a simple declarative sentence in the Russian language can be referred to all the six types of word orders whereas a simple declarative sentence in the English language can be presented only in SVO. Though, we should always remember that there are no pure analytic or synthetic languages, I would like to explain this fact by language typology. So, Russian, as a synthetic language, forms words by affixing a given number of dependent morphemes to a root morpheme. Here word order is less important as individual words express the grammatical relations. In the Russian language a high degree of concordance (agreement between different parts of the sentence) is essential. Consequently, morphology in Russian, as well as in all synthetic languages, is more important than syntax. However, English, as probably one of the most analytic of Indo- European languages, shows a low ratio of morphemes to words. Here sentences are composed of independent root morphemes. Grammatical categories are mainly indicated by word order. So, in the English language, context and syntax are more important than morphology. According to Chomskian generative grammar, both the following Russian sentences- Саша обучает Анну - Анна обучает Сашу - and their English equivalents - Sasha teaches Anna – Anna teaches Sasha - have the same lexical elements but different meaning. However, the sentences Саша обучает Анну and Анна обучаема Сашей / Sasha teaches Anna and Anna is taught by Sasha have the same meaning but different grammar forms. Here it should be noted that the Russian version Анна обучаема Сашей (Anna is taught by Sasha) is less usual in the Russian spoken language as there is a more 211 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA convenient form with the same meaning: Анну обучает Саша (OVS), which can be formed with the help of morphemes. The creation of generative grammar by Chomsky lead to the strengthening of principles of rationalism in linguistics. Since then generative grammar has been undergoing considerable evolution. As Chomsky believed that grammar should be considered independent from semantics, his researches provoked disputes among linguists over the interrelation between semantics and syntax. For example, the semantic- syntactic theory proposed by Wallace Chafe in his work «Meaning and the Structure of Language» received wide publicity. Chafe named his conception a model of language competence. He didn‘t object to the differentiation between language competence and its performance but, unlike Chomsky, Chafe disapproved of the strict separation of the theory of competence from researches in the usage of language units. Chafe believed that linguistic conception should describe the real process of expression production. In this case, as ideas are first born, first semantic structures are created and the ways to combine them into a large scale of different configurations. Let‘s see how ‗ ideas‘ can form syntactic structures on the level of mere expressions highly used in the Russian language :

For example, if a person is offered a cup of tea, the possible answers may be:

(Question) - Хочешь чаю? - Wanna a tea?

(Answers) - Да, давай! -Yeah! - Не (нет). -Nope. - Да нет наверное. -Nope.

Here the third Russian version suggests the following English translation (if word for word): ‗Yes no probably‘. I would like to note that the change of word order in the third Russian expression can construct new variations of the same meaning: - Да нет (Yes no) – surely no; - Нет да (No yes) – No (used in arguments); Да нет + наверное (Yes no + probably) ‗probably no‘ - No. A question can be brought up whether a syntactic structure or a semantic structure makes the three expressions highly popular among Russian speakers in the presented word order. However, it should also be mentioned that in the Russian language, they pay a special attention to the intonation used during the conversation. So, disregarding the word order in all the three expressions, we can surely say that ‗no‘ is always accented. However, according to Chafe, any sentence can be formed around a ‗verb‘ which is the main component of a sentence as it defines the presence and character of a ‗noun‘. The linguist believes that ‗verb‘ is present in almost all (if not all) marginal expressions of a language. So, Chafe suggests six verb groups: 1- state, 2- process, 3- action, 4- process- action, 5-ambient state, 6- ambient action. Here are some examples:

English Russian State: The wood is dry. Дрова сухие. Process: The wood dried. Дрова сохли. Action: She sang. Она пела. Process-action: He dried the wood. Он сушил дрова. Ambient state: It‘s warm. Тепло. Ambient action: It‘s snowing. Идет снег.

As we see, in all the sentences, both English and Russian, ‗verb‘ is present. In Russian ‗State‘ and ‗Ambient state‘, the presence of verb ‗to be‘ is implied. However:

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English Russian What a beautiful day! Какой чудесный день! How nice! Как славно! Well, what about it? Ладно, что насчет этого? So long! Пока!

Now we can say that ‗verb‘ can be omitted (but still implied) in both English and Russian in ‗State‘ and ‗Ambient state‘ groups. In his work ‗Discourse and Context‘, Van Dijk T.A. introduced a notion of ‗contextual models‘ which reflect relevant properties of the communicative environment in episodic memory and control discourse formation and comprehension. According to the author, contextual models contain a set of categories common in every culture and universal data (space-time, aims, participants, actions). Otherwise, people could hardly understand difficult social situations. At the same time, these contexts are socially anchored and consider the ideology, knowledge, grammar, rules, norms, values, etc. of a definite discursive community. According to Van Dijk‘s conception, contextual models are not semantic but pragmatic and control the way to adapt an expression to a communicative situation via a communicant‘s subjective interpretation of the social environment. So, with the theory of social structures and linguistic theories of discourse and language structures the relations between semantics and syntax can be explained. Let‘s imagine a situation in an office with a broken photocopier. The worker has to inform his boss/co-worker/friend about the failure. The worker is expected to use three different expressions depending on the person he will talk to (English/Russian):

(Formal to his boss)- The photocopier is malfunctioning again. / Фотокопировальная машина вновь неисправно работает. (Relaxed to his co-worker)- The photocopier’s broken down again./Ксерокс опять сломался. (Informal to his friend)- The photocopier’s on the blink again. / Ксерокс опять глючит.

As it is seen in the given examples, the same ideas can be formed in different syntactic constructions depending on such conditions as environment, culture, personal relations, etc. In a less formal environment not only vocabulary but also syntactic structures are simplified. It can be explained by the fact that while speaking people try to deliver as much information as they can and economize their time on complicated syntactic structures even selecting idiomatic expressions for a better clarity among the members of their environment. Moreover, in case of a misunderstanding that may occur during the conversation, interlocutors can clarify the context immediately. As it is inappropriate to lead a listener/interlocutor to a misunderstanding during formal conversations, negotiations, and so on, it is demanded that the necessary syntactic structures be used with no omissions, shortenings, etc., which may even define a speaker‘s linguistic style. However, it remains unclear how situational communicative models interact with the situations they were formed in. It is believed that contextual models are related with a number of objective restrictions.

RESULTS AND DISCUSSION Generalizing the notions of syntactic structures and semantics of a language it can be said that their interrelations mainly depend on the morphological characteristics of a language. So, if a language is considered analytical, the rules of syntactic structures are strict: every part of sentence is assigned its own place, which is different in, for example, 213 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA synthetic languages. So, word order on English is of much greater importance than in Russian. Due to a big number of inflexions, word order in Russian is rather free as the inflexions indicate the function of each word in a sentence. As English words have hardly any inflexions and their relation to each other is shown by their place in the sentence and not by their form, word order in English is fixed. The position of different parts of the sentence cannot be changed at will, especially that of the subject and the object. For example, in the English language, a simple declarative sentence has the following word order: the subject, the verb, objects, and adverbial modifiers. However, unlike Russian sentences, certain types of English sentences require the inverted order of words (verb- subject): English Russian Is he at home today? Он сегодня дома? There is nothing I can do about it. Я не могу ничего с этим поделать. Hardly had he come in when this happened. Только он вошѐл, как это случилось Etc.

In the English language, the inverted order of words is also widely used when a word or a group of words is put in a prominent position for stylistic purposes. In this case inversion is not due to the sentence structure but to the speaker‘s or writer‘s desire to produce a certain stylistic effect. However, in the Russian language, the same stylistic effect can be produced either by means of rising and falling intonation or inversion (the accented part of a sentence is placed first).

CONCLUSIONS AND FUTURE WORK It can be concluded that syntactic structures can really carry meanings and reflect linguistic styles. Such meaningful structures are frequently helpful, sometimes even indispensable, in the written form of a language. For example, in the English language, emphasis may be reached by the prominent position of some part of the sentence with or without inversion (without placing the verb before the subject). For example: So it’s you that phoned me last night. It is not in your sister that he is interested. In his works, Noam Chomsky tried to create a set of universal grammar principles as he believed that languages should be learnable. So, in a variety of languages of different typologies, the importance to study and classify syntactic structures cannot be belittled. However, it must be borne in mind that in many cases syntactic structures are not only to present a set of words but also to show the attitude of a speaker or a writer to the shared information.

REFERENCES

[1]. Edward Kako and Laura Wagner 2001. Trends In Cognitive Sciences Vol.5 No.3. The semantics of syntactic structures.pp102-108. [2]. Noam Chomsky 2002. Syntactic Structures. [3]. Van Dijk T. 2008. Discourse and Context. A Sociocognitive Approach. [4]. Wallace Chafe 2008. Aspects of Discourse Analysis. [5]. Wallace Chafe 1970. Meaning and the Structure of Language.

ABOUT THE AUTHORS Liana Ramazanova, PhD, Azerbaijan National Academy of Sciences (AzNAS), Baku European Lyceum, 1073, 5 M.Arif, Baku, Azerbaijan, E-mail [email protected]

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ENGLISH FOR SPECIFIC PURPOSES IN CAREER OPPORTUNITIES

Rodica Silvia STAN

Abstract. The constant progress of international communication in various fields and the globalization of economy involve career-specific terminology in order to coordinate research and to deliver professional services. This is why the demand for English for Specific Purposes (ESP) is expanding in technical universities. The current article points out that ESP should be taught as a subject matter important to students, integrated in their real professional world. It will enhance the students' interest in their domain and will motivate them in their professional work. Key words: Globalization, Career, Analysis, Terminology, Interaction, Communication

INTRODUCTION The development of global economy involves a wide landscape of career opportunities and implies an increasing number of well-prepared professionals. Students and specialists have to become proficient in a foreign language, usually English, in order to meet a predictable range of communicative needs. Knight, Lomperis, van Naerssen & Westerfield [10] divide language learners who need ESP into two categories: 1. Language learners who are in the process of developing expertise in their fields need English communication skills as tools in their training. 2. Language learners who are already experts in their fields need English communication skills as tools in their work. Most students of technical universities are aware of the fact that they should become successful members of the global workforce. The best environment to achieve this purpose is the language course offered by the universities the students belong to. In most European universities this course starts in the first year, but the difference consists in its duration: one to four years.

MATERIAL AND METHODS The design of an ESP is usually based on the specific needs of learners of a particular discipline. There are various purpose-oriented ESP courses, such as EST (English for Science and Technology), EAP (English for Academic Purposes), EBP (English for Business Purposes), EMP (English for Medical Purposes), EOP (English for Occupational Purposes). In designing ESP courses, several authors have outlined the way of conducting needs analyses to ascertain the learners' target needs and learning needs, which are then integrated into the syllabus. In the 1980s, the basic characteristics of ESP, taken into consideration by most ESP designers, were as follows:

- conceived to meet specified needs of the learner; - related in content to particular disciplines, occupations and activities; - focused on the language appropriate to those activities in syntax, lexis, discourse, semantics, and analysis of this discourse; - in contrast with General English [14] In the 1990s the definition of the variable characteristics of ESP was modified: " ESP may be related to or designed for specific disciplines"; "ESP may use, in specific teaching situations, a different methodology from that of General English"; "ESP is likely to be designed for adult learners, either at a tertiary level institution or in a professional work situation. It could, however, be for learners at secondary school 215 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA level"; "ESP is generally designed for intermediate or advanced students". [3] Consequently, a change can be noticed in how the range of ESP has been extended and has become more flexible in its definition. ESP course designers of the 1990s have removed the absolute characteristic that 'ESP is in contrast with General English' and added more variable characteristics. They assert that ESP is not necessarily related to a certain area. According to L. Anthony, it is not clear where General English courses end and ESP courses begin; numerous non-specialist ESL instructors use an ESP approach in which their syllabi are based on analyses of learners' needs and their own personal, specialist knowledge of using English for real communication. Many ESP programs are examples of such curriculum development and course design [1]. At the beginning of our millennium, Gatehouse integrates General English language content and acquisition skills when developing the curriculum for health sciences (2001). Another ESP instructor, T. H. Xenodohidis states that "the goals should be realistic, otherwise the students would be de- motivated" [15]. The integration of a considerable amount of General English along with the necessary terminology into the syllabi is based on the needs assessment from students, who consider it realistic. In order to face an ESP course the students need a previous course of General English. The ESP course should be designed in such a way as to integrate the subject matter area important to the students' real concerns. General English and ESP diverge both in the nature of the learner and in the aim of instruction. As a general rule, in General English all four language skills: listening, reading, speaking, and writing are stressed equally, while in ESP it is the needs analysis that indicates which language skills are most needed by the students, and the syllabus is designed accordingly. An ESP course might, for example, emphasize the development of reading and writing skills in students who are preparing to graduate from management or business administration, or it might promote the development of speaking skills in students who are preparing to work in public relations. Joining General English and subject matter is highly motivating within an ESP course because students, whatever their main field of study is ( engineering, agriculture, medicine, accounting, business management, economics, computer science, tourism), are able to apply what they learn in their specialty courses to their English classes. Being able to use lexical items and grammatical structures correctly, they can both understand and create meaningful contexts. Thus, their motivation increases and the students become more and more confident.

RESULTS AND DISCUSSION The design of an ESP course is usually based on the specific needs of learners of a particular discipline. The designers of the course conduct needs analyses to ascertain the students' target needs and learning needs and then integrate the required linguistic elements and skills into the syllabus. The pre-course needs analyses are used to obtain information and data concerning the need for communication skills in order to give directions for the course design and syllabus development. Questionnaires are given to students in order to gain insights into their perceived needs, preferences and gaps. Pre- course tests for reading, speaking, and listening are taken by all the potential course participants every year before the language course begins. The results of the tests show the instructors can establish the language level of the students, which is helpful in the design of the course. In view of the needs of students, ESP courses should be designed to provide students with opportunities and resources to pursue their interest in communication skills in English; to improve their performances in the classroom; to prepare themselves for their business internships and careers. In our opinion, by the end

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of the ESP course the students should be able to: communicate with foreign peers; read professional literature, academic journals; write technical and academic articles; attend lectures, conferences, symposia; perform technical or business negotiation in English; read and write instructions; visit and receive foreign peers. The students usually have a language background when they proceed to ESP study. Most of them studied English for 10 or 11 years in primary, secondary and high school. Except for having learnt English for at least 10 years at school some of the students had working experience abroad for several years. An ESP course is therefore designed for intermediate or advanced level. Romanian universities generally include the language course in the first or in the first two years of Bachelor level. Students are organized in groups of 20-25 and are aged from 18 to 21. Since ESP is an approach to language teaching in which all decisions as to content and methodology are based on the student's rationale for learning, the role of the ESP instructor is essential to the success of the ESP program. In terms of their subject-matter specialties the ESP instructor conceives a wide variety of questionnaires. Minimum standards must be established in the areas of language level, motivation, and prior education and experience. Most importantly, these standards must be strictly enforced at the time of placement. If the number of students allows more groups of the same level, the best method is to work with homogeneous groups. First year students have to learn both the language and the content at the same time, thus, most first year groups are homogeneous. Second year students, on the other hand, possess some professional knowledge, but it often happens that some of them struggle to catch up with the general language proficiency and find the content activities to be overwhelming. Tests and questionnaires are given to students in order to gain insights into their perceived needs, wishes, preferences, and gaps. Alongside these, information was sought about their professional areas, work experience, personal background and other relevant details that would play some role in shaping the program. Members of the teaching staff, administrative personnel (workers, technicians who perform activities in the laboratories, workshops or greenhouses where the students will do their placement for the practice period), from whom we can determine the objective communication needs, are also interviewed. The tests include the cheching of listening, reading and speaking skills and are taken by all the potential course participants every year before the language course begins. From the results of the tests, we can ascertain the language level of the learners, which is helpful in course design and materials selection. The next step is to connect general English with technical English. Several courses are organized with the language input from general to specific, taking into consideration all the skills. For example, in order to check the speaking abilities, topics are sequenced in three stages. In the first stage, general topics are used, such as personal data, nature, easy conversation, and story telling. The second stage progresses towards covering general topics, such as talking about Romanian and European manners, old and modern technology, EU standards. The third stage of topic selection includes designing projects and business presentations. Since the last stage assignment involves professional areas, the students' needs of terminology, of technical English varies greatly so that the 'common core or nucleus' (Robinson 1991) of special English has to be taught in such a way as to break the gap between general and technical English. In order to accomplish all these the appropriate materials should either be written or addapted. Hutchinson and Waters [8] assert that there are three possible ways of applying materials: using existing materials, writing materials and adapting materials. According to Robinson, [12] these come in the form of textbooks and in-house materials. Taking into consideration the aims and objectives of the university the students belong to, the course

217 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA design and course syllabi, all the types of materials - published textbooks, tailor-made materials and written materials – are usually employed in the program. Published textbooks have the advantages of saving time, costing less than in-house materials, having greater availability. There is insufficient time and there are little human resources to write all the necessary textbooks for all the sections who need an ESP course. Consequently, some courses are taught with published textbooks to exploit the syllabus objectives. In order to meet the students' needs and expectations, the selection of textbooks takes into consideration the following ideas: the selection of textbooks with appropriate contents relevant to learners' level of English and professional interests; avoidance of extremely difficult textbooks because students would be confrunded with difficult language added to the complexity of terminology, and, consequently lose interest in the text; paying attention to both verbal and visual information of the textbooks. Alongside published textbooks, written textbooks are also used; such materials are more specific for unique learning situations, and for more suitable methodology for the intended students. However, they are more expensive and time consuming to produce than published textbooks. Besides, they will appear more difficult to deal with in both contents and language if the authentic materials are not carefully or cautiously organized, or if the data are not properly collected. These integrated materials proved to be successful in terms of assisting the students' professional needs and linguistic development.

CONCLUSIONS AND FUTURE WORK The endeavours to conceive an ESP project proved to be successful both on campus and at future workplaces. This research led to several conclusions: course designers of any ESP program should firstly explore the potential students' actual needs, wishes, gaps, and requirements. The course designers should help students identify their learning needs since sometimes the learners do not have a clear picture of their own capacity; though ESP students are usually learners of the intermediate or advanced level and in specific disciplines, they still need to improve their knowledge of general English. The ESP course provides various types of support to meet the learner‘s specific, identified needs and objectives. The modules conceived within the course provide learning opportunities, which address the needs of students that cannot always be met in the classroom. The ESP approach enhances the students' interest in their domain and motivates them to use professional publications in English, to interact with specialists from other countries, to get involved in professional international events and to express themselves freely and correctly in English.

REFERENCES [1]. Anthony, L. 1997. ESP: What does it mean? ON CUE. http://interserver.miyazaki- med.ac.jp/~cue/pc/anthony.htm [2]. Chamot, A. U. , Barnhardt, S., El-Dinary, P. B. and Robbins, J. 1999. The Learning Strategies Handbook. New York: Longman. [3]. Dudley-Evans, T., and St John, M. 1998. Developments in ESP: A multi-disciplinary approach. Cambridge: Cambridge University Press. [4]. Falchikov, N. 2001. Learning together: Peer tutoring in higher education. London, UK: RoutledgeFalmer [5]. Good, T.L., & Brophy, J. 1995. Contemporary educational psychology. White Plains, NY: Longman. [6]. Holliday, A. 1995. Assessing Language Needs Within An Institutional Context: An Ethnographic Approach. English for Specific Purposes, 14

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[7]. Holme, R. 1996. ESP Ideas. London: Longman. [8]. Hutchinson, T. and A. Waters. 1987. English for Specific Purposes: A Learning- centred approach. Cambridge: Cambridge University Press. [9]. Johns, Ann M. & Dudley-Evans, Tony. 1991. English for Specific Purposes: International in Scope, Specific in Purpose. TESOL Quarterly 25:2, 297-314. [10]. Knight, K., Lomperis, A., van Naerssen, M. & Westerfield, K. 2010. English for Specific Purposes: An Overview for Practitioners and Clients (Academic and Corporate). PowerPoint presentation submitted to Alexandria, Virginia: TESOL Resource Center. Retrieved at http://www.tesol.org/s_tesol/trc/uploads/Other/119485/1564_Knight_ESPPPTforTRC. pdf [11]. Liu, L. 2010. Conversations on leadership: Wisdom from global management gurus. San Francisco, CA: Jossey-Bass. [12]. Robinson, P. 1991. ESP Today: A Practitioner's Guide. Hemel Hempstead: Prentice Hall [13]. Strevens, P. 1978. New Orientations in the Teaching of English. Oxford: OUP [14]. Strevens, P. 1988. ESP after twenty years: A re-appraisal. In M. Tickoo (Ed.), ESP: State of the Art (pp1-13). Singapore:SEAMEO Regional Centre [15]. Xenodohidis, T. H. 2002. An ESP Curriculum for Greek EFL Students of Computing: A New Approach. ESP World, Issue 2, Vol. 1. http://www.esp- world.info/index.html

ABOUT THE AUTHOR Rodica Silvia Stan, Professor of English for Specific Purposes, University of Agricultural Sciences and Veterinary Medicine, 3-5 Mănăştur Street, 400372 Cluj Napoca, Romania, phone:+40 264 596384; E-mail: [email protected]

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OPPORTUNITIES AND PROSPECTS OF IMPROVEMENT OF DIFFERENTIATION OF LANDS IN UKRAINE

A.N. Lychogrud

Abstract: Data of the analysis of patent development condition are provided in a land surveying. The prospect of protection of development on delimitation of lands on state and municipal in Ukraine is shown by patents for inventions. The characteristic of patent development is provided in this area. Key words: Invention, Land Management, Land Surveying.

INTRODUCTION Works on differentiation of lands on state and municipal in Ukraine became today very actual, but their implementation is connected with a number of problems. Among them the special place is taken by the problems caused by lack of experience of work on differentiation, imperfection of methodical and legislative base, imperfection of processing methods and ways of work [1, 2, 3]. Foreign experience in a certain measure can provide progress in this area. But the main changes need to be carried out by improvement of the process of differentiation of lands in Ukraine. Thus it is necessary to pay much attention to improvement of methodical and technological making works on differentiation of lands, increase of efficiency of works, first of all, by increase of accuracy of land use planning works. In this direction to the most rational the effort on development of new ways of a land surveying, more effective from the point of view of productivity and accuracy of performance of work [4, 5] is. One of the most perspective directions of improvement is development of new ways of the land surveying which purpose consists in reduction of expenses of time and funds for implementation of works on a land surveying of the land plots. This direction has big patent prospect, and new ways of a land surveying can be applied in the course of performance of work on delimitation of lands both in Ukraine, and behind its borders.

MATERIAL AND METHODS Object of researches are the land plots which are subject to differentiation on state and municipal according to decisions of the Verkhovna Rada and the Government of the country about differentiation of lands [6]. Research problems consist in optimization of works on differentiation of lands in Ukraine by technology improvement, including by development of new methodical and technical solutions. The program of researches includes consideration of the questions concerning directly development of technical solutions at the level of possible inventions, the works directed on improvement on differentiation of lands. In work are used standard in land management, geodesy and cartography of a technique of researches [3, 5]. Performance of work was preceded by patent search on depth till 50 years on the main states patentees.

RESULTS AND DISCUSSION The results of the conducted patent researches which have captured more than 600 patented in geodesy, cartography and land management of decisions showed that for today from the point of view of possible patenting the whole segment of land management concerning first of all ways of work practically isn't considered. Thus, a number of paramount questions and level of relevance of possible new development is established. Their number includes questions of rational use of opportunities of geodetic devices and computer facilities, optimization of a configuration of the land plots, optimization of

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sequence of performance of land use planning works and a number of other not less important questions. For today practically all these questions aren't worked sufficiently from the point of view of patenting that opens great opportunities for patenting and the subsequent use of inventions. All this defined the direction of the researches conducted by us by results of which three decisions at the level of possible inventions for today are developed. Decisions are directed first of all on improvement of differentiation of lands in Ukraine by optimization of works and reduction of material inputs and costs of time of work. First of all the question concerning optimization of performance of work on differentiation of lands on state and municipal was considered. It was developed at the level of the possible invention and "The way of a land surveying of the land plot" which use can reduce at a repeated land surveying of the land plot costs of work of 40-60%, and of 15-60% - funds for work on differentiation of lands (Table 1, 2) is given on patenting.

Table 1. Costs of time of a land surveying of the land plot. The area is 34,7 hectares Method Works Expenses of time, hour preparatory work 13,0 establishment and fixing of boundary signs 12,0 Basic measurements 12,0

processing of field materials 14,0

drawing up the cadastral plan 44,0 total 95,0 Stated preparatory work 13,0 establishment and fixing of boundary signs 12,0 measurements 22,0 processing of field materials 16,0 drawing up the cadastral plan 46,0 total 109,0

Table 2. Costs of time of a repeated land surveying of the land plot on condition of change of its area and a configuration Method Works Expenses of time, hour preparatory work 13,0 establishment and fixing of boundary signs 12,0 Basic measurements 12,0

processing of field materials 14,0 drawing up the cadastral plan 44,0 total 95,0 preparatory work 8,0 establishment and fixing of boundary signs 4,0 Stated measurements 5,0 processing of field materials 5,0 drawing up the cadastral plan 26,0 total 48,0

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The essence of development consists in possibility of a repeated reuse of materials of geodetic shooting of the land plots divided into making parts provided that these making parts didn't change the area and a configuration. Retake carry out only for making parts of the land plot, the configuration and which area changed. Such approach allows quickly and at the minimum material inputs and expenses of work to carry out land use planning works at change of a configuration of the land plots. When carrying out researches also there was a question of combination of materials of the geodetic shooting executed with a different accuracy by different geodetic devices. For today it is one of the most difficult questions of a land management [7]. Here the only decision orientation to the minimum accuracy of measurements can with be greatest possible exact correction of external points of geocontours and their binding to a geodetic network. This question demands carrying out separate in-depth studies and is beyond the tasks set in the real work. However, taking into account these difficulties probably considerable reduction of material inputs and time expenses when using the proposed solution. There is open also a question of a binding of the land plot to a geodetic network on what the accuracy of establishment of external borders of the land plot substantially depends. For today it too one of difficult questions of modern land management of Ukraine [3, 4]. However, the most difficult question is the question of financing of works on differentiation of lands on state and municipal. Proceeding from it, the proposed solution allows to use more rationally financing of works on differentiation of lands and to accelerate their carrying out. Production check of a way, showed its efficiency when performing repeated works on a land surveying of the land plots. Application in practice of development allows to save considerably time for work, and it is essential to reduce expenses. It is necessary to pay attention also that the most rational is simultaneous use of a way at delimitation of the next land plots that can reduce considerably costs of time of establishment of external borders of sites and is considerable increase work accuracy. That is, simultaneous performance of work within territorial and administrative units is desirable. It can cut down twice at least expenses on establishment and fixing of external borders of the land plots that together with economy at a repeated land surveying land a site significantly reduces the cost of performance of work on delimitation of lands. Besides, for today are developed as well other ways of a land surveying of the land plots which pass for today production check and will be presented after pronouncement of the conclusion about their practical suitability. This development is also directed on improvement of a land surveying of the land plots and devoted to a problem of increase of efficiency of work on differentiation of lands on state and municipal in Ukraine.

CONCLUSIONS AND RECOMMENDATIONS Results of the conducted researches showed that the direction of the works connected with differentiation of lands on state and municipal for today practically isn't protected by patents for inventions. It represents undoubted interest and for today is actual. By results of patent researches development and protection of a significant amount of inventions is possible. As for data directly provided in article then it is possible to draw the following conclusions: - developed "The way of a land surveying of the land plot" allows at a repeated land surveying on condition of change of borders and the area of the land plot to reduce costs of work of 40-60%, and of 15-60% - funds for work on delimitation of lands; - efficiency of use of development is reached by division of the land plot into components that allows to carry out geodetic shooting at a repeated land surveying only in parts with a changed area and a configuration; - use of development most rationally when carrying out complex land management

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within administrative units that allows to save in addition on allocation and fixing of external borders of the land plots.

REFERENCES [1]. Martin A.G., Pidruchniy A.Sh. 2008. Differentiating of state and municipal property: organizationally methodical mechanism. Organization of the use of land and cadastre, № 3. pp. 26-36. [2]. Sulin М.А. Land management. 2005. SPb.: Lan‘, 448 p. [3]. Tretyak A.M. Scientific bases of land management. 2002. Кyiv.: CLRU, 342 p. [4]. Tretyak A.M., Drugak V.М., Tretyak R.А. 2004. Interadministrative-territorial formations. Кyiv.: CLRU, 85 p. [5]. Shevchenko T.G., Moroz О.І., Trevogo І.S. 2006. Geodetic devices. Lviv: Lviv polyequipment, 464 p. [6]. The Land Code of Ukraine. 25.10.2001 р. № 2768-III. – Visnyk VR Ukraine, 25.01.2002 р. № 3. [7]. Perepylytsya М. 2009. How to overcome what brakes to establishment of limits of settlements? Zemleystr. visnyk, № 2. pp.42-43.

ABOUT THE AUTHOR

A.N. Lychogrud Kyiv National University of Construction and Architecture

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Plenary paper

USAGE OF RENEWABLE ENERGY RESOURCES IN SERBIA

Larisa Jovanoviš

Abstract: Serbia is rich in renewable energy resources: solar, wind, biomass and geothermal energy. Energy Policy of the Republic of Serbia is fully based on the principles of sustainable development. This paper reviews the specifics of energy policy in Serbia and possibility of renewable energy usage. Key words: renewable energy sources, energy policy, biomass, solar energy, wind energy, geothermal energy, Republic of Serbia

INTRODUCTION All countries have the difficult problem: how to reduce the use of fossil fuels and emission of harmful gases - causing greenhouse effect, global warming, climate changes, and the occurrence of acid rain, reducing the ozone layer, natural disasters and in addition keep the level of technological and economic development. The only way is to decrease the use of fossil fuels and to develop technology of renewable energy sources. Replacing fossil fuels with renewable sources has a major impact on the successful solutions of global environmental problems. Today, usage of renewable resources is becoming more and more important, especially for the reasons of environment preservation and usage of resources already available on own territory. The potentials for the use of renewable energy sources (RES) are high in the Republic of Serbia and estimated to be over 4,5 mln. tonnes of oil equivalent (toe) by year. From biomass is obtained 63%, from solar energy – 14%, from hydroenergy – 14%, from wind energy – 5%, from geothermal energy 4% [7]. With the ratification of the Energy Community of Southeast Europe countries (14th July 2006) Republic of Serbia, among other things, accepted the obligation to adopt and implement a plan of applying the Directive 2001/77/EC about promoting the production of electrical energy from renewable energy sources. In September 2008, the European Parliament adopted a package of legislation on climate change which aims to provide a reduction in emissions of greenhouse gases by 20%, improving energy efficiency by 20% and share of renewable energy from 20% in total energy consumption in the EU - till 2020, in comparison with 1990. The new Directive 2009/28/EC on renewable energy sets binding national targets which the members of the European Union need to achieve through the promotion of renewable energy in the sectors of electrical energy, heating and cooling and in the transport sector, to ensure that by 2020 the renewable energy is at least 20% of total energy consumption in the European Union. Member countries are obliged adopt, publish and inform the European Commission about the National Action Plans for the accomplishment of the instructed goals and to ensure that procedures for approval, certificates and permits are simplified in order to clear obstacles for developing a market for renewable energy. The aim of the Republic of Serbia, by the end of 2020, is to increase the participation of energy produced from renewable resources by 20.0 %, compared to the total national consumption of electrical energy in 2007 [6].

USAGE OF BIOMASS RESOURCES Possibilities for use of biomass are numerous (biofuels production, biogas plants).

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Biofuels are fuels from renewable resourses like plants - and animals sources. Biofuels have chemical and physical properties that are close to fuels from fossile fuels. They can be used to exchange petrodiesel in egsisting engines.The biofuels are much cleaner for the environment and it has potential to solve other problems of environment protection (like waste management) or if the sourse of the biofuels are algae that are grown on water that has to be cleared. These sources provide the fuel which price will depend more on microeconomic than on macroeconomic factors. In this way, economies of many countries will be protected from external economic crises. In addition to the remains and waste, there are a large number of plant species that it is possible to raise: from the fast growing trees with the annual yield of 17 tons per hectare, and to the green algae with a yield of 50 tons per hectare. Biofuels, which are most widely used are biodiesel and bioalcohols with ethanol accounting for more than 90% of global production of biofuels [11]. Ecological standards of developed countries according to Kyoto Protocol are growing due to which production of biodiesel receives a greater stimulus. The reasons for the sudden increase in production of biodiesel fuel are the necessity of the struggle with air pollution and climate changes, growth and fluctuations of oil prices, as well as the need for ecologically clean fuel. It is proven that biodiesel does not damage the diesel engine. Contrary, his high lubricant properties compared with regular diesel prevent spending clips, cylinders and precision parts for injection pumps. The most important raw material for the production of biodiesel is oil seed rape with 82.8% of the world production. In addition, there is significant production of biodiesel from sunflower oil with 12.5% of the world production (the remaining 4% cover other cereal oils, for example, corn). In Serbia are developed own technology, engineering and technical procedures for transesterification of oils and fats obtained by extrusion of grain crops. There is also developed finalization of by-products, such as meal, glycerol, salt and soap into quality products for animal feed and raw material for chemical industry [10]. Plants for the production of biodiesel by batch method, with capacity up to 1000 t, are in preparation for the construction in Sombor, Melenci, Knjaţevac and Backa Topola. In the case of discontinuous transesterification, oils and fats are added into reactor where by mixing and heating to the set temperature (usually till boiling alcohol) one obtains methyl ester and glycerol. The amount of added methoxide in the first batch of transesterification is 80%. After separation of phases, the top layer of biodiesel is blended and heated again with the addition of 20% of the remaining methoxide. After separation (decantation) crude biodiesel is passed through an ion-exchange column, and finally filtered through 5 microns filter. The process is conducted without the participation of water. Oil for biodiesel production is obtained from grain using extruding process. Further transesterification results in byproducts such as glycerol and soaps. Usage of those byproducts results in a cost competitive biodiesel versus the petrol diesel. The analysis indicates that the discontinuous biodiesel production is more effective than the continuous one. The biogas power plant is designed in accordance with the existing energy policy that recognizes producers of energy from renewable sources as producers with privileged status and guarantees the purchase of energy at subsidized prices [1,2]. The investor company revised its own energy policy and aligned it with the priorities of sustainable energy policy of Republic oof Serbia.The support system for producers of electricity from renewable resources in the Republic of Serbia has been established by the following legislative acts Energy Law: 225 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

 Decree on conditions for obtaining the status of privileged power producers and criteria for evaluating compliance with these conditions (3rd September 2009),  Decree on measures of incentives for the production of electricity using renewable energy resources and combined production of electrical and thermal energy (Official Gazette of RS from 01/12/2009 No. 99/2009 year). Within the farm "Sava Kovacevic" in Vrbas (october 2012) is finished the construction of plant for production of biogas, which later converts into heat and electricity, worth 200.000 EUR [8].

WIND ENERGY RESORCES IN SERBIA Wind energy is environmentally acceptable renewable energy source. Serbia has not yet windgenerator farms, but there are plans for their construction on the Vlasina, in Bela Crkva and on some other locations. One of the main objectives of the project "Strengthening of the Center for renewable energy sources in the Agency for Energy Efficiency" is to show that Serbia has the wind energy resources and to identify areas that have prospects for the production of electricity using wind. In Šajkaš (municipality Titel), Veliko Gradiste and Negotin posts 50 m high are set for measuring wind potential. For the economical production of electrical energy by modern windgenerators is required annual average wind speed of 5 m/s and more, determined on the basis of ten minute average speed at a height of 50 m above the ground. Serbia by this criterion has significant wind potential. Area with suitable wind is 471.25 km2. Best wind is registered on Midţor (7.66 m/s), Suva Planina (6.46 m/s), Vršački Breg (6.27 m/s), Tupiţnica (6.25 m/s) and Krepoljin (6.18 m/s). In addition, there is a significant number of sites, with a total area of 244 km2, in which over 50% of the time in the year, the wind blows with an average speed over 6 m/s [3]. For climate protection and the promotion of risk management system in the Spatial Plan of Serbia 2010-2020 were established the priority strategic activities for mitigation of climate change. Ministry of Infrastructure and Energy has prepared the Strategy for implementation of Clean Development Mechanism (CDM) in the energy sector of Serbia. National Strategy for the inclusion of Serbia into CDM, for the sectors of waste management, agriculture and forestry was adopted in February of 2010 ("Official Gazette of RS", No. 30/10). The Strategy provides information on overall and priority resources for the implementation of CDM projects in the Republic of Serbia (National Environmental Protection Programme, 2010). In November 2010 adopted the first report of the Republic of Serbia to the United Nations Framework Convention on Climate Change. In February 2012 the project "Wind Farm Cibuk 1" (Municipality of Kovin, 171 MW) was recorded as the first CDM project. Design documents (PDD) for projects: "Wind Farm Plandiste 1", "Wind Farm II Oro", "Wind Farm Cibuk 1", and "Reduce methane leaks in the distribution network of Srbijagas" were submitted to Designated National Authority of the Republic of Serbia (DNA), to implement the Clean Development Mechanism. Official data indicate that the price of electric energy produced by windgenerators, for specific locations, is competitive with conventional sources. This trend will continue in the near future.The first wind farm Chibouk at Kovin, Vojvodina will be build by 2014 [5].

UTILIZATION OF GEOTHERMAL ENERGY IN SERBIA Serbia is rich in geother mal energy and has a great natural and market potential for large investments in the area [4,5]. The existence of large hydrogeothermal convective system with the reservoir of the thermal low-mineralized water (temperature between 75 and 120°C) is important for

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heating. Geothermal energy could be efficiently utilized for various purposes in the Republic of Serbia [9]. Horticultural hotbed gardens and greenhouses are usually growing nearly thermal springs. Thermal water is desirable for hotbeds heating. Mercur hotel (Vrnjacka Banja),Vrujci Hotel (near Valjevo) and Selters spa, located on the hills of Kosmaj mountain, are the spa that use geothermal energy. In Selters spa instead of fuel oil, thermal water is used for heating of rooms. In this way, apart from annual savings of 250 tones of fuel oil, an important step in preservation of natural environment is at the temperature of above 50°C and will to heat up approximately 30.000 m2.

SOLAR ENERGY Hotel "Olimp", Zlatibor, is using solar energy for heaving of water for the last three years. Solar heaters can heat such a quantity of water that it is enough to heat the hotel swimming pool. According to people from hotel's technical department, at any moment approximately five tons of warm water is available. During especially sunny days, this capacity is rising above seven and a half tones. Hotel "Elite" is also using renewable resources of energy. From the design stage of hotel building the care has been taken about preservation of energy. While solar energy is being used to heat the water, energy of warm underground waters is being used for central heating. Solar company Danone Belgrade on 06 July 2012 started work on the installation of solar collectors in elementary school, "Dr. Jovan Cvijic" in Smederevo. Setting the first solar energy collector is a realization of a larger project of the city of Smederevo that includes greater use of RES in public facilities, and preserving the environment.

CONCLUSIONS The necessity of fast solving global environmental and economic problems lead to expansion in the study and use of renewable energy sources (biomass, geothermal energy, wind energy, solar energy). Serbia is rich in energy resources that are renewable The largest contribution of all renewable energy sources in the nearest future is expected from biomass. Biomass, as well as its products: synthetic biofuel and biogas, are not only potentially renewable, but are sufficiently similar to fossil fuel. So that the complete replacement is possible. Biomass and its products (biofuels and biogas) are fuels from renewable resourses like plants and animals sources.Biofuels have chemical and physical properties that are close to fuels from fossile fuels. Wind energy potentials in Serbia, if the zone with medium speed greater than 5 m/s, allows the construction of 1,316 MW windgenerator's capacity, which is about 15% of current installed capacity of all kinds. These capacities, assuming a low coefficient of capacity of 0.2 (in the world range from 0.25 to 0.4) would be able to produce about 2.3 TWh per year. By applicable international criteria, the wind energy resource of Serbia, whose activation would significantly increase the installed capacity, diversity of energy sources and reduce dependence on imports of energy. Investigations of mineral and thermomineral water sources carried out in Serbia have indicated high quality mineral waters that can be used for medical purposes. Mineral and thermomineral sources in health resorts in Serbia represent natural resources with large possibilities for the development of tourism, healthtourism, agriculture and other economic activities. Special attention should be paid to the required multidisciplinary researches for the purpose of the relevant criteria necessary for usage of every type of thermomineral water. 227 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA

Intensive exploitation of the renewable resourses must become one of the important goals of economical development in Serbia. Since the production of electricity from renewable resources in most cases is more expensive than energy production from fossil fuels, the so-called support systems has been introduced, i.e. financial and nonfinancial measures of incentives to invest in facilities that use renewable energy .

REFERENCES [1]. Golušin, M., Dodiš, S., Vučuroviš, D., Jovanovic, L., Munitlak Ivanoviš, O. 2012. Sustainable energy management in industry of Republic of Serbia. Biogas power plants advantages. Industrija 40, No 4, pp. 107-124. [2]. Golušin, M., Dodiš, S., Vučuroviš, D., Ostojiš, A., Jovanoviš, L. 2011. Exploitation of biogas power plant - CDM Project, Vizelj, Serbia. Journal of Renewable and Sustainable Energy 3, No 5, 052701, pp. 1-11. [3]. Ivanov, M., Jovanoviš, L., Martinoviš, D. 2009. Wind energy use in Serbia. Ecologica 54, pp. 107-112. (in Serbian) [4]. Jovanoviš, L. 2002. The possibilities of exploitation of Serbian thermomineral waters. In: D.P. Antiš and J. Lj. Vujiš (eds.), Environmental Recovery of Yugoslavia (ENRY 2001), Belgrade, pp. 747-751. [5]. Jovanoviš, L. 2012. Renewable energy resources in Serbia. International Symposium "Achievements and Prospects in Biotechnical Engineering Systems" (ISBTEH 2012), Bucharest. [6]. Jovanoviš, L. 2012. Environment protection policy and sustainable development: progress of Serbia in the process of EU integration. In: P. Teixeira, A.P. Duarte, S. Redţepagiš, D. Eriš, S. Andrejeviš (eds.), European Integration Process in Western Balkan Countries, Faculty of Economics of the University of Coimbra, Coimbra (Portugal), Ch. 33, pp. 648-671. [7]. Jovanoviš, L. 2013. L'analyse comparative de l'utilisation des ressources énergétiques renouvelables en Serbie et d'autres pays de l'Europe du Sud-Est. Int. Conf. "Les Relations entre l‘Union Européenne, la Russie et les Balkans", Nice. [8]. Jovanoviš, L., Cvijiš, L., Tomiš, A., Radosavljeviš, M., Anđelkoviš, M. 2013. Progress of the Republic of Serbia in the preparation of CDM projects. Ecologica 69, pp. 67-72. (in Serbian) [9]. Jovanoviš, L., Stojadinoviš, D. 2003. Managing of water resources in urban areas of Serbia. Proc. of 4th European congress on regional geoscientific cartography and information systems, Bologna, vol. 1, p. 216. [10]. Jovanoviš, S., Jovanoviš, L. 2009. Perspectives of application of biodiesel. Ecologica 54, pp. 123-127. (in Serbian) [11]. Jovanoviš, S., Radovanoviš, A., Jovanoviš, L. 2009. The methods of the bioethanol production in the world. Ecologica 54, pp. 117-122. (in Serbian)

ABOUT THE AUTHOR Prof. dr Larisa Jovanoviš, Alfa University, Palmira Toljatija 3, 11070 Belgrade, Serbia, E-mail: [email protected], [email protected]

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Plenary paper

APPLICATION OF THE MULTI-CRITERIA DECISION ANALYSIS FOR ASSESSING THE STUDENT’S PROGRAMMING TASK PERFORMANCE

I. Zheliazkova and O. Kir

Abstract: The MCDA is one of the most widely applied approaches for support of human decision making in problem solving is planning, management, education, and so on. In this paper the application and implementation of the MCDA model to support the instructor’s decision making about the student’s test-like exercises involving programming tasks. Identification of the options, objectives and criteria is described. Scoring and weighting in the decision context are considered. Tool for programming tasks is shortly presented too. Finally, the received results are examined.

Key words: Multi-Criteria Decision Analysis, Programming Task Perfomance, Student’s Assessment, Tool for Data Mining.

INTRODUCTION Recently Multi-Criteria Decision Analysis (MCDA) approach [23] presents a set of methods for disaggregating a complex decision problem into subproblems, measuring the degree to which the alternatives achieve criteria, weighting them, and reassembling the subproblems. Here the weights have a sense of the degree of criteria preference importance. The applications have different decision socio-economics contexts, e.g. for strategic management [3, 5], marketing, physical, urban and regional planning [2, 12, 13, 18], agricultural mapping [15], cultural heritage building [4]. Another, their advantage is a clearer and more readily justifiable audit trail, e.g. data evidence that enables a researcher to trace the path of past actions or decisions. The original MCDA method was derived by Keeney and Raiffa, 1976 [9] from the Multi-Attribute Value Theory to support decision making with multiple objectives/goals. It is an intuitive and compensative method that in a formulaic way ranks the alternatives from the most preferred to the least preferred ones. According to the independent preference axiom the chosen criteria must be mutually independent, e.g. the scores assigned to all alternatives on one criterion are unaffected by those on the other criteria. This idea is simpler and less restrictive than the real-world or statistical independences. The method also assumes that the decision maker‘s preferences and decisions are consistent. The group of the recent MAVT methods are easy to be understood and computationally less to be implemented even by means of EXCEL. Besides them the MCDA was extended with other groups of methods based on boolean functions, fuzzy sets, common sense rules and so on [6, 7, 8, 16]. The methods derived form MAVT compute the overall/total score for each alternative using several aggregation operators [2]. The most common of them is simple Weighted Averaging (WA) that computes the weighted average (Arithmetical or Geometrical) of the criterion score for each alternative. The Ordered Weighted Averaging (OWA) operators introduced by Yager, 1988 [20] are another group of aggregating operators. It includes, as particular cases the WA, and as extreme situations the MAX and MIN operators. If the weights are obtained by a non-monotonic quantifier the OWA operator implements linguistic statements such as ‗Excellent, ‗Very Good, ‗Good‘, and so on. The compensation operator introduced by Zimmermann, 1995 [17] uses a tuning operator for representation of thus more or less compensative situations. A different approach is obtained using a fuzzy expert system, developed by Von Alrock, 1995 [17] but its design is not a simple task, since many efforts need for definition of the interface rules.

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Nowadays, Goal Programming (GP) introduced by Charnes and Cooper, 1961 is also a group of models belonging to the MCDA approach [1]. It is transparent and closer in nature to the programming task performance. In the 70‘s an interesting study was carried out concerning the goals and team‘s programming [19]. Each of five teams was assigned one and the same task for programming but with different goals for the program optimization: amount of labour, number of operators; memory volume; program readability, and output clarity. Each team achieved its goal but none of the teams achieved different goals equally well. In a recent and exhaustive survey on goal programming [1] following directions for future research are listed: (1) Combine the GP methodology with other ones from the discrete multi-criteria field, in order to increase the number of applications in the decision contexts; (2) Increase and improve the use of heuristics in the GP models, given that they allow more flexibility to model real problems and to solve them in a competitive time; (3) Favors the use of interactivity of these models with the aim of better supporting real decision making processes; Develop and use integrated schemes in real applications that can, in certain cases, introduce in a more effective way the decision maker‘s preferences into the model; Work in the development of theoretical applications in order to strengthen the framework of the GP models, and to provide it with the necessary maturity and multiple real applications. Taking into account these directions the author‘s team has applied the MCDA for the personal authoring and teaching team‘s assessment in e-testing and e-lecturing environments respectively [22, 21]. This paper deals with the application and implementation of the WA and OWA models to support the instructor‘s decision making about the student‘s programming tasks assessment by a data mining tool. This new decision context is identified in the next section through a decision tree. The third section presents short description of the tool‘s user interface and programming language. The fourth section deals with the model implementation which received results are examined in the fifth section. The last section presents the paper research finding and conclusions.

IDENTIFICATION OF THE DECISION CONTEXT In each university system (traditional or computerized) student‘s assessment is recognized as a decision-making problem based on several criteria and connected with the instructor‘s and course team‘s assessment. The number and type of these criteria vary among different universities, specialities, courses, and even among a course team. The set of criteria concerning the student‘s exercise tasks, lectures, work and other assignments vary too. Usually in the real practice the number of the taught students, e.g. alternatives is great. Hereafter for clarity of the decision context a single criterion is called indicator, the number of indicators is called sub-criteria, and the set of sub-criteria is called criterion. The decision tree model on fig. 1 shows up to down the disagregation of the criteria, sub- criteria and indicators to applying the WA and OWA models for the student‘s assessment. Different members of the course team play the role of decision makers at the levels and even for the tree nodes at a given level. The specific of the decision context requires considering the student‘s assessment as a MCDA problem. The decision problems frequently raised are: determine the best student, select a subset of students, satisfying a given set of criteria, rank or categorize them in teams with similar attitudes, knowledge, and skills. It is supposed that a test-like exercise includes several tasks for programming. Each task performance takes into account several indicators. The results of the model present both student‘s related scores and traditional marks referred to the criteria, sub- criteria, and indicators.

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In the general term of criteria the preference score for student i on criterion j at level k k k k k = 1 can be represented as the proportion ps ij  soj / sij where sij is the student‘s k measured value, and s0j the expert‘s measured value, e.g. in the range between zero k and one. If Nk is the selected number of criteria at level k, w j the weight for each criterion, then the overall weighted score for each student (Si) at the level k+1 will be

Nk Nk k1 k k k written as S i  w j .ps ij (i = 1,…, n), w j 1, e.g. the sum of weights assigned j1 j1 for each criterion at the level k must be equal to one (k = 1,2,3,4);. Programming this family of linear additive models the student‘s weighted scores consequently are computed for each parent node using the scores computed for its children nodes and the weights assigned by the decision maker, e.g. for indicators, sub- criteria, criteria and course respectively. For the first level the indicators‘ numerical values serve as input data set. Their assignment is one of the critical points of each application.

Level 4 Student‘s Course mark

Level 3 Lecture Exercise Course Criteria work

Level 2 Task1 Taskj TaskM Sub-criteria

Level 1 Indicator1 Indicatori IndicatorN Indicators Level 1 . Fig.2. The levels of the decision three

The known applications concerning hierarchical decision processes like the considered are based on questionairing e.g. implicit elicitation from expert‘s judgments [14], applying complex optimisation algorithms on experimental data or data mining techniques. The reported application uses a tool for programming of data mining for this purpose. Note, that the proposed model can be usefully applied even for one student, one lecture, exercise, and work assignment. The reported case study is limited only to exercises each one including several programming tasks.

TOOL FOR PROGRAMMING OF DATA MINING This tool developed by the author‘s team has been put into the teaching practice of the master degree students specialty ―Computer Systems and Technologies‖. During test- like exercises of the course ―System for Business Modeling‖ the students perform different tasks for economical and educational data mining applying table presentation, visualization, statistical and multi-criteria decision analysis. Till now the tool was used for socio-economics data mining with input data sets gathered from literature, traditional and web-based teaching environments. In [11] the technological scheme for solving a real-world problem by means of the tool is presented as a sequence of steps. Its formulation in free-text has to be clear, precise, and compact. Depending on the problem context some steps can be omitted others 231 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA repeated or interpreted as sub-problem solving. To perform each step the student has to know the syntax and semantics of the offered SESSIONSCRIPT language. It is high level powerful and expressive and at the same time with a simple and easy syntax. The full specification of the recent language version is given in [10]. In order to enhance the SESSIONSCRIPT language learning a colour coding scheme has been accepted. The screen windows of the tool are seen on fig. 2.

Fig.1. The screen windows of the tool for data mining The left side window contains the script code of the program written by the student, and the right side window the table with the input data set. The description of the problem solved in a free text format can be seen switching from ―Table‖ to the ―Description‖ tabs (not shown here). The student is also recommended to save it in a standard text file serving as a common catalogue of the problems already solved by the members of the course team. The user can choose also the menu-command Window|Variables to view the names and values of the system variables and the program variables. The visualization allows choosing different kinds of diagrams, such as bar, pie, line, and point viewed in separated windows. In order to view the corresponding diagram the table name has to be chosen from the menu-item View|Bars. For the needs of this kind of tasks a power command for visualization of a family of lines in a common coordinate system had been implemented in the tool, as well as some kind of the OWA and correlation operators. One of its most attractive features is generation of the student‘s report for programming task performance including several indicators in different dimensions and numerical scales.

IMPLEMENTATION OF THE MSDA PROCEDURE This procedure ensures precise and objective measuring the student‘s task performance, program implementation of the WA operator, OWA for conversation of the overall score into the traditional six-point scale with the desired accuracy, as well as conducting a sensitive analysis.

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As the main goal of a task programming is to reach some expert‘s performance the results of which have to be compared with the student‘s ones. His role was played by the instructor which performance gives the following indicator‘s values with prefix E_: number of keywords (E_KEY = 379); number of correct lines (E_CLIN) = 26); number of incorrect lines (E_ILIN = 0), and task duration (E_DUR) = 180 sec). The student‘s faculty number served as the name of the .xls file generated by the tool after each task performance. Table 1 presents a student‘s report with the measured indicators, their values and dimensions. The four indicators initially chosen for the student‘s performance are denoted with prefix S_ as: S_CLIN the number of correct lines in the student‘s task; S_ILIN the number of the student‘s incorrect lines; S_KEY the number of the keywords and S_DUR the time undertaken.

Table 1. The student‘s report STARTING TIME OF THE SESSION 10:51:31 The data collected together with the TABLE NAME TABLE1 weights assigned to the chosen TABLE DESCRIPTION indicator served as input data for the NUMBER OF CORRECT LINES 24 lines program implemented in the tool‘s NUMBER OF INCORRECT LINES 2 lines language. Table 2 presents the input ERROR FREQUENCE 0.0769 data set, e.g. average indicators‘ values NUMBER OF KEYWORDS 327 keywords for all exercise tasks performed by each FINISHING TIME OF THE SESSION 11:07:07 student. Obviously, they are consistent. DURATION OF THE SESSION 0h 15m 36s In the model the indicators CORRECTS RATE 20 keywords/min DATE Tuesday March 19 2013 and INCORRECTS lines are integrated 11:07:18 - Tuesday March 2013 into a single indicator, and the three weights were chosen equal to 1/3.

So, the applied simple WA model can be written as RES = W1*(E_CLIN/(S_CLIN+S_ILIN)) + W2 *(E_KEY/S_KEY)* + W3 *(E_DUR/S_DUR). Table 3 contains all computations results concerning the WA and OWA models. Column RES presents the overall student‘s scoring received under the selected indicators and relatively to the expert‘s performance. Column MARK corresponds to converting RES to the discrete six-point scale traditional for education in Bulgaria with accuracy till 0.25. More roughly the assessment scale for MARK is: 0 ≤ RES ≤ 0.4 – ―Poor” (2); 0.4 < RES ≤ 0.55 – ―Satisfactory” (3); 0.55 < RES ≤ 0.70 – ―Good” (4); 0.70 < RES ≤ 0.85 – ―Very good” (5); 0.85 < RES ≤ 1.0 – ―Excellent‖ (6); 1.00 < RES ≤ 1.15 – (7); 1.15 < RES ≤ 1.30 – (8); 1.30 < RES ≤ 1.45 – (9). The experience accumulated during the last decade by Zheliazkova‘s research group has pointed out that such a non-linear scale leads to marks acceptable by both instructors and students.

RESULTS EXAMINATION The number and weights of the indicators could be changed for better differentiation of the students according to their programming skills. That‘s why RES and MARK were considered carefully to answer the question: ‖Do other indicators and/or weights affect the overall ordering of the students?‖. A very sensitive and variable indicator is obviously the duration for the programming task performance. Due mainly to this indicator some students‘ assessments reach MARK = 9, e.g. these students turned to be faster than the expert‘s performance of the instructor.

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Table2. Input data set Table 3. Received results

The adaptation of the model to the instructor‘s preferences is achieved considering four single indicators with weights equal to 0.25. The computed results (RES1 and MARK1) in the range of 2.5 and 6.5 are even more acceptable than the initial ones. Instead to relate the student‘s indicators values with the expert‘s ones it is also reasonable to relate the first ones with the statistical mean for the group of students. The group‘s computed values are: G_KEY = 338,08; G_CLIN = 22,7; G_ILIN = 2,00; G_DUR = 937,15 sec. Fig. 3 shows that as a whole the group‘s overall scoring is close to the expert‘s one which is equal to 1. However, the new student‘s results (column RES2 and MARK2) are unacceptable for the decision maker, as MARK2 is in the range of 4 and 15,75.

Fig. 3. Relation to the expert‘s and mean of the group Fig. 4. Correlation table

To decrease the initial number of the indicators the rate of programming (S_RATE = S_KEY/S_DUR) and frequency of errors (S_FRQ = S_ILIN/(S_CLIN+S_ILIN) were considered as possible coalitions of two indicators. The computed RES3 and MARK3 (not given here) were closer to poor performance for all students. This is explained with

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the confliction between these coalitions, e.g. S_RATE has a positive impact on the successful programming skills while the impact of S_FRQ is negative. To confirm the validation of the implemented WA and OWA models table 4 was created containing all possible correlation coefficients between RES, RES1, and RES2. The degree of correlation between the initial WA model implementation and the above- discussed its variants is very high. A good correlation (not given in table 4) with the discrete six-point marks given by another instructor for exercises with task programming in DELPHI also was found. Another goal of the results examination can be to search for other important factors not captured in the model. For example, in order to adapt to some instructor‘s preferences the number of the system and program variables, could be added to the initial list of four indicators and this extension of the model needs and additional case study.

FINDINGS AND CONCLUSIONS A new successful application of the multi-criteria decision analysis integrated with the goal programming is reported in this case study. Its implementation by means of a tool for programming of data mining allows quickly and precisely to assess the student‘s programming tasks performance. The case study confirms that such an application supports the instructor‘s deeper reasoning about the assessment process at hand and with more confidence. The student‘s motivation and activity in achieving his/her best exercise performance also is stimulated. Although the proposed procedure was implemented for task and exercise nodes it serves as a template for applying the MCDA to other nodes of the decision tree, as well as for programming in other languages and tools.

ACKNOWLEDGMENTS The authors of the paper are grateful to the master degree students specialty ―Computer Systems and Technologies‖ at Rousse University. During the first semester of the 2012/13 academic year in the framework of the course ―System for Business Modeling‖ they do their best to carry out test-like exercises with programming tasks in the tool for data mining.

REFERENCES [1] Caballero, R., Gomez, T., Riuz, F. 2009. Goal Programming: Realistic Targets for the Near Future, Journal of MCDA, Vol. 16, pp. 79-110; http://onlinelibrary.wiley.com/doi/10.1002/mcda.442/full [2] Despic , O., Simonovic S. P. 2000. Aggregation Operators for Soft Decision Making in Water Resources, Journal Fuzzy Sets and Systems, Vol. 115, pp. 11-33. [3] Eden, C. and Ackermann, F. 1998. Making Strategy: the Fourney of Strategic Management, Sage Publishers. [4] Giove, S., Rosato, P., Breil, M. 2010. An Aplication of Multi-Criteria Making to Built Heritage. The Redevelopment of Venice Arsenal, Journal of MCDA, Vol. 17, Issue 3-4, pp. 85-99, http://onlinelibrary.wiley.com/doi/10.1002/mcda.455/full [5] Goodwin, P. and Wright, G. 1983. Decision Analysis for Management Judgement, Second Edition, John Wiley, Chichester. [6] Grabish, M. 1997. K-order Additive Discrete Fuzzy Measures and their Representation, Journal Fuzzy Sets and Systems, Vol. 92, pp. 167-189.

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[7] Grabish, M., Lebreuche, C, Vansnik, J. C. 2003. On the Extention of Pseudo-Boolean Functions for the Aggregation of Interaction criteria. European journal of Operational Research, Vol. 148(1), pp. 28-47. [8] Kasprzyk, J. 1987. Towards ‗Human Consistent‘ Decision Support System through commonsense-Knowledge Based Decision Making and Control Models: a Fuzzy Logic Approach, Computers and Artificial Intelligence, Vol. 6, pp. 97-122. [9] Keeney, R. L., & Raifa, H. 1976. Decisions with Multiple Objectives: Preferences and Value Tradeoffs, John Willey, New York reprinted, Cambridge University Press, 1993. [10] Kir, O., Zheliazkova, I. I. 2011. A Script Language for Data Mining, Proceedings of the Second International Scientific Conference Dedicated to 50th Anniversary of Yuri Gagarin’s Flight, Shoumen, pp. 91-101. [11] Kir, O., & Zheliazkova, I. I. 2011. Prediction of Educational Data Mining by Means of a Postprocessor Tool. International journal of Information Technologies & Knowledge, 5(2), pp. 190-199. [12] Nijkamp, P. and Van Delft, A. 1977. Multi-Criteria Analysis and Regional Decision Making, Martinus Nijhoff, Leiden. [13] Nijkamp, P., Rietveld, T., and Voogd. 1990. Multi-Criteria Evaluation in Physical Planning, North Holland, Amsterdam. [14] Saaty, T. 1980. The Analytical Hierarchy Process, John Willey, New York. [15] Stirling, A. and Mayer, S. 1999. Rethinking Risk: A Pilot Multi-Criteria Mapping of a Genetically Crop in Agricultural Systems in the UK, (SPRU Report No. 21), Science Research Unit. [16] Vincke, P., Gassner, M., Roy, W. 1989. Multicriteria Decision Aid, Wiley, New York. [17] Von Altrock, C. 1995. Fuzzy Logic and Neurofuzzy Applications Explained. Prentice Hall PTR: Upper Saddle River, NJ. [18] Voogd, H. 1893. Multi-Criteria Evaluation for Urban and Regional Planning, Pion, London. [19] Weinberg, G.M., Schulman, E. L., 1974. Goals and Performance in Computer Programming, Journal Human Factors, 16(1), pp. 70-77. [20] Yager, R. R. 1988. On Ordered Weighted Averaging Aggregation Operators in Multicriteria Decision Making. IBEE Transactions on Systems, Man and Cybernetics, Vol. 18, pp. 183-190. [21] Zheliazkova I., Borodzhieva, A. N. 2013. Collaborative Authoring and Measuring the Author‘s Team Assessment in E-Learning. Proceedings of the 9th International Scientific Conference ―E-learning and Software for Education‖, Bucharest, 25-26 April. (to be published). [22] Zheliazkova I., Borodzhieva, A. N. 2013. Personal Authoring and Measuring its Author‘s in an E-testing Environment – A Case Study. Proceedings of the 9th International Scientific Conference ―E-learning and Software for Education‖, Bucharest, 25-26 April (to be published). [23] Multi-criteria Analysis: A Manual, January, 2009, Department for Communities and Local Government, London, https://www.gov.uk/government/uploads/system/uploads/.../1132618.pdf

ABOUT THE AUTHORS I. Zheliazkova, University of Rousse, Studentska street 8, 7017 Rousse, Bulgaria, E-mail: [email protected]. O. Kir, University of Rousse, Studentska street 8, 7017 Rousse, Bulgaria, E-mail: [email protected]

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ENVIRONMENTAL APPLICATIONS OF ZEOLITE-BASED NANOMATERIALS. HOSPITAL WASTEWATER TREATMENT INVESTIGATION

C.I.Covaliu, L.Toma, G. Paraschiv, S.Şt. Biriş, I.R.Bunghez

Abstract: Hospital wastewater represents a major source of pollution with chemicals. Residues of pharmaceuticals can be found in all wastewater treatment plant (WWTP) effluents, due to their inefficient removal using the conventional techniques. One of the most dangerous class of chemicals is represented by cytostatic agents due to their proven carcinogenicity, mutagenicity and embryotoxic properties. For these reasons we have chosen for study the doxorubicine cytotoxic agent removal from a synthetic wastewater. Doxorubicine is used in clinical practice for treatment of leukemias, as well as cancers of the bladder, breast, stomach, lung, ovaries, thyroid and others. From all nanomaterials that could have potential for an efficient hospital wastewater treatment we have chosen as nanoadsorbents zeolites within an innovative technique. Key words: hospital wastewater treatment, zeolite nanoadsorbents, environmental protection.

INTRODUCTION Hospitals discharge considerable amounts of chemicals in their wastewaters which resist to normal wastewater treatment and end up in surface waters where they can influence the aquatic ecosystem and humans which are exposed to serious risks by the drinking water, produced from surface water. Most important chemical polluants in hospital wastewater are antibiotics, cytostatic agents, anesthetics, disinfectants (due to their major use in hospital practice), platinum, mercury (in composition of diagnostic agents and as active ingredients of disinfectants), rare earth elements (gadolinium, indium, osmium) and iodinated X-ray contrast media [1]. Other pharmaceuticals which have been detected in wastewater treatment plants effluents from other countries include lipid regulators, analgesics, antibiotics (cf. above), antidepressants, antiepileptics, antineoplastics, antipyretics, antiphlogistics, antirheumatics, b-blockers, broncholytics, b2-sympathomimetics, estrogens (cf. below), secretolytics, vasodilators and X-ray contrast media [2,3]. From the total amount of 10 000 tones of antibiotics consumed annually in Europe, roughly half are utilized in human medicine. A percent of 26% of the antibiotics used for human purposes, are used in hospitals [4]. A part of antibiotics and their corresponding metabolites reached in the wastewater being excreted with urine and faeces. A survey achieved in a university hospital in the period 1998– 2000 showed that antibiotic use increased by 16% for the surgical procedures and by 20% for the medical services. Generally, the antibiotic use is expressed as defined daily doses per 100 patient (DDD/100), (World Health Organization, WHO). Comparing the worldwide hospital antibiotic use, values of 60 DDD/100 for surgical services and 80 DDD/100 in medical services are consistent [5]. It was reported a 16–78 DDD/100 for six Central Eastern European countries [6]. Some authors revealed that in hospital wastewater were detected various antibiotics such as: fluoroquinolones, erythromycin, gentamycin, trimethoprim, sulfamethoxazole, roxithromycin and clarithromycin in hospital wastewater, each having the concentrations below 0.01 mg/L [7]. It was revealed that ciprofloxacin concentration in hospital wastewater was 3– 10mg/L, being above the concentration consider not harmful [8]. In the literature are presented some previously undetected fluoroquinolones in European wastewater treatment effluents [9]. The risk of indirect exposure to antibiotics is evaluated in various studies centered on the presence of antibiotics in surface waters and/or its implications regarding drinking 237 RESEARCH PEOPLE AND ACTUAL TASKS ON MULTIDISCIPLINARY SCIENCES 12 – 16 JUNE 2013, LOZENEC, BULGARIA water production technology [10, 11, 12]. As a result to the consumption of these polluted waters was observed a genotoxic effect which correlates with the findings of Kummerer et al. in the Closed Bottle Test (CBT) [13]. The biodegradation of ciprofloxacin, ofloxacin and metronidazole could not be detected by the authors. In this test, the genotoxicity of these chemicals was not eliminated. All of the researches done in this field highlight the efforts made to evaluate the risk of antibiotics presence in human body. In conclusion, the risk cannot be estimated correctly due to the lack of concentration level data in hospital wastewater. Iodinated contrast media (ICMs) The hospital source is obvious in case of substances such as iodinated X-ray contrast media, which are used for X-ray imaging of soft tissues. The worldwide ICM consumption was of 3460 tonnes in 1993 [14,15]. About 100 g of X-ray contrast media is used for one medical treatment which represents about 30 g of absorbable organic iodinated compounds (AOI) [16]. During their passage through the body, the AOI are biologically inert and stable towards metabolism. They are eliminated almost completely within a day after administration, ending up in the WWTP, where they are poorly removed [17]. ICMs persist in the environment in the aqueous phase due to their high hydrophilicity, rather than sorb onto organic material or accumulate in organisms [18]. Because its long term effects and impact on environment are unknown, there is a risk connected to their spread. This fact calls for more research on the subject and requires precautionary measures. Estrogens Because a feminization of male caged fish at discharge sites of wastewater treatment plants was observed, the estrogens are of particular interest [19]. The most important sources of estrogenic activity in wastewater samples are the natural estrogens estradiol (E2), estrone (E1) and estriol (E3) together with the synthetic estrogen ethinylestradiol (EE2) [20]. Estrogens are eliminated in urine as sulfate or glucuronide bound complexes by both male and female mammals. On a daily basis, women excrete on average approximately 32, 14 and 106 mg of conjugated E1, E2 and E3, respectively while pregnant women excrete about 100 times this amount [21]. One can assume that elevated concentrations of E1, E2 and E3 can be expected in hospital wastewater. Other chemicals The antimanic/antiepileptic drug carbamazepine tested on ceriodaphnids indicates a toxicity lower than 25mg/L. Risk quotients calculated for French and German WWTP effluents are higher than unity suggesting that these effluents represent a threat for aquatic life. This finding is corroborated by the fact that there is a continuous input of drugs in the environment, hence yielding chronic toxicity effects [22]. The researche of Stackelberg et al. [23] determine that carbamazepine was detected in the final drinking water of a US water production plant, although granular activated carbon (GAC) filtration was used for water treatment. Also, the studies highlight the presence of other drugs and antibiotics (erythromycin metabolite, sulfamethoxazole, acetaminophen, codeine, trimethoprim) in the surface water used for drinking water production. This finding calls for a component specific analysis in order to make a risk analysis of drinking water production. From experimental data resulted that only a mixture of 4 lipid regulators and 4 b- blockers of which only 2 are considered as harmful based on toxicity categories regulated by European Directive 93/67/EEC and on EC50 values, prove to be very toxic while each individual drug was only present at a low concentration of 2mg/L [24]. Because of their carcinogenicity, mutagenicity and embryotoxic properties, the cytostatic agents represent a danger. Gadolinium (Gd) and recently indium (In) complexes are non-biodegradable and are used in MRI (magnetic resonance imaging).

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These studies show that hospital wastewaters are an important source of toxic agents, dangerous to human and they are 2–10 times more harmful than domestic wastewater. The nanomaterials can be use to resolve the challenges faced by environmental scientists, engineers and policy makers. Some of environmental applications of nanotechnology are contaminant remediation, wastewater treatment, sensing and energy storage. This is why in this paper we propose a specific treatment of a wastewater containing as drug doxorubicine using zeolite nanomaterials. The study represents a preliminary research upon the absorption potential of the doxombicine by the two tested (evaluated) zeolite samples S1 and S2.

MATERIAL AND METHODS The treatment of synthetic wastewaters was studied on zeolite nanomaterials. The synthetic wastewater used for this study contains doxorubicine as cytotoxic drug. The zeolite nanomaterial samples (S1 and S2) were prepared by sol – gel method and their average particle size is 200 nm for S1 (Na-ZSM-5) and 220 nm for S2(NH4-ZSM-5), respectively. In this regard, the tests were conducted by contacting the two ZSM-5 zeolite powder samples with the synthetic solution of 50 % doxorubicine simulating hospital wastewater using a 1:5 molar ratio. The experiments were done at room temperature (21oC) under vigorous mixing of the reaction mixture (650 rpm) during 6, 12 and 24h.

RESULTS AND DISCUSSION The zeolite nanomaterial samples (S1 and S2) tested for the retaining the doxorubicine from synthetic wastewater have the average particle size of 200 nm for S1 and 220 nm for S2, respectively. These morphological data were obtained by transmission electron microscopy (TEM) analysis Fig. 1.

(a) (b) Fig.1. The images of S1 and S2 samples obtained by TEM analysis The percentage of doxorubicine incorporation on two zeolite samples are presented in table 1.

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Table 1. The doxorubicine incorporation percentage Sample/ 6 h 12 h 24 h % doxorubicine

S1 15 26 35

S2 18 28 40

The higher percentage of doxorubicine incorporation was obtained for S2 zeolite (40%). The percentages of doxorubicine incorporation obtained for the two tested samples at three contacting time values were slightly different. CONCLUSIONS AND FUTURE WORK The preliminary tests done in the present study support the potential of two zeolite tested as adsorbents in wastewater treatment process and sustain conducting complex studies for finding the parameters that influence the wastewater treatment process and the actual application of these adsorbents in hospital wastewater treatment plants.

REFERENCES [1]. Kümmerer, K. 2001. Drugs in the environment: emission of drugs, diagnostic aids and disinfectants into wastewater by hospitals in relation to other sources – a review. Chemosphere 45, 957–969. [2]. Sacher, F., Lange, F. T., Brauch, H. J. & Blankenhorn, I. 2001. Pharmaceuticals in groundwaters – analytical methods and results of a monitoring program in Baden- Württemberg, Germany. J. Chromatogr. A 938, 199–210. [3]. Ternes, T. A. 2001. Analytical methods the determination of pharmaceuticals in aqueous environmental samples. TrAC 20(8), 419–434. [4]. Kümmerer, K. 2001. Drugs in the environment: emission of drugs, diagnostic aidsand disinfectants into wastewater by hospitals in relation to other sources – a review. Chemosphere 45, 957–969. [5]. de With, K., Bergner, J., Bühner, R., Dörje, F., Gonnermann, C., Haber, M., Hartmann, M., Rothe, U., Strehl, E., Steib-Bauert, M. & Kern, W. V. 2004. Antibiotic use in German university hospitals 1998–2000 (Project INTERUNI-II). Int. J. Antimicrob Agric. 24, 15–20. [6]. Cizman, M., Beovic, B., Krcmery, V., Barsic, B., Tamm, E., Ludwig, E., Pelemis, M., Karovski, K., Grzesiowski, P., Gardovska, D., Volokha, A., Keuleyan, E., Stratchounski, L., Dumitru, C., Titov, L. P., Usonis, V. & Dvorak, P. 2004. Antibiotic policies in Central Eastern Europe. Int. J. Antimicrob Agric. 24, 1–6. [7]. Ohlsen, K., Ternes, T., Werner, G., Wallner, U., Löffler, D., Ziebuhr, W., Witte, W. & Höcker, J. 2003 Impact of antibiotics on conjugational resistance gene transfer in Staphylococcus aureus in sewage. Environ. Microbiol. 5(8), 711–716. [8]. Giger, W., Alder, A. C., Golet, E. M., Kohler, H. P., McArdell, C. S., Molnar, E., Siegrist, H. R. & Suter, M. 2003 Occurrence and fate of antibiotics as trace contaminants in wastewaters, sewage sludges, and surface waters. Chimia 57, 485–491. [9]. Andreozzi, R., Raffaelle, M. & Nicklas, P. 2003. Pharmaceuticals in STP effluents and their solar photodegradation in aquatic environment. Chemosphere 50, 1319–1330. [10]. Sacher, F., Lange, F. T., Brauch, H. J., Blankenhorn, I. 2001 Pharmaceuticals in groundwaters – analytical methods and results of a monitoring program in Baden- Württemberg, Germany. J. Chromatogr. A 938, 199–210. [11]. Snyder, S. A., Westerhoff, P., Yoon, Y., Sedlak, D. L. 2003 Pharmaceuticals, personal care products, and endocrine disruptors in water: implications for the water industry. Environ. Engng. Sci. 20(5), 449–469.

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[12]. Webb, S., Ternes, T. A., Gibert, M., Olejniczak, K. 2003 Indirect human exposure to pharmaceuticals via drinking water. Toxicol. Lett. 142, 157–167. [13]. Kümmerer, K., Al-Ahmad, A. & Mersch-Sundermann, V. 2000 Biodegradability of some antibiotics, elimination of the genotoxicity and affection of wastewater bacteria in a simple test. Chemosphere 40, 701–710. [14]. Engels-Matena, U. 1996 Der Abbau von iodierten Röntgenkontrastmitelndurch den Weißfäulepilz Trametes versicolor. Germany. PhD thesis. University of Hamburg-Harburg, 145 [15]. Kalsch, W. 1999 Biodegradation of the iodinated X-ray contrast media diatrizoate and iopromide. Sci. Total Environ. 225,143–153. [16]. Doll, T. Frimmel, F. H. 2003 Fate of pharmaceuticals – photodegradation by simulated solar UV-light. Chemosphere 52, 1757–1769. [17]. Steger-Hartmann, T., Länge, R., Schweinfurth, H., Tschampel, M., Rehmann, I. 2002 Investigations into the environmental fate and effects of iopromide (ultravist), a widely used iodinated X-ray contrast medium. Wat. Res. 36, 266–274. [18]. Kalsch, W. 1999 Biodegradation of the iodinated X-ray contrast media diatrizoate and iopromide. Sci. Total Environ. 225, 143–153. [19]. Purdom, C. E., Hardiman, P. A., Bye, V. J., Eno, N. C., Tyler, C. R., Sumpter, J. P. 1994 Estrogenic effects of effluents from sewage treatment works. Chem. Ecol. 8(4), 275– 285. [20]. Daughton, C. G. & Ternes, T. A. 1999 Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ. Health Persp. 107(6), 907–938. [21]. D‘Ascenzo, G., Di Corcia, A., Gentili, A., Mancini, R., Mastropasqua, R., Nazzari, M. & Samperi, R. 2003 Fate of natural estrogen conjugates in municipal sewage transport and treatment facilities. Sci. Total Environ. 302, 199–209. [22]. Ferrari, B., Paxeus, N., Lo Giudice, R., Pollio, A., Garric, J. 2003 Ecotoxicological impact of pharmaceuticals found in treated wastewaters: study of carbamazepine, clofibric acid, and diclofenac. Ecotoxicol. Environ. Safety 55, 359–370. [23]. Stackelberg, P. E., Furlong, E. T., Meyer, M. T., Zaugg, S. D., Henderson, A. K. & Reissman, D. B. 2004 Persistence of pharmaceutical compounds and other organic wastewater contaminants in a conventional drinking-water-treatment plant. Sci. Total Environ. 329, 99–113. [24]. Hernando, M. D., Petrovic, M., Fernandez-Alba, A. R., Barcelo, D. 2004 Analysis by liquid chromatography-electrospray ionization tandem mass spectrometry and acute toxicity evaluation for b-blockers and lipid-regulating agents in wastewater samples. J. Chromatogr. A 1046, 133–140.

ABOUT THE AUTHORS C.I. Covaliu, Lecturer at University Politehnica of Bucharest, Faculty of Biotechnical Systems Engineering, 313 Splaiul Independentei, 060042, Bucharest, Romania,E-mail: [email protected] L.Toma, Assitant at University Politehnica of Bucharest, Faculty of Biotechnical Systems Engineering, 313 Splaiul Independentei, 060042, Bucharest, Romania, G.Paraschiv, professor at University Politehnica of Bucharest, Faculty of Biotechnical Systems Engineering, 313 Splaiul Independentei, 060042, Bucharest, Romania, S.Şt. BIRIŞ, assistant professor at University Politehnica of Bucharest, Faculty of Biotechnical Systems Engineering, 313 Splaiul Independentei, 060042, Bucharest, Romania,

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I. Tudose, N. Ghiţeanu, C. Popa and R.Micu, students at University Politehnica of Bucharest, Faculty of Biotechnical Systems Engineering, 313 Splaiul Independentei, 060042, Bucharest, Romania; Ioana Raluca Bunghez, scientific researcher at Scientific Research and Technological Development in Chemical and Petrochemical Industry Institute- INCDCP-ICECHIM, 202 Splaiul Independentei Street, Bucharest, Romania

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