ANTIOXIDANT ACTIVITY AND MINERAL CONTENT OF ROCKET (ERUCA SATIVA) PLANT FROM ITALIAN AND BULGARIAN ORIGINS
Georgi Matev1,2, Petya Dimitrova2, Nadezhda Petkova3, Ivan Ivanov3 and Dasha Mihaylova4*
Address(es): 1 Executive Environment Agency (ExEA), Regional Laboratory - Plovdiv, Bulgaria. 2 Department of Analytical Chemistry and Physicochemistry, University of Food Technologies, Plovdiv, Bulgaria. 3 Department of Organic Chemistry and Inorganic Chemistry, University of Food Technologies, Plovdiv, Bulgaria. 4 Department of Biotechnology, University of Food Technologies, Plovdiv, Bulgaria.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.756-759
ARTICLE INFO ABSTRACT
Received 3. 5. 2018 Rocket plant (Eruca sativa) is a green leafy vegetable with significant levels of bioactive active components. Although, this plant is Revised 31. 7. 2018 known in Bulgaria, scanty data concerning chemical composition of representatives with Bulgarian origin is available. The aim of this Accepted 7. 8. 2018 study was to evaluate and to compare the biological potential of Italian and Bulgarian rockets in terms of their antioxidant activity, total Published 1. 10. 2018 phenolic content and total hydroxycinnamic acid derivatives. The micro- and macro elements were evaluated as well. The highest content of total phenolic content was detected in Bulgarian rocket – 4.45 mg gallic acid equivalent per g dry weight, while in Italian samples dominated the total hydroxycinnamic acid derivatives – 1.52 mg chlorogenic acid equivalent per g dry weight. The antioxidant Regular article activity was not significantly different in both samples. The presence Co and Al was reported for the first time, as in our study their values were higher in a rocket from Italian origin (75.6 and 595 mg/kg). Italian rocket contained arsenic (0.07 mg/g) in comparison with Bulgarian samples where it was not found. The results revealed the sample with Italian origin as a significant better source of antioxidants with potential beneficial effects. Bulgarian rocket was evaluated with smaller amount of some metals as nickel, arsenic, chromium, manganese, aluminum and sodium, but the concentrations of Pb (0.58 mg/kg) and Cd (0.27 mg/kg) were higher than accepted levels by EU.
Keywords: antioxidant activity, micro- and macro elements, rocket salad, total phenolic compounds
INTRODUCTION Organization approves maximum permission levels in raw plant materials for some elements, such as cadmium (0.3 mg/kg), arsenic (1 mg/kg), and lead (10 With the development of functional foods, the interest of scientists, consumers mg/kg) (Carvelho et al., 1997). and industrialists in the raw plant materials rich in biologically active substances Eruca sativa (Brassica eruca L.) is an edible annual plant, commonly known as has increased. Moreover, consumers require more accurate information about rocket salad or arugula (rucola, rucoli, rugula, colewort, and roquette). It is a food and drinks quality. green leafy vegetable, member of Brassicaeae family that originates from Several biologically active compounds in leafy vegetables were reported to Mediterranean countries. Rocket is much appreciated vegetable in Europe, North possess beneficial health effects. Vegetables are considered as rich sources of America, Argentina and South Africa. It has been cultivated in Central and nutrients such as carbohydrates, proteins, vitamins, calcium, iron and Western Asia for seed oil production (Yaniv et al., 1998). This plant is consumed concentration of trace elements (Prakash and Pal, 1991; Jimoh and Oladiji, in several ways as a flavoring in salads, spice and in boiled or baked dishes (Kim 2005). Most of them contain various pharmaceuticals agents and they are highly et al., 2006). Several recipes provide the preparation of pureed, sauces and pesto beneficial in treatment of various diseases. Some minerals as calcium, from its leaves (Tripodi et al., 2017). phosphorus, iron, magnesium, copper and potassium in vegetables maintain the The researchers reported the nutrient composition and concentration of acid/base balance in body tissues. They help for absorption of vitamins, proteins, phytochemicals in this plant (Bouis, 1996). In this respects, the representatives fats and carbohydrates (Kalita, 2007; Hussain et al., 2010; Hussain et al., from Brassicaeae family play significant role in human diet and they are rich 2011). sources of bioactive compounds such as glucosinolates, flavonoids, phenolic Phenolic compounds were reported to possess different bioactivities. They are acids, fibers, vitamins and carotenoids (Martinez-Sanchez et al., 2006; Jin et considered as antioxidants, antibacterial, antimutagenics, anti-inflammatory and al., 2009; Durazzo et al., 2013). The consumption of rocket plant increases antiallergic agents. Such activities contribute significantly for the consumers’ constantly due to its low prices, easy cultivation, short harvest time, high mineral well-being. In point of fact, the consumption of foods rich in antioxidants content and many health promoting substances (Kawashima and Valente- prevents from diabetes, cancer, cardiovascular or neuronal diseases (Zhang et al., Soares, 2003; Esiyok et al., 2010). E. sativa was treated as a plant with powerful 2015). active components that might be effective in increasing human health and as a Hydroxycinnamic acid derivatives (cаffeic acid derivatives - CADs) are naturally novel preventing cancer agent (Michael, 2011). occurring substances and they are considered as dietary phenolic compounds. However, the quality and nutritional potential of rocket plant available on The various biological activities like antioxidantive, hepatoprotective, anxiolytic, Bulgarian market are faintly investigated and controlled. The imported plants can insect repellent, antidiabetic and anticholesterolemic ones were investigated be a potential source of desease or present a treat for consumers. Because of that, (Sharma, 2011). the screening of their bioactive compunds and mineral content in important for Metals have an impact on human health. Some elements, such as Cu, Zn and Mn, human health. The microelements content in rocket leaves were not detailled are essential micronutrients with requirements that not exceed daily intake more invastigated, especially Bulgarian plants. than a few milligrams per day (Ajasa et al., 2004; Atlabachew et al., 2011). On Therefore, the current study aims was to evaluate the bioactive compounds and the other hand, the presence of toxic metals in medicinal plants is harmful for the mineral content in the rocket plants leaves (E. sativa) available on the Bulgarian human health (WHO, 1998). Therefore, for consumer’s safety, the World Health
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market and to compare the samples from Italian and Bulgarian origins in terms of Analysis of micro- and macro-elements antioxidant activity and mineral content. Mineralization of rocket leaves (0.5 g) was performed in a heat-controlled MATERIAL AND METHODS microwave system with 9 mL of 65 % HNO3 in a closed vessel system. The microwave-assisted acid digestion was carried out on Milestone ETHOS PLUS Plant material with MPR-300/12S medium pressure rotor and heating in two stages (5 and 10 min at 180°C up to 1000 W power, respectively). The microelements including some Eruca sativa leaves from Italian and Bulgarian origin were purchased fresh from heavy metals accumulated in leaves (Ni, Cu, Zn, As, Cd, Pb, Cr, Mn, Co, Al, Na the local market in Plovdiv (Bulgaria). The plant material was then air-dried, and K) of the investigated E. sativa samples were analyzed using ISP-MS Agilent ground and stored at close-tight containers until further uses. 7500 (G3272B) (Agilent Technologies, Inc., Japan). The results were expressed in mg/kg using calibration standards according to BSS EN ISO 17294-2 Extraction procedure (2016).The reference material used was SRM 1547 - Peach Leaves of National Institute of Standards and Technology (Gaithersburg, USA). Each dried and ground plant material (1 g) was extracted with 50 mL hexane: petroleum ether mixture (1:1, v/v) for 1 h and then was filtrated. The residue was RESULTS AND DISCUSSION dried and subsequently extracted with 50 mL 50% ethanol in order to dissolve the total hydroxycinnamic acid (caffeic acid) derivatives for 30 min at 30 °С in The frequent consumption of E. sativa and the control of its quality contribute to ultrasound water bath (ultrasonic bath SIEL UST 5.7 150 (Gabrovo, Bulgaria) the current research. On Bulgarian market was available rocket plants with Italian operating at 45 kHz). and Bulgarian origin. Because of the scanty data for chemical composition of the rocket leaves, the biological activity and nutrient composition of the both plants Determination of total phenolic content (TPC) were assessed and compared. The total phenolic content was studied in order to evaluate the biological The TPC was analyzed using the method of Kujala et al. (2000) with some potential of the samples (Table 1). The results revealed that the plant from modifications. Each extract was mixed with Folin-Ciocalteu`s reagent and 7.5 % Bulgarian origin contained higher value for total phenolic compounds – 4.45 ± Na2CO3. The mixture was vortexed and left for 5 min at 50ºС. After incubation at 0.14 mg GAE/g dw compared twith the Italian plant material (3.62 ± 0.16 mg room temperature, the absorbance was measured at 765 nm against control. The GAE/g dw). Other authors reported rocket leaves and flowers as sources of TPC was expressed as mg gallic acid equivalents (GAE) per g dry weight (dw) significant amount of phenolic compounds (23.07 ± 0.11 and 19.9 ± 0.3 mg plant material. GAE/g dw, respectively) (Sadiq et al., 2014) and (0.9 and 4.7 mg/g fresh weigh) (Heimler et al., 2007). The significant differences in the values were due to the Determination of total hydroxycinnamic acid derivatives use of young, fresh plants for consumption and analyses. However, our results indicated that rocket leaves was a source of phenolic compounds. This finding Total hydroxycinnamic acid content (including caffeic acid derivatives) was supported its use in most of the regions where people consume this vegetable as a expressed as chlorogenic acid according to the European Pharmacopoeia 8th whole plant (leaf, flowers, stem and seed) or various combinations in the form of edition (2014). The extract (1 mL) was added to 2 mL 0.5 M HCl, 2 mL Arnow’s fresh salad. reagent (10 % sodium nitrite and 10 % sodium molybdate in distilled water), 2 In order to compare both plants from Italian and Bulgarian origin, the mL 2.125 M NaOH and 3 cm3 of distilled water. Each sample was measured hydroxycinnamic acid (caffeic acid) derivatives content as a part from phenolic against control sample prepared by above mentioned procedure without Arnow’s compounds were also evaluated. E. sativa of Italian origin consisted of higher reagent and with additional volume of 2 mL distilled water. Absorbance was read total dihydroxycinnamic derivatives 1.52 ± 0.30 mg CAE/g dw, almost twice at 525 nm. The content of each plant was calculated and expressed as mg higher than E. sativa of Bulgarian origin (0.91 ± 0.20 mg CAE/g dw) (Table 1). chlorogenic acid equivalents (CAE) per g dw plant material. Total hydroxycinnamic derivatives presented about 41.44% of total phenolic compounds for the Italian origin plants and 20.22% for the Bulgarian ones. It is Determination of antioxidant activity known that caffeic acid and its esters such as caffeic acid phenyl ester and octyl caffeate are potential antioxidants that possess some important anti-inflammatory ABTS•+ radical scavenging assay action (da Cunha et al., 2004). The radical scavenging activity of the extracts against 2,2´-azino-bis(3- To evaluate the antioxidant activity of the rocket plant materials several assays ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) was estimated according to Re et based on different principles were applied (Table 1). Among them, the FRAP al. (1999). The results were expressed as TEAC value (μM TE/g dw). method showed the highest values followed by the ABTS and the CUPRAC methods. However, despite of the higher total phenolic content in the Bulgarian Ferric-reducing antioxidant power (FRAP) assay rocket, the antioxidant activity tested by the three assays was lower when the The FRAP assay was carried out according to the procedure of Benzie and values of analyses were compared. The antioxidant potential in respect of FRAP Strain (1999) with a slight modification. The FRAP reagent was prepared freshly assay was 35.85 ± 0.65 and 29.82 ± 0.75 μM TE/g dw, respectively. The high and it was warmed at 37°C before use. The absorbance was measured at 593 nm antioxidant activity was also demonstrated for rocket plants by DPPH and FRAP and the results were expressed as μM TE/g dw. methods (Heimler et al., 2007; Durazzo et al., 2013). This could be due to the phenolic compounds having different antioxidant activity with each single Cupric ion reducing antioxidant capacity (CUPRAC) assay method. The extraction procedures strongly influenced on the composition of the The CUPRAC assay was carried out according to the procedure of Ak and extracts and also influence on the antioxidant activity results (Halliwell, 1997; Gülcin (2008). Absorbance against a blank reagent was measured at 450 nm after Schwarz et al., 2001; Trojakova et al., 2001). In addition, the effect of the 30 min. The results were expressed as μM TE/g dw. antioxidant compounds in a food matrix may be significantly different than the activity of a purified extract (Brewer, 2011).
Table 1 Total phenolic content (TPC, mg GAE/g dw), total hydroxycinnamic acid derivatives (THAD, mg CAE/g dw) and antioxidant activities (TEAC, μM TE/g dw) in the E. sativa extracts TEAC TEAC TEAC Samples TPC THAD FRAP ABTS CUPRAC Italian E. sativa 3.62±0.16 1.52±0.30 35.85±0.65 32.07±0.64 25.03±0.22 Bulgarian E. sativa 4.45±0.14 0.91±0.20 29.82±0.75 28.76±0.26 19.74±0.25
As it was known mineral deficiencies can lead to many health disorders. The Manganese content in Italian rocket was approximately 2.5 higher than in mineral composition can influence on the antioxidant activity as some metal ions Bulgarian rocket. act as pro-oxidants. Minerals are presented usually in all parts of body and are Arsenic was detected only in Italian rocket and absent in Bulgarian plant. essential for life (WHO and FAO, 2004; Soetan et al., 2010). The detailed Moreover, in the current study the presence of Co (cobalt) (0.1 mg/kg) and Al information in respect of micro- and macro-elements composition of the (aluminum) in rocket was established for the first time. The amount of Al in the investigated E. sativa samples was summarized (Table 2). The results revealed Italian sample (595.0 mg/kg) was almost 8 times higher than the Bulgarian that the most frequently found metals were K and Na as macro-elements in sample (75.60 mg/kg). However, the differences among the two samples were accordance with Villatoro-Pulido et al. (2012), Barlas et al. (2011), Tripodi et observed. Zinc was evaluated in significantly higher amount (6 times) in the al. (2017). Rocket salad nutritional values for 100 g of fresh leaves (based on Bulgarian plant (91.05 mg/kg) than the Italian sample (15.07 mg/kg). USDA Nutrient Database) include K, Ca, Mg and Na as major elements in rocket The established quantities of manganese (Mn) (144.5 – 365.5 mg/kg) were 10 leaves, followed by P, Fe and Zn (Tripodi et al. 2017). In addition, among the times higher than those reported in other studies (Bukhsh et al., 2007; Barlas et micro-elements Mn, Al and Zn were in the highest content for the both samples. al., 2011; Villatoro-Pulido et al., 2012). In addition, the concentrations of copper (Cu) and chromium (Cr) were approximately 2 to 4 times lower (3.0 – 8.0
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mg/kg and 0.6 – 1.4 mg/kg, respectively) in comparison with the results of (EU) food standards (EC, 2006) as follow: Pb – 0.3 and Cd 0.2 mg/kg, Bukhsh et al. (2007) and Villatoro-Pulido et al. (2012). respectively. Cd may accumulate in the human body and induces kidney However, the geochemical properties of the soil, aerial, and/or aquatic disfunction, skeletal damage and reproductive deficiencies (Commission of the environment as well as the capacity of plants to accumulate elements selectively European Communities, 2001). Although Cd is a highly toxic metal occurred from their surroundings are known factors influencing the elements content in naturally in soil, it was distributed in the environment because of human plants. It had to be noted that the established Pb and Cd content did not exceed activities (Alam et al., 2003). Concentrations of metals analyzed in the samples the values in the standards for maximum levels of heavy metals (mg/kg of fresh could be related to their concentration in the corresponding soils. Therefore, to product) for leafy vegetables and fresh herbs with respect to E. sativa with Italian protect humans from harmful effects of heavy metals, the daily intake should not origin. Regarding the Bulgarian origin plant the values were exceeding the exceed the levels approved by the authorities. maximum allowed concentrations (MACs) approved by the European Union
Table 2 Analysis of the micro-elements (Ni, Cu, Zn, As, Cd, Pb, Cr, Mn, Al and Co) and macro elements (Na and K) in the investigated E. sativa extracts, mg/kg Elements Bulgarian E. sativa Italian E. sativa References Microelements, mg/kg Nickel (Ni) 0.38±0.06 0.78±0.10 0.17±0.10a 21.00±5.60a Copper (Cu) 8.37±0.90 3.58±0.40 10.10±0.40b 5.37c 1.12±0.09a Zinc (Zn) 91.05±0.65 15.07±0.60 42.10±0.40b 64.86c Arsenic (As) ND 0.07±0.01 - Cadmium (Cd) 0.27±0.03 0.12±0.01 0.0053a Lead (Pb) 0.58±0.03 0.28±0.01 - Chromium (Cr) 0.56±0.06 1.43±0.10 4.72±0.03a 10.60±2.80a Manganese (Mn) 144.50±7.00 365.50±18.00 5.34±0.12b 40.58c Cobalt (Co) 0.10±0.01 0.13±0.01 - Aluminum (Al) 75.60 ±7.30 595.00 ±60.00 - Microelements, g/kg Sodium (Na) 2.51±0.25 4.12±0.41 1.90±0.04b; 0.79c Potassium (K) 47.26± 4.72 43.56± 4.35 49.60±1.00b; 51.40c Legend: ND – not detected; a – Bukhsh et al. (2007); b – Villatoro-Pulido et al. (2012); c – Barlas et al. (2011)
However, the intake of heavy metal-contaminated vegetables may pose a risk to ATANASOVA, E., ATANASSOVA, I., SIMEONOVA, Ts., NENOVA, L., the human health. Food contamination with heavy metal is one of the most TEOHAROV, M., STOIKOVA, M. (2015) Assessment of the nutritional status important aspects of food quality assurance. The accumulation of heavy metals in of rocket salad (Eruca sativa) in technogenically affected soils. Soil Science vegetables depended on some factors as climate conditions, soils, water Agrochemistry and Ecology, 49(1), 65-71. irrigation, maturity of the plants, harvest and post harvest conditions (Atanasova ATLABACHEW, M., CHANDRAVASHI, B. S., REDI, M. 2011. Profile of et al., 2015). major, minor and toxic metals in soil and khat (Catha edulis Forsk) cultivars in Many reports were connected with growing rocket plants in contaminated with Ethiopia. Trends in Applied Sciences Research, 6(7), 640-655. toxic metals soils (Atanasova et al., 2015). Saleh (2001) claimed that rocket had http://dx.doi.org/10.3923/tasr.2011.640.655 the potential to accumulate minerals from soils in significant amounts. The AK, T., GÜLCIN, I. 2008. Antioxidant and radical scavenging properties of increase of Cd and Pb levels in soils was observed their accumulation in plant curcumin. Chemico-Biological Interactions, 174(1), 27-37. without further toxic effect, but improvement in biomass, chlorophylls content http://dx.doi.org/10.1016/j.cbi.2008.05.003 and enzyme activity of the plant. Shobha and Kumar (2016) reported for E. ALAM, M. G. M., SNOW, E. T., TANAKA, A. 2003. Arsenic and heavy metal sativa form Mysore district Cd content of 0.1 mg/kg and Pb content of 0.81 contamination of vegetables grown in Samta Village, Bangladesh. Science of The mg/kg. Total Environment, 308(1-3), 83–96. http://dx.doi.org/10.1016/S0048- According to Demirezen and Aksoy (2006) heavy metal levels in vegetables in 9697(02)00651-4 Turkey were within safe limits for Cu, Zn, Ni and exceeded for Cd and Pb. BARLAS, N. T., IRGET, M. E., TEPECIK, M. 2011. Mineral content of the Hussain et al. (2013) established for E. sativa leaves from Pakistan the micro- rocket plant (Eruca sativa). African Journal of Biotechnology, 10(64), 14080- elements (ppm; Fe, Cu, Mn, Cr, Cd, and Pb) and macro-elements (Mg and Na) 14082. http://dx.doi.org/10.5897/AJB11.2171 content as follow Fe - 7.96 ± 0.12; Cu- 3.97 ± 0.17; Mn - 0.75 ± 0.01; Cr - 0.94 ± BENZIE, I. F., STRAIN, J. J. 1999. Ferric reducing/antioxidant power assay: 0.03; Cd - 0.09 ± 0.00; Mg- 25.65 ± 0.21; Na- 40.56 ± 0.24. The values for Pb Direct measure of total antioxidant activity of biological fluids and modified and Cd were below the health-based guidance values (EU standards), as the Na version for simultaneous measurement of total antioxidant power and ascorbic and Mg had the highest concentrations. acid concentration. Methods in Enzymology, 299, 15-27. http://dx.doi.org/10.1016/S0076-6879(99)99005-5 CONCLUSION BOUIS, H. E. 1996. Enrichment of food staples through plant breeding: a new strategy for fighting micronutrient malnutrition. Nutrition Reviews, 54(12), 131– The current research evaluated rocket as rich sources of bioactive compounds. 137. http://dx.doi.org/10.1016/S0899-9007(00)00266-5 The evaluation of antioxidant activity and presented dihydroxycinnamic BREWER, M. S. 2011. Natural Antioxidants: Sources, Compounds, Mechanisms derivatives revealed the potential application of this plant as source of natural of Action, and Potential Applications. Comprehensive Reviews in Food Science antioxidants. The levels of micro- and macroelements in rocket plant with Italian and Food Safety, 10(4), 221–247. http://dx.doi.org/10.1111/j.1541- and Bulgarian origin were determined. The results suggested that significant 4337.2011.00156.x differences existed in the element concentrations between both samples. The BSS EN ISO 17294-2:2016. Water quality - Application of inductively coupled presence of Co and Al was established for the first time. The obtained results for plasma mass spectrometry (ICP-MS) - Part 2: Determination of selected elements the E. sativa of Bulgarian origin showed that concentrations of some metals (Pb including uranium isotopes. and Cd) exceeded the recommended maximum acceptable levels proposed by BUKHSH, E., MALIK, S. A., AHMAD, S. S. 2007. Estimation of nutritional EU. 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Food Chemistry, 103(1), 204-209. http://dx.doi.org/10.1016/j.foodchem.2006.08.007 KAWASHIMA, L. M., VALENTE-SOARES, L. M. 2003. Mineral profile of raw and cooked leafy vegetables consumed in southern Brazil. Journal of Food Composition and Analysis, 16(5), 605-611. http://dx.doi.org/10.1016/S0889- 1575(03)00057-7 KIM S. J., KAWAHARADA, C., ISHII, G. 2006. Effect of ammonium nitrate nutrient ratio on nitrate and glucosinolate contents of hydroponically-grown rocket salad (Eruca sativa Mill.). Soil Science and Plant Nutrition, 52(3), 387– 393. http://dx.doi.org/10.1111/j.1747-0765.2006.00048.x KUJALA, T. S., LOPONEN, J. M., KLIKA, K. D., PIHLAJA, K. 2000. Phenolics and betacyanins in red beetroot (Beta vulgaris) root: distribution and effect of cold storage on the content of total phenolics and three individual compounds. Journal of Agricultural and Food Chemistry, 48(11), 5338-5342. http://dx.doi.org/10.1021/jf000523q MARTINEZ-SANCHEZ, A., MARIN, A., LLORACH, R., FERRERES, F., GIL, M. I. 2006.Controlled atmosphere preserves quality and phytonutrients in wild rocket (Diplotaxis tenuifolia). Postharvest Biology and Technology, 40(1), 26– 33. http://dx.doi.org/10.1016/j.postharvbio.2005.12.015 MICHAEL, H. N., SHAFIK, R. E., RASMY, G. E. 2011. Studies on the chemical constituents of fresh leaf of Eruca sativa extract and its biological activity as anticancer agent in vitro. Journal of Medicinal Plants Research, 5(7), 1184–1191. PRAKASH, D., PAL, M. 1991. Nutritional and anti-nutritional composition of vegetable and grain Amaranthus leaves. Journal of the Science of Food and Agriculture, 57(4), 573-585. http://dx.doi.org/10.1002/jsfa.2740570410 RE, R, PELLEGRINI, N., PROTEGGENTE, A., PANNALA, A., YANG, M., RICE-EVANS, C. 1999. Antioxidant activity applying an improved ABTS
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THE HEAVY METAL CONTENT IN SELECTED KIND OF SPICES
Ľuboš Harangozo1, Marek Šnirc1, Július Árvay1, Daniel Bajčan1, Judita Bystrická1, Pavol Trebichalský1, Ján Kovarovič1, Ivona Jančo
Address(es): Ing. Ľuboš Harangozo, PhD., 1Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Chemistry, Tr. A. Hlinku 2, 949 76 Nitra, phone number:+4213764134376
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.760-764
ARTICLE INFO ABSTRACT
Received 13. 6. 2018 Hazardous (heavy) metals are widely spreaded in our environment, which is endangered by these hazardous metals that infiltrate water, air, soil, plants and subsequently in the food chain. This has a major impact on human health and animal health, because these metals Revised 31. 7. 2018 can cause serious health problems. For this reason, it is very important to control the occurrence of heavy metals in the environment, Accepted 7. 8. 2018 water, air and soil, and then eliminate them to the permissible limit values. Published 1. 10. 2018 Spices have been used in the past as natural flavors, used to highlight or create flavor. Nowadays, spices are commonly found in different trade chains and different brands. Consumer information is a very important decision-making factor when purchasing, but there Regular article are not always, on the packaging, all the information on harmful substances. For this reason, it is important to clarify the content of the risk metals, as it can prevent health problems and difficulties, and consumers can choose a healthy food (spice). Regarding this fact, the aim of this study was to closely determine the amount of hazardous (heavy) metals in selected kinds of spices (basil, black pepper, marjoram, sweet red paprika) that can cause serious health issues, if the limited levels of these heavy metals described in the Codex Alimentarius of Slovak republic are overstepped. The analyzed spices were purchased from three local markets. The amount of lead (5.59 mg.kg-1) and cadmium (1.38 mg.kg-1) were exceeding the highest acceptable limits stated in the Codex Alimentarius SR (Cd 0,50 mg.kg-1, Pb 5 mg.kg-1) in all three samples of basil. The content of cuprum and zinc was under the highest acceptable limits stated in the Codex Alimentarius of the Slovak Republic. According to these findings, we would recommend more inspections focused on the level of heavy metals in spices.
Keywords: risk mineral compounds, culinary herbs, limit, Codex Alimentarius
INTRODUCTION Pharmacological studies have also showed anti-cancer and antioxidant effects (Selvendiran et al., 2006). Interestingly, piperine also plays an important role in The spices are obtained from spice plants. They are used as a whole spice plants increasing the bioavailability of many drugs (Chithra et al., 2014) by increasing or just a part of the root plant like underground or aboveground. They are used to intestinal absorption, suppressing drug metabolism in the human body, in improve sensory properties, but also have many health benefits to human pulmonary and liver tissues through inhibition of CYP3A4 and P-glycoprotein organism. Species have been used in the past to overburden the odor of foods (Makhov et al., 2012). Piperine as a pungent alkaloid has been identified as the (Habán et al., 2001). Culinary herbs have always been used for their most effective adjuvant to increase the efficacy of tumor necrosis induced organoleptic properties, because they have a distinctive aroma and a pleasant and apoptosis ligand (Sattarinezhad et al., 2015). delicious flavor. Aromatic herbs (basal, coriander, mint, oregano, rosemary, etc.) Capsicum annum L. is an important agricultural crop, not only for its economic are widely used in the food industry, distilleries, candy processing (as aroma), importance, but also for its role in the food, and its unique flavor and aroma. and even as preservatives (Cozzolino et al., 2016). Sweet paprika is studied extensively for its high content of bioactive substances Spices are also used in the pharmaceutical industry. Consumers demand natural with antioxidant activity, such as carotenoids, vitamin C and A, phenolic products and therefore synthetic substances are replaced by natural ones. One of substances (Garcia-Mier et al., 2015) and micro and macro elements. Sweet the alternatives of natural active ingredients are herbs, spices and their extracts as paprika is a rich source of antioxidants in food (Saini and Keum, 2016). a substitute. Their effects were tested primarily on humans, but also on laboratory The experimental proven potential of red paprika is in reducing oxidative stress animals (Zotte et al., 2016). (Cervantes-Pay et al., 2014), inflammation (Spiller Numerous studies have documented antioxidant, anti-inflammatory and et al., 2008), fat intake and body weight (Kawabata et al., 2006). immunomodulatory effects of species that may be associated with The majoran (Origanum majorana L.) belongs to the family Lamiaceae. Old the prevention and treatment of some diseases (Zheng et al., 2016). Egyptians, Greeks and Romans also used it. It has come In folk medicine, they were used for a number of biologically active substances to attention recently, mainly for its useful physiological function and and beneficial effects on health, for example, some antioxidants from species, antimicrobial activity (Roby et al., 2013). It is used fresh or dried in food, such as curcumin from curcumin, eugenol from cloves and capsaicin from beverages, perfumes and cosmetics, for its pleasant and highly desirable aroma paprika (Srinivasan, 2014). (Jiang et al., 2011). Essential oils of spices have been used as preservatives in It has been experimentally proven the management of cellular oxidative stress studies, due to their antimicrobial activity against pathogens transmitted by food based on antioxidant properties and their ability to block (Marques et al., 2015). the production of reactive oxygen species and interference with signaling Ocimum basilicum L. is a rich source of a certain class of secondary metabolites, pathways (Rubio et al., 2013). known as phenolic acids. Basil is rich in rosmarinic, caftaric Black pepper (Pipper nigrum L., family Piperaceae) is one of the most widely and cichoric acids and has antioxidant effects that have a number of beneficial used spices in the world, known for its acrid and penetrating component piperine. effects on human health. Piperine is in principle a bioactive compound, which has been reported to have Purple varieties of basil also contain anthocyanins, a group of red-blue plant anti-carcinogenic, antiasthmatic, antimicrobial, anti-inflammatory and anti-viral dyes, belonging to one of the largest classes of secondary metabolites, called effects (Meghwal and Goswami, 2013). flavonoids. Anthocyanins have anti-inflammatory, anti-carcinogenic and neuroprotective effects that are beneficial to humans (McCance et al., 2016).
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Heavy metal contamination is very dangerous from a health point of view, due RESULTS AND DISCUSSION their carcinogenicity and toxicity (Baraka et al., 2007). These metals enter to the human body through oral administration, inhalation, skin and hair contact The following graphs show the content of the risk metals in selected types of (Eastman et al., 2013; Censi et al.,2011). In the body they cause acute and spices, obtained from markets A, B and C, and then their comparison with the chronic toxicity, damage of the blood component, lung, kidney, liver, and central Codex Alimentarius (CA) of the Slovak Republic. Results are shown in figures 1- nervous and immune system, and sometimes they lead to letalite (Zeng et al., 7, which indicated the average values of each element and limit value according 2016; Vallverdu-Coll et al., 2015). to Codex Alimentarius. Lead (Pb) is a systemic toxic substance that causes serious damage to virtually of all organs, but mainly affects the central nervous system, especially brain development (Duruibe et al., 2007). Lead is a poison that also causes a reduction 30,00 in hemoglobin formation and kidney dysfunction (Nadeem et al., 2006). Pb (II) 25,00 has been identified as one of the most toxic heavy metals because its effects are detrimental to the bloodstream, kidney and human reproductive system (Yu et 20,00 1 market A al., 2011; Huang et al., 2010). - Cadmium accumulates in the human body in the kidneys, liver, breasts, prostate, 15,00 market B
but also in the brain and lungs (Notarachille et al., 2014). Cadmium is dangerous mg.kg to humans for its toxicity; it has undesirable effects on kidney function and also 10,00 market C increased exposure to cadmium results in an increased risk of cancer (Satarug et al., 2010). 5,00 limit value Exposure to cadmium has also shown significant effects on human and animal health, including chemical pulmonary inflammation, bone deformity and renal 0,00 impairment (Bertin and Averbeck, 2006). Excessive exposure to Cd2+ may lead black paprika basil marjoram to a disease that is affecting the cardiovascular system and GIT (Shah and pepper Nahakpam, 2012). Figure 1 The copper content in selected spices from three markets in mg.kg-1,
compared to limit in CA of the Slovak Republic.
MATERIAL AND METHODS The highest copper content was found in basil from market A (20.11 mg.kg-1), in
samples from market B and C was copper content approximately in the same The spices – black pepper (Piper nigrum L.), red sweet paprika (Capsicum level (17.40 mg.kg-1). The copper content in sweet red paprika was annuum L.), basil (Ocimum basilicum L.), marjoram (Origanum majorana L.) approximately 10 mg.kg-1 in all tested samples. The copper content in black were bought in the three local markets (A, B, C) (totally 20 samples). The pepper samples ranged from 10.93 mg.kg-1 (A) to 12.44 mg.kg-1 (B). In obtained results were compared with the Codex Alimentarius of the Slovak marjoram, the copper content ranged from 12.83 mg.kg-1 (A) to 16.81 mg.kg-1) Republic. All spices were grown out of the Slovak Republic. (B). There were observed statistically significant differences in copper content
between marjoram producers. Also, there were observed statistically significant Determination of heavy metals (by AAS) differences in copper content between black pepper samples from tested
producers (Table 3). All the tested samples were bellow the limit value according By analytical weight were weighted 1 g samples of dried spices. Mineralization to Codex Alimentarius. Bielicka-Gieldon and Rylko (2013) reported a copper of samples was in a mixture of distilled water content in black pepper of 10.6 ± 0.03 mg.kg-1, in sweet paprika12.6 ± 0.05 with concentrated nitric acid in a ratio 1:1. The weighed samples were put into mg.kg-1, which is in accordance whit our results. Valtcho, Lyle, and Baoshan Teflon vessels with 5 cm3 of distilled water (2006) reported a copper content in basil 22.5 mg.kg-1. Significant differences of with 5 cm3 of concentrated nitric acid. Closed vessels with the samples were the copper content were also found in marjoram and black pepper in market A, as mineralized (Tab. 1) by microwave digestion unit MARS X-press (USA). opposed to markets B and C. Increasing copper content in selected market spices
is in the following order: sweet paprika ˂ black pepper ˂ marjoram ˂ basil. Table 1 Experimental conditions for element determination by FAAS.
Initialization Duration Power Power Temp. Stage Time time (W) (%) (°C) 90,00 (min) (min) 80,00 Initialization 800 90 15 160 0 70,00 Mineralization 800 90 0 160 20 60,00
Cooling – – – – 20 1 market A - 50,00 After mineralization were analyzed samples filtered through quantitative filter 40,00 market B -2 mg.kg paper MUNKTELL (Germany) grade 390.84 g.m (green) 30,00 market C to volumetric flasks (50 cm3). 20,00 Flasks were refilled with distilled water to the mark and after that was the limit value determination of heavy metals by VARIAN AA 240FS (Australia). The detection 10,00 limits and sensitivity are displayed in table 2. 0,00 black paprika basil marjoram Table 2 Detection limit and sensitivity of the investigated elements. pepper -1 -1 Element Detection limit (mg.l ) Sensitivity (mg.l ) -1 Cd 0.001 0.01 Figure 2 The zinc content in selected spices from three market in mg.kg , compared to limit in CA of the Slovak Republic. Pb 0.02 0.1
Cu 0.002 0.03 The lowest zinc content was detected in black pepper, approximately 12.4 mg.kg- Zn 0.006 0.008 1for all the three markets. The highest content of zinc was observed in basil and Co 0.005 0.05 ranged from 34.86 mg.kg-1 to 48.65 mg.kg-1. In the case of sweet red paprika, the Cr 0.003 0.04 content of zinc ranged from 14.58 mg.kg-1 to 19.51 mg.kg-1. The lowest content Ni 0.008 0.06 of zinc in marjoram was observed in the samples from market not work A (26.9 Mn 0.003 0.02 mg.kg-1), the other samples had approximately the same values around 32.1 Fe 0.005 0.04 mg.kg-1. All the tested values were bellow the limit value according to Codex Analysis determination has not a deviation more than 3%, the gas flow: air: 13.5 Alimentarius. There were observed statistically significant differences between l.min-1, acetylene 2.0 l.min-1. the producers in zinc content in basil and sweet red pepper (Table 3). Bielicka- Gieldon and Rylko (2013) reported the zinc content in black pepper 15.9 ± 0.1 Statistical analysis mg.kg-1, which is comparable to our achieved results. The highest zinc concentration was measured in basil, with the highest content in market network For testing the normal division data were used the Shapiro-Wilk test and the B (48.65 mg.kg-1). The increasing content of zinc in selected market network Kolmogorov-Smirnov test. As a result, there were chosen non- spices is in the following order: black pepper ˂ sweet red paprika ˂ marjoram ˂ parametric test, there was used a Kruskal-Wallis test with a significance level of basil. p-0.05 to testing the statistical hypotheses. To describe the data set was used the arithmetic mean and the standard deviation. All calculations were performed using MS Excel 2016 and XLSTAT (Addinsoft, 2014).
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1,60 3,50 1,40 3,00 1,20 2,50
market A 1
1,00 1 - - 2,00 market A 0,80 market B 1,50
mg.kg 0,60 market B market C mg.kg 0,40 1,00 market C 0,20 limit value 0,50 0,00 0,00 black paprika basil marjoram black paprika basil marjoram pepper pepper Figure 3 The cadmium content in selected spices from three markets in mg.kg-1, Figure 5 The chromium content in selected spices from three markets in mg.kg-1, compared to limit in CA of the Slovak Republic. compared to limit in CA of the Slovak Republic.
The highest amount of cadmium was measured in basal from market A (1.38 Compared to these four spice types, the lowest chromium content was recorded mg.kg-1), which was eight times more than was measured in black pepper and in the black pepper and sweet red paprika. The chromium content in the sweet red paprika of all three markets. The content of cadmium in basil in all marjoram from market C (3.04 mg.kg-1) is more than three times higher as the three markets exceeded the limit of Codex Alimentarius of Slovak Republic. The average amount of chromium in black pepper and sweet red paprika of all three cadmium content in marjoram ranged from 0.37 mg.kg-1 (A) to 0.47 mg.kg-1 (B). market networks. In the case of basil, the chrome content from market network A There were observed no statistically significant differences between the (2.02 mg.kg-1) and market network C (3.05 mg.kg-1) is approximately the same. producers in cadmium content. The cadmium content in sweet red pepper was The rising content of chromium in selected species from market A is in the approximately 1.12 mg.kg-1 in all three markets. Bojňanská, Frančáková and following order: black pepper ˂ sweet red paprika ˂ basil ˂ marjoram. There Pavlisová (2002) analyzed a cadmium content of 0.44624 mg.kg-1 in maize and were observed statistically significant differences between the producers in compared to our measured values, it coincides with all three markets. Valtcho, chromium content in marjoram, black pepper and sweet red paprika (Table 3). Lyle, and Baoshan (2006) reported a cadmium content in basil 0.7 mg.kg-1.
Seven Hungarian spice paprika samples (7/22) were contaminated with cadmium, the average concentration was 0,086 ± 0,002 mg.kg-1 In case of the Serbian (2/5) 9,00 and Peruvian samples (2/2), these values were 0,170 ± 0,001 and 0,254 ± 0,001 8,00 mg.kg-1, respectively. Cadmium contamination was not detectable in the Spanish, 7,00 Chinese and Bulgarian samples (Molnar et al., 2018). The increasing content of 6,00
cadmium in selected species of market network is in the following order: black 1 - pepper ˂ sweet red paprika ˂ pepper ˂ marjoram ˂ basil. 5,00 market A 4,00 market B mg.kg 3,00 6,00 2,00 market C 5,00 1,00
4,00 market A 0,00 1 - black paprika basil marjoram 3,00 market B pepper
mg.kg 2,00 market C Figure 6 The nikel content in selected spices from three markets in mg.kg-1, 1,00 limit value compared to limit in CA of the Slovak Republic. 0,00 black paprika basil marjoram The lowest average values of nickel were recorded in sweet red paprika of all pepper markets (1.75- 2.16 mg.kg-1). The highest nickel content was measured in marjoram in network market C (7.69 mg.kg-1). The nickel content in black pepper -1 Figure 4 The lead content in selected spices from three markets in mg.kg , ranged from 1.70 mg.kg-1 (A) to 4.97 mg.kg-1 (B). There were observed compared to limit in CA of the Slovak Republic. statistically significant differences between the producers in nickel content in marjoram, and basil (Table 3). Bielicka-Gieldon and Rylko (2013) reported a The lowest content of lead in market A, B, C was recorded in black pepper (0.05, nickel content in sweet red paprika of 1.29 ± 0.14 mg.kg-1 and the nickel values -1 0.04, 0.04 mg.kg ). The highest lead content was measured in basil, which even that we measured in the sweet pepper were slightly higher. The increasing exceeded the highest permissible levels for all three markets, compared to the content of nickel in selected species of network market A is in the following Codex Alimentorius of the Slovak Republic. In comparison to the lead content in order: black pepper ˂ sweet red paprika ˂ marjoram ˂ basil. The increasing basil, in at all three brands its content is three times higher than in sweet red content of nickel in selected spices of market B is in the following order: sweet paprika. Increasing lead content in selected market network spices is in the red paprika ˂ basil ˂ marjoram ˂ black pepper. The increasing content of nickel following order: black pepper ˂ sweet red paprika ˂ marjoram ˂ basil. The lead in the selected species of market C is in the following order: sweet red paprika ˂ -1 -1 content in sweet red pepper ranged from 0.90 mg.kg (C) to 1.50 mg.kg (A). In black pepper ˂ basil ˂ marjoram. -1 case of marjoram, the content of lead ranged from 3.02 mg.kg (A) to 4.78 mg.kg-1 (C). Valtcho, Lyle, and Baoshan (2006) reported a lead content in basil Leaves from 26.8 to 33.6mg.kg-1. Lead was detected in 1 out of 22 Hungarian 3,00 spice paprika samples, at a level of 0,334 ± 0,0013 mg.kg-1, below the legal limit 2,50 (Molnar et al., 2018). There were observed statistically significant differences
between the producers in lead content in marjoram, basil and sweet red pepper 2,00 1 (Table 3). - market A 1,50
market B mg.kg 1,00 market C 0,50 0,00 black paprika basil marjoram pepper
Figure 7 The cobalt content in selected spices from three markets in mg.kg-1, compared to limit in CA of the Slovak Republic.
Cobalt content in black pepper and sweet red paprika was found in small quantities. The highest cobalt content, at almost the same level (approximately
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2.3 mg.kg-1) in all markets was recorded in basil. Cobalt content in marjoram DURUIBE, J. O., OGWUEGBU, M. O. C., EGWURUGWU, J. N. 2007. Heavy from market A (0.80 mg.kg-1) was three times lower than in market C (2.41 metal pollution and human biotoxic effects. Journal of Physical Sciences, vol. 2, mg.kg-1). Increasing cobalt content in selected species of market A was recorded 112-118. ISSN: 1992-1950. in the following order: black pepper ˂ sweet red paprika ˂ marjoram ˂ basil. EASTMAN, R. R., JURSA, T. P., BENEDETTI, C., LUCCHINI, R. G., SMITH, Increasing cobalt content in selected species of market B was in the following D. R. 2013. Hair as a biomarker of environmental manganese exposure. order: black pepper ˂ sweet red paprika ˂ marjoram ˂ basil. Increasing cobalt Environmental science & technology, vol. 47, 1629-1637. 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EFFECTS OF SOLVENT TYPES AND CITRIC ACID CONCENTRATIONS ON THE EXTRACTION OF ANTIOXIDANTS FROM THE BLACK RICE BRAN OF ORYZA SATIVA L. CV. HOM NIN
Anek Halee1, Piyawan Supavititpatana2, Khanitta Ruttarattanamongkol3, Nitipong Jittrepotch3, Kamonwan Rojsuntornkitti3 and Teeraporn Kongbangkerd*3
Address(es): 1Division of Food Science and Technology, Faculty of Science and Technology, Kamphaeng Phet Rajabhat University, Kamphaeng Phet 62000, Thailand. 2Division of Food Science and Technology, Faculty of Food and Agricultural Technology, Pibulsongkram Rajabhat University, Phitsanulok 65000, Thailand. 3Department of Agro-Industry, Faculty of Agriculture, Natural Resources and Environment, Naresuan University, Phitsanulok 65000, Thailand.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.765-769
ARTICLE INFO ABSTRACT
Received 4. 5. 2018 Thai colored rice has been reported as a potential source of antioxidants. This research aimed to study the effects of different solvents on Revised 10. 8. 2018 the antioxidant extraction of Hom Nin rice bran. The solvents (water, methanol and ethanol) combined with citric acid at concentrations 3 Accepted 10. 8. 2018 of 0, 0.05 and 0.1 mol/dm were used for the extraction. The antioxidant activities and the contents of phenolics, flavonoids and 3 Published 1. 10. 2018 anthocyanins were determined. The results showed that the methanolic extract with 0.1 mol/dm of citric acid gave the highest yield whereas phenolics, flavonoids and anthocyanin were 43.72%, 86.63 mg of GAE/g DM, 14,667.48 µg CE/g DM and 6.18 mg/g DM, respectively. Moreover, the antioxidant activities as ABTS, DPPH and FRAP were 25.76, 29.02 and 140.57 µmol TE/g DM, Regular article respectively. In addition, the total phenolic contents of the extract indicated a highly positive correlation with antioxidant activity.
Keywords: Extraction, Antioxidant, Hom Nin rice bran, Citric acid
INTRODUCTION the cost of extraction. The temperature used for extraction needs to be determined due to some instability of the antioxidant compounds with heat (Benchahem et. Rice (Oryza sativa L.) is the most important cereal crop cultivated in the world, al., 2015). For those antioxidants that react to alkaline and neutral solutions, which is the staple food for more than half the world’s population (Danielski et acidic aqueous solvents have been used for extracting, so to disrupt the cell al., 2005; Lu et al., 2008; Xia et al., 2006). It is believed to provide more health membranes and at the same time dissolve the water-soluble antioxidative benefits than any other carbohydrate-based foods since rice contains several compounds. Usually, citric acid, hydrochloric acid, and acetic acid are chosen for nutrients and antioxidant compounds (Vijittra et al., 2011). In addition, colored acidulating the extraction solvent but using citric acid is also safe for human rice consumption has been rapidly incremental (Kong and Lee, 2010). It has consumption (Li et al., 2012; Mateus and Freitas, 2009). The most commonly further been reported that colored rice has a higher antioxidant content in its seed used solvents for antioxidant extraction are ethanol or methanol and water coat or pericarp than non-colored rice (Yodmanee et al., 2011). (Garcia et al., 1998). The objectives of this study were to investigate the effects The main antioxidants in the colored rice seed coat are flavonoids, mainly of extracting solvents and the concentration of citric acid on phenolic compounds anthocyanins, a subgroup of phenolic compounds (Abdel-Aal et al., 2006). as well as to evaluate the antioxidant activity from the Hom Nin rice bran extract. Furthermore, black rice has been known to provide health benefits that can Thus, the natural antioxidants and also the color from the extracts of by-product reduce the risk of chronic diseases; such as, cancers and cardiovascular problems materials could then be possibly used to substitute synthetic antioxidants and
(Xia et al., 2006), diabetes and its complications (Walter and Marchesan, 2011) colors. since it contains various phytochemicals, especially phenolic compounds (Shen et al., 2009). It has also been shown that black rice has a profitable contribution MATERIAL AND METHODS to nutritional and therapeutic values in comparison to white rice (Chen et al., 2006 ;Choi et al., 2007). Rice samples Hom Nin rice is a kind of black rice and it contains a higher nutritional value than that of other strains of rice including protein, vitamins and minerals (Suzuki et Freshly milled Hom Nin rice bran (after milling up to 24 hours) was collected al., 2004). In addition, the contents of vitamin B, niacin, vitamin E, calcium, from the Nongpingkai Rice Mill Group, Amphoe Mueang, Kamphaeng Phet magnesium, iron and zinc were higher when compared to white rice and it could province, Thailand. The bran was sifted to separate the rice grains from the bran, also possibly be a significant potential source of anthocyanins. Anthocyanins in vacuum packed in aluminum foil bags, and stored at -20°C. foods provide advantages in anti-cancer prevention, liver protection, prevention of heart disease, decrease of dyslipidemia, reduction of coronary heart disease Chemicals and reagents and improvement of visual acuity (Chen et al., 2006; Lee, 2010; Mazza and Miniati, 1993). (+)-Catechin hydrate, 2,4,6-tripyridyl-s-triazine (TPTZ), 2,2-diphenyl- 1- Rice bran is a by-product from rice milling and comprises about 10% of the total picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic rice grain, which is normally used as animal feed. It also contains the same acid (Trolox), gallic acid, and 2,2’-azino-bis (3- ethylbenzothiazoline-6-sulphonic nutrients and antioxidative compounds as brown rice including tocopherols, acid) diammonium salt (ABTS) were purchased from Sigma–Aldrich (St. Louis, tocotrienols, phytic acid, and tricin as well as pigments (Vijittra et al., 2011) in MO, USA). Folin-Ciocalteu’s reagent, 2-diohenyl-1-picryhydrazyl (DPPH), which most of them are stored in the bran or pericarp of the rice kernel, and the ethanol, methanol, potassium chloride, sodium acetate, and sodium carbonate different pigmented compounds are related to distinct colors; such as, red, purple were purchased from Merck (Darmstadt, Germany). Aluminum chloride hydrate, and black (Hu et al., 2003). potassium persulfate, hydrochloric acid and acetic acid were purchased from Extraction of antioxidants has become an alternative method for developing black Ajax Finechem Pty Ltd. (Auckland, New Zealand). All chemicals and solvents rice waste to be a more valuable product. However, the antioxidant stability of were of an analytical reagent grade. the extract is a matter of great concern as it affects the extraction process and also
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Sample preparation Ferric reducing antioxidant power (FRAP)
Hom Nin rice bran was sieved through a 100 mesh (150 µm) sieve and heated by The ferric reducing antioxidant power (FRAP) assay was determined using a a hot air oven at 100๐C (Memmert UNE 500, Germany) for 15 minutes to modified method of Benzie and Strain (1996). The FRAP reagent was inactivate the endogenous lipase and then cooled to room temperature (Juliano, performed by using 300 mM of acetate and glacial acetic acid buffer (pH 3.6), 10 1985). The rice bran was defatted with hexane by using the ratio of the bran and mM of TPTZ (4,6- tripyridyl-s-triazine) solution in 40 mM HCl and 20 mM of solvent at 1:10 (w/v) for two hours in a shaker at 100 rpm (room temperature). ferric chloride. The working FRAP reagent was freshly prepared by mixing the The homogenized mixture was filtered through Whatman No. 1 filter paper and three solutions together in the ratio of 10:1:1 and the reagent was incubated at re-extracted twice using the same conditions and then dried in a hood for 12 37oC in a water bath (Memmert WB 22, Germany). Three milliliters of the FRAP hours to remove the solvent residual. Then, the rice bran was packed in reagent was mixed with 100 μl of the sample and incubated at 37oC for 30 aluminum foil bags and stored at -20๐C (Kim et al., 2013). minutes. The absorbance was read at 593 nm using a spectrophotometer (Thermo Scientific Genersis10, USA) and expressed as micromoles of the Trolox Extraction of the Hom Nin rice bran powder equivalents per gram of the DHRB dry matter (μmol TE/g DM).
Five grams of defatted Hom Nin rice bran (DHRB) were extracted by 0, 0.05 and ABTS radical scavenging assay 0.10 mol/dm3 of citric acid in water, 99.9% methanol and absolute ethanol (1:10 w/v) with a rotary shaker (Gemmy Orbit Shaker VRN-480, Taiwan) at 100 rpm The ABTS assay was determined according to Arnao et al. (2001) with some for two hours at room temperature. After pouring out the supernatant, the modifications. The ABTS radical cation was generated from 7.4 mM of the ABTS solution with 2.6 mM of potassium persulfate. The mixture was allowed to precipitates were re-extracted using the same procedure. Both supernatants were •+ then combined and the methanol and ethanol were evaporated by using rotary stand in the dark at room temperature for 12-16 hours before use. The ABTS evaporator (Buchi R 205, Switzerland) at 40๐C and 100 mbar before freeze solution was diluted with methanol to obtain an absorbance of 1.10±0.02 at 734 nm. One hundred μl sample solutions were mixed with 3 ml of the ABTS•+ drying and the dried extracts were then used for phytochemical analyses solution and allowed to stand in the dark for 30 minutes at room temperature. The (Graciele et al., 2011; Nontasan et al., 2012; Tananuwong and Tewaruth, absorbance was then measured at 734 nm using a spectrophotometer (Thermo 2010). For freeze drying, the samples were previously frozen and then put into a Scientific Genersis10, USA) and the results were expressed as micromoles of the chamber (Christ alpha 1–4 LD plus, Germany) at -55 ๐C under pressure of 0.05 Trolox equivalents per gram of the DHRB dry matter (μmol TE/g DM). bar and were maintained under these conditions for 48 h. Total anthocyanin content Extraction yield The total anthocyanin content was determined using the pH-differential method, The extraction yield is a measure of the solvent efficiency to extract specific which was slightly modified from Finocchiaro et al. (2010). The DHRB sample components from the original material. In the case of DHRB, this would provide was diluted with 0.025 M of the potassium chloride buffer, pH 1.0 and separately some understanding about the extractability of the antioxidant activity under with 0.4 M of the sodium acetate buffer, pH 4.5 and left for 15 minutes before different solvents. Thus, this could be calculated according to the method of taking the absorbance measurements. The absorbance in each buffer was Zhang, Bi, and Liu (2007) as follows: measured at λmax (520nm) and at 700 nm using a spectrophotometer (Thermo Scientific Genersis10, USA). Distilled water was used as a blank. Extraction yield (%) = (weight of the freeze-dried extract x 100) / (weight of the The total anthocyanin content was calculated as equivalent to cyanindin-3- original sample) glucoside according to the following equation:
Preparation the extract solution for assays Total anthocyanin content = (ΔA × MW × DF × 1000) / e)
For the determination of the bioactive compounds and antioxidant activity, the Where extracts were diluted with water using the ratio of 1:19 (extract : solvent, w/v) ΔA= (Absλ520-Absλ700) pH 1.0 - (Absλ520-Absλ700) pH 4.5 (Zhang et al., 2007). MW (molecular weight) = 449.2 g/mol for cyanidin-3-glucoside DF = dilution factor Determination of the total phenolic content e = is the molar extinction coefficient, equaling 26,900 L/mol cm for cyanidin-3- glucoside. The total phenolic content of the DHRB extract was determined using the method 1000 = conversion factor from g to mg. of Folin- Ciocalteu’s reagent as described by Singleton et al (1999). Briefly, 100 μl of the extracts solution were mixed with 3 ml water, water-diluted Folin- The total anthocyanins were expressed as mg of cyanidin-3-glucoside equivalents Ciocalteu’s reagent (1: 10 v/v), and 2 ml of sodium carbonate (7.5% w/v). The per 1 gram of rice bran. mixture was kept in the dark at room temperature for two hours. The absorbance was measured at 760 nm using a UV-Vis spectrophotometer (Thermo Scientific Statistical analysis Genersis10, USA). Gallic acid was used as a standard and expressed as milligrams (mg) of gallic acid per gram of DHRB dry matter (mg/g DM). The measurements were carried out in three replicates, and all data were subjected to the analysis of variance (ANOVA). Significant differences among Determination of the total flavonoid content the treatments were analyzed by Duncan’s multiple range test (DMRT) at a 95% confidence level (p<0.05). The total flavonoid content was determined by Yang et al. (2009) with some modifications. 100 μl of the sample was mixed with 1.25 ml of deionized water RESULTS AND DISCUSSION and 75 μl of 5% of sodium nitrite and then incubated in the dark at room temperature for six minutes. 150 μl of 10% of the aluminum chloride solution Effects of the solvent type and citric acid concentration on the yields and was added and allowed to stand in the dark at room temperature for five minutes antioxidant activity of DHRB before the addition of 0.5 ml of 1.0 M sodium hydroxide. The absorbance was measured at 510 nm using a UV-Vis spectrophotometer (Thermo Scientific To obtain the yields and antioxidant activity with the lowest changes of the Genersis10, USA). compared to the catechin standard. The total flavonoid functional properties of the extract required, the extraction technique is one of the content of the sample was expressed as mg of catechin equivalents per gram of most important stages (Zhu et al., 2010). In this experiment, the effects of the DHRB dry matter (mg CE/g DM). solvent type on the yields of extract are summarized in Table 1. The solvents used for the extraction of DHRB showed statistically significant DPPH radical scavenging assay different yields (P<0.05). The DHRB extract yields using different solvents with various citric acid concentrations were between 2.41 to 43.72%. The yields of the The DPPH method was determined as described by Brand-Williams et al. DHRB extracted by using methanol were higher than those of water and ethanol, (1995) as modified by Zhang et al (2007). Briefly, 100 μl of the sample was and the extraction yields rose with the increasing concentration of citric acid from added to 3 ml of 100 µM DPPH radical solution, which was freshly prepared. 0 M to 0.1 M (P<0.05). Although, a higher yield was observed when using 0.1 The reaction mixture was agitated and allowed to stand at room temperature in M. of citric acid with water and methanol, there was no significant difference the dark for 30 minutes. The absorbance at 517 nm was used to measure the (P≥0.05). Since, the viscosity of the solvent affected the extraction’s efficiency, concentration of the remaining DPPH using a spectrophotometer (Thermo the viscosity of methanol was lower than that of water and ethanol (0.59, 0.89 Scientific Genersis10, USA). The DPPH free radical scavenging activities of the and 1.07 centipoises) (Alam et al., 2018). Therefore, higher extraction yields in extracts were expressed as µM of the Trolox equivalents (TE) per gram of the general were obtained by the less viscous solvents (Wijekoon et al., 2011). DHRB dry matter using a standard curve of Trolox (μmol TE/g DM).
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The antioxidant activity of the extracts from DHRB determined as ABTS, DPPH viscosity and stronger polarity, as it could spread into a plant’s cells faster and and FRAP are presented in Table 1. It was found that both the solvent type and provide a higher extraction capacity. Antioxidants in most plants are normally the citric concentration affected the ABTS value of the DHRB extract polar substances and due to the polar nature of the water molecule itself, significantly (p<0.05). Therefore, it was likely that the efficiency of the antioxidants are generally able to dissolve in water. The antioxidant activity of antioxidant activity was then increased with increasing citric acid concentration, the phenolic compounds depended on a number of hydroxyl categories and which was the same trend as the increase of total phenolic contents (Figure 1). properties to catch electrons among carboxylic acid in a molecule capable of Table 1 shows the efficiency of the antioxidant activity of the extract of DHRB. providing a decrease in hydrogen (Moongngarm, 2012) , as well as functional The results reveals that the DPPH values were significantly influenced by the groups; such as, groups bearing polar molecules. The obtained results when using type of solvent and the concentration of citric acid (p<0.05). It has also been water for the extraction of antioxidants had the same trend as the number of noticed that the efficiency of antioxidants was decreased when using water with a phenolic compounds (Figure 1). The antioxidants in rice are mainly phenolic higher citric acid concentration, whereas the extraction with ethanol provided compounds and are immobilized in cellulose, lignin and proteins; such as, ferulic higher antioxidant efficiency when using citric acid at a higher concentration. acid which contains 93% of the total volume (Jirum, and Srihanam, 2011; Li, The decrease of DPPH values of DHRB when using water with higher acid et al., 2012). The acidic extraction could somehow facilitate the release of concentration may be due to the free form of phenolic compounds obtained from phytochemicals by breaking the plant’s cells’ walls and the performance of the water extraction were destroyed by the acid (Ammar et al., 2015; Bridgers et extraction would depend upon the solvent and its concentration (Rayle and al., 2010; Rayle and Cleland, 1992). It can be noted that at atmospheric Cleland, 1992; Vadivel and Brindha, 2015). Moreover, the DHRB extract pressure, the dielectric constant (index of polarity) of water (80.1) is higher than obtained from different solvents would be composed of several chemical that of ethanol (24.5) (Cuevas et al, 2014) so that the polyphenols can be compounds which exhibited different antioxidant activity. Extraction with water dissolved more in water. By the way, the increase of polarity of the acidified tended to decrease the antioxidant activity when using a higher concentration of ethanol seemed to improve the solubility of polyphenols which then provided citric acid, while using alcohol with higher citric acid provided higher antioxidant better antioxidant activity. activity. As such, the results of this present study were in accordance with The efficiency tests for the antioxidant activity of the extracts from DHRB by Jianmei et al. (2004) who reported the antioxidant extraction of peanut skin using a FRAP assay also found that using different solvents with different citric using water and 80% of ethanol concentration and found that extraction by using acid concentration affected the FRAP values (p<0.05). In addition, the extraction water gave better antioxidant activity than that of ethanol. Furthermore, the with water, ethanol and methanol would likely diminish the antioxidant activity ABTS values were 4.10 and 3.39 μmol of the Trolox equivalents per 1 gram of when a higher citric acid concentration was used due to the above mentioned the dried sample, respectively. Pinelo et al. (2004) extracted antioxidants from reasons. the bark of almonds with water, methanol and ethanol and found that the Considering the antioxidant activity values obtained from the DHRB extract from methanolic extract had better antioxidant activity than that of water and ethanolic the three assays, the results showed that the solvent type and the citric acid extracts, respectively. Moreover, Anwar et al. (2013) investigated the methanolic concentration had a great impact on the overall antioxidant activity. Methanol and ethanolic extracts for the antioxidant activity of cauliflowers, which were could be used to extract phenolic compounds and also antioxidants better than dried using a different drying condition and found that the methanolic extract water and ethanol, respectively. Alcohol is normally used in antioxidant gave better antioxidant activity. extraction and is better than water because of its smaller molecular size, less
Table 1 Yields and antioxidant activities of DHRB extracts by using different extracting solvents determined by DPPH, FRAP and ABTS assays from Antioxidant activity (µmol Trolox equivalents/g) Yield of powder Solvent extract (%) ABTS DPPH FRAP Water 22.73±0.51d 24.18±1.44 bc 29.47±0.58 b 153.40±2.72 c 0.05 M of citric acid with water 32.95±2.32 b 23.41±0.39 bc 23.42±0.14 d 92.65±1.92 f 0.1 M of citric acid with water 41.34±2.56 a 22.84±0.59 c 22.47±0.36 e 65.14±1.36 h Methanol 8.77±0.69 f 25.73±0.57 a 30.56±0.27 a 160.89±0.72 b 0.05 M of citric acid with Methanol 25.41±1.00 c 25.76±0.55 a 29.82±0.48 b 189.21±0.66 a 0.1 M of citric acid with Methanol 43.72±1.29 a 25.76±0.55 a 29.02±0.82 c 140.57±0.47 d Ethanol 2.41±1.03 g 18.63±0.21 d 9.26±0.32 g 155.75±2.71 c 0.05 M of citric acid with Ethanol 15.40±0.27 e 24.57±1.18 ab 8.63±0.17 g 98.67±0.26 e 0.1 M of citric acid with Ethanol 22.18±1.69 d 25.78±1.05 a 11.29±0.23 f 70.48±0.91 g *Different letters within the same row indicate statistical differences (one-way ANOVA and Duncan test, P< 0.05). Values are mean ± S.D of triplicate determinations
Effects of the solvent type and citric acid concentration on the total phenolic (temukut) and found the methanolic extract provided a higher phenolic content of DHRB compound than that of water. Pinelo et al. (2004) conducted the extraction of phenolic compounds from pine sawdust using different solvents; i.e., water, Figure 1 shows that the type of solvent and concentration of citric acid methanol and ethanol and found that methanolic extract had the highest phenolic significantly affected the amount of the phenolic compounds in DHRB (p<0.05). compounds followed by ethanolic and water extract, respectively. Jianmei et al. The methanolic extract gave the highest phenolic compound followed by the (2004) extracted phenolic compounds from the skin of peanuts using water, water and ethanolic extracts, respectively. By using methanol and ethanol for the methanol and 80% of ethanol concentration and found that the methanolic extract extraction, it could be shown that the number of phenolic compounds were likely provided the highest phenolic compounds followed by ethanolic and water to rise with an increasing citric acid concentration while the extraction using extracts as 90.1, 89.9 and 56.7 mg of gallic acid/g, respectively. In addition, the water presented a lower number of phenolic compounds but when using a lower results from this present study were consistent with Bahar et al. (2009) who citric acid concentration, the obtained phenolic compounds were higher. studied the effect of ethanol with various amounts of 30% of citric acid on the Consequently, it seemed that alcohol and water were opposed to each other in extraction of phenolic compounds from olives and found that using 10 ml of interacting with the citric acid concentration. DHRB extracted by methanol with citric acid gave the highest phenolic compounds and when using a higher amount 0.1 M of citric acid concentration had the highest phenolic compounds of 86.63 of citric acid, the phenolic compound was reduced. This was because phenolic mg of gallic acid/g and the highest phenolic compounds extracted by water only compounds normally found in most plants comprise three different groups; i.e., a and ethanol with 0.1 M of citric acid were 52.54 and 13.35 mg of gallic acid/g, free form, conjugated form and bound form. The bound form is the group, which respectively. Almost all of the phenolic compounds in plants are mainly in the is mainly found in the layer of lignin extracted by using acidic or alkaline form of a water-soluble form (Moongngarm, 2012), and the extraction of hydrolysis. The free form is the second group, but the structure is rarely stable so phenolic compounds often uses organic solvents since they can be dissolved by when using an acidic extraction of phenolic compounds, the structure would be the same principles. Additionally, the organic solvents used for the extraction of partially destroyed. However, when using acid at a higher concentration, the phenolic compounds usually included methanol and ethanol (Jirum and plant’s cells’ walls were ruptured; hence, the more active ingredients were Srihanam, 2011), and ethanol was normally used because it is safer than other released. The results of this present study revealed that each solvent had a certain types of organic solvents. Thus, the obtained results were consistent with the citric acid concentration for the optimum extraction condition as well (Adom experiments of Arab et al. (2011) who studied the type of solvent used in the and Liu, 2002; Choi et al., 2007; Zhou et al., 2004). extraction of phenolic compounds from two rice cultivars (Fajr and Tarem) and found that the methanolic extract gave the highest phenolic compounds followed by ethanolic and ethyl acetate extract, respectively. Tan et al. (2013) studied the use of water and methanol in the extraction of phenolic compounds from rice
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consistent with the study of Li et al. (2012) who reported the use of microwave 100 a assisted extraction of anthocyanins from grape peels with citric acid. The results 90 showed the factors that mostly affected the anthocyanin content were the 80 concentration of citric acid, the extraction time, the power of the microwave, and b 70 the ratio between the sample and the solvent, respectively. Moreover, the 60 c anthocyanin content was increased with increasing citric acid concentration. d 50 e f 7 40 a 30 6 b g mg of gallic acid/g gallicofmg 20 h h 5
10 glucoside/g
- 3 0 - 4 c
d
0.1M water
water 3
water
0.1M 0.05M
0.05M e
0.1M
Ethanol Ethanol Ethanol
0.05M f
Methanol Methanol Methanol 2 g Figure 1 Total phenolic contents of DHRB extracts by using different extracting h h solvents. *Different letters indicate statistical differences (one-way ANOVA and Duncan cyanidinofmg 1 test, P< 0.05). 0
Effects of the solvent type and citric acid concentration on the flavonoid
0.1M
water water
content of DHRB water
0.1M
0.05M
0.05M
0.1M
Ethanol Ethanol Ethanol
0.05M
Methanol Methanol Methanol The study found that the type of solvents and the concentrations of citric acid statistically affected the amount of flavonoids extracted from DHRB (p<0.05). Figure 3 Anthocyanin contents of DHRB extracts by using different extracting The treatments extracted by using methanol gave the highest flavonoids followed solvents. *Different letters indicate statistical differences (one-way ANOVA and Duncan by those with water and ethanol, respectively. The extraction using methanol and test, P< 0.05). ethanol tended to increase the flavonoid contents when increasing the citric acid concentration while the flavonoid contents of the water extract was decreased CONCLUSION with an increase of the citric acid concentration. The extraction using methanol with 0.1 M of citric acid concentration gave the highest flavonoid content of The results of this study showed that the yield of phenolic and flavonoid contents 14.67 mg catechin equivalents/g, and other solvents provided a flavonoid content as well as the efficiency of antioxidant activity had been affected by the solvent in the range between 2.52-13.66 mg catechin equivalents/g. The obtained results types and citric acid concentration used for antioxidant extraction from colored were the same as the phenolic contents, as the flavonoid antioxidants were in the rice, and the DHRB methanolic extract with 0.1 M. of citric acid gave the highest phenolic compound group (Liu, 2004). antioxidants. The extraction using water and ethanol without acid found a similar efficiency of antioxidant activity. However, using citric acid in the extraction 16,0 b a showed a trend of decreasing antioxidant activity and the anthocyanin content b 14,0 from DHRB extracted by using acidified water tended to increase with an c increasing citric acid concentration. 12,0 10,0 Acknowledgement: This work was financially supported by the Doctoral 8,0 Research Scholarship, Faculty of Agriculture, Natural Resources and Environment, Naresuan University. The authors would like to thank all staffs of d d 6,0 e the faculty for maintenance and operation of the scientific laboratory and f 4,0 g equipment. 2,0
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INFLUENCE OF PARTIALLY HYDROLYZED GUAR GUM AS SOLUBLE FIBER ON PHYSICOCHEMICAL, TEXTURAL AND SENSORY CHARACTERISTICS OF YOGHURT
Deepak Mudgil*
Address(es): Department of Dairy and Food Technology, Mansinhbhai Institute of Dairy and Food Technology, Mehsana, Gujarat-384002, India.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.794-797
ARTICLE INFO ABSTRACT
Received 17. 6. 2018 The effect of partially hydrolyzed guar gum fortification on physicochemical, textural and sensory characteristics of yoghurt was Revised 22. 8. 2018 investigated. Fiber fortification was done at 1-5% levels to study the effect of soluble fiber fortification on the quality of yoghurt. Fiber Accepted 28. 8. 2018 addition significantly influenced the textural characteristics of yoghurt such as hardness, adhesiveness, cohesiveness, springiness and Published 1. 10. 2018 gumminess (p<0.05). Water holding capacity and viscosity was highest in yoghurt sample with 3% fiber while syneresis was lowest for the sample (p<0.05). The viscosity of the yoghurt samples increased with the fiber addition upto 3% level fortification, thereafter it decreased (p<0.05). Inclusion of soluble dietary fiber at 3% level in milk produced yoghurt with improved texture, physicochemical, Regular article higher nutritive value and desirable sensory characteristics.
Keywords: soluble fiber; syneresis; yoghurt; texture; sensory
INTRODUCTION gum, gum acacia, oat fiber and soy components can be used for fiber fortification in yoghurt. A lot of research work has been carried out on soluble fiber Yoghurt also known as Dahi in India is well known fermented dairy product in fortification of bakery products, processed foods, beverages etc, but research on the Indian sub-continent and forms an important part in Indian diet. Yoghurt is fiber fortification in yoghurt is very limited. However, the effect of dietary fiber known with different names in different countries such as Leben (Iran), Jugurt on Misti Dahi quality is reported in literature (Raju and Pal, 2014). Partially (Turkey), Roba (Iraq), Shosim (Nepal) etc (Tamime and Robinson, 2007). hydrolyzed guar gum is a low viscosity water soluble product which can be Yoghurt is so popular in India that a meal is considered incomplete without prepared by enzymatic hydrolysis of native guar gum (Mudgil et al., 2014). consuming it towards the end of the meal. In southern parts of India, yoghurt is Partially hydrolyzed guar gum contains magnificent amount of soluble fiber eaten with rice and is popularly known as curd rice and is used to prepare several (Mudgil et al., 2012a; Mudgil et al., 2012b). Native guar gum is generally used culinary dishes. The beneficial aspects of yoghurt and other fermented dairy as stabilizer and thickener in various food products such as tomato ketchup, ice products are well documented in the literature (Yadav et al., 2008; Bhat and cream, beverages etc (Mudgil et al., 2011). Partially hydrolyzed guar gum is low Bhat, 2011). Few lactobacilli were also isolated from yoghurt which produced molecular weight galactomannan having low viscosity, colorless, tasteless, bacteriocins effective against human pathogens (Bhattacharay and Das, 2010). odorless in nature and hence do not affect the product characteristics (Mudgil et Thus, the therapeutic properties of yoghurt are well known, hence yoghurt is used al., 2016a; Mudgil et al., 2016b; Mudgil et al., 2016c; Mudgil et al., 2016d; in several of Ayurvedic formulations which is a traditional Indian medicinal Mudgil et al., 2018). In present study, partially hydrolyzed guar gum was system (Devi et al., 2010). Yoghurt is prepared from buffalo milk as well as cow selected for soluble fiber fortification of yoghurt and its effect on the milk. Yoghurt prepared from buffalo milk is white in colour and possesses firm physicochemical, textural and sensory characteristics of yoghurt. body and slightly granular texture, whereas yoghurt prepared from cow milk is les firm and smooth in texture. Hence mixed milk is generally used for making MATERIALS AND METHODS yoghurt. These differences in final product quality are attributed to inherent variations in the protein make up of both the milks (Aneja et al., 1965). The Raw materials and ingredients organized sector dairies are major players in yoghurt market. It is estimated that 6.9% of total milk produced in India is utilized for yoghurt making in India Pasteurized standardized milk with 4.5% fat and 8.5% SNF was procured from (Tamime and Robinson, 2007). Yoghurt is also used as a vehicle to incorporate Dudhsagar Dairy, Mehsana, India and stored in refrigerated conditions until use. probiotics into the health conscious consumers because of the fact that it is A freeze-dried direct vat set (DVS) yogurt culture (RST-744 & CHN-11) consumed by people daily (Yadav et al., 2008). containing a mixed strain of thermophilic and mesophilic homofermentative Yoghurt is good source of calcium, phosphorus, vitamin B2, vitamin B12, bacterial culture was obtained from Chr. Hansen Inc. (Milwaukee, WI). The pantothenic acid-vitamin B5, zinc, potassium, protein, iodine and molybdenum. culture was stored at −18°C until use. Partially hydrolyzed guar gum which is an All these nutrients make yoghurt a nutritious and health-supportive food. Yoghurt enzymatically hydrolyzed guar gum with low viscosity was procured from Lucid has been attributed nutraceutical, therapeutic and probiotic effects, such as Colloids Ltd., India. digestion enhancement, immune system boosting, anti-carcinogenic activity and reduction in serum cholesterol. Yoghurt is deficient in iron, vitamin C & dietary Preparation of yoghurt fiber like milk and milk products. Ever-growing consumer demand for convenience, combined with a healthy diet Control yoghurt sample was prepared using pasteurized standardized milk of 4.5 and preference for natural ingredients has led to a growth in functional beverage % fat and 8.5% SNF. The milk was heated to 42 °C on a bench-top stirring hot markets. Yogurt is considered a healthy food and incorporating dietary fiber will plate (Nova Instruments Pvt. Ltd.). Milk was inoculated with RST-744 (0.1 make it even healthier. In the present study, soluble dietary fiber fortification of unit/litre) & CHN-11 (0.01 unit/litre) culture blend and mixed thoroughly in the yoghurt was carried out to make it a complete food. The beneficial role of dietary milk. The milk was transferred to 50 ml, 100 ml & 250 ml cups with lids (Fig.1). fiber in human nutrition has led to a growing demand for incorporation of novel The samples were incubated at 42 °C in incubator (Patel Instruments Ltd, India) fiber into foods (Mudgil and Barak, 2013). There is little information about for 7 hrs. For fiber fortified yoghurt, soluble dietary fiber was added to the fiber fortification in cultured dairy products. Various fibers like psyllium, guar standardised milk at 1-5% levels. To allow good dispersion of the fiber in milk,
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the fiber was sprinkled in vortex of milk using laboratory stirrer at 800 rpm. The were calculated from the obtained profiles using the software provided by Stable milk was processed further similarly as control yoghurt. After incubation, Microsystems. yoghurts were immediately cooled in an ice water bath and stored at 10°C for 16 h. All the yoghurts were prepared in triplicate and the results were expressed as Sensory evaluation mean. Sensory evaluation of control yoghurt and fiber fortified yoghurt was done using 9-point Hedonic scale. 25 panelists were selected on the basis of their previous experience and knowledge of sensory evaluation of dairy and dairy-associated products. Color and appearance, body & texture, flavor and overall acceptability were evaluated by panel. All samples were removed from the refrigerator 1 h before the beginning of every evaluation session. Serving temperature range for samples was 10 to 12°C. Each yogurt was presented in a 100-g plastic cup fitted with lid and labelled with a 3-digit code. Order of presentation of samples was randomized. Water and expectoration cups were also presented to each panelist to rinse their mouths between samples. Evaluation was divided into 3 sections: visual, texture, and flavor evaluations. For visual attributes, the surface of each yogurt was examined in terms of free whey. After that, texture and flavor evaluations were conducted.
Statistical analysis Figure 1 Process for preparation of yoghurt
The experimental data collected was analyzed for significant differences with the Titratable acidity and pH help of analysis of variance (ANOVA) conducted using SPSS 16.0 software.
The titratable acidity values of yoghurt samples were determined after mixing 10 RESULTS AND DISCUSSION g of yoghurt sample with 10 ml of hot distilled water (90°C). Phenolphthalein was used as an indicator. The mixture was then titrated with 0.1 N NaOH to an Titratable acidity and pH end point of just appearance of faint pink color. pH of yoghurt samples were measured using glass electrode digital pH meter. All the measurements were Titratable acidity of control and fiber fortified yoghurt ranged between 0.76-0.78 made in triplicate and the results were expressed as mean. (% lactic acid) as presented in Table 1. Titratable acidity of yoghurt showed an
increasing trend with increase in level of fiber fortification. pH of yoghurt Syneresis samples ranged from 4.40 to 4.43. The results of titratable acidity and pH showed
no significant changes in control yoghurt and fiber fortified yoghurts. However, Syneresis in yoghurt samples was determined using drainage method as described slight increase in the titratable acidity and lower pH values could be attributed to by Chawla and Balachandran (1994) with slight modifications. In present enhanced levels of lactic acid development in yoghurt samples. The results were study, after about 16 h of storage under refrigeration, the set yoghurt cups were in concordance with the study reported in literature (Hashim et al., 2009). taken out and tempered at 25°C for 2 h. The contents of the yoghurt cups were loosened with the help of a spatula from the sides and directly emptied into a Table 1 Physicochemical properties of control and fiber fortified yoghurt glass funnel with a Whatman No.1 filter paper. The funnel was placed on a Dahi Titratable pH WHC (%) Syneresis Viscosity graduated glass cylinder (17×2.5 cm) of 50 ml capacity. The funnel with cylinder Acidity (ml/50 g) (S-62, 5 was kept in a room maintained at 25°C. The quantity of whey collected in (%) rpm, 20°C) graduated glass cylinder after 2 h of drainage was measured and considered as Control 0.76±0.02a 4.43±0.04a 43.07±0.03b 26±0.5d 982±35a a a c c d syneresis, expressed as percent whey separated. Y1 0.76±0.02 4.43±0.02 44.54±0.06 22±0.3 1590±25 a a d b e Y2 0.76±0.03 4.42±0.05 45.02±0.04 20±0.3 1872±20 a a f a f Viscosity Y3 0.76±0.02 4.41±0.04 53.73±0.05 18±0.5 2191±35 a a e b c Y4 0.77±0.04 4.41±0.03 48.30±0.03 20±0.4 1555±25 a a a e b Apparent viscosity (expressed in cPs) of yoghurt samples were measured using Y5 0.78±0.03 4.40±0.05 39.98±0.04 31±0.3 1238±40 viscometer (Brookfield, USA). Viscometer was auto zeroed in the air after fixing Values are mean ±S.D. of determinations made in triplicate. The values followed by different superscripts are significantly different at P< 0.05, Y1= 1% fiber, Y2=2% fiber, Y3= 3% fiber, the spindle (ASTM Disk Spindle S-62 at room temperature). Yoghurt samples Y4=4% fiber, Y5= 5% fiber were tempered to 20°C and stirred gently 20 times in clockwise and anticlockwise direction using a spatula and filled into a beaker for measuring the Syneresis viscosity. The spindle S-62 was selected for viscosity measurement of yoghurt based on the instructions described in supplier’s instruction manual. The Whey separation or syneresis is considered as a defect on the surface of yoghurt, viscosity of stirred yoghurt samples was measured at 5 rpm, after starting rotation set yoghurt and other set-style fermented dairy products and is defined as the and results were expressed in cPs. Apparent viscosity reading was recorded after expulsion of whey from the network which then becomes visible as surface whey 60 seconds rotation of the spindle. Viscosity was measured for each sample in and negatively affects consumer perception. The syneresis value of control triplicate and the results were expressed as mean. yoghurt sample (Y1) was 26 ml, while it was 31 ml for yoghurt sample (Y5) with 5% soluble fiber. In the present study, the syneresis in yoghurt samples decreased Water holding capacity with increase in level of fiber fortification upto 3% (18 ml) then showed a sharp increase. The fortification of partially hydrolysed guar gum (PHGG) as soluble The water holding capacity of yoghurt samples was determined using a modified fiber significantly decreased whey separation in all yoghurt samples. This effect method as reported by Sodini et al. (2004). Ten grams of yoghurt samples were could be attributed to the gelling capacity of the PHGG soluble fiber and its high taken in centrifuge tubes and then centrifuged at 1,250 × g for 10 min at 5°C in a ability to interact with the milk constituents (mainly proteins), and stabilize the refrigerated high-speed centrifuge (Remi Centrifuge, Mumbai). The amount of protein network, preventing free movement of water. Functionality of whey expelled (g) during centrifugation (W) was weighed, and WHC calculated hydrocolloids in yogurt is demonstrated by their ability to bind water. However, as follows: WHC (%) = (10 − W)/10 × 100. The measurement was carried out in at higher concentration of partially hydrolyzed guar gum soluble fiber, there is triplicate and the results were expressed as mean. higher tendency of whey separation or syneresis because it affects the network and textural properties. Texture profile analysis of yoghurt Viscosity Yoghurts were analyzed for textural characteristics such as hardness, adhesiveness, cohesiveness, springiness and gumminess. Texture Profile Analysis Viscosity is resistance to flow of a fluid. It is a desirable characteristics in (TPA) was carried out using Texture Analyzer, TA-XT2i (Stable Micro Systems, yoghurt as it contributes to mouthfeel and physical properties such syneresis, Surrey, UK). The texture profile analysis was analyzed by performing two whey separation and water holding capacity. Yogurts fortified with soluble fiber sequential compressions using a flat-end cylindrical plunger (25-mm probe) had the higher viscosity values as compared to control yogurt, which showed the separated by a rest phase of 30 s that generated plot of force versus time. Samples lowest viscosity. Viscosity values of yoghurt samples recorded were ranges were compressed upto 70% of their original length. Crosshead speeds of 4.0, 1.0 between 982-2191 cPs. Fiber fortification upto 3% level in yoghurt samples and 1.0 mm/s were maintained for pretest, test and post test settings, respectively. increased the viscosity values whereas above this concentration there was The speed of obtaining the data was 200 pps. Five independent observations were observed a decrease in viscosity of yoghurt samples. While lower concentration made. Hardness, adhesiveness, cohesiveness, springiness and gumminess values of soluble fiber supported the network formation and contributed to high viscosity in yoghurt samples.
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Water holding capacity yoghurt is due to higher water holding capacity of soluble fiber which also provided strength to yoghurt coagulum network and aids in more water retention. Water holding capacity of yoghurt is considered as an indicator of its ability to At 5% fiber concentration, the water holding capacity of yoghurt reduced to retain serum in the gel structure. The ability of yoghurt to exhibit minimal whey 39.9% which is even lesser than control curd sample. The lower water holding separation is an important factor with respect to consumer point of view as whey capacity at higher percentage of fiber was partially due to the unstable gel separation negatively affects consumer perception (Lee and Lucey, 2010). Water network of yogurt, in which the weak colloidal linkage of protein micelles could holding capacity of yoghurt fortified with fiber increased to 53.73 % as compared not entrap water within its three dimensional network (Donkor et al., 2007). to 43.07 % of control yoghurt sample. This increase in water holding capacity of
Table 2 Texture profile analysis of control and fiber fortified yoghurt Sample Hardness (g) Adhesiveness (g.s) Cohesiveness Springiness Gumminess (g) Control 45.5±0.27c 43.8±0.18c 0.48±0.08a 0.72±0.02a 21.8±0.10a e c a b c Y1 53.9±0.32 43.6±0.20 0.52±0.06 0.78±0.04 28.0±0.12 e c a b c Y2 54.2±0.24 43.7±0.16 0.53±0.10 0.79±0.05 28.7±0.14 d c a c c Y3 49.9±0.22 43.6±0.18 0.58±0.12 0.85±0.04 28.9±0.11 b b a d c Y4 47.4±0.18 42.8±0.10 0.59±0.10 0.91±0.03 27.9±0.14 a a a d b Y5 38.5±0.20 42.1±0.12 0.60±0.14 0.92±0.04 23.1±0.16 Values are mean ±S.D. of determinations made in triplicate. The values followed by different superscripts are significantly different at P< 0.05, Y1= 1% fiber, Y2=2% fiber, Y3= 3% fiber, Y4=4% fiber, Y5= 5% fiber
Texture profile analysis Sensory evaluation
Texture profile analysis of food sample in a texture analyzer imitates the Sensory evaluation of control and fiber fortified yoghurt is presented in Table 3. conditions in the mouth by compressing a product twice (Bourne, 1978). Results Compared to control yoghurt sample, the sensory characteristics of yoghurt for texture profile analysis parameters such as hardness, cohesiveness, fortified with fiber markedly improved except in case of Y5 sample in which fiber adhesiveness, springiness and gumminess are presented in Table 2. Results of concentration was highest i.e.5%. Lower values for sensory characteristics of Y5 texture profile analysis revealed that textural properties of yoghurt significantly sample can also be co-related with pH, acidity, water holding capacity, syneresis improved on addition of soluble fiber. Hardness is used to estimate the maximum and viscosity of curd. Sensory evaluation as well as overall acceptability results force of the first compression and is considered as a critical parameter for of the yoghurt samples showed that Y3 yoghurt sample (3% fiber level) was the evaluation of textural characteristics of food. The highest hardness was measured most acceptable sample by judging panel members. Y3 sample scored highest in Y2 yogurt sample (54.2 g), while the lowest one was observed in Y5 and among all samples with respect to color and appearance, body and texture, flavor control yoghurt sample (38.5 g & 42.5 g, respectively). Textural and rheological and overall acceptability. characteristics of coagulated yogurt are generally determined by their internal structure. Tight and rigid internal molecular structure results in a firm protein gel. CONCLUSION The microstructure of yoghurt consists of a three-dimensional network of casein particles containing spherical molecules of different sizes (Fiszman et al., 1999). Partially hydrolyzed guar gum as soluble fiber can be used as yoghurt improver Results suggest that fiber fortification in yoghurt upto 4% level give strength to as it improved textural, physicochemical and desirable sensory characteristics. network architecture. Adhesiveness is commonly calculated as the area of a Fiber fortification of yoghurt enhances the viscosity and water holding capacity negative peak. Adhesiveness value was highest for control yoghurt sample (43.8) of yoghurt samples. Moreover, it reduced the syneresis of yoghurt which can be and lowest for Y5 sample (42.1) which demonstrates that less force was required an additional advantage to the yoghurt manufacturers. Fiber fortified yoghurt to remove the material adhered to the mouth during eating yoghurt samples samples showed non-significant increase in acidity as compared to control fortified with fiber as compared to control yoghurt sample. Fiber fortification sample but at acceptable level. Sensory evaluation results revealed that Y3 leads to decrease in adhesiveness of yoghurt samples. This may be due to the yoghurt sample fortified with 3% soluble fiber was having highest acceptability. gummy nature of partially hydrolyzed guar gum which reduces the adhesiveness It is concluded that fortified yoghurt with 3% soluble fiber produced an characteristic in yoghurt. Cohesiveness of yogurt samples were increased with acceptable product with improved characteristics. increased level of fiber fortification which indicates that fiber fortification supports and enhances the strength of internal bonds of all samples. Gumminess REFERENCES is the multiplication of hardness and cohesiveness. With the increased values of cohesiveness and variable values of hardness, gumminess of yoghurt samples Aneja, R. P., Mathur, B. N., Chandan, R. C. & Banerjee, A. K. (2002). first increased and then decreased. Springiness of control and fiber fortified Technology of Indian Milk Products. New Delhi: Dairy India publication, 42 p. yoghurt samples showed an increasing trend with respect to fiber fortification ISBN 9788190160308 levels. The springiness of Y5 (0.92) and Y4 (0.91) yogurt samples were higher Bhat, Z. F. & Bhat, H. (2011). Milk and dairy products as functional foods: A than of the other samples indicating that yoghurt samples fortified with fiber review. International Journal of Dairy Science, 6(1), 1-12. returned more easily to its original shape after the deforming force was removed. http://dx.doi.org/10.3923/ijds.2011.1.12 Bhattacharya, S. & Das, A. (2010). Study of physical and cultural parameters on Table 3 Sensory characteristics of control and fiber fortified yoghurt the bacteriocins produced by lactic acid bacteria isolated from traditional Indian Sample Color & Body & Flavour Overall fermented foods. American Journal of Food Technology, 5(2), 111-120. Appearance Texture Acceptability Bourne, M. (1978). Texture profile analysis. Food Technology, 32, 62–72. Chawla, A. K. & Balachandran, R. (1994). Studies on Yoghurt from buffalo
b b a b milk: Effect of different solids not fat content on chemical, rheological and Control 7.0±0.45 7.1±0.10 7.1±0.14 7.05±0.17 sensory characteristics. Indian Journal of Dairy Science, 47, 762-765. Devi, M. C. A., Rao, K. J. & Ravindra, M. R. (2010). Role of milk and milk b b a b Y1 6.9±0.30 7.5±0.22 7.0±0.12 7.13±0.15 products in traditional medicinal systems. Indian Dairyman, 62(3), 114-119. Donkor, O. N., Henriksson, A., Vasiljevic, T. & Shah, N. P. (2007). Rheological b b,c b c Y2 7.1±0.25 7.9±0.15 8.0±0.18 7.66±0.12 properties and sensory characteristics of set-type soy yoghurt. Journal of Agricultural and Food Chemistry, 55(24), 9868-9876. d d c d https://doi.org/10.1021/jf071050r Y3 8.5±0.30 9.0±0.17 9.0±0.20 8.83±0.15 Fiszman, S. M., Lluch, M. A. & Salvador, A. (1999). Effect of addition of gelatin
c c b c on microstructure of acidic milk gels and yoghurt and on their rheological Y4 7.5±0.22 8.1±0.11 8.0±0.15 7.86±0.10 properties. International Dairy Journal, 9(12), 895–901. https://doi.org/10.1016/S0958-6946(00)00013-3 a a a a Y5 6.0±0.14 6.5±0.15 7.0±0.15 6.50±0.11 Hashim, I. B., Khalil, A. H. & Afifi, H. S. (2009). Quality characteristics and consumer acceptance of yogurt fortified with date fiber. Journal of Dairy Values are mean ±S.D. of determinations made in triplicate. The values followed by different Science, 92(11), 5403-5407. https://doi.org/10.3168/jds.2009-2234 superscripts are significantly different at P< 0.05, Y1= 1% fiber, Y2=2% fiber, Y3= 3% fiber, Lee, W. J. & Lucey, J. A. (2010). Formation and physical properties of yogurt. Y4=4% fiber, Y5= 5% fiber Asian Australian Journal of Animal Science, 23(9), 1127–1136. http://dx.doi.org/10.5713/ajas.2010.r.05 Mudgil, D. & Barak, S. (2013). Composition, properties and health benefits of indigestible carbohydrate polymers as dietary fiber: A review. International
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Journal of Biological Macromolecules, 61, 1-6. https://doi.org/10.1016/j.ijbiomac.2013.06.044 Mudgil, D., Barak, S. & Khatkar, B. S. (2012a). Soluble fibre and cookie quality. Agro Food Industries Hi Tech, 23(3), 15-17. Mudgil, D., Barak, S. & Khatkar, B. S. (2012b). Process optimization of partially hydrolyzed guar gum using response surface methodology. Agro Food Industries Hi Tech, 23(1), 13-15. Mudgil, D., Barak, S. & Khatkar, B. S. (2011). Effect of hydrocolloids on the quality characteristics of tomato ketchup. Carpathian Journal of Food Science and Technology, 3(1), 39–43. Mudgil, D., Barak, S. & Khatkar, B. S. (2014). Optimization of enzymatic hydrolysis of guar gum using response surface methodology. Journal of Food Science and Technology, 51(8), 1600-1605. https://doi.org/10.1007/s13197- 012-0678-z Mudgil, D., Barak, S. & Khatkar, B. S. (2016a). Optimization of textural properties of noodles with soluble fiber, dough mixing time and different water levels. Journal of Cereal Science, 69, 104-110. http://dx.doi.org/10.1016/j.jcs.2016.02.015 Mudgil, D., Barak, S. & Khatkar, B. S. (2016b). Optimization of bread firmness, specific loaf volume and sensory acceptability of bread with soluble fiber and different water levels. Journal of Cereal Science, 70, 186-191. https://dx.doi.org/10.1016/j.jcs.2016.06.009 Mudgil, D., Barak, S. & Khatkar, B. S. (2016c). Effect of partially hydrolyzed guar gum on pasting, thermo-mechanical and rheological properties of wheat dough. International Journal of Biological Macromolecules, 93, 131-135. https://doi.org/10.1016/j.ijbiomac.2016.08.064 Mudgil, D., Barak, S. & Khatkar, B. S. (2016d). Development of functional yoghurt via soluble fiber fortification utilizing enzymatically hydrolyzed guar gum. Food Bioscience, 14, 28-33. http://dx.doi.org/10.1016/j.fbio.2016.02.003 Mudgil, D., Barak, S. & Khatkar, B. S. (2018). Partially hydrolyzed guar gum as a potential prebiotic source. International Journal of Biological Macromolecules, 112, 207-210. https://doi.org/10.1016/j.ijbiomac.2018.01.164 Raju, P. N. & Pal, D. (2014). Effect of dietary fibers on physico-chemical, sensory and textural properties of Misti Dahi. Journal of Food Science and Technology, 51(11), 3124-3133. https://doi.org/10.1007/s13197-012-0849-y Sodini, I., Remeuf, F., Haddad, S. & Corrieu, G. (2004). The relative effect of milk base, starter, and process on yogurt texture: a review. Critical Reviews in Food Sciences and Nutrition, 44(2), 113-137. https://doi.org/10.1080/10408690490424793 Tamime, A. Y. & Robinson, R. K. (2007). Tamime and Robinson’s Yoghurt: Science and Technology. (3rd ed.). Cambridge: CRC Press, 1 p. ISBN 9781845692131 Yadav, H., Jain, S. & Sinha, P. R. (2008). Oral administration of dahi containing probiotic Lactobacillus acidophilus and Lactobacillus casei delayed the progression of streptozotocin-induced diabetes in rats. Journal of Dairy Research, 75(2), 189-195. http://doi.org/10.1017/S0022029908003129
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EFFECT OF ADDED RAPE HONEY ON CHOSEN PHYSICOCHEMICAL AND TEXTURAL PROPERTIES AND ANTIOXIDANT ACTIVITY OF YOGURTS DURING STORAGE
Zuzana Remeňová*1, Margita Čanigová1, Miroslav Kročko1, Viera Ducková1
Address(es): Ing. Zuzana Remeňová, 1Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Animal Products Evaluation and Processing, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, +421 37 641 4309.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.802-804
ARTICLE INFO ABSTRACT
Received 26. 3. 2018 This study evaluated chosen physicochemical properties, antioxidant activity and textural properties of yogurts during refrigerated Revised 6. 9. 2018 storage. The yogurts were divided into four groups as follows: control group without any additions of honey (K) and yogurts samples Accepted 6. 9. 2018 with the different addition 1, 3 and 5 % (w/v) of rape honey (A, B and C). These samples were analysed during fourteen days of storage Published 1. 10. 2018 at cooling temperature (6 ±1 °C). Yogurt samples were analyzed for dry matter content, fat content, pH values, lactic acid percentage, antioxidant activity and textural properties (firmness and cohesiveness). Dry matter content increased gradually with increased addition of honey. On the contrary, the rising addition of honey caused a decrease of fat content. The pH values of yogurt samples decreased Regular article during storage. The lowest firmness and cohesiveness was observed in the sample with highest addition of rape honey.
Keywords: yogurt, honey, physicochemical parameters, antioxidant activity, textural properties
INTRODUCTION MATERIAL AND METHODS
Yogurt is milk fermented with symbiotic starter cultures of lactic acid bacteria Yogurts production Streptococcus thermophilus and Lactobacillus delbrueckii subs. bulgaricus, which shall be in a viable state, active and still present in the product through the Yogurts were made and assessed in Department of Evaluation and Processing of end of shelf life (Yilmaz and Gökmen, 2017). The pH value decreased to the Animal Products, Slovak University of Agriculture in Nitra. Semi-skimmed milk level 4.6 – 4.7 by fermentation with lactic acid bacteria, which convert lactose obtained from trade network was used for yogurts production. The milk was into lactic acid (Sert et al., 2017). Yogurt is renowned for its health benefits, heated on 40 – 42 °C, mixed with skimmed milk powder and again heated to 80 – nutritional value and digestibility. It contains protein, B vitamins and calcium 82 °C for two minutes. Yogurt culture (Laktoflora®, Milcom a. s., Czech (O´Sullivan et al., 2016; Nguyen et al., 2017). Yogurt is gel that is composed of Republic) was added into the cooled milk and inoculated milk was dosed into denatured protein and milk fat globule. The fat content in yogurts influences the sealable glasses. This product was marked as control sample (K). To all firmness of gel network structure. The low fat content can leads to less desirable experimental samples (A, B and C) was added rape honey. These experimental textural properties, flavour and aroma (Yu et al., 2016). samples of yogurts were divided to three groups: first group of samples (A) Yogurt has its own peculiar flavour. The flavour of yogurts has played an additionally contain of 1 % (w/v) of honey, second groups of samples (B) important role in their acceptance by consumer. Sweeteners, flavourings and additionally contain of 3 % (w/v) of honey and third groups of samples (C) other ingredients are added to modify the taste and aroma of yogurts. Also there additionally contain of 5 % (w/v) of honey. The fermentation of milk was set at has been an interest in fortification of yogurt to improve its nutritional value and 42 – 43 °C during four hours. The final products were stored at 6 ±1 °C during 14 health benefits. The ambition to provide tasty and also nutritive rich food days. Each experiment was evaluated at least three times and the given results are increased with the development of technologies and the growing competition represented as the mean values. (Routray and Mishra, 2011; Singh et al., 2012). Honey has been used in foods and drinks as sweetener and flavouring agent. Its Physicochemical analysis main producers are China, Turkey, Argentina, Ukraine and other. Yogurts with honey are not widely available in trade network (Meo et al., 2017). Blossom Dry matter content, fat content, pH values and lactic acid percentage of yogurts honey and honeydew honey are the main honey types in term of its origin. There were determined. The analyses were performed at least in duplicate. Dry matter are unifloral and multifloral types of blossom honey. Unifloral honey is obtained content and fat content were determined in the 1st day following the yogurts from profuse crops, for example Brasicca napus. This type of honey is called production. The dry matter content was determined with a gravimetric reference rape honey (Kňazovická et al., 2015). According to Codex Alimentarius "Honey method by drying to constant weight at 102 ± 2 °C. The fat content was is the natural sweet substance produced by honey bees from the nectar of plants determined with Gerber´s acidobutyrometric method (Cvak et al., 1992). The pH or from secretions of living parts of plants or excretions of plant sucking insects values of the yogurts samples were measured by pH meter Orion Star A211 on the living parts of plants, which the bees collect, transform by combining with (Thermo Fisher Scientific, USA). The pH measurement was carried out in the 1st, specific substances of their own, deposit, dehydrate, store and leave in the honey 7th and 14th day following the yogurts production. Electrophoretic analyser EA comb to ripen and mature" (Codex Stan, 2001). Honey is popular because of its 102 (Villa Labeco, Slovak Republic) was used for capillary isotachophoretic high nutritive value and due to its beneficial effects on human health. It has determination of lactic acid in yogurt samples. The electrolyte system was antioxidant, bacteriostatic, anti-inflammatory and antimicrobial properties. created by the leading electrolyte (LE) and the terminating electrolyte (TE). Honey contains approximately 80 % of carbohydrates, glucose and fructose are Driving current 250 µA was used for isotachophoretic analysis. Lactic acid the main contributors, 15 – 17 % of water, 0.1 – 0.4 % of proteins, ash, amino percentage was determined in the 1st, 7th and 14th day following the yogurts acids, vitamins, minerals, enzymes, organic acids and phenolic compounds production. (Kasprzyk et al., 2018). The objective of our study was to evaluate chosen physicochemical and textural properties of yogurts with the different additions of rape honey and compare them with a control samples without any addition of honey.
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Antioxidant activity Table 3 Lactic acid percentage in yogurts stored at 6 ± 1 °C during 14 days Day of storage Sample Antioxidant activity of yogurt samples by 1, 1-diphenyl-2-picrylhydrazyl radical 1. day 7. day 14. day (DPPH) inhibition was determined by an assay modified from Zainoldin and K 0,91 1,05 1,25 Baba (2009). The decrease in absorbance was monitored at 517 nm until a A 1,03 1,03 1,25 constant reading was obtained. The readings were compared with the controls B 0,99 1,03 1,27 which contained distilled water instead of yogurt water extract. The results were C 0,91 0,96 1,27 expressed as the trolox equivalent antioxidant capacity (TEAC). Antioxidant Legend: K – yogurt containing added without rape honey, A – yogurt containing added rape activity of yogurt samples was determined 7th and 14th day following the yogurts honey at 1 % (w/v), B – yogurt containing added rape honey at 3 % (w/v), C – yogurt production. containing added rape honey at 5 % (w/v)
Textural properties Antioxidant activity
Texture properties were measured on a texture analyser TA.XT Plus (Stable Fermented dairy products are the excellent sources of bioactive peptides. They Micro Systems LTD., UK). The texture analyser was used to measure chosen provide numerous peptides with antioxidant properties (Rahmawati and textural properties and that firmness and cohesiveness. The texture analysis was Suntornsuk, 2016). Honey is rich in antioxidants and the components in honey carried out on the 1st, 7th and 14th day following the yogurts production. The test responsible for its antioxidative effect are phenolic compounds. The composition was carried out in a plastic container with 50 mm diameter, using a 35 mm back and antioxidant capacity of honey depend on the floral source used to collect extrusion disk probe and 5 kg load cell. Temperature of samples was 15 °C. Test nectar and other factors. The higher antioxidant activity was found for darker speed of probe was 1 mm.s-1 and a distance reached in the yogurt sample was 30 honeys (Lachman et al., 2010). The rape honey added into the yogurts had mm. The course of measurement was recorded through the curves by Texture antioxidant activity 1,053 mg TEAC.g-1. Antioxidant activity of yogurts (Table 4) Exponent software. depends on the kind of honey, which is added into the yogurt samples. The higher addition of honey led to higher antioxidant activity of yogurt samples. In the RESULTS AND DISCUSSION samples K, A and B was observed the sharp decline (9,857 – 10,142 µg TEAC.g- 1) of antioxidant activity during 7 days. The least decline of antioxidant activity Physicochemical analysis (4,467 µg TEAC.g-1) was determined in the sample with the highest addition of honey. Our findings are comparable with those of Perna et al. (2014), who Dry matter content and fat content in yogurts are shown in table 1. The highest determined a higher antioxidant activity in yogurts with addition of chestnut and dry matter content was determined in the samples with highest honey addition sulla honey. Yogurts with chestnut honey addition showed higher antioxidant (C). Dry matter content was increased by the presence of rape honey. Glušac et activity compared to the samples with sulla honey, because total phenolic and al. (2015) showed similar trend for yogurts with same addition of whey protein flavonoid contents were higher in chestnut honey. concentrate and different addition of acacia honey. They caused an increase in dry matter content of yogurts. Table 4 Antioxidant activity of yogurts stored at 6 ± 1 °C on the 7th and 14th day The lowest fat content was observed in the yogurt samples with highest addition of storage of honey. The rising addition of honey amount caused a decrease of fat content. µg TEAC.g-1 Sample These findings are in line with results by Rashid and Thakur (2012), who found 7. day 14. day that the fat content decreased gradually with increased addition of honey. K 12,095 2,238 A 13,153 3,151 Table 1 Dry matter content and fat content in yogurts B 14,071 3,929 Dry matter content C 15,954 11,487 Sample Fat content (%) (%) Legend: K – yogurt containing added without rape honey, A – yogurt containing added rape K 15.21 1.48 honey at 1 % (w/v), B – yogurt containing added rape honey at 3 % (w/v), C – yogurt A 15.52 1.48 containing added rape honey at 5 % (w/v) B 17.13 1.47 C 18.09 1.45 Textural properties Legend: K – yogurt containing added without rape honey, A – yogurt containing added rape honey at 1 % (w/v), B – yogurt containing added rape honey at 3 % (w/v), C – yogurt Changes in textural properties of yogurts during storage are shown in figure 1, 2 containing added rape honey at 5 % (w/v) and 3. In the storage period, the lowest firmness and cohesiveness were found in the samples with highest addition of honey. On the last day of storage, the highest The highest pH value on the first day of storage was determined in the sample firmness and cohesiveness were measured in the control sample without honey with highest addition of honey (Table 2). On the last day of storage, the sample addition. Honey addition led to the lower firmness and cohesiveness of yogurts. without any addition of rape honey showed lowest pH value. The pH values of all These results are comparable with those of Shleykin et al. (2015), who found analysed yogurt samples decreased during storage. These findings are consistent that increasing concentrations of honey (10 %, 20 % and 30 %) leading to lower with those of Varga (2006), who reported a decrease in pH values of yogurts firmness of yogurts. with different honey additions during six weeks storage.
Table 2 The pH values in yogurts stored at 6 ± 1 °C during 14 days Day of storage Sample 1. day 7. day 14. day K 4.63 4.20 3.95 A 4.59 4.08 3.99 B 4.60 4.20 4.01 C 4.75 4.25 3.97 Legend: K – yogurt containing added without rape honey, A – yogurt containing added rape honey at 1 % (w/v), B – yogurt containing added rape honey at 3 % (w/v), C – yogurt containing added rape honey at 5 % (w/v)
Yogurt is obtained by lactic acid fermentation and therefore the yogurts contain the different percentage of lactic acid (Table 3). Lactic acid percentage of all samples increased during storage. In all of analysed samples, the highest content Figure 1 Textural properties of yogurts 1st day of storage of lactic acid was determined after 14 days of storage. Our findings are consistent Legend: K – yogurt containing added without rape honey, A – yogurt containing added rape with those of Rotar et al. (2015), who reported a growth of lactic acid percentage honey at 1 % (w/v), B – yogurt containing added rape honey at 3 % (w/v), C – yogurt in yogurts with the same level of polyfloral honey (3 %) and the different levels containing added rape honey at 5 % (w/v) of goji berries (3, 5 and 7 %) during 21 days of refrigerated storage. They found that the content of lactic acid increased during storage. In the study of Varga (2006), acacia honey had no effect on lactic acid levels of the final products. There were no significant differences among control and honey-containing yogurts in lactic acid levels during storage.
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in yogurt. Innovative Food Science & Emerging Technologies, 37, 293-299. https://doi.org/10.1016/j.ifset.2016.07.031. Perna, A., Intaglietta, I., Simonetti, A., & Gambacorta, E. (2014). Antioxidant activity of yogurt made from milk characterized by different casein haplotypes and fortified with chestnut and sulla honeys. Journal of Dairy Science, 97(11), 6662-6670. https://doi.org/10.3168/jds.2013-7843. Rahmawati, I. S., & Suntornsuk, W. (2016). Effects of Fermentation and Storage on Bioactive Activities in Milks and Yoghurts. Procedia Chemistry, 18, 53-62. https://doi.org/10.1016/j.proche.2016.01.010. Rashid, A., & Thakur, E. S. N. (2012). Studies on Quality Parameters of Set Yoghurt Prepared By the Addition of Honey. International Journal of Scientific and Research Publications, 2(9), 1-10. Rotar, A. M., Vodnar, D. C., Bunghez, F., Cǎtunescu, G. M., Pop, C. R., Jimborean, M., & Semeniuc, C. A. (2015). Effect of Goji Berries and Honey on Figure 2 Textural properties of yogurts 7th day of storage Lactic Acid Bacteria Viability and Shelf Life Stability of Yoghurt. Notulae Legend: K – yogurt containing added without rape honey, A – yogurt containing added rape Botanicae Horti Agrobotanici Cluj-Napoca, 43(1), 196-203. honey at 1 % (w/v), B – yogurt containing added rape honey at 3 % (w/v), C – yogurt https://doi.org/10.15835/nbha.43.1.9814. containing added rape honey at 5 % (w/v) Routray, W., & Mishra, H. N. (2011). Scientific and Technical Aspects of Yogurt Aroma and Taste: A Review. Comprehensive Reviews in Food Science and Food Safety, 10(4), 208-220. https://doi.org/10.1111/j.1541-4337.2011.00151.x. Sert, D., Mercan, E., & Dertli, E. (2017). Characterization of lactic acid bacteria from yogurt-like product fermented with pine cone and determination of their role on physicochemical, textural and microbiological properties of product. LWT - Food Science and Technology, 78, 70-76. https://doi.org/10.1016/j.lwt.2016.12.023. Shleykin, A. G., Barakova, N. V., Petrova, M. N., Danilov, N. P., & Argymbaeva, A. E. (2015): The influence of sugar syrup, honey and cereals on the rheological properties of yogurt. Scientific Journal ITMO University, 2, 24- 34. Singh, M., Kim, S., & Liu, S. X. (2012). Effect of Purified Oat β-Glucan on Fermentation of Set-Style Yogurt Mix. Journal of Food Science, 77(8), E195- E201. https://doi.org/10.1111/j.1750-3841.2012.02828.x. Varga, L. 2006. Effect of acacia (Robinia pseudo-acacia L.) honey on the Figure 3 Textural properties of yogurts 14th day of storage characteristic microflora of yogurt during refrigerated storage. International Legend: K – yogurt containing added without rape honey, A – yogurt containing added rape Journal of Food Microbiology, 108(2), 272-275. honey at 1 % (w/v), B – yogurt containing added rape honey at 3 % (w/v), C – yogurt https://doi.org/10.1016/j.ijfoodmicro.2005.11.014. containing added rape honey at 5 % (w/v) Yilmaz, C., & Gökmen, V. (2017). Formation of tyramine in yoghurt during fermentation – Interaction between yoghurt starter bacteria and Lactobacillus CONCLUSION plantarum. Food Research International, 97, 288-295. https://doi.org/10.1016/j.foodres.2017.04.014. Rape honey addition into the yogurts leads to higher dry matter content. The Yu, H., Wang, L., & McCarthy, K. L. (2016). Characterization of yogurts made honey addition caused a decrease of fat content in final yogurts. The different with milk solids nonfat by rheological behavior and nuclear magnetic resonance addition of honey had no significant effect on the pH values of yogurts. The spectroscopy. Journal of Food and Drug Analysis, 24(4), 804-812. rising addition of rape honey caused less desirable textural properties of yogurts. https://doi.org/10.1016/j.jfda.2016.04.002. Zainoldin, K. H., & Baba, A. S. (2009): The effect of Hylocereus polyrhizus and Acknowledgments: This work was supported by AgroBioTech Research Centre Hylocereus undatus on physicochemical, proteolysis, and antioxidant activity in built in accordance with the project Building ,,AgroBioTech" Research Centre yogurt. International Journal of Biological, Biomolecular, Agricultural, Food ITMS 26220220180. and Biotechnological Enginee
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Codex Standard: 2001. Codex Standard for Honey 12-1981. Adopted in 1981. Revisions 1987 and 2001. Cvak, Z., Peterková, Ľ., & Černá, E. (1992): Chemické a fyzikalně chemické metódy v kontrole jakosti mléka a mlékárenských výrobků. Praha : VÚPP. Glušac, J., Stijepić, M., Đurđević-Milošević, D., Milanović, S., Kanurić, K., & Vukić, V. (2015): Growth and viability of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in traditional yoghurt enriched by honey and whey protein concentrate. Iranian Journal of Veterinary Research, 16(3), 249-254. Kasprzyk, I., Depciuch, J., Grabek-Lejko, D., & Parlinska-Wojtan, M. (2018). FTIR-ATR spectroscopy of pollen and honey as a tool for unifloral honey authentication. The case study of rape honey. Food Control, 84, 33-40. https://doi.org/10.1016/j.foodcont.2017.07.015. Kňazovická, V., Mederiová, B., Haščík, P., Trnková, M., & Kačániová, M. (2015). Quality evaluation of unifloral and multifloral honeys from Slovakia and other countries. Journal of Microbiology, Biotechnology and Food Sciences, 4, 82-86. https://doi.org/10.15414/jmbfs.2015.4.special3.82-86. Lachman, J., Orsák, M., Hejtmánková, A., & Kovářová, E. (2010). Evaluation of antioxidant activity and total phenolics of selected Czech honeys. LWT - Food Science and Technology, 43(1), 52-58. https://doi.org/10.1016/j.lwt.2009.06.008. Meo, S. A., Al-Asiri, S. A., Mahesar, A. L., & Ansari, M. J. (2017). Role of honey in modern medicine. Saudi Journal of Biological Sciences, 24(5), 975-978. https://doi.org/10.1016/j.sjbs.2016.12.010. Nguyen, P. T. M., Kravchuk, O., Bhandari, B., & Prakash, S. (2017). Effect of different hydrocolloids on texture, rheology, tribology and sensory perception of texture and mouthfeel of low-fat pot-set yoghurt. Food Hydrocolloids, 72, 90- 104. https://doi.org/10.1016/j.foodhyd.2017.05.035. O´Sullivan, A. M., O´Grady, M. N., O´Callaghan, Y. C., Smyth, T. J., O´Brien, N. M., & Kerry, J. P. (2016). Seaweed extracts as potential functional ingredients
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POLLEN CAN - TESTING OF BEE POLLEN FERMENTATION IN MODEL CONDITIONS
Vladimíra Kňazovická*1, Zuzana Mašková2, Eva Vlková3, Roman Švejstil3, Hana Salmonová3, Eva Ivanišová4, Martina Gažarová5, Iveta Gamráthová6, Monika Repková6, Marián Tokár4, Viera Ducková1, Miroslav Kročko1, Margita Čanigová1, Miroslava Kačániová2
Address(es): Ing. Vladimíra Kňazovická, PhD. 1Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Animal Products Evaluation and Processing, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 4428 2Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Microbiology, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 4432 3Czech University of Life Sciences Prague, Faculty of Agrobiology, Food and Natural Resources, Department of Microbiology, Nutrition and Dietetics, Kamýcka 129, 165 00 Praha 6 – Suchdol, Czech Republic, phone number: +420 22 438 2755 4Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Storing and Processing of Plant Products, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 4421 5Slovak University of Agriculture in Nitra, Faculty of Agrobiology and Food Resources, Department of Nutrition, Trieda Andreja Hlinku 2, 949 76 Nitra, Slovakia, phone number: +421 37 641 4210 6students at Food Technology program (MSc.), Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.805-811
ARTICLE INFO ABSTRACT
Received 13. 6. 2018 The aim of the study was to simulate bee work with pollen by its fermentation in model conditions and evaluate the final product, which Revised 6. 9. 2018 is called - pollen can. Bee pollen grain has thick wall, which is reason of its poor digestibility in organism. Bee pollen after fermentation Accepted 6. 9. 2018 is more suitable for human organism because of easily-available nutrients. Fresh bee pollen was added into the solution water-honey Published 1. 10. 2018 (with/without yoghurt) and was fermented. We tested 3 types of pollen and 3 types of honey in 3 ways of fermentations, which differed in starter addition, access of oxygen during fermentation and its time. The physico-chemical and microbiological parameters were measured in raw materials and products. Pollen can contained approximately 40% of water, 5% of fat and pH was approximately 4.5. Regular article These physico-chemical properties were influenced notably by pollen as raw material. The highest water content was found in rape pollen and consequently rape pollen cans. TPC (total plate count) of pollen can ranged from 4.10 to 7.10 log CFU.g-1 after fermentation and depend on type of pollen and fermentation process. We observed decrease of bacteria from Enterobacteriaceae family and microscopic filamentous fungi after fermentation in all 3 testing performances, whereas counts of sporulating aerobic microorganisms, yeasts and preliminary LAB (lactic acid bacteria) were stable, in comparison to pollen as raw material. Bacilli and clostridia from group of sporulating bacteria were recorded and identified. Value of pH less than 4.5 is important due to avoid clostridia germination.
Keywords: beekeeping, protein, better digestibility, lactic acid bacteria, microscopic fungi
INTRODUCTION with intestinal tract (Vidová et al., 2013). Two agents are important for intestinal microbiota – probiotic and prebiotic. It has universal validity – for people and After a period of fascination with highly processed products, the return to natural animals. Kačániová et al. (2013) found increase of lactobacilli and enterococci foods, whose nutritional value is confirmed by the results of scientific research, is and decrease of Enterobacteriaceae family in intestine of broiler chickens after currently observed around the world, mainly in the developed countries. Each bee pollen addition into the feed mixture. product is pharmacologically active and may, therefore, be the source of many There are three types of pollen: native plant pollen, bee pollen (pollen loads active substances. Of particular importance the new products are derived from formed by bee foragers) and comb pollen (pollen processed and stored in the bee products with specified pharmacokinetics and pharmacodynamics, which hive). Bee pollen is partially processed by the bees in outdoor conditions and may be the basis for many new forms of drugs or dietary supplements (Kieliszek collected by the beekeeper. Bees do not consume pollen as collected by foragers et al., 2018). – they store pollen pellets in the comb cells and add honey, nectar or glandular Honey is the main source of saccharides for bee colony and pollen is the chief secretions to the stored mass and then it is called comb pollen (or perga or bee source of protein not only for bees but also for many other solitary insects and bread) (Herbert et Shimanuki, 1978). Comb pollen undergoes lactic acid insects living in colonies (Linskens et Jorde, 1997). According to Campos et al. fermentation and can be thus preserved (Bogdanov, 2017). (2010), it contains high concentration of reducing sugars, essential aminoacids, Some countries such as Brazil, Argentina, Switzerland or France have legally unsaturated/saturated fatty acids and mineral substances as Zn, Cu, Fe and high recognized bee pollen as a food supplement presenting identity and quality K/Na ratio and significant quantities of several vitamins: provitamín A, vitamin E standards, as well as the limits for each parameter to be analyzed (de Arruda et (tocopherol), niacin, thiamine, folic acid and biotin. Bee pollen is considered as al., 2017). In Slovakia, there are not legislative limits for pollen, there are only “only perfectly complete food” (Kostić et al., 2015). The old Egyptians describe general legislative for dietary supplements. According to Bogdanov et al. (2017), pollen as “a life-giving dust” (Bogdanov, 2017). The amount of nutrition- international proposal of chemical standard for pollen loads has been recently relevant components is largely dependent on the botanical source of pollen made as follows: water content – not more than 8%, total protein content – not (Campos et al., 2010). Pollen is known because of wide range of its biological less than 15%, sugar content – not less than 40% and fat – not less than 1.5%. effects. Pollen increases immunity against harmful physical, chemical and According to Roulston et Cane (2000), there are differences in digestibility biological agents, which is helpful e. g. for patients with infections of the upper among pollen types in connection with pollen wall porosity, thickness and respiratory tract, or pneumonia or leukemia, because pollen increases organism's composition, although hummingbirds digest pollen very poorly, most animals, immunity to infections (Kieliszek et al., 2018). Immune system of 80% is joined including those that do not regularly consume pollen, can digest 50-100% of
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ingested grains. Kieliszek et al. (2018) stated that pollen must be thoroughly indigenous bacterial microbiota, dominated by Lactobacillus and Bifidobacterium chewed because its nutrients are used by an organism only on a level of 10-15% phylotype related to Lactobacillus kunkeei, Bifidobacterium asteroides and B. in raw form. Linskens et Jorde (1997) published that under the influence of coryneforme. Endo et Salminen (2013) analyzed FLAB (fructophilic lactic acid enzymes of gastrointestinal tract digestion of pollen proteins, polysaccharides and bacteria) in the samples from bee hive, including bee pollen and they found lipids take place and the unbound elements, i. e. monosaccharides, amino acids, Lactobacillus kunkeei as dominant FLAB. Lamei et al. (2017) used the - term vitamins and fatty acids are subjected to the normal process of resorption. Zhang honeybee specific Lactic Acid Bacteria (hbs-LAB) originating from the honey et al. (2017) studied the influence of fermentation on pollen morphology and crop (also known as honey stomach or honey sack). These bacteria probably play stated that fermentation is an efficient method for pollen wall breakdown, but its significant role in pollen fermentation. mechanism remains relatively unclear. Zhou et al. (2018) evaluated the digestion Despite the fact that pollen stores are rich supplies of nutrient which makes it fit and fermentation in vitro of polysaccharides from bee pollen of Goji (Chinese for consumption it must be recommended only with restraint because of its wolfberry – Lycium chinense) and found enhancing of short-chain fatty acids allergenic potential (Linskens et Jorde, 1997). Pollen proteins are considered to production and modulating of gut microbiota composition via increasing the be the allergens (Bogdanov, 2017). Some proteins are breakdown during the relative abundances of genera Prevotella, Dialister, Megamonas, fermentation and potentially have lower allergenic effect. However, each allergic Faecalibacterium, Alloprevotella and decreasing the numbers of genera person probably can have own answer of organism. The field about allergy of Bacteroides, Clostridium XIVa, Parabacteroides, Escherichia/Shigella, pollen after fermentation could be searched in the future. Phascolarctobacterium, Parasutterella, Clostridium sensu stricto and The objective of our study was to test the production of pollen can, i. e. the bee Fusobacterium. pollen fermentation in model conditions, at 3 various ways – with/without access There is a necessity of fast processing of fresh bee collected pollen because of of oxygen in initial fermentation caused only by microorganisms naturally high water content. High humidity of fresh bee pollen (about 20-30 g/100 g) is an occurred in pollen and without access of oxygen in initial fermentation caused by ideal culture media for microorganisms like bacteria and yeasts (Bogdanov, microorganisms from pollen and yoghurt (added as starter). 2017). The fresh bee pollen can be frozen, dried or lyophilized. Interesting way of pollen processing is its fermentation, which is partial simulation of comb MATERIAL AND METHODS pollen production. Krell (1996) described the process of fermentation as pollen can (“home bee bread”) production after Dany 1988 with addition of pollen to Material solution of water and honey and possible addition of lactic acid bacteria. The bee bread has already been processed by the bees for storage with the addition of We used water, fresh bee pollen, honey and yoghurt (in one variant) for pollen various enzymes and honey, which subsequently ferments; this type of lactic acid can development. There were three different types of pollen and honey. fermentation is similar to that in yoghurts (and other fermented milk products) Characterization of raw material is in the table 1. Pollen was kept in the freezer and renders the end product more digestible and enriched with new nutrients until the production testing (at about 1 week). (Krell, 1996). Microorganisms, especially lactic acid bacteria, have an important role in the process of pollen transformation. Olofsson et Vásquez (2008) detected the
Table 1 Raw materials for pollen can production Sample Origin / Obtaining Geographical origin Year POLLEN A early spring (from plants before Brassica napus blooming) / directly from beekeeper Male Krstenany (Slovakia) 2017 B rape (Brassica napus) / directly from beekeeper Male Krstenany (Slovakia) 2017 C late spring (from plants after Brassica napus blooming) / directly from beekeeper Dolny Pajer* - (Slovakia) 2017 HONEY 1 blossom / directly from beekeeper Male Krstenany (Slovakia) 2017 2 rape (Brassica napus) / directly from beekeeper Male Krstenany (Slovakia) 2017 3 blended (Tilia cordata + honeydew) / directly from beekeeper Dolny Pajer* - (Slovakia) 2017 YOGHURT cow whole milk / commercial yoghurt from small producer eastern part of Slovakia 2018 * unpolluted area surrounded by forest with cottages and only a few inhabitants
Fermentation – production of pollen can 6 75 ml 3 – 45 g - C – 300 g 3rd experimental variant In the table 2, there is a reception of all examined types of pollen can. We 7 75 ml 1 – 45 g 1 g A – 300 g proceeded after Dany 1988, as described by Krell (1996): Water was boiled, and 8 75 ml 2 – 45 g 1 g B – 300 g then cooled (30 ± 2 °C). Honey was added. Solution was carefully mixed. 9 75 ml 3 – 45 g 1 g C – 300 g rd Yoghurt was added (for 3 experimental variant) and mixed. Pollen was added and mixed to obtain homogenous material. Then products were bottled. For There were tested 3 types of fermentation (I, II, III). Descriptions of tested successful fermentation, air space above the food was set on 20-25% of total variants are shown in the table 3. We used for fermentation only microorganisms volume. naturally occurred in the pollen (and partially in honey) for variant I and II. We used the yoghurt as an additional easily-available starter of fermentation for Table 2 Detailed receipt for tested variants of pollen can variant III. The fermentation process consisted of initial and next fermentation. Pollen can Water Honey Yoghurt Pollen The differences were in realising of initial fermentation and time of next 1st experimental variant fermentation, which was undertaken without access of oxygen in airtight bottles. 1 75 ml 1 – 45 g - A – 300 g In variant I and III, initial fermentation was according to Krell (1996) - without 2 75 ml 2 – 45 g - B – 300 g oxygen - because of LAB stimulating. Variant II was traditional, according 3 75 ml 3 – 45 g - C – 300 g Hajdušková (2006), where bottles were covered by linen cloth; consequently 2nd experimental variant oxygen was present during the initial fermentation. 4 75 ml 1 – 45 g - A – 300 g 5 75 ml 2 – 45 g - B – 300 g
Table 3 Design of fermentation Initial fermentation Next fermentation Total Experimental conditions fermentation variant material with temperature time (time) microorganisms temperature time O2 access
I pollen 30 ± 1 °C 2 days no RT (23 ± 2 °C) 16 days 18 days
II pollen RT (23 ± 2 °C) 5 days yes RT (23 ± 2 °C) 13 days 18 days III pollen + yoghurt 30 ± 1 °C 2 days no RT (23 ± 2 °C) 6 days 8 days RT – room temperature
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Physico-chemical analysis Basic microbiological analysis
The raw materials and tested variants of pollen can were analysed from physico- Raw materials (pollen, honey and yoghurt) as well as examined pollen cans (after chemical quality point of view. In the honey, we tested water content (by total fermentation) were tested from microbiological point of view. We used refractometric method), free acidity (by titration to pH 8.3), electrical dilution plating method for quantitative detection of total plate count (TPC), conductivity (by conductometric method) and hydroxymethylfurfural - HMF (by sporulating aerobic microorganisms (SAM), Enterobacteriaceae family, Reflectoquant®, Merck, Germany). The first three parameters were measured in preliminary lactic acid bacteria (LAB) and microscopic fungi (MF). Description accordance to IHC (2009). In pollen and pollen can, we found water content of individual parts of microbial examination is recorded in the table 4. (detected by drying at 100 ± 2 °C for 4 h to constant weight), free acidity (by Preliminary LAB means that they were found by dilution plating method with titration to pH 8.3), pH (by pH-meter) and fat content (by fat extractor observation of colony morphology, without next biochemical or molecular- ANKOMXT15 Technology, USA). genetic confirmation.
Table 4 Microbial examination of raw materials and products Cultivation Microbial group Medium Inoculation temperature time O2 requirement TPC GTY pouring 30 °C 2-3 days aerobic SAM NA 2 pouring* 25 °C 3 days aerobic Enterobacteriaceae VRBG pouring 30 °C 1-2 days aerobic preliminary LAB MRS pouring** 37 °C 3 days aerobic** MF DG 18 pouring 25 °C 5-7 days aerobic TPC – total plate count, cultivated on GTY – agar with glucose, tryptone and yeast extract (HiMedia®, India); SAM – sporulating aerobic microorganisms, inoculated by *pouring – after heat shock (at 80 °C for 10 min), cultivated on NA 2 – nutrient agar no. 2 (HiMedia®, India); Enterobacteriaceae family, cultivated on VRBG – violet red bile glucose agar (HiMedia®, India); LAB – lactic acid bacteria, inoculated by **double-pouring (decrease of oxygen in medium), cultivated on MRS – de Man, Rogosa and Sharpe agar (HiMedia®, India); MF – microscopic fungi, cultivated on DG 18 – Dichloran Glycerol agar - with chloramphenicol (HiMedia®, India)
Statistical processing of results be a valid species level identification; and values between 2.0 and 1.7 represent reliable genus level identifications. Individual measurements were performed at least 2 times with average expression. Results for experimental variants were processed in MS Excel 2007 RESULTS AND DISCUSSION as mean ± sd (standard deviation). Data from microbiological analysis were calculated as log CFU.g-1. Significant difference was assessed if it was at least Quality of raw materials 1.00 log CFU.g-1. Physico-chemical quality of honey samples is recorded in the table 5. All samples Identification of aerobic and anaerobic sporulating bacteria met requirements of Council Directive 2001/110/EC that has validity in EU countries including Slovakia. Honey had low water content and low HMF content Sample of pollen can (no. 9) was tested at Czech University of Life in Prague in and low free acidity. That indicates ripe unheated honey of high quality from term of sporulating bacteria detection. Firstly, we performed the heat shock (80 beekeeper of good beekeeping practise without formic acid treatment during the °C/10 min) of basic sample solution. We inoculated dilutions from 10-1 to 10-3 by honey production. Electrical conductivity confirms honey origin. At present, pouring of TPY agar (Oxoid, UK) with addition of glucose at 1 g.l-1. Plates were measurement of electrical conductivity is the most useful quality parameter for cultivated under the aerobic conditions at 30 °C for 1 day and under the anaerobic the classification of unifloral honeys, which can be determined by relatively conditions at 37 °C for 1 day. Then, isolates were transferred into liquid medium inexpensive instrumentation (Bogdanov et al., 2004). There is usually a positive for 24 h under the appropriate conditions. After 24 h, bacteria were visualised by correlation among the colour, mineral content and electrical conductivity of microscope with phase contrast and they were identified based on ribosomal honey (da Silva et al., 2016). Honey, used in tested pollen can, had electrical protein analysis on an Autoflex speed matrix-assisted laser desorption/ionisation conductivity to 0.8 mS.cm-1. According to beekeeper, sample 1 and 2 were time-of-flight (MALDI-TOF) mass spectrometer (Bruker, Daltonik, Germany) blossom and sample 3 was blended (honeydew and blossom origin). The according to Salmonová et al. (2018), i. e. extracted by ethanol-formic acid determination of electrical conductivity is the fastest method for routine honey extraction, transferred onto an MTP 384 polished steel BC target (Bruker, control as a criterion of the differentiation between blossom and honeydew Daltonik, Germany) and overlaid with HCCA matrix solution (Bruker, Daltonik, honeys (Bogdanov et Martin, 2002). Kukurová et al. (2008) stated that Germany). Measurements of each strain were performed in duplicate. electrical conductivity of forest honey in Slovak Republic was 0.55-1.00 mS.cm-1 Identification was considered as successful if the score value (degree of similarity before 2004. Forest honeys from Slovakia are often honeydew or blended in term to the reference spectrum) was at least 2.0 and was identical at species level. of their origin, but the strict requirement for electrical conductivity of blended According to Wieser et al. (2012), score value provides information about the honey is not known nowadays. validity of the identification; the score value above 2.0 is generally considered to
Table 5 Physico-chemical quality of honey Electrical conductivity Sample Water content [%] Free acidity [meq.kg-1] HMF [mg.kg-1] [mS.cm-1] Honey 1 16.3 27.15 0.33 10.80 Honey 2 16.5 13.70 0.12 16.50 Honey 3 16.4 27.75 0.37 11.90 mean ± sd (n = 3) 16.4 ± 0.1 22.87 ± 7.94 0.27 ± 0.13 13.07 ± 3.02 HMF - hydroxymethylfurfural
Physico-chemical quality of pollen samples is in the table 6. Fresh bee collected mainly polar and neutral fats (mono-, di- and triglycerides), as well as small pollen contains about 20-30 g water per 100 g (Campos et al., 2010). Rape amounts of fatty acids, sterines and hydrocarbons (Bogdanov, 2017). Range for pollen (B) had the highest water content (more than 30%) and it was the most fat content in pollen is wide, from 1.1% to 25.0% (Bíro et al., 2010). Expression acidic, what indicated the lowest pH and the highest free acidity. According to of fat and other nutrients as % DM (percentage from dry matter content) is more Bíro et al. (2010), rape pollen is suitable for animal nutrition because its nutrients suitable and precise, because of variable water content in fresh as well as are easily and highly digestible; rape pollen is typical by medium content of dehydrated pollen. Taha (2015) found fat content from 1.82% to 5.38% DM in nitrogen compounds, relatively low content of fiber and high content of nitrogen- pollen from Saudi Arabia. Average fat content in rape pollen was 3.92% DM, free extract, i. e. easy-digestible saccharides. There are some risks joined with however typical time for growing and blooming of rape is January in Saudi pollen consumption, too. Rape bee pollen at concentrations of 0.50% and 0.75% Arabia (Taha, 2015). Fuenmayor et al. (2014) found fat content from 2.80 to positively affected the body weight gain of female rats; however this higher 9.70% DM in Colombian bee pollen. Results of fat in tested pollen are in pollen consumption in feed reduced the triglycerides serum content in female rats accordance to the published ranges. According to Herbert et Shimanuki (1978), and increased the cholesterol serum content in male and female rats (Gálik et al., the chemical composition of pollen varies with plant species, environment during 2016). Fat content was variable. The highest value was reached in early spring pollen development, age of plant when pollen developed, nutrient status of the pollen (A) and the lowest value in late spring pollen (C). There are considerable plant, methods of pollen extraction and storage. differences of the fat composition, depending on the botanical origin; there are
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Table 6 Physico-chemical quality of pollen Sample Water content [%] pH Free acidity [meq.kg-1] Fat [% DM*] Pollen A (n = 2) 21.93 5.71 346.00 5.75 Pollen B (n = 2) 31.45 4.83 391.50 4.77 Pollen C (n = 2) 20.16 5.75 334.00 2.42 mean ± sd 24.51 ± 5.43 5.43 ± 0.52 357.17 ± 30.33 4.55 ± 1.41 * % DM – percentage in dry matter
In the table 7, the microbiological quality of raw materials is recorded. In the comparing with pollen samples in our study, because Brazilian bee pollen honey, we found low microbial counts, below 2.00 log CFU.g-1. We did not find samples were dehydrated. There is only limited number of studies about quantity microscopic fungi. In ripe honey of high quality, we can observe mainly inactive of microorganisms in fresh pollen; however there are many studies about forms of microorganisms, what was confirmed in our study. The difference of identified microorganisms from pollen. Endo et Salminen (2013) found TPC and evaluated microbial groups between the honey and pollen was fructophilic LAB in the pollen. These bacteria have positive influence on health approximately 4 log CFU.g-1. The difference of microbiological quantity between of bees, animals and humans. On the other hand, we found high number of honey and pollen is the same as in last research (Kňazovická et al., 2011), where filamentous microscopic fungi. Average value is above 4.00 log CFU.g-1. From TPC as well as TPC of anaerobic microorganisms were at level 5 log CFU.g-1 in hygienic point of view the microbiological safety is the main quality criterion, fresh bee pollen. According to de Arruda et al. (2017), who tested 62 samples of especially the absence of pathogenic germs and fungi in the pollen (Bogdanov, Brazilian pollen, microbial counts were at level 1-2 log CFU.g-1 for TPC, 2017). microscopic fungi, coliform bacteria and staphylococci. The values are lower
Table 7 Microbiological quality of raw materials [log CFU.g-1] preliminary TPC Enterobacteriaceae SAM Yeasts MFF LAB Honey (n = 3) 1.20 ± 0.17 < 1.00 1.30 ± 0.30 < 1.00 < 1.00 < 1.00 Pollen (n = 6) 5.96 ± 0.38 5.05 ± 0.83 2.48 ± 0.28 5.00 ± 0.88 5.03 ± 0.76 4.70 ± 0.51 Yoghurt (n = 1) 4.95 1.00 2.97 5.06 4.16 < 1.00 TPC – total plate count, SAM – sporulating aerobic microorganisms, LAB – lactic acid bacteria, MFF – microscopic filamentous fungi
Quality of fermented products more precise. Fat values were similar before and after fermentation. Mostly, the values decreased. Exception was sample 1, where the increase was recorded. In the table 8, there are results of physico-chemical analysis of pollen can. In Herbert et Shimanuki (1978) found 4.90% fat in bee-collected pollen from USA general, we recorded increase of water content by 60%, increase of free acidity and 5.40% fat in perga from the same region. There are considerable differences by 40%, decrease of pH by 17% and decrease of fat by 1-2% (as fat in DM) in of the fat composition, depending on the botanical origin Bogdanov (2017). The pollen can comparing to pollen as a raw material. Water content was higher highest values are found in dandelion (Taraxacum officinale) and mustard because of its addition. In all experimental variants of fermented pollen, pH (Brassica sp.), and the lowest in birch (Betula sp.) brush and buckthorn decreased. Low pH has protective function. Spoilage of bee bread is avoided by (Rhamnus sp.) (Herbert et Shimanuki, 1978). The next factor, influencing the the low pH, caused by lactic and acetic acid fermentation and alcoholic fat content and composition, is microbiota during the fermentation. Ether extracts fermentation, and by the presence of natural products with antibiotic properties may include fats, fatty acids, some vitamins, pigments, higher alcohols, waxes, produced by resident microbiota of stored bee pollen (Menezes et al., 2018). The sterols and saturated hydrocarbons (Solberg et Remedios, 1980 cit. Roulston et order of pollen from the most acidic to less acidic was following: before Cane, 2000). Zhou et al. (2018) observed the degradation of polysaccharides and fermentation B ˂ A ˂ C and after fermentation C ˂ B ˂ A. In pollen C, increasing of short-chain fatty acids during fermentation of pollen grains by gut fermentation was intensive according to pH values. The most acidic products microbiota. Kačániová et al. (2018) isolated bacteria from gut of local Slovakian were from 2nd experimental variant. Free acidity measurement was a little bit bees and identified them, as follows: Lactobacillus gasseri, L. amylovorus, L. problematic. The values are of wide range. We recommend automatic titrator kunkeei, L. fructivorans and Paenibacillus larvae. with pH detection for next trials. For this trial, we considered the pH values as
Table 8 Physico-chemical quality of pollen can Water content [%] Dry matter [%] Free acidity [meq.kg-1] pH Fat [% DM] st 1 EV (- O2) 1 37.07 62.93 415.00 5.08 6.98 2 47.32 52.68 658.00 4.43 3.14 3 33.46 66.54 762.00 3.81 0.85 mean ± sd (n = 3) 39.28 ± 7.19 60.72 ± 7.19 611.67 ± 178.08 4.44 ± 0.64 3.66 ± 3.10 nd 2 EV (+ O2) 4 41.23 58.77 466.50 4.51 4.55 5 41.48 58.52 563.00 4.50 3.01 6 34.87 65.13 550.00 3.94 0.57 mean ± sd (n = 3) 39.19 ± 3.75 60.81 ± 3.75 526.50 ± 52.37 4.32 ± 0.33 2.71 ± 2.01 rd 3 EV (with starter, - O2) 7 37.31 62.69 325.50 5.06 5.31 8 44.34 55.66 382.00 4.64 2.79 9 36.92 63.08 315.50 4.60 0.69 mean ± sd (n = 3) 39.52 ± 4.18 60.48 ± 4.18 341.00 ± 35.86 4.77 ± 0.25 2.93 ± 2.31 EV – experimental variant, DM – dry matter
Zhang et al. (2017) observed the fermentation by Ganoderma lucidum and (4) At the late stage of fermentation (day 8), all of the bonds were abscised, Saccharomyces cerevisiae on rape pollen grain and suggested some hypothesis absolutely releasing the entire contents. The content and fragments of the pollen for explaining the mechanism of the pollen wall rupture, as follows: wall remained scattered in the fermentation broth. (1) In fermented infancy (days 0-1), the fermentation species could secrete Microbial counts in pollen can after fermentation are recorded in the table 9. protease and other enzymes to remove the coating of pollen and to expose the Botanical origin of pollen seems to be the considerable factor in experimental germinal apertures and the porous exine; variants I and II in term of microbial counts, because we found TPC and count of (2) In the earlier stage of fermentation (days 2-5), cellulose, pectinase, and other preliminary LAB at level 6.00-7.00 log CFU.g-1 in the sample 2 and 5 (from rape enzymes from the species could easily permeate through the inner pollen to pollen - B) as well as absent bacteria from Enterobacteriaceae family and absent destroy the intine. At this time, some bond structures form at the pollen interior, microscopic filamentous fungi or their presence near the detection limit. In rape and these structures could completely immobilize the pollen content. pollen after fermentation (sample 2 and 5), increase of preliminary LAB (with (3) In the middle of the fermentation process (day 6-7), cellulose and pectinase counts over 6.00 log CFU.g-1) could inhibit the growth of bacteria from could abscise the bonds, leading to the separation of the exuviae. The content Enterobacteriaceae family (counts equal or below 1.00 log CFU.g-1). LAB were then overflowed at the exine hole; frequently isolated from gut of bee, nowadays, they are confirmed from pollen, too (Olofsson et Vásquez, 2008; Lamei et al., 2017). According to Krell (1996)
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and Bogdanov (2017) fermentation at 30 °C for first days without oxygen helps Enterobacteriaceae family was detected at 3.00 log CFU.g-1. In Slovakia, there to better development of lactic acid fermentation. The hypothesis was not are not the legislative regulations for pollen and the research studies could make confirmed by our study. We did not find differences in LAB counts in term of help for set the limits in the future. For example, de Arruda et al. (2017) fermentation performance. Specific group of LAB - FLAB prefer aerobic rather published the standard established by the Argentinean Food Code is 1.5 × 105 than anaerobic conditions for growth and cannot grow on glucose under CFU.g-1 (i. e. 5.18 log CFU.g-1) for aerobic mesophiles (equivalent to TPC) in anaerobic conditions (Endo et Salminen, 2013). Other samples from dehydrated bee pollen. According the results, similar or higher value could be the experimental variant I and II (1, 3, 4, 6) have lower and similar values of limit for TPC in fermented pollen from Slovakia and related countries, because mentioned microbial counts. The counts of SAM as well as yeasts had similar pollen is food with relatively high counts of microorganisms, which occur values in experimental variants I and II. In experimental variant III (with addition naturally and probably most of them are positive for human health, however it is of yoghurt), we found less differences in term of pollen of botanical origin. In necessary to eliminate the activities of pathogenic bacteria and microscopic sample 8 (from rape pollen B), the count of microscopic filamentous fungi was filamentous fungi. lower comparing with sample 7 and 9. The presence of bacteria from
Table 9 Microbiological quality of pollen can after fermentation [log CFU.g-1] Preliminary TPC SAM Enterobacteriaceae Yeasts MFF LAB st 1 EV (- O2) 1 4.34 2.53 2.81 ˂ 1.00 4.48 2.67 2 7.10 2.37 ˂ 1.00 6.58 4.96 ˂ 1.00 3 4.91 2.78 3.06 3.61 4.83 1.99 mean ± sd (n = 3) 5.45 ± 1.46 2.56 ± 0.21 2.94 ± 0.18 5.10 ± 2.10 4.76 ± 0.25 2.33 ± 0.48 nd 2 EV (+ O2) 4 4.10 2.57 1.95 2.62 4.93 1.49 5 6.88 2.31 1.00 6.44 4.58 1.15 6 4.77 2.92 2.79 4.42 5.90 2.67 mean ± sd (n = 3) 5.25 ± 1.45 2.60 ± 0.31 1.91 ± 0.90 4.49 ± 1.91 5.14 ± 0.68 1.77 ± 0.80 rd 3 EV (with starter, - O2) 7 5.15 1.60 3.92 2.94 4.85 3.70 8 4.76 2.19 3.48 4.84 4.48 1.60 9 4.59 3.39 2.85 4.72 4.41 3.48 mean ± sd (n = 3) 4.83 ± 0.29 2.39 ± 0.91 3.42 ± 0.54 4.17 ± 1.06 4.58 ± 0.24 2.93 ± 1.15 EV – experimental variant, TPC – total plate count, SAM – sporulating aerobic microorganisms, LAB – lactic acid bacteria, MFF – microscopic filamentous fungi
Identification of sporulating bacteria cultivated from pollen can people and people with weakened immunity; and food with pH below 4.5 is considered as safe food, because clostridia (especially Clostridium botulinum) are Sporulating bacteria, isolated from pollen can, were identified (table 10). The not able to multiply and produce botulotoxin under the pH 4.5. most frequent sporulating aerobic bacteria was Bacillus licheniformis (4 isolates), then B. megaterium (2 isolates) and 1 isolate was identified as B. sonorensis (figure 1). Bacillus licheniformis and B. megaterium are frequently present all over the world and their strains are used in biotechnology (de Clerck et de Vos, 2004; Eppinger et al., 2011). Bacillus sonorensis is named after the Sonoran Desert (USA), where the organism was collected (Palmisano et al., 2001). From sporulating anaerobic bacteria, we found 2 species Clostridium perfringens (2 isolates) and C. baratii (3 isolates, figure 2). De Arruda et al. (2017) did not find sulphite-reducing clostridia in 62 samples of Brazilian bee pollen. Spores of bacilli and clostridia are normal part of nature. They are found e. g. in the soil, dust, air. However, vegetative clostridia of some species are pathogenic and can produce the toxins. Clostridium perfringens spores are spread in the soil and intestinal tract of animals and humans, in vegetative form and under specific conditions, the bacteria can produce more than 15 toxins (Lindström et al., 2011). Clostridium baratii can produce botulotoxin in rare cases and result in infant botulism (Khouri et al., 2018). On the other hand, Stefka et al. (2014) demonstrated that the allergy-protective capacity is conferred by a clostridia- containing microbiota, where clostridia regulate innate lymphoid cell function and intestinal epithelial permeability to protect against allergen sensitization.
Table 10 Identification of sporulating bacteria cultivated from pollen can Figure 1 Isolate 1, cultivated from pollen can – Bacillus sonorensis (40×10×1.5 isolate no. species identification score-value* by phase contrast) AEROBIC 1 Bacillus sonorensis 2.219 2 Bacillus licheniformis 2.313 3 Bacillus licheniformis 2.261 4 Bacillus licheniformis 2.299 5 Bacillus licheniformis 2.152 6 Bacillus megaterium 2.094 7 Bacillus megaterium 1.909 ANAEROBIC 8 Clostridium perfringens 2.271 9 Clostridium perfringens 2.408 10 Clostridium baratii 2.205 11 Clostridium baratii 2.306 12 Clostridium baratii 2.205 *Identification results with score values above 2.0 are considered to be correct for determination of the respective species (Wieser et al., 2012).
According to Grieger et Vařejka (1990), food contains clostridia at dose less than 2.00 log CFU.g-1 cannot cause the illness, because the counts are reduced Figure 2 Isolate 11, cultivated from pollen can – Clostridium baratii (40×10×1.5 during the transport by gastrointestinal tract, but risk groups are children, older by phase contrast)
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De Arruda et al. (2017) suggested that the main physico-chemical and DHINGRA, S., LUKYANCHUK, V., BALL, B., UL-QAMAR, R., SEIBEL, J., nutritional composition of bee pollen in natura combined with the presence of BREMER, E., JAHN, D., RAVEL, J., VARY, P. S. 2011. Genome sequences of microorganisms naturally available in this product are conditions that have to be the biotechnologically important Bacillus megaterium strains QM B1551 and monitored for the good practices of bee pollen collection, transport, packaging DSM319. Journal of Bacteriology, 193(16), 4199-4213. and processing. LAB produce antibacterial compounds as organic acids, http://dx.doi.org/10.1128/JB.00449-1 hydrogen peroxide, diacetyl, benzoate and bacteriocins (de Vuyst et Vandamme, FUENMAYOR, C. B., ZULUAGA, C. D., DÍAZ, C. M., QUICAZÁN, M. C., 1994 cit. Olofsson et Vásquez, 2008). COSIO, M., MANNINO, S. 2014. Evaluation of the physicochemical and functional properties of Colombian bee pollen. Revista MVZ Córdoba, 19(1), CONCLUSION 4003-4014. GÁLIK, B., BÍRO, D., ŠIMKO, M., JURÁČEK, M., CAPCAROVÁ, M., Bees collect the pollen and ferment it in their hives. Bee pollen is better KOLESÁROVÁ, A., ROLINEC, M., TOMAN, R., KANKA, T. 2016. The effect digestible for bees and humans after processing. Fermentation is one of the of dietary bee pollen intake on growth performance and biochemical indicators of possible ways for better availability of high quality nutrients for the body. In the rats. Acta Veterinaria Brno, 85(1), 99-104. study, we tested the production of pollen can by 3 variants of fermentation with 3 http://dx.doi.org/10.2754/avb201685010099 different types of honey and pollen. The main factor influenced the pollen can GRIEGER, C., VAŘEJKA, F. 1990. Mikrobiológia poživatín živočíšneho was pollen as raw material, which differs in term of water, fat and pH. Tested pôvodu. Bratislava : Príroda, 288 p. ISBN 80-07-00388-6. variants of fermentation process seem to be suitable in term of physico-chemical HAJDUŠKOVÁ, J. 2006. Včelí produkty očima lékaře. Praha : ČSV, 50 p. ISBN quality. Pollen naturally contains microorganisms in relatively high numbers. 80-903309-2-4 Fermentation resulted in quantity decrease of bacteria from Enterobacteriaceae HERBERT, E. W., SHIMANUKI, H. 1978. Chemical composition and nutritive family and microscopic filamentous fungi, comparing of pollen and pollen can in value of bee-collected and bee-stored pollen. Apidologie, 9(1), 33-40. all 3 tested variants. Counts of SAM, yeasts as well as TPC were kept like in IHC. 2009. Harmonised methods of the International Honey Commission. 63 p. pollen before fermentation. Count of preliminary LAB was also kept on average, KAČÁNIOVÁ, M., ROVNÁ, K., ARPÁŠOVÁ, H., HLEBA, L., PETROVÁ, J., but it depended on pollen type. Risk microbial groups are sporulating bacteria HAŠČÍK, P., ČUBOŇ, J., PAVELKOVÁ, A., CHLEBO, R., BOBKOVÁ, A., and microscopic filamentous fungi, mainly for children, older people and people, STRIČÍK, M. 2013. The effects of bee pollen extracts on the broiler chicken's who have problems with immune system. Sporulating bacteria from the genus gastrointestinal microflora. Research in Veterinary Science, 95(2013), 34-37. Bacillus and Clostridium were isolated and identified from pollen can after heat http://dx.doi.org/10.1016/j.rvsc.2013.02.022 shock. They occurred only in inactive form of spores. Bacilli and clostridia are KAČÁNIOVÁ, M., GASPER, J., TERENTJEVA, M., KUNOVÁ, S., KLUZ, M., commonly present in honeys or other foodstuffs or natural materials. The risk is PUCHALSKI, C. 2018. Antibacterial activity of bees gut lactobacilli against germination of certain clostridia species during the storage. Generally, spores of Paenibacillus larvae in vitro. Advances Research in Life Sciences, 2(1), 7-10. clostridia can begin to germinate at pH more than 4.5. Therefore, pH seems to be http://dx.doi.org/10.1515/arls-2018-0020 essential for considering the safety of the product. In practise, we suggest to KIELISZEK, M., PIWOWAREK, K., KOT, A. M., BŁAŻEJAK, S., modify the fermentation process considering the pH. In next research, we plan to CHLEBOWSKA-ŚMIGIEL, A., WOLSKA, I. 2018. Pollen and bee bread as new observe the pollen can quality during the shelf life in term of basic physico- health-oriented products: A review. Trends in Food Science and Technology, chemical and microbiological analyses and continue with the pollen research to 71(2018), 170-180. http://dx.doi.org/10.1016/j.tifs.2017.10.021 expected result in simple recommendation about production and shelf life of KHOURI, J. M., PAYNE, J. R., ARNON, S. S. 2018. More Clinical Mimics of pollen can for beekeepers and bee products processors. Infant Botulism. The journal of paediatrics, 193(Feb 2018), 178-182. http://dx.doi.org/10.1016/j.jpeds.2017.09.044 Acknowledgments: This work was supported by the Grant 05-GA SPU 17 KŇAZOVICKÁ, V., KAČÁNIOVÁ, M., MILUCHOVÁ, M., GÁBOR, M., “Testing of pollen can production and shelf life” and by AgroBioTech Research DOVIČIČOVÁ, M., MELICH, M., TRAKOVICKÁ, A. 2011. Honey and Centre built in accordance with the project Building “AgroBioTech” Research microorganisms : Monitoring of microorganisms in Slovak honey by molecular- Centre ITMS 26220220180.We are thankful to beekeeper Jozef Švercel for biological methods in relation to physico-chemical properties. Saarbrűcken : material of high quality and really great discussion and cooperation at field of bee Lambert Academic Publishing, 192 p. ISBN 978-3-8473-2310-5. products processing. Thanks belong to Regina Bányiová and Natália Karapinová KOSTIĆ, A. Ž., BARAĆ, M. B., STANOJEVIĆ, S. P., MILOJKOVIĆ- for assistance with laboratory analyses and to Erasmus+ programme for OPSENICA, D. M., TEŠIĆ, Ž. L., ŠIKOPARIJA, B., RADIŠIĆ, P., opportunity to mobility and cooperation. PRENTOVIĆ, M., PEŠIĆ, M. 2015. Physicochemical composition and techno- functional properties of bee pollen collected in Serbia. LWT – Food Science and REFERENCES Technology, 62(2015), 301-309. http://dx.doi.org/10.1016/j.lwt.2015.01.031 KRELL, R. 1996. Value added products from beekeeping. Rome : Food and BÍRO, D., GÁLIK, B., JURÁČEK, M., ŠIMKO, M., TOMAN, R. 2010. Agriculture Organization of the United Nations. ISBN 92-5-103819-8. Obnôžkový peľ vo výžive zvierat. In BROVARSKYI, V., BRINDZA, J. et al. KUKUROVÁ, K., KAROVIČOVÁ, J., KOHAJDOVÁ, Z., BÍLIKOVÁ, K. Včelí obnôžkový peľ. Nitra : SPU v Nitre, 275-281, ISBN 978-966-8302-31-2. 2008. Authentication of honey by multivariate analysis of its physico-chemical BOGDANOV, S., MARTIN, P. 2002. Honey Authenticity. Mitteilungen aus parameters. 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Detection and Identification of a Novel ALMEIDA-MURADIAN, L. B. 2017. Microbiological quality and Lactic Acid Bacterial Flora Within the Honey Stomach of the Honeybee Apis physicochemical characterization of Brazilian bee pollen. Journal of Apicultural mellifera. Current Microbiology, 57(4), 356-363. Research, 56(3), 231-238. http://dx.doi.org/10.1080/00218839.2017.1307715 http://dx.doi.org/10.1007/s00284-008-9202-0 DE CLERCK, E., DE VOS, P. 2004. Genotypic diversity among Bacillus PALMISANO, M. M., NAKAMURA, L. K., DUNCAN, K. E., ISTOCK, C. A., licheniformis strains from various sources. FEMS Microbiology Letters, 231(1), COHAN, F. M. 2001. Bacillus sonorensis sp. nov., a close relative of Bacillus 91-98. http://dx.doi.org/10.1016/S0378-1097(03)00935-2 licheniformis, isolated from soil in the Sonoran Desert, Arizona. International ENDO, A., SALMINEN, S. 2013. Honeybees and beehives are rich sources for Journal of Systematic and Evolutionary Microbiology, 51(2001), 1671–1679. fructophilic lactic acid bacteria. Systematic and Applied Microbiology, 36(6), http://dx.doi.org/10.1099/00207713-51-5-1671 444-448. http://dx.doi.org/10.1016/j.syapm.2013.06.002 ROULSTON, T. H., CANE, J. H. 2000. Pollen nutritional content and EPPINGER, M., BUNK, B., JOHNS, M. A., EDIRISINGHE, J. N., digestibility for animals. Plant Systematics and Evolution, 222(2000), 187-209. KUTUMBAKA, K. K., KOENIG, S. S. K., CREASY, H. H., ROSOVITZ, M. J., SALMONOVÁ, H., KILLER, J., BUNEŠOVÁ, V., GEIGEROVÁ, M., RILEY, D. R., DAUGHERTY, S., MARTIN, M., ELBOURNE, L. D. H., VLKOVÁ, E. 2018. Cultivable bacteria from Pectinatella magnifica and the PAULSEN, I., BIEDENDIECK, R., BRAUN, CH., GRAYBURN, S., surrounding water in South Bohemia indicate potential new
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Gammaproteobacterial, Betaproteobacterial and Firmicutes taxa. FEMS Microbiology Letters, 365, 1-10. http://dx.doi.org/10.1093/femsle/fny118 STEFKA, A. T., FEEHLEY, T., TRIPATHI, P., QIU, J., MCCOY, K., MAZMANIAN, S. K., TJOTA, M. Y., SEO, G.-Y., CAO, S., THERIAULT, B. R., ANTONOPOULOS, D. A., ZHOU, L., CHANG, E. B., FU, Y.-X., NAGLER, C. R. 2014. Commensal bacteria protect against food allergen sensitization. Proceedings of the National Academy of Sciences of the United States of America, 111(36), 13145-13150. http://dx.doi.org/10.1073/pnas.1412008111 TAHA, E-K. A. 2015. Chemical composition and amounts of mineral elements in honeybee-collected pollen in relation to botanical origin. Journal of Apicultural Science, 59(1), 75-81. http://dx.doi.org/10.1515/JAS-2015-0008 VIDOVÁ, M., HRONSKÁ, H., TOKOŠOVÁ, S., ROSENBERG, M. 2013. Importance of prebiotic and probiotic: the role of galactooligosacharides as prebiotic additives: a review. Potravinarstvo, 7(1), 28-35. http://dx.doi.org/10.5219/251 WIESER, A., SCHNEIDER, L., JUNG, J., SCHUBERT, S. 2012. MALDI-TOF MS in microbiological diagnostics-identification of microorganisms and beyond (mini review). Applied Microbiology and Biotechnology, 93(3), 965-974. http://dx.doi.org/10.1007/s00253-011-3783-4 ZHANG, Z., CAO, H., CHEN, CH., CHEN, X., WEI, Q., ZHAO, F. 2017. Effects of fermentation by Ganoderma lucidum and Saccharomyces cerevisiae on rape pollen morphology and its wall. Journal of Food Science and Technology, 54(12), 4026-4034. http://dx.doi.org/10.1007/s13197-017-2868-1 ZHOU, W., YAN, Y., MI, J., ZHANG, H., LU, L., LUO, Q., LI, X., ZENG, X., CAO, Y. 2018. Simulated Digestion and Fermentation in Vitro by Human Gut Microbiota of Polysacchyrides from Bee Collected Pollen of Chinese Wolfberry. Journal of Agricultural and Food Chemistry, 66(4), 898-907. http://dx.doi.org/10.1021/acs.jafc.7b05546
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AN OPTIMIZED ALTERNATIVE FOR PHENOLIC COMPOUND-EXTRACTION OF STRAWBERRY BAGASSE AGRO- INDUSTRIAL RESIDUES
Antonio Carlos Santos Felix1, Cleber Galvão Novaes2, Maísla Pires Rocha1, George E. Barreto3, Marcelo Franco4, Baraquizio Braga do Nascimento Junior1, Lisandro Diego Giraldez Alvarez1*
Address(es): Lisandro Diego Giraldez Alvarez, 1Grupo de Pesquisa Aromas e Análise de Alimentos, Departamento de Ciências e Tecnologias (DCT), Universidade Estadual do Sudoeste da Bahia (UESB), Jequié, Bahia, Brasil. 2Grupo de Pesquisa Laboratório de Química Analítica, Departamento de Ciências e Tecnologias (DCT), Universidade Estadual do Sudoeste da Bahia (UESB), Jequié, Bahia, Brasil. 3Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeriana, Bogotá, D.C., Colombia. 4Departamento de Ciências Exatas e Tecnologia (DCET), Universidade Estadual de Santa Cruz (UESC), Ilhéus, Bahia, Brasil.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.815-820
ARTICLE INFO ABSTRACT
Received 7. 4. 2018 The optimum extraction conditions for highest recovery of total phenolics content and antioxidant capacities were analyzed for Revised 24. 8. 2018 strawberry (Fragaria ananassa) using response surface methodology. Phenolic compounds have been used in food technological Accepted 7. 9. 2018 processes, and thus it can contribute to the prevention of some degenerative diseases and used as antioxidant or antimicrobial. The Published 1. 10. 2018 assessment of the concentration of total phenolics, as well as their capacity to scavenge ABTS and the antioxidant capacity, determined by the modified DPPH method, were investigated based on distinct combinations of time, temperature, and solvents concentration. It was investigated that the optimum condition for getting the highest antioxidant yield was obtained under water-acetone (80:20, v/v) at Regular article 36 ºC and 36 min. We have found that the maximum yield of total phenolic was 1707.66 ± 38.43 (mg GAE/100 g), preserving the antioxidant capacity which was measured by using DPPH and ABTS assays, showing an EC50 of 1085.20 ± 32 (g fruit/g of DPPH) and 66.67 ± 2.4 (µM trolox/g fruit) respectively. This method is also easier and cheaper than other methods to perform polyphenols extractions since does not require expensive reagents or high quantities of organic solvents.
Keywords: antioxidants, strawberry, optimization, phenolic compounds
INTRODUCTION and cereals, green and black teas, coffee beans and red and white wines (Alu’datt et al., 2017; Magalhães et al., 2017). Recently, phenolic compounds have been The strawberry (Fragaria ananassa) belongs to the Rosaceae family and is one used in food technological processes, and thus it can contribute to the prevention of the most consumed and currently investigated non-climacteric fruit (Brito de of some degenerative diseases and used as antioxidant or antimicrobial agents Figueirêdo et al., 2015). The ripe fruit, which has a pleasant flavor, is consumed (Dzoyem et al. 2017). Polyphenols may exert an indirect antioxidant effect, by fresh as natural drinks, eat with or without the peel and seed (Tomadoni et al. protecting endogenous antioxidant enzymes in the human body (Pradeep & 2016). Sreerama, 2018; Zhang et al., 2015), moreover these compounds prevent The health benefits of strawberries have been linked mostly to their biological amyloid β-protein oligomerization and synaptic dysfunction by site-specific activities in the prevention of cardiovascular disease, inflammation, oxidative binding (Ono et al., 2012). Related to this, the interest in food phenolics has stress, obesity, and diabetes. Thanks to its high amount of folate, vitamin C, as increased due to their antioxidant and free radical-scavenging abilities (Santos well as phytochemicals, attention has been given to strawberry consumption Felix et al., 2018), anti-inflammation, modulation of signal transduction and anti- effects (Scalzo et al. 2005). Consumption of antioxidant-rich strawberries in proliferation activities (Banothu et al. 2017). healthy volunteers diminished mortality rate of mononuclear cells and increased It should be pointed out that the effectiveness of nutraceutical compounds in erythrocytes resistance to hemolysis (Tulipani et al. 2014). Cranberry and preventing diseases depends on the bioactivity, bioavailability and stability of the strawberry polyphenols compounds improve insulin sensitivity in insulin- active substances. Thereby, the “medical use” of these agents need food resistant, non-diabetic adults (Paquette et al., 2017). Anthocyanin-rich formulations and processes able to maintain the active molecular structures until strawberry supplementation positively improved lipid profiles by significantly their consumption and release in the physiological target (Vieira da Silva, decreasing of total cholesterol concentrations, triglycerides and low-density Barreira, & Oliveira, 2016). The bioactive phenolic compounds feature lipoprotein cholesterol (LDL) (Alvarez-Suarez et al., 2014). Numerous studies different polarities and chemical properties that are related to their structures. The and health benefits related to the strawberries were described in previous review. extraction process is affected by the polarity of the bioactive compounds; However, clinical trials on cancer chemoprevention are still limited (Afrin et al. therefore, the matrix type, time, and temperature used in the process can 2016). influence the efficiency of extraction of phytochemicals (Andrade et al. 2015; Residues of the industry juices can trigger serious environmental problems, these Rufino et al. 2010). residues, consisting of seeds, peels, husks, whole pomace, among others, are The extraction process is an important step for isolating and identifying generated every year in the form of wastes, and are poorly valorized (S. Martins polyphenols. Phenolic compounds are the major phytochemicals in strawberry et al., 2011). Recently, increased attention has been given to these materials as fruits, mainly represented by anthocyanins (41%), flavan-3-ols (28%), cinnamic abundantly available and cheap renewable feedstock for the production of acid conjugates (13%), ellagitannins (14%), flavonols (3%) and ellagic acid compounds like the polyphenols (Sójka et al. 2013; Tumbas Šaponjac et al. conjugates (1%), which have been considered to be the most important 2015; Zhu et al. 2015). Phenolic compounds are plant secondary metabolites contributors to the various biological potentialities of strawberries (Zhu et al., commonly found in plants and derived products such as berries, apples, citrus 2015). fruit, cocoa, grapes, onions, olives, tomatoes, broccoli, lettuce, soybeans, grains Response surface methodology (RSM) are techniques used for optimization of
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processes and explores the relationships between explanatory variables and ABTS+ assay response variables (Bezerra et al. 2008). It is widely used to optimize conditions for extracting active compounds from herbs (Lai et al. 2013; Ji et al. 2012). The The ABTS+ assay was based on a method developed by Evans Re et al with use of RSM is useful to determine optimum values of the independent variables modifications (Re et al., 1999). ABTS+ radical were produced by reacting 7 mM to achieve better response, and enables the user to investigate the interaction ABTS stock solution with 145 mM potassium persulfate and allowing the among variables, being more efficient than the traditional process using the one- mixture to stand in the dark at room temperature for 14 h before use. The ABTS+ variable-at-a-time method (OVATM). Also, the application of RSM provides a solution was diluted with ethanol until an absorbance of 0.7 ± 0.02 at 734 nm was faster and less expensive procedure to obtain optimal values of chemical and reached. 30 µL of the extract were added in 3.0 mL of diluted ABTS+ solution. instrumental variables affecting the extraction, in accordance with the principles After the addition of 30µl of the extract, samples absorbances were recorded at 6 of the green chemistry (Altemimi et al. 2015; De Souza et al. 2014). Hence, in min after mixing. The results were expressed as µM trolox/g fruit. this study two factorial designs (mixture and three level factorial) were applied to optimize the best proportion of solvents, time and temperature for the extraction RESULTS AND DISCUSSION of phenolic compounds from strawberry (Fragaria ananassa) bagasse agroindustrial residues. Free radical scavenging capacities were also measured Application of the experimental designs in the extraction process using DPPH and ABTS radical cations. In this study, we have determined the optimum values of the independent MATERIALS AND METHODS variables to achieve the maximum response for the extraction of total phenolics compounds, preserving their antioxidant activity in strawberry (Fragaria Chemicals and reagents ananassa) bagasse agroindustrial residues. The assessment of the concentration of total phenolics, as well as their capacity to scavenge ABTS and the antioxidant Gallic acid monohydrate (98 %), sodium carbonate P.A, Folin & Ciocalteu’s capacity, determined by the modified DPPH method, were investigated based on phenol reagent and Trolox (2,5,7,8-tetramethylchroman- 2-carboxylic acid) were distinct combinations of time, temperature, and solvents concentration. purchased from Sigma-Aldrich (São Paulo, SP, Brasil). Acetone P.A., distilled The maximum yield of total phenolic were 1707.66 ± 38.43 (mg GAE/100 g). water, and ethanol 95 % P.A. were obtained from Vetec (Rio de Janeiro, RJ, The antioxidant capacity which was measured by using DPPH and ABTS assays, Brasil). Methanol 99,8 % P.A. was purchased from Chemis (São Paulo, SP, showing an EC50 of 1085.20 ± 32 (g fruit/g of DPPH) and 66.67 ± 2. 4 (µM Brasil). DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS, 2,2 azino bis (3- trolox/g fruit) respectively. ethylbenzo thiazoline 6 sulfonic acid), diammonium salt were purchased from Actually, relevant attention has been given to the health benefits provided by Sigma-Aldrich (São Paulo, SP, Brasil). polyphenols derived from plants. Berries are rich in nutrients, vitamins, dietary Sample fibers, and minerals, as well as polyphenols (Nile and Park 2014). The content The strawberry bagasse agro-industrial residues sample employed for the of flavonoids, micro-nutrients such as folate, vitamin C, and minerals determine development of the extraction method were obtained from local industry the potential health effects of strawberries (Giampieri et al., 2012). In spite of (Frutisol, Jequié, Bahia, Brazil). Residues was dried in an air-circulating oven at that, it is not always possible to extract all the target compounds with a unique 50°C for 72 h and then powdered in a grinder and stored at 20°C until extraction solvent. Therefore, different solvents are often required to extract species of and analysis. varying polarities in compound mixtures. The mixture consisting of water and organic solvent may facilitate the extraction of species that are soluble in water Extraction total phenolic contents and/or organic solvent (Boeing et al. 2014). Indeed, in this study, a mixture design was performed to optimize a better combination involving three solvents The polyphenols of strawberry bagasse agro-industrial, 10-fold volume (w/v) of different polarities (acetone, ethanol and water) in the extraction of phenolic were extracted with 50 mL of a mixture (ethanol, acetone, and water) for 2 h at compounds from strawberry. 35°C in a shaking incubator (Shaker SL 222, Solab) at 200 rpm. After completion The evaluated response was the total phenolic content extracted. In this study, we of the extraction time, the crude extract was centrifuged at 5000 rpm (Fanem- applied two response surface methodology techniques to determine the optimum Tecnal, São Paulo, Brasil) for 10 min. The extract was filtered for removal of values of the independent variables to achieve the maximum response for the solids particles, and the supernatant was collected and analyzed for apparent extraction of total phenolics compounds: (1) design matrix and (2) 3K factorial phenolic content, ABTS and DPPH radical scavenging activity. design. The response expressed as total phenolic content were calculated by the Initially, a mixture design was performed to optimize the best proportion of the adapted Folin–Ciocalteu method and the results achieved are shown in Table 1. three solvents used. After this preliminary evaluation a three level factorial design (TLFD) was performed involving the variables time and temperature. The factors Table 1 Design matrix and results for optimization of the extraction of optimized by mixture design remained constant during execution of the 3k compounds phenolic. factorial designs. All the experiments were also performed randomly. The Mixture design experimental dominions as coded and real values for the factors and the response Variables Total Phenolic* (Total Phenolic) obtained are shown in Table 1. Experimental designs and the Run Water ® Acetone (%) Ethanol (%) (mg GAE/100 g) statistical analysis was conducted using the statistic software 10 (Statsoft) with (%) 95% of confidence level. All determinations were carried out in triplicate (n=3) 1 100.0 0.0 0.0 125.43 ± 5.55 and the data recorded as mean and standard deviation. 2 0.0 100.0 0.0 1067.45 ± 10.7 3 0.0 0.0 100.0 218.68 ± 9.85 Determination of total phenolic contents 4 50.0 50.0 0.0 935.20 ± 8.54 5 50.0 0.0 50.0 334.66 ± 13.44 Total phenolics content was determined according to the adapted Folin–Ciocalteu 6 0.0 50.0 50.0 595.93 ± 7.75 method (Rebaya et al., 2015). Briefly, the extracts (0.5 mL) were mixed with 2.5 7 66.7 16.66 16.7 504.72 ± 6.65 mL of Folin–Ciocalteu reagent (1:10) and 2 mL of sodium carbonate solution 8 16.7 66.7 16.7 695.12 ± 12.54 (4%). The mixture was stirred and kept at room temperature for 2 h in the dark. 9 16.7 16.7 66.7 643.06 ± 12.05 Then, the sample absorbance was measured by spectrophotometer (MARTE 10 (CP) 33.3 33.3 33.3 707.83 ± 10.40 SPECTRO 560) at 750 nm against a blank. Aqueous solutions of gallic acid were 11 (CP) 33.3 33.3 33.3 691.04 ± 9.15 used for calibration. The results were expressed as milligrams (mg) of gallic acid 12 (CP) 33.3 33.3 33.3 693.76 ± 7.25 equivalents per 100 g of residue (mg GAE/100 g). All measurements were 3k factorial design performed in triplicate. Variables Total phenolic* Run Temperatur Time (min) (mg GAE/100 g) Determination of antioxidant activity e (°C) 1 (-1) 30 (-1) 25 751.85 ± 77.46 DPPH (free radical-scavenging) assay 2 (-1) 30 (0) 35 1110.26 ± 51.05 3 (-1) 30 (+1) 45 996.26 ± 94.09 The antioxidant capacity was determined by the modified DPPH method (Brand- 4 (0) 60 (-1) 25 996.04 ± 104.81 Williams et al. 1995), which is based on the quantification of free radical- 5 (CP) (0) 60 (0) 35 1122.08 ± 39.50 scavenging. A methanol solution containing 0.06 mM DPPH was prepared. After 6 (CP) (0) 60 (0) 35 1053.2 ± 13.01 adjusting the blank with methanol, an aliquot of 100 µL of fruit extract was added 7 (CP) (0) 60 (0) 35 826.42 ± 5.57 to 3.9 mL of this solution. The reduction in absorbance at 515 nm was measured 8 (+1) 90 (-1) 25 1052.83 ± 35.00 at 1 min intervals for the first 10 min, and then at 5 min intervals until 9 (+1) 90 (0) 35 1004.45 ± 8.50 stabilization. The antioxidant capacity was expressed as the concentration of 10 (+1) 90 (+1) 45 910.26 ± 2.50 antioxidant required to reduce the original amount of free radicals by 50% (EC50) 11 (0) 60 (+) 45 1061.18 ± 36.50 and the values expressed as g fruit/g DPPH. CP – Central Point; *Mean ± Standard Deviation,
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The extraction of phenolic compounds is dependent on the solvent used and their extraction solution (Boeing et al., 2014). In addition, with a high ratio of water polarity. In this work, were used ethanol, water, and acetone, which present a (80%) in the solvent mixture, we can infer that our extraction procedure can be a dipolar moment (µr) of 1.69 Debye (D), 1.85 D and 2.88 D, respectively (C. R. clean alternative to procedures involving only organic solvents or mixtures Martins et al. 2013). The highest value of phenolic contents was obtained with containing a high proportion of organic solvents. This behavior was supported by water and the lowest with acetone or ethanol (Table 1: Run 1 and 3). According the response surface plots and polynomial equations (Fig. 1). The surface to Fig. 1a and 1b, an increase of water concentration resulted in enhancement of presented in the Fig. 1 (a) has a maximum as critical point. The proportions of the the polyphenol content. Figure 1 shows the effect of the interaction of different components that generate the better response in the extraction are the coordinates solvents concentration and extraction power. Boeing et al. (2014) found that of this point. Therefore, the optimum conditions for the total phenolics extraction acetone is the least efficient solvent when used pure, but showed good results were water/acetone mixture (80:20, v/v) without the presence of ethanol. The when combined with water due to increased solvation provided by the presence surface presented in the Fig. 2 (b) is the contour graphic that is generated by the of water (Boeing et al., 2014). Moreover, a constrained mixture design was used projection of the surface shown in Fig. 2 (a) in the two-dimensional plane. to investigate the best proportion of the three solvents used for the extraction. The matrix of the experimental design and the response (Total Phenolic - TP) When working with mixtures variables, this freedom of combination between obtained are shown in Table 1 (n = 3). The surfaces response can be described for levels does not exist, because they cannot vary without taking into consideration Eq. (1), resulting from an adjustment of the quadratic model: the levels of other variables. In this case, the variables are the components of a mixture, and the sum must be constant and equal to 1 (Novaes et al., 2016). TP = 119.46(A) + *1003.98(W) + *272.83(E) + *1282.51(AW) + 812.95(AE) - 140.92(WE) Eq. (1)
The values marked with * are considered significant; A – Acetone, W – Water and E – Ethanol. The coordinates of the maximum point are found by means of the first derivative of the mathematical function (Lemos et al. 2009). Analysis of variance (ANOVA), P-value significant levels and the analysis of the residues generated between the predicted values and the observed values were used to check the significance of the effects, at the 95% confidence level (p < 0.05).
Figure 1 (a) Response surface and (b) Contour plot for optimization of the total phenolic contents of strawberry extracts as a function of acetone, ethanol and water concentration.
By applying the Lagrange criteria, the critical point of the mixture design is characterized as a saddle point. However, the coordinates of the saddle point will not show the optimum conditions of extraction (Ferreira et al., 2007). Therefore, in order to find the optimum conditions for the extraction, a visual inspection of the surfaces was performed, by analyzing the response surfaces presented in the Fig. 1 is possible to observe that polyphenols content increased parallel to water concentration up to almost 80%, which was further confirmed by the contribution of each factor displayed by the polynomial equation obtained. Furthermore, we Figure 2 (a) Response surfaces and (b) Contour plot of the total phenolic were able to find that a high ratio of water (80%) in the solvent mixture with contents of strawberry extracts as a function of time and temperature acetone (20%) enhanced the extraction yield. According to Alothman et al (2009), acetone-water mixtures are good solvent combinations for the extraction Effects of time and temperature on the extraction yield of polar antioxidants (Alothman et al. 2009). These results are interesting, since it is the first attempt to identify the best solvents used for the extraction of It is known that high temperatures improve the efficiency of the extraction; antioxidant compounds from three different berries cultivated in southern Brazil. however, the excessive temperature may degrade phenolic compounds. Previously, Boeing (2014) found that the highest total phenolic contents for Therefore, extraction temperature and time were another parameters studied in strawberry extracts were obtained for acetone/water (50/50, v/v) and (70/30, v/v) the extraction procedure. After the preliminary evaluation by mixture design, a
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three level factorial design (TLFD) was used to screening new variables that antioxidant capacity of the extracts is their preservation. In spite of that, the significantly affect polyphenol extraction. TLFD presents three levels (−1, 0, and results from this study demonstrated that strawberry bagasse agroindustrial +1) and is generated by the expression 3k. The experimental matrix is composed residues could be considered a potential source of phenolic bio-active compounds of all three level combinations of these levels (Novaes et al., 2016). Table 1 with strong antioxidant capacity. With our optimal conditions of extraction, we showed the matrix of the experimental design of the two factors expressed as were able to obtain a high antioxidant capacity comparing with others works. For coded and real values and the response (Total Phenolic - TP) obtained (n=3). The example, De Souza et al (2014) found an EC50 of 3778.94 ± 333.88 (g fruit/g of ratio of water (80%) and acetone (20%) was kept according to the results DPPH) and 7.87 ± 0.87 TEAC (µM/g fruit) when the extracts were obtained with obtained in design 1. The function that represents the relationship between the methanol/water (50:50, v/v) at room temperature for 1 h (De Souza et al., 2014). time (Ti), temperature (Te) and Total Phenolic (TP) for polyphenol extraction Although there exist some studies with extraction times ranging from a few from strawberry is presented by Eq. (2): minutes to several hours (Boeing et al. 2014), in this work we have found parameters that can obtain not only the maximum extraction of total phenolic, but TP = 1033.82 + 36.43(Ti) - 52.72(Ti)2 + 55.85(Te) - 109.52(Te)2 - 193.42(Ti)(Te) also an effective antioxidant capacity by using a short period of time (36 min). Eq. (2) Although we worked with bagasse agroindustrial residues, it should be remarked that antioxidants can perform protective roles against free radicals through a This model fits the experimental data. The quadratic model allowed one to locate variety of different mechanism like catalytic systems to neutralize or divert the optimal conditions and generated the corresponding response surfaces (Fig. 2 reactive oxygen species (ROS) (Hwang et al., 2015). It should be pointed out (a)) and contour plot (Fig. 2 (b)). The surface presented in the Fig. 2 (b) is the that in biological conditions the antioxidant capacity could change, however, contour graphic that is generated by the projection of the surface shown in Fig. 2 many reports support the potential protective effects of various polyphenol-rich (a) in the two-dimensional plane. The Fig. 2 a-b shows the effect of the foods against chronic disease, including cancer, cardiovascular disease and interaction of the extraction time and temperature variables in the extraction of neurodegeneration. All of these biological activities can be ascribed to the wide the polyphenols. Response surface plots showed that polyphenols extraction range of bioactive compounds present in the strawberry (Giampieri et al., 2017). increased under experimental conditions optimized. The contribution of each According to Del Rio, to evaluate if polyphenols cause these effects, well- factor is displayed by the polynomial equation obtained. The critical points were powered and well-controlled human intervention trials are necessary (Del Rio et calculated by solving the equation system formed by the partial derivatives of the al., 2013). Related to this, positive results were obtained and confirmed in in vivo function (Martendal et al. 2007): studies on animals and humans, showing the effects of strawberry phenolics on oxidative stress in both physiological and pathological situations. Tulipani et al. ∂TP/∂(Ti) = 0 = 36.43 – 105.44(Ti) – 193.42(Te) Eq. reported that strawberry intake increases blood fluid, erythrocyte and (3) mononuclear cell defenses against oxidative challenge, thus suggest that regular ∂TP/∂(Te) = 0 = 55.85 – 219.04(Te) – 193.42(Ti) Eq. consumption of strawberries may improve human defenses against oxidative (4) challenges (Tulipani et al., 2014). Strawberry consumption alleviates (Ti) = 66 min and (Te) = 36 °C doxorubicin-induced toxicity by suppressing oxidative stress (Giampieri et al., 2016). The critical points also can be observed by visual inspection of the charts The recovery of antioxidants reflects not only the need for biofunctional depicted in Fig. 2. In this way, for the evaluated experimental domain, an ideal compounds, which may also be interesting from a technological point of view as experimental condition is obtained when using intermediate values of time and valuable components of nutraceuticals in food and pharmaceutical preparations temperature. Under these experimental conditions optimized (water/acetone or cosmetics industry, but can also exploit the food production chain (Van Der mixture (80:20, v/v); time = 36 min and temperature = 36°C), the extraction of Goot et al., 2016). polyphenolic compounds from strawberry was 1707.66 ± 38.43 (mg GAE/100 g). The model generated by mixture design was able to establish better conditions for CONCLUSION the extraction of compounds from strawberry. The phenolic content of the strawberry was higher than that reported by De Souza (De Souza et al., 2014) To sum up, the importance of this work is that we were able to find optimal who found 621.92 ± 15.51 (mg GAEs/100 g) by extracting sequentially with conditions producing the maximum extraction yield of polyphenolic compounds methanol/water mixture (50:50, v/v) at room temperature for 1 h. Moreover, with strong antioxidant capacity. It was investigated that the optimum condition Patras et al. (2009) reported 855.02 ± 6.52 (mg GAEs/100 g) when using with for getting the highest antioxidant yield was obtained under water/acetone unprocessed purées (Patras et al. 2009). To evaluate the significance of the mixture (80:20, v/v) at 36ºC and at 36 min of time. This method is also easier and model and the effect of the main parameters, analysis of variance (ANOVA) was cheaper than other methods to perform polyphenols extractions since does not assessed (Table 2). The p-value for lack of fit of the model was 0.7535 (p > 0.05, require expensive reagents or high quantities of organic solvents. Fruit sources not significant). The results demonstrated that the mathematical model is well like waste strawberry may bring new natural products into the food industry with suited to the obtained values. safer and better antioxidants qualities against oxidative damage thus suggesting a new interesting target to search for a novel technological extraction method not yet patented. Table 2 Analysis of variance (ANOVA) for response surface quadratic model.
Factor SS d.f. M.S. F-value p-value Acknowledgments: The authors are grateful to Conselho Nacional de Ti(L) 1990.7 1 1990.7 0.083184 0.8002 Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Ti(Q) 1760.0 1 1760.0 0.073545 0.8117 Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação de Te(L) 4678.8 1 4678.8 0.195510 0.7016 Amparo à Pesquisa do Estado da Bahia (FAPESB) for their financial support and fellowships. Te(Q) 7596.1 1 7596.1 0.317411 0.6299 Ti by Te 37411.3 1 37411.3 1.563272 0.3376 Conflict of Interest Statement: The authors of the paper do not have a direct Lack of Fit 31011.3 3 10337.1 0.431947 0.7535 financial relationship with the commercial identities mentioned in this paper that Pure Error 47862.8 2 23931.4 might lead to a conflict of interests. Total SS 135126.7 10 Ti - Time; Te - Temperature; L - Linear; Q - Quadratic; SS - sum of squares; df - degree of REFERENCES freedom; MS - mean square. Afrin, S., Gasparrini, M., Forbes-Hernandez, T. Y., Reboredo-Rodriguez, P., Effects of conditions on antioxidant activity Mezzetti, B., Varela-López, A., … Battino, M. (2016). Promising Health Benefits of the Strawberry: A Focus on Clinical Studies. Journal of Agricultural and Food The antioxidant capacity of extracts obtained in the optimal conditions producing Chemistry, 64(22), 4435–4449. https://doi.org/10.1021/acs.jafc.6b00857 the maximum extraction yield of polyphenolic compounds, water/acetone Alothman, M., Bhat, R., & Karim, A. A. (2009). Antioxidant capacity and mixture (80:20, v/v); time = 36 min and temperature = 36°C, was measured by phenolic content of selected tropical fruits from Malaysia, extracted with using DPPH and ABTS assays, showing an EC50 of 1085.20 ± 32 (g fruit/g of different solvents. Food Chemistry, 115(3), 785–788. DPPH) and 66.67 ± 2.4 TEAC (µM/g fruit), respectively. https://doi.org/10.1016/j.foodchem.2008.12.005 The DPPH assay is employed to test the ability of compounds to act as free Alvarez-Suarez, J. M., Giampieri, F., Tulipani, S., Casoli, T., Di Stefano, G., radical scavengers, and frequently used to evaluate the antioxidant capacity of González-Paramás, A. M., … Battino, M. (2014). One-month strawberry-rich foods. When a solution of DPPH• radical is mixed with an antioxidant substance, anthocyanin supplementation ameliorates cardiovascular risk, oxidative stress its color turns from purple to yellow. ABTS assay estimates more accurately the markers and platelet activation in humans. Journal of Nutritional Biochemistry, antioxidant capacity of foods, particularly those containing hydrophilic, lipophilic 25(3), 289–294. https://doi.org/10.1016/j.jnutbio.2013.11.002 and highly pigmented compounds (Floegel et al., 2011). It is worthwhile noting Andrade, R. A. M. de S., Maciel, M. I. S., Santos, A. M. P., & Melo, E. de A. that the solvent extraction of antioxidants can be improved increasing, for (2015). Optimization of the extraction process of polyphenols from cashew apple example, the solvent temperature; however, one of the major problems of the
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PHYSICAL, CHEMICAL AND NUTRITIONAL EVALUATION OF Arachis hypogaea L. SEEDS AND ITS OIL
Akshata Salve1, Shalini Arya*1
Address(es): Dr. Shalini Arya, 1Food Engineering and Technology Department, Institute of Chemical Technology, Mumbai, Matunga, Mumbai - 400019, India.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.835-841
ARTICLE INFO ABSTRACT
Received 26. 3. 2018 Peanut is an important crop grown worldwide. High yielding Indian peanut varieties namely SB-11 (Boria), JL-24 (Disco), TLG-45 Revised 1. 8. 2018 (Latur), grown under local soil and climatic conditions, were compared for their chemical composition and nutritional quality. Due to Accepted 7. 9. 2018 the insufficiency of the scientific information available, study aims at generating systematic and segregated data on the proposed peanut Published 1. 10. 2018 varieties. SB-11 variety contained maximum protein (42.09%) whereas, JL-24 exhibited highest oil content (47.00%) and sensory acceptability. Mineral compounds were present in significant and comparable amounts using ICP- AES in all the varieties. Functional properties like water holding capacity (2.90g/g), oil binding capacity (1.50g/g) was recorded highest for SB-11 and water solubility Regular article index (40%) for JL-24 variety, which facilitates its incorporation in various food products. TLG-45 variety was estimated to have highest total flavonoid (0.51mg/g), DPPH (1.81mg/g) and ABTS (0.24mg/g) radical scavenging activity, whereas SB-11 with highest total polyphenol content of 2.05 mg/g. Difference in the fatty acid composition of oil from peanut varieties was analysed by GCMS. Acid value, iodine value, saponification value, peroxide value, low absorbance and no measurable aflatoxin in all varieties substantiated that they are excellent for human nutrition, especially during malnourishment, incorporation for food enrichment or functional food development. This study can thus be helpful to researchers and industries globally, indicating use of these studied varieties for export, applications in food and oil industries.
Keywords: peanuts; functional properties; physico˗chemical composition; antioxidant; fatty acid composition; GC-MS
INTRODUCTION MATERIAL AND METHODS
Peanut (Arachis hypogaea L.) or “groundnuts” are the edible seeds of a legume. Materials Peanut belongs to the family fabaceae of bean/legume. India is the second largest producer of peanut in world, with total production of approximately 7.131 Fresh peanut varieties namely SB-11, JL-24 and TLG-45 belonging from million tons per year after China’s 13.42 million tons (Arya et al., 2016; USDA, different states of India, were purchased from Agricultural Produce Market 2016). Peanut consumption all over the world varies in large proportions hence Committee (APMC), Mumbai, India, where they are transported for distribution the commercial products too are variant and generally localized. Peanuts are throughout India. Folin-Ciocaleteu reagant, sodium carbonate anhydrous, sodium consumed around the world as raw and in variety of products. Peanuts are valued hydroxide was purchased from SD Fine Chemicals, Mumbai, India. Vanilline, for their high value oil content. Apart from oil, by-products of peanut contains petroleum ether was purchased from HiMedia, Mumbai, India. HCl, gallic acid, many other functional compounds like proteins, fibers, vitamins, minerals, sodium acetate, potassium chloride, sodium sulphate, potassium per sulphate, antioxidants etc, (USDA, 2015). Literature has revealed that peanuts are excellent were purchased from SD Fine Chemicals, Mumbai, India. 2, 2-azinobis (3- source of compounds like resveratrol, phenolic acids (Ma et al., 2014), ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), 1, 1-diphenyl- flavonoids and phytosterols (Chukwumah et al., 2009) that block the absorption 2-picrylhydrazyl (DPPH), trolox, catechin were obtained from Sigma–Aldrich, of cholesterol from diet. Peanuts contain all the 20 amino acids in variable India. Hexane, methanol and water of HPLC grade were purchased from Merck proportions and is the biggest source of the protein called “arginine” (USDA whereas, ethanol and conc. sulphuric acid from SD Fine Chemicals. All other 2015). These bioactive compounds have been known for their disease preventive chemicals and reagents used in the present study were of analytical grade. The properties and are believed to promote longevity (Arya et al., 2016). standards of esters were obtained from Sigma Aldrich (USA). Apart from the above health benefits; peanuts are rich in monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs) (Veličkovska & Mitrev, Chemical composition 2016). Additional beneficial nutrients indigenous to peanuts include vitamin E, L-arginine, soluble and insoluble fibre, as well as water and lipid-soluble natural Peanut varieties were subjected to proximate analysis such as moisture, fat, phenolic antioxidant. These nutrients act synergistically with the numerous protein, ash by AOAC Method, (AOAC, 1995) and total carbohydrates content protective bioactives, making peanuts a desirable nutrient-dense plant-based food was calculated using following formula: (Zhang et al., 2011). Total carbohydrate (% dry weight) = {100 - moisture (%) - protein content (% In India, about 50% of all childhood deaths are attributed to malnutrition dry weight) - fat (% dry weight) – ash (% dry weight)} (Sharma et al., 2014). Protein deficiency is the main cause of malnutrition. Vitamin and mineral deficiencies also affect children’s survival and development. Sample preparation Beside all the above facts, systematic and scientific database on different peanut varieties and peanut based products of Indian origin is missing. Hence in the Peanuts were stored under cool and dry condition prior to analysis. Soxhlet present research efforts were to perform a detailed characterization, comparison extraction was carried out using petroleum ether as solvent. 25 g of peanuts of and nutritional assessment of Indian peanut varieties namely SB-11, JL-24 and each variety were finely crushed and tied in a cellulose thimble. The apparatus TLG-45 which are locally called as Boria, Disco and Latur respectively. was run for 6 h and solvent was eliminated by distillation. Defatted samples were dried and stored at -20ºC until analysis.
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Fat extraction centrifuge tube and 15 ml distilled water was added to the tube and vortexed for 2 min to form a suspended mixture. This mixture was then centrifuged at 4000 rpm Fat extraction from peanut varieties for chemical analysis of oil was done using a at 20°C for 20 min. The supernatant was then discarded and the difference in mechanical oil press equipment (Adeeko & Ajibola, 1990). weight between dry flour and wet pellet was taken as water binding capacity (Nicholls et al., 1995). Sensory analysis Water binding capacity (g/g) = (Weight of sediment)/(Weight of sample)
Sensory analysis of raw peanut varieties and a control of branded supermarket Oil binding capacity peanut sample (Satyam) of which variety was unknown was carried out according to the method of Lawless and Heymann (2010) using 5-point rating The oil binding capacity (OBC) of flours was analysed using protocol by scale in laboratory at ambient conditions. Semi-trained twelve panellists were Adeleke and Odedeji, (2010).Defatted peanut sample – 1 g was taken in a selected between age group of 23 to 30 years. Sensory panellists were asked to centrifuge tube and 15 ml of oil was added to the tube and vortexed for 2 min to give score between 1-5, for all peanut varieties in comparison with control for form a suspended mixture. This mixture was then centrifuged at 4000 rpm at different parameters like colour, aroma, texture, taste and overall acceptability. 20°C for 20 min. The supernatant was then discarded and the difference in weight between dry flour and oil pellet was taken as oil binding capacity Preparation of extract (Nicholls et al., 1995). Oil Binding Capacity (g/g) = (Weight of sediment)/(Weight of sample) Defatted powders of all three peanut varieties (1 g) were extracted for 3 hour with 10 ml of methanol/water (80/20) solvent on an orbital shaker set at 180 rpm (30 ± Water soluble index 10C). The sample suspension was centrifuged at 8,000 rpm for 15 minutes at 10˚C, the supernatant was collected, filtered and was stored at –20˚C. Analysis The water soluble index (WSI) of flours was analysed using protocol described was completed within two days. by Kaur and Singh, (2005). Defatted peanut sample (2.5 g) was dispersed in 30 ml of distilled water, using a glass rod, and cooked at 90°C for 15 min in a water Determination of total phenolic content bath. The cooked paste was cooled to room temperature and transferred to centrifuge tubes, and then centrifuged at 3000g for 10 min. The supernatant was The total phenolic content (TPC) was measured by Folin-Ciocalteau method as decanted for determination of its solid content into a pre weighed evaporating explained by Hussain et al. (2012). Gallic acid was used as a standard for total dish and the sediment was weighed. The weight of dry solids was recovered by phenolics. 0.2 ml methanolic extracts were added with 1 ml of Folin–Ciocalteau evaporating the supernatant overnight at 110°C. Triplicate determinations were reagent (diluted in a ratio of 1:10 with distilled water) and 0.8 ml sodium carried out. WSI were calculated by the equation: bicarbonate (7.5%) was added to this mixture. This mixture was allowed to stand Water soluble index (%) = (Weight of dissolved solids in supernatant)/(Weight of in dark for 30 min at room temperature and absorbance was measured at 765 nm sample) 100 using Shimadzu UV-180 spectrophotometer. The standard curve was linear between 0 and 250 μg/ ml gallic acid. Results were represented as mg of GAE/g Foaming capacity (FC) wet basis. Foaming capacity was determined by Yu et al. (2007). 1% defatted sample was Determination of total flavonoid content prepared in de-ionised water and adjusted to pH 7.4 with 1.0 N NaOH and 1.0 N HCl. 100 ml (V1) of peanut flour concentrate was blended using a high speed The total flavonoid content (TFC) was measured by vanillin-HCL method as blender for 3 min and then poured into 250 ml graduated cylinder and the volume explained by Rebecca et al. (2010). Methanol extracts (0.5 ml) were dispensed of foam (VF) was immediately recorded. Foaming capacity was calculated using into 2.5 ml of vanillin reagent (8% HCl in methanol/ 4% vanillin in methanol, following equation: 1:1, v/v) and were incubated for 20 minutes at room temperature. The absorbance Foaming capacity = VF/V1 was taken at 500 nm on a Shimadzu UV-180 spectrophotometer. The standard curve was linear between 0 and 250 μg/ ml catechin. The flavonoids were Chemical analysis of oil represented as mg of CE/g wet basis. Iodine value DPPH free radical-scavenging assay Iodine value was estimated as per titre metric method of Pearson (1981). Oil The ability to scavenge DPPH free radicals was determined based on the method sample – 2 g was weighed in a 250 ml dry stopper bottle and 10 ml carbon tetra of Sonawane and Arya, (2015) with little modification in DPPH concentration. chloride was added to it. 20 ml of Wij’s solution was added and then mixture was 0.1mM of DPPH prepared in methanol was diluted to set the absorbance at 0.7 allowed to stand for 30 min in the dark. 15 ml of 10% potassium iodide and 100 (±0.02) at 517 nm. 3.9 ml of diluted DPPH was added to 0.1 ml of sample extract ml of distilled water was added to the solution and then titrated with 0.1M in test tube and it was vigorously shaken and kept in dark room for incubation sodium thiosulphate solution using starch as an indicator before the end point. (15 min) and absorbance was measured at 517 nm using Shimadzu UV-180 Along with oil samples blank was also prepared. Iodine value was calculated spectrophotometer. The standard curve was linear between 0 and 250 μg/ ml using following formula: Trolox. Results are expressed in mg of TE/g on wet basis. The percent Iodine value (Wij’s) = [(V2-V1)1.269)/(weight of sample (g)] scavenging was calculated by following formula; Where, V2= titre value for blank Scavenging % = 100 (Ablank – Asample/Ablank) V1= titre value for sample Where, Ablank is the absorbance of the DPPH solution and Asample is the absorbance of the extract solution. Acid value
ABTS Assay free radical-scavenging assay Titre metric method of Pearson (1981) was used for acid value determination. Oil sample – 5g, 75 ml hot neutral alcohol was added in a conical flask with few Antioxidant activity was measured using the modified ABTS method of Re et al. drops of phenolphthalein as an indicator. Mixture was titrated with 0.1 M NaOH (1999). The ABTS reagent was prepared freshly and used within 2 days. The with continuous shaking until solution turns pink. Acid value was calculated reagent was made by mixing 7mM ABTS + 2.45 mM potassium persulfate and using the formula: incubated for 16 hours at 37ºC. The ABTS cations diluted with ethanol to set O.D Acid value = (V(5.6))/(Weight of sample) at 0.7 (±0.02) at 734 nm (1:30 v/v). 3.9 ml (absorbance of 0.700±0.02) was added Where, V= titration end point value to the 0.1 ml extract of sample and mixed thoroughly and absorbance was measured at 734 nm immediately after 6 min. The standard curve was linear Peroxide value between 0 and 50 μg/ml Trolox. Results were expressed in mg of TE/g wet basis. Peroxide value was determined as per AOAC (Helrich, 1984). Oil sample – 2 g Estimation of functional properties and 1 g of potassium iodide were mixed with 20 ml of solvent mixture i.e. glacial acetic acid and chloroform (3:2 v/v) in a beaker. This was then placed in a boiling Water binding capacity water bath for 30 seconds. The content was poured in a flask containing 20 ml (5%) iodide solution. The beaker was rinsed with 25ml distilled water and The water binding capacity (WBC) of peanut was analysed using protocol by mixture in the flask was titrated with 0.002 N sodium thiosulphate using starch as Adeleke and Odedeji, (2010). Defatted peanut flour sample – 1g was taken in a
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an indicator. Along with oil samples blank was also prepared. Peroxide value was using a split mode, with the split ratio of 10:1. The mass spectrometer was set to obtained using following formula: scan in the range of m/z 35–600 with electron impact (EI) mode of ionization. Peroxide value = (2(V1-V2) mEq/kg)/(weight of sample) Where, V2= blank titre value Mineral and heavy metal analysis of peanut varieties by ICP-AES V1=sample titre value Mineral composition of peanut varieties was determined using Inductively Saponification value Coupled Plasma- Atomic Emission Spectroscopy (ICP-AES) with make of SPECTRO Analytical Instruments GmbH, Germany and Model ARCOS, Saponification value was determined according to Pearson (1981). Oil– 2 g was Simultaneous ICP Spectrometer. Ash of sample was obtained by AOAC Method, weighed into a conical flask and 25 ml of 0.5 N alcoholic potassium hydroxide (AOAC, 1995). Obtained ash samples were dissolved in aqua regia while heating was added and this solution was refluxed for 1h, cooled and titrated with 0.5 N on hot plate. Dissolved samples were diluted using de-ionized water and were HCl using phenolphthalein as indicator. A blank was prepared alongside and analysed in ICP-AES (Özcan & Seven, 2003). value was calculated using following formula: Saponification value = (56.1N (A-B))/(Weight of sample) Aflatoxin analysis Where, N= Normality of HCl A= Volume of HCl for blank AgraStrip Total Aflatoxin Test (4 ppb cut off) kit according to Luis and B= Volume of HCl for sample Kemerait (2015) was used for aflatoxin estimation, which is validated screening 56.1= Equivalent weight of KOH method for determination of total aflatoxin at 4 ppb cut off level. Tests were performed in duplicate for conformation. Fatty acid composition of oil from peanut varieties by GC˗MS Statistical analysis Fatty acid methyl esters (FAMEs) were prepared from cold pressed oil by modified method of Tarmizi et al, (2013) and Mishra et al, (2014). Oil sample – Statistical analysis was performed by analysis of variance (ANOVA) using IBM 20 mg was dissolved in 1 ml HPLC grade hexane. To this mixture 1ml of HPLC SPSS 24. All data were expressed as mean from triplicate samples ± standard grade methanol and 300 µl H2SO4 were added for trans esterification reaction deviation. Duncan test was used and differences were considered statistically which was then incubated at 70°C for 1 hour in a heating oven. After the significant at p < 0.05 level. reaction, samples were allowed to cool to room temperature. 1ml of HPLC grade water was added to the mixture and the samples were mixed for 30 seconds using RESULTS AND DISCUSSION vortex. Samples were allowed separate and the upper organic layer was collected, 0.5 g sodium sulphate was added to it to absorb remaining moisture and was Proximate analysis filtered through 0.2 µm syringe filter. These samples were analysed for fatty acid profiling using Gas Chromatography (GC), model Agilent Technologies 7890 Proximate analysis includes evaluation of moisture, ash, crude lipid, total protein equipped with Mass Spectrometer (MS) Jeol model AccuTOF GCV. Separation content and total carbohydrate content of three Indian peanut varieties. It is was performed on a capillary column HP5 capillary column (30m × 0.25mm id., inferred from Table 1 that peanuts had 36-42% protein and therefore are a good film thickness 0.25 micron). The carrier gas was helium with flow rate of 1 source of protein. Peanuts are characterized by high oil and protein contents and ml/min. The oven temperature was held for 10 min at 100°C and subsequently low carbohydrates and ash. Knowledge of these components is important in the increased to 280°C at 6°C/min. A sample volume of 1 μl in hexane was injected end products of the industry (Ahmed & Young, 1982).
Table 1 Proximate analysis of Indian peanut varieties Oil Ash Fibre Protein Carbs Variety Moisture % % % % % % SB-11 5.80 ± 0.07a 41.53 ± 0.08b 3.22 ±0.05b 6.11 ± 0.23c 42.09 ± 0.12c 1.37 ± 0.03b JL-24 5.92 ± 0.05a 47.00 ± 0.11a 3.80 ± 0.08c 3.72 ± 0.03a 38.61 ± 0.07b 1.81 ± 0.05a TLG-45 7.08 ± 0.09b 46.10 ± 0.21a 2.48 ± 0.03a 3.83 ±0.04ab 36.89 ± 0.06a 2.85 ± 0.05c All the values are means ± standard deviations (n = 3). Values within a given column followed by the same superscript letters are not significantly different (p > 0.05) according to ANOVA and Duncan’s studentised range test; carbs – carbohydrates
The highest protein (42.09%) and fibre (6.11%) contents were observed in SB-11 Sensory analysis of raw peanut varieties variety followed by JL-24 (38.61%, 3.72%) and TLG-45 (36.89%, 3.83%) respectively. The differences in the protein content was observed among varieties which could be due to the size variation of kernel. Our observations are in accordance with Prathiba and Reddy, (1994) who reported correlation between kernel size and its nutritional composition where, protein content was indirectly proportional to kernel size whereas oil content was independent of kernel size. However, no significant difference in fibre content was observed among JL-24 and TLG-45 varieties, dietary fibre consumption contributes to an increase in faecal bulk thus increased rate of intestinal transit. Oil (47%) and ash (3.80%) content were highest for JL-24 variety. The discrepancy range of oil percentage (41.53- 47.00%) was analogous to that of other species of wild peanuts as previously reported by Stalker et al. (1989), although the studied species and environmental conditions were different. Carbohydrate and moisture content was JL-24 (Disco) TLG-45 (Latur) SB-11 (Boria) highest for TLG-45 i.e. 2.85% and 7.08% respectively. Ash percentages were 2.48-3.80%, and they exhibited significant differences among TLG-45 and other Figure 1 Indian peanut varieties two varieties. Highest crude protein content (47.00%) and the least oil content (41.53%) was observed in SB-11which is ideal for products which require more protein and less oil like bread, nufu (peanut tofu), biscuits, etc. This will prove substantial in reducing malnutrition especially in young and growing children due to protein deficiency. Substantial genetic variability exists for chemical composition and nutritional traits which could be utilised for various food preparations and selection for breeding purpose.
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holding capacity (Table 2). Although no significant difference was observed in 25 WHC of all the varieties under study, OBC was highest for SB-11(1.50g/g) followed by TLG-45 (1.33g/g) and JL-24 (1.16g/g). Proteins rich foods with high 20 oil and water binding are desirable for use in meats, sausages, breads, and cakes, while proteins with high emulsifying and foaming capacity are good for salad ab* b* b* dressing, sausages, bologna, soups, confectionery, frozen desserts and cakes (Yu 15 a* a" a" et al., 2007). Peanuts are good source of protein which is the reason for low oil a" binding capacity (Prinyawiwatkul et al., 1993; Wu et al., 2009). Water a 10 a^ a^ a^ solubility index was highest for JL-24 > TLG-45 > SB-11. The formation of
Sensory score Sensory a^ foam is analogous to the formation of emulsion. In the case of foam, water b' molecules surround air droplets, and air is the non-polar phase. Theoretically, the 5 b' b' a' amphipathic character of protein makes them the good foaming agents that work a b ab ab at air–water interface to prevent bubble coalescence. Data in Table 2 suggest that 0 defatted peanut flour is not a good foaming agent, with a maximum FC of only SB-11 JL-24 TLG-45 control 1.1 for TLG-45 and minimum (1.03) for JL-24. Therefore, defatted peanut flours Peanut varieties may not be suitable in food system that requires foaming such as cake and ice cream. Functional properties are the intrinsic physiochemical characteristics which may affect the behaviour of food systems during processing, product Colour Texture Mouthfeel Taste Overall Accecptability formulation and storage (Menon et al., 2015). Many foods in India are made up of wheat flour so peanut flour can be incorporated in such foods to add functional Figure 2 Sensory analysis of raw peanut varieties properties to it.
Note: The bars on the columns represent standard deviation and the letters on the Bioactive constituents of peanut varieties same column with different superscripts indicate significant difference (p<0.05) Analysis of bioactive constituents of peanut such as total polyphenol content, Sensory analysis of raw peanut varieties namely SB-11, JL-24 and TLG-45 was total flavonoids, DPPH and ABTS scavenging capacity was performed (Table 2). carried out according to the method of Lawless and Heymann (2010) and were Phenolics and flavonoids are an important constituent of peanut quality because compared to super market peanut sample of which variety was unknown of their contribution to the taste, colour and nutritional properties (Francisco & (Control). Disco variety showed maximum acceptance in terms of all parameters Resurreccion, 2012). Peanuts are rich in antioxidants, and the composition of viz., colour, texture, mouthfeel, taste and overall acceptability compared to SB- antioxidants in the methanolic extract is complex (Wang et al., 2007). Total 11, TLG-45 and control sample (Fig 2). Lowest sensory scores were obtained by phenolic content was found highest in SB-11(2.05 mg/g) followed by TLG-45 SB-11, which could be due to the smallest kernel size and comparatively dull (1.88 mg/g) and JL-24 (1.81 mg/g) respectively. Polyphenols help in chelating colour (Fig 1) making it less appealing to the panellists. Bigger kernel size and metal ions such as copper and iron which act as pro-oxidants. Thus we can say bright reddish pink colour of TLG-45 variety was very appealing however, JL-24 polyphenols are secondary antioxidants. TLG-45 had high levels of DPPH radical had highest score for taste among other varieties. The redness and hue angle of scavenging activity (1.81 mg/g) followed by SB-11and JL-24. Similar trend was peanut skin are correlated with total polyphenol content and antioxidant capacity also observed for ABTS scavenging activity with no significant difference among indicating that the hue angle of the peanut skin may be used as a biomarker of JL-24 and TLG-45. As polyphenols and antioxidants protect cell constituents total polyphenol content (Chukwumah et al., 2009). No significant difference in against oxidative damage and, therefore, limit the risk of various degenerative the texture and mouthfeel of TLG-45 and SB-11 was observed when compared to diseases associated with oxidative stress. Lipid soluble polyphenols and control. In peanuts the taste of bitterness and the tactile sensation of astringency antioxidants are expelled with the oil. This is the reason that whole peanuts will is primarily due to the polyphenol and flavonoids (Francisco & Resurreccion, have higher DPPH scavenging activity compared to defatted peanut flour. 0.51 2012), which are known health promoting bioactives (Oliveira et al., 2014). mg/g of total flavonoid was found in TLG-45 variety which is highest among all Sensory acceptability of peanuts is influenced by these bioactives and can also varieties and is attributed to its reddish skin colour. There was no significant reveal a lot about its nutritional constituents. difference found in total flavonoid contents among SB-11(0.24mg/g) and JL-24 (0.25mg/g). Total flavonoid content has a strong correlation with total Functional properties of peanut varieties proanthocyandins and hence can be used as indicator for proanthocyanidin content of peanuts (Chukwumah et al., 2009). It is known that there is reduction Table 2 Functional properties and biochemical constituents of Indian peanut in flavonoid during processing as flavonoids are heat labile (Odriozola-Serrano varieties et al., 2008), therefore total flavonoids in whole raw peanuts will be more than Variety SB-11 JL-24 TLG-45 defatted or heat treated peanuts.
Functional properties Mineral composition of Indian peanut varieties WHC (g/g) 2.90 ± 0.67a 2.88 ± 0.13a 2.76 ± 0.08a OBC (g/g) 1.50 ± 0.10a 1.16 ± 0.05b 1.33 ± 0.05c Study of mineral composition is of interest due to the pro-oxidant activity and a b c health benefits associated with minerals (Parcerisa et al., 1995). Mineral content WSI (%) 19.33 ± 0.57 40.00 ± 1.73 35.00 ± 1.00 of Indian peanut varieties are given in the table (Table 3), significant differences FC 1.06 ± 0.03ab 1.03 ± 0.01a 1.10 ± 0.02b were observed between mineral compositions of varieties. All varieties were found rich in Na, K, Ca, P, Fe, Zn, Cu, Mg and Mn. TLG-45 variety was Biochemical observed to have highest levels of Na, K, P, Zn, Cu and Mg compared to SB-11 constituents and JL-24. Ca and Mg levels were high in SB-11 variety whereas JL-24 was TPC (mg/g) 2.05 ± 0.11a 1.81 ± 0.26a 1.88 ± 0.22a richest in Fe content, which are very close to the RDA and are in accordance with TFC (mg/g) 0.24 ± 0.06a 0.25 ± 0.02a 0.51 ± 0.08b the results reported by (Gyu-Seong, 1993). Heavy metals like Pb and Ni were DPPH (mg/g) 1.46 ± 0.02a 1.03 ± 0.02b 1.81 ± 0.07c found in TLG-45 variety which are slightly exceeding the FSSAI permissible a bc c limits of 2.5 ppm and 5 ppm respectively. Heavy metals are usually absorbed ABTS (mg/g) 0.21 ± 0.01 0.22 ± 0.00 0.24 ± 0.02 from soil by crops and due to contamination during post-harvest practices and TPC- total polyphenol content, TFC- total flavonoid content, DPPH- 1-diphenyl-2- amount of absorption depends on factors like pH of soil and type of heavy metals picrylhydrazyl radical scavenging activity, ABTS- 2-azinobis (3-ethylbenzothiazoline-6- sulphonic acid) diammonium salt scavenging activity present (Ali et al., 2013). Heavy metal contaminants may also get incorporated All the values are Means ± standard deviations (n = 3). Values within a given column due to the equipments used and poor crop harvesting practices (Godfray et al., followed by the same superscript letters are not significantly different (p > 0.05) according to 2010) which could be the reason for its presence in varieties under study. Cr was ANOVA and Duncan’s studentised range test. found to be 0.75 µg/g in SB-11, no traces of Cd and Se were found in any of the variety. JL-24 was also nil for Pb and other heavy metals i.e. Cr was found to be Determination of functional properties like WHC, OBC, WSI and FC is within the permissible limits. This study shows that mineral composition varies necessary for product formulation and in food processing. These properties are from variety to variety and different geographical conditions as they are grown in affected by the intrinsic factors of protein such as molecular structure and size, different states of India. The mineral elements examined were substantial in and many environmental factors including the method of protein separation, pH, reducing malnutrition especially in young and growing children. Thus Indian ionic strength, and presence of other components in the food system. Peanuts peanut varieties under study are mineral rich and are excellent for human were found to have high fibre content which is the reason for its higher water nutrition.
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Table 3 Mineral and heavy metal analysis of peanut varieties using ICP-AES (mg/100g) Element SB-11 JL-24 TLG-45 RDA Copper(Cu) 1.83 ± 0.01a 1.40 ± 0.01 b 1.85 ± 0.03 a 0.9 mg Zinc(Zn) 7.20 ± 0.01 a 5.78 ± 0.06b 8.09 ± 0.01 c 11-8 mg Iron(Fe) 8.27 ± 0.05 a 11.73 ± 0.07 b 9.09 ± 0.03 c 18-8 mg Sodium(Na) 79.45 ± 0.34a 62.85 ± 1.10b 295.10 ± 2.93c 2300 mg Manganese(Mn) 3.88 ± 0.01 a 3.67 ± 0.04b 4.24 ± 0.03c 1.8-2.3 mg 1293.23 ± Potassium(K) 1373.15 ± 3.67 a 1511.45 ± 16.47c 4.7g 11.24b Calcium(Ca) 167.23 ± 1.17 a 131.37 ± 0.59b 150.35 ± 1.75c 1000 mg Magnesium(Mg) 589.12 ± 4.37 a 530.94 ± 1.34 b 507.45 ± 4.99 c 310-400mg Phosphorus(P) 794.78 ± 0.92 a 704.8 ± 0.86b 829.57 ± 3.35c 700 mg Selenium(Se) ND ND ND 0.05mg 2.5 µg/g * Lead(Pb) (µg/g) 1.80 ± 0.09 a ND 5.00 ± 0.25 b
1.5 µg/g * Cadmium(Cd) (µg/g) ND ND ND
5 µg/g * Cromium(Cr) (µg/g) 0.75 ± 0.00a 0.73 ± 0.03 a ND
All the values are Means ± standard deviations (n = 3). Values within a given row followed by the same superscript letters are not significantly different (p > 0.05) according to ANOVA and Duncan’s studentised range test. Note: * FSSAI permissible limits for heavy metals ND: Not detected
Physico-chemical properties of oil extracted from peanut varieties Fatty acid composition of oil from peanut varieties by GCMS
Indian daily diet constitute vegetable oil as a major component. Demand for any Table 5 Fatty acid composition of oil from peanut varieties by GC˗MS oil in the market is based on its functionality, economy and acceptability. Three SB-11 JL-24 TLG-45 Fatty acids Indian peanut varieties analysed above show that the JL-24 variety has highest oil (g/100g) (g/100g) (g/100g) content of 47% followed by TLG-45 and SB-11(Table 4), hence are suitable for Saturated commercial oil production. TLG-45 variety showed highest absorbance at 470nm Palmitic acid 17 16.79 11.12 by Spectrometric analysis. Chemical analysis revealed that the iodine value, Stearic acid 4.66 5.04 4.16 peroxide value and saponification values were highest for JL-24, whereas acid Behenic acid 3.7 3.25 3.63 value was highest for TLG-45. Kirk and Sawyer (1991) indicated that high Unsaturated iodine value denotes high degree of unsaturation of oil due to oxidation and Linoleic acid 28.46 25.75 18.12 degree of heat treatment during oil processing. Peroxide values for all varieties Oleic acid 44.54 44.37 50.21 were between 0.13- 0.26 mEqKg-1 which was less than the standard peroxide Other 1.63 4.79 12.76 value (10 mEqKg-1) for deterioration of vegetable oil, indicating slow oxidation of these oils. Peroxide values between 20 and 40 mEqKg-1 results in rancid taste (Akubugwo & Ugbogu, 2007). Acid values are used to measure the extent to which glyceride has been decomposed in the oil by lipase and other factors such as light and heat (Demian MJ, 1990). Acid value is indicator of condition and edibility of oil. Locally produced oils have higher saponification values compared to refined oils. According to Denniston et al, (2004) high saponification values indicate presence of greater number of eater bonds, suggesting that fat molecules were intact. The study shows that oil from all three Indian peanut varieties have high shelf life, nutritional value and industrial applications.
Table 4 Physico-chemical properties of oil extracted from peanut varieties Parameters SB-11 JL-24 TLG-45 Colour Bright yellow Light yellow Pale yellow a. SB-11 Odour Agreeable Agreeable Agreeable Absorbance (470nm) 0.15 ± 0.00a 0.14 ± 0.00a 0.12 ± 0.01b Iodine value (wij’s) 4.61 ± 0.26a 6.34 ± 0.25b 4.86 ± 0.07a 1.79 ± 0.22a 2.01 ± 0.19a Acid value (%) 1.93 ± 0.06a
Peroxide value 0.23 ± 0.05ab 0.26 ± 0.05b 0.13 ± 0.05c (mEqKg-1) 191.67 ± 368.38 ± Saponification value 4.28a 4.28b 199.15 ± 5.61c
All the values are Means ± standard deviations (n = 3). Values within a given row followed by the same superscript letters are not significantly different (p > 0.05) according to ANOVA and Duncan’s studentised range test. b. JL-24
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three saturated (palmitic acid, stearic acid and behenic acid) and two unsaturated (linoleic and oleic acid) FAMEs in all three varieties having similar impact spectra. The determined fatty acid composition were in accordance with the reported composition of the peanut oil although there are reports of presence of arachidic, eicosenoic and lignoceric fatty acids (Nelson et al., 2000) in traces which could not be detected in studied Indian varieties. Oleic and linoleic acids were the major components of peanut oil from all varieties. All varieties showed lower linoleic and higher oleic acid concentrations. Oleic was predominant in TLG-45 with 50.21%. Similar fatty acid composition was observed for SB-11 and JL-24 variety except linoleic acid percentage. Whereas palmitic, oleic, stearic and behenic acid did not exhibit substantial difference among SB-11and JL-24. However wide variation of palmitic (11.12%), linoleic (18.12%) and oleic acid (50.21%) concentrations were found in TLG-45 variety. This differences among c. TLG-45 varieties investigated in this work were probably due to variation in environment, genotype and growing location because all samples of species were cultivated in Figure 3 GCMS total ion chromatograms of fatty acid composition of Peanut the same year and growing season (Brown et al., 1975). However, incorporation varieties (a) SB-11, (b) JL-24, (c) TLG-45 of peanut oil into food products or incorporation of oil-containing peanut meals could alter shelf life, nutrition value and flavour of the food. Behenic acid GCMS analysis was used to study the fatty acid composition of the oil from concentration was least in all varieties (around 3%). Savage and Keenan (Savage peanut varieties. Five major peaks were observed in the total ion chromatogram & Keenan, 1994) reported considerable compositional and quantitative changes of all three peanut varieties (Fig 3). Each FAME peak was identified and in the fatty acids and lipids of peanut during various stages of growth and quantified by referring to the library match software (NIST MS search 2.0) the maturity. obtained fatty acid compositions are shown in Table 5 and corresponding chromatograms are shown in Fig 3. GCMS analysis confirmed the presence of
Aflatoxin analysis of peanut varieties Samples Observation Result Inference JL-24 1.Test line (faint) Negative Two lines are visible, this indicates 2.Control line the sample contains total aflatoxin less than 4 ppb.
SB-11 1.Test line (present) Negative Two lines are visible, this indicates 2.Control line the sample contains total aflatoxin less than 4 ppb.
TLG-45 1.Test line (present) Negative Two lines are visible, this indicates 2.Control line the sample contains total aflatoxin less than 4 ppb.
Aflatoxin contamination in food grains is now well recognized as a public health in salad, cooking, frying etc. TLG-45 variety can be concluded superior among hazard (Goldblatt, 1971) as they are highly toxic and carcinogenic secondary other varieties attributed to its mineral rich composition, highest TFC, DPPH and metabolites of concern in food safety (Achar et al., 2009). Peanuts are frequently ABTS radical scavenging activity. All varieties exhibited functional properties contaminated by the fungal species Aspergillus flavus, which can produce like water holding capacity, besides excellent oil binding capacity and water aflatoxin. This infection can occur during transportation or storage of peanuts. solubility index of SB-11 and JL-24 respectively. These functional and Infection and aflatoxin concentration in peanut can be related to the occurrence of beneficial components that exhibit wide biological and practical applications that soil moisture stress during pod-filling when soil temperatures are near optimal for are of great interest to the food industry. There is also a greater need of spreading A. flavus. In similar environments these associations could be supportive to awareness that peanut can prevent undesired supplementations through non predict the risk of aflatoxin contamination in peanuts (Craufurd et al., 2006). dietary sources on regular consumption in Indians, particularly. Overall we and On estimation of peanut varieties for aflatoxin, a visible line in test and control concluded that each variety under study exhibited its own peculiar superiority in zones of the strip indicated that total aflatoxin content in all varieties is less than terms of its properties and nutrition, hence depending upon the application 4 ppb (negative) for JL-24, SB-11 and TLG-45 variety (Table 6), thus were presented study can assist in selection of suitable peanut variety and also regarded as safe for consumption. The maximum level of total aflatoxin in indicating the use of these varieties for export and other oil extraction usages. foodstuffs in European community is 4ppb (Commission Regulation (EC) No 1525/98). It is intriguing that certain varieties of peanuts support low toxin Acknowledgments: Authors acknowledge the University Grants Commission, production, whereas other varieties support maximal production. This difference New Delhi, Government of India, for providing financial support under UGC- is possibly related to certain basic biochemical characters such as protein BSR Fellowship Award No. F.4-1/2006(BSR)/5-62/2007(BSR). (Nagarajan & Bhat, 1972) or possibly vitamin E (Detroy et al., 1971) besides cultivar practices. REFERENCES
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INFLUENCE OF pH AND EXTRACTION CONDITIONS ON ANTIOXIDANT PROPERTIES FROM PURPLE STICKY RICE (ORYZA SATIVA.L.GLUTINOSA)
B.T. Sun1, T. Kongbangkerd1, K. Rojsuntornkitti1, N. Jittrepotch*1
Address(es): Nitipong Jittrepotch, 1 Department of Agro-Industry, Faculty of Agriculture Natural Resource and Environment, Naresuan University, Phitsanulok, Thailand, Phone: (+66) 55 96000 Ext. 2747.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.847-852
ARTICLE INFO ABSTRACT
Received 6. 5. 2018 This study aimed to determine the effect of different extraction conditions on antioxidant activity of purple sticky rice (Oryza sativa L. Revised 11. 9. 2018 Glutinosa). The extractions conditions were ultrasonic 30, 40 and 50 minutes and microwave extraction at 300, 400 and 500 W with Accepted 18. 9. 2018 various pH 3, 4 and 5. The antioxidant activities were determined in terms of phenolic compound, total monomeric anthocyanins, Published 1. 10. 2018 flavonoid, ABTS and DPPH assays. The results showed that microwave extraction at pH 3 and the power 500 W had the highest extraction yield 69.85% (DW), total phenolic compound 1318.92 mg GAE/100 g, flavonoid 627.74 mg QE/g. DPPH scavenging ability was 85.97% and ABTS 413.36 mg/g. Cyanidin-3-glucoside was the main anthocyanin that found in purple sticky rice in the range of Regular article 72.18% to 90.21%. The highest percentage of cyanindin-3-glucoside was microwave extraction at 500 W. These results suggested that microwave extraction at pH 3 and 500 W was the best condition of extraction.
Keywords: anthocyanin, ultrasonic extraction, microwave extraction, antioxidant activity, purple sticky rice (Oryza sativa L. Glutinosa)
INTRODUCTION sites in the matrix (Eskilsson and Björklund, 2000). However, Routray and Orsat (2012) reported that the efficiency increases with the increase in Rice is the most important cereal crop and the stable food source being consumed temperature until an optimum temperature is reached and then starts decreasing by over half of the world’s population (Gomez, 2001).In the traditional growing to the further increase in temperature: this happens because the selection of ideal areas of Asia, various colors of rice are red, purple, black, brown, yellow, and the extraction temperature is directly linked with the stability. Ultrasound-assisted green. They have known the color of rice has been preferred in the past for their extraction could be used as a tool to overcome the drawbacks of conventional special features such as medicinal value and exclusive taste (Ahuja et al., 2007). solvent extraction methods and to improve some benefits of the solvent Several varieties of rice contain pigment particularly in brown, red and black rice extraction process. The application of ultrasound generates cavitation, which is have been widely cultivated in Thailand and generally used as an ingredient in the generation of bubbles in the system (Lieu and Le, 2010). snack, dessert, wine, food supplement, pharmaceuticals and cosmetic (Phengrat In this study was designed to find the effect of different extraction condition of and Jearakongman, 2009; Tananuwong and Terawuth, 2010). Oryza sativa ultrasonic extraction and microwave extraction in various pH on antioxidant L. glutinosa (Purple sticky rice) composes of 80% endosperm which consists of activity, anthocyanins, phenolic compound and flavonoid of purple sticky rice storage carbohydrate and its bran is rich in the source of Vitamin B and E, (Oryza sativa L. Glutinosa). mineral, oil, and phytochemicals (Zhang et al., 2004). Purple sticky rice (Oryza saitva L. var. glutinosa) is an indigenous Thai glutinous rice strain characterized MATERIAL AND METHODS by purple pigments in the husk and the pericarp. The color is determined by a number of distinct anthocyanins (Abdel-Aal et al., 2006). Sampling collection Anthocyanin which belongs to flavonoid is responsible for the attractive colors of flowers, fruits, and grains in nature (Kong et al., 2003). It has been widely The sample of purple sticky rice has been purchased from the local market from reported that anthocyanin plays an important role in reducing a risk of oxidative Phitsanulok province, Thailand. The rice was ground and and sieved 0.3 mm. All damage and is a kind of the potential drug candidates to treat cancer and the samples were stored at -18 ± 2 0C in aluminium bag until analyzed. cardiovascular diseases (Chen et al., 2012). Anthocyanins undergo structural alterations, depending on the pH and ionic strength of the aqueous environment Rice extraction and adjustment to low pH is found to enhance the efficiency of the anthocyanin extraction. The purple sticky rice (Oryza sativa L. glutinosa) powder was extracted by Extraction yield of anthocyanins is depending on the value of pH and in an ethanol extraction method. One gram of rice powder was extracted by combining acidified solvent with strong acid media have a high yield of extraction because with 8 mL of ethanol absolutes in ratio 1:8 w/v (Abdel- Aal et al., 2006) and acid can change the native form of anthocyanins, the glucoside bond of monoside divided into two part as: anthocyanins could be destroyed (Kapasakalidis et al., 2006). Its antioxidant activity decreases as pH increases from pH 2 to 7 (Sukhapat et al., 2004). Ultrasonic extraction In Microwave accelerate extraction, the process acceleration and high extraction yield may be the result of a synergistic combination of two transport phenomena: The purple sticky rice was placed into sonication bath for extraction in 30, 40 and heat and mass gradients (Chemat et al., 2009). Microwave power and 50 min at room temperature and then filter in a vacuum in Whatman paper No. 1. temperature are interrelated because high microwave power can bring up the Finally, evaporate at temperature 40 oC in 15 min and stored at temperature -18 temperature of the system and result in the increase in the extraction yield until it ± 2 °C in the dark bottle color. becomes insignificant or declines (Chemat et al. 2009; Hu et al., 2008; Xiao et al., 2008). In addition, when microwave extraction is performed in closed vessels, the temperature may reach far above the boiling point of the solvent, leading to better extraction efficient by the desorption of solutes from actives
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Microwave extraction Activity (%) = Ac-At/Ac x 100
The purple sticky rice was placed into microwave oven at powers 300, 400 and Where: At was the absorbance of samples and Ac was the absorbance of ethanol 500 W for 30 minutes at ambient temperature and then filter in a vacuum in DPPH solution Whatman paper No. 1. Finally, evaporate at temperature 40 oC in 15 min and stored at temperature -18 ± 2 °C in the dark bottle color. Anthocyanin detection
Extraction yield Anthocyanin content was determined by reverse phase HPLC. Anthocyanin separation was achieved by gradient elution using 0.1% formic acid as solvent A, The method was determined by Leung et al. (2006) which the yield was and methanol as solvent B. The elution scheme was modified due to differences calculated from the following equation in instrumentation, column selection and to improve on peak resolution: isocratic 40% B, 0-30 min; linear gradient from 40% B to 90% B, 40-45 min; isocratic Weight of purple sticky rice extract 90% B, 45-50 min; gradient to 98% B, 50-55 min; to 100% B, 55-60min; flow Percent Yeild 100 Weight of purple sticky rice rate 0.5 mL/min; injection volume 20 µl and the column that have been used a Zorbax SB-C18 column (150 mm x 4.6 mm, 3.5 mm, Agilent Technologies,
Santa Clara, CA, USA) (Hao et al., 2015). Phenolic compound content
The total phenolic content of each extracted rice was determined by using the Data analysis colorimetric method described by Singleton and Rossi (1965). First, crude All experiment had been conducted triplicate and expressed as mean standard. extracts 0.1 mL was added with 2 mL H2O were reacted with Folin-ciocalteu Data were analyzed by program with deference mean value by using an analysis reagent 1 mL in the ratio of water (1:1) for 5 min. Secondly, neutralized with 10% w/v sodium carbonate 1mL with 1 mL water. It was kept at dark room of the variance (ANOVA). Significant differences (P0.05) between means were temperature 60 min and measured the absorbance at 765 nm. Gallic acid was identified using Duncan range test procedures. used as the standard and total phenolic content was expressed as mg of Gallic acid equivalents (GAE) per 100 g of dry weight (DW) of the sample. RESULTS AND DISCUSSION
Flavonoid content Percentage Yield
Flavonoid was determined by sodium nitrite aluminum dichloride system Extraction efficiency is affected by the chemical nature of phytochemicals, the (Zhishen et al.,1999). Firstly, mix extracted rice with 2 mL distilled water, with extraction method used, sample particle size, the solvent used, as well as the 1 mL sodium nitrate 5% and kept 5 min. Then add 0.15 mL of aluminum chloride presence of interfering substances (Stalikas, 2007). The yield of extraction 10% and kept 5 min. After that, added 1 mL of sodium hydroxide 1 M and kept 5 depends on the solvent with varying polarity, pH, temperature, extraction time, min before measured absorbance 415 nm. The results were expressed as mg of and composition of the sample. Figure 1 showed that the extraction yields range quercetin (QE) per 1 g of dry weight (DW). between 69.36 to 69.64% of dry weight for ultrasonic extraction and 69.63 to 69.84% of dry weight for microwave extraction. The highest extraction yield was Total monomeric anthocyanin (TAC) found using microwave extraction with 500 W at pH 3 (p<0.05). In this figure reported that increasing microwave power the percent yeild could be increased The total anthocyanin content (TAC) was determined by the pH-differential because the microwave power could also influence the yield of antioxidants in method (Giusti and Wrolstand, 2005). First, transfer 1 mL extracted solution the microwave extraction process. On the one hand, an increase in microwave into 10 mL volumetric flask for preparing two dilutions of the sample, one power could accelerate the solvent’s movement, cell rupture and diffusion of adjusts volume with potassium chloride buffer, pH 1.0, and the other with sodium extractives into the solvent, thereby increasing the extraction efficacy. Moreover acetate buffer, pH 4.5. Let these dilutions equilibrate for 15 min. Measure the for ultrasonic extraction, increasing duration time could be increase the percent absorbance of each dilution at the 510 and 700 nm (to correct for haze), and a yeild of extraction beacuse ultrasound also exerts a mechanical effect, allowing blank cell filled with distilled water. Calculate the absorbance of the diluted greater penetration of solvent into the tissue, increasing the contact surface area sample (A) as follows: between the solid and liquid phase and ultrasonic extraction time could affect the extraction as well, the yield of extraction will be increased by increasing the time for extraction (Galhiane et al., 2006). As a result, the solute quickly diffused A = (A510 – A700) pH 1.0 – (A510– A700) pH 4.5 from the solid phase to the solvent ( Rostagno et al., 2003). The highest Calculate the monomeric anthocyanin pigment concentration in the original extraction yield was found using microwave extraction with 500 W at pH 3 sample using the following formula: (p<0.05).
Monomeric anthocyanin pigment (mg/l) = (A x MW x DF x 1000)/ (ε x 1) 70 69,9 Where MW is the molecular weight MW = 449.2, DF is the dilution factor, and ε 69,8 is the molar absorptivity 69,7 calculate pigment content as cyanidin-3-glucoside, ε = 26,900 69,6
Antioxidant activity 69,5 69,4 pH 3 69,3 ABTS pH 4 69,2 For ABTS assay, the procedure followed the method of Re et al., (1999) with % yeild Percent 69,1 pH 5 some modifications. The stock solutions included 7.4 mM ABTS solution and 69 2.6 mM potassium persulfate solution and kept 12 h at room temperature in dark. 68,9 The solution was diluted methanol to obtain an absorbance of 0.70 ± 0.02 units at 734 nm using the spectrophotometer. Extracted rice 0.1 mL were allowed to react with 0.9 mL of the ABTS+ solution for 2 h in the dark condition. Then the absorbance was taken at 734 nm using the spectrophotometer. The standard curve was linear between 25 and 600 μM Trolox. Results were expressed in mg Trolox equivalents (TE)/g. Figure 1 Percent yield of purple sticky rice (Oryza sativa L. glutinosa) extract using different extraction condition DPPH Total Phenolic compounds (TPC) For DPPH assay was performed based on the method described by Brand- Williams et al., (1995). A 1 mL volume of the sample extract was added to 1 mL According to Adom and Liu, (2002) reported that phenolic compounds in rice of the DPPH solution (dissolved in ethanol), and the mixture was placed in the has been existing as both free and bound forms. Bound phenolic acids are dark at room temperature for 30 min. Subsequently, the absorbance at 515 nm typically involved in cell wall structure. They are conjugated or cross-linked with was recorded. The percentage of radical scavenging ability was calculated by lignin components and are mainly found in bran fraction (Zhou et al., 2004). using the formula: Total phenolic content of different extraction condition of purple sticky rice (Oryza sativa L. Glutinosa) are showed in Figue 2.Total phenolic content has
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been in the range of 691.89-1318.92 mg GAE/100 g for ultrasonic extraction and 691.89-2389.19 mg GAE/100 g for microwave extraction. As shown in Figure 2, 700 an increase in exposure power there was significant increase in total phenolic 600 compound in purple sticky rice. Maximum total phenolic compound 2389.19 mg GAE/100 g was recorded at microwave power at 500 W. The highest 500 concentration was pH 3 with microwave extraction (p<0.05). Microwave power can have the influence on the total phenolic content when the power of 400 microwave extraction has increased. In microwave extraction, microwave power pH=3 was a key variable affecting the release of phytochemicals from different 300 matrices by rupturing cell the wall, and also had the ability to modify equilibrium 200 pH=4 and mass transfer conditions during extraction. Increasing the microwave power Flavonoid mg QE/g mg Flavonoid pH=5 accelerated purple sticky rice extraction (Ghasemzadeh et al., 2017). Most 100 phenolics present in plant tissues are soluble in polar solvents and can be extracted using methanol containing a small amount of hydrochloric or formic 0 acid. These results similar to Seawan et al., (2014) reported that the power level of microwave effected the total phenolic content of Homnin black rice and Munpu red rice.Moreover, for ultrasonic extraction, increasing duration time the total phenolic compound could be increase because of the structural destruction and the decomposition of polyphenols (Carrera et al., 2012; Makris et al.,
2007; Sun et al., 2011). When extraction time increased the variance of Figure 3 Flavonoid content of purple sticky rice (Oryza sativa L. glutinosa) extraction yield is relatively rapid and reaches a maximum at 20 min. Ultrasound extract using different extraction condition with mechanical agitation effect, cavitation effect, and thermal effect can improve the mass transport and facilitate the release of compounds from the Total monomeric anthocyanin extracted materials (Carrera et al., 2012). On one hand, the low acid pH value of the extraction solution can stop the oxidation of phenolics, while the use of small Total monomeric anthocyanin in ultrasonic extraction and microwave extraction temperatures may preserve anthocyanin stability (Li and Jiang, 2007, Laleh et is shown in figure 4. For ultrasonic extraction total monomeric anthocyanin has al., 2006). Microwave extraction showed the highest extraction efficacy of total been in the range of 98.99-183.24 mg/g and the highest concentration was pH 3 phenolic compound followed by ultrasonic extraction (p<0.05). The highest in ultrasonic 50 min which 183.24 mg/g. For microwave, extraction has been in concentration was pH 3 with microwave extraction (p<0.05). the range between 90.17-614.52 mg/g and the highest concentration was pH 3 in microwave extraction 500 W. Acidity of the solvent had greater effects on total 3000 monomeric anthocyanin contents. At the similar extraction duration, acidic solvent provided the extract with significantly higher amount of total monomeric anthocyanin than neutral solvents (p0.05). This could be due to the higher 2500 stability of anthocyanin in acidic solution (Abdel-Aal and Hucl, 2003). Dranca et al., 2016 has been reported that high yield of total monomeric anthocyanins 2000 could be the effect of extraction time of ultrasonic extraction, long period of extraction more effect of high yield of extraction. Abdel-Aal et al., 2014, has been shown that increasing of microwave power could increase the yield of 1500 pH =3 extraction. This observation is in agreement with Golmohamadi et al, (2013) where they discovered that total phenolic content and total monomeric 1000 pH =4 anthocyanin were significantly increased by 11.97% and 12.6% after 30 and 20 min sonication at 20 kHz and 400 W ultra-sonication conditions. pH=5 500 900
Phenolic compound mg GAE/100g mg compound Phenolic 800 0 700 600 500 pH=3 400 Figure 2 Total phenolic content of purple sticky rice (Oryza sativa L. glutinosa) 300 pH=4 extract using different extraction condition 200 pH=5 Flavonoid content 100
Flavonoid consist of a 15-carbon skeleton that was organized in two aromatic mg/g anthocyanins monomeric Total 0 rings (A- and B-rings) interlinked by a three-carbon chain (structure C6-C3-C6) and are recognized for both their ability to donate electrons and to stop chain reactions. These activities are attributed to the phenolic hydroxyls, particularly in the 3′OH and 4′OH of the three-carbon chain (Ramarathnam et al., 1989; Hudson et al., 2000; Kim et al.,2010; Cho et al., 2013). Flavonoid contents in purple sticky rice with different extraction were shown in Figure 3. For ultrasonic Figure 4 Total monomeric anthocyanin content of purple sticky rice (Oryza extraction, the flavonoid content were between 90.32-464.52 mg QE/g and the sativa L. glutinosa) extract using different extraction condition highest flavonoid condition content was found by using ultrasonic extraction time of 50 min with the extraction at pH 3 microwave, extraction has been between Antioxidant of DPPH and ABTS 489.68-627.74 mg QE/g and the highest concentration is pH 3 in microwave extraction 500 W which was 627.74 mg QE/g.Similar to phenolic acid, The DPPH free radical (DPPH) does not require any special preparation, while flavonoids are synthesized by the phenylpropanoid metabolic pathway. The high the ABTS radical cation ABTS must be generated by enzymes or chemical concentration flavonoid was depending on low pH because of stability effect on reactions (Arnao, 2000). Another important difference is that ABTS can be both flavilium salt and anhydrobase (Goto et al., 1991). Zhou et al., (2004) dissolved in aqueous and organic media, in which the antioxidant activity can be reported the high yield of extraction of flavonoid were depending on extraction measured, due to the hydrophilic and lipophilic nature of the compounds in time. In the present study, the accelerated extraction of flavonoids by increasing samples. In contrast, DPPH can only be dissolved in organic media, especially in microwave power was related to the direct effects of microwave energy on bio- ethanol, this being an important limitation when interpreting the role of molecules by ionic conduction and dipole rotation which result in power hydrophilic antioxidants. Both radicals show similar bi-phase kinetic reactions dissipated inside the solvent and plant material and then generate molecular with many antioxidants.The antioxidant activity of purple sticky rice has been movement and heating (Gfrerer et al., 2005). determined using the ABTS and DPPPH assay and showed very strong antioxidant activity. Figure 5 showed the antioxidant for by using ultrasonic and microwave extraction and value of ABTS express as Trolox/g. The trends seen in antioxidant yields were reiterated in these activities. The increasing with
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extraction time for ultrasonic extraction and the power for microwave extraction Table 1 Anthocyanin profile of purple sticky rice extract using difference increased ABTS. For ultrasonic extraction the ABTS value has been in the range conditions, expressed as percentage % of five anthocyanins in purple sticky rice. of 240.41-348.61 mg/g of ABTS. The highest was found at pH 3 with 50 min of Percentage MvGL DpGL CyGL PtGL PnGL extraction time and for microwave, extraction ABTS value has been in the range pH 3 300 W 9.17 8.40 72.18 3.02 7.23 of 246.51-413.36 mg/g of ABTS and the highest concentration was pH 3 with microwave extraction 500 W. These results matches well our observations on pH 3 400 W 1.96 5.02 85.67 0.99 6.36 anthocyanin and total phenolic contents. The DPPH scavenging ability highest of pH 3 500 W 0.25 0.48 98.46 0.16 0.65 purple sticky rice showed in Figure 6. Ultrasonic extraction has been in the range pH 4 300 W 5.89 9.28 71.04 4.24 9.54 of 44.70-81.4% and the highest concentration was pH 3 in ultrasonic 50 min and for microwave extraction and DPPH scavenging ability has been in the range of pH 4 400 W 3.66 5.33 82.24 2.02 6.75 58.06-85.79% and the highest concentration was pH 3 in microwave extraction pH 4 500 W 3.84 3.81 89.31 0.20 2.83 500 W.The power level of microwave affected the ABTS and DPPH activities. pH 5 300 W 7.49 11.11 75.09 2.20 4.10 The antioxidant activities of purple sticky rice were increased with the increase of pH 5 500 W 2.89 7.08 83.11 1.95 4.98 microwave power (p0.05). The different of effect of microwave power level might due to different active compound in sample. (Seawan, et al., 2014). pH 5 500 W 0.40 4.95 87.44 0.54 6.67 pH 3 30 min 14.99 3.82 76.33 1.14 3.71 4500 PH 3 40 min 2.93 4.59 90.43 0.62 1.41 4000 pH 3 50 min 3.08 5.02 90.21 1.66 0.03 3500 pH 4 30 min 6.41 8.04 77.17 1.10 7.27 3000 pH 4 40 min 4.72 6.64 80.62 0.79 7.22 2500 pH 4 50 min 1.42 4.04 87.91 0.73 5.89 2000 pH=3 pH 5 30 min 4.76 4.66 87.64 0.14 2.80
ABTS mg/g ABTS 1500 pH=4 pH 5 40 min 4.45 4.26 87.59 0.18 3.50 pH 5 50 min 2.19 4.61 89.91 0.23 3.05 1000 pH=5 MvGL: Malvidin-3-glucoside, DpGL: Delphinidin-3-glucoside, CyGL: Cyanidin-3- 500 glucoside, PtGL: petunidin-3-glucoside and PnGL: Peonidin-3-glucoside.
0 Table 1 showed anthocyanin profile of each sample, expressed as percentage of five anthocyanins in purple sticky rice. The major of anthocyanin in purple sticky rice is cyaniding-3- glucoside that contains between 70-90% of total amount of anthocyanins. According to Escribano-Bailón et al, (2004) and cyanidin-3- glucoside is the major anthocyanin, ranging from 80-100% of total contents in pigmented rice cultivars. Yawadio et al, (2007) identified cyanidin-3-glucoside Figure 5 ABTS of purple sticky rice (Oryza sativa L. glutinosa) extract using and peonidin-3-glucoside in black rice, while Sompong et al, (2011) confirmed different extraction condition that cyanidin-3-glucoside and peonidin-3-glucoside are the most abundant 35000 anthocyanins in black rice extracts. For microwave, extraction has been shown that cyaniding-3-glucoside was in the range of 72.17-98.45% and the highest 30000 percent was pH 3 in power 500 W that contains 98.45%. For ultrasonic extraction 25000 have been shown that cyaniding-3-glucoside is in the range of 76.32-90.20% and the highest percent was pH 3 in 50 min that contain 90.20%. The highest amount 20000 of cyaniding-3-glucoside is pH 3 in power 500 W. Many studies report that black 15000 pH=3 and red rice have 2 main compounds of anthocyanin such as cyanidin-3- glucoside and peonidin-3-glucoside in which cyaniding-3-glucoside contain 93% DPPH mg/g DPPH 10000 pH=4 of the quantified anthocyanin. These generally found a pigment of fruits, vegetables, and colored rice have important roles in reducing the risk of cancer 5000 pH=5 and other chronic diseases because of their free radicals scavenging capacities 0 (Wang and Stoner, 2008; Elisia and Kitts, 2008). In acidified solvent extractions, strong acid media should be avoided because the acid may change the native form of anthocyanins, the glycoside bonds of 3-monoside anthocyanins could be destroyed, and acylated anthocyanins might be degraded by hydrolysis reaction (Kapasakalidis et al., 2006).
Figure 6 DPPH scavenging activity of purple sticky rice (Oryza sativa L. CONCLUSION Glutinosa) extract using different extraction condition Experiments demonstrate that low pH has influenced the extraction of antioxidant Anthocyanins activities of purple sticky rice (Oryza sativa L. Glutinosa) and the high yield of extraction in this study was pH 3. Moreover, duration time is effected by ultrasonic extraction and high antioxidant of extraction will be increased when increasing duration time of extraction. On the other hand, microwave power can effect in yield of extraction by increasing high power of microwave. Microwave extraction in power 500 W in 30 min composes of the highest yield of extraction and the greatest amount of phenolic content, flavonoid, total monomeric anthocyanins, ABTS, DPPH and anthocyanin detection. It is the best conditions for extraction. Acknowledgments: This research was funded by Naresuan University. The authors are grateful to the Department of Agro-industry, Faculty of Agriculture Natural Resource and Environment, Naresuan University.
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EFFECTS OF TWO ROOTSTOCKS (PISTACIA VERA L. AND PISTACIA ATLANTICA DESF.) ON THE YIELD, MORPHOLOGY, CHEMICAL COMPOSITION, AND FUNCTIONAL PROPERTIES OF TWO PISTACHIO VARIETIES (“MATEUR” AND “ACHOURY”)
Samiha Ouni1, Azza Chelli Chaabouni2, Luis Noguera-Artiaga3, Francisca Hernández4, Hmida Ben Hamda5, Imen Ouerghui2, Ángel A. Carbonell-Barrachina*3, and Ali Rhouma6
Address(es): 1Faculty of Sciences of Tunis, university campus 2092, El Manar, Tunis, Tunisia. 2National Institute of Agricultural Research of Tunisia (INRAT), 2049 Ariana, Tunisia. 3Universidad Miguel Hernández de Elche (UMH), Escuela Politécnica Superior de Orihuela (EPSO), Department of Agro-Food Technology, Research Group “Food Quality and Safety”, Ctra. Beniel, km 3.2, 03312-Orihuela, Alicante, Spain. 4UMH, EPSO, Department of Plant Sciences and Microbiology, Research Group “Plant Production and Technology”, Ctra. Beniel, km 3,2. 03312-Orihuela, Alicante, Spain. 5Unity of Agricultural Experimentations of INRAT, Mornag, Tunisia. 6Laboratory of breeding and Protection of Olive Genetic Resources, Olive Tree Institute, 2049 Ariana, Tunisia.
*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.853-856
ARTICLE INFO ABSTRACT
Received 4. 7. 2018 The low diversification of rootstocks can be a problem in reducing the adaptation of pistachio to the Tunisian climatic and soil Revised 7. 8. 2018 conditions. Nowadays, the most used rootstock is Pistacia vera; however, the autochthonous Pistacia atlantica could be a good Accepted 4. 9. 2018 alternative. The aim of this work was to study the effect of two different rootstocks Pistacia vera and Pistacia atlantica on the quality Published 1. 10. 2018 and functionality of two pistachio cultivars (the local variety “Mateur” and the Syrian one “Achoury”). The different parameters studied were: morphological (size, weight), chemical (minerals and fatty acids) and functional (total phenolic content, and antioxidant activity: ABTS+, DPPH•, and FRAP). The pistachios are a good source of minerals (especially Ca and K 19.9 and 9.8 g kg-1, respectively, and Fe Regular article 54.5 mg kg-1), polyunsaturated fatty acids (oleic and linoleic acids), and phenolic compounds (16.0 g GAE kg-1). The combination P. atlantica and variety “Mateur” led to proper values of tree yield, nut dehiscence, morphology (length and width), and oleic acid content. Thus, it can be concluded that the use of the local rootstock P. atlantica led to promising results and can be a good option due to its higher adaptation to the local climatic and soil conditions.
Keywords: total polyphenol content, fatty acids, fruit morphology, mineral content, antioxidant activity
INTRODUCTION these reasons suggest that grafting is a relevant agronomic practice which requires further attention. Pistachio (Pistacia vera L.) fruit are one of the most liked nut worldwide. Halvorsen et al. (2006) determined the antioxidant concentrations of 1113 food Pistachio species are drought and salt tolerant and that is why they are widely products and ranked pistachio nuts among the first 50 items with the highest cultivated in saline, dry, and hot areas of the Middle East, Mediterranean antioxidant capacity; this is probably due to the fact that pistachio nuts are a rich countries and United States (Demiral et al., 2009). Now, pistachio is among the source of phenolic compounds (Rodríguez-Bencomo et al., 2015). Moreover, most spread nut tree crop in Tunisia occupying an overall area of 44000 ha, the pigments responsible for pistachio purple and green kernel color have been mainly concentrated in the south and center regions of the country (Sarra et al., attributed to lutein derivatives and anthocyanins (Dreher, 2012). Pistachio 2015). The high nut quality, price, demand, and tolerance to abiotic stresses have kernels are a good source of fat (50–60%) and contain high quantity of encouraged its plantation in large scale in semi-arid areas during the last four unsaturated fatty acids (linoleic, linolenic and oleic acids), essential for human decades, including Tunisia (Chelli Chaabouni et al., 2014; Ghrab and Gouta, diet (Carbonell-Barrachina et al., 2015; Maskan and Karataş, 1998). 2005). Pistachio is mainly propagated by grafting, and many wild Pistacia The aim of this study was to investigate the effects of rootstock on fruit species (Pistacia integerrima L., P. atlantica Desf., P. terebinthus L., P. vera production and quality parameters (minerals, fatty acids, and total phenolic L…) are used as rootstocks for Pistacia vera, which is the only cultivated species content) and antioxidant capacity of two pistachio cultivars (Mateur and for industrial use. Pistachio industry in Tunisia is based on a main cultivar Achouri), grown under rainfed conditions in the northeast of Tunisia. Mateur, with P. vera being its rootstock. Thus, the low diversification of pistachio rootstocks in Tunisian orchards and the low adaptation of some MATERIAL AND METHODS cultivars are among the major problems affecting crop sustainability. It has been clearly demonstrated that the rootstock will drastically affect the three Plant material, growing conditions and experimental design vegetative growth. The use of different rootstock or hybrid seedlings have been shown to significantly influence some key parameters of the tree growth, such as This study was carried out at the INRAT Unity of Agricultural Experimentation nutrient contents, yield, alternate bearing, resistance to cold and salt stress, and of Mornag (Tunisia), which has semi-arid climatic conditions, with a mean shell splitting (Ashworth, 1985; Walker et al., 1987), and also to key parameters temperature range of 10-27 °C, and 450 mm of annual precipitation. 22 year-old of the nut quality, including weight, size, color, and mineral composition trees were cultivated under rainfed conditions. Two pistachio cultivars were (Carbonell-Barrachina et al., 2015). Their effects were also seen on plant considered in this study (i) the Tunisian “Mateur” and the Syrian “Achoury”, and blooming, vegetative growth, fruit quality and total production (Giorgi et al., both were grafted on: (i) Pistacia atlantica or (ii) Pistacia vera rootstocks. 2005; Weibel et al., 2003; Young and Houser, 1980; Zarrouk et al., 2005). All Monitoring was performed on 5 trees of comparable size and vigor, chosen from each treatment (rootstocks cultivars combination). Fruits were harvested
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approximately at the end of August, at their commercial maturity, and the yield in 2014 than in 2015, which was indicative of an alternate bearing phenomenon. per tree was measured at two consecutive seasons, 2014 and 2015. During the 2015 season, the “Mateur” variety grafted on P. atlantica rootstock had a significantly higher fruit yield than the “Achoury” scion/rootstock Pomological parameters combinations; however, no significant effects of the rootstock on the yield were observed in the 2014 season. The in-hull and in-shell fruit fresh weights, in-shell dry weight and nut Although the data was not always statistically significant, it is implied that the dehiscence were measured for 3 replicates of 100 fruits (3 100) per treatment. local “Mateur” variety was slightly more productive than the Syrian “Achoury” The nut and kernel sizes were measured with a digital caliper for 25 fruits per cultivar, and seemed to be better grafted on P. atlantica rootstock. These results treatment following IPGRI International Plant Genetic Resources Institute agreed with the findings reported by Carbonell-Barrachina et al. (2015). These (1997) descriptors. authors reported a significant effect of rootstock on the tree yield with higher yield being recorded for the “Kerman” cultivar grafted on P. atlantica than for P. Mineral analysis integerrima and P. terebinthus. They also found a significantly higher tree yield in 2012 season (40.1 kg) compared to that registered in 2013 season (12.0 kg), Mineral content was quantified according to Carbonell-Barrachina et al. clearly due to an alternate bearing, a characteristic phenomenon of this type of (2002). Approximately 0.5 g ground pistachios were digested and to assess crop. Ghrab and Gouta (2005) reported a great variation of “Mateur” and precision and accuracy, the protocol and equipment were validated by using in “Ohadi” pistachio cultivars tree yield over fifteen year of study (1983-1997), and each batch: (i) the GBW07603 (bush, branches and leaves; Institute of they established a link between the production and the annual rainfall. Moreover, Geophysical and Geochemical Exploration of China) certified reference material, Johnson and Weinbaum (1987) found that production can vary 3 to 5 times 1 blank, and 1 spiked-sample in each batch. Calibration curves were used for the between “off" and "on" years. quantification of minerals and showed good linearity (R2 ≥0.998). This analysis It is difficult to establish a clear behavior of the nut splitting as affected by the was run in triplicate. rootstock, because the rate of nut dehiscence changed from year to year for both varieties under study (Table 1). “Achoury” exhibited a significantly higher nut Fatty acids profile dehiscence (76-84%) than “Mateur” (30-33%) in 2014; however, no effect of the rootstock (P. vera and P. atlantica) was noticed for this parameter for both Fatty acid methyl esters (FAMEs) were prepared according to the method varieties under study. On the other hand, in the following season (2015), a described by Carbonell-Barrachina et al. (2015) and identical chromatographic significantly higher nut dehiscence (89%) was recorded for the “Mateur” set-up and conditions were used. 50 mg of extracted oil (using sonication) were pistachios grafted on P. atlantica. Besides, it seemed that the rootstock had no used and identification of FAMEs was made by comparison with authentic effect on the dehiscence of the “Achoury” nuts. Current results supported those standards from Sigma-Aldrich. This analysis was run in triplicate, and results by Loudyi (2001), who reported greater variation of “Mateur” nut splitting from were expressed as % of the total area. year to year. The difference between both varieties can be explained by their maturity degree and adaptation to climatic and drought conditions. The level of Total polyphenols content (TPC) cold temperature during the vegetative dormancy of the pistachio trees seems to have an effect on the nut dehiscence of “Mateur” trees (Oukabli, 1998). It is well The TPC was measured using the Folin–Ciocalteu colorimetric method as known that shell splitting can be enhanced by management practices, especially previously described by Gao et al. (2000), and using an extract obtained after by properly managing the irrigation water (Goldhamer et al., 1987). using ~1 g of grinded pistachio and a solution of 80 % aqueous methanol (MeOH) and 1 % HCl. Quantification was conducted by using a gallic acid Table 1 Average yield (kg/tree) and nut dehiscence rate (%) as affected by calibration curve, and results were expressed as mg of gallic acid equivalents pistachio variety and rootstock, on seasons 2014 and 2015. (GAE) per 100 g of dry mass. This analysis was run in triplicate. Yield (kg) Nut dehiscence rate (%) Variety Rootstock 2014 2015 2014 2015 Antioxidant activity Ϯ Mateur P. vera 8.41 3.29 ab 32.52 b 60.12 b P. The same extract used for the TPC analysis was also used for the analysis of the 9.14 4.44 a 30.97 b 89.01 a free scavenging activity DPPH• method as described by Brand-Williams et al. atlantica (1995), with a modification in the reaction time (10 min were used in the current Achouri P. vera 7.45 0.78 b 83.84 a 67.06 b study). This analysis was run in five replications, and results were expressed as P. -1 + 7.16 1.05 b 76.54 a 68.33 b mmol trolox kg dry matter, dm. The ABTS radical cation and FRAP methods atlantica were also used as described by Re et al. (1999) and Benzie and Strain (1996), ANOVA test‡ NS ** ** * respectively. Calibration curves were used for quantification of the three methods Ϯ 2 Values (mean of 5 replications) followed by the same letter, within the same column, were of antioxidant activity and showed good linearity (R ≥ 0.998). The analyses were ‡ -1 not significantly different (p 0.05), Tukey's least significant difference test. NS = not run in five replications, and results were expressed as mmol Trolox kg dm. significant at p< 0.05; *, and **, significant at p< 0.05 and 0.01 respectively.
Statistical analyses Morphological parameters
The data was subjected to one-way analysis of variance (ANOVA) and later to Data presented in Table 2 indicated that whole and de-hulled fruit size parameters Tukey’s multiple-range test to compare the means. Differences were considered were significantly different for each variety, with “Mateur” pistachios being statistically significant at p<0.05. All statistical analyses were done using bigger that those from the “Achoury” variety. Fruits from both varieties under StatGraphics Plus 5.0 software (Manugistics, Inc., Rockville, MD). study, showed values of length/with (l/w) higher than 1.80. A previous study (Caruso et al., 1998) claimed that in Italian, Greek, and Tunisian pistachio varieties, the nuts were elongated (l/w > 1.80), but the Iranian, Turkish and, to a RESULTS AND DISCUSSION minor extent, Syrian ones were ovoid (l/w = 1.50–1.80). However, it is difficult to establish these trends because the physical characteristics can be influenced by Yield production location and the particular year of alternate bearing (Seferoglu et al., 2006), or even by the pollinator (Ak, 1998). The yield per tree of both “Mateur” and “Achoury” cultivars during 2014 and 2015 seasons is shown in the Table 1. Trees exhibited significantly higher yields
Table 2 Fruit morphology (mm) as affected by pistachio variety and rootstock Whole fruit (mm) De-hulled fruit (mm) Edible kernel (mm) Variety Rootstock Length Width Thickness Length Width Thickness Length Width Thickness Mateur P. vera 25.44 aϮ 13.44 b 12.28 a 21.18 a 11.73 a 10.51 a 16.64 b 8.21 b 7.96 ab P. atlantica 25.35 a 13.84 a 12.40 a 20.98 a 11.89 a 10.44 a 16.94 a 8.83 a 8.04 a
Achouri P. vera 23.43 b 12.31 c 11.26 b 19.80 c 10.89 b 9.84 b 15.61 c 8.36 ab 7.64 c P. atlantica 23.68 b 12.24 c 11.31 b 20.12 b 10.98 b 9.92 b 16.38 b 8.06 a 7.75 bc
ANOVA test‡ ** ** ** *** ** *** * * ** Ϯ Values (mean of 5 replications) followed by the same letter, within the same column, were not significantly different (p 0.05), Tukey's least significant difference test. ‡NS = not significant at p< 0.05; *, **, and **, significant at p< 0.05, 0.01 and 0.001 respectively.
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Mineral content demonstrated in future studies with more replications that the use of P. atlantica increased the mineral contents, although no statistically significant differences The mineral contents of pistachio kernels are shown in Table 3. Values recorded were found in the current study. It is difficult to clearly establish which were the in “Achoury” kernels were, in general, significantly higher than those of the effects of the pistachio cultivar/variety and the rootstock on the mineral contents, “Mateur”; the lowest values were found for the “Mateur” fruits grafted on P. and in this way Küçüköner and Yurt (2003) found no significant differences in vera. No statistical significant differences among samples were recorded for the Turkish pistachio nuts for Cu and Mg. While, Carbonell-Barrachina et al. Mg content. The most important conclusion of this section is that the worst (2015) only found P. atlantica nuts contained higher amounts of Fe, Cu and Zn, combination rootstock and variety was P. vera and “Mateur”, which led to the than the other rootstocks under analysis (P. integerrima and P. terebinthus). lowest contents of Ca, K, Fe, Cu, Mn, and Zn. There seemed to be a trend to be
Table 3 Mineral content in pistachio samples as affected by variety and rootstock Macro-elements (g kg-1) Micro-elements (mg kg-1) Variety Rootstock Ca Mg K Fe Cu Mn Zn Mateur P. vera 18.0 bϮ 4.8 9.3 b 51.4 b 13.7 b 7.4 b 34.8 b P. atlantica 19.7 ab 4.9 9.7 ab 52.6 ab 15.3 ab 8.9 ab 35.3 b
Achouri P. vera 19.8 ab 4.9 10.1 a 55.6 ab 15.8 a 9.4 a 40.5 a P. atlantica 22.1 a 5.0 10.2 a 58.5 a 16.1 a 9.9 a 37.6 ab
ANOVA test‡ * NS ** ** ** *** ** Ϯ Values (mean of 5 replications) followed by the same letter, within the same column, were not significantly different (p 0.05), Tukey's least significant difference test. ‡NS = not significant at p< 0.05; *, **, and **, significant at p< 0.05, 0.01 and 0.001 respectively.
Fatty acid profile 2003). The highest and lowest content of oleic acid were found in “Achoury” variety grafted on P. atlantica (69.58 %) and “Mateur” variety grafted on P. vera Five fatty acids [palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (65.58 %), respectively. These results agreed with those of Chahed et al. (2008), (C18:0), oleic acid (C18:1), and linoleic acid (C18:2)] were detected in all who reported that the oleic acid ranged from 54.2 to 76.8 % in “Mateur” kernels pistachio samples (Table 4). Palmitic acid and palmitoleic acid contents were cultivated in four areas with different climatic conditions; the lowest amount of similar in all samples, with no significant differences among samples, and with oleic acid (54.2 %) was found in the area with semi-humid climate compared to C16:0 being higher (mean of 10.37 % for all samples) than C16:1 (mean of 0.85 the other three areas with semi-arid to arid climates. %). Data showed no statistically significant differences for palmitic, palmitoleic, On the other hand, the combination of P. vera and “Mateur” variety led to the and stearic acids; whereas, significant differences between varieties were found highest content of linoleic acid. Although both unsaturated fatty acids have for oleic and linoleic acids. The oleic acid was the predominant compound in interesting health effects, a higher linoleic acid content can be responsible for both studied varieties, reaching a mean value for all studied samples of 67.63 %. faster oxidative alterations of pistachio oil. This finding agreed with previous studies (Chahed et al., 2008; Satil et al.,
Table 4 Fatty acid profile (%) in pistachio samples as affected by variety and rootstock Fatty acids (%) Variety Rootstock C16:0 C16:1 C18:0 C18:1 C18:2 Mateur P. vera 10.46 0.89 2.03 65.58 bϮ 21.03 a P. atlantica 10.23 0.80 2.59 68.70 ab 17.68 b Achouri P. vera 10.50 0.90 2.54 66.64 ab 19.44 ab P. atlantica 10.29 0.80 2.72 69.58 a 16.61 b
ANOVA test‡ NS NS NS * ** Ϯ Values (mean of 5 replications) followed by the same letter, within the same column, were not significantly different (p 0.05), Tukey's least significant difference test. ‡NS = not significant at p< 0.05; * and **, significant at p< 0.05 and 0.01, respectively.
Total polyphenolic content and antioxidant activity role in everyday life due to their unquestionable beneficial effects on living organisms that enable them to overcome, for instance, oxidative injuries, A number of studies have shown that the presence of phenolic compounds in modulating biological pathways and membrane functionality, showing anti- food and especially in fruits can be of particular importance for consumers, inflammatory, anti-infective, antifungal, antiviral, antibacterial and antioxidant because of their beneficial health properties (Chong et al., 2013). Current data activities (Barreca, Bellocco, Laganà, et al., 2014; Barreca, Bellocco, Leuzzi, showed that total polyphenolic compounds values ranged between 1556 and 1629 et al., 2014; Smeriglio et al., 2014). mg GAE 100 g-1 fresh weight, fw (Table 5). There were no significant differences The only method reporting differences among the studied samples was ABTS+. between the different variety/rootstock combinations. The ABTS+ activity was significantly higher in the “Achouri” as compared to the The antioxidant potential of pistachio fruit can be affected by many factors, “Mateur” ones; however, no statistically significant differences were found for including maturity stage. Pistachio nuts are a rich source of phenolic compounds, the rootstock (Table 5). and have recently been ranked among the first 50 food products highest in antioxidant potential (Tomaino et al., 2010). Antioxidants play a fundamental
Table 5 Antioxidant activity (mmol Trolox kg-1 fresh weight, fw) and total polyphenol content (mg GAE 100-1 g fw) in pistachio samples as affected by variety and rootstock FRAP DPPH ABTS TPC Variety Rootstock (mmol Trolox kg-1 fw) (mg GAE 100-1 g fw) Mateur P. vera 220 49.9 12.5 bϮ 1581 P. atlantica 219 49.5 12.2 b 1556
Achouri P. vera 229 52.9 15.6 a 1629 P. atlantica 223 50.4 15.3 a 1615
ANOVA test‡ NS NS *** NS Ϯ Values (mean of 5 replications) followed by the same letter, within the same column, were not significantly different (p 0.05), Tukey's least significant difference test. ‡NS = not significant at p< 0.05; *** significant at p< 0.001.
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CONCLUSION fruit quality, and nutrition in the peach (cv. 'Suncrest'). Scientia Horticulturae, 107(1), 36-42. https://doi.org/10.1016/j.scienta.2005.06.003 The morphological, chemical, and functional parameters depended more on the GOLDHAMER, D. A., PHENE, B. C., BEEDE, R., SHERLIN, L., MAHAN, S., pistachio variety (“Mateur” or “Achoury”) than on the rootstock (Pistacia vera or ROSE, D. 1987. Effects of sustained deficit irrigation on pistachio tree P. atlantica). The replacement of the most popular P. vera by P. atlantica in performance. In: California Pistachio Industry Annual Report—crop year 1986– “Mateur” fruits had positive effects on yield, nut dehiscence rate, and oleic acid, 1987, (pp. 61-66). Fresno, CA: California Pistachio Commission. while in “Achoury” pistachios showed positive effects on the morphology, and HALVORSEN, B. L., CARLSEN, M. H., PHILLIPS, K. M., BØHN, S. K., oleic acid. Thus, it can be concluded that the use of the autochthon rootstock, P. HOLTE, K., JACOBS JR, D. R., BLOMHOFF, R. 2006. Content of redox-active atlantica, can be a good alternative for Tunisian pistachios orchard, because its compounds (ie, antioxidants) in foods consumed in the United States. American use improved or maintained the global quality parameters as compared to the Journal of Clinical Nutrition, 84(1), 95-135. other studied rootstock, P. vera. IPGRI INTERNATIONAL PLANT GENETIC RESOURCES INSTITUTE. 1997. Pistachio descriptors (Pistacia vera L.). Retrieved from Acknowledgments: This work was co-funded by the Tunisian Institution of https://www.ifpri.org/publisher-source/international-plant-genetic-resources- Agricultural Research and Higher Education (IRESA) and Faculty of Sciences of institute-ipgri Tunis. Luis Noguera-Artiaga was funded by FPU grant from the Spanish JOHNSON, R., WEINBAUM, S. 1987. Variation in tree size, yield, cropping government (FPU014/01874). efficiency, and alternate bearing among'Kerman'pistachio trees. 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