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Influence of some physical treatments and natural on the diversity of in certain Egyptian

By

Mohamed Nagah Zayed Ibrahiem Abu El-Naga B.Sc. ( - chemistry), Fac. Sci., Al-Azhar Univ., Egypt, 2003.

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

IN

Microbiology

Department of Botany & Microbiology Faculty of Science Al-Azhar University

Cairo

2009

SUPERVISION SHEET

Influence of some physical treatments and preservatives on the diversity of microorganisms in certain Egyptian juices

By

Mohamed Nagah Zayed Ibrahiem Abu El-Naga B.Sc. (Microbiology - chemistry), Fac. Sci., Al-Azhar Univ., Egypt, 2003.

SUPERVISION COMMITTEE

Prof. Dr. Backry Mohamed Haroun Prof. of Microbiol., Fac. of Sci., Al-Azhar University.

Prof. Dr. Mohie El-Din Zohier El-Fouly Prof. of Microbiol., National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority.

Dr. Hala Ahmed Hussein Assistant Prof. of Microbiol., National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority.

Acknowledgment

Praise and gratitude is to ALLAH SOBHANAHU WATAALA, the God of all creatures, for guiding me to the right way.

I wish to express my appreciation to Dr. Backry Mohamed Haroun, Professor of Microbiology, Botany and Microbiology Department, Faculty of Science, Al-Azhar University, for helpful advice, encouragement and for sharing in the supervision.

Sincere thanks and deep gratitude to Dr. Mohie El-Din Zohier El-Fouly, Professor of Microbiology, National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority, for helpful advice, encouragement, sharing in the supervision and lively interest he showed during the entire thesis.

I wish to express my grateful acknowledgment to Dr. Hala Ahmed Hussein, Assistant Professor of Microbiology, National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority, for suggesting the subject of study, progress of the work, helpful advice,

encouragement, sharing in the supervision and lively interest she showed during the entire thesis.

I would especially like to thank Prof. Dr. El-Said Ghazal, Professor of Microbiology, and the head of Botany and Microbiology Department, Faculty of Science, Al-Azhar University, for offering facilities, continuous encouragement and sustainment.

Also, I would like to express my appreciation and thanks to Prof. Dr. Bothaina M. Youssef Professor and Head of Microbiology Department at NCRRT.

Sincere thanks to Prof. Dr. T. M. El-Mongy, Professors of of National Center for Radiation Research and Technology, for help through out conducting my experiments.

Finally, special thanks to Said El-Sayed Desouky, Assistant lecturer, Botany and Microbiology Department, Faculty of Science, Al-Azhar University, for his cooperation and great help in this study.

Last, but not least I wish to express my grateful thanks, to all Members of Microbiology Department, and to all my Colleagues at Microbiology Department, NCRRT.

DEDICATION

I dedicate this work to my Parents, as well as to my Sisters and my Brother and a special dedication for my Mother for her great support.

Mohamed Nagah

CONTENTS

CONTENTS Page INTRODUCTION ...... 1 REVIEW OF LITERATURE...... 4 1. Juices and soft ...... 4 2. Food additives ...... 7 2.1. Introduction...... 7 2.2. Types of additives……………………………………………… 8 2.2.1. Preservatives...... ……………………………………… 8 2.3. Risks of additives…………………………………………… 9 2.4. Balancing risks and benefits…………………………… 11 2.5. Categories of Risk………………………………………………… 14 2.5.1. Phenyl ethylamine………………………………………………… 16 2.5.2. Azo and Benzoates…………………………………… 16 2.5.3. ………………………………………………………………… 17 2.5.4. ……………………………………………………………………………… 18 2.5.5. Other Agents………………………………………………… 20 3. The use of natural ……………………………………… 22

3.1. Natural preservatives...... 25 3.1.1. …………………………………………………………………… 25 • Mechanism of action……………………………………. 31 3.1.2. Organic ………………………………………………………… 36 3.1.2.1. Mode of action………………………………………………… 38 3.1.2.2. Resistance mechanisms…………………………………… 42 3.1.2.3. Citric ………………………………………………………… 48 3.1.2.4. ………………………………………………………… 49 3.1.3. and their essential oils……………………… 53 3.2. Physical treatments…………………………...... 58 3.2.1. Gamma irradiation ……………………………………… 58 3.2.1.1. Types of radiation ……………………………………… 58 3.2.1.2. Living cell and radiation……………………………… 60 3.2.1.3. Dose survival curves…………………………………… 61 3.2.1.4. The decimal doses (D10 value) 61

3.2.1.5. Effect of radiation on microorganisms…… 61 3.2.1.6. Direct action ………………………………………………… 62 3.2.1.7. Indirect action…………………………………………………… 63 3.2.1.8. Effect of radiation on molecules… 64 3.2.2. Heat treatment………………………………………………………… 67 3.2.2.1. Introduction...... ………………………………….... 67 3.2.2.2. Thermal technologies………………………………… 67 3.2.2.3. ……………………………………………… 68 3.2.2.4. The decimal reduction time……………………… 68 3.2.2.5. Mechanism of action………………………………… 69 MATERIALS AND METHODS.... ………………… 71 • Materials……………………………...... …………… 71 1. Samples………………………………………………………...... 71 2. Source of samples………………………………...... ………… 71 3. Samples sorts……………………...... …………………………… 72 4. Source of preservatives……………………...... …………………… 72 5. Irradiation source…………………………………………………...... …… 72 6. Isolation media………………………………………………...... 73 • Methods…………………………...... …………………………… 76 1. Microbial counts ……………………………...... ……………… 76 2. Isolation of ……………………………...... …………… 76 3. Isolation of ...... 76 4. Identification……………………………………...... ……………… 77 5. Selection of most common isolates……………………………………. 78 6. Effect of some natural preservatives………...... ………………… 78 7. Effect of some physical treatments……………………...... ……… 79 8. Effect of the combination treatments…………...... …………… 80 9. Storage…………………………………………………………...... … 81 RESULTS AND DISCUSSION………………………..... 83 1. Isolation and identification……………………………….. 83 2. Single treatments……………………………………...... ………….. 93 2.1. Physical treatments……………...... ………………………. 93 2.1.1. Gamma irradiation…………………………………...... … 93 2.1.2. Pasteurized temperature……...... …………………… 99 2.2. Natural treatments…………………...... ………………………………… 105 2.2.1. Nisin……………………………………….……………… 105 2.2.2. ………………………………………… 112 2.2.2.1. …………………………… 112

2.2.2.2. Lactic acid…………………………… 117 2.2.3. Cinnamon……………………………………………… 124 3. Combination treatments …………………. 130 3.1. Combination with Gamma Irradiation….. 130 3.1.1. Combination of Gamma irradiation with citric acid 131 3.1.2. Combination of Gamma irradiation with lactic acid 134 3.1.3. Combination of Gamma irradiation with cinnamon 138 3.1.4. Combination of Gamma irradiation with Nisin 142 3.1.5. Comparison between gamma irradiation single and combined treatments 147 3.2. Combination with Pasteurized temperature 149 3.2.1. Combination of pasteurized temperature with Nisin 149 3.2.2. Combination of pasteurized temperature with citric acid 152 3.2.3. Combination of pasteurized temp with lactic acid 154 3.2.4. Combination of pasteurized temp with cinnamon 156 3.2.5. Comparison between the selected natural preservatives alone 160 and in combination with pasteurized temperature.

3.3. Combination of natural preservatives…………… 163 3.3.1. Combination of Nisin with citric acid and lactic acid……………… 163 3.3.2. Combination of Nisin with cinnamon………………………………………… 170 3.3.3. Comparison between the selected natural preservatives in single treatment and in combination with nisin. 174

4. Combination of gamma irradiation with 177 pasteurized temperature…………………………………………. 5. Storage………………………………………………………………………………… 179 6. General Discussion………………………………………………………………… 182 SUMMRAY…………………………………………………………… 188 CONCOLUSION AND RECOMMENDATION 198 REFERENCES…………………………………………………… 199 ARABIC SUMMARY……………………………………………… 1

Table LIST of TABLES Page

(a) Properties of some organic acids used as additives. 42 Screening and isolation of microorganisms (1) 86 contaminated certain Egyptian juices. Identification of contaminated microorganisms isolated (2) 90 from certain Egyptian juices. Effect of different doses of gamma irradiation on the (3) 96 viable counts of the selected organisms. Effect of pasteurized temperature 90 C ْ at different time (4) 102 on selected organisms. Effect of different conc. of nisin on the selected (5) 109 organisms. Effect of different conc. of citric acid on P. aeruginosa and (6) 114 S. aureus. (7) Effect of different conc. of citric acid on Debaryomyces sp. 115

(8) Effect of different conc. of lactic acid on Debaryomyces sp. 118 Effect of different conc. of lactic acid on P. aeruginosa and S. (9) 119 aureus. Effect of different conc. of cinnamon on S. aureus and (10) 126 Debaryomyces sp. (11) Effect of different conc. of cinnamon on P. aeruginosa 127 Effect of different doses of gamma irradiation in (12) 137 combination with lactic acid. Effect of different doses of gamma irradiation in (13) 138 combination with citric acid. Effect of different doses of gamma irradiation in (14) 140 combination with cinnamon. Effect of different doses of gamma irradiation in (15) 145 combination with nisin. D10 - values of gamma irradiation for single and (16) 148 combined treatments. Effect of pasteurized temperature 90 C ْ for 30 sec in (17) 151 combination with different conc. of nisin

Effect of pasteurized temperature 90 C ْ for 30 sec in (18) 153 combination with different conc. of citric acid. Effect of pasteurized temperature 90 C ْ for 30 sec in (19) 155 combination with different conc. of lactic acid. Effect of pasteurized temperature 90 C ْ for 30 sec in (20) 159 combination with different conc. of cinnamon. Sub-lethal values of natural alone and in (21) 161 combination with pasteurized temperature. Effect of different conc. of citric acid in combination (22) 165 with nisin. Effect of different conc. of citric acid in combination (23) 167 with Nisin 200 µg/ml on Debaryomyces sp. Effect of different conc. of lactic acid in combination with (24) 168 nisin Effect of different conc. of lactic acid in combination with (25) 169 Nisin 200 µg/ml on Debaryomyces sp. Effect of different conc. of cinnamon in combination with (26) 172 nisin Sub-lethal values of selected natural preservatives alone and in (27) 175 combination with nisin. Effect of storage for recovery of Debaryomyces sp. (28) 180

Fig. LIST of FIGURES Page

(a) Structure of nisin and unusual amino acids. 28 A schematic diagram of the stress response of a yeast (b) 47 cell challenged with weak organic acids. (1) Percentages of isolated microorganisms. 92 Effect of increasing doses of gamma irradiation on (2) 97 the selected organisms. Effect of pasteurized temperature 90 C ْ at different (3) 103 time on the selected organisms. General model of the `barrel-stave' mechanism of pore (5) 108 formation by peptides Effect of different conc. of nisin on the selected (4) 110 organisms. Effect of different conc. of citric acid on P. (6) 115 aeruginosa and S. aureus. Effect of different conc. of citric acid on (7) 116 Debaryomyces sp. Effect of different conc. of lactic acid on Debaryomyces (8) 118 sp. Effect of different conc. of lactic acid on P. aeruginosa (9) 120 and S. aureus. A schematic impression of the stress response of a (10) 122 yeast cell challenged with weak organic acids. Effect of different conc. of cinnamon on S. aureus and (11) Debaryomyces sp. 127

(12) Effect of different conc. of cinnamon on P. aeruginosa 128

Effect of different doses of gamma irradiation in (13) 136 combination with lactic acid. Effect of different doses of gamma irradiation in (14) 134 combination with citric acid. Effect of different doses of gamma irradiation in (15) 146 combination with cinnamon.

Effect of different doses of gamma irradiation in (16) 146 combination with Nisin. D10 - values of gamma irradiation for single and (17) 148 combined treatments Sub-lethal values of natural preservative alone and in (18) 162 combination with pasteurized temperature Effect of different conc. of citric acid in combination (19) 166 with nisin Effect of different conc. of citric acid in combination (20) 167 with Nisin 200 µg/ml on Debaryomyces sp. Effect of different conc. of lactic acid in combination with (21) 169 nisin Effect of different conc. of lactic acid in combination with (22) 170 Nisin 200 µg/ml on Debaryomyces sp. Effect of different conc. of cinnamon in combination with (23) 173 nisin. Effect of different conc. of cinnamon in combination with (24) 173 nisin on S. aureus. Sub-lethal values of selected natural preservatives alone and in (25) 176 combination with nisin.

Abbreviations

ACSH → American Council on Science and Health. AMU→ Atomic Mass Unit. ATP → . BHA → Butyl Hydroxyanisole. BHT → Butyl Hydroxytoluene. CDCP → Centers for Disease Control and Prevention. CFU → Colony Forming Unit.

CO60 → Cobalt 60. Conc. → Concentration. D → Dalton.

D10 → Decimal Reduction Dose. DHA → Dehydroalanine. DHSS → Department of Health Social Security.

Dt → Decimal Reduction Time. E300 → Number. EDTA → Ethylene DiamineTetracetate. FDA → Food and Drug Administration. FPCFN → Food Protection Committee of the Food and Board. GRAS → Generally Recognized as Safe. H → Hour. HSP → Heat Shock . IAEA → International Atomic Energy Agency. IFT → Institute of Food Technologists.

IgE → Immunoglobulin E. INS → International Numbering System. kGy → kilo Gray.

LD50 → Lowest Dose (in mg/kg or g/kg of body). LPS → Lipopolysaccharide. mg → Milligram. MIC → Minimum Inhibition Concentration. Min → Minute. MRS → de Man Rogosa and Sharpe. NAS/NRC → National Academy of Science/National Research Council. OM → Outer Membrane. PDA → Potato Dextrose . PL → . PMF → Proton Motive Force. RAST → Radio-allergo sorbent Test. ROS → Reactive Species. TDT → Thermal Death Time. W/V → Weight Per Volume. ΔΨ → Membrane Potential. μg → Microgram.

INTRODUCTION

Introduction begin to lose their quality from the moment they are harvested through changes resulting from physical, chemical, enzymatic, or microbiological reactions. prevents deteriorative reaction, extending the and assuring its safety. Microorganisms and are the main agents responsible for food spoilages and therefore the targets of preservation techniques. The "ideal" method of food preservation has the following characteristics: • It improves shelf life and safety by inactivating spoilage and pathogenic microorganisms. • It dose not leave residues. • It is cheap and convenient to apply. • It encounters no objection from consumers and legislators. One of the major advances in human history was the ability to preserve food. It was the perquisite to man setting down in one place instead of moving from place to place in the never ending hunt for . The earliest preservation technologies developed were , , chilling and heating. The use of various compounds such as and spices to preserve food was also used in ancient times. 1

Unfortunately, the gradual use of a wider range of chemicals for preservation for long time led to several problems and hence consumers have developed some suspicion of the use of chemical additives. On the other hand, consumers have fewer reservations about physical treatments, although one of the oldest technologies smoking is now suspected of being carcinogenic. Another more recent physical treatment which is also much under debate is irradiation. Many studies have shown it to safe and it has been approved for use in in several countries because it has proved to be the best way to kill and other pathogenic bacteria. Recent debate about preservation techniques has focused on not only safe but also preserves the intrinsic nutritional and sensory qualities present in raw and fresh food. Combined treatments are advantageous principally because many individual treatments alone are not adequate to ensure or stability. In some circumstances combined treatments allow a milder use of single treatments with consequent improvement in . Combining non-thermal methods with other food preservation techniques can enhance the lethal effect of non- thermal processing, reduce the severity of non-thermal treatment needed to obtain a given level of microbial

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inactivation and or prevent the proliferation of survivors following treatment. So the purpose of this article is to study some successful combinations of given thermal and non-thermal technologies with other natural preservatives to ensure the safety of some juices commonly produced in food Egyptian factories. To achieve this aim, the work plane was: 1- Isolation and identification of the micro flora of some Egyptian fruit juices (pulp – end product) before and after pasteurization, with and without preservatives. 2- Study the effect of nisin, lactic acid, citric acid, cinnamon, radiation and temperature on the isolated organisms under study. 3- Study the effect of combining treatments of irradiation with other food preservatives on isolated microflora. 4- Study the effect of combining treatments of pasteurization with other food preservatives on isolated juice microflora. 5- Study the effect of combining treatments of nisin with other food preservatives on isolated juice microflora. 6- Study the storage of juice by use the best natural preservatives and physical treatment obtained from the previous experiments.

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LITERATURE REVIEW

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Review of Literature 1. Fruit Juices and soft Drinks: Due to their intrinsic properties in particular their low pH value and low and oxygen contents, fruit juices and soft drinks impose an adverse environment for most microorganisms. On the other hand, these beverages are excellent substrates for supporting growth of certain microorganisms such as and, to a lesser extent, and (Deak, 1980). Of these beverages, fruit juices contain the highest amounts of nitrogenous compounds and and, hence, are most susceptible to yeast spoilage.

The stability of fruit-based beverages and drinks is also influenced by the type of fruit juice or concentrate used in their formation (Deak, 1986). Low- pH fruit juices, especially cider and , have been associated with food born diseases (Birkhead et al., 1993; CDCP, 1995; CDCP, 1999). One major factor in the occurrence of juice- related outbreaks is the lack of a kill step such as pasteurization during juice processing. In fact, un- pasteurized fruit juices have been reported to be the vehicle in numerous outbreaks of Salmonellosis and Escherichia

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coli 0157:H7 infections (Besser et al., 1993; CDCP, 1995; CDCP, 1999). In the past, low pH juices were not considered to pose a risk because of the poor survival of like Salmonella at the pH of these products (Goverd et al., 1979). However, pathogens such as monocytogenes and E.coli 0l57:H7 have been shown to tolerate adverse conditions, including acidic conditions and in the case of L. monocytogenes, low temperatures (Ita and Hutkins, 1991; Benjamin and Datta, 1995; Conner and Kotrola, 1995). Therefore, the avoidance of the presence of pathogens on the surface of a fruit before juice is squeezed from it may be important in preventing food borne disease associated with these types of foods. The potentials for cross contamination from the rinds to the juice may differ for different products. juices are not exempt from cross- contamination if the surfaces of the raw are contaminated. However unlike the case for apple juice processing where by are milled, there by providing intimate contact between juices and peel, little contact between peel as a significant source of microorganisms in citrus juice (Parish, 1998).

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Freshly squeezed or extracted fruit juices require processing to prevent spoilage. Chilling in most commonly used to extend shelf life of un-pasteurized products. Murdock and Hatcher (1975) showed that the rate of yeast growth in orange juice decreases as temperature decreases. rouxii grew at temperatures between 1.7 to 10 Cْ with a generation time of 19.6 to 8.6 h. Fruit juices can also be chemically preserved, pasteurized, frozen, concentrated, and or irradiated to prevent spoilage. Preservation of concentrated orange juice can be achieved by the addition of dioxide (230 mg/ l) or by (800mg/l) (lioyd, 1975). The spoilage yeast Z. balii often exhibits resistance to . Due to its pH, light pasteurization (66 Cْ for 10 s) is sufficient to inactivate most microorganisms in orange juice (Sadler et al., 1992 ) other studies have shown that pasteurization juice may undergo spoilage due to yeast or growth within five weeks of refrigerated storage (parish and Higgins, 1989). The effect of temperature and juice composition on survival of yeasts has been the subject of numerous studies. Citric acid influence the thermal resistance of spoilage yeasts. Heat resistance increases with an increase in concentration of orange juice (Juven et al.,

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2. Food additives 2.1. Introduction : According to the FPCFN, food additives may be defined as follows:

A substance or mixture of substances, other than a basic foodstuff, which is present in a food as a result of any aspect of production, processing, storage, or packaging. The term does not include chance contaminants. Since prehistoric times, chemicals have been added to foods to perform special functions. Although basic foods contain no additives, as foods are processed for conversion into a variety of products, an increasing number of additives are generally used. Technological advances in food processing have increased the variety and use of these additives. Today, more than 2500 different additives are intentionally added to foods to produce a desired effect. The use of these additives is a well-accepted practice but is not without controversy (Branen et al., 2002).

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2.2. Types of additives :

Additives can be divided into six major categories: preservatives, nutritional additives, flavoring agents, coloring agents, texturizing agents, and miscellaneous additives. Several additives commonly serve more than one function in foods. In Europe and other parts of the world, the E system, developed by the (formally the European Economic Community), provides a listing of several commonly used additives (Jukes, 2001).

Since preservatives are the main object of the present research, some details will be given here.

2.2.1. Preservatives: There are basically three types of preservatives used in foods: antimicrobials, , and antibrowning agents. These additives are grouped under the category of preservatives in the INS system (Branen et al., 2002). • The antimicrobials, with E and INS numbers ranging from 200 to 290, are used to check or prevent the growth of microorganisms. Antimicrobials play a major role in extending the shelf-life of numerous and convenience foods and have come into even greater use in recent years as microbial food safety.

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• The antioxidants (INS 300–326 and E300–E326), are used to prevent and/or oxidation in food products. They are used primarily to prevent autoxidantion and subsequent development of rancidity and off-. They vary from natural substances such as vitamins C and E to synthetic chemicals such as BHA and BHT. The antioxidants are especially useful in preserving dry and frozen foods for an extended period of time.

• Antibrowning agents are chemicals used to prevent both enzymatic and non-enzymatic browning in food products. (E300), citric acid (E330), and sodium (E221) are the most commonly used additives in this category. These additives are classified as either antioxidants or preservatives in the INS system.

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2.3. Risks of additives :

Despite the benefits attributed to food additives, for several years there have also been a number of concerns regarding the potential short- and long-term risks of consuming these substances. Critics of additives are concerned with both indirect and direct impacts of using additives. As for many of the benefits mentioned, there is not always adequate scientific proof of whether or not a particular additive is safe. Little or no data are available concerning the health risks or joint effects of the additive cocktail each of us consumes daily (Branen et al., 2002).

The indirect risks that have been described for additives are the converse of some of the benefits attributed to their use. While it is accepted that through additives a greater choice and variety of foods have been made available, there is no question that additives have also resulted in the increased availability of food products with a low density of nutrients. These so-called junk foods, which include many snack items, can in fact be used as substitutes in the diet for more nutritious foods. Recently the food has attempted to address this criticism by adding nutritional additives to snack items so that these foods are a source of selected vitamins and minerals (Branen et al., 2002).

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Of greater concern than the indirect risks are the potential direct toxicological effects of additives. Short-term acute effects from additives are unlikely. Few additives are used at levels that will cause a direct toxicological impact, although there have been incidents where this has happened. Of particular concern are the hypersensitivity reactions to some additives that can have a direct and severe impact on sensitive individuals even when the chemicals are used at legally acceptable levels. Toxicological problems resulting from the long-term consumption of additives are also not well documented. and reproductive problems are of primary concern, although there is no direct evidence linking additive consumption with their occurrence in humans. There are, however, animal studies that have indicated potential problems with some additives. Although most of these additives have been banned, some continue to be used, the most notable being (Branen et al., 2002).

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2.4. Balancing risks and benefits : Due to the difficulties in precisely defining the risks and benefits of individual additives, a legal rather than a scientific decision is commonly made regarding the safety of a . In such a decision, the potential risks must be weighed against the potential benefits. A common example of this balance is saccharin. Although there is no direct evidence that saccharin, in the low amounts consumed in foods, causes cancer in humans, risk evaluation in rats indicates a potential for cancer in humans. On the benefit side, saccharin is an excellent non- caloric sweetener that is useful for diabetics and those inter- ested in reducing consumption of calories. Many consumers feel that the benefits of having saccharin available as a sweetening agent outweigh the risks (Branen et al., 2002). The potential risks of additives are well recognized, but the beneficial role these additives play in food production, processing, and utilization is also felt to be essential to the maintenance of our current food systems. With the convenient, tasty, and nutritious foods demanded, or at least desired, by consumers and the increasing overall demand for foods as populations increase, food additives will continue to play an important and essential role in food production. There will, however, continue to be concern regarding the

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potential risks associated with long-term consumption of small amounts of these chemicals and the possible interactive toxicological effects. As methods improve for evaluating these toxicological effects, some additives may be banned. At the same time, the same information may be used to develop safer new additives or techniques for using existing additives in a way that will lessen risk (Branen et al., 2002). New technology is likely to have a profound impact on the use of food additives in the future. Of these, recombinant DNA biotechnology may have the greatest effect on the future development and use of food additives. Recombinant DNA biotechnology is already routinely used for production of additives through bioprocessing, including organic acids, preservatives, enzymes, microorganisms, vitamins, and minerals (IFT, 2000). Biotechnology may also decrease the need for food additives. Plants have been produced through recombinant DNA with increased shelf-life and nutritional value, thus decreasing the need for a variety of additives. Although it is expected these recombinant DNA methods will be accepted in the future, there are currently several questions being raised regarding the risks and benefits of these products as well.

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2.5. Categories of Risk

What are the risks associated with our food supply? This can be a very confusing question because of our inability to adequately measure the risks associated with different components. Roberts (1981) has categorized the major hazards associated with foods, including additives, into five groups, ranked in order of importance: (1) food borne hazards of microbial origin, (2) nutritional hazards, (3) environmental contaminant hazards, (4) food borne hazards of natural origin, and (5) food and color additive hazards. The public perception of the risks associated with foods is often in the reverse order of the list above (Oser, 1978). Therefore, it is important to examine each of these areas to gain a better understanding of the total food safety problem. Ranked below the other four food hazards is the risk obtained from food additives. The GRAS would be included in this classification. Although GRAS ingredients are not legally food additives, the public perceives no distinction. This class includes thousands of substances. Any potential hazard to humans from a certain food additive depends on the toxicity of the food additive and the level at which the additive is ingested. The four most widely used direct food additives, which account for 93% by weight of all the direct food additives, are , , corn syrup, and

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dextrose (Roberts, 1981 and Clydesdale, 1982). Oser (1978) and Roberts (1981) speculated that the problem with the public perception of food ingredients is that these food borne substances must be proven ‘‘safe.’’ It is impossible to ensure the complete safety of any substance for all human beings under all conditions of use. Therefore, any uncertainty about the safety of a food additive can result in the public suspicion of a much greater risk. The recently approved ingredient (brand name Olean), a non- caloric replacer, is just one example where debate continues about the safety of its use in foods (ACSH, 1998).

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The categories of additive risks are: 2.5.1. Phenyl ethylamine Ingestion of phenyl ethylamine may lead to headaches. It is present in small amounts in many and and in very low concentration also in chocolate. Three milligrams of phenyl ethylamine provoked headaches during 12 h. In 16 of 38 patients who had suffered from headaches after ingestion of chocolate (Moneret-Vautrin, 1983). However, it seems unlikely that one could eat enough chocolate to reach 3 mg of phenyl ethylamine.

2.5.2. Sodium is used as an and agent in various foodstuffs, such as cooked . The authorized and widely accepted daily dose is 0.2 mg/kg. Daily consumption commonly exceeds this figure, particularly in children. Moneret-Vautrin et al. (1980) have shown that an oral challenge test with 30 mg of sodium nitrite may cause urticaria, intestinal disorders, or headache. The mechanism by which sodium nitrite acts are unclear. It may cause cellular anoxia and inhibit the protective enzymatic activities of the intestinal mucosa. This may lead to increased permeability of the mucosa to other

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antigens. In addition, sodium nitrite may in some way or other enhance the effect of histamine present in many foods.

2.5.3. Azo Dyes and Benzoates

In the late 1950 it was discovered that food colorants may provoke asthma (Lockey, 1959). Of them, tartrazine has been most studied, and its role in asthmatic reactions was established in the 1960 (Chafee and Settipane, 1967). It is still added to many pharmaceutical products as well as to foods and soft drinks in several countries. Many adverse reactions reported have been associated with , pills, capsules, and . and other benzoic acid derivatives can also produce respiratory symptoms. The tartrazine molecule possesses similarities to other azo compounds, benzoates, pyrazole compounds, and the hydroxyaromatic acids, which include salicylate and aspirin (Miller, 1985).

2.5.4. Sulfites

Sulfating agents include (SO2), sodium sulfite, and the potassium and sodium salts of bisulfite and metabisulfite. Sodium and potassium bisulfite and metabisulfites are converted into sulfurous acid in solutions and sulfur dioxide itself. Sulfites are antioxidants that are used as preservatives in foods and drugs, especially 18

in injected and inhaled medications, and as antimicrobial and sanitizing agents in the production of (Settipane, 1984). They are used on dehydrated and dried , in fruit drinks, and also in many other products (Przybilla and Ring, 1987). Prenner and Stevens (1976) were the first to report a patient who experienced an anaphylactic reaction after ingestion of sulfites in foods. After that, several reports suggesting anaphylactoid reactions to sulfite agents were published (Stevenson and Simon, 1981).

Inhaled sulfur dioxide may produce bronchoconstriction at 1 ppm in asthmatics (Koenig et al., 1980). It seems that sulfites cause adverse respiratory reactions more often than food colorants and benzoates. These reactions are obviously nonspecific in nature, similar to the response to metacholine and histamine (Bush et al., 1986). Reactions to sulfites may be severe and not readily recognized if not suspected.

3. Nitrites

Nitrite salts (KNO2 and NaNO2) have been used in curing for many centuries. Meat curing utilizes salt, , spices, and ascorbate or erythorbate in addition to nitrite. The reported contributions of nitrite to meat curing include

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characteristic color development, flavor production, texture improvement, and antimicrobial effects (IFT, 1987).

Nitrite has been shown to have variable effects on microorganisms other than . C. perfringens growth in laboratory medium at 20°C was inhibited by 200 µg/ml nitrite and 3% salt or 50 µg/ml nitrite and 4% salt at pH 6.2 (Gibson and Roberts, 1986).

The mechanism of nitrite inhibition of microorganisms has been studied for 50 years (Tompkin, 1983). The inhibitory effect of nitrite on bacterial sporeformers is apparently due to inhibition of outgrowth and during cell division (Genigeorgis and Riemann, 1979; Cook and Pierson, 1983).

The lethal dose of nitrites in humans is 32 mg/kg body weight or 2 g (Burden, 1961). Prolonged ingestion of sodium nitrite or has been shown to cause methemoglobinemia, that is, excessive production of abnormal hemoglobin, especially in infants (NAS/NRC, 1981).

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2.5.6. Other Agents

‘‘Chinese restaurant asthma’’ has been suggested to be caused by monosodium l-glutamate (Schaumburg et al., 1969; Allen and Baker, 1981 and Allen et al., 1983). There are, however, studies in which double-blind provocation tests have produced no asthmatic symptoms at all (Ghezzi et al., 1980; Grattini, 1982 and Morselli and Grattini, 1970). Numbness of the neck, headache, facial pressure, and chest pain have also been claimed to be symptoms of Chinese restaurant syndrome in some individuals. Tarasoff and Kelly (1993) made a critical review of the glutamate literature and performed a very strict double-blind per oral challenge test with 1.5, 3.0, and 3.15 g per person. They found no difference between the responses to placebo and glutamate, and they stated the ‘‘Chinese restaurant syndrome’’ to be anecdotal. Just recently, Yang et al. (1997) recorded headache, muscle tightness, numbness and tingling, general weakness, and flushing from glutamate more often than from placebo, 2.5 g of being a threshold dose for positive responses. The dilemma on the effects of glutamate thus seems to continue. Occupational asthma induced by inhalation of dust from spices such as powdered coriander,

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curry, and paprika has been described (Toorenenberger and Van Dieges, 1985).

Enzymes are frequent causes of IgE-mediated nasal, bronchial, and skin symptoms. α- is a usual additive which has been found to cause both immediate and delayed contact in bakers. Morren et al. (1993) tested 32 bakers, 7 of whom reacted to a-amylase in scratch- chamber test at 20 min. RAST was positive in five of them. Two patients showed also a delayed reaction in the scratch- chamber test. Larese et al. (1993) made skin prick tests with A-amylase in 226 bakers and pastry makers. Seventeen (7.5%) reacted to it. Rhinitis, conjunctivitis and asthma were the most common allergic symptoms.

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3. The use of natural antimicrobials

Introduction: Food antimicrobials are usually chemical compounds added to or present in foods that retard microbial growth or kill microorganisms. The functions of food antimicrobials are to inhibit or inactivate spoilage microorganisms and pathogenic microorganisms. The latter function has increased in importance in the past 10-15 years as food processors search for more and better tools to improve food safety (Davidson, 2001). Most of the traditional, currently approved food antimicrobials have limited application due to pH or food component interactions. For example, organic acids function at low concentrations is only effective in high acid foods (generally less than pH 4.5-4.6). This is because the most effective antimicrobial form is the undissociated acid which exists in majority only at a pH below the pKa of the compound. All regulatory-approved organic acids used as antimicrobials have pKa values less than 5.0 which means their maximum activity will be in high-acid foods. For food products with a pH of 5.5 or greater, there are very few compounds that are effective at low concentrations. Another factor leading to reduced effectiveness among chemical food antimicrobials is

23

food component interactions. Most chemical food antimicrobials are amphiphilic. As such, they can solubilize in or be bound by or hydrophobic in foods making them less available to inhibit microorganisms in the food product (Zeuthen and Bøgh-Sørensen, 2003).

Interest in natural antimicrobials is also driven by the fact that international regulatory agencies are generally very strict about requirements for toxicological evaluation of novel direct food antimicrobials. In many parts of the world, toxicological testing of new synthetic compounds could take many years and many millions of dollars to obtain approval. For some types of food additives a payback may be possible (e.g., artificial sweeteners), but for food antimicrobials it is less likely that obtaining approval would be profitable (Zeuthen and Bøgh-Sørensen, 2003).

An argument often used to justify natural antimicrobials is that they will produce `green' labels, i.e., one with few or no `synthetic' additives in the ingredient list. While this rationale may be true, it must be remembered that many of the antimicrobial compounds approved for use in foods today come from natural sources. If a truly effective antimicrobial was discovered from a natural source, it may

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be more economically feasible to synthesize it than to extract it from a natural source. This justification also leads consumers to the mistaken belief that food additives currently in use are potentially toxic and should be avoided. In addition to potential benefits associated with natural antimicrobials in foods, there are a number of potential concerns that need to be examined with respect to food safety. For example, if an antimicrobial is to be used exclusively to inhibit a pathogenic , it must be uniformly effective, stable to storage, and stable to any processes to which it is exposed. Standardized assays for activity need to be developed to ensure that the antimicrobial compounds retain potency. Finally, producers and users of natural antimicrobials that make claims for efficacy of use will be likely to be liable for any claims they make. In short, natural antimicrobials have excellent potential but probably will not produce miracles (Zeuthen and Bøgh-Sørensen, 2003).

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3.1. Natural preservatives:

3.1.1. Nisin Nisin was first recognized in 1928 by Rogers and Whittier and was later isolated, characterized, and named by Mattick and Hirsch (1947). The compound is a peptide produced by a strain of the dairy starter culture Lactococcus lactis ssp. lactis. The structure and amino acid content of nisin was determined by Gross and Morell (1971). The molecular weight of nisin is 3.4 kDa (Gross and Morell, 1967); however, it usually occurs as a dimer with a molecular weight of 7000 AMU (Jarvis et al., 1968).

Nisin is a 34 amino acid peptide which contains the unusual amino acids DHA, dehydrobutyrine, lanthionine, and 0-methyl-lanthionine (Delves-Broughton and Gasson, 1994). The latter amino acids are found in other inhibitory peptides produced by gram positive bacteria. The group of anti-microbial peptides containing these amino acids are called ‘‘lantibiotics’’ (Chatterjee et al., 2005). A variant of nisin, called nisin Z, is produced by some strains of L. lactis ssp. lactis and contains an asparagine in place of a histidine at residue 27 (fig. a). Lantibiotics are gene-encoded peptides that contain intramolecular ring structures, introduced through the thioether containing lanthionine and 26

methyllanthionine residues. The overwhelming majority of the lantibiotics shows antibacterial activity. Some lantibiotics, e.g. nisin, are characterized by a dual mode of action. These peptides form a complex with the ultimate cell wall precursor lipid II, thereby inhibiting cell wall biosynthesis. The complexes then aggregate, incorporate further peptides and form a pore in the bacterial membrane. Recent results show that complexing of lipid II is widespread among lantibiotics; however, pore formation depends on the overall length of the peptide and the lipid composition of the test strain membrane. (Bierbaum and Sahl, 2009).

Nisin by itself has a narrow spectrum affecting only gram positive bacteria, including Alicyclobacillus, , Clostridium, Desulfotomaculum, Enterococcus, Lactobacillus, Leuconostoc, Listeria, Pediococcus, Staphylococcus, and Sporolactobacillus (Branen et al., 2002). It does not generally inhibit gram negative bacteria, yeasts, or molds.

Nisin activity is affected by a number of environmental factors, including pH, inoculum size, and interaction with food components (Harris et al., 1991 and Ukuku and Shelef, 1997). Activity generally increases with decreasing

27

pH and decreased initial numbers of microorganisms. The presence of food components such as lipids and protein influence nisin activity (Scott and Taylor, 1981). Nisin was less active against L. monocytogenes in (Jung et al., 1992) and (Dean and Zottola, 1996) with increasing fat concentrations. This was probably due to binding of nisin to fat globules (Jung et al., 1992); this binding was overcome by adding emulsifiers (e.g., Tween 80). Thomas et al. (1998) demonstrated that sucrose fatty acid esters (sucrose palmitate and sucrose stearate) enhanced the activity of nisin against Bacillus cereus, , Lactobacillus plantarum, and , but had no effect on the activity of nisin against any gram negative bacteria.

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Fig. (a) Structure of nisin and unusual amino acids. Primary structure of nisin as determined by Gross (1977). ABA, Amino butyric acid; Ala-S-Ala, lanthionine; ABA-S-Ala, β-methyllanthionine. D-Stereo configuration (*) is shown for the α-carbon

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In most cases nisin is sporostatic rather than sporocidal (Delves-Broughton et al., 1996). Morris et al. (1984) reported the existence of sulfhydryl groups in the membrane of germinated B. cereus spores. Sulfhydryl agents such as S-nitrosothiols inhibit outgrowth of germinated spores by interacting with the sulfhydryl groups in the membrane. At low concentrations, nisin inhibited spore outgrowth and competed with sulfhydryl agents for membrane sulfhydryl groups, indicating that the membrane sulfhydryl groups may be the target for nisin. Morris et al. (1984) hypothesized that the dehydroalanine (DHA) groups on nisin may react with the membrane sulfhydryl groups. In light of the results of Hansen (1994), where alterations of the 5-dehydroalanine group of subtilin eliminated activity against spores, it is becoming clearer that the DHA groups may in fact play a very important role in the sporostatic activity of nisin.

The application of nisin as a food preservative has been studied extensively (Hurst, 1981 and Hurst and Hoover, 1993). Nisin was first used as a food preservative by Hirsch et al. (1951). Somers and Taylor (1987) studied the use of nisin to prevent C. botulinum outgrowth in

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process spread formulated to have higher than normal moisture content and/or lower salt content. Nisin was an effective antibotulinal agent at 12.5–250 µg/g. The higher nisin levels allowed for the safe formulation of cheese spreads with higher moisture content and lower salt concentration. Delves-Broughton (1990) reported that nisin levels of 6 to 12.5 µg/g controlled non- Clostridium botulinum spoilage in process cheese.

Nisin has been recommended for use in canned products to prevent the outgrowth of Clostridium botulinum when less severe sterilization conditions are desired or required (Denny et al., 1961). Other researchers have reported decreased survival for pathogenic or spoilage microbes in juice products supplemented with nisin or other antimicrobials (Kisko and Roller, 2005 and Komitopoulou et al., 1999).

In addition, Delves-Broughton et al. (1992) demonstrated that nisin may be used to increase the shelf- life of pasteurized liquid whole eggs. They found that 5 µg/ml nisin added to eggs prior to pasteurization increased the refrigerated shelf-life by 11–14 days. In addition, nisin prevented growth of the B.

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cereus, which grew well in with no added nisin. Nisin has generally been considered nontoxic. The oral LD50 in mice is 6950 mg/ kg body weight, which is similar to that of common salt (Hara et al., 1962). Nisin was found to have no effect on animals in studies of sub chronic or chronic toxicity, reproduction, or sensitization. It has also been shown that nisin does not produce cross- resistance in microorganisms to therapeutic antibiotics (Branen et al., 2002).

Mechanism of action: Nisin has no antimicrobial effect on yeasts and filamentous fungi. These organisms each have a rigid cell wall, a complex structure consisting of glucan cross- linked with chitin and cell wall protein (Brul et al., 1997 and Klis et al., 1997). The processing of mannoproteins is complex and has been partially characterized in yeasts (Lu et al., 1994 and Kollar et al., 1997). A similar mechanism has been suggested for filamentous fungi (Brul et al., 1997). Because mannoproteins are generally considered one of the key wall components which determine cell porosity (Cid et al., 1995 and Klis et al., 1997), they may represent a major barrier preventing free

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Under normal circumstances produced by Gram-positive bacteria do not have a bactericidal effect on gram-negative species. However, in some cases activity against gram-negatives can be observed on disruption of the outer membrane as reported for nisin (Stevens et al., 1991). It has been established that the primary target for many of these small cationic peptides is the cytoplasmic membrane of sensitive cells (Kordel and Sahl, 1986; Van BelKum et al., 1991 and Moll et al., 1996). Where they act to dissipate the proton motive force (PMF) through the formation of discrete pores in the cytoplasmic membrane and thus deprive cells of an essential energy source (Montville and Bruno, 1994). The PMF which is composed of a chemical component (the pH gradient and pH) and an electrical component (the membrane potential; Δψ), drives ATP synthesis and the accumulation of ions and other metabolites through PMF – driven transport system in the membrane. Collapse of the PMF induced by bacteriocin action leads to cell death through cessation of energy-requiring reactions. However, recent work has shown that the activity of nisin is dependent on the concentration of lipid II (undecaprentyl – pyrophosphory-

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MurNAc – (pentapeptide- GlcNAc) in the membrane of sensitive cells (Breukink et al., 1999 and Breukink and de Kruijff, 1999).

It was demonstrated that in vitro nisin inhibited bacterial cell wall biosynthesis (Reisinger et al., 1980). Subsequently, it has been shown that nisin kills bacterial cells by interfering with basic energy transduction occurring at the cytoplasmic membrane (Ruhr and Sahl, 1985; Kordel and Sahl, 1986 and Sahl et al., 1987). It was found that pores formed in the membrane by the nisin molecules allowed the diffusion of small compounds since no transport system for ATP has been reported (Sahl et al., 1987 and Abee et al., 1994). The increase in membrane permeability result in the collapse of the PMF ; in the case of nisin, both the ΔpH and the Δψ are completely dissipated leading to a rapid cessation of all biosynthetic processes (Sahl et al., 1987; Okereke and Montville, 1992; Bruno et al., 1992 and Bruno and Montville,1993).

In contrast, the inhibition of cell wall biosynthesis is a comparatively slow process. Thus, pore formation is considered the primary mode of action of nisin. The

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interaction of nisin with membrane components of sensitive cells is considered a vital step in its mode of action. It has been reported that even in the absence of an energized membrane, nisin can associate tightly with lipid bilayers through electrostatic interaction with the phospholipids head groups (Driessen et al., 1995).

The degree of association of nisin with the membrane is largely dependent on the type of lipids present, and most importantly, the charge carried by those lipids. Several groups have demonstrated that due to the cationic nature of nisin, its activity in vitro is most efficient when a high percentage of anionic, or negatively charged, membrane lipids are present (Garcia-Garcera et al., 1993; Martin et al., 1996 and Breukink et al., 1997).

More recent studies have substantiated this evidence, confirming the ability of nisin to insert into lipid monolayers in an anionic lipid-dependent (Demel et al., 1996). Therefore, the composition of membranes is likely to be an important determination in the sensitivity of different bacterial species to nisin.

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Breukink et al. (1997) reported that this initial interaction with anionic phospholipids is mediated by the C-terminal domain of the peptide, since this region contains the bulk of positive charge carried by the nisin molecule. It is believed that nisin also mediates inhibition of cell wall biosynthesis, by forming a complex with the bactoprenol-bound peptidoglycan precursor, lipid II (Reisinger et al., 1980).

Brotz et al. (1998) suggesting that nisin may use lipid II as a’docking molecule’ for binding to specific membranes. Breukink et al. (1999) demonstrated that increasing the concentration of lipid II in isolated model membranes in the range of 0.001-0.1 mol percent increases the sensitivity of the membrane to nisin.

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3.1.2. Organic acids • Introduction Organic acids have a long history of being utilized as food additives and preservatives for preventing food deterioration and extending the shelf life of perishable food ingredients (Ricke, 2003). Although the antibacterial mechanism(s) for organic acids are not fully understood, they are capable of exhibiting bacteriostatic and bactericidal properties depending on the physiological status of the organism and the physicochemical characteristics of the external environment (Ricke, 2003). Given the weak acid nature of most of these compounds, pH is considered a primary determinant of effectiveness because it affects the concentration of undissociated acid formed (Davidson, 2001). It has been traditionally assumed that undissociated forms of organic acids can easily penetrate the lipid membrane of the bacterial cell and once internalized into the neutral pH of the cell cytoplasm, dissociate into anions and protons (Davidson, 2001). Generation of both these species potentially presents problems for bacteria that must maintain a near neutral pH cytoplasm to sustain functional macromolecules (Ricke, 2003). Export of excess protons requires consumption of cellular adenosine triphosphate

37

(ATP) and may result in depletion of cellular energy (Davidson, 2001).

The effective use of organic acids in food products is governed in part by the desired end product. contribute a variety of functional properties that enhance the quality of food. The proper selection of an acid depends on the property or combination of properties of the desired acid as well as cost. Acids are most commonly used for flavor and tartness, and the buffering ability of the salts of some acids can modify and smooth out these characteristics. Acidulants all share a characteristic sour flavor; however the degree of flavor profile varies significantly depending on the final pH of the product (Hartwig and Mc Daniel, 1995). These differences can be used in the formulation of foods with specific sensory qualities that may be unrelated to any antimicrobial effects of the individual acids.

In their synergistic capacity, acids used in conjunction with antioxidants prevent rancidity or complex with heavy metals that might initiate oxidation or browning reactions. Acids stabilize color, reduce turbidity, change melt characteristics, prevent splattering, or enhance gelling.

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They can also act as leavening or inversion agents, emulsifiers, nutrients, and supplements (Branen et al., 2002).

A major use of acidulants is as an antimicrobial agent and this use is receiving much attention today in a desire to achieve safer food products. With many foods, the incorporation of acids into the product at sufficiently high levels ensures a commercially sterile product. The target levels necessary for this purpose, however, can overwhelm the sensory properties of the food and thereby prevent the use of an acid. When used with other preservation processes, such as or heating, acids extend shelf-life for almost indefinite periods from an antimicrobial standpoint. The judicious use of an acidulants for its antimicrobial properties is counterbalanced by the desired sensory characteristics that predicate the amount of acid that is utilized. It is in the product development and safety testing phases that the addition of the acid with its inherent properties can be evaluated to produce a product desirable to the consumer.

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3.1.2.1. Mode of action The most common classical preservative agents are the weak organic acids, for example acetic, lactic, benzoic and sorbic acid. These molecules inhibit the outgrowth of both bacterial and fungal cells. Sorbic acid is also reported to inhibit the germination and outgrowth of bacterial spores (Sofos and Busta, 1981 and Blocher and Busta, 1985).

The inhibiting effect of organic acid is based on their pKa, the antimicrobial activity of their non-dissociated form, and the specific effects of each acid. Less direct antibacterial activities include interference with nutrient transport, cytoplasm membrane damage resulting in leakage, disruption of outer membrane permeability, and influence on macromolecular synthesis (Velázquez et al., 2009). In solution, weak acid preservatives exist in a pH- dependent equilibrium between the undissociated and dissociated state. Preservatives have optimal inhibitory activity at low pH because this favours the uncharged, undissociated state of the molecule which is freely permeable across the plasma membrane and is thus able to enter the cell. Therefore, the inhibitory action is classically believed to be due to the compound crossing the plasma membrane in the undissociated state.

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In yeasts, it has also been proposed that the actual inhibitory action of weak acid preservatives could be due to the induction of an energetically expensive stress response that attempts to restore homeostasis and results in the reduction of available energy pools for growth and other essential metabolic functions (Holyoak et al., 1996 and Bracey et al., 1998).

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The principal mode of action of organic acids is the release of protons from carboxylic groups, thus lowering the pH of their environment. Proton release means dissociation of the acid molecule, with the degree of dissociation depending on the pKa value. Strong acids tend to be fully dissociated and thus produce a strong drop in pH, while weak acids are only partially dissociated. This has two important consequences: (i) organic acids with two or three carboxylic groups react on changes of pH by changes in protonation of their carboxylic groups, i.e., they may `buffer', and (ii), in their undissociated state, some of these acids can pass through lipophilic cell membranes. Consequently, on a molar basis, the antibacterial efficacy of weak acids is stronger than that of strong acids. If the undissociated acid is not soluble in lipids, it is unlikely to have any inhibitory effect on microorganisms because the cell membrane is impermeable to protons and to polar compounds such as citric acid and malic acids, unless it contains specific carrier proteins. On the other hand, lipophilic undissociated acids will penetrate the cell membrane and acidify the cytoplasm. Table (a) reviews properties of selected organic acids. Kation effects are reported also. Protons may

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change the conformation of polar membrane proteins. Binding metal ions (Fe3+, Cu++, Mn++, Ca++, Mg++) by chelation is relevant only for di- and tricarboxylic acids, especially citric acid (Stratford, 1999).

Table (a): Properties of some organic acids used as additives (Smulders, 1987; Stratford, 1999, Saltmarsh, 2000 and Naidu, 2000).

weak Acid COOH* pKa Acid.** taste spec. effect**** acid'***

Membrane diffusion and Lactic 1 3.66 2.5 No Yes reduction(specific anion effect) marked Citric 3 5.7/4.3/2.9 2.6 generic no Chelation taste * Number of carboxylic groups; ** pH fall of a 1% (W/V) bacteriological peptone solution (pH 6.2), when acids (0.5% (W/V), compared on a weight basis, Stratford, 1999) are added; this represents the strength of the effect on `environmental pH'; *** Lipophilic character; ability to depress intracellular pH; **** Effects other than acidulants.

3.1.2.2. Resistance mechanisms Microbial resistance to weak organic acids can involve various mechanisms. For bacteria, significant knowledge exists on their intrinsic, non-inducible resistance mechanisms against these compounds (Russel, 1991). Gram-positive bacteria do not possess an outer membrane 43

and the exclusion limit of the cell wall of vegetative cells of Bacillus megaterium has even been calculated to be as high as 30,000 D (Lambert, 1983). Hence preservatives can easily enter these cells and their intrinsic resistance is relatively low. In gram-negative bacteria, resistance mechanisms are more complicated since these organisms possess an inner and an outer membrane. The latter membrane has a clear role in modulating the accessibility of a cell to preservatives and other small molecules (Vaara, 1992 and Helander et al., 1997); the lipopolysaccharide layer is of crucial importance in this respect (Helander et al., 1996). In some cases microorganisms are able to degrade the added preservatives by making use of specific enzymes. An example of this is the degradation of methyl para (4)-hydroxybenzoate by P. aeruginosa (Hugo and Foster, 1964).

Recently, inducible resistance mechanisms in micro- organisms have been more extensively studied. For Salmonella typhimurium, it is known that cells encounter many potential stress factors in their ‘‘natural’’ habitat, e.g., the extremely low pH in the stomach or the presence of large amounts of partially hydrophobic weak organic acids in the intestine.

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Indeed, this organism is known to possess an acid tolerance response which consists of a complex defence system that allows cells to survive pH values as low as pH 3 (Park et al., 1996). Interestingly enough, this stress response also conveys cellular resistance against the weak acids butyric, acetic and (Baik et al., 1996). Furthermore, in a recent study with Escherichia coli O157:H7, resistance towards benzoic acid was observed upon induction of an acid tolerance response with a strong acid at pH 2.0 (Lin et al., 1996).

Also, certain gram-positive bacteria, e.g., Listeria monocytogenes, are known to induce an acid tolerance response at a challenge pH of 3 after prior exposure to a mild-acid at pH 5.0 (Davis et al., 1996). No data are currently available on whether this response gives protection to weak organic acids commonly used in the .

In fungi, similar resistance mechanisms may be found. The enzymatic degradation of sorbic acid to pentadien by certain fungal species is well documented (Samson et al., 1995). The resistance of spoilage yeasts to weak organic acid preservatives has been extensively studied and is

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known to depend on the H+ -pumping P-type membrane ATPase (Holyoak et al., 1996). Studies by Kubo and Lee (1998) have shown that compounds that inhibit the plasma membrane H+ -ATPase synergistically enhance the activity of sorbic acid.

Piper et al. (1997) showed that the long term stress response of yeasts to weak organic acids also involves the induction of an integral membrane protein, Hsp30, which downregulates the increased activity of the membrane ATPase. It was deduced from these observations that Hsp30 was acting as a molecular ‘‘switch’’ downregulating the activity of the membrane ATPase in order to conserve cellular energy pools which would otherwise be consumed by the attempting to restore homeostasis (Braley and Piper, 1997).

Studies by Henriques et al. (1997) have shown that Saccharomyces cerevisiae is able to actively extrude [C- 14] labelled benzoic acid, suggesting that there is an efflux system, presumably membrane localised, and that removes accumulated an-ions from inside the cell. Supporting this, data gathered by Piper et al. (1998) has demonstrated the existence of a multidrug resistance

46

pump, the ATP binding cassette transporter, Pdr12, which actively extrudes preservative anions from the cell. Our current understanding of the mechanisms that are involved in weak acid resistance in yeasts are summarised in Fig. (b). As indicated on the diagram, simply pumping preservative anions out of the cell could create a futile cycle where the anions reassociate at the lower external pH and reenter the cell. However, this assumes that any rate of diffusion across the plasma membrane remains the same and that the cell makes no effort to alter membrane composition or structure to reduce the access of the toxic compound.

Recently, have shown that adapted yeasts reduce the diffusion coefficient of preservatives across the plasma membrane such that passage of weak acids into the cell is reduced. Therefore, efflux of protons and anions by the H+ -ATPase and Pdr12 respectively would not create a futile cycle if there is a concurrent reduction in the ability of the compounds to diffuse across the cell membrane and enter the cytosol (Brul and Coote, 1999).

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Fig. (b): A schematic diagram of the stress response of a yeast cell challenged with weak organic acids (Piper et al., 1998). Shown are, a transporter, and the membrane located Pdr12 multidrug resistance pump active against anions of acetic, sorbic and benzoic acid, and the plasma membrane P-type H + -ATPase.

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3.1.2.3. Citric acid Citric acid generally is not used as an antimicrobial, but it has been shown to possess activity against some molds and bacteria. Reiss (1976) found that 0.75% citric acid slightly reduced growth and greatly reduced production by Aspergillus parasiticus. With Aspergillus versicolor, growth was inhibited at the same level, but toxin production was prevented by 0.25% citric acid. In contrast, 0.75% citric acid did not influence the growth or toxin production of Penicillium expansum (Reiss, 1976). Citric acid was observed to be more inhibitory to Salmonella than lactic or hydrochloric acids (Subramanian and Marth, 1968). In a related study, Thomson et al. (1967) found that as little as 0.3% citric acid could reduce the level of viable Salmonella on poultry carcasses. , shrimp puree, tomato puree, and shrimp and tomato puree acidified to pH 4.2 and 4.6 with citric acid showed no significant growth or toxin production by C. botulinum after 8 weeks at 26°C (Post et al., 1985). Minor and Marth (1970) showed that Staphylococcus aureus was inhibited 90% and 99% in 12 h at pH 4.7 and 4.5, respectively, by citric acid. Citric acid was found to be particularly inhibitory to flat-sour organisms isolated from tomato juice, but the inhibition

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was pH-dependent (Branen, et al., 2002).

The mechanism of inhibition by citrate has been theorized to be related to its ability to chelate metal ions. Branen and Keenan (1970) were the first to suggest that inhibition may be due to chelation, in studies with citrate against Lactobacillus casei. Chelation was also indicated as the reason for inhibition of S. aureus (Rammell, 1962) and Arthrobacter pascens (Imai et al, 1970) by citrate. In contrast, Buchanan and Golden (1994) found that while un-dissociated citric acid was inhibitory against Listeria monocytogenes, the dissociated molecule protected the microorganism. They theorized that this protection was due to chelation by the anion. The U.S. FDA has classified citric acid as GRAS for miscellaneous and general purpose use (Branen et al., 2002).

3.1.2.4. Lactic Acid Lactic acid (pKa = 3.79) is a primary end-product of the lactic acid bacteria and serves to assist in preservation of many fermented dairy, vegetable, and meat products. It is used as a food additive primarily for pH control and flavoring. The antimicrobial activity of the compound is variable. For example, in cold pack cheese formulated with

50

lactic and and inoculated with Salmonella Typhimurium, no increase in destruction of the organism was found (Park et al., 1970). In contrast, lactic acid was found to be four times as effective as malic, citric, propionic, and acetic acids in inhibiting growth of Bacil- lus coagulans in tomato juice (Rice and Pederson, 1954). Lactic acid inhibited spore forming bacteria at pH 5.0 but was much less effective against yeasts and molds (Woolford, 1975). Staphylococcus aureus was inactivated by 90 and 99% in 12 h at pH 4.9 and 4.6, respectively, by lactic acid (Minor and Marth, 1970). Based upon molar concentration, pH, and activity of un- dissociated acid, lactic acid was one of the most effective organic acids against the growth of Yersinia enterocolitica (Brackett, 1987). Lactic acid at concentrations between 0.3% and 4% has demonstrated to be effective in the in vitro reduction of Y. enterocolitica (Virto et al., 2005). Addition of 5.0% lactic acid in ground patties stored at 4°C for 10 days reduced coliforms by 3.9 logs (Podolak et al., 1996). Oh and Marshall (1993) reported that there was little interaction between lactic acid and against L. monocytogenes. The MIC value of lactic acid alone was 0.5%, but was lower when 1.25% ethanol was combined with 0.25% lactic acid. When

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2.5% ethanol was combined with 0.25% lactic acid, the extent of inhibition was not more than that of the most active single compound alone. Different concentrations of lactic acid alone or in combination with other chemicals have been shown to be effective in the elimination of bacterial pathogens (Akbas and Olmez, 2007).

Snijders et al. (1985) and Smulders et al. (1986) advocated the use of lactic acid for surface decontamination of fresh meats, slaughter byproducts, and poultry. Lactic acid at 1–2% was reported to reduce Enterobacteriaceae and aerobic plate counts by 0.3–2.7 log on beef, , pork, and poultry. The compound also delayed growth of spoilage microflora during long-term storage of products. Visser et al. (1988) confirmed the antimicrobial effectiveness of 2.0% (v/v) L-lactic acid on fresh veal tongues and demonstrated that the compound decreased the growth rate of spoilage organisms during -packaged storage at 3°C. Presumably lactic acid applied as antimicrobial to foods functions similarly to other organic acids and has a primary mechanism involving disruption of the cytoplasmic membrane proton motive force (Eklund, 1989).

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In contrast, there has been some controversy concerning the mechanism of lactate salts used at high concentrations. These salts have been shown to have little effect on product pH. Therefore, most of the lactate remains in the less effective anionic form. Initially it was thought that the high concentration of the salts may reduce water activity sufficiently to inhibit microorganisms (Debevere, 1989).

However, Chen and Shelef (1992) and Weaver and Shelef (1993) using cooked meat model systems and , respectively, containing lactate salts up to 4% concluded that water activity reduction was not sufficient to inhibit Listeria monocytogenes. In addition, Papadopoulos et al. (1991) reported that water activity was not lowered in cooked beef top rounds injected with various levels of . Inhibition may be due to the presence of sufficient undissociated lactic acid, possibly in combination with a slightly reduced pH and water activity, to cause inhibition of some microorganisms.

Lactic acid was probably one of the first acids used in food due to its wide distribution in nature as a product of . The U.S. FDA has approved lactic acid as

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GRAS for miscellaneous and general purpose usage with no limitation upon the concentration used. It may not be used in infant foods and formulas. The U.S. Department of allows lactic acid in meat products at the lowest concentration necessary for the intended purpose (Branen et al., 2002).

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3.1.3. Spices and their essential oils Spices and their essential oils have varying degrees of antimicrobial activity. Among the spices, cloves, cinnamon, oregano, thyme, sage, rosemary, and vanillin have the strongest antimicrobial activity. The major antimicrobial components of clove (Syzygium aromaticum) and cinnamon (Cinnamomum zeylanicum) essential oils are eugenol (2-methoxy-4-(2-propenyl)- )) and cinnamic aldehyde (3-phenyl-2-propenal), respectively. Smith-Palmer et al. (1998) determined that the 24h. minimum inhibitory concentrations of cinnamon and clove essential oils against , Escherichia coli, Salmonella Enteritidis, Listeria monocytogenes, and Staphylococcus aureus were 0.05, 0.04-0.05, 0.04-0.05, 0.03, and 0.04%, respectively, in an agar dilution assay. Cinnamic aldehyde or (600 mg/liter of air) significantly reduced Salmonella populations on alfalfa used for sprouting and did not affect germination (Weissinger et al., 2001). Mixtures of cinnamon and clove oils were capable of suppressing the growth of major spoilage microorganisms of intermediate moisture foods (Huiyun Zhang et al., 2009).

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Many spices, , and plant extracts have strong medicinal, preservative, and antioxidant properties (Zaika, 1988). The antimicrobial activity of these ingredients is attributed to their essential oils, which are lipophilic and penetrate through the membrane to the interior of the cell and perform the inhibitory activity at the target site (Smith-palmer et al., 1998). Cinnamon effectively inhibits the growth of bacteria (with gram- positive being more sensitive than gram –negative), yeasts, and molds (Shelef, 1983).

Bullerman (1974) determined that 1.0% cinnamon in raisin bread inhibits growth and production by A. parasiticus. Lòpez-Malo et al. (2002) confirmed growth inhibition of the related mold, A. flavus, by eugenol, thymol ((5-methyl-2-(1-methylethyl) phenol)) and carvacrol ((2-methyl-5-(1-methylethyl) phenol)). Smid and Gorris (1999) reported that cinnamic aldehyde inhibited growth of both bacteria and fungi and increased shelf life of treated packaged tomatoes. Cinnamon oil and clove oil are both natural preservative and flavouring substances that are not harmful when consumed in food products. There have been a number of reports of substances in each of

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cinnamon and clove oils that inhibit the growth of molds, yeasts and bacteria. Both cinnamon oil and clove oil added at 2% in potato dextrose agar (PDA) completely inhibited the growth of seven mycotoxigenic molds (A. flavus, A. parasiticus, A. ochraceus, Penicillium sp. M46, P. roqueforti, P. patulum, and P. citrinum) for various times up to 21 days (Azzouz and Bullerman, 1982) and could also inhibit the growth of yeasts (Conner and Beuchat, 1984). Suksrikarm (1987) similarly reported that cinnamon oil and clove oil could separately inhibit many other microbes including Lactobacillus sp., Bacillus thermoacidurans, Salmonella sp., Corynebacterium michiganense, Pseudomonas striafaciens, Clostridium botulinum, Alternaria sp., Aspergillus sp., Canninghamella sp., Fusarium sp., Mucor sp., and Penicillium sp. Soliman and Badeaa (2002) found that <500 ppm of cinnamon oil can inhibit A. flavus, A. parasiticus, A. ochraceus and Fusarium moniliforme on PDA and August (1978) reported that high concentrations of cinnamon oil and clove oil could also inhibit the asexual spores of fungi. Mixtures of cinnamon and clove oils are therefore an interesting alternative to use of other chemical preservatives and appear well suited to use in active packaging systems.

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The greater effectiveness of the cinnamon bark oil on S. Enteritidis and E. coli O157:H7 in fruit juices was associated to the media pH, being more effective in media with lower pH. Burt (2004) reported that the bacterial susceptibility to the antimicrobial effect of essential oils appears to increase with a decrease in the pH of the food, because at low pH the hydrophobicity of an essential oil increases, enabling it to more easily dissolve in the lipids of the cell membrane of target bacteria. Although, the mechanism of action of cinnamon oil on the microbial cells is still unclear, Wendakoon and Sakaguchi (1995) and Burt (2004) have indicated that the interaction of carbonyl group of the cinnamaldehyde, main compound of cinnamon oil from bark, on the cell proteins embedded in the cytoplasmatic membrane appear to inhibit the action of the enzymes amino aciddecarboxylases, which are necessary for the amino acids biosynthesis and biodegradation.

On the other hand, Oussalah et al. (2006) reported a release of the cell constituents, a decrease of intracellular ATP concentration and a decrease in intracellular pH due to an increase in the permeability of cell membrane when cinnamon oil was applied up to 0.1%.

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Nonetheless, these authors did not observe an apparent change on the cell surface (by electron micrographs) when cinnamon oil was added. In the same way, Gill and Holley (2004 and 2006) reported a rapid decrease of cellular ATP but no increase of extracellular ATP when 0.15 or 0.6% of cinnamaldehyde was used. Thereby, the mechanism of action of the cinnamon oil and its main compound to inactivate microorganisms according to Gill and Holley (2004 and 2006) and Oussalah et al. (2006) appear to be related to the cell membrane from which a slight disruption seem to occur causing dispersion of the proton motive force by leakage of small ions without leakage of larger cell components, such as ATP, which are subsequently degraded by the ATPase enzyme.

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3.2. Physical treatments 3.2.1. Gamma irradiation Gamma radiation is a physical phenomenon in which energy travels through spaces as a wave motion without the aid of traveling medium. The exposing materials (living cells, foods, medical products) to gamma radiation in such away that a precise and specific dose in absorbed termed "gamma irradiation"

3.2.1.1. Types of radiation I. Non , having longer wave length and lower energy such as: UV, visible light, infrared, microwave and radio waves. The amount of energy carried by UV radiated is not large and its lethal activity is of a relatively lower order. Its penetration ability is much smaller than gamma radiation (Alper, 1990).

II. Ionization radiation, having short wavelength and contain enough energy to ionize the molecules in their paths. Ionizing radiation is a convenient tool for studying the fundamental of life. There are no phenomena in nature that do not experience the modifying action of ionizing

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radiations because their energy is always superior to that of intermolecular bonds. This is why radiobiology inevitably reflects all the fields of biology to some extent. Accordingly, the set of objects studies by radiobiology is exceedingly diverse. It includes macromolecules phages, viruses, protozoa cell tissues, plants, animals, cells tissues humans, populations, and biogenesis (Yarmenko, 1988).

Types of ionizing radiation used in peaceful application. All ionizing radiation are divided in their nature into electromagnetic and particulate one. Electromagnetic radiation include x-rays from x-rays tube, the gamma-rays of radioactive, and the x-rays of electron accelerators produced when highly accelerated electrons are directed at a special x-rays target. Visible light and radio waves also electromagnetic radiation but they do not ionizing matter because they are characterized by a longer wavelength "lower frequently". The depth of penetration of an ionizing radiation depends on one hand on the nature of the radiation, the charge of the particles forming it and their energy, and on the other hand, on the composition and density of the irradiated substance (Ivanov et al., 1986).

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Gamma radiation from (CO60) is the most widely used in practices because of costs and high penetrating.

3.2.1.2. Living cell and radiation The exposure of cells to ionizing radiating sets off a chain of reaction giving rise to chemical and then to metabolic or physiological changes. So, irradiation presents an additional stress to the cell, which tends to disturb their organization (Lawerence, 1971). Irradiation effects have been shown to be occurring with proteins, enzymes, nucleic acids, lipids and , all of which may have marked effect on the cell (Habbs and Macellan, 1975). Generally, radiation occurs over abroad time scale which extends from his radiation easily physical process to the very late biological effect (Edward, 1990). It has been reported that on number of intracellular constituents may be responsible for the high radiation resistance in some radio resistance strains. This may include certain chemical compounds such as mercopto- alkyl amine (Anderson et al., 1956), sulphydryl compounds (Bridges, 1964), amino acids (Work, 1964) and proteins (Mareson and Stelow, 1987).

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3.2.1.3. Dose survival curves Dose survival curves illustrate the relationship between numbers of surviving organisms and radiation dose. In practice the necessary data are usually obtained by exposing number of equal size population to increasing radiation doses and counting the number of survivors (Ley, 1973).

3.2.1.4. The decimal reduction doses (D10 value). The resistance or sensitivity of microorganisms is

measured by the so-called D10 value. The D10 value is the doses required to reduce the initial population to 90 %. When the dose survival curve is straight line it is possible

to read the D10 value from the graph by reading off the dose required to reduce a surviving fraction throughout

one log cycle (Erdman et al., 1961). The D10 value is defined as the dose of ionizing radiation required to reduce a given microbial population by a factor of 10 or 90% or by one logarithmic cycle.

3.2.1.5. Effect of radiation on microorganisms Ionizing radiation imparts their energy to molecules in a manner that depends on the atomic number of constituent atoms and not on the molecular.

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Configuration as in the case with UV interaction of radiation with matter occurs in a more or less random manner in a biological material in living cell. It is to be expected that certain sites or system will be more readily damaged than others (IAEA, 1973 and Giusti et al., 1988). The primary chemical changes in target molecules can be produced both by direct and indirect effect of radiation. The result would be ionization and or excitation in atoms of these molecules and formation, with the important biological system of which leads to its death or inactivation (Roger et al., 1998).

3.2.1.6. Direct action The direct effects are changes that appear as a result of the absorption of radiation energy by the molecules of interest. In such processes the energy by is directly deposited in the target molecule of biological system without the intervention of radical species derived from water radiolysis, or other system of the environment. The target theory states the effect of ionizing or in very near to some particular molecules or structure is responsible for the measured effect, the production of on effective in the target is often called hit. The target may be a whole cell, part of cell, or critical molecules. Generally,

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the measured effect in a system may be cell death or inability to grow or to divide in the simplest from of the target theory, one hit is sufficient to product the measured effect in associated organisms with very small radiation doses, the number of effected targets will be directly proportional to the amount of radiation (Roots et al., 1985 and Roger et al., 1998).

3.2.1.7. Indirect action

The indirect effect of radiation on target molecule is produced by way of intermediary radiation products, the photon may interact with water, the predominant molecule in the mammalian cell to produce free radicals, these free radicals are relatively short- lived, they can interact with biologically important material causing a determined effects, or conversely can react innocently to revert to their former state (Yuring et al., 2000). A free radical is an electrically neutral molecule, which has unpaired electron in the outer orbit. The steps of the formation of free radicals could proceed through various ways the most simple of which is as follow (Casarett, 1968). Cell death ( defined for proliferation cells as loss of reproductive capability ) is predominatly induced by double-strand breaks in DNA, separated by not more than a few base

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pairs, which can generally not be repaired by the cell ( Hall and Giaccia, 2006).

Reactive oxygen species (ROS), including

hydrogen peroxide (H2O2), superoxide, and hydroxyl radical, are toxic to cells due to their ability to damage DNA and especially Proteins containing Iron-sulfur clusters or sulfur atoms (Imlay, 2003). Mn (II) can act

catalytically as scavenger of either superoxide (O-2) or

hydrogen peroxide (H2O2) (Archibald and Fridovich, 1982 and Stadtman et al., 1990).

3.2.1.8. Effect of radiation on water molecules: Note: the dot [•] means exciting (has excess energy) or unpaired electron in the radical . 1- H2O → H2O (excitation)

. . . 2- H2O →H + OH (splitting)

3- H2O →H2O+ + e- aq (ionization)

4- H2O + e- aq →H20- The positive and negative free radicals of water ions are both unstable and each dissociates to form stable ions and free radicals. . 5- H2O+ → OH + H+ 66

- - . 6- H2O → OH + H

The stable [H+, OH-] ions combine to form water

7- OH-+ H+ →H2O . . But the primary free radical [H , OH , e- aq] are very

reactive and during the course of diffusion, they distribute the absorbed energy to solute molecules either organic or inorganic with high efficiency and may give rise to secondary free radical ( somewhat less reactive) and molecules which in turn are capable to attaching macromolecules. . If there is O2 in the environment [H and e- aq] react

with it. . . 8- O2+ H → H O2

9- O2+ e- aq→ O2- The primary free radicals can also react with each other. . . 10- H + H →H2

. . 11- H + OH → H2O

. . 12- OH + OH → H2O2

H2O2 may also form by:

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. . 13- HO2 + HO2 → H2O2+ O2

The ionized water molecules [H2O+] may react with another natural water molecule to form: . 14- H2O+ + H2O→H3O -+ OH

The products of water radiolysis could be summarized in the follow . . 15- (H , OH , H3O +, e- aq, OH-, H+, H2O2)

Radiation may cause alteration in DNA:

a) Rupture of the hydrogen bonds which link the base pairs adenine-thymine, cytosine- guanine, loss of base or change of abase of (for example deamination).

b) Induction of break in either or both of the DNA chains, between and groups.

c) Formation of links between adjacent thymine of residues on a chain, to from a dmire.

d) Cross-linking between the singlestrands of helix, or between a strand and the protein (histone) associated with DNA in the chromosome (IAEA, 1970).

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3.2.2. Heat treatment 3.2.2.1. Introduction The delivery of an adequately lethal thermal process is central in the preservation of food by the application of heat, both to prevent a risk to and to achieve a commercially reliable shelf life for the food. Conventional heat treatment systems must reliably deliver the required thermal process and at the same time accommodate many products, process and package variables and their interactions with each other and the thermal process. By its very nature, thermal processing has a detrimental effect on some food quality attributes and the food processor must design the thermal process to balance the unavoidable needs of commercial sterility with the commercial desire to present a high-quality product (Branen et al., 2002).

3.2.2.2. Thermal technologies The thermal process is required to achieve commercial sterility that is to render the food free from viable micro-organisms, including those of known public health significance, capable of growing in the food at temperatures at which the food is likely to be held during distribution and storage (DHSS, 1994).

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3.2.2.3. Psateurization

Pasteurization is the treatment of packaged foods to temperatures below 100 Cْ over a given time to eliminate pathogenic microorganisms which may gow under certain storage conditions (Hackney et al, 1991). It is a heat treatment which kills pan of the vegetative microorganisms present in the food and relies heavily on handling and storage conditions to further minimize bacteria growth (Karel et al, 1975).

3.2.2.4. The Decimal Reduction Time

The thermal death of specific microorganisms does not follow a linear path with a finite time to complete death. Rather, it follows a logarithmic relationship (Stumbo, 1973). If a semi-logarithmic plot was made between the numbers of one type of bacteria surviving a single lethal heat and time, an approximately linear relationship would be observed with a negative sloping line. The Iength of time required to reduce the numbers of microorganisms by one log scale, by 90%, is referred to as the decimal reduntion time and is given the symbol D. This is qual to the negative inverse of this semi-logarithmic or"survivor" curve slope.

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One D time increment at the lethal temperarure reduces the population of microorganisrns by 90%. The next D will reduce the remaining population by 90% and so on. Sterilization processes usually hold the product at the lethal heat for the equivalent of 12 D time increments while pasteurization processes usually involve 4 - 6 D time increments. If D has a value of 0.20 minutes then 12D, a sterilization process, wouId be 2.4minutes, reducing 106 bacteria to 10-6, read as 1 in a million chance of 1 bacteria surviving. This would be read as a 12 D reduction in the target microorganism if it was held at the sterilization temperature for 2.4 minutes (Trenholm, 1998).

3.2.2.5. Mechanism of action Heat can damage protein, lipids, and nucleic acids and destabilize membranes (such as oxidation of sulfydryl groups of the membrane-binding bound proteins) (Gould, 1989). However, heat induces primarily blebbing and vesiculation of the outer membrane, leading to bacterial cell inactivation, and consequently increases its permeability to hydrophobic compounds (Tsuchido and Takano, 1988). Heating-induced membrane destabilization sequentially initiates indirect DNA damageas a consequence of increased nuclease activity in

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bacteria and is often the critical injury, resulting in death of the cell (Gould, 1989).

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MATERIALS & METHODS

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Materials

(1) Samples :

Ten juice samples from each product were tested according to the production of the company. During two years from summer 2006 to summer 2007 for microbial contaminations. Five samples were tested from the end product for the manufacture and five samples from concentrated juices (raw materials). Complete microbiological analysis was done (bacteriological, yeast estimation). The raw materials was free from any preservatives while the end product contained sodium benzoate , citric acid with concentrations 0.1 %, 0.3 % ( wt/v) respectively also, the end product was pasteurized .at 90 Cْ for five minutes, approximately

(2) Source of samples:

The end product were taken from United Food Industries Company – bader city(A) and sterilized by pasteurization, while concentrated juices (raw materials) were taken from Cairo Agro-Processing Company- Al- Obour city (B).

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(3) Samples sorts:

No. of Samples Type Company Preservatives Sterilization samples Apple 10 Pulp A Guava 10 Pulp A without preservatives Pulp A Mango 10 End Sodium benzoate (0.1%) and citric acid B product (0.3%) Pulp A without preservatives Orange 10 End Sodium benzoate (0.1%) and citric acid B Pasteurization product (0.3%) End Sodium benzoate (0.1%) and citric acid Cocktail 10 B product (0.3%)

(4) Source of preservatives: • Lantibiotic nisin standard supplied from Biomedical Company U.S.A with activity approximately 1000- units/mg powder. • Cinnamon (ground state) supplied from commercial source. • Citric acid supplied from . • Lactic acid (local) supplied from El- Naser Company.

(5) Irradiation source:

Irradiation was carried out using cobalt 60 irradiation source (Gamma chamber 4000 India, located at

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National Center for Radiation and Technology) with dose rate 8.33 KGy/min. at the time of experiment).

(6) Isolation media: i. Isolation media for bacteria a. Nutrient Agar (NA) medium (Barrow and Feltham, 1993):

Nutrient agar medium contained the following (g/l): Beef extract, 3; peptone, 5; , 5; and agar, 15. All ingredients were dissolved in distilled water and completed up to 1000 ml. pH was adjusted at 6.8.

b. Nutrient Broth (NB) medium (Barrow and Feltham, 1993):

Nutrient agar medium contained the following (g/l): Beef extract, 3; peptone, 5; sodium chloride, 5. All ingredients were dissolved in distilled water and completed up to 1000 ml. pH was adjusted at 6.8.

c. M.R.S Agar

This medium was supplied from Oxoid (CM361) (62 g/l), pH was adjusted at 6.8.

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d. Violet Red Bile Agar

This medium was supplied from Oxoid (CM107) (38.5 g/l), adjusted at pH 6.8.

e. Baired-parker Agar

This medium was supplied from Oxoid. Suspend 63 g in one litre of distilled water and boil to dissolve the medium completely. Dispense into tubes or flasks and sterilize by autoclaving at 121 C for 15 minutes. Cool to 50 C and aseptically add 50 ml of Egg Yolk-Tellurite SR54. Mix well before pouring, adjusted at pH 6.8.

f. Peptone water

This medium supplied from oxoid (15g/l), adjusted at pH 6.8. ii. Isolation media for yeast a. Sabauroud's Agar

Sabauroud's agar medium contained the following (g/l): glucose, 40; peptone, 10; agar 15. All ingredients were dissolved in distilled water and completed up to 1000 ml, adjusted at pH 7.

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b. Sabauroud's broth

Sabauroud's agar medium contained the following (g/l): glucose, 40; peptone, 10. All ingredients were dissolved in distilled water and completed up to 1000 ml, adjusted at pH 7. c. YM Agar( -malt extract agar)

YM agar (yeast extract-malt extract agar) contained the following (g/l): yeast extract, 3; malt extract, 3; peptone, 5; glucose, 10. All ingredients were dissolved in distilled water and completed up to 1000 ml, adjusted at pH 6.

.All previous media were sterilized at 121 Cْ for 15 min

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Methods

(1) Microbial counts

Ten milliliters of each fruit juice was transferred into 90 ml of sterilized saline solution to get the 1:10 dilution, which serially diluted in sterilized saline solution to 104. 0.1 milliliter volume of the dilutions was transferred onto various Plate count agar.

(2) Isolation of bacteria

Five milliliters of juice was added by sterile pipette to enrichment medium 50 ml nutrient broth double strength as enrichment media for 24h. at 37 Cْ . Isolates were obtained in pure culture from enriched cultures by streaking on a suitable medium, such as Nutrient agar, Violet Red Bile Agar, and Baired-parker Agar, M.R.S Agar (for this medium another layer is added to obtain anaerobic condition.

(3) Isolation of yeast

Five milliliters of juice was added by sterile pipette to enrichment media 50 ml sabauroud's broth (double strength) for 24h. at 37 Cْ . And after incubation period it was cultured by added 0.1 ml over agar surface by sterile 79

(4) Identification a- Identification of bacterial isolates

Bacterial isolates were identified according to According to the Keys of Bergey's Manual of Determinative Bacteriology (Buchanan and Gibbson, 1974), and Cowan and Steel's Manual for the Identification of Medical Bacteria (Barrow and Feltham, 1993) and Bergey's Manual of Systematic Bacteriology, Vol. (2) (Sneath, 1986), Bergey's Manual of Determinative Bacteriology 9th ed. (Hensyl, 1994). b- Identification of yeast isolates

Yeast isolates were suggested to follow the next genera according to keys of (Barnett et al., 1990), confirmed and renamed by (Barnett et al., 2000) and (Kurtzman and Fell, 2000).

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(5) Selection of most common isolates:

Three isolates were selected, gram positive isolate, gram negative isolate, and yeast isolate. Selection of most common isolates was according to its existing percentage of its dominance in samples, and fast growth.

(6) Effect of some natural preservatives:

Series containing different concentrations of:

• Nisin (25-50-100-200-250) µg/ml. • Cinnamon (0.2-0.3-0.4-0.5-0.6-0.7-0.8-1-1.2-1.5-1.7-2- 3-4) % (W/V). • Citric acid (0.05-0.1-0.15-0.2-0.3-1-1.5-2.0-2.5-3-3.5- 4.0) % (W/V). • Lactic acid (0.001-0.01-0.05-0.1-0.15-0.2-0.3-1.5-2.5-3- 3.5-4) % (W/V).

Were added to a broth medium individually, inoculate with the organism and incubate at 37cْ for 48h, 1 ml of each treatment and control were serially diluted in sterile saline, cultured over agar surface on a suitable medium. .All plates were incubated at 37 Cْ for 72h

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(7) Effect of some physical treatments: i. Irradiation

The selected isolates were irradiated at different doses (0.25-0.5-1-2-3-4-5-6-7) kGy on broth medium. Immediately after irradiation, 1 ml of each treatment and control (non-irradiated spore suspension) were serially diluted in sterile saline. The count of yeast was enumerated on Sabauroud's dextrose agar. All plates were incubated at 37 Cْ for 72h. The growing colonies were counted and irradiation dose was plotted against log of the number of survivors divided by the initial count before irradiation (log N/N0).

D10 values of the tested isolates were calculated from the regression linear equation (Lawerence, 1971).

Y = a ± b x

D10 value = - 1/b

− − ∑ xy − n x y b = − 2 2 ∑ x −n x

Where:

a : log of microbial count when x equal zero.

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b : regression factor.

y : log of surviving fraction.

x : dose level in KGy.

n : number of calculated points.

x-: ∑x/n

y-: ∑y/n ii. Pasteurized temperature

The selected isolates were pasteurized at temperature Cْ for different times (0.25-0.5-1-3) minute. 0.1 ml of 90 the pasteurized cells were cultured over agar surface on a ْ.suitable medium and incubated for 48h. at 37 C

(8) Effect of the combination treatments:

The concentration of the natural preservatives which start to give distinct reduction for the growth in single treatment was applied in combination with physical treatments in one treatment in broth medium. 1 ml of each treatment and control (non treated ) were serially diluted in sterile saline, 0.1 ml of the treated cells were

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cultured over agar surface on a suitable medium and ْ.incubated for 24h. at 37 C

(9) Storage

The storage was performed for more resistant organism by applying two experiments as follow:

1-experiment (1)

Was perfomed by applying the combination between the following treatments:

a- Nisin was used in concentrations (50, 100 and 200). b- Citric acid 2% (W/V).

.(.c- Pasteurized temperature 90 ْ C for (30, 60 and 120 sec

Where, the nisin was added to Sabauroud's broth (double strength) with citric acid, then it was inoculated with 1ml of the organism broth (fresh culture broth with count 45 x ْ.then it was pasteurized, finally it incubated at 37 C ,(104 This was perfomed for each conc. of nisin with its time of pasteurized temperature and citric acid. Storage time was for 90 days, during this period 0.1 ml of the treated cells were cultured every week over agar surface on a double

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2-experiment (2)

Was perfomed by applying the combination between the following treatments:

a- Gamma irradiation (0.2 kGy). .b- Pasteurized temperature 90 Cْ for 30 sec

Where Sabauroud's broth (double strength) was inoculated with 1ml of the organism broth (fresh culture broth with count 45 x 104), then it was pasteurized and after that treated withgamma rays (0.2 kGy), finally it incubated at C.ْ Storage time was for 90 days, during this period 0.1 37 ml of the treated cells were cultured every week over agar surface on a double strength medium of Sabauroud's agar ْ.and incubated for 24h. at 37 C

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RESULTS & & DISCUSSION

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RESULTS AND DISCUSSION

1–Isolation and identification

The development of food preservation process has been driven by the need to extend the shelf- life of foods. Several food preservation systems such as heating, refrigeration and addition of antimicrobial compounds can be used to reduce the risk of outbreaks of food poisoning microorganisms. However, these techniques frequently have associated adverse changes in organoleptic characteristics and loss of nutrients.

The objectives of this study were divided into three parts; the first considered the main microorganisms contaminated certain juices usually consumed in Egypt before and after the preservation processes. The selected juices were apple, guava, mango, and orange. The second was to use some physical and natural agents for preservation the above juices instead of using chemical agents or high temperature degree with pressure. The physical agents were gamma radiation and pasteurized temperature while the natural agents were nisin, citric acid, lactic acid, and cinnamon. The third objective was to investigate the application of combined treatments from 87

the above agents aiming to prolonging the shelf life of the juice products and applying the minimum doses of these agents.

As shown in table (1), ten samples of each type of chosen juices were collected represented the raw materials and the end product: the samples were taken during years 2006 and 2007 from United Food Industries Company and Cairo Agro-Processing Company in Egypt. The samples represented apples, guava, mango, orange, and cocktail (mixture of apple pulp, mango and juice). The end products of apple juices and guava juices were found to be free from microorganisms. It worth to mention that these end products juices were treated with sodium benzoate combined with citric acid with the dose permissible by the Egyptian authorities (i.e. 0.1% (W/V) for sodium benzoate and 0.3% (W/V) for citric acid ).

On the other hand, positive results of contamination in the end product of mango, orange and guava were observed in spite of pasteurization and the addition of sodium benzoate and citric acid indicating the insufficient of this treatment.

From the same table, all guava juice and apple juice or cider samples were free from yeast, or bacteria, 88

the lowest contamination was recorded in cocktail samples which contained total microbial loads ranging from 12 x 10 cfu/ml to 18 x 102 cfu/ml, followed by apple pulp samples which contain microbial load between 15 x 10 to 45 x 102 cfu/ml. While the total bacterial load for mago pulp and orange pulp samples ranged from 104 to 105 cfu/ml. while for guava (pulp and juice), mango juice and orange juice samples the bacterial load was ranged from 102 to 103, while yeast load was in range104 cfu/ml for the same samples. This is agreement with Hatcher et al., (1992) which reported that the microbial populations of the raw juices are within the range of 102 –105cfu/ml. However, the high bacterial and mould counts may be indication of improper and may perhaps be a result of poor quality fruit being used (Lateef et al., 2004).

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Table (1): Screening and isolation of microorganisms contaminated certain Egyptian juices

Average total count Microorganisms Sample Type State pH ± 0.1 -1 cfu m Bacteria Yeast

Cider*** 2.7 Nil - - Apple Raw material** Pulp 3.0 15 x 10 - 45 x 102 + -

Juice End product* 3.8 Nil - -

2 Cocktail Juice End product* 3.5 12 x 10- 18 x 10 + -

2 3 Guava Pulp Raw materials** 4.2 15 x 10 - 52 x 10 + +

2 3 Juice End product* 4.2 15 x 10 - 52 x 10 + +

4 5 Pulp Raw materials** 3.5 23 x 10 - 35 x 10 + -

Mango 2 3 Juice End product* 4.0 12 x 10 - 45 x 10 + + 4 5 Pulp Raw materials** 3.5 17 x 10 - 28 x 10 + - Orange 2 3 Juice End product* 3.8 22 x 10 - 38x 10 + +

*End product with sodium benzoate and citric acid ***Cider = Oily product from apple **Raw materials without any preservatives -Cocktail = apple + mango + peach

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Fresh apple juice is expected to be safe because its acidic pH (usually ranging from 3 to 4) affects growth and multiplication of pathogens. However, several outbreaks have occurred due to consumption of un-pasteurized apple juice and cider (Goverd et al., 1979; Parish, 1997 and Sado et al., 1998). Some outbreaks of Salmonella food poisoning have been traced to un-pasteurized apple cider and orange (Goverd et al., 1979).

Regarding the pulp of the previous fruits (raw material), all of them as expected were contaminated (table 1) either with bacteria or yeast. End product of cocktail juice samples were contaminated with yeast only. On the other hands, the pH of previous samples was found to be ranged from 3.0 to 4.2 as clearly shown in table (1).

Martinez – Gonzales et al. (2003) reported that, the low pH fruit juice samples, especially apple cider and orange juice, have been associated with food borne diseases. Lateef et al. (2004) reported that the acidic juices are (pH 3.0 and 3.65) a good condition for the growth of yeasts.

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Forty two colonies were taken from contaminated samples according to their morphological shape, four from apple pulp, twelve from guava pulp, five from mango pulp, one from mango end product, ten from orange pulp, and seven from orange end product and three from cocktail juice.

As clearly shown from table (2), the four colonies obtained from apple pulp were identified as two strains of Micrococcus sp. and two strains of S. aureus. The twelve colonies of guava pulp were; seven S. aureus, one Staphylococcus warneri, three Debaryomyces sp., and one Pichia sp. The five colonies of mango pulp were; two Staphylococcus auricularis and three Staphylococcus epidermidis while the only colony isolated from mango end product was identified as Kluveromyces sp. The five colonies of orange pulp were; two Bacillus sp. (Endo spore former), six P. aeruginosa and two Citrobacter frundii. While the seven colonies isolated from orange end product were identified as Streptococcus pedococcus. The three colonies of cocktail were identified as; two Debaryomyces sp. and one Pichia sp..

92

Lateef et al. (2004) isolated strains of Saccharomyces cerevisia, Saccharomyces sp., Rhodotorula sp; Bacillus cereus, subtilis, E. coli, S. aureus, Streptococcus pyogenes and Micrococcus sp. from orange juice. Debaryomyces hansenii isolated from orange concentrate (Deak and Beuchat, 1996). Debaryomyces hansenii and Zygosaccharomyces rouxii on syrups, fruit concentrates and jams (Andrews et al., 1997).

93 Table (2): Identification of contaminated microorganisms isolated from certain Egyptian juices Juice micro flora Samples Type Total Isolated microorganisms No. of isolates

Micrococcus agilis 2 Apple Pulp* 4 Staphylococcus aureus 2

Staphylococcus aureus 7

Staphylococcus warneri 1 Guava Pulp* 12 Debaryomyces sp. 3

Pichia sp. 1

Staphylococcus epidermidis 3 Pulp* 5 Mango Staphylococcus auricularis 2 End prods. ** 1 Kluveromyces sp. 1 Bacillus sp. ( endo-spore former ) 2

Pulp* 10 Pseudomonas aeruginosa 6 Orange Citrobacter frundii 2 End prods. ** 7 Streptococcus pedococcus 7

Debaryomyces sp. 2 Cocktail End prods. ** 3 Kluveromyces sp. 1 Total 42

* Pulp = raw juice with out any preservatives. ** End product = juice with sodium benzoate preservative.

94

Youssef et al. (2002) isolated strains of Candida tropicalis, Hanseniaspora uvarum, Rhodotorula glutins, and Z. rouxii from un-steamed and un-irradiated mango pulp.

Mean while Abd El-karem and Farag (1996) found that Candida tropicalis, Zygosaccharomyces rouxii and Rhodotorula graminis were occurred in mango juice and the first was the predominant (54% (W/V)).

Lee et al. (2002) reported that the thermo acidophilic spore-forming Alicyclobacillus acidoterrestris were isolated from commercial pasteurized apple juice in the United Kingdom, Germany, and the United States.

Dellaglio et al. (2005) isolated two strains of gram negative bacteria, rod-shaped, non-spore-forming bacteria; Gluconacetobacter swingsii and Gluconacetobacter rhaeticus from apple fruit juice in the region of the Italian Alps.

95

According to table (2) the 42 identified colonies were divided to; 15 of genus Staphylococcus, 6 of genus Pseudomonas, 7 of genus Streptococcus, 2 of genus Micrococcus, 2 of genus Citrobacter, 2 of genus Bacillus, 5 of genus Debaryomyces, 2 of genus Pichia and one Kluveromyces. In addition fig (1) shows the percentages of these identified organisms.

25.0 21.4

20.0 16.7

14.3

15.0 11.9

10.0 7.1

Percentages % Percentages

4.8 4.8 4.8 4.8 4.8

5. 0 2.4 2.4

0.0

s . s . s s i a i . . . eu sp di sp li ri er s di sp sp sp r p. i s gi la rn no n s a s .au re rm lu a cu a ge fru ce hi ce h St de il us ri .w ro r y ic y ap pi ac cc au ae te om P om St .e B o . ta . ac ry er ph oc ph S ud ob a uv ta icr ta se tr eb l S M S P Ci D K Types of microorganisms

Fig. (1): Percentages of isolated microorganisms

96

For further studies, three different strains of above isolates were chosen. The selection was according to their common contamination in all samples and their characteristics; S. aureus represented gram positive bacteria, P. aeruginosa represented gram negative bacteria, and Debaryomyces sp. chosen to represented yeast strains.

97

3. Single treatments 2.1. Physical treatments 2.1.1. Gamma irradiation Irradiation is a non-thermal food preservation process that has been reexamined in recent years. Irradiation increases the shelf-life of foods and ensures their innocuousness because most microorganisms in vegetative form are extremely sensitive to irradiation at low doses (Radomyski et al, 1994). However, in some circumstances combined treatments are more satisfactory since the dose required for complete sterilization can induce undesirable changes in food flavor or is higher than permitted levels (Thakur and Singh, 1995).

As shown in table (3) illustrated by fig. (2), it is clear that the viable counts of all the selected isolates decreased by increasing the dose of radiation. The dose response curve of S. aureus, P. aeruginosa and Debaryomyces sp, showed straight-line curves (type

A). The D10 values, of the tested strains were 0.75 kGy, 0.69 kGy, and 1.48 kGy, for S. aureus, P. aeruginosa, and Debaryomyces, respectively. It is clear from the results that S. aureus and P. aeruginosa were more sensitive to the irradiation

98

treatment than Debaryomyces strain. Also dose levels 4 and 5 kGy was completely enough to destroy the viable cells of P. aruginosa and S. aureus, respectively. While 7 kGy was needed to obtain the same effecting case of Debaryomyces strain. It is worth to mention here that the initial viable counts of the tested strains, used in the present experiment, was ranged between 107and 108 cfu/ml. In practice these counts never exceed than 105 cfu/ml, hence, the dose level needed to eliminate the resistant strain (i.e Debaryomyces strain) will not exceed 5.5 kGy.

Several investigators have reported that fungi are typically more resistant to radiation than are bacteria

(ƠConnor and Mitchell, 1991; Monk et al., 1994). D10 values, i.e., the amount of radiation necessary to Kill 90 % of the initial microbial count, have been reported for yeasts and molds in the range of 1 to 3 kGy (Lescano et al.,

1991 and Narvaiz et al., 1992), as opposed to D10 of 0.3 to 0.7 kGy for pathogenic bacteria on produce and juices (Buchanan et al., 1998; Rajkowski and Thayer, 2000 and Niemira et al., 2001).

99

Frazier and Westhoff, 1988 stated that the lethal dose for yeasts, S. aureus and P. aeruginosa were at 4 to 9, 1.4 to 7.0, and 1.6 to 2.3 KGy, respectively. Lawerence (1971) mentioned that the loss of proliferate capacity is usually covered by damage with in DNA molecules which controlling all aspects of , structure and development. In the mean time, high doses of gamma radiation were proved to be inhibitory for both growth and enzymatic activities of microorganisms, (Sadi, 1987).

Doses KGy 0 01234567 -2

-4 o -6 Log N/N

-8

-10

Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces spp -12 Fig. (2): Effect of increasing doses of gamma irradiation on the selected organisms.

100 Table (3): Effect of different doses of gamma irradiation on the viable counts of the selected organisms* Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Doses kGy Average Log Average Log Average Log Log N** Log N** Log N** count cfu m-1 N/No count cfu m-1 N/No count cfu m-1 N/No Initial count 98 x 106 7.99 0 18 x 106 7.25 0 41 x 106 7.61 0 ± 0.045 A ± 0.056 A ± 0.089 A 0.5 10.2 x 106 7.0 - 0.98 98 x 104 5.99 - 1.26 89 x 105 6.94 - 0.66 ± 0.021 B ± 0.022 B ± 0.072 B 1 11.5 x 105 6.06 - 1.93 48 x 103 4.68 - 2.57 20 x 104 6.30 - 1.31 ± 0.028 C ± 0.033 C ± 0.074 C 2 85 x 103 4.92 - 3.06 37 x 102 3.56 - 3.68 25 x 104 5.39 - 2.21 ± 0.029 D ± 0.042 D ± 0.03 D 3 98 x102 3.99 - 4.0 15 x 10 2.17 - 5.07 10.4 x 104 5.01 - 2.59 ± 0.024 E ± 0.033 E ± 0.015 D 4 12 x 102 2.07 - 5.91 -*** -*** -*** 12 x 103 4.07 - 3.53 ± 0.013 F F ± 0.025 E

5 -*** -*** -*** 84 x 102 3.92 - 3.68 G ± 0.024 F 6 94 x 10 2.97 - 4.63 ± 0.022 G 7 -*** -*** -*** H

D10 0.75 kGy 0.69 kGy 1.48 kGy * This experiment was counted after 24h incubation from irradiation. *** The dash means no distinct growth. ** Means with the same letter are not significantly different.

101

In general, the gram–negative bacteria, including most of the common organisms (e.g., Pseudomonas), are more sensitive to irradiation than are the gram-positive organisms (e.g., lactic acid bacteria and micrococci) and yeasts are more radiation resistant than typical spoilage bacteria (Ingram, 1975). Generally, Pseudomonas sp. lacks the inducible error-prone DNA repair system (Miller and Kokjohn, 1990; Karentz, 1994 and Joux et al., 1999).

Daly et al. (2004) propose that Mn (II) accumulation facilitates recovery from radiation injury. It was further noted that the intracellular concentration of Mn in S.aureus was ~50-100um (Horsburgh et al., 2002).

In yeast enhanced radiation resistance is generally believed to be the result of an increase in the capacity or efficiency of a cell to repair its DNA (Bryant, 1976 and Mitchel and Morrison, 1984), DNA being the critical target for ionizing radiation- induced lethality. The recombination DNA repair system is considered to be the major DNA repair pathway that confers resistance to killing by ionizing radiation in yeast cells (Mitchel and Morrison, 1984). Irradiation of concentrated juice in the range of 0.5-2.0 kGy slightly altered some sensory

102

characters; these changes increased at higher doses (Zegota et al., 1988).

2.1.2. Pasteurized temperature Pasteurization is widely used in the food and dairy industry both to minimize the risk from pathogens and to extend product shelf life. To ensure a secure and steady food supply, food needs to be preserved and stabilized so it can be stored for extended periods. Extending food products shelf life requires inactivation of the spoilage or toxic microorganisms they contain and avoiding their subsequent proliferation. Food preservation thus implies exposure of microorganisms to hostile conditions. Heating is one of the most practical efficient and inexpensive methods of disinfection and sterilization of food.

Results in table (4) and figure (3) showed that, exposured the three organisms, Staphylococcus, pseudomonas, and Debaryomyces to 90 Cْ sharply decreased their counts. The initial count was 98 x 105, 98 x 105, and 55 x 105 cfu m -1 for Staphylococcus, pseudomonas, and Debaryomyces respectively. The Dt values (the decimal reduction time i.e. that time of heating

103

Also, TDT (thermal death time) was 157.4 sec. for Staphylococcus, 208.0 sec. for pseudomonas and 224 sec. for Debaryomyces. The lethal time was at 120 sec for three organisms. The initial count was decreased to 1.84 log for Staphylococcus at sub lethal time 60 sec, to 2.87 log for pseudomonas at sub lethal time 60 sec, and to 3.54 log for Debaryomyces at sub lethal time 60 sec.

By comparing between three treated strains, it was observed that Staphylococcus was highly sensitive to pasteurized temperature followed by Pseudomonas and then Debaryomyces.

According to Moats (1971), Cell death due to the exposure to high degree of temperature results from the denaturation or inactivation of numerous critical sites (or a large number of the same site) in cells.

104

Rosenberg et al. (1971) demonstrated the existence of a good numerical correlation between some thermo-dynamic parameters in protein denaturation and in death rates of bacteria yeast, viruses and drosophila.

Hurst, (1977) and Gould, (1989) summarized some of the mechanisms by which heat may cause cellular inactivation: (i) damage of DNA, (ii) inhibition of protein synthesis, (iii) damage of cell membrane, and (iv) inactivation of critical metabolic enzymes.

Heat can damage protein, lipids, and nucleic acids and destabilize membranes (Gould, 1989). Heating- induced destabilization, such as oxidation of sulfydryl groups of the membrane – binding bound proteins, resulting in cell death in addition into direct DNA damage (Yatvin and Grummer, 1987).

105

Tim / sec. 0 0 10203040506070

-1

-2

-3 Log N/No Log N/No -4

-5

Staphalylococcus aureus Pseudomonas aeruginosa Debaryomyces spp -6 Fig. (3): Effect of pasteurized temperature 90 C ْ at different time on the selected organisms.

106 Table (4): Effect of pasteurized temperature 90 Cْ at different time on selected organisms

Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Time/Sec. Average Log Average Log Average Log -1 Log N* -1 Log N* -1 Log N* count cfu m N/No count cfu m N/No count cfu m N/No

Initial count 98 x 105 6.99 0 98 x 105 6.99 0 55 x 105 6.74 0 ± 0.045 A ± 0.014 A ± 0.046 A

24 x 104 5.38 - 1.61 32 x 104 5.50 - 1.48 95 x 104 5.97 - 0.76 15 ± 0.032 B ± 0.055 B ± 0.038 B

55 x 102 3.74 - 3.25 29 x 103 4.46 - 2.52 118 x 103 5.07 - 1.66 30 ± 0.014 C ± 0.058 C ± 0.022 C

1.84 2.87 2 3.54 60 7 x 10 - 5.14 75 x 10 - 4.11 35 x 10 - 3.19 ± 0.088 D ± 0.055 D ± 0.031 D

-** -** -** 120 -** -** -** -** -** -** E E E

t 13.1 Sec. 17.3 Sec. 18.6 Sec. D

TDT 157.4 Sec. 208.0 Sec. 224.0 Sec.

* The same letter is not significantly different. ** The dash means no distinct growth. TDT = thermal death time (Dt * 12).

107

Many factors influence the heat resistance of non-spore forming microorganisms. Time and temperature of incubation dramatically affect the heat resistance of both gram-negative and gram-positive bacteria.

Beuchat (1978) speculated that growth at high temperature results in the production of thermo stable membranes, which results in increased thermo tolerance. A factor that has received considerable attention recently is sub lethal heat shock (Mackey and Derrick, 1986), which induces the rapid synthesis of heat shock proteins (Lindquist and Craig, 1988). Factors other than heat, such as hydrogen peroxide (Morgan et al., 1986) and glucose starvation (Jenkins et al., 1988), also induce the rapid synthesis of heat shock protein. Evidence is accumulating that heat shock proteins are a major determinant of bacterial thermo tolerance (Jenkins et al., 1988). An important factor in thermo tolerance may be the effect of superoxide (O2-) and hydrogen peroxide (H2O2) on the recovery of bacteria injured by heat (Knabel et al., 1990).

108

Iandolo and Ordal, (1966) reported that heating at 55Cْ caused the release of intracellular constituents from cells of S. aureus. Release of intracellular constituents could also occur as a result of cell lysis (Pethica, 1958).

It is believed that these temperatures trigger physiological responses that lead to the synthesis of special proteins Known as heat shock proteins (HSPs) (Knabel et al., 1990 and Lindquist, 1986).

109

2.2-Natural treatments 2.2.1-Nisin Nisin is an antimicrobial peptide produced by lactococci and has been used in consumer products for many years (Taniguchi et al., 1994). Although this lantibiotic is inhibitory to microorganisms, it is harmless to humans (Hurst and Hoover, 1993). Nisin is the first antimicrobial peptide with generally "recognized as safe" status in the United States for use in ; in addition, its use in various food products is allowed in several countries (Delves-Broughton, 1990).

As shown in table (5) illustrated by figure (4), the effect of Nisin on S. aureus was examined, when Nisin concentration increases the count decreased. At the sub lethal conc. 200 ug/ml the initially bacteria count 6.17 was decreased to 1.39, the lethal conc. was at 250 ug/ml.In the same time as shown in table (5) and fig. (4), nisin has no effect on both Debaryomyces sp and P. aeruginosa.

110

Crandall and Montville, (1998) reported that the lethality of nisin is due to its effect on the cytoplasmic membrane of vegetative cells and that is the primary site of action of Nisin. The primary mechanism of nisin believed to be the formation of pores on the cytoplasmic membrane which result in depletion of proton motive force and loss of cellular ions; amino acids, and ATP. Nisin binds electro statically to the negatively charged phospholipids and increases the permeability of the membrane by pore formation, resulting in rapid efflux of essential intracellular small molecules (Abee et al., 1994; Breukink et al., 1997; Montville et al., 1999). The efflux of cellular constituents resulted in a complete collapse of the proton motive force and subsequently in cell death (Wincowski et al., 1994). Nisin uses lipid II (undecaprenyl- pyrophosphoryl-MurNAc-(pentapeptide)- GlcNAc) as a receptor molecule to increase its antimicrobial efficacy dramatically (Breukink et al., 2003). However, recent work has shown that the activity of Nisin is dependent on the conc. of lipid II in the membrane of sensitive cells (Breukink et al., 1997;

Breukink and de Kruijff, 1999).

111

Pore formation by the `barrel-stave' mechanism, used by a number of cytolytic pore-forming (Ojcius and Young, 1991), has been predicted (Fig. 5). This model involves the initial accumulation of the peptide at the membrane surface through ionic interactions with the phospholipids head groups. The presence of these peptides induces significant thinning of the membrane in these areas, due to localized displacement of the phospholipids. On application of a ΔΨ, the molecules adopt a transmembrane orientation. As lantibiotics are small peptides that can span the membrane only once, so it is assumed that several molecules associate with the membrane to form a pore. Whether this aggregation of molecules occurs prior to insertion, or in the membrane after insertion is unknown. It has been demonstrated that at high pH, nisin monomers aggregate outside the membrane, significantly reducing biological activity (Garcia-Garcera et al., 1993).

Hasper et at. (2006) has been reported that, nisin binds to the pyrophosphate moiety of lipid II and removes lipid II from its functional location, thereby inhibiting cell wall synthesis, and it induces the formation of lipid II-nisin hybrid pores in the cytoplasmic membrane ().

112

Fig. (5): General model of the `barrel-stave' mechanism of pore formation by peptides (Jack et al., 1997).

Ruhr and Sahl, (1985) reported that the addition of Nisin to susceptible cells (Staph. cohnii, Bacillus sub, Micrococcus luteus, Strepto zymo) resulted in a rapid efflux of small cytoplasmic compounds such as K+ and amino acids. In addition Nisin inhibited proline and glutamine uptake by membrane vesicles of susceptible cells. The loss of ions resulted in a rapid decrease in the membrane potential, ΔΨ the effects of Nisin in depleting proton motive force components, ΔΨ and Δ pH ,were confirmed in L. monocytogene (Bruno et al., 1992) and C. sporogenes (Okereke and Montville, 1992).

113

Conc. u g/ml 0 0 50 100 150 200 250 300

-1

-2

-3 Log N/No

-4

-5

Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces spp -6 Fig. (4): Effect of different conc. of nisin on the selected organisms.

114 Table (5): Effect of different conc. of nisin on the selected organisms

Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Conc. μg/ml Average Log Average Log Average count Log -1 Log N* -1 Log N* -1 Log N* count cfu m N/No count cfu m N/No cfu m N/No Initial 6.17 6.99 6.61 15 x 105 0 98 x 105 0 41 x 105 0 count ± 0.045 A ± 0.0 A ± 0.0 A 5.63 6.99 6.61 25 43 x 104 - 0.54 98 x 105 0 41 x 105 0 ± 0.060 B ± 0.0 A ± 0.0 A 4.74 6.99 6.61 50 55 x 103 - 1.43 98 x 105 0 41 x 105 0 ± 0.064 C ± 0.0 A ± 0.0 A 3.41 6.99 6.61 100 26 x 102 - 2.76 98 x 105 0 41 x 105 0 ± 0.046 D ± 0.0 A ± 0.0 A 1.39 6.99 6.61 200 25 - 4.77 98 x 105 0 41 x 105 0 ± 0.079 E ± 0.0 A ± 0.0 A -** 6.99 6.61 250 -** -** 98 x 105 0 41 x 105 0 F ± 0.0 A ± 0.0 A

* The same letters are not significantly different. ** The dash means no distinct growth.

115

On the other hand, Brul et al., (1997) and Klis et al., (1997) reported that nisin has no antimicrobial effect on yeasts. These organisms have a rigid cell wall, a complex structure consisting of glucan cross- linked with chitin and cell wall proteins. The processing of manno-proteins is complex and has been partially characterized in yeasts (Lu et al., 1994 and Kollar et al., 1997). Because manno- proteins are generally considered one of the key components, which determine cell wall porosity (Cid et al., 1995 and Klis et al., 1997) they may represent a major barrier preventing free permeation of Nisin through the cell wall and thus access to the cytosolic membrane.

The inability of nisin to attack Gram-negative bacteria is due to the protective outer membrane (OM), which covers the cytoplasmic membrane and peptidoglycan layer of Gram-negative cells. This asymmetrical membrane contains Glycerophospholipids in its inner leaflet, but the outer leaflet is built of lipopolysaccharide (LPS) molecules. LPS, which are composed of a lipid part and a complex heteropolysaccharide with partly anionic character, form a tight layer endowed with a hydrophilic surface (Nikaido, 1996). As a result, OM is a penetration barrier that excludes hydrophobic substances and macromolecule.

116

Nisin, as a hydrophobic macromolecule, is unable to traverse a normal OM and thus cannot reach its target of action.

2.2.2- Organic acids Weak acid preservative are generally considered safe antimicrobials, consistent with the long history and widespread use of these compounds for the preservation of foods and beverages. The preservative molecule diffuses into the cell until equilibrium is reached in accordance with the pH gradient across the membrane resulting in the accumulation of anions and protons inside the cell (Booth and Kroll, 1989).

There for, inhibition of growth by weak acid preservative has been proposed to be due to a number of actions including , membrane disruption (Stratford and Anslow, 1998., Bracey et al., 1998) , inhibition of essential metabolic reaction ( Krebs et al., 1983), stress on intracellular pH homeostasis (Bracey et al., 1998), and the accumulation of toxic anions ( Eklund , 1985).

117

2.2.2.1- Citric acid From the tables (6 & 7) illustrated by figures (6 & 7), the growth curve of both P. aeruginosa and Debaryomyces sp. were belonged to type B (i.e it has a shoulder at the begging of the curve that means, it has resistance to citric acid at low concentration), followed by exponential rate death. The increase in citric acid concentration after shoulder part was paralleled to the decrease in the number of survival bacterial cells. While the growth curve of S. aureus was belonged to type A (i. e. a straight-line curve with a logarithmic decrease was obtained). The S. aureus organism count was decreased sharply by increasing the conc. of citric acid.

The sub lethal conc. 0.15% (W/V) decreased the count from 6.92 log to 2.81 log; the lethal conc. was at 0.2% (W/V). On the other hand, the sub lethal conc. of p. aeruginosa was 0.2% (W/V), while lethal conc. at 0.3% (W/V). The initial count 7.06 log decreased to 2.44 log. With Debaryomyces sp. the sub lethal conc. was at conc.3.5% (W/V) where the initial count 6.57 log decreased to 1.39 log, and lethal dose was at 4.0% (W/V) of citric acid. By comparison between the three organisms, it was observed that, the highest sub lethal

118

conc. was 3.5% (W/V) then 0.2% (W/V) and 0.15% (W/V) for Debaryomyces sp., p., and S. aureus respectively. The growth count was decreased 4.11 log cycles, 4.62 log cycles and 4.62 log cycles for each S. aureus, p. aeruginosa and Debaryomyces sp., respectively at the sub lethal conc. of citric acid.

119

Table (6): Effect of different conc. of citric acid on P. aeruginosa and S. aureus

Staphylococcus aureus Pseudomonas aeruginosa Conc.% Average count cfu Average count Log N* Log N/No Log N* Log N/No m-1 cfu m-1

5 6.92 5 7.06 Initial count 85 x 10 0 115 x 10 0 ± 0.045 A ± 0.014 A 6.02 6.64 0.05 105 x 104 - 0.90 44 x 105 - 0.41 ± 0.030 B ± 0.028 B 4.11 5.25 0.1 13 x 103 - 2.81 18 x 104 - 1.80 ± 0.030 C ± 0.029 C 2.81 3.72 0.15 66 x 10 - 4.10 53 x 102 - 3.33 ± 0.151 D ± 0.140 D -** 2.44 0.2 -** -** 28 x 10 - 4.61 E ± 0.058 E -** 0.3 -** -** F

* The same letters are not significantly different. ** The dash means no distinct growth.

120

Conc. % 0 0 0.05 0.1 0.15 0.2 0.25 -0.5

-1

-1.5 -2

-2.5

Log count -3 -3.5

-4

-4.5 S. aureus P. aeruginosa -5 Fig (6): Effect of different conc. of citric acid on P. aeruginosa and S. aureus

Table (7): Effect of different conc. of citric acid on . Debaryomyces sp Average count cfu Conc.% Log N* Log N/N m-1 o 6.57 Initial count 38 x 105 0 ± 0.089 A 5.95 1 90 x 104 - 0.62 ± 0.020 B 4.83 1.5 68 x 103 - 1.74 ± 0.023 C 3.96 2 93 x 102 - 2.61 ± 0.022 D 3.53 2.5 18 x 102 - 3.32 ± 0.050 E 3 26 x 10 2.41 - 4.16 ± 0.038 F 3.5 25 1.39 - 5.18 ± 0.061 G 4 -** -** -** F * The same letters are not significantly different. ** The dash means no distinct growth.

121

Conc.%

0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

-1

-2 o -3

Log N/N -4

-5

-6 Fig. (7): Effect of different conc. of citric acid on .Debaryomyces sp

122

2.2.2.2 - Lactic acid It shown from tables (8 & 9) and figures (8 & 9), the growth curves of S. aureus, p. aeruginosa and Debaryomyces sp., shown a straight-line curve with a logarithmic decrease was obtained. The growth curve of S. aureus was decreased sharply by increasing the conc. of lactic acid. Where the count was decreased to log 1.59 at conc. 0.1% (W/V) of lactic acid, the initial count was 6.98 log, where 5.99 log cycles were decreased.

The sub lethal conc. was 0.2% (W/V) for P. aeruginosa; the initial count was decreased 4.76 1og cycles. While the Debaryomyces sp. were decreased 4.48 log cycles and the sub lethal conc. was at 4% (W/V) and count 2.44 log, where the initial count was 6.86 log (cfu/m-1).

By comparing between three organisms, the lactic acid was decrease S. aureus 5.39 log cycles and P. aeruginosa 4.78 log cycles and Debaryomyces sp. 4.48 log cycles at sub-lethal conc. 0.1% (W/V), 0.2% (W/V) and 4% (W/V), respectively. This shows that, the Debaryomyces sp. was relatively resistant to lactic acid followed by P. aeruginosa in comparing by S. aureus.

123

Table (8): Effect of different conc. of lactic acid on Debaryomyces sp.

-1 Conc.% Average count cfu m Log N* Log N/No

Initial 6.92 85 x 105 0 count ± 0.069 A 5.56 1.5 37 x 104 - 1.36 ± 0.041 B 4.96 2 93 x 103 - 1.96 ± 0.039 D 4.20 2.5 16 x 103 - 2.72 ± 0.048 E 3.63 3 43 x 102 -3.29 ± 0.021 F

4 28 x 10 2.44 - 4.48 ± 0.045 G

4.5 -** -** -** H * The same letters are not significantly differ ent. ** The dash means no distinct growth

Conc.% 0 00.511.522.533.544.5 -0.5 -1 -1.5

-2

-2.5

Log N/No Log N/No -3

-3.5

-4 -4.5 -5 Fig. (8): Effect of different conc. of lactic acid on Debaryomyces sp.

124

Conc. % 0 0 0.05 0.1 0.15 0.2 0.25

-1

-2

-3 Log count -4

-5

S. aureus P. aeruginosa -6 Fig (9): Effect of different conc. of lactic acid on P. aeruginosa and S. aureus.

125 Table (9): Effect of different conc. of lactic acid on P. aeruginosa and S. aureus

Staphylococcus aureus Pseudomonas aeruginosa Conc.% Average count cfu Average count cfu Log N* Log N/No Log N* Log N/No m-1 m-1

Initial count 96 x 105 6.98 0 98 x 105 6.99 0 ± 0.045 A ± 0.014 A

5 6.68 0.001 48 x 10 - 0.30 Not tested ± 0.045 A

0.01 47 x 104 5.64 - 1.31 29 x 105 6.46 - 0.52 ± 0.042 B ± 0.048 B

0.05 62 x 102 3.06 - 3.18 Not tested ± 0.082 C

0.10 40 1.59 - 5.38 26 x 103 4.41 - 2.57 ± 0.064 D ± 0.047 D

0.15 -** -** -** Not tested E

2.23 0.20 17 x 10 - 4.76 ± 0.098 E -** 0.30 -** -** F

* The same letters are not significantly different. ** The dash means no distinct growth.

126

By comparing between the lactic acid and citric acid and its antimicrobial activity against the treated microorganisms it was showed that: (i) with S. aureus the sub lethal conc. of lactic acid and citric acid was at 0.10% (W/V) and 0.15% (W/V) respectively, lethal conc. of lactic acid was 0.15% (W/V) and citric acid was 0.2% (W/V). (ii) With P. aeruginosa the sub lethal conc. of lactic acid and citric acid was the same at 0.20% (W/V), lethal conc. 0.3% (W/V). (iii) With Debaryomyces sp. the sub lethal conc. of lactic acid and citric acid was at 4.0% (W/V) and 3.5% (W/V) respectively, lethal conc. of lactic acid was 4.5% (W/V) and citric acid was 4% (W/V).

From the previous comparison, the sub lethal concentration of citric acid was less than lactic acid with S. aureus and Debaryomyces sp., and the same conc. with P. aeruginosa.

127

Acid tolerance can be viewed in terms of efflux ability. The mechanism by which organic acids inhibit microorganisms involves passage of the un-dissociated form of the acid across the cell membrane lipid bilayer. Once inside the cell, the acid dissociates because the cell interior has a higher pH than the exterior. Protons generated from intracellular dissociation of the organic acid and other organic acids via several mechanisms. They use the enzyme H+-ATPase along with ATP (adenosine triphosphate) energy to remove excess protons from the cell. Inhibition and/or inactivation of yeasts may be due to eventual loss of cellular energy or inactivation of critical cellular functions due to low intracellular pH (Fig. 10). To prevent energy depletion, a membrane protein may be induced for decreasing ATPase activity and thus conserve energy (Brul and Coote, 1999). To circumvent the problem of extruded anions and protons reentering the cell upon recombining in the extra cellular medium, adapted yeasts apparently reduce diffusion of the weak acids, most likely by altering cell membrane structure to reduce passage of the acids into the cell (Brul and Coote, 1999).

128

Fig. (10) A schematic impression of the stress response of a yeast cell challenged with weak organic acids (Piper et al., 1998).

It is clear from the above results that S. aureus was the most sensitive of treated organisms to both citric acid and lactic acid followed by P. aeruginosa while Debaryomyces sp. was the highest resistant strain.

Gram- positive bacteria (such as S. aureus) do not possess an outer membrane and the exclusion limit of the cell wall of vegetative cells (Lambert, 1983), hence preservatives can easily enter these cells and their intrinsic resistance is relatively low. In gram- negative bacteria (such as P. aeruginosa), resistance mechanisms

129

are more complicated since these organisms possess an inner and outer membrane; the latter membrane has a clear role in modulating the accessibility of a cell to preservatives and other small molecules (Vaara, 1992 &

Helander et al., 1997).

The actual inhibitory action of weak acid preservatives on yeast could be due to the induction of energetically expensive stress respond that attempts to restore homeostasis and results in the reduction of available energy pools for growth and other essential metabolic functions (Holyoak et al., 1996, Bracey et al., 1998). Indeed, lactic acid was recently shown to permeabilize Gram-negative bacteria efficiently and causes LPS release (Alakomi et al., 2000) and citric acid is metal chelating (Verhoff, 1986).

130

2.2.3- Cinnamon In recent years, because of negative perception of synthetic additives, consumers are demanding natural and fresh-like food products with guaranteed safety and long shelf- life. For this reason, there is a renewed interest in the use of spices, and plant extracts as alternative food processing method. Several authors (Yousef and Tawil, 1980; Deans and Ritchie, 1987; Baratta et al., 1998; Smith- palmer et al., 1998) reported that cinnamon was one of the inhibitoriest against pathogens such as Salmonella sp.., Y. enterocolitice and S. aureus. Ground cinnamon (0% (W/V) and 0.3% (W/V) was added to apple juice to study the sensitivity of Salmonella typhimurium, Yersinia Enterocolitica and S. aureus (Yuste and Fung, 2003). Yousef and Tawil (1980) and Smith-palmer et al., (1998) found that the minimum bacteriostatic conc. was equivalent to the minimum bactericidal conc. for S. aureus.

Tables (10 & 11), fig (11 & 12) showed that, the growth of Debaryomyces sp. had shoulder at the beginning of the curve till conc. 0.6% (W/V), followed by exponential rate death indicating the resistance of

131

Debaryomyces sp.. to cinnamon at low concentrations. The increase in cinnamon concentration after shoulder part was paralleled to the decrease in the number of survival yeast cells. The sub lethal was at conc. 1.7% (W/V).

Mean while, the growth curves of both S. aureus and P. aeruginosa, showed straight-line curve. S. aureus was effect by increasing the conc. of cinnamon and the sub lethal conc. was at 1% (W/V). While, the sub lethal conc. for P. aeruginosa was at conc. 3% (W/V). By comparing between the treated organisms it was found that, S. aureus was highly sensitive to cinnamon than P. aeruginosa and Debaryomyces sp., while P. aeruginosa was more resistant to cinnamon than Debaryomyces sp.

132

Conc. 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8

S. aureus Debaryomyces spp. -1

-2

-3

Log count Log

-4

-5

-6 Fig. (11): Effect of different conc. of cinnamon on S. aureus and Debaryomyces sp.

133 . Table (10): Effect of different conc. of cinnamon on S. aureus and Debaryomyces sp Staphylococcus aureus Debaryomyces sp. Conc.% Average count cfu m-1 Log N* Log N/No Average count cfu m-1 Log N* Log N/No 7.0 5.84 Initial count 102 x 105 0 70 x 104 0 ± 0.016 A ± 0.069 A 6.51 0.2 33 x 105 - 0.49 Not tested ± 0.123 B 5.62 0.3 Not tested 42 x 104 - 0.21 ± 0.084 A 5.79 0.4 63 x 104 - 1.20 Not tested ± 0.038 C

4.67 5.54 0.6 47 x 103 - 2.33 35 x 104 - 0.3 ± 0.017 D ± 0.044 B 2.95 0.8 9 x 102 - 4.05 Not tested ± 0.04 E 1.43 4.32 1.0 27 - 5.57 21 x 103 - 1.52 ± 0.036 F ± 0.034 C

-** 1.2 -** -** Not tested G 3.07 1.5 12 x 102 - 2.76 ± 0.026 D 2.49 1.7 31 x 10 - 3.35 ± 0.036 E -** 2 -** -** F * The same letters are not significantly different. ** The dash means no distinct growth.

134

Table (11): Effect of different conc. of cinnamon on P. aeruginosa

Average count cfu Conc.% Log N* Log N/N m-1 o Initial 6.74 55 x 105 0 count ± 0.014 A 5.55 0.5 36 x 104 - 1.18 ± 0.065 B 4.63 1 43 x 103 - 2.10 ± 0.067 C 3.46 2 29 x 102 - 3.27 ± 0.067 D 3 86 1.93 - 4.80 ± 0.055 E 4 -** -** -** F

* The same letters are not significantly different. ** The dash means no distinct growth.

Conc.% 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

-1

-2

-3

Log N/No N/No Log

-4

-5

-6 Fig. (12): Effect of different conc. of cinnamon on P. aeruginosa 135

Cinnamon effectively inhibits growth of bacteria, yeasts and molds (Smith-palmer et al., 1998). Its essential oil fraction contains cinnamaldehyde and eugenol as major antimicrobial compounds (Shelef, 1983). Confirming our results Shelef (1983) and Zaika (1988) stated that gram-positive bacteria are more sensitive to cinnamon than gram-negative bacteria. This is probably due to the outer membrane of gram-negative bacteria, which is highly hydrophilic and acts as a strong barrier (Smith-Palmer et al., 1998).

The antimicrobial activity of cinnamon due to its essential oils, which are lipophilic and could penetrate through the membrane to the interior of the cell and perform the inhibitory activity at the target site (Ramos- Nino et al., 1996 and Smith-Palmer et al., 1998).

Smith-Palmer et al. (1998) determined that the 24h. minimum inhibitory concentrations of cinnamon and clove essential oils against Campylobacter jejuni, Escherichia coli, Salmonella Enteritidis, Listeria monocytogenes, and S. aureus were 0.05, 0.04-0.05, 0.04-0.05, 0.03, and 0.04% (W/V), respectively, in an agar

136

dilution assay. Cinnamic aldehyde or thymol (600 mg/liter of air) significantly reduced Salmonella populations on alfalfa seeds used for sprouting and did not affect germination (Weissinger et al., 2001). In conclusion the inhibitory effect of cinnamon on Gram positive bacteria, Gram negative bacteria, yeast organisms was observed, which suggests the potential use of this in the food industry for enhancement of safety furthermore, sensory attributes given by cinnamon may appeal to consumer . These, if foods and beverages are manufactured under sanitary conditions so that they have relatively low microbial conditions so that they have relatively low microbial counts, naturally occurring food grade ingredients with antimicrobial properties can give substantial protection and replace some artificial preservations (Yuste and Fung, 2003).

137

3. Combination treatments

The antimicrobial effect of preservation treatment can be optimized by combining them as hurdles in an overall preservation strategy by placing a number of sub-lethal stresses (i.e. hurdles) on a microbial cell, the organisms expends energy to metabolic exhaustion and death (Leistner, 2000). Hurdles targeting the same elements within the cell have an additive inhibitory effect only, where as synergistic effects may result from disturbing several functions of the cell (Leistner, 1992; 1994). Proper application of combined methods gives stable products, prevents the undesired side effect of each individual treatment, saves energy and lowers the required concentration of added preservatives (Pokorn, 1994; Abd El-karem and Farag, 1996).

3.1. Combination with Gamma Irradiation

According to Leistner (2000), attacking various cellular targets will have a synergistic effect by making the organism strain every possible repair mechanism and the activation of stress shock proteins also, becomes more difficult. 138

Gamma irradiation is considered the only known technique capable of ensuring the hygienic quality of raw food in a fresh or frozen state (Loaharanu, 1995). So combining this treatment by other preservatives will give synergistic effects on undesirable microorganisms contaminating food and beverages.

3.1.1. Combination of Gamma irradiation with lactic acid

From the previous experiments, Lactic acid was used in conc. 0.01% (W/V), 0.1% (W/V), 1.5% (W/V) for S. aureus, P. aeruginosa, and Debaryomyces sp., respectively, at this conc. lactic acid start to give distinct reduction for the growth count as shown previously in single treatments tables (8 & 9).

As shown in table (12) illustrated by fig (13), it is clear that, the dose level 2 kGy combined with lactic acid was sufficient to eliminate both S. aureus and P. aeruginosa while Debaryomyces sp. neede 3 kGy . The

D10 values of the previous organisms were 0.27, 0.40 and 0.47 kGy for S. aureus, P. aeruginosa and

139

Debaryomyces sp., in the same sequence comparing by 0.75, 0.69 and 1.48 kGy for the same organisms in the single treatments with gamma rays, respectively. These results clearly indicated the synergistic effect of the combined gamma rays by lactic acid. Also, the dose levels needed for eliminated the tested organisms were reduced from 5 kGy to 2 kGy for S. aureus and from 4 kGy to 2 kGy for P. aeruginosa, whiledestorying the cells of Debaryomyces sp. neede only 3 kGy instead of 7 kGy in the case of single treatment.

Doses kGy 0 00 .511.522.5

-1 Staphylococcus aureus Pseudomonas aeruginosa

-2 Debaryomyces spp.

-3

Log N/No N/No Log -4

-5

-6 Fig. (13): Effect of different doses of gamma irradiation in combination with lactic acid.

140 Table (12): Effect of different doses of gamma irradiation in combination with lactic acid Organism Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Lactic acid 0.01 0.1 1.5 conc. % Average count Log Average count Log Average count Log Doses kGy Log N* Log N* Log N* cfu m-1 N/No cfu m-1 N/No cfu m-1 N/No Initaial count 25 x 106 7.39 - 65 x 106 7.81 - 55 x 105 6.74 - ± 0.030 A ± 0.011 A ± 0.032 A Control 48 x 105 6.68 0 26 x 103 4.41 0 37 x 104 5.56 0 lactic acid ± 0.045 B ± 0.047 B ± 0.048 B

0.25 Not tested 98 x 102 3.99 - 0.42 Not tested ± 0.022 C

0.5 11 x 103 4.04 - 2.63 26 x 102 3.41 - 1.0 13 x 103 4.11 - 1.45 ± 0.050 C ± 0.037 D ± 0.05 C

1 25 x 10 1.39 - 4.28 14 x 10 2.14 - 2.26 82 x 10 2.91 - 2.65 ± 0.042 D ± 0.05 E ± 0.057 D

2 -** -** -** -** -** -** 9 .95 - 4.61 E F ± 0.024 E

3 -** -** -** F

D10 0.27 kGy 0.40 kGy 0.47 kGy * The same letters are not significantly different. ** The dash means no distinct growth.

141

3.1.2. Combination of Gamma irradiation with citric acid Citric acid was used in conc. 0.05% (W/V), 0.1% (W/V), 1.5% (W/V) for S. aureus, P. aeruginosa, and Debaryomyces sp., respectively, at this conc. citric acid start to give distinct reduction for the growth count as shown previously in tables (6 & 7).

It was obvious from table (13) and Fig (14), a straight-line curve with a logarithmic decrease was obtained. The death-value was at 2 kGy for S. aureus, 3 kGy for P. aeruginosa, while Debaryomyces sp.. requires

4 kGy to destroy its cells. D10 was 0.32, 0.58, and 0.75 kGy, respectively for the same organism's combaring by single treatments 0.75, 0.69 and 1.48 kGy, respectively.

On the other hand, by comparing between lactic acid and citric acid in combination with gamma irradiation, lactic acid was more active against the treated organisms than citric acid and the D10 values where distinct decreased from single treatment. This may due to lactic acid has lipophilic character; ability to depress intracellular pH. In addition to membrane diffusion and water activity reduction ' specific anion effect', while citric acid lack this character and acting as chelating agent 142

(Smulders, 1987; Stratford, 1999, Saltmarsh, 2000 and Naidu, 2000)

Ross et al. (2003) reported that the lethal effects of ionized radiation are not greatly enhanced by mild acidification. The addition of naturally occurring antimicrobial has proven to be an effective hurdle when combined with non-thermal processing techniques (Ross et al., 2003).

Doses kGy 0 0 0.5 1 1.5 2 2.5 3 3.5 -0.5

Staphylococcus aureus -1 Pseudomonas aeruginosa -1.5 Debaryomyces spp. -2

-2.5 Log count -3 -3.5 -4 -4.5

-5 Fig. (14): Effect of different doses of gamma irradiation in combination with citric acid.

143

Table (13): Effect of different doses of gamma irradiation in combination with citric acid Organism Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Citric acid conc. 0.05 0.1 1.5 % Average count cfu Log Average count Log Average Log Doses KGy Log N* Log N* Log N* m-1 N/No cfu m-1 N/No count cfu m-1 N/No

Initial count 32 x 106 7.50 - 65 x 106 7.81 - 41 x 105 7.50 - ± 0.031 A ± 0.030 A ± 0.047 A 5.95 Control 10.5 x 105 6.02 0 18 x 104 5.25 0 90 x 104 0 citric acid ± 0.030B ± 0.029 B ± 0.020 B

0.5 18 x 102 3.25 - 2.76 83 x 103 4.91 - 0.33 98 x 103 4.99 - 0.96 ± 0.094 C ± 0.070 C ± 0.038 C

1 50 1.69 - 4.32 14 x 103 4.14 -1.1 18 x 103 4.25 - 1.69 ± 0.064 D ± 0.058 D ± 0.013 D

2 -** -** -** 24 x 10 2.38 -2.87 78 x 10 2.99 - 3.06 E ± 0.042 E ± 0.015 E

3 -** -** -** 45 1.65 - 4.3 F ± 0.008 F -** 4 -** -** G

D10 0.32 kGy 0.58 kGy 0.75 kGy * The same letters are not significantly different. ** The dash means no distinct growth.

144

3.1.3. Combination of Gamma irradiation with cinnamon

From the previous experiments it is clear that dose level 0.75, 0.69 and 1.48 kGy was needed to reduce one log cycle of viable cells of S. aureus, P. aeruginosa, and Debaryomyces sp., respectively (table 3). On the other hand, low concentrations of cinnamon did not affect the viable counts of the tested organisms. Concentration of 0.4% (W/V) of cinnamon started to decrease the viable counts of S. aureus indicating its sensitivity to cinnamon comparing with the Debaryomyces sp., P. aeruginosa whose started to be affected by cinnamon after concentration 1.0 %(W/V) and 0.5%(W/V), respectively tables (10 & 11). So, in the present experiment concentration of 0.4, 0.5 and 1.0 %( wt/v) of cinnamon were used combined with gamma rays. As shown in table (14) and fig. (15), it is clearly that combined treatments reduced the D10 value of the tested organgisms to 0.31, 0.57 and 1.02 kGy for S. aureus, P. aeruginosa, and Debaryomyces sp., respectively. Hence, the dose levels of gamma rays needed to eliminate the previous organisms decreased from 5 to 2 kGy, from 4 to 3kGy and from 7 to 4 kgy for S. aureus, P. aeruginosa, and Debaryomyces sp., in the same sequence. 145

Shelef (1983) and Zaika (1988) stated that Gram-positive bacteria are more sensitive to cinnamon than Gram-negative bacteria. This is probably due to the outer membrane of gram-negative bacteria, which is highly hydrophilic and acts as a strong barrier (Smith-Palmer et al., 1998). The primary mode of action of ionizing radiation is via oxygen and hydroxyl radicals (Niemira, 2003).In suspensions with a high antioxidant capacity, these radicals can be neutralized before doing damage to bacterial cell membrane, protein structures, and nucleic acid strands, thereby protecting the bacteria and reducing the efficacy of the process (Sommers, 2003). Doses kGy

0 0 0.5 1 1.5 2 2.5 3 3.5

-0.5 Staphylococcus aureus Pseudomonas aeruginosa -1 Debaryomyces spp.

-1.5

-2

Log N/No N/No Log -2.5

-3

-3.5

-4

-4.5 Fig. (15): Effect of different doses of gamma irradiation in combination with cinnamon. 146 Table (14): Effect of different doses of gamma irradiation in combination with cinnamon

Organism Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Cinnamon conc. 0.4 0.5 1.0 % Average Log Average count Log Average count cfu Log Doses kGy Log N* Log N* Log N* count cfu m-1 N/No cfu m-1 N/No m-1 N/No

6 6 7.39 5 Initial count 32 x 10 7.50 - 25 x 10 - 55 x 10 6.74 - ± 0.031 A ± 0.02 A ± 0.046 A

Control 63 x 104 5.79 0 36 x 104 5.55 0 21 x 103 4.32 0 Nisin ± 0.038 B ± 0.065 B ± 0.034 B

3 3 4.77 2 0.5 28 x 10 4.44 -1.35 59 x 10 -0.78 98 x 10 3.99 -0.37 ± 0.03 C ± 0.064C ± 0.007 B 1 72 x 10 2.85 -2.94 98 x 102 3.99 -1.56 38 x 102 3.57 -0.74 ± 0.017 D ± 0.043D ± 0.017 C 2 -** -** -** 15 x 10 2.17 -3.38 53 x 10 2.72 -1.59 E ± 0.027 E ± 0.033 D -** 3 -** -** 35 1.54 -2.77 F ± 0.030 E 4 -** -** -** F

D10 0.31 kGy 0.57 kGy 1.02 kGy

* The same letters are not significantly different. ** The dash means no distinct growth.

147

3.1.4. Combination of Gamma irradiation with nisin

From the previous experiment, application of nisin did not affect either P. aeruginosa or Debaryomyces sp. On the other hand nisin at concentration 25 µg/ml decreased the viable count of S. aureus approximately one log cycle in single treatment table (5). Several investigators has reported about the synergistic effect ofcombined treatments on microorganisms.the object of the present experimentwas to study the effect of gamma radiation combinedwith nisin on the S. aureus besides its its effect on the cell wall of both of P. aeruginosa and Debaryomyces sp. which may permit the nisin to enter their cells and hence assist to destroy the cells. In the case of S. aureus, gamma radiation was combined with 25 µg/ml of nisin. While, 200 µg/ml of nisin was used in case of P. aeruginosa and Debaryomyces sp.

Data presented in table (15) and fig. (16) clearly showed that, cmbined treatment of gamma rays and nisin sharply affcte the viable count of S. aureus and dose level

3 kGy was sufficient to destroy its cells, giving D10 value 0.48 kGy, while it was 0.75 kGy in case of single treatment of gamma rays.

148

As expected from the previous experiment, the results clealy indicated that gamma rays affected the cell wallsof both P. aeruginosa and Debaryomyces sp. which permit the nisin to enter theircells andgave synergistic effect on destroying the cellsof both organisms. In the case of P. aeruginosa the data showed that the combined treatment decreased its D10 values from 0.69 to 0.45 kGy and hence dose level 3 kGy wasquiet sufficient to eliminate its cells from themedium instead of 4 kGy in the single treatment.

The same trend was occurred in case of Debaryomyces

sp.; hence its D10 value was decreased from 1.48 kGy in single treatment to 0.68 kGy in combined with nisin. Also, dose level 4 kGy eleminat its cells insteated of 7 kGy in the single treatment with gamma rays alone.

It is clear from the previous data that, the highly resistant of P. aeruginosa and Debaryomyces sp. to nisin become more sensitive to the natural preservative and this may be due to the injuring by irradiation. Irradiated bacteria may be more easily lyses than non- irradiated bacteria (Andrej Trampuz et al., 2006). Both direct and indirect reactions between ionizing radiation and cellular components occur in direct

149

proportion to the amount of energy that is absorbed. Since 50 to 70% of the bacterial cell mass is water, it absorbs much of the radiation. As a result, hydroxyl radicals and hydrated electrons (which are important in irradiation- induced cell inactivation) are produced (Kim and Thayer, 1996). These radicals damage cell membranes beside protein structures and nucleic acid strands (Niemira and Solomon, 2005). And hence increase the permeability of the cell wall which permite to the preservative to enter the cell.

150

Doses kGy

0 0 0.5 1 1.5 2 2.5 3 3.5 -0.5 Staphylococcus aureus -1 Pseudomonas aeruginosa Debaryomyces spp. -1.5

-2

-2.5 Log N/No -3 -3.5 -4

-4.5

-5 Fig. (16): Effect of different doses of gamma irradiation in combination with Nisin.

151

Table (15): Effect of different doses of gamma irradiation in combination with nisin

Organism Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Nisin conc. 25 200 200 µg/ml Average count Log Average count Log Average count Log Doses kGy Log N* Log N* Log N* cfu m-1 N/No cfu m-1 N/No cfu m-1 N/No Initial count 45 x 106 7.65 - 98 x 105 6.99 - 41 x 104 6.61 - ± 0.062 A ± 0.028 A ± 0.032 A Control 43 x 104 5.63 0 98 x 105 6.99 0 41 x 105 6.61 0 Nisin ± 0.064 B ± 0.0 A ± 0.0 A 0.5 64 x 103 4.80 - 0.82 64 x 104 5.80 - 1.18 91 x 104 5.95 - 0.65 ± 0.039 C ± 0.021 B ± 0.050 B 1 65 x 102 3.81 - 1.82 64 x 103 4.80 - 2.18 26 x 104 5.41 - 1.19 ± 0.034 D ± 0.026 C ± 0.053 C 2 53 1.72 - 3.9 32 x 10 2.50 -4.48 74 x 102 3.86 - 2.74 ± 0.046E ± 0.058 D ± 0.006 D 3 -** -** -** -** -** -** 23 x 10 2.36 - 4.25 F E ± 0.003 E 4 -** -** -** F

D10 0.48 kGy 0.45 kGy 0.68 kGy

* The same letters are not significantly different. ** The dash means no distinct growth.

152

• General Discussion

Gamma irradiation enhances the shelf-life of foods and ensures their innocuousness because most microorganisms in vegetative form are extremely sensitive to irradiation at low doses (Radomyski et al., 1994). The lethal effect of ionizing radiation is primarily due to DNA damage, which destroys the reproductive capabilities and other functions of the cell (DeRuiter and Dwyer, 2002).

On the other hand, 'minimal processing' is a concept describing approaches to food safety and preservation that are designed to retain the natural and as-fresh properties of foods (Manvell, 1997). Hence, gamma irradiation can be used in low doses (which cause injuring to microbial cell) in combination with other prsevatives.

However, in some circumstances combined treatments are more satisfactory since the dose required for complete sterilization can induce undesirable changes in food flavor or is at higher than permitted level (Thakur and Singh, 1995). Effective Combinations of two or more preservation hurdles may be chosen once the modes of action and

153

cellular targets of each treatment are known (Leistner, 2000). As mention previously, nisin, cinnamon, citric acid and lactic acid acts on cell membrane of microbial cell. So, the combination of these preservatives with gamma irradiation it implies more stress will acts on the same target of the microbial cell this refers to hurdles (by placing a number of sub lethal stresses on microbial cell which agree with Leistner, (2000). Hurdles targeting the same elements within the cell have an additive inhibitory effect only, whereas synergistic effects may result from disturbing several functions of the cell (Leistner, 1992, 1994).

154

3.1.5. Comparision between gamma irradiation single and combined treatments

Table (16) and Fig. (17) shows that, the D10 of gamma irradiation in single treatment for S. aureus was 0.75 kGy which decreased to 0.32, 0.27, 0.31 and 0.48 kGy by combination with citric acid, lactic acid, cinnamon and nisin, respectively.

Also, the D10 of P. aeruginosa was 0.69 kGy which reduced to 0.58 kGy by combination with citric acid, reduced to 0.40 kGy by combination with lactic acid, lowered to 0.57 kGy by combination withcinnamon, and reduced to 0.45 kGy by combination with nisin.

The D10 of Debaryomyces sp. was 1.48 kGy which reduced to 0.75 kGy by combination with citric acid and reduced to 0.47 kGy by combination with lactic acid. Also, this dose was decreased to 1.02 and 0.68 kGy by combination with cinnamon and nisin, respectively.

155

Table (16): D10 - values of gamma irradiation for single and combined treatments

Debaryomyces S. aureus P. aeruginosa Treatment sp. D10 value kGy

Radiation alone 0.75 0.69 1.49

Radiation and citric 0.32 0.58 0.75 acid Radiation and lactic 0.27 0.40 0.47 acid Radiation and 0.31 0.57 1.02 cinnamon

Radiation and nisin 0.48 0.45 0.68

1.6 1.48 Staphylococcus aureus Pseudomonas aerogenosa Debaryomyces spp.

1.4

1.2 1.02 0.69 1

0.75 0.75

kGy 0.8 0.68 10

D 0.58 0.57 0.45 0.6 0.47 0.48 0.4

0.4 0.32 0.31 0.27

0.2

0

alone c acid c acid amon nisin iation d citri d lacti d cinn on and Rad tion an tion an tion an adiati Radia Radia Radia R Treatment

Fig. (17): D10 - values of gamma irradiation for single and combined treatments. 156

3.2. Combination with Pasteurized temperature

Thermal treatment leads either to pasteurization, when only vegetative microorganisms are destroyed or sterilization, when spores are also destroyed. Food preservation implies exposure of microorganisms contaminated the food to hostile conditions. Synergies are particularly interesting because combination of processes i.e., , are a promising means of enhancing safety whilst retaining food quality. In combination commonly pasteurized temperature, .Cْ for 30sec was used 90

3.2.1. Combination of pasteurized temperature with Nisin

Data presented in table (17) clearly indicated that, S. aureus was highly sensitive to both high temperature and nisin. The initial count log 7.53 sharply decreased to log 3.74 by temperature alone while combined it with 25 μg/ml nisin, completely desteroyed all the organism cells. Although both of P. aeruginosa and Debaryomyces sp. revealed highly resistance to nisin even at higher concentration, but combined treatment 157

with pasteurization assisted to eliminate them from the medium. Application of 50 μg/ml of nisin combined with pasteurized temperature (90 Cْ for 30sec) completely inhibited the cells of Debaryomyces sp. while P. aeruginosa needed 150 μg/ml of nisin to destroy the cells in the medium as clearly shown in table (17). Allan et al., 1988 demonstrated HSP in P. aeruginosa following a temperature shift from 30 to 45 Cْ that led to the synthesis of at least a new 17 proteins within approximately 1 min.

Heat can damage protein, lipids, and nucleic acids and destabilize membranes (Gould, 1989). Heating- induced destabilization, such as oxidation of sulfydryl groups of the membrane – binding bound proteins, Yatvin and Grummer, 1987). This disturbance in the cell especially in the cell membrane may due to the entrance of nisin to the cell and induced its death.

158

Table (17): Effect of pasteurized temperature 90 Cْ for 30 sec in combination with different conc. of nisin Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp.

Average Average Average Conc. ug/ml Log Log Log count cfu Log N* count cfu Log N* count cfu Log N* N/No N/No N/No m-1 m-1 m-1

Initial count 34 x 106 7.53 - 25 x 106 7.39 - 54 x 105 6.73 - ± 0.043 A ± 0.03 A ± 0.013 A

Control 55 x 102 3.74 0 29 x 103 4.46 0 118 x 103 5.07 0 temperature ± 0.014 B ± 0.058 B ± 0.022 B

25 -** -** -** Not tested 15 x 102 3.17 - 1.89 C ± 0.040 C

50 20 x 102 3.30 - 1.16 -** -** -** ± 0.037 C D

100 15 1.17 - 3.28 ± 0.050 D

150 -** -** -** E

* The same letters are not significantly different. ** The dash means no distinct growth.

159

3.2.2. Combination of pasteurized temperature with citric acid

It is clear from table (18) that, pasteurized temperature at 90 Cْ for 30sec. greatly affected all the tested organisms. Pasteurization treatment alone decrease the viable count of S. aureus more than 4 log cycle while it decrease the viable count about 2 log cycle in case of P. aeruginosa and Debaryomyces sp..combined the pasteurized temperature with only 0.1 % (W/V) of citric acid, completely destroyed all the cells of S. aureus while 0.15 %(W/V) of citric acid was needed to eliminate P. aeruginosa. The rest viable cells of Debaryomyces sp. revealed relativily high resistance to the combined treatment and 3 % (W/V) of citric acid was applied to annihilate its cells.

160 Table (18): Effect of pasteurized temperature 90 Cْ for 30 sec in combination with different conc. of citric acid

Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Average Average Average Conc. % (W/V) Log Log Log count cfu Log N* count cfu Log N* count cfu Log N* N/No N/No N/No m-1 m-1 m-1 Initial count 65 x 106 7.81 - 25 x 106 7.39 - 55 x 105 6.74 - ± 0.038 A ± 0.020 A ± 0.013 A Control 55 x 102 3.74 0 29 x 103 4.46 0 118 x 103 5.07 0 temperature ± 0.014 B ± 0.058 B ± 0.022 B 1.82 0.05 67 -1.91 Not tested Not tested ± 0.064 C -** 1.61 0.1 -** -** 41 - 2.84 Not tested D ± 0.031 C -** 0.15 -** -** Not tested D 1 64 102 3.8 - 1.26 ± 0.027 C 2 17 x 10 2.21 - 2.84 ± 0.044 D 2.5 21 1.32 - 3.74 ± 0.021 E 3 -** -** -** F

* The same letters are not significantly different. ** The dash means no distinct growth.

161

3.2.3. Combination of pasteurized temp with lactic acid

From table (19), lactic acid combined with pasteurized temperature revealed to be more effective for elimination the tested organisms than citric acid. In the case of S. aureus and P. aeruginosa, 0.05% (W/V) and 0.15 %( wt/v) were sufficient to completely destroy their viable cells while only 0.5% (W/V) of latic acid was needed to eliminate Debaryomyces sp. compared with 3 % (W/V) of citric acid

The data of combination of pasteurized temperature with citric acid and lactic acid shows that, the lactic acid more effective with low conc. this may due to lactic acid has lipophilic character; ability to depress intracellular pH. In addition to membrane diffusion and water activity reduction ' specific anion effect', while citric acid lack this character and acting as chelating agent (Smulders, 1987; Stratford, 1999, Saltmarsh, 2000; Naidu, 2000).

162 Table (19): Effect of pasteurized temperature 90 Cْ for 30 sec in combination with different conc. of lactic acid

Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Conc. % Average Average Log Average (W/V) Log N* Log N/No Log N* Log N* Log N/No count cfu m-1 count cfu m-1 N/No count cfu m-1

6 5 7.39 5 Initial count 21x 10 7.32 - 35 x 10 - 65 x 10 6.81 - ± 0.048 A ± 0.03 A ± 0.013 A Control 55 x 102 3.74 0 29 x 103 4.46 0 118 x 103 5.07 0 temperature ± 0.014 B ± 0.058 B ± 0.022 B

0.001 35 x 10 2.54 - 1.19 Not tested Not tested ± 0.021 C

2 3.87 0.01 24 1.38 -2.36 75 x 10 - 0.58 Not tested ± 0.053 D ± 0.031 C

0.05 -** -** -** Not tested Not tested E 1.65 0.1 45 - 2.8 48 x 10 2.68 -2.39 ± 0.033 D ± 0.026 C

0.15 -** -** -** Not tested E

0.5 -** -** -** D

* The same letters are not significantly different. ** The dash means no distinct growth.

163

3.2.4. Combination of pasteurized temp with cinnamon It is clear from table (20) that, pasteurized temperature at 90 Cْ for 30sec. greatly affected all the tested organisms. Pasteurization treatment alone decrease the viable count of S. aureus more than 4 log cycle while it decrease the viable count about 2 log cycle in case of P. aeruginosa and Debaryomyces sp.. Combined the pasteurized temperature with only 0.5 %(W/V) of cinnamon, completely destroyed all the cells of S. aureus while 0.6 % (W/V) of citric acid was needed to eliminate both of P. aeruginosa and Debaryomyces sp.. And by comparing with single treatment of cinnamon (tables 11, 12) it showed that, the lethal concentration of cinnamon was reduced from 1.2, to 0.5 for S. aureus while 2, and 4%(W/V) were reduced to 0.6%( wt/v) for P. aeruginosa and Debaryomyces sp., respectively.

Shelef (1983) and Zaika (1988) stated that Gram- positive bacteria are more sensitive to cinnamon than gram-negative bacteria. This is probably due to the outer membrane of gram-negative bacteria, which is highly hydrophilic and acts as a strong barrier (Smith-Palmer et al., 1998).

164

According to Moats (1971), cell death results from the denaturation or inactivation of numerous critical sites

(or a large number of the same site) in cells. Sub-lethally injured bacterial cells are reported to become sensitive to bactericins of lactic acid bacteria (Kalchayanand et al., 1992 and Ray, 1993).hence the pasteurized temperature increases the sensitivity of both P. aeruginosa and Debaryomyces sp. to cinnamon

Hurst, 1977 and Gould, 1989 reported that heat can damage protein, lipids, and nucleic acids and destabilize membranes. However, heat induces primarily blebbing and vesiculation of the outer membrane leading to bacteria cell inactivation, and can sequentially increases its permeability to hydrophobic compounds (Scheie and Ehrenspeck, 1973 and Tsuchido and Takano, 1988).

Heating induced membrane destabilization sequentially initiates indirect DNA damage as a consequence of increased nuclease activity in bacteria and is often the critical injury , resulting in death of the cell (Sedgwick and Bridges, 1972; Gomez, 1977; Kadota et al., 1978 and Gould, 1989). Because multiple changes may occur during the inactivation of bacteria by heat,

165

thermal inactivation is not linear (Hurst, 1977 and Gould, 1989). Similarly the mechanism of inactivation of bacteria by heat, it may also occur with yeast.

166

Table (20): Effect of pasteurized temperature 90 Cْ for 30 sec in combination with different conc. of cinnamon

Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Conc. % Average Average Average count cfu Log N* Log N/No count cfu Log N* Log N/No count cfu m-1 Log N* Log N/No m-1 m-1 6.74 6.65 5 5 6.85 5 Initial count 56 x 10 ± 0.049 - 72 x 10 - 45 x 10 ± 0.118 - ± 0.020 A A A 3.74 5.07 Control 2 3 4.46 3 55 x 10 ± 0.014 0 29 x 10 0 118 x 10 ± 0.022 0 temperature ± 0.058 B B B 1.93 2.81 2.78 0.3 86 ± 0.049 - 1.8 66 x 10 - 1.64 58 x 10 ± 0.057 -2.3 ± 0.033 C C C -** 1.34 0.5 -** -** 22 - 3.11 Not tested D ± 0.025D

0.6 -** -** -** -** -** -** E D

* The same letters are not significantly different. ** The dash means no distinct growth.

167

• General disscusion Exposure of heat-stressed microbial cells generally results in cytoplasmic membrane disfunction characterized by a release of nucleic acids, amino acids, ATP, and K+ (Beuchat et al., 1997). In the same manner the primary action site of nisin against vegetative cells is considered to be the cytoplasmic membrane, with nisin acting as a voltage-dependent polarizer (Ruhr and Sahl,

1985 and Delves-Broughton and Gasson, 1994). Also, lactic acid was recently shown to permeabilize Gram- negative bacteria efficiently and causes LPS release (Alakomi et al., 2000) and citric acid is metal chelating (Verhoff, 1986). As, mentioned previously gram-negative bacteria, are normally insensitive to lysozyme and nisin because their outer membrane prevents access to the sites of action of these agents. Similarly, in yeast are insensitive to Nisin and may make adaptation to make organic acids as mentioned previously in single treatment. Hence, the sensitivity of P. aeruginosa, Debaryomyces to nisin, organic acid were increased during inactivation by pasteurized temp according to the mode of action of pasteurized temp which mentioned previously. Effective combination of two or more preservation hurdles may be

168

In application manner, it is feasible to produce a safe apple cider by combining heat with pH modification and preservative addition. Dock et al, (2000) investigated the effect of pH and preservatives on the heat resistance of E. coli O157:H7 in apple cider. The D- values at 50Cْ of 65min in apple cider were reduced to 13.9, 13.2 and 7.0 min in apple cider with addition of 0.5% (W/V) , 0.1% (W/V) sorbate and 0.1% (W/V) benzoate, respectively.

169

3.2.5. Comparison between the selected natural preservatives alone and in combination with pasteurized temperature. 1- S. aureus From table (21) and fig. (18), The sub-lethal value of nisin was 200 μg/ml which decreased to 25 μg/ml by combination with pasteurized temperature, the sub-lethal value of citric acid was 0.15 % (W/V) which reduced to 0.05% (W/V) by combination with pasteurized temperature, the sub-lethal value of Lactic acid was 0.1 % (W/V) which lowered to 0.01% (W/V) by combination with pasteurized temperature, and the sub-lethal value of cinnamon was 0.15 % (W/V) which decreased to 0.05% (W/V) by combination with pasteurized temperature.

2- P. aeruginosa From table (21) and fig. (18), there is no effect of nisin on P. aeruginosa which enhanced by combination with pasteurized temperature and the sub-lethal conc. decreased to 100 μg/ml, the sub-lethal conc. of citric acid and lactic acid was 0.2 %(W/V) which decreased to 0.1 %(W/V) by combination with pasteurized temperature, also, the sub-lethal conc. of cinnamon was 3 %(W/V) and

170

by using pasteurized temperature it was decreased to 0.5 % (W/V).

3- Debaryomyces sp. From table (21) and fig. (18), there is no effect of nisin on Debaryomyces sp. but it enhanced by combination with pasteurized temperature and the sub-lethal conc. decreased to 25 μg/ml, the sub-lethal conc. of citric acid and lactic acid was 3.5 and 4 %(W/V) which decreased to 2.5 and 0.1 %( wt/v) by combination with pasteurized temperature, also, the sub-lethal conc. of cinnamon was 1.7 %(W/V) and by using pasteurized temperature it was lowered to 0.3 % (W/V).

171

Table (21): Sub-lethal values of natural preservative alone and in combination with pasteurized temperature*

Debaryomyces S. aureus P. aeruginosa Treatment sp. Sub-lethal value Nisin alone 200 ** ** Temperature + 25 100 25 Nisin Citric acid 0.15 0.2 3.5 alone Temperature + 0.05 0.1 2.5 Citric acid Lactic acid 0.1 0.2 4 alone Temperature + 0.01 0.1 0.1 Lactic acid Cinnamon 1 3 1.7 alone Temperature + 0.3 0.5 0.3 Cinnamon * Nisin conc. = μg/ml, other preservatives conc. = % (W/V). ** There is no effect of nisin alone on P. aeruginosa or Debaryomyces sp.

172

300300 300 Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces spp.

250 200

200

150 100

Conc. % Conc.

100 25 25 4 0.1 2.5 50 3.5 0.2 0.1 1.7 0.3 0.5 0.2 0.1 0.01 3 0.1 0.3 0.15 0.05 1

0

e in e id e id e n on is on ac on ac on mo al N al c al ic al a n d id tri id ct n nn isi .an c Ci c a o Ci N p c a d c a L am d m tri an ti nd n n te i p. ac .a in . a st. C m L p C p a te em em P t. . t . t as st st P Pa Pa

Fig. (18): Sub-lethal values of natural preservative alone and in combination with pasteurized temperature

173

3.3. Combination of natural preservatives Natural antimicrobial compounds available for use in food processing and shelf life extension include enzymes (lactoperoxidose, lactoferrin, Avidin, lysozyme), microbial preservatives produced from starter cultures (Nisin, Lactacin/Lactococin/Lacticin/, , Variacin), and plant sources (herbs, spices, extracts, essential oils and their isolated components) (Valero and Francés, 2006).

3.3.1. Combination of Nisin with citric and latic acid. From the previous experiments (single treatment), nisin alone has no effect on both of Debaryomyces, and Pseudomonas while it wasvery effective in case of S. aureus. The object of the present experiment was to investigate the effect of nisin combined with citric acid or lactic acid, hoping that the acids may affect the cell membrane of both Debaryomyces, and P. aeruginosa which permite the nisin to enter their cells. Concentration of 25 μg/ml of nisin was used in case of S. aureus which reduce its counts by one log cycle as previously mentioned, while 200

174

μg/ml in case of Debaryomyces, and P. aeruginosa was used in combined with different concentration of citric acid or lactic acid. As shown in tables (22 & 23) and Figures (19 & 20), only 0.05 % (W/V) of citric acid beside 25 μg/ml nisin was sufficient to annihilate the viable cells of S. aureus, while 0.2 % (W/V) of citric and 200 μg/ml nisin were needed to eliminate P. aeruginosa (it worth tomention that 0.3 % (W/V) of citric acid alone was needed to destroy the cells of P. aeruginosa in the single treatment). Regared the Debaryomyces, 3.0 % (W/V) of citric acid was applied beside 200 μg/ml nisin to reach its cell death (it worth tomention that 4 % (W/V) of citric acid alone was needed to destroy the cells of Debaryomyces in the single treatment). In case of lactic acid tables (24 & 25) and Figures (21 & 22), 0.1% (W/V) was used beside 25 μg/ml of nisin to eliminate S. aureus and 200 μg/ml of nisin to eliminate P. aeruginosa (comparing with 0.3 % (W/V) required in single treatment). Concentration 3.5 % (W/V) of lactic acid beside 200 μg/ml nisin were applied to eliminate Debaryomyces (comparing with 4.5 % (W/V) required in single treatment).

175

Conc. %

0 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 -0.5 -1 -1.5 -2

-2.5 Log count -3 -3.5 -4 Staphylococcus aureus Pseudomonas aeruginosa -4.5 Fig. (19): Effect of different conc. of citric acid in combination with nisin

176 Table (22): Effect of different conc. of citric acid in combination with nisin

Organism Staphylococcus aureus Pseudomonas aeruginosa Nisin conc. µg/ml 25 200 Log Conc. % Average count cfu m-1 Log N* Log N/No Average count cfu m-1 Log N* N/No

Initial count 98 x 105 6.99 - 98 x 105 6.99 - 0.017 A ± 0.014 A

Control 4 5 6.99 43 x 10 5.63 0 98 x 10 0 Nisin ± 0.060 B ± 0.0 A

0.01 89 x 102 3.94 - 3.04 Not tested ± 0.032 C

0.03 15 x 10 2.17 - 4.81 Not tested ± 0.050 D

0.05 -** -** -** 48 x 104 5.68 -1.30 E ± 0.045 B

0.1 22 x 103 4.34 -2.64 ± 0.094 C

0.15 56 x 10 2.74 -4.24 ± 0.032 D

0.2 -** -** -** E

* The same letters are not significantly different. ** The dash means no distinct growth.

177

Table (23): Effect of different conc. of citric acid in combination with Nisin .200 µg/ml on Debaryomyces sp Log Conc.% Average count cfu m-1 Log N* N/No 0 41 x 105 6.61 0 ± 0.012 A 6.61 Control nisin 41 x 105 0 ± 0.0 A

1.0 30 x 104 4.47 -1.13 ± 0.016 B

2.0 51 x 102 4.70 -2.90 ± 0.027 C

2.5 88 x 10 2.94 -3.66 ± 0.020 D

3.0 -** -** -** E

* The same letters are not significantly different. ** The dash means no distinct growth. Conc. % 0 00 . 511 .522 .533.5 -0.5

-1

-1.5

-2

-2.5 Log N/No N/No Log -3

-3.5

-4

-4.5 Fig. (20): Effect of different conc. of citric acid in combination with Nisin 200 µg/ml on Debaryomyces sp..

178

Table (24): Effect of different conc. of lactic acid in combination with nisin Organism Staphylococcus aureus Pseudomonas aeruginosa Nisin conc. µg/ml 25 200 Log Conc. % Average count cfu m-1 Log N* Log N/No Average count cfu m-1 Log N* N/No

Initial count 121 x 105 7.08 - 98 x 105 6.99 - ± 0.011 A ± 0.014 A

Control 4 5 43 x 10 4.74 0 98 x 10 6.99 0 Nisin ± 0.060 B ± 0.0 A

0.01 Not tested 88 x 104 5.94 ± 0.062 C

0.02 25 x 103 4.39 -1.23 Not tested ± 0.030 C

0.04 38 x 102 3.57 -2.05 Not tested ± 0.062 D

0.05 Not tested 25 x 102 3.39 -3.59 ± 0.025 D

0.06 150 2.17 -3.45 Not tested ± 0.050 E

0.1 -** -** -** -** -** -** F E

* The same letters are not significantly different. ** The dash means no distinct growth.

179

Conc. %

0 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07

-0.5

-1

-1.5

-2 Log count -2.5

-3

-3.5 Staphylococccus aureus Pseudomonas aeruginosa

-4 Fig. (21): Effect of different conc. of lactic acid in combination with nisin

Table (25): Effect of different conc. of lactic acid in combination with Nisin .200 µg/ml on Debaryomyces sp

Log Conc.% Average count cfu m-1 Log N* N/No Initial count 41 x 105 6.61 0 ± 0.01 A Control 41 x 105 6.61 0 nisin200 µg/ml ± 0.0 A

1.5 29 x 103 3.46 -2.15 ± 0.012 B 2.5 13 x 10 2.11 -4.49 ± 0.086 C 3 5 0.69 -5.91 ± 0.106 D 3.5 -** -** -** E

*The same letters are not significantly different. ** The dash means no distinct growth.

180

Conc. % 0 00.511.522.533.54

-1

-2

Log N/No Log N/No -3

-4

-5

-6

-7 Fig. (22): Effect of different conc. of lactic acid in combination with Nisin .200 µg/ml on Debaryomyces sp

3.3.2. Combination of Nisin with cinnamon It is obvious from table (26) and Figures (23 & 24) that, the addition of cinnamon with nisin, the count decreases sharply by increasing the conc. of cinnamon and accelerate the death of S. aureus, Pseudomonas, and Debaryomyces. The lethal-values were at 2% (W/V) for both Pseudomonas, Debaryomyces (comparing with 4 % (W/V) and 2 % (W/V) in single treatments, respectively) and at 0.4% (W/V) for S. aureus.

181

Cinnamon contains approximate 0.5-2.0 % Essential oil (Snyder, 1997). Cinnamon oil contains 50-60 % cinnamaldehyde and 4-7% eugenol (Matan et al., 2006). Although essential oils components are used as flavoring in the food industry, nowadays they represent a highly interesting source of natural antimicrobials for food preservation due to their antimicrobial and antioxidative activity (Beuchat, 1994). Cinnamon effectively inhibits growth of bacteria, yeasts and molds (Smith-palmer et al., 1998).

Shelef (1983) and Zaika (1988) stated that Gram- positive bacteria are more sensitive to cinnamon than Gram-negative bacteria. This is probably due to the outer membrane of gram-negative bacteria, which is highly hydrophilic and acts as a strong barrier (Smith-Palmer et al., 1998).

The mechanism of action of the cinnamon oil and its main compound to inactivate microorganisms according to Gill and Holley (2004, 2006) and Oussalah et al. (2006) appear to be related to the cell membrane from which a slight disruption seem to occur causing dispersion of the proton motive force by leakage of small ions without

182

leakage of larger cell components, such as ATP, which are subsequently degraded by the ATPase enzyme. Hence, the antimicrobial effect of nisin combined with cinnamon is enhanced due to the formation of pores on the cell membrane, which may favor more easily the diffusion of the nisin to cell inside and cause damage in the cell functions.

183 Table (26): Effect of different conc. of cinnamon in combination with nisin Organism Staphylococcus aureus Pseudomonas aeruginosa Debaryomyces sp. Nisin conc. ug/ml 25 200 200 Average count Log Average count Log Average count Log Conc. % Log N* Log N* Log N* cfu m-1 N/No cfu m-1 N/No cfu m-1 N/No Initial count 95 x 105 6.97 - 98 x 105 6.99 - 41 x 105 6.61 0 ± 0.009 A ± 0.012 A ± 0.003 A

Control 4 5 5 43 x 10 4.74 0 98 x 10 6.99 0 41 x 10 6.61 0 Nisin ± 0.060 B ± 0.0 A ± 0.0 A 0.2 48 x 102 2.68 -1.95 Not tested ± 0.015 C 0.3 12 1.07 -4.55 Not tested 61 x 104 5.78 -0.82 ± 0.079 D ± 0.003 B 0.4 -** -** -** Not tested E 0.5 65 x 103 4.3 -2.17 ± 0.030 B 3 4.54 0.6 Not tested 35 x 10 -2.06 ± 0.087 C

2 2 3.41 1 35 x 10 3.5 -3.44 26 x 10 -3.16 ± 0.043 C ± 0.011 D

1.84 1.5 Not tested 7 x 10 -4.76 ± 0.020 E -** 2 -** -** -** -** -** D F

* The same letters are not significantly different. ** The dash means no distinct growth.

184

Conc. % 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 -0.5

-1

-1.5

-2

-2.5 Log count -3 -3.5 -4

-4.5 Pseudomonas aeruginosa Debaryomyces spp.

-5 Fig. (23): Effect of different conc. of cinnamon in combination with nisin

Conc. % 0 0 0.2 0.4 0.6 0.8 1 1.2 -0.5

-1

-1.5

-2

Log N/No N/No Log -2.5

-3

-3.5

-4

-4.5 Staphylococcus aureus

-5 Fig. (24): Effect of different conc. of cinnamon in combination with nisin on S. aureus. 185

• General discussion In Gram–positive cocci (such as S. aureus) there are no specific receptor molecules or permeases to prevent preservatives penetration. Consequently, preservatives enter these cells readily and intrinsic resistance is therefore low (Russell, 1991). In contrast, the outer membrane of Gram-negative bacteria (such as P. aeruginosa) plays an important role in limiting the entry of preservatives into the cell (Nikaido and Vaara 1985). The cell surface of smooth, wild-type gram-negative bacteria is hydrophilic in nature; deep rough (hepatoseless) mutants are much more hydrophobic because of the appearance of phospholipids (PL) patches on the cell surface. In wild-type bacteria, low molecular weight hydrophilic molecules (such as lactic acid) cross the outer membrane via the aqueous porins. On the other hand, lipopolysaccharide (LPS) molecules prevent ready access of hydrophobic preservatives to the PL (Russell, 1991).

Lactic acid was recently shown to permeabilize Gram- negative bacteria efficiently and causes LPS release (Alakomi et al., 2000) and citric acid is metal chelating (Verhoff, 1986). Like EDTA which destabilize and cause

186

lipopolysaccharide release from the outer membrane by complexation of bridge-forming bivalent cations such as Mg2+ and Ca2+ (similarly citric acid act in the same manner) (Hauben et al, 1996). Inhibition of the growth of the outer membrane gram-negative bacteria can be achieved by simultaneous treatment with nisin and an agent which modifies and chelates the outer membrane, such as EDTA (Stevens et al., 1991). These findings are consistent with notion that nisin acts on the cytoplasmic membrane.

Phillips and Duggan, (2002) reported that a compound like citric acid makes gram negative bacteria more sensitive to Nisin. Similarly, in yeast because its sensitivity to acids (single treatments) make it sensitive to nisin (combined treatments).

Nisin speeded up the inactivation, with cinnamon and generally contributing to the suppression of pathogens. The higher of nisin dose needed to greater killing effect (Yuste and Fung, 2003). Kalchayanand et al, (1992) observed an increase in nisin effectiveness when gram negative bacteria were subjected to sub lethal stresses (Including exposure to acids). Further more, Nisin

187

is more stable and more effective of acidic pH (Deans and Ritchie, 1987 and Ray, 1992).

In the same mechanism, the combination of Nisin and cinnamon accelerates death of Salmonella Typhimurium and E.coli 0157:H7 in apple juice and give substantial protection to the product (Yuste and Fung, 2003). In a similar purpose, it may be occurring with both Pseudomonas and Debaryomyces. This suggests the potential use of such combination in the food industry for enhancement of safety.

188

3.3.3. Comparison between the selected natural preservatives in single treatment and in combination with nisin. 1- S. aureus As showen in table (27) and fig. (25), the sub-lethal value of citric acid was 0.15 % (W/V) and by using nisin it was reduced to 0.03 %( wt/v). The sub-lethal conc of lactic acid 0.1 %( wt/v) was reduced to 0.06 %( wt/v) by using nisin. While, cinnamon was reduced from 1 %( wt/v) to 0.3 %( wt/v).

2- P. aeruginosa Table (27) and fig. (25) showed that, the sub-lethal value of citric acid and lactic acid was 0.2 % (W/V) and by using nisin it was reduced to 0.15 and 0.05 %( wt/v), respectively. While, cinnamon was reduced from 3 %( wt/v) to 1 %( wt/v).

3- Debaryomyces sp. From Table (27) and fig. (25), the sub-lethal value of citric acid and lactic acid was 3.5 and 4 % (W/V) and by using nisin it was decreased to 2.5 and 4 %( wt/v). While, cinnamon was lowered from 1.7 %( wt/v) to 1.5 %( wt/v).

189

Table (27): Sub-lethal values of selected natural preservatives alone and in combination with nisin*

S. aureus P. aerogenosa Debaryomyces sp. Treatment Sub-lethal value % Nisin conc. ** 25 200 200 Citric acid 0.15 0.2 3.5 Nisin + citric 0.03 0.15 2.5 acid

Lactic acid 0.1 0.2 4

Nisin + lactic 0.06 0.05 3 acid

Cinnamon 1 3 1.7

Nisin + 0.3 1 1.5 Cinnamon

* Nisin conc. = μg/ml, other preservatives conc. = % (W/V). ** There is no effect of nisin alone on P. aeruginosa or Debaryomyces sp.

190

4 4 Staphylococcus aureus 3.5 Pseudomonas aerogenosa

3.5 Debaryomyces spp. 3 3 3 2.5 2.5

2 1.7

Conc. % Conc. 1.5

1.5 1 1

1

0.2 0.2 0.05 0.15 0.3 0.5 0.15 0.03 0.1 0.06

0

one acid one acid one mon id al tric id al tic n al nna ac d ci ac d lac amo d Ci itric n an actic an inn an C Nisi L isin C isin N N Treatment

Fig. (25): Sub-lethal values of nisin alone and in combination with the selected natural preservatives.

191

4. Combination of gamma irradiation with pasteurized temperature

A combination of irradiation has been proposed for required lethality while preserving food quality, there by reducing the detrimental effects of heat or irradiation alone on food while the inactivation effect of heat treatment followed by irradiation is additive (Kim and Thayer, 1996), a synergistic effect has been observed when heat treatment is applied after irradiation or when both treatments are simultaneously applied (thermo radiation).

This experiment was performed by heating suspension of yeast cells in broth medium to pasteurized temperature 90 Cْ for 15 sec followed by gamma irradiation at dose 0.25 KGy, and results showed, nil count in all dilutions, at control 98 x 104cfu ml-1.

The results suggest that irradiation enhanced heat sensitivity of Debaryomyces sp. originated from oxygen- independent intracellular damages such as DNA damages at radiation doses of 0.2 to 1.0 KGy as suggested by previous experiments.

192

However, the denaturation or dissociation of broken DNA strands from the cellular membrane by heating following irradiation makes them unrepairable. Alternatively, (i) heating may alter DNA polymerase and topoisomerase I as in yeast cells (Borehan et al., 1990), or (ii) irradiation may induce functional changes in the cytoplasmic membrane in addition to the heating-induced desbilization, such as oxidation of sulfydryl groups of the membrane-binding bound proteins, resulting in cell death in addition to direct DNA damage (Yatvin and Grummer, 1987).

Also, it is possible that heat and radiation act on different sites but also share some targets such as DNA to cause additive or synergistic effect on cells (Bridges et al., 1969 and Yatvin et al., 1986). Alternatively, heated cells could be more sensitive to irradiation either because the heat-labile recovery capacity of bacterial cells as affected (Bridges et al., 1969) or because recovery from heating- induced damage by irradiation is abolished, preventing protein synthesis as a consequence of DNA damage in vegetative bacterial cells (Mukherjee and Bhattacharjee, 1970)

193

Pallas and Hamdy (1976) observed that the D10- value (dose inactivating 90% of population) of S. aureus decreased from 0.098 t 0.053 KGy, while the irradiation . temperature increased from35 to 45 Cْ

5. Storage The Debaryomyce sp. was selected for storage, due to it was more resistant to physical and natural treatments than Staphylococcus and Pseudomonas, two experiment were performed as follow:

5.1. Combination between nisin and citric acid and pasteurized temperature.

As shown in table (28), the nisin was used in concentration 200 µg/ml in combination with temperature Cْ for 60, 120 sec. and at citric acid concentration 90 2.0%, it was more effective for more than 90 days without any growth. While, when the time of temperature was decreased to 30 sec., the Debaryomyces can be recovery and give count 12 x 10 cfu ml-1. Also, when the conc. of nisin was decreased to 100 or 50 µg/ml at 2 % citric acid ,and pasteurized temperature 90 ْ C for 30 or 60 or120 sec

194

Table (28): Effect of storage for recovery of Debaryomyces sp.*

Pasteurized temperature90ْ C for time/sec Nisin conc. Citric acid Storage period μg/ml conc. % 30 60 120 Average count (cfu/ml)

6 days 50 45 x 104 78 x 103 28 x 102

6 days 100 2.0 33 x 103 46 x 102 38 x 10

90 days 200 22 x 10 Nil Nil

*Treated with pasteurized temperature, nisin and citric acid.

5.2. Combination between gamma irradiation and pasteurized temperature. It was performed by heating broth medium inoculated with yeast cells to pasteurized temperature 90 ,(Cْ for 30 sec followed by gamma irradiation (0.25 kGy results showed, nil count in all serial dilutions and it was effective for more than 90 days at control 98 x 104cfu ml-1.

195

This synergistic effect of thermoradiation has also been observed in the inactivation of microorganisms in several foods. It has been reported that combining low- temperature (50 C)ْ heating with low-dose irradiation is highly effective for controlling spoilage organisms in apple juice (Urbain, 1986).

196

SUMMARY

197

Summary

1. Isolation and identification From forty two isolates from different juice samples, it was select three isolates (Staphylococcus aureus, Pseudomonas aeruginosa, and Debaryomyces sp.) to study the effect of physical treatment and natural treatments as follow:

2. Single treatments 2.1. Physical treatments 2.1.1. Gamma irradiation The lethal dose for S. aureus was 5 kGy; P. aeruginosa was more sensitive to gamma irradiation than S. aureus. The lethal dose was at 4 KGy, while Debaryomyces sp. was highly resistant to irradiation treatment and more survive than Pseudomonas aeruginosa or Staphylococcus aureus where, the lethal dose was at 7 kGy.

ْ(Pasteurized temperature (90 C .2.1.2 The lethal time for S. aureus was reached to

120 sec. Dt 13.1 sec., while the lethal time of P.

aeruginosa was reached to 120 sec. and Dt was 17.3 sec.,

198

Debaryomyces sp. was more survival and sub-lethal time reached to 120 sec. and Dt was13.1 sec.. Also the TDT was 157.4, 208.0 and 224.0 sec., respectively for the previous organisms.

2.2. Natural treatments 2.2.1. Nisin The lethal conc. for S. aureus was at 250 µg/ml, While, nisin alone has no effect on P. aeruginosa or Debaryomyces sp.

2.2.2. Citric acid The lethal conc. of S. aureus was 0.2 % (W/V), the lethal conc. of P. aeruginosa was 0.3 % (wt/v), while For Debaryomyces sp. was the most resistant to citric acid than P. aeruginosa or S. aureus. Where, the lethal conc. reached to 4.0 % (W/V).

2.2.3. Lactic acid The lethal conc. of S. aureus was 0.15 % (W/V), the lethal conc. of P. aeruginosa was 0.3 % (W/V), while Debaryomyces sp. was highly survive to lactic acid, where the lethal conc. was 4.5 % (W/V).

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2.2.4. Cinnamon The lethal conc. of Staphylococcus aureus was 1.2 % (W/V), the lethal conc. for P. aeruginosa was 4.0 % (W/V), While, for Debaryomyces sp.. the lethal conc. was at 2.0 % (W/V).

3. Combination treatments 3.1. Combination with Gamma rays The lethal dose for S. aureus was 5 kGy; this dose was reduced to 3 kGy by combination with nisin 25 µg/ml. The lethal dose decreased to 2 kGy by combination of gamma irradiation with citric, lactic acid and cinnamon at conc. 0.05% (W/V), 0.01% (W/V) and 0.4 % (W/V), respectively.

P. aeruginosa was more sensitive to gamma irradiation than S. aureus. The lethal dose was at 4 KGy, and by combination with nisin (200 µg/ml) or citric acid 0.1 % (W/V) or with cinnamon 0.5 % (W/V), it was decreased to 3 kGy. The lethal dose was highly reduced to 2 KGy by combination with lactic acid 0.1 % (W/V).

Debaryomyces sp. was highly resistant to irradiation treatment and more survive than Pseudomonas aeruginosa or Staphylococcus aureus

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where, the lethal dose was at 7 kGy which decreased to 4 kGy by combination with nisin 200 µg/ml or with citric acid 1.5 % (W/V) or cinnamon 1 % (W/V). Also, this lethal dose lowered to 3 kGy with lactic acid 1.5 % (W/V).

3.1. Combination with pasteurized temperature 3.1.1. Combination with Nisin The lethal conc. for S. aureus was at 250 µg/ml, which highly reduced to 25 µg/ml by combination with pasteurized temperature 90 C,ْ 30 sec. While, nisin alone has no effect on P. aeruginosa or Debaryomyces sp., by ,combination with pasteurized temperature 90 Cْ 30 sec lethal conc. effective at 150 µg/ml for P. aeruginosa and to 50 µg/ml for Debaryomyces sp.

3.1.2. Combination with citric acid The lethal conc. of S. aureus was 0.2 % (W/V), which lowered to 0.1 % (W/V) by combination with pasteurized .temperature 90 Cْ 30 sec The lethal conc. of P. aeruginosa was 0.3 % (wt/v), which highly decreased to 0.15 % (W/V) by combination .with pasteurized temperature 90 Cْ 30 sec For Debaryomyces sp. was the most resistant to citric acid than P. aeruginosa or S. aureus. Where, the lethal

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conc. reached to 4.0 % (W/V). This conc. decreased to 3.0 ْ W/V), by combination with pasteurized temperature 90) % C for 30 sec.

3.1.3. Combination with lactic acid The lethal conc. of S. aureus was 0.15 % (W/V), by combination with pasteurized temperature 90 ْ C 30sec, the lethal conc. of lactic acid reduced to 0.05% (W/V). The lethal conc. of P. aeruginosa was 0.3 % (W/V). This conc. decreased to 0.15% (W/V) by combination with .pasteurized temperature 90 ْ C for 30 sec For Debaryomyces sp. was highly survive to lactic acid, where the lethal conc. was 4.5 % (W/V), which lowered to 0.5 % (W/V) by combination with pasteurized .temperature 90 Cْ 30 sec 3.1.4. Combination with cinnamon

The lethal conc. of S. aureus was 1.2 % (W/V) and by .combination with pasteurized temperature 90 Cْ for 30 sec it was reduced to 0.4 % (W/V). The lethal conc. for P. aeruginosa was 4.0 % (W/V), by ,.combination with pasteurized temperature90 Cْ for 30 sec it was reached to 0.6 % (W/V).

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While, for Debaryomyces sp. the lethal conc. was at 2.0 % (W/V), by the combination with pasteurized temperature 90 Cْ for 30 sec, the sub-lethal conc. of cinnamon decereased to 0.6 % (W/V).

3.2. Combination of Nisin with nature preservatives 3.2.1. Combination with citric acid The lethal conc. of S. aureus was 0.2 % (W/V), which decreased to 0.05 % (W/V) by combination with nisin (25 µg/ml). The lethal conc. of P. aeruginosa was 0.3 % (wt/v), which decreased to 0.2 % (W/V) by combination with nisin 200 µg/ml. For Debaryomyces sp. was the most resistant to citric acid than P. aeruginosa or S. aureus. Where, the lethal conc. reached to 4.0 % (W/V). This conc. decreased to 3.0 % (W/V), by combination with nisin 200µg/ml.

3.2.2. Combination with lactic acid

The lethal conc. of S. aureus was 0.15 % (W/V), which reduced to 0.1 % (W/V) by combination with nisin 25 µg/ml.

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The lethal conc. of P. aeruginosa was 0.3 % (W/V), which reduced to 0.1 % (W/V) by combination with nisin 200 µg/ml. For Debaryomyces sp. was highly survive to lactic acid, where the lethal conc. was 4.5 % (W/V), which decreased to 3.5 % (W/V) by combination with nisin 200 µg/ml.

3.2.3. Combination with cinnamon

The lethal conc. of S. aureus was 1.2 % (W/V) and by combination with nisin 25 µg/ml, it was lowered to 0.5 % (W/V). The lethal conc. for P. aeruginosa was 4.0 % (W/V) and by combination with nisin 200 μg/ml it was decreased to 2 % (W/V). While, for Debaryomyces sp. the lethal conc. was at 2.0 % (W/V) and by combination with nisin 200 μg/ml the lethal conc. redeuced to 2.0 % (W/V).

5. Storage The Debaryomyce sp. was selected for storage due to it was more resistant to physical and natural treatments

than Staphylococcus and Pseudomonas

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5.1. Nisin was used in concentration 200 µg/ml in combination with temperature 90 Cْ for 60, 120 sec. and at citric acid concentration 2.0%, it was more effective for more than 90 days without any growth. Pasteurized temperature 90 Cْ for 30 sec followed .5.2 by gamma irradiation (0.25 kGy) were performed, results showed, nil count in all serial dilutions and it was effective for more than 90 days.

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CONCOLUSION & & RECOMMENDATION

206

CONCOLUSION AND RECOMMENDATION

The best results obtained from our experiments for preserving the fruit juice were occure by combing the pasteurized temperature 90 Cْ for 30 sec. with gamma irradiation (0.2 kGy). Or using 200µg/ml of nisin in combination with 2 % (W/V) of citric acid and pasteurized .temperature90 Cْ for 30 sec

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