Influence of some physical treatments and natural food 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. 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 natural food 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 Food Microbiology 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. Fruit Juices and soft Drinks .......................................... 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 Dyes and Benzoates…………………………………… 16 2.5.3. Sulfites ………………………………………………………………… 17 2.5.4. Nitrites……………………………………………………………………………… 18 2.5.5. Other Agents………………………………………………… 20 3. The use of natural antimicrobials……………………………………… 22 3.1. Natural preservatives................................................... 25 3.1.1. Nisin…………………………………………………………………… 25 • Mechanism of action……………………………………. 31 3.1.2. Organic acids ………………………………………………………… 36 3.1.2.1. Mode of action………………………………………………… 38 3.1.2.2. Resistance mechanisms…………………………………… 42 3.1.2.3. Citric acid ………………………………………………………… 48 3.1.2.4. Lactic Acid………………………………………………………… 49 3.1.3. Spices 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 reduction 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 water 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. Pasteurization……………………………………………… 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 bacteria……………………………............................…………… 76 3. Isolation of yeast............................................................ 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. Organic acid ………………………………………… 112 2.2.2.1. Citric acid …………………………… 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