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    THẠC SĨ Purification of lysozyme from shell liquor of eastern oysters (crassostrea virginica) and its use in antimicrobial films to preserve smoked fish

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  6. Purification of lysozyme from shell liquor of eastern oysters (crassostrea virginica) and its use in antimicrobial films to preserve smoked fish

    ACKNOWLEDGMENTS

    I would like to thank my major professor Dr. Jack Losso and committee members Dr.
    Jerome La Peyre, Dr. Marlene Janes, Dr. Witoon Prinyawiwatkul and Dr. Qing-Gang Xue for
    their guidance and support throughout this study.
    The financial support of the NOAA Saltonstall-Kennedy Grant Program and the Gulf
    Oyster Industry Program, which made the present work possible, is highly acknowledged.
    Special thanks to Al Sunseri and his personnel at P & J Oyster Company (New Orleans, LA)
    for providing the samples for the whole study.
    I would like to take this opportunity to thank my parents and parents-in-law for their
    encouragement and all my friends especially Sajida Begum, Tracy Dupard, Madhavi
    Chintalapati, Evi Menelaou, and Aisha Abushelaibi for their assistance and moral support
    over the last two years. Special thanks go to Rishipal Bansode, Sonja Jones, and Dr. Richelle
    Beverly for their suggestions, which have been of great help at all times. Acknowledgements
    are due to Dr. James Geaghan and Sangeeta Singh who helped me understand statistics.
    Most importantly, I would like to thank my husband Proyag Datta for being there by
    my side in all the difficult moments. His remarkable support and encouragement has made
    this study possible.
    iii

    TABLE OF CONTENTS
    ACKNOWLEDGMENTS ii
    LIST OF TABLES v
    LIST OF FIGURES . .vi
    ABSTRACT vii
    1. INTRODUCTION . 1
    2. LITERATURE REVIEW 4
    2.1. Properties of Lysozyme 4
    2.1.1. Definitions 4
    2.1.2. Sources of Lysozyme . .4
    2.1.3. Evidence of the Presence of Lysozyme in Oysters 5
    2.1.4. Structure of Lysozyme (Hen Egg White Lysozyme) . 7
    2.1.5. Mechanism of Lysozyme Action .8
    2.1.6. Inactivation of Bacteria 9
    2.2. Antimicrobial Properties of Lysozyme (Hen Egg White Lysozyme) . .10
    2.2.1. Action against Gram-Positive Bacteria 11
    2.2.2. Action against Gram-Negative Bacteria . .12
    2.3. Lysozyme Use in the Food Industry . . . 13
    2.4. Antimicrobial Films and Fish Products Preservation .14
    2.4.1. Foodborne Pathogens Associated with Fish and Fish Products 14
    2.4.2. Cold Smoked Salmon .14
    2.4.3. Edible Film Coatings . 17
    2.4.4. Listeria monocytogenes in Seafood .18
    2.4.5 Salmonella in Seafood . . .20
    3. PURIFICATION OF LYSOZYME FROM SHELL LIQUOR OF THE EASTERN
    OYSTER, CRASSOSTREA VIRGINICA AND ITS POTENTIAL USE AGAINST
    BACTERIA CAUSING FOOD POISONING AND FOOD SPOILAGE 22
    3.1. Introduction 22
    3.2. Materials and Methods .23
    3.2.1. Materials 23
    3.2.2. Methods .24
    3.2.2.1. Seasonal Variation of Protein and Lysozyme Concentration in
    Oyster Shell Liquor . . . 24
    3.2.2.1.1. Collection of Oyster Shell Liquor . .24
    3.2.2.1.2. Protein Concentration and Lysozyme Activity Determination 24
    3.2.2.1.3. Purification of Lysozyme from Oyster Shell Liquor . 25
    3.2.2.1.4. Molecular Weight and Purity of Oyster Shell Liquor Lysozyme 25
    3.2.2.1.5. Statistical Analysis .25
    3.2.2.2. Large Scale Purification of Lysozyme from Oyster Shell Liquor . 26
    iv
    3.2.2.2.1. Sample Processing .26
    3.2.2.2.2. Strong Cation Exchange Chromatography 26
    3.2.2.2.3. Weak Cation Exchange Chromatography 27
    3.2.2.2.4. Molecular Weight and Purity of Oyster Lysozyme .27
    3.2.2.3.Determination of Minimum Inhibitory Concentration (MIC)
    of Oyster Lysozyme and Hen Egg White Lysozyme . .27
    3.2.2.3.1. Preparation of Bacterial Cultures . 27
    3.2.2.3.2. Preparation of Lysozyme Solutions .27
    3.2.2.3.3. MIC Assays 28
    3.3. Results . 29
    3.3.1. Seasonal Variation of Protein and Lysozyme Concentration in Oyster
    Shell Liquor . 29
    3.3.2. Large Scale Purification of Lysozyme from Oyster Shell Liquor .32
    3.3.3. Determination of MIC of Oyster Lysozyme against Foodborne Pathogens 35
    3.4. Discussion .36
    4. CONTROL OF LISTERIA MONOCYTOGENES AND SALMONELLA ANATUM ON
    THE SURFACE OF SMOKED SALMON COATED WITH EDIBLE COATINGS
    CONTAINING OYSTER LYSOZYME AND NISIN 40
    4.1. Introduction . .40
    4.2. Materials and Methods .41
    4.2.1. Culture Growth Conditions . 41
    4.2.2. Preparation of Edible Coatings . .42
    4.2.3. Preparation of Antimicrobials and Fish Samples. 42
    4.2.4. Determination of the Most Effective Film Coating for Enhancing
    the Antimicrobial Activity of Oyster Lysozyme . . .42
    4.2.5. Effect of Oyster Lysozyme Incorporated into Calcium Alginate Coatings
    against Listeria monocytogens and Salmonella anatum during 35 days
    Storage .43
    4.2.6. Statistical Analysis . 44
    4.3. Results .44
    4.4. Discussion .51
    5. SUMMARY AND CONCLUSIONS 58
    REFERENCES . .59
    APPENDIX 1: DETERMINATION OF THE MOST EFFECTIVE FILM COATING FOR
    ENHANCING THE ANTIMICROBIAL ACTIVITY OF OYSTER LYSOZYME .64
    APPENDIX 2: EFFECT OF OYSTER LYSOZYME INCORPORATED INTO CALCIUM
    ALGINATE COATINGS AGAINST LISTERIA MONOCYTOGENES AND SALMONELLA
    ANATUM DURING 35 DAYS STORAGE. 65
    VITA 66

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