INFLUENCE OF LYSOZYME ON EGG WHITE QUALITY DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by OWEN JAY COTTERILL, B. S., M. S. The Ohio State University 19SU Approved by* Adviser i TABLE OF CONTENTS INTRODUCTION ................................................ 1 REVIEW OF LITERATURE........................................ h .Composition of Egg W h i t e ............. 5 Gross structure ............... 5 Chemical composition ............ 6 W a t e r ................................... • . • 6 Proteins • .................... ....... 7 Minerals ....... .......... ...... 10 Carbohydrates •••..•• • 10 L i p i d ........................................ 10 Vitamins . ........ 11 froperties of Egg White Proteins •••••••••••* 12 Physical ••••.«••• 12 Biological ............... lii Conalbumin ............. Ill Ovomucoid lU Avidin . .................................. 1$ Ovomucin .................................. 15 Lysozyme ............. 16 Factors Affecting Egg Whits Quality • • 19 Temperature .................................. 19 Humidity.......................................... 19 ii pH .... ...................................... 20 Disease 25 Breeding 26 Nutrition •••••... .............. ••••• 26 Season of year and age of b i r d ................... .. 27 Thermostabilization . .............. 27 Mechanism of Egg White Thinning...................... .. 30 Tiyptic digestion ..................... 30 Fibrous structure 32 Sulfhydryl groups . • ............................ 36 I^rsozyme-ovomucin interaction ••••••••••« 36 EXPERIMENTAL................................................ 1*2 Lysozyme Standard 1*2 Lysozyme Determination ............ Li* Measurement of Egg White Quality •••••*.••••* 52 Solids, Total Nitrogen, and Protein Determination • • « • 52 E g g s .................................................. 52 PART I RELATIVE LYSOZYME ACTIVITY IN FRESH EGGS HAVING LOW AND HIGH INTERIOR QUALITY............................................ 53 Review of L i t e r a t u r e ............................ .. 53 Experimental and Results .......... .. • 56 Lysozyme activity of eggs from individual birds • • 56 iii lysozyme activity of eggs from different strains of b i r d s .............................................. 57 Relationship of lysozyme activity to initial interior quality 57 Discussion 59 S u m m a r y ................................................ 60 PART II THE EFFECT OF THERMOSTABILIZATION OK LYSOZYME ACTIVITY .... 61 Review of Literature................................. • 61 Experimental and Results•.•••••••••••.••• 66 Lysozyme activity of thermostabilized eggs • • • • • 67 Thermostabilization temperatures higher than 130° for 15 minutes • 68 Age of eggs and lysozyme destruction........... 69 Shorter treatment times and higher temperatures • • • 70 Discussion............................. 71 Summary ........ .........................» • 73 PART III LYSOZYME-OVOMUCIN INTERACTION ................................ 75 R e v i e w ............................................... 75 Materials and Methods • • .................... ••••• 79 Thick wh i t e ..................................... 79 iv Crude ovomucin ............... 79 Buffer............................................ 80 lysozyme........................................ .. 80 Setting up each trial ...... 80 Thermostabilization .............................. 8l Experimental and R e s u l t s ............................ .. 82 Preliminary observations on this interaction .... 82 Effect of pH and thermostabilization on lysozyme- thick white interaction........................ .. 83 pH ....... .......................... 83 pH and thermostabilizati o n ................ .. 6U Effect of pH and thermostabilization on lysozyme- crude ovomucin interaction.......... ............. 85 p H .......................................... 85 pH and thermostabilization.................. 86 Effect of pH and dilution on thick white, crude ovomucin and l y s o z y m e ............................ 86 Effect of different thermostabilization temperatures at different pH levels............................ 87 Thick white ................ ........... .. 87 Crude ovomucin .......................... 88 Effect of sulfhydryl g r o u p s ...................... 88 Thick wh i t e .................................. 88 Crude o v o m u c i n ............................ .. 89 V Discussion................................................ 91 The influence of pH . .......................... 91 The influence of heat .......... 93 The influence of sulfhydryl g r o u p s ................ 9k S u m m a r y .................................................. 96 PART IV INFLUENCE OF AMMONIA ON EGG WHITE QUALITY...................... 97 Introduction and R e v i e w ............. 97 Experimental and Results.................................. 98 Quality and pH of whole e g g s ........................ 98 Extinction coefficient and pH of white and pH of yolk 99 Discussion.............................................. 100 S u m m a r y ................................................. 101 GENERAL D I S C U S S I O N ...................... ................... 102 GENERAL SUMMARY...................................... 108 TABLES . ................................................... Ill FIGURES....................................................... 122 REFERENCES ................................................... 132 ACKNOWLEDGEMENTS............................................. tkS AUTOBIOGRAPHY................................................. li*6 1 INTRODUCTION The importance of shell egg quality may be stressed by the fact that about 85 percent of all eggs produced reach the point of consumption in the shell. The factors used to determine quality in shell eggs may be divided into two general groups; (1 ) those affecting the condition of the shell, and (2 ) those affecting the nature and composition of the interior contents* The latter group is often referred to as Interior Quality or more simply abbreviated as I* Q. In deter­ mining I. Q., the physical consistency of egg white warrants special consideration* The determination of quality in a foodstuff is often linked with some physical change in the appearance of the product* One such example is that of egg white thinning* The nature of the thick white gel in the shell egg is one of the more important factors used to determine the apparent I* Q. and the ultimate economic value of this food product in market channels* The physical condition of the white is used as a "built-in*' indicator which generally designates the functional and eating qualities of the product. The importance of egg white thinning in determining the commercial grade of shell eggs is manifested by observing the position and definition of the yolk shadow when the egg is candled* If the white is thick and cloudy, then the yolk shadow will be centered and diffuse. Any thinning or clearing of the white will tend to make the yolk shadow more defined and off center. The environmental factors which affect the thinning of egg white have been the subject of many investigations. The influences of storage temperatures and loss of carbon dioxide have been demonstrated many times, A part of this thesis will deal with a discussion of the environmental and other factors that affect egg white quality. Various means have been employed to retard the loss of quality resulting from egg white thinning. Correct application of these materials and methods are very affective in minimizing the loss of quality* In one instance the I, Q., as measured by the condition of the white, can actually be improved. However, the most effective of these methods is not accepted in ccnuaercial practice at the present time. The development of new methods for shell egg preservation or the improvement in existing practices is hampered by the lack of knowledge concerning the mechanism or nature of the changes taking place during the thinning of the white. The current theories which have a bearing on this subject will be reviewed. One of these theories is based on the interaction of two egg white proteins, namely, ovomucin and lysozyme. The main objective of this study was to determine the possible role of lysozyme in egg white thinning and some of the conditions which affect its interaction with ovomucin. This study was divided into four parts t (1) Relative lysozyme activity in fresh egF;a having low and high interior quality; (2 ) 3 Effect of thermostabilization on lysozyme activity; (3) Lysozyme- ovcmucin interaction, and (U) Effect of ammonia on egg white thinning* A general review of the literature and discussion of materials and methods will be presented independent of the above parts. The more specific review and discussion of procedures will accompany each part. The discussion of results and summary will be included with each part. A general discussion and summary will be presented at the end of the text. h RE VIE* OF LITERATURE Seme general characteristics of egg white will be considered before attempting to discuss the more fundamental aspects of this material* First consideration will be devoted to the composition and some of the properties of many of the constituents of egg white# Next, the factors which affect the physical nature of the white will be presented* Finally, major emphasis will be given some of the theories which have been presented to explain the mechanism of egg white thinning* Many excellent reviews have summarized various