Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 Functionality of egg yolk lecithin and protein and functionality enhancement of protein by controlled enzymatic hydrolysis Guang Wang Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, and the Food Science Commons Recommended Citation Wang, Guang, "Functionality of egg yolk lecithin and protein and functionality enhancement of protein by controlled enzymatic hydrolysis" (2007). Retrospective Theses and Dissertations. 15093. https://lib.dr.iastate.edu/rtd/15093 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Functionality of egg yolk lecithin and protein and functionality enhancement of protein by controlled enzymatic hydrolysis by Guang Wang A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Food Science and Technology Program of Study Committee: Tong Wang, Major Professor Pamela J. White Donald C. Beitz Iowa State University Ames, Iowa 2007 Copyright © Guang Wang, 2007. All rights reserved. UMI Number: 1446129 UMI Microform 1446129 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ii TABLE OF CONTENTS CHAPTER 1. GENERAL INTRODUCTION 1 LITERATURE REVIEW 1 THESIS ORGANIZATION 9 REFERENCES 10 CHAPTER 2. EMULSIFICATION PROPERTIES AND OXIDATIVE STABILITY OF EGG YOLK LECITHIN 13 ABSTRACT 13 INTRODUCTION 14 EXPERIMENTAL PROCEDURES 16 RESULTS AND DISCUSSION 20 ACKNOWLEDGEMENTS 26 REFERENCES 27 CHAPTER 3. EFFECT OF CONTROLLED ENZYMATIC HYDROLYSIS ON FUNCTIONALITIES OF EGG YOLK PROTEIN 42 ABSTRACT 42 INTRODUCTION 43 MATERIALS AND METHODS 45 RESULTS AND DISCUSSION 50 ACKNOWLEDGMENTS 54 REFERENCES 55 CHAPTER 4. GENERAL CONCLUSIONS 65 ACKNOWLEDGEMENTS 67 1 CHAPTER 1. GENERAL INTRODUCTION LITERATURE REVIEW Egg yolk is a good source of choline, omega-3 fatty acids, and natural antioxidant for humans, as well as a high quality protein. Egg yolk contains 31.8-35.5% lipids, 15.7-16.6% proteins as illustrated in table 1, almost all vitamins (A, B, D, E, K) except for vitamin C, and multiple minerals [1, 2]. Yolk lipids are almost all associated with proteins among which phospholipids (PLs) comprise about 30% of total lipids [1]. Egg yolk PLs, also known as egg yolk lecithin (EYL), are mainly composed of phosphatidylcholine (lecithin, PC, 66-76%) and phosphatidylethanolamine (cephalin, PE, 15-24%) [3]. EYL has been extensively used in foods, pharmaceuticals, and cosmetics because it is a good emulsifier, and it is usually believed to be well-tolerated and non-toxic in the native form. Native yolk proteins mainly exist as lipoproteins that are separated into a plasma fraction (soluble materials upon centrifugation) and a granular fraction (precipitants upon centrifugation). Egg yolk protein (EYP) is almost denatured after total lipid extraction where ethanol and hexanes are applied [4]. It is critically important to determine and improve its functional properties in order to make the further processing more economically feasible for industry. This review will place emphasis on EYL and lipid-free EYP, which has been subjected to ethanol and hexanes extraction. Three aspects of EYL and EYP will be covered: 1) 2 emulsification properties of EYL; 2) oxidation stability of EYL; 3) controlled enzymatic hydrolysis of protein and its application on lipid-free EYP. Table 1 Composition of egg yolk [1, 2, 3] Water Protein Lipid Carbohydrate Ash 48-52% 15.7-16.6% 31.8-35.5% 0.2-1.0% 1.1% Plasma 78% TAG 66% Granular 22% PLs 28% Phosvitin 16% Cholesterol 3% Lipovitellins 70% LDL 12% LDL: low density lipoprotein; TAG: triacyglycerol; PLs: phospholipids Emulsification properties of phospholipids An emulsion comprises two immiscible liquids, with one of the liquids finely dispersed as small droplets in the other. The liquid making up the droplets in an emulsion is known as the dispersed phase, whereas the other liquid surrounding the dispersed phase is defined as the continuous phase. Oil and water are commonly used liquids in making emulsion. Emulsion can be conventionally categorized into two classes, e.g., oil in water (o/w) and water in oil (w/o) systems according to the relative spatial distribution of the oil and aqueous phase [5]. Most food-based emulsions are o/w formation where the oil is the dispersed phase and the water is the continuous phase, such as beverage, mayonnaises, and salad dressings. Margarine is an example of w/o emulsion. Because most oils in a food matrix contain medium- and long-chain fatty acids and are highly hydrophobic (water-hating), the surface tension is quite high if the two liquids are directly emulsified without any emulsifiers. Typically, separation into two distinct layers happens immediately after homogenization. Surface active materials, known as emulsifiers or 3 surfactants, such as fatty acids, PLs, protein, polysaccharides, are usually used as amphiphilic materials, and they could reduce surface tension, thereby kinetically stabilize emulsions for a relatively long shelf-life. As emulsifiers, PLs function by coating on oil droplets and reducing surface tension between immiscible phases. Similar to other emulsifiers, various interactions are involved in stabilizing PL-stabilized o/w emulsions, such as Van der Waals force, steric interaction, hydration interaction, electrostatic force, and hydrophobic interaction [5]. Emulsification properties of purified individual PL originating from both soybean and egg yolk have been investigated under various conditions [6, 7]. However, EYL contain several types of PL and the interaction among PL class composition should be taken into account when it is applied into food as a whole. As mentioned earlier, EYL is mainly composed of PC and PE, which are at 66~76% and 15~24%, respectively. Lysophospholipid (LPC, 3-6%), sphingomyelin (SM, 3-6%), and phosphatidylinositol (PI, 0.6%) are also found in EYL as minor components [8]. EYL have different PL class compositions from soy lecithin (SL) (Chapter 2, Table 1). SL contains less proportion of PC (33%) and PE (14.1%) but more PI (16.8%) and phosphatidic acid (PA, 6.4%). Among these phospholipids, PC and PE are zwitterions, whereas PI and PA are anions as illustrated below: Phosphatidylcholine (PC) 4 Phosphatidylethanolamine (PE) Phosphatidylinositol (PI), sodium salt Phosphatidic acid (PA), sodium salt The electrostatic repulsion is usually the dominant force in the emulsion stabilized by charged PLs, such as PI and PA in SL. It was also reported that zwitterionic PLs stabilize emulsion mainly through hydration interaction due to the electronic characteristics of zero net charge, though electrostatic interaction was also observed [9, 10]. Hydration repulsion of PC and PE becomes predominant as the distance between the droplets gets closer [11, 12]. Van der Meeren [9] studied the emulsion stability with a mixture of the anionic PA in the zwitterionic PC and found that the emulsion stability was improved by partly replacing PC with PA when the amount of PC used was not high enough to form a stable emulsion. This study indicates that anionic PA and PI are good at stabilizing emulsions compared with zwitterionic PC and PE at lower concentrations. These emulsions stabilized mainly through electrostatic repulsion, such as PI and PA stabilized ones, are susceptible to change of ionic strength, pH, surface charge density, and 5 surface potential. For those stabilized through hydration interaction, such as PC and PE, they are usually sensitive to temperature change [5]. Lecithin oxidation in bulk systems and emulsions Lipid oxidation has long been recognized as a major cause of deterioration of fatty foods. Several mechanisms have been proposed and widely accepted to explain lipid oxidation by autoxidation, photooxidation, and enzyme-catalyzed oxidation. Lipid oxidation results in off- flavor (rancidity) and changes in texture and other sensory characteristics of fatty foods. It also affects physical properties, such as smoke point and viscosity. The oxidation products, hydroperoxides and their secondary decomposites, such as short-chain volatile compounds, ketodienes, and epoxy compounds, are potentially toxic compounds and may cause chronic disease, such as atherosclerosis when consumed [13]. Three steps involved in lipid oxidation are initiation, propagation, and termination. The abstraction of hydrogen atom from allylic or bisallylic position has been found to be the rate- determining step in the oxidation process [14]. Because the bond dissociation energy for monoallylic C-H is ~10 kcal/mol higher than that for bisallylic C-H, which is ~85 kcal/mol [14], oxidation rate is largely determined by the amount of di- and poly-unsaturated double bonds present in the lipid. Research has demonstrated that fatty acids with two conjugated double bonds oxidize 10 times faster than do mono-unsaturated fatty acids; a further increase in each activated