SYNTHETIC MEMBRANE FORMATION AS APPLIED TO A SIMULATED EGG YOLK By James David Mingus AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fuifiiiment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1978 In. ..::> ,C1 ABSTRACT SYNTHETIC MEMBRANE FORMATION AS APPLIED TO A SIMULATED EGG YOLK By James David Mingus Contemporary emphasis on cholesterol as an undesirable dietary ingredient for some individuals has nurtured the commercial development of fabricated liquid egg substi- tutions. Utilization as conventionally cooked eggs is primarily restricted to the "scrambled" style. The objec- tive of this study is to replace the natural egg yolk with a functional, low cholesterol simulated yolk, which resem- bles the appearance and functionality of natural egg. Sodium alginate can be reacted with polyvalent ions such as calcium to produce food matrix systems, and modified to simulate various food textures. When yolk-resembling emulsion, containing calcium salt is inserted into an algin solution of approximately equal density for a short period of time, film formation giving artificial yolk is obtained. This encapsulation resembles the appearance of the yolk vitelline membrane. An acceptable emulsion was formulated using fresh liquid egg white, vegetable oils, calcium salts, flavoring, coloring, emulsifier (lecithin), and a protein stabilizer James David Mingus (whey protein concentrate). ACKNOWLEDGEMENTS The author wishes to express sincere thanks and appreciation to his major professor, Dr. J.R. Brunner, for his continual encouragement, advice, guidance (and patience), toward completion of this research and manu- script. The author also wishes to acknowledge the efforts of guidance committee members, Dr. L.L. Bieber, Dr. J.N. Cash and Dr. L.E. Dawson, for their advice, aid in obtaining materials and references, and for their review of the manu- script. A special thanks to Ursula Koch for her continual assistance in the lab. Others who have aided in research endeavors with their time and/or equipment include: Dr. R.F. McFeeters, Dr. M.A. Uebersax, Kit Uebersax, and Dr. M.E. Zabik, and the occu- pants of office 336, Food Science Building. A special thanks of appreciation, for their continued interest and encouragement belongs to the author's family (especially parents) and the many friends from the Okemos Church of the Nazarene, (in particular John Riebow and Rev. and Mrs. Ed Comandella). Finally, the author wishes to express gratitude to his wife, Peggy, for her love, encouragement, and countless hours to completion of this project. 1'1 TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES LIST OF APPENDIX TABLES INTRODUCTION. LITERATURE REVIEW Low Cholesterol Egg Products Emulsion Stability . Protein Crosslinking and Film Formation. Sulfur Compounds . . . . Ruminant Feeds Polyelectrolytes Wool and Fiber Reactions . . Enzyme Immobilization and Glutaraldehyde Tannins. . . Phosphates and Starch Crosslinkers Polysaccharide Films . . Applications of Algin in Foods EXPERIMENTAL. Exploratory Methods and Materials. General Methods and Materials. Oil Selection and Drop Spreading Emulsion Stability, Texture and Taste. Studies. Crosslinker Studies. . Polysaccharide Films and Emulsion Interaction. pH and Crosslinker Mixing Effects. Polysaccharide and Salt Solutions. Artificial Yolk Emulsion Manufacture Polysaccharide Film Formation. . . Whey Dialysis to Increase Protein Concen- tration Emulsion Stability after Salt Incorporation: Egg White and "Yolk" Binding Taste Panel. . N-a_.a._:_a__a_.a Ammmmwmum NNN NhN 32 RESULTS. Evaluation of Exploratory Observation Emulsions Protein Crosslinkers. Selection of the Oil Phase. Oil- -Drop Spread . Emulsification Potential of Lecithin and Protein Solids. . . Effect of pH on Oil- -Drop spread . . Centrifugation Verification of Oil- -Drop Spread Test. . . . . . . . . . . . . . Emulsion Stability. . . Additional Stability Studies. Coagulated Emulsion Texture Compressibility Taste Relative Viscosities. Yolk Weights. Crosslinkers. . Polysaccharides . Algin Selection Salt Selection. . Alginate Concentration. . . . Emulsion Stability and Function After Calcium Salt Addition . Acid and pH Effects on Emulsions Containing CBICium Acetate . . . . Yolk Rupture and Yolk Index Comparisons Artificial Yolk- -Egg White Adherence Taste Panel . . DISCUSSION Selection of Emulsion Ingredients Oil . . Other Ingredients Lecithin. Whey Solids . . Citric Acid . . . Liquid Egg White. Emulsion Stability. Centrifugation. . . Microscopic Analysis. . Correlation Between Stability and Textural Studies . . . . . . . . . . Relative Viscosities. Texture Comparisons Between Hard- Boiled Yolks and Heat- -Coagulated Emulsions . . Size Comparisons of Yolk and Formed Emul- sions . . . . . . . . . . . . . iv Emulsion Encapsulation with Alginate and Com- parison with Authentic Yolks. . Alginate Films. . Calcium- -Alginate Reaction Encapsulated Emulsion . Films . . . Yolk Membrane Strength. Yolk Index. . . Cooked Simulated Yolks. . . Stability of Emulsions with Safflower Oil and Whey Isolate. . . . . Taste Panel Responses . . Stability of Stored Samples CONCLUSION . . . . APPENDIX BIBLIOGRAPHY Table II III IV VI VII VIII LIST OF TABLES Typical Bifunctional Protein Crosslinking Reagents (Uy and Wold). . Examples of Ruminant Feed Crosslinking Compounds . . . . . Compounds Used for Whey Precipitation (Sternberg, 1975) . . . . . . . Solutions and Conditions Used for Oil Drop Spread Tests. . . . . . . . . . Emulsion Conditions and Methods of Mixing Used to Study Stability, Texture and Taste PY‘operties. . . . . . . Emulsion Ingredients and Special Conditions Used to Establish the Effect of Salts and Homogenization Treatments on Stability. All emulsion Listed Contained 1% Lecithin and 0.2% Egg Flavoring. . . . . Artificial Yolk Pressure and Yolk Index Evaluations Summary of Taste Panel Evaluation Scores For Soft Poached Eggs with Emulsion "Yolks" . . . . vi Page l4 T7 '18 37 38 48 75 76 Figure 10 ll 12 LIST oF FIGURES Metal forms used to fabricate simulated hard boiled "yolks" for texture studies. Method of inserting emulsion into an algin solution to form "yolk" . . . . . A formed "yolk" with calipers and height gauge used to measure yolk index. . Centrifugally induced phase separation in formulated emulsion as influenced by ingre- dients and pH . . . . . . . Compressibility of coagulated emulsions as influences by ingredients and pH. Stability increasing effects of some emul- sion ingredients and emulsification vari- ables . . . . . . . . . . . . Stability comparison of emulsions using all of the oils originally tried in the drop- spread tests. . . . . Homogenization influence on particle size and emulsion stability in comparison with authen— tic yolk. . . . . . . . . . . Allo- Kramer Shear Press peak profile com- paring a hard boiled yolk and coagulated emulsion. . . . Comparison of raw yolk vitelline membrane and algin films , . . . . . Poached eggs. Fried eggs. vii Page 34 47 '47 57 62 73 73 84 88 91 93 94 Table Al A2 A3 A4 A5 A6 Figure A1 B LIST OF APPENDIX TABLES Results of oil spread studies on solutions with potential use in artificial yolk emulsions. Results of oil drop spread studies on solutions, containing egg white with additional solids and varying lecithin percentages. Potential crosslinker or film forming compounds selected for reaction with emulsion at initial and various levels of adjusted pH Summary of crosslinker responses (reactions include No Response (NR), Coagulation (C), Pre- cipitation (P), and Film Formation (FF) Relative viscosity comparisons of emulsions and egg yolks based on emptying times from a 20 ml pipette . . . . . . . . . . . . . . Chattilon pressure and yolk index values for artificial egg yolks with varied emulsification and formation timesa. . . Microscopic Evaluation of Emulsions TASTE PANEL FORM. viii Page 100 . 101 103 105 108 110 114 116 INTRODUCTION There has been a nutritional appeal for low choles- terol and saturated lipid diets to reduce atherosclerotic diseases, generating the production of low cholesterol food substitutes. As a major source of dietary cholesterol, the egg yolk has been widely condemned, thus reducing egg Con- sumption for persons desiring to restrict dietary choles- terol. Numerous substitute egg products have been fabricated, but few as functional, whole, liquid centered, multiple-use, egg yolk replacements. This study addresses the feasibility of developing an artificial egg yolk by encapsulation of an oil-liquid emul- sion in a membrane-like film. A two phase project was developed. The first, was to establish a stable emulsion, possessing yolk-like appearance and functionality. The second, and most important, was to encapsulate the emulsion, producing the appearance of a freshly separated, intact egg yolk. The primary object of this study, therefore, was to evaluate the practicality of a simulated whole yolk, and to select ingredients appropriate for compounding such a pro- duct. LITERATURE REVIEW Controversy exists over the status of cholesterol and saturated lipids as undesirable ingredients in the diet. McGill and Molt (l976), summarize the widely accepted belief that high dietary cholesterol is a major determinant of high serum cholesterol in the U. S. population and the belief that mild dietary manipulations, limiting eggs, animal fats, and dairy products, can reduce cholesterol intake signifi- cantly. Low Cholesterol Egg Products The emphasis on cholesterol and saturated fats as undesirable dietary ingredients has nurtured the develop- ment of numerous substitute egg products. Because the lipid fraction of the egg is concentrated in the yolk, major emphasis centers on reducing, substituting or modifying the yolk. Both United States and foreign patent literature con- tain numerous examples of low cholesterol egg products. However, a review of'U.S. patents will suffice since few, if any, foreign patents contain novel ideas. Most patents deal with either whole egg or egg yolk replacements for use in baking or for "scrambled style" cooked eggs. Gorman (l965) was the first to recognize the need for dietary considerations in an egg formulation. His product utilized dried egg albumin, agglomerated nonfat milk solids, grain or root flours and polyunsaturated oils. On mixing with water a scrambled-type product was obtained. Although Jones' (1969) major purpose was to find a functionally and bacteriologically superior low-fat product, he recognized cholesterol reduction as an advantage in egg formulations. He reduced the yolk fraction of his product with milk solids, vegetable proteins, a hydrophobic thixo- trophic material - such as carboxymethyl cellulose - and increased egg white content. Melnick (l97l) described a solvent extraction method designed to reduce cholesterol and unsaturated fats in yolks, while Levin (l97l) proposed a lipid solvent - extraction method to be used on whole raw eggs. An artificial egg yolk formulation which is claimed to be indistinguishable in taste from actual egg yolk was developed by Perret (l974). He incorporated amino acids and an inorganic sulfide into an albumin and oil emulsion, adding emulsifying and stabilizing ingredients to achieve a slightly alkaline pH. This product was usable as a replace- ment in recipes or as scrambled eggs. Strong (l974) des- cribes a scrambled-style product produced from liquid egg white, corn oil, nonfat dry milk solids, preservatives and coloring agents. The mixture was pasteurized and, emulsi- fied to attain stability. To simulate whole egg appearance, B-carotene and xanthophyll were added. In l974, Seeley updated a patent of Ziegler, Seeley and Holland, (l97l) which used a mixture of nonfat milk solids, potato flour, and water, with carboxymethyl cellu- lose and citric acid added to whole eggs, to yield a stable egg product. Seeley reduced yolk content by adding non egg ingredients to a level that doesn't greatly impair texture or flavor, yet results in a low calorie, low cholesterol product. When Strong and Redfern (l975), incorporated xanthan gum alone or in conjunction with guar gum or carboxymethyl cellulose to an egg white, oil, milk solid and lecithin emulsion, they obtained a low cholesterol egg product with improved texture and stability. Colors and flavors were added as desired. Glaser and Ingersen (l975), developed a method for stabilizing a liquid scrambled - egg product. Since most low cholesterol egg products are sold in the frozen state, their aim was to produce a stable liquid for- mulation. They succeeded by combining egg white, polyun- saturated vegetable oils and a fatty acid lactylate alkali metal salt at about 0.05 - 1.0%, with other additives and emulsifiers. Math and Newbold (l976) employed ultracentrifugation of egg yolks to separate three yolk fractions, one of which contained a proportionately high concentration of choles- terol. The other fractions were used as yolk replacements in fabricated foods. A process for producing both artificial egg white and yolk which can be used to regulate lipid and cholesterol levels was described in a patent by Johnson (l972). The white was made primarily of water, oil, and gelling agents, such as low methoxy pectin and dicalcium phosphate. The yolk was comprised of water, proteins, coloring, flavoring, and binding agents similar to those incorporated into the white. Moulding gives convenient egg forms such as hard boiled, deviled, poached, or fried. These can be consumed either cold or warmed. . Perhaps the closest system to a natural appearing egg was invented by Glasser and Matos (1976). A yolk like emulsion was layered into a form and then covered with reconstituted powdered egg white. This is sealed and frozen, to be cooked later without prior thawing. Major ingredients in the yolk include dried egg white, an unsaturated oil, water and added proteins. Flavor and physical properties were adjusted by using sodium hexametaphosphate, sodium caseinate, and starch. These ingredients were stabilized by combining hydrophilic and hydrophobic emulsifiers to simulate a lecithin-cholesterol balance. Also, gums such as guar gum, gum arabic or a cellulosic derivative impart a proper yolk-like glossy sheen. Besides a sunnyside-up formed egg, the yolk emulsion gives satisfactory results in formulations. Water content can be adjusted to impart the desired runny or dry consistency. Although each patent portrays some characteristic feature(s), actual commercial egg replacements exhibit marked similarities. Common items for baking formulations include soy or wheat flour or starch, lecithin, egg white or vegetable protein, and artificial color. Varying with intended use, other ingredients are gums, lactose, preser- vatives, glycerin, and spice or flavor enhancers. Examples of commercial products available include: Monark Egg Cor- poration (Mogold-CEB and Mogold-Tex#lOR), Stauffer Corpo- ration (Triet 2C and Triet TMERB), and Precision Foods Company (Artiyolk). Claiming 96% cholesterol removal, the Fanning Chemical Company and Viobin Corporation produce defatted egg powders to be utilized either in scrambled egg mixes or in cooking preparations. These are essentially fat-free egg prepara- tions. Similarly, scrambled breakfast egg products contain many common ingredients. Most are comprised of 80% liquid egg white, a protein source such as milk solids, soy iso- late or egg white solids, and vegetable oils. As of July, l976, the following companies offered such products: Draper Products (Egg Delight), Fleishmann (Egg Beaters), Avoset Food Corporation (Second Nature), General Mills (Scramblins) and Miles Laboratories (Scramblers). All are cholesterol free except Egg Delight, which is advertised as "low cho- lesterol". And all are frozen products except Second Nature, which is a refrigerated product. Second Nature can be used as baking replacement for whole eggs and is fortified with vitamins and minerals. Emulsion Stability According to Glaser and Ingerson (1975), a major dis- advantage of most egg substitute products is their poor physical stability; partly due to the inability of egg white to combine with oil to form a stable emulsion. This charac- teristic necessitates the distribution of most simulated egg product in frozen form. In a system where numerous components are incorporated, as an egg emulsion, the emulsion becomes quite complex. It is much more complicated than the simple oil micelle, where according to Walnack, Barrington, and Faller (l960), emulsi- fier is adsorbed to the oil droplet decreasing interfacial tension and giving each droplet a similar net electrostatic charge, thus repelling one another. Although much is known concerning the behavior and properties of emulsion systems containing only two or three components under given condi- tions (i.e. mixing, temperature, concentration), most food systems are multicomponent. Increasing complexity decreases predictability of emulsion characteristics. In the past researchers adopted an Edisonian approach (Wolnick gt 11., 1960), to obtain satisfactory, stable emulsions. Trial and error selection of emulsifiers for most food products is based on experience and pilot plant tests (Krog and Laurid- sen, 1976). Because this approach is time consuming and expensive, an ideal approach is to use model systems for objective measurements of functional properties (Kinsella, 1976). But the number and complexity of food systems make it difficult to validly extrapolate data from model systems to specific food application. The trial and error approach can be altered somewhat, as discussed by Smith 33 11. (1977), by using computer plotting and response methodology, but ingredients and processing variables still must be limited. Krog and Lauridsen (1976) states that most food emulsions are not governed by simple emulsion or emulsifier evalua- tions such as the common hydrophile-lypophile balance (HLB) system due to narrow classification and the lack of accoun- ting for complex formations from such components as protein and starch. Simulated egg yolk formulations fit these characteristics. A brief look at factors and ingredients that might influence egg emulsions is necessary to obtain a feel for their tendency toward instability. The type and amount of lipid and emulsifier in a food emulsion significantly affect emulsion stability. Their influence, however, can be mini- mized and predicted by knowing what type of emulsifier or oil is needed for desired conditions or effects. Oil- emulsifier matches are made much easier by following recom- mendations presented in the emulsifier literature and selecting those which work best in water-oil (w/O) or oil- water (O/W) systems. Proper hydrophile-lypophile balance, pairing emulsifiers and oil; and such characteristics as phase inversion temperature (PIT) and desired texture or viscosity also influence ingredient selection. In general ions (particularly cations), destabilize emulsions, with polyvalent ions showing much greater desta- bilizing effects than monovalent ions (Friberg, 1976). However, Princen (1972) notes that, occasionally, stability may be increased by addition of electrolytes. For example, anionic emulsifiers in contact with cations in O/W emul- sions may form soaps which then form stable W/O emulsions. Petrowski (l976) observes that salts may have markedly variable affects on proteins in emulsions. They influence ion binding, ionic strength, and alter solvent properties. The salting-in or salting-out characteristics affect emul- sion stability. Petrowski (1976) states that heat effects on an emul- sion are basically twofold. Upon manufacture, heat is advantageous for both sterilization and emulsification, but promotes destabilization once the emulsion is formed. Krog and Lauridsen (1976) describe three main func- tions of food emulsifiers: 1) The reduction of surface tension at oil interfaces, induces and stabilizes emulsification, resulting in phase equilibrium. 2) Interaction with starch and protein components in foods modify texture and rheological properties. 3) The crystallization of fats and oils is modified. They note also that the method of emulsifier incorpo- ration significantly influences optimum results in many 10 types of food products. Petrowski (1976) points out the importance of droplet size as a major consideration in emulsion stability. The larger the droplet size the greater the attractive forces and the less stable the emulsion. Emulsions destabilize in the order of drop aggregation or floculation, creaming (density differences), and coalescence. When mixing, it must be determined whether or not particle size reduction for stability is more important than possible adverse shearing or denaturation. Occasionally more than one necessary agent used in food preparation will show antagonistic emulsifying proper- ties, with competing O/W and W/O tendencies and thus create an overall instability. Perhaps the greatest area of interest for emulsion technology in relationship to egg emulsions is that of the role of proteins. These play a significant part in the characteristics and functionality of an emulsified food system. Kinsella (1976) gives a good summary of the func- tionality and variables involved in emulsions and particu- larly those involving proteins. In solution proteins adsorb and absorb other food ingredients, including volatile components, flavors, lipids and water. They influence viscosity, film formation, ad- hesion, and fiber formations. Emulsion characteristics and binding properties of proteins respond according to condi- tions of temperature, pH, ionic strength, chemical or 11 enzymatic modification, presence and type of carbohydrates and lipids, mechanical agitation and protein particle size, and concentration. Ionic strength and pH influence both surface properties and surface area of protein molecules, regulating counter ions, conformation and degree of hydra- tion. Additional factors important to the water-binding capacity of proteins in solutions vary with protein source and composition, previous processing and the nature of polysaccharides present. Functionally, proteins no longer in their native environment, vary according to source, method of isolation, precipitation, concentration, modification (enzymatic or chemical) dehydration, and associated environmental condi- tions (temperature, pH, ionic strength). Where several discrete proteins are involved (which usually is the case) each exhibits characteristic properties, thus system func- tionality and reproducability depend on strictly controlled "make" conditions. Most proteins appear to retain optimum emulsifying properties at the pH where they are most soluble. Generally globular proteins in their native conformation, where polar amino acids are exposed to the aqueous phase, favor solu- bility and thus this conformation is usually preferred for emulsification applications. Rand (1976) discusses the affects of oils on proteins. In direct contact with lipids, proteins, that in water 12 exhibit an optimum free energy with their hydrophilic groups inside in the tertiary conformation, may turn inside out or become denatured and exhibit entirely different functional properties. These interactions may develop tenaceous lipid protein bonds, stabilized by ionic, polar, hydrogen, and hydrophobic interactions. Friberg (1976) states that polymers such as proteins, when used as emulsifiers, reach saturation adsorption at extremely low concentration in solution. And, when emul- sions flocculate, proteins stabilize the anisotropic skins which develop between the droplets. In summary Petrowski (1976) implies, when emulsifiers are used with such compounds as proteins, that no unambi- guous generalization can be made regarding the effect of emulsifier concentration on critical micelle concentration and its resultant effect on emulsion stability. Protein Crosslinkinngnd Film Formation As previously described, several attempts have been made to develop a substitute which would appear to have an intact yolk, but none with the versatile appearance or function of fresh egg. To accomplish this, one might encapsulate the emulsion in a transparent film or mem- brane. Although numerous compounds have been used to develop synthetic membrane or film structures, few have even remote feasibility as food ingredients. Perhaps the most desirable method of obtaining an encapsulated emulsion would be to cross-link emulsion 13 surface oriented proteins. An idea of the multitude of crosslinker functional groups and resulting compounds usable in protein reactions can be obtained from Table I (Uy and Wold, 1977). Several possible reactive systems, whether currently food related or not, will be discussed. A major condition for most crosslinkers described is their ability to react in aqueous environment at ambient temperatures and under moderate pH conditions. Most agents find some utilization in such industries as animal feeds, pharmaceuticals, tex- tiles, and foods. Sulfur Compounds Needles and Whitfield (1969) describe a method used for coatings and sizings for paper and fabrics in which a colla- gen-protein is bound together in the presence of water solu- ble per-sulfate. The crosslinking reaction can be carried out at room temperature with the addition of a reducing agent. Shapiro and Gazit (1977) noted that at neutral pH and physiological temperatures bisulfite will catalyze the transamination of cytosine (nucleic acids) and amines. Both a and e aminos of L-lysine are reactive. In practical application, Jensen (1959) described the crosslinking of a monomolecular layer of fibrinogen to yield a semi-permeable protein membrane for use in model cell membrane studies. A thin layer of fibrinogen in saline is carefully floated on an aqueous surface. The addition of 14 Table I. Typical Bifunctional Protein Crosslinking Reagents (Uy and Wold . ‘ Functional Groups (x,y) in Bifunctional Reagents (x-R-x, x-R-y) 8. 9. 10. Functional Group ‘\ Aromatic \\C-F // -sozc1 -COOC5H4NOZ -cou3 ODSC'THZ -COO-N ;Ec-ca O 2 -COCH 2 Br(I) -CO-CHN Aromatic/C412 ~CHO 'fi - on @NHZ Reacts with Lys, (Tyr. Cys) Lys Lys Lys Lys Cys, Met, His, Lys Asp, Glu (Cys) Tyr, His Examplesa A. al> B. A. p.p'-difluoro-m,m'-di- nitrodiphenylsulfone (1); l,5-dif1uoro-2,4- dinitrobenzene (2). . l-Fluoro—Z-nitro-é-Azi- dobenzene (3). . Phenol-2,4-disulfony1 chloride; a-naphthol- 2,4-disu1fony1 chloride (4). Adipate bis-(p-nitro— phenyl ester (5); car- bonyl bis(methionine p- nitrophenyl ester (6). Tartaryl diazide; Tar? tryl bis-(glycylazide) (7). . Succinate bis-(hydroxy- succinimide ester) (8). . N-(Azidonitropheny1)Y- aminobutyrate hydroxy— succinimide ester (3). . 1,3-dibromoacetone (9) . p—azidophenacyl bromide (10) See also 7-8 and 10-3. 1,1-bis-(diazo acety1)- 2-pheny1ethane (11). l-diazoacetyl-l-bromo-Z- phenylethane (ll). Bis diazo benzidine (12). Lys (Cys,His,Tyr)A. Glutaraldehyde (13). Lys Ar Polymethylene (n-3-12) di-imidates (14); Di- Table 11. 12. 13. 14. -N a c . o Lys -N - c - s Lys Aromatic\\C-N (b) / 3 15 Cys (Lys) Nonspecific methylsuberimidate (15). B. Ethyl (chloroacetimi- date-(l6). A. Hexamethylene diiso- cyanate (17). B. Toluene Z-isocyanate, 4-isothiocyanate (18). B. See above (11B). A. Bis(maleidomethy1)ether (l9); N,N'-phenylene- dimaleimides (20). B. (See Trommer‘gg‘gl., this volume). A. N,N'-Bis(p-Azido—o-ni— trepheny1)l,3 diamino- 2-pr0panol (21). B. See above (13, 68). aThe examples have been selected to illustrate homofunctional re- agents (A) and heterofunctional reagents (B) with recent references either to reagent preparations or applications. For a more com- plete survey of reagents and applications see Uy and Wold (l976). hv bPhotoactivated: -Nj-->~§ nitrene is the reactive species. References to Table 3: \JO‘UIL‘MNH c O O O O 0000 Macleod and Hill (1970) Crow and Fried (197$) Yagub and Guire (1974) Herzig'ggpgl. (1964) Brandenburg (1972) Busse and Carpenter (1974) Lutter gg‘al. (1974) See also Wetz gswal. Lindsay (1971) Husain and Lowe (1970) Hixson and Hixson (1975) (1974) 11. Husainlgg‘al. (1971) 12. Silman 35.31. (1966) 13. Josephs 35.21. (1973) 14. Hucho 35-31. (1975) 15. Tinberg 55.31. (1976) See also Wang and Richards (1975) 16. Olomucki and Diopoh (1972) 17. Snyder 33.21. (1974) 18. Schick and Singer (1961) 19. Freeberg and Hardman (1971) 20. Chang and Flaks (1972) 21. Guire (1976) 16 cysteine to the water yielded a two dimensional crosslinking of the proteins to form a stable film. Ruminant Feeds Crosslinking systems are used to protect proteins in feeds from microbial utilization in the near neutral envi- ronment of the rumen; but are available to the animal under the acid condition of the abomasum and intestines. Table II lists examples of a broad range of compounds utilized in patents by Miller, 1972; Miller, 1973; Wildi and Miller, 1973; and Scott and Hills, 1975. Most reactions can be carried out at neutral pH and room temperatures. Polyelectrolytes Sternberg and Hershberger (1974) reviewed the use of polyelectrolytes to precipitate protein. These include amylopectin sulfate, dextran sulfate, carboxymethyl cellu- lose, anionic hydrocolloids and heteropolyacids. As an example, polyacrylic acid precipitates many proteins between pH 3 and 6; above and below this point they are released. The polyacrylates can be recovered by precipitation with a suitable polyvalent metal salt such as calcium chloride after protein removal. Then the calcium is removed with acid. This principle was used by Sternberg (1975), to precipitate and recover cheese whey proteins. Suitable polyacids are listed in Table III; additional information on these and other precipitants are discussed by Sternberg, Chiang, and Eberts (l976). 17 ousaxas canyons ahead ovhnovamuuusam owm.m~o.n .wca azaleas» no need one a . .uaxozsm .oeagoegaauoe. oc»:oe~< eggs: as: aaoom ..:Hw a .cmm modem .0 —.u .ocumAH o:«Ed a mvco>nom x: 1.x unmannedgoaha Auto‘s Nua.w~h.m .o:«e¢.fl.~mcueuoa oucou hadoacoz a .ocanumuonzo Hugues—us mocaudmo~¢= head“: rokw... 22252.5. 122: 32:: 2234.; who duodenum ..:Am :9 Ounce .zwiuzm. w .onmuauconauo< ocox~< unfiuux ca .coa 0 ea .wcaman ocuww u ocauvhzcu caumzan movaheagcm mwu.o~s.n 5:13 8 Adams—hon. 53.31923 cacao: Eon 3.395 you“; uuouoodc . ovuuoamoha mucosa: oucouavoou as. on:.c~5.n .93 («3:36 :5» =9 cameo x.»o-w.umuv.n .oumuod 93¢: .mhoumoocol .33:- oca .cmm «o ceded .Hoohum ocouazum oacouhuoo< can and“: . c a o: as a «a u no .mwso an . ~auuoa Aaaoo< oo~.-e.n 25.3 v 1558. $7.36 3 152:. .5383 538.3 2:2: . so movuheazca .9 go hoszomoo 3232...: .3 one. o: \ aw.c oquGEoconaguo aquaxonhmo ouco>~om 1. 1» .hoshnomoeo: coauuaaamcz omm.mow.n msoozaa :0: w oeuuehgcm odoumaauom conduoshnom scan“: at IIIIIIIIIuflmdmmuumll awaaomoz Ho .maduc ludmddflmmwluo iuqaflqumoui ummmma cause ucoduuccoo o>qaoaoz monnemxu an undue ecu acoaam egos“ mwumowmm mezzoneoo wcdxcqqmmouu comm cannula: ho nounemxu .mm oflpma 18 Table III. Compounds Used for Whey Precipitation (Stern- berg, 1975). Compound, General Title Trade Name Manufacturer Polyacrylics Carbopol 934, B.F. Goodrich 940, 941, 960, 961. Goodrite K-702, K-714 Ethylene-maleic EMA ll, 21, 22. Monsanto Chemical anhydride copolymers 31, 61 Company Methylvinylether- Gantrez AN 119, General Aniline and maleic anhydride AN 139, AN 169 Film Corporation A look at commercial literature yields an understanding of the way these polymerized crosslinkers perform. Gantrez (GAF Corporation, 1965), for example, coacervates with proteinaceous material at low pH in aqueous media but redissolves when the pH is raised. Crosslink formation is via primary valence bonds with polyfunctional compounds. Wool and Fiber Reactions Another area of continued research on crosslinkers is being conducted by the wool and fiber industries to modify texture, strength and shrink properties of these products. Several reviews (Ziegler gt_al., 1975; Friedman, 1977; and Tillin gt 11., 1977) discuss chemicals, reactions, and effects, based on wool crosslinking studies. Some compounds which have been grafted successfully to wool fibers are acrylic acid and other acrylate compounds (Pavlath, 1974) as well as Zinc acetate, which reacts with 19 both amino and carboxyl groups (Koenig gt 1., 1974). ~ Enzyme Immobilization and Glutaraldehyde Perhaps the greatest input into protein crosslinking and chemical modification emanates from studies on enzyme immobilization. Olson and Stanley (1974) review some common crosslinkers including glutaraldehyde, tannic acid and formaldehyde. Numerous chemicals and processes have been developed with varying degrees of success. Broun (1976) also discusses chemical crosslinking of proteins and enzymes; and again glutaraldehyde is a major crosslinker discussed due to its satisfactory and reproducible results. Glu- taraldehyde is a water soluble dialdehyde. Its reaction with proteins (Olson and Stanley, 1974) in solution, pro- gress with time; insolubilization is most rapid for most proteins at their isoelectric point (Jensen, Tomimatsu, and Olson, 1971). Essentially reactivity is with the 8 amino group of lysine. Effective reactions occur with either high concentrations of a lysine-rich protein or lower lysine proteins in combination with high lysyl residue proteins such as bovine serum albumin (Broun, 1976). Reactions can be controlled by attaching proteins either to insoluble supports (Wirth and Tixier, 1974) or on a solid, internally insolubilized compound (Jansen gt_al., 1971). Atallah and Hultin (1977) crosslinked glucose oxi- dase and catalase with glutaraldehyde in the presence of BSA to obtain a bifunctional enzyme system with increased 20 thermal stability. Korn, Keairheller, and Filachione (1972) discussed the glutaraldehyde/protein response. In reaction (at acid or neutral pH) glutaraldehyde covalently attaches primarily with lysine or hydroxylysine at ratios of about four moles of glutaraldehyde to one of lysine. The reaction is com- plicated, yielding at least three protein bonded products besides glutaraldehyde polymers. They used u.v. absorption spectra studies and observed several possible polymer structures. Also, there is a direct correlation between the amount of unreacted glutaraldehyde and the amount com- bined with proteins. Under alkaline conditions, polymeri- zation is different and protein response less effective. Habeeb and Hiromota (1968) noted that in addition to lysine and terminal amino group responses with glutaralde- hyde the sulfhydryl group of cysteine, the phenolic ring of tyrosine, and the imidazol ring of histidine also may be partly reactive. Working with papain, Ottesen and Svensson (l97l) noticed that mild increases in temperature, pH, and glu- taraldehyde concentration increased the tendency to form insoluble derivatives. Simplistically, the glutaraldehyde reaction sequence may follow the scheme: Protein - NHZ + OHC (CH2)3 CH0 ;_—2 Protein - NH - CHOH (CH2)3 - CHO ‘l__.- H20 Protein - N = CH (CH2)3 CH0 —-> Protein - NHCHZ (CH2)3 CHZOH 21 Although it's possible for more than one molecule of glu— taraldehyde to react with each amino group, for steric reasons it appears that only one usually does. The pro- posed lysine derivative obtained upon hydrolysis is: H 1 HOOC - C - (CH2 ~ NH - (CH2) I NH - CH OH 4 2 2 Tannins With greater application to food, but with less defined structure and reactivity are the tannins. Strumeyer and Malin (1975) presented an elementary discussion on the nature and properties of tannins. These complex phenolic compounds are divided into hydrolyzable and condensed tannins. The hydrolyzable are readily cleaved by enzymes and relatively dilute acids into simple sugars such as glucose and a phenolcarboxylic acid such as gallic acid. Condensed tannins are comprised primarily of polyphenolics which resist enzyme and mild acid degradation. Although very common in nature. the elucidation of tannin reactivity and chemistry seems relatively incomplete. Loomis and Battaile (1966) review the interaction of tannins with proteins" From tanning experiments, it becomes appar- ent that only the peptide bonds are required for formation of tannin-protein complexes, through hydrogen bonding. The hydrolyzable tannins, especially,exert pH-reducing effects, producing hydrogen bonds between the un-ionized carboxyl 22 groups of the tannins and protein hydroxyl groups. Oliver and Boyd (1972) list three types of bonding mechanisms which occur between tannins and other polymers: 1) Hydrogen bonds between tannin phenolic groups and receptor groups (-NH, C0-, or OH) on proteins or other polymers. 2) Ionic interactions between anionic groups of the tannins (ionized phenolic or carboxylic groups) and the cationic groups on protein (8 amino of lysine). 3) Covalent bonds between quinones (form part of tannin structure or are produced by oxidation) and reac- tive groups of the reacting polymer. These bonds are especially important to final reaction stability. Loomis and Battaile (1966) identify possible protein covalent bond sites as free amino groups, sulfhydryls, and the imino group of proline. Phosphates and Starch Crosslinkers In addition to proteins, crosslinking has been studied in starches, using both phosphates and other reactive chemi- cals. Ellinger (1972) sites work of researchers who used phosphates to bind proteins. Ortho and meta-phosphates were bound to hydroxyl groups of amino acids. Others have used phosphates as protein precipitants, and with acidifi- cation under proper conditions this can form an edible coating (Ellinger, 1972; and Gordon, 1945). 23 Phosphates have been used for protein stabilization and viscosity control for milk, eggs, and gelatin, and for precipitation of gelatin and blood plasma. An example is the use of sodium hexametaphosphate or polyphosphoric acid to prepare industrial gelatin films (Stauffer Chemical Company, 1975). Low levels of phosphates tend to promote protein emulsification and stabilization, whereas higher levels coagulate proteins (F.M.C. Corporation). This characteris- tic is utilized in manufacture of firm puddings and in whey protein recovery. Much of the phosphate reaction technology is derived from the starch industry, where two types of reactions can be forced to occur. This first is phosphate esterification to a single starch molecule and second by phosphate cross- links between starch molecules. Different reactive com- pounds promote different responses. Other starch reactive molecules have possible use due to similarity between starch and protein binding sites, i.e. hydroxyls. Epichlorohydrin in the presence of NaOH and mild heat over a period of time crosslinks dextran mole- cules by bonding hydroxyl groups with a glyceryl bridge (Floden, 1962). In most starch reactions, crosslinks with phosphorus oxychloride, epichlorohydrin, or sodium trimethaphosphate, can be initiated with sodium chloride, with reactions occuring at room temperature in solution (Radley, 1968). 24 Polysaccharide Films Another possible method of encapsulating an emulsion is to coat it with a polysaccharide gum. The gums which work best are alginates and pectates, in the presence of a polyvalent ion such as calcium. Alginates are the most versatile and useful, and as a result of their unique properties numerous recent artificial food products have been developed. Rees (1972) and Wylie (1973) describe the alginic acid and pectic acid similarities that make them different from other polysaccharides, and readily reactive with polyvalent ions. The similarity is based on their uronic acid units and regular 1:4 glycosidic linkages. Altinates are composed almost entirely of L-guluronic (G) and D-mannuronic (M) residues. Most alginate is pro- duced as sodium alginates. In contrast, Rees (1972) clas- sifies pectins as D-galacturonic acid units exhibiting weaker and less predictable reactivity with polyvalent cations than those of alginates. Weaker pectin-ion bonds are due to varying degrees of methyl esterification and foreign residues in the basic backbone chain, such as L- rhamnose. Frequently, side chains of neutral sugar residues and other imbellishments limit reactivity. Algin structures (McDowel, 1975) are generally un- branched block polymers, (short polysaccharide units), divided into categories of M blocks, G blocks, and MG blocks, each 20 to 50 residues long. Blocks are alternately or randomly distributed. For film and gel forming properties 25 the most important variable is the percent of G blocks, which range from 0 to 60%, depending mainly on plant spe- cies and to some extent on part of plant from which derived or stage of growth. Available calcium ions bind preferen- tially with the G blocks by cooperative binding, possibly due to the fact that G blocks are stiffer than M blocks which, in turn, are stiffer than MG blocks. Presumably the G block reaction with each other and with calcium is energetically more favorable than with the other two blocks (Rees, 1972). The algins exhibit linear extended structures, like corrugated ribbons (rather than carrageenan-like spiral structures). They aggregate to form interstices, coordi- nating calcium ions in a favorable way without altering the crystal conformation of polyuronic acid (particularly the poly-guluronic acid fraction). Calcium concentrations in excess of the amount necessary to combine with G blocks will react with other parts of the alginate molecule . (McDowell, 1975). Where this occurs gel strength increases over a period of hours, indicating a slow rearrangement. Excess calcium and acid causes chain rearrangement and syneresis. Calcium ions fit into folds of the uronic acid and are coordinated with oxygen atoms in neighboring chains, sta- bilizing the entire structure which firmly holds the calcium ions. In a partly reacted calcium gel, alginates which are still sodium ionized tend to repel each other, but calcium 26 serves to stabilize the entire aggregation. Most protein reactivity with algins is electrostatic (McDowell, 1975), however reactions may occur if proteins have a positive charge (are at a pH below their isoelectric point), since the alginate molecule in solution reacts as an anion (Alginate Industries Limited, 1975). A great deal of technical information is furnished by the Kelco Co. Inc. (ref.). The following discussion summa- rizes much of this information. Gels with a large proportion of polyguluronic residues form rigid, brittle gels that tend to undergo syneresis. Polymannuronic gels form elastic gels with less tendency toward syneresis. The general reaction mechanism is: 2Na alginate + Ca++-9 Ca alginate + 2Na + Gel reactivity can be controlled by several methods: Calcium salt selection to vary solubility for desired tem- perature or pH; sequestrant utilization to adjust calcium solubility and thus gellation time and final texture; acid incorporation to control reaction rate and setting time; and other ingredients addition to algin, such as sugar to produce softer gels. Usually texture and setting time are controlled by calcium availability and algin composition. Greater metal ion concentration reduces setting time and sequestrants increase it. Normally, algin gels are compatible with 27 starches, sugars, water-soluble gums and proteins. Other algin variations are ramified in particular characteristics; high molecular weight and greater concen- tration produce stronger gels. Highly refined algins yield crisper gels and greater clarity. Finer mesh algin powders produce smoother gels especially in quick set application. Due to commercial availability, cost and acceptability for food use, calcium salts are the most practical and common gelling agents. Calcium forms the strongest and most stable gels at 30 - 40% of the stoichiometric cross- link value of 7.2 g/100 g of algin. Greater concentrations increase syneresis by precipitating calcium alginate. Salts that perform best include calcium chloride, cal- cium acetate and calcium lactate. Calcium chloride is optimal but imparts a bitter taste unless the product is rinsed to remove free ions. Calcium acetate may impart an acetic acid taste; while a greater concentration of salt is required to provide the desired amount of calcium. Calcium gluconate is usable but has a low percentage of calcium per molecular weight. Other calcium salts such as sulfate, citrate, tartrate, carbonate, and phosphates have poor solubility and except under modified conditions such as high acidity the calcium is unavailable for reaction. Applications of Algin in Foods Besides its' use as a thickener, the gelling ability of algin has led to numerous applications in food coatings, as 28 texture modifiers or in synthetic food systems. Glicksman (1975) describes a U.S. system for producing artificial caviar. Sodium alginate containing color, flavor, salt and texture modifying ingredients are extruded as droplets into a calcium salt bath. The caviar appearance and texture is created by the instantaneous formation of an insoluble cal- cium alginate membrane around the droplet. Texture is varied by type and concentration of alginate, calcium salt, droplet size and soaking time. Although this type of pro- duct has not been commercialized in the U.S., the Soviets have pursued it to production. Slonimsky _t‘_l. (1973) de- scribes a somewhat similar method utilizing a gelatin pro- tein solution containing pectinate or alginate. Droplets are formed in an oil bath, fixed in a bivalent metal salt solution, then treated in a vegetable tannin solution to impart desirable caviar similarities. Luh, Flink and Karel (1976, 1977) reviewed and de‘ scribed fabrication of simulated foods when calcium ions are reacted with alginate or pectates. In most of these appli- cations the food matrix is obtained by introducing poly- saccharide gel structure into an aqueous solution of calcium ions. Due to calcium ion migration into the food system, uniform texture is obtained throughout the structure rather than as an encapsulated product. Luh gt :1. (1976) de- veloped a simulated fruit gel acceptable for freeze drying. They developed a weak algin-gelatin gel which allowed for solidification and shaping before the algin was crosslinked. 29 Final gel structure was obtained by varying the concentra- tion of algin, sucrose, and calcium in the gelling bath. To demonstrate that various modifications can be made to impart desired textural differences to fabricated food matrices, Luh at 11. (1977) studied the rheological pro- perties of algin gels as influenced by various ingredient modification and gelling conditions. Other methods of producing alginate products have been introduced. Szcesniak (1968) patented a process for inter- acting an alginic acid solution with solutions containing an alkaline earth metal salt. First, an insoluble film was formed around the algin, then a calcium solution was adjusted to obtain a uniform cellular structure throughout to simulate a particular fruit or vegetable. Unilever (1972) developed a process that simulates fruit pieces by quickly and simultaneously mixing combinations of an algi- nate or pectate solution, fruit pulp, a suitable insoluble calcium salt such as dicalcium hydrophosphate with an edible acid. Wood (1974) describes an artificial fruit of hetero- geneous eating texture in which particles of a gelling agent like agar, carrageenan, gelatin or starch are dispersed in an alginate or pectinate solution. The solution around the particles is then gelled by introducing calcium ions at a predetermined ratio. Unilever (1974) described a method for mixing an algi« nate or pectate solution containing fruit pulp or puree with a calcium ion insufficient to cause gelling on rapid 3O mixing, but capable of gelling under shear-free conditions. A method for preserving fresh foods (meats in particu- lar) was patented by Earle (1968). An aqueous algin disper- sion containing at least one mono or disaccharide was subjected to an aqueous gelling solution, containing an effective amount of water-soluble calcium for sufficient time to bond the coating to the food product without impar- ting bitter taste. For specific meat preservation proper- ties, Earle and McKee (1976) patented a method whereby aqueous algin is applied to the meat followed by a suitable gelling solution to form a continuous film. Using Flavor--Tex® (malt dextrin and sodium alginate), Williams, Oblinger and West (1978) and Lazarus gt al. (1977) demonstrated the control of shrinkage and microbiol conta- mination of meat products with an algin film. The film is formed over the meat, dipping first in Flavor-Tex and then in a calcium chloride-carboxymethyl cellulose solution. Earle (1975) also applied the coating principle to vegetables and in particular onions. A cold water-insoluble, amylaceous material was applied directly to raw onion which was then dipped in an algin solution. The product is then dipped in a calcium solution to complete film formation. Another method of coating (Unilever, 1971a ) for frozen food products was obtained by emersing in a solution of partially reacted calcium or aluminum alginate (which may also contain xanthan gum) followed by drying in a stream of cold air to accelerate hardening. 31 By adding calcium or aluminum salt directly to fruit puree or other product to be encapsulated, Unilever (1971b) obtained liquid centered droplets. The product was brought into contact with a stream of alginate or pectate solution as free falling droplets. Excess pectate or alginate was gelled by recontact with calcium or aluminum solution. Algin can also be incorporated into spun protein fibers and the fibers insolubilized by reaction with polyvalent ions (Glicksman, 1975). Other patents, particularly those from Japan, demon- strated the continued application of alginate as food coat- ings. In most instances, a solid food was dipped into a solution containing alginic acid or sodium alginate followed by reaction in a polyvalent metal salt solution. Variations were based on solutions applied before the algin treatment or on flavors or other compounds incorporated with the algin before salt treatment, and finally in method of drying. EXPERIMENTAL Exploratory Methods and Materials As a research initiation exploratory trials were per- formed with ingredients commonly used in egg substitute formulations to determine coagulation characteristics; and film-forming potential with common protein crosslinkers and egg white precipitants. Corn oil was mixed with liquid egg white. at 32% of the final weight in a Waring blender or homogenized three times at approximately 1000 psi. Five m1 aliquots were coagulated in pyrex test tubes in boiling water at 15 second intervals from 15 sec. to 210 sec. A corn oil-lecithin mixture was mixed with egg white in 5% increments from O - 35% and homogenized twice at 1000 psi. These preparations were stored at 4 C for several days to detect phase separation, then acidified to approximately 6.0 and restored for further observation. Based on a fresh yolk average composition of 50% water, 16% protein, 1.0% salts and 33.0% lipids (Powrie, 1972), a model system was formulated containing 100 9 liquid egg white, 18.8 g whey protein concentrate (Stauffer Chemical Co., ENRPRO 50), 1.2 g calcium phosphate, 40.6 9 corn oil and 17 g lecithin. After mixing for 30 seconds at high 32 33 speed in a Waring blender, the emulsion was held at 4 C for four days, then adjusted to pH 6.3-6.4 (approximate pH of natural yolk) with 7% acetic acid. Following twelve addi- tional days of cold storage, coagulation was observed in test tubes as previously described for heating periods of up to four min. Additional emulsions were made under similar conditions modifying the percentage of various ingredients. Milk- derived proteose peptone (Cante and Moreno, 1975) was also incorporated up to 0.5% as an emulsifier. Gelatin was incorporated up to 6% as a protein additive and potential crosslinking enhancer. A meat-ball press with 3.5 cm internal diameter and a volume capacity of 22.5 ml was adapted to form a coagulated emulsion of yolk shape and approximate size (Fig. l). The hole on one side of the apparatus was sealed with a flat head screw (to the inside) and a nut. The opposite hole was used for inserting emulsion. The middle of the sphere was sealed with plastic tape before emulsion addition and the top was sealed with either tape or a piece of rubber held in place with a clamp. Heating was performed at various time intervals in boiling water and the contents inspected for general appear- ance and degree of coagulation. Crosslinking solutions tried included tannic acid (0.05-10%) and glutaraldehyde (0.05-25%). Reactions were observed with egg white or model emulsion using 5-10 m1 samples in 20-50 ml of reactive PLEASE NOTE: Dissertation contains glossy photographs that will not reproduce well on microfilm. Filmed best way possible. UNIVERSI1Y MICROFILMS Figure l. 34 Metal forms used to fabricate simulated hard boiled "yolks“ for texture studies. 0n the left is the modified meat-ball press and to the right, the milled aluminum form. Coagulated simulated yolks from the milled from are shown whole and halved at the top. Actual similar size yolks from hard boiled eggs are shown in the lower section of the picture. 35 solutions in beakers. Other potential film formers tried include ethanol and albumin reactive compounds such as alu- minum potassium sulfate, hydrogen peroxide, urea, zinc acetate, and succinic acid. Some combinations of the above agents were also tested. Reaction times were varied from ten minutes to several hours. General Methods and Materials The sources for ingredients, chemicals, and equipment used in numerous experiments throughout the project will be listed with only one procedure, usually the first. Most work was performed at ambient temperatures. However, when not in use or in preparation for use, emulsion ingredients, emulsions, and most solutions were stored at 4 C. Emulsion ingredient concentrations are based on w/w ratios. Concen- trations of crosslinkers, polysaccharides, and other solu- tions are w/v, and were prepared with distilled water. Oil Selection and Drop Spreading Commercial oils were purchased and analyzed for emul- sifying characteristics, adapting the simple method de- scribed by Becker (1960). Twenty five milliliters of various emulsion ingredients and mixtures, dissolved in water, were placed in disposable petri dishes. Each oil sample (0.5 ml) was pipetted onto the center of the liquid surface. Oil droplets were allowed to spread for ten mi- nutes and approximate diameter of the oil droplet spread was measured. Oils used included Mazola (corn oil), 36 Pompeian (olive oil), Wesson (a soy blend with cottonseed and polyglycerides), Crisco (soy oil), Planters (peanut oil), Hollywood (safflower oil), and Hollywood Blend (soy, peanut, walnut and safflower oils). Table IV lists the liquid solutions used for oil drop spread tests to deter- mine emulsification potential of the oils. Solutions were mixed using a Tekmar Super Dispax agitator, model SD45K with a G-301 generator operated at approximately 60 on the speed control for one minute or until mixing appeared complete. Samples were weighed on a Mettler 800 9 capacity K7t top loading balance. To verify these screening evaluations 50 ml portions of liquid egg white were mixed with 10 m1 of various oils (peanut, olive, soy, and soy blend) and mixed on the Dispax at speed 40 for 10 sec. Fifteen milliliter alliquots were removed and centrifuged for five min. at ~500xg and recentrifuged for 15 min. at ml,200xg. The effects of pH on oil drop spread was observed by adjusting 30 m1 egg white samples to whole number pH values from 4 to 11 i 0.05 with either sodium hydroxide or citric acid, and peanut oil applied as previously described. Emulsion Stability, Texture and Taste Studies After selection of an oil, emulsions were made to study texture, stability and taste characteristics, by changing various emulsion properties. Emulsion formula- tions, and method of mixing are listed in Table V. 37 mcouammlmmoouoga u acumen; was: mvvapp a~.mm we we mommucmugmq saw: pan pro aaozuwz man: u some m~o>mp copmva=Em m>p$ an meowuovce> mcaux_e sacs .mmgaust zucmza mo Page» < a «Nam opacogm :_ xpco mmwga> a o.m .o.p .uaa m.o+m.o .m.o .o case—om; "mo mpm>mp cwpewmpaem gu_z zoom om o o o Loam: o o op o zamz 5.0. o o o. ma_=z was um_aa o cl o o mul_om see: c a.em a.em a.wm m»_;z out u.=cas mmmcmx .mxmao» .mcoom saoexom e m N — o.n"mmg=pxpz =o_mp:su .m cease napu wool o.~p Azouzv app: ago uamcoz .o .saao ao=m_um atapsoa-=mz -.- sale: new evacls .8 mm xm—ocucmu go a xmpogucmusmxom pmgucmu o.~ =.:u_omg .m 1.. -1- swam: umppvumvo .< Any cowumsoqgoocu acmpuwgmeu mueovvosmcm can meopuzpcm smvpaazm mummw ummcam coca Fwo soc com: meowawvcoo use m:o_u=_om .>H m—nmh Table V. 38 Emulsion Conditions and Methods of Mixing Used to Study Stability, Texture and Taste properties Emulsion Formulationa Method of Mixing A. . Lecithin, 1%; peanut Emulsions described in Table IV, F, but with peanut oil added to 31.7% of the Emulsion. Lecithin, 1%; peanut oil, 34%; egg white, 65%; pH adjustments to 4.08, 5.07, 6.95, 8.24, 8.55, 9.00, 9.66, 10.42 Lecithin, 1%; peanut oil, 32%; whey solids, 10.75%; egg white; 56.25%; pH adjustments to 5.3, 6.15, 6.9, 7.2, 8.1, 8.95, 9.95, 11.00 oil, 32%; whey solids and egg white together, 67%; with whey at 0, 2.5, 5, 7.5, 12.5, 15 and 20%; pH to 6.0 i 0.1 l) Lecithin, 1%; whey solids, 10%; oil and albumin toge- ther, 89%; with oil from O to 60% at 5% increments; pH adjusted to 6.0 i 0.1 2) Same as above with oil varied from only 15 to 40% Lecithin, 1%; oil, 32%; whey solids, 8.5%; pH adjusted egg white, 58.5%; pH adjus- ted to 5.7, 6.1, 6.5, 6.95, 7.25, 7.65, 7.9 . Lecithin, 1%; oil, 32%; whey solids, 8.5%; pH adjus- ted egg white yielding final pH values of 6.05, 6.15, 6.25, 6.35, 6.4, 6.6, 6.7, 6.75, 6.85 10.0, adjusted Dispax, speed 35-40 for 15 sec. Dispax, speed 35, until visibly well mixed Dispax, speed 30, 30 sec.; speed 70, 45 sec. Dispax, speed 40, 30 sec. Dispax, speed 35 - 40, until visibly well mixed Dispax, speed 35 - 40 until well mixed Dispax, speed 35 - 40 until well mixed 39 Table V' (cont‘d.). V Viv Emulsion Formulationa Method of Mixing H. Lecithin, 1%; oil, 32%; whey Dispax, speed 40, l min., solids, 8.5%; egg white, then homogenized once at 57.0%; calcium chloride di- 500 psi hydrate, 1.0%, and 0.5% of the following stabilizers: additional lecithin; carboxy- methylcellulose, (CMC); 7LF and 7MF, and 00 slow set pectin (Hercules, Inc.); sodium hexametaphosphate and sodium tripolyphosphate (FMC Corp.); Sodium trimeta- phosphate (Stauffer Chemical Co.). All samples were pH adjusted to approximately 6.85 a) Emulsion pH's were adjusted with 10% sodium hydroxide or 10 or 20% citric acid 4o Emulsion characteristics were assessed by some or all of the following methods: 1) Centrifugation - model CL International table top centrifuge; 15 ml samples at 1,200xg for 15 min. Egg white separated at the bottom from the remainder of the emulsion, was observed. 2) Measurements of pH and adjustments - Chemtrix, type 60A, with a single reference glass electrode; or an Instru- mentation Laboratory Inc. Model 245 pH/mv electrometer. 3) Microscopic inspection - American Optical Co. Mi- crostar Series 10 microscope with fluorolume illuminator, lens setting at 0, light setting 8 and 45x eye piece. Emul- sion samples were inspected for both predominant and overall droplet size ranges. 4) Visual separation - observation of samples stored overnight or for several days, to detect phase separation. 5) Texture studies - a) Large eggs were placed in cool water and brought to a boil for 18 min., quickly cooled, until cool enough to handle, shelled and the yolks removed. 6) Small eggs were prepared the same way with the exception that boiling was ended at 14 min. c) Samples prepared as described in Table V, A, were coagulated for seven min. in the metal sphere described in the preliminary methods sec- tion. d) Other samples, as described in Table V, were formed for 5.5 min. in an aluminum sphere, (fabricated by the MSU machine shop) comprised of two 2.9 cm diameter, overlapping hemispheres, total capacity, 12.5 ml, and clamped together 41 with a three prong extension clamp. Samples were poured into a quarter inch hole at the top and sealed with a flat head screw (Fig. 1). These are comparable to small egg yolks obtained from eggs ranging in size from 17 - 21 oz. per dozen. Texture comparisons were obtained using an Alla-Kramer shear press, Model SP12 with recording attach- ment, a 100 1b. ring, and aluminum CS-l standard shear com- pression cell. The instrument was operated with the range setting at 50 for samples described in condition A, Table V, and at range 20 for the other coagulated emulsion samples (Table V) and for egg yolks. 6) Taste - shear press samples were tasted after shearing to observe flavor and mouth feel characteristics. 7) Authentic yolks of small size and of some fabri- cated samples were weighed to substantiate size comparison for textural studies. 8) Viscosity - a 20 m1 volumetric pipette with part of the tip removed was mounted vertically to a ring stand. To provide relative comparisons of emulsion and yolk vis- cosities, the pipette emptying time was recorded. Yolks were from fresh eggs (2 days old) and eggs stored 4 and 8 weeks. Crosslinker Studies In addition to unadjusted solutions, potential film forming crosslinkers were assessed by adjusting the simu- lated egg yolk emulsion to pH values of from 3 to 12 in 42 whole number values. Most pH adjustments were made with 10% sodium hydroxide and 10 or 20% citric acid. Thirty-five to fourty-five milliliters of crosslinker solutions were poured into 3.5 inch petri dishes. Five milliliters of the simu- lated emulsion, containing typically, 1% lecithin, 10% whey solids, 59% liquid egg white, and 31% oil, was layered onto the surface of the crosslinker solution. Periodic observations were made for up to a minimum of one hour. Type of interaction and, specifically, incidence of film formation were noted. Chemicals and suppliers, solution concentration and pH ranges, and literature refer- ences, where applicable, are listed in the Appendix. Polysaccharide Films and Emulsion Interaction Development of polysaccharide and polyvalent cation interactions to develop optimum film formation around emul- sion globules was studied by varying parameters of polysac- charides and salts; their concentrations, combinations, reaction times and effects of pH adjustment. Several approaches were used to determine optimum film formation potential and effectiveness with algin and pectin solutions. These include: 1) Pectinate and alginate mixed with emulsion before addition to polyvalent cation salt solution. 2) Addition of salts at various concentrations and mixtures, then insertion into polysaccharide solutions. 3) pH adjustment of emulsions and of algins before 43 interaction. pH and Crosslinker Mixing Effects The influence of pH on algin reactivity was tested by fabricating emulsions with and without 0.5% sodium alginate. Portions of each were adjusted to pH 5.8 and 6.5. Emulsions with alginate were studied in pH-adjusted water; and those without algin in pH adjusted 0.5% alginate. Emulsions were reacted in petri dishes in the manner described for cross- linker studies applying emulsion to pH adjusted solution. Combination effects of GRAS (Generally Regarded as Safe, F.D.A.) crosslinkers and egg white reactive salts were also tested by mixing 15 ml of each of two solutions in 50 ml beakers. Eight ml of emulsion (without added salt) was then injeCted with a syringe into the mixtures which in- c1ude: l) Tannin (2%) and sodium polypectate (1.8%). 2) Tannin and sodium alginate (1.5%). 3) Alginate and polypectate. 4) Potassium aluminum sulfate (2%) and tannin. 5) Ammonium aluminum sulfate (2%) and tannin. 6) Ammonium aluminum sulfate and potassium aluminum sulfate. Polysaccharide and Salt Solutions Algin and pectin solutions ranging from 0.5 to 5.0% were formulated either by dry addition or after mixing with glycerine - as dispersing agent and plasticizer, to 1.5 44 times the polysaccharide weight, to water during stirring with a magnetic stirrer. Occasionally, mild heat was applied while mixing to aid solubilization. Samples were then held at 4°C until uniformly dispersed. Pectins used include LM AB pectin and DD slow-set pectin (Hercules, Inc.), and 1.8% sodium polypectate solu- tion in 0.02 M citrate buffer, pH 5.0 and 0.005% sorbate (courtesy Dr. R.F. McFeeters). Algins used were sodium alginate, Type "CA" (Meer Corporation), and Kelgin XL sodium alginate and Kelmar KR potassium alginate (Kelco Co.). U.S.P, N.F. or reagent grade salts used in emulsions or as solutions to determine effectiveness in film formation and taste include: aluminum sulfate - A12 (504) . 18H20, aluminum potassium sulfate - ALK (504)2 ' lZHZO, aluminum ammonium sulfate ALNH4 (SO4)Z, calcium acetate - Ca (C2 H3 02)2 ° H20, calcium chloride dihydrate - Ca C12 o2H20, calcium gluconate - Ca (C6H1107)2, calcium lactate - Ca C6H1006’ calcium phosphate dibasic - CaH P04- 2H20, and calcium phosphate monobasic - Ca (HZPO Calcium chloride 4)2' dihydrate was the most frequently used salt and the one used as a control in comparing other salts. Artificial Yolk Emulsion Manufacture Artificial yolk emulsions for algin encapsulated yolks were manufactured with various salt concentrations and pH adjustments. The range of ingredients and method of 45 formulation were: salts as listed above up to 2.0%, leci- thin 1.0 - 1.3%, whey solids 8.0 - 8.5%, oil (peanut) 32 - 33%, liquid egg white up to 58.5%, 30% B carotene in vege- table oil (Hoffman LaRoche, Inc.) sufficient to give the desired color up to 0.05%, artificial egg flavor (Stepan Chemical Co.) 0.2%. pH adjustments were accomplished with either acetic or citric acid. Small amounts of CMC were incorporated into selected emulsions to aid spherical for- mation in algin solutions. Sample mixing was accomplished with either - or a com- bination of the Tekmar Super Dispax, a model M62 electro mortar and pestle (Torsion Balance Co.) until completely mixed or by means of a homogenizer operated at up to 1400 psi. Most homogenizations were at 500 - 1000 psi. Polysaccharide Film Formation Emulsions were injected into algin or pectin solution in 50 ml beakers by means of plastic syringes (discardit, Arthur H. Thomas Co.), with the tips removed, leaving a 8" diameter hole on the end. Injections of emulsion into solutions were made with the tip of the syringe at or just below the liquid surface, Figure 2. Steady pressure was applied to the plunger until all emulsion was injected. Eight to fifteen ml samples were used - 12 ml is about the volume of a small yolk. Reaction time was also varied, ranging from one to thirty minutes. Upon removal from film-forming solutions, "yolks" were allowed to drip to 46 remove excess solution before further evaluation. Encapsulated emulsions were analyzed by measuring the yolk index (height of the yolk divided by the average width) and compared with actual yolk values (Funk, 1948; Sharp and Powell, 1930), Figure 3. Real yolks used for comparison were separated from the white before testing. Yolk and emulsion membrane rupture strengths were compared by a method similar to that suggested by Haugh (1937). A Chatillon puncture tester, Model DPD-SOO, (John Chatillion and Sons, New York) was mounted to a ring stand. A 3.2 cm diameter acrylic disc was mounted on the bottom of the pressure rod. Yolks in petri dishes were slowly raised against the disc by means of a "little jack" (Precision Scientific Co.). The pressure gauge was read in 0.1 units at the point of membrane rupture. Whey Dialysis to Increase Protein Concentration ENRPRO - 50 whey protein isolate was dialyzed in re- generated cellulosic tubing exclusion limit 10,000 to 12,000 M.W. (American Cyanamide Corp.) in distilled water. Two hundred grams of whey solids were mixed with either 600 or 1200 ml of water. The dialysis reaction was allowed to run for about five days with at least two water changes per day. Emulsion Stability after Salt Incorporation In order to evaluate the effect of salts and homogeni- zation treatments on emulsion stability, "yolks" were manu- factured according to the scheme listed in Table VI. 47 Figure 2. Method of inserting emulsion into an algin solu- tion to form "yolk". Figure 3. A formed "yolk" with calipers and height gauge used to measure yolk index. 48 ingredient vari- ations Table VI. Emulsion Ingredients and Special Conditions Used to Establish the Effect of Salts and Homogeniza- tion Treatments on Stability. All emulsionslisted contained 1% lecithin and 0.2% egg flavoring. Treatment Homogenization Ingredients (%) A- P” adjusted to 5.1 2 x 500 psi calcium acetate -l.2 with 1N acids whey solids - 8.3 1) control, 3.0 m1 liquid egg white - H20 added 57.9 2) 3.6 ml lactic oil (peanut) - 31.4 acid 3) 3.0 ml lactic acid 4) 3.1 ml citric acid 5) 3.0 ml phospho- ric acid 8. l) unadjusted pH 2 x 800 psi calcium acetate - 1.2 2) pH adjusted to whey solids - 8.5 6.85 with 10% liquid egg white - ‘citric acid 57.1 oil (peanut) - 32.0 C. Homogenization l) 2 x 500 ps1 calcium acetate - 1.0 variations 2) l x 500 ps1 whey solids - 8.5 and 1000 p51 liquid egg white - 59.3 oil (peanut) - 30.0 D. All of oils used in 2 x 700 psi calcium acetate -1.0 original oil tests whey solids - 8.5 1) After 4 days liquid egg white - cold storage 57.3 2) After stirring oil (peanut) - 32.0 and salting 3) pH adjusted to 6.0 4) pasteurization at 60°C in water bath (took up to 2 hrs. in covered containers) E. Whey isolate and 500 psi and calcium acetate -1.0 safflower oil as 1250 psi whey isolate - 8.5 liquid egg white - 57.3 oil (safflower) - 32.0 49 Egg White and "Yolk" Binding To aid in adherence of the formed yolk to egg white the following methods were used: 1) 2% caltium carrageenan (Marine Colloids, Inc.) as a crosslinker or as a dip after reaction in alginate. 2) 2% sodium carrageenan in the same manner. 3) Incorporation of 1% calcium or sodium carrageenan with 2.25% alginate. 4) Algin-glycerin mixed into liquid egg white as the film forming solution. 5) 1% and 2% dried egg white mixed with 2.25% alginate. 6) AL (SO4)3 - 18H20 at 0.11% mixed with the egg white outside the formed yolk. 7) Calcium acetate at 1% level in egg white. The formed yolks were placed in egg white for several hours or overnight and then poached over steam to observe for desired adherence between egg white and "yolks". Taste Panel The simulated yolk emulsion was manufactured using whey isolate and safflower oil (Condition 0., Table VI). B-caro- tene coloring yielding 8 on the Roche yolk color fan was added by incorporating 1 g of 30% B-carotene to 9 g of safflower oil, mixing and adding this to the oil fraction of the emulsion. The emulsion was homogenized at 500 then 1000 psi. Yolks were formed using approximately 15 ml of emulsion in 2.25% Meer algin with 2% added powdered egg 50 white, for 4 min. Yolks were removed from algin, allowed to drip for a short time, then placed in beakers containing the separated white from an egg. After overnight storage in the cold room eggs were poached for 3% to 4 min. Yolks were served with a slice of buttered toast in the Food Science taste panel room to 33 randomly selected students, professors, and staff of MSU. Michigan State Uni- versity Committee on Research Involving Human Subjects approved consent forms specifying ingredients used, were presented to panel members along with evaluation score sheets. The score sheets were used to establish degree of like and dislike for overall yolk appearance, yolk flavor, and general yolk acceptability. Yolk texture and color were also evaluated. All scores were based on ranking from 1 to 7. Copies of these forms are in the Appendix. RESULTS Evaluation of Exploratory Observation Emulsions Fresh liquid egg white emulsified with 32% corn oil demonstrated instability even after being homogenized three times at 1000 psi, and began to separate after one day of cold storage. Mildly stirring orshaking the emulsion yielded quick resuspension. When heat coagulated in test tubes, some oiling off was observed as solidification ap- proached completion. However, most of the oil is firmly held within the framework of coagulated egg white, without significantly weakening the coagulum. Emulsion samples prepared with lecithin as part of the lipid fraction also separated under storage conditions, regardless of the amount of lipid in the system. Reducing the pH to 6.0 did not improve stability. In the model system much less separation occurred during cold storage, perhaps partly due to increased vis- cosity from added solids. Separation which did occur con- sisted primarily of egg white at the bottom of the containers. Coagulation in test tubes revealed continuous firming throughout the emulsion without oil loss. Varying emulsion ingredients, followed by coagulation in the metal form 51 52 resulted in the following observations: 1) The greater the percentage of egg white in the emulsion, the firmer the coagulum. 2) Gelatin, even at very low concentrations produced samples too viscous to work with, 3) Lecithin and milk proteose«peptone fractions resulted in soft coagulums. 4) Addition of whey solids enhanced emulsion stability, oil retention and uniform gel structure. Protein Crosslinkers Tannin solutions reacted readily with egg white or the formulated emulsions, forming a fragile film at the protein- tannin interface. As tannin concentration (above about 0.5%) and reaction time (longer than 10 min.) were increased, the film thickened and became brittle. Glutaraldehyde con- centrations above 0.5%, likewise showed reactivity with the emulsion forming a film strong enough to encapsulate the emulsion, but had a leathery look and texture. The extent of interaction and film thickness were time and glutaraldehyde concentration dependent. 0f other albumin—reactive compounds, only aluminum sulfate, aluminum potassium sulfate, succinic acid, and 95% ethanol, exhibited film-forming potential. All of these films, however, were quite fragile and/or brittle. Combinations of reactants did not improve the quality of the films with the possible exception of glutaraldehyde and tannin mixtures. In this instance a more elastic membrane 53 was formed. It was also observed that higher concentra- tions of crosslinkers, or the addition of thickeners such as carboxymethylcellulose or glycerin aided in the formation of spherical glubules when the emulsion was extruded into crosslinker solutions. Selection of the Oil Phase Oil-Drop Spread With the oil drop spread test, it is believed that the less the drop spreads the better the solution emulsifying properties. Based on this concept peanut oil was selected as the oil of choice for further emulsion preparations. 0f the oils investigated peanut oil yielded the most consistant results, ranking as one of the best three in most of the trials. It responded favorably with formulations con- taining 1% lecithin and whey solids; both of which were selected for continued emulsion studies. Also it was noted that peanut oil had less tendency to continue spreading on the surface of solutions after measurements were made, than the other oils. Each oil demonstrated some advantages under specific circumstances except olive oil, which consistantly displayed the least potential for emulsification. Emulsification Potential of Lecithin and Protein Solids The best overall emulsification properties appeared to be in solutions containing 3% lecithin, where very little difference was noted in drop-spread between oils. At 0 and 54 0.5% lecithin the best emulsification properties were dis- played by solutions comprised entirely of egg white as the protein reactant. Solutions containing protease-peptone as an emulsifier in conjunction with lecithin exhibited optimum results with solutions containing non-fat dry milk. At 1% lecithin both milk and whey solid solutions show more promise for emulsification than those with only egg white. Oil-drop spread measurements are listed in the Appendix. Effect of pH on Oil-Drop Spread Peanut oil spreading on pH-adjusted egg white was minimum at pH values of 5 and 6 with a 1.9 cm diameter spread. Below pH 5 and above pH 7 the drop size increased, indicating that pH adjustments could be significant for emulsion stability. Centrifugation Verification of Oil-Drop Spread Test When placed on liquid egg white soy and olive oil had exhibited the greatest oil drop spread; soy blend and peanut oil least. To confirm oil-drop spread results these four oils were emulsified at 17% with egg white. Exact separa- tion volumes were difficult to measure due to phase color similarity, but greater separation was evident for emulsions containing olive and soy oils than those containing peanut oil and soy blend. This correlated with the findings of the oil-drop spread test. 55 Emulsion Stability For emulsions listed in Table V, (emulsion conditions) centrifugation was the primary method of analysis. Observa- tions of emulsions stored at 4 C, and microscopic evaluations were used to verify these results. In most emulsions, phase separation occurred between liquid egg white and the rest of the system, not between oil and emulsion. Stability of emulsion conditions listed in Table V are presented in Figure 4. Emulsions with dried egg white as a solid additive (Figure 4A) are less stable than those with milk or whey solids. The emulsions formulated with whey solids showed the best physical stability. Differences based on emulsifier levels were not as clearly differentiated by phase separation as by the oil spread test. The effect of pH over a wide range is demonstrated by data in Figure 4B. Characteristics of the emulsion contain- ing only oil, lecithin, and pH-adjusted egg white demonstrate the instability of emulsions formulated without stabilizers. When whey solids were added at a 10.75% level, the stability improved. Emulsions with incorporated whey solids show enhanced stability at lower values of pH, those without added solids show instability. Figure 4C demonstrates the stability enhancing effects of whey solids. The same is true of increases in oil concen- tration (Figure 40). However above 50% incorporation, oil began separating from the emulsion. The effect of adjusting Figure 4. 56 Centrifugally induced phase separation in formulated emulsion as influenced by ingre- dients and pH. (See Table V for formula- tions). A - lecithin percentage as emulsifier in 'T'II'TIUC') emulsions containing liquid eg white with -- added dried egg white (0), NFDM (E1), whey solids (A), and an emul- sion with only reconstituted egg white in addition to oil and emulsifier (0); pH adjustment over a wide pH range with and without whey solids; varying concentrations of whey solids; varying concentrations of peanut oil; pH adjustments over narrow pH ranges, and calcium chloride incorporation along with 0.5% added stabilizers, control - 1% lecithin (a) additional lecithin (b) sodium hexametaphosphate (c) sodium tri- metaphosphate (d) sodium tripolyphosphate (e) carboxymethylcellulose (CMC) 7LF (f) CMC-7MF (g) and slow-set pectin (h). 57' . . . . . I 05+o.51PP‘- 3 ‘ Z LECITHIN (15 es n4 .5 .2 .1 aszv zc_p<¢~.4.n.»am¢azou Fig. 5 Cont. IIPsumrcuL ao 15 20 84 . mw O .xao»\uu¢om .as. »~.4_a_wau¢a:gu 3 PH Stuntman 5 10*- ..xso»\mu¢oe .as. »p.s.a_awu¢ezou 64 The effects of pH and added whey solids are evidenced by Figure 58. Without added solids, an egg white, oil and lecithin emulsion was quite soft. A 10.75% whey addition significantly increased the compressibility of the emul- sions, and raising the pH also increases the compressibility. Increasing the level of whey solids incorporated into emulsions elevated compressibilities (Figure 5C). When fresh yolk averages were considered as optimum for compres- sibility; whey solid incorporation at about 8.5% was con- sidered ideal. Compressibility also increased with an increase in oil percentage. Figure 50 demonstrates the effects of increasing the oil concentration, with whey solids held constant at 10%. Increases in compressibility appear to level off at about 30% oil. Compressibility values for pH adjusted emulsions with 8.5% whey and 32% oil are shown in Figure 5E. This graph demonstrates that pH even in narrow ranges plays a signifi- cant role in textural properties. And also indicates that the best ranges for comparison with yolks are between pH 6 and 7; probably optimum at pH 6.1 and pH 6.8 or 6.9. The addition of calcium chloride appears to both lower the compressibility below a desirable level, and to negate any effects which the phosphate or polysaccharide compounds might impart (Figure 5F). 65 Taste Tasting samples that were used in the Kramer analysis helped establish guidelines for further adjustments of for- mulation. Emulsions comprised primarily of egg white did not display the mealy texture of yolks; and emulsions incorporating milk were mushy. Whey solid addition most closely approximated yolk texture. Lecithin at a 3% level resulted in a sticky, mushy mouth feel. Below pH 6 emul- sions were slightly sour and above pH 9 somewhat rubbery. The most desirable flavor considering crumbliness and a less distinct whey influence in taste were between pH 6.5 and 7.5. The mouth-feel and flavor of whey modified emulsions was optimum in the 7.5 - 10% range. However, the higher the concentration of whey the more sweet and whey flavored the yolks became. When percent oil was lowered below 20%, the formed yolks were mushy and bland. Above 35% oil con- centration, the texture and flavor became oily and somewhat harsh. The addition of calcium chloride caused the emul- sions to become mushy and slightly astringent. Relative Viscosities Viscosity comparisons were primarily between real yolks and pH adjusted emulsion or emulsions with added stabilizers. Real yolks when containing a small amount of egg white, had an average pipette emptying time of 1.9 min. Fresh yolks with all white removed were timed between 66 6.4 and 9.1 min. Yolks from eggs stored eight weeks had an average time of 4.9 min. Emulsion viscosity, in general, increased at lower pH values. At pH 7.9 the dropping time was 0.60 min., but at pH 5.7 was 10.3 min. The addition of carboxymethylcellulose and pectin increased the dropping time to over 30 min. Additional lecithin also increases viscosity, but the rate of increase depends on other emulsion conditions. The emulsion with apparent viscosity nearest that of fresh yolk contains 1% CaClz and 1.3% lecithin. Compiled results are listed in the Appendix. Yolk Weights The weights of coagulated emulsion samples with pH adjustment between 5.7 and 7.9 ranged from 11.20 to 12.36 g with an average of 11.73 9. For the pH range 6.05 to 6.85, weights were 11.54 g to 12.10 g with an average of 11.86 9. Those with calcium chloride and stabilizers incorporated into emulsion weighed 12.23 to 12.65 g (ave. 12.47 9). For actual yolks, one dozen yolks from fresh eggs ranged from 7.88 g to 16.75 g and averaged 12.55 g. Weights for 10 week old eggs were 9.78 g to 14.31 g (ave. 11.64 9). Based on both weight and size there is a good correlation between coagulated emulsion and yolks, thus the two may be compared for compressibility. 67 Crosslinkers The crosslinkers responding with the most potential were tannic acid, glutaraldehyde, aluminum potassium sulfate and aluminum ammonium sulfate. Of all the compounds used only glutaraldehyde supported the emulsion without rupture while remaining at an acceptable thickness. Glutaraldehyde is not, however, a GRAS food constituent, precluding it from direct food use.‘ A summary of potential crosslinker results is listed in the Appendix. Polysaccharides Algin Selection In view of the lack of adequate or desirable reactivity with crosslinkers, another method of encapsulation was investigated, utilizing polysaccharides (algins and pectins) that develop solid gels or films in the presence of appro- priate cations. Since the results of algin and pectin response are based primarily on observations, a summary of these observations is included. When pectin or algin were incorporated into emulsion, the emulsion became quite viscous. And when the emulsion is placed in contact with calcium ions the resulting film became quite thick as the calcium migrated inward, even after emulsion was removed from the calcium solution. When calcium chloride or other suitable salt was incorporated into the emulsion, then placed in algin or pectin solution, a desirable encapsulating film was formed. 68 Without calcium or other suitable cations, algins and emulsions do not react as a result of the pH adjustment, unless the pH is lowered below 4. At this pH the response appeared to be more of a coagulation than an emulsion - algin interaction. At low calcium ion levels (i.e. 0.15%) a lowered pH of either algin or emulsion aids in film for- mation. But. unless allowed to react until quite thick the films were fragile. At higher calcium ion concentrations (i.e. p.3%), pH effects became less significant. Slow-set pectin doesn't yield desirable film forma- tion, irregardless of calcium content. The low-methoxy pectin and sodium polypectates produced films with excellent yolk membrane-like sheen, but readily ruptured, except when the encapsulating film was judged to be too thick. Kelco algins responded much like the pectins. Low viscosity alginate would not support the emulsion and the potassium alginate formed nice appearing films, which split under very mild pressure. Therefore, most encapsulation was done with the Meer Co. type "CA" sodium alginate. Mixtures of pectins and algins only weaken the film potential of the algin. Also, mixtures using tannins, potassium, or ammonium aluminum sulfate in conjunction with pectin or algin do not improve the film response of any of the solutions, or show any advantage over alginate alone. Glycerin aids somewhat in the dispersal of algin upon mixing, but has little effect on film formation. 69 Salt Selection Both reactivity and taste had to be considered when selecting polyvalent cations to incorporate into emulsions. Cations that are both algin reactive and GRAS food additive are limited to the calcium and aluminum salts. All of the aluminum salts employed yield adequate films, but at salt levels sufficient for reactivity an objectionable astrin- gency developed. Calcium phosphates impart the least objec- tionable taste, but do not readily ionize at the pH condi- tions compatible with emulsion or algin utilization. Calcium gluconate ionized too slowly to produce a thin, strong film and imparts an undesirable flavor. Calcium lactate yielded a gritty textured emulsion and produced a thick film before attaining strucutral integrity. Initially, calcium chloride dihydrate was found to be the salt demon- strating superior results, when incorporated into emulsions. Finally, however, calcium acetate was observed to display equal or superior results both in cation availability and taste. When placed in contact with boiling water, the film surrounding an encapsulated emulsion becomes thinner and turns a milky white, but does not soften. At least 1% salt (by weight in an emulsion), in the form of calcium chloride dihydrate or calcium acetate was necessary to provide avail- able calcium adequate for a supportive encapsulation, without the film becoming too thick. 7O Alginate Concentration For adequate emulsion globule and film formation, algi- nate concentration should be greater than 2.0%. A 2.25% solution demonstrated the most desirable encapsulations. Algin concentrations below 2.0% produced elongated struc- tures when the emulsion was injected into algin - containing solution. The best "yolks" were formed when emulsion was injected just at or below the algin surface followed immedi- ately by an addition of algin to layer over the top of the emulsion. Emulsion inserted much below the algin surface (i.e. 1 cm) left a protrusion that readily ruptured; and if the algin was greater than 2.5% in concentration a large flat spot developed on top upon injection, or an irregularly formed "yolk" resulted. Emulsion Stability and Function After Calcium Salt Addition Mechanical emulsification with the Dispax, incorporated air in amounts sufficient to cause the emulsions to float in the algin solution before a uniform film could be obtained. Homogenized samples performed better. Acid and pH Effects on Emulsions Containing Calcium Acetate When calcium acetate was selected as the preferred salt, emulsions formulated according to conditions and from ingredients listed in Table VI, were centrifuged to ascertain the effects of calcium salt, pH, and homogenization 71 interactions on stability. Different acids didn't vary significantly in their effects on emulsion stability; and in the presence of calcium acetate, actually lowered the stability. Centrifugation of emulsions adjusted to pH 6.0 resul- ted in greater egg white separation (3.5 to 3.7 ml separa- tion) than that of unadjusted emulsion at pH 7.3 (3.3 ml separation). This was a reversal of the pH effects observed on emulsions, before calcium salt was incorporated. Figure 6 demonstrates that increased homogenization pres- sures and the use of dialyzed whey and safflower oil aided stability. Stability changes based on various oils in emulsions with calcium acetate, are demonstrated in Figure 7; the influence of pH adjustment and pasteurization are also shown. Yolk Rupture and Yolk Index Comparisons Emulsions can be encapsulated with alginate at a level that yields comparable membrane strengths to that of authen- tic yolks. Both egg yolks and artificial yolks demonstrate a wide range of rupture strengths. The Chattilon values from real yolks ranged from 6.0 to 18.3 for a dozen large eggs; three of which were one week old, the rest fresh. The artificial yolks ranged from 0 to 88.7 depending on time of reaction, method and shape of yolks formed, and pH and concentration of alginate solution. It can be seen Figure 6. Figure 7. 72 Stability increasing effects of some emul- sion in redients and emulsification vari- ables. ?Table VI): A 1.2% calcium acetate, pH 6.85 homoge- nized twice at 800 psi. 8 - 1.2% calcium acetate, no pH adjustment, homogenized twice at 800 psi. C - 1% calcium acetate, homogenized at 500 and 1000 psi. 0 - 1% calcium acetate, incorporation of safflower oil and dialyzed whey, homo- genized at 500 psi and 1250 psi. Stability comparison of emulsions using all of the oils originally tried in the drop- spread tests; 1% calcium acetate is incorporated and the emulsions are homoge- nized twice at 700 psi. Oils are: a) peanut oil, b) safflower oil, c) soy oil, d) olive oil, e) Hollywood blend, f) corn oil and g) soy blend. Conditions or treatments include: after 3 days storage (0); after remixing with a ma netic stirrer (0); pH adjustment to 6.0 A0; followed by pasteuri- zation (D) . 73 d OJ 0.4 Cu Cr N m c «u e c D m L c I N d 0 J. m b u c c U cm .0 Lt a a a r . L p F . . . 5 4 5 Z l. O 5 4 3 21 I 5 4 3 A14 .1 O 35 5352.5 WEE. may 35 28:52.3... 3:... 8m. .35 zoEifim “5:: 8m (.0 7 7 m. u m 3.. F P 011.. 74 from Table VII that films with acceptable resistance to rupture were formed in very short periods of time. However, film formation is usually not adequate unless reaction is allowed to continue for at least three minutes. Most of the emulsions listed contain 1% calcium chloride. Emul- sions with calcium acetate at a 1% level respond similarly. The pressures required to break similarly formed "yolks" often were widely varied. Measured yolk index values for artificial yolks are also listed in Table VII. A more com- plete set of pressure and yolk index data are listed in the Appendix. Artificial Yolk-Egg White Adherence When egg white was incorporated into the alginate solu- tion used to encapsulate emulsion it was found that the resulting film adhered to egg white much better than for other methods tried. Calcium or aluminum ions incorporated into egg white, migrated into the algin increasing the algin firmness. Carrageenan solution, either as a dip after film formation or incorporated with algin as a film forming ingradient, did not aid in algin-egg white interaction. These observations were made when yolks were placed in egg white and poached or fried. Taste Panel laStepanel results as an indication of acceptance of an artificial egg yolk product are listed in Table VIII. 75 Table VII. Artificial Yolk Pressure and_Yolk Index Evalu- ationsa Method of Time of Algin Yolk Chattilon Reading Emulsification Film Formation Index (total Pressure = (min.) Reading x 10 g) Homogenized 15 .491 42.3 2 x 500 psi 10 .477 24.0 pH 7.45 8 .463 11.9 8 .422 16.3 7 .414 20.0 6 .430 16.7 6 .457 7.0 Homogenized 6 .556 0b 2 x 500 psi 6 .498 13.6 pH 6.85 4 .496 6.6 CMC added 4 .488 19.2 Homogenized 4 .527 10.9 2 x 700 psi 4 .471 4.8 pH 6.9 ' 3.5 .488 24.1 CMC added 3.5 .492 17.1 3 .468 23.1 2.5 .418 15.4 2.5 --- 0 2 .461 21.9 1.5 --- 0 1.5 .456 a) Emulsions contain 1% CaClz - 2H20, injection samples are from 12-15 m1 and formed in 2.5% alginate. b) 0 is assigned to emulsions which were already leaking before pressure was applied. 76 mmugm>< ¢.m ¢.m —.¢ m.¢ o.m mm mm mm mm —m mmmcoammz .mbop o cwsu xsm> .N N :_;u Agm> .5 guns Agm> P p o mxwpmpo .m pgmp_ etc» P x_mumgmnoz .o o xpmumgmuoz .o Apmumgmvoe m m e mx_pm_o .o gnaw. gas» a s_u;mPFm .m A spbgm2_m .m >_b;mme N o— N wxw_m_o .m m FMQVQXF .e m paovaah .q «xv—mpc so: xgmv xuwcu n p m mxpp Lunavwz .e m s_u;mv_m .m m spasm._m .m m m m xpugawpm m¥_4 .m xsmc xopsu o xpmumgmcoz .N —p xpmacgmuoz .N v a m apmuugmcos mxpd .N N xsmv asm> .p p xowgu agm> .— o F e 50:5 xgm> mxwg .P xup__a moan gopou mgauxmu smunmuu< go>mpy sgmmaam xpo> xpo> xpo> xpo> x—oa ngmcmu ppasm>o :owusnmgamwc mgoom tam mapsommpou :owuaapm>m zmx P°>.: copmpzsm saw: mmum cmzumom “com com museum comum=~m>m .mcmm mummw mo zsuesam .-~> mpnmh 77 Since no comparison was made with real yolks, a statistical interpretation was not applied. Responses were widely varied, and although most panelists did not indicate a neither like not dislike score, it was the overall average. DISCUSSION The development of an artificial egg yolk was under— taken to provide an alternative to low cholesterol scrambled- style egg replacements. To develop a substitute with the appearance of an intact egg yolk required both the develop- ment of a stable yolk-like emulsion and surrounding yolk- size portions of emulsion with an edible membrane or film. Such a product might be fabricated either by inserting emulsion into an edible sack or by evolving a film around the emulsion. It was decided that forming the film around the emulsion would perhaps be more feasible. Exploratory experiments indicated that a reasonably stable, liquid yolk-like emulsion could be formulated. Other observations indicated that, if an appropriate cross- linking or film forming agent could be found, emulsion globules could be macro-encapsulated, resulting in a non rigid spherical product. Selection of Emulsion Ingredients Oil Peanut oil, containing 29.3% polyunsaturated glycer- ides, was selected as the most desirable oil for incorpo- ration into artificial yolk emulsions. But because a major 78 79 purpose for developing an artificial egg yolk was to appeal to individuals wishing to decrease cholesterol and satu- rated fatty acid consumption, other oils were studied. Safflower, corn, soy, and cottonseed are more desirable, since they contain 72.1, 52.9, 52.1 and 50.0 percent poly- unsaturates, respectively (Church and Church, 1975). In comparison the lipid fraction of natural yolks contain only 9.3% polyunsaturated glycerides (Cotterill, 1973). In general, other oils including safflower, resulted in equal or greater emulsion stability than observed with peanut oil. Other Ingredients Whey solids and lecithin were selected as emulsion ingredients for the following reasons: Both have been utia lized extensively in egg emulsion systems, are readily available and imparted appropriate functionality to emul- sions. Major emphasis was directed to the encapsulation tech- nology, utilizing only ingredients which were readily accessible and acceptable. Lecithin Lecithin is native to egg yolk and has been promoted because of its potential to bind dietary cholesterol. As a commercial compound, lecithin is a mixture of phospholipids including phosphotidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol (Central Soya, Chemurgy Div.). 80 This mixture is actually more comparable to egg yolk phos- pholipids than is phosphatidyl choline alone. A one percent (w/w) concentration of lecithin in the emulsion was most satisfactory based upon the following observations: a) Centrifugation results indicated that higher con- centrations of lecithin did not significantly increase emul- sion stability; b) Textural properties of the emulsion were optimal at this level; and c) Slight variations above and below the one percent level greatly increased or decreased emulsion viscosity. Whey Solids It was apparent early in the project that a thickener was needed to achieve emulsion stability. Whey solids, particularly ENRPRO-50 (Stauffer) protein concentrate was selected as the most satisfactory product for the following reasons: a) Viscosity was enhanced b) Desirable modifications in the texture of coagu- lated emulsions were produced; and c) Emulsion stability was enhanced. Citric Acid Citric acid is frequently employed to make pH adjust- ments in food systems and was used for this purpose in this study. It is highly soluble, imparts less harsh flavor than most acids, loosely sequesters and lessens the 81 astringency of salts, and may aid in emulsification (Gard- ner, 1975). Liquid Egg White Liquid egg white was utilized throughout the project to provide the aqueous phase in the formulated emulsions. It is employed in nearly all simulated egg products, contribu- ting the essential characteristics of heat coagulation to the ingredient system. Emulsion Stability Centrifugation Centrifugation appears to be an adequate method of studying emulsion stability to determine the effect of both ingredients and method of homogenization on final stability. It is quick, simple, and quantitative, and correlates with observations made on emulsion stability as a result of short term storage at 4 C. There are some latent disadvantages to this method for determining stability. Phase separation is often difficult to observe where color differences between the separated egg white and emulsion are not distinct. An oil rich layer at the top, and an oil poor layer at the bottom may develop without being visible (Puski, 1976). ‘In addition desta- bilization mechanisms may be altered during the centrifuga- tion process and produce atypical results (Petrowski, 1976). However, these problems did not appear to limit the method 82 for detecting the characteristics of stability. Centrifugation also indicates the amount of air in- corporated into the emulsion by comparing initial and final volumes. And ingredients which denature proteins may be identified if a precipitant is present. Microscopic Analysis Groves and Freshwater (1968) state that emulsion sta- bility and viscosity are dependent upon particle size or particle size distribution of the dispersed phase. Measuring changes in particle distribution reveal slight changes in emulsion stability, even before phase separation is visualized. Therefore, comparing droplet distribution and their change with time should give the most accurate indication of emulsion stability. However, in this study microscopic analysis was not found to be an adequate indicator of emulsion stability. This may be attributed to: a) Different ingredients produced equal stabilities with different droplet sizes due to altered viscosity and emulsification effects; b) Occasionally, the small size of emulsion samples may not have been representative; c) Results were reported in terms of relative obser- vations rather than as a direct measurement; and d) Specific numbers of particles or a specific emul- sion area was not considered. 83 Microscopic analysis did make it possible to visualize the destabilizing processes. Particle floculation was fre- quently observed, particularly in less stable emulsions. Also visual comparisons of emulsions, differing only in the method of mixing, are valid indicators of emulsion stabili- ty. An example of this is the effect of homogenization pressures on emulsion stability (Fig. 8). The observed decrease in droplet size with homogenization at 1000 psi resulted in increased stability; an observation substan- tiated by centrifugation. The stability of native yolk is also illustrated. Under centrifugation conditions identical to those employed on the formulated emulsions no phase separation was apparent; only a slight amount of precipitant was detected. Correlation Between Stability and Textural Studies Centrifugation and Kramer texture analysis were the primary evaluations in selecting the proper levels of vari- ous ingredients. Emulsion viscosity, and the organoleptic characteristics of coagulated samples were also considered. After the selection of whey and lecithin as previously described, the effect of pH adjustments was considered. Initially the pH range was limited to between 6.0 and 9.0. Above and below this range, viscosity, texture, and taste were not acceptable. At this point 8.5% (w/w) whey solids was selected as most ideal from the standpoint of taste and texture. 84 mflmcxm m. U n :osomm=¢~mnso= dzwdcmznm o: umxndndm msnm man macdmfios mama‘ddak fl: ooaumxamoz is”: acasmanfio «oar. my :osocm:+~ma «:‘om m” moo ems” av soaocm=¢~ma oanm ma moo um‘ mag oanm ma dooo ems“ nv «oar. 45m .mmmsa d: n3m :uumx s¢u3a :msa nowzmx Sm Mm : a: cssnm om m. 85 Stability was not optimum, yet was acceptable as a compro- mise between stability, flavor and texture. It most closely approximates the characteristic crumbliness of yolks. Increases in oil concentration also increased stabili- ty, but at cencentrations of over 40%, with constant whey solids content, emulsions became overly viscous. The tex- ture of coagulated emulsion samples, containing 30-40% oil, were similar indicating that above 30% oil, whey solids became the major factor contributing to compressibility. In terms of flavor, less than 25% oil in emulsion produced a soft or mushy mouth feel; over 35%, an oily taste was apparent. A final oil content of 32% was selected in com- bination with 1% lecithin to simulate the lipid content of natural yolks. A final set of pH adjustments indicated that the most desirable stability and compressibility traits were obtained in the range of 6.05 and 6.85. As the texture of coagulated emulsions approached yolk-like texture, the distinctive flavors imparted by whey and/or egg white were minimized. When one percent CaC12°H20 was incorporated into emulsions, compressibility was substantially reduced. The addition of ph05phates or polysaccharides as potential stabilizers did not improve either texture or stability. Relative Viscosities Emulsion Viscosities were affected by minor changes in composition, pH, and method of emulsification. Yolks were 86 affected by age and the amount of adhering white. Viscosity measurements were not a major consideration in selecting or modifying emulsion ingredients. They did, however, demon- strate that slight adjustments particularly in the level of lecithin added, would alter the fluidity of emulsions. Desired Viscosities (i.e. near that of fresh egg yolk) could be obtained. Texture Comparisons Between Hard-Boiled Yolks and Heat- Coagulated Emulsions Although the texture of egg yolk depends largely on the method of cooking, hard-boiled yolks were compared with heat-coagulated emulsions. The rationale for this compari- son was as follows: 1) This represents the most reproducible method of comparing yolks and emulsions on a size and shape basis. 2) The texture of hard-boiled yolks was more consis- tant from egg to egg than was observed for other cooking methods. 3) The possibility of developing a yolk substitute for tube-style hard-boiled eggs became apparent. 4) Adjustments in the composition of the emulsion for- mulation to approximate the texture of yolks could be pre- dicted. Shear press data indicated the major comparison between yolks and coagulated emulsion was the compressibility para- meter. The shapes of graph peaks, however, indicated 87 differences between textures of samples possessing identical compressibilities. Coagulated emulsions with a high egg white content had a tendency to yield split peaks; a characteristic encountered only occassionally in yolks or other emulsion formulations. For both formed emulsions and real yolks, relatively narrow peaks were obtained. Ten-week old yolks, and emulsions with calcium chloride and polysaccharides or polyphosphates added demonstrated a flatter peak profile. The shoulder at the trailing side of the peak resulted from shear blade drag against the shear cell (Szcazesniak, Humbaugh and Block, 1972). This was an indication of sample adherence or stick- iness. That authentic egg yolk and coagulated emulsions may be adjusted to very similar peak profiles and conse- quent texture may be seen in Fig. 9. Size Comparison of Yolk and Formed Emulsion Comparison between yolks and formed emulsions was based on texture relative to shape and surface area. The inside surface area of the sphere used for most coagula- tions was approximately 26.0 cmz. Assuming that authentic yolks are spherical and the fresh yolk density is 1.035 (Berquist, 1973), the surface area for a fresh hard boiled yolk of 12.55 9 -average yolk weight for fresh small eggs - would be 25.5 cmz. This value is within 2% of the area for the formed emulsions. Therefore differences in compressi- bility between yolks and formed emulsion primarily indicate Figure 9. 88 Allo-Kramer Shear Press peak profile com- paring a hard boiled yolk and coagulated emulsion. 0n the left is the peak profile of coagulated emulsion with 1% lecithin, 8.5% whey solids, 32% oil, 58.5% liquid egg white, and pH adjusted to 6.1; Compressi- bility is 8.3. The profile at the right is from an egg yolk with a compressibility (7.9) about average for fresh yolks. 89 textural differences. The emulsion yolks contain some incorporated air which seems to affect texture readings., It is of interest to note that real yolks once broken and coagulated in the spherical form yielded a much firmer, rubbery coagulation than those excised from hard-boiled eggs. Emulsion Encapsulation with Alginate and Comparison with Authentic Yolks Alginate Films After it became apparent that none of the protein crosslinkers were satisfactory for film or membrane formation, polysaccharides were investigated for encap- sulation of emulsions. Due to their unpredicatable com- position, pectins demonstrated limited potential for desirable film formation. 0f the algins used for this study, the Meer type "CA" sodium alginate displayed the most satisfactory results. Calcium-Alginate Reaction At a given algin concentration, film thickness and strength are based primarily on time and the calcium ions availability in the emulsion. Calcium ions migrate from the emulsion into the algin as a function of sample compo- sition, temperature, and time (Luh 33 31., 1976). The film thickens as ions continue to migrate. Therefore, suffi-. cient calcium ions must be present at the emulsion-algin 90 interface to establish a thin, strong film, Calcium ace- tate at 1% (w/w) incorporation in emulsion was selected as the salt most capable of providing an adequate film forma- tion, yet minimizing an objectionable taste. Encapsulated Emulsion Films The film necessary to encapsulate emulsions was much thicker than the yolk vitelline membrane. It is, however, transparent enough to allow for a good yolk-like color and sheen with properly colored emulsions. It became translu- cent and blended well with egg white upon heating (Fig. 10). Yolk Membrane Strength The Chattilon pressure tests on yolks and encapsu- lated emulsions demonstrated that algin films can be formed with rupture strengths comparable to those of fresh yolks. Under proper conditions films can be formed quickly which are sufficiently thin and of adequate strength. Yolk Index The yolk index, i.e. yolk height divided by average width, was employed as an indicator of freshness in eggs; the higher the value the fresher and more desirable the yolk. A summary of yolk index values was prepared by Funk (l948).\ Fresh yolks were found to have average index values of 0.48 when measured in their natural position in the egg. and 0.43 when separated from the albumen. When Figure 10. 91 Comparison of raw yolk vitelline membrane and algin films. Both yolk and encapsula- ted emulsion were broken and the membranes rinsed with water. At the top is the vitelline membrane. The white algin film has been heated in boiling water. To compare these to appearance with yolk inside see Fig. 3. 92 properly formed, simulated yolks compared with these values, particularly with values of yolks remaining in their natural environment. This demonstrates that in addition to good color and membrane strength, adequate physical shape of emulsion yolks is obtainable. Cooked Simulated Yolks Once yolks with adequate algin films were obtained, their response as simulated eggs was examined. Simulated yolks were added to egg whites previously separated from fresh eggs and poached or soft fried and compared to natural eggs. Examples of these processed eggs are shown in Figures 11 and 12. Two major problems in physical appearance were evident: l) the simulated yolks did not adhere well to the egg white, and 2) the liquid centers had a slightly curdled appear- ance. At this point whey solids were substituted with whey isolate resulting in yolks possessing a smoother appear- ing liquid center when soft-cooked. Dried-egg white incor- porated into the alginate was found to effectively increase the attachment between egg white and algin. Finally, peanut oil was replaced with safflower oil to increase the polyunsaturated lipid fraction. Figure 11. 93 Poached eggs. The artificial egg is in the center of both pictures. The real egg at the left was separated and then returned before poaching. It shows comparable lack of adhesion between white and yolk to that of the artificial yolk. The bottom picture with yolks displays the difference in color hue and the curdled appearance of the liquid center. These yolks were manufactured before textural improvements were made. Figure 12. (J u Fried eggs. These pictures better demon- strate the tendency for the artificial yolk (at the bottom of each picture) to separate from the egg white. The incon- sistency of emulsion yolk is also apparent for the artificial egg yolk. 95 Stability of Emulsions with Safflower Oil and Whey Isolate Yolks manufactured with calcium acetate, whey protein isolate and safflower oil showed stability equal or su- perior to that of yolks manufactured with undialyzed ENRPRD-SD and peanut oil. A major difference in stability was encountered; decreasing the pH of the new emulsion decreased stability rather than the previously observed increase. Kramer texture evaluations were not performed on emul- sions with the new ingredient formulation. It had previously been observed that calcium chloride addition eliminated otherwide similar textures between yolks and coagulated emulsion. The new emulsion partially achieved the require- ments for a better appearing liquid emulsion when manufac- tured as liquid-centered, cooked eggs. Taste Panel Responses Perhaps the best test of success or failure for the algin encapsulated yolks was the response of the taste panel. Because panel members were asked to compare an artificial yolk product with an ideal for authentic poached eggs, high scores were not anticipated. The scores were widely distributed with essentially as many panelists expressing varying degrees of "like" as those expressing degrees of "dislike". In most cases a neither like or dislike answer was avoided by panelists. Overall scores were good enough to indicate that with minor improvements 96 in yolk flavor and consistancy as well as algin film tex- ture and thickness, yolks could be much more acceptable. This would be true particularly for individuals seeking to restrict consumption of natural eggs, yet desiring eggs as part of their diet. The comments on the evaluation forms were as useful as the scores. Initial appearance before breaking the yolk was quite well received. However, after breaking the mem- brane several persons indicated that a better color and sheen was needed. The liquid yolk looked "mustardy". The color imparted by B-carotene can easily be adjusted or modified with slight changes in concentration, and/or the addition of xanthophylls or other colorings. The glossy yolk appearance perhaps could be improved by better homo- genization or the utilization of a protein source, i.e. such as vegetable proteins, with coagulation characteristics similar to egg yolk. Some individuals noticed the calcium acetate as a slightly "bitter" or "chemical" flavor. Otherwise "bland- ness" of flavor was the primary expression. Calcium acetate might be masked and the blandness overcome by addition of an appropriate egg flavoring other than the one utilized. With the realization that it is impractical and diffi- cult to reproduce the flavor and texture of a natural yolk, the responses of individuals disliking the product were balanced by those expressing good acceptability as a sub- stitute product. 97 Texture responses were also widely varied. The pres- ence of the algin film imparted a "chewiness" or slight "toughness" unnatural to vitelline membranes. Simulated yolks were also slightly difficult to puncture and did not always display a yolk-like fluidity from the liquid center. Thinner films might be achieved by using one or a combination of the following methods: 1) selection of another alginate more specific to the desired response, 2) slight acidification of the alginate to enhance calcium reactivity, 3) rinsing the yolk in water upon removal from the algin bath to reduce adhering and unreacted alginate. Allowing the yolk to drip momentarily and then placing it directly into egg white was not a completely satisfactory procedure. Some incorporation of air was noted producing a spongi- ness in the coagulated portion of the emulsion. Most of the defects might be alleviated by careful mixing of ingredients and homogenization. Perhaps the use of a commercial de- foaming agent would be helpful. Although the overall consensus was that liquid centers are too thick or pudding-like, some panelists felt they were not viscous enough. No doubt this disparity was based on differences in individual perferences for poached eggs. Very minor adjustments in egg white or lecithin content can be employed to adjust viscosity. 98 Stability of Stored Samples Emulsion stability did not appear to be a major prob- lem for the artificial yolk systems. Yolks identical to those used in the organoleptic evaluations were examined after three weeks of storage at 4 C. All had maintained good structural integrity and showed little if any emulsion separation. They did, however, tend to float in the egg white. More conclusive evidence of stability is revealed by the emulsion containing whey protein isolate, calcium ace- tate and Wesson oil (i.e. a soy blend). This preparation was stored seven months at 4 C following pH adjustment to 6.0 and pasteurization. No visual signs of separation occurred during this time and a yolk possessing a satisfac- tory appearance was formed. Instability problems which develop with emulsions might be eliminated by use of addi- tional emulsifiers such as mono—diglycerides. CONCLUSION The manufacture of an artificial egg yolk for appli- cation in "poached" or "fried egg" usage appears to have commercial potential. As a feasibility study in macro- encapsulation of a lipid-water emulsion, the research ob- jectives were met. Minor manipulations in ingredient formulation, homo- genization or pH adjustment could be utilized to optimize emulsion characteristics. In addition to the goal of a non-rigid, self-contained liquid yolk, it was found that "hard-boiled" yolk texture could be duplicated. 99 APPENDIX A 100 o._ N._ m._ m._ o._ m._ m._ =.;u.um_ um 55.: no.m_ AN m.m o.m ¢.~ N.N m.~ o.w o.m u~.cp AP meowuzpom zmcz N.“ ..o o.m o.m m.¢ o.“ m.m Amu_—om uxpv zamz _.m m.m m.~ ~.¢ N.~ o.m m.m muvgz mom upzc_4 o.— e._ e._ m._ m._ m._ e._ copuapom cpzuwumF up m.m o.m oJm ~.m mew m.m 0.x gmumz uwpppumwo ummgam aogu ppo mo Asuv Loamamwo gmzo_ummm . ucmpm “scams mom ucopm m>__o ccoo coozzppo: mom mcopa:_om m__o pmwumeHLm :_ mm: pawucmuoa ;u_z mcopu=_om no mco?mp=Em xpoz mmwuzgm nmmgqm __o no mupzmmm ._< o—nMP 101 Loan: :P o.~ m.m o.~ ~.N c.~ N.m m.m mews: mam woven mvaom mag: new m.m 5.. m._ c.N 5., ¢.~ a._ move: mam n_=c_s zomz new N._ c.~ m._ o.N P.N N.N o.~ aa_;z mom u,=c_s mg_=3 cvga ~.N o.N m.~ o.m ¢.N o.m m.~ mum sec wen aways; -_ums N_ Lupe: cw N.N m.N m.~ o.~ m.~ m.m ¢.~ mgwgz mam capes mu_Fom aux: new ¢.m m._ ~.~ m.m m.~ o.m P.m wuwsz mom cpzc_4 zomz new m.~ m.m N.m o.m m.e m.m N.e ours: mam ww=c_4 mu_;z cwgupumb V.N o.~ m._ m._ N.N m._ o.~ mam see new uL=c_s am.c Lopez :_ m.~ o.~ ~.~ ~.N N.N o.~ p.m apps: mom woven map—om xmgz can o.m _.m m.~ m.~ m.m o.¢ o.e mews: mum cwacpg zomz ucm m.e o.e m.m m.m m.m m.o m.m mppsz mum uwchb maps: gm_w_m_:su m.~ m.N o.~ m.~ m.~ m.¢ N.N mam see use uwsc_s oz vmmgqm aogu __o we AEoV gmumamwo Lmzopw acm—m paced; mom ucwpm m>_po :Lou imam woo: zom ixppoz meowuapom cmpmpm_:sm m._o mommpcmugmq cwzuwump mcrzgm> new mop—om _mcowuvcua guy: mu—zz mam mcwcwaucoo .m:o_u=_0m :o mchaum cmmgam acct ~po mo mupammm .N< «pea» 102 Loan: 2' N._ m._ a._ N._ m._ m._ m._ mo_;z mom um_za mowpom awn: oco m._ N._ m._ m.~ w.F o.~ m._ moon: mom o¢=o_o zoez oea m._ m._ m._ m._ m._ m._ m._ mo_;z mam ooao.o mu.;z =P;o_ums A.” ~._ ~._ 5.. N._ N._ N._ mam see new noses; gm Loam: cw m.~ m.m m.~ m.m o.m m.e v.m move; mam om_ta movpom mos: oco _.m m.~ ¢.~ m.~ N.~ o.~ N.¢ «use: mam c.3o.o weapama :amz oco omomuogo m._ k._ m._ m._ m._ o.~ m.. au_:z and oozes; am.o more: a =*;o_omo ¢.N ¢.N P.~ m.~ ~.N o.~ o.m mam see new opaa.s Rm.o oomcam oogo __o yo AEoV LmuoEowo Lozopm oco—m uncooa xom coo—m o>w_o cgou -wom oooz xom ixp_o= meowuapom Lowwme=Eu m_Po .A.o.o=ouv ~< «pan» 103 Table A3. Potential crosslinker or film forming compounds selected for reaction with emulsion at initial and various levels of adjusted pH Initial pH, pH Manufacturer range and percent Refer- Compound or Supplier level of use ences Polymers Carbopol B.F. Goodrich 3.3-l%, 3.8-0.l% Stern- (Carboxypoly- Chemical Co. 3 to 12, 0.1% berg methylene (1975) polymer) 934 940 941 EMA Monsanto Co. 3.0-1%, 3.6-0.5% Stern- (Ethylene 4.4-0.2% berg Maleic 3 to 12, 0.2% (1975) Anhydride) 3.1-1%. 3.3-0.5% Miller 91 4 to 12.0-0.2% (l972a) 81 3.0 to 12.0-l% 61 Gantrez AN GAF Corp. 2.8-l%, 3.15, 4 Stern- (polymethyl to 12.0.5% berg vinyl ether 2.7-l%,3.15, 4 (1975) maleic to 12-0.5% anhydride) 139 169 Goodrite K B.F. Goodrich 3.6,3 to 12-1% Stern- (polyacrylic 3.4.2 to 12-1% berg acid) (1975) 702 732 Starch Modifiers Acetic Anhyé Fisher Scientific 2.5,3 to 12-2% Whis- dride Co. tler Acetic Anhy- Pfaltz & Bauer, 2.7,3 to 12- (1964) dride with Inc 25% Acetic anhy- adipic anhy dride (polymer) Acrolein Epichloro- hydrin Pfaltz & Bauer, Inc. Eastman Organic Chemicals dride, 0.12% adipic a"hydride 7.4,3 to 12-2% 3.6,3 to 12,3% 104 Table A3 (cont'd.). Initial pH, pH Compound Manufacturer range and percent Refer- or Supplier level of use ences Propylene Aldrich Chemical 7.55.3 to 12-5% Oxide Company Sodium tri- Stauffer Chemi- 6.55, 3 to 12-2% metaphosphate cal Co. Sodium tri- FMC Corp. 9.1.3 to 12-2% polyphosphate Succinic anhy- Pfaltz & Bauer, 0.3% epichloro- dride & epi- Inc. hydrin & 4.0% chlorohydrin succinic anhydride Succinic anhy- Pfaltz & Bauer 2.65.3 to 12-2% Miller dride . ’ -_ ‘ (1973b) Vinyl acetate Eastman Organic 3.3.2 to 12-2% Chemicals Other Protein Reactive Compounds Ammonium Per- J.T. Baker 3.4.3 to 12- Needles & sulfite and Chemical Co. 1.84% (NH4)25208 Whitfield sodium sulfite 0.2% Nag $03 (1969) Aluminum am- Mallinckrodt. 3 to 12-2% monium sulfate Inc. Aluminum po- Mallinckrodt. 3 to 12-2% tassium sulfate Inc. Ethanol 3,5.10.20.40,60. 80, and 100% Glutaraldehyde Pfaltz & Bauer, 7.2-1.0 to 25% Inc. 3 to 12-1% Maleic anhy- Fisher Scientific 1.6.3 to 12-2% Miller dride Co. (1973b) Sodium hexa- FMC Corp. 9.1.3 to 12-2% metaphosphate Succinic acid Tannic acid Mallinckrodt. Inc. 0.1. Baker Co. 2.7.3 to 12-2% 5.7.3 to 12-1% other percentages to 10% at unad- justed pH. 105 woven -zsco ovowoo oco mowgo x o.m-~.~ cowopooz gogopm ->;:< owuoo< Aocopo oo_goxzco owovoo woven ax__v =o_oa_=maoo axo_.mu x m-m.N -»;=< u_umu< meowmwooz zogoum uzmwpm mp omcoomog oprepwm mmoogocp .zo saw: monomgooo x x aim pomspocH mmmz mHPLooou omcoamog oxFPE—pm azmwpm x x oim uoogpocm NoNz mapgooow coraosgoo ucogoomcosu mePsppm osom goo x x cum apoaongou an amp z< Nosucoo .:o =_ ommmgocp za—z mammogooa x x ¢.m osomv .uoog_o:~ mo~ z< Nogpcow o.m o>ono omcoomog oz x o.m .o ono oncoomo» oz x o.m apoooagou Fm <=m zo :F omooso:_ zap: mammoguoo x x m.m sz—V uoogpo:_ pm osos upon sage z_g~aoga o.m Ia on x o.m can» .zmgz _¢m .oaontao omcoomog xpco .zo cw omomgo x o.m mo cowpou_a_o com poooagoo 1:. zap: omcoomos ommomgooo x «in logo uoog_o:_ emm poooosou msosxpoo an a u «2 omcoomoc oncoommg . copuoo . mucoeeoo Lo :owuooos umopoogm Fpoao coo» moczoaaou cop m>som to moons oomoooso .u no In go :o co «coggzu .Aumv :owuosgou Ep_u new .on cowumuwovomgm .on cowuopzmoou .Azzv omcoomom oz oozpoc_ moowuomogv momcoomog Lox=_pmmogo mo xgossam .o< opaop 106 .oomoosocp Io mo mmmoogooo ocoganE do zumcogum =o_u-._ .ocogosos xsoo xgozuooz x _m_sm -poossm oschm ooxzoopogopzpo app» >xo3-opuu_ga Ago> x x moopioo Louooguxm go>mpm pozozwu o :o um mx__E—_$ mace—am anm “mos .:o_uosgo$ ocogasos zoo: x x elm m . ao.o op a: woven x min., Lopm_ooz zogoum -z;:< u_:_oo=m mo_go»:co upcwoosm cu_z x o.m-m.m me$Pooz zogoum cpgozzozo_;o_am ouozomoza Pms_=_z x m-m mgmmno Go mono; oomooogo :o go :o no acmsgou .z.o.u=ouv_e< a_aah 107 Acopomu.a o~_mogm use .copaosgoo spy; x x oim -_oosov mgomno :o so In so acogszu .z.o.o=ouv ¢< a_a~h 108 Table A5. Relative viscosity comparisons of emulsions and egg yolks based on emptying times from a 20 ml pipette Y015a31a5131510n Emptying Time (Min.) Trial 1 2 3 4 Ave. 2.”. 7.9 0.61' 0.60 0.60 7.65 0.65 0.64 0.64 7.25 0.95 0.88 0.94 0.93 6.95 1.24 1.0 1.06 1.10 6.5 3.91 3.8 3.90 6.1 7.40 12.90 10.2 5.7 10 29 10.3 6.85 2.34 2.12 1.83 2.10 6.75 2.50 1.93 2.09 2.15 6.7 1.81 1.86 1.84 6.6 2.07 2.18 2.12 6.4 4.11 4.10 4.10 6.35 3.90 3.98 3.94 6.25 5.33 5.26 5.30 6.15 4.69 4.38 4.54 6.05 4.40 3.57 3.19 3.72 Stabilizer 1% Lecithin 1.57 1.70 1.63 1.5% Lecithin 17 03 16.06 17.50 16.86 Sodium Hexameta- phosphate 3.25 3.13 3.19 Sodium Trimeta- phosphate 2.54 2.49 2.51 Sodium Tripoly- phosphate 6.34 7.25 4.78 6.12 CMC-7LF 19.77 19.92 19.85 CMC-7MF - - 30+ Slo Set Pectin - - 30+ CaClz and Lecithin Variation 0% CaC12 1.3% Lecithin - 1.63 1.53 1.53 1.56 pH 6.85 1% C3C12 1.30 Lecithin - 7.32 10.22 6.95 8.94 8.36 pH 6.85 1% CaCl 1% Leci hin - 0.84 1.00 0.92 pH 6.9 Table A5 (cont'd.). 1 O9 Yolk or Emulsion Emptying Time (Min.) Variable Trial 2 3 4 Ave. Yolks With some egg white .87 1.96 1.88 1.90 Fresh after several hours at room temp. .97 9.20 6.36 6.65 7.30 Fresh ' .92 8.95 8.93 9.12 8.98 4 wks. old .40 6.44 6.43 6.45 6.43 8 wks. old .16 4.96 4.54 4.98 4.91 110 Table A6. Chattilon pressure and yolk index values for artificial egg yolks with varied emulsification and formation timesa Special Algin Time of Algin Chattilon Reading or Emulsifica- Film Formation Yolk (Total Pressure = tion Conditions (Min.) Index Reading x lO'g) Dispax 20 .335 2.6 pH 6.1 20 .339 6.0 0.5 20 .309 O CaClz 20 .460 5.8 2.0% algin 25 .380 21.9 25 .353 2.5 25 - 0 Homogenized 1400 psi 15 .398 0 20 .474 10.9 25 .382 36.1 24 .507 46.0 23 .509 22.6 Homogenized 2 x 500 psi 15 0.491 42.3 pH 7.45 10 0.477 24.0 8 0.463 11.9 8 0.422 16.3 7 0.414 20.0 6 0.430 16.7 6 0.457 7.0 Homogenized pH 6.85. CMC added 6 .556 0 ~ 6 .498 13.6 4 .496 6.6 4 .488 19.2 Homogenized pH 6.9, CMC added 4 .527 10.9 4 .471 4.8 3.5 .488 24.1 3.5 .492 17.1 3 .467 23.1 2.5 .418 15.4 2.5 - 0 2 .461 21.9 1.5 - 0 1.5 .456 0 111 Table A6 (cont'd.). Special Algin Time of Algin Chattilon Reading or Emulsifica- Film Formation Yolk (Total Pressure = tion Conditions -(Min.) Index Reading x 10'9) Homogenized. pH 6.9. CMC added Algin at pH 4.6 3 .426 31.1 3 .438 13.4 3 .496 19.6 Algin at pH 4.8 3 463 16.0 3 .472 88.7 5 .469 45 Algin at pH 10.4 3 .483 28.2 3 .407 7.8 3 .476 20.6 Homogenization pH 6.8. 2 x 500 psi 10 .425 19.4 1% Lecithin 5 - 0 3 .366 7.0 3 .391 7.0 Lecithin 1.5% 5 .406 19.1 4 - 0 4 .423 17.0 3 .480 4.0 3 .462 17.8 Sodium Hexametaphos- phate 15 - 0 10 .379 2 5 5 - 0 Sodium Trimetaphosphate 15 - 0 15 0 10 0 Sodium Tripolyphosphate 15 .345 3.3 15 - 0 CMC-7LF 5 .447 4.0 5 .481 1.4 4 - 0 4 .427 6.1 6 .469 20.0 112 Table A6 (cont'd.). Special Algin Time of Algin Chattilon Reading or Emulsifica- Film Formation Yolk (Total Pressure = tion Conditions (Min.) Index Reading x lO’g) CMC-7MF 6 - 0 6 .451 23.6 5 .500 5.0 4 .470 5.0 3 - 0 Pectin 8 .507 7.0 7 - 0 6 .472 0 5 - 0 5 - 0 1:2% Ca Acetate Glycerin in Alginate 20 - 15.9 15 - 17.8 10 - 7.5 5 - 2.0 a) Unless otherwise indicated. enca sulated emulsion volumes are 10-15 ml (most 12 m1) contain 1% CaClz and are manufactured in 2.5% algin. 113 Figure A1. Microscopic Evaluation of Emulsions The dark area represents the predominant range of droplet size. The shaded part. where added, extends to the maximum droplet size. Each graph is headed by the ingre- dient or condition modification by which emulsion was evaluated. For % lecithin emulsions: A-Liquid egg white with whey solids B-Reconstituted dry egg white C-Liquid egg white with added dry egg white D-Liquid egg white with NFDM. For stabilizers 0.5% of each compound added: a-control (1% lecithin total) b-additional lecithin c-sodium trimetaphosphate d-sodium tripolyphosphate e-carboxymethylcellulose (CMC) 7LF f-CMC - 7MP g-slow set pectin 114 A ‘9 10 “.5 0.1» y 3 1 41 4 q 1 fiwfioamom 79$ at... 5:23 223:5 Z Lscm-mv z LECITHIN «Joe #7.. 5:33. 223:5 3 ll”:,na_‘° nuaflfluaagg.“ x 0n. 2 0n. 11 =_=__===_=_=_=____=l _========_==_== ______=_________________ ...m...... 5050 wfioaomfimfimii pa 34:1.“ 53%... 3.3.5 oummmuuumm 7.9 uuummzu ".0 umunumv.” 'PH PH PH 1 O 6 wwwmwwwwo 34g of.“ 3:23 2233 A13... was 3323 233:5. 741012315 10 24-5 SrAsILz =2 %‘ WHEY soups APPENDIX B 116 o;m__ sgm> . o;m_P spmoatmooz . u;m_~ zpuzmwpm . N o m poowoxp .o xzmo spo;m_.m .m xgoo xpmoosoooz .N xgoo >Lo> .— sopoo xpo> :qu zgm> .N cvgo spmoatmooz .o cvgu apugmppm . Foo—oak . zoos» spmoatmooz . m e 385;“ z_»;mp.m .m N xu.;o sta> .P oszoxou ape» mogoom =o_uo=—o>m zoos xso> xpoumuoooa spasm._m oxw_mpo so: oz?— mx—pmwo . oxppmwo . oxw—mpc . »_o;mz.m meA . apoaosooos ox“; . N o m sonuzmz .v m N P zoos asm> oxvz . xppppnouooooo zpo» _ogo=ou Lo>m.:.— v— _.O> mocmsooaoo xpox pposo>o .oomogzooco «so mucoesoo Am .mom oogoooa oz» mo :oPugoo x—oz any o» museum quPz AN ouoo .xopmn cowaoom mopomoz Lao» cw gazpoo ogoom copuozpo>m goooso oz» socm mum—googooo «mos m_ pom» no» zovzz Lonszc on» oompa .mmo oozomoo uyom o mo uaoocoo goo» :o oomom AP ”meowuozsumcu zmom 4mz .zox xcmsh umpcmssou gopoo xpo> mgzuxou zpo> zuwpwnouamooo xpoz pogococ go>opw xpo> oocogooooo xpoz apogo>o "owuozpo>m mgouoou .z.o.»=ouv m x~ozuaa< 118 Appendix B (cont'd.). Information and Consent Form This form and explanation are presented to conform to current university rules regarding use of human subjects for research purposes. The following ingredients have been incorporated into an artificial egg yolk and cooked with fresh egg white in the form of a soft poached egg: Fresh egg white Whey protein Concentrate (Enpro-SO) A commercial high polyunsaturated oil Lecithin Calcium Acetate Glycerin Sodium Alginate B-carotene coloring Commercial egg flavoring All ingredients are commercial or GRAS list food items. Taste panel work is necessary to determine potential accep- tability of a low cholesterol natural yolk replacement. There are no known or anticipated risks in eating this product, nor are there any specific or implied individual benefits from its consumption. Individual results will be kept in strict confidence. Appropriate precautions have been taken to insure freshness and cleanliness in preparation of these ingre- dients. I, , have been informed of the nature ofiand the ingredients used to compose the product for which I am being asked to serve as a taste panelist. Descriptions of any possible discomfort and/or risks as well as any possible benefits have been given. I agree to serve on this panel which will be conducted . but am free to withdraw my consent and to discontinue par- ticipation in the project at any time. I realize that although my individual results will be kept in strict con- fidence, compiled results may be published. Signed Date 12/77 BIBLIOGRAPHY Anonymous. Alginate Industries Limited. 1975. Alginates- Versatile Food Additives. London Central Soya Chemurgy Division. Technical Service Manual: Lecithin. Fanning Chemical Company. Inc. Product Bulletin. Tech- nical Data. Egg Oil, Defatted Egg Powder. Sol-U-Tein. FMC Corp.. Industrial Chem. Div. Functions of phos- phates in foods, Philadelphia. PA. Food Processing. 1975. Eggs. 35 (4):58-59. Food Processing. 1976. Egg substitutes. 37(7):62-63. Food Product Development. 1975. New product, Second Nature. 9(5):12,l4. GAF Corp. 1965. Gantrez® AN poly(methyl vinyl ether/ maleic anhydride). Technical Bulletin 7543-017. Dyestuff and Chemical Division, New York. Kelco Co. (Division of Merck and Co., Inc.). 1977. Kelco algin/hydrophilic derivatives of alginic acid for scientific water control. 2nd. Ed. Kelco Co. 1973. Industrial algin gels. Technical Bulletin. I#23. Kelco Co. 1977. Kelco algin for industrial uses. Technical Bulletin. I#8. Kelco Co. Additional data about calcium salts-Bulletin. Monark Egg Corporation. Product Bulletin. Mogold-CEB and Mogold-Tex#10R. Precision Foods Company. Product Bulletinle. Stauffer Chemical Co., Food Ingredients Div. 1975. The function of phosphates in food products. Food Release No. l. Westport. Conn. 119 120 ----- Stauffer Chem. Co. Technical Bubletins-TrietTM ERB. TrietTMERB Applications. and Triet ZC. Atallah. M.T. and H.O. Hultin. 1977. Preparation of solu- ble conjugates of glucose oxidase and catalase by cross-linking with glutaraldehyde. J. of Food Science. 42:7-10. Becker, P. 1960. Spreading. HLB. and emulsion stability. 0. of the Soc. of Cos. Chemists. ll(6):325-332. Berquist, D.H. 1973. Egg Dehydration. p. 190-223. In W.0. Stadelman and 0.0. Cotterill. Egg science and technology. AVI Publishing Co. New York. Broun. G.E. 1976. Chemically aggregated enzymes. In K. Mosboch. Immobilized enzymes. Methods in enzy- mology. (44):263-280. Cante. 0.0. and V. Moreno. 1975. Edible polyunsaturated emulsions-use of proteose peptone. U.S. Pat. 3.887.715. Church. C.F. and H.N. Church. 1975. Food values of portions commonly used. 0.B. Lippincott C. Philadelphia. 197 p. Cotterill. 0.0. 1973. Egg products industry. p. 126-131. In W.J. Stadelman and 0.0. Cotterill. Egg science and technology. The AVI Publishing Co., Westport. Conn. Earle. R.D. 1975. Method for providing a continuous film of algin containing coating material surrounding a raw onion product. U.S. Pat. 3,865,962. Feb. 11 Abstr. in Offic. Gaz. U.S. Patent Office 931:871. Earle. R.D. Method of preserving foods by coating same. U.S. Pat. 3,395,024. July 30 Abstr. in Off. Gaz. U.S. Patent Office 852:1213. Earle. R.D. and R.H. Mckee. 1976. Process for treating fresh meats. U.S. Pat. 3,991,218. Nov. 9 Abstr. in Off. Gaz. U.S. Patent Office 952:829. Ellinger, R.H. 1972. Phosphates as food ingredients. CRC Press. The Chemical Rubber Co., Cleveland. Ohio. 190 p. Ellinger. R.H. 1972. Phosphates in food processing. p. 617- 780. In T.E. Furia, Handbook of food additives. 2nd. Ed. CRC Press, Cleveland. Flodin. P. 1962. Dextran gels and their applications in gel filtration. Pharmacia, Uppsala, Sweden. 85 p. 121 Friberg, S. 1976. Emulsion Stability. Ch. 1. In S.F. Fri- berg, Food emulsions. Marcel Dekker, Inc.. New Y Funk. E.M. 1948. The relation of the yolk index determined in natural position to the yolk index as determined after separating the yolk from the albumin. Poultry Sci. 27:367. Gardner, W.H. 1975. Acidulants in food processing. p. 225-270. In T.E. Furia, CRC Handbook of food additives. 2nd Ed. CRC Press, Cleveland. Glaser, E. and P.F. Ingerson. 1975. Egg substitute product. U.S. Pat. 3.928.632. Glasser. G.M. and H. Matos. 1976. Low-cholesterol egg product and process. U.S. Pat. 3,941,892. Glicksman. M. 1975. Carbohydrates in fabricated foods. p. 68-88. In G.E. Inglett, Fabricated foods. AVI Publ. Co., Westport. Conn. Gordon. W.G. 1945. Method for the separation of protein from animal matter containing protein in water-soluble form. U.S. Pat. 2.377.624. Gorman. W.A. 1965. Egg product. U.S. Pat. 3,207,609. Groves. M.J. and D.C. Freshwater. 1968. Particle-size analysis of emulsion systems. J. of Pharm. Sci. 58 (8):1273-129l. Habeeb. A.F.S.A. and R. Hiromoto. 1968. Reaction of pro- teins with glutaraldehyde. Archives of Biochemistry and Biophysics. 126:16-26. Haugh. R.R. 1933. A new method for determining the quality of an egg. U.S. Egg Poultry Mag. 39:27.49. Jansen, E.F.. Y. Tomimatsu, and A.C. Olson. 1971. Cross- linking of a-Chymotrypsin and other proteins by reaction with glutaraldehyde. Archives of Biochemistry and Biophysics. (l44):394-400. Jensen. E.V. 1959. Sulfhydryl-disulfide interchange. Sci- ence. 130:1319-1323. Johnson, E.W. 1972. Simulated cooked egg. U.S. Pat. 3.640.732. Jones, R.E. 1969. Low-calorie egg product. U.S. Pat. 3,475,180. 122 Kinsella, J.E. 1976. Functional properties of proteins in foods: a survey. Critical Reviews in Food Science and Nutrition. 7:219-280. Knight. J.W. 1969. The starch industry. Pergamon Press. New York. 189 p. Koenig, N.H. and M. Friedman. 1977. Comparison of wool reactions of selected mono and bifunctional reagents. p. 355-382. In M. Friedman. Protein crosslinking: biochemical and molecular aspects. Advances in Experi- mental Medicine and Biology. Vol. 86A. Plenum Press. New York. Koenig, N.H.. M.W. Muir, and M. Friedman. 1974. Reaction of wool and zinc acetate in dimethylformamide. Textile Research Journal. 44(9):7l7-7l9. Korn, A.H.. S.H. Feairheller and E.M. Filachione. 1972. Glutaraldehyde: nature of the reagent. 0. Mol. Biol. 65:525-529. Krog, N. and 0.8. Lauridsen. 1976. Food emulsions and their associations with water. p. 67-139. In S. Friberg. Food emulsions. Marcel Dekker, Inc. New York. Lazarus, C.R.. R.L. West, J.C. Oblinger. and A.Z. Palmer. 1976. Evaluation of a calcium alginate coating and a protective plastic wrapping for the control of lamb carcass shrinkage. J. of Food Science. 41(3):639-641. Levin. E. 1971. Stable Dried Defatted Egg Product. U.S. Pat. 3,607,304. Loomis, W.D. and 0. Battaile. 1966. Plant phenolic com- pounds and the isolation of plant enzymes. Phytochemis- try. 5:423-438. Luh. N., J.M. Flink, and M. Karel. 1977. Fabrication. characterization. and modification of the texture of calcium alginate gels. J. of Food Science. 42(4): 976-981. Luh. N., M. Karel. and J.M. Flink. 1976. A simulated fruit structure suitable for freeze dehydration. J. Food Science. 41:89-93. Melnick. D. 1971. Low cholesterol dried egg yolk process. 0.5. Pat. 3.607.304. McDowell. R.H. 1975. New developments in the chemistry of alginates and their use in food. Chemistry and Industry 9(3):391-395. 123 McGill. H.C. and G.E. Mott. 1976. Diet and coronary heart disease. p. 376-391. {In Nutrition Reviews. present knowledge in nutrition. 4th Ed. D.M. Hegsted. Chair. The Nutr. Foundation. Inc.. New York. Miller, R.E. 1972. Protein cross-linked with polymerized unsaturated carboxylic acid. U.S. Pat. 3.685.998. Miller. R.E. l973a. Protein-acrolein acetal complex rumi- nant feed material. U.S. Pat. 3.711.290. Miller. R.E. l973b. Crosslinked protein with acid anhydride as a ruminant feed material. U.S. Pat. 3,720,765. Miller, R.E. 1973c. Alkene-nitrile-protein complex as a ruminant feed material. U.S. Pat. 3,726,971. Miller. R.E. l973d. Halosilane-protein crosslink as rumi- nant feed material. U.S. Pat. 3,726,972. Nath. K.R. and M.W. Newbold, 1976. Fractionated egg yolk product. U.S. Pat. 3,958,034. Needles, H.L. and R.E. Whitfield. 1969. Crosslinking of collagens employing a redox system comprising persul- fate and a reducing agent. U.S. Pat. 3.427.301. Olson, A.C. and W.L. Stanley. 1974. The use of tannic acid and phenol-formaldehyde resing with glutaraldehyde to immobilize enzymes. In A.C. Olson and C.L. Conney, Immobilized enzymes in food and microbial processes. Plenum Publ. Corp. New York. Ostrander, 0., C. Marlinse, J. McCullough. and M. Childs. 1977. Egg substitutes: use and preference with and without nutritional information. 0. of the Am. Diet. Assoc. 70:267-269. Ottesen. M. and B. Svensson. 1971. Modification of papain by treatment with glutataldehyde under reducing and nonreducing conditions. Comptes Rendum Des Travaux Du Laboratoire Calrsberg. 38(11):l7l-185. Pavlath. A.E. 1974. Grafting wool through free radical formation on the protein. Textile Research Journal. 44(9):658-664. ' Perret, M.A. 1974. Synthetic egg composition. U.S. Pat. 3,806,608. Petrowski, G.E. 1976. Emulsion stability and its relation to foods, p. 309-359. In C.0. Chichester. E.M. Mrak, and B.F. Stewart, Advances in Food Research. Vol. 22. 124 Powrie. W.D. 1973. Chemistry of eggs and egg products. p. 61-90. In W.0. Stadelman and 0.0. Cotterill. Egg Science and Technology. AVI Publ. Co. Inc.. Westport. Conn. Princen, L.H. 1972. Emulsion technology, p. 77-128. In R.R. Myers and J.S. Long, Vol. 1 Film forming compo- sitions, Part III. Puski. G. 1976. A review of methodology for emulsification properties of plant proteins. Cereal Chemistry 53(5): 650-655. Radley, J.A. 1968. Starch and its derivatives. 4th Ed. Chapman and Hall, Ltd.. London. 558 p. Rand, R.P. 1976. On the association of lipids and proteins. p. 277-294. In S. Friberg. Food emulsions. Marcel Dekker, Inc. New York. Rees. D.A. 1972. Polysaccharide gels- a molecular view. Chemistry and Industry. 16:630-636. Riberea-Gayon. P. 1968. Plant Phenolics. Oliver-Boyd. Edinburgh. p. 169-197. Scott. T.W. and G.D.L. Hills. 1975. Feed supplements for ruminants comprising lipid encapsulated with protein- aldehyde reaction product. U.S. Pat. 3,925,560. See1ey, R.D. 1974. Low Fat Egg Product. U.S. Pat. 3.843.811. Shapiro. R. and A. Gazit. 1977. Crosslinking of nucleic acids and proteins by bisulfite. p. 633-640. In Fried- man. Protein crosslinking: biochemical and molecular aspects. Advances in Experimental Medicine and Biolo- gy. Vol. 86A. Plenum Press. New York. Sharp, P.F. and C.K. Powell, 1930. Decrease in interior quality fo hen's eggs during storage as indicated by the yolk. Ind. Eng. Chem. 22:909-910. Slonimsky. G.L.. V.B. Tolstuguzov. V.A. Erhova, and 0.8. Izjumov. 1973. Granular protein containing food pro- duct resembling the natural caviar of sturgeon. salmon or other fish, and a method of preparing same. U.S. Pat. 3.717.469. Smith. L.M., M.B. Carter, T. Dairiki. A. AcunaBonilla, and W.A. Williams. 1977. Physical stability of milk fat emulsions after processing as evaluated by response surface methodology. Agricultural and Food Chemistry 25(3):647-653. 125 Sternberg. M. and D. Hershberger. 1974. Separation of proteins with polyacrylic acids. Biochim. Biophys. Acta. 342:195-206. Sternberg. M.. J.P. Chiang, and N.J. Eberts. 1976. Cheese whey proteins isolated with polyacrylic acid. J. of Dairy Science. 59(6):1042-1050. Sternberg, M. 1975. Recovery and purification of proteins. U.S. Pat. 3,883,448. Strong. D.R. 1974. Egg Product. U.S. Pat. 3,840,683. Strong. D.R. and S. Redfern. 1975. Egg Product. U.S. Pat. 3.911.144. Strumeyer. D.H. and M.J. Malin. 1975. Condensed tannin in grain sorghum: isolation, fractionation, and charac- terization. J. Agric. Food Chem. 23(5):909-9l4. Szcesniak. A.S. 1968. Artificial fruits and vegetables. U.S. Pat. 3,362,831. Jan. 9 Abstr. in Offic. Gaz. U.S. Patent Office. 846:561. Szczesniak. A.S. P.R. Humbaugh. and H.W. Block. 1970. Behavior of different foods in the standard shear- compression cell of the shear press and the effects of sample weight on peak area and maximum force. 0. Texture Studies. 1:356-378. Tillin. S.. R.A. O'Connell. A.G. Pittman, and H.W. Ward. 1977. The effects of ethylene glycol on wool fibers. p. 383-390. In M. Friedman. Protein crosslinking: biochemical and molecular aspects. Advances in Experimental Medicine and Biology. Vol. 86A. Plenum Press. New York. Tominatsu, Y., E.F. Jansen. W. Gaffield. and A.C. Olson. 1971. Physical and chemical observations on the a- chymotrypsin glutaraldehyde system during the formation of an insoluble derivative. 0. of Colloid and Inter- face Science. 36(1):51-64. Unilever, N.V. 1971a. Frozen Food Products. Netherland Pat. 7,105,266. Abstr. in Food Sci. and Tech. Abstr. 4(5):208. Unilever. N.V. 1971b. Encapsulated foods. Netherland Patent 7,103,849. Abstr. in Food Sci. and Tech. Abstr. 4(2):395. 126 Unilever, N.V. 1972. Method for manufacturing food pro- ducts. Netherlands Pat. 7.115.640. Abstr. in Food and Sci. Abstr. 4(12):143. Unilever, Ltd. 1974a. Simulated fruit. Brit. Pat. 1(369.198. Abstr. in Food Science and Tech. Abstr. 7 6 :98. . Unilever, Ltd. 1974b. Imitation fruit preparation. Brit. PIt) 1.369.199. Abstr. in Food Sci. and Tech. Abstr. 7 6 :98. Uy. R. and F. Wold. 1977. Introduction of artificial cross-links in protein. p. 169-186. In M. Friedman, Protein crosslinking: biochemical and molecular as- pects. Advances in Experimental Medicine and Biology. Vol. 86A. Plenum Press. New York. Walnak, B., L.F. Barrington. and L. Faller. 1960. Emul- sions and foams. p. 69-72. In Physical functions of hydrocolloids. No. 25 of the Advances of Chemistry Series. American Chemical Society. Washington. D.C. Wildi, 8.5. and R.E. Miller. 1973. A protein-acetylenic ester comples to retard digestion in the rumen. U.S. Pat. 3.718.478. Williams. S.K.. 0.L. Oblinger, and R.L. West. 1978. Evalu- ation of a calcium alginate film for use on beef cuts. 0. of Food Sci. 43(2):292-296. Wirth. P.C. and R. Tixier. 1974. Fixation of nitrogenous materials. U.S. Pat. 3,836,433. Wood, F.W. 1974. Artificial fruit of heterogeneous eating texture. U.S. Pat. 3.892.870. July 1 Abstr. in Offic. Gaz. U.S. Pat. Office. 936:277. Wurzburg. 0.B. 1964. Aceetylation. p. 286-288. In R.L. Whistler. Methods in carbohydrate chemistry. Vol. IV Academic Press. New York. Wylie. A. 1973. Alginates as food additives. Royal Society of Health Journal. 93(6):309-314. Ziegler, H.F.. R.D. Seeley, and R.L. Holland. 1971. Frozen Egg Product. U.S. Pat. 3,565,638. Ziegler. K.. I. Schmitz. and H. Zahn. 1977. Introduction of new crosslink into protein. p. 345-354. In M. Friedman. Protein crosslinking: Biochemical and molecular aspects. Advances in Experimental Medicine and Biology. Vol. 86A. Plenum Press, New York.