LIPID STABILITY OF COOKED, DICED AND FROZEN EGGS Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY PARVIN HOOJJAT 1977 i I I I w IIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIII 3 1293 10469 9941 -| 5: hi CO 2C? CIOQO ABSTRACT LIPID STABILITY OF COOKED, DICED AND FROZEN EGGS BY Parvin Hoojjat Lipid oxidation in commercially prepared cooked- diced frozen egg samples was evaluated. Before freezing, one-half of the samples were treated with a commercial antioxidant, Tenox 2®. After freezing in a CO:2 tunnel the samples were packed in vacuum or air and stored at three different storage temperatures: -23il° and -12i2°C, 'for 6 months and 511°C for 15 days. Fat and moisture content, lipid oxidation and panel evaluations were determined initially and during storage. Total plate counts were determined after 6 months storage for those samples which were held at -23° and -12°C, and every 3 days for those which were held at 5°C for 15 days. The percentage of fat was lower and moisture was higher for cooked-diced frozen eggs than for the fresh cooked egg yolk at 0 day and after 6 months in storage. Parvin Hoojjat Lipid oxidation, as determined by TBA values, was relatively low in cooked egg products. Values for all of the samples fluctuated up and down through storage. Samples containing antioxidant were slightly less oxi- dized than the control especially for those products held at -23°C. Egg products held at -12°C exhibited a dif— ferent oxidation pattern. TBA values declined from time of storage to 3-4 months in storage then increased during storage months 5 and 6. The interfering pigments were separated chromato- graphically before measuring the absorbance of the TBA complex associated with oxidized lipids. The values were approximately 0.5 lower than before this clean-up pro- cedure. Flavor differences and acceptability were deter- mined by a group of randomly selected panelists. The control air packed sample had the least and the antioxi- dant vacuum packaged samples had the greatest difference from the reference at 0 day for those which were stored at -23°C. After 6 months in storage, they had almost the same difference from the reference (moderate). Those samples held at -l2°C were initially moderately different from reference sample and after 6 months they had large differences from reference. The microbial analysis after 6 months in storage showed that samples at -12°C had higher bacterial counts than those stored at -23°C. Parvin Hoojjat The results of TBA values, for those samples which were stored at 5°C for 15 days, showed that antioxidant treated samples had higher TBA values than the controls after 5 days storage. Egg salads were made with thawed eggs and were compared with fresh egg salad. Moderate differences in flavor (from the reference) were found and they were less acceptable than the fresh egg salads. No bacterial growth was found in this group of cooked frozen eggs. LIPID STABILITY OF COOKED, DICED AND FROZEN EGGS BY Parvin Hoojjat A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1977 ACKNOWLEDGMENTS Appreciation is expressed to Dr. L. E. Dawson for his guidance, advice, and encouragement throughout my graduate program. Appreciation is also expressed to Dr. C. M. Stine, Dr. R. J. Evans, and Dr. A. E. Reynolds, members of my committee. Appreciation is also extended to my friends for their assistance in the preparation of this thesis. Thanks are expressed to my parents and family who provided motivation and support to make this achievement possible. ii TABLE ACKNOWLEDGMENTS . . . . LIST OF TABLES . . . . LIST OF FIGURES . . . . INTRODUCTION . . . . . LITERATURE REVIEW . . . OF CONTENTS Chemical Composition of Eggs. Autoxidation of Lipids. Microbiological Properties Uncooked Frozen Egg Products. Precooked Frozen Eggs . Preparation of Hard-Cooked Eggs New Product Development EXPERIMENTAL METHOD. . . Source and Commercial Handling of Eggs of Antioxidant Preparation and Application Freezing . . . . . Analytical Procedures . Moisture . . . . Fat . . . . . Lipid Oxidation . . Bacterial Analysis . Sensory Evaluation . . Statistical Analyses . RESULTS AND DISCUSSION. . Long Storage Period. . The TBA Test . . . Analysis of the Results Taste Panel Results. . Microbiology of Eggs . iii Page ii Short Storage Period at 5°C TBA Test Sensory Evaluation . Microbial Counts. SUMMARY AND CONCLUSION. APPENDIX . REFERENCES. iv Page 58 58 61 66 7O 73 74 Table l. 10. 11. LIST OF TABLES Moisture, fat and solids content of yolk por- tions of cooked-diced whole eggs during 6 months of storage . . . . . . . . . The TBA values of yolk lipids during 6 months of storage at -23°C . . . . . . . . The TBA values of yolk lipids during 6 months Of storage at -120C 0 o o o o o o o The TBA values of the cooked frozen eggs, during 6 months of storage at -23°C, after the interfering color was removed by cellu- lose column . . . . . . . . . . . TBA values of the cooked frozen yolk, during 6 months storage at -12°C, after the inter- fering color was removed by cellulose column . . . . . . . . . . . . Analysis of variance of TBA values obtained from the yolks of cooked-diced frozen whole egg held up to 6 months at two temperatures. . . . . . . . . . . Mean flavor difference scores between refer- ence and treated cooked-diced eggs during 6 months of storage . . . . . . . . Mean acceptability scores of treated cooked- diced eggs during 6 months storage . . . Total bacterial counts of frozen-cooked diced eggs after 6 months in storage. . . The TBA values of the frozen-cooked-diced eggs during 2 weeks storage at 5°C . . . The TBA values of the frozen-cooked-diced eggs during 2 weeks storage at 5°C (after passing through the column). . . . . . Page 31 34 35 42 43 46 48 53 57 59 62 Table Page 12. Flavor difference scores between egg salads made from fresh eggs and frozen thawed eggs 0 O O O I O O O O O O O O O 65 13. Mean acceptability scores of the egg salad made from cooked frozen eggs in comparison with fresh egg salad. . . . . . . . . 67 14. Total plate counts of frozen diced cooked eggs during 18 days of storage at 5°C . . . 68 vi LIST OF FIGURES Figure 1. The absorption spectra of the colors produced with TBA by oxidized egg samples . . . . 2. TBA values of frozen cooked-diced eggs held up to 6 months at -23°C . . . . . . . 3. TBA values of frozen cooked diced eggs held up to 6 months at -12°C . . . . . . . 4. TBA values of frozen-cooked-diced eggs (after removing the interfering color) which had been held up to 6 months at -230C 0 o o o ’ o o o o o o o o o 5. TBA values of frozen cooked-diced eggs (after removing the interfering color) which had been held up to 6 months at -120C 0 o o o o o o o o o o o o 6. Flavor difference between reference and con- trol or antioxidant treated frozen cooked eggs held up to 6 months at -23°C. . . . 7. Flavor differences between reference and con- trol or antioxidant treated frozen cooked eggs held up to 6 months at -12°C. . . . 8. Acceptability scores of frozen cooked diced eggs held up to 6 months at —23°C. . . . 9. Acceptability scores of frozen cooked diced eggs held up to 6 months at ~12°C. . . . 10. TBA values of frozen cooked diced eggs after thawing and holding at 5°C up to 2 weeks . 11. TBA values of frozen cooked diced eggs (after removing the interfering color) after thaw- ing and holding up to 2 weeks at 5°C. . . vii Page 33 37 39 44 45 50 51 S4 55 6O 63 INTRODUCTION The frozen egg industry began in the late 18905 when White and Keith independently conceived the idea of breaking the egg from the shell and freezing the egg meats (Pennington, 1948). These two men combined their ideas and the frozen egg industry was started. Today the frozen egg industry utilizes the most modern equip- ment, practices, the latest methods of plant sanitation, .and breaks high quality shell eggs. To consistently pro- duce top quality frozen eggs, it is essential that strict attention he paid to initial high-quality shell stock, plant sanitation, rapid rate of freezing, and holding of the frozen product at a low temperature (Kahlenberg & Gorman, 1968). Freezing does not appreciably alter the physical characteristic of uncooked, liquid egg white. However, the viscosity of egg yolk and whole eggs increase when they are exposed to temperatures below their freezing point (Davis, Hanson, 1952). Sugar, salt and other addi- tives have been used by industry to limit gelation in yolks, Palmer et al. (1970). They also observed that heating thawed egg yolk at 45 to 55°C for one hour partially reversed this gelation. When cooked eggs are frozen the problem area is reversed from that encountered in the freezing of uncooked eggs. The white becomes rubbery and granular and separates into small clumps or layers, but the yolk remains unchanged during freezing (Woodroof, 1946). In a survey of the 0.5. food manufacturing industry, it was determined that, on a liquid equivalent basis, bakeries, the largest user of eggs, purchased about 58% in dried form (Enochian & Saunders, 1963). The largest consumers of frozen salted yolk in 1961 were the mayonnaise and salad dressing manufacturers, with 42.1 million pounds. Frozen sugared yolk, to the extent of 33.5 million pounds, was utilized by bakeries, baby food processors, and ice cream manufacturers. The principle users of frozen plain yolk were the noodle companies and baby food manufacturers. The quality of the cooked egg white varies with factors that affect ice crystal size. Damage was reduced by super cooling before freezing, or by the addition of finely divided calcium carbonate (Davis et al., 1952). Eggs have been used in various food products, especially to improve the method of preparing products having cooked eggs therein, and which is adaptable to large volume production techniques. The resulting food products must be capable of being safely stored for an extended period of time without deterioration of the texture or appearance of the products (Miller et al., 1966). The lipid components of the yolk are the most important factors from the standpoint of shelf life of egg yolk solids. As usual, in substances of this kind, the shelf life can be extended by refrigeration, gas packing, low moisture drying, glucose removal or combi- nations of these treatments (Forsythe, 1957). The oxidation of egg lipids and the interaction of their oxidation products with other egg constituents cause chemical and physical changes associated with the deterioration of dried egg (Lightbody & Fevold, 1948). Kline et a1. (1964) recently measured 2-thiobarbituric acid reactive substances (TBRS) in dried whole egg samples and found good correlation between the amounts of distillable TBRS and loss of organoleptic quality for powdered eggs stored at low temperatures. The work on lipid oxidation of frozen egg is very limited, or we can say, no work has been reported in this area. Freezing reduces the number of bacteria in egg products. Winter and Wilkin (1947) reported reductions as high as 99%. Some increase in numbers occurs upon defrosting (Winter & Wrinkle, 1949). The primary objective of this study was to investigate the storage stability of hard-cooked, diced and frozen eggs, especially the effects of storage time and temperature, the use of an antioxidant and specific packaging treatments. A second objective was to study the lipid stability and microbial count of similar egg products after defrosting and holding in_a refrigerator at 5°C. The combination of these two studies should provide information on the stability and shelf life of cooked diced, whole eggs for most product users. LITERATURE REVIEW Chemical Composition of Eggs Liquid whole egg consists of 64% white (albumen) and 36% yolk. The white contains approximately 12% solids, which is predominantly protein, with a small amount of minerals and sugars and only a trace of fats. Yolk on the other hand contains about 50% solids, nearly two-thirds of which is fat and one-third protein, the latter being generally a very different nature from egg white protein. The lipid composition of egg yolk is essentially as follows (see page 6). The egg as a whole is an excellent source of pro- tein and the protein is of the very highest biological efficiency. The yolk supplies fat and the low carbo- hydrate content makes it very desirable for special diets. From a mineral standpoint eggs are valuable for phosphorus, magnesium, potassium, sulfur, iron, copper, and many trace elements. Eggs are a good source of A and B vitamins, vitamin D, choline, pantothenic acid, inositol, and folic acid. The only major vitamin deficiency in the egg is vitamin C (Forsythe, 1957). .Awm.mv .oum .mmoamonomumo can Awm.av cwnuflomHOmma .Amm.av «Homfiomawnmm .Aofimfla Hmuou mo ma.vv Honmummaono mnm3 ucmmmum mucmsuwumcoo Hmsuo venom uoz as «m m.m mm m.- m a.m anamsmmo venom uoz m.mH om m.v ea m.mm m.H n.om casuwomq m.m v.0 m.va m.vm m.~ m.~a mm n.o Am.omv mowmaa Ionmmosm momma momma m.HH mm m.m m.oa m.~m e em.o mumumm Houmum v m.a m.m m.mv m.v m.m o.mm m.H v.0 mowom wuuwm moum moons m.o m.HH me o m.m m.w~ m.o No mood Inoomamwua m.o H «A as m.e m.h m.n~ 5.0 iooao moaned Hmuoa euom mama mama Huma "ma ouwa ouma ouva cwqu amuoa mowed pmumnsummsb mowoa vmumusumm no a mcfiqu Mao» mom mo nowuflmomaoo Autoxidation of Lipids The property of undergoing spontaneous oxidation on exposure to air is by no means limited to the lipid of foods. It is exhibited by substances of various chemical types. Among the constituents of foods susceptible to oxidation are the fatty acids in their various forms of combination (Lea, 1962). Oxidation can occur only in the fatty acid portions of the triglyceride molecule because the presence of a double bond is necessary for oxidation to occur under ordinary conditions. Oxidation of unsaturated fatty acids can take place by autoxidation, which is quite slow under normal conditions, or enzymatically, which may be very rapid. The autoxidation of an organic substance, RH, involves a free radical chain process which is described in its early stages by the following simplified reaction scheme. This theory has been reviewed by Swern (1962). Initiation: iv ti n . RH aCt a 0:: free radicals ROOH (RooH)2}‘_—* Free radicals (e.g. R-, RO°, RD .' HO', etc.) 2 Propagation: R' + 02 ———> R02- R02' + RH -—9 R: + RooH Termination: R. + R. R: + R02° stable (nonradical) end products R02 + R02 ° A hydrogen atom escapes from the alpha methylenic carbon atom, leaving an unstable free radical. Oxygen readily adds to the position left by the hydrogen atom, producing an unstable peroxide free radical. The peroxide free radical (R02°) captures a hydrogen atom producing a hydroperoxide which is fairly stable at low temperatures. This is the overall mechanism for the autoxidation of mono-saturated and nonconjugated polyunsaturated fatty esters under normal conditions. A shift of double bonds on these products has been found to produce isomers (Farmer et al., 1943). Fatty esters containing conjugated double bonds are more resistant to autoxidation. This is probably due to the close proximity of the double bonds which give added stability by enabling the electrons to dissipate excess energy through what is called resonance. In the monoethenoic and nonconjugated polyethenoic fatty esters, the movement of the electrons is considered to be blocked by the CH2 group, and hence an electron which has excess energy may expend it by breaking away from the remainder of the molecule. When it does so, it takes a proton with it, which is equivalent to the removal of a hydrogen atom, leaving a free radical (Koch, 1956). The peroxides and hydroperoxides formed during oxidation are not responsible for the rancid odor in fats as they are odorless (Lundberg, 1962). The rancid flavor and odors are due to many secondary substances such as aldehydes, ketones and hydrocarbons. These are formed by peroxide decomposition through different reactions (Lundberg, 1962; Pokorny, 1971). Among the different methods used for the determi- nation of rancidity in food products, the 2-thiobarbituric acid (TBA) procedure has gained the most popularity. In 1944, Kohn and Liversadge observed that animal tissues which had been incubated aerobically gave a red color with TBA. This red color was found to be the result of a complex formed from the oxidation products of unsaturated fatty acids and TBA. They suggested that the TBA reactive material was blocked by semi-carbazine or phenylhydrazine. The compound responsible has been shown (Sinhuber et al., 1958) to be the dicarbonyl compound, malonalde- hyde. The red pigment was found to result from the condensation of the two moles of TBA with one mole of malonaldehyde (Sinhuber & Yu, 1958). Aqueous malonalde- hyde can be converted to its volatile isomers by acidifi- cation only, but acid and heat are necessary to release 10 malonaldehyde from its bound state in protein (Kwon & Brown, 1965). Maximum recovery of TBRS in the steam distillate from oxidized food was obtained only at acidic pH (Tar- ladgis et al., 1960). There is a general agreement that the absorption maxima of these pigments is in the range of 532-538nm, however, with certain foods and tissues, instead of the usual pink or red color an orange or yellow shade results. Wilbur et a1. (1949) and Biggs and Bryant (1953) attributed the yellow color to carbohydrate interference and they showed that this yellow color had an absorption maximum at about 450-460nm. The separation of these colors from red color of TBA reaction has been achieved by absorption chromatography and elution from cellulose (Caldwell & Grogg, 1955; Yu & Sinhuber, 1962). Of the several methods employed to combat ran- cidity in edible animal fats and fat containing foods, the use of antioxidants has been found to be the most effective and efficient. Antioxidants are chemical compounds which can absorb the energy of the activated fat molecule and pre- vent the formation of a chain reaction. They may react either with the free fatty acid radical or with the free hydroperoxide radical, in both cases the active free radicals are deactivated and free antioxidant radicals are produced which are not able to initiate another 11 autoxidation chain. They react with other free radicals or can be further oxidized to quinones (Everson et al., 1957). Phenolic antixodants are very active, but they also may promote autoxidation under unfavorable con- ditions (Pokorny, 1971). It has been shown that antioxidants, particularly those with simple phenolic structure, are quite volatile. They are lost rapidly, not only through steam distillation from frying fat, but by vaporization at room temperatures. In fact, some food processors depend on this vaporization to obtain complete distribution of an antioxidant in food products (Stuckey, 1962). The effectiveness of an antioxidant, the amount of the antioxidant, and the quality of fat, are all factors in determining product stability. Microbiological Properties The bacterial flora of frozen egg products is important because it influences the wholesomeness of the products, their keeping quality and their functional properties in the preparation of food. Lepper et a1. (1944) and Hillig et a1. (1960) reported that a direct microscopic count exceeding 5,000,000 bacteria per g with detectable amounts of formic, acetic or succinic acids, or more than 7 mg lactic acid per 100 g of egg demonstrated the presence of decomposed eggs. 12 Haines (1939), Gibbons et al. (1944), Winter et a1. (1946) and others have presented data which indicate that more than 80% of freshly laid eggs are bacteriologi- cally sterile. Nielson and Garnatz (1940), Schneiter et a1. (1943), Winter and Wrinkle (1949a) and others have shown that freezing and storage of liquid egg products destroys nearly all of the coliform bacteria and more than 90% of the other bacteria present at the time of freezing. How- ever, the few remaining bacteria may multiply at a rapid rate during defrosting and subsequent holding before use. Frozen eggs sometimes have higher bacterial counts . than the liquid products did before they were frozen. Such occurrences as these indicate that the eggs were not frozen quickly enough or that they were not frozen at a temperature low enough to prevent bacteria from multiplying and spoiling the product (Winter & Wrinkle, 1949b). They also concluded that defrosting at low temperatures retard bacterial increases. Pasteurization of liquid whole eggs at 61 to 62°C (143 to 144°F) for 3.7 to 4 min. reduces the standard plate count of bacteria more than 99%, destroys nearly all the coliforms and gram positive cocci, and the pathogenic gram negative bacteria. Nielson and Garnatz (1940) obtained a sharp drop in the bacterial content of frozen egg containing 14% salt 13 when held at -18°C. Frozen egg with 10% sugar failed to show a reduction in bacterial count until after 114 days. Uncooked Frozen Egg Products Most frozen egg products are marketed as ingre- dients for use in other food products. Plain egg white, yolk and whole egg as well as a variety of blends of yolk and white, which may include other food ingredients, are frozen (Cotterill, 1973). Variations in these basic products have resulted from differences in user specifi- cations and additional products such as frozen fortified whole eggs, quick whipping frozen white, frozen sugared yolks, and frozen salted yolks are now available (Dawson, 1969). . The freezing of egg white has little visible effect on its properties, apart from shrinking the thick white to some extent, and thus increasing the volume of thin white in a mixture of the two (Brooks & Taylor, 1955). Frozen whole eggs, frozen yolks and frozen whites are equal to fresh eggs for use in leavening and thickening qualities, according to tests made on angel food cakes, sponge cakes, butter cakes, and baked custards. Frozen eggs stored 6 months are as desirable as those stored 6 weeks (Erikson & Boyden, 1955). Clinger et a1. (1951) stated that cakes made from frozen egg white had nearly as good cake volumes as those made from fresh egg white. 14 Pearce and Lavers (1949) stored frozen white and frozen yolk for three months at -lO°F (-23°C), after thawing, the products were mixed with an equivalent amount of fresh yolk and fresh white, respectively, and the volume of the foams (after addition of sugar) and of sponge cake prepared from the mixtures were measured. The freezing and storage of egg white had little or no effect on foam volume. The use of frozen egg yolk increases efficiency in commercial emulsion production. However, the utility of freezing pure egg yolk is limited by the irreversible gelation reaction (Moran, 1925) which egg yolk undergoes as a result of greatly increased viscosity. Such egg products are difficult to combine with other ingredients. In addition to increased viscosity, thawed whole eggs tend to have a curdled appearance and exhibit separation of a dark, unattractive liquid after thawing. The influence of freezing and thawing egg yolk on its emulsifying capacity has been studied but results have been variable. Miller and Winter (1951) found that frozen yolk was a more efficient emulsifier in mayonnaise than fresh yolk. However, Kilgore (1935) reported that more frozen than fresh yolk was required to produce mayonnaise of acceptable viscosity. Gelation is easily controlled. Sodium chloride and sucrose (10% commercially) are most commonly used 15 (Thomas & Bailey, 1933). The only disadvantage of either of these ingredients is that future use may be restricted to specific food products. LOpez et a1. (1954) reported that treatment of yolk with proteolytic enzymes (papain, trypsin, and rhozyme) inhibited gelation. Adding 5.5 ml distilled water per 100 g yolk partially inhibited gel- ation (Meyer & Woodburn, 1965). Mechanical treatment such as homogenization, colloidal milling or excessive mixing also reduces the viscosity of frozen yolk (Thomas & Bailey, 1933; Pearce & Lavers, 1949; Lopez et al., 1954; Marion, 1958). Egg yolk gelation is affected by rate and temper- ature of freezing and thawing as well as by storage time and temperature. The freezing point of egg yolk is about -1°C, yet, gelation does not occur until a temperature of -6°C is attained. Gelation of plain egg yolk takes place most rapidly at -l8°C (Palmer et al., 1970). Rapid freezing in liquid nitrogen and acetone-CO2 mixture pro- duced less viscous defrosted yolk than did slow freezing (Lopez et al., 1954). Apparently, smaller ice crystals are formed and less dehydration of the protein occurs (Powrie et al., 1963). Jaax and Travnick (1968) postu- lated that the heat of pasteurization may alter the sus- ceptibility of egg yolk to gelation. Decreased gelation can also be effected by increasing the defrosting rate (Lopez et al., 1955). 16 Precooked Frozen Eggs When cooked eggs are frozen, the problem area is a reverse of that encountered in the freezing of uncooked eggs. Freezing causes cooked egg white to become tough or rubbery and water to separate from the clumps or layers. Thus, hard cooked eggs cannot be used when the product is to be frozen (Woodroof, 1946). Cooked egg yolk, on the other hand, can be frozen and stored at -18°C for at least a year without significant texture change (Woodroof, 1946). Davis et a1. (1952) investigated the effects of cooking time, temperature, pH, additives, and freezing rate on cooked frozen eggs. They concluded that freezing cooked egg white caused a series of events to occur during freezing. Water in the gel structure migrated to ice nuclei which grow through the gel so that its elastic tension is released by cleavage, resulting in irreversible contraction. After thawing, pores filled with water were found in the gel from which the water could easily be pressed out, whereas the gel proper had hardened from contraction and the irreversible loss of water. They also concluded that factors which reduced ice crystal size, such as super cooling or the addition of finely ground CaCoB, reduced this damage. Lowering the pH had no effect unless the egg white was supercooled prior to freezing. l7 Bengtsson (1967) concluded that for cooked egg white ultra fast freezing with freezing rates at or above 8 cm/hr resulted in a marked improvement in sensory properties and water holding ability compared to con- ventional air blast freezing at -35°C, whereas, the quality of the yolk was largely unaffected by freezing rate. A comparison between thawing in the refrigerator at +5°C, in warm.water and by microwave heating showed no important effect due to thawing method. Frozen storage up to 6 months at temperatures in the range of ~20 to -40°C did not seem to diminish the advantage of very high freezing rate (Bengtsson, 1967). Hawley (1970) prevented the synersis of cooked frozen egg white by adding 2 to 4% of H20 binding carbo- hydrate such as algin, agar or starch. Subsequently, this product can be used in cooked egg rolls. Davis et a1. (1952) observed that a yolk-white ratio of 40:60 to 80:20 when diluted with 20% H20 and adjusted to pH 6.0 to 7.0 before cooking should be suitable in frozen foods. Preparation of Hard-Cooked Eggs When eggs are cooked in the shell the following criteria are suggested: (1) shell does not break during cooking, (2) shell peels off easily and does not adhere to the coagulated egg albumen, and (3) the yolk should be well centered and free of any dark ring, i.e., 18 temperature of the egg, pH of albumen, temperature of heating medium, length of cooking period, strength of shell and quality of eggs are some of the factors which influence these criteria (Irmiter et al., 1970). Swanson (1959), Meehan et a1. (1961) and Fry et a1. (1966) have suggested peelability was most directly related to egg white pH. Egg albumen becomes more alkaline with time and loss of C02, and hard-cooked eggs become easier to peel. Meehan et a1. (1961) reported that eggs with albumen pH values of 8-9 or higher will peel well when hard cooked. Fresh untreated eggs require about 48 hours of normal aging before satisfactory peel- ing after cooking. Storage temperature and storage pretreatment of the eggs are important. Hard et a1. (1963) found that storage at 72°F (22°C) hastened the peelability of eggs over storage either at 13°C or at 0°C. They treated I shell eggs before storage by C02 atmosphere plus oiling; oiling with mineral oil; coating with silicone grease and found that, for all storage temperatures, CO2 + oiling and silicone grease were most effective in maintaining poor peelability properties. Peelability can be increased by exposing egg to NH3. Fresh eggs that were exposed to NH3 exhibited an increase in the percentage of thin albumen to a value characteristic of old eggs and an increase in thin, thick and adhering albumen 19 pH values to approximately 8.9 to 9.1 and peeling ease increased (Reinke et al., 1973). Hard-cooked eggs prepared by the boiling water method were easier to peel and the shelled egg had a better appearance than hard-cooked eggs prepared by the cold water method (Irmiter & Dawson, 1970). The dark circle or green color around the yolk of hard—cooked eggs is caused by formation of ferrous sulfide (Tinkler & Soar, 1920). The ring can be pre- vented or reduced by minimizing cooking time and imme- diately cooling the cooked eggs by immersion in cold running water. The Poultry and Egg National Board recently recommended (Anonymous, 1967) a procedure for hard cooking eggs which included the puncturing of the shell at the large end of the egg. The writer claimed this procedure "when used on eggs less than one hour from the nest, produced hard-cooked eggs which could be easily and beautifully peeled." New Product Development The per capita consumption of eggs in the United States has steadily declined for several years. Darrah and Reid (1963) conducted a survey in Syracuse, New York, of 1,000 families and found that the main reason for the decrease in consumption of eggs was the lack of con- veniences. 20 The Poultry and Egg National Board (1962) pub- lished a brochure entitled, "A market challenge to food manufacturers.” In this brochure, several new egg products are described including ”instant scrambled eggs, egg cookies, frozen fried eggs, cantonese eggs, orange eggs, egg dip and egg loaf" (Baker et al., 1966). Details for manufacturing these new egg products can be found in the brochure. Several new products utilizing eggs have been developed and market-tested at Cornell University. Four of these products are instant frozen french toast (Reid et al., 1960), hard-cooked egg roll (Jack, 1964), chiffon pie (unpublished) and frozen western egg (unpublished). Instant frozen french toast was a very popular new product. The product was sold pre-baked and frozen, and the only preparation done by the consumer was to place the frozen product in an ordinary toaster for one minute. The hard-cooked egg roll is definitely a con- venience item and has proven to be very popular at the institutional level. Strawberry and lemon chiffon pies were developed to create an additional use for dried egg albumen. The frozen Western egg was developed as a con- venience item both for the housewife and the institu- tional trade. It can be taken from the freezer, placed on the grill or in a frying pan, and in 6 to 8 minutes the cooked product is ready to eat. There was no problem 21 with bacterial count and oxidative rancidity using the 2—thiobartiburic acid test throughout the normal storage period (Baker et al., 1966). By changing the form in which eggs are offered to consumers and making them more convenient to use, many of the objections can be eliminated or reduced in importance. EXPERIMENTAL METHOD In this investigation the lipid stability and bacterial counts of control and antioxidant sprayed, cooked, frozen eggs were determined for long-term storage (6 months) at -23°C and at -12°C, and for short-term storage (18 days) at 5°C. The preparation of the samples and the analytical procedures used were the same for both storage conditions. Source and Commercial Handling of Eggs Grade A eggs were obtained from a commercial pro— cessor and were cooked, peeled, diced and frozen in the processing plant using its specific processing procedures, which are outlined below. Eggs were cooked in water for 23 min. at 99°C. They were removed from the water and cooled in water prior to shell removal. The egg shells were removed by a mechanical shell remover (Farm Bureau Services, Grand Rapids, MI) and the hard-cooked, peeled eggs were held for three days at 3°C in an egg preservation solution containing citric acid and sodium benzoate. Dicing was done by a mechanical dicer. 22 23 Pre aration and Application 0 AntIOxidant The antioxidant Tenox 2® (20% butylated hydroxy- anisole, 6% propylgallate, 4% citric acid and 70% propyl- ene glycol) was further diluted with propylene glycol (1:99 v/v) and applied to the diced eggs by spraying to yield a final concentration of .04% on a fat basis of the whole egg. The antioxidant was applied to the eggs, prior to freezing, at the processing plant with a chroma- tographic reagent sprayer, delivering a constant mist for one minute. Eight thousand grams of cooked, diced eggs in five lots of 1600 g each were scattered on 16 x 22” stainless steel trays and the antioxidant was sprayed over the eggs. An empty tray was used as a cover on the eggs and trays were flipped upside down to expose the unsprayed egg surface. These were then sprayed in the same manner. The same quantity of cooked, diced eggs was divided into five trays, but not treated with the antioxidant and were designated as "control." Freezing Immediately following the antioxidant application, both the treated and control samples were put into the ultra-freeze cryogenic freezer tunnel (Chemetron Co., Chicago, IL). CO2 was sprayed over the eggs during the five-minute time the trays were moving through the 24 tunnel. The initial freezing temperature was —68°C, and at the end of freezing period -73°C. After freezing, the trays carrying the eggs were packed with dry ice in cardboard boxes and transported to the food science laboratory, where the experimental analyses were done. Samples of 100 g each were heat sealed with and without air in LKD Super-All Vak pouches using a Model C14 vacuum sealer (International Kenfield Distributing Co., Parkridge, IL). Samples were frozen and stored at -23°C and at -12°C for periods up to 6 months. The eggs at -12°C were allowed to thaw completely once at the end of one month. Egg samples for short time refrigerated holding were held at 5°C before analysis. Analytical Procedures Appropriate egg samples, before storage and at specified times during storage were evaluated using the following procedures. Moisture. Moisture was determined by A.O.A.C. Methods (1970, 24.003b). Triplicate 4 g to 5 g samples were weighed into tared aluminum moisture dishes and dried to a constant weight at 100°C (18 hours) in a forced air oven. At the end of this drying time, the sample dishes were placed in a dessicator to cool before weighing. Moisture was expressed at percent weight loss 25 after drying. These samples were then used for the fat determination, as described below. {33. The fat content was determined by ether extraction using a Goldfish apparatus (A.O.A.C., 1970, 24.005b). The dried samples used for the moisture determination were continuously extracted for three hours using anhydrous ethyl ether. The tared beakers contain- ing the extracted material were dried one hour at 100°C in a forced air oven and placed in a dessicator before weighing. The weight of the extracted material was used to calculate crude fat on a fresh weight basis. Lipid Oxidation. The 2-thiobarbituric acid (TBA) analyses were carried out according to the procedure of Tarladgis, et a1. (1960), with slight modifications. Samples were analyzed each month during frozen storage and every two days for the refrigerator stored samples. TBA reagent (0.02 M 2-thiobarbituric acid in 90% redistilled glacial acetic acid) was prepared by the following method: 0.7208 g TBA and 25 ml distilled water were mixed in a 250 ml volumetric flask. About 200 m1 redistilled glacial acetic acid were added. The TBA was brought into solution by gently warming the flask in water bath. When the TBA was dissolved, the reagent was cooled to room temperature and made to volume with redistilled glacial acetic acid. This was stored in the refrigerator until used. 26 The liquid solution including TBA reagent appeared to contain an abnormal pigment which might interfere with the accuracy of determining spectrophotometric readings. Separation of the red from yellow pigment seemed necessary prior to these spectrophotometric color determinations, and such a separation was accomplished using the method of Caldwell and Grogg (1955). Adsorption columns were prepared by plugging one end of 9 mm x 250 mm glass columns (Kontes Chromaflex column, Kontes of Illinois, Evanston, IL) with glass wool and filling the columns under a nitrogen pressure of 10 to 15 psi to a packed height of 50 mm above the plug with Whatman CF Chromatographic cellulose powder (Sargeant welch, Scientific, Detroit, MI). The frozen eggs were held at room temperature for one hour to thaw, then albumen and yolk were manually separated for the TBA analyses. The TBA analyses were done only using yolk parts, because the naturally occur- ring egg white is almost devoid of fatty material. Ten 9 of yolk were blended two minutes at high speed with 50 ml distilled water in a semi-micro stainless steel jar fitted to a Waring blender base. The slurry was quan- titatively transferred using 47.5 ml distilled water to a 500 ml round bottom flask, containing 2.5 m1 HCL:H O 2 (1:2, v/v), antifoam and several glass beads. Fifty ml distillates were collected from each of four distillations 27 per sample. Ten 9 of distillate were then reacted with 10 m1 TBA reagent in capped culture tubes (200 mm x 25 mm) in a boiling water bath for 35 min. The tubes were then cooled 10 min. in cold tap water. For those samples analyzed after chromatographic separation, aliquots of 7 ml were transferred to adsorp- tion columns and forced through with nitrogen at 9 psi pressure. Each column was washed with one ml distilled water three times. The adsorbed red fraction was eluted with aqueous pyridine into 10 ml volumetric flasks, which had been placed under each column. The adsorbance of each sample was read at 530 nm against a reagent blank, using a Beckman Model DB spectrophotometer (Beckman Instruments, Inc., Fullerton, CA). TBA no. (mg of malonaldehyde/100 9 sample) was calculated using a con- stant of 7.8. Bacterial Analysis. Total plate counts were determined for the eggs subjected to long storage at -23°C and at -12°C, after 6 months and for the cooler held eggs every three days. For the cooler held eggs, the samples were obtained frozen, repacked in Whirlpak bags and held in the refrigerator at 5°C. The total plate count was determined every three days for 18 days. The following procedure was used: 50 g of whole eggs were placed into 450 m1 of 0.1% peptone diluent and blended for two minutes in a Waring blender. The total 28 plate counts were determined by pour plate procedures using plate count agar (Difco) with incubation at 30°C for 48 hours. Sensory Evaluation Flavor difference and acceptability tests were used for sensory evaluations in this experiment. The samples were randomized before they were presented to the panel members. Frozen storage samples were removed from the freezer, and placed in a refrigerator (5°C) the day before evaluations at 0 day and after 2 and 6 months storage. During each evaluation time the panelists were presented with four different treated samples and one fresh sample. To evaluate flavor changes in thawed egg samples, they were removed from freezer and held at 5°C and evaluated at 0 day and after 14 days. The panelists were presented with egg salad instead of plain diced eggs, since this is a primary recommended use of such products. For both groups, the panelists were asked to determine the degree of flavor difference from fresh control samples and the acceptability of the treated samples. Score sheets were designed so that samples could be scored 0 to 4 (no difference to extreme dif— ference) and hedonic score cards were used to record acceptability 0 to 4. (See Appendix.) 29 Statistical Analyses Analysis of variance package program (AOV) was performed by using an M.S.U. computer program identified as MSU Agricultural Experimental Station STAT series, description No. 14, programmed for the CDC 6500 at MSU Computer Laboratory. RESULTS AND DISCUSSION Long Storage Period Moisture, fat and solids content of the separated yolk portion of cooked and diced whole egg are reported in Table 1. Fat content averaged 25%, moisture 60% and total solids 40%. Liquid egg yolk contains 31.9, 49.5 and 50.5% and cooked eggs contain 32, 48 and 52%, respectively (Forsythe, 1957). The percentage of lipids in the yolk portion of cooked, diced and frozen whole egg was higher and moisture lower in the products held at -23°C than in those products held at -12°C and subjected to a thaw and refreeze pro- cedure. These differences are significant and result from absorption of liquid by the yolk material when the albumen thawed and released moisture. The lower lipid resulted from this dilution effect of the moisture, since values were calculated on a wet basis. In addition, the yolk pieces containing absorbed moisture, lost their physical chunky texture and were more difficult to separate accurately from the mixture of diced albumen and yolk. Moisture and lipid contents are inversely related, as expected. 30 31 mm do mm mm me am Had oz man mm mm mm mm mm mm om> oz NH: um mm mm mm mm mm Had mm» «a: mm mm on mm mm mm om> new man manmwum> we on mN He mm ow had oz mm: we vm mN mv mm mm oo> oz mm: we mm mm av mm mm de mm» mm: me mm am no hm hm om> mm» mm: unnumcou moeaom m munummoz a use a momaom » musumeoz w umm m pamoaxo loo omnxomm Iaumd musumnmosoa o H . momuoum A.ozv GEAR momuoum unmaumous .mmmnoum mo nausea w weaken mama oaon3 omowolomxooo mo ma0fluuom xaoz mo usouaoo mowaom can you .musumwoz .H manna 32 As shown in Table 1, the treatments did not influence fat and moisture contents. Storage time had some effect and after 6 months of storage the moisture content was slightly lower. The percent fat, moisture and solids content of fresh cooked eggs were 32, 48 and 52, respectively. The difference in the moisture content between the fresh and frozen thawed egg is probably because the yolk and white in the fresh eggs were separated without dicing, so there was no transfer of moisture from albumen to yolk. The percentage fat of the fresh egg yolk was higher than from frozen-diced for the same reason as mentioned above. The TBA Test The amount of malonaldehyde formed in egg yolk lipids was used as a comparative measurement of rancidity. This amount was determined by TBA test. Sinnhuber and Yu (1958) reported a main absorptic maxima at 532 — 535 nm and secondary maxima at 305 and 245 nm for the pigments -formed in the reaction mixture. In the case of egg, the main absorption maximum was at 530 nm (Figure 1). The TBA values for samples, after treatment and storage up to 6 months, are presented in Tables 2 and 3. TBA values were relatively low, and did not reach levels usually associated with detection of rancidity in foods (about 2.0). This shows that the yolk lipids from cooked eggs, because of their composition or characteristic ABSORBANCY 33 0.14 ' 0.12 0.10 0.08 0.06 0.04 0.02 I 4 n n L J 420 440 460 480 500 520 540 WAVELENGTH (nm) Figure 1. The Absorption Spectra of the Colors Produced with TBA by Oxidized Egg Samples 34 Table 2. The TBA values of yolk lipids during 6 months of storage at -23°C. Treatment Time (month) Antioxidant Control Air Vacuum Air Vacuum Pack Pack Pack Pack TBA Values 0 1.14a 1.51 1.36 1.24 1 1.28 1.38 1.64 1.47 2 1.58 1.17 1.49 1.43 3 1.34 1.30 1.46 1.58 4 1.42 1.24 1.46 1.30 5 1.32 1.17 1.21 1.18 6 1.26 1.31 1.36 1.46 Mean 1.33 1.30 1.43 1.38 aEach value shows the results of 8 different values (2 replicates, 2 samples per replicate and 2 distillations per sample). 35 Table 3. The TBA values of yolk lipids during 6 months of storage at -12°C. Treatment Time (month) Antioxidant Control Air Vacuum Air Vacuum Pack Pack Pack Pack TBA Values 0 1.23a 1.50 1.31 1.37 1 1.33 1.31 1.32 1.25 2 1.20 1.29 1.08 1.04 3 1.07 1.16 0.90 1.01 4 1.10 1.01 1.05 1.09 5 1.02 0.95 1.18 1.09 6 1.19 1.09 1.14 1.18 Mean 1.16 1.19 1.14 1.15 aEach value shows the results of 8 different values (2 replicates, 2 samples per replicate and 2 distillations per sample). 36 are not subject to serious lipid oxidation as measured by TBA values and under conditions of this experiment. The same results were obtained by Baker et a1. (1966). They stored several new products utilizing eggs (instant frozen french toast, hard—cooked egg roll, chiffon pie and frozen western egg) at -30°C. Bacterial counts and TBA tests were made over 3 months storage period at -30°C. During this period there was no problem with bacteria or oxidative rancidity. The data in Table 2 show that the samples with an antioxidant were slightly less oxidized than the control after 6 months of storage, although it has a higher TBA number in some cases. The air packed sample with anti- oxidant was less oxidized than the control, but the vacuum packed were almost similar for the treated and control samples. As the data show, the vacuum packed samples with an antioxidant had relatively high TBA values on the first day, but other samples were almost normal. After 6 months in storage the antioxidant vacuum packed samples had lower TBA values than those evaluated the first day. The control samples (vacuum and air packed) and the samples with antioxidant treat- ments which were air packed had higher TBA values after 6 months storage than the first day, thus they were slightly oxidized. These data are also plotted in Figure 2. Table 3 shows the results of the lipid oxi- dation in yolks which were stored at -12°C. TBA VALUE 37 H _ o—o Antioxidant - Air ' >————< Antioxidant - Vacuum 1.0I' .——-c Control - Air >——-—4 Control - Vacuum 0.9L 1 L ‘ * ' J 0 1 2 3 4 5 6 S TORAGE TIME ( Mo.) Figure 2. TBA values of frozen cooked-diced eggs held up to 6 months at -23°C. 38 As the results show, the TBA values of the samples stored at -12°C are lower than those from pro- ducts stored at -23°C. This was the opposite of what was expected. Up to the first month of storage, the results were normal but after thawing and refreezing the TBA values were lower for samples held up to the 4th month, after which time they started to increase (Figure 3). As the figure shows, the samples containing the antioxidant (Tenox 2) and stored vacuum packed were slightly less oxidized than the other treated samples after 4 months. As shown in Figures 2 and 3, in all cases there was a decrease in TBA values after the peak was formed. These results agree with those obtained by Tarladgis and Watts (1960). Studies in their laboratories on TBA values of cooked meats and fishery products over the past several years have frequently shown a fall of TBA from earlier higher values. They concluded that malonaldehyde production during the oxidation of pure unsaturated fatty acids under controlled conditions follows very closely their oxygen uptake, reaching a peak at the same time the oxygen uptake starts declining. It becomes evident, therefore, that the malonaldehyde precursor does not accumulate as a stable end product, but that after reaching the peak, more precursor is destroyed than produced. Similar results were obtained by Maleki TBA VALUE 1.6 °——' Antioxidant — Air >—--< Antioxidant - Vacuum 'L5 ' '--—' Control - Air ’-""_‘ Control - Vacuum L4 133 III’FVV' . L2 - 1.1 1 _ ’ V( 1.0 - v . 0.9 I l J n n 0 1 2 3 4 5 6 STORAGE TIME (M0.) Figure 3. TBA Values of Frozen Cooked Diced Eggs Held up to 6 Months at -12°C. 40 (1974). Maleki (1974) studied the pattern of rancidity in linoleic acid and corn oil using the TBA method. He showed that malonaldehyde was not a stable end product of rancidity and therefore, the TBA test is not a reliable means of quantitative measurement of degree of rancidity in food products in long-term storage. Privett et a1. (1964) concluded that the TBA values are inconsistent in detection of oxidation flavor deterioration in egg yolk powder. Privett et a1. (1964) compared the peroxide (on extracted fat), carbonyl and thiobarbituric acid values (on steam distillates of whole egg powder), and ultraviolet absorbancy of volatiles at 280 nm, with organoleptic evaluations. The ultraviolet absorbancy method correlated better with organoleptic deterioration over a wider range of conditions than did the other chemical techniques. According to Reinhard and Johansson (1973) aldehyde products of lipid oxidation gave rise to the formation of two pigments. One is the pigment with maximum absorbance at 530 nm, which is the conventional application of the TBA test, is generally taken as an index of lipid oxidation. The other is a yellow pigment with a maximum peak at 450 nm, which is not generally used as a measure of lipid oxidation. Relative amounts of TBRS were corrected for interference by absorbance at 450 nm according to the 41 method of Caldwell and Grogg (1955) for cereal and baked products. The results are shown in Tables 4 and 5. It is obvious that these values are lower than those results before passing through the column and it shows that there may be some interfering pigments which were separated by column treatment. The results are plotted in Figures 4 and 5. By comparing Figures 2 and 4 (TBA values before and after column) it is obvious that, although the values in Figure 4 are lower, differences are relatively constant and need for this extra clean-up step is not apparent. As the curve shows, the samples containing an antioxidant are slightly less oxidized (lower TBA) than the control. As the results show, little or no oxidation occurred in egg yolk after 6 months in storage and in some cases the values are less than those from eggs on the first day, thus some other variables may have interfered with the results; these results also show that malonaldehyde is not a stable end product of rancidity and it changes as the condition of experiment changes. Analysis of the Results The analyses of variance of TBA values are reported in Table 6. Normally obtained TBA values showed no significant differences due to the antioxidant added or to the package treatment. The TBA values from the egg yolk stored at -23°C were significantly (p < 0.05) 42 Table 4. The TBA values of the cooked frozen eggs, during 6 months of storage at -23°C, after the inter- fering color was removed by cellulose column. Treatment Time . . (month) Antioxidant Control Air Vacuum Air Vacuum Pack Pack Pack Pack TBA Values 0 0.71 0.86 0.98 0.70 1 0.81 0.93 1.07 1.00 2 1.01 0.82 0.88 1.04 3 0.80 0.77 1.00 1.10 4 0.81 0.67 0.77 0.87 5 0.79 0.79 0.67 0.76 6 0.67 0.66 0.72 0.92 Mean 0.80 0.79 0.87 0.91 43 Table 5. TBA values of the cooked frozen yolk, during 6 months storage at -12°C, after the interfer- ing color was removed by cellulose column. Treatment (gigih) Antioxidant Control Air Vacuum Air Vacuum Pack Pack Pack Pack TBA Values 0 0.69 0.84 0.66 0.85 1 0.65 0.85 0.91 0.92 2 0.89 0.80 0.85 0.64 3 0.86 0.56 0.56 0.59 4 0.61 0.80 0.50 0.67 5 0.65 0.64 0.70 0.72 6 0.51 0.74 0.51 0.55 Mean 0.66 0.75 0.67 0.71 TBA VALUE L1 10 (19 (18 (17 C16 (15 (14 44 M " ' Fix ,. o——o Antioxidant — Air >——-—< Antioxidant - Vacuum .. |———o Control - Air p——-< Control - Vacuum l I l l n I C) 1 .2 3 ‘4 5 6 STORA GE TIME(M0) Figure A. TBA values of frozen-cooked-diced eggs (after removing the interfering color) which had been held up to 6 months at -23°C. TBA VALUE 45 O——0 Antioxidant - Air >--—-4 Antioxidant - Vacuum I--—I Control - Air >--¢ Control - Vacuum Figure 5. 2 3 . 4 5 6 STORAGE TIME(M0.) TBA values of frozen cooked-diced eggs (after removing the interfering color) which had been held up to 6 months at 46 .Hm>ma wmo.o we» on unseemecmflm i .sEsHoo 0m0asaaoo an uoHoo msflummnmuca mo coaumummmm momma medam> one «o wumsom one: a .musomooum Hmsuoc an omcwmuno 0u03 moans mmsHm> «m9 m0 mumsom noose w~.o mo.o musumnmmsma mmmxomm mmmnoum unmowxowucm av.o «mo.o ommuouw wusumummama mmmxomm mm.o «ma.o wmmuoum muaumumesma pampwxowucm m~.o HmH.o momsouw mmmxomm usmowxowusd HN.o e~.o mmmuoum musumnmosma om.o «ma.o ommuoum coexomm 5H.o «v0.0 momuoum usmpwx0wuod hv.o «hm.o momuoum « « mo.o oo.o musumumm809 mmmxomm unmonoHucd ve.o ma.o ousumummsma momxomm ~m.o v~.o musumumefima unmowxowuca ha.o oo.o omnxomm unmoflxowucd ew.m ma.v musumummewe eao.o .oo.o «magnum ~>.o ¢H.o pamprOwucd mummmm one: QANV «ma maav.¢ma ma maanum> ma manmwum> mocmwum> mo mousom ucoocmmoo unoccommo .mousumnmmfimu 03M um nausea m ou an pawn mom maogz sououm ocean nomxooo mo mxaoz on» Scum omswmuno m00Hm> «m9 m0 moamaum> mo mwmzams< .o manna 47 higher than frozen eggs at -12°C and allowed to thaw during storage. The same significant results were obtained for TBA values after column treatment. A number of significant interactions were found mostly involving product storage temperature and package treatment among values obtained by normal TBA procedure, but no interactions were found among values obtained when TBA reaction solution was passed through the cellu- lose column. It was expected that interactions might occur between the temperature, experimental conditions and storage time over 6 months period. Further studies are suggested to determine which set of TBA values most accurately reflect actual flavor changes identified as rancid. Taste Panel Results Flavor changes and acceptability of eggs were determined 3 times during the 6 months in storage. Eggs were thawed at 5°C for 24 hours, then served at room temperature (see page 28). Each sample was tasted by 8 panelists in two replicate trials. Table 7 shows the mean flavor differences (from references) for fresh and stored products. The higher the values, the greater the difference between treated and fresh samples. All samples of cooked-diced and frozen eggs dif- fered from unfrozen controls, but did not differ among treatments. Before storage, no flavor difference (from 48 .Ahuwaflnmnonm mo Hmbmd mo.ov uEOHoMMHo hausmowmwcmwm no: one Hones: 05mm may we oesoHHOM m00am> « fl .mamemm wosmuommu Eoum oucmuommap umoumeum usmmmummu mucosa: meumq . k m~.~ mo.~ mo.H osHo> one: comm.~ oaH.m oooAH.~ oom.H NH- oz oz m mam.H oom.H osq.H ooH.H mm- oz oz 5 ooo-.~ coho.~ ooomm.~ eHoH «H. mm» oz o ooHo.H o¢¢.H ooom.H mm.H mm: mow oz m oom~.~ ooao.~ oma.~ omm.H «H: oz mo» 4 ooe~a.H oomA.H oo~A.H oho.H mm: oz mm» m om«.~ oom.~ ooom.~ oma.H NH- no» mo» N oooomm.H nama.H oeao.H o-.~ mm- mm» mm» H «mononmmmm scum mosoummmwo coo: 0o us one cm x one: o N o mlgwma Esoom> unamwdb .oz Amnugoev mafia omenoum mucmfiumoua .mmmuoum mo nausea m mewnso ammo omowo nomxooo omummuu one mosmnmmou somsumn mouoom mosmummmao uo>mHu one: .5 manna 49 reference) were detected by panelists, except treatment No. 1. No reason for this significant difference in flavor of products before storage is known. After 2 and 6 months storage, those products which had thawed and were refrozen (variable temperature -12°C) were generally less desirable than those frozen and held at -23°C at constant temperature. The results are shown in Figures 6 and 7. Before storage (zero day) only the egg treated with antioxidant-vacuum packed varied moderately from the reference (Figure 6). This difference decreased after 2 months of storage and remained constant through 6 months of storage. The control air packaged eggs had a slight flavor difference from references before storage and then increased slowly to 6 months of storage. Figure 7 shows flavor differences for eggs stored at -12°C (variable temperature). The flavor differences among treatment were not significant at zero day. These differences increased and by 2 months of storage, all products had greater differences from unfrozen reference eggs. From 2 to 6 months of storage, flavor differences (from reference) increased only slightly. By comparing Figures 6 and 7, it is obvious that the flavor difference from fresh cooked samples is higher for those which were stored at -12°C after 2 and 6 months storage. FLAVOR DIFFERENCE Figure 6. 50 O--. Antioxidant - Air D———-4 Antioxidant - Vacuum o————c Control - Air ..__—¢ Control - Vacuum I l I 2 4 6 STORAGE TIME (Ma) Flavor difference between reference and control or anti- oxidant treated frozen cooked eggs held up to 6 months at -23°C. No Difference Slight Difference Moderate Difference Large Difference Extreme Difference uncunahuo II II II II II FLAVOR DIFFERENCE 51 o————O Antioxidant - Air t—————< Antioxidant - Vacuum f.- .—. Control - Air ,____‘ Control - Vacuum STORAGE TIME (M0,) Figure 7. Flavor differences between refer— ence and control or antioxidant treated frozen cooked eggs held up to 6 months at -12°C. 52 The acceptability of frozen cooked-diced eggs is reported in Table 8. The higher values represent lower acceptability scores. No significant differences were found between treated and fresh (unfrozen) eggs before storage, but after 2 months the samples which had thawed and refrozen (-12°C) were less acceptable than those samples stored at -23°C. As Figure 8 shows the mean acceptability values of products at —23°C were about 2 (neither like or dislike) except sample 1 (anti- oxidant-vacuum), which approached a value of 3 (dislike moderately). Product acceptability changed very little from 2 to 6 months storage. Figure 9 shows the accepta- bility values for products at -12°C. Products at -12°C (variable temperature) were less acceptable than those stored at -23°C (constant temperature). In general, the cooked eggs which had been frozen and thawed were rated low by taste panelists. The texture of cooked eggs after freezing and thawing was responsible for those low scores. The white (albumen) was rubbery, granular and watery. Similar results were obtained by Davis et a1. (1952). According to Davis this damage is due to the mechanical effects of ice crystals formed. In freezing, water in the gel structure migrates to ice nuclei which grow through the gel so that its elastic tension is released by cleavage, resulting in irrevers- ible contraction. After thawing pores filled with water 53 Table 8. Mean acceptability scores of treated cooked- diced eggs during 6 months storage. Treatments Storage Time (month) . Temper- Antl- No. oxidant Vacuum atgre 0 2 6 Mean Acceptabilitya 1 Yes Yes -23 2.67 2.06 2.33 2.35 2 Yes Yes -12 2.50 3.06 3.17 2.91 3 Yes No -23 2.00 2.17 2.56 2.24 4 Yes No -12 2.00 2.94 3.06 2.67 5 No Yes -23 2.00 2.28 1.83 2.04 6 No Yes -12 2.39 2.89 3.00 2.76 7 No No -23 1.83 1.61 2.05 1.83 8 No No -12 2.28 2.56 3.33 2.27 Mean 2.21 2.44 2.67 a0 - like very much; 4 - dislike very much. ACCEPTABILITY SCORE 54 4.. .'—0 Antioxidant - Air >————< Antioxidant - Vacuum I——-I Control - Air 3 - v———¢ Control - Vacuum 1 - O I J_ 1 (J 2 4 6 STORAGE TIME (MO) Figure 8. Acceptability scores of frozen cooked diced eggs held up to 6 months at -23°C. Like very much Like slightly Neither like nor dislike Dislike moderately Dislike very much thI—‘O II II II II II ACCEPTABLITY SCORE 55 o————o Antioxidant — Air D-—-—< Antioxidant - Vacuum 1.. I———. Control - Air ~——-—« Control — Vacuum O . l O 2 4 6 b mL STORAGE TIME (Ma) Figure 9. Acceptability scores of frozen cooked diced eggs held up to 6 months at -12°C. Like very much Like moderately Neither like nor dislike Dislike moderately Dislike very much waI-‘C II II II II II we] be fre ing Woc unc ref watt tern; been so c of s that Micr< \ 6 mor that 56 were found in the gel from which the water could easily be pressed out. Cooked diced yolk changed less during freezing than albumen and was slightly mushy, and accord- ing to the panelists, it lacked flavor of fresh yolk. Woodroof (1946) reported that cooked yolk is practically unchanged after freezing. The texture of thawed and refrozen eggs (-12°C) was less desirable and was more watery than those which were frozen and held at constant temperature (-23°C). The freezing damage had apparently been caused by mechanical action of the ice crystals and so could be reduced by factors favoring the formation of small ice crystals (Davis et al., 1952). They found that super cooling had such an effect. Microbiology of Eggs The bacterial counts from frozen cooked egg after 6 months of storage are shown in Table 9. Results show that samples held at -12°C had higher bacterial counts than those held at -23°C. The antioxidant treated eggs also had higher bacterial count than control eggs. This increase in bacterial count may be due to the higher storage temperature or the thawing and refreezing treat— ment of eggs. Winter and Wrinkle (1949b) reported that, upon defrosting some increase in bacteria numbers occur. Pennington et a1. (1916) confirm that high temperature defrosting increases the number of bacteria. 57 Table 9. Total bacterial counts of frozen-cooked diced eggs after 6 months in storage. Treatment Storage Temperature No. 0223:;t Vacuum -23°C -12°C Total Plate Counts 1 Yes Yes 2.7 x 104 9.8 x 103 2 Yes No 2.5 x 102 2.2 x 102 3 No Yes 0 2.1 x 103 4 No No 0 7.8 x 104 58 According to Schneiter et a1. (1943) frozen eggs of good quality are able to withstand at least two com- plete thawings and refreezings without significant change in bacterial content or without acquiring abnormal appearance of odor. Winter and Wrinkle (1949) reported that freezing temperatures slightly below the freezing point of water destroy more bacteria than lower temper- atures. The reason for the lower number of bacteria in the control (-23°C) compared to the antioxidant treated eggs is not known. Short Storage Period at 5°C TBA Test. To determine the lipid stability of treated and control eggs after freezing and then holding in a refrigerator, frozen eggs were held at about 5°C for 2 weeks. TBA values were determined 3 times each week, and results are shown in Table 10. It was assumed that TBA values above 2.0 would be associated with the development of rancid odors or flavors. All TBA values were relatively low and they were less than that usually associated with rancidity. This shows that little lipid oxidation occurred in the egg yolk during this storage time as measured by TBA values. As shown in Figure 10, TBA values fluctuated up and down with a general trend up through 3-5 days 59 Table 10. The TBA values of the frozen-cooked-diced eggs during 2 weeks storage at 5°C. Treatment Time Antioxidant Control (daYSI Air Vacuum Air Vacuum Pack Pack Pack Pack TBA Values 0 1.19 1.39 1.12 1.38 3 1.26 1.31 1.24 1.21 5 1.44 1.44 1.15 1.26 9 1.33 1.23 0.96 1.05 11 1.32 1.24 0.98 1.08 13 1.24 1.25 1.03 1.03 Mean 1.30 1.31 1.08 1.17 TBA VALUE 60 O——0 Antioxidant - Air {,5 - b——-0 Antioxidant - Vacuum l—-—' Control - Air 1.5 " >—--1 Control -— Vacuum 1.4 1.3 ~ \ 1.2 w 1.1 +- 1.0 . 0.9 . l L . L SI. 3 5 7 9 11 STORAGE TIME (days) Figure 10. TBA values of frozen cooked diced eggs after thawing and holding at 5°C up to 2 weeks. 61 storage and then down through the remainder of the 2-week period. The large scale used to plot the data over- emphasized the small changes in values. A difference in values for control and antioxidant treated egg yolk is apparent, but is only at a magnitude of 0.1 TBA value and is not a significant difference. No explanation is known for the apparent higher values on antioxidant treated egg products except that eggs were sprayed with the antioxidant. This process could have introduced a higher level of oxygen into the product. Table 11 and Figure 11 show the TBA values after the TBA reaction solution was passed through the cellulose column. These values are all lower than the values obtained before this filtration procedure and had greater fluctuations. The TBA values of antioxidant air packed eggs increased slightly after 3 weeks, but remained constant through 2 weeks of storage. TBA values for all other samples fluctuated greatly. This procedure was used to remove possible interfering substances, however, these results suggest that this clean-up procedure did not produce more uniform results. Sensory Evaluation Sensory evaluations for this group of cooked frozen eggs were determined before and after 2 weeks refrigerated storage. Frozen cooked eggs stored at 5°C 62 Table 11. The TBA values of the frozen-cooked-diced eggs during 2 weeks storage at 5°C (after passing through the column). Treatment Time . . (days) Antioxidant Control Air Vacuum Air Vacuum Pack Pack Pack Pack TBA Values 0 0.58 0.64 0.72 0.80 3 0.69 0.73 0.71 0.59 5 0.68 0.95 0.52 0.81 9 0.63 0.56 0.76 0.57 11 0.66 0.80 0.67 0.55 13 0.66 0.69 0.51 0.60 Mean 0.64 0.64 0.65 0.64 TBA VALUE IJ - >---< LO" >—-—-< 0.9 - 0.8 63 o-——o Antioxidant - Air Antioxidant - Vacuum ..__. Control - Air Control — Vacuum O 3 5 STORAGE TIME(day5) Figure 11. TBA values of frozen cooked diced eggs (after removing the interfering color) after thawing and holding up to 2 weeks at 5°C. 64 for 2 weeks were used in making egg salad, and evaluated for flavor differences and acceptability in comparison with egg salads made from freshly cooked eggs. Table 12 shows mean flavor differences from fresh egg salad found by the panel. The higher the values, the greater the difference between reference and treated samples. All mean values were close to 2.0, a moderate difference from the fresh egg salad. Packag- ing and antioxidant treatments had little uniform influence on flavor changes and mean flavor difference values changed little during the 2-week storage of eggs. The use of mayonnaise and mustard improved the flavor of cooked frozen eggs, however, no large flavor differences were detected by the panelists. The major objection to the eggs according to the panelists was the poor texture of frozen white and yolk. The acceptability scores before and after two weeks storage at 5°C are shown in Table 13. The higher values represent lower acceptance. As shown, the acceptability increased slightly for products after 2 weeks in storage. No real flavor differences were found between the salad made from fresh eggs and those which were made from cooked frozen eggs after holding the eggs for 2 weeks at 5°C. Bengtsson (1967) investi- gated the effects of freezing rates on the quality of frozen cooked egg white and fried eggs, from very low 65 Table 12. Flavor difference scores between egg salads made from fresh eggs and frozen thawed eggs. Treatments Storage Time (days) No. Antioxidant Vacuum 0 l4 Flavor Differencea 1 Yes Yes 1.94 1.89 2 Yes No 2.11 2.06 3 No Yes 1.67 1.44 4 No No 2.00 1.61 Mean 1.80 1.75 aLarge numbers represent greatest difference from reference sample. 66 freezing rates up to the ultrafast freezing by liquid nitrogen spray. He concluded that high freezing rates would make it possible from, quality point of view, to market fried eggs, sliced cooked eggs, baked custard, etc. for use in salad, in precooked meals and as sandwich decorations. Microbial Counts The total plate counts of bacteria found in frozen cooked diced eggs, which were held for 18 days at 5°C are shown in Table 14. No growth of bacteria was detected, except in the control sample after 18 days. Nielson and Garnatz (1940), Schneiter et a1. (1943), Winter and Wrinkle (1949) and others have shown that freezing and storage of liquid egg products destroys nearly all of the coliform bacteria and more than 90% of the other bacteria present at the time of freezing. Winter and Wrinkle (1949) concluded that there was no appreciable increase in the bacterial count of the egg held at 32°F for as long as 72 hours. As the results in Table 14 show, the defrosting temperatures were suitable for preventing bacterial growth. According to the U.S. Department of Agriculture (McFarlane et al., 1945), frozen eggs may be held at a maximum air temperature of 40°F for a maximum of 72 hours, or at a temperature between 50 and 75°F for a maximum 67 Table 13. Mean acceptability scores of the egg salad made from cooked frozen eggs in comparison with fresh egg salad. Storage Time Treatments (days) No. Antioxidant Vacuum 0 l4 Accountabiligya 1 Yes Yes 2.33 2.39 2 Yes No 2.50 2.33 3 No Yes 2.56 2.33 4 No No 2.50 2.33 Mean 2.47 2.35 a0 = like very much; 4 = dislike very much. 68 Table 14. Total plate counts of frozen diced cooked eggs during 18 days of storage at 5°C. Time (day) Anti-Vac Anti-Air Control—Vac Control-Air Total Plate Counts 1 0° 0 o o 4 0 0 0 0 6 0 0 0 0 8 0 0 0 0 ll 0 0 0 0 4 4 18 0 0 2.0 x 10 1.5 x 10 aNo growth detected at 10"3 dilution. 69 of 24 hours to temper or partially defrost them. The defrosted product should be cooled to 45°F or less and held at that temperature. SUMMARY AND CONCLUSION Commercially prepared cooked—diced eggs were frozen in a C02 tunnel (the initial temperature was —68°C and at the end of freezing period -73°C). Before freezing, one-half of the eggs were treated with a commercial anti- oxidant, Tenox 2® The eggs were packed either in air or vacuum, stored at three different temperatures (-23, -12, +5°C) and examined for evidence of lipid oxidation and microbial counts during storage time. Fat, moisture and solids contents were determined for those samples which were stored at -23°C and -l2°C. The fat content was lower and moisture was higher than that of freshly cooked eggs. The eggs held at -23°C had higher fat and lower moisture contents than those stored at -12°C, presumably due to moisture transfer between albumen and yolk during a short thaw period. . TBA values were relatively low, and they did not reach a level associated with rancidity detection (about 2.0) by sensory means. The values fluctuated up and down during storage of the eggs. Values for those products held at -23°C had a slight increase in most samples 70 71 through 1-2 months storage, then a general but slight decrease through 3-5 months and an increase after 6 months of storage. The combination of antioxidant and vacuum packaging generally resulted in lowest TBA values after one month of storage. In those samples stored at -12°C, TBA values declined from time of storage through 3-4 months, reach- ing a low value of 1.0, then increased. The eggs treated with the antioxidant followed by vacuum packaging exhibited lower TBA values after storage for 4, 5 and 6 months. Interfering colors were removed by a column separation technique from the TBA reactive solution before spectrophotometric determinations for each product analyzed. These TBA values were lower than normal values and fluctuated, in a similar manner. There appeared to be no benefit gained from this clean-up procedure. Panel sensory evaluations were determined 3 times during storage for those eggs stored at -23 and -12°C. All samples of cooked, diced frozen eggs differed from unfrozen control eggs and they were less acceptable by the panelists. The eggs stored at -12°C were less acceptable than those at -23°C. According to the panelists there were no flavor differences, however, the low scores were influenced by undesirable texture characteristics of the products. The white was rubbery, 72 granular and watery and the yolk was slightly mushy and it lacked flavor of fresh yolk. Bacterial analyses were determined for eggs after 6 months of storage. Those samples held at -12°C had higher bacterial counts than those stored at -23°C. The control air and vacuum packed cooked frozen eggs held at -23°C showed low numbers of bacteria after 6 months. TBA values for those cooked frozen eggs held at 5°C for 2 weeks fluctuated up and down with a general trend up through 3-5 days storage, then down through 5-11 days and up again 11-13 days. The antioxidant treated sample had higher values than the controls. No explanation for these higher values is apparent. The antioxidant application may have introduced a higher level of oxygen with the product. Sensory evaluations for egg products held at high storage temperatures (5°C) were determined before and after two weeks of storage. Egg salads made from the frozen thawed eggs were less acceptable than from freshly cooked eggs, being moderately different from fresh egg salads. Mean flavor difference values changed little during two weeks storage of eggs. For microbial analyses, the cooked frozen eggs were stored at 5°C for 18 days. No growth of bacteria was detected except in the control samples after 18 days of storage. APPENDI X Date: I Name: DICED EGG FLAVOR DIFFERENCE EVALUATION Instructions 1. Determine by flavor comparisons with the reference sample the degree of flavor difference for each coded sample: a. If you do not detect any flavor difference, please check the line opposite the word "none." b._ If in your judgment any flavor difference exists, place a check on the line opposite the term which best describes the degree of flavor difference. 2. Indicate the descriztion of flavor which best describes the difference. 3. Indicate the degree of acceptability for each coded sample. 1. .Qggree of Flavor Difference Ref. I.I —I I -_I I I I -I 0 None _____. .____. 1 Slight _____. .____. 2 Moderate __ ._.._.. 3 Large _____. _____ 4 Extreme 2. Desgrigtion of Flavor Difference Rancid _____. _____ Chemical .____. .____. Salty state m Other _____ .____. 3. Accggtabiligz 0 Like very much ______ Like moderately Neither like or dislike ._, Dislike moderately Dislike very much nbUNI-I' III HOTE: The reference sample should be tasted as often as necessary to determine the degree of flavor difference for each sample. Thank you! 73 REFERENCES REFERENCES Anonymous. 1967. A world of information about eggs. Bulletin E-23. Poultry and Egg National Board. Association of Official Agricultural Chemists. 1970. Official Methods of Analysis. 12th Ed. Assoc. Offic. Agr. Chem., Washington, D.C. Baker, R. C., L. B. Darrah and J. M. Darfler. 1966. The use of eggs for new products. Poultry Sci. 45:1011. Bengtsson, N. 1967. Ultrafast freezing of cooked egg white. Food Tech. 21:1259. Biggs, D. A. and L. R. Bryant. 1953. The TBA test for butterfat oxidation. Can. J. Tech. 31:138. Brooks, J. and D. J. Taylor. 1955. Food Investigation. Report 60. Her Majesty's Stationery Office, London 55 . Caldwell, E. F. and B. Grogg. 1955. 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