FOOD HYDROCOLLOIDS ELSEVIER Food Hydrocolloids 12 (1998) 409-415 Emulsifying characterization of hens egg yolk proteins in oil-in-water emulsions Yoshinori Mine * Department of Food Science, University of Guelph, Guelph, Ontario, Canada NIG 201 Received 20 August 1997; accepted 24 February 1998 Abstract Emulsifying properties of egg yolk as a function of pH and oil volume were studied. Egg yolk proteins formed larger emulsion particles at pH 3 and the mean droplet size of the emulsions was decreased with increasing pH. A linear relationship between tur- ity and mean droplet size of egg yolk emulsions could not be obtained. This may be due to the floculation of the emulsions. Egg yolk proteins formed thicker multilayers at low oil volume, however total protein adsorption ratio against original proteins was 5S— 165%, independent to protein and oil concentration. Electrophoretic analysis of the egg yolk emulsion revealed that the main com- ponents to adsorb at the interface was glanular lipovitellins, even though its emulsifying property was lower than that of plasma because of poor solubility at low ionic strength (0.1 M NaCI) at pH 7. These results indicate that the main contributor for egg yolk emulsion is granules and it can affect the emulsifying properties of egg yolk at different pH values. © 1998 Elsevier Science Ltd, All rights reserved, 1. Introduction Hens egg yolk is an important emulsifying ingredient in the manufacture of mayonnaise, salad dressing and cakes. Egg yolk contains about 52.3~53.5% water, 31.8- 35.5% lipids and 15.7-16.6% proteins. The egg yolk lipid fraction contains approximately 66% triacylgly- cerol, 28% phospholipids, 5% cholesterol and minor amounts of other lipids (Powrie & Nakai, 1985), Fac- tors affecting emulsifying characteristics of hens ege yolk was reviewed by Cunningham and Varadarajula (1973). The emulsifying components in egg yolk are phospholipids, lipoproteins and proteins (livetin and phosvitin). It was reported that lipoproteins in egg yolk are the most important substances but the emulsifying efficiency of the lipoproteins was reduced by addition of phospholipids (Cunningham, 1975; Shell, Olsen, & Kremers, 1935; Yeadon, Goldblatt, & Altschul, 1958). Pasteurization causes little or no damage to the func- tional property and did not affect the formation of stable mayonnaise (Palmer, Ijichi, Cimino, & Roff, 1969). Emulsifying capacity of egg yolk was reduced on spray drying (Varadarajula & Cunningham, 1972). * Corresponding author. Tel: $19 824 4120; fax: $19 824 6631; ymine@uoguelphes (0268-008X/98/S19.00 © 1998 Elsevier Science Ltd, All rights reserved PII: $0268-005X(98)00054-X Emulsifying capacity and heat stability in mayonnaise was improved by preparing the emulsion with pancrea- tic phospholipase fermented egg yolk (Dutilh & Groger, 1981). Hens egg yolk can be separated by centrifugation into plasma, the supernatant, and granule, the pre- cipitate (Li-Chan, Powrie, & Nakai, 1995). Plasma is composed of 85% low-density lipoproteins (LDL) and 15% livetin. Granules contain mainly 70% high-density lipoproteins (HDL), 16% phosvitin and 12% LDL (McCully, Mok, & Common, 1962). Several studies, have concentrated on the emulsifying properties of ez yolk constituents. LDL is considered to be the most important contributor to the emulsifying properties of egg yolk (Mizutani & Nakamura, 1984; Vincent, Pow- rie, & Fennema, 1966). LDL has better emulsion stabi- lizers than granules and livetin in water (Davey, Zabik, & Dawson, 1969). The poor emulsion property of granules is due to low solubility at low ionic strength (Causeret, Matringe, & Lorient, 1991). Stability of egg, yolk emulsions can be improved by acetylation and succinylation of egg yolk lipoproteins (Tsutsui, Matsu- moto, & Obara, 1980). Mayonnaise is a typical oil-in- water emulsion stabilized with egg yolk. Egg yolk lipo- proteins and phospholipids are major constituents of the protective layers. Electron microscopy observation suggested that mixture of lipovitellin, livetin are the410 ¥. Mine{Food Hydrocolloids 12 (1998) 409-415 major constituents of the protective layers (Tung & Jones, 1981). However, a few results deal with emulsi- fying properties of egg yolk at various conditions such as pHs, oil volume and salts. Studies on the interaction of each egg component (HLD, LDL, phovitin and live- tin) or adsorption behaviour at the interface are meagre. The objective of this study was concerned with emulsi- fying characterization of egg yolk at various conditions and adsorption behaviour of each components as a mixture of proteins in egg yolk in oil-in-water emulsion, . Materials and methods materials 2.1. Materials Fresh eggs were obtained from the University of Guelph’s Arkell poultry farm and separated into egg white and yolk. The egg yolk was rolled on a filter paper to remove adhering fragments of the egg white, the membrane punctured and the liquid yolk allowed to flow into a suitable container. Plasma was prepared from the method of Raju and Mahadevan (1974). The liquid yolk was diluted with 3 vols of 0.1 M NaCl, mixed on a magnetic stirrer and centrifuged (8 000g; 60min; 4°C), The precipitate (crude granules) was washed twice with 3vols of 0.05M NaCl, centrifuged as before and the final precipitate dispersed in 1.5 M NaCl solution containing 0.05% sodium azide. The protein content of each fraction was determined according to the modified Lowry method (Markwell, Maas, Bieber, & Tolbert, 1978). Soybean oil was purchased from Sigma Chemi- cals Co. (St. Louis, MO). 2.2. Determination of emulsifying properties The egg yolk, plasma and granule preparation were diluted with various buffers (100mM acetate buffers for pH 3 and 5, 100 mM imidazole buffer for pH 7 and 100mM borate buifers for pH 9, containing 0.1M NaCl, respectively) to give various egg yolk concentra- tions from | to 3% (w/v). Emulsions were prepared by homogenising 3m! of each protein solution with various volumes of soybean oil (from 2.5 to 40%, w/v) for I min at a speed of 12,000rpm using a Polytron PT 2000 homogenizer (Kinematica AG, Switzerland) equipped with a 10mm diameter mobile head. Emulsifying activ- ity was determined using the turbidimetric method of Pearce and Kinsclla (1978). Immediately after emulsion formation, an aliquot was removed and diluted 1:250 a 0.1% sodium dodecyl sulphate (SDS) solution (pH 7). Absorbance was measured at 500nm using a spectrophotometer (Model UV-1201, Shimadzu, Kyoto, Japan). The droplet size distribution (432) of the emulsions was determined using light scattering by a Mastersizer X (Malvern Instruments Ltd, Malvern, UK). The emulsions were centrifuged at 20°C at 3000¢ for 30min and the cream washed twice with Sml of appropriate buffer with each washing followed by cen- trifugation; the subnatants were pooled together for protein determination. The sera were filtered through 0.2m filters and the protein contents of the filtrates determined according to the method of Markwell et al (1978). A protein content of the cream was estimated as a difference between protein concentration of the serum and total protein present in the cream was divided by the specific surface area (from Mastersizer result) of its emulsion to give the amount of protein bound per m?, 2.3. Electrophoresis ‘The washed cream was treated with a solution of 10% (w/v) SDS in 0.1M Tris-HCI buffer, pH 7, containing 5% (v/v) 2-mercaptoethanol (ME). The treated creams were vortexed vigorously for 3min, heated in a boiling water bath for Smin, cooled to room temperature and centrifuged at 16,000g for Smin. The protein compo- sition of the supernatant was determined by SDS-poly- acrylamide gel electrophoresis (SDS-PAGE) on 4-15% gradient gels using the Bio-Rad Mini Protean IT electrophoresis cell at a constant voltage of 20mA/gel. The gels were first stained using the modified Coomassie blue method for phosphoproteins (Itoh, Kubo, & Ada- chi, 1986), i.e. 0.05% Coomassie blue in a solution of 0.1M aluminium nitrate/25% isopropanol/10% acetic acid/1% Triton X-100, in order to stain the phosvitin molecules. The second staining used the normal Coo- massie blue procedure to stain the other proteins; the gels were destained in a solution containing acetic acidj ‘methanol/water (1:8:12, v/v/v). The gels were scanned on a Sharp JX-330 scanner (Sharp Electronics, Tokyo, Japan) and the Rf of each band was determined using the Pharmacia Image Master (Pharmacia Biotech, Que- bec, Canada) to estimate the molecular weight. 3. Results and discussion The changes in mean droplet size of emulsions stabi- lized with 2% egg yolk as a function of pH and oil volumes (10-30% , w/v) are shown in Fig. 1. The results from the mean droplet size of emulsions showed a decrease of droplet size with increasing pH and with decreasing oil volume used to make the emulsions, The emulsions at pH 3 formed larger droplet emulsions (> 10pm) at all oil volume, however, the emulsions at pH 5-9 showed $-6ym droplet size at 10% oil volume ig. 2 shows micrographes of emulsions stabilized with, 2% egg yolk and 10% oil volume at different pH values. A larger droplet emulsions were observed at pH 3 and emulsions with smaller droplets were formed at pH 9 than pH 5 and 7. The results indicate that egg yolks are¥. Mine) Food Hydrocolloids 12 (1998) 409-415, au 15, 1 30% oil volume HE 20% oil volume =e D1 10% oil volume S10 =z 2 z $ 3 5 3 = o L 3.0 5.0 7.0 9.0 pH Fig. 1. Changes of mean droplet ize of emulsions a a funion of 0 ‘olume a dflret pi values: Emulions were prepared wth 2% (9) ‘egg yolk. The values are an average of triplicate measurements better emulsifiers at pH 9 than neutral or acid pH values. At low pH regions (3 and 5), emulsions droplets were flocculated immediately after the preparation of emulsion and creaming rate was faster than that of PH 9 The ioelectroric point of egg yolk proteins is pH 5.5-5.8 (Powrie & Nakai, 1985). The stabilization of emusic dependent on the force of electrostatic repulsions, between the adsorbed protei ial protein film (Dickinson, 1988), The decrease in the number of negative charges (COO-) decrease electrostatic repul sions presumably result in flocculation and creaming of 12 yolk emulsion at acidic pH values. The main con- stituents of egg yolk as an emulsifying agent are LDL, HDL, phosvitin and fivetin (LicChan et al, 1995; Vincent et al., 1966). The contribution of pro to emulsifying activity is higher than that of phospholipids (Kiosseoglou & Sherman, 1983; Mizutani & Nakamura, 1984). The ability of proteins to act as emulsifiers depends upon their ability to adsorb at interface, greatly reduce the interfacial t a cohesive film, It was reported that acidification of egg yolk did not affect the formation of stable mayonnaise (Palmer et al, 1969). Effect of pH and salt on emulsifying against coalescence/flocculation is greatly son the inter rapes of egg yolk emulsions at (a) pH 3, (b) pH 5, (e) pH 7.0 and at) pH 9. Emulsions were prepared with 2 (o)y) egg yolk and412 Y, Mine) Food Hyd activity of LDL was small (Mizutani & Nakamu 1984). The change of emulsifying properties of phosvi- tin, a one of major proteins in egg yolk with pH was slight (Chung & Ferrier, 1992). However have concerned the emulsifying properties of separated constituents from egg yolk. The present results suggest that emulsifying properties of egg yolk constituent mix: ture show a different profile from that of each compo- nent. Fig. 3 shows the changes of emulsifying activity nd mean droplet size of emulsions as a function of e} yolk and oil volumes. The emulsifying activity increased with increasing egg yolk concentration from | to 3% at all oil levels except at 2.5% oil volume. The emulsifying activity of 1% egg yolk emulsion increased slightly from 2.5 to 15% oil volume emulsions, but decreased over volume egg yolk emulsions. On the other hand, the emulsifying activity of 2 and 3% egg yolk emulsions inereased from 2.5 to 10% oil volume with 2.0 egg yolk emulsion and 30% oil volume with 3% egg yolk emulsion and pleateued, respectively (Fig. 3a), However, the results from mean droplet size of emul- sions (Fig. 3b) showed that mean droplet size of emulsions remained increasing from 2.5 to 40% oil contents at all levels, except at 3% egg yolk with 30. 40% oil content. Larger particle size was formed at lower egg yolk concentration. The turbidimetric technique has these studies 30% of oi ids been adapted for evaluating the emulsifying properties of proteins, because of a linear relationship between turbidity (adsorbance at S00nm) and interfacial area of an emulsion (Pearce & Kinsella, 1978). The higher the turbidity, the more effective was the emulsifiers. This technique has been well known as a simple method for measuring properties of food proteins However, the present data showed that emulsifying activity of egg yolk emulsion is not always reflected the characterization of emulsions. The factor affecting the turbidity measurement may be more complicated com- ponents of egg yolk derived from phospholipids-protein interactions than other food proteins such as casein and, WPI. Rapid creaming of coarse emulsion during mea. surement may affect th 1 500nm. Thus, turbid tic method is not suitable for the precise evaluation of egg yolk emulsion The surface coverage and total adsorption ratio of cee yolk proteins against the original proteins used for preparing emulsions at different egg yolk and oil volume are shown in Fig. 4. There was decrease surface protein at all levels of egg yolk content with increasing oil volume from 2.5 to 40%. The surface coverage of emulsions containing 15-40% oil showed sim tein concentration (3.5-5.5mgim?) at all e pro- yolk levels, 5% oil but it increased with increasing egg yolk at @ 125 Br 093 yoke 100 ME > 003 yok 8 & ‘3% 099 yolk 8 os e D say 2 2 so = oso 8 2 B25 Ges ! B 00 ooo » 2 § © 1% m w ‘2 § © 1% % 4 _ Es E 5 ]E toa yom 5 zg S E | aeenayok 5 eo @ 2 g 3% 09g yok § © |D s%e00y = wo & 5: a 5 S 2 5 le Eo. 2505S a it volume (*4) Oit volume (%) Fig 3. Changes of emulsifying acity and mean droplet size of Fig. 4, Surface concentration and total protin adsorption ratio of eggY. Mine|Food Hdrocollods 12 (1998) 409-415 an emulsion. These results suggest that egg yolk proteins form thicker film at low oil volume or higher egg yolk concentration, resulting in formation of smaller droplet It is possible to discuss when interfacial area is completely covered. additional egg yolk prot round the film and form multilayers, The centrifugation steps involved in the procedure to determine the protein, surface coverage did not wash off adsorbed multilayers, Interestingly, the total adsorption ratio of proteins at interface was constant at all levels ranging from $5 to 65% of original proteins, independing to oil contents in emulsions. This suggests that there is some components in egg yolk proteins which do not adsorb at an oil-in- water interface. SDS-PAGE analysis has advantage to monitore the occurrence of preferential adsorption between the indi- vidual components of egg yolk proteins. The composi- tion of egg yolk proteins adsorbed onto interface and proteins in serum was analyzed by SDS- 5). The egg yolk is composed many polypeptides with molecular weight from 19 to 225kDa. (Fig. Sa) Nine major polypeptides is present in plasma fraction (LDL + livetin) (Fig. Sb) and 4 lipovi- tellins with molecular weight of 215, 105, 81 and 30 kDa and phosvitin (38-48 kDa) are found in granule fraction (mainly HDL and phoyvitin) (Fig. 5c). It has been reported that LDL consists of about 6 major polypep- tides that range in molecular weight from 10) to 180kDa and several unidentified minor polypeptides (Raju & Mahadevan, 1974; Yamauchi, Kurisiki, & Sasago, 1976). Here, the author used the names of lipoproteins for LDL components and lipovitellins for HDL ones \ en -—e3- 3S Mw. ba) - 205 — ee 116 7 84 66 abe @ eae Fig. 5. SDS-PAGE pattems of proteins from egg yolk components and the cream isolate fom egg yolk stabilized emulsions. Lane a yolk; lane b, sma: lane egranui; lanes d a Jind 7 emulsions, lanes F and g. serum from pH 3 and 7 emulsions, . ercam from pl respectively With their molecular masses which were ealeulated from, Ry of each band using the scanner. From the SDS- PAGE analysis, preferential adsorption of egg yolk components was observed at the interface. The main adsorption proteins at interface was from granule con- stituents, namely lipovitellins with molecular weight of 105, 81, and 30kDa. The major LDL lipoproteins were not adsorbed preferentially at the interface and they remained in serum including some granule proteins, which may be due to the excess of egg yolk proteins in serum used to make the emulsions. However, some lipoproteins with high molecular weight from 66 to 225kDa were found at the interface, but the one with Jow molecular weight (<48 kda) did not adsorb at the terface. The phosvitin (38 48kDa) in granule fraction and livetin (S7 kDa) from plasma did not adsorb at the terface. No preferential adsorption of egg yolk pro- teins was observed between pH 3 and 7 (Fig. Sd-e). It can be possible to explain why maximum adsorption tio of egg yolk proteins was lower compared to other proteins [for example, the value of whey proteins is 85 87%, (Hunt & Dalgleish, 1994)]. It was reported that all constituents of egg yolk (HDL, LDL, phosvitin and jerface in pre~ jolk (Chung & Ferrier, 1991; Kiosseoglou & Sherman, 1983; Davey et al., 1969). Egg yolk apoprotein exhibit a higher adsorbing capacity than phosvitin and fivetin because of its flexible struc- ture and a greater surface hydrophobicity (Kiosseoglou & Sherman, 1983). Contribution of proteins of e to emulsifying activity is higher than that of phospholi- pids (Davey et al. 1969; Kiosscoglou & Sherman, 1983a; Mizutani & Nakamura, 1984), HDL lipovitellins of granules were less efficient than LDL apoproteins of plasma (Anton & Gandemer, 1997). Kisscoglou and Sherman also proposed that eg yolk lipoprotein breakdown at the interface and then apoproteins adsorb at the interface whereas neutral lipids coalesce with oil droplet from the interfacial tension-time behaviour and the viscoelastic properties of egg yolk lipoproteins at the groundnut oil-water interface (Kiosseoglow & Sherman, 1983b). However, they did not show any direct evidence to support their hyposesis and little was known about the adsorption behaviour and interaction of plasma and granule fraction from egg yolk at an oil-in-water emul- sion. The results from SDS-PAGE indicate that the ‘major contributor as an emulsifier to cover oil surface is HDL. LDL, livetin and phosvitin have lower affinity to oil-in-water at the interface. In other words, LDL is not ‘major contributor of egg yolk emulsions as reported by ‘many investigators (Mitutani & Nakamura, 1984; Raju & Mahadevan, 1974; Varadaralulu & Cunningham, 1972: Vincent et al., 1966). Subsequently, egg yolk was fractionated by cen- trifugation into granules and plasma. The emulsifying Property of each fraction was compared at various oil livetin) can adsorb at an oil-in-water i paring emulsions with44 Y. Mine|Food Hydrocolloids 12 (1998) 409-815 20: [E99 you Pasa OD cranue Mean droplet size (d,.) (um) 25 5 10 15 30 40 Ol volume (%) Fig. 6 Mean droplet size of emulsions stabilized with egg yolk plasma and granule as a function of oil volume, Emulsions are pre pared with 2% of exch fraction in 100mM imidazole-HC1 buffer, pH 7 containing 0.1M NaCl. The values are an average of triplicate mea volumes (Fig. 6) The emulsions were prepared in 100 mM imidazole buffer, pH_ 7 containing 0.1M NaCl Plasma formed a smaller emulsion than that of egg yolk at all oil levels in the presence of 0.1M NaCl. In con- trast, granule showed a larger droplet size and its mean, droplet size was 2-3 times larger than that of plasma emulsions. These differences of particle sizes of plasma, and granules may be closely related to their solubility in serum used in this study. At low ionic strength, granules have low solubility which may explain its poor emulsi fying properties (Causeret et al., 1991). Yolk and gran- ule require at least 0.3M NaCl to become solubility at 80% at pH 7, whereas plasma was solubilized at any ionic strength (Anton & Gandemer, 1997). It was reported that granule provided a lower emulsifying activity than plasma in distilled water (Dyer-Hurdon & Nnanna, 1993). In distilled water, granules are in a complex form of HDL and phosvitin which is insoluble and consequently cannot adsorb at the interface (Kios- scoglou & Sherman, 1983). However, granules and plasma showed similar emulsifying activities at 0.5M NaCl where granules are highly soluble (Anton & Gan- demer, 1997). In this light, the main factor affecting the difference of particle sizes of plasma and granule emul- sions can be their solubility in the buffer used here. The solubility of granule proteins is low at pH 4-6 at low ionic strength, but increased with increasing pH, or ionic strength (0.58M NaCl), At the pH 6.3 and low ionic strength (0.3 M NaCl), the mixture of lipovitellins. phosvitin and LDL were associated with insoluble granules and dissociation and solubilization occurred at acidic pH (<4.2), alkalization or when the concentration of NaCI reached to 0.58 M (Causeret et al., 1991). These characterizations of granules may affect the emulsifying properties of egg yolk at different pH values. However, it is still questionable why main surface composition of egg yolk is lipovitellins from granules even though the solubility of granules was low at 0.1M NaCl It is assumed that granule lipovitellins are hydrophobic pro- teins and they can interact with oil surface wherever it is, insoluble and complex form. The lipovitellins from HDL may be more efficient to form thicker multilayers around oil droplets. The ionic strength may affect the adsorption behaviour of egg yolk components at differ- ent pH values. In this present work, the author did not analyzed. phospholipids and cholesterol derived from, LDL and HDL at the interface. The role of these com. ponents in egg yolk as an emulsifier is not clear from this work, however, the interaction of phospholipids and proteins in egg yolk is also one of great interests of egg yolk functionality. 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