preparation of cooked egg white, egg yolk, and whole egg gels for scanning electron microscopy

Preparation of Cooked Egg White, Egg Yolk, and Whole Egg Gels for Scanning Electron Microscopy

S.A. WOODWARD and O.J. COTTERILL

ABSTRACT Cooked egg white, egg yolk, and whole egg gels, fixed with glutaraldehyde or unfixed, were frozen at - 35°C or at - 95°C and freeze- dried. Alternatively, unfrozen gels fixed with glutaraldehyde, osmium-thiocarbohydrazide-osmium, or osmium-tannic acid-uranyl acetate were dehydrated in ethanol and critical point-dried from carbon dioxide. Egg yolk and whole egg gels were defatted. Freeze-dried and critical point-dried gels were examined by scanning electron microscopy. Freezing and freeze-drying introduced artifacts due to ice crystal damage, with egg white gels distorted most, and egg yolk gels distorted least. Gels fixed only by glutaraldehyde shrank by 50% during critical point drying. Further fixation by osmium tetroxide and uranyl acetate stabilized gels against shrinkage. Removal of fat from egg yolk and whole egg was essential for observation of protein matrices.

INTRODUCTION

ONE IMPORTANT FUNCTIONAL PROPERTY of egg white (EW), egg yolk (EY), and whole egg (WE) is the ability to form gels upon heating. The physical attributes of the heat- formed gel, such as texture and water-binding ability, are highly dependent on the gel microstructure (Hermansson, 1982). It is therefore important to assess structural characteristics in order to understand the textural characteristics of gels. Scanning electron microscopy (SEM) is an excellent tool for evaluating gel microstructure because of the high resolution available. In preparation of samples for SEM, water must be removed due to the high vacuum conditions required in the electron micro- scope (Postek et al., 1980). For this reason, the sample must be chemically fixed or stabilized to withstand dehydration without changes in the gel structure.

Although scanning electron micrographs of egg gels have been published previously, relatively little attention has been focused on the method of preparation. Most micrographs of egg gels have been obtained from freeze-dried samples (EW gels: Kalab and Harwalker, 1973; Beveridge et al., 1980; John- son and Zabik, 1981; WE gels: O’Brien et al., 1982). How- ever, freezing and freeze-drying produce artifacts in most protein gel structures because of ice crystal formation (Hermansson and Buchheim, 1981; Davis and Gordon, 1984). Montejano et al. (1984) prepared EW gels for SEM using a method which combined fixation and freezing. Samples were fixed in glutaraldehyde, frozen and fractured in liquid nitrogen, thawed and further fixed in glutaraldehyde, dehydrated in ethanol, and critical point-dried. This procedure apparently minimized structural damage due to freezing; however, no comparisons were made to non-frozen samples. Beveridge and Ko (1984) re- cently published two micrographs of WE gels prepared for SEM by glutaraldehyde and osmium tetroxide (0~0~) fixation, followed by ethanol dehydration and critical point drying. Cooked EY gels have not been studied previously by SEM.

The purpose of this study was to compare different techniques for the preparation of egg protein gels for SEM eval-

Author Cotterill is affiliated with the Dept. of Food Science and Nutrition, l-14, Room 107, Univ. of Missouri, Columbia, MO 6521 II Author Woodward’s present address is Dept. of Poultry Science, Univ. of Florida, Gainesville, FL 32611.

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uation. The use of suitable SEM preparation methods should lead to more accurate information as to the microstructure of egg gels.

MATERIALS & METHODS

Preparation of egg gels Day-old eggs were obtained from Single Comb White Leghorns of

a single strain. Albumen was separated from yolk, pooled, and blended in a Waring Blendor at low speed with a small Erlenmeyer flask held in the vortex to minimize incorporation of air. Yolks were rolled on wet cheesecloth to remove adhering albumen and chalazas. Yolk membranes were punctured and the yolk liquid was collected and stirred with a glass rod. WE was prepared by blending eggs at low speed as described for albumen. The pH of EW, EY and WE was adjusted to 9.0, 6.0, and 7.0, respectively, with I .OM HCI or NaOH added dropwise. Gels were formed by heating liquid samples in 50 mL beakers at 85°C for 30 min, cooled to 20°C, and cut with a razor blade into I.5 x 4 X IO mm strips.

Freezing and freeze-drying Gel strips (EW, EY and WE) were prepared by four treatments,

comparing fixed and unfixed gels frozen at two temperatures. Samples (ca IO strips per treatment) were fixed in 2% glutaraldehyde in 0. IM phosphate buffer for 2 hr. Both fixed and unfixed samples were then frozen on the cold plates of a RePP Model I5 laboratory freeze-dryer (VirTis Co., Inc., Gardiner, NY) at -35”C, and freeze-dried for 24 hr at a plate temperature of 24°C with the water vapor condenser at - 60°C and a vacuum of 50 mTorr. A second set of fixed and unfixed strips was submerged in liquid hexane (- 95°C) for a few seconds, immersed in liquid nitrogen (- 196”C), and freeze-fractured (Rebhun, 1972). Fractured gel pieces were then freeze-dried in a TIS-U-DRY freeze-dryer (FTS Systems, Inc., Stone Ridge, NY) at a plate temperature of - 55°C for 48 hr with the water vapor condenser at - 95°C and a vacuum of 5 mTorr.

Freeze-dried EY and WE pieces were subsequently defatted in chloroform (Kalab, 1981), placed in absolute ethanol and critical point- dried with CO2 as the transition fluid. Samples were mounted on copper tape and/or silver paint and sputter-coated with ca 500 A gold. The samples frozen at - 35°C were examined in a JEOL S 1 -SEM at 45” tilt and an accelerating voltage of IO kV, while the samples frozen at -95°C were examined in a JEOL JSM-35 SEM at 0” tilt and 20 kV.

Fixation and critical point drying of unfrozen samples EW and EY gels were treated by three different fixation methods:

(I) glutaraldehyde, (2) glutaraldehyde followed by osmium-thiocarbohydrazide-osmium (OTO) post-fixation, and (3) glutaraldehyde followed by osmium-tannic acid-uranyl acetate (OTU) post-fixation. WE was treated only by the glutaraldehyde-OTU fixation procedure.

Glutaraldehyde fixation. Gel pieces were fixed for 3-4 hr at 20°C in 2% glutaraldehyde in 0. IM phosphate buffer, pH 6 (EY) or 7.5 (EW). EW samples were not processed above pH 7.5 to prevent glutaraldehyde polymerization (Hayat, 1970).

OTO procedure. The method of Postek and Tucker (1977) was followed. Fixed gel pieces were first cut to 0.5 X 1.5 X 4 mm to expose a fresh inner surface and post-fixed in 1% 0~04 for 2 hr at 20°C in veronal acetate buffer, pH 6 (EY) or 7.5 (EW). Samples were rinsed three times in deionized water and placed in a filtered, saturated I % thiocarbohydrazide solution for 30 min. After rinsing six times in deionized water, samples were placed in 2% 0~0, in deionized water for I hr.

OTU procedure. This method, developed by Wollweber et al.

SCANNING ELECTRON MICROSCOPY OF EGG GELS. . .

(1981), was designed to prevent shrinkage of specimens during sub- sequent critical point drying. Samples previously fixed in glutaraldehyde and post-fixed in 0~0, were washed thoroughly in veronal acetate buffer and placed in 1% tannic acid in Verona1 acetate at pH 6 for 1 hr at 20°C. After three rinses with deionized water, samples were placed in 0.5% many1 acetate in deionized water for 1 hr at 20°C and then rinsed again.

Samples from all three fixation treatments were stored overnight in deionized water at 4”C, and then dehydrated successively in 20, 40, 60, 80, 95, and three changes of 100% ethanol. EY and WE were defatted overnight in chloroform at 20°C and returned to ethanol. Samples were critical point-dried with CO* as the transition fluid, mounted, sputter-coated with ca 450 A gold-palladium and examined in a JEOL JSM-35 SEM at 20 kV. An average of five samples from each treatment was observed in order to obtain representative micrographs.

RESULTS

Egg white gels Freezing of EW gels produced extensive ice crystal damage.

The EW gel frozen at - 35°C (Fig. 1 A) had pockets of 15-30 km surrounded by thin protein walls. Spherically shaped structures l-5 pm in diameter were attached to the network by protein strands. Pore openings from the gel frozen at -95°C (Fig. 1B and C) were 0.1-2.0 pm across, about 90% smaller than those in the more slowly frozen sample. The structure was a rather uniform network of interconnected protein strands.

Samples fixed only by glutaraldehyde shrank to about 50% of their original size during the critical point drying step. The gel structure (Fig. 1D) consisted of numerous small globules 0.1-0.2 pm in diameter, closely compacted together and interconnected by protein strands. Pore openings 0.1-0.2 p,rn

across were present, but the overall structure was compact. The OTO-processed gel (Fig. IE) had a more open structure, with globules and strands forming a weblike network. Pores ranged in size from 0.1-0.8 pm, and the strands were much finer than those present in freeze-dried EW gels. The OTU- processed gel (Fig. IF) was similar in particle and pore size to the OTO-treated gel, with small particles interconnected by numerous strands.

Egg yolk gels Slow-frozen EY gels, plain and defatted, are shown at low

magnification in Fig. 2A and B, respectively. The lipid-containing sample consisted of spherically shaped particles 1-15 pm in diameter. Structural details were obscured by a viscous covering. The appearance of this material is probably related to freeze-induced gelation of yolk, which involves the complexing of lipoproteins into large aggregates (Powrie et al., 1963). Defatting the sample exposed a yolk sphere in a protein matrix (Fig. 2B). The irregular surface contained numerous voids and large cavities, suggesting extensive damage due to freezing. The surface of an EY gel frozen at -95°C before defatting (Fig. 2C and D) was covered by spherically shaped structures 0.3-2.0 pm in diameter, trapped in a fibrous matrix. These structures were tentatively identified as yolk granules based on their size and number (Chang et al., 1977). The void spaces were oriented diagonally across the micrograph, indi- cating limited ice crystal damage. The EY gel prepared by glutaraldehyde fixation (Fig. 2E) was similar in appearance to the freeze-dried gel (Fig. 2D). Both treatments allowed lipid removal, which exposed the underlying granules and support- ing protein matrix. However, neither sample was free of arti-

Fig. I-Micrographs of cooked EW gels prepared for SEM by various methods. (A) Frozen at -35°C and freeze-dried; (B) Frozen in liquid hexane (-95”(J), immersed in liquid nitrogen (- 19s”C), and freeze-dried; (C) Higher magnification of B; (D) Fixed in glutaraldehyde and critical point dried; (E) Fixed in glutaraldehyde followed by OTO post-fixation and critical point drying; (F) Fixed in glutaraldehyde followed by OTU post-fixation and critical point drying.

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SCANNING ELECTRON MICROSCOPY OF EGG GELS.. .

Fig. 2-Micrographs of cooked EY gels prepared for SEM by various methods. (A) Frozen at -35°C and freeze-dried; (B) Frozen at -3SC, freeze-dried, defatted and critical point dried; (C) Frozen in liquid hexane (-9SC), immersed in liquid nitrogen (- 196’C), freeze-dried, defatted and critical point-dried; (D) Higher magnification of C; (E) Fixed in glutaraldehyde, defatted, and critical point- dried; (F) Fixed in glutaraldehyde, post-fixed by OTO, defatted and critical point-dried.

Fig. 3-Micrographs of cooked WE gels prepared for SEM by various methods. (A) Frozen at -35°C and freeze-dried; (6) Frozen in liquid hexane (-95”C), immersed in liquid nitrogen I- 19sOC). freeze-dried, defatted and critical point-dried; (C) Fixed by glutaraldehyde, post-fixed by OTU, defatted and critical point-dried.

facts, as the freeze-dried sample was distorted by ice crystal formation and the gel fixed in glutaraldehyde underwent some shrinkage during critical point drying. The EY gel prepared by the OTO treatment (Fig. 2F) had no pores but exhibited clusters of small globules from 0.1-0.4 pm in diameter. Based on their size, these globules may include low-density lipoproteins (LDL), clusters of LDL, and myelin figures, which are all present in yolk plasma (Chang et al., 1977; Garland and Powrie, 1978). Micrographs of EY gels prepared by the OTU

procedure were similar to those of OTO-treated gels and therefore are not shown. Both the OTO and OTU treatments effec- tively prevented appreciable shrinkage of EY gels, but they also fixed some of the lipid-containing particles in the gels, obscuring the protein matrices. Whole egg gels

The slow-frozen WE gel (Fig. 3A) had a coarse protein structure covered by an apparent lipid layer. Large pore open-

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Fig. 4-Comparison of whole and fractured spheres from EY and WE prepared for SEM by freeze-drying or the OTU fixation procedure. (A/ Sphere from EY gel frozen in liquid hexane, immersed in liquid nitrogen, freeze-dried, defatted and critical point-dried. (B) Sphere from OTU-processed, defatted EY gel. (Cl Sphere from WE gel frozen in liquid hexane, immersed in liquid nitrogen, freeze-dried, defatted and critical point-dried. ID) Sphere from OTU-processed, defatted WE gel. (E) Interior of sphere from EY gel fractured in liquid nitrogen, freeze-dried, defatted and critical point-dried. lF) Interior of sphere from EY gel prepared by OTU fixation, defatted and critical point-dried.

ings of IO-20 pm across were present due to ice crystal damage. Freezing WE at -95°C resulted in an open network structure (Fig. 3B) with protein strands enclosing numerous ice crystal pockets that were 90% smaller than those in the more slowly frozen WE gel. The unfrozen gel processed by the OTU method (Fig. 3C) consisted of a uniform array of irregularly shaped globules from 0.05-0.2 pm in size. The surface was quite uneven and irregular and lacked the presence of pores or openings. The structure of this gel resembled that of EW gels prepared by the same fixation method.

Egg yolk and whole egg spheres

Whole and fractured spheres from EY and WE are shown in Fig. 4 in order to compare the effects of freeze-drying and the OTU treatment on the structural appearance of spheres. The spheres were lying in cavities probably formed by the removal of lipid and the shrinkage of spheres. Freeze-dried spheres (Fig. 4A, C and E) had surface pores where lipid had been removed, while OTU-treated spheres (Fig. 4B, D, and F) had no pores. A freeze-dried EY sphere 30 pm in diameter (Fig. 4A) had a fairly smooth surface interrupted by pores. The surface of the EY sphere prepared by OTU fixation (Fig. 4B) was covered by small particles 0.1-0.2 pm in size. A granule protruded from the adjacent cavity wall. The freeze- dried WE sphere (Fig. 4C) had a portion of its surface broken away. The interior of this I5 pm sphere was similar to its surface, with numerous 0.5 pm diameter granules connected by protein strands. The 7 pm WE sphere fixed by the OTU

method (Fig. 4D) had a rather smooth surface interrupted by small particles and granules. The 35 pm EY sphere in Fig. 4E was fractured during liquid nitrogen freezing. It contained about a dozen smaller spheres ranging in size from 3-7 pm embed- ded in a protein matrix. The interior of the 40 pm EY sphere in Fig. 4F contained spherical particles and granules ranging from 0.1-3.0 pm in size. The protein matrix was not evident due to the presence of lipids or lipid-containing particles.

DISCUSSION

THE PREPARATION of egg protein gels for SEM by freeze- drying produced structural artifacts due to ice crystal formation. Damage was most severe in EW gels, less severe in WE gels and only slight in EY gels. The formation of numerous large ice crystals in EW and WE gels was related to their high water content of 88 and 75%, respectively (Cotter-ill and Glauert, 1979). EY gels, with high lipid levels and only 50% water, were damaged to a much lesser degree by freezing, as only minor crystal damage was detected. Ice crystal pockets were reduced in size but not eliminated by freezing in liquid hexane/ liquid nitrogen. Fixation with glutaraldehyde prior to cry- ofreezing did not prevent artifact formation, but it made gel pieces somewhat brittle, as was reported by Hermansson and Buchheim (1981) in the preparation of soy protein gels.

Davis and Gordon (I 984) stated that fast-freezing techniques produce artifacts in gel systems because of slow freezing rates. Hermansson and Buchheim (198 I) explained that the networks formed in gels by ice crystallization can easily be mistaken for

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SCANNING ELECTRON MICROSCOPY OF EGG GELS. . .

the original gel structures. Most of the previously published SEM micrographs of egg gels were obtained by freeze-drying of samples, and the resulting structures contained evidence of ice crystal damage. Because such artifacts occur when egg protein gels are prepared for SEM by improper freezing techniques, alternative preparation methods are essential.

Freezing-induced gelation may have further affected the structure of EY and WE gels. Both raw EY and WE undergo increases in viscosity upon freezing, and the effects are espe- cially pronounced at slow freezing rates (Cotterill, 1977). Has- iak et al. (1972), in a SEM study of raw EY microstructure, found that slow freezing gave EY a coarse network appearance. A similar coarseness was evident in the slow frozen EY and WE gels which had not been defatted in this study. The viscosity increase caused by freeze-induced gelation is based on the complexing of lipoproteins to form an insoluble gel (Powrie et al. ,. 1963). It is possible that the lipoproteins in EY and WE gels would complex in a similar manner to produce the coarse structural appearance which was observed in this study.

Micrographs of egg gels prepared by fixation and critical point drying showed gel structures which were consistent with those of other heat-formed protein gels prepared by similar fixation procedures (Hermansson, 1979, 1982; Hermansson and Buchheim, 1981). The WE gel prepared in this study by the OTU method resembled the WE gel prepared by glutaraldehyde and 0~0~ fixation in the study of Beveridge and Ko (1984).

The structure of EW and EY gels fixed only in glutaraldehyde was distorted by shrinkage of gels during critical point drying, while the OTO and OTU procedures adequately prevented gel shrinkage. Wollweber et al. (1981) reported 45% shrinkage of cells fixed in both glutaraldehyde and 0~0~. Sub- sequent treatment in tannic acid and uranyl acetate limited shrinkage to 5%. The authors attributed the enhanced structural stability to the mordanting effect of tannic acid in binding 0~0~ and uranyl ions.

The presence of fat in gels tends to obscure protein matrices and may also cause charging artifacts in the electron micro- scope (Kalab, 1981). Lipid removal appeared to be most complete in EY and WE gels which were freeze-dried or fixed only in glutaraldehyde. Post-fixation of gels apparently prevented complete removal of fat by the binding of lipid-containing particles, such as LDLs, to the gel matrix by 0~04. Although fixation by OTO and OTU obscured the protein matrices, these methods allow a comparison between the structure of lipid- free and lipid-containing gels.

The spheres of WE and EY compared favorably with the structures elucidated by Bellairs (1961). Both the freeze-drying and OTU procedures were useful in providing insight to the structure of spheres. The protein network was best seen in freeze-dried gels which were defatted. However, this network was undoubtedly distorted by the freezing process. Further efforts should be made to devise a procedure which would

prevent the formation of artifacts due to freezing or shrinkage, while allowing the fat to be removed from lipid-containing gels.

REFERENCES Bellairs, R. 1961. The structure of the yolk of the hen’s egg as studied by

electron microscopy. I. The yolk of the unincubated egg. J. Biophys. Biochem. Cytol. 11: 207.

Beveridge, T., Amttield, S., Ko, S., and Chung, J.K.L. 1980. Firmness of heat induced albumen coagulum. Poultry Sci. 59: 1229.

Beveridge, T. and Ko, S. 1984. Firmness of heat-induced whole egg coagulum. Poultry Sci. 63: 1372.

Chang, C.M., Powrie, W.D., and Fennema, 0. 1977. Microstructure of egg yolk. J. Food Sci. 42: 1193.

Cotter-ill, O.J. 1977. Freezing egg products. Ch. 11. In “Egg Science and Technologv.” 2nd ed. W.J. Stadelman and O.J. Cotter-ill (Ed.). AVI Pub- -_ lishing Co., Inc. Westport, CT.

Cotterill, O.J. and Glauert, J.L. 1979. Nutrient values for shell, liquid/ frozen, and dehydra conversion factors, I

ted eggs derived by linear regression analysis and ‘oultry Sci. 58: 131.

Davis, E.A. and Gordon, J. -1984. Microstructural analyses of gelling systems Food Technol. 38(5): 99.

Garland, T.D. and Powrie, W.D. 1978. Isolation of myelin figures and low- density-lipoproteins from egg yolk granules. J. Food Sci. 43: 592.

Hasiak, R.J., Vadehra, D.V:, Baker, R.C., and Hood, L. 1972. Effect of certam physical and chenncal treatments on the microstructure of egg yolk. J. Food Sci. 37: 913.

Hayat, M.A. 1970. “Principles and Techniques of Electron Microscopy: Bio- logical Applications,” Vol. 1. Van Nostrand Reinhold Co., New York.

Hermansson, A.M. 1979. Aggregation and denaturation involved in gel formation. In “Functionality and Protein Structure,‘: A. Pour-El (Ed). ;ES Symposium Senes 92. American Chemrcal Socrety, Washmgton,

Hermansson, A.M. 1982. Gel characteristics - structure as related to texture and water-binding of blood plasma gels. J. Food Sci. 47: 1965.

Hermansson, A.M. and Buchheim, W. 1981. Characterization of protein gels by scanning and transmission electron microscopy. A methodology study of soy protein gels. J. Colloid Interface Sci. 81: 519.

Johnson, T.M. and Zabik, M.E. 1981. Gel&ion properties of albumen pro- teins, singly and in combination. Poultry Sci. 60: 2071.

Kalab, M. 1981. Electron microscopy of milk products: A review of techniques. Scanning Electron Microscopy 1981/111: 453.

K&b, M. and Harwalker, V.R. 1973. Milk gel structure. I. A plication of scanning electron microscopy to milk and other food gels. s, Dairy Sci. 56: 835.

Montejano, J.G., Hamann, D.D., Ball, H.R., Jr,, and Lamer, T.C. 1984. Thermally induced gel&ion of native and modrfied egg white - Rheolog- ical changes during processing; final strengths and microstructures. J. Food Sci. 49: 1249.

O’Brien, SW., Baker, R.C., Hood, L.F., and Liboff, M. 1982. Water-holding capacity and textural acceptability of precooked, frozen, whole-egg ome- lets. J. Food sci. 47: 412.

Postek, M.T., Howard, KS., Johnson, A.H., and MeMichael, K.L. 1980. “Scanning Electron Microscopy. A Student’s Handbook.” Ladd Research Industries, Inc., Burlington, VT.

Postek, M.T. and Tucker, SC. 1977. Thiocarbohydrazide binding for bo- tanical specimens for scanning electron microscopy: A modification. J. Microscopy 110: 71.

Powrie, W.D., Little, H., and Lopez, A. 1963. Gel&ion of egg yolk. J. Food Sci. 28: 38.

Rebhun, L.I. 1972. Freeze-substitution and freeze-drying. In “Principles and Techniques of Electron Microscopy: Biological Applications,” Vol. 2, M.A. Hayat (Ed). Van Nostrand and Reinhold Co., New York.

Wollweber, L., Stracke, R., and Gothe, U. 1981. The use of a simple method to avoid cell shrinkage during SEM preparation, J. Microscopy 121: 185.

Ms received 2/13/85; revised 5113185; accepted 6/24/85.

This work was supported in part by the American Egg Board, 1460 Renaissance Drive, Suite 101, Park Ridge, IL 60068, and by a Ralston Purina Food Science Fellow- ship. Thanks are due to Dr. M.F. Brown, Dr. D.A. Kinden, and Preston Stogsdil for their technical assistance.

Missouri Agricultural Experiment Station Journal Series No. 9191.

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preparation of cooked egg white, egg yolk, and whole egg gels for scanning electron microscopy

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