Factors Affecting Alkaline Coagulation of Egg White1

PHOSPHATASE ACTIVITY OF FOLLICLES Effect of gonadotropic hormone on ovarian follicles and serum vitellin of fasting hen. Proc. Soc. Exp. Med. 88: 502-504. Morris, T. R., and A. V. Nalbandov, 1961. The induction of ovulation in starving pullets using mammalian and avian gonadotropins. Endocrinol. 68: 687-697. Nishihara, M. T., 1961. Hexokinase and phosphatase activities of intestinal mucosa following

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thyroidectomy and thyroid administration. Endocrinol. 68: 850-854. Romanoff, A. L., 1943. Growth of avian ovum. Anat. Rec. 85: 261-267. Smith, A. H., 1959. Follicular permeability and yolk formation. Poultry Sci. 38: 1437-1450. Warren, D. C , and R. M. Conrad, 1939. Growth of the hen's ovum. J. Agric. Res. 58: 875-894.

Factors Affecting Alkaline Coagulation of Egg White 1

(Received for publication February 6, 1962)

I

T HAS been reported in a patent authored by Blick and Hopkins (1956) that an alkaline egg white gel, when liquified and neutralized, could be added to normal egg white to improve its foaming properties. It was stated that the utilization of this product resulted in angel cakes of desirable texture and was especially beneficial when used in egg whites having very poor whipping qualities. Only limited information was presented relative to factors effecting the formation of the alkaline egg white gel. Thus, it was decided to study the coagulation of egg white at high pH values and to further evaluate the use of this product as an additive with respect to egg white functional capacity. Few references could be found in the literature concerning the coagulation of whole egg white; however, considerable work has been done using individual egg white proteins, particularly ovalbumin. Anson and Mirsky (1931) used acids, salts and heat in order to study the denaturation of egg albumin and hemoglobin solutions and their data suggest that these proteins formed 1

Contribution from the Missouri Agricultural Experiment Station. Journal Paper Series 2405. Approved by Director.

thixotropic gels. Jirgensons (1936) made a rather complete study of the thixotropic properties of ovalbumin gels using n-propyl alcohol and salt solutions. Myers and France (1940) used concentrated acetic acid and found that the viscosity of egg albumin solutions increased with time and frequently formed solid gels. The fact that egg white is capable of forming gels was reported by Donnelly (1936) who observed that when egg white was added to sodium hydroxide, firm gels resulted. He also noted that egg white gelled by alkali would undergo self-liquefaction coincidental with the formation of hydrogen sulfide. MATERIALS AND METHODS

Egg white. All egg white used in this study was obtained from 3-day-old eggs stored at approximately 10°C. The eggs were broken out and the egg white was separated from the yolks carefully. The combined whites were then blended in a Waring blendor, placed in polyethylene containers, frozen and held at 0°C. until needed. pH measurements. All pH determinations were made using a Beckman glass electrode with a 12 mm. bulb of Type "E" blue glass especially made for pH values

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F. E. CUNNINGHAM AND O. J. COTTERILL Poultry Department, University of Missouri, Columbia

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F. E. CUNNINGHAM AND 0. J. COTTERIXL

face tension of the samples. Prior to measurement, all samples were allowed to come to 70°F. Formol titration. Formol titrations were carried out on samples of egg white adjusted to various alkaline pH values to obtain an estimate of the amount of amino acids present in solution. The samples were first made neutral to phenolphthalein, then a definite volume of 40% formaldehyde solution was added and each sample titrated with 0.1002 N sodium hydroxide to a pink end point. For calculations, 1 ml. of 0.1 N NaOH was considered as being equivalent to 1.4 mg. of amino acid nitrogen. Preparation of samples for angel cake. Previously frozen egg white was allowed to thaw overnight at room temperature and to reach a temperature of 70°F. before preparing the samples. A definite volume of the additive was mixed with 200 ml. of the prepared egg white and stirred on a magnetic mixer for 2 minutes. The samples were then held for 2 hours at 70°F. before preparing the angel cakes. Preparation of the additives. Several methods were employed in the preparation of the material herein referred to as the additive. In general, 3 N NaOH was added to 100 ml. of egg white until the system reached the desired pH value. In some cases, the mixture was held in a liquid state, in other instances it was caused to form a solid gel. Those systems forming gels were held at room temperature until self-liquefaction occurred. After holding the samples for 24 hours, they were neutralized to a pH of 8.9 with 3.0 N HCl. In some cases, H 2 0 2 was added during the neutralization process. RESULTS AND DISCUSSION

Time and pH of gelation. In the determination of pH values, alkali was added as quickly as possible to the egg white while constantly stirring with a magnetic

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between 7 and 14 at temperatures from 5 to 40° C. The use of this type of electrode tends to minimize sodium ion error. Viscosity. The viscosity of the various systems was measured using an OstwaldCannon Fenske type viscosimeter. The viscosimeters used were calibrated with both distilled water and an aqueous-glycerol solution of known viscosity at 21°C. All viscosity measurements were made immediately following the addition of the alkali to the egg white. Optical density. Optical density measurements were made using a Bausch and Lomb Spectronic 20 colorimeter at a wave length of 550 millimicrons and at room temperature. Electrophoresis. Each of the prepared egg white-additive solutions used in this experiment was analyzed electrophoretically using a Spinco Model R paper electrophoresis cell. The analytical procedure used has been described by Evans and Bandemer (1956). In every case buffers of pH 8.6 were used. Only visual analysis was made of the completed paper strips. Angel cake baking. The methods used in preparing the cakes for these experiments were described in detail by Gardner (1960) and were essentially the same as those outlined by the NCM-7 sub-committee on methodology which used the same ratio of cake ingredients reported by Slosberg et al. (1948). Eight experimental cakes and one control cake were prepared during each baking period. After baking, the cakes were allowed to cool overnight in an inverted position. After 24 hours the volume of the cakes was measured by the rape seed method as outlined by Bennington and Geddes (1938). The cake volume ratio is expressed as the ml. of cake obtained per gram of batter used. Surjace tension. A Du Noiiy tensiometer, calibrated against double distilled water, was employed for the determination of sur-

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COAGULATION OF EGG WHITE TABLE 1.—The effect of pll on the time required, for formation of alkaline gels of egg white Observed pH 11.3 11.6 12.0 12.1 12.2 12.3 12.4 12.5 12.6 12.7

TABLE 2.—The effect of alkali concentration on gelation time and pH of a constant volume ofet

Gelation time (sec.) NaHO (molarity) 15,000 10,000 1,000 480 340 180 90 10

1.0 2.0 3.0 4.0 5.0

Gelation time (sec.)

Final pH

150 73 30 10 6 4 5 2 2 1 1

— —

12.4 12.4 12.5 12.6 12.2 12.5 12.6 12.5 12.6 12.6 12.7

50,000 25,000 50,000

300 180 160 120 100 35

much as 150 percent by volume and still form firm gels. The addition of an alkaline reagent to egg white on a weight basis results in a gelation time curve typical of that depicted in Figure 1. The addition of from 2 to 4% by weight of sodium hydroxide to small volumes of egg white results in very rapid complete gelation. Lesser amounts of the reagent, below 1% by weight, cause the gelation time to increase sharply. Gelation of egg white has been effected by as little as 0.35% of sodium hydroxide by weight but only after an extended period of time. That temperature of the system plays a

2.0

3.0

NaOH (%)

FIG. 1. The effect of sodium hydroxide concentration on the gelation time of egg white.

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mixer. In general, egg white undergoes gel formation at pH values greater than 12.0. An erratum was noted in a paper by Cotterill et al. (1959) which indicated that egg white formed a gel at pH 11.5. Gelation time, however, of a given volume of egg white is effected by the volume and strength of the alkali reagent used, the rate with which the reagent is mixed with the egg white and, to a lesser degree, the temperature of the system. The relationship between the time required for complete gelation and the pH to which the system is adjusted is shown in Table 1. Quickly changing the pH to above 12.8 causes immediate gelation of egg white. In this investigation, gelation was not considered complete until there was absolutely no flow when the sample was subjected to the force of gravity. The effect of different concentrations of alkali on the time and pH required to gel a given volume of egg white is shown in Table 2. Usually only 20 ml. of egg white was used per trial, however, the values of time and pH remained fairly constant when the volumes of reagents were doubled and tripled. The values presented in Table 2 are averages compiled from many determinations. Although 0.1 M and 0.2 M sodium hydroxide raised the pH of egg white to above 12.0 the samples failed to form gels due to excessive dilution. When 0.5 M NaOH is used, egg white can be diluted as

0.1 0.2 0.3 0.5

Volume used (ml.)

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F. E. CUNNINGHAM AND 0. J. COTTERILL

20

Temp. (°C.)

FIG. 2. The effect of temperature on the gelation time of egg white.

role in the time required for gelation is shown in Figure 2. For a given volume of egg white and alkali reagent, less time is required for complete gelation when the temperature of the system is between 10 and 30°C. When the egg white is cooled or heated, slightly longer times are required for gelation to occur. The various conditions under which egg white can be caused to form gels were somewhat unexpected. Certain gels, such as those of gelatin, are quite stable and generally unaffected by drastic changes in factors such as pH, concentration, etc. It was supposed that egg white gels, like other denatured protein gels, would be formed only under more specific conditions. It has been proposed that such gels are formed of a network structure formed from polypeptide chains associated via attractions which act throughout the entire molecular length (Ferry, 1948). Ferry (1948) also states that in order for denatured protein gels to form there must exist a correct balance of attractive and repulsive forces between polypeptide chains and that in the case of gels formed with alkali, the coulombic forces caused by the high negative charges

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10

probably provide the necessary repulsive balance. The self-liquefaction of alkaline gels. Donnelly (1936) observed that alkaline egg white gels would undergo self-liquefaction accompanied by liberation of hydrogen sulfide and an increase in the formation of free amino acid groups. These observations were confirmed by this study. It was also noted that certain prepared gels underwent self-liquefaction quicker than others. It was found that when egg white was gelled at pH values of 12.6 to 12.8, self-liquefaction occurred more rapidly than when the gels were formed at a pH value of 12.2 to 12.5. When gels were formed at pH 12.1 to 12.2, liquefaction required several weeks and even then was not complete. Gently heating the gels to between 40 and 50°C. hastened the liquefaction process. The alkali treatment of egg white itself did not cause a noticeable liberation of hydrogen sulfide; however, the slightest addition of acid in an attempt to neutralize the liquefied mass resulted in the formation and liberation of large amounts of the gas. Formol titrations of liquefied alkaline egg white gels were conducted in the following manner. The pH of 10 ml. portions of egg white was adjusted to the desired value using 3.0 M sodium hydroxide. Those samples which formed gels were allowed to liquefy and stand for 24 hours. At the end of that time, 20 ml. of water and sufficient quantities of dilute acid were added to readjust the pH to 8.6 as determined with a Beckman pH meter. Half of the samples were titrated using phenolphthalein as the pH indicator and the other half were titrated using the pH meter without the addition of phenolphthalein. Twenty ml. of a 40% formaldehyde solution, previously neutralized, was then added to each sample and the solution back titrated with

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COAGULATION OF EGG WHITE TABLE 3.—Formol titrations of egg white treated with various amounts of alkali

Treatment Control

Alkali added

PH

Amino 0.1 M NaOH/gm. nitrogen/gm. egg white egg white

8.6 8.6 8.6

.487 .490 .488

6.82 6.87 6.84

11.3 12.0 12.4 12.6 12.8 13.0

.503 .510 .519 .520 .525 .532

7.04 7.14 7.26 7.28 7.36 7.44

pH

Surface tension* (dynes/cm.)

9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5

54.9 55.0 55.0 54.6 55.1 55.0 55.0 54.9 55.0

* An average of three measurements.

there are many reports dealing with increases in viscosity accompanying protein denaturation. They state that increases in intrinsic viscosity implies an increase in effective solute volume, brought about by either an increase in molecular asymmetry or in the degree of hydration, or both. They believe that this change in viscosity is, at least, in accord with the concept of an unfolding of the molecules. Alkaline egg white, within certain but as yet ill-defined limits, possesses thixotrophic properties. Bull (1951) stated that almost any gel will show a certain degree of thixotrophy; however, certain gels

FIG. 3. The effect of pH on the viscosity of egg white.

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0.1002 N. sodium hydroxide. The results of these titrations are given in Table 3. The non-alkaline treated egg white or controls averaged 6.84 mg. of amino nitrogen per gm. of egg white. The alkaline treated egg white contained more amino nitrogen per gm. as the pH increased indicating that the protein had undergone some hydrolysis. Surface tension oj alkaline egg white. The surface tension values for egg white ranging in pH from 8.8 to 12.5 are given in Table 4. As the egg white samples were made more alkaline, sodium hydroxide of increasing concentration was used so that the dilution factor remained constant. A control was prepared using sodium chloride as the diluent. There was essentially no difference in the surface tension of alkaline treated egg white due to pH. Viscosity oj alkaline egg white. The treatment of egg white with an alkaline reagent brings about a drastic change in the relative viscosity. As illustrated in Figure 3, increasing the pH of egg white from its normal value to around 11.5 causes only a slight but gradual rise in viscosity. Beyond pH 11.5, however, the system gives rise to an apparently high viscosity which proceeds to such an extent that the system no longer resembles a liquid. According to Fox and Foster (1957)

TABLE 4.—The effect of increasing alkali concentration on the surface tension of egg white

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F. E. CUNNINGHAM AND 0 . J. COTTERILL

.40 -

.10-

' 9.0

1 10.0

1 11.0

-J 12.0

13.0

PH

FIG. 4. The effect of alkaline pH on the optical density of whole egg white.

possess this property to a high degree. He denned thixotrophy as a reversible, isothermal gel-sol transformation. When certain gels are shaken, they undergo a change to a liquid, gelling again when the shaking is stopped. As in the case of alkaline egg white, gelatin sols will liquefy if shaken before the structure is too firmly set (Weiser, 1949). Alkaline egg white, at a pH of 12.0 to 12.2, held at constant temperature overnight will form what appears to be a gel and yet when shaken it regains a tendency to flow. Within a short period of time after the shaking is stopped, it solidifies again. Studies as to the effect of concentration upon this property have not been completed. Attempts were made to form alkaline gels using concentrated solutions of lysozyme without success. Ovomucin solutions, however, formed gels readily upon addition of sodium hydroxide. Egg white with the ovomucin fraction removed also formed alkaline gels. Optical density of alkaline egg white. Cotterill et al. (1959) presented data which indicated that the optical density of egg

Considering the present results coincident with the previously cited work of Cotterill et al. (1959) it can be stated that there exists two minima and three maxima in the optical density-pH curve of egg white under the conditions employed in the two experiments. The maximas apparently correspond to the precipitation of different egg white protein fractions. Electrophoresis of alkaline egg white. An electrophoretic study of alkaline egg white was made to determine the effect of pH on the mobility of the various egg white proteins. Egg white was adjusted to the desired pH values with 1.0 N NaOH and allowed to stand for five hours. First the samples were analyzed without pH readjustment and, secondly, the samples were neutralized to pH 8.9 before analysis. There was no difference in the electrophoretic patterns. The results of the second trial are shown in Figure 5. A typical electrophoretic pattern of egg white was obtained at pH 9.0. As the pH was increased above 10.0 the mobility of the ovomucoid fraction increased, reaching a maximum dis-

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0 I 8.0

white varied with pH. They constructed an optical density-pH curve for egg white covering the pH range from 1.0 to 10.0. The present work extended this optical density-pH study in the alkaline range up to pH 12.0. The data presented in Figure 4 show the general effect of increasing concentration of sodium hydroxide on the turbidity of fresh blended whole egg white. The optical density remains relatively constant between pH 8.0 and 10.0, but beyond this value it increases rapidly until the pH is in excess of 11.0 where a maximum occurs. This increase in optical density suggests that precipitation of one of the egg white proteins takes place between pH 10 and 11. Beyond the maximum, the curve declines to its original value at approximately pH 12.

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COAGULATION OF EGG W H I T E

9.5

10.0

10.5

10.8

11.0 PH

11.2

11.4

11.6

11.8

12.0

FIG. S. The effect of pH on the separation of egg white protein by filter paper electrophoresis. A—ovalbumin 0—ovomucoid C—conalbumin M—immobile fraction L—lysozyme

tance traveled at about pH 11.6, and then declined as the pH value approached 12.0. The electrophoretic behavior of ovomucoid closely coincided with the optical densitypH curve shown in Figure 4. The migration of conalbumin was generally not affected by the use of the alkaline reagent until the pH value approached 11.8. At this pH the typical conalbumin band lost its identity. At pH values greater than 11.8, conalbumin apparently migrated with the ovomucoid fraction. At pH 12.0 the mobility of ovalbumin decreased and the intensity of the conalbumin and ovomucoid fractions also decreased. The lysozyme fraction was little affected by pH; however, its mobility decreased slightly at pH values approaching gelation. Additives used in angel cakes. A summary of the results obtained when the various additives were added to egg white for the preparation of angel cakes is pre-

sented in Table 5. Some of the figures shown in Table 5 are averages of as many

TABLE 5.—The effect of various preparations used as additives on whip time and angel cake volume

Description of preparation of additive

Whip time* (sec.)

Yolk-free Egg White A. Control 77 B. Alkaline egg white, gelled, liquefied, neutralized 70 C. Treatment B plus H2O2 66 D. Alkaline egg white, nongelled, neutralized 129 E. Treatment D plus HaOa 91 46 F. Egg white plus H 2 0 2 Yolk-Contaminated Egg White 73 G. Control (with 0.1% yolk) H. Alkaline egg white, gelled, liquefied, neutralized L09 54 1. Treatment H plus H2O2 142 J. Egg white plus H2O2

Cake volume ratio* (ml./gm.) 5.61 5.54 5.66 5.34 5.61 5.70 4.68 3.51 4.98 4.16

* Average of five to 12 observations per treatment.

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9.0

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Using egg white contaminated with 0.1% yolk gave somewhat similar results. Only those additives containing H 2 0 2 produced any promising results. The use of additive I at the 1 to 2% level decreased the beating time and increased angel cage volume. The use of H 2 0 2 as an additive at the 0.01 and 0.02% levels also decreased beating time and increased cake volume. Therefore, while it is true that egg white alkaline gels, when liquefied, neutralized and with added hydrogen peroxide (to oxidize hydrogen sulfide), can successfully be used as an additive to normal egg white to improve its functional properties, the beneficial effects are derived from the use of hydrogen peroxide and, apparently, not from the egg white gel itself. The use of

H 2 0 2 alone, at suitable concentration, has been shown to be an additive capable of improving the functional properties of normal egg white. SUMMARY

A study was conducted on factors affecting the gelation of egg white in the alkaline pH range and on the utilization of derivatives of the resultant product as an additive in normal egg white. At pH values in excess of 11.9, egg white forms a translucent gel which, after a period of time, undergoes self-liquefaction. The time required for gel formation is affected by pH, rate at which the alkali is added, temperature, volume of the reagents and strength of the alkali. The time required for liquefaction of the gels is pH dependent. The amount of amino nitrogen per ml. of solution is proportional to the increase in pH as estimated by formal titrations. Surface tension is not affected by the addition of sodium hydroxide within the pH range of 9.0 to 12.0. Alkaline egg white, within certain limits of pH and concentration, possesses thixotropic properties. The viscosity of alkaline egg white rises very rapidly with increasing pH, above pH 11.5, until the sol-gel transformation occurs. The optical density of alkaline egg white is relatively constant between pH 9.0 and 10.0, increases rapidly until a maximum is attained between pH 11,1 and 11.3, and then declines to its original value at pH 12.0. As the pH of the egg white system is increased above 10.0, the mobility of the ovomucoid fraction is increased as determined by paper electrophoresis. The distance migrated by ovomucoid is a maximum between pH 11.6 and 11.8. At pH values near 12.0 the conalbumin fraction becomes nonmobile and the mobility of lysozyme is decreased by approximately 50%. Attempts were made to utilize various

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as twelve separate determinations. The additives described were added to normal egg white in concentrations varying from 0.006 to 10.0%. The use of additive B (liquefied and neutralized gels) had no beneficial influence an angel cake volume, failed to influence the beating time and tended to decrease foam volume slightly. Additive C (additive B plus H 2 0 2 ), however, increased angel cake volume, decreased beating time and increased foam volume. Optimum additive concentration appeared to be in the range of 1 to 3 % . Additive D (non-gelled and neutralized) had no beneficial effect at any concentration tested. Additive E (additive D plus H 2 0 2 ), as with the other additive containing H 2 0 2 , produced the most effect in the concentration range of 1 to 3% and tended to reduce foam volume although producing satisfactory angel cakes in satisfactory beating times. Additive F (egg white with H 2 0 2 ) was the most beneficial additive used in these experiments. In the concentration range of 0.008 to 0.03% the use of hydrogen peroxide alone increased angel cake volume, decreased beating time, and increased foam volume and stability.

COAGULATION OF EGG WHITE

forms of liquefied and neutralized egg white gels as additives to improve the functional performance of normal. egg white. Only those additives which contained hydrogen peroxide showed promising results. REFERENCES

of egg white proteins by paper electrophoresis. Agri. Food Chem. 4 : 802-811. Ferry, J. D., 1948. Protein gels. Adv. in Protein Chem. 4 : 2-78. Fox, S. W., and J. F. Foster, 1957. Introduction to Protein Chemistry. John Wiley and Sons, Inc., New York. Gardner, F. A., 1960. Chemical modification of egg white function. Ph.D. Thesis, University of Missouri, Columbia. Jirgensons, B., 1936. The gelatenization of albumin in salt-containing aqueous propyl alcohol—thixotrophy and synerisis of the albumin-propyl alchol gel. Kolloid—Z. 74: 300; Chem. Abs. 30: 5096. Myers, W. G., and W. G. France, 1940. A study of some properties of thixotropic gels containing egg albumin as the disperse phase. J. Phy. Chem. 44: 1113-1125. Slosberg, H. M., H. L. Hanson, G. F. Stewart and B. Lowe, 1948. Factors influencing the effects of heat treatment on the leavening power of egg white. Poultry Sci. 27: 294-301. Weiser, H. B., 1949. Colloid Chemistry. John Wiley and Sons, Inc., N.Y.

The Haugh Unit as a Measure of Egg Albumen Quality1 E. J. EISEN, B. B. BOHREN AND H. E. MCKEAN Purdue University Agricultural Experiment Station, Lafayette, Indiana (Received for publication February 8, 1962)

T

HE PROBLEM of improving the egg albumen quality in the fowl has been extensively studied. Vital to a statistical study of any trait is an objective definition and a simple, accurate, normally distributed, quantitative measurement. Although a definition of albumen quality known to satisfy all of these requirements has not been provided, many methods of measuring albumen quality have been reported in the literature. Brant, Otte and Norris (1951) have reviewed the relative merits and disadvantages, in relationship to market 1

Journal Paper No. 1876 of the Purdue University Agricultural Experiment Station.

grades, of the various albumen quality measurements, which include: the percentage of thick albumen (Hoist and Almquist, 1931, 1932), the albumen height (Wilgus and Van Wagenen, 1936), the albumen index (Heiman and Carver, 1936; Heiman and Wilhelm, 1937), the albumen area index (Parsons and Mink, 1937), the Van Wagenen visual score (Van Wagenen and Wilgus, 1934), and the Haugh unit score (Haugh, 1937). The use of Haugh unit scores has been generally accepted as a measure of albumen quality in egg quality studies in recent years. The formula given by Haugh (1937) for

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Anson, M. L., and A. E. Mirsky, 1932. Effect of denaturation on the viscosity of protein systems. J. Gen. Physiol. I S : 341-350. Bennington, D. S., and W. G. Geddes, 1938. An improved wide-range volume measuring apparatus for small loaves. Cereal Chem. 15: 235-246. Blick, P., and E. W. Hopkins, 1952. Preparation of egg white additives. U. S. Patent, 2,752,248. Bull, H. B., 1951. Physical Biochemistry. 2nd Ed., John Wiley & Sons, Inc., New York. Cotterill, 0. J., F. A. Gardner, F. E. Cunningham and E. M. Funk, 1959. Titration curves and turbidity of whole egg white. Poultry Sci. 38: 836-842. Donnelly, J. L., 1936. On the liquefaction of sodium hydroxide-protein gels. Kolloid—Z. 77 : 343-345. Evans, R. J., and S. L. Bandemer, 1956. Separation

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