INDUSTRIAL AKD ENGINEERIXG CHEMISTRY
594
will be taken by the Federal Government and the several states to regulate the industry so that the present adequate crab supply will be perpetuated.
Composition and Nutritive Value A preliminary study was made by Watson and Fellers ( I S ) in 1936. Earlier Fellers and Parks (4) had reported on the chemical composition and other characteristics of Pacific Coast crabs. A few mineral and iodine analyses have been made by Coulson ( 2 ) and by Kilson and Coulson (10). Representative data are reported in Table I. The protein content is high and of apparently high quality, inasmuch as young rats on a diet containing 9 per cent protein from crab meat made average weight gains of 5 grams a week for a 6week feeding period. The crab meat was used in this experiment as the sole source of protein in the diet.
TABLEI. ANALYSIS OF CASNEDBLUECRABMEAT Composition, yo------,-hlineralcontent, P . P M.Moisture 79.00 K 1880 * Dry matter 21.00 Ca 1330 Protein ( N X 6.25) 18.00 hI g 120 Total ash 22.20 20 P380 Ether ext. ifat) n0 . 4400 F zn (fat) FeP 20 Ext. mati matter (by difference) 0.40 cu 13 Ca1./100 grams 77 I 0.46 Alkalinity of ash 11.7 Vitamin Content per 100 Grams 0.012 mg. (24 I. I.?.) Ascorbic acid Thiamin 230 T (70 I. U.) 150 y (60 Bourquin-Sherman units) Riboflavin ~~
VOL. 32, NO. 4
One of the chief sources of interest in this food lies in its high mineral value-calcium, iron, copper, and iodine. The iodine content is among the highest ever reported for any food. The high alkalinity of the ash is likewise notable. The crab meat is a moderately good source of vitamins B1 (thiamin) and BQ (riboflavin) and also contains a small amount of vitamin C (ascorbic acid).
Literature Cited Conant, J. B., Chow, B. F., and Schoenbach, E. B., J . Biol. Chem., 101, 463 (1933).
Coulson, E. J., U. S.Bur. Fisheries, Investigational Rept. 25, 3 (1935).
Fellers, C. R., U. S.Patent 2,155,308 (Jan. 7, 1936). Fellers, C. R., and Parks, C. T., Univ. Wash., P u b . Fisheries, 1, No. 7, 145 (1926). Harris, M. M., Am. J . H y g . , 15, 260 (1932). Harris, S.G., U. S.Patent 2,155,308 (April 18, 1939). Howe, D. W., Ibid., 1,927,123 (Sept. 19, 1933). Hunter, A. C., Am. J. Pub. Health, 24, 199 (1934). Jarvis, N. D., Canner, 83, No. 13, 9 (1936). Nilson, H. W., and Coulson, E. J., U. S. Bur. Fisheries, Investigational Rept. 41, 6 (1939).
Oshima, K., U. S. Bur. Fisheries, Investigational Rept. 8, 6 (1931).
U. S. Dept. Agr., Notices of Judgment under the Food and Drugs Act, 1936-39. Watson, V. K., and Fellers, C. R., Trans. A m . Fisheries Soc., 65, 344 (1935). PRESENTED before the Division of Agricultural and Food Chemistry a t the 98th Meeting of the American Chemical Society, Boston, Mass. Contribution 353 of the Massachusetts Agricultural Experiment Station.
Behavior of Ovomucin in the Liquefaction of Egg White A. K. BALLS AND SAM R. HOOVER Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture, Washington, D. C.
No evidence has been found for the existence of active or activatable proteinase in egg white. The liquefaction of egg white is not accompanied by a diminution of the amount of ovomucin present. VOMUCIN occurs in the thick white of hens’ eggs in
0
far higher concentration than in the thin white, according to McNally (11). He found that the mucin nitrogen is 5.7 per cent of the total nitrogen of the thick white and 0.66 per cent of the total in the thin. We have found that removal of this protein from the thick white gives a watery solution of low viscosity that still contains about 95 per cent of the protein initially present. It is therefore probable that the ovomucin is responsible for the characteristic properties of thick as opposed to thin white. From previous work in this laboratory (6) i t was concluded that the breakdown of the thick white is caused by proteolysis of the mucin by a tryptic type of enzyme naturally occurring in the egg white. This conclusion was disputed by van Manen and
Rimington (12) who were unable to demonstrate proteolysis in the thick white, and confirmed by Hughes, Scott, and Antelyes (9). I n view of these conflicting results the problem of the liquefaction of zgg white has been reinvestigated in this laboratory for the past three years. Attempts to demonstrate proteolysis by using a variety of methods were unsuccessful. The few positive results obtained were of small magnitude, little more than the limits of error of the various methods. A study of the method used in the original work (titration with sodium hydroxide in hot alcoholic solution according to FVillstatter and WaldschmidbLeitz) showed that the precipitation of the egg white in the hot alcohol trapped appreciable quantities of the ammonia-ammonium chloride buffer used. The precipitate was much more finely divided in the aliquots incubated for some minutes with the buffer solution and did not render so much of the buffer nontitratable. Therefore titration values were increased by incubation. This result explains the apparent proteolysis observed by Balls and Swenson (5) and by Hughes et al. (9). It is also consistent with the negative findings of van Manen and
APRIL, 1940
INDUSTRIAL AND ENGINEERING CHEMISTRY
Rimington ( l a ) . These authors employed the formaldehyde titration technique. We have used this technique as well as methods based on milk clotting, blood clotting, protein precipitable by trichloroacetic acid, and gelatin liquefaction. The last method is delicate and simple, and was therefore used in most of the later work. Thick white and a number of preparations made with a view of eliminating the inhibitor of trypsin present in egg white (6) all gave consistently negative results. Bmong the variables investigated were: dilution of egg white with two volumes of water (12); activation by enterokinase (5) and by cysteine; autolysis of isolated mucin; precipitation of the egg white with trichloroacetic acid (16), and analysis of the supernatant liquid and of the eluate of the precipitate with water, 0.02 h'hydrochloric acid, enterokinase solution, 50 and 80 per cent glycerol solutions, ammoniaammonium chloride buffer atJp H 8.4, 0.005 N hydrogen peroxide, etc. Attempts were also made to demonstrate a proteinase by precipitation with safranine (IS), by adsorption on alumina C y , and by partial coagulation with heat (14). The p H used for the digestions was usually 7-8; some of the extracts were checked at p H 5 and a few between 8 and 9. The methods of testing for proteolysis were those used in various publications from this 1aborat)ory( 2 , 3 , 4 ); in most cases they are adaptations of those of J. H. Korthrop and his group, as referred to in the papers cited. In view of the large amourit of trypsin inhibitor present in egg white, it does not seem possible a t this time to decide for or against the presence of some proteolytic enzyme. But the negative results obtained in these experiments contradict the conclusions of Balls and Swenson (5) and confirm the position taken by van Manen and Rimington ( l a ) , Sharp (16), and others that proteolysis is not the cause of the disappearance of thick white. This conclusion is amply verified by the finding reported here that the ovomucin does not disappear during the change from thick white to thin. One of the most sensitive tests for proteolysis of any particular protein is the alteration of its solubility in various salt solutions and at different p H levels. Ovomucin is precipitated b y acidification to p H 5 or by dilution with two volumes of water at pH 6-8. It was observed in this laboratory that ovomucin could be salted out by 0.3 saturation with ammonium sulfate. The protein was found t o disperse to a flocculent swollen gel in the absence of salt and in salt solutions to a translucent gel that also swells. Since Eichholz (7) discovered the property of precipitation upon dilution, various modifications have been used to precipitate ovomucin and wash the precipitate. The following technique was worked out and used in this study.
Mucin Analysis The sample of egg white was acidified to pH 6.9-7.1 with 1 N acetic acid while stirring. Three volumes of water were added with stirring, and the mucin precipitate was well broken up with a stirring rod; then it was centrifuged 10 minutes at a relative centrifugal force of 2000 times gravity. After the supernatant liquid was decanted, the precipitate was stirred with 200 cc. of 0.01 N sodium chloride containing 1 cc. of glacial acetic acid and centrifuged again in the same field for 10 minutes. The washing was repeated once with 200 cc. of 0.01 N sodium chloride and then once with distilled water. The final wash water did not show more than a bare opalescence when trichloroacetic acid was added t o 5 per cent concentration. The fina! recipitate was washed into a tared crucible and in a vacuum of 25-28 inches. The compact predried a t 50 cipitate obtained by this procedure had a negligible ash, less than 1 mg. from 0.25 gram of dry protein. Ten dozen white leghorn eggs of uniform size (23-24 ounces per dozen) were obtained from a producer the morning they were laid. Four eggs were separated on the Holst-Almquist screen, each being allowed to drain well. Mucin was determined in the pooled thick white of these four eggs and in the thin white separately. Four more determinations were run, each on the
595
whole white from two eggs. Thus the points plotted in Figure 1 for mucin in the thick and thin white and for the percentage of thin white are the mean of four eggs; the mucin content of the whole white is the mean of twelve eggs. The results were calculated as grams per 100 cc. for convenience. The total nitrogen content of the egg white did not change appreciably (less than 5 per cent) during the experiment. The pH was taken with a standard glass electrode instrument. The chalazas were picked out and discarded in all cases. The eggs were held a t 30" C. and a relative humidity of 45-50 per cent, and were sampled periodically over a period of 17 days. At the last sampling the thick white had almost entirely disappeared and the yolk membrane was very weak.
I
I
I
1
GAYS
FIGURE 1. MUCIS COXTESTOF EGGSHELDAT 30' C.
The mucin content of the egg white as a whole remained constant over this period of time. I n the separation of the thick from thin white there are particles of thick white that break off and pass through the screen. The separatioii of the two fractions is of necessity arbitrary, and in eggs incubated for a week or longer a t 30" C. the fraction passing the screen seems to become thicker as the time of drainage passes (2 minutes were allowed). Therefore, the main value of the data for the mucin contents of the thick and the thin white is to show that the mucin in the thin approaches that of the whole egg as the thick white liquefies. The increase observed in the mucin content of the thick white amounted to about 40 per cent of the original value. This is possibly due to incomplete removal of the chalazas, for it was observed that the chalazas broke and were difficult to pick out of the thick white in the later separations. Because of the small amount of thick white remaining, such an effect would make a negligible error in the over-all figure. TABLE I. RESULTS OF DIGESTING 100 Cc. OF THICKWHITE WITH COMMERCIAL TRYPSIN FOR 18 HOTRSAT 30" (2. Trypsin Used Mg./cc.
0.2 1.0
Initial Mg. 531 03 1
Mucin Content -FinalI n thick I n thin Mo. 'MQ. 135 206 0 35
Total M Q
341 35
.
Digestion
Liquefaction
%
%
36 93
100
67
8.
The following experiment was run as a control of the method used: Thick white that had been separated on the screen was incubated with commercial trypsin at 30" C. The p H maintained was that of the thick white-viz., 9.00. After 18 hours the fractions were separated on the screen and the mucin content of each was determined (Table I).
596
INDUSTRIAL AND ENGINEERING CHEMISTRY
VOL. 32, NO. 4
The process did not proceed to any considerable extent. We believe that this slight effect was Thick Nitrogen -Mucin Contentpossibly nonenzymic. Koga was unable to Type of Eggs White Content pH I n thin I n thick Total or ereptic action by the demonstrate proteolysis % MQ./cc. % % %" formaldehyde titration method. The experiments Fresh 56 18.7 8.10 0.13 0.51 0.35 + 0.025 Fresh 61 18.7 8.49 0.20 0.50 0.37 0.010 referred t o in the first section of this paper indicate Middle Western, ailed, stored that there is little or no active proteinase present. 5 rno. 43 15.8 8.79 0.14 0.55 0.33 * 0.014 Eaatern,exptl,storage8mo. 47 18.0 9.12 0.22 0.48 0.33 * 0.009 The liquefaction of the thick egg white is due Plus and minus standard deviation. to a breaking up of the mucin fibers. It can be retarded by maintaining the pH around 8 (16). This is the DH amroximated bv the vacuumThe higher concentration of enzyme completely liquefied carbon dioxide oiling process- developed here (27). Howthe thick white. The supernatant liquid from the preever, it must be remembered that the breakdown of the cipitation of this sample was quite milky; this turbidity, thick white is rapid a t higher temperatures, as shown by however, was not decreased when the supernatant liquid was Wilhelm and Heiman (18) and confirmed in this work. The filtered through S. and S. paper No. 1450. The lower coneggs that had been stored 7 months compared favorably with centration of enzyme liquefied 67 per cent of the thick white strictly fresh eggs held only 3 to 4 days a t a temperature no and digested 36 per cent of the mucin initially present. The higher than is often encountered during the laying season. liquefaction of thick white by commercial trypsin is thus seen Therefore, necessary emphasis must be placed on rapid chillto differ from the liquefaction observed in the shell. ine and handling of eggs that are to be processed or stored. Eggs from commercial storage were analyzed for mucin by the technique used in the previously described experiments. Literature Cited I n Table I1 results on two samples of strictly fresh eggs are Almquist, H . J., Givens, J. W., and Klose, -4., IXD. ENG.CHEM., included for comparison. The eggs which had been stored 26, 847 (1934). for 8 months were an experimental run put up in the winter Balls, A. K., and Hale, W. S.,Cereal Chem., 15, 622 (1938). Balls, A. K., and Hoover, S. R., J . Bid. Chem., 121, 737 (1937). and were of excellent quality (8). The oiled eggs were also Balls, A. K., and Lineweaver, Hans, Food Research, 3, 57 of satisfactory quality. The results of analysis for total (1938). mucin are slightly lower than those on the strictly fresh eggs. Balls, A. K., and Swenson, T. L., IND.ENQ.CHEM.,26, 570 If the results were calculated on a total nitrogen basis, this (1934). Balls, A. K., and Swenson, T. L., J . BioZ. Chem., 106,409 (1934). slight difference would be eliminated. Because of the lack Eichholr, A., J. Phusiol., 23, 163 (1898). of conclusive data concerning the transfer of water and difHoover, S. R . , J . Assoc. Oficial Agr. Chem., 21, 496 (1938). fusible nitrogen through the yolk membrane, a decision as t o Hughes, J. S., Scott, H. M., and Antelyes, J., IXD. ENQ.CHEM., the more valid basis of calculation cannot be made a t this Anal. Ed., 8, 310 (1936). Koga T., Biochem. Z., 141, 430 (1923). time. The authors believe that the conclusion is justified McNally, E., Proc. Soc. E x p t l . Bid. Med., 30, 1254 (1933). that substantially all of the mucin is present in these storage Manen, E. van, and Rimington, C., Onderstepoort J . Vet. Sci. eggs. Animal Ind., 5 , 329 (1935). The mucin content of the various fractions is consistent Marston, H. R., Biochem. J . , 17, 851 (1923). Northrop, J. H., and Kunitz, M., J . Gen. Physiol., 16, 267 with the data of Almquist, Givens, and Klose (1). TABLE11. MUCINCONTENT OF FRESH AND STORAGE EGGS
(1932).
Discussion Although i t is impossible from the results given to decide whether Droteolvtic enzymes occur in eae: white. the evidence favoring their Occurrence is now meager* KO@' (Io) found a slight fibrinolysis by using carmine-dyed fibrin as a substrate.
--
Ibid., 16, 313 (1932). Sharp, P. F., Food Research, 2 , 4 7 7 (1937). Swenson, T. L., Ibid., 3, 599 (1938). Wilhelm, L. A., and Heiman, V., U.S. Egg Poultry Mag., 44, 661 (1938). CONTRIBUTION 450 from the Food Research Division, Bureau of Agricultural Chemistry and Engineering.
