September, 1932
I N D U S T R I l L AND E S G I N E E R I N G CHEMISTRY
1077
liver oil contains less unsaturated fatty acid, and therefore be taken with impunity and accepted as a valuable accessory mav be expected to oxidize less easilv than cod-liver oil. to human nutrition. 6. Tolerance tests on rats gave evidence that halibut-liver LITER.4TUKE CITED oil produced no undesirable effects. Khen the young rats (whether on a normal diet or a vitamin A-free diet) were 4 m . Med. .-issoc., "Xew and Nonofficial Remedies, 1931," p. 259. given, over periods of 50 to 100 days, as much as 10.000 Brode and Magill, J . Bid. Chem., 92, 87 (1931). times the daily vitamin X and 400 times the daily vitamin D Carr and Price, Biochem. J . , 20, 497 (1926). requirements to correct the induced deficient symptoms, they Holmes, J . A m . Pharm. Assoc., 20, 588 (1931). grew rapidly and del-eloped no apparent indications of any McCollum et al., J . Bioi. Chem., 51, 41 (1922). Moore, Biochem. J . , 25, 275 (1931). pathology. The level of blood phosphorus and serum calcium Morton and Heilbron, Ihid., 22, 987 (1928). remained a t normal. The excess of vitamin A was itored to a Norris and Church, J. Biol. Chem., 85, 477 (1930). large degree in the liver as a nutritional reserve. Pharmacopeia of the United States, 10th Decennial Rev., p. 7. Halibut-liver oil, as far as the authors are aware, is the 469, Lippincott, 1928. Proc. Am. Drug -Wig. Assoc., p. 281 (1931). richest knoim natural source of vitamins A and D. The high Rept. of Brit. Pharmacopeia Comm., March, 1931. vitamin d content of this oil is particularly valuable because Schmidt-Nielsen and Schmidt-Kielsen, Biochem. J., 23, 1153 this vitamin exerts a direct influence on the growth and de(1929). \-elopment of the young, and may be a n aid toward the estab- (13) Steenbock and Black, J . B i d . Chem.;64. 263 (1925). lishment of better resistance of the body to infections in genR E C E I ~ E 4pril D 7, 1932 Presented before t h e Division of Medicinal to ~halibut-liver oil ~by the experieral, ~ ~the tolerance ~ h ~ ,Chemistry a t t h e 83rd Meeting of the American Chemical Society, New Ormental animals-even in massive doses-indicates that it can leans, L a , x i a r c h 28 to 4pril 1, 1932
Optical Activity of Commercial Caseins S. P. GOULD,Bureau of Dairy Industry, Department of Agriculture, Washington, D. C.
S
tions or p h y s i c a l tests. The EVERXL i n v e s t i g a t o r s The spec$c rotations of fifteen commercial of this paper thought author h a v e d e t e r m i n e d the caseins and a Hammarsten casein have been that differences in specific rospecific rotation of pure determined us a means of establishing the tation might furnish informacasein, but whileeach apparently molecular identity of these samples. .'2lost of h a s obtained r e p r o d u c i b 1e tion to e x p l a i n these differthe differences found among these values for inences in coating characteristics, r e s u 1t s , there is considerable particularly adhesive strength. difference between the values dicidual caseins appeared to be due to experiIt was a l s o c o n s i d e r e d that r e p o r t e d . Much of this dimental dificulties. The average value for the the effect of using an excess of v e r g e n c e may be due to the hydrochloric acid group agreed reasonably well the p r e c i p i t a t i n g a c i d , of difuse of strong alkali as a solwith that f o r the lactic acid caseins; that of the v e n t , w h i c h causes hydroylferent acids, or of time and sulfuric acid group was distinctly less. I t is sis, or to other fundamental temperature of heating in the c h a n g e s in the casein, which process of manufacture might believed that the differences observed are not would bring about a variation b e s t u d i e d by this method. suficiently large to indicate appreciable deAt the same time, the desirain the rotation. Long (3) decomposition of the casein molecule in any sample termined the specific r o t a t io n bility of using i t as a regular except in the case of the sulfuric group. No o f H a m m a r s t e n casein, b u t analytical procedure to detercorrelation between adhesive strength and specific his results are not comparable mine the purity of commercial with those of the author since caseins and their s u i t a b i 1i t y rotations could be established. Further exhe used mostly strong alkalies for specific industrial purposes periments showed that an excess of the precipitatas solvents, whereas the writer could be appraised. ing acid changed markedly the specific rotation employed sodium acetate of casein; also that overheating as high as 90" C . solution. Zaykowsky (5) has XETHODS USED during drying did not aflect noticeably the optical investigated v e r y extensively the optical activity of various activity. When Hammarsten c R s e i n a m o u n t s of p u r e casein disdissolved in any of the comsolved in alkaii, acid, and salt solutions of different concen- mon solvents was used, no difficulty was experienced in obtrations. He concluded, among other things, that the specific taining solutions clear enough to polarize. However, in rotation varies with the concentration of the casein and the the case of the commercial caseins, although they were diskind of solvent employed. Hewitt ( 2 ) reports the value solved in a large number of acid, alkaline, and neutral solvents, [n]E& = - 105' for casein but, since it is not clear what few were found which gave clear solutions. Filtration, solvent was used, a comparison is not possible. Csonka and when first tried, proved such a slow process as to be impracHorn (1) have studied the effect of sodium hydroxide on pro- tical. Later it was found that the addition of Filter-Cel teins, including casein, and report the specific rotations of increased enormously the speed of filtration. In selecting the cleavage products. These rotations show a decrease of a solvent, it was important to choose one which would not about 50 per cent from the usual values for the unchanged cause appreciable hydrolysis under the conditions of the casein. experiment. Zaykowsky's results (6) indicated that 10 per Unpublished work on fifteen commercial caseins has cent sodium acetate solution would meet this requirement; brought out differences in paper-coating characteristics which it was therefore chosen for this work. '0 far have not been accounted for by analytical determinaThe following procedure was adopted for all the caseins:
INDUSTRIAL AND ENGINEERING
1078
5 grams of c. P. sodium acetate were dissolved in 40 to 42 cc. of water, 1 gram of casein was added, and the mixture allowed t o stand for 2.5 hours or more. It was heated for 20
minutes on the steam bath. If the casein particles were not then all in solution-a fact which was difficult t o determinethe heating was continued until it was judged that only insoluble salts remained undissolved. -4 Gooch crucible was prepared with an asbestos mat 2.0 to 2.9 cm. thick. A thin layer of Filter-Cel was placed on the mat, and a larger quantity was mixed with the casein solution. It was then sucked through the Gooch crucible three or more times until the solution was clear enough to polarize. The residue was washed, the filtrate and washings were added to a volumetric flask, and the solution was made up to 50 cc. This solution, containing 2 per cent of casein and 10 per cent of sodium acetate, was polarized at 30" C.
CHEMISTRY
Vol. 24, No. 9
The effect of overheating during drying next was investigated in the following way: X sample of Hammarsten casein was moistened with water. Then it was dried for 5 hours a t 51" to 55' C. (the usual commercial procedure) and finally overheated by holding for 4 hours more at 85" to 90" C. The product was hard and tough, and therefore very difficult t o grind and dissolve. Severtheless, no significant = change in the specific rotation was noted; for [.I3$" -83.8". This reading and the two given above should be compared with that for the original Hammarsten caseini. e., [ T ] ~ $ ' = -81.7". DISCUSSION OF RESULTS
Schering-Kahlbaum's Hammarsten casein was polarized first in order to establish a standard for comparison. Xext the specific rotations of fifteen commercial caseins, secured from widely distributed plants and including hydrochloric, sulfuric, and lactic acid caseins, were determined The specific rotations were calculated on the basis of waterfree and ash-free casein, the moisture and ash determinations having been made previously. The results are shown in Table I. It should be mentioned that the sulfuric caseins were very difficult to polarize because, even after repeated filtrations, the solutions were still somewhat opalescent, and as a result, rather indistinct readings were obtained. Zaykowsky (5) found it impossible to polarize a solution of casein dissolved in 0.1 N sulfuric acid because of the opalescence developed. The readings for the commercial sulfuric caseins are included in the table nevertheless, although they are known not to be as accurate as the others. All specific rotations given are averages of five or more readings. TABLEI. SPECIFICROTATIONS AND ADHESIVE STRENGTHS OF CASEINS HYDROCHLORIC CASEINS-SOLFURICCASEIKS- -LACTIC C ~ S E I N S -
b I % O
3 4 5
-72.6 -85.9 -77.5 -80.4 -87.0
6
-92.2
1 2
7 8
-72.6 -83.7
9 9 9
9 10 10 11 9
9 10
-61.7 -72.2
10 11
11 12
13 14 15
-81.3 -75.4 -74.4 -80.7 -89.9
9 9 10 11 11
Av. -81.5 -66.9 -80.3 a Determined b y the method described by Sutermeister (4) in which t h e adhesive strength is inversely proportional to the figures.
For Hammarsten casein, the average value obtained was = -81.7'. Zaykowsky reported [n]':' = 83.59" for 2 per cent of pure casein dissolved in a 10 per cent sodium acetate solution. However, as Hewitt ( 2 ) pointed out, the rotation should be levo. Next a study was made of the effect of an excess of different acids on the casein molecule. To a sample of Hammarsten casein was added a considerable excess of hydrochloric acid; a second sample was treated in like manner with lactic acid. Both were held a t 37" C. for 2 hours, this treatment causing the caseins to dissolve. After that, sodium bicarbonate was added until p H values of 4.2 and 4.4, respectively, were reached. The precipitated caseins were filtered and washed, and then dried a t 51" to 55" C. for 5 hours. The dry samples were ground, dissolved, and polarized. The hydrochloric acid casein gave [el3$' = -61.7", and the lactic acid casein [ ~ x ] ~ , O "= -56.5", values considerably lower than the initial rotation. Tests showed, however, that the strengths of the acid-treated caseins were the same as those of the untreated samples. [0(I3,O'
Some variation was found among the readings of some of the caseins of each group, but in many cases this was probably due t o experimental difficulties. The average values for the hydrochloric and lactic samples were practically checks, whereas the average for sulfuric caseins was distinctly lower. However, as explained before, it was very difficult to obtain sharp readings for the sulfuric samples. Differences in adhesive strengths of commercial caseins cannot be explained by differences in specific rotations of the caseins. Conversely, differences in specific rotation do not necessarily involve differences in adhesive strength. Long contact with an excess of hydrochloric and lactic acids a t low pH values, though not affecting adhesive strength, decreased markedly the specific rotation. This might indicate a fundamental change in the casein molecule, or a direct combination with the acids giving substances of different rotations. An excessively high drying temperature, such as 85' to 93" C., apparently does not affect the casein molecule, since no significant change in specific rotation was observed. I n commercial practice then, there would be little danger of injuring the casein molecule as long as the temperature was kept a t least below 90" C. However, if 55" C. were exceeded, grinding would be made more difficult and solution slower. As to the desirability of using the foregoing method as a regular analytical procedure for determining the purity of commercial caseins, or its adaptability to specific industrial purposes, there is some question. When applied to caseins of high purity such as samples 3, 4, 8, and 11, a fair degree of reproducibility can be attained, but with the average casein, the reproducibility is not so precise. The reason for this lies in the difficulty of determining whether all the casein is in solution, as it is hard to distinguish between undissolved casein particles and insoluble salts. I n spite of this limitation, however, the marked deviation of any casein from normal can easily be detected by the polariscope. This fact is evident from Table I, and from the study of the effect of acids on casein. It is confirmed also by the work of Zaykowsky ( 5 ) and of Csonka and Horn ( I ) , whereby it is shown that any profound change in the casein molecule, such as that brought about by hydrolysis with sodium hydroxide, will produce specific rotation values differing widely from those of the original casein. I n view of the comparative difficulty of the method, i t is not adapted perhaps to the regular routine testing of commercial casein. It should be valuable, though, as a supplementary test in case further information is needed about any particular lot of casein; or in research work when it is necessary to determine whether the molecule has been altered by the manufacturing process or by any unusual condition. ACKNOWLEDGMENT The author wishes to thank E. 0. Whittier for his advice and assistance.
September, 1932
INDUSTRIAL AND ENGINEERING CHEMISTRY
LITERATURE CITED (1) Csonka, F. A., and Horn, M. J., J.Biol. Chem., 93,677 (1931). (2) Hewitt, L. F., Biochem. J., 21, 216 (1927). (3) Long, J. H., J.-4m. Chem. Soc., 27, 363 (1905).
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(4) Sutermeister, E., “Casein and Its Industrial Applications,” p. 275, Chemical Catalog, 1927. ( 5 ) Zaykowsky, J., Biochem. Z.,137, 562 (1923). RECEIVED April 11, 1932
Quantitative Variations in Vitamin A Content of Butter Fat G.
s. FRAPS-4ND RAYTREICHLER
Texas l g r i c u l t u r a l Experiment Station, -4gricultural a n d Mechanical College of Texas, College Station, Tex.
A
?;UMBER of workers (4-6,lI)have studied the variation in the vitamin A content of milk and of butter fat, although few have made quantitative measurements. It is recognized that vitamin A comes directly or indirectly from the vitamin A in the food eaten by the cow. Some variations of the vitamin A in butter fat which have been observed could not be attributed to destruction during the preparation of the samples of butter. Drummond, Coward, and Watson ( 2 ) concluded that the feed of the cows a t any season is the predominant factor influencing the vitamin A in the milk and butter. Sherman and Smith (10) state that butter usually contains about 30 to 50 units of vitamin ,4 per gram. Nelson and Jones ( 8 ) found that butter fat has one-fifteenth the potency in vitamin A of a good grade of cod-liver oil. Scheunert (9) stated that cows fed beets and grain produced butter containing about half as much vitamin A as cows fed beets, grain, and silage. McLeod, Brodie, and MacLoon ( 7 ) concluded that the vitamin A in the milk from a dairy herd fed indoors was comparatively constant throughout the year. It is generally believed that the butter fat contains practically all the vitamin A of milk ( I ) . All the work was done upon butter fat prepared from the butter by melting it, by allowing the water, casein, and salt to settle, and by filtering. The samples were kept in an electric refrigerator. The method of estimating the vitamin A was a modification of the Sherman and Munsell procedure, described fully elsewhere (3) and there termed the “unit” method. The unit is a gain of about 24 grams in 8 weeks, by rats previously depleted of vitamin A. A I N BUTTERFAT Seasonal variations were studied on samples of butter secured a t intervals of a month or longer from the college creamery. The butter was prepared from cream purchased from neighboring farmers. The cows generally mere fed corn, cottonseed meal, farm roughage, and such pasturage as was available. Practically no silage was fed, and the pastures were scanty during dry periods or cold winter weather. The approximate quantity of vitamin A in the sixteen samples of butter fat is shown in Table I. Details of the work are given in Table 11. The vitamin A content varied from approximately 17 to 50 units to the gram. The richest sample contained three times as much vitamin A as the poorest. While the variations were relatively high, all the butter was rich in vitamin A. The last two samples were taken during a severe drought, when there was no green pasturage. I n spite of this, one sample was among the highest in vitamin 1,and the other was little below the average. RELATION O F SEASON TO VITa4MIN
TABLEI.
VITAMIX
-4 I 9
DATE UXITJ I K SAMPLE RECEIVED 1 GRAM 29303 March 7 1928 20 29417 April 8, i928 17 29498 25 May 4, 1928 29547 June 4, 1928 25 29955 July 4, 1928 33 30165 20 Aug. 2, 1928 30269 20 Sept. 3, 1928 33541 33 Oct. 8, 1928 30313 N J V .5, 1928 25
CREAMERY
BUTTER
FAT
DATE UNIT?I N SAMPLE RECEIVED 1 GR.AV 31006 Dec. 14, 1928 40 25 31074 Jan. 8, 1929 20 31112 Feb. 4, 1929 25 31148 March 4. 1929 25 April 2, 1929 31186 31310 May 10, 1929 50 31311 May 10, 1929 40 31865 Aug. 26, 1929 40 25 32069 Sept. 28, 1929
Sample 31310 was from cows fed a balanced ration contnining cottonseed meal, while sample 31311 was from cows receiving a ration high in cottonseed meal. Both samples are high in vitamin A.
RELATIOX OF VITAMIN A
CONPENT TO
FEED
The effect of the ration on vitamin A in butter fat was studied on samples of butter furnished by 0. C. Copeland, of the Division of Dairy Husbandry, from cows which had been fed the same ration for about 15 months. One group of cows received cottonseed meal, cottonseed hulls, sorghum silage, and Sudan grass pasture, A second group received the same ration without the pasturage, and a third group received only cottonseed meal and hulls. The results are given in Table 11. The butter fat from the cows on pasturage contained approximately 33 units of vitamin A to the gram. This corresponds approximately to the vitamin A in butter fat of high content from the farm cream of this locality (Table I). After 15 to 16 months the cows that received silage, but did not have access to pasture, produced butter fat varying from 2 to 12 units in vitamin A, but averaging about 4 units of vitamin A to the gram. The cows that had received only cottonseed meal and hulls for 15 to 16 months produced butter fat containing only 2 units of vitamin A to the gram. It is seen from the figures that pasture grasses are highly effective in maintaining a high content of vitamin A in butter fat. The sorghum silage did not furnish much vitamin A to the butter, whereas the cottonseed meal and hulls fed alone produced butter still lower in vitamin A. It is probable that these cows had depleted their bodily store of vitamin A, and that the vitamin A in the butter fat was the quantity they were able to put in from the feed received. The units of vitamin A produced daily by each cow were calculated from the number of units in each gram of butter fat and the quantity of butter produced daily, with the results given in Table 111. The cow receiving silage produced about 1960 units a day, or about six times as much as the cow not receiving silage. The cow receiving pasturage in addition to silage produced about 17,030 units of vitamin A a day, or about sixty times as much as the cow receiving no
