August 1948
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
residual solids left in the starch fermentations are soluble, nonvolatile solids, the small.amount of suspended material, which is readily removed by filtering with filter-aid, consisting of bacterial cells and traces of suspended protein. The soluble solids content of the filtrates from either starch fermentations or corn mash fermentations are approximately the same, and about 80% of these soluble residual solids are carbohydrate in nature. Hence, the major advantage of fermenting cornstarch, aside from recovery of valuable by-products during processing for the separation of the starch, would be greater ease of .filtration prior to final recovery of the 2,3-butanediol by either distillation or solvent extraction methods, both of which have been successfully employed for obtaining this product from fermentation beers. ACKNOWLEDGMENT
Whis work was supported by a grant from the Industrial Science Research Institute of the Iowa State College for studies on the fermentative utilization of agricultural products. LITERATURE CITED
(1) Adams, G. A,, Can. J . Research, 24F,1-11 (1946). (2) Adams, G.A.,and Leslie, J. D., Zbid., 24F, 12-28 (1946). (3) Ibid., 24F,107-16 (1946). Ibid., 23B,1-9 (1945). (4) Adams, G.A.,and Stanier, R. Y., (5) Aasoo. Offioial Agr. Chem., Offioial and Tentative Methods of Analvsis. 5th ed.. 1940. (6) Blackwood,’A. C., and Ledingham, G. A,, Can.J.Research, 25E, 180-91 (1947).
1445
(7) Blom, R. H., and Efron, A., IND.ENG. CHEM.,37, 1237-40 (1945). ,---, (8) Breden, C. R., and Fulmer, E. I., I o w a State CoZZ. J . Sci., 5, 133-53 (1931). (9) Fratkin, S. B.,and Adams, G. A,, Can. J . Research, 24F,29-38 (1946). (10) Fulmer, E. I., Plant Physiol., 1, 67-76 (1926). (11) Fulmer, E.I.,Christensen, L. M., and Kendall, A. R., IND. ENG. CHEM.,25, 798-808 (1933). (12) Fulmer, E. I., Nelson, V. E., and Sherwood, F. F., J. Am. Cheni. SOC.,43, 191-9 (1921). (13) Harden, A,, and Walpole, G. S., Proc. Roy. SOC.(London), B77, 399-405 (1906). (14) Johnson, M.J.,IND. ENG.CHEM.,ANAL.ED.,16,626-7(1944). (15) Katznelson, H.,Can. J . Research, 22C.235-40 (1944). (16) Ibid., 22C,241-50 (1944). (17) Ibid., 24’2, 99-108 (1946). (18) Katznelson, H., andLochhead, A.G., Ibid., 22C,273-9 (1944). (19) Kluyver, A. J., and Scheffer, M. A., U. S. Patent 1,899,156 (Feb. 28,1933). (20) Kozelka, F. L.,and Hine, C. H., IND.ENG.CREM.,ANAL.ED., 13,905-7 (1941). (21) Langlykke, A.F., and Peterson, W. H., Zbid., 9, 163-6 (1937). (22) Ledingham, G.A.,Adams, G. A,, and Stanier, R. Y.,Can. J. Research, 23F,48-71 (1945). (23) Rose, D.,and King, W. S., Ibid., 23F,79-89 (1945). Adams, G. A., and Ledingham, G. A,, Ibid., 23F, (24) Stanier, R. Y., 72-8 (1945). (25) Underkofler, L. A.,Guymon, J. F., Rayman, M. M., and Fulmer, E. I., I o w a State CoZZ. J . Sci., 17,377-9(1944). RECBIYED June 30, 1947. Presented before the Fermentation Section, Division of Agricultural and Food Chemistry, at the 111th Meeting of the AYPJRICAN CHEMICAL S O O I ~ Y ,Atlantic City, N. J.
Beta-Carotene from Sweet Potatoes W, C. SHERMAN’ AND C. J. ICOEHN2 Alabama Agricultural Experiment Station, Auburn, Ala.
purification does not lend itHE present investigaA simplified method for the commercial preparation of self well to large scale operation relates to the preppure &carotene from Porto Rico sweet potatoes is detions. aration of pure crystalline scribed. This preparation is suitable for use in fortifying It has been shown (4, 6) @-carotene and an oil conhuman foods and as a spectrophotometric and biological that the principal pigment of centrate of @-carotene by a standard for research purposes. Its preparation involves the Porto Rico variety of process that is commercially direct crystallization from sweet potato oil and does not sweet potato is p-carotene. feasible. This process is require further purification by chromatographic adsorpVillere et al. (8) prepared conadapted to large scale protion means or diphasic extraction. The residual sweet centrates from sweet potatoes duction at a cost far below potato oil, as a by-product, still contains enough p-carowhich contained 72 to 90% the prevailing price. During tene to have a vitamin A activity of over 10,000 I.U. (intercarotene. It was pertinent World War 11, when the national units) per gram. This constitutes a more potent to the present investigation world’s production of fish souroe of vitamin A than most commercial fish liver oils. to determine if carotene oils was drastically curcould be prepared with suftailed, a shortage of concenficient purity for human food and for use as a laboratory trated sources of vitamin A for humans resulted. Adequate provision of vitamin A in animal feeds, moreover, became a standard without resorting to chromatographic purification. It was found in this laboratory that the Porto Rico variety serious problem. Research was undertaken in this laboratory , to ilevelop a method of preparation of @-carotenefor human and of sweet potato is rich in carotene and for practical purposes devoid of pigments other than @-carotene. It was chosen, thereanimal consumption that would be pure enough for use as a fore, as the source of p-carotene in the present study. laboratory standard and that could compete commercially with fish oils as a source of vitamin A. METHOD OF PREPARATION OF @-CAROTENE Carrot roots, the most common commercial source of @-caroOne hundred and sixty kilograms of raw sweet potatoes were tene, contain not only large amounts of xanthophylls, which do cut into shreds of about 3-mm. thickness by means of a powernot have vitamin A activity, but also the less active alpha and driven shredder. The shredded material was dried t o about 7% gamma isomers of carotene. The complete separation of these moisture content in a continuous-flow rotary dryer and ground t o isomers from the beta form is difficult and has been shown by 40-mesh particle size in a laboratory mill. The powdered material was placed in a steam-jacketed percolator and extracted 4 Zscheile et al. (9) t o require repeated chromatographic adsorptimes with 55-liter quantities of hot Skellysolve B, which retions and recrystallizations. The chromatographic method of moved most of the pigment. The combined Skellysolve extract was placed in a large still and reduced to about 6 liters in volume 1 Present address, Ralston Purina Company, St. Louis, Mo. by distillation. The partially concentrated extract was placed 2 Present address, O 5 c e of Surgeon General, U. 8. Army, Washington. in an all-glass still and reduced t o about 9@0ml. under reduced D. c.
T
IN D U S T R I A L A N D E N G I N E E R IN G C H E M I S TR Y
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piessure in an atniuspheie ul nitlogen. The concentrated exdract, which still contained about 360 ml. of Skellysolve, was eransferred t o 250-ml. centrifuge bottles, stoppered, and allowed to stand at 3" C. for several days t o crystallize the carotene. T h e bottles were centrifuged and the supernatant liquid was decanted from the crystals. The crystalline material was washed twice with cold n-hexane (b.p. 68" to 71' C., sp. gr. 0.6846, donated for use in this study by the American Mineral Spirits Company of New York) and transferred to a 50-ml. centrifuge tube. The combined crystals were extracted with eight successive 50-ml. quantities of boiling hexane. Each extract was centrifuged quickly and decanted into other 50-ml. centrifuge tubes while still hot. The hexane extracts were cooled to 3' C. to crystallize the carotene. The crystals, which formed in the first two extracts, coniained visible colorless impurities and were kept separate. The crystals in the other six extracts were uniform. They were combined, dried in vacuum and sealed in glass tubes under nitrogen (crop I, weight 484 mg.). The mother liquors from the carotene crystallizations were combined with the original mother liquor and the solvent was completely removed on a hot water bath under reduced pressure. The residue, contained in a 6-liter round-bottomed flask, was saponified by boiling for 1hour with 2000 ml. of anhydrous methanol and 200 grams of potassium hydroxide. The saponificate was diluted with a n equal volume of water and was exhaustively extracted with Skellysolve B. The Skellysolve solution of the carotenoid pigments was thoroughly washed with water and extracted three times with a n e ual volume of 90% methanol. The methanol extracts were comiined and extracted once with Skellysolve B. This Skellysolve extract was extracted once with c 9 0 ~ methanol o and added to the main Skellysolve fraction. The methanol extracts were combined and set aside for further examination (xanthophyll frrcction). The carotene fraction of the saponificate was transferred to a &liter flask and evaporated t o dryness under reduced pressure. T h e residue, which weighed 85 grams, was dissolved in carbon disulfide and was precipitated with 5 volumes of methanol. This supernatant liquor was decanted off and discarded. The precipitated carotene was dried in vacuum and extracted nine times with 50-ml. quantities of boiling hexane. The extracts were transferred to 50-ml. centrifuge tubes and cooled to 1 " C. for crystallization. The first three batches were contaminated with a white precipitate and were kept separate. The last six batches, which were of better quality, were combined and were recrystallized once from n-hexane (rrop 11,weight 473 mg.). SPECTRAL ABSORPTION AND CHROMATOGRAPHIC PROPERTlhS OF CAROTENE
Absorption data for the crystalline carotene preparations were obtained by means of a Beckman spectrophotometer and are expressed in terms of the specific absorption coefficient, a :
was 98.5% pure at the 540-micron niaximurn and 97.5% pure ttt 478 microns or an average of 98%. The absorption spectra, expressed as specific absorption coefficients, of crop I and crop I recrystallized, are given in Figure 1. The curves are typical of p-carotene and give no indication of contamination with acarotene or other colored impurities. T o test the purity of the carotene further, i t was subjected to chromatographic analysis according t o the method of Strain ( 7 ) ; this is reported to allow the isolation of as little as 0.029?, of a-carotene from carotene preparations. The carotene was dissolved in carbon tetrachloride and passed through a column packed with magnesium oxide (Micron brand) and Hyflo Superc e l l to 1 by weight. Crops I and I recrystallized, thus chromatographed, gave no indication of an a-carotene band. Two distinct bands formed: a narrow band of very strongly adsorbed pigment which was brown in color formed atthetop of the column; a wider band of p-carotene formed and slowly passed down the column. The bands were separated and eluted from the adsorbent Rith methanol:hexane, 1 to 1 b y volume. The eluted pigments were washed with water, dried with sodium sulfate, and diluted to volume with hexane. The brown pigment comprised 0.24 and 0.28% and the p-carotene 99.76 and 99.72%, respectively, of the total pigments of crops I and I recrystallized. Crop I1 was similarly chromatographed and showed no evidence of a-carotene. The colored impurity comprised 1.48% and the &carotene 98.52% of the total pigments. The solution of brown highly adsorbed pigment was analyzed .pertrophotometrically. The absorption curve (Figure 1) , derived from density values, was similar in shape to that of carotenoid pigments in general, but the absorption occzlrred a t lower wave lengths than that of a- or p-carotene. Absorption maxima were at 428 and 450 microns with a minimum a t 245 microns in hexane solution. This pigment is not, the neo-8carotene isolated by Beadle and Zscheile ( 1 ) because of its chromatographic adsorption properties and its spectral absorption at relatively lower wave lengths. It may, however, be some other cis-isomer of carotene. I n view of the possible commercial value of sweet potato oil, the crude extract, after chilling to remove crystalline carotene, was analyzed for carotene content. This crude extract contained
300
r
IO
iY
=
log10 -cl
Vol. 40, No. 8
1
t
where lo = intensity of light transmitted by holvent-filled cell; I = intensity of light transmitted by solution-filled cell; c = concentration in grams per liter; and I = thickness of solution in centimeters. The specific absorption coefficients of carotene, crops I and I1 and I recrystallized, at points of maximum absorption are given in Table I. Crop I, which was crystallized only once after separation from t h e crude extract and had received no purification by chromatographic means, was approximately 97% pure based on the absorption values of Zscheile et al. (9) for pure pcarotene. After one more recrystallization crop I recrystallized
TABLEI. ABSORPTIONCOEFFICIENTS OF P-CABOTEND FROM SWEETPOTATOES
,
Preparation Crop I Crop I, recrystallized Crop I1
(Hexane solution) Absorption Coefficient _____--_ 430 mp 251.6 254.0 246.0
478 mw 219.7 222.0 217.0
1
CROP I RECRYSTALLIZED COLORE0 I M P U R I T Y I
400
l
I
I
I
1
1
1
450
1
500
W A V E LENGTH m&
Figure 1. Absorption Spectra of @-Carotene Prepared from Sweet Potatoes and the Colored Impurity Which Occurred with the Carotene in n-Hexane
August 1948
INDUSTRIAL AND ENGINEERING CHEMISTRY I
538 grams of oil with a carotene content of 7.04 mg. per gram of oil. Chromatographic analysis showed that 86% of the total pigment was p-carotene, which passed down the column with no indication of an a-carotene band. The brown band of strongly adsorbed pigment at the top of the column contained 9.4% of the total pigment; the remaining 4.6y0 was contained in a narrow yellow band, which separated from the brown band at the . top after considerable washing with hexane.
I
I
.I
I
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I
I
I
I
I
I
I
I
I
I
1
STEROLS AND XANTHOPHYLLS IN UNSAPONIFIABLE MATTER
It was observed that the white precipitate in the unsaponifiable matter made the crystallization of carotene difficult because it greatly increased the solubility of carotene in all solvents ordinarily used for crystallization. Most of this white material was extracted in the first hexane extract of the unsaponifiable matter. The second and third extracts contained small quantities of the interfering substance, but subsequent fractions were essentially devoid of it. No satisfactory method has been found for separating and purifying the relatively large amount of carotene which accompanied this white material in the first three extracts of the unsaponifiable matter. A small quantity of the white material was purified for the purpose of identification. The first three hexane extracts of the unsaponifiable matter were combined and the solvent was removed under reduced pressure. The residue was crystallized by dissolving it in a minimum volume of boiling ethanol and cooling. These crystals were recrystallized five times from hot ethanol to obtain colorless, waxy, needle-shaped crystals. These crystals gave a positive Lieberman-Burchard test for sterols and exhibited strong absorption in the ultraviolet region of the spectrum with maxima a t 252 and 272 microns in ethanol solution (Figure 2). For the determination of the xanthophyll content of sweet potatoes, the xanthophyll fraction was dilutea with an equal volume of water and exhaustively extracted with diethyl ether. The ether solution was dried with sodium sulfate evaporated t o dryness, and made to volume with anhydrous ethanol for the spectrophotometric determination of xanthophyll. The xanthophyll fraction, which comprised all the xanthophyll in the original sample, contained 0.54 mg. of xanthophyll or 0.012y0 of the total pigment of the sweet potatoes.
I 225
WAVE
285
LENGTH mfi
Figure 2. Absorption Spectrum of a Crystalline Sterol Fraction Isolated from Sweet Potato Tubers in Ethanol
As soon as 'the samples weGe sufficiently dried, they were removed from the dryers, ground in a laboratory mill, and immediately analyzed for moisture and carotene contents. The results, summarized in Table 11, show very little difference in the percentage of carotene destroyed by the different methods of dehydration. No destruction of carotene occurred in the raw sweet potatoes dehydrated by the fuel-oil dryer until the moisture content was reduced t o below 25y0. The preliminary blanching of the sweet potatoes caused a marked increase in the destruction of carotene during dehydration. The samples of raw and blanched sweet potatoes were left in the electric oven and fueloil dryer for the same lengths of time, and, although the blanched samples were not dried t o as low a moisture content as the raw samples, they retained appreciably less carotene. Similarly, the blanched samples required more than twice as much sun drying as the raw samples, and, although they still contained more moisture, the carotene content was considerably less. ANALYSIS O F SWEET POTATO LEAVES
DESTRUCTION OF CAROTENE OF SWEET POTATOES BY DEHYDRATION
It was of importance to study the destruction of carotene by different methods of dehydration and t o determine the effect of preliminary blanching of sweet potatoes on carotene losses. For these tests, Porto Rico sweet potatoes were washed and sliced with a hand slicer t o about 3/32 inch in thickness. The slices were mixed and divided into two equal portions; one of these was dehydrated raw and the other was blanched by steaming f o r % minutes. The raw and blanched samples were each divided into three equal portions for dehydration. The following methods of dehydration were,employed:
255
.
I n view of the absence of a-carotene and the extremely low content of xanthophyll in the sweet potato tubers, it was of interest to identify the carotenoid pigments present in sweet potato leaves. For the analysis, fresh sweet potato leaves were blended in a Waring Blendor with %yoethanol-hexane, 4 to 3 by volume, after which the pigments were completely extracted by repeated extractions with hexane. The extract was saponified a t room temperature with alcoholic potassium hydroxide and thoroughly washed with water. The xanthophylls were removed from the carotene fraction by diphasic fractionation between 90% methanol and hexane. The leaves were found t o contain 6.12 mg. of carotene per 100 grams, fresh basis. The carotene fraction, in hexane solution, was used for the spectrophotometlric determination of total carotene and for chromatographic analysis. A distinct band of a-carotene formed on passage through a column packed with magnesium oxide-Super-cel which comprised about 15% of the carotene fraction; the rest of the pigment was contained in the p-carotene band, The xanthophyll contained in the methanol extract comprised approximately one half the total carotenoid pigments of the sweet potato leaves.
SUNDRYING.The slices were spread out on brown paper and placed in the sun. The raw slices were dry in 10 hours, whereas the blanched slices required 24 hours. DRY HEAT-ELECTRIC OVEN. The TABLE 11. EFFECTOF BLANCHING ON DESTRUCTION OF CAROTENE DURINQ slices were spread out and dried with DEHYDRATION OF SWEET POTATOES dry heat in a forced-draft oven at 75 C. for 240 minutes. Carotene Content Dry Destruction, yo CONTROLLED HUMIDITY-FUEL OIL Moisture, % Baqis, Mg./l 00' G. UnDRYING. The slices were spread out in Sweet Potato Sample Unblanched Blanched Unblanched Blanched blanched Blanched a cabinet and dried by a forced circulaFresh 74.20 74.20 14.02 14 02 6.75 8 30 10 69 7 68 23 8 4i 2 tion of moist hot air of controlled humid(electric oven) 6.05 6.35 10.73 7.15 23.4 48.9 ity heated to 93 O C. by means of a fuelControlled humidity fuel oil) 24 90 14.02 oil burner. The samples were left in the Controlled humldlty {fuel 011) 4 06 6:47 9 11 5 80 3g.O 58 6 dryer for 180 minutes.
Ey
*
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INDUSTRIAL AND ENGINEERING CHEMISTRY DISCUSSION
The ease with which p-carotene of high purity can be obtained by a method involving few operations is made possible by the fact that the pigment of the Porto Rico sweet potato is so predominantly @-carotene. The only pigment, other than pcarotene, present in the crystalline material which was recrystallized once from the crude oil (crop I), was the trace (0.24%) of a pigment which was strongly adsorbed and appeared browu on a magnesium oxide column. There was no apparent removal of this pigment by recrystallization. However, it has been repeatedly observed in this laboratory that the strong affinity of this pigment for magnesium oxide makes it readily possible to remove it quantitatively from carotene preparations by means of one chromatographic separation. It is of interest that crop I was approximately %'yoas pure as the purest preparation of @-carotenemade by Zscheile et al. (9) after only one recrystallixation and no chromatographic separations. I n order to produce a product of constant absorption properties, the latter workers (9) used a commercial preparation of carrot root crystalline carotene, which had undoubtedly been recrystallized several times in manufacture. This product was then subjected to four further recrystallizations and four chromatographic separations. Chromatographic separations are time-consuming and if used to isolate @-carotene,they greatly limit production. From the data prcsented in Table 11, it may be calculated that the sweet potatoes used in this work contained 36.17 mg. of @-carotene per kg. of fresh material. Thus, the starting material contained 5.7872 grams of p-carotene of which 0.9570 gram (crops I and 11) or 16% was recovered in crystalline form of 95a/c or higher purity. I n the sweet potato oil, after removal of crop I, 56% of the original p-carotene could be recovered in the form of a marketable product. Most of the loss encountered was attributable to destruction duiing dehydration. Because the efficiency of solvent recovery is an important item in the cost of production of p-carotene, the present method has the advantage that only one solvent, undiluted, is employed; tjhe solvent can be recovered by simple distillation. I n the procedure of Villere et al. (8)in which acetone extraction is employed, the solvent is diluted in one step and mixed solvents are used in another. I n order to recover these solvents, refractionation is nrcessary with consequent loss of solvents. The high degree of purity of the p-carotene heiein described makes it desirable for enriching food with vitamin A activity for human consumption. This is particularly true of oleomargarine and butter produced during the winter; these are at present colored with coal tar dyes or annatto extract that do not have vitamin A activity. Butter made from milk produced in the winter contains much less vitamin A activity than that produced in the summer because of a lack of carotene in the feed ( 6 ) . If p-carotene, the natural pigment in butter, were used to color winter butter, it would be possible to restore the vitamin A content t o nearly the summer level. The present cost of pure @-carotene,however, makes this impractical. There is a demand for p-carotene of high purity by laboratories engaged in nutritional or biochemical research or control work. The p-carotene described in this paper is of sufficient purity to be used as a spectrophotometiic, colorimetric, or biological standard. The p-carotene which crystallized froin the sweet potato oil was purer than that which crystallized from a solution of the unsaponifiable matter in hexane. This was caused by'the presence of a sterol, which crystallized out with the p-carotene from the unsaponifiable niatter and from which it was difficult to separate. This sterol greatly increased the solubility of p-carotene in hexane. For these reasons it does not appear to be commercially feasible to attempt to recover all the p-carotene from the oil. After as much @-carotene as will crystallize out without saponification has been removed from the sweet potato oil, the oil still contains over 10,000 I.U. of vitamin A activity per gram; this is q u c h more than commercial cod liver oil contains and the
Yol. 40, No. 8
oil would find a ready market as a vilamiu A-rich supplement for animal feeds. The international unit of vitamin A is by definition equivalent the vitamin A activity of 0.6 microgram of &carotene. N o s t cod liver oils contain approximately 1700 to 2000 U.S.P. XI1 units of vitamin A per gram. The U.S.P. unit of vitamin A was originally standardized to be equal tc the I.U., but recent data indicate t>hatthe U.S.P. XTT unit is less than the 1.U. io
Although the tubers 01 the Porto Ricu bweet potato art different from carrot roots in that they do not contain appreciable amounts of pigments other than 6-carotene, the leaves are similar in that they contain appreciable amounts of a-carotene and large amounts of xanthophylls. Ezell and Wilcox (9) reported that p-carotene comprised 81.76% of the pigment of the Portci Rico sweet potato tuber. Their method of chromatographir analysis is open to question, however, because they determined total pigmenth before chromatographing; after chromatographi~ig they determined only carotene and riot the other pigrnrxnt s. Their method, therefore, did not take into account destruction of carotene during chromatographing or the difficulty of making a quantitative transfer of the \\hole solution to the chromatograph tube. The fact that no a-carotene was found in the Porta Rico variety of sweet potato tubers confirms the work of Kemmerer et al. (8). It appears that the impurity, A, described by these investigators is the same bs the strongly adsorbed pigmriit noted in the present work. Extraction of the fresh sweet potatoes requires large volumes ot relatively expensive solvents, such as alcohol or acetone, which are necessary for preliminary dehydration. Although appreciable quantities of carotene are destroyed by the heat dryirrp process, it is economical to start with the dried sweet potatow which can be extracted vr-ith relatively small amounts of lesexpensive hydrocarbon solvents. SURZlClAKY
A simplified method for preparing pure crystalline p-carotena from sweet potatoes is described. The pure @-caroteneis suitable for use in fortifying human foods and as a laboratory standard. The sweet potato oil, from mhich a portion of the 6-caroterxchas been crystallized, still contained enough @-caroteneto furnish over 10,000 I. U. of vitamin A activity per gram. There was rub a-carotene in the oil and only slight traces of xanthophyll. A crystalline sterol from sweet potato tubers wias isolated aiid described. Blanching of sweet potatoes preliminary to drying caused a marked increase in the destruction of carotene during dehydmtion. Sweet potato leaves nere similar t o other leafy material in ronterit of carotene and xanthophyll. LX'I'ERATUHE CITED
(1) Beadle, B. W.,and Zscheile, F. P., J. B i d . Chem., 144, 21 -83 (1 942), (2) Ezell, B. D., and Wilcox, M aS., Science, 103,193-4 (1946). (3) Kemmerer, A. R.,Fraps, G. S., and Meinke, W. W., Food Research, 10,66-71 (1945). (4) Lease, E. J., Proc. Assoc. Southern A y r . Workers (42nd annual convention, Atlanta), 162 (1941). (5) Matlack, M. B., J . T u s h . A c a d . Sci., 27,493-6(1937). (6) Maynard, 1 2 . A., et al., U. S. Dept. Agr., Misc. Pub. 571, 1-14 (1945).
(7) Stiain, H. H., J . BioZ. Chem., 127,191-201 (1939). (8) Villere, J. F., Heinzelmann, D. C., Pominski, J., and Wakeham, H. R. R.,FoodZnd., 16,76-8(1944). (9) Zscheile, F. P., White, J . W., Jr., Beadle, B. W., and Roach, J~l i Plant Physiol., 17,331-46(1932).
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RECEIVED March 27, 1947. Puhliahed w i t h lJie ai)proval of the I>irw1
