Fortification of Oil, Fat, and Flour STABILITY OF ADDED CAROTENE AND EFFECT OF ANTIOXIDANTS P. W. MORGALI, L. W. BYERS2, AND E. J. MILLER Michigan Agricultural Experiment Station, East Lansing, Mich. porated in foods with a n oil solution of crystalline carotene (1S), accelerated carotene decomposition tests were made with both crystalline carotene-in-oil and dehydrated alfalfaleaf unsaponifiable concentrate (7, 11). Various antioxidants were added to some of the preparations to determine their effectiveness.
The fortification of foods with vitamins serves as a nutritional benefit only in so far as the vitamins have not decomposed before the food is consumed. Therefore stability tests to determine the most stable conditions for a given vitamin are of utmost importance. Carotene (provitamin A) has been considered for increasing the vitamin A value of foods. By an accelerated test the stability of crystalline carotene in various oils, both with and without added antioxidants, is compared with that of a carotene concentrate prepared from alfalfa-leaf meal. The stability of the same carotene preparations in Crisco, lard, oleomargarine, white flour, soybean meal (solvent- and expeller-extracted), and soybean flour, is given. In most preparations the alfalfa carotene concentrate was the more stable. Soybean lecithin improved the stability of carotene in soybean flour.
ACCELERATED DECO.MPOSITION TEST FOR CAROTENE
To hasten the decomposition of carotene, which may be very slow at room temperature, two thermostatically controlled electric ovens a t 45' and 60" C. were used. Weighed samples, in laboratory glassware and exposed t o the air, were placed in the
ovens, and a t selected intervals duplicates were removed and analyzed for carotene by the method of Petering, Wolman, and Hibbard (1%). Samples were prepared by dissolving a weighed amount of crystalline carotene (90 per cent beta, 10 per cent alpha, S. M. A. Corporation) in low-boiling petroleum ether, adding a definite amount of Wesson oil, and evaporating off the petroleum ether by heating at 50" C. under nitrogen and reduced pressure. Heat and vacuum were continued for 20 minutes after the solvent had apparently been removed. The solution was then analyzed for carotene by the Petering, Wolman, Hibbard method (12). Samples of this stock solution of carotene in Wesson oil (containing 1.2 mg. of carotene, or 2000 I. U. of vitamin A, per gram of oil) were then subjected to the accelerated decomposition test. A series of runs was made t o determine the effect of the amount of surface exposed to the air on the carotene stability. The surface exposed varied from 31 to 630 sq. mm. per 0.1 gram of sample. The results showed that the amount of surface exposed to the air under the conditions of our test was not a critical factor in carotene decomposition. The following procedure was used in the subsequent stability tests: A 0.2-gram sample of carotene in oil was weighed into glass vials 12 mm. in diameter and 25 mm. high. Larger samples and vials were used for the fat and flour preparations. For convenience the vials were placed into holes in hardwood blocks.
ECENT trends in nutrition have been toward the addition of vitamins t o the common foods (1). Milk has been fortified with vitamin D and margarine with vitamin A. In 1941 enriched flour and bread were produced which contain, in addition t o the usual ingredients,. proper amounts of thiamine, nicotinic acid, and iron as specified b y the Food and Drug Administration. Considerable interest has been shown in the possibilities of fortifying various foods, such as margarine, soybean flour, etc., with carotene as a source of vitamin A. Carotene (provitamin A) is found widely distributed in nature and in some instances, notably carrots and dehydrated alfalfa-leaf meal, in sufficient concentration and availability to warrant the development of processes for its commercial isolation (2, 7, 1'1). With our imports of fish liver oils containing vitamin A practically cut off as a result of the war, we may be forced t o obtain much of our vitamin A supply from domestic sources. Since vitamin A and carotene, as well as most of the other vitamins, are subject to destruction under various conditions, it is essential that they should not be added to foods in which they are not relatively stable. Many leafy-green plants contain vitamin E and other antioxidants (9)which are concentrated along with the carotene and sterols in the unsaponifiable Concentrate. Therefore, to compare the stability of these preparations when incor-
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STABILITY IN OILS
Crystalline carotene in both Mazola and Wesson oil showed approximately the same stability as an alfalfa carotene concentrate in the same two oils. Figure 1 presents the stability of crystalline carotene in Wesson oil, Since alfalfa-leaf meal is known to contain vitamin E which has antioxidant activity, and possibly other antioxidants also, it might be expected that alfalfa carotene concentrate would be more stable than crystalline carotene in the same solvent. To clarify this point, antioxidants were added to a solution of crystalline carotene in Wesson oil to determine their effect on the carotene stability. A solution of crystalline carotene in Wesson oil was prepared as previously described except for the addition of the antioxidant before the solvent was removed. The antioxidants were hydroquinone, phenyl-a-naphthylamine, phenyl+ naphthylamine (14, 1 4 , and a-tocopherol added at 0.01 per cent level based on the weight of oil. The hydroquinone and or-tocopherol were also used at the 0.1 per cent level. The data (Figure 1)show that only hydroquinone was effective in retarding carotene decomposition; other antioxidants were ineffective. The same result was obtained with alfalfa
Present address, Purdue University, Lafayette, Ind. Dow Chemical Company, Midland, Mich.
a Present address,
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trate should also show greater carotene stability than crystalline carotene, STABILITY IN EDIBLE FATS
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NO ANTIOXIDANT ADDED 0.1% HYDROQUINONE ADDED QOI% HYDROQUINONE ADDED 0.1 % ALPHA-TOCOPHEROL ADDED 0.01'4 ALPHA -TOCOPHEROL ADDED 0.01 Yo PHENYL- ALPHA- NAPHTHYLAMINE ADDED 001 fflENYL BETA NAPHTHYLAMINE ADDED
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200
300
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Figure 1. Effect of Added Antioxidants on Stability of Carotene in Wesson Oil
Accordingly, alfalfa carotene concentrate and crystalline carotene were added to certain food products to compare the stability. Commercial samples of Crisco, lard (Armour's Star pure lard), and oleomargarine (not fortified with vitamin A, John F. Jelke Company) were obtained and prepared for the test as follows: Wesson oil stock solutions of both crystalline carotene and alfalfa carotene concentrate were prepared as before. To 200 grams each of Crisco, lard, and oleomargarine, sufficient Wesson oil stock solution was added to bring - 500 the fat to the equivalent of 10,000 units of vitamin A per pound. After thorough mixing Crystalline at 34" C., 2.2-gram samples were weighed directly into vi& and $aced in an oven maintained a t 45' C. At appropriate intervals samples were taken and analyzed for carotene. The results are given in Figure 2. The addition of small amounts of antioxidants to material which already contains considerable antioxidants, such as Crisco and oleomargarine, does not add to the carotene stability in that material. I n lard, a slight protective action was observed with the alfalfa concentrate. These results agree with the findings of Olcott et al. (8, 9,10) that "inhibitols" are effective for animal fats but not for the vegetable oils from which they come. Stability tests were run on the same samples for 6060 hours a t room temperature and in a refrigerator at 8" C. in 2-ounce sample bottles with screw tops. Again Crisco was superior to the other two samples, and kept over 90 per cent of its carotene in both the crystalline carotene and alfalfa concentrate at both temperatures. At room temperature oleomargarine contained about 50 per cent for both preparations, while with lard the alfalfa concentrate improved the carotene stability from 30 to 67 per cent. I n the refrigerator test, lard and oleomargarine were practically identical (about 80 per cent remaining) with the alfalfa concentrate slightly higher in each case.
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carotene concentrate in Wesson oil. Presumably Wesson oil contains a sufficient quantity of natural antioxidants so that the addition of the phenylnaphthylamines, or a-tocopherol, or the natutal antioxidants in alfalfa, in the quantities used have practically no effect in retarding the decomposition of carotene dissolved in it. To determine the presence or absence of antioxidants in a given preparation, a solvent which was free of antioxidants would have to be used. Therefore to show that the alfalfa carotene concentrate contains antioxidants, a similar series of runs were made using a mineral oil solvent (Petrofol, United Drug Company); it is understood that mineral oil is not satisfactory as a medium for administering carotene for its provitamin A activity due to the poor absorption attendant upon its use (4). Crystalline carotene in mineral oil without added antioxidant remained quite stable for about 230 hours and then underwent almost complete decomposition by 280 hours at 45" C. By comparison, crystalline carotene in Wesson oil at 280 hours contained about 65 per cent of its carotene (Figure 1). The phenylnaphthylamines and a-tocopherol a t the 0.01 per cent level and the a-tocopherol and hydroquinone at the 0.1 per cent level effectively protected the carotene in STABILITY IN FLOUR AND MEAL mineral oil so that about 88 per cent remained in each case after 476 hours. The 0.001 per cent a-tocopherol had A white, bleached flour (Gold Medal brand, enriched), and dropped to 39 per cent in the same time, while the 0.01 per two soybean meals, one solvent-extracted and the other excent hydroquinone was only slightly better than the unpropeller-extracted, were obtained. Stock solutions of crystaltected carotene. The 0.1 per cent hydroquinone sample protected the carotene very well and, when analyzed after 838 hours, was found still to contain 81.8 per cent of its original carotene. The alfalfa carotene concentrate contained 89 per cent of its carotene after 865 hours, which is even better than the protection afforded by 0.1per cent hydroquinone, the best antioxidant used. It thus appears evident that the alfalfa carotene concentrate does contain naturally occurring antioxidants which undoubtedly protect the carotene from decomposition during its extraction and concentration. Although these antioxidants do not add to the carotene stability when in Wesson oil, because of the antioxidants already present in that solvent, they do become effective stabilizers in solvents TIME IN HOURS which are naturally deficient in antioxidants. Therefore, when added to foods relatively poor Figure 2. Stability of Crystalline Carotene and Alfalfa Carotene in antioxidants, the alfalfa carotene concenConcentrate in Lard, Crisco, and Margarine
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60" C. Three-gram samples were weighed into open vials, and a t appropriate time intervals the entire amount was used for the carotene determination. Figure 4 shows the results. I n both tests sample 4 proved most stable. Also, soybean lecithin improved the carotene stability in each preparation. These same samples were run a t room temperatures for 5424 hours (226 days) in metal cans about half full and closed with a metal lid. Again sample 4 was best and contained 94.5 per cent of its carotene after 5424 hours. Sample 3 was next (91.3 per cent), followed by sample 2 (83 per cent) and 450 SOYBEAN MEAL sample 1 (75 per cent). At room temperature sample 3 proved more stable than sample 2, but in the accelerated tests a t 45" C. this was not true. The alfalfa carotene concentrate used in 0 CRYST, CAROTENE these tests contained some xanthophyll, and ALFALFA CONC since the xanthophyll content mas readily 20 obtainable during the progress of the tests 200 400 600 800 1000 1200 ( 7 ), the xanthophyll stability was determined TIME IN HOURS under the same conditions as the carotene. Figure 3. Stability of Crystalline Carotene and Alfalfa Carotene The original xanthoohvll content of samtdes Concentrate in White Flour and Soybean Meals 3 and 4-of Figure 4 w & 15 mg. per pound of flour. At both 45" and 60" C. the addition of soybean lecithin improved the stability of line carotene and alfalfa carotene concentrate were added to xanthophyll approximately as much as it did that of the caro150-gram portions each of the white flour, solvent-extracted tene. The sample containing lecithin in the 45" C. test still soybean meal, and expeller-extracted soybean meal to give a contained 51.5 per cent of its xanthophyll after 1895 hours. potency of 60,000 I. U. of vitamin A per pound of flour or meal. Petroleum ether was added, the mixture was stirred DISCUSSION thoroughly, and the petroleum ether was removed as before. Crystalline carotene, to be suitable for stability tests, must After thorough mixing, a carotene determination was run in not have been exposed to air for any appreciable time before duplicate, and each preparation was divided into equal porit is used in a test, as it oxidizes rapidly (5). Partially detions and placed in open beakers. One sample was stored at composed carotene accelerates carotene decomposition in room temperature in a dark closet, and the other was placed oils; although this is not so pronounced in vegetable oils, such in an oven a t 45" C. Three-gram samples were taken for analysis. The results are shown in Figure 3. Stability as Wesson, it is striking in an oil free of antioxidants. A crystalline carotene sample which had been exposed t o air for tests were run at room temperature for those samples containsome time was completely decomposed in less than 24 hours ing the alfalfa concentrate. The order of stability was the a t 45" C. in mineral oil, whereas 300 hours were required with same as a t 45" C.; solvent-extracted soybean meal mas best (90 per cent), then the white flour (77 per cent), and last the expeller-extracted soybean meal (34 per cent), a t the end of 6060 hours. Caroteniaed soybean flour was available commercially, and a comparison was made of the stability of the carotene in this product with the stability of our alfalfa carotene concentrate in the same flour. The ArcherDaniels-Midland Company prepared several caroteniaed flours; a solvent-extracted soybean flour containing less than 1 per cent residual fat or ether extractable material was used for all preparations, and all were carotenized to a potency of 60,000 I. U. of vitamin A (as carotene) per pound of flour: Sample 1, soybean flour with a carotene-inoil preparation added; sample 2, soybean flour with carotene-in-oil and 2.5 per cent of a technical grade soybean lecithin added; sample 3, soybean flour with alfalfa carotene A 3, ALFALFA CON concentrate added; and sample 4, soybean flour with alfalfa carotene concentrate and 2.5 per cent of technical grade soybean TIME IN HOURS lecithin added. All four products were subjected to the Figure 4. Stability of Crystalline Carotene and Alfalfa Carotene Concentrate in Soybean Flour, with and without Added Lecithin same decomposition tests a t both 45" and
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crystalline carotene from freshly opened vials. This test was frequently used on carotene samples to ensure their suitability for the stability tests. Whereas the addition of small amounts of hydroquinone (0.002 and 0.005 per cent based on the weight of oil) has been reported ineffective in protecting carotene in Wesson oil (S), at the higher levels (0.01 and 0.1 per cent), the amount of carotene remaining was increased from 43 to 73 and 81 per cent, respectively. The stability of carotene in sweet potato flour under various conditions of storage has been reported (6); at room temperature and in loosely stoppered bottles sweet potato flour had approximately 25 per cent of its original carotene after 120 days, while sweet potato flour plus 10 per cent crude cottonseed oil had 61 per cent under the same conditions. I n corpparison, the four soybean flour samples, under similar conditions of temperature and air exposure, contained from 81 to 96 per cent carotene after 169 days. Although the soybean flour samples contained only 60,000 I. U. of vitamin A (as carotene) per pound and the sweet potato flour contained almost twice that amount, it appears that carotene is more stable in soybean flour than in sweet potato flour. CONCLUSIONS
The stability of carotene is greatly influenced by the presence or absence of antioxidants contained in the solvent. I n solvents free of antioxidants, the addition of antioxidants has a strong stabilizing effect; in solvents like Wesson oil, which contain naturally occurring antioxidants, little additional effect is observed. Carotene is much more stable in solvent-extracted than in expeller-extracted soybean meal. Soybean lecithin improves the stability of carotene in soybean flour. Alfalfa carotene concentrate showed better stability in many of these products than crystalline carotene, an indica-
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tion that the naturally occurring antioxidants present in the concentrate are effective in these food products. ACKNOWLEDGMENT
This research was supported by the Horace H. Rackham Research Endowment of the Michigan State College of Agriculture and Applied Science for studies on the industrial utilization of agricultural products. I n connection with the carotene stability tests on the soybean flour preparations, we wish to thank the Department of Biological Research of the Archer-Daniels-Midland Company for supplying the soybean flour and incorporating the carotene concentrate in it. LITERATURE CITED
(1) American Chemical Society, Symposium on Nutritional Restoration and Fortification of Foods, IND.ENG. CHEM.,33, 707 (1941). (2) Barnett, H. M., News Ed. (Am. Chem. Soc.), 19,778 (1941). (3) Bauman, C. A., and Steenbock, H., J. Biol. Chem., 101, 561 (1933). (4) Dutcher, A. R., Harris, P. L., Hartzler, E. R., and Guerrant, B., J . Nutrition, 8,269 (1934). ( 5 ) Fraps, G. S., and Kemmerer, A. R., News Ed. (Am. Chem. Soc.), 19,846 (1941). (6) Mitchell, J. H., and Lease, E. J., 5. C. Agr. Expt. Sta., Bull. 333 (1941). (7) Morgal, P. W., Petering, H. G., and Miller, E. J., IND.ENQ. CHEM.33, 1298 (1941). (8) Olcott, H. S., and Mattill, H. A.,Chem. Rev., 29,257(1941). (9) Olcott, H.S., and Mattill, H. A., J . Am. C h m . SOC.,58, 1627 (1936). (10) Ibid., 58,2204 (1936). (11) Petering, H.G.,Morgal, P. W., and Miller, E. J., IND.ENG. CEEM.,32,1407 (1940). (12) Petering, H. G.,Wolman, W., and Hibbard, R. P., IND.ENG. CHEM.,ANAL.ED., 12, 148(1940). (13) Quackenbush. F. W.,Cox, R. P., and Steenbock, H , J. Bid. Chem., 145, 169 (1942). (14) Semon, W. L.,Sloan, A. W., and Craig, David, IND. ENG.CHEM., 22, 1001 (1930). (15) Smith, P. I., Soap,15, 21 (1939). APPROYBID by Station Director.
Article 600 (n. s.).
ALCHEMIST By David Teniers
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No. 151 in the Berolzheimer series of Alchemical and Historical Reproductions presents the eighteenth Teniers subject we have brought. Since it is known that the elder Teniers produced a t least one alchemical painting and that the younger Teniers painted seventeen such pictures, it is believed that this completes the series. The location of the original in this case is not known. It seems much cruder than the other Teniers paintings and various parts appear t o be copied from some of those previously reproduced. Distillation apparatus does not appear as it does in the others.
D. D. BEROLZHEIMER 50 East 41st Street New York, (17) N. Y. The lists of reproductions and directions for obtaining copies appear as followa: 1 to 96, January, 1939, page 124; 97 to 120 Janua 1941 p a p 114; 121 to 144, January, 1943, page 106. An iddition3 reproduotlon appears each month.
