STABILITY of CAROTENE Added to Solid Carriers
The addition, in pellet form, of carotene to certain dry carriers without protective measures results i n very rapid loss. With protective measures (such as favorable storage conditions, the addition of certain oils and of very small amounts of antioxidants, and a reasonably low but significant concentration of carotene), the retention of carotene is greatly improved and suggests that pelleted mixtures containing extracted carotene may prove feasible as a supplementary feed.
EMANUEk BICKOFF AND KENNETH T. WILLIAMS \Vestern Regional Research Laboratory,
U. S.Department of Agriculture, Albany, Calif.
F
ORMERLY large quantities of cod liver and shark liver oils were added to feeds to supplement the vitamin A normally present. The present scarcity and increase in price may limit the use of such oils as feed supplements, but oil solutions of carotene may be of value in supplanting the liver oils in animal feeding. Although some work has been reported on the stability of vitamin A of fish liver oils incorporated into mixed feeds, very little has been published on the stability of added carotene in mixed feeds. Some of the factors affecting the stability of carotene in solutions have been investigated. Sakamura (18) reported that stability varies with the solvent, and that when oil is used as the solvent, moderate unsaturation IS necessary for the best stability of carotene. Baumann and Steenbock (1) studied the effects of light, temperature, concentration, and chemical stabilizers; carotene stability appeared to be best in refined cottonseed oil. Hume and Smedley-Maclean (9) showed that decolorization of carotene solutions is much more rapid in esters such as ethyl oleate than in mineral or edible oils. They also shqwed that the addition of an antioxidant, such as hydroquinone, retards the loss in color. A recent paper (16) showed that the tocopherols are effective antioxidants for carotene in ethyl linoleate. McDonald (IO), Turner ( % I ) , and others have shown that, with decrease in temperature, carotene loss is decreased; Scheunert (17) reported that storage in the absence of air further retards carotene loss. Tauber (do), as well as Sumner and Sumner ( l a ) , presented evidence which indicates that loss of carotene in fats may result from a coupled enzymic oxidation. Fraps and Kemmerer ( 5 ) found that carotene dissolved in cottonseed oil and added to mixed feeds lost from 24 to 70% of its value in 16 weeks of storage. They found that the presence of skim milk powder, wheat gray shorts, or yeast, all of which might be assumed to contain stabilizing agents, did not increase the stability of carotene to any practical extent and recommended that a liberal allowance be made for losses during storage. Since it is essential that these mixed feeds retain adequate vitamin A potency and since the addition of liberal excesses constitutes an economic waste, the factors affecting the stability of carotene added to feeds should be determined. The present investigation was undertaken as an approach to the problem and to determine some of the factors that influence loss of carotene. It is hoped this report will stimulate further work. The variables studied included : carrier, added oil (type and concentration), concentration-of carotene, and added antioxidants. PREPARATION O F MIXTURES
The materials used as carriers for the carotene in this investigation included oat flour, potato flour, commercial wheat flour, rice bran, and Avenex (16),a commercial oat flour intended especially for use as an antioxidant. The oat flour was prepared in this
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laboratory; hulls were removed and the oats were finely ground The potatoes were sliced, air-dried, and powdered. Crystalline carotene (S.M.A. Corporation, 90% beta and 10% alpha) was used. It was dissolved in reagent chloroform and the solution added to the carrier being tested. The solvent was removed in a current of air with continuous stirring to ensure uniform distribution of carotene in the carrier. When an antioxidant was to be incorporated, it was dissolved in a suitable solvent and added to the mixture of carrier and carotene, and Ohe solvent was removed as indicated above. I n certain experiments a n oil was added as a 207, solution in petroleum ether subsequent to the addition of carotene and antioxidant, the solvent was again removed as described above, and the last traces were removed by keeping the sample under high vacuum for several hours. Tests were run in which the solvents were evaporated in a stream of nitrogen to prevent possible oxidation during the evaporation, but these tests indicated that carotene stability was not increased by this modification. The experimental mixtures were formed into one-gram cylininch in height) to expose inch in diameter and drical pellets a uniform surface t o the air. Two pellets were used for the initial carotene analyses, and the others were stored in large test tubes loosely covered with cotton plugs at 37" C. in darkness. CAROTENE DETERMINATION. The pellet was added to 10 ml. of petroleum ether (30-60' C.) and crushed with a glass stirring rod. This readily $issolved all the added carotene and other soluble pigments. r h e crushed pellet and the petroleum et'her solution were transferred to a chromatographic column containing dibasic calcium phosphate ( I d ) in order to remove the noncarotene pigments. The carotene was washed through the column with additional petroleum ether. Pressure was applied to thc column, and the solution and wash solvent were received in a 100-ml. volumetric flask. After dilution to a suitable volume, t.he percentage of light transmission was measured with an Evelyn photoelectric colorimeter, using a X o . 420 Evelyn filter. The carotene concentration was determined from a previously constructed transmission-concentration curve. When series of five pellets, prepared from a rice-bran mixture, were analyzed for carotene after 30-day tora age, they were found to agree within 2%. This deviation included inequalities in the preparation of pellets together with variations involved in carrying out the analysis. Subsequent work (2) showed that the dicalcium phosphate column does not remove all the noncarot'ene pigments from a petroleum ether solution containing dissolved oil. Therefore, in the older storage samples the carotene values obtained are somewhat high. However, the accuracy is sufficient to permit comparative studies. RESULTS OF STABILITY TESTS
The nature of the carrier to which the carotene was added greatly influenced the subsequent stability of the carotene. Table I shows the results obtained with a group of cereal carriers. In all cases 50% or more of the added carotene disappeared after 30-day storage. The rate of carotene loss was even greater in the mixtures prepared from carriers which had been extracted with petroleum ether; it was particularly pronounced in the mixtures prepared with extracted rice bran. To each of a series of mixtures of rice bran (extracted) and carotene was added 10% by weight of an oil. One group of oils (Table 11), including coconut paring, palm, cottonseed, and corn oil, contained natural antioxidants (1.4) which may have been responsible for the carotene stability, since each of these was equal to or better than mineral oil. The best retention of caro-
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tene occurred in the mixtures prepared with corn oil. Although the above tests indicate that carotene is less stable in cottonseed than in corn oil, later tests with commercial samples of the two oils have given comparable stability values for dissolved carotene during equal storage periods. A second and larger group of oils (Table 11) retained little carotene after one month of storage under the test conditions. Figure 14 . shows the effect of different initial carotene contents upon the rate of breakdown of added carotene in the pelleted mixtures. Increasing the initial quantity of carotene in the pellets caused rapidly increasing losses. However, regardless of the initial carotene content, the loss was so great in all cases that no carotene remained in any of the mixtures, prepared from mineral oil and extracted rice bran with an oil ratio of 1 to 10, after 120 days of storage. Figure 2 shows the increasing protection afforded the carotene
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I
by increasing amounts of mineral oil incorporated in the mixtures until an optimum protection is afforded at an oil-to-carrier ratio of 1to 4. Even though carotene stability can be increased by the utilization of an optimum oil concentration in the mixtures, this factor alone is not enoDgh to prevent carotene loss. Figure 1B illustrates the rate of loss of carotene from mixtures containing varying initial carotene concentrations and optimum oil content 'for carotene retention. Two supplemental antioxidants were used in this investigation, hydroquinone and diphenylamine. Hydroquinone has long been recommended as an antioxidant for carotene (9). In the prepared mixtures it did have some value (Table I and 11), especially for those made with vegetable oils which exhibited an ac-
Figure 1. Stability of Added Carotene in Pelleted Mixtures of Mineral Oil and Extracted Rice Bran
.60b .40
A. 1 part mineral oil to 10 parts bran B . 1 part mineral oil to 4 parts bran C. Same as A plus 0.5% diphenylamine D . Same as B plus 0.5% diphenylamine
'
A
20
50
100
200
IS0
250
300
350
MILLIGRAMS OF OIL P E R GRAM OF BRAN
.80
B
Figure 2. Effect of Oil Content on Carotene Stability in Pelleted Mixtures of Mineral Oil and Extracted Rice Bran Original Concentration of carotene, 1 mg. per gram pelleted mixture; stored 30 days
TABLEI. RETENTIONOF ADDEDCAROTENEIN PELLETS OB VARIOUS SOLIDCARRIERS (FLOUR AND BRAN)
30 DAYS
60 C
z w
Solid Carrier Containing 1 Mg. Carotene per G. Rice bran Avenex Oat flour Whole wheat flour Powdered potato
Carotene Retained after 30-Day Storage a t 37' C., % Carrier Extd. with Pet. Untreated C&er Ether 1 mg. hydroWithout Without 1 mg. hydrohydroquinone, per hydroquinone. per g. carrier quinone g. carrier quinone 52 1 37 50 27 1 26 35 13 14
29
11 11
11 13
..
..
12 9
..
OF ADDED CAROTENE IN PELLETED TABLE 11. RETENTION MIXTURESOF RICE BRANAXD.OIL
Ceroteneb Retained after 30Day Storage a t 37" C., % 5 mg. hydroWithout quinone per g. hydroquinone rice bran
Peroxide Acid Oil Used, 100 Mg./ G. Rice Brana No. No. 20 44 White mineral 44 46 0:9 Corn 35 45 4.0 8.3 Palm 23 55 1.3 Coconut paring 2.0 18 31 0.9 0.12 Cottonseed, neutral 12 44 2.1 4.4 Tucum, crude 7 59 0 0.4 Methyl laurate 5 40 6.5 14.6 Palm kernel 5 25 0.7 Linseed 0.9 5 27 14 2 0 Soya salad 5 25 12.8 12.1 Teaseed 5 31 20.8 6.2 Avocado 5 32 5.9 9.2 Peaah seed 5 26 0.12 5.8 Sesame salad 11.1 5 38 4.7 Orange seed 5 46 0 8 0 . 2 Pure coconut 5 41 5.2 8 7 Walnut 5 Rice bran was extracted with petroleum ether, boiling a t 30-60' C. b Original carotene concentration, 1 mg. per gram mixture,
..
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TABLE 111. EFFECTS OF VARIOUS CONCENTRATIONS OF HYDROQUINONE ON CAROTENE RBTENTION IN PELLETED MIXTURESOF RICE BRAN^ AND MINERALOIL, STORED 30 DAYSAT 37" C. .Mg. Hydrohfe. Hydroquinone per YoCarotene quinone per % Carotene G . Rice B r a n after Storage, G. Rice B r a n after Storage* 0 42 15 49 5 44 25 55 10 45 50 62 0 Extracted with petroleum ether, mineral oil added (100 mg. per gram). b Original carotene concentration, 1 mg. pel gram mixture.
celerated destruction of carotene over that of mineral oil. The effectiveness of hydroquinone as an antioxidant can be increased by increasing its concentration (Table 111). I n a study of antioxidants, diphenylamine was found to be superior to hydroquinone (28) in mineral oil. When diphenylamine was incorporated into the mixtures prepared with rice bran and mineral oil a t the optimum oil level, no loss of carotene occurred in the pelleted mixtures containing initially 0.4 mg. per gram or less of carotene; there was only slight loss a t higher initial carotene concentrations even after 4 months of storage (Figure 1D). I n mixtures containing less than the optimum oil content the antioxidant is less effective, especially in the mixtures containing the higher initial carotene content (Figure 1C). T o determine whether diphenylamine is as effective in mixtures prepared with edible oils and extracted rice bran as in mineral oil-bran mixtures, a series of mixtures was prepared with mineral oil and a similar series with corn oil. Diphenylamine was added and then carotene a t approximately 0.25, 0.40, or 1.10 mg. per gram of mixture, and the samples were stored for 30 days. I n the corn oil mixtures the carotene stability was less than in those prepared with mineral oil. This difference was more marked a t the higher carotene concentration (Figure 3). Carotene retention in mixtures prepared with pure coconut oil was similar to that in the corn oil preparations in 30-day storage tests. DISCUSSION
The data indicate that many factors influence the stability of carotene added to flour or bran. When carotene is distributed over the surface of such finely divided dry carriers, a large surface is exposed directly to the air and rapid loss of carotene may result by oxidation, When an inerteoil is added to the mixture, carotene stability is markedly increased. The oil may exert its favorable effect by making the pellet more dense and thus retarding free circulation of air. The oil may also serve as a mutual solvent for the carotene and any antioxidant naturally present in the carrier (6, 7'). The antioxidants would exert their effect in proportion to their solubility in the oil as well as to any specific antioxidant activity they might have. There is also the possibility that the carrier contains certain pro-oxidants or enzymes that would act to accelerate carotene breakdown (18, 19). No study has been made of the possible presence of enzymes in the various carriers used in this investigation. Carotene stability in mixtures prepared with vegetable oils varies considerably with the type of oil as well as the previous treatment of the oil, I n addition a given sample of oil may change on standing so that results obtained at different times with the same oil may not be consistent. The marked variation in carotene stability in the mixtures prepared with different oils could not be correlated with the peroxide value nor the acid number of the oil measured at the time of preparation (Table 11). Baumann and Steenbock (1) also observed that carotene stability in oils could not be correlated with acidity as the dominant factor. Mineral oil, which was assumed to be relatively inert, mas included in this study as a reference oil. It is known that mineral oil interferes with the utilization of carotene by the animal (4), and the studies with mineral oil were intended only to aid in determining the effect of different variables (such as carotene,
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oil, and antioxidant concentrations) in the mixtures. It was hoped that the knowledge so obtained might be applied to the stabilization of carotene in mixtures prepared with vegetable oils or with oils which naturally contain carotene as well as antioxidants. The natural fats or oils, either present in the flour or bran or added to the mixture, may undergo autoxidation (8). The oxidation products formed in this process (for example, peroxides) might then act to accelerate the breakdown of the carotene ( 1 1 ) . It is possible that the chief value of an added antioxidant in these cases is to prevent the formation of peroxides from the oil, and only secondarily t o act as an antioxidant for the carotene molecule itself. D iphenlyamine was most effective in the rice bran mixturee that contained 1part of added oil to 4 parts of extracted bran. In the mixtures prepared with corn oil and rice bran, the diphenylamine was less effective than in those of mineral oil and bran; the reason i i I I I is not apparent. The 0.20 0.40 0.60 0.60 1.00 120 ORIBWiAL CONCENTRATION OF CAROTENE (MOa/G) breakdown products of the oil may hasten Figure 3. Retention of Carotene the destruction of the i n Pelleted Mixtures of Extracted added antioxidant. Rice Bran with Mineral Oil and with Corn Oil There m a y be a Stored in darkness 30 days at 3 7 O C. synergistic action be150 mg. of oil per gram of bran tween diphenylamine and antioxidants in the flour or bran, although in tests not reported here, diphenylamine proved very effective in stabilizing carotene dissolved in organic solvents such as petroleum ether or benzene. Tests on the toxicity of diphenylamine are in progress at the Pharmacological Laboratory, Stanford University School of Medicine (S), and this antioxidant should not be added to food or feed while it3 physiological and toxicological effects are still in doubt. *e Since the addition of carotene to dry carriers results in a rapid loss of added carotene, protective measures as well as the most favorable storage conditions must be utilized in stabilizing the carotene. The addition of a liberal excess is not practical, because the rate of loss increases with increase in carotene content. Cold storage or storage in nitrogen atmospheres would be effective, but facilities are expensive and are not always available. The addition of oils such as corn, cottonseed, or palm to the carrier, however, results in a marked increased in carotene stability. The optimum oil-to-carrier ratio for carotene stability results in A feed having a high oil content, too high for animal consumption except as a supplement. Antioxidants added to the pelleted supplementary feed will prove effective in preserving the carotene over an extended period. SUMMARY
When carotene was added to various flours or rice bran and the mixtures formed into I-gram pellets and stored a t 37" C. exposed to air, a loss of 50 to 87% of the carotene occurred during the first 30 days. The amount of loss varied with the type of carrier as well as with the amount of carotene added to the mixture. I n a series of mixtures prepared with carotene and rice bran (pytroleum-ether-extracted) to which various oils (vegetable or mmeral) were added, a considerable variation in carotene stability resulted, depending upon the type of oil. I n mixtures
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INDUSTRIAL AND ENGINEERING CHEMISTRY
prepared with oils which contain natural antioxidants (corn, palm) there was a better retention of the carotene than in the mixtures of mineral oil and rice bran. I n most of the mixtures prepared with vegetable oils, carotene loss was more rapid than in comparable mixtures prepared with mineral oils. In mixtures prepared with rice bran (extracted), to which mineral oil was added, the stability of carotene was increased with increase in oil content, until a level of 275 mg. of oil per gram of bran was reached. Increasing the oil beyond this point caused no further increase in carotene stability. Diphenylamine a t a concentration of 0.5% was effective in stabilizing the carotene in the pelleted mixtures of rice bran and mineral oil, especially in the mixtures containing the optimum oil content for carotene retention (275 mg. of oil per gram of bran). Thus, pelleted mixtures of rice bran and mineral oil (approximately 1 part oil to 4 parts bran) containing 5 mg. of diphenylamine per gram of mixture, retained 80% of added carotene after 4-month storage a t 37“ C. Similar samples to which no diphenylamine was added retained only 2% added carotene after similar storage. Diphenylamine protected carotene in pelleted mixtures prepared with corn oil almost as well as in corresponding mixtures prepared with mineral oil a t low carotene levels (0.2 mg. per gram), but when the original carotene concentration was about 1 mg. per gram, the carotene oxidized more readily in samples prepared with corn oil than in corresponding samples prepared with mineral oil. LITERATURE CITED
(1) Baumann, C . A., and Steenbock, H., J.B i d . Chem., 101,561-72 (1933). (2)‘Bickoff,Emanuel, and Williams, K. T., IND.ENG.CHEM., ANAL. ED., 15,266-8 (1943).
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DeEds, Floyd, private communication. Dutcher, R. A., Harris, P. L., Hartder, E. R., and Guerrant, N, B., J . Nutrition, 8, 269-83 (1934). Fraps, 0.S., and Kemmerer, A. R., Texas Agr. Expt. Sta., Bull. 557, 1-28 (1937).
Green, T. G., and Hilditoh, T. P., J . SOC.Chem. Ind., 56, 23-6T (1937).
Gyorgy, Paul, Tomarelli, Rudolph, Ostergard, R. P., and Brown, J. B., J. ExptZ. Med., 76,413-19 (1942). Holm, G. E., Greenbank, G. R., and Deysher, E. F., IND. ENQ. CHEM., 19,156-8 (1927).
Hume, E. M., and Smedley-Maclean, I., Lancet, 218, 290-2 (1930).
McDonald, F.G., J,Biol. Chem., 103,455-60 (1933). Mattill, H. A., J . Am. Med. Assac., 89,1505-8 (1927). Moore, L.A,, IND.ENG.CHEIM., ANAL.ED.,12, 726-9 (1940). Nakamura, Mitsuo, J. SOC.Chem. Ind. Japan, 40, Suppl. Binding 203-5 (1937). Oloott, H. S.,and Mattill, H. A., J.Am. Chem. SOC.,58, 1627-30 (1936).
Peters, F. N., and Musher, S., IND.ENQ.CRBM.,29, 146-51 (1937).
Quackenbush, F, W.,Cox, R. P., and Steenbock, H., J . Bwl. Chem., 145, 169-77 (1942).
Scheunert, A., and Schieblich, M., Bioahm. Z.,263, 454-7 (1933). (18) Sumner; J. B.,and Sumner, R. J., J. B i d . Chem., 134, 531-3 (1940). (19) Sumner, R.J.,J . Biol. C h m . , 146,215-18 (1942). (20) Tauber, H., J . Am. Chem. SOC.,62, 2251 (1940). (21) Turner, R.G., J . Bid. Chem., 105,443-54(1934). (22) Williams, K. T., Bickoff, Emanuel, and Van Sandt, Walter, Science, 97,96-8 (1943).
ANALYSIS of FILTRATION DATA is to deal with the first step only-filtration A filtration problemis usually accompanied D. R. SPERRY analyses of slurries. A brief description by a sample of the slurry to be filtered. In 189 Main Street, of test procedure, definition of filtration attacking - such a problem the first step terms, and a simple derivation of the basic should be afiltration analysis of the slurry. filtration formula are given. A graphical interpretation The next step is to use the analysis to determine the filtraand a worked example based on actual data from tungstic tion conditions necessary to meet the requirements of the acid slurry are included. A simple method of finding C, problem, such as pressure, thickness of cake, length of the latus rectum of the parabolic time-discharge curve is cycle, amount and kind of filter aid, etc. A third step offered,and analyses of twenty widely differing slurries are consists of selecting the type and design of filter to fulfill given, as well as a biblography on theories of filtration. the needs of the second step. The purpose of this article
HIS article does not pretend to deal with the details of test making, but a general outline of the method used in testing is necessary to a complete understanding of the making of slurry analyses. First, the conditions under which the test is to be made must be decided upon. A series of pretest cut-and-try runs are made in a small, quickly opened-and-closed filter similar to that pictured in Figure 1and shown in cross section in Figure 2. By repeated short tests, the proper constant pressure, tempersr ture, amount and kind of filter aid, etc., can be found. Next the main test is made under the selected conditions in a filter with vertical filter bases similar to that shown in Figure 3. An agitated pressure vessel or monte-jus is used to feed the filter. The air pressure in the monte-jus is kept constant by a pressure regulator. Time and corresponding discharge readings must be carefully and accurately made. By fixing upon the conditions of the test in advance, application of the general filtration formula is simplified since most of the variables thus become constants which can be grouped together.
T
slurry in contact with one side of a “porous layer” a t a sufficiently higher pressure than that on the other side to cause the liquid to flow through the capillary channels thereof, none of which are large enough to permit passage of the solids.
To understand better the phenomenon of filtration as well aa the meaning of terms used, the following definition is offered: Liquid filtration consists of the separation of the solids and liquid from a mixture of solids and liquid, called “slurry”, by placing the
The liquid assing through the porous mass is called “filtrate”, the solids left%y the filtrate are called “cake”. The porous layer consists of three parts: (1) the porous mass used to initiate filtration, called “filter base”, usually cloth or aper; (2) the solids on the filter base a t the start of the run; and p3) the solids (cake) deposited by the filter during the run. Figure 4 shows the position of these parts. Parts 1 plus 2 constitute a constant resistance; part 3 is a variable resistance. Slurry may be considered a mixture, material to be filtered, influent, or refilt. Filtrate is sometimes called “effluent” or “liquid withgawn”. Cake may be called “solids removed” or “mud”. Filter base means any porous layer used solely to initiate filtration. If fine enough, filtration starts a t once. Usually, however, some solids pass through and filtration does not start until enou h solids are caught to prevent others from passing through. A h t e r as used in this article, means a device in which the process o! filtration takes place. All filters have three rincipal parts: a filter base, a support for the filter base which aEows the filtrate to drain way, and a chamber t o hold the slurry in contact with the unsupported side of the filter base and to permit pressure to be built u p to cause filtration. The word ‘filtration” means constant-pressure liquid filtration, unless otherwise specified. It is assumed that the feeding of
DEFINITION OF TERMS
