Nutritive Value of COTTON, PEANUT, and SOY SEEDS In contrast to cereal grain milling, the process applied to oil-bearing seeds conserves the vitamin B complex values in the same edible milling fraction with the protein. The hull is the only waste product as far as food uses are concerned. Judging by comparison of assays on seed and seed flour, the removal of the oil leaves the cake with an increased concentration of watersoluble vitamins. From a food standpoint the oil has calorie and culinary value with little if any known specific nutritional properties. Flours of cottonseed, peanut, and soybean are concentrated sources of high quality protein especially suitable for supplementing white flour. Results so far obtained indicate moderate differences in amounts of the three seed flours required for producing normal growth. Soybean flour is superior in protein quality, cottonseed flour in riboflavin content; assays on peanut flour indicate an outstanding nicotinic acid value. As substitutes for both the protein and the vitamins of meat, a mixture of the three seed flours offers possibilities. MONG the available materials high in protein and
vitamin B complex which have not yet been utilized to more than a fraction of their possibilities are the seeds of cotton, peanut, and soybean. Peanuts and certain varieties of edible (i. e., directly edible) soybeans make up a very small part of the American diet. All three seeds are the basis of large industrial operations for vegetable oils. The press cake or expeller residue from such operations yields enormous amounts of practically dehydrated, low-fat concentrates of protein. I t s use in the past has been principally as animal feed and to some extent as fertilizer. Improvements in process have in recent years yielded products quite palatable and adequately sanitary for consumption as wholesome food for man. I n the form of flour and grits they are now available in commercial quantities for use in the human dietary. A general description of properties and process has been given for cotton by McMath (18) and Council on Foods ( 3 ), for peanut by Harrell(8) and Payne (18 ) ; for soybeans by the Soybean Nutritional Research Council (9). The latter discusses the feeding of various farm animals but as yet has no section on application to the nutrition of man. As early as 1910 Fraps (6) used cottonseed flour as an enriching component in bread making, and Johns and coworkers have published studies of the nutritional quality of bread made with supplements of peanut flour (11)and soy-
THEODORE F. ZUCKEK AND LOIS ZUCBER Columbia University, New York, N. Y.
bean flour (11). Soybean preparations (curd) aswell as the beans themselves have been used as human food in the Far East for centuries. Adaptations to western food habits are discussed in detail by Schroeder (29). A synopsis ( 7 ) of the German Army Soya Cook Book 1935 indicates further developments. The soybean curd used in China and other eastern countries is apparently the only instance of an oilseed product, including the protein fraction, being used as such for food. All other uses employ the product as a supplement in some other food article. Two plans may be followed: Fraps, Johns et al., and others utilize large amounts in baked goods (25 per cent is apparently the highest amount that can be used without disturbing the baking quality of ordinary flour); the German Army Cook Book and Schroeder recommend the incorporation of small amounts of soybean flour in almost every article of the menu. The present report is aimed a t showing the effect of graded amounts of the seed flours as supplements to white flour. Suitable salt mixtures and adequate supplies of the fatsoluble vitamins are incorporated in the diets so that the variables are reduced to protein and the vitamins of the B complex. The ability of the diet to support the maximal growth rate in rats is used as criterion, since deficiency in any of the nutritional factors under question results in a reduced growth rate. The method used is more nearly related to the determination of protein requirement than to biological value technique. Maynard (17) discusses and illustrates the various methods of protein investigation, and McCollum et al. (16) discusses the difficulties in interpreting biological values. The criterion employed has the advantage over others in current use that it allows data to be collected a t the level of nutrition which is approximately optimal for the test animal, and this is the nutritional level at which we wish to operate in human feeding. An important requirement is a well defined growth standard which will not be exceeded by dietary means and to which the degree of adherence on any diet to be tested can be quantitatively determined. GROWTH STANDARDS
As shown elsewhere (IS), all the carefully recorded growth data on healthy albino rats, both large and small strains, indicate that when they are put on well chosen liberal diets, there is an upper limit t o the growth process. This optimal growth can be put into straight line form if the log of the weight is plotted against the reciprocal of age in any appropriate units. Means for strains of different characteristic body sizes come out in such a plot as parallel straight lines with slope determined by sex, intercept by inherent size. Dietary deficiencies result in suboptimal growth curves which, by their shape, are frequently characterisic of the type of deficiency. For instance, with a moderate protein deficiency the curvilinear log reciprocal plot tends to ap868
INbUSTRIAL AND ENGINEERING CHEMISTRY
Figure 1. Mean Female Growth on Various Levels of Cottonseed Flour Compared with Optimal Growth of Stock Diet Controls and the Previously Published Colony Average for 1936-37 on Some 600 Animals In the log reciprocal plot the referenee points for the log seals have been arbitrarily shifted to separate the various curves. The 5 and 10 per eent cottonseed flour diets are clearly deficient; the 16 per cent curve shows a slight tendency toward. a growth deficit which may or may not be real.
proach the straight line optimal growth as time goes on (spontaneous realimentation). This can be attributed to decreasing protein requirement as the animal grows older.
from spring wheat, Techanny Milling Corporation; crude blood fibrin, Armour Glue works, ground in a hammer mill and repeatedly extracted with hot 95 per cent ethanol.
EXPERIMENTS WlTH RATS
Young rats from the stock colony weaned a t 4 weeks were selected for the experiments on the basis of meeting set standards of weight and growth rate in the 3-4 week age period. Such selection is indispensable for obtaining consistent results (compare Eppright and Smith, 6). Experimental groups initially comprised twelve animals, half of each sex, but some groups as finally reported are a little smaller owin6 to the elimination of rats showing respiratory involvement with consequent erratic growth. The diets were fed ad libitum from 4 to 14 weeks of age when the experiment was terminated. Weighings were made to the nearest gram a t biweekly and later at weekly intervals. Common to all the seventeen diets was the addition of 5.5 per cent of a salt mixture (Rf) modified to give, respectively, 0.6 and 0.4 gram of added calcium and phosphorus per 100 grams of diet. A uniform addition of the fat-soluble vitamins gave each diet at least 50 U. S. P. units of D in 0.2 gram of cod liver oil and 750 units of A activity in 0.1 gram of carotene solution. A stock solution of these two factors in cottonseed oil was made up so that 2 grams of the solution could be weighed out per 100 grams of diet. Each of the seed flours was studied a t levels of 5, 10, and 16 per cent in the diet; with cottonseed flour a 24 per cent level was also introduced. In each case the diet was made up to 100 per cent with white flour. To test for adequacy of B complex factors, additional groups were run with the addition of 4 per cent of rice bran extract. To test for adequate rotein, check experiments were conducted with the addition o f blood fibrin to make the total estimated rotein content approximately constant; this required 4, 7, a n t 1 0 per cent of added fibrin, res ectively, in the diets containing 16, 10,and 5 per cent cottonseefflour. The products used were from the following sources: cottonseed flour, McMath process Proflo, Traders' Oil Mill Company; peanut flour, McMath-Howard process PDP, from the same source' soybean flour, No. 1, A. E. Staley Manufacturing Company; 'rice bran extract, Vitab type 11, National Oil Products Company; white flour, Power Plus, a standard patent flour made
Cottonseed flour Soybean flour Peanut flour Rice bran extract
10.4 5.4 6.0
10.2 4.1 3.0
Micrograms per gram 85 25.5 29 15.0 High (a&. 200) Micrograms per ca. 275 2000
The peanut values refer to the product used in this work. The difference between these figures and those of 12 and 5 , respectively, for thiamine and riboflavin cited by Payne (18) is accounted for by the absence or presence of the skins which are known t o be high in these factors. T h e product with skins has apparently been discontinued on account of flavor difficulties. The description as otherwise given b y Payne refers t o the product without skins. Various analytical figures obtained in this laboratory on some of the diet constituents may be of interest;
White flour Cottonseed flour Peanut flour Soybeanflour Fibrin Ricebranext.
0.130 1.26 0.56 0.58 0.096 0.54
0.014 0.20 0.070 0.24 0.18 0.03
0.039 0.65 0.36 0.25 0.015 0.16
45 32 3 18
2.47 9.0 9.7 6.8 13.7 1.60
9.6 6.3 7.6 8.6 7.1 37
6.1 3.6 4.8 3.4 4.9
The soybean flour was higher in fat than the other two; a higher nitrogen content would be found in a soybean flour
INDUSTRIAL AND ENGINEERING CHEMISTRY
defatted to a corresponding extent. Nitrogen, calcium, and phosphorus determinations were used principally to check on the constancy of the several batches of each diet mixed. The nitrogen figures cannot properly be calculated to protein because of lack of information on the amount of nonavailable nitrogen in the seed flours and the rice bran extract. For instance, varying percentages of betaine have been isolated from cottonseed meal. The seed flours as a class may differ markedly from white flour and from fibrin in the proportion of nonavailable nitrogen, and they may differ markedly among themselves. This makes no particular difference in the present study, which is a comparison of the three products as they are available for use and not of the relative values of the protein mixtures found in the seeds. I n view of the known uncertainty of physiological availability of the soybean carbohydrates ( I ) , and a similar possible difficulty with regard to the raffinose of cottonseed, it appears to be a useless refinement to attempt t o equalize dietary composition on calorie content. Irrespective of the merits of such equalizing, there is a practical interest in the data presented here, based on percentage of a given supplement in the diet. GROWTH RESULTS
A few experiments will be described in detail as illustrative of procedure and results. Figure 1 shows the growth curves of fernale rats on a graded series of diets containing cottonseed flour a t levels of 5, 10, and 16 per cent, coinpared with the colony growth standard established some years ago and with a contemporary small group of controls run on the same stock diet. I n the plots made on ordinary cross section paper (lefthand side of the figure), there is apparently as much difference between the two groups of animals on the stock diet as between any other two neighboring groups. I n the log reciprocal plot of the same data (right-hand side), it is clear that
Vol. 35, No. 8
of cottonseed flour, we expect the addition of more protein, or the addition of a B complex concentrate, or both, to improve the growth. Figure 2 covers these points. Supplements to the 16 per cent level lead to insignificant improvement. With the 10 per cent level, rice bran extract leads to no change while protein in the form of fibrin does bring the curve into the optimal range. Thus the 10 per cent level supplies optimal B complex but not optimal protein. Similar experiments a t the 5 per cent level show up deficiencies in both protein and B complex. The same experiments carried out with male rats confirmed the adequacy of the 10 per cent level in B complex. However, as Figure 3 shows, even the 16 per cent level diet optimal for females is far from optimal for males. The deficiency is not of B vitamins since the addition of the B complex concentrate does not improve the growth. It will also be seen that the addition of 4 per cent protein, either in the form of added fibrin or of added cottonseed flour, does lead to optimal growth. It has not been previously emphasized that the protein requirement of male rats is greater than that of females. The difference is apparently due to the greater potential growth rate of the male. RELATIVE VALUEOF SEEDFLOURS. I n Table I each diet is evaluated by a single figure for each sex which represents the weights attained in per cent of the optiinal weights, as an average of the percentage a t each weighing day. The values for comparable diets range fairly consistently so as to make soybean a little better than cotton, and cotton a little better than peanut. A more highly defatted soybean preparation could be expected to increase the difference somewhat. The superiority of the soybean is attributable to protein quality. As a general B complex source, the cottonseed flour is superior, since it provides optimal B complex at
Mean Female Growth on Protein and B Complex Supplements at Two Cottonseed Flour Levels
The protein supplement definitely improves the growth a t the 10 per cent cottonseed flour level; at both levels the B complex supplement makes the growth slightly worse if anything. The 4 per cent Vitab substitutee for 4 per cent flour and thereby lowers the protein by about 0.6 per cent; the Vitab itself conhibutes some nitrogen which is probably largely nonprotein. If this is a valid interpretation, it contributes more evidence that the 16 per cent level is just border line as far as protein is concerned.
the two stock diet groups differ only in inherent size, both lying on straight lines of the same slope. Also, the 16 per cent level diet seems to be practically optimal and will not be materially improved by either more protein, more B complex, or a higher level of cottonseed flour. With the lower levels
the 10 per cent level whereas the soybean flour (on the basis of experiments just now terminating) is definitely below B complex requirements when fed a t the 10 per cent level (approximately 5 per cent soybean protein). Hayward et al. (10) state that soybean flour providing 18 per cent of protein
INDUSTRIAL AND ENGINEERING CHEMISTRY
in the diet furnishes adequate B complex. Even a t the 16 per cent level the peanut flour may be suboptimal in B complex, judging from the general appearance of the growth curves and also from the assay values given above. It
said to be essential for growth but are not required maintenance. It is not unlikely that the more rapid characteristic species growth, the more carefully must adequacy of amino acids be guarded. By such token
for the the we
6 2 4 % C.S. F l o u r
4% F i b r i n
4% Vita b
30 Rge i n days
Mean Male Growth on Some of the Cottonseed Flour Diets
The normally much Y t e r growth rate of the males (cf. Figure I), whieh is not accompanied by any greater food intake in comparison with that of a female of t e same body weight, results in the male having a higher requirement of protein on a percentage basis in the diet. The 16 per cent level is definitely inadequate.
should be noted, however, that peanut flour is an unusually rich source of nicotinic acid, an essential factor in human nutrition. Unfortunately the rat does not require an external source of nicotinic acid, and nicotinic acid thus comes outside of the scope of this investigation. Recent experiments of Jones et al. (IS)place the three seed flours in the same order with regard t o quality of protein. I n his experiments the protein quality is the only variable, adequate B complex being supplied from other sources.
may be bending over backward in using the rat as a test object in discussing human requirements, but we are playing safe.
TABLEI. DIETEVALUATION Per Cent 24 16 10 5
Usually it is stated that the protein requirement of man (including infancy) is satisfied when 10 per cent of the calories are derived from a good quality protein. More rapidly growing animals, such as the rat and the chick, require about double this amount when young. Differences in total amount of protein required may appear to obscure the problem of protein quality when we compare one species with another. The greatest demand for amino acids is for tissue building. Older rats (14) do just as well on 8 per cent protein in the diet as on larger amounts. Later in life the requirements are also less exacting with regard to the essential amino acids. This is exemplified by the fact that certain amino acids are
-----FemalesSoyCottonbean meed 100.0 96.8 97.7 88.7 82.6 79.8 73.a
Cottonseed level, % ' Sex
Basal Basal f Vitab Basal fibrin Basal fibrin
-16F 97.7 96.6 100.7 99.4
90.0 82.4 72.2
86.0 78.1 64.9
Cottonseed Peanut 97.0 86.3 79.9 72.7 69.1 63.8 62.1
-10M 86.3 85.3 99.5 99.4
82.5 72.7 82.5 74.7 100.0 97.3
No growth (initial wt. just maintained)
F 101.1 46.2
7--5--F M 73.5 63 8 80.4 69 0 100.3 84.7
M 99.1 37.3
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 35, No. 8
There are several reasons for questioning the completeness and correctness of the above discussion of the B complex factors in the seed flours in relation to human requirements. The human requirement figures are not only tentative, but also do not include all the known B complex vitamins. They cannot cover any unidentified factors whose importance is indicated by recent rat work. Some question can be raised as to the assay figures based on the new and relatively untried microbiological assay methods. A recent paper (20) on the riboflavin assay, one of the best worked out of these methods, studies a change in technique Khich lowers values for soybean meal to about half of those obtained by the previous standard technique. It is possible that the three seed flours may be ranked in approximately the correct order even if the assay methods are faulty, because of their similar constitutions, but comparison with articles such as meat or milk may be questionable. Therefore it is of real importance that feeding tests on the rat show that the seed flours do make important contributions to the B complex requirement (unknown factors as well as known), and that the three seed flours rank as B sources in the same order as indicated by the assays. I n seeking substitutes for the animal protein foods, it cannot be too strongly emphasized that animal protein sources are relied on in our American dietary, not only for good quality protein but also for B complex vitamins. Nicotinic acid and riboflavin are the two B factors for whose supply animal protein sources are known to be especially important. The three vegetable seed protein sources studied provide good protein and important supplies of B complex factors; in general quality, all three are comOF FOOD VALUESFROM COTTONSEED parable, but the soybean is outstanding in protein quality, TABLE11. COMPARISON the cottonseed as a source of riboflavin, and the peanut as a FLOUR AND OTHERSUBSTANCES source of nicotinic acid. NionP a n t n_ ”
With regard to the amino acid distribution in the diets of young rats, containing mixtures of wheat and seed protein, a t levels of total protein comparable with those required with high quality single proteins (egg protein or casein) all the amino acid requirements are fully met, provided the seed protein represents about half the total. Probably the conclusion stated in this way is directly applicable to man with a considerable margin of safety. For the various known B complex factors, tentative requirements for man have been set up (4); it seems that these , together with the assay data, would allow a complete discussion of the seed flours as B complex sources without further experimental work. It mould appear, for instance, that if approximately half of the daily protein requirement of an adult was provided from cottonseed flour, a third to a half of the requirements for thiamine, riboflavin, and nicotinic acid would be met, using Elvehjem’s tentative standards. The soybean flour would rank a little lower in all respects, whereas the peanut flour would rank lower in thiamine and riboflavin, but would more than supply the nicotinic acid requirement. Cottonseed flour a t this level supplies approximately 10 per cent of the daily requirement of 2500 calories; comparison can also be made (Table 11) with the contributions of other food articles when fed at the same 10 per cent level, using the data assembled by Williams (22). The cottonseed flour ranks remarkably >\-ell in comparison with other important dietary articles.
10% qf 2500 Calories as. Milk Whole wheat Lean beef Liver Cottonseedflour
ProWeight, tein Grams Grads 360 60
125 195 65
6.7 25 40
Thismin, Mg. 0.15 0.33
Riboflavin, Mg. 0.7 0.11 0.45 5.0 0.7
Mg. 0.2 3.8
thenic Acid, Mg.
1.1 12.0 1.7
The procedure by which cottonseed flour becomes available is of interest on several scores. Originally cotton was grown simply for the fiber. When it was found that a valuable oil could be obtained from the seed, the press cake a t first was a waste product which soon, however, vias utilized in stock feeding as a source of protein. By careful control of the process ( 3 , 8, 26), a wholesome food product high in protein and B complex vitamins was prepared. I n a sense, grain milling and oil seed milling are complementary processes. Both grains and oil seed store a large nonprotein energy content, in the one case as carbohydrate and in the other as f a t ; and in the separations involved in milling, the watersoluble vitamin potency goes with the protein fraction. From wheat the principal edible portion is used for its calorie value and contains a relatively low amount of protein of not particularly good quality; little of the B complex contained in the grain accompanies this fraction. I n cottonseed milling, a large yield of oil is first expressed and marketed as such. The remainder contains large amounts of relatively good quality protein together with nearly all the water-soluble vitamin fraction. Through the removal of the oil, both protein and vitamins are concentrated in comparison to their content in the seed. The vitsmin asssy figures for whole cottonseed as determined by Cheldelin and eo-workers (a) are, in micrograms per gram, 3.2 of thiamine, 2.3 of riboflavin, 11 of pantothenic acid, and 16 of nicotinic acid. The cottonseed flour represents 15 per cent by weight of the cottons:ed.
(1) Adolph, W. H., and Kao, H.-C., J . Nutrition, 7, 395 (1934). (2) Cheldelin, V. H., Lane, R. L., and Williams, R. J., ,4m. Chem. Soc., Buffalo, 1942. (3) Council on Foods, J . Am. M e d . Assoc., 105, 800 (1935). (4) Elvehjem, C. A., IND.ENG.CHEM.,33, 707 (1942). ( 5 ) Eppright, E . S., and Smith, A. H., J . Nutrition, 14, 21 (1937). (6) Fraps, G. S., Texas Agr. Expt. Sta., Bull. 128 (1910); 163 (1913). (7) German Army Soya Cook Book 1938, Soybean Digest, Dec., 1941. ( 8 ) Harrell, T. J., Proc. 1st Ann. Meeting Natl. Peanut Council, Pensacoh, 1941, 61. (9) Hayward, J. W., et al., “Composition and Nutritive Properties of Soybeans and Soybean Oil Meal”, Chicago, Soybean Nutritional Research Council, 1940. (10) Hayward, J. W., Steenbock, H., and Bohstedt, G., J . Nutrition, 11, 219 (1936) (11) Johns, C. O., and Finks, A. J., Am. J . Physiol., 55, 455 (1921). (12) Johns, C. O., and Finks, A. J., J. Bio2. Chem., 42, 569 (1920). (13) Jones, D. B., and Divine, J. P., Am. Soybean Assoc., Lafayette, Ind., 1942. (14) McCay, C. M.,Maynard, L. A., Sperling, G., and Osgood, H. S., J . Nutrition, 21, 45 (1941). (15) McCollum, E. V., Orent-Keiles, E., and Day, H. G., “Newer Knowledge of Kutrition”, 5 t h ed., Chap. 6, New York, Macmillan Co., 1939. (16) MoMath, C. M., Proc. Cotton Research Congr., Waco, Texas, 1, 243 (1940). (17) Maynard, L. A., “Animal Nutrition”, 1st ed., pp. 309-13, 33445, New York, McGraw-Hill Book Co., 1937. (18) Payne, D. S., Chem. Eng. News., 20, 1173 (1942). (19) Schroeder, F., E m u h r u n g , 3, 245, 281 (1938). (201 Stronn. F. M.. and Camenter. L. E.. ISD. ENG.CHBM..ANAL. ED.: 14,909 (1942). (21) Wesson, L. G., Science, 75, 339 (1932). (22) Williams, R. J., J . Am. M e d . Assoc., 119, 1 (1942). (23) Zucker, T. F., et al., J . Nutrition, 22, 123 (1941); Growth, 5, 399, 415 (1942); J . Gen. Physiol., 25, 445 (1942). ~I
PRESENTED before the Division of Biological Chemistry at the 104th Meeting of the ADIERICAX CHEMICAL SOCIETY, Buffalo, N. Y .