ISDUSTRIAL All-D EXGIiVEERIiVG CHEMISTRY
726
the results obtained with some of the materials which are presented in Table 11, it will be noted that wherever a small amount of diluent is added to the original product the mobilometer usually showed an appreciable difference in the time of flow, thus indicating the sensitivity of this apparatus t o small differences in the consistency of the product experimented with. If pressure-flow curves are desired for a product, a number of different readings are made with different loads, taking care that the product remains a t a fairly constant temperature. Should it be desired to reproduce a certain consistency, a standard pressure or load
Vol. 19, No. 6
which is best suited for the product should be used and the time in seconds of the rate of flow of the product be noted. All these above products were run at 20” C. (68’ F.) * 1O C. To each load should be added the weight of the brass plunger, a perforated disk attached and a weight pan, which was 74 grams. The load, or pressure, denotes the amount of weight added to the brass plunger. These tables should give interesting information upon what weights should be used for various products-that is, what weight will show the greatest difference in time of flow and be applicable to a wide range of consistencies.
Heat and Moisture as Factors in the Destruction of Gossypol in Cottonseed Products‘ By Willis D. Gallup DEPARTMENT OF AGRICULTURAL CHEMICAL RESBARCH, OKLAHOMA EXPERIMENT STATION, STILLWATER, OKLA.
Cotton seeds contain a toxic substance called gossySince this might come about pol, the destruction of which appears justifiable espemanufacturers of cotin a variety of ways it was cially if the seeds are to beusedasa cattlefood. Astudy tonseed oil have known thought well to mention some was therefore made of the effects of heat and moisture that during the expression of of the properties of this subin bringing about the destruction of this substance the oil from the cotton seeds stance. within the seeds. there is removed, along with Properties of Gossypol Cotton seeds were heated dry in an electric oven, the oil and other impurities, heated in the presence of excess moisture (autoclaved), Gossypol occurs as a colorcertain substances which imand germinated. The time of the first two treatments ing matter in the internal part to the oil a dark redwas varied between wide limits and the decrease in glands of the cottonseed kerdish color. One of these subgossypol determined by chemical and biological methnels, and probably throughstances has recently aroused ods. out the cotton plant. It is considerable interest in view Heating the seeds in a dry condition effected achange quite insoluble in petrolic of its toxic properties, which in the form of the gossypol but only slowly reduced ether, but soluble in diethyl have been demonstrated by their toxicity. Heating the moist seeds in the autoe t h e r , chloroform. carbon Withers and Carruth2 in an clave rapidly destroyed the gossypol and produced a t e t r a c h l o r i d e , ethylene dieffort to explain the toxicity non-toxic product. Germination was not effective in chloride, acetone, and carbon of cottonseed meal. Marchreducing the toxicity of the seeds. disulfide. The amount found lewski3 had previously isoin “hull-free” kernel< by esl a t e d the same compound from the “foots” of Eottonseed oil and gave it the name traction with diethyl ether \-aries from 0.4 to 1.2 per cent. “gossypol.” Following the work of Carruth there was much According to Carruth6 its molecular weight is about 532, i t discussion in the literature as to the real cause of the dele- is very unstable in alkaline solution, and decomposes a t about terious effects produced in animals by feeding large quan- 176” C. to a dark mass. It combines with aniline and some tities of Cottonseed meal, and Schwartse and Alsberg4 quite other compounds with the - S H 2 grouping to form a less definitely established a relationship between the toxicity of soluble compound, and Carruth used this property as a means of its quantitative determination. cotton seeds and their gossypol content. Gossypol is readily oxidized in alkaline solution by hydrogen Since the meal consists of cottonseed kernels which have gone through a heating and pressing process to remove the peroxide and other oxidizing agents and its solution in organic larger portion of the oil, these investigators offer indirect solvents turns greenish black on the addition of iron salts. evidence as to the cause of the toxicity of the cottonseed I t may be isolated easily from cotton seeds by first removing meal. However, there is still considerable question as t o the oil with petrolic ether followed by extraction with dithe toxicity of the cottonseed meal as determined by its ethyl ether, which removes the gossypol and considerable gossypol content, since we know that the process of manu- coloring matter. After evaporating the ether completely facture has a very definite effect upon the gossypol which a t a low temperature, the gossypol is liberated by adding was contained in the original seeds, and Sherwood5 has petrolic ether. The purification of this product is best shown that not only is the amount lowered but it is possibly effected by dissolving it in a mixture of ether and 80 per cent changed in form. The present problem involves the elimi- acetic acid and allowing it to stand for several days. The nation of gossypol so that it no longer remains in the press gossypol crystallizes out in combination with acetic acid, cake to impair its feeding value after the oil has been removed. from which it is liberated by dissolving in ether and washing several times with water. The water removes the acetic 1 Received February 4 , 1927. Published with the permission of the acid and the free gossypol is recovered on evaporation of Director of the Oklahoma Experiment Station, C. T. Dowell, who initiated the study of factors in0uencing the gossypol content of cottonseed meal in the ether. Schwartze and Alsberg7 found gossypol purified this laboratory. in a similar manner, when fed to experimental animals, 2 J . Agu. Reseauch, 5, 261 (1915). t o produce “nearly all the manifestations reported as chara J . g r a k f . Chem., 60, 84 (1899). J Am. Chem. S o c , 40, 647 (1918). 4 J . Agr. Research, 2 8 , l i 3 (1924).
OR s o m e t i m e t h e
F
6
5
I b i d . , 32, 793 (1926).
7
J . .-1gv. Resenrch, 28, 191 (1924).
June, 1927
acteristic of the cottonseed intoxication of the farm animal." They further studied its pharmacological action. Purpose of Investigation
During some inrestigations on the feeding value of cottonseed meal and methods of treating it such that it might be used more extensively and with less precaution as a stock feed, the writer and his collaborators became convinced that autoclaving the meal was of considerable benefit.*j9 The temperature attained and time of heating are both important factors in this process. Tlie present invest'igation ws carried out to show some of the effects of heat and inoist'ure upon gossypol and to throw some light on the problems connected with t,he preparation of the cottonseed meal, both factors being considered necessary to produce the best meal from the feeding standpoint. Although the toxicity of gossypol has been definitely established, little is known concerning the toxicity of its decomposition products, and since these appear to be the form in which gossypol is found in t'he meal, it is important8that we know more about their physiological effects. A study of this type has been begun in this laboratory, and the results are forthcoming. The severe treatment to which cotton seeds are subjected in the oil mill is the probable cause of the change gossypol undergoes and the reduced toxicity of the meal. The results of the experiments described herein show the importance of some of the conditions which prevail during this process. -1 generally accepted hypothesis is that during the cooking of the seeds previous to pressing out the oil some of the gossypol is entirely destroyed and some is changed to another form of gossypol having about the same molecular weight and properties except that it is no longer soluble in the ordinary fat solvents and therefore cannot be removed by the usual methods. This form of gossypol, which is quite insoluble in ether but soluble in hot aniline, will be called d-gossypol throughout this paper. Sherwood5 determined by chemical methods the gossypol and d-gossypol content of a large number of cottonseed meals which had been prepared by different processes, and found the gossypol content to vary from 0.007 to 0.150 per cent and the d-gossypol content to vary from 0.335 to 1.076 per cent. He further states that "seventy-five per cent or more of the gossypol present in the cottonseed kernels is converted, in the manufacture of cottonseed meal, into the less toxic d-gossypol." The main factors in the preparation of cottonseed meal which seem to bear directly upon its gossypol content are the t'emperature a t which the seeds are heated, the time of such heating, the amount of moisture present, and the amount of oil expressed. Occasionally a meal has been found entirely free of gossypol as determined by Carruth's method modified by Schwartze and AlsberglO for cotton seeds, but in all cases there has been present a substance with properties similar t o those of gossypol which is believed to be one of its decomposition products, probably the tkgossypol. Previous work in this laboratory had indicated that heat alone was not sufficient to destroy the gossypol, although no determinations for this substance had been made. Osborne and hIendelll found cotton seeds to be toxic to rats even aft'er heating the whole kernels a t 110" C. for 22 hours, although these investigators did not make chemical determinations of the gossypol content of the products fed. The writer desired to know if heating under more favorable conditions would destroy the gossypol and to what extent water would aid in this process. 8
10
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I S D C S T R I A L A S D ESGISEERISG CHEMISTRY
Dowell and hfenaul, J. A i r . Research, 26, 9 (1923) Gallup, J . D a i r y Sci., 9, 359 (1926). J . A g i . Research, 15, 285 ( 1 9 2 3 ) . J . B i d . Chem., 29, 289 (1917).
The plan of t,his experiment was to subject cotton seeds to the act'ion of heat and moisture both separately and combined, if possible, within limits which would not be det'rimental to the final product and yet be sufficient to destroy t'he gossypol and produce a feed of good quality. Chemical determinations were to be made of the gossypol content of t'he seeds before and after such treatment and thc products fed to experimental animals t o note the effects upon growth as an index of toxicity. Chemical Determinations of Gossypol Content before and after Treatment
The cotton seeds.. which were of one variety, were delinted with sulfuric acid, washed, air-dried, and divided int'o four portioiis. One portion was kept moist a t about 40" C. until the seeds had sprouted. A second portion was soaked in water for 4 hours and then autoclaved for different periods of time under 20 pounds (1.4 kg.) steam pressure. ,1third portion was finely ground, spread in tliiii layers, and placed in an electric oven a t 110" C. for the same and longer periods of time. In this manner cottonseed products were obtained which had been subjected to the action of moisture alone, heat and moisture, and heat alone. These were sampled after being brought' to room t'emperature and determinations made for gossypol and d-gossypol. Table I-Gossypol TIMEOF
HEATING
a n d d-Gossypol Content of Cotton Seeds Treated in Various Ways FIXAL ANILINE- ANILINE~ I O I S T U R EGOSSYPOL d-GosswoL dCONTENT COMPOUKDCOMPOUNDGOSSYPOLGOSSYPOL Per cent Grams Grams Per cent Per cent
GROUNDCOTTONSEEDSHEATEDDRYAT~~~~C.
10 15 20 30 1 2 4 8 16
min. min. min. min. hour hours hours hours hours
2.40 1.97 1 .6.i 1.15
0.42 0.11 None None None
0.0931 0.0147 0.0145 Trace None None h'one None None
0.0914 0.20R9 0.1503 0.1643
0.1602 0.1468 0.1238 0,1180 0.0760
0.137 0.021 0.021 ...
min.
43.70
min.
44.24
hour hours
46.85 48.01
0.0560 0.0393 None None
0.0818 0 0510 0 0326 Kone
n
0
... ... ... ...
0.082 0.058
...
...
24:1
237 0.217 0.185 0.174 0.112
...
W H O L E COTTON S E E D S S O A K E D 4 H O U R S A N D ALrTOCLAVED A T PRESSURE
10 30 1 2
0 133 0.809 0.222
20
LBS.
0.091 0 075 0.048
...
O R I G I N A L COTTON S E E E S
Unheated Unheated, germinated
7.31
0.1860 None determinations
0.2i5
...
pi0
Soaking the seeds in the second port,ion was for the purpose of softening the hulls and allowing the seeds to take up considerable water, so that when they were autoclaved the steam was able t o penetrate the kernel. During the procedure there is no packing and very little loss of oil as is the case if the seeds are first ground. The seeds were ground in the third portion to expose the gossypol and increase the opportunities for oxidation or other chemical reactions t.0 take place. The initial moisture content of the seeds, which was 7.51 per cent, was decreased during the dry-heating process to the amount's shown in Table I. This small amount is believed from other studies to be of little consequence in bringing about the destruction of gossypol. The autoclaved seeds, however, contained 45.25 per cent moisture, which was varied as shown during the periods of autoclaving and in such amounts was instrumental in bringing about the rapid destruction of gossypol. The results of the gossypol and d-gossypol determinations on these products using 50-gram samples are given in Table I. The gossypol determinations were made by Carruth's method as modified by Schwartze and Xlsberg,lo and the d-gossypol was determined by the met'hod used by S h e r ~ o o d . ~ The gossypol content of the aniline-gossypol precipitates
INDUSTRIAL A N D ENGINEERING CHEMISTRY
728
was determined by using the factor 74 in assuming the composition of this precipitate to be CmHZsOg.2C6H5NH2. The d-gossypol was calculated in the same way, as it has been shown6 that the composition of the two precipitates is practically the same. No determinations were made upon the germinated seeds, as the feeding experiments showed no decrease in toxicity between the germinated and the original seeds.
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T a b l e 11-Rats o n C o t t o n s e e d P r o d u c t Diets DURATION OF RATION OF COTTONSEED RATS KO. EXPT. TREATMENT GROWTHLIVED DIED Days XXXIV 30 Ground, heated dry 1 None ., 4 hour, 110' C. XXVIII 50 Ground, heated d r y 8 None 1 3 hours, l l O o C. XXIX 60 Ground, heated dry Poor 4 ... 16 hours, 110' C. V 100 Soaked 4 hours: autoFair 4 ... claved wet 30 min., 20 lbs. pressure I1 60 Soaked 4 hours; autoGood 4 ... claved wet 1 hr., 20 Ibs. pressure I11 60 Soaked 4 hours: autoVery 4 claved wet 2 hours, good 20 lbs. pressure XXXIII 40 UntreatedNone ... 4 Germinated xxxv 40 None .,. 4
Z~GMBER
.
The average growth curves of the animals are shown in Figure 1. Those animals receiving in their diet cotton seeds which had been autoclaved for 2 hours in the presence of excess moisture made the best growth and were in the best condition throughout the experiment. The animals receiving the germinated seeds and seeds heated dry a t 110' C. for one hour showed no improvement over those animals receiving the untreated seeds. None of these animals made any gain in weight, but all slowly declined and died, following along the general growth curve for the rats on the untreated seeds. Heating the seeds a t 110" C. for 16 hours decreased their toxicity, and the rats fed on this product are still alive after 90 days although their growth is below normal. Conclusion
F i g u r e 1-Growth
Curves of R a t s on C o t t o n s e e d P r o d u c t Diets
From the results in Table I it appears that through the agency of heat gossypol is readily changed to d-gossypol, even though the time of heating is short. Longer heating tends to destroy the d-gossypol. This change is most apparent after 15 minutes' heating, and in only one instance when a large quantity of ground seeds was used was there an appreciable amount of gossypol left in the seeds that had been heated in the electric oven for one hour. The amount of d-gossypol was decreased very slowly and could be detected after 16 hours of such heating. Whether or not gossypol is destroyed by heat after first being converted to d-gossypol is still a question. Heating the seeds in the presence of excess moisture, as in autoclaving, destroyed both the gossypol and the d-gossypol in relatively short periods of time. The optimum time of such heating under these conditions was one hour. These results are based upon chemical determinations for the two compounds and represent the average of no less than two determinations for each compound. Test on Rats
Some of the products described above were incorporated in diets of young rats to observe the effect upon growth as a further check upon the destruction of the toxic principle. The cottonseed products were used in a ration made up as follows: Cottonseed product 35, wheat 60, CaC03 1, NaCl 1, and cod-liver oil 3 parts. Such a ration had been previously employed for rats over short periods and good growth and reproduction12 were obtained when using both cotton seeds and cottonseed meal which had been autoclaved for one hour, When the germinated seeds were fed, they made up 50 per cent of the ration, thereby decreasing the wheat to 45 per cent. This change, however, did not affect the general results. The results of this feeding are shown in Table 11. 12
Unpublished work.
The destruction of gossypol, the toxic principle in cotton seeds as indicated by feeding tests and chemical determinations of both gossypol and d-gossypol, is brought about slowly by heat if the seeds are first ground and spread out in thin layers. The time of such heating necessary to produce a non-toxic product is much longer than that employed a t the oil mill. The toxic principle is rapidly destroyed by steaming the seeds as in autoclaving in the presence of excess moisture. Under the conditions employed, one hour of such treatment was sufficient to reduce the toxic principle such that it could not be determined by the usual chemical methods. Germination does not decrease the toxicity of the seeds. Acknowledgment
The author desires to express his appreciation of the interest taken in this work by V. G. Heller, who sprouted the cotton seeds and did that portion of the feeding work which had to do with the germinated seeds. Thanks are also due to N. B. Guerrant for his helpful suggestions and advice. Pulp Mill Waste Utilized in Tanning-The possibility of converting a waste product of the paper industry into a necessary material for the leather industry has been revealed as a result of research conducted by the Bureau of Standards. It has found that leather filled with materials derived from waste liquors is equally as durable as leather lilled with such tanning materials as chestnut wood and quebracho wood extracts. The bureau then investigated the suitability of sulfite cellulose extracts (treated and concentrated waste liquors) for use in the actual tanning operations. Chemical examinations of various extracts showed them to contain usable amounts of materials which would firmly combine with hide to form leather. The color imparted t o leathers by the use of these materials was satisfactory. Actual tanning tests produced leather of good strength, wearing quality, and appearance. The investigation shows that sulfite cellulose extracts can be satisfactorily used for tanning when blended with other materials such as quebracho wood and chestnut wood extracts. Both the tanner and the paper manufacturer would benefit by this utilization.
