Determination of free gossypol in chemically treated cottonseed meals containing dianilinogossypol

Summary A method is proposed for the determination of free gossypol in chemically treated cottonseed meals containing dianilinogossypol. The procedure includes a rapid qualitative test for detecting the presence of dianilinogossypol in cottonseed meals. Investigation of the properties of dianilinogossypol showed that it was appreciably soluble in water-free solvents, such as acetone, methyl ethyl ketone, or chloroform, but only slightly soluble in 70% acetone, an efficient solvent for the extraction of free gossypol. Both 70% acetone extracts of chemically treated meals and pure dianilinogossypol in the same solvent exhibited significant changes in optical density due to slow hydrolysis of dianilinogossypol. Dilution of aqueous acetone extracts with 80% isopropyl alcohol produced changes in optical density even greater than those occurring in undiluted extracts, introducing serious errors in the free gossypol determination. The addition of a small amount of aniline to the aqueous acetone for extraction stabilized the extract against changes in optical density and yielded constant values for free gossypol over a period of several hours. Use of aqueous acetone without aniline gave values which increased with the time interval between extraction and analysis. Extrapolation of these values to zero time gave results in good agreement with those obtained by the use of aqueous acetone containing aniline. The intense background color in extracts of chemically treated meals introduces a source of error when photoelectric colorimeters yielding non-linear calibration curves are used for analysis. A modification in the customary method of calculation eliminates this source of error. One of the laboratories of the Southern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture.     

Cottonseed extraction with mixtures of acetone and hexane

Cottonseed flakes were extracted with mixtures of n-hexane and acetone, with the concentration of acetone varying between 10 and 75%. Adding small amounts of acetone (≤25%) to n-hexane significantly increased the extraction of free and total gossypol from cottonseed flakes. Sensory testing detected no difference in the odor of cottonseed meals produced either by extraction with 100% n-hexane or by extraction with a 10∶90 (vol/vol) mixture of acetone/hexane. More than 80% of the free gossypol was removed by the 10∶90 mixture of acetone/hexane, whereas pure n-hexane extracted about 47% of the free gossypol from cottonseed flakes. A solvent mixture containing 25% acetone removed nearly 90% of the free gossypol that was removable by extraction with pure acetone; the residual meal had only a minimal increase in odor. In contrast, cottonseed meals produced by extraction with pure acetone had a much higher odor intensity. The composition of the cottonseed crude oil was insignificantly affected by the acetone concentration of the extraction solvent. The results indicate that mixtures of acetone and n-hexane can be used as extraction solvents to produce cottonseed crude oil without the concomitant development of odorous meals.     

Preparation of gossypol from cottonseed pigment glands

Summary Cottonseed pigment glands, produced from whole cottonseed meal and defatted cottonseed meal by the gland flotation process, have been investigated as a raw material for the production of gossypol. Methods based on the previously reported properties of gossypol and cottonseed pigment glands have been developed for the relatively rapid isolation of gossypol from pigment glands. Extraction of gossypol from pigment glands with acetone followed by precipitation of gossypol acetic acid from the extract was found to be the preferable method for obtaining pure gossypol in good yields. The precautions which must be applied in order to produce gossypol on a large scale by the acetone-acetic acid method are discussed together with methods for the purification and preservation of gossypol. Presented at the Fall Meeting of the American Oil Chemsits' Society, October 20–22, 1947. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Application of the antimony trichloride-spectrophotometric method to the determination of gossypol in cottonseed and cottonseed products

Summary A method is described which permits application of the antimony trichloride spectrophotometric method to the determination of gossypol in a variety of cottonseed products. Gossypol is determined by means of the following series of operations: 1. extraction of gossypol from cottonseed or cottonseed products by use of chloroform or aqueous ethanol; 2. isolation of gossypol from the extracts by use of aqueous alkali; and 3. application of the antimony trichloride-spectrophotometric test. Data are presented to show the results obtained by application of this procedure to the determination of gossypol in pigment glands, raw cottonseed meats, cooked cottonseed meats, hydraulic- and screw-pressed meals, solvent-extracted meals, gland-free meals, and oils, both expressed and solvent-extracted. Presented at the 39th Annual meeting of the American Oil Chemists' Society in New Orleans, May 4–6, 1948. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Note on acetone-soluble material in cottonseed meals

Summary In connection with a study of methods of eliminating traces of “free” gossypol from cottonseed meals a procedure for isolation of gossypol from the 70% acetone extracts of the meals by transfer to benzene solution has ben developed. A procedure is also given for quantitative determination of the isolated gossypol. Analysis of four samples of cooked cottonseed meats and meal by the method showed that in each instance substances other than gossypol are measured by the A.O.C.S. method for “free” gossypol in meals containing low concentrations of residual “free” gossypol. This procedure promises to provide another analytical tool for the study of residual material in processed cottonseed meal that causes egg discoloration when fed to laying hens. One of the laboratories of the Southern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture.     

Effect of fractionation and treatment on the acute oral toxicity and on the gossypol and gossypurpurin content of cottonseed pigment glands

Summary Twenty-one preparations from cottonseed pigment glands were tested for their acute oral toxicity in 1,208 fasted rats and for their content of extractable gossypol and gossypurpurin. LD₅₀ studies on six samples of pure gossypol were performed on 167 fasted rats. There was no correlation between the toxicity of the various samples of cottonseed pigment glands and their extractable gossypol or gossypurpurin content. Samples containing very large amounts of extractable gossypol were less toxic than many samples with considerably lower extractable gossypol content. Various fractionation procedures carried out on the same lot of cottonseed pigment glands caused wide alterations in their toxicity, from the extreme of very marked toxicity for the water-soluble, acetone-soluble fraction (LD₅₀ ca. 700 mg./kg.) to no detectable toxicity for the acetone-insoluble residue (LD₅₀<6,000 mg./kg.). There was a decreased toxicity of subsequently prepared pigment glands with increased time of storage of the cottonseed in a silo. Storage of the pigment glands themselves at 7°C. however had little effect on their toxicity even after 26 and 32 months. The procedures causing greatest detoxification of cottonseed pigment glands, given in the order of increasing effectiveness, were: heating in the presence of water < extraction with ethanol < extraction with acetone. In the fasted rat the acute oral toxicity of pure gossypol was less than that of 17 preparations from cottonseed pigment glands having extractable gossypol contents ranging from as little as 24 to as much as 90%. Report of a study in which certain phase were carried on under the Research and Marketing Act of 1946. Presented at fall meeting, American Oil Chemists' Society, Chicago, Oct. 31, Nov. 1–2, 1949. This work was conducted with Swift and Company under a memorandum of understanding with the Bureau of Agricultural and Industrial Chemistry. The preparation of the samples of gossypol and cottonseed pigment glands and the analyses for gossypol and gossypurpurin content were carried out in the Southern Regional Research Laboratory; all of the animal experiments were performed in the Research Laboratories of Swift and Company. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Spectrophotometric determination of total gossypol in cottonseed meal and cottonseed meats

Summary An improved method has been developed for the determination of total gossypol in cottonseed and cottonseed meal. The sample is heated with aniline to convert the gossypol to dianilinogossypol, which is extracted with chloroform and measured spectrophotometrically. The values for total gossypol are slightly higher and more accurate and precise as determined by the proposed method because of more complete extraction than by a recent p-anisidine method or the revised A.O.C.S. Tentative Method Ba 8-55. The advantages of the proposed method are its simplicity, accuracy, reproducibility, and expeditiousness. Published with the approval of the Director of Research, N. C. Agricultural Experiment Station, as paper No. 758 of the Journal Series.     

The determination of aflatoxins in cottonseed products

A rapid and simplified procedure is proposed for the determination of aflatoxins B₁, B₂, G₁, and G₂ in cottonseed products. The method involves extraction of aflatoxins essentially free of lipid contamination by equilibrium extraction with 70% acetone. Interfering gossypol pigments are removed from the aqueous acetone extract by precipitation as insoluble lead salts. Aflatoxins in the centrifugate are quantitatively separated from excess lead salts, residual pigments and carbohydrates by extraction into chloroform to yield final extracts for thin-layer chromatographic (TLC) analysis on silica gel which are low in total solids and pigmentation. The procedure is sensitive to about 1 ppb in cottonseed meats and 4 ppb in cottonseed meal. Honorable mention, Bond Award competition. Presented at the American Oil Chemists’ Society, Chicago, October 11–14, 1964. A laboratory of the So. Utiliz. Res. and Dev. Div., ARS, USDA.     

Binding of gossypol under conditions of complete rupture of the pigment glands

Summary The “free” gossypol of cooked cottonseed meats is composed of gossypol and of “gossypol-like pigments,” which are soluble in 70% aqueous acetone. The “gossypol-like pigments” may account for 30 to 100% of the “free” gossypol as determined by the A.O.C.S. method. Thorough comminution, followed by prolonged cooking, results in reduction of all the pigments in cotton-seed meats. “Free” gossypol is not completely eliminated, but the gossypol level, as determined by the benzene transfer method, may be reduced to zero. “Gossypol-like pigments” are converted into gossypol by the action of hot aqueous hydrochloric acid. Good agreement is observed between the data for “total” gossypol, as determined through the use of the A.O.C.S. oxalic acid method and through the use of hot aniline. One of the laboratories of the Southern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.     

Effect of temperature on the content of pigments of stored cottonseed

Summary Three pure-bred varieties of cottonseed,G. hirsutum, which were planted and grown under similar environmental conditions were stored at 38°, 77°, and 85°F. After determining the initial contents of lipids, nitrogen, moisture, gossypol, and gossypurpurin each lot of seed was stored at the different temperatures and analyzed periodically with respect to changes in pigmentation. The content of gossypurpurin was found to increase during storage in all of the samples. Its increase was proportional to the temperature and length of storage. On the other hand, gossypol decreased during storage of all samples. The antimony trichloride test for gossypol was found to be applicable only to extracts prepared from fresh cottonseed. During storage of the seed another yellow-colored pigment(s) developed which could be separated from gossypol by alkaline extraction of the original chloroform extract of the stored seed. The alkali extractable portion of the chloroform extract gave a red-colored antimony trichloride reaction product characteristic of gossypol. It is postulated that at least a fraction of the non-acidic pigment(s) in the crude chloroform extracts obtained from stored cottonseed is diaminogossypol. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Effect of time and temperature of storage on the free and total gossypol content of cottonseed meals and of mixed diets

Summary With different types of cottonseed products and cottonseed products to which gossypol is added, there is a loss or disappearance of gossypol upon storage, as determined analytically. The rate of disappearance of gossypol varied for the different type cottonseed and was increased at higher temperatures and over the longer periods of storage. When gossypol is incorporated into different types of animal diet materials, there is a loss or inactivation of some portion of the added gossypol as determined analytically immediately after preparation of the gossypol-diet mixtures, and a further additional loss or inactivation upon storage. The factors which may contribute to this loss or inactivation of gossypol are: a) the components of the diet materials; b) the temperature and length of time of storage; c) the concentration of gossypol added and the final percentage of gossypol in the mixture. One of the laboratories of the Southern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture.     

Lysine,gossypol, and nitrogen solubility in chemically treated cottonseed meals

The effects of treatment of commercial prepress-solvent extracted and direct-solvent extracted cottonseed mares with several chemical agents and solvents were studied. The analytical results for “free” and “total” gossypol of the finished meals show that treatment with aliphatic amines, followed by extraction with a suitable solvent, removed large proportions of the “free” as well as “bound” gossypol. This reduction of “free” and “bound” gossypol was accompanied, in some experiments, by an increase in the nitrogen solubility and available lysine, as compared with the results obtained with the untreated air-dried marcs. The available lysine contents of the treated mares was significnatly correlated with the nitrogen solubility in 0.02N aqueous NaOH. One of the laboratories of the Southern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.     

Ethanol vapor deactivation of gossypol in cottonseed meal

Most cottonseed cultivars contain gossypol, a polyphenolic antinutritional compound. “Free” gossypol is a physiologically active form of gossypol, which is toxic to young- and nonruminant animals. To utilize solvent-extracted cottonseed meal as a general feed, gossypol must be either removed or deactivated to a minimum level specified for each class of animal. Normally, deactivation is carried out prior to oil extraction; however, the desired level of deactivation is not always attained. A new supplemental method of deactivation has been found by using either ethanol or isopropanol vapors on solventextracted meal. In a bench-top set-up, ethanol vapor reduced free gossypol from 0.115 to 0.053%, and a further reduction to 0.026% has been observed with the addition of ferrous sulfate. The supplemental deactivation method can, in most cases, reduce free gossypol to significantly safer levels for feeding, thus increasing utility, and possibly demand, for cottonseed meal as a general animal feed protein source. Presented in part at the AOCS Annual Meeting, Atlanta, GA, May 8–12, 1994.     

Cottonseed extraction with a new solvent system: Isohexane and alcohol mixtures

A solvent system, consisting of isohexane and 5 to 25% alcohol, either ethanol (EtOH) or isopropyl alcohol (IPA), was tested for extracting gossypol and oil from cottonseed. The test results indicate that this new solvent system not only is effective in removing free and total gossypol but also is as efficient as n-hexane when extracting oil. The amino acid analysis of cottonseed meal, produced by the new solvent system, is similar to that produced by commercial n-hexane. Present commercial cottonseed extraction and downstream processing of cottonseed oil refining may need little change to adopt this new solvent system. This new solvent system may lead to a solution to the gossypol problem of cottonseed extraction.     

Adsorptive gossypol removal

Gossypol is extractable from cottonseed by using aqueous ethanol. The equilibrium between undissolved gossypol in cottonseed and that dissolved in the solvent determines the residual gossypol. To move the equilibrium toward extraction from the seeds, the dissolved gossypol needs to be removed from the gossypol-solvent-oil mixture. Gossypol removal from the mixture by adsorption on alumina, silica and molecular sieve 5Å was tested. Experimental results indicated that gossypol was more selectively adsorbed than triglycerides by these adsorbents. Alumina and silica had higher gossypol adsorption capacities than molecular sieve 5Å.     

Acidic ethanol extraction of cottonseed

Ethanol (EtOH) is being evaluated as an alternate solvent to hexane for the extraction of glanded cottonseed. Hot EtOH, needed for efficient oil and aflatoxin extraction, binds gossypol to protein. However, this binding can be minimized by acidifying aqueous EtOH with a tribasic acid, such as phosphoric or citric. While this solvent extracts oil and gossypol, it does not affect EtOH’s ability to extract aflatoxin. The defatted cottonseed meals produced from this process contained 0.03% total gossypol (which is lower than meal prepared by most other processes) and the aflatoxin content was reduced from 69 to 2.9 ppb. These are preliminary results and additional research is needed to determine commercial feasibility. The removal of essentially all gossypol from an extracted meal has the potential to expand the use of cottonseed meal as a feed, increasing its value to both the cotton farmer and the seed processor. Presented in part at the 40th Oilseed Processing Clinic, March 4, 1991, New Orleans, LA.     

3-amino-1-propanol as a complexing agent in the determination of total gossypol

Summary A method is proposed for the determination of total gossypol in cottonseed meals, crude oils, and soapstocks based on a rapid extraction of gossypol by neutralized 3-amino-1-propanol in dimethylformamide to form a stable complex, followed by colorimetric analysis of an aliquot of the extract by means of an aniline reaction. A determination can be completed in about 2 hrs. and with minor modification in 1 hr. compared to about 7 hrs. for current methods. Results obtained by the proposed procedure on meals, oils, and soapstocks are in essential agreement with those found by use of other accepted methods. Desirable features, such as stability of reagents and extracts and a high degree of reproducibility, suggest that the procedure will satisfy the requirements for a rapid and simplified method for the analysis of all cottonseed products for total gossypol. One of the laboratories of the Southern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.     

Improved objective fluorodensitometric determination of aflatoxins in cottonseed products

Modifications in the extraction solvent, lead acetate, and silica gel extract purification steps, and the TLC development conditions of the aqueous acetone procedure of Pons et al. [JAOAC49, 554–562 (1966)] for the estimation of aflatoxins in cottonseed products, provides an improved method with essentially quantitative recovery of aflatoxins added to typical cottonseed materials. Both the accuracy and precision of aflatoxin estimates are significantly improved by the incorporation of an objective fluorodensitometric measurement of aflatoxins on TLC plates. Presented at Joint AOCS-AACC Meeting, Washington, D.C., March 31–April 4, 1968. So. Utiliz. Res. Dev. Div., ARS, USDA.     

Removal of gossypol from cottonseed by solvent extraction procedures

Summary The relative efficiencies of organic, polar solvents and of solvent-water pairs for use in the extraction of gossypol and related compounds from cottonseed flakes were determined in a specially devised glass laboratory extractor. Of the solvents tested a butanone-water pair containing 10% of water by volume was the most effective, and chlorine-substituted hydrocarbons were the least effective. Under equilibrium conditions maximum extraction of gossypol was obtained with a butanone solvent containing 2.5% of water by weight. The rate of extraction of gossypol from cottonseed meal with butanone-water pairs increased with increase in the amount of water in the system and with increase in temperature of the extraction system. The greater amounts of water in the extraction system resulted in swelling and packing of the flakes and in a decrease in extraction efficiency. Flakes extracted at 26°C. contained 0.08% free gossypol and those extracted at 71°, 0.054%. This decrease may be due, in part, to the reaction of gossypol with the protein to form bound gossypol. Report of a study carried on under the Research and Marketing Act of 1946. This paper is No. 9 in the series on “Processing of Cottonseed” from the Southern Regional Research Laboratory. References to other papers in this series are: J. Am. Oil Chem. Soc.24, 97–108 (1947); J. Am. Oil Chem. Soc.24, 276–283 (1947); J. Am. Oil Chem. Soc.24. 362–369 (1947); J. Am. Oil Chem. Soc.26, 28–34 (1949); Oil Mill Gaz.54 (2), 12–15 (1949); Cotton Gin and Oil Mill Press51 (9), 18–20 (1950); Official Proc. Natl. Cottonseed Products Asso,55, 32–34, 36 (1951); and J. Am. Oil Chem. Soc. (in press), “The Effect of Screw Press and Hydraulic Press Processing Conditions on Pigment Glands of Cottonseed”, by D. M. Batson. F. H. Thurber, and A. M. Altschul. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Improved method for determining gossypol in crude cottonseed oils

Summary The p-anisidine method for the determination of gossypol in crude cottonseed oils has been reinvestigated and modified to make it applicable to all crude oils obtained by the newer methods of processing cottonseed. The modifications included a change in the composition of the solvent, a higher reaction temperature, and the use of a more concentrated panisidine reagent. The modified method was found satisfactory where different colorimeters and spectrophotometers were used for measuring the color developed. Comparison of aniline and p-anisidine as reagents for the analysis of gossypol pigments showed that the presence of modified gossypol in some crude oils resulted in an over-correction for background absorption and led to significant errors when aniline was used as the color development agent. One of the laboratories of the Southern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture.