**Effect of selected fungi on the reduction of gossypol levels and nutritional value during solid substrate fermentation of cottonseed meal

ZHANG Wen-ju†1,2, XU Zi-rong†‡1, SUN Jian-yi1, YANG Xia1

(1Key Laboratory of Molecular Animal Nutrition of Ministry of Education, College of Animal Science, Zhejiang University, Hangzhou 310029, China)(2College of Animal Science and Technology, Shihezi University, Shihezi 832003, China)‡ Corresponding Author†E-mail: zhang-wj1022@tom.com; zrxu@zju.edu.cn Received Aug. 24, 2005 revision accepted Feb. 21, 2006

Abstract: The objective of this work was to investigate the effect of six individual strains of fungi on the reduction of gossypol levels and nutritional value during solid substrate fermentation of cottonseed meal (CSM). Six groups of disinfected CSM substrate were incubated for 48 h after inoculation with either of the fungi C. capsuligena ZD-1, C. tropicalis ZD-3, S. cerevisae ZD-5, A. terricola ZD-6, A. oryzae ZD-7, or A. niger ZD-8. One not inoculated group (substrate) was used as a control. Levels of initial and final free gossypol (FG), crude protein (CP), amino acids (AA) and in vitro digestibility were assayed. The experiment was done in triplicate.

Key words: Fungi, Free gossypol, Solid substrate fermentation, Cottonseed meal, Detoxification, Nutritional valuedoi:10.1631/jzus.2006.B0690             CLC number: Q819; S38

References:

[1] AOAC, 1984. Official Methods of Analysis, 13th Ed. Association of Analytical Chemists, Washington, DC.

[2] AOAC, 1999. Official Methods of Analysis, 16th Ed. Association of Analytical Chemists, Washington, DC.

[3] AOCS, 1989. Free and Total Gossypol Methods, Official and Tentative Methods of the AOCS, 4th Ed. American Oil Chemists’ Society, Chicago.

[4] Barraza, M.L., Coppock, C.E., Brooks, K.N., Wilks, D.L., Saunders, R.G., Latimer, G.W., 1991. Iron sulfate and feed elleting to detoxify FG in

cottonseed diets for dairy cattle. J. Dairy Sci., 74(10):3457-3467.

[5] Berardi, L.C., Goldblatt, L.A., 1980. Gossypol. In: Liener, I.E. (Ed.), Toxic Constituents of Plant Foodstuffs, 2nd Ed. Academic Press, NY, p.183-237.

[6] Canella, M., Sodini, G., 1977. Extraction of gossypol and oligosaccharides from oil seed meals. J. Food Sci., 42(4):1218-1219.

[7] Cherry, J.P., Gray, S., 1981. Methylene chloride extraction of gossypol from cottonseed products. J. Food Sci., 46(6):1726-1733.

[8] Damaty, S., Hudson, B.J.F., 1975. Preparation of low gossypol cottonseed flour. J. Sci. Food Agric., 26(1):109-115.

[9] Francis, G., Makkar, H.P.S., Becker, K., 2001. Antinutritional factors present in plant-derived alternate fish feed ingredients and their effects in fish. Aquaculture, 199(3-4): 197-227.

[10] Nagalakshmi, D., Sastry, V.R.B., Agrawal, D.K., 2002. Detoxification of undecorticated cottonseed meal by various physical and chemical methods. Anim. Nutr. Feed Technol., 2(2):117-126.

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Title:**Method for removal of gossypol from cottonseed meal by the use of urea in a borate containing buffer Document Type and Number:United States Patent 5277909 Link to this page:http://www.freepatentsonline.com/5277909.html Abstract:Plant material may be treated for the removal of gossypol therefrom to provide a protein-rich product and/or oil relatively free of gossypol. The

process includes the steps of: a. grinding the plant material to form a meal, b. adding an amine and a buffer to the meal and mixing to form a slurry and react the gossypol in the plant material with the amine and buffer to form a gossypol/amine/buffer complex, c. allowing the slurry to form a crystal layer of the complex above a layer of the meal having gossypol removed therefrom, d. separating the crystal layer from the layer of meal. The gossypol containing complex recovered also possesses insecticidal activity.

Inventors:Schmidt, John H. (Leland, MS, US)Wells, Randy (Raleigh, NC, US)Bailey, Jack C. (Leland, MS, US)Application Number:572070 Filing Date:08/24/1990 Publication Date:01/11/1994 View Patent Images:Images are available in PDF form when logged in. To view PDFs, Login  or  Create Account (Free!) Referenced by:View patents that cite this patent Export Citation:Click for automatic bibliography generation Assignee:The United States of America, as represented by the Secretary of (Washington, DC) Primary Class:424/776 Other Classes:530/377 International Classes:A61K 035/78, A61K 035/80 Field of Search:424/195.1 530/377 US Patent References:4340676 Jul, 1982 Bourque 435/232.4546004 Oct, 1985 Rhee 426/656.Other References:

The Merck Index 9th Ed., Merck & Co. Rahway N.J. 1976 #4377. J. H. Schmidt & R. Wells, "Recovery of Soluble Proteins from Glanded Cotton Tissues with Amines," Anal. Biochem. 154: 244-249 (1986). E. E. King, "Extraction of Cotton Leaf Enzymes with Borate," Phytochemistry 10: 2337-2341 (1971). S. P. Clark et al., "Removal of Gossypol from Cottonseed Meats with Aliphatic Amines," Oil Mill Gazeteer 69: 16-21 (1965). W. G. Bickford et al., "The Antioxidant and Antipolymerization Properties of Gossypol, Dianilinogossypol, and Related Materials," J. Am. Oil Chem. Soc. 31: 91-93 (1954). J. H. Schmidt & R. Wells, "Evidence for the Presence of Gossypol in Malvaceous Plants Other than Those in the Cotton Tribe," J. Agric. Food Chem. 38: 505-508 (1990). Primary Examiner:Wityshyn, Michael G. Assistant Examiner:Gitomer, Ralph Attorney, Agent or Firm:Silverstein; M. Howard, Fado; John D., Deck; Randall E. Claims:We claim:

1. A process for the removal of gossypol from cottonseed comprising the steps of:

a. grinding cottonseed,

b. adding an amine and a buffer to said meal and mixing to form a slurry and react gossypol in said cottonseed with said amine and said buffer to form a gossypol:amine:buffer complex,

c. allowing said slurry to form a crystal layer comprising said complex, said crystal layer being formed above a layer of the meal having gossypol removed therefrom,

d. separating said crystal layer from said layer of meal.

2. A process as defined in claim 1 wherein said amine is selected from the group consisting of urea and ammonium sulfate.

3. A process as defined in claim 2 wherein said amine is urea and said complex comprises gossypol:urea:buffer.

4. A process as defined in claim 1 wherein said buffer contains borate and said complex comprises gossypol:amine:borate.

5. A process as defined in claim 4 wherein said buffer is Tris/sodium tetraborate buffer.

6. A process as defined in claim 1 wherein said step of allowing the slurry to form a crystal layer comprises heating said slurry.

7. A process as defined in claim 1 wherein said amine is urea, said buffer contains borate, and said complex comprises gossypol:urea:borate.

Description:BACKGROUND OF THE INVENTION

1. Field of Invention

The invention relates to a method for the treatment of plant material to provide a protein-rich product and/or oil relatively free of gossypol, and a gossypol-containing complex possessing insecticidal activity.

2. Description of the Prior Art

Gossypol is a known poisonous pigment found in plant material such as cottonseed and cotton leaves, and it has been the object of schemes for removal therefrom.

Previous attempts to remove gossypol from cottonseed meal have included extraction with hexane, ethers, alcohols, ketones, metal salts, or aliphatic or aromatic amines.

Schmidt et al. [Analytical Biochemistry 154, 244-249 (1986)] disclosed that Tris/borate buffer containing urea was effective for the removal of gossypol from cotton leaf tissue in cotton leaf protein studies. However, while this process allowed the removal of gossypol from plant material, it did not provide a convenient process allowing mechanical separation of the urea/gossypol/borate complex from the gossypol-free material, or recovery

of a purified complex possessing insecticidal activity.

SUMMARY OF THE INVENTION

We have now invented a method for the treatment of plant material to provide a protein-rich product relatively free of gossypol. Plant material is prepared for treatment by grinding until a powdered or granular meal is obtained. The meal is then mixed in a slurry with an amine and a buffer to react with gossypol in the meal and form a gossypol/amine/buffer complex. After settling, the slurry is held, and preferably heated, at a sufficient temperature and time to form a crystal layer above the meal. This crystal layer contains at least a substantial amount of the gossypol from the plant material in the complex, and leaves meal relatively gossypol-free below it. Formation of this crystal layer further allows for mechanical separation of the gossypol/amine/buffer complex from the meal. Optionally, oil which is also free from gossypol can be extracted from the treated meal using organic solvents as is conventional in the art.

Any gossypol-containing plant material can be treated using the process of the invention. The process is particularly advantageous for the removal of gossypol from cottonseed, cotton leaves, okra, or meal therefrom.

The gossypol/amine/buffer complex recovered after separation from the plant material possesses insecticidal activity and may be used as an insecticide.

In accordance with this discovery, it is an objective of this invention to provide a method for removing gossypol from plant material yielding a protein-rich product and/or oil relatively free from gossypol. In particular, it is an objective to provide a process for removing gossypol from plant meal, especially cottonseed meal.

It is a further objective of this invention to provide a gossypol-containing complex and a method of using the same as an insecticide.

DETAILED DESCRIPTION OF THE INVENTION

According to the process of the invention, plant material to be treated is ground to form a powdered or granular meal. Preferably the material is ground to a fine powder for enhanced contact of the gossypol therein with an

amine and buffer as described below. An amine and a buffer are then added to the meal and mixed to form a slurry. Mixing is continued for a sufficient time at a sufficient temperature to react gossypol in the meal with the amine and buffer to form gossypol/amine/buffer complex. After reaction, the slurry is allowed to settle and is held, preferably while heated, at a sufficient temperature and for a sufficient time to form a crystal layer of the gossypol/amine/buffer complex above the meal. This crystal layer contains at least a substantial amount of the gossypol from the plant material, leaving meal relatively gossypol-free in the lower layer. The crystal layer is then mechanically separated from the meal, for example by scraping, and the remaining gossypol-free meal is recovered for use as a protein-rich feed or food product. Optionally, oil which is also free of gossypol can be extracted from the treated meal using organic solvents as is conventional in the art.

The crystal layer containing the gossypol/amine/buffer complex may be discarded or retained as substantially pure product or incorporated into a composition for use as an insecticide as described below. In addition, the complex may be further treated or purified to increase insecticidal activity. For example, the recovered complex may be solubilized in a solvent such as water and dialyzed against pure distilled water. The complex will pass through the tubing walls and may be recovered and dried as pure complex. Further still, recovered complex may be heated to about 90.degree. C. to increase insecticidal activity. While not wishing to be bound by theory, it is believed that the heating results in the formation of stronger bonds between the components of the complex.

Any plant material containing gossypol can be treated using the process of this invention. Plant material from the tribes Gossypeae or Hibiscadeae may be treated, and particularly cottonseed, cotton leaves, okra, or the meal therefrom.

The amine and buffer employed may be varied and may be readily determined by one skilled in the art. Suitable amines must be capable of reacting with a carbonyl group of gossypol and include, for example, ammonium sulfate and particularly urea. Suitable buffers have ionic groups or metal-containing ionic groups capable of hydrogen bonding with gossypol, and include solutions of weak acids and/or their salts. Preferred buffers include those containing boric acid and/or borate, particularly Tris/sodium tetraborate buffer. In this preferred embodiment, a gossypol/urea/borate complex is formed.

Treatment conditions also may be varied. Suitable urea concentrations range between about 2 and 10M, while the buffer may have an ionic strength between about 0.01 and 1M with 0.1M being preferred. The effective range of pH for the complexing reaction is between about 7.0 and 8.0, with 7.6 providing for peak activity. The temperature of the complexing reaction may be between about 0.degree. C. and 60.degree. C., although the rate of reaction is slower below 25.degree. C. Formation and drying of the crystals after the complexing reaction may also proceed within this same temperature range. However, for substantially faster crystal formation and drying, heating of the slurry is preferred, particularly between about 55.degree. C. and 60.degree. C. One skilled in the art will recognize that the reaction time and the crystal formation and drying time will vary with temperature and may be readily determined.

As noted above, the gossypol/amine/buffer complex may be retained for use as an insecticide. Depending on the pest species, concentration of agent, and method of application, the subject complex acts to control insect pests by one or more mechanisms including, for instance, death inducement, growth regulation, sterilization, as well as interference with metamorphosis and other morphogenic functions. Accordingly, the level of active agent is administered in an amount effective to induce one or more of these responses as predetermined by routine testing. Where the ultimate response is pest mortality, an "effective amount" or a "pesticidally effective amount" is defined to mean those quantities of agent which will result in a significant mortality rate of a test group as compared to an untreated group. The actual effective amount may vary with the species of pest, stage of larval development, the nature of the substrate, the type of vehicle or carrier, the period of treatment, and other related factors.

To be effective, the agent must be applied to the locus of, or the vicinity of, the pest to be controlled. When the agent is intended as a stomach poison, it is applied in conjunction with its carrier to the pest diet. In the case of plants, the composition will typically be applied to the leaf surfaces or else systemically incorporated. Alternatively, when the agent is to be applied as a contact poison, any method of topical application, such as direct spraying on the pest or on a substrate which is likely to be contacted by the pest, would be appropriate. In any of these embodiments, the agent may be incorporated with a bait as will be recognized within the art.

When applied as 2% solution on test plants, the complex lowered the populations of Heliothis by at least 15% greater than controls. The complex has also demonstrated insecticidal activity against boll weevils compared to controls.

The following example is intended only to further illustrate the invention and is not intended to further limit the scope of the invention which is defined by the claims.

EXAMPLE

Cottonseed meal was prepared for treatment by grinding the seed until a fine powder was obtained. The powdered meal was then mixed in a slurry with 8M urea in 0.1M Tris/sodium tetraborate buffer (pH 7.6) at a ratio of 1 g meal to 5 ml buffer. Mixing was continued for 1 hr to allow reaction of the gossypol with the urea and borate and formation of gossypol/urea/borate complex. After reaction, the slurry was allowed to settle and then heated to 55.degree. C. for 24 hr. A crystal layer containing the gossypol/urea/borate complex formed above the meal during this step, which was subsequently separated from the remaining meal.

It is noted that the foregoing detailed description is given merely by way of illustration and that modifications and variations may be made therein without departing from the spirit and scope of the invention. For example, the process may be used to remove toxic carbonyl compounds from other source materials, especially but not exclusively those containing aromatic carbonyls with near-neighbor hydroxy functions. Further, oil can be extracted before gossypol removal, such as after grinding the plant material, but such oil would of course not be free of gossypol.

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Title:**Removal of toxins from cottonseed Document Type and Number:United States Patent 4747979 Link to this page:http://www.freepatentsonline.com/4747979.html Abstract:A method is disclosed for removal of toxins and oil from cottonseed by contact of the toxin/oil containing cottonseed with a chlorinated hydrocarbon solvent and a protic or aprotic solvent. Batch, semicontinuous and continuous methods of contacting are also described.

Inventors:Gimber, Gerald A. (Lake Jackson, TX, US)Haschke, Elliot M. (Lake Jackson, TX, US)Application Number:701846 Filing Date:02/14/1985 Publication Date:05/31/1988 View Patent Images:Images are available in PDF form when logged in. To view PDFs, Login  or  Create Account (Free!) Referenced by:View patents that cite this patent Export Citation:Click for automatic bibliography generation Assignee:The Dow Chemical Company (Midland, MI) Primary Class:554/14 Other Classes:426/430, 426/630 Field of Search:426/430, 630 260/412.4 US Patent References:1260656 Mar, 1918 Bollmann 426/430.

1653201 Dec, 1927 Bollmann 260/412.2475419 Jul, 1949 Battistella 260/412.2484831 Oct, 1949 Hutchins et al. 260/412.2680754 Jun, 1954 Stapelberg 260/412.3721569 Mar, 1973 Steinkraus 260/412.4008210 Feb, 1977 Steele et al. 426/430.4062984 Dec, 1977 Lindquist 426/430.4279811 Jul, 1981 Gray et al. 426/430.4460504 Jul, 1984 Rubin et al. 260/412.Other References:Fore et al, "J. Am. Oil Chemists Soc.", vol. 47, No. 1, pp. 17-18, (1969), Reprint. Vix et al, "Chem. Eng. Progress Symp. Series", vol. 65, No. 93 (1969) pp. 49-56 (Reprint). Primary Examiner:Evans, J. E. Attorney, Agent or Firm:Baker; Glwynn R., Ancona; A. Cooper Parent Case Data:CROSS REFERENCE TO THE RELATED APPLICATION

This application is a continuation-in-part of application Ser. No. 627,014 filed July 2, 1984, now abandoned. Claims:We claim:

1. A method for simultaneous toxin (gossypol and aflatoxin) and oil removal from cottonseed meal which comprises

contacting the cottonseed with a solvent mixture of from 80 to 99.5 percent by weight of a chlorinated hydrocarbon solvent having one to two carbon atoms and at least one chlorine atom and from about 20 to about 0.5 percent by weight of at least one other solvent selected from the group consisting of a C.sub.1 to C.sub.4 alcohols, acetone, ethyl ether, dimethyl formamide, dimethyl sulfoxide, which other solvent may be saturated with water,

at a temperature from room temperature to the boiling point of the solvents.

2. The process of claim 1 wherein said contacting is in a countercurrent

manner with respect to movement of said cottonseed.

3. The process of claim 1 wherein said contacting is by percolation of solvent mixture through a bed of said cottonseed.

4. The process of claim 3 wherein said percolation is carried out in a series of percolation zones and said solvent moves from one zone to the next in a counter current manner.

5. The process of claim 1 wherein said cottonseed is mildly agitated during said contacting.

6. The method of claim 1 wherein said cottonseed is subjected to repeated contact with agitation to said solvent mixture.

7. The method of claim 1 wherein the cottonseed is flaked, ground or expanded prior to contact with the solvent mixture.

8. The method of claim 1 wherein said solvent mixture is from 0.5 to about 20 percent by weight of methanol based on the chlorinated solvent.

9. The method of claim 1 wherein said solvent mixture is from 0.5 to about 20 percent by weight of ethanol based on the chlorinated solvent.

10. The method of claim 1 wherein said solvent mixture is from 0.5 to about 20 percent by weight of dimethyl formamide based on the chlorinated solvent.

11. The method of claim 1 wherein said solvent mixture is from 0.5 to about 20 percent by weight of dimethyl sulfoxide based on the chlorinated solvent.

12. The method of claim 1 wherein said solvent mixture is from 0.5 to about 20 percent by weight of isopropyl alcohol based on the chlorinated solvent. Description:BACKGROUND OF THE INVENTION

Various solvents have been employed to extract oil from cottonseed meal. Hexane is the most extensively used solvent in the cottonseed processing industry. While cottonseed oil is very soluble in hexane, the toxic

components such as aflatoxin and gossypol are not, and thus much of this toxic material remains in the extracted meal following hexane extraction, lowering the value of the meal. The gossypol is in the form of free gossypol, which is toxic, and bound gossypol, in combination with lysine, which is not toxic but decreases the protein content of the meal. The aflatoxin remaining with the meal is a toxic residue which reduces the value of the meal

It would be useful to have a process which simultaneously removes oil, aflatoxin and gossypol resulting in an increase in the value of extracted cottonseed meal.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention the meal, (flake, ground meal or expanded) obtained from cottonseed is contacted with a solvent mixture composed of a chlorinated hydrocarbon solvent and either an aprotic or protic solvent. The treatment is carried out in a batchwise, semi-continuous or continuous manner, at ambient temperature or an elevated temperature up to the boiling point of the solvents. The contacting maybe carried out by contacting the solvent with the meal batchwise or in counter current percolation flow through a bed, deep or shallow with or without mild agitation, e.g. mechanical, ultrasonic or the like. It is of course understood that the solvent mixture may be vaporized and passed through a mass or body of flake or meal condensing onto the meal dissolving the toxins into the condensate. The method of contacting is not critical so long as the meal is essentially contacted with a sufficient amount of solvent to maintain the solvent substantially below its saturation point, with respect to the toxins and oil, in the final contact.

The toxins (e.g. gossypol and aflatoxin) in the solvent/oil solution can be removed by conventional refining, which are well understood process steps.

Solvents which have been satisfactorily employed in combination to extract the toxins and oil are those chlorinated hydrocarbon solvents having from one to two carbon atoms and, of course, at least one chlorine atom. Exemplary of such solvents are methyl chloride (CH.sub.3 Cl), methylene chloride (CH.sub.2 Cl.sub.2), chloroform (CHCl.sub.3), carbon tetrachloride (CCl.sub.4), 1,1,1-trichloroethane (C.sub.2 H.sub.3 Cl.sub.3), 1,1,2-trichloroethylene (CH.sub.2 CHCl), perchloroethylene (CCl.sub.2 CCl.sub.2). It is to be understood that methylene chloride, and 1,1,1-

trichloroethane are the preferred solvents as they are more suitable for use in removing gossypol from cotton meal which is to be used as a nutrient product. Methyl chloride, chloroform, trichloroethylene, and carbon tetrachloride each dissolve gossypol and aflatoxin but are not suitable for use in treating any product which has the possibility of use as a food because of physical properties; protein interaction with the solvent forming toxic or suspect toxic properties leading to public uncertainty that all of the solvent and these protein interaction products can be removed.

Suitable aprotic and/or protic solvents are the lower C.sub.1 to C.sub.4 alcohols (methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secondary butyl, and tertiary butyl alcohols), alkyl ethers, ketones, as well as dimethyl formamide, dimethyl sulfoxide and the like. Again, because of public sentiment lower alcohols are preferred. However, dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO) are the most efficient solvents in combination with methylene chloride.

Commercial cotton seed extraction processes include the conditioning of the raw cottonseed material (often referred to in the trade as meats) with moisture prior to flaking, a procedure whereby the meats are compressed into a flake-like body. The conditioning is required to give the flakes a modicum of integrity during further processing. Thus, any commercially acceptable extraction process practiced in an economically viable manner will subject the solvent to the presence of water in the flake. The following description of the present invention contemplates the presence of water and its pick-up by the solvent from the flake. In the later examples data is presented in which the solvent is saturated with water prior to use to simulate the condition in a commercial operation where the solvent becomes saturated by repeated contact with the water wet flake or by steam distillation of the solvent from the product and or meal. Some of the protic solvents such as ethanol contain dissolved water, e.g. 5% in the case of ethanol, but when in combination with the chlorinated hydrocarbon solvent the system will still pick-up some additional water. Thus, applicants have found that chlorinated solvent/alcohol solutions, particularily ethanol containing 5% water, can pick up and hold water an excess of the amount initially present in the ethanol. The amount of extra (dissolved) water picked up increases as the amount of alcohol in the solvent is increased. However, addition of water in excess of saturation point of the chlorinated solvent/ co-solvent may reduce the effectiveness of the solvent system, due to the presence of free water. When free water is present, it adheres to the flakes

forming a somewhat impermeable cake, which reduces solvent penetration resulting in decreased extraction efficiencies. Thus, the presence of water in the extraction process, so long as it is not free water, is not detrimental and is especially advantageous with some of the combinations herein disclosed.

The proportions of the two classes of solvents used in accordance with the present invention are not critical but for economic and time efficiency from about 0.5 to about 20 percent by weight of the aprotic or protic solvent is generally preferred.

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLE 1

Cottonseed meal containing approximately 0.8% free gossypol was extracted in a soxhlet extractor to remove free gossypol. The solvents used were:

1. MeCl.sub.2 (100%)

2. MeCl.sub.2 +MeOH (6% Vol.)

3. MeCl.sub.2 +Acetone (10% Vol.)

*MeCl.sub.2 =methylene chloride; MeOH=methyl alcohol

The cottonseed meal was placed in a thimble and installed in the soxhlet apparatus. Low heat was applied which allowed a slow thimble immersion cycle rate, simulating the percolation process commonly used in the industry. Extracted meal was dried and analyzed for residual free gossypol.

Results are as follows:

______________________________________ Residual Free Gossypol (% Wt.) Total Total Total Cycles Time Cycles Time Cycles Time Solvent 3 (min) 5 (min) 10 (min) ______________________________________

MeCl.sub.2 0.21 70 0.12 105 0.083 210 MeCl.sub.2 /6% 0.096 70 0.063 105 0.041 210 MeOH MeCl.sub.2 /10% 0.20 70 0.15 105 0.063 210 Acetone ______________________________________

EXAMPLE 2

Cottonseed meal was extracted with various solvent blends for oil and gossypol removal. This method employed agitating the samples on a wrist action shaker for extraction. The blends used were:

1. 7% MeOH in MeCl.sub.2

2. 5% EtOH* in MeCl.sub.2

3. 10% Ethyl ether in MeCl.sub.2

4. 10% DMF in MeCl.sub.2

5. 14% DMSO in MeCl.sub.2

6. MeCl.sub.2 (control)

*EtOH=95% ethanol 5% water

Flaked cottonseed meal containing 0.8% free gossypol was placed in an 8 oz. bottle, covered with solvent, and secured to a wrist action shaker for 15 minutes of agitation. The miscella was poured off, fresh solvent added and the process repeated. A total of four 15 minute extractions were performed on each sample. The samples were desolventized in an oven at 60.degree. C. for one hour then analyzed for free gossypol.

Data is as follows:

______________________________________ Solvent Free Gossypol (Wt. %) ______________________________________ MeOH/MeCl.sub.2 0.051 EtOH/MeCl.sub.2 0.058 Ethyl ether/MeCl.sub.2 0.140 DMF/MeCl.sub.2 0.0236 DMSO/MeCl.sub.2 0.0123 MeCl.sub.2 0.135 ______________________________________

Industrial application of the present invention can conveniently be carried out by contacting in a moving or fixed bed of cottonseed (flakes, expanded or ground material) with a countercurrent flow of the solvent blend in an apparatus whereby the material is in contact with the solvent blend for from 5 to 600 minutes, preferably about 60 minutes. Batchwise operations can be employed with equal success, that is a quantity of the solvent is circulated through the bed until its toxin content is equal to or near the solvent's saturation point. Optional extraction techniques such as ultrasonic assisted extractions, mixing, etc. will also work. A preferred extraction technique is the conventional percolation technique or percolation like technique now used for hexane extraction wherein initial aliquots of flakes are contacted with oil rich solvent, and such so contacted flake aliquot is successively contacted with successively less oil rich solvent, the final contact being with oil free solvent. The solvent can be recovered by distillation techniques and reused. The recovery may be carried out simultaneously with the contacting in adjacent conventional distillation equipment, or periodically, depending upon the nature of the operation.

The process of course can be conveniently carried out in any of the myriad of equipment conventionally employed by the industry and the solvent recovered in stills and reused.

The toxins may be isolated from the oil/solvent solution for purification or removed via conventional refining for disposal.

EXAMPLE 3

A comparison of oil extraction from cottonseed meal employing either hexane or MeCl.sub.2 was performed using a 6".times.6".times.69" column simulating a deep bed counter current percolation extractor. The extraction was performed in a semi-countercurrent fashion, with fresh flakes in the bed being percolated with consecutive miscella (oil & solvent) solutions of decreasing oil content, followed by a rinse with fresh solvent. Miscella solutions were prepared by adding cottonseed oil to the solvent, to create desired miscella oil concentration. In the extraction sequence, the entire miscella solution of a given stage was allowed to percolate down through the bed of flakes, with miscella exiting the bottom recycled back to the head of the column for 10 minutes of additional percolation. This miscella was then stored in a separate container. Then the next miscella of the series (lower oil content) was contracted with the same bed of flakes in an identical manner, and so on, with the final percolation solvent being initially oil free.

In the MeCl.sub.2 extraction, fresh flakes were first percolated with miscella (MeCl.sub.2 +oil) containing 6.8% oil, followed by successive miscellas of 3.3% oil and 1.0% oil, and a clean MeCl.sub.2 final percolation, simulating a four stage countercurrent extraction. Solvent to flake ratio (wt) was held at 2:1 for each percolation stage.

The hexane extraction consisted of 5 stages, with initial miscella oil concentration of 10, 7, 5, 3.0 and 0.0% oil in hexane, respectively, performed in a manner identical to the MeCl.sub.2 extraction. The solvent to flake ratio (wt) for the hexane extraction was 1.1 to 1, due to the lower density of hexane. Results are as follows.

______________________________________ Initial Oil in Flakes Residual Oil in Meal (% wt) (% wt) ______________________________________ MeCl.sub.2 29.8 1.17 Hexane 29.8 1.57 ______________________________________

EXAMPLE 4

Flaked cottonseed meal was soaked in fresh solvent in a 125 ml separatory funnel, with the solvent solution drained off through the stopcock. Eight successive 15 minute soak periods were performed with each solvent blend on its respective flake sample. Extracted meal samples were desolventized to a bulk temperature of 80.degree. C. in a vacuum oven, over a 45 minute period. Results are presented on an as-is basis.

______________________________________ Extracted Meal Toxin Residuals Free Feed Analysis Aflatoxins Gossypol Solvent (B.sub.1 + B.sub.2, ppb) % ______________________________________ Feed Analysis 194 .426 MeCl.sub.2 140 .048 MeCl.sub.2 + .5% EtOH.sup.1 123 .043 MeCl.sub.2 + 2.1% EtOH 2 .034 MeCl.sub.2 + 5.0% EtOH 1 .012 MeCl.sub.2 + 10% EtOH 1 .003 Feed Analysis 183 .449 MeCl.sub.2 + 2% EtOH.sup.1 + .5% H.sub.2 O 1 .022 MeCl.sub.2 + 2% EtOH + 1% H.sub.2 O .5 .118 Feed Analysis 207 .494 MeCl.sub.2 152 .045 MeCl.sub.2 + .52% MeOH.sup.1 63 .052 MeCl.sub.2 + 2.1% MeOH .6 .016 MeCl.sub.2 + 2% MeOH + .32% H.sub.2 O

.5 .011 MeCl.sub.2 + 5.2% MeOH .5 .012 Feed Analysis 195 .456 MeCl.sub.2 139 .030 MeCl.sub.2 + .5% DMSO.sup.1 147 .033 MeCl.sub.2 + 2% DMSO 139 .006 MeCl.sub.2 + 2% DMSO, H.sub. 2 O sat. 2 .018 MeCl.sub.2 + 5% DMSO 2 .006 Feed Analysis 213 .422 MeCl.sub.2 122 .067 MeCl.sub.2 + .05% ISOH.sup.1 131 .056 MeCl.sub.2 + 2% ISOH 118 .037 MeCl.sub.2 + 2% ISOH, H.sub.2 O Sat. 3 .035 MeCl.sub.2 + 5% ISOH 158 .015 ______________________________________ .sup.1 EtOH = 95% ethyl alcohol 5% water; MeOH methyl alcohol; DMSO = dimethylsulfoxide; ISOH = isopropyl alcohol.

EXAMPLE 5

Toxin contaminated cottonseed meals were cracked in a flaking mill. For conditioning, tweIve percent moisture was added and the meals were cooked to 180.degree. F. After cooking and flaking to 0.008-0.012 inches, final moisture content was 10% by weight. Conditioned Cottonseed flakes were extracted in a pilot scale, Crown Iron works shallow bed percolation extractor. Solvent to flake ratio was 2:1 by weight and extraction was at room temperature. Extracted cottonseed flakes were desolventized in a conventional pilot scale desolventizer. Results reported below are the desolventized meals on an as-is basis.

______________________________________ Free Aflatoxin B.sub.1 + B.sub.2 Gossypol Oil Solvent (ppb by wt) (% wt) (% wt) ______________________________________ MeCl.sub.2 61 0.135 1.19 MeCl.sub.2 + 5% EtOH 26 0.056 0.65 Feed Flake Analysis 262 0.694 28.0 ______________________________________

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**R. J. Hron Sr.1  , P. J. Wan1 and M. S. Kuk1

(1)  SRRC, ARS, USDA, 70179 New Orleans, Louisiana(2)  SRRC, P.O. Box 19687, 70179 New Orleans, LA

Received: 20 September 1995  Accepted: 3 July 1996  

Abstract  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.

Key Words  Alcohol - cottonseed - deactivation - ethanol - feed - ferrous sulfate - gossypol - isopropanol - vapor

Presented in part at the AOCS Annual Meeting, Atlanta, GA, May 8–12, 1994.

References secured to subscribers.

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**Summary  In a series of four tests in which three deliberately chosen toxic cottonseed meals were treated with aqueous solutions of salts and alkalies, it was found that the best detoxifying effect was obtained with sodium hydroxide, followed very closely by potassium and ammonium hydroxides. Dry heat treatment alone did not detoxify, and mortality was high. Treatment with moisture plus heat gave partial detoxification. Of the 22 chemically-treated cottonseed meal samples tested, those treated with alkalies showed the best weight gains, the order of decreasing effectiveness being

. The residual toxicity of treated cottonseed meals cannot be explained on the basis of their free gossypol content as analyzed for meals with high values

gave better growth performance than some with lower levels of free gossypol. There were also very marked differences in final body weight after 8 weeks of feeding six different treated cottonseed meal samples having practically the same free gossypol

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**Titre du document / Document titleA study on the reduction of gossypol levels by mixed culture solid substrate fermentation of cottonseed meal

Auteur(s) / Author(s)WENJU ZHANG ; ZIRONG XU ; JIANYI SUN ; XIA YANG ;

Résumé / AbstractThe objective of this work was to study the effect of mixed culture solid substrate fermentation of C. tropicalis ZD-3 with A. niger ZD-8 on detoxification of cottonseed meal (CSM), and to investigate the effect of fermentation period, proportion of CSM in substrate, sodium carbonate, minerals and heat treatment on the reduction of free gossypol levels during mixed culture solid substrate fermentation of CSM. Experiment 1: Three groups of disinfected CSM substrate were incubated for 48 h after inoculation with either of the fungi C. tropicalis ZD-3, A. niger ZD-8 or mixed culture (C. tropicalis ZD-3 with A. niger ZD-8). One non-inoculated group was used as the control. Levels of initial and final free gossypol (FG), CP and in vitro CP digestibility were assayed. The results indicated that mixed culture fermentation was far more effective than single strain fermentation, which not only had higher detoxification rate, but also had higher CP content and in vitro digestibility. Experiment 2: CSM substrates were treated according to experimental variables including fermentation period, proportion of CSM in substrate, sodium carbonate, minerals and heat treatment, Then, the treated CSM substrates were inoculated with mixed

culture (C. tropicalis ZD-3 with A. niger ZD-8) and incubated at 30°C for 36 h in a 95% relative humidity chamber. After fermentation ended, FG and CP content of fermented CSM substrate was assayed. The results showed that the appropriate fermentation period was 36 h, and the optimal proportion of CSM in substrate was 70%. Addition of sodium carbonate to CSM substrate was beneficial for fermentative detoxification. Heat treatment could facilitate fermentative detoxification, and supplementation with minerals was instrumental in reducing gossypol levels during mixed culture solid substrate fermentation of CSM.

Revue / Journal TitleAsian-australasian journal of animal sciences  (Asian-australas. j. anim. sci.) ISSN 1011-2367 

Source / Source2006, vol. 19, no9, pp. 1314-1321 [8 page(s) (article)]

Langue / LanguageAnglais

Editeur / PublisherAsian-Autralasian Association of Animal Production Societies, Kwachon-Shi,Kyunggi-Do, COREE, REPUBLIQUE DE (1988) (Revue)

Mots-clés d'auteur / Author KeywordsFungi ; Free Gossypol ; Detoxification ; Fermentation ; Cottonseed Meal ; Sodium Carbonate ; Minerals ;

Localisation / Location

INIST-CNRS, Cote INIST : 27305, 35400015714588. cottonseed meal by Candida tropicalis ZD-3 during solid substrate fermentation

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**Wen-Ju Zhanga, b, Zi-Rong Xua, , , Shun-Hong Zhaob, Jun-Fang Jianga, Yan-bo Wanga and Xiang-Hua Yana aKey Laboratory of Molecular Animal Nutrition, Ministry of Education, Animal Science College, Zhejiang University, Hangzhou 310029, PR ChinabCollege of Animal Science and Technology, Shihezi University, Shihezi, Xinjiang 832003, PR China Received 6 January 2006;  revised 11 May 2006.  Available online 19 May 2006.

Abstract

The objective of this work is to optimize the process parameters for detoxification of gossypol in cottonseed meal (CSM) by Candida tropicalis ZD-3 during solid substrate fermentation (SSF). The maximum detoxification efficiency of gossypol was achieved by employing the substrate, which consists of 70% of CSM, 20% of corn flour and 10% of wheat bran. The optimum fermentation conditions for gossypol detoxification are incubation period of 48 h, incubation temperature at 30 °C, inoculum level 5% v/w, moisture content of solid substrate 50% and pH in nature. Adding minerals solution to CSM substrate benefit fermentation detoxification.

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**0160A process to lower the free gossypol content of cottonseed cakes

The recent "Guideline for Edible Cottonseed Protein Flours and Related Products" (Food and Nutrition Bulletin, vol. 2, no. 3) provided up-to-date information on cottonseed products and processes for their production.

A technical article describing a very simple process to lower the free gossypol content of cottonseed cakes-H. Mayorga, J. Gonzalez, J.F. Menchu, and C. Rolz, "Preparation of a Low Free Gossypol Cottonseed Flour by Dry and Continuous Processing," J. Food Sci., 40: 1270 (1975) was unfortunately overlooked by the experts who prepared the Guideline. The process was developed at the Instituto Centroamericano de Investigación y Tecnología Industrial (ICAITI) and tested on a pilot-plant scale. Unfortunately, because of the fixed price structure existing in Central America for cottonseed products, the industrial consortium that had decided to undertake production on a commercial scale shelved the project until the situation changes. However, we have received inquiries about it from many parts of the world and believe that a description of the process could be of interest to your readers as complementary information to the Guideline.

A laboratory study was carried out to determine the kinetics of gossypol reduction in cottonseed meal mixed with 0.5 per cent ferrous sulphate and 1 per cent calcium hydroxide at several temperatures. It was found that the reaction followed first-order kinetics and that the variation of the rate constant with temperature could be described well with the Arrhenius equation. A similar study was done for the thermal available Iysine destruction in cottonseed meal samples heated for 1 hour at 100°, 125° ,150°, and 170° C. It was found that the available Iysine content decreased significantly after heating for 40 minutes at 170°C. No significant difference was found at lower temperatures.

A pilot plant was built for the continuous processing of cottonseed meal to achieve gossypol reduction. Two arrangements for solids were tested, one using a bag collector and the other using a cyclone separator. The arrangement using the cyclone worked well, while the other method was less satisfactory. Several runs were made at different temperatures and flow rates. It was found possible to obtain a homogeneous product with a final gossypol content of 0.04 per cent. Trying to reduce it further by increasing the temperature is not recommended because of a higher lysine loss. Gossypol reduction was a function of temperature, residence time, initial gossypol content, and amount of chemical additives, but not of meal flow rate.

The dry continuous process has the following advantages over the new technologies that the Guideline makes reference to (liquid cyclone, acetone, membrane, aqueous extraction):

i. it can be adapted to existing commercial processes;ii. the investment is minimal relative to the alter natives; andiii. it does not need special solvents, special equipment, or a new plant.

Carlos RolzHead, Applied Research DivisionInstituto Centroamericano de Investigatión y Tecnología IndustrialGuatemala

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**Iron Sulfate and Feed Pelleting to Detoxify Free Gossypol in Cottonseed Diets for Dairy Cattle

M. L. Barraza 1, C. E. Coppock 1, K. N. Brooks 1, D. L. Wilks 1, R. G. Saunders 1, and G. W. Latimer Jr. 1

1 Texas A&M University, College Station 77843

Thirty-two lactating cows were fed ad libitum diets with 1) 18.7% soybean meal, 2) diet 1 plus 500 ppm supplemental Fe from FeSO4.H2O, 3) 15% whole cottonseed, or 4) diet 3 plus 500 ppm Fe from FeSO4.H2O. Dry matter intakes were similar except for cows fed diet 2, which was lower. Cows fed whole cottonseed diets ingested 23 g/d of free gossypol per cow. Free gossypol apparently excreted was lower than its intake. Iron excretion was similar to Fe intake. Blood metabolites and productive performance did not differ among the diet groups. No signs of gossypol toxicity were observed.

Twelve neonatal Holstein male calves were fed a commercial milk replacer for 4 wk, then were allowed ad libitum access to diets with 1) 27% soybean meal, 2) 50% whole cottonseed, or 3) diet 2 plus 500 ppm Fe from FeSO4.H2O. Dry matter intakes were similar but slightly lower for calves fed diet 3. Daily individual intakes of free gossypol from diets 2 and 3 were 2 g, which was lower than the expected 4 g due to an apparent effect of pelleting. Blood metabolites did not differ among the groups, and calves averaged

about .6 kg of daily gain on these diets. A follow-up study showed that pelleting reduced free gossypol by as much as 70% in whole cottonseed and by 48% in cottonseed meal. Pelleting represents a mechanism to decrease the toxicity of gossypol in cottonseed products.

Key Words: detoxifying • gossypol • cattle

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**Article

Detoxification and nutritional evaluation of solvent extracted Egyptian cottonseed mealFikry I. El-Nahry 1, Dr. Nargis S. Bassily 1, Seham A Tharrwat 2

1Nutrition Institute, Ministry of Health, 16, Kasr El-Aini Str., Cairo2Faculty of Home Economics, Helwan University, Cairo EgyptABSTRACT

Attempts for degossypolization of the solvent extracted Egyptian cottonseed meal (CSM) and evaluation of its nutritional value as a possible protein supplement for human feeding were undertaken. Three methods were applied for degossypolization: Steaming, addition of iron(II)-sulphate dihydrate, and steaming after addition of iron(II)-sulphate dihydrate. Steaming was done for varying periods of time. Nutritional evaluation was undertaken by chemical analysis and by feeding experiments. Chemically, the third method for degossypolization was the most effective. Feed efficiency and protein efficiency for diets containing 15 and 10% levels of CSM protein indicated that all treatments used for detoxification caused a decrease in diet and protein quality. Diets containing iron-supplemented CSM at the same level were the best of all. Mixing CSM with casein to form a diet containing 10% protein increases the protein quality of both casein and CSM.

Received: 30 August 1992 DIGITAL OBJECT IDENTIFIER (DOI)

10.1002/food.19830270607  About DOI

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**Studies on the Chronic Oral Toxicity of Cottonseed Meal and Cottonseed Pigment Glands

Anthony M. Ambrose and Dorothy J. Robbins

Pharmacology Laboratory, Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture, Albany, California

The results of these experiments point to the conclusion that cottonseed meal free of pigment glands is devoid of any toxic principle as judged by the growth of rats and histopathological examination of visceral organs. Our results would indicate that hexane-extracted meal is not completely devoid of toxic principles or appetite-depressing factors, as judged by inhibition in the growth of rats on a diet containing 15% or more of the meal. This inhibition in growth cannot be accounted for completely by the slight difference in food consumed between rats on this diet and the controls or rats on a lower dietary intake of hexane-extracted meal. It would appear at first sight that the toxicity of hexane-extracted meal (15% level) may be due to the gossypol present in the meal, since 0.256% of pigment glands (equivalent to 0.096% gossypol) in the diet of rats produced an inhibition in growth comparable to that produced by the diet containing 15% of hexane-extracted meal. However, it is doubtful if gossypol is the chief responsible

constituent, since rats on the 0.256% pigment gland diet processed from seeds stored for approximately 6 months were not similarly affected. This difference in toxicity of the two samples of pigment glands used may be accounted for on the basis of storage time before processing for removal of the pigment glands. The gossypol content of the two pigment-gland diets was extremely small, 0.096 and 0.0934%, respectively, as compared to 0.16% for the diet containing 15% of hexane-extracted meal. Comparable

chronic toxicity studies of gossypol were not made, because of the unavailability of enough gossypol at the time the studies with cottonseed meal and pigment glands were being carried out.

Paired feeding experiments with cottonseed pigment glands and gossypol would tend to confirm the above observations that decrease in growth is not entirely due to reduced food intake, but that gossypol plays an important role in depressing growth.

From the acute and subacute oral toxicological data obtained, it is concluded that the toxicity of cottonseed pigment glands and gossypol is low. Single oral doses of pigment glands of up to 3,000 mg/kg, or gossypol in doses of up to 800 mg/kg each, did not appear to produce any toxic symptoms.

The repeated administration of cottonseed pigment glands in doses of 2,000 mg/kg three times at 7-day intervals caused the rats to lose weight, depressed appetite, and produced diarrhea. Similarly, the repeated oral administration of gossypol in doses of 500 mg/kg at 5-day intervals had an effect similar to that of the pigment glands except that it did not produce diarrhea. Lower doses of pigment glands or gossypol were without effect.

The experimental data raise the question of whether or not chemical assay of cottonseed pigment glands for gossypol is a reliable index, since a difference has been observed in the toxicity of pigment glands processed at different times, while the gossypol content was approximately the same.

Since the completion of this work a paper by Lillie and Bird (’50) on chicks has appeared in which our findings on the toxicity of cottonseed pigment glands and gossypol are confirmed.

**Detoxification of Gossypol in Raw Cottonseed and the Use of Raw Cottonseed Meats as a Replacement for Soybean Meal in Diets for Growing-Finishing Pigs1

Romulo Rincon2, F. H. Smith3 and A. J. Clawson3 ,4, North Carolina State University, Raleigh 27650

Abstract Ratios of added iron to free gossypol in corn-raw cottonseed diets in the range of .5:1 to 1:1 reduced free gossypol level in the liver of rats enough to alleviate symptoms of toxicity (reduced feed intake, weight loss and eventual death). When iron to gossypol ratios within this range were used, rate of gain and feed efficiency were significantly superior to that

obtained when ratios either higher than 1:1 or lower than .5:1 were used. Higher ratios of iron to gossypol 1.5 and 2:1 resulted in excessive amounts of dietary iron.

It was observed that the use of warm water (50 C), in preparing the ferrous sulfate-cotton-seed meats mixture, improved the combination of iron and

gossypol to the extent that free gossypol in the redried meats was reduced from 1.17 to .01%.

The addition of propionic acid as a mold inhibitor apparently reduced the binding of gossypol and iron as evidenced by higher free gossypol values in cottonseed meats treated with a ferrous sulfate solution. However, liver residues of gossypol in rats were not affected by the presence of propionic acid in the diets in which the cottonseed meats were used.

The substitution of uncooked and unextracted cottonseed meats (treated with ferrous sulfate) for soybean meal (in the range of 20 to 80% on an equal protein basis) was without effect on performance of growing-finishing pigs (25 to 90 kg). Accumulation of gossypol in the pigs livers was low and there was a trend for this level to be reduced as cottonseed level in the diet was increased (increasing dietary cottonseed level also increased total dietary iron since ferrous sulfate was added to the cottonseed meats at a 1:1 weight ratio to free gossypol).

Footnotes 1 Paper No. 5397 of the Journal series of the North Carolina Agricultural Experiment Station, Raleigh. The use of trade names in this

publication does not imply endorsement by the North Carolina Agriculture Experiment Station of the products named, nor critisism of similar ones not mentioned.

2 Present address: Venezuelan Ministry of Agriculture, Maracoibo,

Venezuela.

3 Animal Science Department.

4 Appreciation is expressed to Cotton Incorporated for support of this project.