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**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 value
doi: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
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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
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Referenced by:
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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
______________________________________

<- Previous Patent (Calcium hypochlorite compositions) | Next Patent


(Cooling apparatus) ->

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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 title


A 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 / Abstract
The 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 Title
Asian-australasian journal of animal sciences (Asian-australas. j. anim. sci.)
ISSN 1011-2367
Source / Source
2006, vol. 19, no9, pp. 1314-1321 [8 page(s) (article)]
Langue / Language
Anglais
Editeur / Publisher
Asian-Autralasian Association of Animal Production Societies, Kwachon-
Shi,Kyunggi-Do, COREE, REPUBLIQUE DE (1988) (Revue)
Mots-clés d'auteur / Author Keywords
Fungi ; 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
a
Key Laboratory of Molecular Animal Nutrition, Ministry of Education,
Animal Science College, Zhejiang University, Hangzhou 310029, PR China
b
College 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; and
iii. it does not need special solvents, special equipment, or a new plant.

Carlos Rolz
Head, Applied Research Division
Instituto Centroamericano de Investigatión y Tecnología Industrial
Guatemala

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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 meal
Fikry I. El-Nahry 1, Dr. Nargis S. Bassily 1, Seham A Tharrwat 2
1
Nutrition Institute, Ministry of Health, 16, Kasr El-Aini Str., Cairo
2
Faculty of Home Economics, Helwan University, Cairo Egypt
ABSTRACT
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.