Toxicology in the Use, Misuse and Abuse of Food, Drugs and Chemicals

Arch. Toxicol., Suppl. 6, 167-174 (1983)

© by Springer-Verlag 1983

Gossypol in Cottonseed Products:

Toxicology and Inactivation

S. Yannai and D. Bensal

Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology,
Haifa 32000, Israel

Abstract. The little attention given to the potential of cottonseeds as food for
monogastric animals and man is due to the presence of toxic principles in
them, expecially gossypol. The injury to livestock associated with ingestion
of cottonseeds results from the unfavorable physiological effect of gossypol
per se, as well as from its adverse effects on the protein nutritive value of the
food (mainly by rendering lysine metabolically unavailable). Various
approaches have been employed to overcome these problems, by removing
the gossypol glands, growing glandless varieties, extraction of gossypol with
solvents, or through inactivation by heat processing, reacting with cations or
with certain organic compounds. In this study the phospholipid fraction
(gums) derived from soybeans, which is relatively rich in phospholipids
containing a free amino group, was used with a view to lowering the tendency
of gossypol to bind with lysine, and at the same time to inactivate the
gossypol by converting it into a bound form. Dehulled and flaked
cottonseeds were mixed with 5% soybean gums and 0.5% NaOH, in a
laboratory cooker resembling the apparatus used in the cottonseed
processing industry. The meat was cooked for different periods and at
several temperatures. Later the oil was extracted with hexane and the meal
was tested for free gossypol, "available lysine" and protein nutritive value, in
comparison with a control batch treated similarly but without gums. The
results proved the superiority of the meal treated with gums, as evidenced by
all the above parameters.
Key words: Gossypol - Protein - Available lysine - Nutritive value -
Phospholipids

Gossypol is a yellow pigment occurring in the genus Gossypium (cotton plant)

and in some other plants of the family Malvaceae. This highly reactive
polyphenolic and polyaldehydic compound is present in most parts of the cotton

The study was supported by Milouot, Ltd., and the Ministry of Industry and Commerce, Israel.
168 s. Yannai and D. Bensa!

plant in special "pigment glands", and provides resistance to certain insects

(Berardi and Goldblatt 1980); hence it may decrease the need for insecticides.
Moreover, gossypol isolated from cotton seeds was found to possess consid-
erable pesticidal activity (Abou-Donia 1976; Jones 1979; A. Nir, personal
communication) .
Unfortunately, gossypol is also toxic for monogastric farm animals,
especially pigs and poultry (Abou-Donia 1976), and even for young calves, until
the rumen is fully developed (Adams et al. 1960; Jones 1979). Therefore,
although cottonseed meal has the potential of becoming a low-cost, high-quality,
protein-rich food for monogastric animals and humans (Cater et al. 1977; Milner
1980), its use has mostly been limited, for many years, to ruminant livestock.
The latter animals species can also tolerate fairly high levels of other toxic
principals occurring in cottonseed meal.
The ill effects in non-ruminants associated with the ingestion of gossy-
pol-containing cottonseed products may be due to the unfavorable physiological
influence of gossypol per se, as well as from its adverse effect on the protein
nutritive value by rendering lysine metabolically unavailable (Abou-Donia
1976). The latter effect is exerted through reaction between the aldehydic groups
of gossypol and the free epsilon-amino groups of lysine (Conkerton and
Frampton 1959).
The mild toxicity symptoms observed in non-ruminants, which result from
chronic exposure to relatively small doses of gossypol, include mainly depressed
appetite, malnutrition, growth retardation, hypoprothrombinemia and diarrhea
(Adams et al. 1960). Acute toxicity is best manifested in circulatory failure; and
subacute toxicity, in pulmonary edema (Abou-Donia 1976). Pigs, which are very
sensitive to gossypol, show an oral LDso of 550mglkg body weight (Lyman et al.
1963), whereas that reported for rats is ca. 2,600 mg/kg (EI-Nockrashy et al.
1963). Other pathological findings included gastritis, enteritis in the lower part
of the duodenum, congestion throughout the intestine and the kidneys,
prominent liver lobules and haemorrhage (EI-Nockrashy et al. 1963), accumu-
lation of fluid in the body cavities, cachexia, hind leg paralysis and inanition
(Alsberg and Schwartze 1919, cited by Berardi and Goldblatt 1980) and decrease
in erythrocyte count due to inhibition of hematopoiesis (Danke and Tillman
1965). The latter effect could be prevented by administration of iron (Tone and
Jensen 1974), a fact that can be ascribed to chelation of liver iron by gossypol,
thus interfering with synthesis of hemoglobin, and possibly of respiratory
enzymes (Skutches et al. 1973) and enzymatic function involving iron as well
(Abou-Donia 1976). Another effect observed was an almost complete blocking
of spermatogenesis in men (National Coordinating Group on Male Antifertility
Agents 1978, cited by Berardi and Goldblatt 1980). A list summarizing the major
antemortem and postmortem symptoms exhibited by nonruminant species, with
the respective references, was compiled by Berardi and Goldblatt (1980). Many
of the physiologically adverse effects exerted by gossypol can be attributed to its
inhibitory action on certain enzymes, by reacting with the substrate or by
combining with the enzyme itself. Both types of action have been shown to be
involved in gossypol-inhibited proteolysis. Interference with enzyme-catalyzed
reactions may also cause uncoupling of respiratory chain-linked phosphorylation
Gossypol in Cottonseed Products: Toxicology and Inactivation 169

(Abou-Donia and Dieckert 1974), possibly by affecting the activity of hepatic

mitochondria oxidative enzymes (Abou-Donia 1976). Thus, the unfavorable
influence of gossypol on protein metabolism is twofold; not only does it inhibit
proteolysis by enzymes of the digestive system (Damaty and Hudson 1979), but
(as is mentioned above) it also renders lysine, which is the first limiting amino
acid in cottonseed protein, unabsorbable through the intestinal mucosa
(Altschul et al. 1958; Panemangalore et al. 1970), thereby further lowering the
protein nutritional quality.

Detoxification and Elimination of Gossypol

Heat processing of cottonseed meats has been employed for p1any years as an
effective treatment for inactivating gossypol (i.e., conversion of the pigment
from the free to the bound form). The resulting product is practically nontoxic;
however, this can only be accomplished at the expense of some of the metabolic
availability of lysine, as is explained above (Adams et al. 1960). Such binding of
free gossypol is very rapid at a high cooking temperature; but it also takes place
during prolonged storage, especially at elevated ambient temperature and
humidity. The protein nutritive value of cottonseed meal was found to increase
and its free gossypol content to decrease with storage time (Yannai and
Zimmermann 1970). It appears that the beneficial effect of heat or prolonged
storage, brought about by inactivation of gossypol, can be accomplished by
several more recent techniques, the most popular of which are solvent extraction
and the "liquid cyclone process" (which involves centrifugal separation of intact
pigment glands in a hexane slurry) (Cater et al. 1977). Both methods are of an
obvious advantage over heat treatments, as the protein nutritional quality of the
resulting meal should theoretically remain unaffected. Yet, the additional cost
involved makes them forbidingly expensive, except, perhaps, when employed
for the production of protein concentrates intended for human consumption.
Other techniques proposed, which have never been fully explored, include
cultivation of gossypol-free ("glandless") varieties, reacting gossypol with
various cations which render the pigment inactive and/or unabsorbable (such as
iron, calcium and other metals), and its destruction by mixtures of steam with
sulfur dioxide, alkali, hydrogen peroxide and other oxidizing agents, and even
fermentation with the fungus Diplodia (Abou-Donia 1976).
In the study reported here a different approach was tried, which does not call
for major changes in the process commonly used in the cottonseed industry. In
this method "gums" produced from soybeans were used. These are rich in
phospholipids containing free amino groups and can therefore react with the
aldehyde groups of free gossypol, thus converting it into a bound form (a Schiff's
base), similar to that formed with amino acids. The gums were added to the
cottonseed meats prior to the cooking operation employed in most processing
plants to facilitate high extractability of the oil, with a view to lowering the free
gossypol content and at the same time achieving a higher "available" lysine
content in the treated meal.
170 S. Yannai and D. Bensal

Table 1. Chemical characterization of the flaked cottonseed meats used

Component Concentration

Moisture 7.6%
Proteine (N . 6.25) 34.4%
Crude fiber 3.2%
Oil 34.2%
Free gossypol 0.84%
Total gossypol 1.01%
Bound gossypol 0.17%
"Available" lysine 3.7 g/lOO g protein
Nitrogen solubility 97.6%

Materials

All experiments were conducted with commercially dehulled and flaked

cottonseeds (meats), the chemical characterization of which is given in Table 1.
The gums (phospholipid fraction), received from an oil manufacturing company,
were obtained by "degumming" crude soybean oil. The laboratory cooker used
for the heat treatments of the meats was designed to process 5 kg batches in a
manner resembling a commercial cooking process. It was divided into two parts
which made it possible to cook two samples of meats at the same time and under
the same conditions. Cooking was performed at different temperatures and for
different time intervals. After establishing the optimal conditions the tested
sample was mixed with 5% (w/w) phospholipids and 0.5% (w/w) NaOH,
whereas the control sample was only mixed with 0.5% (w/w) NaOH. (Addition
of NaOH greatly increased the gossypol elimination efficiency.) The meats were
also mixed with an appropriate amount of water, to bring the moisture content
up to the desired level in each case. Oil extraction was carried out with hexane
and the resulting meals were subjected to chemical analyses and a biological
test.
The experimental animals employed were weanling rats of the Charles River
C.D. strain from our stock colony. Each experimental group consisted of four
males and four females, which were housed in individual metal screen cages, to
permit determination of individual food intake. The diets and tap water were
offered without restriction for 10 days.

Methods

Free gossypol was determined by the method of Pons and Guthrie (1949), using
para-anisidine as reagent; or by the method described in the Official and
Tentative Methods of the AOCS (1964), using aniline as reagent. Total gossypol
was determined by the method of Pons et al. (1958). "Available" lysine was
determined as described by Kakade and Liener (1969). "Chemical Index" was
determined by the method of Lyman et al. (1953).
Gossypol in Cottonseed Products: Toxicology and Inactivation 171

The protein nutritive value for rats was estimated by determining the protein
efficiency ratio (P.E.R.), which is defined as the gain in weight per gram of
protein consumed.

Results and Discussion

The free gossypol and available lysine contents of the cottonseed meals used in
the biological test, one being a commercial product and the other one heated at a
much lower temperature but with 5% phospholipids, are presented in Table 2.
As can be seen from the table the free gossypol content of the latter meal was
lower and its available lysine higher than those of the former meal.
The proportions of the protein-contributing constituents of the diets are
shown in Table 3 and the detailed compositon of the diets in Table 4. The results
of the biological experiment are given in Table 5 and their statistical analysis in
Table 6. It is evident from Table 5 that while a level of 20% of the commercial
meal in the diet did not cause a significant decrease in P.E.R., the diets
containing 40% and 60% of this meal (meal A) had a statistically significantly
lower protein nutritive value than the diets containing corresponding amounts of
the experimental meal (meal B). A similar picture is also revealed by the growth

Table 2. The protein, free gossypol and "availbale" lysine contents of the cottonseed meals used in
the biological experiment

Treatment before Commercial meal A (control) Meal B (experimental)

oil extraction Heating at 110° for 1 h, Heating at 90° for 1 h,
without added phospho- with 5% soybean phospho-
lipids, 17% moisture lipids and 0.5% NaOH,
20% moisture

Protein (%) 50 47
Free gossypol (%) 0.057 0.044
"Available" lysine
(g/100 g protein) 2.60 3.06

Table 3. Source of protein and amino acid supplements in the diets used

Diet for Source of protein Supplements

group no.

1 Soybean meal 100% methionine

2 Soybean meal 80% + cottonseed meal A 20% methionine
3 Soybean meal 60% + cottonseed meal A 40% methionine
4 Soybean meal 40% + cottonseed meal A 60% methionine
5 Soybean meal 40% + cottonseed meal A 60% methionine + lysine,
histidine, threonine
6 Soybean meal 60% + cottonseed meal B 40% methionine
7 Soybean meal 40% + cottonseed meal B 60% methionine
172 S. Yannai and D. Bensal

Table 4. Detailed composition of the diets given to the different groups

Ingredient (%) Group

1 2 3 4 5 6 7

Soybean meal 21.3 17.0 12.8 8.5 8.5 12.8 8.5

Cottonseed meal A 4.0 8.0 12.0 12.0
Cottonseed meal B 8.5 12.8
Methionine 0.12 0.12 0.12 0.12 0.12 0.12 0.12
Lysine 0.4
Histidine 0.125
Threonine 0.25
Standard ingredients a 15.5 15.5 15.5 15.5 15.5 15.5 15.5
Corn starch 63.08 63.08 63.58 63.88 63.105 63.08 63.08

a Soybean oil 8%, mineral mix 4%, vitamin mix 1%, choline chloride 0.5%, powdered cellulose
2%

Table 5. Weight gain and protein efficiency ratio (P.E.R) of rats

given the different diets (means ± SD)

Group no. Weight gain (g) P.E.R.

1 40.7 2.87 ± 0.30

2 36.9 2.78 ± 0.56
3 33.9 2.10 ± 0.46
4 29.9 1.95 ± 0.28
5 31.7 2.61 ± 0.32
6 36.6 3.01 ± 0.24
7 30.8 2.30 ± 0.Q3

Table 6. Significance levels of the differences in P.E.R. values

between the groups compared

Groups compared Calculated t Significance level

1 and 2 0.403 p > 0.05

1 and 3 3.98 p <0.01
1 and 4 6.32 p < 0.01
1 and 5 1.67 p > 0.05
1 and 6 1.03 p > 0.05
1 and 7 19.55 p <0.01
3 and 6 6.86 p < 0.01
4 and 7 3.21 p < 0.01
4 and 5 4.32 p < 0.01
3 and 4 0.785 p > 0.05
6 and 7 33.17 p < 0.01

rate of the rats fed the different diets. These results prove the superiority of meal
B, which was cooked in the presence of added phospholipids. Yet, although the
P.E.R. of the diet containing 40% of meal B was comparable to that of the diet
with soybean meal as the only source of protein, the one with 60% of meal B
appeared to be of a considerably lower protein nutritive value. It is of interest to
Gossypol in Cottonseed Products: 'Toxicology and Inactivation 173

Table 7. The contents of gossypol in the cottonseed meats and meals, and "available" lysine and
"chemical index" of the meals subjected to different cooking, with and without added phospholipids
(PL)

Cooking Total Gossypol forms in meals "Available" "Chemical

conditions gossypol lysine index"
in meats Free Total Bound in meals in meals
(%) (%) (%) (%) (gl100 g
protein)

5 Min at 100°,
without PL 1.14 0.151 1.64 1.49 2.97 40.0
5 Min at 100°,
with PL 1.08 0.089 1.15 1.06 3.27 62.4
60 Min at 110°,
without PL 1.14 0.116 1.65 1.53 2.87 39.0
60 Min at 110°,
with PL 1.09 0.066 1.25 1.18 3.04 53.7

note that the P.E.R. of the diet containing 60% of the lower quality meal (meal
A) supplemented with lysine, histidine and threonine was not much lower than
that of the diet containing only soybean meal.
The results summarized in Table 7 show that cooking of cottonseed meats for
as little as 5 min at 100° C with added phospholipids can eliminate more of the
free gossypol than cooking for 60 min at 110° C without added phospholipids.
Addition of phospholipids also improved the available lysine and chemical index
values, under both ordinary and mild cooking conditions. The lower total
gossypol contents of both samples cooked with added phospholipids are due to
partial extraction of the gossypol bound to phospholipids by hexane, and indeed
the oil produced from these meats showed much higher gossypol levels than the
oil extracted from the meats processed without added phospholipids.
In conclusion, cooking cottonseed meats with added phospholipids can
detoxify most of the gossypol, while maintaining a relatively high available lysine
and chemical index. The beneficial effect of added phospholipids was also
reflected by a considerably better performance (in terms of growth rate and
P .E.R.) of the animals. The cost of the required amount of phospholipids from
soybean oil is quite low and will not have much influence on the price of the
meal. Moreover, most of the used phospholipids can be separated and recycled,
the gossypol can be liberated and used for various purposes, and the extra profit
may cover the additional expenses.

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