EFFECT OF MOISTURE AND OF TREATMENTS WITH ACID AND
ALKALI ON RATE OF FORMATION OF FREE FATTY
ACIDS IN STORED COTTONSEED'
M. L. KARONT AND A. M. ALTSCHUL
(WITH SEVEN FIGURES)
Introduction
Cottoniseed deteriorates upon1 storage, with the result that the contenit
of free fattv acids in the seed oil is increased, the color of the oil in the seed
is darkened, the seed protein is partially denatured, and viability is decreased. This increase in free fatty acid conitent is reflected in a higher
refining loss during the processing of the crude oil and the darkened color
of the oil reduces its desirability as ani edible product. AIALOWAN (7, 8),
FREYER (3), anid ROBERTSON anid CAMPBELL (14) have investi(gated the effects
of methods of storage, moisture content, and temperature of the seed on the
formationi of free fattv acids during the storaoe of cottonlseed. SIMPSON
(16, 17) has made a thorough study of the effect of storage coniditions on the
viability- of cottonseed, and RUSCA and GERDES (15) have observed the effect
of the drying of cotton prior to ginning uponi the subsequeint storage behavior of the seeds. All of these invest.igators have concluded that, with
iniereasino moisture content of the seed and higrher temperature of storage,
there is a greater tendency- for the formation of free fatty acids in the seeds
and a corresponding loss in t.heir viability.
It has become evidenit, however, both from studies of the behavior of
individual lots of seed and from a statistical study of the cottoinseed in the
Delta reoion (9), that moisture content an(d temperature are niot the only
factors influencing the quality of stored seed. This meanis that two samples
of cottonseed which have the same moisture contenit anid whieh are stored
at the same temperature will not necessarily develop free fatty acids at the
same rate.
The presenit report covers the results of the first of a series of investigations undertaken at this laboratory in anl effort to ascertaini the nature
and mechanism of the various chemical systemas which are responsible for
the chanoes in cottonseed during" storaoe. It was fouLnid necessary to establish a quantitative expression for the course of the deteriorationi so that the
results of separate experiments could be eompared direetlv. Such an
expression was developed as a consequeniee of ani inivestigationi of the kinetics
of the formationi of free fattv acids in cottoniseed, anid it has beenl applied
1 The storage of cottonseed. I. The first of a selies of papers o0l the niature anid
mechanism of the chemical system operative durinig stor age of cottoniseed. The second
paper in this series has appeared wlhile the first was in press; ALTSCHUL, A. M., KARON,
M. L., KYAMIE, LILLIAN, and CARAVELLA, MAIZIE. The stor age of cottonseed. II. The
effect of ammonia treatment on the free fatty acids and color of the seed oil, and on the
rate and degree of heating of the seed. Oil and Soap 20: 258-262. 1943.
310
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
KARON AND ALTSCHUL: FREE FATTY ACIDS IN COTTONSEED
311
to the evaluation of the effect of moisture and of treatment with acid and
alkali on the rate of hydrolysis of the glycerides under conditions where
other factors such as temperature and variety of seed were maintained
constant.
Methods
The moisture content of the intact cottonseed was determined by the
official method of the American Oil Chemists' Society (1). Percentage of
moisture was expressed on the basis of the dry weight.
--CHROMEL
COIL
FIG. 1. Titration assembly for determination of free fatty acids.
I
Since the pH of cottonseed is an empirical quantity, dependent to a great
extent on the method of determiniation, it is essential that any method used
should give reproducible results and reflect changes taking place during
storage. A method based on the suspension of a definite weight of cottonseed meats in a definite volume of water gave the most consistent results
and otherwise was found to comply with the requirements.
Fresh meats were obtained by cracking cottonseeds in a Bauer mill and
separating the meats from the hulls and lint by screening. The separated
kernels were ground in a Wiley mill and 0.25 gram of the freshly ground
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
312
PLANT PHYSIOLOGY
meats was thoroughly homogenized in 10 ml. of distilled water by the use
of a glass homogenizer (12). The pH was determined as sooni as the
homlogenization was completed; otherwise inconsistent and generally lower
results were obtained. A glass electrode wi-as used for all pH measurements.
The determinations of free fatty acids were made on the oil which had
been extracted with Skellysolve F (boiling range, 30° to 600 C.) from the
iueats prepared as described above. Three ml. of oil, freed from solvent,
were transferred by use of a calibrated pipette ilnto 50 ml. of hot. neutralized
ethyl alcohol and were titrated with 0.05 N sodium hydroxide solutioln,
usingy thymol blue as the indicator. Whenever an oil with a very high content of free fatty acids was titrated, a stronger alkali solution (0.5 N)
was used. In order to provide for proper stirring and heating of the emulsion of alcohol and oil dcuring the titration, the assembly shown in figure 1
was used. The stirring, was accomplished by an air-driven stirrer operating
in one arm of the cell, and heat was applied by means of a chromel resistance
wvire wound around the bottom bend. A potential of 20 volts applied to
three feet of no. 25 chromel wire provided sufficient heating.
It is possible to estimate the content of free fatty acids in cottonseed
oil without determining the total acid content of the oil. This is standard
practice whenever a large number of analyses are made. The official
American Oil Clhemists' Society method, used on a large scale for commercial testing of seed, assumes that the average molecular weight of the free
fatty acids in cottonseed is the same as that of oleic acid. This asstumption
is not strictly justified but, since the actual average molecular weight of
the fatty acids in cottonseed oil varies within narrow limits, it is valid for
comparative purposes. Therefore, the free fatty acids are reported as percentage oleic acid and are calculated as follows:
Percentage free 1 28.2 x normality of alkali x ml. alkali
fatty acids
weight of ol
(as oleic acid) J
Duplicate determinations on each oil were found to agree within 0.01
per cent. for oils having a content of free fatty acids of the order of 10 per
cent. The greatest source of error in the titrations was found to be variation
in the individual seeds of any lot. By careful sampling it was possible to
reduce this error to a minimum; duplicate determinations made on two
different samplings of the same seed agreed within 0.1 per cent. The results
reported here represent the average of several determinations.
w
Effect of moisture
The mnajority of the experiments oln the effect of moisture on the storage
of cottonseed were conducted with the "Delfos 3506" variety grown at the
Delta Experiment Station in Stoneville, Mississippi. Two other varieties of
seed, "D. and P. L. 048" grown at the Louisiana State Experiment Station
at Calhoun, Louisiana, ancd "Acala 111" from the Texas Experiment Station
at LIubbock, Texas, wNere used for parallel experiments. The experimental
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
313
KARON AND ALTSCHUL: FREE FATTY ACIDS IN COTTONSEED
results obtained with the latter varieties indicate that the effect of moisture
is not confined to cottonseeds of any one variety or those produced in a particular locality. Only the work on the Delfos seeds, however, is reported
here in detail. These seeds were obtainied from cotton harvested and g,inned
in the fall of 1941 and had an original moisture content of 10.5 per cent.
when received in December of that year.
Lots of 3 pounds each of the cottonseed were conditioned to the desired
moisture content and stored in air-tight, 2-quart glass jars at 250 C. The
moisture content of the seed was changed under conditions which minimized
any other activity during the process, and did not permanently injure the
seed. An increase in moisture was obtained by spreading the seeds in a
5Sr
I
z
0 10
I1
_
/
c5
/
/
PRET
CONTENT
MOISTURE
( ~~~~~~~~~~~PERCENT)
o*
*
<
0/
/
*
@-®
0-0
0
10 5
513 0
~~~155
18 0
50J
1001520 5
L~5
0
LENGTH
OF STORAGE (DAYS)
FIG. 2. Effect of moisture conitent of cottonseed oni rate of formatioin of free fatty
acids.
thin layer in a room maintained at 30 C. and 100 per cent. relative humidity
until the desired moisture content was reached. One three-pound lot of
seed was dried at room temperature in vacuum desiccators until its moisture
content dropped to 7.5 per cent.
Analyses of the stored seeds were made at regular intervals. The results
of the determination of the free fattv acids are shown in graphical form in
figure 2.
Each curve represents seed of a particular moisture conteiit and the
points on the curve represent the content of free fatty acids of the seed at
various times during a storage period of 200 days. Mold formation was
visible after 100 days oni the seeds of high moisture content (18 an-d 20.5
per cent.) and the experiments with these lots were, therefore, discontinued
at that point.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
314
PLANT PHYSIOLOGY
It is conceivable that the effect during storage of cottonseed produced
by variations in the artificially controlled moisture content is not analogous
to that which would be produced by similar variations in moisture content
of seeds within the cotton boll. This is probably true when seeds which were
originally in a moist condition are dried to a lower moisture content, since
certain changes may have taken place which cannot be completely reversed
after the seeds are dried. When, however, seeds with an original moisture
content of 10 per cent. and in a practically dormant condition are conditioned to some higher moisture content, the resulting increase in physiological activity must be considered as the sole result of the added water.
It was observed that the pH of cottonseed containing moderate percentages of moisture (15.5 per cent. or less) had decreased after 250 days of
storage but not after 100 days; whereas that of seed containing high moisture had decreased after 100 days (table I). The decrease in pH paralleled
the formation of free fatty acids in the seed, as shown in figure 2. This
effect apparently is dependent upon the moisture content of the seed. Free
TABLE I
INFLUENCE OF MOISTURE CONTENT AND LENGTH OF STORAGE ON PH OF STORED COTTONSEED
MOISTURE CONTENT OF SEED
0
LENGTH OF STORAGE IN DAYS
100
No
7.5
10.5
13.0
15.5
18.0
20.5
pH
7.05
7.00
7.00
6.90
7.00
7.02
pH
7.05
7.00
7.00
6.90
6.51*
6.19*
250
pH
6.70
6.70
6.60
6.26
.........
.........
*-The pH of the high-moisture seeds again increased after 100 days' storage coincident with the appearance of mold growth. It would seem, therefore, that the products
of the mold metabolism are alkaline and tend to counteract the natural tendency of the
seeds to increase their hydrogen-ion concentration on storage.
fatty acids, formed during storage, are not themselves responsible for the
lowering of the pH, for it was observed that prior removal of petroleum
ether-soluble constituents from the meats had no effect on the pH value.
It is not possible to state definitely the cause of the lowering of pH, but it
is probably the formation of water-soluble acidic products through respiration and fermentation. Similar decreases in pH during the storage of corn
and soybeans have been previously reported (2, 13).
Effect of treatments with acid and alkali
The possibility of changing the pH of cottonseeds by exposing them to
the fumes of volatile acids or bases is evident from the following results
obtained with Delfos seed. In one experiment, the seeds were treated with
ammonia for one hour until the pH rose to 7.9. The limiting factor determining the rate at which the pH is raised is probably the rate of diffusion
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
315
KARON AND ALTSCHUL: FREE FATTY ACIDS IN COTTONSEED
of the vapor through the small openinig in the seed coat (18). In practice
it was found that it was onily niecessary to saturate the Iiiit anid atmosphere
surrounding the seeds with ammonia gas and let them remaini sealed under
these conditions until the pH had riseln to the desired level. The same statement applies to acid treatmenit of the seed. In another experiment, the seeds
were exposed to dry hydrogen chloride gas until the pH dropped to 4.6.
Inasmuch as the acid vapors eaused disintegration of the linit and even some
destruction of the seed coat, it is apparent that there is a faster diffusion
of the gas into the seed. Following treatmenit with acidic or basic vapors,
the seeds were coniditioned to approximately 18 per cenit. moisture conitent
and stored in air-tight jars together with the other samples previously
mentioned.
z
A10
0
MOISTURE CONTENT
(PERCENT)
180
O
*
0
50
100
LENGTH OF STORAGE
ORIGINAL
p1H
70
807
150
200
250
(DAYS)
FIG. 3. Effect of treatmenit of cottonseed with acid and alkali oni the rate of formation of free fatty acids.
The rates of free fatty acid formatioin during storage of the acid anid
alkali-treated seeds are compared in figure 3 with that for an initially
neutral seed coniditioned to the same moisture content. In both cases, the
treatment very effectively reduced the rate of formationi of free fatty acids
as compared to the conltrol. In addition, no mold formation was apparenit in
the acid- or alkali-treated seed during the eintire storage period. The color
of the oil extracted from the ammonia-treated seed was considerably lighter
than that of oil from the control. Oni the other hand, the acid treatmenit of
cottonseed resulted in a verv pronounced darkening of the extracted oil.
Duringc 250 days of storage the pH of the acid-treated seeds remained
conistanit, whereas the pH of the alkali-treated seeds gradually dropped from
the originlal value of 7.9 to 7.0. The effect of treatment with acid or alkali
does not necessarilv mean that there is an optimum pH for the hydrolysis
of the oil in the seed. The changes iniduced by these treatmenits were of a
permanient nature and could not be reversed by bringingr the pH baek to
neutralit.v.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
316
PLANT PHYSIOLOGY
TABLE II
GERMINATION
TESTS ON STORED COTTONSEED
EXPERIMENT
MOISTURE
LENGTH OF
CONTENT
STORAGE
A
B
C
D
10.5
18.0
18.0
17.0
%
days
200
100
200
200
PH AT START FREE FATTY
CONTENT
OF STORAGE ACID
OF SEEDS
7.0
7.0
7.9*
4.6t
GERMINA-
TION
%
%
1
11
3
3
100
88
0
0
* Treated with ammonia gas before storage.
t Treated with hydrogen chloride gas before storage.
.......
FIG. 4. Germination tests on treated and untreated cottonseed. A, Control;
free fatty acid; C, Alkali treated; D, Acid treated.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
B, High
KARON AND ALTSCHUL: FREE FATTY ACIDS IN COTTONSEED
317
Evideence that the acid or alkali treatment permanently affected the
seeds was obtained by mean-s of germinationi tests. These tests were colnducted as suggested by TOOLE and DRUMMON (19). The seeds were prewetted and then place between folds of moistened towel paper. They were
kept at 31° C. for eight hours, and at 28° C. for the remainder of each
twenity-four hours. This cycle was maintained througyhout the test. The
results after 53 hours are shown in table II and figure 4.
The content of free fatty acids is not the sole factor in determining the
viability of seeds, as is shown by the high percentage of germination of the
seed eontaining 11 per cent. of free fatty acids. It is evident that treatment
of the seeds with acid or alkali completely destroys their ability to germinate,
along with their ability to form free fatty acids. Thus, it is quite logical to
assume that. viability and ability to form free fatty acids are kindred properties and, as a conseqnence, inhibition of one seriously inhibits the other.
Kinetics of formation of free fatty acids
The rate of hydrolysis of the fats in cottonseed was not uniform during
the period of storage, but rather increased as the length of storage continued.
More free fatty aeids were formed in the second of two equal storage periods.
This was especially true for the seeds of higher moisture content where the
amount of free fatty acids formed in the second fifty days greatly exceeded
that formed in the first fifty-day period. Such behavior is indicative of an
autocatalytic type of reaction. If this is true, the kinetics of t.his reaction
should conform to the general equation for an autocatalytic reaction which
may be expressed as:
dP =
dt k(A) (B)
(1)
where
P = the product of the reaction,
A = a quantity which increases as the reaction progresses,
B = a quantity which decreases as the reaction progresses, and
k = the rate constant.
In terms of experimentally determined quantities, the equation for the
rate of hydrolysis of the glycerides in cottonseed may be provisionally
written as (5)
dF
-d
=
k(F) (100-F)
(2)
where
F = the percentage of free fatty acids formed, and
100-F = the percentage of residual unhydrolyzed fat.
Upon integration, equation (2) becomes:
F
ln 100-F -ln
100-Fo =lOOkt
where F0 thle original conitenit of free fatty acids of the seed.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
(3)
318
PLANT PHYSIOLOGY
Equation (3) by transposition and conversion to common logarithms
yields:
F
100
Fo
F
(4)
log1F
=2 3 kt + log
From the nature of equation (4) it is evident that a plot of log 100-F
a straight line with a slope equal to 2 k.
against t should give
tn
~~~~~~~~~~2.3
When the data in figure 2 are plotted in such a fashion, the points thus
obtained fall practically on straight lines as is shown in figure 5. The nu-0.5
I
I
MOIS.TURE CONTENT
(PERCENT)
10.5
*
13.0
15.5
50
100
-1.0
~~
180
~~~~~~~~0
@p
20.5
-1.5
0
20
-25
LENGTH OF STORAGE (DAYS)
F
FIG. 5. Effect of varying moisture content on log 100-F /t
merical value of the rate constant, k, calculated from the slopes of the curves
in figure 5, varies as a function of the moisture content of the seed (table III)
and quantitatively characterizes the course of free fatty acid formation in
cottonseed. This relationship between k and the formation of free fatty
acids is best demonstrated by reconstructing theoretical curves from the
values of k shown in table III and comparing these curves to the actual
experimental results. In order to accomplish this purpose, it is necessary
to convert equation (3) into a form from which the value of F may be readily
calculated for any time, t. Transposition of equation (3) yields:
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
KARON AND ALTSCHUL: FREE FATTY ACIDS IN COTTONSEED
In
F
100-F
=lOOkt
Fo
319
(5)
100-Fo
whieh in the exponenitial form becomes:
F
100-F
elookt
(6)
Fo
100-Fo
The equat.ion from wlhieh the value of F may be calculated for any time, t,
is obtained by solviilo for F.
Fo0
F =100
lookt
100-Fo
(7)
1±100-F0eoOk
+ lOF.e
TABLE III
EFIFECT OF MOISTURE CONTENT ON RATE CONSTANT, K
PERCENTAGE
MOISTURE CONTEN T
SLOPE
(RECIPROCAL DAYS)
K X 10S
(RECIPROCAL DAYS)
10.5
13.0
15.5
18.0
20.5
0.00085
0.00085
0.0044
0.0138
0.0163
1.96
1.96
10.1
31.8
37.4
The conitent of free fatty- acids of the seeds at the beginningw of the storage
period was 0.5 per cenit.; thus the value of Fo used in equationi (7) becomes
0.3. A summarv of the cale-Plations used in reconstructingy the cottonseed
storage curves show-n in figure 6 is (iven ini table IV. The solid linles in
figure 6 represent the theoretical curves calculated as described above, while
the poinits are the same experimental points showni in figure 2.
The assumptionis expressed in equation (2) have led t.o oiie successful
initerpretation of the results of this storage experimenit although it has niot
provided an explanationi for the results found by other inivestigators who
have shown that seeds with the same moisture content may develop free fatty
acids at different rates. Experiments are in progress to determine whether
factors other than moistture and pH could be responsible for these variations.
The eonstant, k, wheni used in conjunetion with equationis (2) and (7)
not only- allows onie to calculate the amount of free fatty acids formed at aniy
time, but also oives the rate at which more free fatty acids are being formed
at that particular time. A similar analysis of the storage of the acid- and
alkali-treated seeds gave values of k for those reactions. It becomes possible.
therefore, to obtain a quantitative representation of the effect of moisture,
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
320
PLANT PHYSIOLOGY
V
z
Cl~ ~
o
¢
~
~
o
~
Clx"
-
So~~~~~~C
o
oc
O .c
>C
o C) cqA In c CC)
Z e
IT
-
O
V
H
0
oC
o
t
t
m t
m
-oee
CD
OH C H
~
HV_
0
z
Cl
o ¢
l
OAeDo:[it
rn
I
o ]
1:4~~
o; 1 ~M
o
C.)
xv
4 oic-io
HX:SuO
vi4L
HX
r-~r
I1
0 1
0IOH8°
1
HH ]t rsi
OC12ii~1
Eq
11~~~~~~C
o o nQ > e tO Oo m
~~~~~~~~~I
1 EC
H 1
H r1
I
CD U:to
+
s
o cl x
zCC
I
CC
Al
CI
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
321
KARON AND ALTSCHUL: FREE FATTY ACIDS IN COTTONSEED
z
U
a<)
C
0
4
ir
4
200
LENGTH
OF STORAGE (DAYS)
FIG. 6. Theoretical curves showiug effect of moisture on rate of formation of free
fatty acids.
INITIAL p H
*
li30
o
*
4.6
7.0
7.9
-
20o
x
10'0
-IC
15
MOISTURE
CONTENT
20
(PERCENT)
25
FIG. 7. Effect of moisture content of cottonseed and of treatment with acid and
alkali oni the value of the rate constant for hydrolysis of the oil in cottonseed.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
322
PLANT PHYSIOLOGY
and of the treatmlent with acid and alkali, on the hydrolysis of cot.tonseed
oil. These effects are represented by the curve shown in figrure 7, where the
points oni the curve represent the effect of moisture, and the isolated points
the effect of acid and alkali treatment. It is apparent from this curve that
treatment of cottonseed with ammoinia has the same effect as does lowering
the moisture conitent of the seed from 18.0 to 15.4 per cent.; and that treatment with acid has the same effect as does lowering the moisture from 17
to 15.1 per cenit. As is shown in figure 7, there is nio apparent difference in
the value of k for seeds with moisture contents of 10.5 and 13.0 per cenit.
It is quite possible that an extended storage investigration over several years
would reveal some difference in the value of k for these two differelnt moisture contents.
Discussion
The fact that equationi (2) satisfactorily represen-ts the course of hvdrolysis of cottonseed oil in the seed does not constitute evidence that the
actual mechanism of the reaction involves catalysis by the free fatty acids
which are formed. Any other variable which affects the rate of hydrolysis
of the oil, and which increases durino storage, would give the same result..
The quanitities, F and 100-F, were used because they were the only measured
variables available for use in equation (1) and niot because there was any
special theoretical significance attached to their use. It is best., therefore,
to consider the storage of cottonseed from the general viewpoint expressed
in equation (1) and try to determine the factors which are responsible for
the apparent autocatalytic nature of the curves. In addition to the concentration of free fatty acids, t.here are two other variables which satisfy
the requirements of the quantity A in equation (1). These are the activity
of lipase or lipases contained in the seed itself, and the activity of lipases
produced by molds and bacteria, growing oni the seed. It has been shown
by LANGTON (6) that the initial part of the saponiificationi of some fats by
magnesium oxide and calcium oxide is autocatalytic. Similar results were
reported by KAUFMANN (4, p. 255) for the hydrolysis of sunflower-seed oil
in water at elevated temperatures. The explanationi which was given is that
the rate of hydrolysis of the glycerides depends oni the degree of emulsification of the oil in water. Inasmuch as the presenice of free fatty acids facilitates the emulsification of the remaining fat, the rate of the hydrolysis of
the remaining glycerides will increase as the concentration of free fatty
acids increases until optimum emulsification has been achieved. It is conceivable that a similar mechanism operates in the intact cottonseed aind that
the state of emulsification of the fat globules in the cells is enhanlced by the
formation of free fatty acids. Such an explanation, however, caniiot. be
reconciled with the observation that exposure of seed to ammonia or hydrogen chloride vapors inhibits the hydrolysis. In fact, just the opposite
effect would be expected because acids and alkalis are knowni to catalyze the
hydrolysis of fats and oils in vitro.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
KARON AND ALTSCHUL: FREE FATTY ACIDS IN COTTONSEED
323
The second possibility is that the hydrolysis of the glycerides is brought
about by enzymes present in the seed. It has been shown that there is an
increase in detectable lipase activity upon germination of cottonseed (10).
This increase in enzymatic activity accounts not only for the increase in
free fatty acids in the germinatinig seed but also explains the autocatalytic
course of the hydrolysis. More lipase was found to be present on the second
day of germinationi than on the first, and consequently more free fatty acids
were formed duringf the seconid day, etc. The storage of cottonseed at an
elevated moisture conitent approximates'a condition between that of a completely dormant seed an-d a oerminating seed. High moisture may cause a
gradual development of lipase activity resulting in an accelerated rate of
formation of free fatty acids. The lipase may be liberated from other protein material by the action of proteolytic enzymes in a manner very similar
to that reported by WILLSTATTER for the activation of castor bean lipase in
dormant seed (20). Althioughli OLCOTT and FONTAINE (11) were unable to
detect lipase in ungerminated cottonseed, it is quite possible that the lipase
is so bouncd to other material in the seed that it cannot be detected unless it
is loosened from the proteins and other substanees surrounding it. The
effect of acid or alkali treatmelnt of seeds may be explainied oni the assumption that these treatments either directly inhibit the lipase activity or interfere with the mechanisms responsible for the liberation or aetivation of
the lipase.
The third possibility- is that the hydrolysis of the glyeerides is catalyzed
by- the lipolytic enzymes produced by molds and bacteria growing on the
seed. Cottonlseed, partieularly if it has a moisture content. of about 17 per
cent., is an exeellent meedium for the growth of these organisms. As was
mentionied above, mold growth was visible after 100 days' storage in cottonseed containing 17 per cenit. moisture. The gradual inerease in the microbial population might be expected to result in an aecelerating( rate of
formation of free fatty acids andl thus produce an apparently autocatalytic
reaction. The effect of acid or alkali treatment of the seed could be readily
explained oni the assumption that such treatment destroys, or materially
inhibits the growth of microoroaniisms. In a recent paper, RAMSTED and
GEDDES (13) have suggested that. the apparent respiration of sovbeans is
due to associated microorganiisms.
On the basis of the experimenital results obtained so far, it is not possible
to determine which of the above explanations is correct, although the last
two involvinig enzymatic hydrolysis appear to be more plausible than the
first onie. More work will be required to permit differentiation between the
effects caused by einzymnes in the seed, ancd by enzymes produced by micro-
organismus.
Summary
1. The effect of the variationi of moisture on the rate of formation of
free fatty acids in eottoniseed has been determined over a moistuLre range
of 7.5 to 20 per cent. (dry basis).
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
324
PLANT PHYSIOLOGY
2. Inerease in moisture content of the seed results in an increased rate
of hydrolysis of the glycerides. The most rapid increase in the hydrolytic
rate was observed to occur when the moisture eontenit was raised from 15
to 18 per cent.
3. Treatment of cottonseed with amilmonia or hydrogen chloride gas was
found to greatly retard the formation of free fatty acids. Treat.ment with
ammonia retarded the darkening of the oil and in fact resulted in an oil
with a much lighter color than that from t.he untreated seed.
4. It has been shown that progress of the hydrolysis of glycerides in
the oil in cottonseed can be expressed quantitatively by use of the differ-
ential equation:
dF
t-
=
k(F) (100-F)
where:
F = the percentage free acids,
100-F = percentage unhydrolyzed fat, and
k = the rate constant.
5. Three possible mechanisms for the hydrolytic splitting of the glycerides of cottonseed oil have been discussed and evaluated on the basis of the
kinetics of this reaction.
The authors wish to thank MR. A. K. SCHWARTZ of the South Texas
Cotton Oil Company and the Directors of the Experiment Stations at Stoneville, Mississippi; Calhoun, Louisiana; ancd Lutibbock, Texas, who kindly furnished the cottonseed samples used in this investigation.
SOUTHERN REGIONAL RESEARCH LABORATORY
NEW ORLEANS, LOUISIANA
1.
2.
3.
4.
5.
6.
7.
LITERATURE CITED
AMERICAN OIL CHEMISTS' SOCIETY, Official aln Tenitative Methods. New
Orleans. 1941.
BAILEY, C. H. Respiration of shelled eorni. Miinnesota Agr. Exp. Sta.
Tech. Bull. 3. 1921.
FREYER, E. Additional data on the relation of moisture content to the
inerease of the free fatty acid contenlt of cottonseed in storage. Oil
and Soap 11: 162-164, 176. 1934.
KAUFMANN, H. P. Studien auf dcem Fettoebeit. Verlag Chemie, G. M.
B. H. Berlin. 1935.
KUNITZ, M. Kinetics of the formatioln of clhvymotrypsin from ervstalline
chymotripsinogen and of trypsin from crystalline trypsinogen.
Enzymologia 7: 1-20. 1939.
LANGTON, H. M. Some problems conniiected with the sapolnificationi of
fatty oils. Jour. Soc. Chem. Ilnd. 42: 51T-57T. 1923.
MALOWAN, J. Some observations on the heatingo of cottonseed. Cotton
Oil Press 4: 47-49. 1921.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.
KARON AND ALTSCHUL: FREE FATTY ACIDS IN COTTONSEED
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
325
Changes in cottonlseed during heat and storage. Cotton
Oil Press 5: 40-43. 1921.
MIELOY, G. S. An hypothesis concerning the role of the enzymes in the
relative value of cottonseed. Oil and Soap 16: 174-178. 1939.
OLCOTT, H. S., and FONTAINE, T. D. Composition of cottonseeds IV.
Lipase of germinated seed. Jour. Amer. Chem. Soc. 63: 825-827.
1941.
, anid
The absen-ce of lipase in cottonseed.
Oil and Soap 18: 123-124. 1941.
POTTER, V. R., and ELVEHJEM, C. A. A modified method for t.he study
of tissue oxidations. Jour. Biol. Chemn. 114: 495-504. 1936.
RAMSTAD, P. E., and GEDDES, W. F. The respiration and storage behavior of soybeans. Minnesota Agr. Exp. Sta. Tech. Bull. 156.
1942.
ROBERTSON, F. R., and CAMPBELL, J. G. The increase of free fatty acid
in cottoniseed. Oil and Soap 10: 146-147. 1933.
RUSCA, R. A., and GERDES, F. L. Effects of artificially drying seed
cotton on certain quality elements of cottonseed in storage. U. S.
Dept. Agr. Cire. 651. 1942.
SIMPSON, D. M. Relation of moisture content and methods of storage
to deterioration of stored cottonseed. Jour. Agr. Res. 50: 449456. 1935.
. Factors affecting the longevity of cottonseed. Jour.
Agr. Res. 64: 407-419. 1942.
, ADAMS, CAROLINE L., an1d STONE, G. M. Anatomical
structure of the cottonseed coat as related to problems of germination. U. S. Dept. Agr. Tech. Bull. 734. 1940.
TOOLE E. II., and DRUMMOND, P. L. The germination of cottonseed.
Jour. Agr. Res. 28: 285-292. 1924.
WILLSTXTTER, R., and WALDSCHMIDT-LEITZ, E. Uber Ricinuslipase.
Zeitschr. physiol. Chem. 134: 161-223. 1924.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1944 American Society of Plant Biologists. All rights reserved.