ANTIOXIDAT[VE PROPERTIES OF CACAO AND
TI-[EIR E F F E C T ON B U T T E R O I L ~
w. S. MUELLER
Depart~Jnent of Dairy Industry
University of Massachusetts, A~nherst
P r e l i m i n a r y work (2) in 1938 revealed t h a t cacao contained substances anti-
oxidative f o r milk fat. Meager e x p e r i m e n t a l d a t a i n d i c a t i n g the effectiveness of
the cacao a n t i o x i d a n t in l a r d , soya bean oil, cod liver oil, a n d coffee beans are
given in a p a t e n t (3). Since the l i t e r a t u r e contained insufficient i n f o r m a t i o n to
d r a w conclusions r e g a r d i n g the effectiveness of cacao a n t i o x i d a n t s in d a i r y
products, this i n v e s t i g a t i o n was u n d e r t a k e n . Possible a n t i o x i d a n t s f r o m cacao
shell are emphasized in the p r e s e n t report.
The effectiveness of the cacao a n t i o x i d a n t s on b u t t e r o i l was t e s t e d b y n o t i n g
the f o r m a t i o n of peroxides, bleaching of the carotenoid pigment, a n d taste a n d
odor of the butteroil. A c c e l e r a t e d tests were used in place of o r d i n a r y storage
conditions for d a i r y products. A l t h o u g h there is some risk of e r r o r in a p p l y i n g
results obtained b y accelerated tests, experience has shown t h a t d a t a thus ob-
t a i n e d u s u a l l y f u r n i s h a reliable guide to behavior d u r i n g storage.
Preparation of b~ttteroil. The b u t t e r o i l for this s t u d y was p r e p a r e d f r o m
sweet cream ( a p p r o x i m a t e l y 40% f a t ) o b t a i n e d from the U n i v e r s i t y creamery.
Cream was c h u r n e d in a Dazey glass churn, a n d the r e s u l t i n g b u t t e r was m e l t e d
a n d washed with w a t e r at 158 ° F. The oil a n d water were l a t e r s e p a r a t e d b y
means of a s e p a r a t o r y funnel. This process was r e p e a t e d u n t i l a r e l a t i v e l y clear
b u t t e r o i l was obtained. The final s e p a r a t i o n of oil a n d water was made b y
c e n t r i f u g i n g in s e p a r a t o r y funnels. The entire p r o c e d u r e was c a r r i e d out a w a y
from d i r e c t sunlight. The b u t t e r o i l contained 94.4% solids a n d was s t o r e d in
amber-colored bottles in the r e f r i g e r a t o r (42 ° F . ) .
Source of cacao. The cacao shell and cocoa powder were obtained f r o m the
W a i t e r B a k e r Company, Dorchester, Mass. The cacao shell was g r o u n d to pass
t h r o u g h a 20-mesh wire screen.
Swift fat stability test. The modifications of the Swift f a t s t a b i l i t y test as
recommended by Riemenschneider et al. (4) were used with the exception t h a t
a l-g. sample of b u t t e r o i l was dissolved in 50 nil. of a m i x t u r e of glacial acetic
Received for publication January 11, 1954.
~Contribution No. 936 from the Massachusetts Agricultural Experiment Station. This
paper reports research undertaken in cooperation with the Quartermaster Food and Container
Institute for the Armed Forces and has been assigned No. 463 in the series of papers approved
for publication. The views or conclusions contained in this report are those of ttle author. They
are not to be construed as necessarily reflecting tlle views or indorsement of the Department
ANTIOXIDATIVE PI~OPEI~TIES OF CACAO ~55
acid and chloroform ( 3 : 2 b y volume). All the peroxide nmnbers in this report
represent the n u m b e r of millimoles of peroxide present per 1,000 g. of fat.
Modified Schaal test. T w e n t y - g r a m samples of butteroil were incubated in
unstoppered test tubes in the d a r k in an electrically heated hot air oven at 212 ° F.
Before placing the test tubes in the oven and once each day thereafter, each
tube was stoppered and then agitated b y inverting it twenty-five times. The
color of the butteroil was determined at various times.
Determination of color of butteroil. The yellow color of the butteroil or the
degree of bleaching was determined by making comparisons with the aid of a
Lovibond tintometer against 19 potassium dichromate dilutions, r a n g i n g from
0.10 to 0.001%. D u r i n g the summer months, fresh butteroil has a color closely
a p p r o x i m a t i n g that of 0.10% potassium dichromate, a concentration of 0.01%
m a y be considered for all practical purposes as being completely bleached.
Taste and odor tests. Taste and odor tests were made by l a b o r a t o r y assistants
and the writer. Odor was determined by smelling the tubes containing the oil,
and taste was determined b y t r a n s f e r r i n g a small amount of oil b y means of
glass stirring rods f r o m the tubes to the month. Since rancid taste and odor are
only relative terms, their absence is indicated in the table by a negative sign and
their presence b y positive signs in three degrees of intensity.
Effect of varying amounts of copper on oxidative spoilage of butteroil. W h e n
the butteroil p r e p a r e d for this s t u d y was aerated at 208.4 ° F. by the Swift test,
the length of time necessary for the determination of its stability was approxi-
mately 50 hours. F o r obvions reasons, it is desirable to reduce the time for
making this determination. I t is well known that a metallic catalyst, such as
copper, will greatly accelerate oxidative spoilage. Five samples of butteroil con-
taining 0, 1, 2, 3, and 4 p.p.m, of added copper ( C u S Q ) , were aerated at 208.4 °
F., and peroxides and color were determined at various times. The time required
to bleach the test butteroil samples to a p p r o x i m a t e l y 0.0.05% potassium dichro-
mate color s t a n d a r d and the corresponding peroxide n u m b e r at the time of
bleaching are listed in Table 1.
Thus, the addition of 1, 2, 3, or 4 p.p.m, of copper to the butteroil greatly
increased its susceptibility to oxidation. Since 4 p.p.m, of copper does not exceed
Effect of varying amounts of copper on oxidative spoilage of butteroil
Amount of Time to bleach to approx. Peroxide No. when bleached
copper added 0.005% potassium dichro- to 0.005~v potassium dichro-
mate color standard mate color standard
o 51½ 10.10
1 121~ 9.39
2 91/~ 9.50
3 6 9.34
4 6 9.41
756 w . s . MUELLER
t h e m a x i m u m a m o u n t f o u n d in some d a i r y p r o d u c t s , t h i s a m o u n t of c o p p e r w a s
a d d e d to b u t t e r o i l t e s t e d b y t h e a e r a t i o n m e t h o d in m o s t i n s t a n c e s i n t h i s s t u d y .
Effect of cacao shell on oxidative spoilage of butteroil with copper as pro-
oxidant. F i n e l y p u l v e r i z e d cacao shell d e p l e t e d of f a t b y p e t r o l e u m e t h e r e x t r a c -
t i o n was a d d e d to b u t t e r o i l i n t h e f o l l o w i n g c o n c e n t r a t i o n s : 0, 0.025, 0.05, a n d
0 . 1 0 % . F o u r p . p . m , of c o p p e r (as C u S Q ) was a d d e d to a l l s a m p l e s , w h i c h
were t h e n a e r a t e d a t 208.4 ° F . , a n d p e r o x i d e n u m b e r s , color, t a s t e , a n d o d o r
were d e t e r m i n e d a t v a r i o u s times. R e s u l t s o b t a i n e d w i t h 0.1% cacao shell a r e
g i v e n in T a b l e 2, a n d i t is e v i d e n t t h a t cacao shell possesses c o n s i d e r a b l e a n t i o x i -
d a t i v e p r o p e r t i e s t o w a r d b u t t e r o i l . The a n t i o x y g e n i c effect of 0.025% cacao
shell was f o u n d to be n e a r l y e q u a l to 0.05%, b u t less effective t h a n 0.1% cacao
Antioxygenic influence of cacao shell in butteroil plus 4 p.p.m, of copper
tIours of Color - - Degree of
aeration at Peroxide % potassium oxidized
208.4 ° F. number dichromate flavor
0 0.00 0.10 --
4 1.08 0.i0 --
5 1.11 0.10 +
8 3.05 0.07 ++
8½ 3.91 0.03 +++
9½ 8.05 0.005 +++
O.lO% cacao shell added
0 0.00 0.10 --
21 1.34 0.10 --
27 1.66 0.10 +
40 3.70 0.06 +
41 6.27 0.02 ++
42 9.80 0.005 +++
Effect of cacao shell on oxidative spoilage of butteroil in absence of added
copper. I t is conceivable t h a t a s u b s t a n c e such as cacao shell m a y d e c r e a s e t h e
p r o - o x i d a n t effect of t h e a c c e l e r a t o r ( C u ) a n d y e t h a v e no d i r e c t effect on t h e
o x i d a t i o n of the n a t u r a l fat. C o n s e q u e n t l y , the a n t i o x i d a t i v e p r o p e r t i e s of such
s u b s t a n c e s c o u l d r e a d i l y be m i s j u d g e d .
S i n c e c o p p e r was u s e d as a n a c c e l e r a t o r in t h i s s t u d y , a t e s t was m a d e to
d e t e r m i n e t h e a n t i o x i d a t i v e effect of 0.05% cacao shell on b u t t e r o i l in t h e absence
o f a d d e d c o p p e r . The r e s u l t s a r e g i v e n in T a b l e 3, a n d it is r e a d i l y a p p a r e n t t h a t
t h e cacao shell has definite a n t i o x i d a t i v e p r o p e r t i e s in the absence of a d d e d
c o p p e r . H o w e v e r , i t s effectiveness is m o r e p r o n o u n c e d w h e n c o p p e r is a d d e d .
Effect of cacao shell, cocoa powder and extracts of these products on bleaching
of butteroil subjected to a modified Schaal oven test. T h e p u r p o s e of i n v e s t i g a t i n g
t h e a n t i o x i d a t i v e p r o p e r t i e s of v a r i o u s e x t r a c t s of cacao shell a n d cocoa p o w d e r
was t w o - f o l d : (a) to i n c r e a s e t h e p o t e n c y of t h e cacao a n t i o x i d a n t b y c o n c e n t r a -
t i o n a n d (b) to o b t a i n t h e a n t i o x i d a n t in a f o r m t h a t c o u l d be m o r e c o m p l e t e l y
incorporated in the butteroil.
ANT~OXIDATIVE PROPERTIES OF CACAO 757
A n t i o x y g e n i v i~fluence o f cacao shell i~t b u t t e r o i l w i t h o u t adde d c o p p e r
Peroxide number Color--% potasium dichromate
H o u r s of
aeration at 0.05% 0.05%
208.4 ° F. Control cacao shell Control cacao shell
0 0.00 0.00 0.10 0.10
42 2.53 2.39 0.08 0.08
50 6.21 2.57 0.02 0.08
53 11.48 2.96 0.001 0.07
I n this experiment, cacao shell and cocoa powder and various extracts of
these products were added to butteroil, and the stability of the butteroil was
determined by the modified Schaal oven test, as described under Procedure.
Rate of bleaching of color was the criterion for determining the antioxygenic
properties of the various substances. The concentrations of the various sub-
stances and their effect on loss of color of butteroil are given in Table 4. I t will
be noted that the aqueous extracts of cacao shell were more potent than the
defatted cacao shell. The temperature at which the aqueous extracts were made
had no significant effect on the potency of the extract. The alcohol extract was
just as effective as the aqueous extracts. The pro-oxidant effect of the ethyl
ether extract may be due to the formation of peroxide during extraction, because
it is difficult to maintain ether peroxide-free while making the extraction. Acetic
acid extract of cacao shell had no autioxidative properties, but the sodimn hy-
l~ffect o f extracts o f cacao she~ and cocoa p o w d e r on b~eacld~g o f b ~ t t c r o i l
s u b j e c t e d to a .modified Se haal oven t e s t
T i m e f o r b l e a c h i n g "~
to a s t a n d a r d of
Concentration 0.01% p o t a s s i u m
Substance tested us e d dichromate
C o n t r o l ......................................................................................... 4
Cold w a t e r (42 ° :F.) e x t r a c t of cacao shell
d r i e d to p o w d e r ................................................................... 0.05 6
H o t w a t e r (145" F.) e x t r a c t of cacao shell
c o n c e n t r a t e d to 8.18% T.S ............................................. 0.05 on d r y b a s i s 6
H o t w a t e r (212 ° F.) e x t r a c t of cacao shell
c o n c e n t r a t e d to 11.20% T.S ............................................ 0.05 on d r y b a s i s 6
H o t w a t e r (212 ° 1~.) e x t r a c t of cacao shell
d r i e d to p o w d e r .................................................................. 0.05 6
I t o t alcohol (174 ° F.) e x t r a c t of cacao
shell c o n c e n t r a t e d to p a s t e ............................................... 0.05 6
H o t e t h y l e t h e r (95 ° F . ) e x t r a c t of cacao shell ...................... 0.05 3
A c e t i c a c i d e x t r a c t of cacao shell ............................................. 0.05 4
Sodium h y d r o x i d e e x t r a c t of r e s i d u e
a f t e r a c e t i c acid e x t r a c t i o n ............................................... 0.05 5
D e - f a t t e d cacao shell ................................................................. 0.05 5
Cocoa p o w d e r ............................................................................ 0.05 6
H o t w a t e r (212 ° F . ) e x t r a c t of cocoa
p o w d e r d r i e d to p o w d e r ..................................................... 0.05 6
Color o f f r e s h b u t t e r o i l is equal t o a p p r o x i m a t e I y 0 . 1 ~ s o l u t i o n of p o t a s s i u m dichromate-
a n d a 0.01% s o l u t i o n equals for m o s t p r a c t i c a l p u r p o s e s c o m p l e t e l y b l e a c h e d b u t t e r o i l .
758 w.s. MUELLER
droxide extract of the residue after acetic acid extraction equaled the cacao shell
in antioxidative properties. Cocoa powder was more potent as an antioxidant
than cacao shell, and their aqueous extracts were of equal potency.
Effect of various substances ou b~dtcroil subjected to tt~e Swift fat stability
test. Defatted cacao shells were extracted in the following order in an attempt
to isolate the cacao-tannin: chloroform, 5 days; ethyl ether, 6 days; and ethyl
acetate, 5 days. According to A d a m (1), chloroform removes the theobromine
and caffeine; ethyl ether removes the eacao-catechin; and ethyl acetate removes
the cacao-tannin and probably phlobaphenes. All of the extracts were eoneen-
trated under partial vacuum and each was added to butteroil at a concentration
of 0.1%. Also, the residue from the three extractions was added to butteroil
at a concentration of 0 . 0 5 ~ . F o u r p.p.m, of copper was added to all samples,
including the control. All samples were aerated at 208.4 ° F., and peroxides,
color, taste, and odor were determined at various times. The aeration time,
peroxide number, and degree of color when a slight oxidized flavor was first
noted and when a very. pronounced oxidized flavor had developed are given in
Table 5. I t will be noted that the chloroform extract and the ether extract had
a pro-oxidant effect. The ethyl acetate extract and the remaining residue after
the three extractions possessed antioxygenie properties, but to a lesser degree
than that of either dried water extract of cacao shell or cocoa powder. This
indicates that not all the antioxidative material was removed from the defatted
cacao shell by successive extractions with chloroform, ethyl ether, and ethyl
Antioxygenic influence of various s~tbstances on butteroil subjected to the S w i f t fat stability test
Very pronounced oxidized
Slight oxidized flavor noted flavor noted
Time Color - - % Time Color - - %
aerated Peroxide potassium aerated Peroxide potassium
Substance added --hours number dichromate - - hours number dichromate
Control 5 2.14 0.09 8 10.42 0.005
Caffeic acid 0.05% ~68 ~ 1.90 0.10 ~68 b 1.90 0.10
Tannic acid 0.05% 69 1,56 0.08 ~ 69 b 1.56 0.08
:NDGA 0.05% 32 2.27 0.10 ~51 h 3.33 0.10
Viobin 0.05% 19 2.10 0.10 22 12.05 0.005
Dried w a t e r extract
of cacao shell 0.05s/( 29 2.25 0.09 39 10.33 0.005
Dried w a t e r extract
of cocoa powder 0.05% 26I/_, 2.20 0.08 361~ 16.31 0.005
of cacao shell 0.10°/~ ~6 ~ 7.62 0.007 6 7.62 0.007
E t h y l ether extract
of cacao shell 0.10% ~6 ~ 7.90 0.005 6 7.90 0.005
E t h y l acetate extract
of cacao shell 0.10% 14 1.26 0.06 151~ 11.52 0.005
Residue a 0.05oA 191~ 3.57 0.09 21~ 11.03 0.009
a M a x i m u m aeration time. Oxidized flavor absent.
b M a x i m u m aeration time. A very pronounced oxidized flavor h a d not developed.
¢ A very pronounced oxidized flavor had developed.
a Residue a f t e r e x t r a c t i n g with solvents in the following order: (1) petroleum ether, (2)
chloroform, (3) ethyl ether, and (4) ethyl acetate.
ANTIOXIDATIVE PROPERTIES OF CACAO 759
I n another experiment, the antioxygenie properties of cacao shell were com-
pared with various antioxidants. A water extract of cacao shell dried to a powder
was selected for making the comparison with the following antioxidants : caffeic
acid, tannic acid, nordihydroguaiaretic acid (NDGA) and Viobin. All sub-
stances were added to butteroil in concentrations of 0.05%. F o u r p.p.m, of
copper was added to all samples, including the control. The samples were
aerated at 208.4 ° F., and peroxide, color, taste, and odor were determined at
various times. The aeration time, peroxide number, and degree of color when
a slight oxidized flavor was first noted and when a very pronounced oxidized
flavor had developed are given in Table 5.
I n general, the dried water extract of cacao shell was more effective in re-
tarding oxidative spoilage than Viobin but less effective than caffeic acid, tannic
acid, and NDGA. However, as j u d g e d by the first appearance of a slight
oxidized flavor, the dried water extract of cacao shell nearly equaled N D G A
ill antioxygenic properties. The water extract of cacao shell had an advantage
in that it did not impart an objectionable flavor to butteroil as did caffeic acid,
tannic acid, and NDGA, in the concentrations used.
The antioxidant properties of cacao shell, cocoa powder, and certain extracts
of these products were determined in butteroil b y the use of two accelerated
tests, namely, the modified Schaal test and the Swift fat stability test. Deter-
minations were made for peroxide values, color, taste, and odor.
This study substantiated the writer's previous (1938) observations that cacao
shell and cocoa powder contain a n a t u r a l antioxidant that is effective in retard-
ing oxidative deterioration of milk fat. The cacao antioxidant was more effective
when copper was added. The cacao antioxidant also was all inhibitor of the pro-
oxidant effect of copper.
The antioxidants in cacao shell were p a r t l y extracted with water or alcohol,
and the concentrated water and alcohol extracts were more potent than cacao
shell or cocoa powder. D r y i n g the water and alcohol extracts to a powder did
not significantly decrease their protective properties. I n general, the dried water
extract of cacao shell was more effective ill retarding oxidative spoilage than
Viobin but was less effective than caffeie acid or tannic acid. The dried water
extract of cacao shell nearly equaled N D G A when the antioxygenic effect was
evaluated by comparing the aeration time to produce the first slight oxidized
flavor. The antioxidants in cacao shell and cocoa powder were not extracted
with fat solvents.
A cacao tannin-like m~terial was isolated from cacao shell and was found to
be less potent as all antioxidant than pure tannic acid.
The cacao inhibitor at a concentration of 0.05% did not impart a foreign
flavor to the butteroil.
Whether the antioxidative materials derived from cacao are nontoxic has
not been determined.
760 w.s. MUELLER
(1) ADAM, W. B. Determination of the Colour-producing Constituents of the Cacao Bean.
~4nalyst, 53: 369. 1928.
(2) MUELLER, W. S. Research Project on Improving the Flavor and Keeping Properties of
Milk and Some of Its Products. Unpublished data. 1938.
(3) MUSHER, S. Retarding Rancidity in Glyceride Oils. U. S. Patent No. 2,176,031. Assigned
to Musher Foundation Inc., New York. 1939.
(4) ]~IEMENSCHNEIDER, t~.. W., TUI~ER, J., AND SPECK, R. W. Modifications of the Swift
Stability Test. Oil and Soap, 20: ]69. ]943.