AN E X T R A C T I O N - T I T R A T I O N M E T H O D F O R T H E D E T E R M I N A T I O N

OF F R E E F A T T Y A C I D S IN R A N C I D M I L K AND C R E A M 1

E. N. F R A N K E L AI~D N . P. T A R A S S U K
Depart~cnt of Dairy Industry, Un~,versity of California, Da.vis

The titration of free f a t t y acids in fat derived from rancid milk and cream
has generally been accepted as the most reliable quantitative method for measur-
ing the extent of lipolysis. However, the method of obtaining fat fSr titration
affects its acid degree. Johnson and Gould (12) found that the solvent extraction
of fat from rancid cream gives a greater recovery of f a t t y acids than does churn-
ing. In a study of the partition of free f a t t y acids among different phases upon
the churning of rancid cream, Tarassuk and F r a n k e l (19) established that about
75% of the added low-molecular-weight f a t t y acids (butyric, caproic, and ca-
prylic acids) are lost in the buttermilk and as much as 30% of the added high-
molecular-weight f a t t y acids (pahnitic and stearic acids) are retained in the
foam phase that is formed in the melting of butter. These limitations of the
acid degree determination of churned fat emphasize the need for a method of
determining free f a t t y acids directly in milk and cream.
Extraction-titration methods have been described for determining f a t t y acids
in rancid milk (15) and artificial emulsions (7, 16, 17), and the water-insoluble
f a t t y acids in cream (1). These procedures are based on the extraction of lipid
material with fat solvents and the subsequent titration of free f a t t y acids in the
solvent extract.
No reliable information is available concerning the recovery of free f a t t y
acids that can be obtained b y solvent extraction methods. The recovery data of
Johnson and Gould (12, 13) and Nashif and Nelson (16) were based on the
extraction of a limited number of f a t t y acids that had been previously added in
solution directly to cream. The origin of f a t t y acids in rancid milk is in the fat
phase. Upon hydrolysis of fat the partitiop of f a t t y acids between fat and water
phases will depend upon the relative solubility of each acid in these phases. This
partition will be f u r t h e r modified by the mutual solubility of the acids. Since
the f a t t y acids (except butyric acid) are slightly soluble or insoluble in water,
their distribution in the water and fat phases when added directly to cream will
not be comparable to their distribution in n a t u r a l rancid cream. Therefore, for
the recoveries studies reported in this paper, milk fat to which known amounts
of f a t t y acids were added was emulsified in fresh skimmilk. These f a t t y acids
were representative of the fatty-acid composition of milk fat. It is considered
that emulsions so p r e p a r e d are more nearly comparable to the n a t u r a l emulsion
of rancid milk than milk or cream to which f a t t y acids have been added directly.
A simple extraction-titration method applicable directly to milk and cream
is described in this paper. This method is based on the solvent extraction pro-

Received for publication November 26, 1954.

1A preliminary report of this work has been made (19).
751
752 E . N . F R A N K E L AND N . P . T A R A S S U K

cedure of Johnson and Gould (12). The development of the procedure and
evaluation of the results are based on determinations of recovery of f a t t y acids
f r o m emulsions p r e p a r e d as described above.

E X P E R I M E N T A L PROCEDURE

Extraction-titration procedure. 10.0 ml. of milk is pipetted into a 50-ml. cen-

trifuge tube and 10 ml. of 95% neutralized ethanol is added. The firmly stop-
pered tube is shaken vigorously for 1 minute, 15.0 ml. of a mixture of 40% ethyl
ether and 60% petroleum ether 2 is added, and the tube is shaken for another
minute. The tube is then centrifuged at a p p r o x i m a t e l y 1,500 r.p.m, for 3 rain-
utes. An aliquot portion of 5.0 ml. is pipetted f r o m the s u p e r n a t a n t clear ether
layer and mixed in a 125-ml. E r l e m n e y e r flask with 15 ml. of 95% ethanol con-
taining five drops of 1% alcoholic phenolphthalein and previously neutralized to
a p i n k color. This m i x t u r e is t i t r a t e d with 0.0250 N alcoholic K O H to the same
color, using a 2-ml. burette g r a d u a t e d to 0.010 ml. The titration reading is mul-
tiplied b y three to obtain the volume of alkali necessary to neutralize the total
ether layer. The results are expressed as " f r e e f a t a c i d i t y " ( F . F . A . ) , i.e., milli-
liters of 1 N alkali required to neutralize an ether extract of milk or cream con-
raining 100 g. of fat. I n order to calculate this value, the f a t is determined in the
samples b y the Babcock method. The " f r e e fat a c i d i t y " is equivalent to the
" a c i d d e g r e e " used for the titration of fat.
The same procedure is applied to cream, except for the use of a smaller
s a m p l e - - o n e containing the same amount of f a t as in 10ml. of 4% milk. The
cream sample is weighed on a moisture and b u t t e r f a t torsion balance (sensitivity
± 30 rag.).
Materials. Fresh, grade A raw milk f r o m the University C r e a m e r y was used
in all experiments. Lipolysis was induced by homogenizing raw milk at tempera-
ture of 35 ° C. and pressure of 2,000 p.s.i. The milk was subsequently cooled to
4 ° C. and stored at this temperature. When lipolysis became sufficient to lower
surface tension to about 36 dynes per centimeter (6, 18), the milk was heated
to 80 ° C. to inactivate the lipase. Rancid cream was p r e p a r e d in the same way
as rancid milk. Samples of v a r y i n g degrees of rancidity were obtained by mix-
ing rancid milk or cream with different proportions of fresh pasteurized milk or
cream of the same f a t content.
Milk fat was obtained f r o m cream either by solvent extraction or by churning
mechanically in Dazey glass churns. The butter was melted in a 60 ° C. oven
until three distinct layers appeared. The u p p e r foam layer was removed and the
clear f a t was decanted and filtered through No. 1 W h a t m a n fluted filter p a p e r in
the oven. The solvent extraction procedure used to obtain milk f a t f r o m cream
was essentially that of Johnson and Gould (12). The fats produced by both
methods were stored at 0 ° C. until analyzed.
Recovery experiments were carried out on milk emulsions made with milk
f a t to which f a t t y acids 3 of different molecular weight were added in varying

2 B o i l i n g r a n g e : 35-60 ° C.
:~ O b t a i n e d f r o m t h e E a s t m a n Kodak Company, Rochester, New York.
 ~ECOVERY OF FREE FATTY ACIDS 753

amounts. The f a t used for this purpose was in some cases p r e p a r e d b y removing
the small amounts of f a t t y acids that are present in fresh milk f a t with the
method of Breazeale and B i r d (3). The acid degree of the " a c i d - f r e e " f a t ob-
tained b y this method generally varied f r o m 0.150 to 0.200. A more effective and
simple method for the removal of f a t t y acids f r o m fresh milk f a t was described
b y Borgstr5m (2), i.e., passing milk f a t in wet ethyl ether t h r o u g h an ion ex-
change column of Amberlite I R A 400. 9 The f a t obtained by this method had an
acid degree of a p p r o x i m a t e l y 0.050. This method was used in one experiment,
where the most complete removal of f a t t y acids was necessary.
The weighed f a t t y acids were added to batches of f a t of 100 g. each which
were then heated in a water b a t h until the f a t t y acids dissolved. These fats were
then emulsified in fresh pasteurized skimmilk at 37 ° C. by passing them through
the homogenizing valve of a h a n d emulsifier, using a positive p u m p , u n d e r a
pressure of 250 p.s.i., to give a 4% milk. The resulting emulsions showed micro-
seopically a f a t globule size distribution comparable to that of normal non-
homogenized milk.
Methods. The acid degree of churned and extracted fats was determined
essentially b y the method of I t e r r i n g t o n and K r u k o v s k y (9)---titrating 5.00 g. of
f a t with 0.050 N alcoholic K O H in the presence of neutralized ethanol at 60 ° C.
Surface tension measurements were made in triplicate with a Cenco-du Noiiy
surface tensiometer at 20 ° C.
The p H of milk was determined with a Beckman p H meter.

RESULTS A~'D DISC~'SSION

Titration of the ether extract. The reliability of the titration of a 5-ml. ali-
quot of ether extract was studied by comparing it to the titration of the entire
ether layer. This latter procedure was carried out b y the use of a siphon similar
to that described b y tIillig (10), and two extractions with 15 and 10 ml. of ether
mixture, respectively. The results of Table 1 show that for rancid milk the
titration of the entire ether l a y e r yielded somewhat higher values t h a n did the
titration of a 5-ml. aliquot of the ether extract; for fresh milk it yielded lower
blank values. However, the titration of the aliquot portion gave more u n i f o r m
results than did the titration of the total ether layer. The titration of a 5-ml.
aliquot portion of the ether extract was therefore chosen because of its simplicity
a n d reproducibility in routine analyses.
A suitable indicator was chosen on the basis of a potentiometric curve estab-
lished with a titration m i x t u r e f r o m rancid milk. F i g u r e 1 shows t h a t phenol-
phthalein and the mixed indicator of Kleinzeller and T r i m (14) provide suitable
end points. Phenolphthalein was chosen because its end point was closer to the
neutralization point ( p H 10.3-10.4) t h a n was that of the indicator of Kleinzeller
and T r i m .
Determination of the proper ratio of ethyl ether to petroleum ether. The
o p t i m u m solvent ratio to use in the present extraction-titration procedure was

Donated by the Rohm and H a a s Company, Philadelphia, Pa.

754 E.N. F R A N K E L A N D N. P. T A R A S S U I ¢

TABLE 1
Comparison between tit'ratio~ o f 5 ml. aliquot o f ether layer and titration of tota~ ether layer
Homogenized milk
Procedures Fresh ]~aneid
(ml. 0.025 N (ml. 0.025 N
NaOH per (% deviation NaOH per ( % deviation
10 ml. mill~) from average) 10 ~nl. milk) from average)
(1) 0.408 3.55 3.622 0.385
I• Titration of 5 rot. (2) 0.378 4.06 3.645 0.248
aliquot of ether layer (3) 0.398 1.01 3.678 1.16
(4) 0.391 0.762 3.600 0.990
Average 0.394 3.636
(1) 0.286 5.92 3.898 0.154
II. Titration of total (2) 0.345 13.5 3.968 1.95
ether layer (3) 0.275 9.54 3.910 0.463
(4) 0.311 2.30 3.790 2.62
Average 0.304 3.892

d e t e r m i n e d on the basis of r e c o v e r y of f a t t y acids f r o m m i l k emulsions. Since

the e t h e r l a y e r o b t a i n e d b y the p r o c e d u r e u n d e r i n v e s t i g a t i o n v a r i e d i n volume
w h e n different ratios of e t h y l ether to p e t r o l e u m e t h e r were used, the p r o c e d u r e
c o n s i s t i n g of t i t r a t i n g the e n t i r e e t h e r l a y e r s d e r i v e d from two e x t r a c t i o n s
described p r e v i o u s l y was used for this e x p e r i m e n t .
T h e above p r o c e d u r e was a p p l i e d to r a n c i d a n d fresh milk a n d to artificial
m i l k e m u l s i o n s m a d e w i t h fresh f a t a n d w i t h f r e s h f a t to which were added
e q u a l m o l a r q u a n t i t i e s , respectively, of l o w - m o l e c u l a r - w e i g h t f a t t y acids (bu-

. . . . I . . . . ~

D2.0

o
I I.0

I0.0
s +

~ 9.0

I• •
qD• • ° ° o0

8.0

7.0 •

I i I r I I I I I I
6.0
0~)0 0.50 1.00
MI. 0.0250 N AlcoholicKOH

Fla. 1. Potentlometric curve of titration mixture obtained from rancid milk (× = end point
of pheno]phtba]ein, ~ = neutralization point, + ----end point of indicator of Kleinzeller and
Trim).
 TABLE 2
Effect of varying the ratio of ethyl ether to pctroleucn ether on the extraction of f a t t y acids
Solvent ratio Artificial emulsions m a d e with : Homogenized milk
Ethyl Petroleum Fresh F a t + butyric, caproie F a t + myristic, p a h n i t i c
ether ether fat ~ and caprylie acids ~ and stearie acids" Fresh Rancid

(Net (% (Net (% (Net

(,ml.) (,rot.) (F.F,A.) (F.F.A.) F.F.A.) b Recovery) (F.F.A.) F.F.A.) b l~eeovery) (F.F.A.) (F.F.A.) F.F.A.) ~
100 0 2.42 5.75 3.33 75.8 5.20 2.78 61.2 2.42 12.5 10.1
75 25 2.42 5.54 3.12 71.1 5.20 2.78 61.2 2.49 12.2 9.71
66.6 33.3 2.21 4.50 2.29 52,2 5.20 2.99 65,8 2.21 11.6 9.39
50 50 0.693 3.12 2.43 55.4 4.92 4.23 93.2 2.21 10.4 8.19
40 60 0.208 2.49 2.28 52.0 4.50 4.29 94.5 0.970 9.70 8.73
33.3 66.6 0.140 2.42 2.28 52.0 4.50 4.36 96,0 0.900 9.54 8.64
25 75 0.140 1.59 1.45 33.0 4.43 4.29 94.5 0.900 9.40 8.50
0 100 0.140 0.692 0.552 12.6 4.43 4.29 94.5 0.900 6.49 5.59
Acid degree
before emulsification
Fresh fat 0.304
F a t + butyric, caproic and caprylic acids 4.39
F a t + myristic, palmitic and stearic acids 4.54
b N e t F . F . A . = F . F . A . - F . F . A , of corresponding emulsion made with fresh fat.
c N e t F . F . A . = F . F . A . - F . F . A , of corresponding fresh milk,
756 E. N. F R A N K E L AND N. P. T A R A S S U K

tyric, caproic, and caprylic acids) and high-molecular-weight f a t t y acids (my-

ristic, pahnitic, and stearic acids). Recoveries in the milk emulsions were calcu-
lated from the acid degree of the fats before emulsification and the free fat
acidity values of the corresponding emulsions.
The results presented in Table 2 reveal that as the ratio of petroleum ether
to ethyl ether increases, recovery of low-molecular-weight f a t t y acids decreases
and recovery of high-molecular-weight f a t t y acids increases, the latter reaching
a constant value when petroleum ether is 60% or more of the solvent mixture.
High free fat acidity values were obtained with the emulsion made with fresh
fat and with fresh homogenized milk when the proportion of ethyl ether was
increased above 40%. This indi.cates that a portion of the aqueous phase of the
milk would be extracted in the ethereal layer, thus contributing to the free fat
acidity. On the basis of these results, a ratio of 40% ethyl ether and 60% petro-
leum ether was considered to provide the highest recovery of f a t t y acids with
the least extraction of the aqueous n o n f a t t y acid constituents that would con-
tribute to the free fat acidity. This solvent ratio, based on recovery of added
f a t t y acids, proved to be the same as that of Johnson and Gould (12), which was
based on acid degree values and percentage recovery of solvent extracted milk
fat from cream.
E l e c t of the p H of rancid milk on the recovery of f a t t y acids. Johnson and
Gould (13) reported that a higher recovery of f a t t y acids from milk or cream
results from acidification to p i t 2 with sulfuric acid before extraction with
solvents. They stated that negative qualitative tests obained for sulfate ions
indicated that no entrained sulfuric acid resulted from this procedure. Nashif
and Nelson (16) also acidified their samples to a pink color with thymol blue
before extraction with solvent.
The effect of p H on the recovery of f a t t y acids was studied on rancid milk
and milk emulsions containing low-molecular-weight and high-molecular-weight
f a t t y acids. A solution of sulfuric acid (25% by volume) was added to measured
samples of milk in the centrifuge tubes to obtain p H values ranging from 5.0 to
1.0. Representative results from several experiments in Table 3 show an increase
in free fat acidity as p H was lowered. W h e n p H was lowered below 5.0, the

TABLE 3
Effect of lowering the pH of rancid milk and mille emulsions with sulfuric acid
on the free fat acidity and recovery of fatty acids
Milk emulsion of fat Milk emulsion of fat
Rancid ~- butyric; caproic, ÷ myristic, palmitic,
pH milk and caprylic acids a and stearic acids b
(F.F.A.) (F.F.A.) (~/v recovery) (F.F.A.) (% recovery)
6.5 20.5 3.62 58.2 6.13 96.0
5.0 22.0 3.87 62.2 6.45 101
4.0 22.3 3.87 62.2 6.72 105
3.0 22.7 4.16 66.8 6.96 109
2.0 23.7 4.38 70.4 7.22 113
1.0 23.8 6.20 99.6 7.74 121
a Acid degree before emulsification: 6.22.
b Acid degree before emulsification: 6.38.
 R E C O V E R Y OF F R E E F A T T Y A C I D S 757

emulsion containing high-molecular-weight fatty acids showed recoveries of

fatty acids that exceeded 100%. This may be the result of a number of factors,
as follows • (a) entrained sulfuric acid in the ether layer ; (b) shift of the phos-
phate equilibrium in milk from I t P O ( - to H2P04- and H~P04, the latter of
which may be carried over in the ether extract; (c) hydrolysis of milk fat by
the added acid. The first possibility could not be verified since it is difficult to
test for small amounts of sulfate in the ether layer. The phosphorus contents of
the fats obtained from rancid milk to which sulfuric acid was added prior to
extraction and the same milk without sulfuric acid were 0.238 and 0.174 rag. per
gram of fat, respectively. The recovery of fat in the ether layer was the same in
both milks. This indicates that the second factor plays some role in the increased
free fat acidity. This contribution of phosphoric acid to the increased free fat
acidity was confirmed by the excessively high free fat acidity value obtained
in fresh mille (5.50) when phosphoric acid (85%) was used to lower the pH of
milk to 2 prior to solvent extraction. In order to determine whether fat hydroly-
sis had occurred in the rancid milk to which sulfuric acid was added (pH 2),
titrations were made of both the ether and aqueous layers obtained after extrac-
tion of this milk, and of the same milk without added sulfuric acid. The differ-
ence between the total acidity of the ether plus aqueous phase in the two milks
(1.0 milliequivalent) in no case exceeded the acidity of the added sulfuric acid
(1.1 milliequivalent). This precludes the possibility that the acid hydrolyzed
the fat.
Since the evidence indicates that the higher free fat acidity obtained in milk
when its p H is lowered to 2 with sulfuric acid before solvent extraction is due
partly to factors other than higher recovery of fatty acids, the adoption of this
procedure in the extraction-titration method was not deemed justified. Whether
there is actually a greater recovery of f a t t y acids as a result of lowering the p H
of milk can be established only by determinations of individual fatty acids in
the ether extract.
Effect of lactic acid in rancid milk on the free fat acidity. It has been re-
ported that the presence of lactic acid in rancid cream gave a greater increase
in acid degree in churned fat than in solvent-extracted fat (12).
Varying amounts of lactic acid (7% by volume) were added to a series of
samples of rancid milk. Determinations were made of free fat acidity by the
extraction-titration procedure and titratable acidity. The results presented in
Figure 2 show that with increasing amount of lactic acid added to rancid milk,
free fat acidity increased negligibly, whereas titratable acidity increased sub-
stautially. Since lactic-acld producing organisms are inhibited in rancid cream
and milk (4, 5, 21), the effect of lactic acid in rancid cream on the fat acidity
is not a serious limitation.
Determination of free fat acidity in milk and cream of varying rancidity.
The relationship between extent of rancidity and free fat acidity was determined
by using rancid milk diluted with fresh milk in varying proportions to vary
rancidity. Results from the proposed extraction-titration procedure were com-
pared with titratable acidity and surface tension determinations. Figure 3
758 E. N. FRANKEL AND N.P. TARASSUK

~5.0 I I t

0.7

0,6

IO.C o.5~

J 0.4 ~,
jJ g
==
j°J 0.3~
5.0

I
Io j o.2-~
o_

• : Free Fat Acidity 0.1

o : Titratable Acidity

0.0 I I ~ 0.0
0.0 0.05 0.I0 0.15 0.20
Percent Lactic Acid Added

FIG. 2. Effect of lactic acid on the free fat acidity and titratable acidity of rancid milk.

shows t h a t free f a t a c i d i t y of m i l k as d e t e r m i n e d b y the p r e s e n t p r o c e d u r e varies

i n direct p r o p o r t i o n to the p e r cent r a n c i d m i l k m i x e d w i t h f r e s h milk.
Several e x p e r i m e n t s were c a r r i e d out to establish a n e x t r a c t i o n - t i t r a t i o n
p r o c e d u r e f o r c r e a m t h a t w o u l d give the same free f a t a c i d i t y as m i l k c o n t a i n i n g
a n equal a m o u n t of free f a t t y acids. A s a m p l e of r a n c i d c r e a m of 2 5 ' ~ f a t was

I0.(
/I
/ -t .50

50.0

T_ 5.( .v-. i_.

= -f /
2:2{
A= l'itratoble Acidity ~. 10 20.0
o: Surface Tension J
I
0.( I L = J00-- 10.0
0.0 25.0 50.0 75.0 I00.0
Percent Rancid Milk Mixed with Fresh Milk
FIG. 3. :Free fat acidity, titratable acidity, and surface tension of rancid milks varying in
degree of rancidity.
 RECOVERY OF F R E E F A T T Y A C I D S 759

diluted with fresh skimmilk to give samples with fat contents v a r y i n g from 5
to 25%. W h e n computing the free f a t acidity, the extent of dilution of the
degree of rancidity by the skimmilk was taken into consideration. I t was ob-
served that when the size of the sample was kept constant (at 10 g.) the free fat
acidity decreased as the f a t content of the samples increased. This indicated a
lowering of the efficiency of the solvent extraction of f a t t y acids as the f a t per-
centage of the samples increased. Therefore, the weight of the samples was so
varied that the amount of f a t was kept c o n s t a n t - - c o r r e s p o n d i n g with the fat
content of 10 ml. of 4% milk. This maintained a constant free f a t acidity, irre-
spective of the fat percentage of the cream samples. This latter procedure was
thus adopted.
This established procedure for rancid cream was compared with the direct
titration of f a t obtained f r o m the same cream by Johnson and Gould's extraction
(12)--without the use of sulfuric a c i d - - a n d by churning'. Samples of v a r y i n g
rancidity were p r e p a r e d by mixing rancid cream with fresh cream in v a r y i n g
proportions. The values for free fat acidity obtained b y the present procedure
were corrected for the titration value contributed by the n o n f a t t y acid constitu-
ents of the creams. The latter value was determined on artificial emulsions of
neutral fat passed through Amberlitc I R A 400 and of fresh skimmilk, containing
the same f a t percentage as the corresponding cream. This value for the blank
varied f r o m 0.13 to 0.16 ml. of 0.025 N alkali per entire ether layer. F i g u r e 4 re-
veals a linear relationship between rancidity and fat acidity as determined by all
three procedures. The similar slopes obtained by the three methods can be
explained by the fact that the same distribution of f a t t y acids existed in the
samples of cream, which varied in degree of rancidity. I t is evident that a
higher sensitivity can be achieved by the method under study t h a n by the titra-
tion of either the solvent extracted or the churned fats.
I i i

5.0 ~ / ~

4.0

3,0

2.0

1.0
o:AcidDegreeofChurnedFat
0.0 i i i
0.0 25.0 50.0 75.0 I00.0
PercentRancidCreamMixedwithFreshCream
FIG, 4. F a t acidity of rancid creams as determined by proposed extraction-titration method
and by titration of the fat obtained from the same creams by solvent extraction and by churn-
ing.
760 E.N. FRANKEL A N D N, P . T A R A S S U K

Higher initial values of free fat acidity for fresh cream were obtained by
the method under study than by the other two procedures. Since these values
are much higher than those usually obtained oll fresh, churned fat, samples of
fresh milk and cream from several sources were analyzed for free fat acidity
by the method under study. Values ranging from 1.50 to 3.50 were obtained with
an average of 2.01. This substantiates the findings of Thomas et al. (22) that
the content of free fatty acids of fresh milk is much higher than has generally
been accepted on the basis of acid degree values of churned fats. However, these
authors did not consider the possible activation of " n a t u r a l l y active" lipase
by cooling raw milk (20). Fur t he r investigation is being made of variations of
free fat acidity in freshly drawn milk.
Recovery of added .fatty acids. The effectiveness of the proposed procedure
to extract individual fatty acids was determined on artificial emulsions made
with " a c i d - f r e e " fat to which varying amounts of fatty acids were added and
with fresh skimmilk, to give a 4% milk. In addition, rancid fat was mixed with
fresh fat in varying proportions and then emulsified in skimmilk to give milks
of varying rancidity. The free fat acidity obtained on controls made with "acid-
f r e e " fat was subtracted from that obtained on the corresponding rancid emul-
sions. The recoveries of individual fatty acids and the fat t y acids from rancid
fat were calculated from the slope of the line determined by the method of least
squares, obtained by plotting free fat acidity of milk emulsions against the acid

15.0

X=I r5

*,4 .~7" "6

5 ~$111__ _

"/"
5.o "i
O.G I I
0.0 5.0 I 0.0 15.0
Acid Degree of Fat Before Emulsification
FIG. 5. Recovery of added f a t t y acids by proposed e x t r a c t i o n - t i t r a t i o n m e t h o d in artificial
m i l k emulsions (1 = b u t y r i c acid, 2 = caproic acid, 3 -- p a l m i t l c acid, 4 : stearic acid,
5 --- oleic acid, 6 = f a t t y acids f r o m cha~aed rancid f a t ) .
 RECOVERY OF FREE FATTY ACIDS 761

degree of the f a t before emulsification. The results of this experiment are pre-
sented in F i g u r e 5. The percentage recovery calculated for butyric, caproic,
palmitic, stearic, and oleic acids and the f a t t y acids f r o m churned rancid f a t
are 29.3, 38.4, 95.6, 99.8, 97.3, and 93.2, respectively. The data for butyric and
caproic acids were calculated on the basis of the initial slope in the range of
acid degree f r o m 0.00 to 5.00.
An a t t e m p t was made to compare the recovery of f a t t y acids according to
whether they are added directly to milk or cream, as done by Johnson and
Gould (12, 13), or to f a t before emulsification with skimmilk. This was only
feasible for b u t y r i c acid, which is relatively soluble in water. The f a t t y acids
above butyric acid in the homologous series are v e r y slightly or not soluble in
water and no homogeneous distribution could be obtained by their direct addition
to milk. A recovery of 28.6% of butyric acid was obtained b y the proposed
extraction-titration procedure when this acid was added as a 0.25 N aqueous
solution directly to milk. This recovery is equivalent to t h a t obtained above by
adding the acid to the f a t phase of an emulsion. Therefore, butyric acid, when
added directly to milk, a p p e a r s to be distributed in the same way as when
added to fat before emulsification with skimmilk.

TABLE 4
t~eproducibility of results
Mean F.F.A. Standard 95% confidence limits
reading ~ deviation for mean F.F.A. reading
9.41 0.193 +0.0815
15.2 0.321 ___0.153
22.8 0.477 ___0.201
a For 24 determinations on each lot of rancid milk.
Reproducibility of results. The free f a t acidity w a s determined t w e n t y - f o u r
times on each of three lots of rancid milk by two technicians. The statistical
analysis of these data is reported in Table 4. The n a r r o w 95% confidence linlits
obtained for the mean free f a t acidity values show that the results are highly
reproducible.
SUMMARY AND CONCLUSIONS

An extraction-titration method for determining free f a t t y acids in rancid

milk and cream is described. Factors affecting the recovery of f a t t y acids b y
this method were studied.
A f t e r evaluating various extraction methods for estimating free f a t t y acids
in milk and cream, it is evident that no method is as yet available t h a t p e r m i t s
quantitative recovery of all the f a t t y acids. I t was demonstrated that the proposed
extraction-titration method makes possible a 95 to 100% recovery of the high-
molecular-weight f a t t y acids added and 52 to 58% of the low-molecular-weight
f a t t y acids added (mixture of butyric, caproic, and c a p r y l i c - - T a b l e s 2 and 3).
The method is simple and can be applied directly to milk and cream.
Although the information on specificity of lipase action in milk is only scanty
in the literature, there is no evidence that either the high-molecular-weight f a t t y
7~2 E . N . FRANKEL AND N. P. TARASSUK

a c i d s or t h e l o w - m o l e c u l a r - w e i g h t f a t t y a c i d s a r e e x c l u s i v e l y h y d r o l y z e d f r o m
m i l k f a t . I t a p p e a r s , t h e r e f o r e , t h a t t h e d i s t r i b u t i o n of t h e p r o d u c t s of h y d r o l y s i s
of m i l k f a t will be r e l a t e d to t h a t of t h e f a t ' s o r i g i n a l f a t t y a c i d c o m p o s i t i o n .
This is s u p p o r t e d b y t h e s i m i l a r f a t c o n s t a n t s o b t a i n e d w i t h e t h a n o l f r a c t i o n s
f r o m f r e s h a n d r a n c i d m i l k f a t s (12) a n d t h e a n a l y s e s of s t e a m - v o l a t i l e a n d
w a t e r - i n s o l u b l e f a t t y a c i d s in r a n c i d f a t s (8). The l o w - m o l e c u l a r - w e i g h t f a t t y
a c i d s ( b u t y r i c , c a p r o i c , a n d c a p r y l i c ) c o n s t i t u t e o n l y !4.7 tools p e r c e n t of the
t o t a l f a t t y acids of m i l k f a t (11). U s i n g t h e r e c o v e r y v a l u e s o b t a i n e d b y t h e
p r o p o s e d e x t r a c t i o n - t i t r a t i o n m e t h o d , it c a n be e s t i m a t e d t h a t a b o u t 9 0 % of the
t o t a l f a t t y acids r e l e a s e d f r o m m i l k f a t a t a n y g i v e n d e g r e e of h y d r o l y s i s can
be d e t e r m i n e d b y this m e t h o d . S i m i l a r c a l c u l a t i o n s a p p l i e d to d a t a o b t a i n e d f r o m
c h u r n e d f a t (19) y i e l d a v a l u e of a p p r o x i n l a t e l y 6 5 ~ f o r r e c o v e r y of t h e f a t t y
acids from milk fat.
I n v i e w of the l i m i t a t i o n s of the a c i d d e g r e e d e t e r m i n a t i o n f o r c h u r n e d fat,
the v a l i d i t y of p r e v i o u s l i p a s e s t u d i e s b a s e d u p o n a c i d d e g r e e of c h u r n e d f a t has
been q u e s t i o n e d (12). A l t h o u g h t h e r e a r e a p p r e c i a b l e differences in recoveries
of free f a t t y acids b y v a r i o u s m e t h o d s , t h e d a t a p r e s e n t e d in t h i s p a p e r i n d i c a t e
a l i n e a r r e l a t i o n b e t w e e n t h e e x t e n t of r a n c i d i t y in c r e a m a n d f a t a c i d i t y as
d e t e r m i n e d b y the r e p o r t e d e x t r a c t i o n - t i t r a t i o n m e t h o d a n d b y the a c i d degree
o f s o l v e n t - e x t r a c t e d a n d c h u r n e d fat. I t is t h e r e f o r e c o n c l u d e d t h a t p r e v i o u s
l i p a s e s t u d i e s b a s e d u p o n a c i d d e g r e e Of c h u r n e d f a t r e m a i n v a l i d w h e n this
v a l u e was used f o r d e t e r m i n i n g o n l y t h e r e l a t i v e e x t e n t of r a n c i d i t y .

ACKNOWLEDGMENT
The help of H. L. Ahter, Department of Mathematics, in the statistical analysis is grate-
fully acknowledged.
REFERENCES
(1) ARMSTI~ONG,T. V., AND HARPER, W. J. A Modified Rapid Solvent Extraction Method for
the Estimation of Water Insoluble F a t t y Acids in Cream. Milk: Prod~tets J., 45 (5) :
37. 1954.
(2) Bo~Gs~a5~, B. Investigation on Lipid Separation Methods. Separation of Cholesterol
Esters, Glycerides and Free Fatty Acids. Acta Physiol. Scand., 25: 111. 1952.
(3) BREAZE4LE,D. F., AND BIRD, E. W. A Study of Methods for the Determination of Acid-
ity in Butterfat. J. Dairy Sci., 21: 335. 1938.
(4) COSTILOW,R. N., AND SPECK, M. L. Inhibition of Streptococcus lactis in Milk by Fatty
Acids. J. Dairy Sci., 34: 1104. 1951.
(5) COSTILOW,R. N., AND SPECK, M. L. Inhibitory Effect of Rancid Milk on Certain Bac-
teria. J. Dairy Sci., 34: 1119. 1951.
(6) DUNKLEY, W. L. Hydrolytic Rancidity in Milk. I. Surface Tension and Fat Acidity as
Measures of Rancidity. J. Dairy Sci., 34: 515. 1951.
(7) DUNKLEY, W. L., AND S~[ITIt, L. M. tIydrolytic Rancidity in Milk. I I L Tributyrinase
Determination as a Measure of Lipase. J. Dairy Sci., 34: 935. 1951.
(S) FRANKEL, E. N. Specificity of Lipase Action in Milk. M.S. thesis, University of Cali-
fornia, Davis. 1951.
(9) HERRINGTON,B. L., AND KRUKOVSKY, V. N. Studies of Lipase Action. I. Lipase Action
in :Normal Milk. J. Dairy Sci., 22: 127. 1939.
(10) HILLIG, F. Determination of Water-Insoluble Fatty Acids in Cream and Butter. J.
Assoc. Offic. Agr. Chemists, 30: 575. 1947.
 RECOVERY OF FREE FATTY ACIDS 763

(11) JACK, E. L., AND }IENDE~SON, ,]-. L. The F a t t y - A c i d Composition of Glyceride Fractions
Separated from Milk F a t . J. Dairy Svi., 28: 65. 1945.
(12) JOHNSON, B. C., AND GOULD, I. A. Milk Lipase System. I I . Comparison of Solvent Ex-
traction and Churning Methods for Obtaining F a t f r o m Milk for Free F a t t y Acid
Measurement. J. Dairy Sci., 32: 435. 1949.
(13) JOtINS0~NT, B. C., AND GOULD, I. A. Milk Lipase System. I I I . F u r t h e r Studies of the
Solvent Extraction Procedure for Obtaining F a t f r o m Milk for Titration. J. Dairy
Sci., 32: 447. 1949.
(14) ]~LEINZELLER, A., AND TlcI~f, A. R. Use of a Mixed Indicator in the T i t r a t i o n of F a t t y
Acids. Analyst, 59: 241. 1944.
(15) LONCIN, M., AND JACQMAIN, D. Contribution ~ l']~tude de la Lipase du Lait. Lc LaiG
31: 233. 1951.
(16) •ASHIlV, S. A., AND NELSON, F. E. The Lipase of Pseudomonas fragi. I. Characteriza-
tion of the Enzyme. J. Dairy Sci., 36: 459. 1953.
(17) S~ITH, L. M., LU~ERT, D. J., AND THOI~lqTON, H. R. E s t i m a t i o n of Lipase in Dairy Prod-
ucts. I. A n E x t r a c t i o n - T i t r a t i o n Method for E s t i m a t i o n of Milk Lipase. Can. J. t~e-
search, lv, 27: 483. 1949.
(18) TARASSUK, N. P. Rancid Flavored Milk: I t s Cause and Control. Intern. Assoc. Milk
Dealers, Assoc. llull., 6: 153. 1939.
(19) TARASSUK, N. P., AND FI~ANKEL, E. N. Evaluation of the Acid Degree of F a t and Ex-
tractlon-Titration Methods for the E s t i m a t i o n of Free F a t t y Acids in Rancid Milk
a n d Cream. (Abs.) J. Dairy Sci., 37: 646. 1954.
(20) TAILdSSUK, N. P., AND RICYIAI~DSOIq, G. A. Inhibition of Lipase Activity in Raw Milk.
Science, 93: 310. 1941.
(21) TARASSUK,N. P., AND SMITH, F. R. Relatiou of Surface Tension of Rancid Milk to I t s
Inhibitory Effect on the Growth and Acid F e r m e n t a t i o n of Strcptococc~s lactis. J.
Dairy Sci., 23: 1163. 1940.
(22) THOMAS, W. R., HAPPEn, W. J., AND GOULD, 1. A. Free F a t t y Acid Content of F r e s h
Milk as Related to Portions of Milk D r a w n . . 7 . Dairy Sci., 37: 717. 1954.