Precision and Sensitivity of a Test for Vegetable Fat Adulteration
of Milk Fat ~
ABSTRACT
A test for routine screening of Moz-
zarella cheese and butter for vegetable fat
adulteration is described. Fat is extracted
and saponified. The potassium salts of the
fatty acids are measured through direct
gas chromatographic analysis. A ratio,
calculated from the concentrations of
butyric and oleic acids, is used to evaluate
the purity of a sample. The test offers
good precision and can detect less than
10% partially hydrogenated vegetable fat.
INTRODUCTION
In recent years, consumers have shown
interest in quality, safety, nutrition, health
aspects, and the labeling of food products. The
dairy industry has perceived this consumer
climate as a marketing opportunity and has
introduced advertising and promotional cam-
paigns such as the "Real Seal" program to help
develop consumer awareness of the natural
nutrition in dairy products. The controversial
implication of such a promotion is that imita-
tion products are less desirable than "real"
products.
Imitation and part-imitation dairy products
have shown continued, impressive growth over
the past several years (4, 14). This fact alone is
enough to cause concern within the dairy
industry. Unfortunately, the problem is com-
pounded by the occasional unscrupulous
manufacturer who, in an attempt to reduce
costs or gain a competitive edge, may substitute
some or all of the milk fat in a dairy product
Received June 8, 1987.
Accepted October 5, 1987.
1Research supported by the Vermont Agricul-
tural Experiment Station, University of Vermont,
Burlington.
1988 J Dairy Sci 71:574-581
J. R. FOX, A. H. DUTHIE, and S. WULFF
Dairy Foods Program
Department of Animal Sciences
University of Vermont
Burlington 05405-0044
with less costly vegetable fat. This practice is
acceptable if the product is correctly labeled;
improperly labeled products, however, have the
13otential to do great harm to the dairy in-
dustry. A program such as "Real Seal", which
makes an implicit promise to the consumer,
should have some system to verify the au-
thenticity of products that producers submit as
candidates for inclusion in the promotion
and also to monitor compliance with labeling
regulations for products containing imitation
ingredients.
Methods to detect misrepresented products
include the sterol acetate melting point meth-
od, gas chromatographic analysis for /3-sito-
sterol, and the Reichart-Meissl and Polenske
values (30). All three methods are recognized as
official by AOAC (1), and in addition, the
Reichart-Meissl and Polenske values often are
considered appropriate for general use as a
screening method. The official methods,
although very sensitive to the presence of fats
of vegetable origin in milk fat (<2%), require
relatively elaborate sample preparation and
handling techniques and interpretation of the
results (35, 36). The Reichart-Meissl and
Polenske methods provide empirical data on the
relative concentrations of groups of fatty acids
(26). Limits of sensitivity for the detection of
vegetable fats could not be ascertained from the
literature. Other proposed methods include
~-tocopherol (vitamin E) assays (25), analysis
of various nonsaponifiables (9), analysis of
thermal properties, various methods and
interpretations of triglyceride analysis (17, 23,
28, 33), and analysis of fatty acids (3, 8, 11,
16, 19, 20, 22, 34, 36). The preferred con-
firmatory regulatory method is the analy-
sis of/3-sitosterol (30).
Current methods that analyze fatty acid
composition almost invariably require the
transformation of fatty acids to either methyl
or butyl esters (19, 29). These compounds,
particularly the short-chain fatty acid methyl
574
 TESTING FOR
esters, are highly volatile, and even with special
precautions, losses of the short-chain fatty acids
occur (12, 22, 29).
A method that converts fatty acids to
potassium salts and then analyzes them by gas
chromatography eliminates many of the prob-
lems associated with other methods of fatty
acid analysis (7, 16). Most notable is the
elimination of the problem of sample losses due
to vaporization. The fatty acid salts are injected
into a gas chromatograph in which the carrier
gas is saturated with formic acid vapor. In the
acidic environment the salts are converted to
free fatty acids and are analyzed as such.
Efficiencies of conversion and recovery have
been documented (7, 16).
Concentrations of individual fatty acids in
milk fat are highly variable (2, 15, 18, 21, 32).
Ratios of concentrations of selected fatty acids
or fatty acid groups have shown some success in
defining normal milk fat in terms of its fatty
acid composition (3, 11, 12, 15, 22, 34, 36,
37); however, none of these references was
primarily concerned with the effect on these
relationships of a foreign fat added to the milk
fat; rather they concentrated on describing the
relationships as they exist in pure milk fat. In
addition, use of the short-chain fatty acids, C4
through C10, in such descriptive relationships
has been largely overlooked, possibly because
of the lack of appropriate, quantitative meth-
odology.
We developed a method that constitutes an
improvement over other available methods in
that it eliminates handling problems associated
with volatile acids, is simple, and has good
sensitivity. The method shows potential for use
as a routine screening method to identify
samples of fat extracted from dairy products
that may be adulterated with vegetable fat (10).
This paper reports an evaluation of the pre-
cision and sensitivity of this proposed method
for detecting vegetable fat in milk fat.
MATERIALS AND METHODS
Commercial brands of butter and Mozzarella
cheese were purchased approximately biweekly
=Vegetable fat samples were provided by Kraft
Incorporated, Industrial Foods Division, Mepphis, TN.
FAT ADULTERATION 575
from retail outlets, and samples were obtained
from cheeses and butter manufactured in the
University of Vermont dairy plant. The pre-
cision of the method was evaluated with five
replicate samples each of pure butter and of
Mozzarella cheese. Cheeses were shredded to
obtain representative samples, and the fat was
extracted from the cheese and butter by the
Mojonnier method (27). The extracted fat from
each sample was divided into four portions, and
each portion was then blended with one of four
types of vegetable fat commonly used in the
manufacture of imitation dairy" foods (pure
soybean oil, two partially hydrogenated soy-
bean oils, and coconut oil) to concentrations of
0, 5.0, and 10.0% vegetable fat. 2 One-half-gram
samples of the fat blends then were saponified
with 6 ml of an isopropyl alcohol-KOH, solution
(25 mg KOH/ml) and held at 100°C in a water
bath for 20 min (24). A large glass bead was
placed over the top of the reaction vial to
moderate the rate of solvent evaporation. At
the completion of the reaction, the excess
alcohol was evaporated under a stream of
nitrogen. Complete saponification was verified
by thin-layer chromatography (5). Fifty milli-
grams of the dried mixture of potassium salts of
the fatty acids were added to 4 ml of 80%
(vol/vol) ethanol containing .25 rag/rot of C7
and 1.0 mg/ml of C17 as internal standards
(38). An aliquot of the solution, approximately
1 ~tl, then was injected into a gas chromatog-
raph, Hewlett-Packard model 5790, equipped
with an automatic sampler, Hewlett-Packard
model 3388. The chromatographic operating
conditions were the following: 1.83 m glass
column, 2 mm i.d., packed with 5% DEGS-PS,
100/120 Supelcoport (Supelco); injection port
temperature 250°C; detector temperature
250°C; helium carrier at 60 ml/min; and oven
temperature 100 to 200°C with an 8C°/min
increase programmed after an initial delay of 2
rain. The chromatograph was modified with a
trap to saturate the carrier gas with formic acid
(7). The concentrations of each of the 10 major
fatty acids, C4 through C18:2, were identified
and quantified according to the method of
Wulff et al. (38).
The sensitivity of the test was evaluated with
Mozzarella cheese fat containing each of four
types of vegetable fat in concentrations of 0,
5%, and 10% vegetable fat. Additionally,
shredded imitation cheese was blended with
Journal of Dairy Science Vol. 71, No. 3, 1988
576 FOX ET AL.

shredded Mozzarella cheese to approximate a
10% substitution of milk fat with vegetable fat;
fat was extracted and analyzed as described.
These data were used to evaluate the various
relationships and identify the most appropriate
ratio to use as an indicator of fat purity or
adulteration. The dependency of each ratio on
the percent added vegetable fat was determined
by linear regression. The regression coefficients
were used to identify the most favorable ratios.
The Shapiro-Wilk test for normal distributions,
Sheffe multiple range test, two sample t tests,
and correlation analysis were used to evaluate
the various ratios (31).
RESULTS AND DISCUSSION
Precision
The precision of the test, with regard to
reproducibility of the recoveries of the 10
major fatty acids, are reported in Tables 1 and
2. Coefficients of variation averaged 2.18% for
the fatty acids recovered from Mozzarella
cheese and .5, 1.43, and 1.24 for butyric,
capric, and oleic acids, respectively. Coef-
ficients of variation for pure butter averaged
2.53%; for butyric, caproic, and oleic acids
individually, they were .77, 1.16, and 2.31%,
respectively. Quantification of lauric acid (C12)
was compromised to various degrees by an
u n k n o w n interfering substance, and linoleic
acid (C18:2) was a poorly resolved peak (6).
The fatty acid compositions of butter and
Mozzarella cheese are similar (Table 3). There
were no significant differences in the con-
centrations of any of the 10 major fatty acids
among the two sources of fat for 12 mo (P>.5).
The following discussion refers, except where
noted, to data for Mozzarella cheese only;
however, it is assumed, based on the data from
Table 3, that the results can be generalized for
both products.
Choice of Ratio and Sensitivity
The ratio of choice must adequately satisfy
two important criteria if it is to be used to
identify an adulterated sample of milk fat.
First, the ratio must be relatively constant
throughout the range of normal milk fat. That
is, milk fat should be defined by this rela-
tionship within an acceptably narrow range of
calculated values for the relationship. Second,
the ratio must be responsive to the addition of
vegetable fats, i.e., the observed value of the
relationship should increase or decrease at a
significant rate as vegetable fat is added to the
pure fat.
The short-chain fatty acids (C4 through C12)
are not commonly found in vegetable fats
(Table 4). Most notable is butyric acid, which
does not occur naturally in any vegetable fat. It
would be advantageous for one or more of
these short-chain acids to compose a portion of

TABLE 1. Precision of the method for five replicate samples of Mozzarella cheese fat and Mozzarella cheese fat
containing 10% vegetable fat.

Cheese fat + 10% partially

Pure cheese fat hydrogenated soybean oil
Fatty acid Total CV Total CV

(%)
C4 4.02 .51 3.52 1.04
C6 2.55 .51 2.25 .82
Cs 1.39 .37 1.21 .84
C10 3.17 1.43 2.79 2.68
C12 4.14 4.77 3.68 5.21
C14 12.18 1.87 10.96 2.44
C16 29.20 1.94 26.86 1.40
C18:o 10.99 1.84 10.90 1.92
C18:1 26.23 1.24 31.15 1.37
Cia:2 6.12 7.34 6.66 3.42

Journal of Dairy Science Vol. 71, No. 3, 1988

 TESTING FOR FAT ADULTERATION 577

TABLE 2. Precision of the method for five replicate samples of milk fat and milk fat containing 10% vegetable
fat.

Milk fat + 10% partially

Pure milk fat hydrogenated soybean oil
Fatty acid Total CV Total CV

(%)
C4 4.15 .77 3.60 1.12
Ce 2.69 1.70 2.34 1.88
Cs 1.40 2.04 1.22 1.12
CIQ 3.23 1.16 2.82 1.22
Cla 3.81 3.60 3.32 2.05
C14 12.53 2.18 10.93 1.71
C16 30.57 1.56 28,31 1.43
C18:o 12.79 3.50 12.67 3.48
C1~:1 24.72 2.31 30.49 1.41
C18:2 4.10 6.44 4.28 6.55

t h e ratio b e c a u s e t h e a d d i t i o n of vegetable fat sensitivity, and overall s u i t a b i l i t y for this

will, in effect, d i l u t e t h e c o n c e n t r a t i o n s o f t h e
s h o r t - c h a i n acids b y an a m o u n t equal t o t h e
c o n c e n t r a t i o n o f t h e a d d e d vegetable fat. Oleic
acid also is an o b v i o u s c h o i c e because it is t h e
largest single c o m p o n e n t of m o s t h y d r o g e n a t e d
vegetable fats a n d is t h e long-chain f a t t y acid
t h a t differs m o s t significantly in c o n c e n t r a t i o n
f r o m l o n g - c h a i n f a t t y acids f o u n d in milk
fat. R a t i o s t h a t i n c o r p o r a t e these guidelines
were evaluated for t h e i r relative precision,
a p p l i c a t i o n . T h e e v a l u a t i o n criteria were 1 ) t h e
variability of t h e r a t i o and 2) t h e p r e d i c t a b i l i t y
o f its r e s p o n s e t o t h e a d d i t i o n o f vegetable fat.
S o m e of t h e possible f a t t y acid r e l a t i o n s h i p s ,
i n c l u d i n g ratios p r o p o s e d b y o t h e r investigators
(11, 13, 34, 36), are p r e s e n t e d in T a b l e 5. In
m o s t cases, t h e n u m e r a t o r is c o m p o s e d of
e i t h e r a single or g r o u p of s h o r t - c h a i n f a t t y
acids a n d t h e d e n o m i n a t o r is m a d e u p of a
single or g r o u p o f long-chain f a t t y acids. T h e

TABLE 3. Fatty acid composition of butter and Mozzarella cheese as percent of total.

Butter 1 Mozzarella cheese 2

Fatty acid Total CV Total CV

(%)
C4 4.09 6.06 4.12 4.03
C6 2.64 4.73 2.63 4.52
Cs 1.47 10.82 1.44 7.85
C10 3.35 9.79 3.24 8.67
C12 3.95 12.81 3.92 11.71
C~4 12.21 6.02 12.10 6.15
C~6 30.30 7.29 30.77 7,93
C18:o 11.67 11.31 12.09 9.76
C18:1 25.94 7.75 25.98 8.49
C18:2 4.36 31.88 1.34 39.48

ln=50.
an = 104.

Journal of Dairy Science Vol. 71, No. 3, 1988

578 FOX ET AL.

addition of a vegetable fat would decrease the

value of the numerator and increase the de-
•--4 ,4- ",O e¢~ nominator. The net effect would therefore be a
decrease in the value of the ratio. The values of
the majority of the ratios for the pure fats were
normally distributed (P<.01, correlation of
.2. normal scores vs. actual scores greater than .99
(N e~ ~0 l~ for all ratios except [C18:1] + [C14]). For
comparative purposes, the ratio values 2 stan-
dard deviations above and below the mean were
considered the upper and lower limits of the
ratio for pure milk fat. These values were used
o. ~. ~ m. ~.
to evaluate the relative sensitivities of the
different ratios.
The concentration of a mildly hydrogenated
soybean oil (Iodine value = 94.3) necessary to
depress the mean ratio by minus 2 standard
deviations, would be considered the minimum
level of detection of the test using a given ratio.
This critical value is used as a comparative
indicator of the maximum sensitivity of the test
for each of the ratios. Of the ratios considered,
the ratio [Ca] + [ C 1 8 : 1 ] varies the least, has an
approximate mean sensitivity of 6.08% added
vegetable fat, and has the best linear fit be-
tween the ratio and percent vegetable fat (r 2
~4 " : " 4 .748). Ratios proposed by Galanos ([C4] ×
100) + ([C12] + [C161 + [C18:1]) (11) and
Treiger ([C18:11 + [C14]) (36) had relative
sensitivities of 14.6 and 9.2% with r 2 of .237
and .418, respectively (Table 5).
o ~.. 2 .tq. The true sensitivity of the test is limited by
r¢~ . . . XO
high normal ratios. A greater concentration of
8
vegetable fat is required to depress the ratio to
4 the abnormal range if the value is high initially.
High normal values of the ratio are, in almost
all cases, the result of low relative concen-
trations of oleic acid. The observed mean value
for oleic acid was 25.98%, as percent of total,
0 0
with a range of 24.26 to 27.45%. There was
very little observed variability in the con-
centration of butyric acid. The observed mean
value was 4.12% with a range of 4.09 to 4.14%.
As the concentration of native oleic acid
decreases, additional vegetable fat will cause a
8e~
large relative change in the concentration of
8 d 4 ~ i Z :
oleic acid and result in an increased rate of
II
change in the value of the ratio. Adding 10%
vegetable fat to milk fat with an average con-
centration of oleic acid will increase the relative
concentration of oleic acid by about 18.0%,
m~
and adding the same vegetable fat to milk fat
<
[., r,. ~ 0 0 0 0
with a low concentration of oleic acid will

J o u r n a l o f Dairy Science Vol. 71, No. 3, 1988

 TESTING FOR FAT ADULTERATION 579

TABLE 5. Values of six selected fatty acid ratios and two individual fatty acids used to evaluate the purity of
a milk fat sample. Each ratio was calculated from the collected data and subjected to regression analysis to
determine the r 2 value for increasing vegetable fat concentration in milk fat using that ratio.

Relative
maximum
Ratio Mean CV R2 sensitivity

(%) (%)
C4/Cls:a .1593 8.16 .748 6.1
C 4 + C6/C1g:1 .2617 9.25 .681 7.8
C4 + Cs/Cls n .2154 8196 .677 7.6
C4 + C1o/C18:~ .2854 11.04 .570 9.3
Ca0/C~2 + C16 + C~s:~ 5.34 9.59 .237 14.6
Clg:~/C14 2.16 13.94 .418 9.2
C4 % of total 4.09 6.06 .551 7.5
C~s:, % of total 25.94 7.75 .499 10.1

increase the c o n c e n t r a t i o n by about 23.0%
(Table 6). The slope of the change in the ratio,
w i t h the addition of vegetable fat, increases
slightly at higher values of the ratio. The result
is that the sensitivity is slightly better for high
values o f the ratio than would be e x p e c t e d by
assuming a slope parallel to that calculated for
all values. Given a ratio at the e x t r e m e high end
o f the normal range o f ratios for pure milk fat,
a p p r o x i m a t e l y .19, and assuming a slope equal
to that calculated for all normal values, the
apparent m i n i m u m sensitivity w o u l d be about
13.4%. However, the slope calculated for this
ratio value is such that the apparent sensitivity
is actually a b o u t 10.5% added vegetable fat.
Most vegetable fats used in the f o o d and
dairy industries are h y d r o g e n a t e d to produce
desired chemical and physical properties of the
fat. One of the consequences of partial hy-
drogenation o f a vegetable oil, such as soybean
oil, is the conversion of linoleic acid, which
contains two unsaturated carbon bonds, to
oleic acid, a m o n o u n s a t u r a t e d fatty acid.
Therefore, with increasing degrees of hydro-
genation, there is an a c c o m p a n y i n g increase in
the relative c o n c e n t r a t i o n of oleic acid (Table
6). The greater c o n c e n t r a t i o n of oleic acid in a
highly hydrogenated vegetable fat will have a
greater effect on the value of the ratio than will
comparable a m o u n t s of a vegetable fat that is
not as highly hydrogenated when added to milk
fat (Figure 1).
Data in Table 7 show the change in the value
of the ratio with the addition of four types of
vegetable fat. With the addition of 10% parti-
all)" h y d r o g e n a t e d vegetable fat (soybean fat 1)
the value of the ratio decreases f r o m .1577 to
.1219, a - 2 3 . 0 % change. The addition of 10%
of a more highly h y d r o g e n a t e d vegetable fat
(soybean fat 2) results in a 27.0% reduction.

TABLE 6. Change in relative concentrations of oleic acid with the addition of 10% partially hydrogenated
vegetable fat to milk fats having high, average, and low normal concentrations of oleic acid.

Percent of total
% Vegetable fat Low Average High

0% 23.00 26.92 30.21

10.0% 28.37 31.74 34.47
% Change 23.34 17.90 14.12

Journal of Dairy Science Vol. 71, No. 3, 1988

580 FOX ET AL.

TABLE 7. Values of ratios with addition of four types of vegetable fats.

Added vegetable fat

0% 5% 10%
Type of fat Ratio CV Ratio CV Ratio CV

Pure soybean 1 .1577 7.2 .1556 6.9 .1418 6.8

Parti~ly hydrogenated soybean 12 .1577 7.2 .1375 7.2 .1219 6.4
Partially hydrogenated soybean 23 .1577 7.2 .1372 7.1 .1150 6.4
Coconut 4 .1577 7.2 .1562 3.4 .1553 8.7

1 Unhydrogenated soybean fat; iodine value = 130.6.

2 Partially hydrogenated soybean fat; iodine value = 94.3.
3 Partially hydrogenated soybean fat; iodine value = 68.6.
4 Iodine value = 5.1.

T h e effect, o n c e again, is t h a t as t h e degree o f little oleic acid. A m o r e a p p r o p r i a t e r a t i o to use

h y d r o g e n a t i o n increases, t h e slope o f t h e line
calculated f o r t h e value o f t h e ratio vs. t h e
a d d i t i o n o f vegetable fat also increases. T h e
l o w e r limits o f t h e value o f t h e r a t i o are m o r e
q u i c k l y e x c e e d e d w i t h t h e a d d i t o n o f this t y p e
o f v e g e t a b l e fat t h a n w i t h t h e a d d i t i o n o f t h e
less h y d r o g e n a t e d vegetable f a t a n d t h e sensi-
tivity is effectively increased.
A d d i t i o n o f 10% c o c o n u t f a t does n o t
significantly c h a n g e t h e value o f this r a t i o
( P > . 0 5 ) . This is n o t u n e x p e c t e d , given t h e f a t t y
acid c o m p o s i t i o n o f this fat ( T a b l e 4). C o c o n u t
f a t is highly s a t u r a t e d a n d c o n t a i n s relatively
for screening f o r t h e a d d i t i o n o f c o c o n u t fat
w o u l d possibly i n c l u d e laurie acid (C12).
CONCLUSIONS
T h e p r o p o s e d test for vegetable fat ad-
u l t e r a t i o n o f fat in Mozzarella cheese a n d
b u t t e r shows g o o d p r e c i s i o n or r e p r o d u c i b i l i t y
a n d a c c e p t a b l e sensitivity f o r d e t e c t i n g t h e
a d d i t i o n of less t h a n 10% partially h y d r o -
g e n a t e d s o y b e a n oil a n d v e g e t a b l e oils similar in
c o m p o s i t i o n to s o y b e a n oil, such as corn a n d
c o t t o n s e e d oil. V e g e t a b l e oils such as c o c o n u t
oil a n d palm k e r n e l oil w o u l d n o t be easily
d e t e c t e d b y this m e t h o d . T h e m e t h o d w o u l d b e
a p p r o p r i a t e for use as a screening test w h e r e
m u l t i p l e samples are r o u t i n e l y t e s t e d .

ACKNOWLEDGMENTS
This s t u d y was s u p p o r t e d in p a r t b y t h e
=~ .u. TYPE I
V e r m o n t A g r i c u l t u r a l E x p e r i m e n t S t a t i o n Pro-
j e c t H a t c h 385, t h e Dairy R e s e a r c h F o u n d a t i o n
~ .~s. ~ . of the United Dairy Industry Association, and
t h e Walker R e s e a r c h F u n d .

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Journal of Dairy Science Vol. 71, No. 3, 1988

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Journal of Dairy Science Vol. 71, No. 3, 1988