Determination of Content of Linseed Oil in Edible Soybean Oil
Transcript of Determination of Content of Linseed Oil in Edible Soybean Oil
Vol.35, No.9 (1986) 725
ORIGINAL
Determination of Content of Linseed Oil
in Edible Soybean Oil
Poorna P. MANANDHAR*, Akihiko NAGAO**
and Megumi YAMAZAKI***Central Food Research Laboratory , Ministry of Agriculture, Nepal (Babar Mahal,
Kathmandu, NEPAL)**National Food Research Institute , Ministry of Agriculture, Forestry, and Fisheries,
(Yatabe, Tsukuba, Ibaraki)
A method for detecting and computing the quantity of adulterant linseed oil in soybean oil was
developed by determining the fatty acid, sterol and tocopherol compositions of component oils and
adulterated mixtures.
In adulterated oil samples, a decrease in linoleic acid and increase in linolenic acid content were
observed with increase in the amount of linseed oil. Analysis of sterol constituents of linseed oil
showed a characteristic component having a retention time of 78 minutes which was also clearly observed
for the oil mixtures. A remarkable decrease in stigmasterol content was observed with increase in
linseed oil. Analysis of tocopherols showed the linseed oil to be richer than soybean oil in ƒÀ-toco-
pherol content and there to be a significant increase in ƒÀ-tocopherol content with increase in linseed
oil.
These findings provide sufficient evidence of the presence of adulterant linseed oil in soybean oil.
The limit of detection was as low as 5•`10% linseed oil in soybean oil.
1 Introduction
In Nepal, soybean oil is becoming increa-
singly popular and acceptable as the cooking
oil. Linseed oil1),23) on the other hand, is not
accepted as cooking oil, and is less expensive
than soybean oil. Hence, as the demand and
high price can lead to the adulteration, there
is a high chances of soybean oil to be adul-
terated with less expensive linseed oil.
There are a number of specific tests for de-
tecting defferent individual vegetable oils in
the oil mixtures2),3). Apart from the usual and
conventional determination of analytical con-
stants of vegetable oils, the analysis of fatty
acid compositions, sterols, glycerides, and to-
copherols had been carried out in different
ways and by different means using paper, thin
layer, column, gas, and liquid chromatographic
techniques of analysis; a lot of efforts had
been and is being exerted in the development
of methodology concerning detection of one
vegetable oil in another oil or oil mixture4)•`20),
But, for the detection of many kinds of spe-
cific adulterant oil, the details are still lacking.
The Codex Alimentarius Committee on Fats
and Oils of the Food and Agriculture Orga-
nisation/World Health Organisation21) also had
taken great deal of interest in this field and
has compiled a list of fatty acid ranges for
several commercial oils and fats. These ranges
are to be used for the authentication of the
different oils and fats. But, the ranges con-
tained in this list are very wide and hence
several oils could be classified under more than
one type of oil20).
In addition, in adulterated oil with low pro-
portions of one or more kinds of oils, the mere
determination of the overall fatty acid com-
positions is not sufficient to reveal the admix-
ture or to identify the adulteration. Hence, a
deeper and wider analysis of other important
constituents of oils are essentially important
for the proper identification and estimation of
the nature and extent of adulteration.
In our previous paper1), a characteristic sterol
component, and linolenic as well as stearic
acid contents of linseed oil were discussed as
the criteria for detection and estimation of 5
•`10% linseed oil when mixed with rapeseed
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726 J. Jpn. Oil Chem. Soc.
oil. The addition of 3•`5% soybean oil to
sunflower oil was detected on the basis of
negligible ƒ¿-tocopherol content of the latter8).
On the other hand, ƒÀ-tocopherol content of
soybean oil had been shown to be nil15) whereas
the linseed oil had appreciable quantity of ƒÀ-
tocopherol1). These analytical figures encour-
aged us to work on the field of detection and
estimation of linseed oil in soybean oil.
In this article, fatty acid, sterol and toco-
pherol compositions, determined by gas chro-
matography and high performance liquid chro-
matography respectively, are discussed as the
possible means of detection and estimation of
even a small proportion of linseed oil as an
adulterant oil in soybean oil.
2 Materials
Eleven soybean oil samples from soybean
seeds of Nepal, Japan, U.S.A. and China ori-
gin were prepared in the laboratory. One
sample each of reliable commercial soybean oil
and linseed oil were mixed together in the
proportion of 100:0; 95:05; 90:10; 85:15;
80:20; 75:25; and 0:100.
3 Methods
Fatty Acid
Fatty acid methyl esters were prepared from
well mixed sample oils as described previous-
ly1). Gas chromatographic analysis of methyl-
esters of fatty acids was performed with Shi-
madzu GC-7 A gas chromatograph equipped
with an FID. The chromatograph was fitted
with a 2.1m glass column, 3.2mm ID, packed
with 5% Advance-DS, coated on chromosorb
W (AW DMCS), (80•`100mesh). The carrier
gas was nitrogen at the flow rate of 50ml/min.
The column was operated isothermally at 190
The detector and injector temperature was
230•Ž.
Sterol
As described previously1), the unsaponifiable
matter from well mixed adulterated sample
oils were recovered and fractionated. The re-
covered unsaponifiable matter of each fraction
was analysed with Shimadzu GC-7 A gas chro-
matograph equipped with an FID on a glass
column, 3m•~2.6mm packed with 3% OV-17
on Chromosorb WAW, DMCS (60•`80mesh).
The column was operated isothermally at 265
•Ž with nitrogen at 50ml/min as carrier gas.
The injector and detector temperature was 300
•Ž. Total unsaponifiable matter of the adulte-
rated as well as laboratory extracted oil sam-
ples were also analysed directly with Shima-
dzu GC-7 A gas chromatograph equipped with
an FID. The conditions of analysis were the
same as discussed above.
Tocopherol
Tocopherol compositions of the adulterated
as well as the laboratory extracted oil samples
were determined by high performance liquid
chromatography. The experimental procedures
as well as the analytical conditions were the
same as described previously1).
Other Constants
Saponification values, iodine values, and re-
fractive indices of the adulterated oil samples
were determined as according to the Standards
of Analytical Method of the Japan Oil Chem-
i sts' Society22).
4 Results and Discussion
Fatty Acid Composition
Fatty acid compositions of the adulterated
and laboratory extracted oil samples are given
in the Tables-1 and 2 respectively. Linoleic
acid (C18:2) content of adulterated oil sample
decreased as the proportion of linseed oil
increased, but the significant decrease was not
observed. The linoleic acid content of soybean
oil ranged from 49% to 57% of the total fatty
acids with the standard deviation of •}2.256,
i.e. soybean oil could have linoleic acid as
47.13% (Table-2). On the other hand, ac-
cording to bailey's Industrial Oil and Fat Pro-
ducts23) the linoleic acid content of soybean oil
ranges from 43 to 56% of the total fatty acids
depending upon the variation in iodine num-
ber. With these analytical data as background,
it can be stated that the addition of more than
15% linseed oil to soybean oil could only be
suspected as adulterated.
Nextly, as shown also in Table-1, the lino-
lenic acid (C18:3) content increased as the
proportion of linseed oil increased while in
Table-2 the linolenic acid content of soybean
oil ranged from 8% to 11% of the total fatty
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Vol.35, No.9 (1986) 727
Table-1 Fatty acid compositions (% of total) of soybean oil-linseed oil mixturesa,
a. Analytical data obtained from GLC analysis of methyl esters of fatty acid in packed glass column. See the text for
operating conditions of GLC.
b. Saponification value., c. Iodine value., d. Refractive index at 40•Ž.
N.B.: Minor components of less than 0.06% are not tabulated.
Table-2 Fatty acid compositions (% of total) of some soybean oil samplesa.
a. Analytical data obtained from GLC analysis of methyl esters of fatty acid in packed glass column. See the text for
operating conditions of GLC.
N.B: Minor components of less than 0.06% are not tabulated.
acids, with the standard deviation of •}1.146.
Bailey's Industrial Oil and Fat Products23) has
quoted the range for this acid content of soy-
bean oil as 5•`11% of total fatty acids. Hence,
the linolenic acid content of soybean oil could
not be more than 12% of total fatty acids.
This indicates that there is a strong possibi-
lity of detection of adulteration level of less
than 10%. Other fatty acids did not show any
usefulness in solving the problem.
Sterol Composition
1) TLC-GLC Method
Sterol compositions of 4-desmethylsterol
fractions of soybean oil-linseed oil mixtures
are shown in the Table-3. Three main ster-
olic constituents e.q. campesterol, stigmasterol
and ƒÀ-sitosterol of retention times (RT) 51,
56, and 63min respectively were observed.
Even though a gradual decrease in stigmasterol
Table-3 Sterol compositions (%) of 4-desmethylsterol
fractions of soybean-linseed oil mixtures by TLC-GLC methoda.
a. See text for operating conditions of Gas Liquid Chro-
matograph
b. 51: Campesterol, 56: Stigmasterol and 63: ƒÀ-Sitosterol.
c. RRT of ƒÀ-Sitosterol (retention time, 63min) taken as
1.00
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728 J. Jpn. Oil Chem. Soc.
content was observed with increase in propor-
tions of linseed oil, no significant difference
was observed even at the adulteration level of
more than 20%. Similarly, campesterol (RT-
51) and ƒÀ-sitosterol (RT-63) contents of soy-
bean oil and linseed oil not differ much. Hence,
no significant conclusion could be drawn from
this analytical data. Further, for the quick and
more effective results, the direct GLC analysis
of total unsaponifiables, which eliminates the
operation of fractionations by TLC was also
carried out.
2) Direct GLC Method
Sterol compositions of soybean oil-linseed oil
mixtures and laboratory extracted soybean oils
are shown in the Table-4 and Table-5.
Table-4 showed a gradual decrease in stig-
masterol content of oil mixtures when the lin-
Table-4 Sterol compositions (%) from unsaponifiables of
soybean-linseed oil mixturesa.
a. Analytical data obtained from direct GLC analysis of unsaponifiables in
packed glasscolumn. See the text for GLC operating conditions.
Table-5 Sterol compositions (%) of unsaponifiable matter of some soybean oil samplesa
a. Data obtained by direct GLC analysis of unsaponifiable matter in packed glass
column. See the text for GLC operating conditions.
seed oil proportion increased. Similar case was
observed with ƒÀ-sitosterol content. On the other
hand, Table-5 showed the stigmasterol con-
tent of soybean oil in the range of 17.65% to
20.71% of total sterol with standard deviation
of •}1.534 which indicates that the stigma-
sterol content of soybean oil could be as low
as 16.48% of total sterol. Hence, with refer-
ence to Table-4, around 10% level of adul-
teration could easily be detected as far as the
stigmasterol content is concerned.
The ƒÀ-sitosterol ranged from 48.93% to 56.18
% of total sterol with standard deviation of
•} 3.602. That means the ƒÀ-sitosterol content
could be as low as 16.48% of the total sterol.
Hence, with reference to the Table-4, this
data could only help to detect the adulteration
level of more than 25%.
The most important feature in Table-4 and Table-5 was the sterolic component of retention time
(RT)-78 which was not shown to be present in soybean oil but its
presence was indicated even at 5% level of linseed oil added to soy-bean oil. Such component was also discussed as the characteristic com-
ponent of linseed oil in our pre-vious paper1). Hence, it seems to be an important criteria to detect and estimate the addition of as low
as 5% of linseed oil to
soybean oil. This charac-
teristic component had
been identified as grami-
sterol and is one of the
components of monometh-
ylsterol fraction of the
unsaponifiable matter1).
Tocopherol Composi-
tion
Tocopherol compositions
of adulterated sample oils
and the experimental soy-
bean oils are shown in
the Tables-6 and 7 res-
pectively. Linseed oil was
shown to be poor in ƒ¿-
tocopherol and ƒÂ-tocopherol
but rich in ƒÀ-tocopherol
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Vol.35, No.9 (1986) 729
Table-6 Tocopherol compositions (% of total) of soybean oil-
linseed oil mixturesa.
a. Data obtained from high performance liquid chromatographic analysis of tocopherol. See the text for operating conditions of HPLC.
Table-7 Tocopherol compositions (% of total) of some soybean oil sample oilsa.
a. Analytical data obtained from high performance liquid chromato-
graphic analysis of tocopherol. See the text for operating condi-tions of HPLC.
content (Fig.-1). The differences in tocopherol
contents of linseed oil and soybean oil could
be very useful in determining the adulteration
of linseed oil in soybean oil. With reference
to Table-7, the ƒ¿-tocopherol of soybean oil
ranged from 3.65 to 13.5% of total tocopherols
with the standard deviation of •}2.982. This
means, the ƒ¿-tocopherol content of soybean oil
could be as low as 0.67% of the total toco-
pherol. Similarly, ƒÀ-tocopherol content of soy-
bean oil could be as high as 4.81%. These
analytical data showed that ƒ¿-tocopherol con-
tent could not be helpful to identify the lower
level of adulteration. Similar case was observed
with ƒÁ-tocopherol and ƒÂ-tocopherol contents but
in the case of ƒÀ-tocopherol content it showed
Peaks 1, 2, 3 and 4 correspond to ƒ¿, ƒÀ, ƒÁ, and ƒÂ to-
copherols. HPLC conditions are given in the text.
Fig.-1 HPLC of soybean oil and
linseed oil tocopherols.
to be quite promising to detect and
estimate the addition of as low as about
5% linseed oil when mixed with soy-
bean oil.
Physical and Chemical Constants
Saponification value, iodine value and
refractive indices of adulterated oil
samples are shown in the Table-1.
Iodine value and refractive index in-
creased with the increase in the proportion of linseed oil. According to Bailey's Industrial Oil and Fat Products23) the average iodine number of soybean oil ranged from 125 to 130. Hence, the addition of even 5% linseed oil to soybean oil could be suspected as adulterated. But, it was also stated that soybean oil of iodine number of as high as 151 and as low as 103 have been reported which makes iodine number a hazy criteria to detect the adultera-tion although it can always be used as refer-ence.
5 Conclusion
Our analyses on fatty acid, sterol and toco-
pherol compositions in adulterated sample oils
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730 J. Jpn. Oil Chem. Soc.
and experimental soybean oils showed that
determinations of linolenic acid, gramisterol
and ƒÀ-tocopherol are sufficient enough to prove
the evidence of adulteration and to estimate
the addition of as low as 5•`10% of linseed
oil to soybean oil. However, it is always ad()
visable to consider some other helpful criteria
such as iodine number, refractive index and
some specific tests like hexabromide test2) as
the additional aids for the extract verification
of adulteration of linseed oil in soybean oil.
Acknowledgment
The participation of one of the authors (P.P.M.)
in this work was made possible by a fellowship by
the United Nations University, Tokyo.
(Received March 19, 1986)
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食 用 大 豆 油 中 の あ ま に 油 の検 出 と推 定
Poorna P. MANANDHAR*・ 長尾昭彦**・山崎 恵 **
*Central Food Research Laboratory, Ministry of
Agriculture (Babar Mahal, Kathmaudu, NEPAL)**農林水産省食品総合研究所(〒305茨 城県筑波郡
谷田部町観音台2-1-2)
大 豆油に混入 ざれ たあまに油の検 出及び混入量 を推定
す るための方 法(脂 肪酸,ス テ ロール及び トコフェロー
ル分析 によ る)を 検討 した。 あまに油混入大豆油 におい
て,あ まに油の割合 が増加 するに従 い脂肪酸組成で はリ
ノール酸 が減 少 し,リ ノ レン酸 が増加 した。一方,ス テ
ロール分析 の結果 は,ス チグマステ ロール が顕著 に減少
した。あまに油の保持 時間78minの ピークは大豆油に
は観察できない ピー クであ ることが分 かった。 トコフェ
ロール分析 は,あ まに油 には大豆油に比較 し β-トコフ
ェロールが多い ごとを示 した。以上 の結果 は大豆油 に混
入 され た偽 和油脂 としてのあまに油の検 出が可能 である
ことを示 し,そ の限界は5~10%と 考 え られ る。
42