第35巻 第5号 (1986) 359
報 文
Detection and Estimation for Composition of Linseed
Oil in Edible Rapeseed Oil
Poorna P. MANANDHAR*, Akihiko NAGAO**,
and Megumi YAMAZAKI**
* Central Food Research Laboratory, Ministry of Agriculture (Babar Mahal, Kathmandu, Nepal)** National Food Research Institute , Ministry of Agriculture, Forestry and Fisheries
(1-2-1 Kannondai, Yatabe-machi, Tsukuba-gun, Ibaraki,)
To develop a method for detecting and estimating the quantity of adulterant linseed oil in rapeseed oil, samples of these oils collected from different sources were analysed, among other things, for fatty acid, sterol and tocopherol composition content. Each oil sample was mixed with the other in the following proportions: 100:0; 95:5; 90:10; 85:15; 80:20; 75:25; and 0:100. All mixtures thus obtained were analyzed in the same manner as that for individual content oils. The saponification and iodine values and refractive indices of the mixtures were also measured.
In the adulterated rapeseed oil samples, the stearic and linolenic acid content increased with that of linseed oil. Also, GLC analyses of sterol constituents of the linseed oil showed characteristic retention time (RT) which were also clearly observed for the oil mixtures.
These observations indicate that the proportions of adulterant linseed oil when mixed 5 to 10%
with rapeseed oil can quite likely be detected. The analysis of tocopherol did not appear to facili-tate detection of linseed oil in any of the mixtures but the values of iodine, and butyro refracto-meter readings helped to indicate if linseed oil was present or not.
1•E Introduction
In Nepal for many centuries, vegetable oils
of locally grown Brassica species (rapeseed and
mustard) have been used as cooking oils. Very
rarely, some other vegetable oils are known to
have been used, but recently, because of the
high demand and short supply of traditional
cooking oil, soybean oil and imported rapeseed
oil are becoming increasingly acceptable among
Nepalese people. However, the oil from linseed
(Linium usitatissimum) is known to be popular
in some parts of Nepal, as the plant is easily
cultivated in those areas.
Linseed oil is easily oxidised and polymeris-
ed by autooxidation and by heat1), so it is not
generally recommended for cooking food. It is
principally used in the paint and varnish in-
dustries, but as this oil is comparatively chea-
per than rapeseed oil or mustard oil, incom-
petent traders often try to adulterate rapeseed
oil or mustard oil, with linseed oil in order to
gain more profit. Detection of the presence of
linseed oil in rapeseed oil or mustard oil has
been a problem for food authorities of Nepal
for a long time, and there is an urgent need
to establish a method for its identification and
quantification when mixed with other vegetable
oils. Until now the hexabromide test2) has
been the usual method used to identify the
presence of linseed oil in other vegetable oil.
However, as this test is positive when the fatty
acids containing more than three double bonds,
which react with bromine to give insoluble
hexabromide or polybromide, are present, it
can not be used as a specific test for linseed
oil. Mahlenbacher3) has stated that hexabro-
mide test, which is sometimes used to quan-
titatively measure insoluble bromide has been
shown to be unreliable. The authors have
also shown this to be the case when rapeseed
oil containing comparatively higher amounts of
linolenic acid reacted positively in the hexa-
bromide test.
Many different methods which are useful in
detecting adulteration of vegetable oils have
been reported3)•`15), but these have not been
specifically for linseed oil when mixed with
23
360 油 化 学
other oils. In most of the literature, detection
of sterol, fatty acid and tocopherol compositions
in adulterated oil have been emphasised16)•`23).
It can be assumed that brassicasterol can be
taken as an indicator for rapeseed oil. Simi-
larly, linolenic acid, the characteristic fatty acid
of linseed oil, is a good index to estimate the
presence of linseed oil in an oil mixture. Erucic
acid can also be used as a tool to identify adul-
teration in high erucic acid rapeseed oil, but
in the case of low erucic acid rapeseed oil, this
is not possible.
In this article, fatty acid, sterol, and toco-
pherol compositions determined by TLC, GLC,
and HPLC are discussed as possible indices for
detection and estimation of small proportions
of adulterant linseed oil when mixed in low
erucic acid rapeseed oil.
2 Materials
Seven kinds of reliable commercial rapeseed
oil of different origin, and four different sam-
ples of linseed oil from Japan and Nepal were
obtained. One sample of rapeseed oil was
adulterated with a linseed oil sample in the
proportion of 100:0, 95:5, 90:10, 85:15, 80:20,
75:25, and 0:100.
3 Methods
3•E1 Sterol Detection
The well mixed sample oil was saponified in
1N KOH (96% ethanol solution) and the unsa-
ponifiable matter was recovered with diethyl
ether. The unsaponifiable matter was fraction-
ated by TLC using the solvent system of hex-
ane: diethyl ether (70:30). Different bands on
the TLC plate were detected under a UV lamp
with the help of rodamine (0.01% in methanol)
solution as spray reagent. Each band was
scraped, extracted with ether and filtered. The
recovered unsaponifiable matter of each fraction
was analysed with a Shimadzu GC-7A gas
chromatograph equipped with an FID on a
glass column, 3.2m•~2.6mm, packed with 3%
OV-17 on Chromosorb WAW, DMCS (60•`80
mesh). The column was operated isothermally
at 265•Ž and the injector and detector tem-
perature was 300•Ž.
Total unsaponifiable matter was also directly
analysed by glass capillary gas chromatography.
The instrument used was a Shimadzu GC-7
AG gas chromatograph equipped with an FID
on a silica capillary column, 25m•~0.25mm,
chemically bonded with OV-1701 (Gasukuro
Kogyo Inc.). The carrier gas was He at a
flow rate of 0.96ml/min and a split ratio of
1/43. The column was operated isothermally
at 270•Ž. The detector and the injector tem-
perature was 300•Ž.
3•E2 Fatty Acid Detection
Fatty acid methyl esters were prepared from
the well-mixed sample oils by refluxing with
sulfuric acid-benzene-methanol [2ml sulfuric
acid in 230ml benzene-methanol (1:3) solution]
at 80•Ž for about 2.5h and then recovered
using diethyl ether. Gas chromatographic ana-
lysis of methyl esters was performed with a
Shimadzu GC-7 AG gas chromatograph equip-
ped with an FID on a BCL column, 30m•~
0.3mm, coated with Silar 5 CP (Shinwakakou
Inc.). The carrier gas was He at a flow rate of
1.07ml/min and a split ratio of 1/40. The
column was operated isothermally at 180•Ž. The
detector and injector temperature was 240•Ž.
3•E3 Tocopherol Detection
Tocopherol contents were determined by high
performance liquid chromatography. About 1.0
g of the well-mixed oil samples were diluted
with 10ml of n-hexane in 10ml capacity volu-
metric flasks then centrifuged at 3000rpm for
4min. The upper layer was filtered and 20ƒÊl
of the filtrate was injected into a Shimadzu LC
5A liquid chromatograph with a silica column.
The analytical conditions were the same as
described previously except for the flow rate,
which was 1.0ml/min24).
3•E4 Other Constants
Saponification values, iodine values, and re-
fractive indices were determined according to
the Standard of the Analytical Method, the
Japan Oil Chemists' Society25).
4 Results and Discussion
4•E1 Sterol Composition
4•E1•E1 TLC-GLC Method
Sterol compositions of desmethylsterol frac-
tions of the rapeseed oil mixtures are shown
in Table-1. Generally, three main sterolic
constituents e.g. brassicasterol, campesterol, and
β-sitosterol of retention times (RT) 44, 52 and
24
第35巻 第5号 (1986) 361
Table-1 Steral composition (%) of 4-desmethylsterol fractions of rapeseed-linseed oil mixtures by TLC-GLC methoda.
a. See text for operating conditions of gas liquid chromatography.
b. 44: Brassicasterol, 52: Campesterol, and 63: ƒÀ-Sitosterol.
c. RRT of ƒÀ-Sitosterol (retention time, 65min) taken as 1.00.
d. SV: Saponification value., e. IV: Iodine value., f. BRR: Butyro refractometer reading
63min, respectively were detected. Results
obtained for sterol compositions of oil mixtures
of varying degrees of adulteration should show
proportional differences in sterol components, but no marked difference in any of the mix-
tures was detected. Hence, no significant con-
clusion could be drawn from this method. Be-
cause a precise method is required to detect
Peaks 1, 2, 3, 4, 5, and 6 correspond to retention
time of 44, 52, 63, 66, 72, and 82 respectively.
Conditions are given in the text.
Fig. -1 Gas-liquid chromatograms of unsaponifi-
ables of rapeseed and linseed oils.
adulteration, the direct GLC analysis of total
unsaponifiable matter with a capillary glass
column, which eliminates the operation of frac-
tionation by TLC, was also carried out for
quick and efficient separation as well as for
obtaining precise results.
4•E1•E2 Direct GLC Method
GLC profiles of the sterol constituents and
Peaks 1, 2, 3, 4, 5 and 6 correspond to retention
time of 44, 52, 63, 66, 72, and 82 respectively.
Conditions are given in the text.
Fig. -2 Gas-liquid chromatograms of unsaponifi-
ables of rapeseed-linseed oil mixtures.
25
362 油 化 学
hence the sterol compositions of experimental
rapeseed, linseed, and adulterated oils are shown
in Figs. -1 and 2 and Tables-2 and 3 respec-
tively. Experimental linseed oils were shown
to contain an average brassicasterol content of
0.32% of the total sterol, whereas the experi-
mental rapeseed oil contained an average 11.37
% brassicasterol. The rapeseed oil adulterat-ed with linseed oil contained varying contents
of brassicasterol, decreasing in order as the
percentage of linseed oil increased, but the limit of estimation could only be estimated at adulteration levels of greater than 10%. Simi-
larly, no significant changes in campesterol and
Q-sitosterol contents of oil mixtures were ob-served but some sterol constituents of RT 66,
72 and 82min were observed to be quite pro-
minent in linseed oil alone. Among them, a
Table-2 Sterol compositions (%) from unsaponifiable matter of experimental sample oilsa.
Rapeseed oil
Linseed oil
a. Data obtained by direct analysis of unsaponifiable matter in capillary glass column. See the text for GLC operating conditions.
Table-3 Sterol compositions (%) from unsaponifiables of rapeseed-linseed oil mixturesa
a. Analytical data obtained from direct GLC analysis of unsaponifiable matter in capillary glass column.
See the text for operating conditions of GLC.
26
第35巻 第5号 (1986) 363
constituent with RT of 72min was found to
be prominent in adulterated oil samples (Fig. -
2). Such observations show that there is a
strong possibility of detecting and estimating
levels of adulterant linseed oil as low as 5%.
But the problem with the direct GLC method
is whether the important peak was a single
component.
Itoh et al.19),20) showed that linseed oil con-
tained campesterol, stigmasterol, ƒÀ-sitosterol
and Ģ5-avenasterol as its main sterols. If the
relative retention time (RRT) for ƒÀ-sitosterol
was taken as 1.00, the RRT for other sterols
were 0.81, 0.88, 1.00, and 1.12. Itoh et al.
also showed that obtusifoliol (RRT 0.95),
gramisterol (RRT 1.13) and citrostadienol (R
RT 1.52) occured as methylsterol.
Fig. -3 indicates GLC profiles for each TLC
fraction of unsaponifiable matter from linseed
oil.
The peak of RT 72min was observed only
in the methylsterol fraction. RRT values, of
Peaks 2, 3, 4, 5, and 6 correspond to retention
time of 52, 63, 66, 72 and 82 respectivery.
Conditions are given in the text.
Fig. -3 Gas-liquid chromatograms of linseed oil unsaponifiable fractions.
0.82, 1.00, 1.05, 1.14, and 1.30 in the main
peaks of unsaponifiable matter were found by
the direct GLC method. From this we con-
cluded that the RT 72min peak was a single
component and was gramisterol.
The direct GLC method was more precise
than the TLC-GLC method because there was
consistency in the sterol data though apart from
gramisterol, they were not very useful.
4•E2 Fatty Acid Composition
Fatty acid compositions in the experimental
rapeseed oil and linseed oil together with values
from literature and for samples of rapeseed oil
adulterated with linseed oil are given in Ta-
bles-4 and 5 respectively. Clear and distin-
guishable peculiarities were observed in the
linolenic acid and stearic acid contents of rape-
seed oil. The linseed oil contained linolenic
acid in the proportion of 55% of the total
fatty acid, whereas rapeseed oil had only 9%
on average. Similarly, linseed oil contained
4.2% stearic acid whereas the rapeseed oil
contained 1.4% on average. In adulterated
samples of rapeseed oil the variation in lino-
lenic acid and stearic acid contents was quite
remarkable and this was sufficient to detect
adulteration at levels even as low as 5•`10%
linseed oil in rapeseed oil.
4•E3 Tocopherol Composition
Tocopherol composition of experimental rape-
seed oil, linseed oil and the samples of rape-
seed oil adulterated with linseed oil are given
in the Tables-6 and 7 respectively. The ƒ¿-
tocopherol content of rapeseed oil was higher
than that of linseed oil. The ƒÀ-totopherol
content in rapeseed oil was lower than that in
linseed oil but there were no significant dif-
ferences in the ƒÁ-tocopherol and ƒÂ-tocopherol
contents in the two oils. In adulterated sam-
ples, the ƒÀ-tocopherol content gradually in-
creased as the proportion of linseed oil in-
creased. The inverse was found for the ƒ¿-
tocopherol content. However, with reference
to the analytical data of experimental samples
of rapeseed oil and linseed oil, the ƒ¿-tocopherol
and, ƒÀ-tocopherol contents were shown to be
nebulous criteria to prove the occurence of
adulteration.
4•E4 Physical and Chemical Constants
A marked increase in iodine value was ob-
27
364 油 化 学
Table-4 Fatty acid compositions (% of total) of some rapeseed and linseed oilsa.
Rapeseed oil 1) Analysis report (experimental samples)
2) Literature report (sample number: 50)
Linseed oil 1) Analysis report (experimental samples)
2) Literature report (sample number: 21)
a. Analytical data obtained from GLC analysis of methyl esters of fatty acid in capillary glass column. See the text for operating conditions of GLC.
Table-5 Fatty acid compositions (% of total) of rapeseed oil-linseed oil mixturesa.
a. Analytical data obtained from GLC analysis of methyl esters of fatty acid in capillary glass column.
See text for operating conditions of GLC.
28
第35巻 第5号 (1986) 365
Table-6 Tocopherol contents (ƒÊg/g) of experimental sample oilsa.
Rapeseed oil
Linseed oil
a. Analytical 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 rapeseed-linseed oil mixturesa.
a. Data obtained from high performance liquid chromatographic analysis of tocopherol. See text
for operating conditions of HPLC.
served in the adulterated samples as the pro-
portion of linseed oil increased. A significant
difference was not observed in saponification
value but the butyro refractometer reading did
show the same difference as the iodine values.
5 Conclusion
Our analyses on sterol constituents with
special reference to gramisterol content and the
fatty acid compositions in experimental oils
mixtures show that adulteration of linseed oil in
rapeseed oil can be estimated even in amounts
as low as 5•`10%. The direct GLC method
was more precise and rapid for analysis of
sterols, so it is concluded that this method
is very useful for detection and quantification
of an adulterant linseed oil in rapeseed oil.
However, it is always valuable to consider
other criteria such as iodine value and butyro
refractometer reading as additional aids to
prove the occurence of adulteration.
Acknowledgment
THE UNITED NATIONS UNIVERSITY, TO-KYO, JAPAN provided a fellowship for Poorna P.
Manandhar. (Received Dec. 2, 1985)
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366 油 化 学
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食用なたね油中のあまに油の検出と推定
Porona P. MANANDHAR*・
長 尾 昭 彦 **・山 崎 惠 *** Central Food Research Laboratory , Ministry of
Agriculture (Babar Mahal, Kathmandu, Nepal)** 農林水産省食品総合研究所
(茨城県筑波郡谷田部町観音台1-2-1)
なたね油 に混入 されたあ まに油の検 出及 び混入量 を推
定す るた めの方法 を検討 した。なたね油に一定割合 のあ
まに油 を混入 した試料 について,脂 肪酸,ス テ ロール,
トコフェロール の組成分析及 びけん化価,ヨ ウ素価,屈
折率 の測 定 を行った。脂肪酸 はあまに油 の添加量 に応 じ
ステア リン酸,リ ノ レン酸 の量 が増加 した。ステ ロール
については,GCの 測定条件下で66, 72, 82minの 保持
時 間に現れ る ピー クは,あ まに油 の存在に より際 立つ こ
とが観察 され た。 これ らのピー クに注 目する と,な たね
油 中に5~10%の あまに油混在の検 出が可能 であった。
トコフェロール分析 は有効ではなかったが,ヨ ウ素価,
屈折率 の測定 は,偽 和油脂の検 出推定 に補足的 な手段 と
しての意義があった。
30