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

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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-

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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.

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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