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

37

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

38

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

39

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

40

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

41

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