Physico-chemical analysis of Mulberry

(Morus alba) seed oil

Tanzima Yeasmin*, Salim Raza, Subodh Kumar Sarkar,

Abdul Aziz Sarkar' and Nurul Absar
Department of Biochemistry and Molecular Biology, University of Rajshahi.
Rajshahi-6205.Bangladesh.
Tele-0721-750041-4109 (Office)
-0721-750041-3758(Res)
Fax -0721-750064
E mail: yeasmin_bio@yahoo.com
'Bangladesh Sericulture Research Training Institute, Rajshahi,Bangladesh.
Total No. Of Tables and No. Of Figures: 5 and 2
Running Title: Analysis of Mulberry seed oil

To whom all correspondence should be addressed

Physico-chemical analysis of Mulberry (Morus alba) seed oil

Abstract
The major chemical composition such as moisture, protein, fat and carbohydrate content of
mulberry seeds were analyzed. Mulberry seeds contained 38% carbohydrate, 32% fat, 15%
protein and 9.2% moisture. Minerals such as calcium and iron were found to be 65mg and 3.2
mg per 100 gm of seeds. Mulberry seeds oil was extracted with petroleum ether. The oil, as
fractionated into mono-, di- and triglycerides and their compositions were determined to be about
2.92%, 3.15% and 90.12% respectively. The fatty acid composition of mulberry seed oil and two
commercial brand vegetable oils were determined by GLC. The amount of unsaturated fatty
acids were 80.97%, 85.61% and 84.78% for MS oil, Phoolkapi and Fresh brand vegetable oil
respectively.
Key words:
Mulberry seed oil, Fatty acids.

Introduction
Mulberry is the only food and nutritional source for Bombyx mori. Mulberry
belongs to the family Moraceae under genus Morus. The nutritional qualities of mulberry seeds
are determined by the physical and chemical properties of the seeds.
Mulberry seed is oval in shape with a nearly flat surface. The seeds are good
sources of oil (32%) and protein (15%). About

tons of mulberry seeds have been produced

annually in our country but till now the oil, a major by product of sericulture industry has not
been used properly in any commercial preparation. This oil might be used after refining or as
crude oil in soap making , cosmetics products etc. even as animal feed.
Although mulberry seeds contained significant amount of oil but no significant
amount of oil but no systematic data is available about quality as well as fatty acid compositions
of its oil. Keeping this view in mind, this study has been undertaken to attain mainly detailed
informations about quality of the oil by determining its physicochemical characteristics as well
as fatty acid compositions.
Materials and Methods
Mulberry seeds were collected from the Sericulture Research and Training Institute,
Rajshahi. The good seeds were dried in the sun, stored and used for experimental purpose.
Phoolkapi and Fresh brands vegetable oil were purchased from local market.
Analysis of mulberry seed
Ash, moisture, protein, lipid, free sugar, starch, iron and calcium contents of
mulberry seeds were determined following the standard methods as described by AOAC (1980),
IUPAC (1979), Jayaraman(1985), Bligh and Dyer (1959), Loomis and Shull (1937) and Bernard
(1965) respectively.

Extraction of oil
The oil was extracted from mulberry seed by solvent extraction process (South
Comb1926) .The solvent was removed under reduced pressure in a rotary evaporator. The oil
obtained from mulberry seeds was purified over a neutral alumina column using ether-diethyl
ether (70:30,V/V) as eluting solvent. The purity of the oil was checked by Thin Layer
Chromatography (TLC) technique.
Analysis of the oil
The specific gravities of mulberry seed oil and two different commercial brand of
vegetables oils were measured at 250C with the help of a pycnometer. Refractive index of the
clear oils (free from moisture and air bubbles) was determined in the Abbe refractometer at 28 0C
following standard IUPAC (1979) method. The smoke point, flash point and fire point were
determined according to the official methods of the American Oil Chemists Society AOCS
(1980) .The pour, cloud and solidification points were measured according to the ASTM standard
method (1952) for the lubricating oils. Lovibond Tintometer (1cm cell) was used for oil color
measurement. Percentage of free fatty acid (as oleic), peroxidase value, iodine number,
saponification value, unsaponification matter (%), saponification equivalent, thiocyanogen value
and hydroxyl number were determined by the standard methods of the AOCS (1980).
Glyceride composition of the oils
Mono-, di- and triglycerides in the oils were determined by column chromatography
on silica gel. The silica gel (20-150mesh,E. Merck, Darmstadt) was made free of moisture by
heating at 200 0C for two hours and cooled at room temperature in a desiccators. The gel was
then hydrated with 5% (w/w) water and allowed to stand for two hours shaking occasionally to
ensure complete moisture distribution. One gram of each oil was dissolved in 15 ml chloroform

and applied to prepared column (20 mm dia, and 300 mm long.). The triglyceride fraction was
eluted with 200 ml of benzene, diglyceride fraction with 200 ml of benzene and diethyl ether
(9:1, v/v) and monoglyceride fraction with 200 ml of diethyl ether. The flow rate was adjusted to
2 ml of effluent per minute. The free fatty acid present in each oil was eluted with the glyceride
fraction. Percentage of free fatty acid in the fraction was estimated by titrating with KOH using
phenolphthalein as indicator according to the method of AOCS (1952). The purity of each
fraction was checked by TLC using hexane-diethyl ether (80:20, v/v) as the solvents and
developing with chromic-sulphuric acid at 1800C.
Fatty acid composition of the oils
Fatty acid compositions of the oils were determined using the techniques of TLC
and Gas Liquid Chromatography (GLC). The fatty acid methyl esters of the oils and those of the
standard fatty acids were obtained by direct esterification.
TLC examination
Analytical TLC was carried out on glass plates (20x20 cm) with a layer of silica gel
G (thickness 0.25 mm). Chloroform, methanol and acetic acid mixture (90:10:1, v/v) was used as
the developing solvent. Components separated on the plates (R f values were previously
calculated for each methyl ester), were made visible by spraying with 1% phosphomolybdic acid
in ethanol followed by heating at1200 for about 10 minutes. Bands of fatty acid
esters were marked and scraped off. Bands were then slurred with diethyl ether, filtered and the
solvent was evaporated under reduced pressure.
GLC examination
A Pye-Unicam gas chromatograph equipped with a flame ionization detector was
used. Fatty acids were separated on a glass column (length 1.5 m, i.d. 2mm), which was packed

with Gas Chorm P (100-120 mesh) and coated with 10% diethylene glycol succinate polyesters.
Nitrogen was used as a carrier gas at a flow rate of 20-30 mL/min. The temperatures of the
injector, detector and column were maintained at 2200C, 1600C and 1800C respectively. Gas
chromatographic peaks were identified by comparison with those of methyl esters of standard
fatty acids in respect of their retention time. Peaks were measured by a Pye-Unicam electronic
integrator. The percentage of each peak was calculated as % of total area of all the peaks.
Results and Discussion
Table 1 represents the biochemical composition of mulberry seed oil. The seeds contain
9.2% moisture, 15.4% protein, 3.8% ash and 32.1% oil. Among the carbohydrates, the amount of
starch and free sugars are found to be 29.52 % and 8.52 % respectively. The seeds contained
significant amount of calcium (0.065 %) and iron (0.032 %). It can be concluded from the
present data that the mulberry seeds are very good sources of major nutrients such as fat,
carbohydrate and protein as well as of some minerals.
No significant differences in physical properties are observed under investigation of the
experimental mulberry seed oil and vegetable oils (commercial brand such as Phoolkapi, and
Fresh)the specific gravity of the experimental mulberry seed oil is found to be much similar to
the vegetable oils, indicating that the oil contained fairly large amount of unsaturated fatty
acids(Table 2). The thermal properties i.e. smoke point, flash point and fire points are
particularly useful in connection with fats and oils for any kind of frying. The temperatures vary
with the amount of free fatty acids present in an oil or fat decreasing with increased fatty acids.
The free fatty acid is directly related with the flash and fire points .If the free fatty acid is
increased, the flash and fire points oil or fat will obviously be decreased. From the Table2and 3.
the free fatty acids of the oils Phoolkapi, Fresh brand and MS are 0.04,0.062 and 2.35. It is

remarkable to note that the flash and fire points of the oils are in the rangePhoolkapi330,362,Fresh brand-328,346, and MS oil-326,330.
The chemical characteristics of mulberry seed oil have been shown in the Table 3. It is
evident from the results, that the chemical characteristics of the oils such as iodine value,
saponification value, % FFA and unsaponifiable matter are 110-115, 173.91, 2.35 and 0.74
respectively. The iodine value of the experimental oil is found to be almost similar to the
vegetable oil, so further reconfirming that like vegetable oils mulberry seed oil also contained to
mainly unsaturated fatty acids. The characteristic chemical values of the locally available
vegetable oils are found to be very much similar to those of mulberry seed oil except %FFA,
which is found to be much higher in mulberry seed oil. The quality of the oils were also analyzed
on the basis of their % FFA content and peroxide values after storage for 12 months at room
temperature. As shown in Fig 1.and 2. the % free fatty acid contents and peroxide value of
mulberry seed oil are found to be similar to those of the two edible oils even after storage for 12
months although % FFA in the mulberry seed oil was found to be higher initially. The glyceride
composition of the experimental oil is compared with the vegetable oil is shown in Table4 and it
is found that all the oils contained major component triglyceride about 90%. On the other hands
non glyceride composition of mulberry seed oil is much higher than that of vegetable oil. Very
much similar values of nonglyceride content was reported in pumpkin seed oil (Subodh, et.al
2001).
TLC and GLC identified the fatty acids analysis of the experimental mulberry seed
oil. The results have been presented in Table. 5. The fatty acids are palmitolic acid, palmitic acid,
stearic acid and oleic acid The unsaturated fatty acids, as estimated by GLC accounted to be
80.18%, 85.79% and 84.61% for MS oil, phoolkapi and fresh brand vegetable oils respectively.

Like vegetable oils, the MS oil also contained higher amount of unsaturated fatty acids. It is
interesting to note that the vegetable oil contained unsaturated fatty acids mainly higher
percentage of linoleic acid followed by oleic acid and then linolenic acid, but the experimental
MS oil contained mainly monounsaturated oil oleic acid (80.18%), followed by palmitoleic acid
(0.79%). While the content of linoleic and linolenic acid are not detected.
Again the major saturated fatty acids found in MS oil are palmitic acid (13.9%) and
stearic acid (5.13%). Almost similar amounts of palmitic acid (12.7%) and stearic acid (6%)
were reported in pumpkin seed oil by Tsaknis et al. (1997). He also reported that the pumpkin
seed oil contained more than 80% of unsaturated fatty acids .

At this moments it is not possible to suggested that whether, MS oil can be used as a
source of edible oil .All the vegetable oils including soybean oil contained higher amount of
polyunsaturated fatty acids i.e. essential fatty acids but MS oil does not contained any
polyunsaturated fatty acids. Further analysis is needed by animal study to confirm whether mono
un saturated fatty acid i.e. olic can also serve as a substitute of essential fatty acids before
suggesting the edibility of mulberry seed oil.
In conclusion, as the MS oil may be trapped as a very good source of oleic acid, so
it can be used widely in the industry for making soap, cosmetics, body massage oil, preparation
of paint etc as well as in making artificial feed for poultry ,fish, cattle etc.

REFERENCES
American Society for Testing and Materials (ASTM) 1952, 128.
Bernard Hawak, 1965, Physiological Chemistry 14h Edn., McGraw Hill Book Company, New
York, ,1264.

Blight, E. G. and Dyer, W. 1959. Total Lipid Extraction and Purification. Can. J. Biochem.
Physiol., 37: 911.
International Union of Pure and Applied Chemistry (IUPAC),Standard Methods for the
Analysis of Oils, Fats and Derivatives, 1979.6th Edn., Pergamon Press , Oxford.
Jayaraman, J.1985.Laboratory Manual in Biochemistry. Wiley Eastern Ltd. India.53-54, 75.
Loomis, W.E. and Shull, C.A., 1937, Methods in Plant Physiology. Mc. Graw Hill Book
Company, New York.
Official and Tentative Methods of the Associations of Agricultural chemists (AOAC) 1960. 9 th
Edn,
Official and Tentative Methods of the American oil Chemists Society (AOCS), 1980, 3rd
Edn., Vol:1,USA.
Official and Methods of Analysis of the Association of Analytical Chemists, 14th Edn.,1984,
USA.
South Comb, J. E. 1926. Chemistry of the oil Industries.2ndEdn, Constable and Company Ltd.
London, 144.
Subodh, K S, Nurul, A. and Maksud, A., 2001. Physico-chemical Properties of Pumpkin
(Cucurbita Maxima) Seed Kernel and its Oil. Journal of the Bangladesh Chemical Scociety,
14(2): 133-140.
Tsaknis, J., Lalas, S. and Lazos, E.S. 1997. Graasaa Y Acetics ,instituode La grsa(CSIC)revista,
48(5):267-272.

Legends To Table and Figure:

Table 1. Analysis of mulberry seed
Table 2. Physical constants of MS oil and market brand vegetable oils
Table 3. Chemical characteristics of MS oil and market brand vegetable oil
Table 4. Glyceride composition of MS oil and market brand vegetable oil (wt %)
Table 5.Fatty acid composition of MS oil and market brand vegetable oil
thin layer chromatography and TLC.

(wt %) by

Fig.1. Hydrolytic deterioration of mulberry seed oil and market brand vegetable oil.
Fig. 2. Oxidative deterioration of mulberry seed oil and market brand vegetable oil.

Table 1.
Constituent

Amount (%)

Moisture
Protein (N 6.25)
Oil
Crude fibre
Ash
Carbohydrate
Polysaccharides
Free sugar
Ash
Calcium
Iron

9.2
15.4
32.1
6.52
3.8
29.52
8.52
3.8
.065
.032

Table 2.
Constants

MS oil

Specific gravity at 25C

Refractive index at 28C
Flash point, C
Smoke point, C
Fire point, C
Cloud point, C
Solidification, C
Pour point, C

0.921
1.4270
320
212
330
1.08
-6.6
-7.6

Phookapi brand
vegetable oil
0.920
1.4722
330
212
362
360
1.10
-8.0

Fresh brand
vegetable oil
0.921
1.4722
328
214
346
1.10
-8.0
-7.0

Table 3.
Characteristics

MS oil

Phoolkapi brand Fresh brand vegetable

vegetable oil
oil

Iodine number
% FFA (as oleic)
Peroxide value (meq. O2/kg
oil)
Saponification value
Saponification equivalent
Unsaponifiable matter (%)
Thiocyanogen value
Hydroxyl number

110-115
2.35
1.45

118.16
0.04
2.52

116.70
0.062
2.68

173.91
322.58
0.79
5.12
8.70

187.24
299.61
0.78
76.00
8.60

190.30
294.79
0.67
76.20
8.60

Table 4.
Glycerides

MS oil Phookapi brand

vegetable oil

Triglycerides
Diglycerides
Monoglycerides
Nonglycerides

90.12
3.15
2.91
3.81

Fresh brand
vegetable oil

90.79
5.21
3.18
0.82

90.00
4.77
4.50
0.73

Table 5.
Fatty acids
Myristic acid
Palmitoleic acid
Palmitic acid
Stearic acid
Oleic acid
Linoleic acid
Linolenic acid
Arachidonic acid
Others

Methods
TLC
GLC
TLC
GLC
TLC
GLC
TLC
GLC
TLC
GLC
TLC
GLC
TLC
GLC
TLC
GLC

MS oil

Phookapi
brand vegetable
oil
7.56
7.42
4.00
3.41
27.20
26.80
52.90
53.32
5.67
0.92

Fresh brand
vegetable oil

TLC

7.72
8.00
4.21
4.00
26.80
26.41
53.44
24.02
4.18
1.02
-

GLC

2.46

2.43

0.79
3.40
5.13
80.18

Pure
soybean oil
0.1
8
.2
4
28
54
8.5
.6

Source : Fatty acid composition of pure soybean oil(Edwards Sargin ,1963CosmeticsScience and Technology
Intersecience Pub)

Fig.1

4
Mulberry seed oil
3.5

'Phoolkapi' soybean oil

'Fressh' soybean oil

% FFA

2.5

1.5

0.5

0
1

10

11

No. of month oil stored

Fig.2

40
Mulberry seed oil
'Phoolkapi' soybean oil
'Fresh' soybean oil

Peroxide Value

35
30
25
20
15
10
5
0

1 2

7 8 9 10 11

No. of month oil stored

12

13

12

13

To
The Executive Editor
Bulletin of Sericulture Research
Bangladesh Sericulture Research and Training Institute
Rajshahi 6100, Bangladesh.
Dear Sir,
Please find enclosed herewith two copies of revised manuscript as per
referrers comments of our original research article entitled Physicochemical analysis of Mulberry (Morus alba) seed oil
for your kind perusal for publication in the esteemed Journal Bulletin of
Sericulture Research.
An acknowledgement of the receipt of the revised paper will be highly
appreciated.
Publication cost 300/ also given with along with manuscript.
Thanking you in anticipation.
Sincerely Yours,
(Dr. Tanzima Yeasmin)
Associate Professor
Department of Biochemistry and Molecular Biology,
University of Rajshahi.
Rajshahi-6205.Bangladesh.
Tele-0721-750041-4109 (Office)
-0721-750041-3758(Res)
Fax -0721-750064
E mail: yeasmin_bio@yahoo.com