ELSEVIER

Applications of Nigella sativa seed

lipase in oleochemical reactions
L. Dandik and H. A. Aksoy

Faculty of Chemistry and Metallurgy, Chemical Engineering Department, Istanbul Technical

University, Istanbul, Turkey

The possible applications of the native lipase of Nigella sativa seed in oleochemical reactions were investigated
in relation to the process parameters. The investigated enzyme-catalyzed reactions were the hydrolysis of used
frying oil and esterifkation of oleic acid with glycerol and methanol. All reactions were carried out under
solvent-free conditions and catalyzed by pressed seed as the lipase source.
The hydrolysis reactions catalyzed by pressed seed were carried out in the presence of two nonionic surfac-
rants which showed similar effects on the reaction.
Esterification of oleic acid with methanol was carried out without removing the produced water. The highest
yield of ester was observed in the esterijication reaction conducted at 45°C using a 1:lS methanol:oleic acid
molar ratio and a pressed seed content of 50% based on total weight.
Esterification of glycerol with oleic acid was carried out with and without co-produced water removal.
Esterifcation of glycerol with oleic acid carried out withoutproduced water removal was conducted at 1:4.5, 2:1,
3:1, and 4:l glycerol:oleic acid molar ratios. The highest conversion of oleic acid was observed in the reaction
carried out at a 4:l glycerol:oleic acid molar ratio at 45°C and a 45% pressed seed content based on oil weight.
The reaction product contained 15.7% triolein, 15.8% oleic acid, 37.3% diolein, and 31.2% monoolein. The best
triolein synthesis was observed at 55°C and a stoichiometric glyceroboleic acid molar ratio of I:3 with 45%
pressed seed content based on total weight with produced water removal. A final triolein yield of 81.7% was
achieved.

Keywords: Hydrolysis; esterification; triolein; diolein; monoolein; methyl oleate; lipase; Nigella sativa seed

Introduction ibly catalyze the esterification reactions. Many commercial

Fat biotechnology was and still is considered to be one of
the most important new technologies. Enzymatic fat hydro-
lysis and acidolysis of oils to change the melting character-
istics are some examples in which fat biotechnology is prac-
ticed on a ton-scale.’
The use of enzymes to split fats was reported by A. W.
Ralston. In 1890, Green and Sigmund established the pres-
ence of a fat-splitting enzyme in castor beans.* The enzy-
matic reactions were studied by many investigators.2-‘0 En-
zymes which hydrolyze esters such as fats and oils to fatty
acids and glycerol were known as lipases. They can revers-
Address reprint requests to Prof. H. Ayse Aksoy, Department of Chemical
Engineering. Istanbul Technical University, Faculty of Chemistry and Met-
allurgy, 80626 Maslak, Istanbul, Turkey
Received 14 August 1995; revised 23 October 1995; accepted 16 Novem-
ber 1995
lipases from Rhizopus arrhizus, Candida rugosa, and As-
pergillus niger were widely investigated in the hydrolysis of
natural fats and oils.3,4 Geotrichum candidum, Pseudomo-
nas cepacia and C. rugosa lipases were used and compared
for their ability to hydrolyze high erucic acid rapeseed oil
for the isolation of free erucic acid.“’
Esterification of oleic acid with glycerol and other alco-
hols were catalyzed by Candida rugosa and Aspergillus
niger, respectively.3
The synthetic reaction between oleic acid and glycerol,
enzymatic hydrolysis of triolein and alcoholysisiglyc-
erolysis, and transesterification reactions were catalyzed by
Lipozyme, Lipozyme IM-20, and SP 382.7--8
The presence of lipases in castor beans, ground Vemonia
galamensis seed, and Nigella sativa seed were documented
in recent literature.2,1 i-i6
The object of the studies described here is to compare
and evaluate the usefulness of pressed N. sativa seed as
lipase source for oil hydrolysis and esterification of oleic
acid with methanol and glycerol.

Enzyme and Microbial Technology 19:277-281, 1996

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Table 1 The characteristics of used frying oil Table 3 Characteristics of surfactants

Species Species Dehydol LS2 Dehydol TA20

Acid value (mg KOH g-l) 0.65 Chemical name C,,-C,,PEO C,,-C,,-2OEO
Iodine value (Hanus) (gl (100 g-l) 125.4 Fatty alcohol Fatty alcohol
Saponification value (mg KOH g-l) 185 Form of delivery Liquid Solid
Unsaponifiable (%) 1.37 Active substance (%) 99.7-100 99.5-100
Water content (%) co.3 co.5
Refractive index (nozO) 1.4758
Density at 70°C (g ml-‘) 0.869-0.956 0.999-I .005
Density (g ml-‘) 0.9208
pH (1%) 6.0-7.5 6.0-7.5
Water content (Karl Fischer) [%I 1.17
Hydroxyl value 196-204 49-53
Cloud point (“C) ~8
Pour point (“C) s5 243
Materials and Methods Turbidity temperature (“C) 38-42 (BDG) 72-76
(10% NaCI)
N. sutiva seeds of Turkish origin were purchased from a herbal
shop in Istanbul, Turkey. The oil content of seeds on a moisture- From Henkel Dehydag CD”
free basis was 22%; moisture was 5.6%. Used frying oil was
purchased from the campus cafeteria of Istanbul Technical Uni-
versity and investigated according to the method of Dandik et ~1.‘~
The characteristics and fatty acid composition of oil are shown in seed content at 50% based on total weight and the temperature at
Table 1 and Table 2. 45°C. Methanol and oleic acid were transferred to a 250-ml stop-
Nonionic surfactants Dehydol LS2 and Dehydol TA20 (alkyl pered Erlenmeyer flask which was placed in a shaker water bath.
polyglycol ethers of saturated fatty alcohols) were purchased from The mixture was shaken and heated. At tbe reaction temperature,
Henkel (Dusseldorf, Germany). The characteristics of surfactants the esterification reaction was initiated by adding the desired quan-
are shown in Table 3. Methanol, oleic acid, glycerol (87%) and tity of pressed seed into the mixture of oleic acid-methanol. The
glycerol (anhydrous) were obtained from Merck (Darmstadt, Ger- reaction was followed by a thin-layer chromatography-flame ion-
many). All the other chemicals used for experiments were analyti- ization (TLC-HD) technique.t5
cal grade (Merck).
In all experiments, ground N. sativa seeds (600-1400 pm) were Esterification of oleic acid with glycerol
pressed with a laboratory type Carver hydraulic press (Fred S.
Carver, Wabash, IN) at ambient temperature under the pressure Esterification of oleic acid with glycerol was carried out with and
program previously published.r6 Thus prepared pressed seeds were without coproduced water removal.
used as lipase source.
Esterification without water removal. Glycerol (anhydrous)/
Hydrolysis
oleic acid at molar ratios of 1:4.5, 4: 1, 3: 1, and 2: 1 were added to
Hydrolysis reactions were carried out in a three-necked flask (l-l) a 250-ml stoppered Erlemneyer flask which was placed in a shaker
equipped with a mechanical stirrer, thermometer, and a tempera- water bath. The mixture was shaken and heated. At the 45°C
ture controller. Used frying oil (220 g). and phosphate buffer (220 reaction temperature, pressed seeds (50% based on total weight)
ml, pH 6) were stirred and heated to the reaction temperature of were added to the mixture. The reactions were followed by a
50°C. At the reaction temperature, the pressed seeds (45% or 60% TLC-FID technique.
based on oil weight) were added to the oil-buffer mixture. The
stirring rate was adjusted to 700 rpm. Samples were withdrawn at
Esterification of glycerol with produced water removal. Enzy-
selected time intervals and placed in a 90°C water bath for 1.5 min
matic esterification reactionsI were carried out in a three-necked
to inactivate the enzyme. Acid values of the dried products were
flask (250~ml) equipped with a sampling pipet, a temperature con-
determined and the hydrolysis degrees were calculated according
troller, and a vacuum pump connection. Glycerol (87%)/oleic acid
to our previously published study.r5
molar ratios were 1:4, 1:3, 1:2, 1: 1, and 2: 1. Pressed seeds contents
The hydrolysis reactions conducted with nonionic surfactants
were 40, 45, and 50% based on the total weight. The reactions
were carried out according to the same procedure. Surfactants
were carried out at 35,45,55, and 65°C and were followed by the
dissolved in the buffer solution (0.01% based on oil weight) were
TLC-FID technique.
added to the oil.

Esterification of oleic acid with methanol

Results and discussion
The esterification reactions were carried out at molar ratios of
1: 1.5, 1:3, 2: 1, and 3: 1 of methanol:oleic acid keeping the pressed Hydrolysis of used frying oil
Table 4 shows the change in the degree of hydrolysis during
Table 2 Fatty acid composition of used oil
the reactions catalyzed with pressed seeds (45 and 60%
based on oil weight) with and without nonionic surfactants
Fatty acid Composition (wt %I
(Dehydol LS2 and Dehydol TA20). When the pressed-seed
content was increased, the initial rate and percent hydrolysis
Palmitic 8.11
increased. The highest initial rate was obtained from the
Stearic 4.85
Oleic 26.59 reaction conducted with 60% pressed seeds. The nonionic
Linoleic 60.45 surfactants show a similar effect on hydrolysis and use of
surfactants did not yield a significant increase in conversion.

278 Enzyme Microb. Technol., 1996, vol. 19, September

 Oleochemical reactions of seed lipase: L. Dandik and H. Aksoy

Table 4 Hydrolysis of used frying oil catalyzed by pressed OA %

seeds (45% and 60% based on oil weight) with and without sur- 100
factants
so -
Degree of hydrolysis (%)
/’
Without surfactants Dehydol LS2 Dehydol TA20
Time :I - U”’
(h) 45% 60% 45% 60% 45% 60%

60- \
1 10.86 17.91 6.23 14.29 7.72 15.84
2 14.28 41.75 12.96 40.21 19.43 43.54
3 38.36 65.16 38.91 70.05 48.30 69.68 50,
i
4 60.50 79.39 61.12 82.71 68.29 84.04
5 71.16 88.23 74.61 91.38 79.31 90.34
40 -
6 82.18 93.30 83.56 94.45 87.04 92.73
7 87.72 95.24 88.62 96.28 90.74 94.56
8 91.49 95.27 93.79 96.87 93.61 97.24 30 -

Esterification of oleic acid with methanol

:,; / , , , , / , , , , /
Table 5 shows the methyl oleate and oleic acid concentra-
tions of the esterification products during the reactions for
different substrate concentrations. The highest conversion 0 6 12 16 24 30 36 42 40 54 60 66 72
of oleic acid to methyl oleate (49.85%) was obtained at a
1: 1.5 methanol:oleic acid molar ratio for a 24 h reaction Time (h)
time. After a 48 h reaction time, the methyl oleate content of
the reaction mixture was decreased to a small extent. Simi- Figure 1 Effect of glycerol:oleic acid molar ratio on the esteri-
fication of oleic acid with glycerol (Oleic acid, OA). 1:4.5, 0; 2:1,
lar results were reported in the synthesis of n-butyloleate.‘8 A; 3:1,*; 4:1,0

Esteri$cation of oleic acid with glycerol

The reactions carried out without water removal were con-
Composition %
ducted at molar ratios of 1:4.5, 2: 1, 3: 1, and 4: 1 anhydrous
glycerol: oleic acid with a reaction time of 48 h. The highest
loo 7-7
oleic acid conversion was observed at the molar ratio of 4: 1 so
glycerol:oleic acid. Figure I shows the oleic acid contents I
of the reaction mixtures as a function of glycerol:oleic acid
80 i
molar ratios and the reaction time. As can be seen, an in-
crease in the glycerol content increased the conversion of
oleic acid. The composition of the ester product in the re-
action carried out at a 4: 1 glycerol:oleic acid molar ratio as
a function of reaction time is shown in Figure 2. The equi-
librium conversion was reached after 48 h and the esterifi-
cation product contained 15.7% triolein (TG), 15.8% oleic
acid (OA), 37.3% diolein (DG), and 31.2% monoolein
(MG).

Table 5 Change in composition of the reaction mixture (wt %)

during N. sativa lipase-catalyzed esterification of oleic acid with
methanol

1:1.5 Me:OA I:3 Me:OA 2:l Me:OA 3:l Me:OA

Time MO OA MO OA MO OA MO OA ,
(h) (%I (%) (%) 1%) (%I 1%) (%I (%) 0 6 12 16 24 30 36 42 46 54 60 66 72

6 33.32 66.68 17.24 82.76 7.99 92.01 3.18 96.82 Time (h)
24 49.85 50.15 22.98 77.02 18.01 82.11 6.43 93.57
48 47.22 52.78 33.73 66.27 - - - -
Figure 2 Change in composition of glyceddes and oleic acid
during lipase-catalyzed esterification. TG, A; OA, 0; DG, *;
Methanol, Me; oleic acid, OA; methyl oleate, MO MG, 0

Enzyme Microb. Technol., 1996, vol. 19, September 279

Papers

TG % Composition %

80 80

80 80

0” ’ ’ ’ ” ’ ’ ’ ’ ’ ”
0 4 8 12 18 20 24 28 32 38 40 44 48 52 0 4 8 12 18 20 24 28 32 38 40 44 48 52

Time (h) Time (h)

Figure 3 Triglyceride contents of the reaction mixtures as a Figure 4 Composition of the product from the reaction con-
function of reaction time with the glyceroboleic acid molar ratio ducted at optimum conditions with the glycerol:oleic acid molar
at I:3 and the pressed seed content at 45%. 35°C. A; 45°C. *; ratio at 1:3, the pressed-seed content at 45%, and the tempera-
55”C, 0; 65X, 0 ture at 55°C. TG, 0; FA, A; 1,3-DG, 0; 1,2-DG, *; I-MG, 0

The esterification reactions of glycerol with produced Conclusions

water removal were investigated at molar ratios of 1:4, 1:3,
1:2, 1: 1, and 2: 1 87% glycerol: oleic acid and 45°C keeping
the pressed-seed content at 40% based on total weight. It
was observed that the stoichiometric ratio is the optimal
molar ratio that produces the highest yield of triolein.16 To
investigate the effect of the pressed-seed content, reactions
were conducted at the stoichiometric glycerol:oleic acid
molar ratio and 45°C at pressed-seed contents of 40,45, and
50% based on total weight. The highest TG yield was ob-
served with the reaction at the 45% pressed-seed content.
The reaction product contained 72.8% TG, 8.2% DG, and
19% OA.
Triolein synthesis was carried out at the optimum
pressed-seed content and glycerol:oleic acid molar ratio at
temperatures of 35, 45, 55, and 65°C. Figure 3 shows the
effect of temperature on the formation of TG as a function
of reaction time. The highest TG content of 81.7% was
reached at 55°C for a reaction time of 48 h.
The variation of product composition from the reaction
conducted at optimum conditions is shown in Figure 4. This
native lipase prefers the esterification on the sn-1 and sn-3
position of the glycerol. But in view of the high triglyceride
content at the end of the reaction, the N. sativa seed lipase
can be described as a nonspecific lipase. Alternatively, the
enzyme catalyzed the isomerization of the enzymatically
formed 1-monoolein (l-MG) or 1,3--diolein (1,3-DG) into
2-monoolein (2-MG) or 1,2--diolein (1,2-DG) and the
freed primary hydroxyl group reacted with oleic acid.
We have shown in our study that various reactions including
ester hydrolysis and synthesis and the synthesis of methyl
oleate and triglyceride can be catalyzed by N. sativu seed
lipase which is a nonspecific lipase. A comparison with the
enzyme-catalyzed reactions from the literature shows that
activity of N. sutivu seed lipase is high and can readily be
used instead of commercial microbial lipases. Further ex-
periments are being carried out to study the isolation and
purification of the N. sutivu seed lipase.
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