Indian Journal of Chemical Technology
Vol. 16, July 2009, pp. 317-321
Enzymatic synthesis of fructose ester from mango kernel fat
P P Dandekar & V B Patravale*
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology,
N.P. Marg, Matunga, Mumbai 400 019, India
Email: [email protected]
Received 6 October 2008; revised 8 May 2009
Fructose ester as a biosurfactant was successfully synthesized from the fatty acid fraction (olein fraction) of Mango
Kernel Fat (MKF). The synthesis was carried out by reaction of sugar and fatty acid fraction in presence of the enzyme
lipase from Candida rugosa which acted as a biocatalyst. Phosphate buffer (pH 7.0) was used as the reaction medium to
realise maximum enzymatic action. The product was separated from the reaction medium by liquid- liquid extraction.
Maximum conversion (36.52 %) was achieved at fructose to olein fraction molar ratio of 1 : 10 (mol/L), with the lipase
concentration of 4g/L, at a temperature of 30oC at the end of 3 days. The presence of an ester band in the synthesized ester
was confirmed by Fourier Transform Infrared (FTIR) analysis. Identity of the product was further confirmed by NMR
studies and a colour identity test for the ester group.
Keywords: Biosurfactant, Fructose ester, Mangifera indica, Mango kernel fat (MKF), Candida rugosa
Surfactants, capable of reducing the surface tension
and interfacial tension at the interfaces between
liquids, solids and gases, contain one or two long
alkyl chains and a polar group which may be anionic,
cationic, amphoteric or nonionic in nature1.
The bulk of the present day surfactants have alkyl
chains in the C12-C18 range. Natural fats and oils thus
form ideal raw materials for surfactant synthesis2.
Today, even though both oleochemicals and
petrochemicals can be used to make surfactants, only
about 21% of the surfactants are made from
oleochemicals3. However, tightening environmental
regulations and increasing awareness for the need to
protect ecosystems have resulted in an increased
interest for searching eco-friendly alternatives to the
chemical surfactants. In this regards, utilization of
biosurfactants, which are environmentally friendly
and highly functional materials, is in a favourable
situation4. Biosurfactants exhibit several distinct
advantages over chemical surfactants5. Amongst the
biosurfactants; Sugar Fatty Acid Esters (SFAEs) have
attracted attention of biotechnological researchers6.
These compounds have a structure typical of
emulsifiers, containing both a polar and a non polar
group in the same molecule. SFAEs exhibit many
advantages such as availability in a wide range of
HLB values, biodegradability, ability to be
metabolized, safety for human consumption, lack of
taste, lack of odor, non-toxicity, non-ionic nature and
non-irritancy to the skin and the eyes making them
compatible with various pharmaceutical and cosmetic
products7-9.
Mango (Mangifera indica, Anacardiacae) tree
occurs throughout India. The stone portion of the
mango fruit consists of an inner kernel- the source of
Mango Kernel Fat (MKF). which by and large
exhibits a fixed composition with dominance in the
percent content of stearic acid (along with small
quantities of other saturated fatty acids constituting
the stearin fraction) and oleic acid (along with small
quantities of other unsaturated fatty acids constituting
the olein fraction). MKF, which is pale yellow or
cream in colour, represents up to 25% of the fruit
which goes to the waste. India alone can produce
30,000 tons of mango kernel fat each year10. These
two aspects reduce per kilogram cost of the fat
substantially. This would also reduce the cost of the
final sugar esters derived from the fat, making the
product economically feasible.
Sugar esters can be synthesized by numerous
methods which involve the use of either chemical or
enzymatic catalysts. Enzymatic synthesis is achieved
by the coupling of sugar and fatty acid with enzyme
lipase as biocatalyst. The use of enzymes enables the
preparation of a wide range of monosaccharide fatty
acid esters often as single regioisomers and with no
requirement for laborious regioselective protection.
The reaction can be carried out under ambient
318
INDIAN J. CHEM. TECHNOL., JULY 2009
temperature conditions with flexibility to modify the
chemical structure of the product to result in
surfactants usable without any hazard in medical,
cosmetic and food industry11.
The present investigation was thus aimed at the
synthesis of fructose ester from the olein fraction of
MKF in presence of the enzyme lipase from Candida
rugosa. Exploitation of MKF for biosurfactant
synthesis was attempted for the first time and this
application of MKF has not been exploited so far. The
present investigation attempted to exploit the benefits
of the enzymatic synthesis along with advantage of
the presence of higher fatty acids in MKF. These
higher fatty acids, which occur more commonly
naturally and are generally more emollient than the
synthetic fatty acids would enable the synthesis of
water dispersible surfactants for use in pharmaceutical
and cosmetic industry.
Experimental Procedure
Materials and Methods
Fructose,
di-sodium
hydrogen
phosphate
(anhydrous), potassium di-hydrogen phosphate
(anhydrous), sodium hydroxide, ethyl acetate,
methanol,
sulphuric
acid,
chloroform
and
concentrated hydrochloric acid were obtained from
SD fine chem. Ltd. Mumbai, India. The enzyme,
lipase from Candida rugosa (Lipase AYS “Amano”),
used was received as a gift from Amano Enzyme Inc.
Nagoya, Japan. MKF sample was received as a gift
from Charbhuja Trading and Agencies Pvt. Ltd.,
Mumbai, India. The separated olein fraction of the
MKF was used as the fatty acid. Freshly distilled
water was used during the experiments. Pre-coated
thin layer plates of silica gel G were procured from
Merck Ltd. Mumbai, India. Commercial sucrose ester
– Ryoto Sugar Ester, S-1670, a gift sample from
Mitsubishi-Kagaku Foods Corporation, Tokyo, Japan,
was used as a standard for the thin layer
chromatography experiments.
Synthesis of fructose ester (esterification reaction)
Fructose, fatty acid, enzyme were mixed in the
buffer solutions and incubated at different
temperatures with continuous shaking at 250 rpm on a
horizontal shaker-incubator (Innovative™ DTC- 72).
For the synthesis in the present investigation,
phosphate buffer (anhydrous di-sodium hydrogen
phosphate, 0.5 g/L and potassium di-hydrogen
phosphate, 0.301 g/L, pH 7.0) was chosen as the
reaction medium. The reaction was carried out at
different temperatures, molar ratios of the sugar to
fatty acid and at different concentrations of the
enzyme. Also the effect of the reaction time on the
synthesis yield was studied.
Determination of yield of fructose ester
The yield of the product of synthesis was
determined in terms of the number of moles of fatty
acid converted as a percentage of moles of initial fatty
acid used.
The reaction was terminated by addition of an
excess of 0.1 N NaOH solution. The sodium
hydroxide solution also neutralized the unreacted
olein fraction in the reaction mixture. The excess base
was then titrated against 0.1 N HCl. The percent
conversion was then determined on the basis of the
unreacted olein fraction left in the reaction mixture.
Effect of different parameters on the yield of fructose ester
The esterification reaction was carried out at
different molar ratios of fructose to olein fraction. The
various molar ratios of fructose to olein fraction used
were 1:1, 1:2, 1:4 and 1:10 (mol/L). The reaction was
carried out at different reaction temperatures of 20,
30 and 40oC, on the basis of the thermostability of the
enzyme. The esterification reaction was carried out
with different enzyme concentrations of 1, 2 and
4 g/L respectively. The esterification reaction was
carried out up to 120 h. After every 24 h, the yield of
the product of synthesis was determined.
Characterization of fructose ester
Thin layer chromatography (TLC)
The standard used for the TLC study was Ryoto
Sugar Ester, S-1670 while the sample used was the
fructose ester synthesized by the enzymatic reaction.
The sample and the standard solutions were prepared
in chloroform at a concentration of 1% (w/v). The
mobile phase employed was ethyl acetate: methanol:
water (20:80:5 v/v). 20-50 µL of the individual
solutions were spotted following which the
chromatogram was developed by allowing the mobile
phase to run along the plate. The plates were placed in
oven at 110oC after spraying with 50% (v/v) solution
of sulphuric acid in methanol as the detecting agent.
The positions of the coloured spots of each of the
components were noted.
Purification of fructose ester
Chloroform was added to the crude reaction
mixture under continuous stirring. This chloroform
layer was further subjected to liquid-liquid extraction
DANDEKAR & PATRAVALE: ENZYMATIC SYNTHESIS OF FRUCTOSE ESTER FROM MANGO KERNEL FAT
with aqueous sodium hydroxide solution. The
chloroform layer, containing the synthesized fructose
ester was again separated, washed with water and then
utilized for further characterization.
Fourier Transform Infra Red (FTIR) studies
The IR spectrum of the purified product was
recorded on a PERKIN ELMER MY 60 Infra Red
spectrophotometer (PerkinElmer Life And Analytical
Sciences, Inc., MA) on the chloroform solution of the
product.
Confirmatory test for ester group
The chloroform solution of the synthesized fructose
ester was taken in methanol. A drop of dilute sodium
hydroxide solution was added along with a drop of
phenolphthalein indicator. The resulting pink
coloured solution was then heated on a water bath for
a few minutes.
NMR studies
1
H- NMR spectra were recorded on JEOL FTNMR,
JNM-LA 300 WB spectrometer (JEOL Ltd., Tokyo,
Japan) at 300 MHz in CDCl3 using TMS as the
internal standard. Chemical shifts were expressed in
parts per million (δ, ppm).
Results and Discussion
Candida rugosa lipase was chosen for the present
investigation because the use of enzyme enables the
preparation of a wide range of monosaccharide fatty
acid esters often as single regioisomers and with no
requirement for laborious regioselective protection11.
Fructose was chosen as the carbohydrate for the
present investigation as literature survey indicated
that the yield of the product with fructose was higher
than that with sucrose or glucose12.
For the synthesis in the present investigation,
phosphate buffer pH 7.0 was chosen as the reaction
medium on the basis of the reported optimum pH for
maximum enzymatic action. This medium would also
eliminate the toxicities associated with the use of nonaqueous solvents12.
The temperatures were chosen in the range of 20o
40 C on the basis of the reported thermostability and
optimum reaction temperature of the chosen
enzyme12.
The reaction was carried out on a horizontal
shaker-incubator to maintain the homogeneity of the
reaction mixture and the homogeneity of the
temperature during the course of the reaction. The
319
effect of various parameters on the yield of the
reaction has been depicted in Figs 1-4.
As seen from the Fig. 1, a molar ratio of fructose to
fatty acid of 1:10 (mol/L) gave the highest percent
conversion (36.52) at the lipase concentration of
4 g/L. This indicated that the esterfication occurred
better when the fatty acid was used in excess. Excess
fatty acid is required so that the water formed during
the reaction is then proportionately minimal. The
water formed during the reaction would dilute the
reaction mixture, thus changing the molar ratios of the
substrates, the concentration of the added enzyme and
the pH of the reaction medium. Thus excess fatty acid
is required to minimize the dilution effect of the water
generated in-situ.
As seen from Fig. 2, the percent conversion
increases between 20-30oC and then declines at 40oC.
The maximum percent conversion (36.52) was
observed at 30oC with the lipase concentration of
4 g/L. A further increase in the temperature may have
adversely affected the percent conversion of olein
fraction into the sugar ester because of the thermal
degradation of the enzyme leading to a sub-optimal
activity.
The lipase concentration also influenced the
product yield. As seen from Fig. 3, at 20oC, the
percent conversion did not show any appreciable
change on increasing the lipase concentrations. This
may have been because the temperature used was not
conducive for the maximum enzyme activity. Lipase
concentrations of 0.5 g/L gave almost similar results
at both 30 and 40oC. However, at higher lipase
concentrations of 2 and 4 g/L, the percent conversion
declined at 40oC. This low conversion may have
Fig. 1
 Effect of molar ratio of substrates on product yield.
Maximum yield of the fructose ester is obtained at the molar ratio
of fructose to olein fraction of 1 mol/L: 10 mol/L with the lipase
concentration of 4 g/L
0.5 g/L
2.0 g/L
4.0 g/L
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INDIAN J. CHEM. TECHNOL., JULY 2009
Fig. 2
 Effect of reaction temperature on product yield.
Maximum yield of the fructose ester is obtained at the temperature
of 30oC with the lipase concentration of 4 g/L
0.5 g/L
2.0 g/L
4.0 g/L
resulted due to the thermal inactivation of the enzyme
at a higher temperature. At 30oC, the percent
conversion exhibited a steady increase with increase
in the enzyme concentration of the reaction mixture.
The maximum percent conversion (36.52) was
observed at the lipase concentration of 4 g/L at 30oC.
Using a lipase concentration of 4 g/L, a
temperature of 30oC and a fructose to fatty acid ratio
of 1:10, the effect of the reaction time on the yield of
the sugar-ester was studied. The percent conversion
increased from 22.25 to 35.88% from day 1 to day 3.
Longer reaction times did not further increase the
yields (Fig. 4). Thus, the maximum percent
conversion was achieved at the end of 3 days.
Thus, it can be concluded that maximum yield of
the sugar ester was achieved at the fructose to olein
fraction molar ratio of 1 : 10, with the lipase
concentration of 4 g/L, at a temperature of 30oC and
at the end of 3 days.
The retention value of the synthesized fructose
ester was found to be identical to that of the standard.
The sample did not show the presence of any other
TLC spots. This may be due to the solvent system and
the detecting agent used during the procedure, which
fails to detect the presence of unreacted starting
materials. Since a single spot was observed in the
TLC, the column purification of the compound was
not attempted. The solvent extraction was used to
extract the compound from the reaction mixture.
Chloroform was chosen as the solvent for solvent
extraction since the compound was completely soluble
in it and also this would simplify its separation from
the aqueous buffer layer, containing the unreacted
fructose and the residual enzyme powder.
Fig. 3
 Effect of lipase concentration on product yield.
Maximum yield of the fructose ester is obtained at the lipase
concentration of 4 g/L at 30oC
0.5 g/L
2.0 g/L
4.0 g/L
Fig. 4
 Effect of reaction time on product yield. Maximum yield
of the fructose ester is obtained at the end of 3 days at the molar
ratio of fructose to olein fraction of 1 mol/L: 10 mol/L with the
lipase concentration of 4 g/L at 30oC
However, chloroform was also found to extract the
unreacted olein fraction from the reaction mixture.
Hence the chloroform layer was further extracted with
aqueous sodium hydroxide solution to neutralize the
unreacted olein fraction and to facilitate its easy
separation from the chloroform layer containing the
synthesized fructose ester. The layer was washed with
water so as to make it totally free of the sodium salt of
olein fraction.
The details of the FTIR peaks (wavenumber; cm-1)
and the corresponding groups of the synthesized
fructose ester are as follows: 3445.3 (OH bond),
2922.6 (C-H bond in –CH2 or –CH3), 1736.8 (C=O
ester bond), 1456.6 (–CH2, –CH3), 1088.2 (C-O ester
bond), 714.6 (the (CH)2 bond). In case of the
confirmatory test for the presence of the ester group,
the solution showed the disappearance of the bright
pink colour on heating on a water bath for a few
DANDEKAR & PATRAVALE: ENZYMATIC SYNTHESIS OF FRUCTOSE ESTER FROM MANGO KERNEL FAT
minutes. This change in colour was obtained due to
the hydrolysis of the ester bond on heating the
solution, thus liberating the free acid. This acid
neutralized the sodium hydroxide solution leading to
the disappearance of the phenolphthalein colour. The
product structure was confirmed by NMR studies,
wherein the chemical shift values (δ, ppm) of various
groups of the synthesized fructose ester were found to
be as follows: (300 MHz, CDCl3): δ 5.35 (d, 2, CH=CH-), 4.15 (m, 5, 2 -CH2O- and H from ring),
3.73- 3.67 (t, 1, H from furanose ring), 2.31-2.26 (t, 1,
H from furanose ring), 2.02-2.00 (d, 2, -COCH2), 1.25
(m, 26, H from aliphatic chain), 0.88 (t, 3, -CH3).
A comparison of present results with previous
attempts of enzymatic syntheses of fructose esters
employing palm fatty acid using Mucor miehei lipase
and tert-butyl alcohol as the reaction medium shows
that in the previous study8, the reaction temperature
was maintained at 55oC. The resulting product had an
HLB of more than 16. Other researchers have
reported a higher yield of fructose ester with the same
fatty acid source by employing Candida antarctica
lipase and glucose as the acyl acceptor and acetone as
the solvent13. Thus the results indicate that the yield of
a specific sugar ester is largely dependent on the
utilization of specific enzymes and reaction mediums.
The product characteristics vary with the nature of the
fatty acids derived from different natural sources.
Hence the comparison of the yields and characteristics
of a specific sugar ester, even from the same natural
source, becomes difficult when the conditions
employed vary and moreover so when the fatty acid is
derived from a different source altogether. The same
holds true here since the nature of the biocatalyst
employed differs from previous studies.
Conclusion
Enzymatic synthesis of Sugar Fatty Acid Ester
(Fructose ester) was successfully carried out using
lipase from Candida rugosa. Maximum yield of the
sugar ester was achieved with phosphate buffer (pH
7.0) as the reaction medium, at fructose to olein
fraction molar ratio of 1: 10, with the lipase
concentration of 4 g/L, at a temperature of 30oC and
321
at the end of 3 days. The FTIR of the compound
showed the presence of an ester band at 1736.8 cm-1.
The presence of ester band was confirmed by FTIR
and NMR studies, and presence of easter group was
confirmed by colour test. The process yield can
further be increased by process improvisation such as
use of the immobilized enzyme for a continuous
reaction, removal of water generated during the
reaction by the use of molecular sieves in the reaction
medium, or by the use of a more appropriate reaction
medium which can solubilize both the phases while
keeping the enzyme at the interface.
Acknowledgement
The authors are thankful to Charbhuja Trading and
Agencies Pvt. Ltd., Mumbai for the gift sample of
MKF, Amano Enzyme Inc.; Japan for the kind gift of
the enzyme and Mitsubishi-Kagaku Foods
Corporation, Japan for the gift sample of Ryoto sugar
Ester, S-1670. Prajakta P. Dandekar is thankful to the
Department of Biotechnology (DBT), Government of
India, for the financial support required during the
investigation.
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