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SYNTHESIS AND CHARACTERIZATION OF SORBITOL BASED

BIOSURFACTANTS FROM RENEWABLE SOURCES BY USING CANDIDA
ANTARCTICA LIPASE-B ENZYME
Amol.S.Patil1, Ghayas.A.Usmani2*, Pawan D.Meshram3
Department of Oil Technology, University Institute of Chemical Technology, North Maharashtra University,
Jalgaon-425001 (MS) India

ABSTRACT
The sorbitol molecule contains six hydroxyl groups able to be esterified with carboxylic acids. Sorbitol esters
were prepared by lipase catalyzed controlled esterification reaction between sorbitol and fatty acids. In this study,
enzymatic reaction was performed in organic solvent mixtures of pure DMSO and Tert. Butanol in 80:20 ratio in
percentage(v/v).The enzymatic synthesis was carried out by the reaction of sorbitol and various fatty acids like oleic,
lauric, palmitic at temperature 45- 500C in orbital shaker incubator at 200rpm for 48h. This synthesis strategy gave
complete conversion of SMO(92%),SMP(82%),SML(85%).In this research, we report advances made in using
renewable substrates for biosurfactant production and their characterization by TLC,1H NMR,13C NMR, HPLC,FTIR
etc.
KEYWORDS: Candida antarctica lipase B, Selective synthesis, Sorbitol, Renewable sources, Characterization.
INTRODUCTION
Surfactants are critical to the cleaning, emulsifying, wetting, and foaming properties of many products.
Surfactants are found in a host of applications, including cosmetics, textiles, pharmaceuticals, foods, ore floatation,
soaps and detergents, pulp and paper manufacturing, lubricating and metalworking, agrichemicals, and petroleum
mining fields.
In most industrial cases, surfactants are used as processing aids and are disposed off following usage. Issues
regarding disposal have created interest in more biologically acceptable alternatives and more focused use of
renewable vs. nonrenewable resources. Surfactants and emulsifiers are indispensable components of daily life. They
are widely used in the pharmaceutical, cosmetic, petroleum, and food industries. Many different types of surfactants
are already being used in industry, but it is important to develop even more new compounds to broaden the spectrum
of specific properties and applications (Cameotra and Makkar, 1998).
Most of these compounds are synthesized chemically and are of petroleum origin. Most are also toxic to the
environment, not easily biodegradable, and their manufacturing processes and byproducts can be environmentally
hazardous. Increased environmental awareness and strict legislation has made environmental compatibility of
surfactants an important factor in their application for various uses (Maier and Soberon-Chavez, 2000).
In the past few decades, biosurfactants (surfactants of microbial origin) have gained attention because of their
biodegradability, low toxicity, ecological acceptability and ability to be produced from renewable and cheaper
substrates (Ishigami 1997; Makkar and Cameotra, 1999). Several structurally diverse surface-active molecules are
producible by a wide spectrum of microorganisms (bacteria, fungi, yeasts). Basically there are six major types of
biosurfactants: hydroxylated and cross linked fatty acids (mycolic acids), glycolipids, lipopolysaccharides,
lipoproteins - lipopeptides, phospholipids and the complete cell surface itself (Desai and Banat, 1997).
Biosurfactants have been tested in environmental applications such as bioremediation and dispersion of oil
spills, enhanced oil recovery and transfer of crude oil, and are thought to be potential candidates to replace chemical
surfactants in future, especially in the food, cosmetic, and health care industries, industrial cleaning of products and in
agricultural chemicals (Banat et al, 2000).
Biological catalysis has many advantages over chemical catalysis. It performs under mild condition that
consequently save energy. The selectivity of enzymatic reaction avoids side reactions and formation of by products
(Sharma R. et al, 2001; Hills G. et al, 2003). The enzymatic synthesis of sugar ester is based on esterification reaction.
Water is one of the products of esterification which plays an important role to reverse the reaction back resulting to
lower the ester yield (Yoo I.S. et al, 2007).
In the present study,the sugar alcohols holding six hydroxy groups of different sterical orientation such as
xylitol, mannitol, sorbitol have been reacted with carboxylic fatty acids like lauric, palmitic, oleic in organic reaction
medium to form sorbitol esters. Sorbitol esters as carbohydrate derivatives are highlighted here. Our basic aim is to
synthesize various commercially important esters by using enzyme as catalyst,and characterize them.
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PART-I
EXPERIMENTAL MATERIALS AND METHODOLOGY
Materials
Commercial grade Candida antarctica lipase-B (Immobilized on Immobead 150, recombinant , Aspergillus
oryzae), has been procured from Sigma Aldrich, and used as biocatalyst for the sorbitol ester synthesis. The enzyme
had an activity of 7000 PLU/G (propyl laurate unit), at 600C; as specified by manufacturer. DMSO and 2-methyl 2propanol (tert.-butanol) were used as solvents for esterification reaction. Molecular sieves (4A 0 x 1.5 mm equilibrium
capacity for water 300 C; 75% relative air humidity, Merck Specialities Pvt. Ltd.) were used as water removal
adsorbent. The fatty alcohol like Sorbitol (D-Glucitol) was provided from Siscos Research Lab.Pvt. Ltd. and Acyl
donor, oleic acid is supplied by Merck Specialities Pvt. Ltd. Lauric acid and palmitic acid (LR grade) manufactured by
S.d. Fine Chem. Ltd. were used for experimental synthesis.
Methodology
Step-1
Synthesis of sorbitol ester from sorbitol and fatty acids
The reactions were carried out by mixing desired amounts of D-Sorbitol and fatty acid in the 20% DMSO
(Dimethylsulophoxide) and 80% Tert. Butanol (Tertiary butyl alcohol).The reaction mixture is taken in volumetric
flask (250 ml) which was closed with stopper and stirred in orbital shaker incubator at 200 rpm. The reaction
temperature is maintained between 45- 500C, very carefully. Molecular sieves and Lithium Chloride are added into
reaction mixture to remove water. After half hour, lipase Candida Antarctica B enzyme is added. The progress of
reaction is monitored by calculating acid values after every 4h.The progress of reaction is also monitored by TLC
method. It has been found that acid value decreases upto 6 after 48h. The conversional yield of sorbitol esters was
calculated from the decrease in the concentration of fatty acids.
Step-2
Purification of sorbitol esters
When the enzymatic reaction reached to equilibrium, usually after 48h of reaction, the solvent was evaporated
using a rotary evaporator. The oily residual phase was resuspended in 3 ml of n- hexane and cooled to 200C in order to
induce precipitation of the non reacted sorbitol.
The unreacted sorbitol was separated by filtration. The filtrate so obtained, was cooled to 150C for 15 min in
rotary vaccum evaporator. In order to induce the precipitation of sorbitol monoesters, the temperature was further
reduced to 40C. The product was identified and characterized by various analytical methods like TLC, FTIR, 13C
NMR, 1H NMR, HPLC etc.
Reaction Scheme
HO
HO

OH HO

O
+R

Lipase B,45- 500 C

R

OH
Mol. Sieve, LiCl

HO

OH HO

O
C

H2O

O
HO

OH

Water

Fatty acid

HO

OH

Sorbitol Ester

Sorbitol
Step-3
Spectral Analysis
The progress of reaction was monitored by analyzing the amount of unreacted fatty acids in the reaction
mixtures by a titrimetric method such as acid value and by TLC. The purified products were analyzed by IR and 1H
NMR spectroscopy. The IR spectra were recorded on Perkin-Elmer Spectrum Version 10.03.06 FT-IR
spectrophotometer and the 1H NMR spectra were recorded on a Bruker Advance II 400 NMR spectrometer in CDCl 3.
The spectra of different esters obtained in this study, were fully compatible with their structures and matched well
with those reported in the literature for corresponding compounds.
Physico-chemical factors affecting esterification reaction
I.
Different physico-chemical factors were observed in order to achieve maximum formation of sorbitol esters.
These were molar ratio of substrates, temperature, organic solvent mixture, and water activity (aw) control.

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II.

III.
IV.

A significant decline in the conversion of sorbitol to ester was observed after 44 h of reaction run at 45- 500C.
This could have been due to the inactivation of the enzyme at higher temperature in the presence of an organic
solvent.
A major problem for synthesis of sugar ester is the low solubility of sugar alcohol in organic solvent. To
overcome this problem, a mixture of DMSO 20% and tert. butanol 80% were used for the synthesis.
The enzyme catalyzed reactions in organic media critically depends on the amount of water in the reaction
medium. The water released during the esterification reaction may disturb the equilibrium and hydrolyze the
ester formed. It was minimized by analyzing moisture content of reaction mixtures by Karl Fischer technique.
The experimental parameters for ester synthesis are given in table 1.

Expt. No.

Table 1. Experimental parameters for ester synthesis

Substrate Ratio (1:2)
Solvent ratio (80:20)
Temperature

Sorbitol and oleic acid

Ter. Butanol :DMSO

45-500C

92.06 %

Sorbitol and lauric acid

Ter. Butanol :DMSO

45-500C

83.45 %

Sorbitol
acid

palmitic Ter. Butanol :DMSO

45-500C

80.21 %

and

% yield

PART-2
RESULTS AND DISCUSSION
The enzyme catalyzed esters have been characterized by using TLC, FTIR, 13C NMR, 1H NMR. Three esters
have been synthesized.
Sorbitol monooleate-SMO
Sorbitol monolaurate-SML
Sorbitol monopalmitate-SMP
The results are briefly tabulated below along with figures.
TLC
The kinetics of sorbitol- fatty acid monoester was observed by TLC. For TLC analysis, 10cm plates were
developed using a modified three step method previously reported for sorbitol esters (Ducret et.al.,1995); The plate
was first run with a mixture of chloroform/methanol/water (85/13.5/1.5,v/v/v) at 2.7cm.The plate was then dried and
run again with a mixture of chloroform / methanol/ acetic acid (97.5/1.5/1, v/v/v) at 5.2cm.The plate was dried again,
and finally run with a mixture of n-hexane/diethyl ether/acetic acid (70/29/1, v/v/v) at 9 cm. The plates were dried and
sprayed with a saturated solution of sulphuric acid in methanol (5%v/v) and heated to 2000C for 10min.Under these
conditions sorbitol, sorbitol monoester, and oleic acid had a Rf of 0, 0.23, 0.82, respectively.
ACID VALUE
The acid values of different esters decreases gradually and reduces upto 2 to 3 only. This indicates the
formation of esters. The results are shown in table 2.
Table 2 Acid value for SMO, SML and SMP monoesters.
Acid value of synthesized monoesters
Sr. No.

Time Interval (in hrs)

SMO

SML

SMP

204

282

222

195

251

201

12

181

238

181

16

176

189

174

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20

153

163

157

24

123

144

138

28

102

124

120

32

85

95

99

36

62

75

65

10

40

43

56

32

11

44

21

28

18

12

48

FTIR
FTIR spectra of synthesized surfactant is given in fig.1. It shows absorption band at 1734cm -1 (C=O
stretching), 2923cm-1and 2857cm-1 (C-H stretching), 1462cm-1(C-H bending), 1045cm-1 (C-O stretching in C-O),
3294cm-1 (OH- symmetric stretching), 721cm-1(C-H rocking). Some extra peaks were obtained, they may have
resulted not only from the synthesized compound but also from the other byproducts and unreacted compounds.

Fig.1 FTIR Spectra of Fatty Esters (a)SMO (b)SML, (c)SMP.

13

C-NMR
The 13C-NMR spectra of synthesized surfactant obtained for the observed peaks is shown in fig.2. The
resonance at 180ppm may be assigned to the carbonyl group of ester. The resonance at 66ppm may be assigned to the
carboxylate methylene group. The various peaks at 9-59ppm may be due to the presence of methyl and methylene
group in synthesized compound. The methylene group adjacent to the ester group was resonating at 39ppm. The peak
accruing at 72ppm may be due to the presence of CH-OH moiety in the compound. The small amount of unsaturated
alkyl chains is evident from the line at 130-131ppm.

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Fig.2 13C NMR Spectra of Fatty Esters (a)SMO, (b)SML, (c)SMP.

1

H-NMR
The 1H-NMR spectra of synthesized surfactant is shown in fig.3. The shift at 0.9ppm and 1.3ppm are due to
the presence of methyl (CH3) and methylene (CH2) group in synthesized compound. The proton with -value at
2.7ppm is due to the CH- proton present in the compound. The proton with -value at 5.5ppm is due to the presence of
hydroxyl group (OH) in the synthesized compound. The -value at 2.1ppm is due to the ester group i.e. CH-COO-R.
Some extra peaks have resulted not only from the synthesized compound but also from other byproducts and
unreacted compounds.

Fig. 3 1 H NMR Spectra of Fatty Esters. (a)SMO, (b)SML, (c)SMP.

HPLC
The HPLC chromatogram of synthesized surfactants is shown in fig.4. The peak Rt 5.55min having area
18.43mV indicates the presence of unreacted acid group R-COOH. The peak Rt 8.03min having area 30.99mV
confirms the presence of hydroxyl i.e. OH group in the synthesized compounds. The Rt 4.76min. having area
662.84mV is due to the ester group i.e. CH-COO-R. Some extra peaks were resulted not only from the synthesized
compound but also from the other byproducts and unreacted compounds.

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Fig.4 HPLC Chromatograms of Fatty Esters.(a)SMO, (b)SML, (c)SMP.

CONCLUSION
From the experimental results and characteristics analysis presented in this work, it is concluded that synthesis
of monoesters of sorbitol, can be done by a simple process by controlling various parameters like solvent mixture
ratio, temperature control, etc. Significant conversion yield of upto 92% of SMO, 83% of SML, 80% of SMP were
obtained in 48h between 45-500C with the help of orbital shaker incubator. Finally, in contrast to the reported multi
step enzymatic process, a single step enzymatic processes is proposed for the synthesis of sorbitol esters. Thus, the
biocatalytic route described here is economical, significant, efficient and environmentally benign for carrying out
esterification reactions of commercially very important and significant biosurfacatants.
ACKNOWLEDGEMENTS
The authors are thankful to the TEQIP Phase-II (Technical Education Quality Improvement Programme)
Govt. of India, UICT, NMU, Jalgaon for financial support for this research work.
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