SYNTHESIS OF BIOSURFACTANTS FROM NATURAL RESOURCES

jfbc_414 747..758

SYNTHESIS OF BIOSURFACTANTS FROMNATURAL RESOURCES

DIPAK PATIL1, ANTONELLA DE LEONARDIS2 and AHINDRA NAG1,3

1Department of ChemistryIndian Institute of Technology

Kharagpur 721302, India

2Department of Agricultural, Food, Environmental and Microbiological Scienceand Technologies (DiSTAAM)

University of MoliseVia De Sanctis, Campobasso, Italy

Accepted for Publication July 28, 2009

ABSTRACT

Role of biosurfactants has shown renewed interest; hence, search forreadily available natural resources of these materials is continuing. Chickenviscera and bahera oil were used as natural sources of oleic acid and itsmethyl ester, respectively, for the preparation of biosurfactants. Enzymaticesterification and transesterification of oleic acid were performed with sorbi-tol, fructose and ascorbic acid using Candida antarctica lipase, and theirisolated yields were compared. Oleyl esters of sorbitol, fructose and ascorbicacid were produced 27, 21 and 12%, respectively. Transesterification reactiongave better yield than esterification reaction. Synthesized products were con-firmed by nuclear magnetic resonance spectroscopy. Radical scavengingactivity of synthesized ascorbyl oleate was compared with ascorbic acid using1,1-diphenyl-2-picryl hydrazyl radical. Ascorbyl oleate showed better antioxi-dant activity than ascorbic acid.

PRACTICAL APPLICATIONS

Fatty acid esters of polyols have important applications because of theirsurface-active properties resulting from the combination of hydrophilic andhydrophobic parts in a single molecule. These compounds are widely used assurfactants in the food, detergent, pharmaceutical and cosmetic industries.

3 Corresponding author. TEL: +91-3222-281900; FAX: +91-3222-255303; EMAIL: [email protected]

DOI: 10.1111/j.1745-4514.2010.00414.x

Journal of Food Biochemistry 35 (2011) 747–758.© 2010 Wiley Periodicals, Inc. 747

Ascorbyl ester can be used as biosurfactant as well as fat-soluble antioxidantfor prevention of oxidation of fat used in foods.

INTRODUCTION

The synthesis of fatty acid esters of polyols like sugar, sugar alcohol andascorbic acid has gained considerable attention in recent years. Incorporationof the polar moiety to fatty acid converts it to an amphiphilic or surface-activecompound classically known as biosurfactant. These compounds are nonionicand biodegradable in nature. These are mostly applied as emulsifiers forfoodstuff, cosmetics and medicine preparations (Fiechter 1992).

Enzymatic synthesis in organic media provides higher selectivity andpurer products because of enzyme specificity (Klibanov 1990). The catalysis isconducted under mild temperature and pH; these conditions minimize the sidereactions compared with the chemical process (Tarahomjoo and Alemzadeh2003). Several chemical methodologies applied to make sugar esters showedlow selectivity and consequently gave rise to mixture of compounds (Cao et al.1999; Polat and Linhardt 2001).

Lipases (triacylglycerol hydrolases, EC 3.1.1.3) are characterized bytheir ability to catalyze the hydrolysis, esterification and transesterification.In recent years, numerous reports on lipase-catalyzed synthesis of monosac-charide fatty acid esters in organic solvents have been reported. Differentmethods for the removal of water produced during the esterification weresuggested, such as addition of molecular sieves (Chamouleau et al. 2001;Adamczak et al. 2005), azeotropic distillation (Yan et al. 2001), and per-vaporation or vapor permeation, a membrane separation technique (Sakakiet al. 2006). There are reports on the synthesis of sugar ester by direct esteri-fication in the presence of a cosolvent (Janssen et al. 1991; Oguntimein et al.1993), and this reaction leads to water production that promotes reversehydrolysis. In addition, when a cosolvent is used, a complementary step isrequired to remove it.

Chicken viscera constitute nearly 30% of the total wastes in poultryprocessing (Ockerman and Hansen 2000; Jamdar and Harikumar 2005) andare found to be rich in oleic acid; likewise, bahera oil contains high amountsof unsaturated fatty acid (Nag et al. 2007) but is not commercially available. Inthis present report, results of enzymatic synthesis of biosurfactants of oleicacid from readily available natural resources are presented. The aim of thisstudy is to produce oleyl esters from natural sources by transesterification witholeic acid methyl ester (OAME) obtained from bahera oil and by directesterification with oleic acid extracted from chicken viscera.

748 D. PATIL, A. DE LEONARDIS and A. NAG

MATERIALS AND METHODS

Plant Material

Bahera (Terminalia belerica Roxb.) fruits (125 numbers; total weight600 g) were collected from the Arabari forest, Midnapur, India. To reducenatural fungal growth, bahera fruits were washed with dilute potassium per-manganate and copper sulfate solution (approximately 1%, w/v) each. Aftersun drying the fruits, seed kernels were collected by cracking the outer shell.Collected kernels were dried again at 80C for 1 h and were grounded intopowder by ball mill.

Animal Material

Chicken viscera were collected from local poultry houses.

Biocatalyst and Chemicals

The enzyme Candida antarctica lipase B (CAL-B), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2-methyl-2-butanol (99%) were obtained fromSigma Aldrich (Steinheim, Germany). d(-)-Fructose (�98%), silica gel (meshsize, 230–400), molecular sieves (4 Å) and all other solvents were purchasedfrom Merck Chemicals Limited (Mumbai, India), and sodium methoxide(98%) was obtained from Loba Chemie (Mumbai, India). L-ascorbic acid(99.7%) and sorbitol (98%) were purchased from Sisco Research Laboratory,Mumbai, India. Double distilled water was used for the experiment.

Fatty Acid Composition of Bahera Oil

The composition of oil was determined (Nag et al. 2007) by a gaschromatography (GC) (Table 1) using capillary column, Supelcowax 10

TABLE 1.PERCENTAGE COMPOSITION OF FFA IN CHICKEN

VISCERA AND BAHERA OIL

Fatty acids Chicken viscera Bahera oil

Oleic (18:1) 38.4 50.2Linoleic (18.2) 17.8 10.8Palmitic (16.0) 16.5 18.2Eicosenoic (22:1) 6.4 –Stearic (18:0) 5.0 8.2Others 15.9 12.6

FFA, free fatty acid.

749SYNTHESIS OF BIOSURFACTANTS

(Bellefonte, PA), of 30 m ¥ 0.32 mm, film 0.5 mm, connected to a personalcomputer in action with the Chrom-Card 1.2 software (Thermoquest Instru-ment, Rodano MI, Italy). Experimental conditions to determine fatty acidswere as follows: carrier gas He at 50 kPa, split injection system with a splittingratio of 1:40, flame ionization detector system, injector and detector tempera-tures of 250C, oven temperature of 240C and injected quantity of 1 mL; fattyacids were converted to esters by cold methylation with a solution of KOH(2N) in methanol.

Extraction of Oleic Acid from Chicken Viscera

Chopped chicken viscera (100 g) were added to a freshly prepared solu-tion of sodium hydroxide (0.75 mol) in a mixture of water (80 mL) and ethanol(120 mL). The mixture was refluxed under argon atmosphere for 2 h at atemperature of 75C. During this period, the reaction was monitored by thinlayer chromatography (TLC) eluted with 1:4 ethyl acetate : hexane. Themixture was cooled to room temperature and carefully acidified by HCl (2 M)to acidic pH (4–5) in the cold water bath. The resulting free fatty acid (FFA)was extracted with 1:1 mixture of hexane : diethyl ether. The organic layer waswashed with water thoroughly to a neutral pH and dried over anhydroussodium sulfate. The organic layer was concentrated by rotary evaporator (yield5.2%, w/w). Extracted FFA contains both saturated and unsaturated fatty acids.Saturated FFA was removed by dissolving the crude FFA in acetone 12% (w/v)and cooled at -18C for 12 h to precipitate the saturated fatty acid, followed byimmediate filtration on Buckner funnel. The filtrate was concentrated, andoleic acid was further separated by vacuum distillation (34%, w/w of crudeFFA).

Fatty Acid Composition of Chicken Viscera

Fatty acid composition of chicken viscera was determined by GC(Table 1). FFA of 50 mg was taken in 2 mL of BF3–methanol reagent andrefluxed for 2 min. After cooling the solution, 1–2 mL of water was added andthen extracted with hexane. Finally, the solvent was evaporated to get themethyl ester. It was diluted with chloroform or hexane, and injected in GC,where the GC oven temperature was 100–220C at 4C/min, inlet temperaturewas 240C, detector temperature was 250C and capillary column was DB 5.

Methylation of Bahera Oil

Methylation of bahera oil was carried out using sodium methoxide. Solu-tion of sodium methoxide (0.4%, w/v) in methanol was prepared. Bahera oil(4 g) was added to the solution of sodium methoxide (12 mL). It was taken in


the ratio of 1:3 (w/v). After addition of sodium methoxide solution to thepreheated oil, turbidity appeared in the reaction mixture. The reaction mixturewas refluxed until the turbidity disappeared (10–12 min), as, at this point,methyl ester was completely dissolved in methanol. Methylated solution waspoured into ice-cold water (15 mL). The oily layer was removed by using aseparating funnel, followed by water wash (15 mL ¥ 4) to remove generatedglycerol and unreacted sodium methoxide. Resulting methyl ester was keptunder vacuum and dried over anhydrous Na2SO4 to remove traces of water.The yield of methylated oil was 62% (2.48 g) based on the starting weight ofbahera oil. Similarly, OAME was separated by treatment with acetone, fol-lowed by vacuum distillation (45%, w/w of crude methyl ester).

Enzymatic Acylation of Fructose

In a 250-mL three-necked round bottom flask, continuous argon flow overa magnetic stirrer (100 rpm) at 65C was kept in temperature-controlled oilbath for 12 h. Reaction mixture consisted of 2 mmol of fructose, 10 mmol ofacyl donor and 300 mg of lipase in 80 mL of 2-methyl-2-butanol (Fig. 1).Sugar ester was confirmed by TLC, performed on silica gel plate 60F254

(Merck) using developing solvent chloroform : methanol : acetic acid : water(80:15:8:2) or with ethyl acetate : hexane (1:1). The plates were sprayed with50% sulfuric acid and heated at 110C for 5 min that confirmed the presence ofsugar ester (Cao et al. 1997).

The product fructose oleate synthesized from oleic acid and OAME wasseparated by column chromatography. The concentrated reaction mass waspreabsorbed on silica gel and was separated by silica gel column. Unreactedoleic acid or methyl oleate was removed by eluting 0–5% ethyl acetate inhexane, and with gradually increasing the solvent polarity (20–25%), fructoseoleate was obtained. The yield was calculated on the basis of fructose(Table 2).

O OH

OH

OH

CH2

H2C OH

HO

O OH

OH

OH

CH2

H2C

HO

C17H33-COOH / C17H33-COOCH3

+ H2O / CH3OH65 °C

O

O

C17H33

FIG. 1. LIPASE-CATALYZED ACYLATION OF D-FRUCTOSE WITH OLEIC ACID/OLEICACID METHYL ESTER


Enzymatic Acylation of Ascorbic Acid

Reaction was carried out with 2 mmol of ascorbic acid, 8 mmol of acyldonor, 200 mg of lipase and 12 mL of 2-methyl-2-butanol (Fig. 2) in 20-mLscrew-capped glass vials. Molecular sieves (250 mg) were added to control thewater generated in the reaction (esterification) mixture. The reaction wascarried out at 55C in an incubator shaker for 24 h and monitored by silica gelTLC plates (60F254) using ethyl acetate/hexane (90%) as developing solvent.Purification of the products has been done by flash chromatography. Columnwas eluted by medium pressure gas flow, which gave rapid separation andminimized the time of contact with silica gel. Column was prepared by makingslurry in 5%, and products were isolated by eluting 40–50% of the elutingsolvent. Yield of isolated esters was calculated on the basis of ascorbic acid(Table 2).

Enzymatic Acylation of Sorbitol

In a 20-mL screw-capped glass vial, 2 mmol of sorbitol was reacted with6 mmol of acyl donor in the presence of 200 mg of lipase and molecular sieves(250 mg) in 12 mL of 2-methyl-2-butanol (Fig. 3). The reaction was carriedout at 60C in an incubator shaker for 12 h and monitored by silica gel TLCplates (60F254) using ethyl acetate as developing solvent. The product was

TABLE 2.YIELD OF ISOLATED ESTERS

Ester Esterification Transesterification

Fructose oleate 16 21Ascorbyl oleate 10 12Sorbitol oleate 19 27

O

OHHO

O

HO OH

C17H33

O

OH

O

OHO

O

HO OH

C17H33 O

H2O

CH3OH

C17H33

O

OCH3

FIG. 2. LIPASE-CATALYZED ACYLATION OF ASCORBIC ACID WITH OLEIC ACID/OLEICACID METHYL ESTER


purified by washing the crude product with hexane, centrifuging (4,000 rpm)to remove excess of oleic acid, similarly dissolving the product in diethylether. Yield of isolated esters was calculated on the basis of sorbitol (Table 2).

Measurement of Antioxidant Properties

The stable DPPH radical was used to determine radical scavenging activ-ity by spectrophotometric measurement (Brand-Williams et al. 1995). Afterscanning the wavelength between 200 and 800 nm in UV-Vis Double BeamSpectrophotometer (UV-1601, Shimadzu, Kyoto, Japan), the maximum absor-bance of DPPH (100 mM) in methanol was found at 515 nm.

Different concentrations of each test samples (30 mL) were added to 3 mLof DPPH solution. These samples were kept in the dark for 30 min at roomtemperature, and then, the decrease in absorption was measured at 515 nm.Blank sample (A0) was prepared by addition of the same amount of methanol,i.e., in the absence of any sample. All measurements were done in triplicate.Radical scavenging was calculated using the following equation.

% InhibitionA A

A0 C

0

=−

×100,

where A0 = absorbance of blank sample and AC = absorbance at con-centration C.

RESULTS AND DISCUSSION

The weight of the dried seed kernel powder of bahera fruit was 54 g. Theoil was extracted from the powdered meal with hexane (boiling point, 65–70C)

OH

OHHO

HOOH

C17H33

O

OOH

OH

OHHO

HOOH

C17H33-COOH / C17H33-COOCH3

60 °C

FIG. 3. LIPASE-CATALYZED ACYLATION OF SORBITOL WITH OLEIC ACID/OLEIC ACIDMETHYL ESTER


six times, using a Soxhlet apparatus. The oil was recovered from hexane afterdistilling hexane in a nitrogen atmosphere. The oil obtained was 23.4 g (3.9%,w/w of bahera fruits).

Separated oleic acid and OAME were confirmed by 1H nuclear magneticresonance (NMR) spectroscopy. Oleic acid: (400 MHz, CDCl3): 5.35(m, 2H),2.34(t, 2H), 2.02(q, 4H), 1.63(t, 2H), 1.28(d, 20H) and 0.87(q, 3H). OAME:(400 MHz, CDCl3): 5.34(s, 2H), 3.66(s, 3H), 2.29(t, 2H), 2.00(d, 4H), 1.59(t,2H), 1.26(b, 20H), 0.87(t, 3H).

Acylation was done with oleic acid and OAME by esterification andtransesterification. During the transesterification process, methanol was gen-erated as by-product, and because of its high polarity, the enzyme activity wasaffected by removing the hydrated layer of enzyme (Gorman and Dordick1992). Elimination of methanol is very significant to shift the equilibriumtoward the ester synthesis. Reaction was carried out at boiling temperature ofmethanol (65C) under continuous argon flow, which assists to remove themaximum methanol generated in the reaction mixture.

The product fructose oleate synthesized from oleic acid and OAME wasconfirmed by 1H NMR spectroscopy (400 MHz, CD3OD): 5.34(m, 2H),4.28(td, 1H), 4.12(m, 3H), 3.99(m, 2H), 3.85(ddd, 1H), 2.34(t, 2H), 2.02(m,4H), 1.61(t, 2H), 1.30(b, 20H) and 0.90(t, 3H). In both the types of acylationof fructose using oleic acid and methyl oleate, two spots of fructose ester werefound on TLC. Previous studies (Schlotterbeck et al. 1993; Coulon et al. 1995)on enzymatic acylation of fructose with stearic acid using Mucor miehei andoleic acid using C. antarctica had shown the formation of two products asmono-acylated esters at C-1, fructofuranose and fructopyranose. The protonsof sugar moiety were observed as multiplets at 4.28(1H), 4.12(3H), 3.99(2H)

FIG. 4. MAGNIFIED NUCLEAR MAGNETIC RESONANCE SPECTRA OF FRUCTOSEOLEATE INDICATING THE MULTIPLETS OF SUGAR MOIETY PROTONS AT 4.28(1H),

4.12(3H), 3.99(2H), 3.85(1H)


and 3.85(1H) (Fig. 4). These multiplets were overlapped while equating theprotons of both b-1-fructopyranose-oleate and 1-fructofuranose-oleate esters.The reaction equilibrium shifted in favor of ester formation with greater extentin transesterification than esterification reaction, because, comparatively,methanol, having lower boiling point than water, escaped from the reactionvessel under continuous argon flow.

Earlier studies reported that silica gel chromatography is unsuitable forpurification of ascorbyl oleate because considerable losses of product occurred(Yan et al. 1999); this could be because of oxidation and discoloration ofproduct (Viklund et al. 2003). In the present work, the purification of ascorbyloleate was successfully carried out using flash chromatography. Column waseluted with the help of medium pressure gas flow, which gave rapid separationand minimized the time of contact with silica gel. Purified ascorbyl oleate wasconfirmed by NMR spectroscopy (1H 400 MHz, CDCl3): 5.35(m, 2H), 4.78(s,1H), 4.39(s, 1H), 4.24(d, 2H), 2.36(t, 2H), 2.05(m, 4H), 1.62(b, 2H), 1.27(b,20H), 0.87(t, 3H).

Esterification of sorbitol has shown traces of diester on TLC withagreement with the previously studied monoesterification of sorbitol(Dubreucqa et al. 2000). Monoester was separated from the unreactedstarting material by chemical treatment and was confirmed by NMR spec-troscopy (1H 400 MHz, CD3OD): 5.33(m, 2H), 4.34(d, 0.5H), 4.20(dd,0.5H), 4.14(m, 1H), 3.90(m, 2H), 3.77(m, 1H), 3.67(dd,1H), 3.61(d, 1H),2.36(q, 2H), 2.04(m, 4H), 1.62(b, 2H), 1.30(b, 20H), 0.89(t, 3H). On com-parison, synthesized esters gave better yield through transesterification thanesterification reaction.

Oxygen radical absorbance capacity has become the current industrialstandard for assessing antioxidant strength of foods, juices and food additives(Cao et al. 1993). During processing and storage of food, oxidation enhancesdeterioration of food quality. Degradation affects mostly lipids, carbohydratesand proteins (Halliwell 1997). Usually, synthetic antioxidants such as butyl-hydroxyanisole or butylhydroxytoluene are used to delay this degradation.These antioxidants are volatile and easily decomposed at high temperaturesand may produce toxic components (Warner et al. 1986; Kaitaranta 1992).Vitamin C, a well-known natural antioxidant, cannot be used directly in foodbecause of its highly hydrophilic nature (Han et al. 1990; Schuler 1990).Therefore, ascorbyl fatty acid esters are very interesting and can be synthe-sized for use as surfactant cum antioxidant.

The inhibition study of ascorbyl esters for same concentrations hadshown slightly higher inhibition than vitamin C as a control (Fig. 5). Ascorbyloleate had shown 26.36%, whereas vitamin C had shown 21.69% of inhibitionafter 30 min, when 30 mL (0.1 mmol) was added in 3 mL of 100-mM DPPHsolution. Ascorbyl oleate has shown better antioxidant activity.


CONCLUSION

Oleic acid isolated from chicken viscera, a waste product of the poultryindustry, as well as bahera oil, which is not commercially exploited, can beutilized to produce value-added products. Lipase plays an important role tocatalyze fatty acid esterification and transesterification for the production ofemulsifier/antioxidant in food products. The transesterification reaction gavebetter yield than esterification under continuous argon flow with fructose andeven in the presence of molecular sieves with ascorbic acid and sorbitol.

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0.02 0.04 0.06 0.08 0.100

5

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% In

hib

itio

n

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FIG. 5. SCAVENGING EFFECT OF ASCORBYL OLEATE IN METHANOL


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SYNTHESIS OF BIOSURFACTANTS FROM NATURAL RESOURCES