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Lipase Mediated Upgradation of Dietary Fats and OilsDr. (Mrs.) Rani Gupta a , Pooja Rathi a & Sapna Bradoo ba Department of Microbiology , University of Delhi South Campus , New Delhi, 110 021, Indiab Biotechnology Department , Center for Chemistry and Chemical Engineering, LundUniversity , SwedenPublished online: 09 Jan 2007.

To cite this article: Dr. (Mrs.) Rani Gupta , Pooja Rathi & Sapna Bradoo (2003) Lipase Mediated Upgradation of Dietary Fatsand Oils, Critical Reviews in Food Science and Nutrition, 43:6, 635-644, DOI: 10.1080/10408690390251147

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Critical Reviews in Food Science and Nutrition, 43(6):635–644 (2003)Copyright C© 2003 Taylor and Francis Inc.ISSN: 1040-8398DOI: 10.1080/10408690390251147

Lipase Mediated Upgradation of DietaryFats and Oils

Rani Gupta,1* Pooja Rathi,1 and Sapna Bradoo2

1Department of Microbiology, University of Delhi South Campus,New Delhi—110 021, India, and 2Biotechnology Department, Center for Chemistryand Chemical Engineering, Lund University, Sweden

* To whom correspondence should be directed: Dr. (Mrs.) Rani Gupta, Department of Microbiology, University ofDelhi South Campus, New Delhi—110021, India, Phone: +91-011-6886559, Fax: +91-011-6885270, E-mail:microzyme@123india.com

ABSTRACT: In the present scenario, fats and oil modification is one of the prime areas in foodprocessing industry that demands novel economic and green technologies. In this respect, tailoredvegetable oils with nutritionally important structured triacylglycerols and altered physicochemicalproperties have a big potential in the future market. In this context, it is well established that lipasesespecially microbial lipases, which are regiospecific and fatty acid specific, are of immenseimportance and hence could be exploited for retailoring of vegetable oils. Further, of the bulkavailable, cheap oils could also be upgraded to synthesize nutritionally important structuredtriacylglycerols like cocoa butter substitutes, low calorie triacylglycerols, PUFA-enriched andoleic acid enriched oils. It is also possible to change the physical properties of natural oils toconvert them into margarines and hard butter with higher melting points or into special low caloriespreads with short or medium chain fatty acids. Today, by and large, fat and oil modifications arecarried out chemically following the method of directed inter-esterification. The process is energyintensive and non-specific. Lipase mediated modifications are likely to occupy a prominent placein oil industry for tailoring structured lipids since enzymatic modifications are specific and canbe carried out at moderate reaction conditions. However, as a commercial venture, lipases are yetto be fully exploited. Once the technologies are established, the demand of lipases in oil industryis expected to increase tremendously in the near future for specific modifications of fats and oilsto meet the changing consumers’ dietary requirements.

KEYWORDS: dietary fats, fat modification, interesterification, lipases, structured triacylglyc-erols, transesterification.

INTRODUCTION

The growing present day calorie consciousnessamong society and the increasing incidences of car-diac problems have made the types of fats and oilsto be used of great concern. Today, requirementof quality oil has changed the scenario towardsmore and more nonhydrogenated, low cholesteroland polyunsaturated fatty acid enriched fats andoils. The growing demand of the desired triacyl-

glycerol composition cannot be always met fromnatural fats and oils. In this endeavor, oil indus-tries have focused mainly on upgrading fats andoils to meet consumers’ demand. This has beenachieved by chemical modification of fats and oilsto alter the composition and physical properties oftriacylglycerol mixture. The process is promotedby sodium metal or sodium alkoxide to catalyzeacyl migration among the triacylglycerol molecules,but this method produces randomly distributed fatty

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acid residues among the triacylglycerols (Kurashigeet al., 1993). Thus, chemical process is randomand produces undesired products. Hence, the needfor higher-quality products with specific modifica-tions suggests enzymatic reactions using lipases.The specificity of lipases leads to a highly pureproduct and provides a wide range of useful and mar-ketable new products (Al-Duri et al., 1995). In addi-tion, enzymatic reactions require mild reaction con-ditions and meet the increasing need of ecofriendlytechnologies by reducing waste generation.

Many new and interesting ideas for employ-ing biotechnology to produce important structuredlipids by lipase mediated lipid modification havebeen investigated (Pabai et al., 1995; Posorske et al.,1988). In this endeavor, lipases occupy the place ofprominence and carry both position specific and acylgroup specific modifications, thus desired modifica-tion of fats and oils can be carried out (Macrae, 1989;Malcata et al., 1990).

Lipases catalyze modification of fats and oilseither by transesterification or interesterification re-actions (Basheer et al., 1995; Kalo, 1988). Trans-esterification involves an exchange of acyl moietiesbetween a triacylglycerol and a fatty acid (acidol-ysis), an alcohol (alcoholysis), or glycerol (glyc-erolysis), and interesterification involves exchangeof acyl moieties between two triacylglycerols. Onthe other hand, the transesterification by chemi-cal method produces randomly distributed productswhile enzymatic reactions are highly specific.

Lipases can be obtained either from animals,plants or microbes. However, microbial lipases havegained importance owing to their multifold proper-ties, easy extraction procedures and unlimited sup-ply (Ghosh et al., 1996; Macrae and Hammond et al.,1985; Saxena et al.,1999; Yadav et al., 1998). Mi-crobial lipases may be divided into three categoriesbased on the substrate specificity, i.e., nonspecific,regiospecific and fatty acid specific. Nonspecific li-pases act at random on triacylglycerol molecule andhence are of little use for fat and oil modification.On the contrary, regiospecific lipases are 1,3 specificlipases and thus by selective hydrolysis of triacyl-glycerols result in 2-monoglyceride and a mixture offatty acids. Application of position specific lipasesis to alter the fatty acid specifically at 1,3 positionwithout affecting 2 position.

POSITION SPECIFIC LIPASES

There are a good number of microbial lipasesavailable commercially, however. The most po-

tential 1,3 selective lipase is from Mucor miehei(Table 1). Besides this, lipases from Rhizomucor,Penicillium and Candida spp. are also regioselec-tive and are used for specific reactions (Macrae andHammond et al., 1985; Rogalska et al., 1993). Theattractive feature of these lipases is the specificitywith respect to the glyceride positions. For example,high-valued specialty fats such as cocoa butter sub-stitutes and human milk substitutes can be obtainedby exploiting lipases with 1,3- positional specificity.Numerous examples of such structured lipids havebeen cited later.

Most lipases show little preference for differ-ent fatty acids. However, there exists few reportsrelated to fatty acid selectivity’s of lipases. Peni-cillium roquefortii lipase (Proq L) favors small-medium chain fatty acids over long chain fatty acidsand Rhizopus oryzae lipase (ROL) favors medium tolong chain fatty acids. Fatty acid selectivity is bestmeasured by a competition environment where thelipase chooses among different substrates (Bergerand Schneider, 1991; Rangheard et al., 1989).

The most important is lipase from Geotrichumcandidum which shows much higher selectivity forunsaturated fatty acids and being specific for a cis�9 bond by a factor of 20 or more; 100 to 1 for oleicacid vs. stearic acid (Baillargeon and McCarthy,1991; Charton and Macrae, 1993; Jensen, 1973).Candida rugosa lipase (CRL) discriminates againsterucic acid (cis �13 C22) and γ -linolenic acid (cis�6, cis �9, cis �12 C18) (Ergan et al., 1991; Traniet al., 1993). Similarly, Sugihara and his co-workers(Sugihara et al., 1997) reported that Rhizopus dele-mar lipase (RDL) discriminates against docosahex-anoic acid (DHA) in esterification with lauryl alco-hol while with short-chain alcohol it showed littlediscrimination. Several lipases moderately favoredpolyunsaturated fatty acids over saturated fatty acidsbut no qualitative data is available. Therefore, bychoosing the lipases from specific sources, it is pos-sible to impart specific properties to the oils.

The position specificity and the fatty acid speci-ficity of lipases have always been of great relevanceto an oil chemist for desired fat and oil modifi-cation (Bradoo et al., 1999). First, such applica-tions have been presented in the patents filed byUnilever (Coleman and Macrae, 1977; Matsuo et al.,1981a, b) and Fuji Oil (Matsuo et al., 1981a, b)for the synthesis of cocoa butter equivalents. To-day a long list of patents is available on enzymaticsynthesis of cocoa butter substitutes (Matsuo et al.,1981a, b; Hargreaves, 1982).

In addition, many other modifications can bedone to obtain fats and oil with desired physical,

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TABLE 1Regiospecific Nature of Some Commercially Available Lipases

Lipase Source of Lipase Regioselective Nature Commercial Source

CAL-B Candida antarctica 1,3-selective Novo Nordisk, BoehringerMannheim

CAL-A Candida antarctica Non- or 2-selective Novo Nordisk, BoehringerMannheim

CRL Candida rugosa Nonselective Amano Pharmaceutical, AltusBiologics

ANL Aspergillus niger Moderately 1,3-selective Amano Pharmaceutical, Rohm,Novo Nordisk

HLL Humicola lanuginosa Slightly 1,3-selective Amano Pharmaceutical, BoehringerMannheim, Novo Nordisk

GCL Geotrichum candidum Nonselective Amano PharmaceuticalPPL Porcine pancreas 1,3-selective Amano Pharmaceutical, Boehringer

MannheimPcamL Penicillium camemberti Highly 1,3-selective Amano PharmaceuticalProqL Penicillium roqueforti Moderately 1,3-selective Amano Pharmaceutical, Boehringer

MannheimROL Rhizopus oryzae Highly 1,3-selective Amano Pharmaceutical, Sigma,

FlukaRML Rhizomucor miehei Moderately 1,3-selective Amano PharmaceuticalRML Rhizomucor javanicus Slightly 1,3-selective Novo Nordisk, Fluka, Amano

PharmaceuticalPFL Pseudomonas fluorescens 1,3-selective Amano PharmaceuticalPCL Pseudomonas cepacia Nonselective Amano Pharmaceutical, Altus

Biologics

chemical and nutritive properties. Modification ofvarious seed oils for obtaining PUFA enriched, lowcalorie, oleic acid enriched triacylglycerols is of spe-cial relevance. The following is a comprehensiveaccount of various lipase mediated modifications offats and oils to upgrade their nutritional value by ob-taining triacylglycerols of desired composition, i.e.,structured triacylglycerols.

STRUCTUREDTRIACYLGLYCEROLS

Structured triacylglycerols (STs) are triacyl-glycerols modified in either the type of fatty acid orposition of fatty acid. The synthesis of structured tri-acylglycerols with position specificity such as cocoabutter substitutes, MLM lipids (Yankah and Akoh,2000) (triacylglycerols with medium chain length atsn-1 and sn-3 and a long chain at sn-2) employ 1,3selective lipases while synthesis of STs enrichedin PUFA (Xu et al., 2000) or oleic acid (Yu andHammond, 2000) require fatty acid specific lipases.

Cocoa Butter Substitutes (CBS)

Preparation of cocoa butter substitutes (CBS)from lower value oils by lipases has received consid-erable attention (Coleman and Macrae, 1977; Changet al., 1990).

Cocoa butter is crystalline and melts between25–35◦C imparting the desirable “mouth feel”. Co-coa butter is predominantly 1,3 disaturated -2-oleyl-glycerides, where palmitic, stearic and oleic acidaccount for more than 95% of total fatty acids.The first patents of Unilever (Coleman and Macrae,1977) and Fuji Oil (Matsuo et al., 1981a, b) forthe lipase catalyzed synthesis of CBS is from palmoil midfraction and stearic acid. Palm oil mid-fraction consists of 1,3-dipalmitoyl-2-mono-oleine(POP). Interesterification with tristearin or acidoly-sis with stearic acid in presence of 1,3 specific lipaseyields 1(3) palmitoyl-3(1) stearoyl-2-mono-oleine(P-OSt) and 1,3-distearoyl-2-monoleine (St-OSt)(Fig. 1).

A number of reformulated vegetable oils arecurrently produced as CBS. Attempts have been

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FIGURE 1. Synthesis of cocoa butter substitute (CBS). P = Palmitate; St = Stearate; O = Oleate.

made to prepare CBS by interesterification ofhydrogenated cottonseed oil, olive oil and subse-quent fractionation (Sridhar et al., 1991). The prepa-ration of CBS has attracted much attention in recentyears due to the availability of 1,3 specific micro-bial lipases that catalyze regioselective interesterifi-cation at the 1- and 3- positions of triacylglycerols.The CBS thus produced have been granted GRASstatus (Sridhar et al., 1991). Other suitable startingoils are sunflower, rapeseed (Adlercreutz, 1994) orolive oils (Chang et al., 1990). There exists lot of lit-erature on lipase mediated synthesis of cocoa buttersubstitutes (Macrae and Hammond, 1985; Macrae,1983; Quinlan and Moore, 1993).

Good potential exists for some solid or semi-solid fats of tree origin in India for their modifica-tion into CBS because of their physical and chemicalcharacteristics as well as their fatty acid and glyc-eride compositions. Prominent among these are Sal(Shorea robusta), Kokum (Garcinia indica), mahua(Madhuca latifolia or M. indica), dhupa (Vateriaindica) and mango (Mangifera indica) fats, whichhave a combined annual production of about onemillion metric tons (Sridhar et al., 1991). Attemptshave been made to modify these fats, except dhupa,to CBS by solvent fractional crystallization, chemi-cal/enzymatic interesterification and/or hydrogena-tion, but they have met with only limited success(Sridhar et al., 1991).

Human Milk Fat Substitutes

Another important triacylglycerol is humanmilk fat substitute. In contrast to the plant oils, tri-acylglycerols of human milk fat contain palmitic

acid exclusively in the 2-position with 1,3 dioleoyl-2-palmitoyl glycerol as the major component(Kazlauskar and Bornscheuer, 1998). Saturatedfatty acids with chain lengths longer than C18 arepoorly absorbed partly because they form insol-uble calcium salts. Thus, digestion of vegetableoil such as P-O-O (mixture of 1,2 dioleoyl-3-palmitoyl glyceride and its enantiomer) by pan-creatic lipase yields the poorly absorbed palmiticacid. On the other hand, human milk contains 1,3-dioleoyl-2-palmitoyl glycerol (OPO). This yieldsoleic acid and 2-palmityl glycerides, both of whichare much more efficiently absorbed. Interesteri-fication of palm top fraction (tripalmitin) witholeic acid in presence of Rhizomucor miehei lipase(RML) at low water activity yields OPO (Fig. 2).Evaporation removes fatty acid and crystallizationremoves PPP.

Synthesis of Low CalorieStructured Triacylglycerols (LCSL)

Due to calorie consciousness and other healthconcerns related to intake of high calorie fats andoil, there is a growing demand for low calorie fatsand oils. The low calorie triacylglycerols containshort chain fatty acids at sn-1 and sn-3 position andsn-2 position is occupied by a long chain fatty acid(MLMs). Shorter chain fatty acids have fewer calo-ries per unit weight than long chain fatty acids. Fur-ther, these triacylglycerols provide rapid delivery ofenergy via oxidation of the more hydrophilic shortchain fatty acids, while at the same time provide anadequate supply of an essential fatty acid from theremaining 2-monoglyceride (Jandacak et al., 1987;

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FIGURE 2. Synthesis of human milk substitute (HMS). P = Palmitate; St = Stearate; O = Oleate.

Shimada et al., 2000). Hence these LCSL find ap-plications in clinical nutrition, e.g., as a concen-trated form of calories for patients with pancreaticdeficiency.

A commercially available LCSL calledcaprenin contains half the calorie of naturaloccurring triacylglycerol. This triacylglycerol iscomposed of one molecule of behenic acid (C22:0)and two molecules of caprylic acid (C8:0), or capricacid (C10:0). These are chemically synthesizedby high temperature reaction of monobeheninwith the free acids (Kleusener et al., 1992) orreaction of monobehenin with a reactive form offatty acids as the anhydride, at lower temperature(Stipp and Kleusener, 1992). Captex, Neobee andSalatrim are a few more commercially availablechemically synthesized MLMs (Jandacak et al.,1987). Enzymatic synthesis overcomes both therequirements of chemical synthesis, i.e., hightemperature and expensive fatty acid anhydrides.McNeill and Sonnet (1995) initially synthesizedcaprucin, comprising of one molecule of erucicacid and two molecules of caprylic acid, that wassubsequently hydrogenated to caprenin. For thesynthesis of these LCSL, lipases specific for theshort chain fatty acids and incapable of hydrolysisof long chain fatty acid are selected.

Interesterification of high oleic acid oil withcapric acid using Rhizomucor miehei lipase (RML)resulted in the synthesis of 69% COC along withCCO and OCC after 30 hours reaction in hexane(Shieh et al., 1995). A two-step procedure, i.e., alco-holysis of triacylglycerol by 1,3 regiospecific lipaseresults in higher yields of 2-monoglyceride (72%)followed by esterification with caprylic acid in sec-ond step. The final product contained more than 90%caprylic at sn-1 and sn-3 positions with 98% unsat-urated fatty acid at sn-2 position.

LCSL were synthesized from a mixture of tri-acetin and tributyrin by Candida cylindracea li-pase in a reversed micelle configuration (Abrahamet al., 1988). Yankah and Akoh (2000) synthesizedtwo different structured lipids by transesterifyingtristearin with caprylic acid (C8:0) or oleic acid(C18:1) in order to obtain nutritional and low calo-rie fats. Their solid fat contents (∼25%) at 25◦Csuggest possible use in spreads or for inclusion withother fats in specialized blends. Wong with his co-workers (Wong et al., 2000) have reported enzy-matic synthesis of medium-chain glycerides (MCG)from capric acid and glycerol using lipase from Can-dida rugosa. In order to synthesize LCSL, Yangwith his co-workers (Yang et al., 2001) carried outa transesterification between triacetin and stearicacid using immobilized lipase Chirazyme R© in asolvent-free system and obtained 88% conversionwithin 4 h.

Triacylglycerols ContainingPolyunsaturated Fatty Acids (PUFA)

The growing interest in the nutritional and med-ical significance of PUFAs has led to the synthe-sis of structured triacylglycerols with high pro-portion of eicosapentaenoic acid (EPA, 20:5n-3)and docosahexanoic acid (DHA, 22:6n-3), essentialPUFAs which are beneficial in cardiovascular andinflammatory diseases (Akoh et al., 1995; Kosugiand Azuma, 1994; Pederson and Holmar, 1995;Yamane, 2000). Physiological effects of the omega-3 fatty acids have been observed in three main areas.In the heart and circulatory system, these acids havebeen shown to have a beneficial effect in the pre-vention or treatment of artherosclerosis, thrombosisand high blood pressure. Beneficial physiological

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effects have also been observed in the treatment ofasthma, arthritis, migraine and nephritis. Besidesthis, potential application has also been proposedin the treatment of breast, prostrate and colon can-cer. However clinical and nutritional studies havebeen retarded by the nonavailability of commercialpreparations of DHA.

PUFAs are most efficiently absorbed as tri-acylglycerols. However, they are highly labilemolecules, thus elevated temperatures and extremesof pH during chemical processes promote side re-actions, oxidation, cis-trans isomerization or dou-ble bond migration in PUFAs (Haraldsson et al.,1993). In this scenario, lipase mediated synthesisof PUFA containing triacylglycerols is suggestedas an alternative. Several lipases favor PUFA oversaturated fatty acids but no quantitative data is avail-able. Substantial enrichment in PUFA content ofthe monoglycerides fraction has been achieved bylipase-catalysed alcoholysis (Hayes and Kleiman,1992; Zaks and Gross, 1990) or hydrolysis (Zaksand Gross, 1990). The most commonly used lipaseis from Geotrichum candidum that favors unsatu-rated cis �9 bond. Soybean oil has been modified byincorporating EPA/DHA to a final content of 10.5to 34.7% using RML (Rhizomucor miehei lipase)(Huang and Akoh, 1994).

For instance, cod liver oil, a well known foodsupplement for generations, is a complicated mix-ture of more than fifty different fatty acids boundinto the natural triacylglycerols of which there isusually 8–9% each of EPA and DHA and 22–24% of the total of n-3 polyunsaturated fatty acid.Haraldsson (2000) has used Lipozyme, an immo-bilized lipase from the fungus Mucor miehei toincrease the content of EPA+DHA to 60–65% incod liver oil. It is possible to prepare triacylglyc-erols containing up to 30% EPA and DHA directlyfrom fish oil with either PUFA (Shimada et al.,2000) or ethyl ester (PUFEE) (Yamane, 2000) con-centrates, either in the absence of any solvent orin a non-polar organic solvent such as hexane us-ing lipase. Shimada with his co-workers (Shimadaet al., 1994) have used lipases from Candida ru-gosa and Geotrichum candidum for the produc-tion of DHA-rich oil from tuna oil since this li-pase has high hydrolyzing ability, but act on DHAvery weakly. Shimada with his co-workers (Shi-mada et al., 1995) have also used Candida rugosalipase (CRL) to incorporate up to 60% arachidonicacid into single cell oil from Mortierella alpina.An attempt has been made to synthesize struc-tured triacylglycerols containing PUFA from nat-ural edible oils using lipases from Rhizopus dele-

mar, Rhizomucor miehei and Candida antarctica(Yamane, 2000).

Interesterification for High OleicAcid Triacylglycerols

The favorable influence of diets high in linoleicacid in lowering blood cholesterol has led to pop-ularization of PUFA enriched oils. Further, wheredietary fats are largely of plant origin, the PUFA con-tent of most of the natural oils is more than the rec-ommended minimum dose of 30%. Further, PUFAintake from other dietary supplements like cereals,pulses, nuts and vegetables also adds to it. Fish oil isalso a natural source of PUFA for non-vegetarians.However, PUFA enriched oils need a little cau-tion, as these oils are susceptible to peroxidation indeep-frying and in vivo giving rise to free radicals,hydroperoxides, endoperoxides and polymers. Allthese peroxidation products are harmful to healthbeing carcinogenic and also cause artherosclerosis(Kubow, 1990). Further, many more negative ef-fects of linoleic and linolenic acids are also wellknown as occurrences of gall stones, alteration inthe composition of cell membranes and also re-duction of high density lipoproteins (HDL) (Vegaet al., 1982). On the contrary, studies have indicatedthat oleic acid is safe and as beneficial as PUFAin reducing low-density lipoproteins (LDL) with-out affecting HDL. Low linolenic soybean and higholeic safflower and sunflower varieties have been ob-tained by genetic selection and chemical mutagene-sis (Purdy, 1985; Yodice, 1990). However, the possi-bility of lipase-mediated interesterification has alsobeen looked into to reduce PUFA in sunflower, saf-flower, soybean and linseed oil and PUFA and LSFAin peanut oil by replacing them with oleate (Sridharet al., 1991; Macrae, 1983; Kaimal and Saroja, 1989;Schuch and Mukherjee, 1989). In this regard, 1, 3-regiospecific lipase from Mucor miehei (lipozyme)proved better than a nonspecific lipase Candida ru-gosa (Sridhar et al., 1991). Lipozyme as a catalystin ester interchange reaction reduced the LSFA sig-nificantly from 6.6 to 2.8% even at low molar ratio(1:1) of peanut oil and methyl oleate (Sridhar et al.,1991). Lipozyme resulted in considerable decreasein linoleic and linolenic acid in sunflower, safflower,soybean and linseed oil and simultaneous increasein oleic acid. This resulted in oxidative stability ofthe modified oil. The interesterified sunflower andsafflower oils with high oleate and low saturate havefurther prospective market and these would find usefor decreasing the cholesterol and LDL levels and

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maintaining the level of HDL. Thus, these wouldbecome popular as cardiac compatible oils.

Directed Interesterification forNonhydrogenated Solid Fats

In recent years, lipase catalyzed acyl exchangereactions of fats and oils have been investigated asa means of either altering the physical properties,producing a margarine or specialty fat or improv-ing the nutritional quality of a fat or oil (Mukherjee,1990). The main source of solid fats in food is hy-drogenated vegetable oils. However, lately there hasbeen some health concerns with regards to the for-mation of trans fatty acids during the hydrogenationreaction. Thus, food industry demands nonhydro-genated solid fats that are low in trans fatty acid.

Interesterification has been applied to edible oilto randomize the fatty acid distribution in triacyl-glycerols to prevent phase separation in fats suchas lard and to generate hard butters by interesterify-ing fully hydrogenated oils and coconut oils. Inter-esterification of a blend of palm stearin and coconutoil was catalyzed by an immobilized Thermomyceslanuginosa lipase for production of margarine fats(Zhang et al., 2001). This type of triacylglycerolsrearrangement is also used to modify the crystal-lization behavior and melting points of solid fats.Solid fats in general are desired to crystallize insmall, smoother, more palatable,β’ crystals whereasthe larger, coarser, β crystals are usually avoided,specifically in the bakery and confectionery indus-tries.

Another potential application of lipases is thesynthesis of zero-trans margarines (Marangoni andRousseau, 1975). The chemical processing for par-tial hydrogenation of oils to increase their meltingpoint also introduces trans-isomers of unsaturatedfatty acids. Natural oils have only cis-isomers andthe trans-isomers may contribute to cardiac prob-lems. Therefore, lipase mediated interesterificationis the desired option for nonhydrogenated solid fats.Such fats will have changed melting characteris-tics while retaining the unsaturation at sn-2 posi-tion. Various processes have been developed in thisdirection for production of solid fats from nonhy-drogenated oils through winterization and directedinteresterification (Hernandez and Lusas, 1997) andmuch more are on their way to commercialization.

Pal with his co-workers (Pal et al., 2001) havemodified butter stearin fraction by blending and in-teresterification with liquid oils like sunflower andsoybean oil using Mucor miehei lipase to offer nutri-

tionally important fat products with enriched con-tent of essential fatty acids like C18:2 and C18:3.These fat products have desirable properties that canbe used in making melange spread fat products withreasonable content of polyunsaturated fatty acids(PUFAs) and almost zero trans fatty acid content.

CONCLUSION AND FUTUREPROSPECTS

The above account well illustrates the poten-tial of lipases to modify fats and oil for the synthe-sis of precise structured triacylglycerols, which areotherwise difficult to be synthesized chemically. Inthis endeavor, 1,3 specific and fatty acyl specific li-pases have contributed the most. Undoubtedly, thepotential of lipases in revolutionizing the food in-dustry is immense, but the major limitation for thecommercial exploitation of lipases is still the eco-nomics of the process and also the understanding oflipase interfacial kinetics that hinder scaling up ofthe processes. Economics of the process can easilybe met by cost effective production of lipases as wellas by developing strategies for immobilization forreusage. Therefore, the thrust area in lipase researchtoday is to develop scale-up strategies besides stan-dardizing procedures for immobilization of com-mercially available, regiospecific lipases. Besidesthis, thermostable lipases should also be looked intofor possible solvent free interesterification reactionsas the food industry discourages the usage of organicsolvents.

Thus, with the use of immobilized enzymes andthe bulk availability of microbial lipases, oleochem-ical industry has a bright future for developing mod-ified nutritive fats and oils with desired physical andchemical properties.

ACKNOWLEDGEMENT

The authors wish to thank the Department ofBiotechnology (DBT), Govt. of India for financialassistance. Dr. P.K. Ghosh, Advisor, DBT, providedvaluable advice during various discussions.

REFERENCES

Abraham, G., Murray, M.A., and John, V.T. (1988). In-teresterification selectivity in lipase catalyzed re-action of low molecular weight triacylglycerides.Biotechnol. Lett., 8:555–558.

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Adlercreutz, P. (1994). Enzyme-catalyzed lipid modi-fication. Biotechnol. Genet. Eng. Rev., 12:231–254.

Akoh, C.C., Jennings, B.H., and Lillard, D.A. (1995). En-zymic modification of trilinolein: incorporation ofn-3 polyunsaturated fatty acids. J. Am. Oil Chem.Soc., 72:1317–1321.

Al-Duri, B., Robinson, E., McNerlan, S., and Bailie, P.(1995). Hydrolysis of edible oils by lipases immo-bilised on hydrophobic supports: Effects of internalsupport structure. J. Am. Oil Chem. Soc., 72:1351–1359.

Baillargeon, M.W. and McCarthy, S.G. (1991).Geotrichum candidum NRRL Y-553 lipase:purification, characterization and fatty acidspecificity. Lipids, 26:831–836.

Basheer, S., Mogi, K.I., and Nakajima, M. (1995). Inter-esterification kinetics of triacylglycerides and fattyacids with modified lipase in n-hexane. J. Am. OilChem. Soc., 72:511–518.

Berger, M. and Schneider, M.P. (1991). Regioselectiv-ity of lipases in organic solvents. Biotechnol. Lett.,13:333–338.

Bradoo, S., Saxena, R.K., and Gupta, R. (1999). Twoacidothermotolerant lipases from new variants ofBacillus spp. World J. Microbiol. Biotechnol.,15:97–102.

Chang, M.K., Abraham, G., and John, V.T. (1990). Pro-duction of cocoa butter-like fat from interesterifi-cation of vegetable oils. J. Am. Oil Chem. Soc.,67:832–834.

Charton, E. and Macrae, R. (1993). Specificities of im-mobilised Geotrichum candidum CMICC 335426lipases A and B in hydrolysis and ester synthe-sis reactions in organic solvents. Enzyme Microb.Technol., 15, 489–493.

Coleman, M.H. and Macrae, A.R. (1977). Rearrangementof fatty acid esters in fat reaction reactants, GermanPatent DE 2705608.

Ergan, F., Trani, M., and Andre, G. (1991). Use of lipasesto discriminate against fatty acids of industrial in-terest. Inform, 2:357.

Ghosh, P.K., Saxena, R.K., Gupta, R., Yadav, R.P.,and Davidson, W.S. (1996). Microbial lipases:Production and applications. Sci. Prog., 79:119–157.

Haraldsson, G.G. (2000). Enrichment of lipids withEPA and DHA by lipase. In: Bornscheur, U.T.Ed., Enzymes in Lipid Modification. Wiley-VCHVerlag Gmbh, D-69469 Weinheim (FRG), 170–189.

Haraldsson, G.G., Gudmundson, B.O., and Almarsson,O. (1993). The preparation of homogeneous tri-acylglycerides of eicosapentaenoic acid and do-cosahexaenoic acid by lipase. Tetrahedron Lett.,34:5791–5794.

Hargreaves, N.G. (1982). Cocoa butter replacement fatcompositions, U.S. Patent 4,364,868.

Hayes, D.G. and Kleiman, R. (1992). Recovery of hy-droxy fatty acids from lesquerella oil with lipases.J. Am. Oil Chem. Soc., 69:982–985.

Hernandez, E. and Lusas, E.W. (1997). Trends in transes-terification of cotton oil. Food Technol., 51:72–76.

Huang, K.-H. and Akoh, C.C. (1994). Lipase-catalyzedincorporation of n-3 polyunsaturated fatty acidsinto vegetable oils. J. Am. Oil Chem. Soc., 71:1277–1280.

Jandacak, R.J., Whiteside, J.A., Holcombe, B.N.,Volpenhein, R.A., and Toulbee, J.D. (1987). Therapid hydrolysis and efficient absorption of triacyl-glycerides with octanoic acid in the 1 and 3 positionand long-chain fatty acid in the 2 position. Am. J.Clin. Nutr., 45:940–945.

Jensen, R.G. (1973). Characteristics of the lipase fromthe mold Geotrichum candidum: a review. Lipids,9:149–157.

Kaimal, T.N.B. and Saroja, M. (1989). Modification ofvegetable oils by lipase catalysed interesterifica-tion,J. Oil Technol. Assoc. India, 21:2–10.

Kalo, P. (1988). Modification of butter fat by interesteri-fication catalysed by Aspergillus niger and Mucormiehei lipases. Meijeritieteellinen Aikakauskiria,46:36–47.

Kazlauskar, R. and Bornscheuer, U.T. (1998). Biotrans-formation with lipases. In Biotechnology. Eds.Rehm, H.-J. and Reed, G., Wiley-VCH VerlagGmbh, D-69469 Weinheim (FRG), 37–191.

Kleusener, B.W., Stipp, G.K., and Yang, D.K. (1992). Se-lective esterification of long chain fatty acid mono-glycerides with medium chain fatty acids, U.S.Patent 5,142,071.

Kosugi, Y. and Azuma, N. (1994). Synthesis of triacyl-glycerol from polyunsaturated fatty acid by immo-bilized lipase. J. Am. Oil Chem. Soc., 71:1397–1403.

Kubow, S. (1990). Toxicity of dietary lipid peroxidationproducts. Trends Food Sci. Technol., 1:67–71.

Kurashige, J., Matsuzaki, N., and Takahashi, H. (1993).Enzymatic modification of canola/palm oil mix-tures: effects on the fluidity of mixture. J. Am. OilChem. Soc., 70:849–852.

642

Dow

nloa

ded

by [

Nor

thea

ster

n U

nive

rsity

] at

11:

31 0

9 O

ctob

er 2

014

Macrae, A.R. (1983). Lipase-catalysed interesterificationof oils and fats. J. Am. Oil Chem. Soc., 60:291–294.

Macrae, A.R. (1989). Tailored triacylglycerols and esters.Biochem. Soc. Trans., 17:1146–1148.

Macrae, A.R. and Hammond, R.C. (1985). Present andfuture applications of lipases. Biotechnol. Genet.Eng. Rev., 3:193–217.

Malcata, F.X., Reyes, H.R., Garcia, H.S., Hill, C.G., andAmundson, C.H. (1990). Immobilized lipase re-actors for modification of fats and oils- a review.J. Am. Oil Chem. Soc., 67:890–910.

Marangoni, A.G. and Rousseau, D. (1975). Engineeredtriacylglycerols: the role of interesterification.Trends Food Sci. Technol., 6:329–335.

Matsuo, T., Sawamura, N., Hashimoto, Y., and Hashida,W. (1981a). Method for producing cocoa buttersubstitute, U.S. Patent 4,268,527.

Matsuo, T., Sawamura, N., Hashimoto, Y., and Hashida,W. (1981b). The enzyme and method for enzy-matic transesterification of lipid, Eur. Patent No.0035883.

Mukherjee, K.D. (1990). Lipase-catalysed reactions formodification of fats and other lipids. Biocatal. 3,277–293.

Mc Neill, G.P. and Sonnet, P.E. (1995). Low-calorie tria-cylglycerides synthesis by lipase-catalyzed esteri-fication of monoglycerides. J. Am. Oil Chem. Soc.,72:1301–1307.

Pabai, F., Kermasha, S., and Morin, A. (1995). Interesteri-fication of butter fat by partially purified extracellu-lar lipases from Pseudomonas putida, Aspergillusniger and Rhizopus oryzae. World. J. Microbiol.Biotechnol. 11:669–677.

Pal, P.K., Bhattacharyya, D.K., and Ghosh, S. (2001).Modifications of butter stearin by blending and in-teresterification for better utilisation in edible fatproducts. J. Am. Oil Chem. Soc., 78:31–36.

Pederson, S.B. and Holmar, G. (1995). Studies of thefatty acid selectivity of the lipase from Rhizomu-cor miehei toward 20: In-9; 5n-3, 22:1n-9 and 22:6n-3. J. Am. Oil Chem. Soc., 72:239–243.

Posorske, L.H., LeFebvre, G.K., Miller, C.A., Hansen,T.T., and Glenvig, B.L. (1988). Process consider-ation of continuous modification with an immobi-lized lipase. J. Am. Oil Chem. Soc., 65:9220–926.

Purdy, R.H. (1985). Oxidative stability of high oleic sun-flower and safflower oils. J. Am. Oil Chem. Soc.,62:523–525.

Quinlan, P. and Moore, S. (1993). Modification of tri-acylglycerides by lipases: process technology and

its application to the production of nutritionally im-proved fats. Inform, 4, 579–583.

Rangheard, M.S., Langrand, G., Triantaphytides, C., andBaratti, J. (1989). Multi-competitive enzymatic re-actions in organic media: a simple test for the deter-mination of lipase fatty acid specificity. Biochim.Biophys. Acta., 1004:20–28.

Rogalska, E., Cudray, C., Ferrato, F.S., and Verger, R.(1993). Stereoselective hydrolysis of triglyceridesby animal and microbial lipases. Chirality, 5:24–30.

Saxena, R.K., Ghosh, P.K., Gupta, R., Davidson, W.S.,Bradoo, S., and Gulati, R. (1999). Microbial li-pases: potential biocatalysts for the future industry.Curr. Sci., 77:101–115.

Schuch, R. and Mukherjee, K.D. (1989). Lipase catalyzedreactions of fatty acids with glycerol and acyl-glycerols. Appl. Microbiol. Biotechnol., 30:332–336.

Shieh, C.J., Akoh, C.C., and Koehler, P.E. (1995). Fourfactor response surface optimization of the enzy-matic modification of triolein to structured lipids.J. Am. Oil Chem. Soc. 72, 619–623.

Shimada, Y., Maruyama, K., Okazaki, S., Nakamura, M.,Sugihara, A., and Tominaga, Y. (1994). Enrichmentof polyunsaturated fatty acids with Geotrichumcandidum lipase. J. Am. Oil Chem. Soc., 71:951–954.

Shimada, Y., Sugihara, A., Maruyama, K., Nagao,T., and Nakayama, S. (1995). Enrichment ofarachidonic acid: selectivity of hydrolysis of asingle cell oil from Mortierela with Candida cylin-dracea lipase. J. Am. Oil Chem. Soc., 72:1323–1327.

Shimada, Y., Sugihara, A., and Tominaga, Y. (2000). Pro-duction of functional lipids containing polyunstau-rated fatty acids with lipase. In Enzymes in LipidModification. Ed. Bornscheur, U.T., Wiley-VCHVerlag Gmbh, D-69469 Weinheim (FRG), 128–147.

Sridhar, R., Lakshminarayana, G., and Kaimal, T.N.B.(1991). Modification of selected Indian veg-etable fats into cocoa butter substitutes by lipase-catalyzed ester interchange. J. Am. Oil Chem. Soc.,68:726–730.

Stipp, G.K. and Kleusener, B.W. (1992). Selective esterifi-cation of long chain fatty acid monoglycerides withmedium chain fatty acid anhydrides, U.S. Patent5,142,072.

Sugihara, A., Nakano, H., Kuramoto, T., Nagao, T.,Gemba, M., and Tominaga, Y. (1997). Purificationof docosahexaenoic acid by selective esterification

643

Dow

nloa

ded

by [

Nor

thea

ster

n U

nive

rsity

] at

11:

31 0

9 O

ctob

er 2

014

of fatty acids from tuna oil with Rhizopus delemarlipase. J. Am. Oil Chem. Soc., 74:97–101.

Trani, M., Lortie, R., and Ergan, F. (1993). Enzymatic syn-thesis of trierucin from high-erucic rapeseed oil.J. Am. Oil Chem. Soc., 70:961–964.

Vega, G.L., Groszek, E., Wolf, R., and Grundy, S.M.(1982). Influence of polyunsaturated fats on com-position of plasma lipoproteins and apolipopro-teins. J. Lipid Res., 23:811–822.

Wong, W.C., Basri, M., Razak, C.A.N., and Salleh, A.B.(2000). Synthesis of medium-chain glycerides us-ing lipase from Candida rugosa. J. Am. Oil Chem.Soc., 77:85–88.

Xu, X., Balchen, S., Jonsson, G., and Adler-Niessen,J. (2000). Production of structured lipids bylipase-catalyzed interesterification in a flat mem-brane reactor. J. Am. Oil Chem. Soc., 77:1035–1041.

Yadav, R.P., Saxena, R.K., Gupta, R., and Davidson, W.S.(1998). Lipase production by Aspergillus and Peni-cillium spp. Folia Microbiol., 43:373–378.

Yamane, T. (2000). Lipase-catalyzed synthesis of struc-tured triacylglycerols containing polyunsaturatedfatty acids: Monitoring the reaction and increas-ing the yield. In Enzymes in Lipid Modification.

Ed. Bornscheur, U.T., Wiley-VCH Verlag Gmbh,D-69469 Weinheim (FRG), 148–169.

Yang, T.H., Jang, Y., Han, J.J., and Rhee, J.S. (2001). En-zymatic synthesis of low-calorie structured lipidsin a solvent-free system. J. Am. Oil Chem. Soc.,78:291–296.

Yankah, V.V. and Akoh, C.C. (2000). Lipase-catalyzedacidolysis of tristearin with oleic or caprylic acidsto produce structured lipids. J. Am. Oil Chem. Soc.,77:495–500.

Yodice, R. (1990). Nutritional and stability characteristicsof high oleic sunflower seed oil. Fat Sci. Technol.,92:121–126.

Yu, L. and Hammond, E.G. (2000). Production and char-acterization of a Swiss cheese-like product frommodified vegetable oils. J. Am. Oil Chem. Soc.,77:917–924.

Zaks, A. and Gross, A.T. (1990). Production of mono-glycerides by enzymatic transesterification. WorldPatent WO 90 /040 333.

Zhang, H., Xu, X., Nilsson, J., Mu, H., Nissen, J.A., andHoy, C.-E. (2001). Production of margarine fats byenzymatic interesterification with silica-granulatedThermomyces lanuginosa lipase in a large-scalestudy. J. Am. Oil Chem. Soc., 78:57–64.

644

Dow

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ded

by [

Nor

thea

ster

n U

nive

rsity

] at

11:

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er 2

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