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JFS: Food Chemistry and Toxicology Synthesis of Low Molecular Weight Flavor Esters Using Plant Seedling Lipases in Organic Media M. LIAQUAT AND R.K.OWUSU APENTEN Introduction L OW MOLECULAR WEIGHT ESTERS (LMWE) ARE COMMON FLA - voring agents for fruit-based products and dairy products (Schultz and others 1967). Flavor losses during food manufacturing processes must be compensated for by additions. Production of LMWE is of commercial interest. There are general demands for new flavors such as green notes represented by C-6 alcohol derivatives (Somogyi 1996). LMWE can be synthesized by organic phase biocatalysis (OPB) to satisfy increasing commercial demands. Esters produced by OPB are thought to comply with the U.S. Food and Drug Administration’s definition of natural. This mode of production makes the food industry less dependent on seasonal, climatic, and geographic variations. Other well-known advantages of OPB include improved enzyme stability, increased reactant solubility in nonaqueous solvents, and the possibility of reverse hydrolysis reactions. Furthermore, side reactions may be diminished and product as well as biocatalyst recovery is easier. Finally, the risk of microbial contamination is reduced. OPB has been extensively reviewed (Dordick 1989; Zaks and Klibanov 1988; Zaks and Russell 1988; Klibanov 1989; Koskinen and Klibanov 1996). Microbial lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) from Mucor miehei, Pseudomomas fluorescens, Rihizopus arrahisuis, R. niveus, or Candida cylindracea have been applied for LMWE synthesis. Both aliphatic and aromatic esters were synthesized in nonaqueous, solvent-free, or biphasic OPB systems (Gandhi and others 1995; Linko and others 1994). Commercially important LMWE were produced in anhydrous organic solvents by transesterification (Akoh and Claon 1994; Yee and others 1995; Yee and Akoh 1996; Rizzi and others 1992). LMWE have also been produced by esterification of acids and alcohols (Claon and Akoh 1993; Manjon and others 1991; Bourg-Garros and others 1997, 1998a, b; Razafindralambo and others 1994; Leszczak and TranMinh 1998; Perraud and Laboret 1995; Tan and others 1996). Immobilized microbial lipases have been used for OPB. These are stable and are easier to recover from the reaction vessel (Langrand and others 1988; Welsh and others 1990; Bourg-Garros and others 1998). The use of enzymes to produce flavor esters in solvent-free systems has also been described (Oguntimein and others 1995; Karra-Chaabouni and others 1998; Kim and others 1998; Leblanc and others 1998). © 2000 Institute of Food Technologists There appear to be no reports describing the use of plant-derived lipases or acetone powders for LMWE synthesis. Seed lipase or acetone powders from castor bean, rape, and Nigella sativa seeds were used for lipid hydrolysis, glycerolysis, and esterification of glycerols or oleic acids (Hassanien and Mukherjee 1986; Dandik and others 1996; Mert and others 1995; Dandik and Aksoy 1996; Tüter 1998; El and others 1998). Lipase from common oilseed rape (Brassica napus) was isolated, partially purified and used as biocatalyst after immobilization (Hills and others 1990, 1991; Hills and Mukherjee, 1990; Ncube and others 1993). Rapeseed lipase also catalyzed hydrolysis of various seed oils and marine oils containing unusual fatty acids (Jachmanián and Mukherjee 1995; Jachmanián and others 1995). Hassanien and Mukherjee (1986) showed that acetone powder from seedlings of N. sativa had the same lipase specific activity as an undialyzed crude homogenate. Preparation of acetone powder led to high recoveries of lipase activity. Procedures for preparing acetone powder are simple, making it quite suitable for technical use (El and others 1998). The aim of this work was to investigate LMWE synthesis using plant seedling lipases. Seedling powders are a potentially inexpensive form of biocatalyst for OPB. The seedlings used were from wheat (Triticum aestivum cv IPM), barley (Hordeum vulgare cv Decanter), oilseed rape (Brassica napus cv Liga), maize (Zea maize cv River), and linola (Linum usitatissmum cv Windermere). LMWE were formed by direct esterification of acetic, butyric, hexanoic acids with ethanol, butanol, iso-pentanol or (Z)-3- hexen-lol in hexane. Results and Discussion L IPASE ACETONE POWDERS MADE FROM 4- D GERMINATING seedlings of barley, wheat, maize, linola, and rapeseed catalyzed the synthesis of low molecular weight flavor esters (LMWE). The reactions were performed with n-hexane as solvent. The reaction products were analyzed using gas chromatography (GC) and GC-mass spectrometry (GC-MS) analysis. The former technique was highly reproducible. Multiple injections from the same reaction vessel produced an average coefficient of variable of 2 to 5%. The overall precision of the synthesis and analysis experiments was about 10%. Hexane was found to be a suitable solvent for ester synthesis in agreement with previous reports (Carta 1991; Gillies 1987). The moisture content of the enzyme powders was deterVol. 65, No. 2, 2000—JOURNAL OF FOOD SCIENCE 295 Food Microbiology and Safety ABSTRACT: Powders from germinated seedlings of wheat, barley, rapeseed, maize, and linola synthesized low molecular weight flavor esters in an organic medium (hexane). Direct esterification of acetic, butyric, and caproic acids, with ethanol, butanol, isopentanol, or (Z)-3- hexen-l-ol was achieved. Of the systems examined, germinated rapeseed showed the highest degree of flavor synthesis. (Z)-3-hexen-1-yl butyrate and (Z)-3-hexen-1-yl caproate were produced with yields of about 96%. Butyl butyrate, isopentyl butyrate, butyl caproate and isopentyl caproate were produced at 80% yield. Linola seedling powder gave yields of ⱕ63% for ethyl acetate and butyl acetate. More moderate (40%) yields were obtained with barley and maize seedling powders. Rapeseed seedling powder is a convenient and inexpensive catalyst for preparing low molecular weight esters in organic media. Key Words: plant lipases, seedling, flavor, synthesis, organic phase biocatalysis Seedling Lipase Flavor Synthesis . . . mined by drying to constant weight overnight at 105 ⬚C. The seedling powders used contained about 8% moisture on a dry weight basis. During organic phase catalysis, enzymes differ in their requirement for water and also in their sensitivity to different solvents. It has been demonstrated that it is the water bound to the enzyme, which determines the catalytic activity rather than the total water content (Zaks and Klibanov 1988). It is generally accepted that with polar organic solvents, more water is held in solution instead of bound to the enzyme. Good solvents for lipase-mediated esterification are those which do not strip water from the enzyme, such as hexane used in this work. These are characterized by high log P (ⱖ 4) values (Dordick 1989). Choice of seedling acetone powder Figure 1 and Table 1 summarize results from esterification studies involving 5 lipase preparations. Reactions involving a total of 4 alcohols and 3 acids were investigated. This combination of fatty acids and alcohols led to the synthesis of 12 unique esters. Rapeseed lipase consistently gave the highest yield under the conditions of this study. (Z)-3-hexen-1-yl butyrate and (Z)-3hexen-1-yl caproate were produced with yields about 96%. The yield for other esters are as follows; butyl butyrate (> 80%), isopentyl butyrate (⬍ 70%), butyl caproate (⬍ 70%) and isopentyl caproate (⬎ 80%). The enzyme powders used in this work could be re-used for at least 3 successive syntheses without lowering the product yield. A yield of 63.8% for ethyl acetate and 49% for butyl acetate, obtained using linola seedling lipase, are also notable (Table 1). For wheat lipase a 61.1% conversion yield was observed for butyl Table 1—Yield (%)* for low molecular weight esters synthesized using plant seedling powders after 72 h reaction Alcohol Cn** Ethanol 2 4 6 Butanol 2 4 6 Isopentanol 2 4 6 (Z)-3-hexenol 2 4 6 Wheat Barley Plant sources*** Maize Linola 35 28.3 17.7 27 24.2 21 38.7 23.3 12.9 63.8 23.3 19.7 61.1 13.3 20.6 24.4 30 21.5 36.9 26 19.3 49.1 25 13.3 34.7 28 21.1 29.7 23.5 22.3 38.8 24.6 29.9 43.3 23.7 11 30.5 27.4 33.8 31.2 25.5 21.8 24.9 28.8 17.2 28.6 28.9 20.2 * Yield (%) = 100 ([Acid]O – [Acid] F) / [Acid]o where subscripts O and F denote initial and final concentrations respectively. **Cn = number of C-atoms in acid; C2 = acetic acid;C 4 = butyric acid; C6 = caproic acid ***Results for Rapeseed lipase are given in Fig 1. Food Microbiology and Safety acetate. With maize seed lipase, the conversion yield was ⱕ 39% to 40% for ethyl, butyl, isopentyl acetate. Caproic acid conversion remained up to 20%. For wheat and barley acetone powders, the conversion yields for acetic, butyric, and caproic acids for most of the esters were less than 35% (Table 1). The lipase activity in oilseeds and certain cereal grains increases with germination (Huang and Moreau 1978; Huang 1990; Mukherjee 1996). At the initial phase of germination seeds contain large amount of lipids and a small amount of water. Catalysis occurs under these conditions in a predominantly organic media with only small amount of water present. Plant lipases have properties that may make them especially suitable for ester synthesis (Ncube and others 1993). The different synthesis yields can be ascribed to at least 3 factors. First, the same weight of lipase preparations was used in each experiment. As these were crude preparations, the specific activity of lipase in the different powders would be different. Secondly, preliminary studies using p-nitrophenol esters showed that the different enzymes had different specificity towards hydrolysis of esters. Despite the limitations of p-nitrophenol esters artificial substrate, such results suggest that the different lipases could have different specificity towards more realistic substrates. Lipase specificity is expected to affect the conversion yields. Rangheard and others (1989) reported that M. miehei lipase was very active on long chain fatty acids. Aspergillus lipase was more active on short-chain fatty acids. Third, the reaction conditions used were not optimized for the different enzymes or esters. The conversion yields varied markedly with different lipase preparations, acids or alcohols used. The observed conversion yields from rapeseed were enough for preparative purposes. Effect of fatty acid chain length Fig. 1—Esters synthesis using rapeseed seedling acetone powder. Reaction media contain 0.25 M each acid and alcohol in 5 mL hexane with 0.25 g (5%w/v) of enzyme. Samples were shaken at 100 rpm at 30 ⬚ C for 72 h. 296 JOURNAL OF FOOD SCIENCE—Vol. 65, No. 2, 2000 The number of carbon atoms in the short-chain fatty acids strongly influenced the conversion yield. With rapeseed seedling powder, yields of 69% to 97% were obtained for butyric (C4) and caproic (C6) acid esters with butyl, iso-pentyl, and (Z)-3-hexen1-yl alcohols (Fig. 1). Significant yields were also observed using maize (ⱕ40%) and linola (50% to 63%) catalyst to synthesize acetic acid esters with ethyl, butyl, and isopentyl alcohol. Barley seed powder exhibited uniform (ⱕ 31%) yields for all of the acids and alcohol tested (Table 1). Langrand and others (1988) reported that R. arrihizus and M. meihei lipases were more active with C6 and C4-C6 acids respectively. Aspergillus and C. Rugosa lipases showed the highest level of ester synthesis with C3 and C4 fatty acids. Using M. meihei lipase little or no ester synthesis was observed with C2 and C3 acids (Gatfield 1986; Posorske 1984). Bourg-Garros and others (1997) esterified (Z)-3-hexen-1-ol with butyric, isovaleric, and caproic acid on a laboratory scale (35g) using immobilized lipase from Mucor miehei (Lipozyme 1M) or Candida antarctica (Novozym 435). Both proved suitable since, in the absence of water trapping, (Z)-3-hexen-1-yl butyrate, isovalerate, or caproate were produced with yields about 95%. However, the yield for (Z)-3-hexen-1-yl acetate production using Lipozyme 1M was less than 2% . Novozym 435 afforded (Z)-3-hexen-1-yl acetate with yields greater than 90% (Bourg-Garros and others 1998a, 1998b). Such results confirm the peculiar behavior of acetic acid and the difficulties in preparing acetates by direct esterification (Bauer and others 1990; Iwai 1980; Langrand and others 1988; Claon and Akoh 1993). The differences in conversion yields may result from a conjunction of several factors: (1) Partial inactivation of lipase resulting from a decline in pH of the enzyme aqueous microenvironment. This is a consequence of the high solubility of acetic acid in water (Langrand and others 1988, Welsh and Williams 1990, Razafindralambo and others 1994). (2) Modification of the polarity of the medium (Dordick 1989). The hydrophilicity of the organic phase increases on increasing acetic acid and alcohol concentration : log P changes from 3.5 (pure hexane) to ⫺0.23 ( acetic acid). Therefore, the partition coefficient becomes less and less favorable for the ester (Manjon and others 1991). (3) The rather large amount of water released during esterification favors the back reaction (hydrolysis) (Langrand and others 1988, 1990). caproate, provided that the enzyme is stable under the conditions used (results not shown). These results are reproducible — all experiments were repeated at least twice. GC-MS results of a standard and synthesized butyl butyrate Effect of alcohol type and chain length. Food Microbiology and Safety Ester synthesis was examined for a range of alcohols. Four primary alcohols were tested: 2 had linear chains, 1 had a linear chain with one double bound (Z)-3-hexen-1-ol, and 1 had a branched chain (isopentanol or isoamyl alcohol). For lipases from wheat, barley, and maize seedlings, the influence of the nature of alcohol on conversion yields was similar for each acid tested. However, lipase preparation from the rapeseed showed a significant improvement in yields when number of carbon atoms in the alcohol was increased (Fig. 1). With lipase from linola, the yield of ester decreased as the number of carbons in the alcohol increased (Table 1). These results are similar to those of Langrand and others (1990) who also observed decreasing yields with increasing alcohol chain length. They reported achieving the highest yield with short chain fatty acids and alcohols using microbial lipases. Time course of reaction Of the 5 lipase preparation tested, 3 (wheat, barley, and maize) showed less than 30% acid conversion yield after 48 h, irrespective of the type of ester synthesized. The only exception to this was ethyl, butyl, and isopentyl acetates in case of linola seeds lipase. Very high yields were observed with rapeseed acetone powder. The time course of lipase-catalyzed synthesis of ethyl butyrate (Fig. 2) shows that most enzymes formed approximately 20% ester after 2 h. For all lipases, there were no large differences in yields obtained for butyl butyrate (6% to 20% after 1 h; Fig. 3). For an unknown reason, synthesis then declined for most enzyme preparations with the exception of rapeseed. After 48 h a yield of up to 40% ethyl and butyl butyrate was observed with rapeseed lipase and conversions reached 80% after 72 h. It may be that rapeseed lipase is more stable than lipases from other seedlings allowing ester synthesis to proceed for longer. Regardless of the rate of reaction, significant yields could be obtained by prolonged incubation (212 h) even for (Z)-3-hexen-1-yl Figs. 2 and 3—Time course showing yields (% molar conversion ) for ethyl butyrate (2) and butyl butyrate (3) produced using various plant seedling acetone powders. Other reactions conditions are same as in Fig. 1. Vol. 65, No. 2, 2000—JOURNAL OF FOOD SCIENCE 297 Seedling Lipase Flavor Synthesis . . . are shown in the Fig. 4. There was a close resemblance between fragmentation patterns. Using such GC-MS results, it was possible to confirm the identity of all esters produced. GC-MS is probably the most powerful technique available for the separation and identification of volatile compounds in complex mixtures. The combination of GC and MS leads to a fingerprint of the compound detected and therefore highly accurate identification. Conclusion P OWDERS FROM GERMINATED SEEDLINGS OF WHEAT, BARLEY, rapeseed, maize, and linola were able to synthesize low molecular weight flavor esters. Synthesis of acetic, butyric and hexanoic acid esters with ethanol, butanol, iso-pentanol, and (Z)-3hexen-l-ol alcohol was achieved. Germinated rapeseed showed the best flavor synthesis. (Z)-3-hexen-1-yl butyrate and (Z)-3hexen-1-yl caproate were produced with yields about 96%. Linola seedling powder gave yields of ⱕ63% for ethyl acetate and butyl acetate whilst moderate yields were obtained with barley and maize seedling powders (40%). Further work is needed to optimize flavor synthesis using seedling powders. Fig. 4—GC-MS spectra for butyl butyrate. (A) standard and (B) Synthesized in this study. Materials and Methods R were kept in sealed bottles at ⫺20 ⬚C until used. EAGENT GRADE CHEMICALS , ACETIC ACID , BUTYRIC ACIDS , caproic acid , ethanol, butanol, isopentanol, (Z)-3-hexen1-ol and esters were obtained from Sigma-Aldrich Co. Ltd. (Poole, England). Hexane was dried over molecular sieves (3A, 8-12 mesh; both from Sigma-Aldrich Co. Ltd.) for at least 24 h prior to use. Seed material and germination Food Microbiology and Safety Seeds were supplied by Nickerson Seeds Ltd. (Lincoln, U.K.). Dry whole seeds were surface sterilized by soaking in 0.1% sodium hypochlorite solution for 30 s. The seeds were then washed thoroughly with running tap water and soaked for 24 h at 26 ⬚C (designated as day 1st) in a dark incubator. Germination was carried out in an incubator. Seeds were placed on moist filter paper towels placed on top of moist perlite (Silvaperl graded horticultural) in shallows plastics trays covered with perforated aluminium foil. The average germination temperature was 26 ⬚C. Samples of seedlings were withdrawn on day 4 for acetone powder preparation. Our studies showed that lipase activity reached a maximum at 4 to 6 d after germination. These results agree with the reports of Huang and Moreau (1978). Acetone powder preparation from seedlings The procedures used were similar to those described by Hassanien and Mukherjee (1986). A batch of germinated seedling was washed three times with distilled water, kept in a fridge at 4 ⬚C for 10 min and then cut into small pieces with scissors. Seedlings were homogenized with 5 volumes of cold acetone (⫺18 ⬚C or less) for 1 min. The acetone extract was separated from the residue by carefully decanting the whole suspension into a Buchner funnel, containing Whatman No. I filter paper, quipped with vacuum filtration. The resulting powder was re-extracted 4 times with 5 volumes of cold acetone and air dried under a hood for 10 h. The light grayish powders 298 JOURNAL OF FOOD SCIENCE—Vol. 65, No. 2, 2000 Direct esterification conditions. Esterification was performed essentially as described by Langrand and others (1990). However, additional amounts of water were not added to the reaction mixture. Into 20-mL screw-capped vials containing 1.25-mmoles alcohol, 1.25mmoles of acid was added 250-mg acetone powder (5% w/v of total reaction volume) as enzyme source. The final reaction mixture was adjusted to 5-mL with hexane previously dried over molecular sieves (3⬚A, 4-8 mesh). The final concentration of reactants were each 0.25 M. Synthesis was performed by shaking reaction vessels at about 100 rpm at a constant temperature of 37 ⬚C. The concentrations of ester formed were determined by withdrawing samples (1-mL) after 1, 2, 5, 24, 48, 72, or 212 h. These were then centrifuged (1300 g for 5 min at room temperature) to remove the residual lipase. Aliquots of 0.5 mL were taken from the supernatant and stored at ⫺10 ⬚C until analyzed (usually within 24 h). The frozen samples were allowed to warm to room temperature and then analyzed by gas chromatography to determine the concentration of ester, alcohol, and acids. Esters synthesis is expressed as percentage molar conversion of acids. All synthesis experiments were performed in duplicate using separate reaction vials. Gas chromatographic analysis The gas chromatography system consisted of Carlo Erba apparatus (Model 5160) equipped with a flame ionization detector. Separation involved a BP-20 fused silica capillary column (SGE, UK, 25 m ⫻ 0.32 mm ID; film thickness 1 micron) operated with helium gas as carrier (2 mL/min, split ratio 1:15). The oven temperature was maintained at 50 ⬚C for 2 min and then increased to 210 ⬚C at a rate of 15 ⬚C/min and held for 4 min. The injector temperature was fixed at 250 ⬚C and detector Esters identification and quantification Esters, alcohol, and acids were identified according to their retention times on chromatograms and from comparisons with results obtained with standards. A calibration graph of known acids concentration versus corresponding peak area was constructed. Various concentrations of acid (0.0125 to 0.25 M) were prepared by diluting in n- hexane and 0.2 L of each was injected in to GC. Injections were repeated twice for each vial. References Akoh C C, Claon PA. 1994. Lipase catalyzed synthesis of terpene esters by transesterification in n-hexane. Biotechnol Letts. 16: 235-240. Bauer K, Garbe D, Surburg H. 1990. Common Fragrance and Flavor Materials. New York: VCH Publishers. Bourg-Garros S, Razafindramboa N, Pavia AA.1997. Synthesis of (Z)-3-hexen-1-yl butyrate by esterification in hexane and in solvent-free medium using lipases from Mucor miehei and Candida antarctica. J. Am. Oil Chem. Soc. 74: 1471-1475. Bourg-Garros S, Razafindramboa N, Pavia AA. 1998a. Optimization of lipase-catalyzed synthesis of (Z)-3-hexen-1-yl acetate by direct esterification in hexane and in a solvents free medium. Enzyme Microbiol Technol. 22: 240-245. Bourg-Garros S, Razafindramboa N, Pavia AA. 1998b. Large scale preparation of (Z)-3hexen-1-yl acetate using Candida antactica-immobilized lipase in hexane. Biotechnol Bioeng. 59: 496-500. Carta G, Gainer JL, Benton AH. 1991. Enzymatic synthesis of esters using an immobilized lipase. Biotechnol Bioeng. 37: 1004-1009. Claon PA, Akoh CC. 1993. Enzymatic synthesis of geraniol and citronellol esters by direct esterification in n-hexane. Biotechnol. Letts. 15:1211-1216. Dandik L, Aksoy HL. 1996. Applications of Nigella sativa seed lipase in oleochemical reactions. Enz. Microbiol. Technol. 19: 277-281 Dordick JS. 1989. Enzymatic catalysis in monophasic organic solvents. Enz. Microbiol. Technol. 11: 194-211. El N, Dandik L, Aksoy A. 1998. Solvent-Free glycerolysis catalyzed by acetone power of Nigella sativa seed lipase. J. Am. Oil. Chem. Soc. 75: 1207-1211. Gandhi NN, Sawant SB, Joshi JB. 1995. Study on the lipozyme-catalyzed synthesis of butyl laurate. Biotechnol. Bioeng. 46: 1-12. Gatfield IL. 1986. In biogeneration of aromas. Parliment T. 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Klibanov AM. 1989. Enzymatic catalysis in anhydrous solvents. Trends Biochem. Sci. 14: 141-144. Koskinen AMP, Klibanov AM, editors. 1996. Enzymatic Reaction in Organic Media. London: Blackie Academic and Scientific. 314 p. Langrand G, Rondot N, Triantaphylides C, Barratti J. 1988. Lipase catalyzed formation of flavor esters. Biotechnol. Letts. 10: 549-554. Gas chromatography-mass spectrometry (GC-MS) analysis. GC-MS (Carlo Erba GC Model 4200, Kratos MS 80 RFA) was used for identification of esters. The GC-MS was equipped with a 38 m ⫻ 0.32 ⫻ 0.5 m film thickness BP-20 column (SGE, UK); Elution was performed as described above. Injections (0.2 l) were made on column. Mass spectra were recorded with an ion source energy of 70 eV. Fragmentation patterns were compared with a library of results for standard esters. Identification of esters by GCMS enabled compositional analysis of the product mixtures. Leszczak JP, Trasn-Minh C. 1998. Optimized enzymatic synthesis of methyl benzoate in organic medium. Operating conditions and impact of different factors on kinetics. Biotechnol. Bioeng. 60: 356-361. Leblanc D, Morin A, Gu D, Zhang XM, Bisaillon J-G, Paquet M, Dubeau H. 1998. Short chain fatty acids esters synthesis by commercial lipases in low-water systems and by resting microbial cells in aqueous medium. Biotechnol Letts. 20: 1127-1131. Linko YY, Lamsa M, Huhtala A, Linko P. 1994. Lipase catalyzed transesterification of rapeseed oil and 2-ethyl-1-hexanol. J. Am. Oil. Chem. Soc. 71: 1411-1414. Manjon A, Iborra JL, , Arocas AL. 1991. Short chain flavor esters synthesis by immobilized lipase in organic media. Biotechnol. Letts., 13: 339-344. Mert S, Dandik L, Aksoy HL. 1995. Production of glycerides from glycerol and fatty acids by native lipase of Nigella sativa seed. Appl. Biochem. Biotechnol. 50: 333342. Mukherjee KD. 1996. Plant lipases in lipid biotransformations. In: Malcata FX, editor. Engineering of/with Lipases. Vol. 317. Nato ASI Series, Series E: Applied Science. The Netherlands: Kluwer AcademicPublishers. p 494. Ncube I, Adlercreutz P, Reed J, Mattiasson B. 1993. Purification of rape (Brassica nupus) seedling lipase and its use in organic media. Biotechnol Appl. Biochem. 17: 327-336. Oguntimein GB, Anderson WA, Moo-Young. 1995. Synthesis of geranoil esters in a solvent free systems catalyzed by Candida antartica lipase. Biotechnol. Letts: 17, 77-82. Perraud R, Laboret F.1995. Optimization of methyl propionate production catalyzed by Mucor meihi lipase. Appl. Microbiol. Biotechnol. 44: 321-326 Posorske LH. 1984. Industrial scale application of enzymes to the fats and oil industry. J. Am. Oil. Chem. Soc. 61: 1758-1760. Rangheard M-S, Langrand G, Triantaphylindes C, Baratti J. 1989. Muliticompetitive enzymatic reactions in organic media: A simple test for the determination of lipase fatty acid specificity. Biochim. Biophys. Acta 1004: 20-28. Razafindralambo H, Blecker C, Lognay G, Marlier M, Watherlet JP, Severin M. 1994. Improvement of enzymatic synthesis yields of flavor acetates. The example of isoamyl acetate. Biotechnol. Letts. 16: 247-250. Rizzi M, Stylose P, Reuss M. 1992. A kinetic study of immobilized lipase catalysing the synthesis of isoamyl acetate by transesterifications in n-hexane. Enz. Microbiol. Technol. 14:709-714. Schultz HW, Day EA, Libbey LW. 1967. Chemistry and Physiology of Flavors. Westport, Conn.: AVI Publishing Company. 566 p. Somogyi L. 1996. The flavor and fragrances industry: serving a global market, Chem. Ind. 13, 170-173. Tan S, Apenten RKO, Knapp J. 1996. Low temperature organic phase biocatalysis using cold- adapted lipase from psychrotrophic Pseudomonas P 38. Food Chem. 57: 415418. Tüter M. 1998. Castor bean lipase as biocatalyst in esterification of fatty acids to glycerol. J. Am. Oil. Chem. Soc. 75: 417-420. Welsh F, Williams RE, Dawson KH. 1990. Lipase mediated synthesis of low molecular weight flavor esters. J. Food Sci. 55: 1679-1682. Yee LN, Akoh CC, Phillips RS. 1995. Terpene esters synthesis by lipase catalyzed transesterification . Biotechnol. Letts. 17: 67-70. Yee LN, Akoh CC. 1996. Enzymatic synthesis of geranyl acetate by transesterification with acetic anhydride as acyl donor. J. Am. Oil. Chem. Soc. 73: 1379-1384. Zaks A., Klibanov AM. 1988. The effect of water on enzyme action in organic media . J. Biol. Chem. 263: 8017-8021. Zaks A, Russell AJ. 1988. Enzymes in organic solvents: properties and applications. J. Biotechnol. 8: 259-270. MS 19990705 received 7/6/99; revised 11/9/99; accepted 12/28/99. The authors would like to thank the Pakistan Government for financial sponsorship to ML. We are also grateful to Mr. Ian Boyes for his technical assistance. Authors Liaquat and Apenten are from Laboratory of Food Biochemistry and Nutrition, Department of Food Science, University of Leeds, Leeds LS2 9JT (UK). Direct inquiries to author Apenten (E-mail [email protected]). Vol. 65, No. 2, 2000—JOURNAL OF FOOD SCIENCE 299 Food Microbiology and Safety temperature at 240 ⬚C. The GC was connected to an integrator (Hewlett Packard 3395 integrator) which recorded the peak areas and retention times in a chromatogram.