Synthesis of Low Molecular Weight Flavor Esters Using Plant Seedling Lipases in Organic Media M

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

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 Յ63% 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

Introduction There appear to be no reports describing the use of plant-de- OW MOLECULAR WEIGHT ESTERS (LMWE) ARE COMMON FLA- rived lipases or acetone powders for LMWE synthesis. Seed li- Lvoring agents for fruit-based products and dairy products pase or acetone powders from castor bean, rape, and Nigella sati- (Schultz and others 1967). Flavor losses during food manufactur- va seeds were used for lipid hydrolysis, glycerolysis, and esterifi- ing processes must be compensated for by additions. Production cation of glycerols or oleic acids (Hassanien and Mukherjee 1986; of LMWE is of commercial interest. There are general demands Dandik and others 1996; Mert and others 1995; Dandik and Ak- for new flavors such as green notes represented by C-6 alcohol soy 1996; Tüter 1998; El and others 1998). Lipase from common derivatives (Somogyi 1996). oilseed rape (Brassica napus) was isolated, partially purified and LMWE can be synthesized by organic phase biocatalysis used as biocatalyst after immobilization (Hills and others 1990, (OPB) to satisfy increasing commercial demands. Esters pro- 1991; Hills and Mukherjee, 1990; Ncube and others 1993). Rape- duced by OPB are thought to comply with the U.S. Food and seed lipase also catalyzed hydrolysis of various seed oils and ma- Drug Administration’s definition of natural. This mode of pro- rine oils containing unusual fatty acids (Jachmanián and Mukher- duction makes the food industry less dependent on seasonal, cli- jee 1995; Jachmanián and others 1995). Hassanien and Mukherjee matic, and geographic variations. Other well-known advantages (1986) showed that acetone powder from seedlings of N. sativa of OPB include improved enzyme stability, increased reactant had the same lipase specific activity as an undialyzed crude ho- solubility in nonaqueous solvents, and the possibility of reverse mogenate. Preparation of acetone powder led to high recoveries of hydrolysis reactions. Furthermore, side reactions may be dimin- lipase activity. Procedures for preparing acetone powder are sim- ished and product as well as biocatalyst recovery is easier. Final- ple, making it quite suitable for technical use (El and others 1998). ly, the risk of microbial contamination is reduced. OPB has been The aim of this work was to investigate LMWE synthesis using extensively reviewed (Dordick 1989; Zaks and Klibanov 1988; plant seedling lipases. Seedling powders are a potentially inex- Zaks and Russell 1988; Klibanov 1989; Koskinen and Klibanov pensive form of biocatalyst for OPB. The seedlings used were 1996). from wheat (Triticum aestivum cv IPM), barley (Hordeum vulgare Microbial lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) cv Decanter), oilseed rape (Brassica napus cv Liga), maize (Zea from Mucor miehei, Pseudomomas fluorescens, Rihizopus arrahisu- maize cv River), and linola (Linum usitatissmum cv Windermere). is, R. niveus, or Candida cylindracea have been applied for LMWE LMWE were formed by direct esterification of acetic, butyric, hex- synthesis. Both aliphatic and aromatic esters were synthesized in anoic acids with ethanol, butanol, iso-pentanol or (Z)-3- hexen-l- nonaqueous, solvent-free, or biphasic OPB systems (Gandhi and ol in hexane. others 1995; Linko and others 1994). Commercially important

LMWE were produced in anhydrous organic solvents by transes- Results and Discussion Food Microbiology and Safety terification (Akoh and Claon 1994; Yee and others 1995; Yee and IPASE ACETONE POWDERS MADE FROM 4-D GERMINATING Akoh 1996; Rizzi and others 1992). LMWE have also been pro- Lseedlings of barley, wheat, maize, linola, and rapeseed cata- duced by esterification of acids and alcohols (Claon and Akoh lyzed the synthesis of low molecular weight flavor esters (LMWE). 1993; Manjon and others 1991; Bourg-Garros and others 1997, The reactions were performed with n-hexane as solvent. The re- 1998a, b; Razafindralambo and others 1994; Leszczak and Tran- action products were analyzed using gas chromatography (GC) Minh 1998; Perraud and Laboret 1995; Tan and others 1996). Im- and GC-mass spectrometry (GC-MS) analysis. The former tech- mobilized microbial lipases have been used for OPB. These are nique was highly reproducible. Multiple injections from the stable and are easier to recover from the reaction vessel (Lan- same reaction vessel produced an average coefficient of variable grand and others 1988; Welsh and others 1990; Bourg-Garros and of 2 to 5%. The overall precision of the synthesis and analysis ex- others 1998). The use of enzymes to produce flavor esters in sol- periments was about 10%. Hexane was found to be a suitable solvent-free systems has also been described (Oguntimein and oth- vent for ester synthesis in agreement with previous reports (Car- ers 1995; Karra-Chaabouni and others 1998; Kim and others ta 1991; Gillies 1987). 1998; Leblanc and others 1998). The moisture content of the enzyme powders was deter-

© 2000 Institute of Food Technologists Vol. 65, No. 2, 2000—JOURNAL OF FOOD SCIENCE 295 li- 39% lipase Յ lipases M. miehei Aspergillus C. Rugosa C. Maize Linola and were more active with C6 Plant sources*** 31%) yields for all of the acids Յ Aspergillus where subscripts O and F denote initial and final o Barley ) / [Acid] M. meihei lipases F Wheat and – [Acid] O 40%) and linola (50% to 63%) catalyst to synthesize ace- Յ -C6 acids respectively. -C6 acids respectively. R. arrihizus The lipase activity in oilseeds and certain cereal grains in- The lipase activity in oilseeds and certain to at least 3 fac- The different synthesis yields can be ascribed The number of carbon atoms in the short-chain fatty acids to 40% for ethyl, butyl, isopentyl acetate. Caproic acid conver- to 40% for ethyl, butyl, isopentyl acetate. barley acetone powders, sion remained up to 20%. For wheat and and caproic acids for the conversion yields for acetic, butyric, 1). 35% (Table less than most of the esters were 1978; Huang 1990; creases with germination (Huang and Moreau germination seeds con- Mukherjee 1996). At the initial phase of Catalysis amount of lipids and a small amount of water. tain large organic media occurs under these conditions in a predominantly Plant lipases have with only small amount of water present. suitable for ester syn- properties that may make them especially thesis (Ncube and others 1993). was used in tors. First, the same weight of lipase preparations the specific each experiment. As these were crude preparations, would be different. activity of lipase in the different powders preliminaryusing p-nitrophenol esters showed studies Secondly, specificity towards hy- that the different enzymes had different of p-nitrophenol esters drolysis of esters. Despite the limitations the different lipases artificial substrate, such results suggest that could have different specificity towards more realistic sub- strates. Lipase specificity is expected to affect the conversion yields. Rangheard and others (1989) reported that pase was very active on long chain fatty acids. 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. acetate. With maize seed lipase, yield was With maize the conversion acetate. Ethanol 246Butanol 246Isopentanol 24 356 28.3(Z)-3-hexenol 17.724 27 61.1 24.26 13.3 21 20.6 24.4 34.7 30 23.3 28 21.5 38.7 21.1 12.9 29.7 36.9 30.5 23.5 27.4 22.3 26 19.3 23.3 33.8 63.8 31.2 19.7 38.8 25.5 49.1 21.8 24.6 29.9 13.3 25 24.9 28.8 43.3 17.2 23.7 11 28.6 28.9 20.2 Effect of fatty acid chain length Table 1—Yield (%)* for low molecular weight esters synthesized us- esters synthesized weight low molecular (%)* for 1—Yield Table reaction after 72 h powders seedling ing plant Alcohol Cn** (%) = 100 ([Acid] Yield * respectively. concentrations acid; C2 = acetic acid;C 4 = butyric acid; C6 = caproic acid **Cn = number of C-atoms in are given in Fig 1. ***Results for Rapeseed lipase strongly influenced the conversion yield. With rapeseed seedling rapeseed With strongly influenced the conversion yield. (C4) and obtained for butyric yields of 69% to 97% were powder, caproic (C6) acid esters with butyl, iso-pentyl, and (Z)-3-hexen- 1-yl alcohols (Fig. 1). Significant yields were also observed using maize ( and alcohol tested (Table 1). Langrand and others (1988) report- and alcohol tested (Table ed that and C4 tic acid esters with ethyl, butyl, and isopentyl alcohol. Barley seed powder exhibited uniform ( C. The Њ 70%) and isopentyl Ͻ —Vol. 65, No. 2, 2000 65, No. —Vol. 4) values (Dordick 1989). Ն 70%), butyl caproate ( Ͻ 80%). The enzyme powders used in this work could 80%). The enzyme powders used in this Ͼ JOURNAL OF FOOD SCIENCE C for 72 h. A yield of 63.8% for ethyl acetate and 49% for butyl acetate, A yield of 63.8% for ethyl acetate and 49% Figure 1 and Table 1 summarize results from esterification 1 summarize results Table 1 and Figure Њ Њ Њ Њ Њ obtained using linola seedling lipase, are also notable (Table 1). also notable (Table are obtained using linola seedling lipase, was observed for butyl For wheat lipase a 61.1% conversion yield be re-used for at least 3 successive syntheses without lowering be re-used for at least 3 successive syntheses the product yield. caproate ( 296 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 Choice of seedling acetone powder Choice of seedling mined by drying to constant weight overnight at 105 at 105 overnight weight to constant by drying mined seedling powders used contained about 8% moisture on a dry moisture about 8% used contained powders seedling differ in catalysis, enzymes During organic phase weight basis. to differ- also in their sensitivity for water and their requirement water bound that it is the It has been demonstrated ent solvents. rather the catalytic activity which determines to the enzyme, It is gen- and Klibanov 1988). water content (Zaks than the total water is organic solvents, more that with polar erally accepted of bound to the enzyme. Good solvents held in solution instead esterification are those which do not strip for lipase-mediated such as hexane used in this work. These water from the enzyme, high log P ( are characterized by studies involving 5 lipase preparations. Reactions involving a to- preparations. Reactions involving a studies involving 5 lipase 3 acids were investigated. This combination tal of 4 alcohols and es- led to the synthesis of 12 unique of fatty acids and alcohols consistently gave the highest yield under ters. Rapeseed lipase and (Z)-3- (Z)-3-hexen-1-yl butyrate study. the conditions of this yields about 96%. The hexen-1-yl caproate were produced with butyrate (> 80%), iso- yield for other esters are as follows; butyl pentyl butyrate ( Seedling Lipase Flavor Synthesis . . . . . Synthesis Flavor Lipase Seedling

Food Microbiology and Safety showed the highest level of ester synthesis with C3 and C4 fatty caproate, provided that the enzyme is stable under the condi- acids. Using M. meihei lipase little or no ester synthesis was ob- tions used (results not shown). These results are reproducible — served with C2 and C3 acids (Gatfield 1986; Posorske 1984). all experiments were repeated at least twice. Bourg-Garros and others (1997) esterified (Z)-3-hexen-1-ol GC-MS results of a standard and synthesized butyl butyrate with butyric, isovaleric, and caproic acid on a laboratory scale (3- 5g) 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, isov- alerate, or caproate were produced with yields about 95%. How- ever, the yield for (Z)-3-hexen-1-yl acetate production using Li- pozyme 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; Cl- aon and Akoh 1993). The differences in conversion yields may re- sult 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 Will- iams 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 Ϫ0.23 ( 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).

Effect of alcohol type and chain length. Ester synthesis was examined for a range of alcohols. Four pri- mary 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, ir- respective of the type of ester synthesized. The only exception to

this was ethyl, butyl, and isopentyl acetates in case of linola Food Microbiology and Safety 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 approxi- mately 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. Re- Figs. 2 and 3—Time course showing yields (% molar conversion ) for ethyl butyrate (2) and butyl butyrate (3) produced using various plant gardless of the rate of reaction, significant yields could be ob- seedling acetone powders. Other reactions conditions are same as in tained by prolonged incubation (212 h) even for (Z)-3-hexen-1-yl Fig. 1.

Vol. 65, No. 2, 2000—JOURNAL OF FOOD SCIENCE 297 C until ana- Њ C and detector Њ C for 2 min and 10 Њ Ϫ C/min and held for 4 Њ C until used. Њ 20 Ϫ A, 4-8 mesh). The final concentration Њ 0.32 mm ID; film thickness 1 micron) C at a rate of 15 Њ ϫ C. Њ The gas chromatography system consisted of Carlo Erba Esterification was performed essentially as described by Esterification was performed essentially The concentrations of ester formed were determined by The concentrations of ester formed were Gas chromatographic analysis were kept in sealed bottles at Direct esterification conditions. Fig. 4—GC-MS spectra for butyl butyrate. (A) standard and (B) Synthe- Fig. 4—GC-MS spectra for butyl butyrate. (A) sized in this study. apparatus (Model 5160) equipped with a flame ionization de- fused silica capillary col- involved a BP-20 Separation tector. umn (SGE, UK, 25 m operated with helium gas as carrier (2 mL/min, split ratio 1:15). The oven temperature was maintained at 50 Langrand and others (1990). However, additional amounts of additional amounts and others (1990). However, Langrand mixture. Into 20-mL water were not added to the reaction alcohol, 1.25- screw-capped vials containing 1.25-mmoles powder (5% w/v of mmoles of acid was added 250-mg acetone The final reaction total reaction volume) as enzyme source. previously dried mixture was adjusted to 5-mL with hexane over molecular sieves (3 was performed by of reactants were each 0.25 M. Synthesis at a constant tem- shaking reaction vessels at about 100 rpm perature of 37 lyzed (usually within 24 h). The frozen samples were allowed to warm to room temperature and then analyzed by gas chro- alcohol, of ester, to determine the concentration matography and acids. Esters synthesis is expressed as percentage molar conversion of acids. All synthesis experiments were performed in duplicate using separate reaction vials. then increased to 210 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 min. The injector temperature was fixed at 250 , , C C. Њ Њ ACIDS

BARLEY , BUTYRIC WHEAT ,

ACID

—Vol. 65, No. 2, 2000 65, No. —Vol. ACETIC , 63% for ethyl acetate and butyl 63% for ethyl acetate SEEDLINGS

Յ Conclusion ) in a dark incubator. Germination was incubator. ) in a dark st CHEMICALS

GERMINATED

Materials and Methods C or less) for 1 min. The acetone extract was sep- Њ GRADE

C for 10 min and then cut into small pieces with Њ 18 FROM

JOURNAL OF FOOD SCIENCE

caproic acid , ethanol, butanol, isopentanol, (Z)-3-hexen- caproic acid , ethanol, butanol, isopentanol, EAGENT The procedures used were similar to those described by Seeds were supplied by Nickerson Seeds Ltd. (Lincoln, U.K.). Seeds were supplied by Nickerson Seeds Ltd. rapeseed, maize, and linola were able to synthesize low mo- rapeseed, maize, and OWDERS

carried out in an incubator. Seeds were placed on moist filter pa- were Seeds carried out in an incubator. per towels placed on top of moist perlite (Silvaperl graded horti- cultural) in shallows plastics trays covered with perforated alu- minium foil. The average germination temperature was 26 Acetone powder preparation from seedlings

Seed material and germination R scissors. Seedlings were homogenized with 5 volumes of cold acetone ( arated from the residue by carefully decanting the whole sus- I fil- Whatman No. funnel, containing pension into a Buchner pow- The resulting quipped with vacuum filtration. ter paper, der was re-extracted 4 times with 5 volumes of cold acetone and air dried under a hood for 10 h. The light grayish powders Hassanien and Mukherjee (1986). A batch of germinated seed- kept in a times with distilled water, ling was washed three fridge at 4 (designated as day 1 Dry whole seeds were surface sterilized by soaking in 0.1% sodi- Dry whole seeds were surface sterilized by were then washed um hypochlorite solution for 30 s. The seeds for 24 h at 26 thoroughly with running tap water and soaked 1-ol and esters were obtained from Sigma-Aldrich Co. Ltd. 1-ol and esters were obtained from Sigma-Aldrich molecular sieves (3A, (Poole, England). Hexane was dried over Ltd.) for at least 24 h 8-12 mesh; both from Sigma-Aldrich Co. prior to use. 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). lecular weight flavor esters. Synthesis of acetic, butyric and hex- lecular weight flavor ethanol, butanol, iso-pentanol, and (Z)-3- anoic acid esters with achieved. Germinated rapeseed showed hexen-l-ol alcohol was (Z)-3-hexen-1-yl butyrate and (Z)-3- the best flavor synthesis. were produced with yields about 96%. Linola hexen-1-yl caproate yields of seedling powder gave acetate whilst moderate yields were obtained with barley and yields were obtained with barley acetate whilst moderate (40%). Further work is needed to opti- maize seedling powders using seedling powders. mize flavor synthesis

298 P Seedling Lipase Flavor Synthesis . . . . . Synthesis Flavor Lipase Seedling are shown in the Fig. 4. There was a close resemblance between close resemblance was a Fig. 4. There in the are shown possi- it was GC-MS results, such Using patterns. fragmentation is prob- esters produced. GC-MS the identity of all ble to confirm separation available for the powerful technique ably the most mixtures. in complex of volatile compounds and identification of the com- leads to a fingerprint of GC and MS The combination highly accurate identification. and therefore pound detected

Food Microbiology and Safety temperature at 240 ЊC. The GC was connected to an integrator Gas chromatography-mass spectrometry (GC-MS) (Hewlett Packard 3395 integrator) which recorded the peak ar- analysis. eas and retention times in a chromatogram. GC-MS (Carlo Erba GC Model 4200, Kratos MS 80 RFA) was used for identification of esters. The GC-MS was Esters identification and quantification equipped with a 38 m ϫ 0.32 ϫ 0.5 ␮m film thickness BP-20 Esters, alcohol, and acids were identified according to their column (SGE, UK); Elution was performed as described retention times on chromatograms and from comparisons with above. Injections (0.2 ␮l) were made on column. Mass results obtained with standards. A calibration graph of known spectra were recorded with an ion source energy of 70 eV. acids concentration versus corresponding peak area was con- Fragmentation patterns were compared with a library of structed. Various concentrations of acid (0.0125 to 0.25 M) were results for standard esters. Identification of esters by GC- prepared by diluting in n- hexane and 0.2 ␮L of each was in- MS enabled compositional analysis of the product mix- jected in to GC. Injections were repeated twice for each vial. tures.

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Chem. Soc. 75: 1201-1206. Food Microbiology and Safety Kim J, Altreuter DH, Clark DS, Dordick JS. 1998. Rapid synthesis of fatty acid esters for The authors would like to thank the Pakistan Government for financial sponsorship to ML. use as potential food flavors. J. Am. Oil. Chem. Soc. 75: 1109-1113. We are also grateful to Mr. Ian Boyes for his technical assistance. Klibanov AM. 1989. Enzymatic catalysis in anhydrous solvents. Trends Biochem. Sci. 14: 141-144. Authors Liaquat and Apenten are from Laboratory of Food Biochemistry Koskinen AMP, Klibanov AM, editors. 1996. Enzymatic Reaction in Organic Media. and Nutrition, Department of Food Science, University of Leeds, London: Blackie Academic and Scientific. 314 p. Leeds LS2 9JT (UK). Direct inquiries to author Apenten (E-mail Langrand G, Rondot N, Triantaphylides C, Barratti J. 1988. Lipase catalyzed formation of flavor esters. Biotechnol. Letts. 10: 549-554. [email protected]).

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