Vol. 65, No. 2, 2000—JOURNAL OF FOOD SCIENCE 295 © 2000 Institute of Food Technologists Food Microbiology and Safety 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 63% 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 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 manufactur- ing 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 pro- duced by OPB are thought to comply with the U.S. Food and Drug Administration’s definition of natural. This mode of pro- duction makes the food industry less dependent on seasonal, cli- matic, 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 dimin- ished and product as well as biocatalyst recovery is easier. Final- ly, 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 arrahisu- is, 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 transes- terification (Akoh and Claon 1994; Yee and others 1995; Yee and Akoh 1996; Rizzi and others 1992). LMWE have also been pro- duced 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 Tran- Minh 1998; Perraud and Laboret 1995; Tan and others 1996). Im- mobilized microbial lipases have been used for OPB. These are stable and are easier to recover from the reaction vessel (Lan- grand and others 1988; Welsh and others 1990; Bourg-Garros and others 1998). The use of enzymes to produce flavor esters in sol- vent-free systems has also been described (Oguntimein and oth- ers 1995; Karra-Chaabouni and others 1998; Kim and others 1998; Leblanc and others 1998). There appear to be no reports describing the use of plant-de- rived lipases or acetone powders for LMWE synthesis. Seed li- pase or acetone powders from castor bean, rape, and Nigella sati- va seeds were used for lipid hydrolysis, glycerolysis, and esterifi- cation of glycerols or oleic acids (Hassanien and Mukherjee 1986; Dandik and others 1996; Mert and others 1995; Dandik and Ak- soy 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). Rape- seed lipase also catalyzed hydrolysis of various seed oils and ma- rine oils containing unusual fatty acids (Jachmanián and Mukher- jee 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 ho- mogenate. Preparation of acetone powder led to high recoveries of lipase activity. Procedures for preparing acetone powder are sim- ple, 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 inex- pensive 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, hex- anoic acids with ethanol, butanol, iso-pentanol or (Z)-3- hexen-l- ol in hexane. Results and Discussion L IPASE ACETONE POWDERS MADE FROM 4-D GERMINATING seedlings of barley, wheat, maize, linola, and rapeseed cata- lyzed the synthesis of low molecular weight flavor esters (LMWE). The reactions were performed with n-hexane as solvent. The re- action products were analyzed using gas chromatography (GC) and GC-mass spectrometry (GC-MS) analysis. The former tech- nique 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 ex- periments was about 10%. Hexane was found to be a suitable sol- vent for ester synthesis in agreement with previous reports (Car- ta 1991; Gillies 1987). The moisture content of the enzyme powders was deter- JFS: Food Chemistry and Toxicology