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 sol- vent-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 Seedling Lipase Flavor Synthesis . mined by drying to constant weight overnight at 105 ЊC. The Table 1—Yield (%)* for low molecular weight esters synthesized us- seedling powders used contained about 8% moisture on a dry ing plant seedling powders after 72 h reaction weight basis. During organic phase catalysis, enzymes differ in Alcohol Plant sources*** their requirement for water and also in their sensitivity to differ- Cn** Wheat Barley Maize Linola ent solvents. It has been demonstrated that it is the water bound Ethanol to the enzyme, which determines the catalytic activity rather 2 35 27 38.7 63.8 4 28.3 24.2 23.3 23.3 than the total water content (Zaks and Klibanov 1988). It is gen- 6 17.7 21 12.9 19.7 erally accepted that with polar organic solvents, more water is Butanol held in solution instead of bound to the enzyme. Good solvents 2 61.1 24.4 36.9 49.1 4 13.3 30 26 25 for lipase-mediated esterification are those which do not strip 6 20.6 21.5 19.3 13.3 water from the enzyme, such as hexane used in this work. These Isopentanol are characterized by high log P (Ն 4) values (Dordick 1989). 2 34.7 29.7 38.8 43.3 4 28 23.5 24.6 23.7 6 21.1 22.3 29.9 11 Choice of seedling acetone powder (Z)-3-hexenol Figure 1 and Table 1 summarize results from esterification 2 30.5 31.2 24.9 28.6 4 27.4 25.5 28.8 28.9 studies involving 5 lipase preparations. Reactions involving a to- 6 33.8 21.8 17.2 20.2 tal of 4 alcohols and 3 acids were investigated. This combination * Yield (%) = 100 ([Acid]O – [Acid] F) / [Acid]o where subscripts O and F denote initial and final of fatty acids and alcohols led to the synthesis of 12 unique es- concentrations respectively. **Cn = number of C-atoms in acid; C2 = acetic acid;C 4 = butyric acid; C6 = caproic acid ters. Rapeseed lipase consistently gave the highest yield under ***Results for Rapeseed lipase are given in Fig 1. the conditions of this study. (Z)-3-hexen-1-yl butyrate and (Z)-3- hexen-1-yl caproate were produced with yields about 96%. The yield for other esters are as follows; butyl butyrate (> 80%), iso- pentyl butyrate (Ͻ 70%), butyl caproate (Ͻ 70%) and isopentyl caproate (Ͼ 80%). The enzyme powders used in this work could acetate. With maize seed lipase, the conversion yield was Յ 39% be re-used for at least 3 successive syntheses without lowering to 40% for ethyl, butyl, isopentyl acetate. Caproic acid conver- the product yield. sion remained up to 20%. For wheat and barley acetone powders, A yield of 63.8% for ethyl acetate and 49% for butyl acetate, the conversion yields for acetic, butyric, and caproic acids for obtained using linola seedling lipase, are also notable (Table 1). most of the esters were less than 35% (Table 1). For wheat lipase a 61.1% conversion yield was observed for butyl The lipase activity in oilseeds and certain cereal grains in- creases with germination (Huang and Moreau 1978; Huang 1990; Mukherjee 1996). At the initial phase of germination seeds con- tain 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 syn- thesis (Ncube and others 1993). The different synthesis yields can be ascribed to at least 3 fac- tors. 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 hy- drolysis of esters.