Flavor Esters Research Articles

Optimization of Lipase Catalyzed Synthesis of Nonyl Caprylate using Response Surface Methodology (RSM)

Nonyl caprylate, a citrus and rose flavor is short-chain ester with fruity notes is widely used in food, cosmetic and pharmaceutical industries. Traditionally, flavor esters are produced by chemical method or extracted from natural sources. However, with the steadily growing demand for natural flavor compounds, the biosynthesis of such esters by lipase under mild conditions has been receiving much attention for producing these valuable products. In this study, enzymatic synthesis of nonyl caprylate in solvent free system, was successfully optimized via Response Surface Method (RSM) based on 5-level, 4-variable of Central Composite RotaTable Design (CCRD). The parameters were reaction time (3-8 hours); reaction temperature (30-50°C); amount of enzyme (10-20 %, w/w) and shaking speed (100-200 rpm). The optimum condition derived via RSM for the reaction was reaction time of 6.6 hours, reaction temperature of 30.08ºC, enzyme amount of 20 % (w/w) and shaking speed of 128.7 rpm. The actual experimental yield was 90.91% under the optimum condition, which compared well with the maximum predicted value of 91.33%. Comparison of predicted and experimental values reveals good correspondence between them, implying that empirical models derived from RSM can be used to adequately describe the relationship between the factors and response in the synthesis of Nonyl Caprylate.

Purification and properties of Amycolatopsis mediterranei DSM 43304 lipase and its potential in flavour ester synthesis

An extracellular thermostable lipase from Amycolatopsis mediterranei DSM 43304 has been purified to homogeneity using ammonium sulphate precipitation followed by anion exchange chromatography and hydrophobic interaction chromatography. This protocol resulted in a 398-fold purification with 36% final recovery. The purified A. mediterranei DSM 43304 lipase (AML) has an apparent molecular mass of 33 kDa. The N-terminal sequence, AANPYERGPDPTTASIEATR, showed highest similarity to a lipase from Streptomyces exfoliatus. The values of K(m)(app) and V(max)(app) for p-nitrophenyl palmitate (p-NPP) at the optimal temperature (60°C) and pH (8.0) were 0.099±0.010 mM and 2.53±0.06 mmol/min mg, respectively. The purified AML displayed significant activity towards a range of short and long chain triglyceride substrates and p-nitrophenyl esters. Hydrolysis of glycerol ester bonds occurred non-specifically. The purified AML displayed significant stability in the presence of organic solvents (40%, v/v) and catalyzed the synthesis of the flavour ester isoamyl acetate in free and immobilized states.

Enzymatic reactions and synthesis of n‐butyl caproate: esterification, transesterification and aminolysis using a recombinant lipase from Geobacillus thermoleovorans CCR11

The recombinant lipase LipMatCCR11 from the thermophilic strain Geobacillus thermoleovorans CCR11 was applied in the synthesis of n‐butyl caproate via transesterification in hexane and xylene. The short chain flavour ester was obtained by alcoholysis from ethyl caproate and n‐butyl alcohol and acidolysis from n‐butyl butyrate and caproic acid. This enzyme was also used in the condensation reaction from caproic acid and n‐butanol. The conversion percentages at equilibrium (Xe) were similar to those obtained with Candida antarctica lipase fraction B (CAL‐B) in the same reaction conditions, while lower conversion velocities (k) were attained. LipMatCCR11 reached high conversion percentages in either hexane or xylene as organic media (> 63%); the enzyme was also able to catalyze the aminolysis reaction of ethyl caproate with benzyl amine in hexane obtaining a conversion percentage > 62%.

An alkaline lipase from organic solvent tolerant Acinetobacter sp. EH28: Application for ethyl caprylate synthesis

A mesophilic bacterium producing a thermostable alkaline lipase was isolated from oil rich soil sample and identified as Acinetobacter sp. EH28. The lipase was partially purified by ammonium sulphate precipitation followed by hydrophobic interaction chromatography with 24.2-fold purification and 57.1 U/ml specific activity. The partially purified enzyme exhibited maximum activity at pH 10.0 and at 50 °C and was highly stable at 50 °C retaining 100% of its activity up to 90 min. It was highly stable and retained more than 80% of its initial activity upon exposure to various organic solvents. The EH28 lipase was used for synthesis of the flavor ester ethyl caprylate in organic solvents, thus providing a concept of application of Acinetobacter sp. lipase in non-aqueous catalysis. Reaction parameters best suited for this esterification reaction were 40 °C reaction temperature, 1.3:1 ratio of caprylic acid to ethanol and cyclohexane as the medium.

Display of Candida antarctica lipase B on Pichia pastoris and its application to flavor ester synthesis

Two alternative cell-surface display systems were developed in Pichia pastoris using the alpha-agglutinin and Flo1p (FS) anchor systems, respectively. Both the anchor cell wall proteins were obtained originally from Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was displayed functionally on the cell surface of P. pastoris using the anchor proteins alpha-agglutinin and FS. The activity of CALB displayed on P. pastoris was tenfold higher than that of S. cerevisiae. The hydrolytic and synthetic activities of CALB fused with alpha-agglutinin and FS anchored on P. pastoris were investigated. The hydrolytic activities of both lipases displayed on yeast cells surface were more than 200 U/g dry cell after 120 h of culture (200 and 270 U/g dry cell, respectively). However, the synthetic activity of CALB fused with alpha-agglutinin on P. pastoris was threefold higher than that of the FS fusion protein when applied to the synthesis of ethyl caproate. Similarly, the CALB displayed on P. pastoris using alpha-agglutinin had a higher catalytic efficiency with respect to the synthesis of other short-chain flavor esters than that displayed using the FS anchor. Interestingly, for some short-chain esters, the synthetic activity of displaying CALB fused with alpha-agglutinin on P. pastoris was even higher than that of the commercial CALB Novozyme 435.

Esterification activity and operational stability of Candida rugosa lipase immobilized in polyurethane foams in the production of ethyl butyrate

Ethyl butyrate is a fruity flavor ester widely used in food and pharmaceutical products. The synthesis of ethyl butyrate in n -hexane, catalyzed by Candida rugosa lipase immobilized in two hydrophilic polyurethane foams (“HYPOL FHP 2002” and “HYPOL FHP 5000”) was performed. In this study, the effects of (i) the immobilization supports, (ii) the initial substrate concentrations and (iii) the water content of the system, on the activity and operational stability of C. rugosa lipase in both foams, during the esterification in continuous packed-bed reactor (PBR) and in repeated batches, were investigated. When low substrate concentrations were used, no deactivation was observed for both biocatalysts, along the continuous 30-d PBR operation. Conversely, under high substrate concentrations, a fast deactivation of the biocatalysts was observed. In consecutive batches, the deactivation was faster for the lipase in the less hydrophilic foam (“FHP 5000”) with a half-life of 53 h against 170.3 h for the other counterpart. Water molecules in the microenvironment did not present a deactivation effect on the biocatalysts. The low operational stability can be ascribed to the inhibitory effect of ethanol, which tends to accumulate inside the foams.

AN ESTEROLYTIC ACTIVITY FROM A WILD EDIBLE MUSHROOM,LYCOPERDON PERLATUM

Lycoperdon perlatum Pers. (Lycoperdaceae, Agaricales, Agaricomycetidae, Agaricomycetes, Basidiomycota, Fungi) was evaluated for its esterolytic potential. Native electrophoresis of the crude extracts showed four bands having Rf values of 0.34, 0.39, 0.52 and 0.59. The esterase showed the highest activity toward a short-chain substrate, p-nitrophenyl acetate. Optimum reaction conditions for L. perlatum crude extract were attained at pH 8.0 and 40C. Esterolytic activity of enzyme extract was stimulated in the presence of Mn2+, Fe2+, Ca2+ and Zn2+ in the reaction mixture. The enzyme activity was stimulated by incubation at pH 6.0 but retained 77% of its original activity at its optimum pH after 24 h. Thermal inactivation was displayed after incubation for 20 min at various temperatures above 30C. At 1 mM final concentration, 2-mercaptoethanol, dithiothreitol, ethylenediamine tetraacetic acid and p-methylphenyl sulfonylfluoride inhibited the esterolytic reaction. These results support that the crude L. perlatum extract possesses an esterolytic activity having properties similar to other esterases. PRACTICAL APPLICATIONS Esterases catalyzing the cleavage and formation of ester bonds are known α/β-hydrolases (EC 3.1.1.X). Esterases are used for the synthesis of flavor esters for the food industry, modification of triglycerides for fat and oil industry and resolution of racemic mixtures used for the synthesis of fine chemicals for the pharmaceutical industry. Therefore, the search for new enzyme sources is important for the development of new enzymes and applications.

Production of aroma esters by immobilized Candida rugosa and porcine pancreatic lipase into calcium alginate gel

Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL) were immobilized into calcium alginate (Ca-Alg) gel beads by means of entrapment and were used to produce three industrially important flavour esters, namely isoamyl acetate (banana flavour), ethyl valerate (green apple flavour) and butyl acetate (pineapple flavour). Immobilization conditions were optimized in terms of sodium alginate (Na-Alg) and CaCl 2 concentrations by determination of the entrapped enzyme amount as well as by esterification of 4-nitrophenol and acetic acid. The best results were obtained at 2.5% Na-Alg and 2.5 M CaCl 2 for CRL while at 2.5% Na-Alg and 2.0 M CaCl 2 for PPL. On carrying out flavour syntheses in solvent-free medium and also in hexane medium, higher ester yields were obtained in hexane medium for all esters and both types of lipases. Ester esterification efficiency increased in parallel with both enzyme concentrations at immobilization medium and the immobilized lipase amount in esterification medium. Maximum ester production was observed between 40 and 50 °C for CRL and PPL. Besides, the effect of substrate concentrations on ester conversion was remarkable. The best ester yield was obtained for isoamyl acetate when immobilized PPL was used.

Enzymatic synthesis of fruit flavor esters by immobilized lipase from Rhizopus oligosporus optimized with response surface methodology

Short chain aliphatic esters have immense applications as flavors in fruit juices, cheeses, baked goods, candies, beverages and ice creams. Two such flavor esters ( n-butyl acetate and n-propyl acetate) were synthesized enzymatically by transesterification of vinyl acetate with alcohols namely n-butanol and n-propanol, respectively, in solvent-free systems. These two synthesized esters occur naturally in various fruits like apple, strawberry, pear, etc. Lipase from Rhizopus oligosporus NRRL 5905 immobilized onto cross-linked silica gel was used for synthesis of the esters. The process parameters (reaction time, enzyme amount, additional water and shaking speed) were studied to achieve the highest yield of the esters. Maximum conversion of n-butyl acetate (50%) and n-propyl acetate (56%) were achieved after 24 h of reaction at 30 °C, at an enzyme concentration of 25% (w/v) of reaction mixture. This process was further optimized using response surface methodology (RSM) based on a three-level, four-variable central composite design (CCD). The optimum molar conversion of 54.6% using 27.5% enzyme concentration at 215 rpm and 26.5 °C for 28 h and 56.5% molar conversion using 29.8% enzyme concentration at 101 rpm and 28.2 °C for 28 h were achieved for n-butyl and n-propyl acetates, respectively. Immobilized lipase could be used for three cycles for synthesis of n-butyl acetate and n-propyl acetate, respectively, with almost 100% retention of molar conversion. The K m and V max values were determined to be 227 mM and 322 μmol/(g-h), respectively, for n-butyl acetate while for n-propyl acetate the respective values were 222 mM and 385 μmol/(g-h).

Semi-continuous enzymatic production and membrane assisted separation of isoamyl acetate in alcohol — ionic liquid biphasic system

Enzymatic production of an important natural flavor ester, isoamyl acetate, was studied by using immobilized Candida antarctica lipase B in [bmim]PF 6 ionic liquid. The reaction was completed by removing water produced in the reaction by zeolite adsorption, and practically 100% yield was achieved. An integrated system was developed for the separation of the isoamyl alcohol‒water‒isoamyl acetate ternary azeotropic mixture, combining pervaporation and osmotic distillation. The Sulzer “Pervap 2256” flat sheet hydrophobic pervaporation membrane was found to be 100% selective for isoamyl alcohol.

Synthesis of flavour esters using free Candida antarctica lipase B in ionic liquids

AbstractThe enzymatic synthesis of flavour esters by trans‐esterification from vinyl esters and alcohols catalysed by free Candida antarctica lipase B (CaLB) in ionic liquids has been studied. The influence of different parameters, such us pH, temperature and ionic liquid nature, on activity and selectivity has been carefully analysed, using as reaction model the synthesis of butyl butanoate. Finally, a broad range of flavour esters were sucessfully synthesized using the optimal operation conditions previously established. Copyright © 2008 John Wiley & Sons, Ltd.

A new irreversible enzyme-aided esterification method in organic solvents

A new irreversible esterification method for carboxylic acids catalyzed by a lipase from Candida antarctica (Novozyme 435) in organic solvents has been developed. The water produced during the process is chemically destroyed by a corresponding ester of acetoacetate, which acts as a sacrificial substrate in this reaction. The flavour esters isobutyl acetate, methyl butyrate, ethyl butyrate and benzyl butyrate were synthesized either in small scale (0.05 mol) or large scale (1 mol). The yields range from 82 to 92% within 24 h at 52 degrees C. Optimal molar ratios of reactants were 1:1:1 (carboxylic acid:alcohol:acetoacetate).

Lipase catalyzed synthesis of flavor esters in non-aqueous media: Optimization of the yield of pentyl 2-methylpropanoate by statistical analysis

In this study, the synthesis of pentyl 2-methylpropanoate employing a commercial lipase from Candida rugosa was investigated, the emphasis being placed on analyzing the effects of various process conditions on the yield of ester. The response surface methodology (RSM) and five-level-five-factor central composite rotatable design (CCRD) were used to evaluate the effects of variables, namely the initial water content, 0.0-2.0% (w/v), the reaction temperature, 35-75?C, the enzyme concentration, 1.0-5.0 g dm-3, the acid/alcohol mole ratio, 1:2-5:2, and the reaction time, 4-48 h, on the yield (%) of ester. The production of pentyl 2-methylpropanoate was optimized and an ester yield response equation was obtained, enabling the prediction of ester yields from known values of the five main factors. It seems that the enzyme concentration, reaction time and acid/alcohol mole ratio predominantly determine the conversion process, while the amount of added water amount had no significant influence on the ester yield. Conversion of around 92 % of the substrate to ester could be realized using a concentration of lipase as low as 4.0 g dm-3 and in a relatively short time (26 h) at 35?C, when a high substrate mole ratio of 2.5 was used.

Solid-state fermentation for ‘whole-cell synthetic lipase’ production from Rhizopus chinensis and identification of the functional enzyme

Major process parameters affecting the production of whole-cell synthetic lipase (WCSL) by Rhizopus chinensis in solid-state fermentation were optimized based on synthetic activity. A combined substrate of wheat flour with wheat bran (3/2, w/w) supported both good biomass growth and enzyme production. The maximum synthetic activity of 24,447 U/kg substrate was reached by selecting a moisture content of 70%, initial pH of 6.5, supplementation of peptone (2%, w/w) as additional nitrogen source and olive oil (2%, v/w) as inducer, after 72 h of incubation. This production represented a 15.27-fold increase of lipase synthetic activity compared to the initial medium. The synthetic lipase was purified by ammonium sulfate precipitation, hydrophobic interaction and gel filtration to homogeneity, with yield and purification fold of 2.6% and 55.2, respectively, and the molecular weight was estimated to be 60 kDa by SDS-PAGE. Validation of functional enzyme was carried out by catalyzing the synthesis of ethyl flavor esters, and the result showed that conversion of ethyl octanoate and ethyl caprate reached 95%.

Novel approach for the synthesis of ethyl isovalerate using surfactant coated Candida rugosa lipase immobilized in microemulsion based organogels

In our study Candida rugosa lipase coated with various surfactants viz. AOT, CTAB, and Tween 80 was immobilized in microemulsion based organogels (MBGs) and investigated for the production of ethyl isovalerate, a fruity flavor ester. Combinations of different surfactant coated lipase and the surfactant used for preparation of MBG were tried for esterification activity. Amongst various combinations used, CTAB coated lipase immobilized in AOT based MBGs exhibited maximum ethyl isovalerate synthesis (0.082 mol/L) in 9 days. The CTAB coated lipase immobilized in AOT based organogel showed eight-fold increase in the esterification activity as compared to the native lipase. We also found that, increase in the concentration of the CTAB led to the decrease in the catalytic activity of lipase immobilized in AOT based organogels. The AOT based organogels containing CTAB coated lipase could be reused five times, without significant loss in esterification activity. However, after fifth cycle the esterification activity decreased to 40% of the initial activity. The activation energy for catalytic activity of the untreated and surfactant coated lipase was found to be 29.8 and 9.15 kJ, respectively, which depicts some structural change in surfactant coated lipase. Thus, the surfactant coated lipase immobilized in MBGs exhibited significantly higher stability and activity in comparison to uncoated lipase immobilized in MBGs. This strategy seems to hold a good promise for the synthetic flavor industry.

Refractive Indices and Surface Tensions of Binary Mixtures of Isoamyl Acetate, Ethyl Caproate, Ethyl Benzoate, Isoamyl Butyrate, Ethyl Phenylacetate, and Ethyl Caprylate with Ethanol at (288.15, 298.15, 308.15, and 318.15) K

Refractive indices and surface tensions for six binary mixtures formed by the flavor esters (isoamyl acetate, ethyl caproate, ethyl benzoate, isoamyl butyrate, ethyl phenylacetate, and ethyl caprylate) with ethanol over the whole composition range were measured at four different temperatures (288.15, 298.15, 308.15, and 318.15) K and atmospheric pressure. Refractive indices were measured using a digital Abbe-type refractometer. Surface tensions were determined using the Wilhelmy-plate method. From experimental data, deviations in the refractive index Δn were calculated both on a mole fraction basis and on a volume fraction basis. The deviations in the molar refraction ΔR and the deviations in the surface tension Δσ were calculated on a mole fraction basis. The binary data of these deviations were correlated as a function of the mole fraction using the Redlich−Kister equation.

Refractive Indices and Surface Tensions of Binary Mixtures of Ethyl Acetoacetate, Ethyl Isovalerate, Methyl Benzoate, Benzyl Acetate, Ethyl Salicylate, and Benzyl Propionate with Ethanol at (288.15, 298.15, 308.15, and 318.15) K

Refractive indices and surface tensions for binary mixtures of six flavor esters (ethyl acetoacetate, ethyl isovalerate, methyl benzoate, benzyl acetate, ethyl salicylate, and benzyl propionate) with ethanol over the whole composition range were measured at four different temperatures (288.15, 298.15, 308.15, and 318.15) K and atmospheric pressure. Refractive indices were measured using a digital Abbe-type refractometer. Surface tensions were determined using the Wilhelmy-plate method. Deviations in the refractive index ΔnD and deviations in the surface tension Δσ from volume fraction average for the binary mixtures were derived from experimental data. The binary data of ΔnD and Δσ were correlated as a function of mole fraction using the Redlich−Kister equation.

Production of flavour esters by immobilized Staphylococcus simulans lipase in a solvent-free system

Ethyl valerate (green apple flavour) and hexyl acetate (pear flavour) are short-chain esters with fruity notes that are widely used in food, cosmetic and pharmaceutical industries. Crude Staphylococcus simulans lipase was immobilized on different supports. The CaCO 3 was selected as the most suitable support offering maximum adsorption capacity. Different reaction parameters for enhancing ester formation were investigated. In fact, higher conversion of ethyl valerate (51%) was achieved by the addition of 20% (w/w) of water to the reaction mixture, using 200 IU of immobilized lipase, a molar ratio valeric acid to ethanol of 1 and at 37 °C. No decrease of the synthesis activity was observed for 10 cycles of use of the immobilized lipase. In the case of the hexyl acetate, the optimum conditions, giving 41% of conversion, are obtained with 10% (w/w) of initially added water, 100 IU of immobilized lipase, a molar ratio acetic acid to hexanol of 1 and at 37 °C. Immobilized lipase could be repeatedly used for five cycles without a decrease of synthesis activity.

Studies on the specificity of Candida rugosa lipase catalyzed esterification reactions in organic media

In this study, the feasibility of the synthesis of various flavor esters catalyzed by a commercial lipase from Candida rugosa was investigated and the process parameters were optimized. Lipase from C. rugosa successfully catalyzed the synthesis of 19 esters. The highest yields, of more than 90 % after 20 h, were observed in the synthesis of short-chain esters, pentyl propanoate, isopentyl butanoate, and butyl butanoate. Increasing the number of carbon atoms of both substrates above 8 caused a significant decrease of the initial reaction rates and the final yields. The enzyme showed surprisingly low affinity towards pentanoic acid and hexanoic acid, compared with the higher homologues, octanoic acid and decanoic acid. In addition to the number of carbon atoms, the structure of the substrates had a significant influence on the enzyme activity. Namely, the activity of the enzyme towards isopropanol was significantly lower compared with n-propanol. Additionally, cis-9-octadecenoic acid was a better substrate than octadecanoic acid, its saturated analogue.

Densities and Viscosities of Binary Mixtures of Ethyl Acetoacetate, Ethyl Isovalerate, Methyl Benzoate, Benzyl Acetate, Ethyl Salicylate, and Benzyl Propionate with Ethanol at T = (288.15, 298.15, 308.15, and 318.15) K

Densities and viscosities for six binary mixtures formed by the flavor esters (ethyl acetoacetate, ethyl isovalerate, methyl benzoate, benzyl acetate, ethyl salicylate, and benzyl propionate) with ethanol over the whole composition range were measured at four different temperatures (288.15, 298.15, 308.15, and 318.15) K and atmospheric pressure. Densities were determined using a vibrating-tube density meter. Viscosities were measured with an automatic Ubbelohde capillary viscometer. Excess molar volumes (V E) and deviations in the viscosity from mole fraction average (Δη) for the binary mixtures were derived from experimental data. The binary data of V E and Δη were correlated as function of the mole fraction by using the Redlich−Kister equation. Furthermore, McAllister's multi-body interaction models are used to correlate the binary kinematic viscosities.