OPTIMIZATION OF ISOAMYL ACETATE PRODUCTION IN A SOLVENT-FREE SYSTEM

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1 bs_bs_banner Journal of Food Quality ISSN OPTIMIZATION OF ISOAMYL ACETATE PRODUCTION IN A SOLVENT-FREE SYSTEM N. YUSOFF AZUDIN, M.D. MASHITAH and S.R. ABD SHUKOR 1 School of Chemical Engineering, Engineering Campus, Universiti Sains Malaysia, Nibong Tebal, Penang 14300, Malaysia 1 Corresponding author. TEL: ; FAX: ; chsyamrizal@eng.usm.my Received for Publication January, 013 Accepted for Publication August 3, /jfq.1060 ABSTRACT Enzymatic esterification of isoamyl acetate using Candida antarctica immobilized lipase was studied and optimized using response surface methodology (RSM) by Design of Experiment software. Isoamyl acetate is a colorless organic compound which is used by food industry as additives for its banana flavor. The production of isoamyl acetate is costly, hence, RSM giving advantages by minimizing the number of experiments for studying parameter effect. The model developed by RSM also simplified the relationship that can be used for practical engineering purposes. The parameters investigated in this study are mass of enzyme (1 14 wt %), reaction temperature (30 50C) and reaction time ( 6 h). As reaction temperature increased at any given mass of enzyme, a yield of ester increased up to optimum temperature 40C and thereafter decreased. The model obtained by RSM indicated the optimum conditions for maximum esterification (100% yields) at 34.8C with 1.04 g mass of enzyme after 0.5 h reaction time, which is in good agreement with the experimental value (104%). PRACTICAL APPLICATIONS Isoamyl acetate is a compound that has a strong fruity aroma similar to banana and pear. It is commonly used as food flavor in the food industry. Apart from that, it is also popular as a solvent for varnishes, paints and nitrocellulose lacquers. The compound is also used in rayon, dyes, artificial pearls, film manufacturing and extraction of penicillin. INTRODUCTION Short-chain esters have high demand in fragrance and flavors, it is widely used in food, cosmetic and pharmaceutical industries (Romero et al. 005). Isoamyl acetate is one of the short-chain esters; a colorless organic compound that can be formed by reacting isoamyl alcohol with acetic anhydride. It has strong banana flavor and the most desired flavor by the food industry as the demand is high over the year (Romero et al. 005; Guvenc et al. 010). Traditionally, banana flavor has been produced by extraction from natural sources, and often it is in short supply. Fermentation has always been the alternative choice for isoamyl acetate production but it is too expensive for the commercial exploitation (Hari Krishna et al. 001). Esterification reactions on the other hand provides economical alternative route to produce isoamyl acetate via synthesis between isoamyl alcohol and acetic anhydride. Natural esterification takes longer period and produced lower yield; hence, there is a need to investigate the optimum parameters to obtain higher ester yield in shorter period of time. Immobilized lipase-based enzyme are widely used as catalyst during esterification reaction to improve the stability and product yield (Ajay and Gross 000; Bi et al. 009). The synthesis of isoamyl acetate in organic solvent has been widely researched (Chulalaksananukul et al. 1990; Akoh and Yee 1998; Janssen et al. 1999; Romero et al. 005; Guvenc et al. 010). Even though higher conversion yields in organic solvents giving it an advantage, the solvent toxicity creates additional problems especially to the person who handles the chemicals. Taking n-hexane as one of the regularly used solvent, it is known for its carcinogenic character. Fink-Grernmels (1999) has reviewed that frequent contact with toxic and carcinogenic chemicals may risk humans to Journal of Food Quality 36 (013) Wiley Periodicals, Inc. 441

2 ISOAMYL ACETATE SYNTHESIS N. YUSOFF AZUDIN, M.D. MASHITAH and S.R. ABD SHUKOR acute intoxication, losses in productivity and reduced in weight gain. Toxicity of n-hexane has been extensively discussed by Agency for Toxic Substances and Disease Registry (Hashim 011). In addition, some organic solvents in use are too expensive to allow profitable commercial scale-up (Güvenç et al. 00). The aim of this study is to find the optimum conditions for enzymatic reaction between acetic anhydride and isoamyl alcohol in a solvent-free system so that scale-up can be done in shorter time and without perturbing about the cost. MATERIALS AND METHODS Equipment The experiments were carried out in a 100-mL stopped rubber shake flask, which was incubated in Incu-Shaker Mini (Benchmark Scientific, Edison, NJ) at 150 rpm. Temperature was set between 30 and 50C. Working pressure was at ambient pressure condition. Materials Isoamyl alcohol was supplied by Merck Co. (Petaling Jaya, Malaysia), while acetic anhydride (reagent grade 98%) was supplied by Sigma-Aldrich (Kuala Lumpur, Malaysia). All substrates were used without any pretreatment. Immobilized enzyme from Candida antarctica, Novozyme 435 (specific activity 10,000 U/g, recombinant, expressed in Aspergillus niger) was supplied by Sigma-Aldrich. Experimental Design A 3-level-3-factor central composite rotatable design (CCRD) was employed in this study. The 3-factor indicates the three experimental parameters (temperature, mass of enzyme and reaction time) in this study and the 3-level indicates the level of each range ( 1, 0 and +1). The list of parameters and levels studied in this experiment were shown in Table 1. Preliminary studies showed that all of the variables are statistically significant and ignoring these variables may affect the design of experiment. This CCRD design consists of two parts: factorial points ( 1, +1) and center points (0, 0). Six replicate runs at the center point (0, 0) of the design were performed to allow the estimation TABLE 1. LIST OF LEVELS OF EXPERIMENTAL VARIABLES Levels Variables Coding Unit Temperature β 1 C Mass enzyme β wt % Reaction time β 3 h 4 6 of pure error. All of the experiments were carried out in the randomized order to minimize the unexplained variability in the observed responses due to irrelevant factor. Synthesis Procedure Isoamyl acetate synthesis was carried out without any organic solvent in 100-mL stopped rubber shake flask with working volume of 15 ml. Acetic anhydride and isoamyl alcohol was added into the flask until 0.1 alcohol to acid ratio. Then 1 wt % of immobilized enzyme from C. antarctica was inserted into the mixture at the conditions of experiment (refer to Table ). The reaction mixture was then incubated in an Incu-shaker mini (Benchmark Scientific) at 150 rpm for 360 min. Samples were withdrawn periodically and analyzed using gas chromatograph until 6 h of reaction time. Experimental procedures were repeated for different amount of enzyme and conditions. Analysis A total of 0.5 ml of the reaction mixture was withdrawn every 0.5 h starting at t = 0 h until t = 6 h of reaction time. Samples were analyzed by gas chromatograph (780A) supplied by Agilent Technologies (Penang, Malaysia), equipped with a hydrogen flame ionization detector and a SGE BP1 (FFAP) column (60 m 0.3 mm 0.5 μm). Helium was used as carrier gas at flow rate of 5 ml/min. After injection of samples, the temperature of oven was kept at 100C and linearly increased to 140C. The rate of temperature increase was set at 70C/min, and was kept at 140C for the remaining time of analysis. Injector and detector temperatures were set at 00 and 50C, respectively. Quantification of data was done by calibration with standards samples. The retention times of peaks were as follows: isoamyl acetate,.6 min; isoamyl alcohol,.38 min; acetic anhydride,.48 min; and acetic acid, 3.1 min. Optimization by Response Surface Methodology The aim of the response surface methodology (RSM) is to find the optimum operational conditions for enzymatic esterification reaction. The data from the experiments were analyzed using Design of Expert (DoE) release software (Start-Ease Inc., Minneapolis, MN) and then interpreted in three main analytical steps which are analysis of variance (ANOVA), a regression analysis and the plotting of contour plot to establish an optimum condition for the enzymatic esterification synthesis. The first step in RSM is to find suitable approximation for the true functional relationship between the set of independent variables. Generally, a first-order model will be 44 Journal of Food Quality 36 (013) Wiley Periodicals, Inc.

3 N. YUSOFF AZUDIN, M.D. MASHITAH and S.R. ABD SHUKOR ISOAMYL ACETATE SYNTHESIS TABLE. CENTRAL COMPOSITE DESIGN AND EXPERIMENTAL DATA FOR 3-LEVEL-3-FACTOR RESPONSE SURFACE ANALYSIS Run Temperature (C) Mass enzyme (g) Reaction time (h) Result Ester (mol/l) Yield (%) fitted to the response values. When the first-order model does not fit, then the model is improved to a second-order model (Montgomery 1991) k k Y = β0 + βixi+ βiixi + βjixixij + ε, (1) i= 1 i= 1 j= 1 where x 1, x,...,x k are the input variables, which influence the response Y; β i(i = 1,,... k), β j(i = 1,,... k; j = 1,,...,k) are unknown parameters; and ε in a random error. The goodness of the models established was determined using coefficient of determinant (R ), together with the absolute average deviation values and ANOVA. RESULTS AND DISCUSSION First-order model developed by RSM does not fit well with the experimental data, hence second-order polynomial model was developed. A second-order polynomial model equation (1) was fitted to obtain isoamyl acetate concentrations, which were defined as mol per liter of reaction mixture. Table shows the list of 0 runs of experiments performed and the results obtained under operational conditions employed. The standard ANOVA and model coefficients are presented in Table 3. The ANOVA test as shown in Table 3, confirmed that the adequacy of quadratic model, since the probability value was less than indicating that the model was statistically significant. Table 4 shows the list of standard value, mean and regression value of the ANOVA test. The R of the k model was , which indicated that the model adequately represents the real process using the selected parameters. The value of adjusted determination coefficient was also very high (adj. R = 0.83) which represent a high significance of the model. The coefficients of independent TABLE 3. ANALYSIS OF VARIANCE FOR QUADRATIC MODEL Source Sum of squares Degree of Freedom Mean square F value Prob < F 3, < β β 1, , < β β β β ββ ββ Residual Lack of fit < Pure error Corrected total 3, TABLE 4. LIST OF STANDARD VALUE, MEAN AND REGRESSION VALUE Standard deviation 5.81 R-Squared Mean Adjusted R-Squared 0.83 Coefficient of variance 7.65 Predicted R-Squared Press,778.1 Adequate precision Journal of Food Quality 36 (013) Wiley Periodicals, Inc. 443

4 ISOAMYL ACETATE SYNTHESIS N. YUSOFF AZUDIN, M.D. MASHITAH and S.R. ABD SHUKOR variables determined for the second-order polynomial model for enzymatic esterification of isoamyl acetate between acetic anhydride and isoamyl alcohol is a Y = β β β β β β 3. 95ββ ββ 3 () where Y is the response, which is the yield of isoamyl acetate and β 1, β and β 3 are the coded values of the rest of variables as in Table 1. The P-values from Table 3 were used as a tool to check the significance of each of the coefficients that are necessary to understand the pattern of the mutual interactions between the variables. P-values less than represent that the corresponding coefficient are significant. Values greater than 0.1 indicate that the model terms are not significant. In this case, β, β and β β 3 are significant model terms with P-value <0.0001, , and , respectively. This suggests that the mass of enzyme has a direct relationship with the production of enzymatic isoamyl acetate. The best way to predict the relationship among responses, parameters and interactions is to analyze the contour plots. Figure 1 shows the three dimensional (3D) surface response plots for difference variations of experimental factors. Each curve represents combinations of two test variables while the other one factor was maintained at zero level. Figure 1a,b,c shows the 3D surface responses plot for isoamyl acetate yield for interaction between mass of enzyme and incubation temperature at reaction time 1 level, zero level and +1 level, respectively. The figures show that isoamyl acetate yield increased with increased mass of enzyme. Increased temperatures increased the ester yields until 40C and decreased afterward. Figure 1a gives the highest yield of isoamyl acetate (99.15%) at 38C and 1.76 g of enzyme. Longer reaction time (Fig. 1b,c) caused the maximum yield decreased. This is due to the reverse reaction between acetic anhydride and isoamyl alcohol. Details on the reaction can be seen on the reaction scheme below b c (a) Main reaction CH3CH ( CH3) CHCHOH + ( CH3CO) O ( Isoamyl alcohol) ( Acetic anhydride) CH3COOCH( CH3) CHCH3+ CH3COOH ( Isoamyl acetate) ( Acetic acid) (b) Secondary reaction CH3CH ( CH3) CHCHOH + CH3COOH ( isoamyl alcohol) ( acetic acid) CH3COOCH( CH3) CHCH3 + HO ( isoamyl acetate) ( water) FIG. 1. 3D SURFACE PLOT FOR ISOAMYL ACETATE YIELD FOR INTER- ACTION BETWEEN MASS OF ENZYME AND TEMPERATURE (a) Reaction time was set to 1 level. (b) Reaction time was set to zero level. (c) Reaction time was set to +1 level. 444 Journal of Food Quality 36 (013) Wiley Periodicals, Inc.

5 N. YUSOFF AZUDIN, M.D. MASHITAH and S.R. ABD SHUKOR ISOAMYL ACETATE SYNTHESIS (c) Overall reaction CH3CH ( CH3) CHCHOH + ( CH3CO) O ( isoamyl alcohol) ( acetic anhydride) CH3COOCH( CH3) CHCH3 + HO ( isoamyl alcohol) ( water) As stated by Cvjetko et al. (01), overall esterification reaction between acetic anhydride and isoamyl alcohol are based on two main reactions; reaction between acetic anhydride and isoamyl alcohol producing acetic acid and isoamyl acetate, and the other reaction is between acetic acid and excess isoamyl alcohol producing another isoamyl acetate and water as the by-product. Accumulation of water molecules in the final product decreases the production of isoamyl acetate production. This condition was found similar to synthesis done by Ghamgui et al. (006) and Luhong et al. (001). This effect fits with the Le Chatelier s principle where the dynamic equilibrium of the reaction will be shifted toward the left-hand side of the reversible reaction as the by-product increases. The more favored side reaction, hydrolysis of isoamyl acetate cause the formation of acetic acid that has inhibitory effect on the enzyme stability, hence lead to the decrease in ester synthesis (Kumari et al. 009). Optimization and Model Validation The optimal conditions were predicted using numerical optimization function in the DoE software. The optimization conditions of all parameter were set as in Table 5. Based on the numerical optimization, the most desirable reaction conditions for optimum isoamyl acetate yield (100%) with minimum reaction time (0.5 h) are at temperature 34.8C and mass of enzyme 1.04 g. The adequacy of the predicted model was validated by additional experimental test under suggested optimum conditions as in Table 6. Experimental results showed that there were no significant differences for percentage yield of ester produces between the predicted and actual values with error values less than 5%. Therefore, this model obtained is reliable to predict the isoamyl acetate yield in solvent free enzymatic esterification synthesis with high accuracy. CONCLUSIONS Production of isoamyl acetate was carried out in a solventfree system by reacting acetic anhydride with isoamyl TABLE 5. LIST OF CONDITIONS FOR NUMERICAL OPTIMIZATION Parameter Unit Goal Lower limits Upper limits Target Temperature C In range Mass enzyme g In range Reaction time h Minimum Yield % Target TABLE 6. MODEL VERIFICATION BY EXPERIMENTAL RESULT Number Temperature (C) alcohol. Enzyme C. antarctica was used to enhance the production rate of ester. Modeling and optimization process of this enzymatic esterification process were done using central composite design in DoE software. The R (0.91) and ANOVA implied that the model satisfactorily represented the real relationship of the main reaction parameters. Isoamyl acetate synthesis was optimized to higher yield with lower mass of enzyme and shorter reaction time. The optimization process produced 100% conversion yield at 34.8C operating temperature with 1.04 g mass of enzyme after 0.5 h reaction time. A validation tests were done to validate the optimization conditions and the yield were satisfied with the theoretical value. ACKNOWLEDGMENTS This study has been supported by Research University Cluster Grants (1001/PSF/861001) and Research University Grants (1001/PJKIMIA/814140). REFERENCES Mass enzyme (g) Reaction time (h) Yield (%) Predicted Actual Error (%) AJAY, K. and GROSS, R.A Candida antarctica lipase B-catalyzed transesterification: New synthetic routes to copolyester. J. Am. Chem. Soc. 48, AKOH, C.C. and YEE, L.N Lipase-catalyzed transesterification of primary terpene alcohols with vinyl esters in organic media. J. Mol. Catal. B: Enzym. 4, BI, F., IQBAL, S., ALI, A., ARMAN, M. and UL-HASSAN, M Synthesis of isoamyl acetate of isoamyl alcohol obtained from fussel oil using immobilized Candida antarctica lipase. J.Chem.Soc.Pak.31, CHULALAKSANANUKUL, W., CONDORET, J.S., DELORME, P. and WILLEMOT, R.M Kinetic study of esterification by immobilized lipase in n-hexane. FEBS 76, CVJETKO, M., VORKAPIĆ-FURAČ, J. and ŽNIDARŠIČ- PLAZL, P. 01. Isoamyl acetate synthesis in imidazoliumbased ionic liquids using packed bed enzyme microreactor. Process Biochem., 47, FINK-GRERNMELS, J Mycotoxins: Their implications for human and animal health. Vet. Q., 1, GHAMGUI, H., KARRA-CHAÂBOUNI, M., BEZZINE, S., MILED, N. and GARGOURI, Y Production of isoamyl Journal of Food Quality 36 (013) Wiley Periodicals, Inc. 445

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