USING CENTRAL COMPOSITE DESIGNS - RESPONSE SURFACE METHODOLOGY TO OPTIMIZE INVERATSE ACTIVITY CONDITIONS FOR FRUCTOSE PRODUCTION

Transcription

1 Conference proceedings of Biotechnology for Green Solutions and Sustainable Environment: 8-87, 010 USING CENTRAL COMPOSITE DESIGNS - RESPONSE SURFACE METHODOLOGY TO OPTIMIZE INVERATSE ACTIVITY CONDITIONS FOR FRUCTOSE PRODUCTION Tran Hong Thang, Phan Thi Chau Loan, Tran Thi Anh Tu, Ho Thi Thuy, Le Thi Vy Hien, Bui Hong Quan * Institute of Biotechnology and Food Technology Ho Chi Minh City University of Industry. Author for correspondence: Tel: buihongquan@gbd.edu.vn ABSTRACT Enzyme invertase (beta fructofuranosidase fructohydrolase, EC ) that hydrolyzes sucrose to glucose and fructose was used to produce high fructose syrup from sucrose syrup in industry. This study has been focusing on optimization activity interaction of enzyme invertase (Invertase from NOVO Nordisk), including three factors: ph, temperature (t o C), ratio enzyme/substrate (E/S). The design of all experiments based on response surface method (RSM) Central Composite Design (CCD). As the result, ph, ratio enzyme/substrate (E/S) were identified as significant factors (p<0.05). Using RSM-CCD, the optimal levels of two factors were found as the most suitable condition follows: at ph = 4; ratio enzyme/substrate (E/S = 0.01). The predicted maximum reducing sugar is mg/mL (initial sucrose 10 % (w/v)). Keywords: CCD, EC , E/S, fructofuranosidase, fructohydrolase, invertase, RSM, sucrose, syrup fructose. INTRODUCTION Invertase (β-d-fructofuranosidase EC ) which converts sucrose into glucose and fructose is common in many higher plants. Invertase is the enzyme that hydrolyzes the o-glycoside bond of sucrose to produce glucose and fructose. Invertase has been founding in bacteria, fungi, and plants. Invertase is an enzyme mainly used for the production of invert sugar in the food industry. They include acid invertase and neutral invertase, which have widely optimum ph range for their activity. Acid invertase is an important industrial enzyme. They have applied in the production of invert sugars which effectively prevent crystallization in a longer time in industrial chocolate. Invertase has ability hydrolyzing under sucrose concentrations lower than 10% (wt./vol.) into a mixture of glucose and fructose and has trans-fructosylating activity under sucrose concentrations higher than 10% (wt./vol.). Invertase has been widely studied, especially in the yeasts Saccharomyces cerevisiae 38A for the continuous alcoholic fermentation (Fontana, Ghommidh, Guiraud, Navarro, 199); Saccharomyces cerevisiae strain FF1837 (MAT a leu 3-11, trp1-89, ura3-5, ade5, conr) and Schwanniomyces occidentalis strain RK7 (UC7647 (ade) (Costaglioli, Meilhoc, Janatova, Klein, Masson, 1997) and in filamentous fungi such as Aureobasidium sp. ATCC 054 (Hayashi et al., 199) and Aspergillus niger (Romero-Gomez et al., 000). Invert sugars can be produced by acid, enzyme hydrolysis or/and thermal means in acid environment. But enzyme hydrolysis is more efficient, convenient, environmental friendly than others in converting sucrose to glucose and fructose. Invert sugar syrup is a valuable sweetener in food, pharmaceutical, distillery and other industrial application because of its functionally more desirable properties They has important applications in beverage for fresh lemon juice and food industries for making jams, preventing crystallization of cream (cream of tartar), artificial honey (invert sugar cream), caramello candy bars. In present study, we have optimized physical and chemical factors in the process of produce using the Response Surface Methodology-Central Composite Designs to obtain the maximum enzymatic activity (high invert sugar) from acid Invertase from NOVO Nordisk. Ho Chi Minh City University of Industry, Viet Nam 8

2 MATERIALS AND METHODS Tran Hong Thang et al. Materials Invertase (00000 SU/g) was separated and purified from the production Saccharomyces cerevisiae (NOVO Nordisk, Copenhagen, Denmark). It was produced by submerged fermentation of yeast and stored in maltodextrin was used in the experiments. Sucrose: The substrate was acquired from Guangdong Guanghua Chemical Co., Ltd. This was used in a 10% (w/v) solution, containing di-sodium phosphate-citric acid buffer, 0.1 M, in the expected ph. Invertase activity assay The reducing sugars produced by sucrose hydrolysis were measured by the Willstaetter - Schudel method. One unit of activity was defined as the amount of enzyme that produces reducing sugar equivalent to 1µmol of glucose per min. Reducing sugar measure The invertase activity of every experiment was assayed as follows: amount of experimental enzyme solution was added to experimental sucrose solution (0.3 M acetate buffer, experimental ph) and incubated for 4 hours at experimental temperature (experimental design). The total volume was 5 ml. The reducing sugars produced by sucrose hydrolysis were measured by the Willstaetter - Schudel method. The Willstaetter - Schudel method Add 1mL analyzed sample to 50 ml Erlen - Meyer flash, 10 ml 0.1N iod and 15mL 0.1N NaOH. The dripping time of NaOH should spend in minutes. After that, the solution was let it undisturbed in darkness. 0 minutes later, take it out and add ml HSO4 N. The remaining iod was titrated by 0.05N Na S O 3 with starch solution as indicator. Glucose was calculated by formula: cos Vo Vt Glu e = ( g / ).0x10 l 3 Where: V o : Volumes of solution 0.05N Na S O 3 using titrate in the blank sample (replacing analyzed sample by distilled water). V t : Volumes of solution 0.05N Na S O 3 using titrate in the analyzed sample. Response Surface Methodology-Central Composite Design Three major factors determine the RSM-CCD was optimized and the value was studied in five levels (-α, -1, 0, +1, + α) (Table 1) in the CCD 0 experiments (Table ) (Castillo, 007). Response function was chosen as the reducing sugar (Y, mg glucose/ml). Modeling is represented by equation : Y = b o + b 1 x 1 + b x + b 3 x 3 + b 11 x 1 + b x + b 33 x 3 + b 1 x 1 x + b 3 x x 3 + b 13 x 1 x 3 where Y was the predict response; b 0 intercept; b 1, b, b 3 linear coefficients; b 1, b 3, b 13 interaction coefficients; b 11, b, b 33 squared coefficients; x 1, x, x 3, x 11, x, x 33, x 1, x 3, x 13 are levels of the factors. The Design expert gave the response curves and they were used for determining the optimum level of the factors for maximal reducing sugar. Ho Chi Minh City University of Industry, Viet Nam 83

3 Conference proceedings of Biotechnology for Green Solutions and Sustainable Environment: 8-87, 010 Table 1. Concentrations of three factors used in RSM CCD RESULTS Factor and name Range Levels studied -α α x 1 : ph x : Temp. ( o C) x 3: E/S Optimizing the value of factors for maximum enzymatic activity Experimental planning was carried out by RSM-CCD. The result was analyzed by Design expert The response value functions of the experiments and model predictions were presented in Table. After analysis of variance (ANOVA), regression equations were used as a model to predict reducing sugar yield obtained. Production of enzymatic activity can be predicted from the model: y = x x x 1 x 3-0,84x + 0,55x 3 Where y is the reducing sugar production of enzymatic activity (mg glucose/ml); x 1 : ph, x 3 : E/S. Regression coefficient (R ) was calculated as This represents 9.88% of data that is compatible with experimental data in the model predictions. Fig. 1: Response surface production of reducing sugar Fig. : Response surface of the solution No. 1 The three dimensional response surface curve was plotted by statistically significant model to understand the interaction of the medium components and the optimum concentration of each component required for optimum reducing sugar production. Analysis of variance (ANOVA) showed that the factor x (temperature) Ho Chi Minh City University of Industry, Viet Nam 84

4 Tran Hong Thang et al. was insignificant, and x 1 (ph), x 3 (E/S) and x 1 x 3, x, x 3 were significant model terms. Therefore, the temperature was taken at center point (45 o C). The interactive effect of two variables (ph and E/S) at constant temperature was depicted in Fig.1. Response surface (Fig. 1) production of reducing sugar showing the interaction of each factor and from this graph we can determine the optimal value of each factor makes to reach maximal response levels. Table. Experimental plan for optimization of reducing sugar production using response surface methodology Run 1-1 Coded and actual value Reducing sugar production (mg glucose/0ml) x 1 ph x Temp. x 3 E/S Observed Predicted α α α α α α Experimental data was deeply analyzed to understand and take full advantage of Design Expert program. The results show that there was 7 solutions (Table 3) when factor value was set at in range study and the response was set maximize. Among 7 solutions, from solution 1 st to 9 th, the reducing sugar yield reached the maximum value (7.134 mg/0ml) with ph lower (4.0 +/- 0.1). Solution 1 st was choice. The optimal levels of two factors were found as the most suitable condition follows: at ph 4; ratio enzyme/substrate (E/S) The predicted maximum reducing sugar is mg/mL (initial sucrose 10 % (w/v)). The reducing sugar decreased with crease in ph. This demonstrates that invertase acts in acidic ph. At 7 th solution, ph and E/S is same to 1 st solution but the temperature differ (41.44 o C). Model predicted reducing sugar was while 1 st is We suggest that the next study needs to expand study range to find out optimal temperature. DISCUSSION In this study, we haven t found out optimal temperature. In our opinion, the reducing sugar is orient increase towards higher temperature. Ho Chi Minh City University of Industry, Viet Nam 85

5 Conference proceedings of Biotechnology for Green Solutions and Sustainable Environment: 8-87, 010 Table 3. Solutions when was set up ph, temperature, E/S: in range; reducing sugar production: maximize Solution ph Temp. E/S Reducing sugar Desirability CONCLUSION The optimal levels of two factors were found as the most suitable condition follows: at ph 4; ratio enzyme/substrate (E/S) Within o C, the temperature has been insignificant difference yet in this study (95%). The predicted maximum reducing sugar is mg/mL (initial sucrose 10 % (w/v)). Acknowledgements We gratefully acknowledge Global biotechnology development Company limited (GBD Co., Ltd.)- Viet Nam provided Invertase obtained from AEC- Viet Nam. The authors thank executive committee of Institute of Biotechnology and Food technology Ho Chi Minh City University of industry create favorable conditions for this investigation. REFERENCES Castillo E Del, (007). Process Optimization A Statistical Approach. Springer Science. New York, USA: Costaglioli, P., Meilhoc, F., Janatova, I., Klein, R., Masson, J., (1997). Secretion of invertase from Schwanniomyces occidentalis. Biotechnol Lett 19(7): Ho Chi Minh City University of Industry, Viet Nam 86

6 Tran Hong Thang et al. Fontana, A., Ghommidh, C., Guiraud, J., Navarro, J., (199). Continuous alcoholic fermentation of sucrose using flocculating yeast. The limits of invertase activity. Biotechnol Lett 14(6): Romero-Gomez, S., Augur, C., Viniegra-Gonzalez, G., (000). Invertase production by Aspergillus niger in submerged and solid-state fermentation. Biotechnol Lett (15): Ho Chi Minh City University of Industry, Viet Nam 87