Response surface methodology for the optimization of kojic acid production by Aspergillus flavus using Palmyra sap as a carbon source

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1 014; (5): 5-57 ISSN: EJBB 014; (5): 5-57 Received: Accepted: Kayitha Bala Durga Devi Payala Vijayalakshmi Bapatla Veerendra Kumar Correspondence: Kayitha Bala Durga Devi Response surface methodology for the optimization of kojic acid production by Aspergillus flavus using Palmyra sap as a carbon source Kayitha Bala Durga Devi, Payala Vijayalakshmi, Bapatla Veerendra Kumar Abstract The present investigation was planned to conduct for obtaining enhanced yields of kojic acid by using response surface methodology. Initially OFAT strategy was applied to study the effect of seven different macro-micro elements on growth and out of seven variables screened, five factors Substrate concentration, Peptone concentration, Time, Temperature, ph were highly influential in favoring the production rate. In the subsequent step, these factors were furthermore optimized using central composite design and response surface analysis. From the multiple regression analysis of the experimental data, a second order polynomial model was resolved. The maximum kojic acid production obtained was 78.6 g/l under static, surface fermentation conditions at an optimized level of Substrate concentration (100 g/l), Peptone concentration (3 g/l), ph (4.0), Incubation time (3d), and Temperature (8 0 C). It was evaluated from the model that the determination co-efficient was (R =0.9561) denoted that 95.61% of the variability in the response could be interpreted by the model. Keywords: Kojic acid, OFAT, Response surface methodology, Aspergillus flavus, fermentation. 1. Introduction Some of the species of the Genus Aspergillus were capable of secreting a notable secondary metabolic i.e., kojic acid chemically called as 5-Hydroxy--Hydroxy-Methyl-4-Pyrone. This compound had advantageous applications in various industries like food, cosmetics, medical, pharmaceutical etc. So many investigations had been done by the earlier researchers to evaluate an effective and economical process of producing kojic acid. The present report criticizes on the way of kojic acid production from inexpensive C source Palmyra sap and on the process of variables optimization through OFAT method and response surface methodology. This way assists in drawing a cost-effective methodology for the production of kojic acid. Literature survey has revealed that no studies have been reported for statistical optimization of kojic acid production by RSM. Bracarense & Takahashi (014) have used fractional-factorial design to produce the bio-active metabolic kojic acid by A. parasiticus using two different synthetic media CYA and YES media. They also studied the antimicrobial activity of kojic acid against S. aureus, E.coli, C. albicans. One-factor-at-a-time method is a traditional method used for the optimization of process variables. However this method was not able to detect the factors which contribute for the optimum response since the effect of interaction among the factors was not taken as a criteria in such strategies Deepak et al. (008). Recently, statistical optimization was commonly employed in the fermentation process because it quickly screens a number of multiple parameters and their interactions, and reflects the function of individual factor or component. It constitutes a set of empirical procedures employed for evaluating the connecting link between the group of controlled experimental parameters and measured response. The biotechnological and biochemical processes concerned to food systems have utilized the RSM model successfully for the optimization of process parameters de Lima et al. (010). The present research use OFAT and RSM for statistical optimization of kojic acid production using a soil fungus A. flavus. ~ 5 ~

2 Based on the preliminary optimization experiments through OFAT method, the most significant factors substrate concentration (or) carbon source concentration, peptone concentration, ph, time, temperature were selected to find the optimal concentrations for higher kojic acid production using CCD and RSM. The robust tree Borassus flabellifer or Palmyra palm or toddy palm or sugar palm is inhabitant to the Indian subcontinent and Southeast Asia. It produces one of the most significant product sap have a dominant sugar sucrose in proportions 10.36%-16.94% which was selected as a carbon source in the present study. The nutritional composition of Palmyra sweet sap (g/100cc) is protein 0.35, total sugar 10.93, reduced sugar 0.96 and other minerals and sugars.. Materials and methods.1 Microorganism The soil isolated fungal organism Aspergillus flavus which was maintained on CZA slants at 4 0 C was used for the production.. Fermentation media and kojic acid fermentation The basal fermentation medium was prepared to contain 50ml of Palmyra sap, 0.5g peptone, 0.5g KH PO 4 and 0.5g MgSO 4.7H O was taken into a conical flask. The design of the fermentation medium was based on Ariff et al. (006). Now 5ml of spore suspension was added to the flask and incubated at 8 0 C for 1d under static conditions. All chemicals used in the study were of analytical grade and purchased from Coastal Chemicals Pvt. Ltd., Visakhapatnam, When the fermentation was finished, the broth was filtered and the mycelia mat was washed with water and kept in a hot air oven at 80 0 C for 1d and the dry weight mass was estimated. The concentration of kojic acid in the supernatant was determined by Fecl 3 method Bentley (1957) and further subjected to crystallization process. The fraction was concentrated till least volume using rotary evaporator and subjected to 0 0 C, where the crystals of kojic acid were formed. The kojic acid crystals were loaded on filter paper and washed several times with benzene. The kojic acid crystals were dissolved in ethyl acetate several times to get rid of impurities and the ethyl acetate solution was dried at room temperature where the pure crystals were appeared Saleh (006). Afterwards the dry weight of the crystal mass was measured..3 Optimization of process parameters by OFAT strategy Experiments were performed by conventional and traditional method called OFAT strategy to choose the appropriate factor for getting maximum yield of kojic acid. The ranges of Substrate concentration (10-100ml), Peptone concentration (1-5g/L), KH PO 4 (0.5-.5g/L), MgSO 4 concentration (( g/L), ph (4-8), Time (11-37d), Temperature ( C) were selected for the optimization of kojic acid production. In order to acquire very higher amounts of kojic acid, the optimal concentrations of the chosen key factors were furthermore determined..4 Further optimization by Response Surface Methodology In the present days, the highly advanced optimization strategy, RSM was usually applied for the microbial production of both primary and secondary metabolites. It was well suited to study the main and interaction effects of various factors and the production of kojic acid. Hence five-factor-three-level was used in the current study. On the basis of the best results obtained in the preliminary optimization experiments, the ranges and the levels of the most significant variables in enhancing kojic acid production were mentioned in the Table- 1. In order to expand the regression equation, the test factors were coded according to the below equation (1). x i = (X i - X i* ) / X i - (1) x i is the coded value of the i th independent variable, X i is the uncoded value of the i th independent variable, X i * is the uncoded i th independent variable at the centre point and X i is the step change value Minitab version 16.0 was used to perform the design matrix. The CCD matrix with five variables X three levels ( ) was represented in the Table-. The matrix comprises 3 runs. A second order polynomial equation () which is shown below contain all linear, quadratic and interaction terms was used to calculate the predicted response. Y = b o + b i X i + b i X i + b ij X i X j - () Y is the concentration of kojic acid (g/l) b o is the intercept b i is the coefficient for linear direct effect b i is the coefficient for quadratic effect and is responsible for curvatures of the model b ij is the coefficient for interaction effect a positive (or) negative significant value indicates possible interactions between the variables. Statistical analysis of the models was utilized to determine the ANOVA i.e., analysis of variance. The quality of fit of the polynomial model was inferred by the coefficient of determination R and its statistical significance was ascertained by the F-test whereas student s t-test was performed to determine the significance of the regression coefficients. Table 1: Independent variables in the experimental plan Independent Variables Substrate concentration (g/100 ml) Peptone concentration (g/l) Incubation time (d) ph Temperature ( 0 C) Symbols X 1 X X 3 X 4 X 5 Coded Levels Results and discussion The results of one-factor-at-a-time method reveals that the key process parameters which effects the kojic acid production ~ 53 ~ were Substrate concentration (100ml), Peptone concentration (3g/L), KH PO 4 (g/l), MgSO 4 concentration (0.5g/L), ph (4.0), Time (3d) and Temperature (8 0 C). The kojic acid

3 production obtained at these optimized conditions was 75.9g/L. 4. Central composite design matrix In order to find the optimized conditions of physicochemical factors and interactions between them a five-variable-threelevel CCD matrix was used in the present study. The concentrations of kojic acid (g/l) for each and every single run along with their experimental and predicted response were illustrated in Table-. The maximum concentration of kojic acid obtained runs of experimental value was 78.6g/L whereas in the predicted response the highest production obtained was 71.9g/L This result corroborated the validity and effectiveness of this model. However it was noted that the same conditions of optimization was observed with CCD like OFAT method. By employing RSM, the below regression equation has been obtained and it accounts for the empirical connecting link between the kojic acid and the test variables in coded units. Y = x x x x x x 1 x 0.09 x 1 x x 1 x x 1 x x x x x x x x 3 x x 3 x x 4 x x x x x x 5 Y is the response i.e., the concentration of kojic acid obtained as function of x 1, x, x 3, x 4 and x 5 test variables. For the above equation, F-test was performed to its statistical significance. Run Order Table : CCD matrix having real values along with the experimental and predicted values of kojic acid concentration substrate conc ph peptone Incubation time Temperature Experimental values of kojic acid (g/l) Predicted values of kojic acid (g/l) The result of the second order response surface quadratic between 0 and 1. If the R value is nearer to 1.0, the stronger model fitting in the form of ANOVA was shown in Table-4. the model was and better it predicts the response. The The F value of the model was and the probability adjusted determination coefficient value was also high value (P ~ 0) showed that the model terms were significant. (0.896) supporting the significance of the model. There was It was indicated by the regression equation was that R was no evidence of lack of fit. The signal to noise ratio was Table-4 which specified the fitness of the model and determined by adequate precision value and it was designates that roughly 95% of the variability in the response The ratio should be greater than 4. Hence the present model can be interpreted by the model. The value of R is in could be used to navigate the design space. ~ 54 ~

4 The F test and the correlating p values along with the parameters were evaluated and showed in Table-4. The calculated coefficients of the regression model were listed in Table-3 and that contains 5 linear, 10 quadratic, 5 interaction terms and 1 block term. For each coefficient term its significance was measured by Student s t-test and p-value. The results from the Table-3 revealed that two linear coefficients (ph, peptone concentrations) and one cross product coefficient (peptone x peptone concentration) showed significant effects on kojic acid production (p<0.05). Table 3: Model coefficients estimated by multiple linear regression (significance of regression coefficients) Term Coefficient Standard error coefficient T-value P-value Constant Substrate conc ph Peptone Incubation time Temperature Substrate conc*substrate conc ph*ph Peptone*Peptone Incubation time*incubation time Temperature*Temperature Substrate conc*ph Substrate conc*peptone Substrate conc*incubation time Substrate conc*temperature ph*peptone ph*incubation time ph*temperature peptone*incubation time Peptone*Temperature Incubation time*temperature Table 4: ANOVA for the entire quadratic model Source DF Seq SS Adj SS Adj MS F P Regression Linear Square Interaction Residual Error Lack-of-Fit * * Pure Error Total Response Surface plots and Contour plots In order to examine the interaction between different parameters and to find the optimal amount of each factor which influences in yielding higher concentrations of kojic acid, response surface plots should be traced out. The 3D surface curves were plotted across any two independent variables while keeping other variable at its control (0) level. Figure 1 indicates the interdependence of kojic acid production on peptone concentration and substrate concentration. The kojic acid production enhanced with increase in peptone concentration to about -3g/L and afterwards the production rate was declined with furthermore increase in peptone concentration. The similar propensity was noticed in Figure. Increase in the incubation temperature C ensued increase in the kojic acid production. This was apparent from Figures 3, 4, 5. Figure 6 presents the dependency of kojic acid production on ph. The effect of ph resembles to peptone concentration. The increase in ph from enhances the production of kojic acid. Increased kojic acid production was identified on Time 3d was depicted in the Figures 7, 8. The optimum conditions established for the production of kojic acid were Substrate concentration (100ml), Peptone concentration (3g/L), KH PO 4 (g/l), MgSO 4 concentration (0.5g/L), ph (4.0), Time (3d) and Temperature (8 0 C). While comparing the predicted values obtained by regression model equation to that of experimental model the results were closely related to each other. To check the validity of the experimental model, production was once again done at optimized conditions in triplicates. The result was compared with the predicted response, so that the validity of the model was proved. Under these optimized parameters the maximum production resulted was 78.6g/L and upon crystallization the broth was concentration and produced 1.94 g/l of dry crystals. ~ 55 ~

5 Fig 1: Response surface plot for the concentration of kojic acid versus peptone and substrate concentration Fig 4: Response surface plot for the concentration of kojic acid versus temperature and ph Fig : Response surface plot for the concentration of kojic acid versus peptone and ph Fig 5: Response surface plot for the concentration of kojic acid versus temperature and substrate concentration Fig 3: Response surface plot for the concentration of kojic acid versus temperature and peptone concentration Fig 6: Response surface plot for the concentration of kojic acid versus ph and substrate concentration ~ 56 ~

6 Fig 7: Response surface plot for the concentration of kojic acid versus incubation time and peptone 8. References 1. Ariff A B, Salleh M S, Gani B, Hassan MA, Rusul G. Aeration and yeast extract requirements for kojic acid production by Aspergillus flavus Link. Enzyme Microbiology and Technology 1996; 19: Bentley R. Preparation and analysis of kojic acid. Methods in Enzymology 1957; 3: Bracarense APA, Takahashi AJ. Modulation of antimicrobial metabolites production by the fungus Aspergillus parasiticus. Brazilian Journal of Microbiology 104; 45(1): Deepak V, Kalishwaralal K, Ramkumarpandian S, Venkatesh babu S, Senthilkumar S R, Sangiliyandi G. Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology. Bioresource technology 008; 99(17): De Lima JBC, Coelho FL, Jonas C. The use of response surface methodology in optimization of lactic acid production: Focus on medium supplementation, Temperature and ph control. Food technology and Biotechnology 010; 48(): Saleh RM, Kabli SA, Al-Garni SM, Mohamed SA. Screening and production of antibacterial compound from Trichoderma species against human pathogenic bacteria. African Journal of Microbiology research 011; 5(13): Fig 8: Response surface plot for the concentration of kojic acid versus incubation time and ph 6. Conclusion The central composite design and Response surface methodology facilitated in resolving the optimized parameters for the excess production of kojic acid from the economical carbon source Palmyra sap through surface fermentation with the soil isolate Aspergillus flavus. The validity of the model was verified by fitting the values of the independent variables into the regression model equation and by conducting experiments using these values. The optimization of the evaluated responses confirmed that the optimum results for kojic acid production 78.6g/L were obtained with Substrate concentration (100ml), Peptone concentration (3g/L), ph (4.0), Time (3d) and Temperature (8 0 C). Each and every point was positioned near the central point of the design. The authors declared No conflict of interest 7. Acknowledgement The authors would like to thank M/s. Orange Life sciences Pvt. Ltd., Visakhapatnam for assisting us to carry out the research work. ~ 57 ~