) and time (x 3. ), temperature (x 2

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1 26 ASIAN JOURNAL OF DAIRY & FOOD RESEARCH MATERIALS AND METHODS Banana fruits: Bananas (Musa sp.) of variety Robusta were procured at the maturity stage 1(all green) from selected banana orchards near Anand city (India). The procured material was sorted, tagged and kept for ripening as per the method suggested by Kulkarni et.al., (2010) up to 7 th stage of maturity (full yellow with brown spots) according to Standard Banana Colour Chart. Commercial enzymes: Commercial pectinolytic enzyme i.e. Pectinex Ultra SP-L (with enzymatic activity PG U per ml, optimum ph 3.5 to 6 and temperature below 50 0 C) and cellulase enzyme i.e. Celluclast 1.5L with cellulose activity 158 U per ml were obtained from Novozymes South Asia Pvt. Ltd., Bangalore Banana pulp clarification process: Banana pulp of maturity stage 7 was clarified by using the commercial pectinolytic enzyme i.e. Pectinex Ultra SP-L and cellulase enzyme i.e. Celluclast 1.5L. Banana fruits were washed, peeled manually and cut into small pieces. Pieces were then pulped using a kitchen blender for 2 min. The pulp was divided into equal portions for enzymatic treatment. For each experiment 500g pulp was subjected to different pectinase enzyme treatment conditions as shown in Table 1. The required quantity ( %) of pectinase enzyme was added to 500g batches of banana pulp and incubated at different temperature ( C) and time ( min) and then treated with 0.05% cellulase enzyme. At the end of the treatment, the enzymes in the pulp were inactivated by heating at 90 0 C for 5 min and immediately cooled to room temperature. At this stage viscosity of the pulp samples was measured. The enzyme treated pulp was centrifuged at 2900g/15 min. The supernatant was filtered through a fine mesh nylon cloth and juice collected. Viscosity of treated pulp, yield and clarity of juice were used as basis for optimization study. Yield: The juice yield was estimated as percentage of the juice obtained based on the initial pulp. Viscosity: The viscosity of clarified banana juice was determined by using a Brookfield Viscometer (Model LVDV-II+, Brookfield Engineeri ng Laboratory, Inc.) at 100 rpm and 30 0 C temperature with spindle No. LV3 and LV4. Clarity: The clarity of the juice was determined by measuring % transmittance at a wavelength of 660 nm using Shimadzu UV VIS spectrophotometer. Distilled water was used as the reference. Experimental design and statistical analysis: Response Surface Methodology (RSM) was used to moderate the experimental designs, statistical analysis and regression model with the help of Design Expert Software Version 8 (Statease Inc.). The Central Composite Design (CCD) with a quadratic model (Box and Draper, 1987) was employed. Three independent variables namely enzyme concentration ( ), temperature ( ) and time ( ) were selected. Each independent variable 3 levels which were coded as (-)1, 0 and (+ )1. A total of 20 different combinations (including six replicates of the centre point each signed the coded value 0) were chosen in random order according to a CCD for three factors (Cochran and Cox, 1957). The experimental design in the coded (x) and actual (X) levels of variables is shown in Table 1. The responses function (y) measured were the yield, viscosity and clarity of the banana juice. These values were related to the coded variables (xi, TABLE 1: The central composite experimental design employed for enzymatic clarification of banana juice Sr. No. Enzyme Concentration (%) Temperature ( 0 C) Time (min) X 1 ( ) X 2 ( ) X 3 ( ) (+ 1) 50(+ 1) 60(-1) 2 0.1(0) 40(0) 120(0) (+ 1) 30(-1) 180(+ 1) 4 0.1(0) 40(0) 120(0) (-1) 50(+ 1) 180(+ 1) (-1) 30(-1) 60(-1) (-1) 30(-1) 180(+ 1) (0) 40(0) 120(0) 9 0.1(0) 40(0) 120(0) (-1) 50(+ 1) 60(-1) (+ 1) 50(+ 1) 180(+ 1) (+ 1) 30(-1) 60(-1) (0) 40(0) 120(0) (+ 1) 40(0) 120(0) (0) 40(0) 120(0) (0) 50(+ 1) 120(0) (0) 40(0) 60(-1) (-1) 40(0) 120(0) (0) 40(0) 180(+ 1) (0) 30(-1) 120(0) x represents the coded level of variables. X represents the actual level of variables.

2 i = 1, 2 and 3) by a second degree polynomial using the equation below. y = b 0 + b 1 + b 2 + b 3 + b 12 + b 23 + b 33 + b b b 33 2 The coefficients of the polynomial were represented by b 0 (constant term), b 1, b 2 and b 3 (linear effects), b 11, b 22 and b 33 (quadratic effects), and b 12, b 13 and b 23 (interaction effects). The analysis of variance (ANOVA) tables were generated and the ef fect and r egr essi o n coefficients of individual linear, quadratic and i nteracti on terms were determi ned. The significances of all terms in the polynomial were judged statistically by computing the F-value and compared with standard significance level of 0.1%, 1% and 5%. The regression coefficients were then used to make statistical calculation to generate contour maps from the regression models. RESULTS AND DISCUSSION The experimental values for responses viz. yield, viscosity and clarity under different treatment conditions are presented in Table 2. The regression coefficients for second order polynomial equations and results for linear, quadratic and interaction terms are presented in Table 3. The statistical ananlysis indicates that the proposed model was adquate, possessing no lack of fit and with satisfactory values of the R 2 for all the responses. The R 2 values for yield, viscosity and clarity were , and respectively. To aid visualization, the response surfaces for yield, viscosity and clarity are shown in Fig. 1 (a-d). Effect of enzyme concentration, temperature and incubation time on yield of banana juice: The enzyme concentration and time (p< 0.01) affected the yield in both linear and quadratic manners (Table 3). Both independent variables showed a positive effect on linear terms but showed a negative effect on its quadratic terms (p< 0.01).Temperature also affected the yield at quadratic terms with a negative effect (p< 0.01). The interaction effect between enzyme concentration and temperature was negative (p< 0.01); whereas it showed a positive interactions between enzyme Vol. 33, No. 1, concentration and time (p< 0.05) and temperature and time (p< 0.05). The interaction effect between enzyme concentration and time to yield as shown in Fig. 1(a) reveals that yield of banana juice increased conti nuously wi th i ncrease of enzyme concentration up to 0.14%, further increase in enzyme concentration slightly decreased the yield. The yield increased slowly with increase of incubation time up to certain level and then it remai ned constant. Vi quez et al. (1981) observed same trend of increased in yield of banana j uice when treated wi th pecti nase enzyme Fig. 1(b) reveals the interaction effect between incubation time and temperature to yield. It was observed that at constant enzyme concentration (0.1%) initially as temperature increased (up to 40 0 C) yield increases but further increase in temperature decreased the yield. This may be due to denaturation of enzymes at high temperature. Effect of enzyme concentration, temperature and incubation time on viscosity of banana pulp: From Table 3 it may be seen that process variables had a negative effect on viscosity at their linear terms showing a significant level of p< 0.01 whereas their quadratic effect on viscosity was found TABLE 2: Effect of enzyme concentration, temperature and time on three dependent variables for pulp of maturity stage7 Factors Responses Run No. Enzyme Concentration (%) Temp (0C) Time (min) Yield (%) Viscosity (cps) Clarity (% T)

3 28 ASIAN JOURNAL OF DAIRY & FOOD RESEARCH TABLE 3: Regression coefficients, R 2 and mean and SD value for dependant variables Regression coefficient Yield (%) Viscosity (cps) Clarity (%) b b * * 6.83* b * 0.28 b * * 4.91* b * b ** b ** * ** b * * * b * 73.55* -6.80* b * 68.44* -6.09** R Mean SD Subscripts 1= Enzyme concentration, 2= Temperature, 3= Incubation time * Significant at 0.01 level ** Significant at 0.05 level *** Significant at 0.1 level a b c d FIG. 1: (a-d) Response surface for a) yield of banana juice at temp 40 0 C b) yield of banana juice at enzyme concentration 0.1% c) viscosity of banana pulp at enzyme concentration 0.1% d) clarity of banana pulp at time 120 min

4 to be positive (p< 0.01). The interaction effect between temperature and time was negative (p< 0.1). Fig.1(c) depicts the interaction effect between incubation temperature and time to viscosity at enzyme concentration (0.1%). The increased incubation temperature from 30 to 40 0 C with incubation time of 60 to 140 min decreased the viscosity. While, with increase of incubation temperature and incubation time over 40ºC and 140 min respectively increased the juice viscosity. The results are in line with observations of Lee et al. (2006) who reported that higher level of temperature (above 40 0 C) slowly increased the viscosity. Effect of enzyme concentration, temperature and incubation time on clarity of banana juice Enzyme concentration and time significantly (p< 0.01) affected the clarity in both linear and quadratic manners (Table 3). Both these independent variables showed a positive effect on linear terms but showed a negative effect on its quadratic terms. Quadratic effect of temperature was also found be negative (p< 0.01). It can be observed from Fig.1(d), increased in temperature (upto 43 0 C) and enzyme concentration (up to 0.12%) was found to increased the clarity but further increased in both these parameters slightly decreased the clari ty. Tadakittisarn et al. (2007) observed same trend of increased in clarity of banana juice. Optimization of process parameters for clarified banana juice: The effect of various process parameters on the responses were studied in the previous section. These process parameters viz. enzyme concentration, temperature and time were optimized for the clarification of banana pulp by Numerical Optimization. The criterions applied for numerical optimization were maximum yield, minimum viscosity and maximum clarity. The criteria set above produced the graphs of optimum enzymatic conditions of the clarification process for respective responses viz. yield, viscosity and clarity as shown in Fig. 2 (a-c) and the desirability graph of the most desirable combination for clarification of banana pulp at stage 7 as shown in Fig.3. Vol. 33, No. 1, Fig. 2(a) shows the contour plots of yield of banana juice as a function of process variables and i t can be observed that as enzyme concentration increases the yield of banana juice gets increases. At optimum conditions of enzyme (0.11%), temperature ( C) and time ( min) the yield ± 1.62 per cent can be obtained. Fig. 2(b) it can be noted that as enzyme concentration increases viscosity decreases. The viscosity of ± cps and clarity of ± 2.86 per cent (Fig. 2c) can be obtained in clarified banana juice at optimum enzymatic conditions. Fig.3 shows the desirability graphs for the most desirable enzymati c combi nati on for clarification of banana pulp at stage 7. The process variables for the best combination of r esponse funct i ons ar e 0.11% enzym e concentrati on, temperature C and incubation time min. The response functi ons were calculated from the fi nal polynomial and the response at this optimized combination were yield (75.11%), viscosity ( cps) and clarity (94.05%). Lee et al., (2006) also optimized the conditions for enzymatic clarification of banana pulp of stage 7 and optimized conditions reported were: 0.084% enzyme concentration, C incubation temperature and 80 min incubation time. Slight variations in optimum conditions were observed as compared to present study may be due to the different varieties of bananas used in both the investigations. CONCLUSION The present study concluded that viscosity of banana pulp, yield and clarity of banana juice are the functions of different enzymatic treatment conditions viz. enzyme concentration, incubation temperature and time. Statistical analysis using RSM appeared to be a valuable tool for optimizing the effects of applied enzymatic treatment conditions on enzymatic clarification of banana pulp. The recommended enzymatic clarification condition for the banana pulp of maturity stage 7 was 0.11% enzyme concentration at C for min to achieve maximum juice yield (75.11%) and clarity (94.05%) and minimum pulp viscosity (372 cps).

5 30 ASIAN JOURNAL OF DAIRY & FOOD RESEARCH FIG 2: (a-c) The contour plots of a) yield of banana juice b) viscosity of banana pulp c) clarity of banana juice as a function of enzyme concentration, temperature and time (at time min) FIG 3: Desirability graph as a function of enzyme concentration, temperature and time (at time min)

6 Vol. 33, No. 1, 2014 REFERENCES Baumann, J. W. (1981). Application of enzymes in fruit juice technology. In G. G. Birch, N. Blakebrough, & K. J. Parker (Eds.), Enzymes and food processing (pp ). London: Applied Science Publishers Ltd. Box, G. E. P. and Draper, N. (1987). Empirical model-building and response surfaces. New York: John Wiley. Cheirsilp, P. and Umsakul, K. (2008). Processing of banana-based wine product using pectinase and a-amylase. Journal of Food Process Engineering, 31: Cochran, W. G., and Cox, G. M. (1957). Some methods for the study of response surfaces. In Experimental designs (2nd ed., pp ). New York: John Wiley and Sons, Inc. FAO (2012). Banana Statistics Available Source: 567# ancor Koffi, E. K, Sims, C. A. and Bates, R. P. (1991). Viscosity reduction and prevention of browning in the preparation of clarified banana juice. Journal of Food Quality, 14: Kulkarni, S.G., Kudachikar, V.B. and Prakash M.N. (2010). Studies on physico-chemical changes during artificial ripening of banana (Musa spp) variety Robusta. J Food Sci. Technol. (online journal DOI /s y) Lanzarini, G. and Pifferi, P. G. (1989). Enzymes in the fruit juice industry. In C. Cantavelli & G. Lanzarini (Eds.), Biotechnology applications in beverage production (pp ). New York: Elseveir Science Publishers Ltd. Lee, W.C., Yusof, S., Hamid, N.S.A. and Baharin, B.S. (2006). Optimizing conditions for enzymatic clarification of banana juice using response surface methodology (RSM). Journal of Food Engineering 73: Tadakittisarn S, Vichai Haruthaithanasan, Penkwan Chompreeda and Thongchai Suwonsichon (2007). Optimization of Pectinase Enzyme Liquefaction of Banana Gros Michel for Banana Syrup Production. Kasetsart J. (Nat. Sci.) 41 : Viquez, F., Laetreto, C., and Cooke, R. D. (1981). A study of the production of clarified banana juice using pectinolytic enzymes. Journal of Food Technology, 16: Yusof, S., and Ibrahim, N. (1994). Quality of soursop juice afterpectinase enzyme treatment. Food Chemistry, 51: