Food Bioprocess Technol (2009) 2:96–100 DOI 10.1007/s11947-008-0119-1
COMMUNICATION
Optimization of Enzymatic Hydrolysis Pretreatment Conditions for Enhanced Juice Recovery from Guava Fruit Using Response Surface Methodology S. Kaur & B. C. Sarkar & H. K. Sharma & Charanjiv Singh
Received: 6 February 2008 / Accepted: 1 July 2008 / Published online: 29 August 2008 # Springer Science + Business Media, LLC 2008
Abstract The effect of enzyme concentration (0.16– 0.84 mg/100 g guava pulp), incubation temperature (36.6– 53.4 °C), and incubation time (0.95–11 h) on juice yield was studied. A central composite rotatable design was used to establish the optimum conditions for enzymatic hydrolysis of guava to obtain maximum juice yield. Significant regression model describing the changes of juice yield with respect to hydrolysis parameters were established with the coefficient of determination, R2 =0.85. Enzyme concentration was the most significant variable affecting the juice yield. The recommended enzymatic treatment condition from the study was at the enzyme concentration 0.70 mg/ 100 g guava pulp, incubation time 7.27 h, and incubation temperature 43.3 °C. Keywords Guava juice . Enzymatic hydrolysis treatment . Response surface methodology . Pectinase
Introduction Guava (Psidium guajava L.) is one of the most important tropical fruits containing high concentration of vitamin A, ascorbic acid, and lycopene (Kumar and Honda 1994). The ripened guava contains total solids 8.0–15.3° Brix, pectin 0.62%, total sugars 10.0–15.3%, reducing sugar 2.05– 6.08%, ascorbic acids 88.2–250.80 mg/100 g, and acidity 0.08–0.54%.(Kumar and Honda 1994; Chatterjee et al. 1992). It is highly perishable, finishing its ripening process S. Kaur : B. C. Sarkar (*) : H. K. Sharma : C. Singh Department of Food Engineering and Technology, Sant Longowal Institute of Engineering and Technology, Longowal, 148 106 Punjab, India e-mail:
[email protected]
in a few days after harvest when kept at ambient temperature (Kashyap et al. 2001). Because of this, it is processed and preserved in different forms such as pulp, jam, juice, squash, nectar, and dehydrated and canned products. It is mainly harvested at the green maturity stage (Bleinroth 1996) and stored at room temperature with a maximum shelf life of 5–6 days. Guava has gained considerable importance because of its high nutritive value (Sharma et al. 1999; Chatterjee et al. 1992), availability at moderate price, a pleasant aroma and good flavor, and has global consumption. The fruit is mainly consumed as fresh. Tropical fruit juices have become important in recent years due to the overall increase in natural fruit juice consumption as an alternative to the traditional caffeine containing beverages such as coffee, tea, or carbonated soft drinks. Preparation of guava juice is being considered as the most promising method of utilization of guava throughout the year (Kaur 2007). Despite vast potential of either pure or mixtures with other juices (Brasil et al. 1995), guava juice has not been exploited for its commercialization. Generally, three methods of juice extraction are employed viz, cold, hot, and enzymatic methods (Sreekantiah et al. 1971). The use of fungal enzyme in fruit juice extraction had shown significant increase in juice recovery as compared to cold and hot extraction methods (Joshi et al. 1991; Solehah et al. 1964). The enzymes, mainly pectinases, assist in pectin hydrolysis, which cause a reduction in pulp viscosity and a significant increase in juice yield (Pilnik and Voragen 1993; Solehah et al. 1964). The enzymatic hydrolysis of pectic substances depends on several processing variables such as type of enzyme, hydrolysis time, enzyme concentration, incubation temperature, and pH (Neubeck 1975; Baumann 1981). These parameters need to be optimized for maximal juice
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recovery. Diwan and Shukla (2005) developed a process for the production of guava juice using purified enzyme at 2% concentration and 20 h incubation time. The limitations of the process is that the purified pectinase enzyme is very costly ($50/10 g) and the processing time(20 h) is high; new treatment combinations were felt necessary to be explored. The objective of the present study is to optimize the hydrolysis pretreatment parameters (enzyme concentration, time of treatment, and incubation temperature) for the maximal juice yield from guava pulp.
Materials and Methods Materials Fully ripe fresh guavas of variety “L-49 var” without any visual blemishes were purchased from a private orchid in District Sangrur, Punjab, India. The fruits were wrapped in paper and stored in refrigerator (4 °C) for a maximum period of 5 days before use. Enzyme Source Commercial pectinase (M/s SRL Research Chemicals, India) from the source organism Aspergillus niger with
Table 1 The central composite rotatable experimental design employed for enzymatic hydrolysis pretreatment of guava pulp
In coded and uncoded levels of three variables and five levels
Experiment no.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
activity 3.5–7 units/mg was used for enzymatic treatment of guava pulp. Experimental Design and Statistical Analysis Response surface methodology (RSM) was adopted in the experimental design as it emphasizes the modeling and analysis of the problem in which response of interest is influenced by several variables and the objective is to optimize this response (Montgomery 2001). The main advantage of RSM is reduced number of experimental runs needed to provide sufficient information for statistically acceptable results. A five-level three-factor central composite rotatable design was employed (Myers 1976). The independent variables were the temperature of enzyme treatment (X1), time of treatment (X2), and used enzyme concentration (X3). The variables and their levels were chosen based on the limited literature available on enzymatic hydrolysis of guava (Diwan and Shukla 2005; Brasil et al. 1995). These were the temperature (X1; 36.59– 53.41 °C), time (X2; 0.95–11.05 h) of the enzymatic treatment, and concentration of enzyme used (X3; 0.16– 0.84 mg/100 g pulp). The pH of the pulp was kept at its natural value (4.0–5.2) and was excluded from the RSM experimental design as the pH range is optimal for the exogenous pectinases (Grassin and Fauquembergue 1995). The three independent variables were coded as −1.682
Coded variables
Uncoded variables
x1
x2
x3
0 +1 −1.682 0 +1 0 −1 0 −1 0 0 0 −1 0 −1 0 +1 +1 +1.682 0
0 −1 0 0 +1 0 −1 0 +1 −1682 0 0 −1 0 +1 +1.682 −1 +1 0 0
0 −1 0 0 −1 0 +1 0 +1 0 +1.682 −1.682 −1 0 −1 0 −1 +1 0 0
Temperature X1 (°C)
Time X2 (h)
Enzyme concentration X3 (mg/100 g)
45.00 50.00 36.59 45.00 50.00 45.00 40.00 45.00 40.00 45.00 45.00 45.00 40.00 45.00 40.00 45.00 50.00 50.00 53.41 45.00
6.00 3.00 6.00 6.00 9.00 6.00 3.00 6.00 9.00 0.95 6.00 6.00 3.00 6.00 9.00 11.05 3.00 9.00 6.00 6.00
0.50 0.30 0.50 0.50 0.30 0.50 0.70 0.50 0.70 0.50 0.84 0.16 0.30 0.50 0.30 0.50 0.70 0.70 0.50 0.50
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Food Bioprocess Technol (2009) 2:96–100
Table 2 Effect of enzyme concentration, incubation temperature, and incubation time on juice yield
(xi, i=1, 2, and 3) by a second degree polynomial equation (Eq. 1) as given below:
S. no.
y ¼ b0 þ b1 x1 þ b2 x2 þ b3 x3 þ b12 x1 x2 þ b13 x1 x3
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Unhydrolyzed sample a
Coded levels
Dependent variable
X1
x2
x3
Juice yield (%)
0 +1 −1.682 0 +1 0 −1 0 −1 0 0 0 −1 0 −1 0 +1 +1 +1.682 0
0 −1 0 0 +1 0 −1 0 +1 −1.682 0 0 −1 0 +1 +1.682 −1 +1 0 0
0 −1 0 0 −1 0 +1 0 +1 0 +1.682 −1.682 −1 0 −1 0 +1 +1 0 0
73.8 69.6 69.8 74.2 73.5 74.5 73.1 73.8 79.3 65.7 77.3 67.4 63.0 73.8 71.0 74.7 72.5 75.9 71.8 74.2 37.43+0.68a
n=3
(lowest level) −1, 0, 1 (middle level), and +1.682 (highest level). The experimental design matrix in coded (x) form and at the actual level (X) of variables is given in Table 1. The response function (y) was related to the coded variables
Table 3 Analysis of variance table [Partial sum of squares] for response surface quadratic model (Eq. 3)
Sum of squares
DF
Source model x1 x2 x3 x12 x22 x32 x1 x2 x1 x3 x2 x3 Residual Lack-of-fit Pure error Cor total R2 Adj R2 Pred R2 PRESS Adeq precision
276.73 5.25 98.28 119.22 11.03 17.03 1.54 5.95 21.45 0.66 6.04 5.61 0.43 282.77 0.978 0.959 0.8470 43.28 29.611
þ b23 x2 x3 þ
b11 x21
þ
b22 x22
þ
b33 x23
ð1Þ
þ"
The coefficients of the polynomial were represented by b0 (constant), b1, b2, b3 (linear effects); b12, b13, b23 (interaction effects); b11, b22, b33 (quadratic effects); and ε (random error). The statistical analysis of the data and three-dimensional (3D) plotting were performed using Design Expert software ‘DE-6’. Enzymatic Treatment and Juice Yield For each experiment, 100 g of pulp was subjected to different enzyme treatment conditions, as given in Table 1. The temperature of the enzymatic treatment combinations was adjusted to the desired level (±0.5 °C) by using a high precision water bath (Seco, Model 129, India). At the end of the enzyme treatment, the suspension was centrifuged at 3,000 rpm for 10 min (Model R8C, Remi Equipments, India) and the supernatant was heated at 90 °C for 5 min to inactivate the enzyme (Rai et al. 2004) using the same water bath. The supernatant thus collected was considered as clear juice (Kaur 2007). The juice yield was then calculated using the following expression: Juice yield; % ¼
Weight of clear juice *100 Weight of sample
Mean square
F value
Prob > F
9 1 1 1 1 1 1 1 1 1 10 5 5 19
30.75 5.25 98.28 119.22 11.03 17.03 1.54 5.95 21.45 0.66 0.60 1.12 0.087
50.90 8.68 162.68 197.34 18.26 28.19 2.55 9.85 35.51 1.09
