Optimization of pre-treatment conditions for maximum pulp recovery with optimum quality from bael fruit ( Aegle marmelos Correa . )

The effect of pH, heating temperature, heating time and crude mass to water ratio on yield, total soluble solids and ascorbic acid content of pulp were studied. A central composite design was used to optimize the conditions of pulp extraction from bael fruit to maximize pulp yield, total soluble solids and maximum retention of ascorbic acid in pulp. The regression model describing the changes of pulp yield, total soluble solids (TSS) and ascorbic acid of pulp with respect to pretreatment conditions were derived with the coefficient of determination (R), 0.9883, 0.9814 and 0.9554, respectively. The models were found to be fit to predict the data under the different ranges of conditions. The pulp yield, TSS and ascorbic acid content of pulp obtained from bael fruit was 93.5 to 245.30%, 11-22°brix and 8.30-11.62 mg/100 g under the designed ranges, respectively. The optimized pretreatment conditions were pH; 3.3, heating time; 0.5 min, heating temperature; 70°C, and crude mass to water ratio; 1:0.8 and the pulp yield, TSS and ascorbic acid content under these conditions were 148.75%, 16.5°brix and 10.486 mg/100 g, respectively.


INTRODUCTION
The bael fruit (Aegle marmelos Correa.family: Rutaceae) is an important indigenous fruit of Asia and is known by different names such as Bael, Bel, Bengal Quince, Bil, Bilva, Bilpatre, Shul, Shaiphal, Vilvum etc.The ripe fruit of bael is sweet aromatic, nutritious and very palatable, being eaten by all classes of people.The excellent aroma of fruit is not destroyed even during processing therefore the fruit has untapped potential for processing.These products being highly nutritive and therapeutically important can be very easily popularized in Indian as well as international markets (Kaushik et al., 2000).
Because of Bael fruit's hard shell, mucilaginous texture and numerous seeds are not popular as fresh fruit.Although, excellent flavor, nutritive and therapeutic value of bael fruits show greater potentiality for processing into value added products (Ram and Singh, 2003).Bael fruit has been attributed with various nutritional and therapeutic properties such as in the cure of chronic *Corresponding author.E-mail: rbsanurag@gmail.com,Tel: +91-9456022578
Bael can be processed into products like preserves, refreshing beverages, powder, leather, squash, nectars, toffee, jam, syrup etc.The basic requirement for preparation of all these products is the pulp of bael fruit.The mucilaginous texture, fibres and numerous seeds make the extraction of pulp tough one.Rapid browning and development of off flavor due to enzyme activity make the extraction more complicated.The bael fruit pulp extracted by passing through the sieve without addition of water results in very sticky pulp.The pulp so obtained is unfit for handling and nearly 10% of pulp is lost during extraction, partly left with the pomace and partly sticking to the sieve (Roy and Singh, 1979;Shrestha, 2000).
This may be due to mucilage content of the pulp.Incorporation of water and application of heat coupled with acid dilute the mucilage considerably and make the pulp possible to extract easily (Roy and Singh, 1979).Kenghe and Potdar (2009) stated that water addition up to the ratio 1:1 does not impair the quality of extracted pulp even facilitate the pulp extraction.Ghosh and Gangopadhyaya (2002) extracted pulp by addition of water to pulp in proportion of 1:1 and 2:1 and centrifugation of the pulp at 4000 rpm for 10 min.Singh and Nath (2004) extracted pulp by adding water in the ratio of 1:1.25, heating at 90°C for 2 min while maintaining the pH 4.2 by 50% citric acid solution.Roy and Singh (1979) extracted pulp by adding 1:1 water, maintain the pH 4.3 by 0.5% citric acid and heating the mixture at 80°C for 1 min and passing through 20 mesh sieve.The yield by the method given by Roy and Singh was 125%.
Although, few workers have reported the development and evaluation of bael products (Verma and Gahlot, 2007;Rakesh et al., 2005;Roy and Singh, 1979), yet, there is a paucity of literature on processing technology of bael fruit pulp.This study was therefore carried out to optimize the pulp extraction method using response surface methodology.

MATERIALS AND METHODS
Fully ripe fresh bael fruits (A.marmelos Correa.) of Kagazi variety without any visual blemishes were procured from Agricultural farm of R.B.S. College, Bichpuri, Agra (India).The bael fruits were broken by hammering and the crude mass (pulp with seeds and fibres) was scooped out with the help of stainless steel spoon.The scooped crude mass was homogenized by blending manually.This crude mass was used to extract pulp.

Selection of relevant variables and experimental ranges
The extraction method involves pre-treatment parameters mainly pH, heating time, temperature and crude mass to water ratio.Therefore, the initial step was the selection of experimental ranges for the pre-treatment parameters as independent variables.The experimental ranges for the independent variables were selected as pH in the range of 3-5, heating time 0.5-2.5 min, heating temperature 70-90°C and crude mass to water ratio in range of 1:0.5-1:1.5 with respect to the reported literature and existing empirical knowledge (Roy and Singh, 1979;Kenghe and Potdar, 2009) (Table 1).

Experimental design and statistical analysis
Response surface methodology (RSM) was adopted in the experimental design.A three-level four-factor central composite face centered design was employed.The independent variables studied were pH (X 1 ), heating time (X 2 , min), heating temperature (X 3 , °C) and crude mass to water ratio (X 4 ), while response variables were pulp yield (%), TSS (°brix) and Ascorbic acid (mg/100 g).The 100 g of the crude mass was taken for every experiment and a total of 30 experiments were conducted.The range and experimental design matrix in coded (x) form and at the actual level of variables is given in Table 2.The response function (Y) was related to the coded variables (xi, i=1, 2, and 3) by a second degree polynomial equation (Equation 1): The

Extraction
For each experiment, 100 g of crude mass (pulp with seeds and fibres) was added with water, pH was adjusted with 10.0% citric acid solution and subjected to various processing conditions as given in Table 2.The temperature of the mixture 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 treatment, the heated mixture was then sieved through 20 mesh screen.All the fibres and seeds were removed during sieving and the extract thus collected was considered as pulp for commercial use.

Evaluation of pulp yield, TSS
The pulp yield was calculated by following the standard method as reported by Singh et al. (2012).The TSS (°Brix) of the pulp was observed by Erma hand refractometer.

Determination of ascorbic acid
The ascorbic acid content of the pulp was determined by standard method given by Ranganna (1997).The method is based on the reduction of 2, 6-dichlorophenol indophenol by ascorbic acid.The dye which is blue in alkaline solution and red in acid solution is reduced by ascorbic acid to a colorless form.
The dye was standardized against standard ascorbic acid solution for determination of dye factor.Then the sample extract was titrated against the dye.The ascorbic acid content was determined by the following equation: Where A is the volume in ml of standard dye used for titration, B is the weight in mg of ascorbic acid equivalent to 1 ml of indophenol  solution, that is, dye factor, V is the volume made up, D is the aliquot taken for estimation and W is the weight of the sample.

Optimization and validation of the model
Design expert version 6.0.10 (Trial version; STAT-EASE Inc., Minneapolis, MN, USA) software was used for regression and graphical analysis of the data obtained.The optimum values of the selected variables were obtained by solving the regression equation and also by analyzing the response surface contour plots.The validity and adequacy of the predictive models was done by experimental analysis at suggested optimum conditions by the design expert.

RESULTS AND DISCUSSION
The different responses under the different set of designed conditions are shown in Table 2. From the data, it is evident that the yield and quality of extracted pulp has been improved significantly from the variations in the pretreatment conditions.

The effect of processing conditions on the yield of extracted pulp
The yield of bael pulp ranged from 93.5 to 245.3% (Table 2).The minimum yield, 93.5% was observed at pH 5, time of boiling 0.5 min, temperature 70°C and crude mass to water ratio 1:0.5.The corresponding condition for maximum % yield (245.30%) was pH 3, time of boiling 2.5 min, temperature 90°C, and crude mass to water ratio 1:1.5.The response surface curves were plotted to explain the interaction of the variables and to determine the optimum level of each variable.The response surface curves for % yield are shown in Figure 1(a-d) and each figure demonstrates the effect of two factors while the other factors were fixed at middle level.Figure 1a presents the interaction effect of pH (X 1 ) and heating temperature (X 3 ) on the yield of pulp.The yield increased as the pH decreased from 5 to 3 and the temperature increased from 70 to 90°C.The yield was 245.3% maximum at the heating temperature of 90°C and pH 3.0.Any decrease in heating temperature from 90°C caused the decrease in yield.High temperature helps in extraction of adhered pulp which possibly resulted to higher yield at the higher temperature.Roy and Singh (1979) and Kenghe and Potdar (2009) reported that heating at 80°C facilitated the extraction of pulp from bael fruit and increased the yield.Chopra and Singh (2001) also reported that heating at 100°C is ideal for easy extraction of pulp from wood apple fruit.Figure 1b reveals the effect of pH and crude to water ratio on the yield of pulp.It shows that the yield followed a linear behavior with increase in water ratio (1:0.5 to 1:1.5) and decrease in pH (from 5.0 to 3.0).The yield of pulp increased up to 245.3% at pH 3.0 and crude mass to water ratio 1:1.5.Any decrease in crude mass to water ratio from its maximum ratio, 1:1.5 caused decrease in pulp yield.
Figure 1c reveals the effect of heating time (X 2 ) and temperature (X 3 ) on the pulp yield.The yield followed a linear behavior with increase in heating time and temperature up to 2.0 min of heating at 90°C.With further increase in time, the yield slightly decreased, which may be due to the loss of water during heating.The maximum yield of 236.11% pulp was observed at 90°C and 1:1.5 crude mass to water ratio (Figure 1d).

The effect of processing conditions on the total soluble solids
The results showed that the TSS ranged from 11 to 22 °brix (Table 3).The minimum TSS was at pH 5, time of boiling 0.5 min, temperature 70°C, and crude mass to water ratio 1:1.5 whereas the corresponding condition for maximum TSS was pH 3, time of boiling 2.5 min, temperature 90°C, and crude mass to water ratio 1:0.5.The interaction effect of pH (X 1 ) and heating temperature (X 3 ) on the TSS of pulp is represented in Figure 2a.TSS increased with increase in temperature and pH up to 90°C and 3.58 pH, respectively.The decrease in temperature from its maximum value, 90°C caused decrease in TSS.High temperature may facilitate extraction of pulp by dissolving the mucilage uniformly thereby may cause increase in total soluble solids (Roy and Singh, 1979).TSS was maximum, 21.09 °brix at pH 3.46 and crude mass to water ratio 1:0.5 (Figure 2b).With further increase in pH and water ratio, TSS decreased.This may be due to less acid available for hydrolysis of mucilage.The mucilage is a gummy substance chemically allied to pectin and plant gums and is comprised of protein, polar glycoprotein, exopolysaccharides, polysaccharides and uranides (Narkhede et al., 2010).
Figure 2c reveals the effect of heating time (X 2 ) and heating temperature (X 3 ) on TSS of pulp.TSS followed a linear behavior and reaches its maximum value, 15.98 °brix at heating time 2.5 min and temperature 90°C.Joshi et al. (2012) suggested that heat treatment helps in complete removal of tamarind pulp adhered to the skin.The maximum TSS, 21.60 °brix of pulp was observed at 89.84°C and 1:0.5 crude mass to water ratio (Figure 2d).Increase in water causes the dilution of pulp and therefore lowers down total soluble solid.This was significant to note that crude mass to water ratio in the proportion of 1:1.5 caused the loss of pulp characteristics.

Ascorbic acid content
The ascorbic acid content of pulp ranged from 8.30 to 11.62 mg/100 g as shown in Table 1.The minimum ascorbic acid was at pH 3, time of boiling 2.5, temperature 90°C and crude mass: water ratio:: 1:1.5.The corresponding maximum retention of ascorbic acid was at pH 3, time of boiling 0.5 min, temperature 70°C, and crude mass to water ratio 1:0.5 under the various processing conditions.The loss of ascorbic acid at higher temperature which was expected is evident.The response surface curves were plotted to explain the interaction and to determine the optimum level of each variable.The response surface plots for ascorbic acid content are shown in Figure 3a-d.
Figure 3a presents the interaction effect of pH (X 1 ) and heating temperature (X 3 ) to the ascorbic acid content of the extracted pulp.The ascorbic acid content was 9.79 mg/100 g at heating temperature 70°C and pH 4.0.Any further increase in heating temperature caused gradual decrease in ascorbic acid content of the pulp.This may be due to the heat sensitivity of ascorbic acid as high temperature aids the oxidation of ascorbic acid and forms dehydro ascorbic acid (Fenemma, 1985).
The ascorbic acid content of the extracted pulp was maximum 10.46 mg/100 g at crude mass to water ratio (1:0.5) and pH 4.0 (Figure 3b).With further increase in amount of water, ascorbic acid content gradually decreased.This may be due to the oxidation of ascorbic acid in water.The ascorbic acid readily oxidizes to dehydroascorbic acid, then to oxalic, and threonic acids in water (Stešková et al., 2006).The ascorbic acid content gradually decreased to minimum level 8.80 mg/100 g at maximum values of heating time 2.5 min and temperature 90°C (Figure 3c).
The ascorbic acid decreased linearly with increasing crude mass to water ratio and heating temperature.The maximum ascorbic acid of pulp 11.62 mg/100 g was observed at minimum heating temperature 70°C and minimum crude mass to water ratio 1:0.5.Minimum ascorbic acid was observed at temperature 90°C and 1:1.33 water ratio (Figure 3d).

Fitting of the model
The parameters of regression equations obtained by fitting of yield, TSS and ascorbic acid were given in Table 4.The fitness and adequacy of the model was judged by the coefficient of determination (R 2 ) which can be defined as the ratio of the explained variation to the total variation.The closer the R 2 value to unity, the better the empirical model fits the actual data.The coefficients of determination, R 2 , for yield, TSS and ascorbic acid are 0.988, 0.981, 0.955, respectively, suggesting a good fit.The adjusted R 2 is a corrected value for R 2 after elimination of the unnecessary model terms.The adjusted R 2 was very close to their corresponding R 2 value in both models.High values of adjusted R 2 0.977, 0.964 and 0.914, respectively for yield, TSS and ascorbic acid also advocated significance of the models for all responses.The coefficient of variation (CV) describes the extent to which the data are dispersed.The coefficient of variation is a measure of residual variation of the data relative to the size of the mean; the small values of CV give better reproducibility.
The small CV values 4.39, 4.31 and 2.38 of the responses yield, TSS and ascorbic acid, respectively revealed that the experimental results were reliable.Fvalue of 90.21, 56.46 and 22.98 for yield, TSS and ascorbic acid, respectively implied that the models were significant (P < 0.001).The corresponding variables would be more significant if the absolute F value becomes greater and the p-value becomes smaller.The F-value for all responses was greater indicating the adequecy of the models to predict different responses at different pretreatment conditions.

Optimization and verification of process variables
The main criterion for constraints optimization was maximum pulp yield, TSS and ascorbic acid.Under the constraints, the optimum treatment conditions were found to be pH 3.28, heating time 0.5 min, boiling temperature 70°C and crude mass to water ratio 0.79.But in practice, it is difficult to maintain the recommended conditions during processing and some deviation is expected.Therefore, optimum conditions were targeted as pH 3.3, heating time 0.5 min, boiling temperature 70°C and crude mass to water ratio 0.80.Under the optimum condition (target constraint), experiments were conducted to verify the adequacy of the models.The experimental values of different responses under the optimum conditions of different variables were very close to the predicted values (Table 5), with the maximum percentage deviation of 3.67.This implied that there was a high fit degree between the observed and predicted values from the regression model.

Conclusion
The present study concluded that bael fruit pulp yield, TSS and ascorbic acid are function of pH, amount of water added and time of heating.Significant regression model describing the variation of pulp yield, TSS and ascorbic acid with respect to the independent variables (pH, heating time, heating temperature and crude mass to water ratio) were established with R 2 >0.9 for all three responses.The pH and crude mass to water ratio were the most significant variable affecting the pulp yield and TSS whereas heating time and temperature were found significant variable affecting the ascorbic acid content of the pulp.The recommended conditions for the pretreatment conditions with respect to optimal quality were observed as pH; 3.3, heating time; 0.5 min, heating temperature; 70°C, and crude mass to water ratio; 1:0.8.

Figure 1 .
Figure 1.Response surfaces of pulp yield as a function of (a) Temperature and pH (b) water ratio and pH (c) temperature and time and (d) water ratio and temperature.

Figure 2 .
Figure 2. Response surfaces of TSS of pulp as a function of (a) Temperature and pH (b) water ratio and pH (c) temperature and time and (d) water ratio and temperature.

Figure 3 .
Figure 3. Response surfaces of ascorbic acid as a function of (a) Temperature and pH (b) water ratio and pH (c) temperature and time and (d) water ratio and temperature.

Table 1 .
Experimental range and levels of the independent variables.

Table 2 .
The central composite face centered design employed for pulp extraction of bael fruit.
*Crude mass was taken as 1 and the amount of water was varied as given in the table.

Table 3 .
Analysis of variance table (partial sum of squares) for response surface quadratic models for yield, TSS and Ascorbic acid of the pulp.

Table 4 .
Regression coefficients of predicted quadratic polynomial models for the responses b p < 0.05, and c p < 0.10.

Table 5 .
Optimization of process variables with respect to pulp yield, TSS and Ascorbic acid.