Extraction and process optimization of bael fruit pectin

Abstract Pectin was extracted at temperature of 100 and 120°C with two different pH conditions each, pH 1.5 and pH 2 along with different extraction ratios, ER1:1, 1:1.5 and 1:2 respectively. Data obtained were analyzed statistically using ANOVA (no blocking) at 5% level of significance, which showed that there was significant difference (p < 0.05) between the pectin extracted at 100°C, pH 1.5 and extraction ratios of 1:1, 1:1.5, and 1:2, respectively, whereas there was not any significant difference between extraction at 120°C, pH 1.5 and extraction ratios of 1:1, 1:1.5, and 1:2 respectively. Also, there was no any significant difference between pectin extracted at 100°C, pH 2, extraction ratios 1:1, 1:1.5, and 1:2 and also at 120°C, pH 2, 1:1, 1:1.5, and 1:2 respectively. Best yield of pectin was found at temperature 120°C, pH 1.5 and ER 1:1.5.


| INTRODUC TI ON
Pectin (derived from Greek meaning "congealed and curdled") is a structural hetero polysaccharide containing in the cell of terrestrial plant. It was first isolated and described by Heneri Bracannot in 1825. Pectin is a purified carbohydrate product obtained from the inner portion of the rind/peels of citrus fruits. It consists chiefly of partially methoxylated polygalacturonic acid. Pectin is capable of forming gels with sugar and acid under suitable conditions. It is formed almost universally in plant cell of all species suitable for use in the production of sugar jellies and industrial production of apple pomace, citrus peels, and sugar beet chips. Pectin extracted from various materials can be different in molecular structure (i.e., molecular weight, degree of esterification, and acetyl content) and therefore possesses different functional properties. Typically, a whole mature fruit contains 3%-7% pectin substances on a dry weight basis and 0.1%-1.1% on a fresh weight basis. The relatively high pectin and low caloric content of citrus fruits make them a good source of soluble dietary fiber (Joslyn, 1980). A valuable by product that can be obtained from fruit wastes is pectin. Pectin designates those water soluble pectinic acid (colloidal polygalacturonic acids) of varying methyl ester content and degree of neutralization which is capable of forming gels with sugar and acids under suitable conditions. The suitability of pectin for different purposes is determined by their character via, anhydrouronic acid content (AUA) methoxyl content, degree of esterification, and acetyl values. Pectin, the gelatinizing agent is natural to fruits and it also occur in many other vegetable products but the amount and quality of pectin depend upon the type of fruits and vegetables maturity and method adopted for the extraction of pectin (GITCO, 1999).
The bael fruit is known for its medicinal properties and is one of the most nutritious fruits. It contains 61.5 g of water, 1.8 g of protein, 1.7 g of minerals, 31.8 g of carbohydrates, and 1.19 mg of riboflavin/100 g edible portion. Its food value is 88 calories/100 gm.
Thus, it is richer than most of the reputed fruits like apple, guava, and mango which have a calorific value of only 64, 59 and 36 respectively. It may be noted that no other fruits have such a high content of riboflavin. Bael fruit can be stored for 10-15 days at normal temperature, whereas fruit harvested at ripe stage can be stored for a week. After extraction of bael pulp used for the preparation of various fruit products viz. preserve, candy, jam, RTS, nectar squash/ leather/slab, powder etc., which can be commercially exploited (ITDG, 2002).
This research is about pectin extraction from bael fruit which has anti-microbial activity. It gives clear edge over commercial citrus pectin for exploitation in food and pharmaceutical. Bael fruit is an under-utilized fruit. Though abundantly found, its products are limited to home scale only. The commercial value is very low. Bael has very strong taste, its consumption as fruit is not generally preferred. It is highly nutritious in terms of vitamin and dietary fiber. The processing of bael products are in a primitive stage. Hence, this research may contribute in proper utilization of bael fruit for production of high quality pectin which can increase its demand and therefore generate income for farmers and distributers.

| MATERIAL S AND ME THODOLOGY
Baels were purchased from local retailers for use in experiments. The experiment required a larger amount of pulp so the individual trials were conducted using different baels of the same variety and similar maturity level. The baels were washed carefully with tap water to remove dirt soil from surface and broken on hard surface. The pulp was extracted by spoon. After pulp extraction, it was dried at 60°C for 24 hr in a cabinet drier followed by grinding into powder by using a blender. The powder was sieved using sieve and packed in low density polyethylene bag (thickness 75 μm). The obtained powder was sealed and stored at 6-10°C for further study.

| Pectin extraction
300 ml of distilled water was measured into a 1,000 ml conical flask and desired temperature was maintained using hot plate or the shaking water bath. A weighed portion of prepared pulp powder (50 gm) was added to the water and measured amount of acid was added to the pulp-water mixture until the desired pH was obtained. The mixture was agitated at a constant temperature until the desired extraction time (60 min) had elapsed. The pHs and temperatures were recorded and the mixture was allowed to cool in an ice water bath until it reached 55°C, the mixture was then centrifuged at 257 g for 10 min.
The filtrate was vacuum filtered using Whatman filter paper and the centrifugation and filtration steps were repeated. The filtered solutions were combined and approximately the volume of alcohol and filtered solution was maintained for different extraction ratios in different conical flasks, precipitation was allowed overnight. The next day, pectin was separated from the alcohol solution using a double layer of muslin cloth and washed three times with 70% alcohol and once with undiluted alcohol to remove any impurities. The pectin was dried in hot air oven until all moisture was removed. Samples were cooled and weighed. The samples were then stored in small plastic sample bags for further study (Pistoia, 1976).

| Characterization of extracted pectin
The dried pectin obtained was subjected to the following tests for characterization.

1.
Color: Visual observation was done for observing color of extracted pectin from the process given by Karki and Thapaliya (2004).

Equivalent weight: Equivalent weight was determined by process
given in Ranganna, 1995. Equivalent wt. is used for calculating the AUA and the degree of esterification.

Methoxyl content (MeO):
Determination of MeO was done by method given in Ranganna, 1995. 4. Moisture content: Moisture content of pectin was determined by hot air oven method given by Ranganna, 2011.

5.
Total AUA: Pectin which is a partly esterified polygalacturonide, contains 10% or more of organic material composed of arabinose, galactose and perhaps sugars. Estimation of AUA is essential to determine the purity and degree of esterification, and to evaluate the physical properties. Total AUA of pectin was obtained by the following formula (Mohamed & Hasan, 1995).
where, z = ml (titer) of NaOH from equivalent weight determination, y = ml (titer) of NaOH from methoxyl content determination, W = weight of sample.

Degree of esterification (DE):
The DE of pectin was measured on the basis methoxyl and AUA content (Owens et al., 1952) and calculated by following formula.

7.
Ash content: Ash content was determined by dry ashing method given by Ranganna, 2011. 8. Pectin grades: Pectin grade was determined by grading method given in Ranganna, 2011. 9. Pectin yield: Yield of pectin was determined according to process given in Ranganna, 2011. The data obtained from the yield of pectin in different conditions were analyzed by two ways ANOVA (no blockings) at 5% level of significance. The yields were analyzed by using LSD method (GenStat 5 Release 7.1 software programme developed by Lawes Agricultural Trust, Rothamsted Experimental Station, 1985). Means of the data were separated whether they are significant or not by using Fisher's LSD (least significant difference) method at 5% level of significance.

| RE SULTS AND D ISCUSS I ON
The present study was carried out to optimize the process of pectin extraction from Bael fruit. The pulp of bael was used for extraction.
Extraction was carried out by alcohol precipitation method at given temperature and pH. Extraction process was varied in temperature, pH and ethanol extraction ratio i.e., temperature 100 and 120°C, pH 1.5 and 2, ER 1:1, 1:1.5, and 1:2 respectively. Finally, the condition of temperature, pH and ER with highest pectin yield was taken as the best one. During the work, the chemical composition of bael pulp, the chemical composition of extracted pectin and yield of pectin at each varying conditions were studied.

| Analysis of fresh bael pulp
The pulp of bael was analyzed for moisture, carotene, TSS, vitamin C, color, and taste and the following results were obtained as shown in Table 1. 3.2 | Extraction yield at different sets of combination of temperature, pH, and extraction ratios 3.2.1 | Extraction yield at 100°C, pH 1.5, and ER 1:1 Three samples were made namely A 11 , A 12 , and A 13 . Pectin was extracted at 100°C, pH 1.5 and ER 1:1 for each of the samples. The mean of yields is considered as A 1 shown in Table 2.

| Extraction yield at 100°C, pH 1.5, and ER 1:1.5
Also at 100°C, three samples were made namely B 11 , B 12 , and B 13 . For each of the sample extraction was done at 100°C, pH 1.5, and ER 1:1.5. The mean of yields is considered as B 1 shown in Table 3.

| Extraction yield at 100°C, pH 1.5, and ER 1:2
Three samples were prepared namely C 11 , C 12 , and C 13 . Extraction was done for each of the samples. The mean of yields is considered as C 1 shown in Table 4.

| Extraction yield at 100°C, pH 2, and ER 1:1
Three samples were prepared namely A 21 , A 22 , and A 23 . Extraction was done for each of samples. The mean of yields is considered as A 2 shown in Table 5.

| Extraction yield at 100°C, pH 2, and ER 1:1.5
Three samples were prepared namely B 21 , B 22 , and B 23 . Extraction was done for each of samples. The mean of yields is considered as B 2 shown in Table 6.

| Extraction yield at 100°C, pH 2, and ER 1:2
Three samples were prepared namely C 21 , C 22 , and C 23 . Pectin extraction was done for each of the samples. The mean of yields is considered as C 2 shown in Table 7.

| Extraction yield at 120°C, pH 1.5, and ER 1:1
Three samples were prepared namely A 31 , A 32 , and A 33 . Pectin extraction was done for each of the samples. The mean of yields is considered as A 3 shown in Table 8.

| Extraction yield at 120°C, pH 1.5, and ER 1:2
Three samples were prepared namely C 31 , C 32 , and C 33 . Pectin extraction was done for each of the samples. The mean of yields is considered as C 3 shown in Table 10.

| Extraction yield at 120°C, pH 2, and ER 1:2
Three samples were prepared namely C 41 , C 42 , and C 43 . Pectin extraction was done for each of the samples. The mean of yields is considered as C 4 shown in Table 13.

| Chemical analysis
The moisture content of extracted pectin was found 16.66%. This observation was comparable to that found by Ismail et al. (2012) who reported moisture content of dragon fruit pectin varied from 11.13% to 17.33%. The ash content was found to be 1.24%. The ash content increases as the pectin yield decreases, indicating that the sugar content and others constituent increases significantly due to ripening of the fruit. Low ash content i.e., below 10% and maximum limit of ash content 10% are one of the good criteria for gel formation (Ismail et al., 2012). Therefore, the ash content found in this experiment indicates the purity of the pectin. The pectin produced in this study can be categorized as low methoxyl pectin (LMP) 13.39%. Also, it has a %DE lower than 50% so it can be considered as rapid set pectin.
The AUA was found to be 73.21 which showed that the pectin was pure. Also, the pectin isolated was of 150 grade with whitish yellow color and disagreeable odor. The chemical analysis of sample B 3 (extracted at 1200C, pH 1.5 and ER 1:1.5) as shown in Table 14.
TA B L E 5 Yield at 100°C, pH 2, and ER 1:1 3.4 | Variation in ER at temperature 100°C and pH 1.5 Here, A 1 , B 1 , and C 1 are same set of samples but subjected to different ER i.e., 1:1, 1:1.5, and 1:2, respectively, shown in Figure 1, between the samples extracted at 100°C and pH 1.5, the % yield is significantly different from each other. As LSD was found to be 0.1775, it shows that sample extracted at ER 1:1.5 is significantly different from sample extracted at 1:1 and 1:2. From figure above, it shows that mean score of sample extracted at ER 1:1, 1:1.5, and 1:2 are 2.413%, 4.407%, and 2.820% respectively. So, sample B 1 (extracted at ER 1:1.5) was found to be best in terms of % yield.

| Variation in ER at temperature 100°C and pH 2
Also here, let A 2 , B 2 , and C 2 be samples extracted at ER 1:1, 1:1.5, and 1:2, respectively, as shown in Figure 2. The sample extracted at 100°C and pH 2, all the samples were identical. There was significant difference between the samples. Thus, % yield was taken as the basis for best sample selection. So, sample B 2 extracted at ER 1:1.5 was taken as best sample. From figure above, it shows that mean score of sample A 2 , B 2 , and C 2 are 2.373%, 4.473%, and 2.313% respectively.
So, sample B 2 has the highest % yield then sample A 2 and C 2 .

| Variation in ER at temperature 120°C and pH 1.5
Samples A 3 , B 3 , and C 3 are pectin extracted at ER of 1:1, 1:1.5, and 1:2 respectively shown in Figure 3. Among the sample extracted at 120°C, pH 1.5, the % yield was similar to each other. There was significant difference between the samples. From the figure it shows that mean score of sample A 3 , B 3 , and C 3 was 6.760%, 16.173%, and 5.327% respectively. So, sample B 3 was found to be best in terms of % yield.

| Variation in ER at temperature 120°C and pH 2
Samples A 4 , B 4 , and C 4 are samples at ER of 1:1, 1:1.5, and 1:2 respectively in above Figure 4. The sample extracted at 120°C and pH 2, the % yield are similar to each other. It shows that sample A 4 extracted at (ER 1:1) was found to be best in %yield. From figure it shows that the mean score of sample A 4 , B 4 , and C 4 was 10.65%, 5.71%, and 4.37% respectively. So, sample A 4 was found to be best in terms of % yield.

| Comparison between the samples
Comparison between the samples B 1 (extracted at pH 1.5 and ER 1:1.5) and B 2 (extracted at pH 2 and ER 1:1.5) was done statistically.
The yield was found to be similar. Between samples B 3 (extracted at pH 1.5 and ER 1:1.5) and A 4 (extracted at pH 2 and ER 1:1) yield was found to be similar. Thus % yield was taken as the best sample selection. So, sample B 3 (extracted at pH 1.5 and ER 1:1.5 and temperature 1,200°C) was taken as the best among the other samples.

| CON CLUS IONS
The best condition for pectin extraction was at pH 1.

ACK N OWLED G EM ENT
We are very much indebted to our parents and family members for their constant encouragement, love and inspiration throughout the work.
Without their moral support, this work would never have seen the light of day. We are grateful to Mr. Som Raj Shrestha for his help in this research work. We do not have any conflict of interest among our authors.

CO N FLI C T O F I NTE R E S T
None declared.