Physicochemical Analysis of Pomegranate of Gilgit Baltistan, Pakistan

Juice can be considered as an important and functional ingredient in food products. The aim of current study was to screen and compare the physico-chemical properties of some indigenous species of pomegranate in Gilgit-Baltistan (GB) Pakistan. Fruits were collected from three tehsil regions of GB i.e. Bagrote, Jalalabad and Heramosh valleys. The fruits were washed, peeled off and arils were separated. Fresh juice was prepared from the arils and physico-chemical properties were evaluated. The pH of juice was found in the range of 2.4 (Sour, Jutial Gilgit) to 3.9 (sweet, Jalalabad). Comparative to other areas, pomegranate species of the Jutial exhibited higher total soluble solids (TSS) as 11.5 (sour) 14.5 (sweet) 14.2 (doom). The proximate reducing sugar analysis showed the higher content of reducing sugars in Sweet >Doom >Sour varieties. Lowest average ash and moisture content was observed in sour and higher was determined in sweet varieties.


Introduction
Pomegranate (Punicagranatum L.) belongs to the Punicacea family (Hardeet al., 1970). It is one of the important and commercial horticultural fruits which is generally very well adapted to the Mediterranean climate (Biale, 1981). It has been cultivated extensively in Iran, India and some parts in the U.S.A (California), China, Japan and Russia. Pomegranate fruits are consumed fresh or processed as juice, jellies and syrup for industrial production (Hodgson, 1917). Different parts of its tree (leaves, fruits and barks) have been used traditionally for their medicinal properties and for other purposes such as in tanning (Rania and Ne´jib, 2007).
Historical evidence reveals that its primary origin is in Iran where it has been grown in every area, both coastal and mountainous areas. It has now been spread to all other regions of the world ( Kumar, 1990). Turkey was analyzed for the chemical components, some of which was found for nutritional and health. Four samples were found to contain glucose syrup one of which was found to have very high amount of hydroxymethylfurfural (152498 mg/kg). Twelve pomegranate (Punica granatum L.) cultivars obtained from different growing regions of Iran were analyzed for their physical and chemical properties. These properties included fruit fresh weight, volume and density, peel thickness, soluble solids (TSS), titratable acidity (TA), EC, pH, vitamin C, ellagic acid content of juice and peel, total antioxidant activity of peel and juice and etc. Fruit weight ranged from 103.38 to 505.00 g and fruit volume from 99.41 to 547.88 cm. Similarly, average fruit density ranged 0.91 g.cm G3to 1.04 g.cm G3 and peel thickness of the fruit was recorded from 1.60 to 6.01 mm. Reducing sugars (Vahid et al., 2009). Different parts of its tree (leaves, fruits and bark skin) have been used traditionally for their medicinal properties and for other purposes such as in tanning (Rania et al., 2007). The edible part of the fruit contains considerable amounts of acids, sugars, vitamins, polysaccharides, polyphenols and important minerals. (Gil, et al.,2000).The pomegranate fruit is widely considered as a "healthy" fruit due to its biological actions, most of them attributed to its phenolic content (Lansky and Newman, 2007). Studies about pomegranate polyphenols have shown prevention of cardiovascular, cancer diseases and neurological damage in humans (Aviram et al., 2002).

Study Area
The present study was conducted in the Department of Agriculture and Food Technology, Karakorum International University Gilgit. The present study was carried out during 2010 growing season pomegranate trees grown in different areas of three districts of Gilgit Baltistan. All treatments were carried out in first week of September to last week of October.

Collection of Sample
Thirty normal size pomegranates were obtained from mature fruits growing in Bagrote, Jalal Abad and Heramoshvallies of district Gilgit. Commercially ripe fresh fruits were harvested during September and October from different mature trees randomly selected to represent the population of the plantation. The fruits was harvested at commercially maturity stage and transported to the Food Technology Laboratory of Karakorum International University Gilgit.

Separation of Seeds
Fruits were cleaned and washed to remove all foreign matters such as dust, then peeling was done manually by knife and grains or aerials were separated by hands.

Juice Extraction and Filtration
The pulp was extracted from the seeds of pomegranate varieties. Electric blender of good quality was used to crush seeds. Extracted juice was filtered by means of muslin cloths to separate juice from the pulp, fiber and seed particles.

Analysis of Physico Chemical Parameters of Pomegranate Juice
Following chemical parameters of juice of different pomegranate varieties was measured.

PH:
The pH values of samples were measured by using pH meter (Inolab) according to AOAC (1990) method No.981-12. Principle: The basic principle of electrometric pH meter is determination of the activity of hydrogen ions by potentiometer measurement using a standard hydrogen electrode and reference electrode. The glass electrode is commonly used. The pH value of an aqueous solution is defined by the equation: Where, AH+ = the activity of hydrogen ions in the solution in g-moles/l. The electromotive force (emf) produced in the glass electrode system varies linearly with pH. This linear relationship is described by plotting the measured emf against the pH of different buffers. Sample pH was determined by extrapolation.

Procedure
The pH meter is standardized with standard buffers of pH 4, and 7 respectively. Before taking each reading electrode is washed with distilled water and then dried with soft tissue paper. The electrode is simply dipped in sample till the pH meter gives final reading that is considered being the pH value of sample.

Total Soluble Solids
Total soluble solids content expressed as Brix was determined by using refracto meter, (Atago 3810-Japan) as described by AOAC (1984) at temperature (20 C). Refractometer, Distilled water, Spatula, Soft tissue paper and Samples were used. Refractometer gives rapid and accurate ratio of TSS present in sample. TSS is expressed in terms of Brix. The representative sample is placed on dry refractometer prism and refractometer is calibrated and readings were taken directly. Refractometer prism is washed with distilled water and dried with soft tissue paper after each reading.

Total Titratable Acidity
Total titratable acidity of samples was determined by following standard AOAC (1984) method by titrating against strong alkali solution. Burrette stand, 50ml Burrette, Conical flasks, volumetric flasks, Filter cloth, Distilled water, Funnel, Graduated cylinder, 10-ml sample were used. NaOH (0.1 N) 2-g pellets of NaOH are dissolved in 1000 ml distilled water, The resulting solution will be 0.1 N NaOH normal solution (Base) were used during analysis. 0.1g phenolphthalene is dissolved in 50ml distilled water and 50ml alcohol to prepare Phenolphthalene indicator. 5g sample is taken in volumetric flask and the volume is made with distilled water up to 100ml. Filter the sample if needed. The diluted sample (10ml) is taken in a conical flask and 2-3 drops of phenolphthalene indicator and titrated against 0.1N NaOH solution which is filled in a burrette. Continue titration till the solution persists pink color. Appearance of pink color is the end point and the color is persisted for 15-20 seconds. Finally the ml of 0.1N NaOH used is recorded for all the samples and acidity is calculated by using the following formula, Acidity = F x T x 0.1N NaOH x 100 x 100 / L x M Where, F is factor of acid (citric acid) which is equal to 0.0064.

Reducing Sugars
AOAC (1984) recommends following procedure for the determination of reducing sugars present in samples. 50ml Burette, Stand, conical flask, volumetric flask, distilled water, filter cloth, 100ml-graduated cylinder, test tube holder, funnel, and 5g sample was used. Fehling Solution A: 34g CuSO 4 .5H 2 O is dissolved in 500ml of distilled water. Fehling solution B: 173g Sodium Potassium Tarter ate and 50g of NaOH is dissolved in 500ml distilled water and as Indicator: Methylene Blue. Fruit grounded sample (5g) is taken and diluted up to 100ml with distilled water. The burrette is filled with this solution. 5ml fehling A and 5ml fehling B with 10ml of distilled water is taken in conical flask and boiled, On boiling it was titrated against the sample solution from the burrette till color changes to dark brown or red. 2-3 drops of methylene blue is added as indicator till dark brown or red color persisted. The ml of sample is then recorded and reducing sugar percentage is calculated by using the following formula.

Ash Content
Ash is the inorganic residue remaining after the complete oxidation of organic matter in foodstuff. AOAC (1990) recommends following procedure for determination of ash content. Balance precisian, china dishes, electrical muffle furnace, oven, spatula, desiccator, test tube holder, and 10-g sample was used during chemical analysis of samples. China dishes were taken and weighted properly and weight is noted down. 10-gm of sample is added in china dish. Then charred the sample or dry in oven at 70 0 C for 2-4 hours. In temperature up to 100 0 C and left it for 24 hours. After 24 hours dry matter is obtained. The sample must be dried in oven till it gives constant weight of sample. After drying the sample 1-gm dry sample is taken in china dish and incarnated in muffle furnace at 550 0 Cfor 12-18 hours. Turn off the muffle furnace after the required time and wait till the temperature inside furnace dropped to at least 250 0 C. Door of muffle furnace is opened carefully to avoid losing ash that may be fluffy. Ash obtained is transferred in desicator for cooling. After cooling ash is weighted and calculated on the basis of following formula: % Ash = Initial Weight -Final weight / Initial weight x 100

Moisture Content
Sample was dried in the oven provided with opening for ventilation and maintained at 130c for 60 minutes. The loss in sample weight is expressed as percentage moisture. China dish, Desiccators, Silica granules, Rubber gloves were used. Perten laboratory mill 3100, Analytical Balance, Accuracy +/-0.0001g, Hot air oven was used. Clean moisture dishes were taken and dried in oven at 130c for 30 minutes. Moisture dishes were taken out and placed in desiccators and weighed soon after they reach at room temperature.10g of well-mixed sample was added to the each moisture dish and recorded the weight. Removed from scale and covered with lid.
Placed the moisture dish in oven uncovered for 60 minutes (60 minutes dry period Begin when oven temperature is usually 130c. Afterward removed the sample from oven and covered with lid. Place the sample in desiccators for cooling. Gloves were used for sample transfer room oven to the desiccators.
Weighed the sample till after reached at room temperature.

Physico Chemical Analysis of Pomegranate
The chemical composition of fruit differs depending on the cultivar, growing region, climate, maturity, cultural practice  Table 1. While our results were in agreement with values (10-16.5 •Brix) reported by (Fadavi et al,. 2005).
The total soluble solids observed in the present study were found to be in typical range as reported by other researchers (Radunić, Mira, et al. 2015).
The minimum acidity of pomegranate juice is 0.35% which was reported by (Mustafa Ozgen et al., 2008).The titratable acidity content varied from 0.33, similar results were also reported by Fadavi et al. (2005).
According to (Melgarejo, 1993), the predominant acid of this fruit is malic acid. In studies carried out with the Mollar variety quantities of malic and citric acid. The content of malic acid ranged between 0.143% and 0.249% and the citric acid between 0.147% and 0.4 % (Sharms et al., 1997).
Reducing sugar: Data in The pH range (2.75-4.14) is appeared as representative range as reported in different studies and the pH values recorded in in the current study were also observed in the same range (Radunić, Mira, et al. 2015).
The moisture content of the seeds up to 81.53% was reported by Amit Parasahar (2010). Evaluations of various important components are also reported previously to highlight the important pomegranate characteristics (Zaouay Faten et al., 2012).

Conclusion
In the current study physico-chemical properties of pomegranate indigenous to Gilgit-baltistan were determined. The evaluated parameter includes the total soluble solids, acidity, reducing sugurs, pH, seed ash and seed moisture. Significant difference in the physicochemical properties such as sugurs, soluble solids, seed moisture and pH were observed. This study is important in exploring the knowledge of indigenous plants and also increases the information of pomegranate associated properties in specified study region. Whereas, difference in physico-chemical characteristics of the indigenous pomegranate varieties showing the genetically diversity among the species of this Gilgit-baltistan (Rajasekar, Dhivyalakshmi, et al. 2102). Furthermore, such studies may help to the producers or companies for selecting the better indigenous pomegranate specie having better composition.