Effects of Propolis and Black Seed Oil on the Shelf Life of Freshly Squeezed Pomegranate Juice

During the last decade, consumers began to pay more attention on the 100% natural, pure and Not From Concentrate (NFC) fruit juice. However, 100% natural fruit juice has shorter shelf life than the concentrated juice, due to the development of yeast and mould. Therefore, present research aimed to study the effects of propolis and black seed oil on the shelf life of freshly squeezed pomegranate juice. According to the results obtained, both propolis and black seed oil have delaying effect on the development of yeast and mould. Moreover, it was also found that combination of these treatments with freezing, increases the efficiency of tested natural treatments, as well as the shelf life.

find alternative natural and healthy methods. In the light of this information, present work aimed to study the effects of propolis and black seed oil on the shelf life of freshly squeezed pomegranate juice.

Meterials and Methods
Pomegranate fruit samples of present study are belonging to the Wonderful culturae, which has been dominating pomegranate trade in the world. This culturae was originated in Florida. Fruit size of this culturae is big with deep-red fruit color. Fruit juice content is high and taste is sweet-tart. Harvesting period is between October and November in the northern hemisphere. Fruit samples of present study were harvested on October 2015, from a 7-years old pomegranate orchard located in Güzelyurt province in Cyprus. Fruits were harvested by hand at commercial maturity (>17% TSS and >1.80 titratable acidity) and immediately after harvest, fruits were transferred to the factory of Alnar Narcılık Ltd. with a ventilated truck. After that, arils were extracted from the fruits by automatic machine and arils were pressed to produce juice.
Crude propolis was gathered by hand from Bağlıköy province in the western part of Cyprus. The propolis exudates collected by bees (Apis mellifera cypria) were primarily from a mixture of wild and cultivated plant species, including pine (Pinus brutia L.), olive (Olea europea L.), eucalyptus (Eucalyptus globulus L.), citrus (Citrus spp.), trifoliums (Medicago spp. and Trifolium spp.), pimpernel (Anagallis arvensis L.), hordeum (Hordeum bulbosum L.), field bindweed (Convolvulus arvensis L.), chrysanths (Chrysanthemum spp.) and locust (Acacia spp.). Preparation of the propolis extract was done according to the method by Krell (1996) with some modifications. The propolis extracts were frozen to -20º C for 1 month, then cut in small pieces, and ground in a chilled mortar. After that, 10% ethanol extracted propolis was prepared by adding 100 g of the propolis to 900 mL of 70% ethanol and agitating for 1 week. Agitating was done with automatic machine by shaking the extract for 1 minute with 60 minutes interval. The mixture was maintained at room temperature during preparation and was subsequently filtered through Whatman 1 filter paper. The extracts were kept at 4ºC in dark storage until use.
Black seed oil is a product of Nigella Sativa plant which is native to Asia. The black seed oil of present study is belonging to the Pelmur Ltd. with a brand name of Biotama. The black seed oil is obtained by the cold-press of black cumin seeds. The purchased black seed oil was 100% pure and was dissolved in ethanol by adding 100 mL of the black seed oil to 900 mL of 70% ethanol and agitating for 1 day.
First of all, pomegranate juice was pasteurized for 15 second at 72ºC and it cooled to 4ºC in 4 minutes. Determination of yeasts and moulds was done as colony forming unit/g. For this 1 mL of each juice was placed on plate surface that contained Sabouraud Dextrose Agar (SDA) and distributed by a sterilized swab. Plates were incubated for 5 days at 25°C. Colonies were counted and expressed as cfu/g. The antioxidant activity of the pomegranate juice was evaluated using the DPPH free radical-scavenging method. Measurements were carried out according to the modified method of Klimczak et al. (2007). A total of 5 mL pomegranate juice was mixed with 5 mL of methyl alcohol (80%) in teflon tubes and then centrifuged (4000 rpm, 10 min, at 4ºC ). Briefly, 0.1 mL of supernatant was added to 2.46 mL of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH; 0.1 mg/L in 80% methyl alcohol) and mixed by vortex. Absorbance of the samples was measured at 515 nm using the spectrophotometer after incubating for 10 min in the dark. Antioxidant activity was expressed as the percentage decline of the absorbance from control group.
Total soluble solids content of the fruits were measured by a hand refractometer. Titratable acidity (g/100 g of citric acid) of juice samples was determined according to AOAC (1990) with WTW pH-meter (Weilheim, Germany). Titratable acidity was determined by titrating 2 mL of fruit juice in 38 mL of distilled water with 0.1 N NaOH to an end point of pH 8.1. Ascorbic acid determination was performed by following the method of Lee and Coates (1999) by using the HPLC method. The HPLC column was maintained at 25ºC and the flow rate was 0.5 mL min -1 . A total of 10 L supernatant was injected into the C18 XTerra (Waters, 4.6 × 250 mm) column. The photodiode array detector was set at 244 nm, and 2% KH 2 PO 4 (pH 2.4) was used as the mobile phase.
Collected data was summarized by using Microsoft Excel and figures and simple tables were prepared with the mean and standard deviations. The data of the experiments was subjected to analysis of variance (ANOVA) with main treatments and storage conditions as factors using SPSS software. Mean separations was done by using Tukeys (HSD) multiple range test at P ≤ 0.05. Significant differences were showed at the tables by using different letters.

Results and Discussions
Results for the development of yeast and mould at the juice samples which were subjected to different treatments are given in Figure 1. control treatment when samples stored 1 year at -18ºC and then taken out to shelf. These result shows that freezing of freshly squeezed pomegranate juice delays the development of yeast and mould for 4 days. Frozen storage is known to have less detrimental effect on the juice.
Application of both propolis and black seed oil had been found to delay the development of yeast and mould. First measurement of colony forming unit for the application black seed oil and propolis were at 21 th and 23 th days, respectively. Similarly Koç et al. (2007) conducted a study about the anti-fungal effects of propolis in 4 different fruit juice (mandarin, orange, apple and white grape). They reported that presence of propolis inhibited the growth of all spoilage yeast at 25ºC . However, that study was conducted for only 48 hours. Anti-fungal activity of propolis was also reported by some other scientist (Özcan, 1999;Oliveira et al., 2006;Senka et al., 2011;Temiz et al., 2013). On the other hand, Hafez (2008) reported that 0.5% black seed oil application on the cucumber had showed protective effect against powdery mildew. In another study, Forouzanfar et al. (2014) reported that black seed oil contains thymoquinone which has high anti-microbial activity. They also noted that black seed oil had a strong antibacterial activity against all the strains of L. monocytogenes. In present study, similar with the control treatment, freezing had been found to delay the development of yeast and mould for both the application of propolis and black seed oil. Yeast and mould development had firstly observed at 33 th day for black seed oil application. This means that freezing helped the black seed oil to increase the shelf life of pomegranate juice for 12 more days. The best result (longer shelf life) had been obtained from propolis application + freezing, where the development yeast and mould was suppressed until 37 th day.

Figure 1. Effects of Propolis and Black Seed Oil on the Yeast and Mould Development
Antioxidants protect the body from the harmful effects of free radicals. Antioxidants include some vitamins (i.e., vitamins C and E), some minerals and flavonoids, which are found in plants.
Pomegranate fruit is among the good sources of antioxidants (Valko et al., 2007). Antioxidant activity  (Table 1). According to the results, antioxidant activity of control treatment was found to be significantly lower than the other treatments. When the samples stored 1 year at -18ºC , it was determined that antioxidant activity continued to decrease. However, at the same time antioxidant activity of control treatment was again found to be lower than the other treatments. Ascorbic acid is among the important components of pomegranate (Miguel et al., 2010). However, it is believed that the storage duration of fruit causes a decline in the concentration of ascorbic acid (Zarei et al., 2011;Kulkarni & Aradya, 2005). As stated by these studies, the ascorbic acid content of pomegranate juice showed a considerable decline during storage in present study ( Table 2). The ascorbic acid in control treatment decreased from 70.3 mg/L to 66.7 mg/L in 15 days of storage at shelf.
The ascorbic acid content of other treatments also showed a decline but for both propolis and black seed oil applications, it was found to be higher than the control treatment. When the samples freeze for 1 year, the ascorbic acid content for control, propolis and black seed oil treatments were found to decrease until 18.7, 23.9, 23.3, respectively. The ratio (maturity index) between Total Soluble Solids content (TSS) and Titratable Acidity (TA) is the main factor determining pomegranate fruit taste and fruit maturity (Cristosto et al., 2000). The Maturity Index (MI) is related to the taste and flavor of fruit. The MI of present study is determined as 9.7 at the first day of juice production (Table 3). determined from the propolis treatment and highest from control. When the maturity index of a food increases, it causes the food to be sweeter. Not only for the pomegranate juice, but for all juice types, consumers do not prefer high changes in the taste. Therefore, it can be concluded that application of propolis reduces this change in the taste. Application of black seed oil has also been found to have significant effect on the maturity index, but lower than the propolis application.

Conclusions
Yeast and the mould are the main reason for the spoilage of freshly squeezed pomegranate juice and thus main cause of the decrease in the shelf life. In present study, propolis and black seed oil applications showed good performance in controlling the development of yeast and mould. The efficiency of both treatments showed considerable increase; when they combined with freezing technique. Results suggested that the shelf life of freshly squeezed pomegranate juice can be extended to 37 days with the application of propolis (1 drop/250 ml) plus freezing. On the other hand, both propolis and black seed oil applications have been found to protect anti-oxidant activity, ascorbic acid content and maturity index, as compared to control treatment.