


































Food Science and Nutrition Studies 

ISSN 2573-1661 (Print) ISSN 2573-167X (Online) 

Vol. 1, No. 2, 2017 

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114 
 

Effects of Propolis and Black Seed Oil on the Shelf Life of 

Freshly Squeezed Pomegranate Juice 

Ibrahim Kahramanoglu1,2* & Serhat Usanmaz1 

1 Department of Horticultural Production and Marketing, European University of Lefke, Lefke, Cyprus 

2 Alnar Narcılık Ltd., Güzelyurt, Cyprus 

* Ibrahim Kahramanoglu, E-mail: ibrahimcy84@yahoo.com 

 

Received: October 23, 2017    Accepted: October 30, 2017    Online Published: November 6, 2017 

doi:10.22158/fsns.v1n2p114        URL: http://dx.doi.org/10.22158/fsns.v1n2p114 

 

Abstract 

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. 

Keywords 

fresh juice, shelf life, colony forming unit, ascorbic acid, maturity index 

 

1. Introduction 

Pomegranate (Punica granatum L.) is predicted to be among the oldest known cultivated fruit crops. 

Result of some scientific studies showed that antioxidant and anti-microbial capacity of pomegranate 

fruit are high, it decreases blood pressure and it can be used against some illness such as cancer and 

diabetic (Aviram et al., 2000; Jurenka, 2008). However, consumption of pomegranate fruit is difficult 

due to the hassle of aril extraction. On the other hand, pomegranate has lots of low quality products as a 

result of sunburn and fruit cracking. Therefore, using low quality products for juice production is 

important for both producers and consumers. The demand for 100% natural, pure and Not From 

Concentrate (NFC) juice is increasing because of the increase in consumer awareness on the negative 

effects of synthetic food additives on human health (AIJN, 2016). However, development of yeast and 

mould cause 100% natural pomegranate juice to have shorter shelf life as in many other fruit juice. 

Although, the shelf life of pomegranate juice can be prolonged by using chemical additives i.e. sodium 

benzoate and potassium sorbate, changes in the consumer preferences has been directed producers to 



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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. 

 

2. 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 -20ºC 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. 

Immediately after that, pomegranate juice was filled in 250 ml bottles. Numbers of main treatments of 

present study are 3, which are: (1) untreated control, (2) propolis application [1 drop/250 ml] and (3) 

black seed oil application [1 drop/250 ml]. All treatments were subjected to 2 different factors, these 

are: (a) shelf life test after filling [storage at 4ºC] (b) shelf life test after 1 year storage at -18ºC [storage 

at 4ºC]. Experiments were set up with 145 samples for each unique treatment. Five samples from each 

unique treatment were subjected to yeast and mould analyzes (colony forming unit-cfu/g) for 2-days 



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intervals starting from the 5th day (totally 29 measurements). Therefore, experiments were continued 

for 61 days in total. Moreover, following tests were conducted for the samples of (i) 0 day after 

squeezing, (ii) 15 days after squeezing, and (iii) 380 days after squeezing [15 days shelf life after 1 year 

freezing storage]. The tests were: (1) antioxidant activity (%), (2) Total Soluble Solids (TSS), (3) 

titratable acidity (%), and (4) ascorbic acid content [mg/L]. 

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 𝜇L supernatant was 

injected into the C18 XTerra (Waters, 4.6 × 250 mm) column. The photodiode array detector was set at 

244 nm, and 2% KH2PO4 (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. 

 

3. 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. It is clear from the figure that yeast and mould development was 

firstly observed at the control treatment at 15th day. Colony forming unit was 72 ± 41 at 15th day and it 

increased with the increase in the storage duration. According to the result it can be concluded that 

freshly squeezed pomegranate juice can be stored for 15th days at 4ºC without any additive (with 

pasteurization at 72ºC for 15 sn). Development of yeast and mould had been observed at 19th day of 



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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 

21th and 23th 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 33th 

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 37th 

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 



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of samples measured as 95.6 ± 0.17 immediately after squeezing. At 15th day it was observed that 

antioxidant activity of all treatments decreased (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.  

 

Table 1. Effects of Propolis and Black Seed Oil on the Antioxidant Activity  

Treatments 
Antioxidant activity (%) 

Day 0 Day 15 Day 15 after 1 year freezing 

Control 95.6 ± 0.17 a 93.3 ± 0.12 b 90.5 ± 0.22 b 

Propolis 95.6 ± 0.17 a 94.4 ± 0.15 a 92.8 ± 0.11 a 

Black seed oil 95.6 ± 0.17 a 94.2 ± 0.04 a 92.5 ± 0.14 a 

Values followed by the same letter or letters within same column are not significantly different at a 5% 

level (Tukeys (HSD) multiple range test). 

 

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. 

 

Table 2. Effects of Propolis and Black Seed Oil on the Ascorbic Acid  

Treatments 
Ascorbic acid (mg/L) 

Day 0 Day 15 Day 15 after 1 year freezing 

Control 70.3 ± 0.45 a 66.7 ± 0.23 c 18.7 ± 0.38 b 

Propolis 70.3 ± 0.45 a 67.8 ± 0.88 a 23.9 ± 0.49 a 

Black seed oil 70.3 ± 0.45 a 67.3 ± 0.11 ab 23.3 ± 0.17 a 

Values followed by the same letter or letters within same column are not significantly different at a 5% 

level (Tukeys (HSD) multiple range test). 

 

 



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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).  

 

Table 3. Effects of Propolis and Black Seed Oil on the Maturity Index 

Treatments 
Maturity index (TSS/TA) 

Day 0 Day 15 Day 15 after 1 year freezing 

Control 9.7 ± 0.24 a 10.3 ± 0.19 a 23.2 ± 0.6 a 

Propolis 9.7 ± 0.24 a 10.2 ± 0.12 a 18.9 ± 0.5 c 

Black seed oil 9.7 ± 0.24 a 10.3 ± 0.10 a 21.8 ± 0.2 b 

Values followed by the same letter or letters within same column are not significantly different at a 5% 

level (Tukeys (HSD) multiple range test). 

 

The maturity index showed slight increase in 15 days of storage at shelf. At that time, no significant 

difference was determined among the treatments. Approximately 2-fold increase had been determined 

at the samples when they freeze for 1 year and then taken out to the shelf for 15th days. Main reason of 

increase in the maturity index is the considerable decrease in the titratable acidity. At 15th day, 

significant differences were calculated for the different treatments. The lowest maturity index was 

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.  

 

4. 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. 

 

 



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