Anti-Oxidant Effects of Pomegranate Juice on Saccharomyces Cerevisiae Cell Growth

Abstract

Background

Pomegranate juice has a number of positive effects on both human and animal subjects.

Material and methods

Four groups were used in this study. i: Control group, ii: H₂O₂ group, iii: Pomegranate juice (PJ) group and iv: PJ + H₂O₂ group. Following the sterilization method for pomegranate juice (10%) and H₂O₂ (6% v/v), Saccharomyces cerevisiae cultures were added and the cultivation incubated at 35°C for 72 hours. Fatty acids and vitamin concentrations were measured using HPLC and GC and the total protein bands profile were determined by SDS-PAGE.

Results

According to our results statistically significant differences have been determined among the study groups in terms of fatty acids and vitamin (p<0,05). Fatty acid synthesis, vitamin control and cell density increased in groups to which PJ was given in comparison with the control group (p<0,05). Pomegranate juice increased vitamins, fatty acids and total protein expression in Saccharomyces cerevisiae in comparison with the control.

Conclusion

Pomegranate juice has a positive effect on fatty acid, vitamin and protein synthesis by Saccharomyces cerevisiae. Accordingly, we believe that it has significantly decreased oxidative damage thereby making a positive impact on yeast development.

Introduction

Pomegranate juice (PJ) has received a high level of scientific interest in recent years. The effects of PJ on protein, fatty acid and vitamin synthesis in yeasts have been examined; hence we believe that this will contribute meaningfully and pave way for further scientific studies. Pomegranate (Punica granatum) juice and fruit has a high level of polyphenol content and very effective antioxidant capability. As a result, it has attracted the attention of researchers in recent years in relation with human health. Increase in the significance of PJ daily derives from its phenolic compound rich content. It is very important that the anthocyanin amount in its polyphenol content lies within the range of 0.2–1.0g/100g, in addition, it has been stated that the anthocyanin count in this content is between 136 and 23 mg/100 ml (Mena et al. 2012; Rinaldi et al. 2013).

Pomegranate is commonly grown in Turkey, Iran, India, China, Afghanistan, Russia, and America (Rajasekar et al. 2012). Pomegranate is immensely of high nutritional value, owing to its extraordinary nutritional properties. Polyphenols are the vital class of pomegranate phytochemicals, including flavonoids (anthocyanins) and tannins (ellagitannins and gallotannins). Studies have reported that ingestion of pomegranate fruits has nutritional and health benefits, involving reduced oxidatlueive stress, and it has anticancer and antibacterial regulator effects (Santos et al. 2012). Saccharomyces cerevisiae is important yeast that has been used for various studies (Kagan et al. 2005; Comitini et al. 2011). H₂O₂ is a reactive oxygen species (ROS) in organism, being permanently produced intracellularly as a product of the metabolism in aerobic organisms and otherwise extracellularly during infection in specialist organisms (Lopes et al. 2004; Cipak et al. 2006; Folmer et al. 2008). The consumption of H₂O₂ by Saccharomyces cerevisiae is to change the synthesis of fatty acid and total protein in plasma membrane (Matias et al. 2007; Folmer et al. 2008). ROS can oxidize nucleic acid, protein, fat and carbohydrates. For example, the oxidative damage to proteins leads to breakdown of amino acid chains decreasing the biologic activity. Under normal physiological situations, oxidative damages are prevented by antioxidant defenses. On the other hand, under abnormal conditions, antioxidant defense system is insufficient and causes oxidative damage in cell. According to a study it has been observed that the consumption of H₂O₂ at lower dose, caused deadly stress in Saccharomyces cerevisiae and lead to negative effect on the synthesis of essential proteins (Costa et al. 2002; Spiteller 2006; Skoneczna et al. 2007; Folmer et al. 2008; Zupan et al. 2009; Chondrogianni et al. 2012; Yu et al. 2012). In recent years scientists showed that the components of the plant has essential effects in organism. Thus the studies have been confirmed that poliphenols, tannins, flavonoids have antimicrobial effects. Pomegranate fruit is rich in anthocyanin, catechin, and tannins and are the stimulating effect of the oxidative stress (Randhir et al. 2005; Zoreky 2009; Pala and Toklucu 2011). These biological activities have been especially imputed to phenolic compounds, such as anthocyanins and ellagitannins (Mena et al. 2012). In addition the researchers indicated that pomegranate is a strong antioxidant (Mena et al. 2012; Santos et al. 2012; Pala and Toklucu 2011).

In this study we investigated the effect of PJ on cell growth, total protein, fatty acid and vitamin contents in Saccharomyces cerevisiae treated with hydrogen peroxide. We believe that the results we obtained in this study will make an important contribution to current literature.

Material and Methods

Research Groups and Saccharomyces cerevisiae Growth Conditions

In this study, measurements were carried out on culture samples grown for 2, 4, 6 hours and overnight (72 hours). Whereas in other analyses, the overnight developed culture samples were used. In this study, four groups were used; i) control group, ii) H₂O₂ group, iii) PJ group and iv) PJ+H₂O₂ group. Saccharomyces cerevisiae growth media used was YEPD in a working volume of 50 mL (2 g yeast extract, 2 g trypton, 2 g glucose). After sterilization, yeast cultures 10 % (v/v) were added to the four group's media and samples were incubated for 72 h at 35°C. At the end of the incubation period, samples were centrifuged (5000 rpm, 4°C for 5 min). The centrifuged pellets were counted and detached for vitamin and fatty acid analysis (Dilsiz et al. 1997; Ozsahin et al. 2009).

Preparation of Extract

Pomegranate fruit (From Alacakaya county of Elazığ city) was crushed in water and the juice was sterilized for further adding of Saccharomyces cerevisiae cultures (10%,v/v).

Application of H₂O₂ Chemical

H₂O₂ was supplemented in H₂O₂ and PJ+H₂O₂ groups with 6% (v/v) ratio.

Extraction of Fatty Acids and Analyses

Cell pellets were homogenized with Hexaneisopropanol mixture. This solution was centrifuged at 5000 rpm for 5 min at 4°C. The supernatant (containing hexane phase) was used for vitamins A, D, E, K and fatty acid analysis (Ozsahin et al. 2009).

Analysis of Fatty Acid Methyl Esters

Methyl esters were analyzed by gas chromatography (SHIMADZU GC 17 Ver. 3, Japan). For this, a 25 m long Machery-Nagel (Germany) capillary colon with an inner diameter of 0.25 µm and a thickness of 25 micron film was used. During the analysis, the colon temperature was kept at 120–220°C, with an injection temperature of 240°C and detector temperature of 280°C. The colon temperature program was adjusted from 120 to 220°C and the temperature increase was determined to be 5°C minG1 until 200°C and 4°C minG1 from 200–220°C. Temperature was kept at 220°C for 8 min and the total continuance was set as 35 min and nitrogen gas was used as the carrier gas. After this process, the fatty acid methyl esters mixtures of the samples were analyzed

Analysis of Vitamins A, D, E, K and Sterol

A 5 mL of the supernatant was added to tubes with caps (25 mL) and 5% 1ml KOH solution was added. This solution was vortexed and kept at 85°C for 15 min. After that, the tubes were chilled to room temperature and 5 mL of natural water was added and mixed. The Hexane phase was vaporized by nitrogen flow. It was dispersed in 1 ml acetonitril/methanol mixture and then was taken to auto sampler vials and analyzed. The analyses were made by HPLC device. (Shimadzu, Kyoto Japan) (Ozsahin et al. 2009).

Analysis of Total Protein by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

After the protein extraction from cell cultures, protein samples were analyzed by SDS-PAGE. After SDS-PAGE, the gel image was evaluated for total protein bands among the groups studied (Laemmli, 1970).

Analysis of Cell density

Culture samples were collected at 2, 4, 6 hours and overnight (72 hours). The measurement has been carried out using a spectrophotometer at 600nm (OD₆₀₀) (Lopes et al. 2004).

Statistical Analysis

For statistical analysis, SPSS 20.0 software was used. The comparison between experimental groups and the control was made using One way ANOVA and LSD tests. Statistically significant differences among the groups have been stated as p<0.05 and the statistically nonsignificant differences have been stated as p>0.05. Standard deviations were indicated as mean ±SD. Each assay was repeated 3 times.

Results and Discussion

Pomegranate juice added to the yeast culture medium increased cell density in comparison to the control at p<0.05 (Table 1, Figure 1) and vitamins (Table 2) and fatty acid synthesis (Table 3) increased significantly in comparison with the group to which no PJ was added at p<0.05 (Examining the SDS-PAGE protein band profiles, a denser banding is observed in pomegranate added groups in comparison with those to which no PJ was added. In addition, banding has been observed to be denser in the group cultured at 35°C (Figure 2).

Table 1.

Cell density of cultivations at 35°C and PJ addition (10%,v/v).

	2h	4h	6h	72h
Control	1,80±0,01a	1,83±0,03a	1,82±0,03a	2,02±0,01a
H2O2	1,62±0,02b	1,61±0,01b	1,71±0,02b	1,89±0,00b
PJ	1,82±0,01a	1,85±0,00a	1,92±0,00c	1,94±0,05c
PJ + H2O2	1,82±0,02a	1,68±0,00c	1,86±0,00d	1,92±0,00d

Means of cell density sharing the same superscript are not significantly different from each other (Anova Post Hoc LSD Test, P<0.05)

Figure 1.

Cell density of Saccharomyces cerevisiae at spectrophotometer OD₆₀₀

Table 2.

% Fatty acid profiles of cultivations at 35 °C and PJ addition (10%,v/v)

	Control	PJ	H2O2	PJ + H2O2
Myristic acid (14:0)	30,42±0,02a	29,01±0,00b	28,01±0,00c	33,21±0,00d
Palmitic acid (16:0)	218,71±0,01a	245,01±0,00b	183,61±0,00c	269,72±0,00d
Palmitoleic acid (16:1)	38,04±0,00a	44,18±0,02b	40,77±0,00c	50,46±0,00d
Stearic acid (18:0)	114,54±0,47a	161,17±0,00b	105,93±0,00c	143,56±0,58d
Monoenoleic acid (18:1)	306,11±0,11a	184,41±0,01b	97,30±0,00c	172,35±0,31d
Dienlinoleic acid (18:2 n6c)	32,59±0,00a	67,80±0,58b	21,75±0,00c	74,88±0,00d
Trienlinoleic acid (18:3)	99,48±0,00a	92,01±0,06b	83,67±0,00c	105,08±0,08d

Means of fatty acids amount sharing the same superscript are not significantly different from each other (Anova Post Hoc LSD Test, P<0.05)

Table 3.

% Vitamin profiles of cultivations at 35°C and PJ addition (10%,v/v)

	Control	PJ	H2O2	PJ + H2O2
K vit. (1)	0,34±0,00a	3,12±0,00b	0,95±0,02c	3,52±0,00d
6K vit. (2)	0,25±0,00a	0,32±0,00b	0,18±0,14c	0,26±0,00a
D vit. (2)	0,58±0,01a	1,04±0,00b	1,58±0,00c	0,96±0,00d
α-Tocopherol	13,11±0,00a	11,59±0,00b	15,84±0,01c	17,16±0,00d
γ-Tocopherol	0,59±0,00a	0,16±0,00b	0,09±0,00c	0,08±0,00d
Ergosterol	9,36±0,00a	8,15±0,00b	10,30±0,08c	10,74±0,01d
Cholesterol	11,84±0,00a	3,27±0,00b	28,13±0,00c	3,68±0,02d
Stigmasterol	54,51±0,00a	49,44±0,00b	74,57±0,00c	55,81±0,00d
β-sitosterol	2,05±0,01a	23,91±0,01b	0,93±0,00c	29,48±0,00d

Means of vitamin amounts sharing the same superscript are not significantly different from each other (Anova Post Hoc LSD Test, P<0.05)

Figure 2.

SDS-PAGE total protein bands profiles for cultivaions at 35 °C and PJ addition (10%, v/v). Lane 1, Control; lane 2, PJ; lane 3, H₂O₂; lane 4, PJ+ H₂O.

According to our findings, there are a set of alterations in membrane structure in H₂O₂ groups in comparison with control group. Similar results were obtained by Folmer et al. (2008). Santos et al. (2012) indicated that in groups with added PJ in comparison with the control, cell population survival density was increased. According to Yan et al. (2011), H₂O₂ has negative effect on β-caroten synthesis in yeast (Saccharomyces cerevisiae) that has preventive effect on oxidative damage. In our results, we observed that in PJ added groups, when compared with control group produced significant increases and so in PJ added groups, in comparison with in added H2O2 group that there are essential increases in cell density (p<0.05) (Table 1). In addition, when cell densities were examined, it has been observed that PJ added groups had higher cell density in comparison with the control (Table 1).

The fatty acid profile from Saccharomyces cerevisiae in this study was: 14:0, 16:0, 16:1, 18:0, 18:1 and 18:2 n-6c, 18:3 with some changes in the composition according to changes in the culture media. We felt that the primary reason in the synthesis of these fatty acids was due to the enzymes that had a role in the synthesis transcribed in Saccharomyces cerevisiae. In various studies, it was discovered that the enzymes, which made fatty acid synthesis in Saccharomyces cerevisiae and other yeasts species were affected by many components in the culture (Ozsahin et al. 2009). In addition, when we looked at the presence of different fatty acid synthesis in comparison with those in H₂O₂ group, we observed that in PJ added group, the fatty acid synthesis was considerably increased (p<0.05) (Table 2). According to Yan et al. (2011), adding H₂O₂ can significantly induce the β-carotene production and given H₂O₂ amount more and more, in culture media measured β-carotene amount declined in parallel with reported that the catalase enzyme activity was decreased. In accordance with Guvenc et al. (2010), different sugar source in nutrition media increased the fatty acid synthesis. Therefore, these circumstances has contributed to the thinking that H₂O₂ has negative effect on fatty acid synthesis of genes (Kajiwara et al. 1997; Cipak et al. 2006; Ozsahin et al. 2009). In our study, we discovered that vitamin synthesis was made from Saccharomyces cerevisiae: the vitamin rates were α-tocoferol, “γ-tocoferol, β-sitosterol cholesterol, stigmasterol ergesterol, D, and K 1,2. The rates of these vitamins in changing media were identified by computation with statistical methods (Perrone et al. 2008; Ozsahin et al. 2009). Once again, we saw that our results of vitamin contents, in added PJ group was more in comparison with control group (p<0.05) (Table 3).

We saw that in added PJ group; SDS-PAGE total protein band profiles had more intensity than in the H2O2 group (Figure 2). Looking at the effect mechanism of the findings obtained in this study, we believe that significant results have been obtained. Further studies should be carried out and common and effective mechanism of PJ could be tested on different living things. We believe that our study has important contributions to scientific literature.

Conclusion

Pomegranate juice has positive effect on fatty acid, vitamin and protein synthesis by Saccharomyces cerevisiae. Accordingly, we believe that it has significantly decreased oxidative damage thereby making a positive impact on yeast development. In accordance with our results, we believe that PJ might have a similar impact on human health when we consider its effect on yeasts. In addition, further studies with PJ tested on different living things can be carried out and thus PJ consumption can be encouraged in accordance with the obtained results. In line with these conclusions, we believe that people can live a healthy life by consuming PJ regularly.