Inhibitory effects of ethanolic extract of two Iranian pomegranates peel cultivars on Staphylococcus aureus and Salmonella typhimurium

In last decades, the antibiotic resistance is considered one of the essential problems. Therefore, uses of waste agricultural products such as pomegranate peel have drawn attention to be used as an effective preservative in food industry. Ethanolic extract of pomegranate peels (Naderi and Mallas) were prepared. Then, the antibacterial effects on two foodborne pathogens (Staphylococcus aureus and Salmonella typhimurium) in meat broth and TSB media at two temperatures levels (4 °C and 15 °C) during storage were investigated. The Minimal Inhibitory Concentration (MIC) values of Naderi and Mallas cultivar for Staphylococcus aureus and Salmonella typhimurium were measured 15.62 and 62.5 mg/ml, and 19.5 and 64.5 mg/ml respectively. The Minimum Bactericidal Concentration (MBC) of Naderi and Mallas cultivar were evaluated 125 and 130 mg/ml for both bacteria. All concentration of Mallas and Naderi Pomegranate Peel Extract (PPE) in meat broth at 4 °C and 15°C inhibited Staphylococcus aureus growth. It was reported that PPE was less effective in decreasing the S. typhimurium growth compared to the S. aureus. Naderi cultivar showed better effects on bacterial inhibition in compared to Mallas cultivar. According to achieved results, it could be suggested to use the ethanolic extract of pomegranate peel as a useful preservative against foodborne bacteria in the food processing industry.


Introduction
The Infectious disease is known as the one of the major causes of death in the world. During last decades, by increasing concerns about antibiotic resistance the uses of natural and conventional products as a preventive agent for bacterial disease have gained much attention. Therefore, many studies Original Article AJAB on antimicrobial activities of herbal medicines have been reported (Amirmohammadi et al., 2014;Asadi-Samani et al., 2014;Bahmani et al., 2014). The presence of antibacterial compounds in plants could have preventive role in the growth of pathogens. Pomegranate (Punica granatum L.) is the oldest functional fruits cultivated in Iran and neighboring countries (Gullon et al., 2016). Pomegranate peel is a waste part of the juice industry which compromise about 50% of the total weight of pomegranate fruit (Fawole et al., 2012). The pomegranate peel is rich in bioactive compounds such as ellagic tannins, flavonols, anthocyanins, catechin, procyanidins, ellagic acid and gallic acid (Fawole et al., 2012;Gullon et al., 2016). These constitute differs from one cultivar to another as well as climate changes, and place of growth (Fawole et al., 2012). It was shown that all parts of pomegranate fruit are useful for the treatment of common diseases (Ross et al., 2001). Presence of tannins and other biochemical compounds such as phenolic compounds in pomegranate fruit introduced this fruit as an antibacterial, antiviral, antioxidant, and antiinflammatory bioactive agent (Fawole et al., 2012;Malviya et al., 2014). Although the pomegranate peel is considered to be effective enough to be used without any enrichments. It was reported that phenolics compounds in pomegranate peel are involved in bacterial cell lysis by precipitation of membrane proteins and inhibition of enzymatic activities (such as glycosyltransferases) (Ismail et al., 2012). Due to various medical effects of pomegranate and its different parts, it could be helpful to investigate the antibacterial effects of its extracts. However, there is a need to examine the inhibitory effects of different varieties of pomegranates peel extract cultivated in Iran against foodborne pathogens. In current study S. aureus and S. typhimurium were chosen because of their outbreaks and emergence of drug resistance strains (Gullon et al., 2016). By knowing the efficacy of antibacterial activity of pomegranate peel extract (PPE), the use of its extract in food industry as natural food additives could be proven. Therefore, the objective of the present study was the investigation of antibacterial effects of pomegranate peel extract of two cultivar (Naderi and Mallas cultivated in Iran) against Staphylococcus aureus and Salmonella typhimurium in red meat extract and TSB at two temperatures (4 °C and 15 °C). Also, the differences in antibacterial activities of two pomegranate cultivars (Naderi and Mallas) were studied.

Material and Methods
Pomegranate fruit and preparation of extract Two cultivars of Naderi and Mallas pomegranate fruits were purchased from local market of Iran. The fresh fruits were cleaned, peeled manually, dried in an incubator (30 -40 °C), and milled. One gram of powdered pomegranate peel was dissolved in 10 ml aqueous ethanol (80%) and placed in a shaker for 24 hours at room temperature. Then extracts were filtered and dried in oven at 37 °C. The crude extract was stored at 4 °C until use (Derakhshan et al., 2018).

Methods of red meat extraction
Fresh beef were purchased from the market and transferred immediately to the laboratory under suitable condition. The meat broth was prepared by mixing the ground meat and homogenizing in distilled water at a ratio of 1 to 10 by stomacher. In order to coagulate the proteins, homogenized samples were cooked and the resulting broth was passed through filter and, then sterilized by autoclave at 121 °C for 15min.

Microorganisms
The Staphylococcus aureus (PTCC 1113) and Salmonella typhimurium (RTCC 1735) were obtained from the Persian Type Culture Collection (PTCC) and the Razi Vaccine and Serum Research Institute (RTCC), Tehran, Iran, respectively. The bacterial strains were cultured on Tryptic Soy Broth (TSB, Merck, Germany) at 37 °C for 20 min and 0.5 Mc Farland dilutions were prepared (Abdollahzadeh et al., 2011).

Determination of antibacterial activity
Separate tubes containing 1 ml of red meat extract and 1 ml of TSB were prepared for each bacterium. The amount of 10 ml of bacterial suspension was added to each test tube to reach the concentration of 10 7 colony forming units (CFU). Thereafter, pomegranate extract was added to reach the dilutions of 0.5%, 1%, 2.5%, and 5%. One tube of meat extract and one tube of TSB without pomegranate addition were considered as controls. All tested groups were incubated at 4°C and 15°C temperatures. The bacterial count was done at 0, 1, 3, 6, 24 and 48 hours after incubation. Then, the samples were cultured on Asian J Agric & Biol. 2020;8(3):341-347.

Minimum inhibition concentration (MIC) and Minimum bactericidal concentration (MBC) assays
The MIC of the pomegranate extract was determined using macro dilution technique wherein the extract was diluted serially in a series of ten test tubes containing TSB (0.5 ml) and all tubes were loaded with 0.5 ml of each bacterium (10 6 CFU/ml) and then incubated at 37 °C for 24 h. The first tube with transparent appearance was considered as a MIC. All analysis was done in triplicate (Boniadian et al., 2014). The transparent tubes were cultured on Muller Hinton agar plates and after 24 h incubation (37 °C) and the tubes with no bacterial growth recorded as MBC (Naziri et al., 2012).

Statistical analysis
Statistical analyses of the results were carried out using SPSS and the bacterial count was analyzed by a one way ANOVA and LSD comparison test.

Minimum Inhibition Concentration and Minimum Bactericidal Concentration
The pomegranate extracts minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) are shown in table 1. It was shown that two cultivar of PPE had antibacterial activity against both bacteria. According to Al-Zoreky (2009) research MIC of S. aureus, was evaluated 2 mg/ml which was estimated lower than results of current study. The MIC of S. aureus in active pomegranate extract was reported 40 and 90 µg/ml by Duman et al. (2009). In Hayouni et al. (2011) study S. typhimurium was considered the second most sensitive microorganism to methanolic extract of pomegranate peel with the MIC of 0.25 mg/ml, which is not in agreement with present study. Fawole et al. (2012) showed the MIC value of Methanolic extract of pomegranate ranges from 0.2 to 0.78 mg/ml. By comparing results of present study with these reports, the MIC values achieved in current research are considerably higher than previous studies. The MIC and MBC values of 50 and 60 mg/ml against Salmonella sp and S. aureus were reported by Gullon et al. (2016). The MBC of present research was evaluated 125 and 130 mg/ml for Naderi and Mallas PPE respectively which was not in line with Gullon et al. (2016) report. It was shown that the variation in results achieved by different researchers is due to differences among pomegranate cultivars, extraction methods, strain sensitivity, and antibacterial procedures (Gullon et al., 2016). According to our result MIC value of two investigated cultivars were varied, which is due to inter-genetic cultivar variability, geo-environmental spatial variation, and chemical heterogeneity (Fawole et al., 2012).  Negi and Jayaprakasha (2003) which was investigated the ethyl acetate, acetone, methanol extract of pomegranate peel. They indicated a high inhibition activity on both Gram-positive and Gram-negative bacteria such as S.aureus, Pseudomonas aeruginosa, and B. aurous, although E.coli had been resistance to extract (Negi and Jayaprakasha, 2003). Al-Zoreky (2009) (Panichayupakaranant et al., 2010). The antibacterial compounds in pomegranate peel which cause destruction in cell wall, cytoplasmic membrane and membrane proteins have essential role in bacterial death (Ibrahium, 2010). The inhibitory effect of PPE on S.typhimurium was more pronounced at lower temperatures (4°C) than higher one (15°C). The inhibition effect of ethanolic extract was shown at lower temperature for S. aureus than for S. typhimurium. These differences might be due to differences in cell membrane permeability of bacteria at various temperatures (Wu et al., 2016). It was shown that PPE in meat broth were more effective than in TSB media which could be attributed to low pH of meat broth. Djenane et al. (2011) infested the importance of meat pH on bacterial activity of essential oil, which probably could be referred to extracts. According to their results, by lowering the pH, the hydrophobicity of essential oil will be increased and their solubility in lipid phase of bacterial membrane facilitated. The current results are in agreement with Durairaj who reported that by increasing the pH value, the antibacterial effects decreased (Durairaj et al., 2009). Natural antibacterial agents such as ellagitannins, punicalagin, ellagic acid and gallic acid and also, the ability of phenolic compounds to precipitation of membrane proteins and inhibition of enzymes such as glycosyl transferase, make pomegranate peel as a powerful antibacterial preservative.

Conclusion
Result of current study, clearly confirmed the effectiveness of pomegranate peel on inhibition of bacterial activity. It was reported that two cultivar of PPE had antibacterial activity against S. aureus and S. typhimurium. It was shown that PPE in meat broth were more effective than in TSB media which could be attributed to low pH of meat broth. In general, it could be concluded the Gram positive bacteria was more sensitive to ethanolic extract of pomegranate peel than Gram negative one. Also, the PPE was less effective in inhibiting the S. typhimurium compared to the S. aureus, which could be due to their outer lipopolysaccharide (LPS) membranes. Also, the inhibitory effect of PPE on S. typhimurium was more pronounced at lower temperatures (4°C) than higher one (15°C). According to achieved results, the antibacterial activity of pomegranate and its use in traditional medicine are proved.