Background and Objectives:One of the important and global challenges of public health is foodborne diseases, which impose huge economic losses on the economy of societies. Among them, pathogenic bacteria play an important role. The negative effects of chemical preservatives on human health and the increasing resistance of many microbes to common antimicrobial agents have necessitated the identification of safe and effective alternatives. Today, the use of low-risk preservatives such as plant essential oils and bacteriocins has received attention. The antimicrobial effects of essential oils depend on the type and amount of their constituent components. The amount of these compounds is affected by factors such as plant type, species, soil composition, plant organ, age, climatic conditions of the growing region, essential oil extraction method, and harvest date. Clove is a plant belonging to the Myrtaceae family and is known by the scientific name Syzygium aromaticum. The chemical components of clove essential oil, especially eugenol, have a high contribution to its antimicrobial properties. Nisin is an antimicrobial peptide produced by Lactococcus lactis. It is the only bacteriocin that has received permission for use as a preservative for food from FAO and WHO. The aim of this study was to identify the chemical components of commercial Iranian clove bud essential oil (distilled with water) and compare its antimicrobial properties with nisin on controlling the growth of some of the most important foodborne disease-causing bacteria in order to identify the most effective antimicrobial compound agent.
Materials and Methods:In this study, dried clove buds were obtained from a local market (Zabol, Iran). Essential oil extraction was performed using a Clevenger apparatus (water distillation) under controlled temperature. After extraction, the type and amount of chemical compounds of clove essential oil were identified using gas chromatography-mass spectrometry (GC-MS). In this study, six bacterial strains include Listeria monocytogenes NCTC 11994, Staphylococcus aureus Bristol A9596, Bacillus cereus ATCC 9634, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Salmonella typhimurium ATCC 14028 were purchased in lyophilized form from the Microbial Collection of the Scientific and Industrial Research Organization of Iran. The strains were activated under controlled conditions using Tryptone Soya Broth (TSB) and Tryptone Soya Agar (TSA) media. Next, the antibacterial effects of the obtained essential oil and nisin were evaluated by agar diffusion methods against target strains with a population of 10⁶ CFU/ml and the results were reported in terms of mm of inhibitory zone. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the antimicrobial agents were determined by microdilution and 96-well plates containing TSB media. The prepared dilutions of the essential oil ranged from 0.3 to 40 mg/ml and nisin ranged from 4.8 to 0.1023 μg/ml. The obtained data were analyzed using SAS version 9.1 software and the means were compared by Duncan's multiple range test at the 5% level.
Results :GC-MS analysis of clove essential oil showed that 93.66% of the total components, including 17 different components, were identified. The most important compounds identified were eugenol (54.27%), trans-β-caryophyllene (21.13%), and eugenol acetate (11.18%). The antibacterial activity of clove essential oil and nisin by agar diffusion method showed a significant effect on the growth of all strains (p˂0.05). The highest and lowest growth inhibitory effects of clove essential oil were recorded against gram-negative strains Escherichia coli (29.6 mm) and Salmonella typhimurium (11.2 mm), respectively. Nisin showed the highest effect on the growth inhibition of gram-positive strains compared to gram-negative strains (p˂0.05). The highest nisin growth inhibition zone was determined against Listeria monocytogenes (14.3 mm) and the lowest value was determined for the gram-negative strain Salmonella typhimurium (8.6 mm) (p˂0.05). The antibacterial agents studied in this study showed different effects on MIC and MBC values. The highest MIC and MBC values ​​were obtained from the effect of clove essential oil on Salmonella typhimurium strain with values ​​of 2500 and 5000 µg/ml, respectively, and the lowest values ​​were obtained against Escherichia coli strain with values ​​of 156 and 156 µg/ml. The lowest nisin MIC value was recorded at a concentration of 63 µg/ml against Staphylococcus aureus, Bacillus cereus and Pseudomonas aerogenesa strains, which indicated the greater sensitivity of these strains.
Conclusion:Nisin and clove essential oil showed significant growth inhibitory effects. The results of the microdilution antibacterial test showed that nisin was more effective compared to clove essential oil (p˂0.05). Clove essential oil was able to effectively control the growth of Escherichia coli strain. The results of this study showed that despite the difference in the amounts of the constituent components of clove essential oil compared to other published scientific reports, it can be considered as an alternative to chemical preservatives. In this regard, it is necessary to pay attention to the target strain and the concentration of the antibacterial agent. However, the final confirmation of the use of the studied compounds requires clinical and applied research in food.