Probiotic potential of Enterococcus species isolated from raw milk and traditional dairy products of Tabriz area

Introduction: Probiotics are living organisms that, if consumed in the necessary amounts, have health effects on the host's body. The importance of these microorganisms and their health-promoting properties has led to the growth of new useful products including probiotic products (Wohlgemuth et al., 2010). Probiotic bacteria are defined as live microbial dietary supplements that can usefully modify the microbial balance of the host intestine (Durack et al., 2018). Enterococci are commonly found in raw milk and traditional fermented dairy products, and milk is an ideal source for the growth of these organisms (Doming et al. 2003; Švec and Franz 2014). Many species of Enterococcus have proteolytic and lipolytic activity and cause special taste and aroma in traditional cheeses; moreover, some Enterococcus strains have the ability to produce bacteriocin during against pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, Vibrio cholerae, as well as Clostridium and Bacillus spp. These characteristics make Enterococci to be considered as a good probiotics candidate; however, they have not yet obtained the status GRAS (Generally Recognized As Safe). Consequently, the presence of Enterococci in dairy products may be hazardous while being useful (Ogier and Serror, 2008). According to previous studies, different species of bacteria have been introduced as probiotics, but most of the introduced probiotics belong to the genus Lactobacillus and Lactococcus. In this regard, some studies have been done on some traditional Iranian dairy products (Narimani and Tarinezhad, 2014) and less attention has been paid to Enterococcus genus, especially native strains isolated from traditional products. The aim of this study was to isolate and identify Enterococci from raw milk and traditional dairy products in Tabriz region and to evaluate their probiotic potential.
Material and methods: Enterococci were isolated by culture method and identified by biochemical tests. For this purpose, 10 g of the sample was mixed with 90 ml of sterile Ringer's solution and placed in a 40 °C water bath for 5 minutes. It was then homogenized by a shaker at 230 rpm for 2 minutes (Koluman et al., 2009). Afterward, 100 µl of the suspensions (and in the case of buttermilk, 100 µl of non-diluted sample) were cultured on Kanamycin Aesculin Azide agar. Cultures were incubated at 42 °C and aerobic conditions for 24-48 hours (Doming et al. 2003). Colonies 1-2 mm in diameter with black halo were selected and cultured on Brain Heart Infusion agar plate (37 °C for 18-24 h) for morphological and screening assays (Garcia-Cano et al., 2014). Since the purpose of the study was to evaluate the probiotic potential of the isolated Enterococcus spp., the species diversity and the source of the isolates (type of dairy product) were considered as the criteria for selection of the strains. Probiotic properties such as acid and bile tolerance, proteolytic and lipolytic activity, susceptibility to antibiotics and virulence factor genes were investigated.
For acid resistance, Enterococcus isolates were cultured in MRS broth (24 h, 37 °C) and then precipitated at 4 °C for 4 min at 4,000 g. 0.1 ml of the precipitate was added to the tubes containing 2 ml of sterile PBS at pHs 1, 2 and 3 (using hydrochloric acid) and mixed. The tubes were then incubated at 37 °C for 120 minutes. Serial dilutions of the samples were prepared and the populations of the survived Enterococcus species were counted at 0, 30, 60 and 120 minutes of incubation using the plate count method. The resistance of the isolates to bile salts was evaluated in broth medium containing 0.1, 0.2, 0.3 or 0.4% bile salts, followed by incubation at 37 °C for 24 h (Lee et al. 2015). For proteolytic and lipolytic activity, the isolates were cultured on Skim milk agar (at 30 °C for 6, 24 and 48 hours and then at 10 °C for 7, 15 days) and Tributyrin agar (for 5 days at 30 °C), respectively (Serio et al. 2010). The antibiotic resistance pattern was conducted by disk diffusion method. The presence of virulence was exploited using PCR assay to detect Esp and Asa1 genes (Wankerwon et al. 2004).
 
Results and discussion: Various species of Enterococcus were isolated from the tested specimens, among which E. faecalis and E. faecium were more abundant. Therefore, 24 isolates consisting of 6 isolates of E. faecium, 5 isolates of E. faecalis, 4 isolates of E. gallinarum, 2 isolates of E. casseliflavus, 2 isolates of E. avium, 2 isolates of E. mundtii and 1 isolate from each species of E. hirae, E. saccharolyticusand E. raffinosus were selected for evaluation of probiotic potential. Based on results, none of the Enterococcus species tolerated pH= 1 and 2; however, all of the isolates survived pH= 3 during the 30, 60 and 120 minutes of incubation. Bile tolerance assay revealed that all the species were resistant to 0.1%, 0.2%, 0.3% and 0.4% of bile and their population was reduced along with the increasing of bile concentration. Moreover, various degrees of proteolytic and lipolytic activities were recorded for the different species and also among strains of individual species. The highest proteolytic activity was observed after 15 days in E. faecalis, E. mondetii and E. hirae; meanwhile, the highest lipolysis activity was found for E. saccharolythicus, E. casseliflavus and E. faecalis. In addition, different degrees of sensitivity was observed in the antibiogram assay. Esp gene was traced within two E. faecalis strains; however, Asa 1 gene was not observed among E. faecium strains.
Conclusion: Based on the findings of the current study, it can be concluded that raw milk and traditional dairy products are a rich source of Enterococcus species that are highly resistant to acid and bile. In addition, most of the isolates tested have proteolytic and lipolytic properties and can, therefore, contribute to the development and development of flavor in traditional fermented products such as cheese. The resistance of Enterococci to different antibiotics indicates the ability of these strains to survive in the gut if antibiotics are used. However, these data need to be interpreted more cautiously, as over-resistance to antibiotics is not an advantage and may have limitations in the selection of these strains. Finally, lack of Esp virulence gene in most isolates of E. faecalis and lack of Asa 1 virulence gene in E. faescium can be a positive sign of non-pathogenicity of these strains, explaining that one or more virulence factors should not be sufficient in Enterococcus. All virulence genes in Enterococcus are identified and studied in isolates of this bacterium. It can be concluded that Enterococcus species could be considered as potential probiotic bacteria, but it is crucial to conduct precise molecular, cellular and animal model investigations in order to assess the safety features of enterococci.