جداسازی و شناسایی سویه Lactiplantibacillus plantarum MOHA1 از ماست محلی و ارزیابی ویژگی‌های پروبیوتیکی، ضدباکتریایی و ایمنی آن

Introduction: Recent attention has been drawn to consuming probiotic-rich foods to enhance health and prevent diseases. Lactic acid bacteria are commonly used in fermenting foods due to their high potential to create aroma and flavor, inhibit pathogens, and prevent spoilage microorganisms. Probiotic strains commonly used belong to Lactobacillus and Bifidobacterium genera, both Gram-positive bacteria. Lactic acid bacteria are recognized for their significant presence in food items and potential use as protective cultures due to their specific attributes. These bacteria are part of the human gut microbiota, playing crucial roles in microbial balance and immune modulation (Alizadeh Behbahani et al., 2019; Alizadeh Behbahani et al., 2023; Alizadeh Behbahani et al., 2020; Fallah et al., 2021a; Fallah et al., 2021b). The primary requirement for developing a probiotic food product is selecting a suitable probiotic strain. Indigenous strains of lactic acid bacteria have gained particular importance in dairy industries due to their compatibility with regional conditions and ability to produce desirable flavors and aromas in fermented products. This research aims to evaluate the probiotic and antibacterial properties of Lpb. plantarum MOHA1 strain, including acid and bile tolerance, antibiotic resistance, cholesterol absorption, anti-adhesive properties, hydrophobicity, auto-aggregation, cell adhesion potential to Caco-2 cells, biogenic amine production, DNase activity, lack of hemolytic activity, and antioxidant activity. Additionally, the strain's antimicrobial efficacy against six foodborne pathogens (Bacillus cereus, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, and Enterobacter aerogenes) was assessed.
Materials and Methods: The research process was comprehensive and rigorous. Firstly, the Lpb. plantarum MOHA1 strain was isolated and identified using molecular methods. Subsequently, the strain was evaluated for a wide range of probiotic properties, including acid resistance (pH 2, 3, and 4), hydrophobicity, and bile resistance (0.3, 0.5, and 0.7). Cholesterol absorption was also assessed. Additionally, the strain underwent evaluation for biogenic amine production and hemolytic and DNase properties. The antioxidant property of the isolated strain (measured using DPPH and ABTS assays) was determined, and its antimicrobial activity against six foodborne pathogenic bacteria (Escherichia coli, Bacillus cereus, Salmonella typhimurium, Enterobacter aerogenes, Staphylococcus aureus, and Listeria monocytogenes) was investigated using disc diffusion and agar well diffusion methods. Furthermore, adhesion potential to Caco-2 cells, anti-adhesion properties, auto-aggregation capacity, and co-aggregation of the strain were also evaluated.
Results and Discussion: The findings of this study have significant implications for the development of probiotic food products. One of the crucial criteria in evaluating the potential of probiotic strains is assessing their resistance to acidic conditions and high concentrations of bile salts. According to the World Health Organization, probiotics must be consumed sufficiently to demonstrate their beneficial effects. Conditions such as acidic pH can disrupt metabolism and reduce the growth and survival of lactic acid bacteria. The acids present in the human stomach degrade biological molecules such as fatty acids, proteins, vitamins, and nucleic acids. In this study, bile salt concentrations of 0.3%, 0.5%, and 0.7% (W/V) were tested to evaluate the resistance of the targeted strain. The results indicated that Lpb. plantarum MOHA1 has good resistance to different concentrations of bile salts. These findings are consistent with other studies that have shown lactobacilli can survive in high bile salt concentrations (Brezgár et al., 2021; Abushelaibi et al., 2017; Boricha et al., 2019; Molavi et al., 2019; Oulahal et al., 2018; Shahata et al., 2016). To assess the viability of Lpb. plantarum MOHA1, this bacterium was subjected to pH levels 3, 2, and 4. The results showed that the strain Lpb. plantarum MOHA1 has desirable viability under the examined conditions. However, the highest strain viability reduction was observed after 3 hours at pH 2. With a decrease in pH from 4 to 2, a significant reduction in the number of viable cells was observed, decreasing from 7.7 to 2.6 Log CFU/mL. Lactobacillus plantarum converts sorbic acid into 1,3-pentadiene through decarboxylation and produces carbon dioxide and ethanol during fermentation. It can also metabolize sugar compounds and produce acidic compounds such as succinic acid, which lowers the pH level (Ghorbani et al., 2018).
Hydrophobicity is one of the important factors for bacteria regarding their adherence to various surfaces. Hydrophobicity depends on various factors such as van der Waals forces, Brownian motion, surface electric charge, and gravitational force. Therefore, this feature should be separately investigated for bacteria (Noshad et al., 2021). The hydrophobicity level of the Lpb. plantarum MOHA1 strain was determined to be 20.53% ± 0.60%. In a comprehensive study by Vosoughi et al. (2018), it was reported that Lpb. plantarum A44 isolated from whey exhibited the highest cellular hydrophobicity (84.5%) among other strains. In the study by Alizadeh Behbahani et al. (2019), the Hydrophobicity potential of the Lpb. plantarum L15 strain was found to be 54%. In the research conducted by Shahrampour et al. (2019), the effect of genetic diversity of Lpb. Plantarum strains isolated from different food sources were investigated for their antimicrobial, antioxidant, and cumulative activities. The results showed that the strain Lpb. plantarum KMC61 had the highest surface hydrophobicity percentage. However, there was a difference in surface hydrophobicity among the ten strains studied, which may be related to the biosynthesis and composition of their cell surface proteins and polysaccharides. The production of biogenic amines, DNase, and hemolytic activity were negative. In this study Cholesterol absorption was 40.30%. Many studies have shown that some lactic acid bacteria in cell culture media can reduce cholesterol concentration. In some studies, changes in the fatty acid pattern inside the cell have been observed following this reduction. In some cases, microscopic examination of the cell has shown deposition and binding of cholesterol to the bacterial surface. The presence of such strains as starter cultures in foods considered a source of cholesterol can be very beneficial (Modani et al., 2013).DPPH radical scavenging activity was 45.80%, and ABTS radical scavenging activity was 48%. The antimicrobial and antioxidant properties of Lpb. plantarum strains have been reported in various studies. Asadi et al. (2009) investigated the technological characteristics and antimicrobial activity of 17 strains of Lpb. plantarum isolated from a type of salted meat, and observed the highest inhibitory activity of Lpb. plantarum strains against Staphylococcus aureus bacteria. Auto-aggregation potential was 37.60%, and co-aggregation potential was 19.10%. The adhesion potential to Caco-2 cells was 11.30%. One of the most important criteria for selecting lactic acid bacteria as probiotics is their safety and tolerance to gastrointestinal conditions and their ability to adhere to the mucosal cells on the intestinal surface.Challenges associated with clinical studies on adhesion testing have led to the widespread use of simulated models at the laboratory scale. In particular, the use of the Caco-2 cell line for evaluating the adhesion of probiotic strains to the digestive system has gained significant attention because, under these conditions, the morphological and functional properties of cells on the surface of small intestinal villi are well-defined (Fallahi et al., 2018). Anti-adhesion potential against E. coli was 36.70% in competition, 35.50% in inhibition, and 22.40% in displacement. Pathogenic bacteria must attach to the intestinal cell wall to cause infection. Therefore, any factors that prevent this activity benefit health and intervene with the development of infection. Probiotic bacteria, through mechanisms such as producing antimicrobial compounds and disrupting or blocking molecules that pathogenic agents use for attachment, prevent the adhesion of pathogenic bacteria (Vosoughi et al., 2022). A study evaluating the probiotic potential of indigenous Lactobacillus strains isolated from Zabol yellow cheese was conducted by Vosoughi et al. (2022). The antimicrobial effect of Lpb. plantarum MOHA1, both acidic and non-acidic, on pathogenic strains has been demonstrated using the agar diffusion and well methods Showed that the greatest inhibitory effect was observed against L. monocytogenes. This bacterium is a facultative anaerobe capable of surviving in both the presence and absence of oxygen and is known to cause a wide range of diseases in humans and animals. The least inhibition was observed in the agar diffusion method using disks against the pathogens S. typhimurium and E. coli, and in the agar diffusion method using wells against the pathogen E. coli. Intestinal Gram-negative bacteria such as E. coli and S. typhimurium are among the major causes of food poisoning and diarrhea.
Conclusion: Strains isolated from local fermented and dairy products are considered valuable sources of probiotics. These strains have high probiotic potential and exhibit significant antimicrobial properties. According to the results of this study, Lpb. plantarum MOHA1 can withstand acidic conditions in the stomach and the presence of bile salts, indicating essential traits for survival and effective performance in the human digestive system. This strain has demonstrated a high ability to inhibit pathogenic bacteria, attributed to the production of antimicrobial compounds that can inhibit the growth and proliferation of pathogens. Additionally, Lpb. plantarum MOHA1 is sensitive to common antibiotics, which can be beneficial in controlling infections and preventing microbial resistance. Other important characteristics of this strain include its ability to adhere to the intestinal wall, surface hydrophobicity, autoaggregation, and co-aggregation. These features enable this strain to adhere to the intestinal epithelial cells and form resilient colonies, which can improve digestive health and enhance the body's immune system. Given these properties, the Lpb. plantarum MOHA1 strain has a high potential for use as a probiotic supplement in fermentation cultures or as a co-culture in the production process of fermented food products. Using this strain can help improve food products' quality and shelf life and enhance consumer health. Further confirmatory tests and comprehensive research can strengthen these strains' practical and commercial applications in the food industry.