بررسی ویژگی‌های پروبیوتیکی، آنتی اکسیدانی و ایمنی سویه Lactiplantibacillus plantarum sps1

Probiotics are defined as microbial supplements that positively impact host health by modulating gut microbiota balance. Their beneficial effects on human health include improving lactose digestion in lactose-intolerant individuals, regulating the immune system, synthesizing B-group vitamins, producing antimicrobial peptides, enhancing immune response, and preventing cell carcinogenesis (Fallah, Mortazavi, Tabatabaei Yazdi, 2018). While most probiotics used in humans belong to the lactic acid bacteria family, not all bacteria in this group qualify as probiotics, as survivability and colonization in the gastrointestinal tract are essential criteria for probiotic selection (Colado, Meriluoto, Salmin, 2007).Lactic acid bacteria, especially Lactiplantibacillus plantarum, are extensively used in food industry for enhancing flavor, texture, and nutritional properties of various foods such as vegetables, meats, dairy products, and grains. They prolong shelf life by producing organic acids, carbon dioxide, and antimicrobial peptides (Huang et al., 2023). These bacteria also play a crucial role in fermentation processes of foods like dairy products, sausages, pickles, and olives (Sidhu & Nehra, 2019 (Lpb. plantarum, known for its adaptability and high distribution in diverse environments, is found in fermented dairy products, human and animal digestive systems, as well as fermented meats, fish, and vegetables (Panel & Herman, 2021). Studies highlight its probiotic properties including immune modulation, liver disease reduction, and potential anticancer effects (Garcia-Gonzalez, Battista, Prete, Corsetti, 2021). Recent research focuses on characterizing new probiotic strains like Lpb. plantarum sps1 isolated from local yogurt, evaluating its probiotic and antimicrobial potential against various foodborne pathogens (Mokoena, Omatola, Olaniran, 2021)
Materials and Methods
The research process was comprehensive and rigorous. Firstly, the Lpb. plantarum sps1 strain was isolated and identified using molecular methods. Subsequently, the strain was evaluated for a wide range of probiotic properties, including acid resistance (pH 2.5, 3.5, and 4.5), 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. 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
Based on the results obtained, the strain exhibits promising probiotic and antimicrobial potential. It demonstrated the highest reduction in strain count after 3 hours at pH 2.5. The strain showed good resistance to all concentrations of bile salts. Its hydrophobicity was 46.5%. It exhibited negative traits for biogenic amine production, DNase activity, and hemolytic activity. Furthermore, it absorbed 42.30% of cholesterol, and its scavenging activities for free radicals DPPH and ABTS were 40.5% and 46.30%, respectively. It also showed auto-aggregation and co-aggregation potentials of 38.60% and 22.40%, respectively. Adhesion potential to Caco-2 cells was 11%, and it displayed competitive inhibition against Salmonella enterica serovar Typhimurium at 44.20%, inhibition at 29%, and displacement at 22.50%. Based on the obtained results, the strain has acceptable probiotic, antioxidant, and immune potential. It can be stated that this strain is effective in improving health and reducing disease risks, and ultimately, it can be utilized as an effective probiotic for practical applications.
one essential characteristic of a probiotic is its ability to survive under gastrointestinal conditions, particularly its resistance to acid and bile salts. Additionally, acid tolerance enables the use and survival of these strains in acidic food products such as yogurt without significant reduction in bacterial population. Probiotic strains, when exposed to the extreme pH of the stomach, may acquire buffering properties through food or other specific molecular carriers. This acid tolerance is also attributed to the synthesis of various polysaccharides, which play a role in protecting the cell membrane (Dardmeh et al., 2023). Noshad et al. (2021) reported that acid resistance is strain-dependent and should be evaluated individually for each bacterium. They also indicated that Lpb. plantarum and Levilactobacillus brevis exhibit higher resistance to low pH compared to Pediococcus. Koshki et al. (2012) found that the best growth of Lpb. plantarum isolated from traditional dairy products in Sabzevar was at pH 4, and the strain was capable of growth in the presence of bile salts. Additionally, Lpb. plantarum S32E, isolated from traditional Iranian skin cheese, showed the highest survival against simulated gastric and intestinal conditions (Abdoli et al., 2018). actors such as non-pathogenicity, lack of association with diarrhea-causing bacteria, genetic stability, inability to transfer antibiotic resistance genes, and adhesion capability are crucial for identifying a probiotic bacterium. One key aspect for selecting a strain as a probiotic is determining its adhesion capability to the host mucosa. This not only prevents the adhesion of pathogenic bacteria but also creates an unfavorable environment for their growth by secreting certain substances. It allows the bacteria to occupy target sites on epithelial cells, preventing their attachment and invasion into the gastrointestinal tract (Gabriela et al., 2019). In a study, Lpb. plantarum was introduced as a highly adhesive probiotic strain (Handa & Sharma, 2016). Lpb. plantarum L33, isolated from Iranian traditional cheese, showed the highest auto-aggregation (49.56%). This study reported the relationship between adhesion capacity and the hydrophobicity of lactic acid bacteria.reactive oxygen species (ROS), such as superoxide ions, hydroxyl radicals, nitric oxide, and peroxynitrite, can react with all cellular biomolecules and, if accumulated, lead to oxidative stress in the body, causing many chronic inflammatory conditions in the gut (Liu et al., 2018). This antioxidant potential could be attributed to the production of metabolites such as exopolysaccharides (EPS) and various antioxidant enzymes. EPS are long-chained molecules secreted by bacteria into their environment, which have been shown to have antioxidant properties (Fang et al., 2018). Antioxidant compounds produced by probiotic bacteria can scavenge reactive oxygen species and reduce the level of oxidative stress in the host, thereby promoting gut health (Kuda et al., 2019). antioxidative potential of lactic acid bacteria as a free radical scavenger.
DNase, capable of degrading DNA by breaking phosphodiester bonds in the backbone of DNA, may play a role in bacterial growth, enhancing biofilm formation, and immune system modulation, thus acting as a virulence factor (Varela-Ramirez, 2017). Biogenic amines are small molecular compounds that form in protein-rich foods, especially fermented foods. Biogenic amines are formed from free amino acids by microbial decarboxylation. Biogenic amines, especially histamine, are associated with some spoilage microorganisms and can be used as freshness and safety indicators for fermented food products (Sun et al., 2023). Cholesterol, as a vital component of the human body, plays a fundamental role in many processes. However, prolonged high blood cholesterol levels may lead to atherosclerosis and pose a significant risk for cardiovascular diseases. Among the Lpb. plantarum strains isolated from goat milk, the highest cholesterol reduction was observed in Lpb. plantarum Lpb02 with 68%, followed by Lpb. plantarum Lpb01 with the second highest reduction at 51%, while commercial strain Lpb. plantarum ATCC 8014 showed a reduction capability of 51% (Islam et al., 2022). The food chain is considered one of the main pathways for the transfer of antibiotic-resistant bacteria between animal and human populations. Fermented dairy products, especially those not subjected to heat treatment before consumption, are identified as a route for transferring antibiotic-resistant bacteria (Mathur & Singh, 2005). In this study, the resistance of Lpb. plantarum sps1 strain to eight common antibiotics was evaluated (Table 2). This strain was sensitive to six antibiotics, resistant to Imipenem, and showed intermediate sensitivity to Gentamicin. Evidence suggests that Lactobacillus species are generally sensitive to antibiotics such as erythromycin, chloramphenicol, and tetracycline (Alizadeh Behbahani et al., 2023). Anti-adhesive properties were evaluated through various adhesion inhibition assays, including competitive, inhibitory, and displacement tests against S. typhimurium. Lactobacillus strains such as Lpb. plantarum TW57-4 exhibited the highest activity against P. aeruginosa, with strains Lpb. plantarum 5H1, Lpb. plantarum 5 L1, and Lcb. rhamnosus GG showing a greater reduction (56%–73%) in Salmonella adhesion (Vasiee, Falah, Mortazavi, 2022; Luz et al., 2021). The displacement capability of strains may be due to competition for common adhesion receptors, production of anti-adhesive compounds, and secretion of antimicrobial agents. Various mechanisms are involved in bacterial anti-adhesive processes, including chemical and physical interactions between bacterial and host or environmental surfaces. Correlation between anti-adhesive results and adhesion may indicate similar mechanisms in both phenomena; however, other studies have reported no correlation between anti-adhesive and adhesion results (Haghshenas et al., 2016).
Conclusion
The strain Lpb. plantarum SPS1 exhibits potential for survival in acidic conditions across various pH levels. It also demonstrates resilience against different concentrations of bile salts, which is advantageous for its survival in environments like the intestine. The strain shows adequate hydrophobicity and auto-aggregation, facilitating interactions with other cells and adherence to different surfaces. These characteristics position it as an effective probiotic that can prevent pathogenic bacteria from adhering to epithelial cells. The strain's ability to produce biogenic amines, lack of DNase activity, absence of hemolytic activity, and antioxidant activity further endorse its potential health benefits. Ultimately, Lpb. plantarum SPS1 proves to be a valuable probiotic strain for improving health and reducing disease risks, making it a promising candidate for practical applications.