Background and Objectives: During thousands of years, human has unconsciously benefited from lactic acid bacteria (LAB) in the production of fermented food. This is due to the potential of these bacteria to release odor and flavor, and inhibit the growth of pathogens and food-spoilage microorganisms. LAB is Gram-positive, catalase-negative, microaerophilic and non-spore forming. In addition to their health-promoting effects, they have numerous technological and microbiological properties in food products. LAB is usually known as the microorganisms highly consumed in food products, which can be utilized as protective culture media, owing to their specific characteristics. The present study aims to examine the probiotic potential (viability in acidic and simulated gastrointestinal conditions, hydrophobicity, auto- and co-aggregation) of the LAB isolated from Behbahan local Doogh. Next, their technological properties (acidification and autolytic activities, and heat resistance) were determined. Finally, the antimicrobial activity of the strains was measured against some food pathogens.
Materials and methods: In this study, the LAB strains of Behbahan local Doogh were identified through 16S rRNA gene replication by a polymerase chain reaction. First, 85 Gram-positive and catalase-negative isolates were categorized using biochemical and sugar fermentation tests. One representative was selected from each group for the molecular identification test. In order to investigate the resistance of the bacteria to acidic conditions, their viability was examined at pH values of 2.5, 3.5 and 5. For determining the LAB viability in the gastrointestinal system, 30 µl of each bacterium was added to 270 µl of simulated gastric juice (2.5 g pepsin/l and 2 g NaCl/l) whose pH had been set at 2.75 and incubated at 37°C for 1.5 h. Then, 30 µl was mixed with 270 µl of simulated intestinal juice (1 g trypsin/l, 5 g bile salt/l, 2 g pancreatin/l, 11 g sodium bicarbonate/l and 2 g sodium chloride/l) whose pH had been adjusted to 8 and incubated at 37°C for 4 h. Afterwards, the bacteria were cultured on MRS agar at 1.5, 2, 3 and 4 h. The results were expressed as the reduction of log CFU/ml relative to time zero (time zero: 8 log CFU/ml). The other probiotic properties of the LAB, including hydrophobicity, auto- and co-aggregation were also investigated. Their technological potential was determined though acidification ability, autolytic activity and resistance to heat. Eventually, the antimicrobial activity of the strains with better probiotic and technological properties was examined against 5 food pathogens (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Micrococcus luteus and Staphylococcus aureus). One-way analysis of variance was conducted using statistical package for the social sciences (SPSS) version 22. Duncan`s multiple range test was employed for mean comparison at 95% confidence level.
Results and discussion: Lactobacillus plantarum, Lactobacillus delbrueckii and Pediococcus pentosaceus were the most resistant strains to the pH values of 2.5 and 3.5, while Lactobacillus pentosus and Enterococcus faecium showed the most pronounced reduction of log CFU/ml. At the pH value of 5, all the bacteria were able to grow without being reduced. Relative to the simulated gastric juice, L. plantarum was the most resistant one with a reduction of zero log CFU/ml and E. faecium was the most sensitive one with a reduction of 3 log CFU/ml. After 3 h of digestion by the simulated intestinal juice, L. plantatum and L. delbrueckii were the most resistant strains with a reduction of 1 log CFU/ml and E. faecium was the most susceptible one with a reduction of 5 log CFU/ml. The hydrophobicity of the strains was reported to be 17.5-58.3%, the highest of which belonged to L. plantarum. The co-aggregation of the LAB varied from 25.3 to 48.2%, the highest of which was associated with P. pentosaceus. In addition, all the strains showed co-aggregation activity against E. coli, and L. plantarum had the highest value of co-aggregation (46.3%). In terms of the technological properties, the results revealed that all the strains were capable of acid reduction up to 0.4 except L. pentosus. During 24 h of incubation, P. pentosaceus and L. delbrueckii caused the largest pH variations. The results of the autolytic activity of the LAB isolates indicated that all the L. pentosus, L. plantatum and L. delbrueckii could be grouped as relatively good. The highest viability and heat resistance belonged to P. pentosaceus and L. delbrueckii and the lowest viability was related to L. pentosus and E. faecium. In general, L. delbruckeii and P. pentosaceus showed the best results in all the technological tests. Moreover, the antimicrobial activity of L. plantarum, L. delbrueckii and P. pentosaceus was investigated against E. coli, P. aeruginosa, S. typhimurium, M. luteus and S. aureus. In all the samples, the acidic cell-free supernatant had a greater antimicrobial effect than the neutralized cell-free supernatant. The neutralized supernatant of L. plantarum and L. delbrueckii had no antimicrobial effect on E. coli. The shortest inhibition zone diameter of the acidic supernatants of L. plantarum, P. pentosaceus and L. delbrueckii was associated with E. coli. Furthermore, S. aureus was the most sensitive strain against the acidic supernatant of L. plantatum and S. aureus, whereas M. luteus had the longest inhibition zone diameter against the acidic supernatants of L. delbrueckii and P. pentosaceus.
Conclusion: Considering the obtained results, L. delbrueckii, L. plantarum and P. pentosaceus had superlative performances and can be used as complementary strains in the production of food products. It is suggested that supplementary tests, including the adhesion ability to intestinal epithelial cells, anti-adhesion ability against specific pathogens, and the lipolytic and proteolytic activity of these three strains be evaluated.