Investigation of probiotic properties of Lactobacillus paracasei isolated from traditional cheese in Iran

Over the past few decades, there has been a surge in interest in the isolation of novel Lactobacillus probiotic strains that exert beneficial effects in the host gastrointestinal tract (GI). probiotics are ‘‘living micro-organisms, which upon ingestion in certain numbers, exert health benefits beyond inherent basic nutrition’’. Most probiotic microorganisms are lactic acid bacteria (LAB), among them lactobacilli represent one of the major microbial groups. They have been introduced in a wide range of food products, including yogurts, cheese and other dairy products as well as non-dairy products, such as fruit juices and fermented sausages. (Bao et al., 2010). In order for a probiotic strain to exert its beneficial effect on the host, it has to be able to survive passage through the host’s digestive tract. So far, research has mainly focused on strains’sensitivity towards low pH, proteolytic enzymes and bile salts. It is considered that the ability of Lactobacillus strains to adhere to the mucosal surfaces of the intestine before they can exert their beneficial effects has long been one of the most commonly encountered criteria for the selection of probiotic strains (Gao et al., 2013). Auto aggregation ability and surface hydrophobicity of bacteria are two independent traits, and their determination has been proposed as an indirect method for evaluating the adhesion ability of bacteria. Also the ability to co aggregate with other bacteria such as pathogens may form a barrier that prevents colonization by pathogenic microorganisms (Collado et al., 2007). The objective of this study was to evaluate the in vitro probiotic potential of the Lactobacillus strain isolated from Semnan traditional cheese (Khiki), and could therefore be potentially used as novel probiotic strain in the food industry.

In this study, the probiotic potential of Lactobacillus paracasei OK561873 isolated from Semnan traditional cheese in previous study by the Department of Food Hygiene, Faculty of Veterinary Medicine, Semnan University was investigated. The isolated bacterium was sequenced based on the 16s rRNA genome and was confirmed as Lactobacillus paracasei confirmed in the BLAST database and registered as a new strain in the NCBI gene bank with code OK561873.Then, to evaluate the resistance of the Lactobacillus isolates to pH ,10 μl of the 24-hour culture of isolate (108 CFU / ml) was mixed with 240 μl of MRS broth at pH adjustments (1.5 and 2.5) in a microwell plate and incubated for 37 h at 37 ° C. Serial dilutions were prepared at 0 and 3 hours and cultured by surface plate in MRS agar medium and incubated for 24 to 48 hours in aerobic conditions at 37 ° C. Finally, the survival percentage of the strain was calculated by bacterial counting. The ability of the strain to grow under acidic conditions in MRS broth at three different pHs 2.5, 3.5 and 4.5 (based on screening experiments) was evaluated, and the growth curve according to the OD values obtained at 630 nm was drawn by ELISA reader (Biotech, USA) during 24 hours. MRS broth containing 0.3, 0.5 and 1% oxgall was used to determine resistance and growth in the presence of bile. The growth curve was drawn using OD630 nm reading in 24 hours by ELISA reader (Biotech, USA). The 24-hour cultured bacterial pellet (108 CFU / ml) was separated by centrifugation (g6000) and washed twice with PBS buffer; then they were suspended in PBS solutions with a pH of 2 and containing pepsin (3 mg/ml) and NaCl (0.5%). After incubation at 37 ° C, live colonies were counted by culture in MRS agar at 0, 60, 90 and 180 minutes. After 3 hours, 1 ml of the previous stage culture was added to 9 ml of intestinal juice containing 1 mg/ml of pancreatic and bile of 0.3% at pH eight, and live bacteria were counted at 0, 2 and 4 hours. To evaluate the antibacterial activity of the isolate, two bacteria, Staphylococcus aureus ATCC 29213 (as an indicator of gram-positive bacteria) and Escherichia coli ATCC 35218 (as an indicator of gram-negative bacteria) were used. The experiment was performed by two methods of spot agar and well diffusion. The hydrophobicity test, is evaluated by measuring the microbial adhesion to hydrocarbons. Auto-aggregation property of probiotic strain can affect the binding capacity of bacterial cells to intestinal epithelial cells, and co-aggregation with pathogens can prevent the colonization of pathogens in the intestine. In brief, four milliliters of the 24-hour culture containing 108 CFU / ml of bacteria were vortexed and homogenized for 10 seconds and then incubated for 5 hours at room temperature. At one-hour intervals, one-tenth of a ml suspension was removed and placed in a new tube containing 3.9 ml of PBS was transferred. Then the absorption of 600 nm was measured using a spectrophotometer.

the viability was evaluated at pH 1.5 and 2.5 on isolated Lactobacillus. No growth was observed at pH 1.5. While at pH 2.5, the number of viable cells was reported to be 6.23 log cfu / ml and showed a good survival rate (75.5%). According to the growth results of Lactobacillus isolate at different pH (2.5, 3.5, 4.5) showed that the growth rate, after the control group, at pH = 4.5 was higher than other groups (P <0.05) But there was no significant difference in other pHs. Lactobacillus paracasei showed good resistance and growth at different concentrations of bile. Growth retardation time of few than 30 minutes was observed, indicating that the isolates were resistant to different concentrations of bile. No significant differences were observed between treatment groups. The results of isolate resistance in the simulated conditions of gastric and intestinal juice showed that at pH 2 and after three hours, the strain survived and only a reduction equivalent to one and a half logarithms was observed. no decrease happened in the intestinal environment, even an increasing growth of strain was observed. The results of the antagonistic effect of Lactobacillus isolate with Staphylococcus aureus and Escherichia coli by spot and well diffusion methods showed that the growth inhibitory effect against the studied pathogens was related to Lactobacillus paracasei with a growth inhibition zone diameter of 2.94±1.39 mm against Staphylococcus aureus and 2.59±1.37 mm against Escherichia coli. There was also no significant difference in the antagonistic effect of lactobacilli isolate against gram-positive and gram-negative bacteria. According to the results, Lactobacillus paracasei isolate showed a high percentage of auto aggregation characteristics and the average percentage during five hours of incubation at 37 ° C was 92.45%. Co-aggregation of Lactobacillus strain with pathogens (Staphylococcus aureus and Salmonella typhimurium) was also investigated. The results are expressed as a percentage reduction after four hours in the adsorption of the mixed suspension compared to the individual suspension and according to the results, the tested strain showed a significant coaggregation with Salmonella typhimurium. Also, the results of hydrophobicity of Lactobacillus paracasei isolate show that this strain can have good hydrophobicity (57. 82%).

The strain isolated from traditional Semnan cheese showed desirable probiotic properties in vitro. Therefore, it is suggested that this isolate, as a new probiotic strain in the food industry, could be a good candidate for further in vitro complementary tests and in vivo research to demonstrate potential health benefits.