There are several criteria for bacteria to recognize as probiotic. One of the most important features is tolerance to low pH and high bile salt concentrations. They must overcome physical and chemical barriers in the gastrointestinal tract to be able to transit through the stomach. Probiotics to be able to survive and grow and could exert their action in the small intestine must be tolerant to high bile salt conditions 22. In this work, the evaluated isolates indicated varying values of viability in pH 3 and 0.3% bile oxgall. The isolates F3, F12, F15, and F7 showed more than 90% viability at pH 3 and 0.3% bile oxgall. High survival rates of LAB especially Lb. plantarum 9,23, and P. pentosaceus and P. acidilactici 24–26 at gastric pH of 3.0 and 0.3% bile concentration have been reported.
The antimicrobial properties of probiotics are attributed to their metabolite productions including organic acids, hydrogen peroxide, and bacteriocin 27. In this study, isolates were investigated against two Gran-positive (St. iniae and St. agalactia) and six Gram-negative (V. harveyi, V. cholera, V. alginolyticus, V. parahaemolyticus, and Cl. cochlearium) indicators. Evaluated isolates showed better antibacterial effects on Gram-positive than Gram-negative indicators, which this could be due to the outer membrane of Gram-negative bacteria. All the 19 isolates inhibited the growth of St. iniae and St. agalactia. Isolates F12 and F15 showed the best results and were able to inhibit the growth of all the 8 evaluated indicators.
Another strange property of probiotics is their ability to form biofilms. It can promote colonization within the inner wall of the intestine and longer lasting quality of LAB, thus maintaining a strategic distance from colonization by pathogenic microorganisms 28. The ability of form biofilm is of great economic importance to food-processing companies, as subsequent microorganisms attached to industrial devices can form highly resistant biofilms, and their strong tendency to adhere to the surface facilitates the separation of biomass during the fermentation process 29. Isolates F3, F7, F12, and F15 showed the strong biofilm formation ability; cause them to be suitable for use as biocontrol agents.
There are many studies which investigated the cholesterol reduction ability of LAB isolates 13,30,31. The reduction of serum cholesterol values is important to prevent the coronary heart disease due to the main risk factor for the progression of coronary heart disease is hypercholesterolemia 32. Isolates F12, F3, and F7 showed remarkable cholesterol removal ability, with values of more than 59%.
For selection of functional LAB isolates, two groups of antibiotics are recommended; first those are inhibitors of cell wall synthesis such as vancomycin and ampicillin, and second those are inhibitors of protein synthesis including erythromycin, chloramphenicol, clindamycin, gentamycin, kanamycin, tetracycline, and streptomycin 33. In this study, the LAB isolates indicated susceptibility to chloramphenicol (89.47%), tetracycline (94.73%), erythromycin (84.21%), ampicillin (84.21%), azithromycin (52.63%), gentamycin (42.11%), streptomycin (57.89%), penicillin (42.11%), vancomycin (52.63%), and ciprofloxacin (42.11%). The results of this study is consistent with the previous results which showed that LAB are susceptible to antibiotics inhibiting the proteins synthesis, such as chloramphenicol, tetracycline, and erythromycin, and are resistance towards glycopepticides antibiotics such as vancomycin 34, and aminoglycosides antibiotics including streptomycin, kanamycin, and gentamycin 35. Several LAB species belonged to especially Lactobacillus, Pediococcus and Leuconostoc have been reported to exhibit high levels of natural resistance to vancomycin, which is useful to separate them from other Gram-positive bacteria 25.
A desirable property of probiotic bacteria is colonization in intestinal wall 13. Hydrophobicity, auto-aggregation and adhesion to epithelial cells could help probiotics to colonize in intestinal cells. The hydrophobicity of the bacterial cell surface is one of the most important factors for probiotics to be colonized in human and animal gastrointestinal tracts. In this study, the cell surface hydrophobicity percentages of the isolates ranged from 3.7 ± 1.6% to 70.2 ± 2.0% with toluene and 2.2 ± 1.6% to 76.4 ± 2.1% with xylene. This result confirms the results of several previous researches reported by 36–38. Four out of 19 isolates showed both considerable cell surface hydrophobicity and high auto-aggregation (> 67%), which indicated good colonization potential 39. According to Vallejo et al. (2008), strains with values of autoaggregation higher than 65% are strongly autoaggregating. Isolates F3, F7, F12, and F15 showed remarkable autoaggregation ability; making these isolates interesting candidates for potential probiotic applications. The capacity to attach to epithelial cells and mucosal surfaces may be a critical feature of bacterial isolates to be utilized as probiotics. Results showed that only isolates F3, F7, F8, F12, and F15 were able to adhere to Caco-2 cells. The adhesion ability of these 4 isolates to Caco-2 cell had also significant coherence to auto-aggregation and hydrophobicity. The adhesion rates of these isolates to Caco-2 cells were between 23% and 38%. It seems that adhesion ability is correlated with auto-aggregation 30. These results also support the correlation between adhesion and auto-aggregation.
In conclusion, several isolates evaluated in this study showed potential probiotic properties. The majority of evaluated isolates showed high antimicrobial activities against potentially pathogenic Gram-positive and Gram-negative indicator bacteria which deserve them to be used as biocontrol agents in fishery. Regarding the interesting probiotic properties, the highest values of autoaggregation and hydrophobicity as well as a high value of coaggregation and a high capacity for biofilm formation, isolates F3, F7, F12, and F15 could be candidates to be used as bio-control agents in fishery to protect against pathogenic microorganisms and improve health care and food safety by avoiding the use of additives. Cluster characterization of identified potential probiotic bacteria based on two above mentioned methods also highlighted the priority of isolates F3, F7, F12, and F15 as bio-control agents in fishery industry. In line with our study, prior finding also highlighted the effectiveness of above-mentioned methods for characterization and prioritization of newly identified isolates 17,40. Also our empirical and cluster analysis based on unsupervised methods prioritized some isolates as biocontrol agent candidates in fishery industry, however; further analysis would be needed.