The higher the hydrophobicity, higher colonization was observed (de Souza et al., 2019). This characteristic makes it the most suitable to not only remove/decrease the adherence of enteropathogens (Monteagudo-Mera et al., 2019), but also need to exert their health benefits on their respective host.Ĭell surface hydrophobicity of a probiotic strain is a measure of its intestinal colonization, that is, adhesion and persistence once they have entered the intestinal cavity. Furthermore, they must possess the quality of intestinal colonization. So for the selection of probiotics, strains have to access acid, bile, and digestive enzyme tolerance and gastrointestinal (GI) transit tolerance. Low pH in the stomach and gastric enzymes along with the presence of bile and intestinal enzymes in the intestine is considered fatal for bacterial strains. The most important criterion for probiotics is to remain viable under harsh environmental conditions of the gastrointestinal tract.
According to FAO (Food and Agriculture Organization, 2006), “Probiotics are the live microorganisms which when administered in an adequate amount, confers health benefits to the host.” They are also categorized into a category of nutraceutical (Hill et al., 2014). The presence of L. acidophilus is reported in indigenously produced fermented milk products locally named as “dahi” of Pakistan, along with other members of lactic acid bacteria (LAB), L. acidophilus is promising, completely characterized, well-documented probiotic member of LAB (Behbahani et al., 2019) and is already included in the generally recommended as safe (GRAS) category. Regarding their functional properties, L. acidophilus WFA1 (KU877440) showed excellent properties of antioxidants and EPS production. It can be concluded from the present study that the mentioned strains of L. acidophilus (WFA1, WFA2, and WFA3) are strongly hydrophobic thus having an ability to survive and colonize under the gastrointestinal tract which confirms their probiotic nature. Regarding functional properties, exopolysaccharide (EPS) production, and antioxidant potential, strain WFA1 showed promising results EPS (1.027mg/ml), DPPH (80.66%), ABTS (81.97%), and reducing power (1.787). Additionally, a cholesterol-lowering assay was conducted and up to 26% reduction in cholesterol was observed by the strains. PCR amplification of bile salt hydrolase gene (bsh) was performed and sequences were submitted to the public database of NCBI and Gene bank under accession numbers, KY689139, KY689140, and KY689141. In the salt aggregation test (SAT), WFA1, WFA2, and WFA3 aggregated at 0.6, 1.0, and 2.0 molar concentrations of ammonium sulfate, respectively. Cellular auto-aggregation varied from 21.72% to 30.73% for WFA3 and WFA1, respectively. WFA1 showed maximum adherence percentage (55.48%) followed WFA2 (55.48%) and WFA3 (51.38%). The results show that strains showed the least adhesion to xylene (54.25%) as compared to dichloromethane (55.25%) and hexadecane (56.65%). In BATH, adhesion of strains against three hydrocarbons namely xylene, dichloromethane, and hexadecane was conducted. To access cell surface hydrophobicity, bacterial adhesion to hydrocarbons (BATH), cellular auto-aggregation, and salt aggregation were performed.
Results for simulated gastric and intestinal tolerance/ resistance revealed that all three strains can resist and survive under the following mentioned conditions. These selected strains were further assessed for their probiotic and functional attributes. Strains of Lactobacillus acidophilus WFA1 (KU877440), WFA2 (KU877441), and WFA3 (KU877442) were isolated from indigenous Dahi (yogurt), screened, and selected based on acid and bile tolerance along with the antimicrobial activity.