Simulated intestinal fluid was prepared by dissolving 0.3% w/v Oxgall bile salts (Sigma-Aldrich) and 0.1% w/v pancreatin (Sigma-Aldrich) in sterile saline solution (0.85% NaCl) and adjusting to pH 8.0 (27 (link)). Aliquots (100 µL) of each inoculum suspension or five drops (200 µL) of Reuflor and Dicoflor were inoculated in 5 mL of simulated intestinal fluid and incubated at 37°C for 0, 30, 60, 120, 240, and 360 min. At each time point, aliquots (100 µL) of the microbial suspensions were serially diluted and seeded on TSH, MRS, and BSM. Plating was performed in triplicate and plates incubated in the conditions reported above. The number of CFU was determined and the CFU/unit dose of each product extrapolated. The experiments were repeated three times in separate days.
Oxgall bile salt
Manufactured by Merck Group
Sourced in Germany
Oxgall bile salt is a laboratory reagent used for the isolation and identification of bacteria. It is derived from the bile of animals and serves as a selective agent, inhibiting the growth of many gram-positive bacteria while allowing the growth of gram-negative bacteria.
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9 protocols using oxgall bile salt
1
Simulated Intestinal Fluid Assay for Probiotic Survival
2
Simulated Gastric and Intestinal Digestion
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Simulated gastric digestion was tested essentially as described in Zarate et al. 2000 [14 (link)]. Simulated gastric juice prepared with the following composition: KCl 7 mmol L−1; NaCl, 125 mmol L−1; NaHCO3, 45 mmol L−1 and pepsin, 3 g L−1, HCl used to adjust the final pH 2 and 2.5 and with NaOH to pH 7. In general, 1 mL of cell suspension containing approximately 108–109 CFU mL−1 of LAB was transferred into 9 mL of simulated gastric juice with different pH 2, pH 2.5, and pH 7. The mixture was incubated at 37 °C for 0, 1.5, and 3 h. After incubation, viable LAB cells counts were determined by plating serial dilutions. Simulated intestinal fluid (SIF) was prepared using 0.1% (wt/v) pancreatin (Sigma) and 0.15% (w/v) Oxgall bile salts (Sigma) in water, and pH (pH 8.0) was adjusted with 5 mol l−1 NaOH. After 180 min of gastric digestion, cells were centrifuged (3000× g, 5 min), washed with PBS, suspended in SIF were incubated. Viable bacterial counts were taken at 0, 1.5, and 3 h by plating serial dilutions on MRS agar.
3
Bile Salt Tolerance of Bacterial Isolates
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The bile salt tolerance of selected isolates was determined by the method described by Argyri et al. (2013) (link) with minor modifications. Overnight cultures of bacteria cells were washed three times with PBS (pH 7.0) to remove impurities and centrifuged (5,000 rpm for 10 min at 4 °C). The cell pellets were re-suspended in MRS broth containing 0.2 and 0.4 % oxgall bile salts (sigma Aldrich, Germany). The cultures were then incubated anaerobically at 37 °C for 24 h. Aliquots were taken after 0 h and 3 h were enumerated by pour plate counts of all samples using 10-fold serial dilutions prepared in 0.1% peptone water. Samples taken at 0 h were used as the control. Tolerance to bile salts was assessed based on viable colony counts on MRS agar in triplicates after incubation at 37 °C for 0 and 3 h, reflecting the average period spent by food in the small intestine.
4
Bile Salt Resistance Evaluation in Bacteria
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The bile salt resistance of selected isolates was determined by the method described by Argyri et al. [16] with minor modi cations. Overnight cultures of bacteria cells were washed three times with PBS (pH 7.0) to remove impurities and centrifuged (5,000 rpm for 10 min at 4 o C). The cell pellets were re-suspended in MRS broth containing 0.2 and 0.4% oxgall bile salts (sigma Aldrich, Germany). The cultures were then incubated at 37 o C for 24 h. Aliquots were taken after 0 h and 3 h, serially diluted, and plated on MRA agar. Samples taken at 0 h were used as the control. Resistance to bile salt was evaluated based on viable colony counts on MRS agar in triplicates after incubation at 37 o C for 0 and 3 h, re ecting the average time spent by food in the small intestine.
5
Assessing Lactobacillus Acid and Bile Tolerance
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The acid and bile tolerance of isolates were checked according to the methods described previously with minor modifications (11 (link)). Acid tolerance was examined in MRS broth and adjusted to a final pH of 2.5 using 1N HCl. Specifically, 1 mL overnight cultured Lactobacillus isolates were inoculated into 9 ml MRS broth previously adjusted to pH 2.5 and cultured anaerobically at 37°C for 3 h. Bile tolerance was analyzed as above that 1 ml of overnight cultured isolates were inoculated into 9 ml MRS broth with 0.3% (w/v) oxgall bile salt (Sigma) and incubated at 37°C for 8 h. Then the plate counting method was used to measure the viable bacteria after incubation and the initial. Finally, the survival rate (%) of acid and bile tolerance was calculated as the percentage of the number of viable bacteria grown on MRS agar after incubation (N1, lg CFU/mL) and the initial number of viable bacteria (N0, lg CFU/mL) according to the formula: survival rate (%) = N1/ N0 × 100%.
6
Evaluating Probiotic Acid and Bile Tolerance
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Tolerance to acid and bile salt was observed according to the previous studies (Yu et al., 2019 (link); Jomehzadeh et al., 2020 (link)). Each strain was incubated in MRS broth at 37°C overnight. Aliquots (0.1 mL) of each active culture were inoculated in 10 mL MRS broth adjusted to pH 2.5 with 0.1 N hydrochloric acid (HCl) and then incubated for 3 h at 37°C. Each strain was inoculated in MRS broth containing 0.3% ox gall bile salt (Sigma-Aldrich, St. Louis, MO, United States) and incubated for 24 h at 37°C to confirm tolerance to bile salt. After incubation, cell suspension was spread on an MRS agar plate, and then viable cell count that survived at low pH and bile salt were enumerated by plate counting. L. rhamnosus American Type Culture Collection (ATCC) 53103 (LGG) was utilized as the control. The survival rate (%) of each strain was calculated using the following formula: (N/N0) × 100, where N and N0 denote viable cell numbers after culturing with low pH or bile salt and initial viable cell numbers, respectively.
7
Evaluating Bile Tolerance of Lactic Acid Bacteria
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The bile tolerance of LAB (lactic acid bacteria) isolates was demonstrated according to the methodology outlined by Ramos et al. (2013) (link). The LAB culture isolates were collected, and their pellets were obtained using the previously described procedure. Following that, the pellets were reconstituted in 5 mL of MRS broth supplemented with 0.3 and 1% (w/v) oxgall bile salt (Sigma Aldrich, India). A control sample was also prepared without the addition of bile salts. Subsequently, the samples were incubated at 37°C and collected at specific time intervals of 0, 2, and 3 h. To determine the viability counts, 100 μL samples were taken and plated onto MRS agar plates after performing appropriate serial dilutions. The number of viable cells was determined and expressed as log10 CFU/mL.
8
Assessing Bacterial Acid and Bile Tolerance
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The bile and acid tolerance of bacterial strains were analyzed as described earlier in literatures with little modifications (Donatien and Hagreacute tou, S.L., Mamoudou, H.D., Breacute hima, D., Mogens, J., , 2012 (link), Klingberg et al., 2005 (link)). Briefly, the acid resistance of the organisms were examined in MRS broth (Himedia, Mumbai, India) adjusted the pH to 2.5 using 1 N HCl. About 0.2 mL of 18 h culture of Lactobacillus isolates (106 CFU/ml) was inoculated into 25 mL of MRS liquid medium and the pH was adjusted earlier. After 18 h incubation, the viable count of bacterial strain was performed using plating technique on MRS agar. The survival rate (%) of the bacterial strain was analyzed using plate count method on MRS agar, after incubation of 0 and 2 h. Lactobacillus isolates that grown in the acid medium were subjected to bile tolerance assay. Lactobacillus strains were inoculated in Erlenmeyer flask containing 50-mL (106 CFU/ml) of 18 h grown culture was inoculated on 50 mL of MRS liquid medium with 0.3% (w/v) oxgall bile salt (Sigma, USA). To the control, oxgall bile salt was not added.
(N/N0) × 100%.
(N/N0) × 100%.
9
Cholesterol Assimilation by L. paracasei
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Initially, 1% overnight culture was incubated in MRS broth (Sigma) containing 0·30% ox gall bile salt (Sigma) and 100 µg ml−1 filter‐sterilized cholesterol (Cholesterol–methyl‐β‐cyclodextrin, Sigma) for 24 h at 37°C. Tubes were then centrifuged at 5500 g for 15 min at 4°C, and 1 ml of supernatant was collected for measurement of residual cholesterol using a colorimetric method (Miremadi et al. 2014 (link)). Cholesterol concentration was measured using a standard curve from 0 to 100 µg ml−1. The experiment was repeated two times with three replicates each. The ability of L. paracasei DTA81 to assimilate cholesterol was calculated as a percentage of cholesterol removal after 24 h.
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