Tolerance test was examined by pH, bile salt, and osmotic pressure. L. reuteri WHH1689 was cultured in MRS at 37°C for 18 hr under aerobic condition. The bacterial cells were collected by centrifugation (10,000 × g for 10 min) and washed twice with 0.01 M PBS (pH 7.2) before being resuspended in 0.85% sterile saline and adjusted using NaOH (0.5 M) or HCl (0.5 M) to different pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0). To evaluate resistance to bile salt, bacterial cells prepared as above were resuspended in different bile salt solution containing 0.2%, 0.3%, 0.4%, 0.5% (wt/vol) bile salt (Sigma). Bacterial suspensions were cultured at 37°C for 3 hr. Sodium chloride has strong water‐reducing activity could effect on osmotic pressure. For resistance to osmotic stress, bacterial culture was collected as above and resuspended in 6.0%, 7.0%, 8.0% sodium chloride solution. Bacterial cells were cultured at 37°C for 24 hr. Stress resistance was assessed by bacterial survival.
Bile salt
Manufactured by Merck Group
Sourced in United States, Germany, China, Italy, United Kingdom, Australia, Poland, India, Sao Tome and Principe, Spain
Bile salts are a class of organic compounds found in bile, a digestive fluid produced by the liver. They act as emulsifiers, helping to break down and solubilize fats in the small intestine to facilitate their absorption. Bile salts are essential for the proper digestion and absorption of lipids.
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Lab products found in correlation
Pepsin, by Merck Group (107 mentions)
Pancreatin, by Merck Group (78 mentions)
α amylase, by Merck Group (37 mentions)
Trypsin, by Merck Group (24 mentions)
Gallic acid, by Merck Group (19 mentions)
Formic acid, by Merck Group (16 mentions)
Sodium chloride (nacl), by Merck Group (15 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (15 mentions)
Sodium bicarbonate, by Merck Group (14 mentions)
Pepsin from porcine gastric mucosa, by Merck Group (13 mentions)
Methanol, by Merck Group (12 mentions)
Hydrochloric acid (hcl), by Merck Group (12 mentions)
Acetonitrile, by Merck Group (12 mentions)
Porcine pepsin, by Merck Group (11 mentions)
Pancreatin from porcine pancreas, by Merck Group (11 mentions)
Potassium persulfate, by Merck Group (10 mentions)
Quercetin, by Merck Group (10 mentions)
Bovine serum albumin (bsa), by Merck Group (10 mentions)
Porcine pancreatin, by Merck Group (9 mentions)
Trolox, by Merck Group (9 mentions)
273 protocols using bile salt
1
Tolerance Evaluation of Lactobacillus reuteri
2
Tolerance Evaluation of Bacterial Isolates
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Tolerance of isolates to low acidity and bile salts were determined in triplicate experiments as described by Cano Roca et al. (2014) . Briefly, exponentially growing cells in MRS broth were washed by centrifugation (4,000 × g at 25 °C for 10 min) and re-suspended in PBS. After serial dilutions, an initial dilution of the bacterial suspension was prepared for plating on MRS agar. To investigate the reaction of the isolates to low pH values, 100 μL of the cell suspension (108 CFU/mL) was added to 900 μL of sterile PBS (pH = 3) in a 1.5-mL microtube. The endurable cell counts were measured after 3 h of incubation at 37 °C. A similar procedure was performed using PBS (pH = 7) as a control. To determine the tolerance of isolates to bile salts, 50 μL of bacterial suspension was added into tubes containing 4,950 μL of MRS broth (Merck, Germany) with 0.4% (wt/vol) of bile salts (Merck, Germany) and incubate at 37 °C for 6 h.
The harvested cells from the acid in both bile salt stress experiments were washed in PBS (pH = 7.4) and cultured on MRS agar and finally counting was performed. Based on 2, 2–4, 4–6 and >6 log reduction in comparison to the initial suspension after 3 and 6 h of incubation in acid and bile salts, isolates were grouped as strongly resistant, resistant, intermediate and susceptible, respectively.
The harvested cells from the acid in both bile salt stress experiments were washed in PBS (pH = 7.4) and cultured on MRS agar and finally counting was performed. Based on 2, 2–4, 4–6 and >6 log reduction in comparison to the initial suspension after 3 and 6 h of incubation in acid and bile salts, isolates were grouped as strongly resistant, resistant, intermediate and susceptible, respectively.
3
Lactobacillus Tolerance to pH and Bile
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The tolerance to different pH values and bile salt concentrations was assessed by inoculating 1 mL aliquots of each tested Lactobacillus strain suspension in 10 mL of PBS (final viable cell counts of approximately 7 log CFU/mL) with pH adjusted to 2.0, 3.0 or 5.0 (using 1 M HCl) or supplemented with bile salts (Sigma-Aldrich Co., St. Louis, USA) at 1.0, 2.0 or 3.0% (w/v). The cells were incubated aerobically at 37°C under stirring (150 rpm). At different incubation periods (1, 2, and 3 h), 1 mL aliquots were removed from each system, serially diluted in sterile peptone water (10−1–10−5) and spread plated onto MRS agar for enumeration of viable cells. After an incubation period of 48 h at 37°C under anaerobiosis (Anaerobic System Anaerogen, Oxoid), the viable cells were counted, and the results were expressed as the log of the colony forming units per mL (log CFU/mL). For controls, Lactobacillus strains were cultivated in PBS at pH 7.2 (adjusted using 1 M HCl) and in MRS without bile salts (Jacobsen et al., 1999 (link); Monteagudo-Mera et al., 2012 (link)).
4
Bile Tolerance Evaluation of Lactic Acid Bacteria
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bile salt tolerance of each LAB isolate was evaluated based on the methods described by Mallappa et al. [26 (link)], with minor modifications. Briefly, 1% LAB culture was inoculated in MRS broth supplemented with 0.3% (w/v) bile salt (Sigma-Aldrich Co., Ltd., USA) and incubated anaerobically at 37 °C for 4 h. Sample aliquots were taken at time 0 and after 4 h of incubation, plated on MRS agar plates, and incubated anaerobically at 37 °C for 48 h to determine the survival rate after exposure to bile salts. Cultures of each LAB isolate without bile salt were used as controls. The survival rate was calculated as follows:
5
Bacterial Tolerance to Harsh Environments
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The tolerance to low pH value and bile salt high concentration was evaluated in phosphate buffer solution (PBS, 0.05 mol/L K2HPO4/KH2PO4) with pH adjusted to 2 using 1 M of HCl or supplemented with 1% (w/v) bile salts (Sigma-Aldrich) after 3 h of exposure (37 ± 1 °C). The results were expressed as log cfu/mL [18 (link)]. A detection limit of 2 log cfu/mL was used in these experiments.
6
Evaluating Bile Salt Tolerance of LAB
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bile salt tolerance of 12 LAB isolates (those referred to in the previous section) was evaluated following a method adapted from Shehata et al. [27 (link)]. In brief, fresh overnight cultures of the isolates (1% v/v) were sub-cultured in 10 mL MRS broth for 20 h at 37 °C. The cells were washed twice in saline (0.85% w/v) and resuspended in MRS broth containing 0.3% (w/v) bile salt (Sigma Aldrich) and incubated at 37 °C for 3 h. This concentration was applied as 0.3% (w/v) is believed to be the average representative of bile salt concentration in the gastrointestinal tract [29 (link)]. Aliquots of 100 µL were removed at constant intervals (t = 0, 1, 2, 3 h) and spread on MRS agar plates; plates were incubated at 37 °C for 72 h to determine total viable cell count (CFU/mL).
7
Tolerance of Probiotic Strain CP9 to Acidic and Bile Environments
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The tolerance of CP9 in acidic and bile salts environment was studied by methodology previously described [24 (link)] with minor modifications. Briefly, for assessing the tolerance of CP9 to acidic environment, 30 μL of the overnight cultures of CP9 were incubated with 70 μL LB broth adjusted to pH 2, 3, and 6.6 (control) using 1 N hydrochloric acid (HCl) in a 96-well microplate for 2 and 5 h. For assessing the tolerance of CP9 to bile salts environments, 30 μL of the overnight cultures of CP9 were incubated with 70 μL LB broth adjusted with 0% (control), 0.3%, 0.5%, and 1% bile salt (Sigma-aldrich, St. Louis, MO, USA) in a 96-well microplate for 1, 3, and 5 h. After the end of each incubation, cell viability and growth were measured spectrophotometrically via the metabolic activity of the cells using Bacterial Counting Colorimetric Assay Kit (BioVision Technologies, Inc., Chester Springs, PA, USA) following manufacturers protocol. Zero time period in all experiments represented the cellular activity of the initial cell concentration at the time of addition of the substrate. Metabolic cell activity and growth were then compared relative to the zero time point within each treatment group.
8
Simulated GI Stress Resistance Evaluation
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Acidic conditions were simulated by acidic MRS broth with pH adjusted to 3.5, 2.5, and 1.5 by adding 1 M HCl (Minelli et al., 2004 (link)). Simulated gastric juices were prepared fresh daily by suspending pepsin (Sigma-Aldrich, Poole, UK) (3 g/L) in sterile saline and adjusting pH to 1.5 with 1 M HCl at 37 °C (Charteris et al., 1998 (link)). Simulated pancreatic juices were prepared fresh daily by suspending pancreatin USP (Sigma-Aldrich) (1 g/L) in sterile saline (0.5% NaCl w/v) with pH adjusted to 8.0 by adding 0.1 M NaOH at 37 °C (Charteris et al., 1998 (link)). Simulated bile salt solution was prepared by adding 0.1%, 0.2%, or 0.3% (w/v) bile salt (Sigma-Aldrich) to MRS broth.
To test simulated gastric and intestinal stresses resistance, 1 mL of fermented soymilk containing FL or IL (cell counts adjusted to approximately 9 log CFU/g) was incubated in the prepared acidic MRS broth, simulated gastric juices, pancreatic juices, and bile salt solution for 1 or 3 h at 37 °C. Survival was evaluated by plate count on MRS agar.
To test simulated gastric and intestinal stresses resistance, 1 mL of fermented soymilk containing FL or IL (cell counts adjusted to approximately 9 log CFU/g) was incubated in the prepared acidic MRS broth, simulated gastric juices, pancreatic juices, and bile salt solution for 1 or 3 h at 37 °C. Survival was evaluated by plate count on MRS agar.
9
Yeast Growth in Simulated Gut Conditions
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In order to analyze cell ability to grow under the stimulated gastrointestinal condition, growth of isolated yeast strains was examined at pH 2 to 2.5, 37 degrees Celsius and 0.3% bile salt media. Cells were incubated at different temperatures (30, 37 and 42 °C) to check for tolerance. The bile salt (Sigma-Aldrich, St. Louis, MO, USA) stock was dissolved in yeast synthetic or yeast nitrogen base YNB media containing 6.7 g/L Yeast nitrogen base and 10 g/L of glucose buffered at pH 7, 5.4 or 2.5. Cells were incubated in 96-well plates (Bioscreen C, Labsystem, Helsinki, Finland). Changes in optical density of cells were measured every hour from 0 to 56 h. Harvesting of cells was done by centrifugation for 10 min at 3000 g at room temperature. Yeast growth was determined as the area under the growth curve (OD600 × h) which were obtained from the bio screen data. Growth of yeast cells in 0.3% (w/v) bile salt (Sigma-Aldrich, St. Louis, MO, USA) YNB at pH 2.5 was compared with growth at pH 5.4 and pH 7, positive controls.
10
Evaluating Acid and Bile Tolerance of Lactic Acid Bacteria
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Resistance to acidic conditions was tested according to the method of Conway et al. (1987) (link). Lactic acid bacteria were cultured in MRS medium overnight and harvested by centrifugation at 6,000 × g for 10 min. Cells were washed twice with phosphate-buffered saline (PBS; pH 7.2) and resuspended in an equal volume of PBS adjusted to pH 3.0 and 2.5 with HCl. Following incubation for 0, 90, and 180 min at 37°C, acid tolerance was evaluated by spreading cells on MRS agar and counting the number of viable cells (Log CFU/ml) after incubation at 37°C for 48 h (Maragkoudakis et al., 2006 (link)). Tolerance to bile salts was evaluated by suspending cells in PBS solution containing 0.3% (w/v) bile salt (Sigma, St. Louis, MO, United States) and incubating at 37°C (Gilliland et al., 1984 (link)). Bile tolerance was measured using the same method used for acid tolerance.
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