To examine the resistance of the isolates at a low pH, fresh bacterial cells were suspended in a Phosphate Buffered Saline (PBS) buffer with pH adjusted to 2.5, as described before [40 (link)]. In brief, resistance was enumerated after incubation at 37 °C in MRS and M17 agar plate (depending on each microorganism) for 0 and 3 h, reflecting the time that food spends in the stomach. To examine the resistance to bile salts, fresh bacterial cells were suspended in a PBS buffer with pH adjusted to 8 containing 0.5% (w/v) bile salts (LP0055, OXOID, Hampshire, UK) consisting mainly of sodium glycocholate and sodium taurocholate [40 (link)]. Resistance was enumerated after incubation at 37 °C in MRS and M17 agar plates (depending on each microorganism) for 0 and 4 h, reflecting the time that food spends in the small intestine. All tests were performed in triplicate.
Lp0055
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The LP0055 is a laboratory incubator designed for general-purpose temperature-controlled applications. It features a microprocessor-based temperature control system to maintain the desired temperature within the incubator chamber.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using lp0055
1
Evaluating Bacterial Acid and Bile Resistance
2
Cholesterol Removal by Lactobacillus Isolates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One percent (1%) of sub-cultured Lactobacillus isolates were inoculated into MRS broth (10 ml) containing 0.3% (b/v) ox gall (Oxoid; LP0055) and 100 mg/L water-soluble cholesterol (Sigma Aldrich; C4951) to prepare cholesterol removal assay by growing cells. For resting cell preparation, 1% of sub-cultured Lactobacillus isolates that were previously washed 3 × with PBS were suspended in 0.05 M phosphate buffer (pH 6.8) supplemented with 0.3% (w/v) ox gall and 100 mg/L cholesterol. Heat-killed cells were prepared from saline-suspended cell pellets of sub-cultured Lactobacillus isolates that were heated at 121°C for 15 min before being added into MRS broth containing 0.3% ox gall and 100 mg/L cholesterol. Incubation was done at 37°C for 20 h in all assays, after which the mixture was centrifuged at 13,000 rpm for 5 min at 4°C. MRS broth with and without the addition of 0.3% ox gall (b/v) and 100 mg/L water-soluble cholesterol were used as a negative and positive control, respectively. The remaining cholesterol in the supernatant was measured using Cholesterol Quantitation Kit (Sigma Aldrich; MAK048).
3
Bile Salt Tolerance of Probiotic Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The bile salt tolerance of the strains was determined according to the method described by García-Ruiz et al. with slight modifications [21 (link)]. L. rhamnosus GG, a widely characterized and intensively investigated probiotic strain, was used as a reference control. Overnight cultures were inoculated (2% v/v) into a MRS medium with or without 1.2% bile salt (w/v) (Oxoid LP0055). The cultures were incubated for 12 h to measure the optical density at 600 nm (OD600) for the preliminary evaluation of bile salt tolerance. To obtain strains for subsequent validation experiments, tolerant and non-tolerant strains were partially selected for gradient bile salt tolerance (0%, 0.3%, 0.6%, 0.9%, 1.2%, 2%, 3%, 4%, 6%, 8%, and 10% bile salt), and the OD600 was measured as previously described. The growth rates of the strains are expressed as the percentage of OD600 under different bile salt concentrations relative to that of the control. All experiments were performed in triplicate.
4
Acid-Bile Tolerance Evaluation of LAB and Yeasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared fresh daily in order to evaluate the acid-bile tolerance of all strains. Overnight cultures of LAB and yeasts on MRS and YPD liquid medium respectively, were harvested by centrifugation (6000 × g for 15 min). The pellets were re-suspended in SGF (pH 2.0) containing pepsin (1 mg/ml), and maintained at 37°C for 2 h. After acid pre-incubation cells were centrifuged again and re-suspended in SIF (pH 7.4) containing pancreatin (1 mg/ml) and bovine bile (0.5% w/v) dissolved in saline, and maintained at 37°C for 3 h (Haller et al., 2001 (link); Cenci et al., 2006 (link)). Pepsin and pancreatin were obtained from Sigma-Aldrich; bile extract, containing conjugated glycholate and taurocholate sodium salts, from Oxoid (LP0055, Oxoid, Ltd., Basingstoke, United Kingdom). After acid-bile challenge samples were removed, serially diluted, and plated on MRS and YPD containing 2% agar (Oxoid, Ltd., Basingstoke, United Kingdom) to determine cell viability. All the strains was tested in triplicate and the survival rate of all the tested microorganisms was estimated by evaluating the ratio between viable cells after the acid or bile challenge and viable cells of the untreated controls. The acid tolerance and acid-bile tolerance percentage was calculated from the L. plantarum and yeasts survival rate.
5
Bile Tolerance Assay for Microorganisms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The bile tolerance of the test organism was determined using the method of El-Naggar (2010) with slight modifications. Sterile MRSB was supplemented with different concentrations (0.5%, 1.0% and 1.5%) of bile salts (Oxoid LP0055 Basingstoke, England). The medium was separately inoculated with standardized cell suspension (1% v/v) and incubated aerobically at 37 °C for 6 h during which period bacterial growth was monitored hourly at 600 nm wavelength using a spectrophotometer (Vis Spectrophotometer 721D Life Assistance Scientific and Medical Institute, UK). The absorbance values obtained was plotted against the incubation time. Culture medium with no bile served as control.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!