The active product B. coagulans MTCC 5856, 2 billion spores per tablet (2 × 109 spore/tablet), was supplied by Sabinsa Corporation, Utah, USA. Tablets were packed in 70 mL HDPE container. Each active had 2 billion spores per tablet i.e., 333.33 mg of B. coagulans MTCC 5856, 222.67 mg of microcrystalline cellulose, 10 mg of starch, 30 mg of sodium starch glycolate and 4.0 mg of magnesium stearate. Viable spore count of B. coagulans MTCC 5856 was determined as per the method described previously [13 ] Briefly, 1.0 g of B. coagulans MTCC 5856 was mixed in sterile saline (0.9 % NaCl, w/v) and then incubated in a water bath for 30 min at 75 °C, followed by immediate cooling to below 45 °C. The suspension was further serially diluted in sterile saline and the viable count was enumerated by plating on glucose yeast extract agar (HiMedia, Mumbai, India) by pour plate method. The plates were incubated at 37 °C for 48–72 h. Analysis was performed twice in triplicate. Average means of spore viable counts was expressed in cfu/g. For the placebo tablet, maltodextrin of equivalent weight was used and formulated in similar shape and size as that of the active, and packed in HDPE containers.
Glucose yeast extract agar
Manufactured by HiMedia
Sourced in India
Glucose yeast extract agar is a solid culture medium used for the growth and enumeration of microorganisms. It contains glucose and yeast extract as carbon and nitrogen sources, respectively, to support microbial growth. The agar provides a solid matrix for colony formation and isolation.
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Lab products found in correlation
Mrs broth, by HiMedia (4 mentions)
Mrs agar plates, by HiMedia (1 mentions)
Bile salt, by Merck Group (1 mentions)
Copper sulfate, by Merck Group (1 mentions)
Trypticase soy agar, by HiMedia (1 mentions)
Scfas, by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
E coli atcc 25922, by American Type Culture Collection (1 mentions)
9 protocols using glucose yeast extract agar
1
Standardized Probiotic Bacillus Formulation
2
Survival of B. coagulans Spores in pH
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The survival of B. coagulans MTCC 5856 spores was studied by the addition of 1 mL of the suspension into a series of 9 mL of sterile phosphate-buffered saline (PBS) at pH 1.5, 3, 4, 5, 6, 7 and 8 (adjusted using 1 N NaOH and 1 N HCl). The incubation temperature was maintained at 37 °C and 1 mL sample was taken at 0, 0.5, 1.0, 2.0, 3.0 and 4.0 h. After incubation, serial dilution was done in sterile saline (0.89 % w/v) and the viable count was enumerated by plating on glucose yeast extract agar (HiMedia). Experiments were performed in triplicate at two different occasions.
3
Bile Tolerance of Bacillus coagulans MTCC 5856
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Bile tolerance of B. coagulans MTCC 5856 cells was determined by the method described earlier (Gilliland et al. 1984 (link); Hyronimus et al. 2000 (link)). Briefly, overnight grown B. coagulans MTCC 5856 in MRS broth (20 μL corresponding to 2 × 106 cfu mL−1) was spotted onto MRS agar plates containing oxgall bile (0.1–1 % w/v) (HiMedia, Mumbai, India). Plates were incubated at 37 °C for 5 days. The minimal inhibitory concentration (MIC) of bile for B. coagulans MTCC 5856 was determined as the lowest concentration totally inhibiting the growth of spots as judged from visual examination of spots. MRS broth (HiMedia) was inoculated with approximately 106 cfu mL−1 of B. coagulans MTCC 5856 overnight grown culture and then supplemented with 0.3 (w/v) and 0.5 % (w/v) oxgall. Samples were incubated for 24 h at 37 °C with shaking at 120 rpm. Growth in control (no bile) and test cultures (0.3 and 0.5 % oxgall) was monitored hourly by measuring absorbance at 600 nm using spectrophotometer (Shimadzu Corporation, Kyoto, Japan). In same set of experiment, the viable count of B. coagulans MTCC 5856 was determined in triplicate on glucose yeast extract agar (HiMedia) at 0 and 24 h by pour plate method (Majeed et al. 2016 ).
4
Turmeric Starch for Bacillus coagulans
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Example 9
A single isolated colony of Bacillus coagulans MTCC 5856 was inoculated into MRS broth (pH 6.0; Himedia) and incubated in shaker at 37±1° C., 120 rpm overnight. To evaluate the growth of Bacillus coagulans MTCC 5856 using turmeric starch as carbon source, MRS media was used to evaluate the growth of Bacillus coagulans MTCC 5856 by replacing the dextrose with 2.5 g/L, 5 g/L, 7.5 g/L, 10 g/L of turmeric starch and 2.5 g/L, 5 g/L, 10 g/L dextrose used as control in different set of experiments. MRS with Parameters like pH, OD (600 nm), Sporulation, Total viable count (TVC) was determined by serial dilution using Glucose Yeast Extract agar (Himedia) at 0 h and after fermentation (48 h). Total carbohydrate was quantified by Anthrone method. Viable count was determined by pour plate method using GYE agar media. Turmeric starch was found to be suitable for the growth of B. coagulans MTCC 5856 as the sole carbon source. However, FOS was slightly better than turmeric starch for enhancing the viable count of B. coagulans MTCC 5856 at 1.0 and 2.0%, but at 0.5% concentration, B. coagulans MTCC 5856 viable count was better compare to FOS (
5
Probiotic Strain Characterization Protocol
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De Man, Rogosa, and Sharpe (MRS) broth, potato soluble starch, glucose yeast extract agar, trypticase soy agar, eosin methylene blue agar (EMB Agar), glucose yeast extract agar (GYEA) were purchased from HiMedia, Mumbai, India. Lysozyme, pepsin, fructooligosaccharide (FOS), pancreatin from porcine, bile salt, copper sulfate, and SCFAs standards (acetic, propionic and butyric acids) were procured from Sigma‐Aldrich (St. Louis, MO, USA). Oxyrase was procured from Oxyrase, Inc, Mansfield, OH, USA. Probiotic bacteria B. coagulans MTCC 5856 is a patented strain of Sami Labs Limited and deposited to Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. E. coli ATCC 25922 was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). trypticase soy agar (TSA; Becton Dickinson) was used for culturing of bacteria, and culture stocks were maintained in aqueous glycerol (15%, v/v) at −80°C.
6
Spore FCM Analysis and Viability
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For FCM analysis and viable count estimation, 10 mg of spores (15X109/g) were suspended in 1 ml of sterile phosphate buffered saline (PBS, pH 7.4)and incubated in a water bath for 30 min at 75°C, followed by immediate cooling to below 45°C. This suspension was taken for either FCM analysis or further serially diluted in sterile PBS and the viable count was enumerated by plating on glucose yeast extract agar (HiMedia, Mumbai, India) as described earlier[33 ].One set of spores were analysed by FCM without the activation step.Each experiment was repeated thrice in duplicates. Average mean of spore viable counts were expressed in log10 CFU.
7
Evaluating Galactomannan from Fenugreek Seeds for B. coagulans Growth
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A single isolated colony of B. coagulans MTCC 5856 was inoculated into MRS broth (HiMedia) and incubated at 37°C with 120 rpm for overnight. Galactomannan from fenugreek seeds alone (0.5%, 1.0%, and 2.0%, w/v), and along with MRS media (devoid of dextrose) (0.5%, 1.0%, and 2.0%, w/v), was prepared. MRS broth and MRS (devoid of dextrose) were prepared separately. Similarly, fructooligosaccharide (FOS) was taken in the study as a reference control to compare with galactomannan from fenugreek seeds. The final pH of all the media was adjusted to 7.0. Five percent of overnight grown B. coagulans MTCC 5856 culture was inoculated to all the flasks and incubated at 37°C with 100 rpm for 24 h. pH values at 0 h of incubation and after 24 h of fermentation were recorded. Samples were serially diluted in sterile saline, and the viable count was enumerated by plating on glucose yeast extract agar (HiMedia) at the 0 h and after 24 h of fermentation as per the previously described method (Majeed, Majeed, et al., 2016 ). The plates were incubated at 37°C for 48–72 h. Each analysis was performed in triplicate at two different occasions. Average mean of viable counts is expressed in log10 cfu/ml.
8
Bacillus coagulans Growth on Natural Fibers
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Example 4
Single isolated colony of Bacillus coagulans MTCC 5856 was inoculated into MRS broth (pH 7.0±20; Himedia, Mumbai, India) and incubated at 37° C. with 120 rpm for overnight. Plant based natural fibers alone (0.5, 1.0, 2.0%, w/v), and in MRS media (devoid of dextrose) (0.5, 1.0, 2.0%, w/v) were prepared. MRS broth and MRS (devoid of dextrose) were also prepared separately. Similarly, Fructooligosaccharide (FOS) was also taken in the study as reference control to compare with plant based natural fibers. The final pH of all the media was adjusted to 7.0. Five percent of overnight grown Bacillus coagulans MTCC 5856 culture was inoculated to all the flasks and incubated at 37° C. with 100 rpm for 24 h. pH values at 0 h of incubation and after fermentation (24 h) were also recorded. Samples were serially diluted in sterile saline and the viable count was enumerated by plating on glucose yeast extract agar (HiMedia, Mumbai, India) at 0 h and after fermentation (24 h). The plates were incubated at 37° C. for 48 to 72 h. Each analysis was performed in triplicate at two different occasions. Average mean of viable counts are expressed in log 10 cfu/ml (
9
Bacillus coagulans Growth on Fiber Media
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Example 4
Single isolated colony of Bacillus coagulans MTCC 5856 was inoculated into MRS broth (pH 7.0±20; Himeclia, Mumbai, India) and incubated at 37° C. with 120 rpm for overnight. Plant based natural fibers alone (0.5, 1.0, 2.0%, w/v), and in MRS media (devoid of dextrose) (0.5, 1.0, 2.0%, w/v) were prepared. MRS broth and MRS (devoid of dextrose) were also prepared separately. Similarly, Fructooligosaccharide (FOS) was also taken in the study as reference control to compare with plant based natural fibers. The final pH of all the media was adjusted to 7.0. Five percent of overnight grown Bacillus coagulans MTCC 5856 culture was inoculated to all the flasks and incubated at 37° C. with 100 rpm for 24 h. pH values at 0 h of incubation and after fermentation (24 h) were also recorded. Samples were serially diluted in sterile saline and the viable count was enumerated by plating on glucose yeast extract agar (HiMedia, Mumbai, India) at 0 h and after fermentation (24 h). The plates were incubated at 37° C. for 48 to 72 h. Each analysis was performed in triplicate at two different occasions. Average mean of viable counts are expressed in log 10 cfu/ml (
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