Microbial analysis was performed at intervals of 5 days until 60 days of ripening and at intervals of 10 days from 70 to 160 days of ripening. The 25 g of cheese was cut aseptically from the cheese block and placed into a filter bag (3M™, St. Paul, MN, USA). Fifty milliliters of 0.1% buffered peptone water (BPW; Becton, Dickinson and Company) was added to the filter bag, and the bag's contents were homogenized for 60 s using a pummeller (BagMixer® 400W, Interscience, St. Nom, France). Homogenates were serially diluted with 0.1% BPW, and the 0.1 ml dilutions were plated on PALCAM agar (Oxoid Ltd., Basingstoke, Hampshire, UK) with 0.4% of PALCAM supplement. The plates were incubated at 30°C for 48 hr for L. monocytogenes enumeration. To enumerate Lactobacillus spp., the dilutions were also plated on Lactobacilli MRS agar (Becton, Dickinson and Company) and incubated at 37°C for 24 hr.
Lactobacilli mrs agar
Manufactured by BD
Sourced in United States
Lactobacilli MRS agar is a culture medium used for the isolation and enumeration of Lactobacillus species. It provides the necessary nutrients and growth conditions for the cultivation of Lactobacillus bacteria.
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Lab products found in correlation
Salmonella shigella agar, by BD (2 mentions)
Macconkey agar, by BD (2 mentions)
Palcam agar, by Thermo Fisher Scientific (1 mentions)
Bagmixer 400w, by Interscience (1 mentions)
Anaeropack, by Mitsubishi (1 mentions)
Columbia blood agar, by BD (1 mentions)
Lb broth, by BD (1 mentions)
Modified gam agar, by Nissui Pharmaceutical (1 mentions)
Porcine bile extract, by Merck Group (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Mucin, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Potassium hydroxide, by Merck Group (1 mentions)
Uric acid, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Calcium chloride dihydrate, by Merck Group (1 mentions)
11 protocols using lactobacilli mrs agar
1
Microbial Analysis of Ripening Cheese
2
Isolation and Characterization of Bile-Resistant Lactobacillus paracasei Strains
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Previously, we isolated three L. paracasei strains (KBL382, KBL384 and KBL385) from the feces of healthy Koreans and confirmed their strong resistance to bile salts or low pH [12 (link)]. All L. paracasei strains were cultured in anaerobic conditions using Lactobacilli MRS agar (Becton, Dickinson and Company, Sparks, MD, USA) with 0.05% L-cysteine-hydrochloride and Anaeropack (Mitsubishi Gas Chemical Company Inc., Tokyo, Japan) at 37 °C for 24 h as described previously [12 (link)]. Bacteria were collected using centrifugation at 1200×g and washed twice using 1× phosphate-buffered saline (PBS). Bacterial concentrations were quantified as colony-forming units (CFUs) through the cultivation, and the bacteria were stored at −4 °C until further use.
3
Isolating Bacteria from Honey Samples
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For isolation of bacteria from honey, 20 ml of each honey sample was diluted to 50% (v/v) with sterile water and centrifuged at 11,000 rpm for 45 min. The supernatant was removed and the sediment was suspended with 1 ml of sterile water. One hundred microliters of each suspension was plated on Columbia blood agar (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) containing 5% sheep blood (CSA), KSBHI agar (Arai et al., 2012 (link)), Lactobacilli MRS agar (Becton, Dickinson and Company) and modified GAM agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 5% sheep blood (GAMSA). For selective isolation of TS-resistant bacteria, all agar media used for the isolation were supplemented with 1 μg/ml of TS. CSA was incubated at 35°C under air plus 5% CO2 conditions for two days, and the other agar media were incubated at 35°C under anaerobic conditions for four days. Colonies grown on the agar media were subcultured for purification. Pure-cultured bacterial isolates were suspended in LB broth (Becton, Dickinson and Company) containing 30% glycerol and stored at −80°C until use.
4
Microbiological Analysis of Sorghum Silage
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The microorganism count on fresh sorghum and silage samples at 30 days after fermentation was done using the plate count method [32 ] and reported as colony forming unit per gram of fresh matter (cfu/g FM). Fresh chopped sorghum and silages (10 g each) were shaken well by hand with 90 mL sterilized distilled water, and serially diluted at 10−1 to 10−5 in sterilized distilled water. Twenty μL from each dilution was spread on agar plates. LAB numbers were counted on Lactobacilli MRS agar (Difco Laboratories, USA) after incubating in an anaerobic jar (A-110, Sugiyamagen Co., Ltd., Tokyo, Japan) at 30°C for 48 h. Coliform bacteria numbers were counted on blue light broth agar (Nissui-Seiyaku Co., Ltd., Tokyo, Japan) after incubating at 30°C for 48 h. Aerobic bacteria and bacilli numbers were counted on nutrient agar (Difco, USA), yeasts and mold numbers were counted on potato dextrose agar (Nissui-Seiyaku, Japan) after incubating at 30°C for 48 h. In this experiment, mold was counted at 48 h of incubation. Yeasts were distinguished from molds or bacteria by colony appearance and cell morphology observation.
5
Characterization of Probiotic Lactobacillus Strains
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The 11 reference strains and probiotic strain LA1063 used in this study were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan), and Synbio Tech Inc., Kaohsiung, Taiwan, respectively, and they were authenticated through 16S rRNA gene sequencing (Supplementary Table S1 ). Strain LA1063 was isolated from feces of healthy Taiwanese adults and was used as a manufacturing strain for the probiotic supplements. The Lactobacillus strains were incubated anaerobically on Lactobacilli MRS agar (Difco Laboratories, Detroit, MI, USA) at 37 °C for 36 h, and fresh cultures were used for further DNA analyses.
6
Quantitative Analysis of Gut Microbial Populations
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One gram of collected sample from the cecum was diluted with 9 mL of sterile peptone water and mixed for 1 min on a vortex stirrer. Samples were serially blended from 10−1 to 10−6, and were injected by 50 μL in 3 selective agar media as follows; Lactobacilli MRS agar (Difco Laboratories, Detroit, MI, USA) for Lactobacillus spp., coliform bacteria for MacConkey agar (Difco Laboratories, Detroit, MI, USA) and Salmonella-Shigella (SS) agar (Difco Laboratories, Detroit, MI, USA) for Salmonella. Plates were then incubated aerobically at 37°C for 24 h (MacConkey agar and SS agar) or anaerobically at 37°C for 24 h (Lactobacilli MRS agar). The viable colonies of the respective bacteria were counted and expressed as the log 10 of colony-forming units (cfu) g−1 of cecal content.
7
In Vitro Human Digestion Model
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Digestive enzymes for the in vitro human digestion model were prepared using various reagents. Sodium chloride, uric acid, urea, hydrochloric acid (HCl), calcium chloride dihydrate, α-amylase, mucin, bovine serum albumin, pepsin, potassium chloride (KCl), potassium hydroxide (KOH), calcium chloride dihydrate (CaCl2·2H2O), pancreatin, lipase, sodium bicarbonate, and bile extract porcine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Buffer solution (pH 7.0 ± 0.02) was purchased from Samchun Pure Chemicals, Co., Ltd., Korea. For cultivation of gut microbiota used in large intestine digestion, Luria-Bertani (LB) agar, lactobacilli DeMan, Rogosa, and Sharpe (MRS) broth, and lactobacilli MRS agar were purchased from Difco Laboratories (Difco, Sparks, MD, USA). Antibiotics, including tetracycline, ofloxacin, and penicillin, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tryptic soy broth (TSB), required for the cultivation of bacterial strains, was purchased from Difco Laboratories (Difco, Sparks, MD, USA).
8
Probiotic Strains and Periodontopathogens Interaction
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Six probiotic strains were tested [Lactobacillus acidophilus LA-5™ (CHR Hansen Holding A/S, Hørsholm, Denmark), L. rhamnosus HN001 Howaru™ (Danisco, Madison, WI, USA), L. reuteri DSM 17938 (BioGaia AB, Lund, Sweden), Bifidobacterium breve 1101A, B. pseudolongum 1191A and B. bifidum 1622A (isolated from feces of healthy children, obtained at the Federal University of Minas Gerais) [24 (link)]. P. gingivalis (W83 and ATCC 33277) were used as periodontopathogens. S. oralis ATCC10557 and S. gordonii DL1 [25 ] were used in multi-species biofilm assays.
Bacteria were cultivated from frozen stocks at −80°C. Lactobacilli were grown on Lactobacilli MRS agar (Difco Laboratories, Detroit, MI, USA) and streptococci were cultivated on Tryptic Soy agar [TSA] (Difco Laboratories), both under microaerophilic conditions at 10% CO2, 37°C. Bifidobacteria were grown on BSM agar (Bifidus Selective Medium, Sigma-Aldrich, St. Louis, MO, USA) at 37°C, under anaerobic conditions (90% N2, 5% CO2 and 5% H2) in an anaerobe chamber (Plas-Labs Model 855, Lansing, MI, USA). P. gingivalis were also grown under anaerobic conditions (90% N2, 5% CO2 and 5% H2) at 37°C on blood agar plates [TSA (Difco Laboratories) enriched with 5% defibrinated sheep blood, 0.5 mg/mL hemin (Sigma-Aldrich) and 1 mg/mL menadione (Sigma-Aldrich)].
Bacteria were cultivated from frozen stocks at −80°C. Lactobacilli were grown on Lactobacilli MRS agar (Difco Laboratories, Detroit, MI, USA) and streptococci were cultivated on Tryptic Soy agar [TSA] (Difco Laboratories), both under microaerophilic conditions at 10% CO2, 37°C. Bifidobacteria were grown on BSM agar (Bifidus Selective Medium, Sigma-Aldrich, St. Louis, MO, USA) at 37°C, under anaerobic conditions (90% N2, 5% CO2 and 5% H2) in an anaerobe chamber (Plas-Labs Model 855, Lansing, MI, USA). P. gingivalis were also grown under anaerobic conditions (90% N2, 5% CO2 and 5% H2) at 37°C on blood agar plates [TSA (Difco Laboratories) enriched with 5% defibrinated sheep blood, 0.5 mg/mL hemin (Sigma-Aldrich) and 1 mg/mL menadione (Sigma-Aldrich)].
9
Isolation and Evaluation of Probiotic Bacteria from Jangajji
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Three jangajji samples containing the basic materials of chili pepper jangajji, perilla leaf jangajji, and garlic jangajji were purchased from the local markets in the Jeonju region (Chonbuk, Republic of Korea). The samples were homogenized at 500 rpm and suspended in sterile phosphate buffer solution (PBS, 50 mM, pH 7.0). Subsequently, the diluted solution was aseptically distributed for 24 h at 37 °C on lactobacilli MRS agar (Difco Laboratories, Detroit, MI, USA) plates with 1% (w/v) CaCO3. Colonies were selected at random and cultivated in MRS broth at 37 °C for 18 h. Lacticaseibacillus rhamnosus GG (ATCC 53103, LGG), a representative probiotic strain, was used as the reference control to evaluate the probiotic properties of the isolated strains. For further analysis, the isolates were stored at −80 °C in MRS broth containing 20% (v/v) sterile glycerol.
10
Bacterial Strain Cultivation and DNA Extraction
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The Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan) served as the source of most of the reference strains employed in this study (Table 1 ). The bacterial strains were cultivated on lactobacilli MRS agar (Difco Laboratories, Detroit, MI, USA) anaerobically for 24 h at 37 °C. Genomic DNA was extracted using the DNeasy kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocols.
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