Mrs agar

Further analyses included the determination of the lactic acid bacteria population in yogurt using the standard plate method, in two parallel replicates for each analysis and three independent replications for each experiment. MRS agar (Merck, Germany) was used to determine the lactobacilli counts, and M17 agar (Merck, Germany) was applied to determine the streptococci counts. The plates of MRS agar were incubated at 37 °C for 72 h under anaerobic conditions (Anaerocult A, Merck, Germany). Plates of M17 agar were incubated at 37 °C for 72 h under aerobic conditions. After incubation, the number of bacteria was converted into colony-forming units in mL (CFU/mL) and expressed as log CFU/mL.

Erythromycin (Sigma), Anaerocult® C Merk, Gas Pack (Merck, Darmstadt, Germany), MRS broth (De Man et al., 1960 ), sodium citrate, peptone water, and MRS Agar were purchased from Merk company (Merck, Darmstadt, Germany). Sodium alginate was provided by Sigma Company (Sigma, Steinheim, Germany). The Plantago major seed was provided by Ahura Med, Marvdasht, Fars, Iran. It was collected from growing plants in Pasargad city, Fars province (South of Iran). Further plant identification was performed by the herbarium of the Fars Research Center for Agriculture and Natural Resources (FRCANR), Shiraz, Iran. A voucher specimen is deposited in the herbarium of the FRCANR, Shiraz, Iran.

Quinoa (Chenopodium quinoa Willd) was provided by the Gansu Academy of Agriculture Science (Lanzhou, China). Lactobacillus plantarum NO.1−16 were isolated from human intestinal flora. GABA, MRS agar, and Monosodium L-glutamate (MSG) were purchased from Sigma Aldrich, Inc. (Shanghai, China). All chemicals are analytical grade unless otherwise stated.

LAB from the samples was isolated as described by Rajoka et al. [15 (link)]. Briefly, the samples were serially diluted (10⁻¹ to 10⁻³) with peptone water (Thermo Fisher Scientific, Carlsbad, CA, USA) and inoculated on de Man-Rogosa Sharpe (MRS) agar (Sigma-Aldrich, St Louis, MO, USA). The inoculated plates were incubated in anaerobic jars at 37°C for 48 h. Single colonies that grew were randomly selected, and each colony was inoculated in 5 mL of MRS broth (Sigma-Aldrich, St Louis, MO, USA) which was then incubated at 37°C for 48 h. The cultures were then streaked on MRS agar and incubated at 37°C for 48 h. Pure isolates were obtained and maintained at -80°C on MRS agar slants containing 40% glycerol pending further experiments.

Freeze-dried granules of Lactobacillus gasseri ATCC 19992 ™ were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). Sodium alginate, Tween^® 80, calcium chloride, de Man Rogosa and Sharp Medium (MRS), MRS agar, Gram stain kit, and all the reagents used to perform the in vitro evaluation were purchased from Sigma-Aldrich^® (St Louis, MO, USA). The commercial vegetable oil used in this study was bought from a local market in Veracruz (Mexico).

The different lactobacilli were isolated from the digestive tracts (ileum and cecum) of 16 antibiotic-free healthy broiler groups of different ages (four levels), breeds (four species) and diet formulas (four levels), as well as from 10 antibiotic-treated commercial broilers (Table 3). Experiments coded from 1 to 16 corresponded to the antibiotic-free broiler group, the “A”-coded experiment represents the group of antibiotic-treated commercial broilers, and the sample origin was designated as “i” for ileum and “c” for cecum. Samples of the ileum or cecum content of each category were homogenized at a ratio of 1:10 (10 g of ileum or cecum content in 90 mL of buffered peptone water (Scharlau-Chemie, Barcelona, Spain)). The homogenate was diluted 10⁷-fold and 0.1 mL was plated onto de Man, Rogosa and Sharpe (MRS) agar (Sigma-Aldrich, Burlington, MA, USA). The plates were incubated anaerobically for 3 to 4 days at 37 °C. In total, 210 randomly selected strains were first characterized by Gram staining, motility and the detection of catalase activity. Gram-positive, catalase-negative bacilli were presumptively considered as Lactobacillus for further identification. Isolates were preserved in MRS broth with 20% glycerol at −70 °C until further use. All strains were revivified by successive streaking on MRS agar prior to performing any assay.