Anaerocult c

Amount of 25 g of each sample was aseptically weighted and homogenized with 225 ml of sodium citrate using a stomacher (Seward Stomacher 400 Circulator, UK). Samples were subsequently diluted (1:10) using sterile peptone water and 0.1 ml from each dilution was sub-cultured on Man Rogosa and Sharpe (MRS) agar (Merck, Germany) used for isolating lactic acid bacteria [6 ]. Plates were incubated at 30°C and 37°C for mesophilic lactic acid bacteria and at 42°C for thermophilic lactic acid bacteria for 48 h. MRS agar plates were incubated anaerobically using the gas pack systems (Anaerocult C, Merck, Germany). To differentiate homo- and heterofermentative strains, all the isolates were tested for gram reaction, catalase production, and carbohydrates fermentation (homofermentatives showed ribose +, sorbitol +, raffinose ±, lactose +, galactose +, maltose +, melibiose ±, rhamnose −, xylose ±, manose +, salicine +, sorbose +, glucose +, and sucrose + pattern, heterofermentatives showed ribose +, sorbitol ±, raffinose ±, lactose ±, galactose ±, maltose +, melibiose ±, rhamnose ±, xylose −, manose +, salicine ±, sorbose ±, glucose +, and sucrose±pattern). Cimon citrate test was also conducted as a differential test [7 ,8 ].

BIOLOG^® AN plates (BIOLOG^® Inc., Hayward, CA, USA) were used to identify the substrate utilization pattern of the isolates [37 ]. The technology can also be used to determine substrate utilization patterns of microbial communities [63 (link)]. In the present study, the BIOLOG^® AN type plate was used to determine the carbohydrate preference of the Lactobacillus strains. The procedure followed the manufacturers’ guide with a minor modification. Both strains were inoculated in de Man, Rogosa and Sharpe medium (MRS, Carl Roth GmbH + Co. KG, Germany) and incubated in anaerobic jars (Merck KGaA, Germany) with Anaerocult C (Merck KGaA, Germany) overnight. The cultures were then washed with Phosphate Buffered Saline (PBS), pH 7.4, for three times and diluted to 10⁷ cells/mL. A total of 100µL bacterial suspension was pipetted into each well of BIOLOG^® AN plate in triplicate. The plates were incubated in anaerobic jars with Anaerocult C at 37 °C for 24 h and optical density was read with a microtiter plate reader (Tecan Infinite200Pro, Germany) at OD_590nm.

The bile salt hydrolase (BSH)-positive strain Lactobacillus acidophilus DRU, the hydrogen peroxide (H₂O₂) producer Lactobacillus acidophilus ATCC 4356, and the reference strain Lactobacillus rhamnosus GG (ATCC 53103) were routinely cultured in de Man Rogosa and Sharpe (MRS, Biolife, Italy) medium plus 100 mg/L of cycloheximide (Merck, Germany) at 37 °C under anaerobic conditions, using Anaerocult C (Merck, Milan, Italy). The haemolytic positive strains Streptococcus pyogenes ATCC 19615 and Streptococcus pneumoniae ATCC 6303 were cultured on Brain-Heart Infusion (BHI, Becton Dickinson GmbH, Germany) at 37 °C under 5% CO₂ conditions. Escherichia coli 555, E. coli ATCC 25922, E. coli ATCC 700414, and Staphylococcus aureus ATCC 6538 were routinely cultured on Trypticase Soy Broth medium (Oxoid, Milan) at 37 °C, under aerobic conditions. Listeria monocytogenes DSM 12464 strain was reactivated in BHI broth at 30 °C. Gardnerella vaginalis ATCC 14018 was cultured on Casman’s medium base added of 5% of rabbit blood (VWR, Milan, Italy) at 37 °C. Candida albicans ATCC 10231, Candida krusei ATCC 14243, Candida glabrata ATCC 90030, Candida parapsilosis ATCC 90018, Candida lusitaniae ATCC 200951 and Candida tropicalis ATCC 13803 were cultured on Yeast Mold Broth (Conda, Madrid, Spain) at 28 °C in aerobic conditions.

The strains were isolated from broiler intestinal samples and taxonomically identified as L. salivarius and L. agilis by 16S rDNA sequencing. Both strains were stored in cryo stock at −80 °C. They were cultivated in de Man, Rogosa and Sharpe (MRS, Carl Roth GmbH + Co. KG, Germany) broth in anaerobic jars (Merck KGaA, Germany) with Anaerocult C (Merck KGaA, Germany) at 37 °C for 24 h. The inoculum was prepared fresh each time before use. MRS agar plates were used to determine the viable cell number after treatment.

Bacteria were enumerated in yoghurt samples by the plate count method. Yoghurt samples in 1 mL aliquots were combined with 9 mL of sterile saline solution (NaCl 0.9%, w/v). Coliforms were enumerated at 30 °C on Violet Red Bile Glucose agar (VRBG) (Merck, Warsaw, Poland). Starter cultures were enumerated on selective media based according to IDF standards [31 ]. Lactobacillus delbrueckii ssp. bulgaricus strains were isolated on MRS agar (Merck, Warsaw, Poland) and adjusted to pH 5.4 by microaerophilic incubation in an anaerobic jar with Anaerocult C (Merck, Warsaw, Poland) at 37 °C for 72 h. Streptococcus thermophilus strains were isolated on M17 agar (Merck, Warsaw, Poland), adjusted to pH 7.2, by aerobic incubation at 37 °C for 48 h. Microbial counts were expressed as log10 of colony-forming units (cfus) per mL of yoghurt.