L 1 l cysteine hcl

The effect of a 1000 μg mL⁻¹ extract solution prepared using de Mann Rogosa and Sharpe Broth (MRS broth, Biokar Diagnostics, Beauvais, France) for lactobacilli or MRS broth supplemented with 0.5 g L⁻¹ L-cysteine-HCl (Sigma, St. Louis, MO, USA) (MRS + CYS broth) for bifidobacteria. This mixture was inoculated using an overnight inoculum and incubated at 37 °C for 24 h (bifidobacteria were incubated in anaerobiosis). At 0, 6, 12 and 24 h, the total viable cells, environmental pH values and organic acid production/sugar consumption were assessed. The total viable probiotic counts were determined using decimal dilutions and plated in either MRS (48 h at 37 °C) or MRS + CYS (48 h at 37 °C under anaerobic conditions) agar. The culture media pH values were measured using a Crison micropH 2002 (Crison Instruments S. A., Barcelona, Spain) pH reader. Sugar consumption/organic acid production was evaluated using an HPLC-RI-UV system, following the analytic conditions described by Sousa et al. [18 (link)]. Positive controls were drawn through inoculation of the respective culture media without extract and non-inoculated culture media (with and without extract) was used as a negative control. Each condition was assessed in considering three independent assays, each considering triplicate of incubations and duplicate plating, pH measurement or HPLC injection.

Overnight inoculums, incubated at 37 °C (bifidobacteria under an anaerobiotic atmosphere comprised of 10% CO₂, 10% H₂, and 80% N₂ using a Whitley D6250 anaerobic workstation (don Whitley Scientific, West Yorkshire, United Kingdom)) were prepared using tryptic soy broth (TSB, Biokar Diagnostics, Beauvais, France) for E. coli, L. monocytogenes and S. enteritidis, de Mann, Rogosa, and Sharpe broth (MRS broth, Biokar Diagnostics, Beauvais, France) for Lactobacillus, and MRS supplemented with 0.5 g L⁻¹ L-cysteine-HCl (Sigma, St. Louis, MO, USA) for Bifidobacterium. The inocula (10 mL) was centrifuged, washed twice and resuspended in 5 mL of sterile PBS as previously described by Valeriano, et al. [10 (link)].