Mrs agar

Antagonism tests were performed using the overlay method [37 (link)]. L. fermentum ATCC 23271 grown on MRS agar was standardized at OD_600nm = 0.1, in MRS broth, and then 5 μL of the inoculum was spotted (about 0.9 cm) on MRS agar (Difco Laboratories, Detroit, MI, USA) and incubated for 48 h under the same conditions. After that period, a 2 mm layer of Muller–Hinton agar (Difco Laboratories) was added onto MRS agar, and the standard pathogen inoculum (1 × 10⁸ CFU/mL) was seeded. Petri dishes were incubated at 37 °C for 24 h under aerobic conditions, and subsequent zones of inhibition were measured.

Bifidobacterium longum subsp. infantis ATCC 15697 and B. breve SC95 were routinely grown for 48 h anaerobically at 37 °C in the semisynthetic de Man-Rogosa-Sharpe (MRS) broth or MRS agar (Becton Dickinson) supplemented with 1 % (wt/vol) L-cysteine. Single colony isolates were inoculated into modified MRS (mMRS) without sugar supplemented with 2 % of filter-sterilized (wt/vol) lactose (LAC) (Sigma Aldrich), 2 % glucose (GLU) (Fisher) or 2 % purified human milk oligosaccharide mixture (HMO) as the sole carbon source. HMO was kindly provided by the laboratory of Dr. Bruce German (Department of Food Science and Technology, University of California, Davis) and purified according to the method described in Gnoth et al. [53 (link)].

Lactobacillus plantarum (Lp) inoculum was developed following the method of Ogodo et al. (2017 ) with slight modifications. A standard culture of L. plantarum inoculum was prepared on MRS agar (Becton, Dickinson and Co.) from stock cultures frozen in MRS broth, from which isolated colonies were selected for further propagation. The L. plantarumbacterium in the frozen cultures was first activated in MRS broth by adding 0.1 ml of frozen culture to test tube which contained 9 ml of MRS broth and incubated for 48 h at 37°C. Then, one loopful was taken and spread‐plated on De Man, Rogosa and Sharpe (MRS) agar, after which incubation was done anaerobically at 37°C for 48 h. The cells were harvested by centrifugation at 5000 gravity for 10 min and washed with distilled water. Inoculum development of Saccharomyces cerevisiae (Sc) was performed according to Vilela et al. (2020 (link)) with slight modification. A stock culture of S. cerevisiae was activated by inoculating the cells into a freshly prepared yeast extract peptone dextrose (YPD) broth containing 2% glucose, 2% peptone and 1% yeast extract. The culture was incubated overnight at 37°C to achieve a significant growth of population, and one loopful was taken and spread‐plated on YPD agar and incubated at 30°C for 3 days.