Lm17 agar

For bacteriological analysis, cheeses were aseptically sampled at Day 1, 14, 28, and Month 3 of ripening. The samples were placed in a sterile stomacher bag, diluted 1:10 with sterile 2% trisodium citrate and homogenized using a stomacher (Iul Instruments, Barcelona, Spain) for 5 min. Independent duplicate samples were taken at each time point and serial dilutions were prepared as required. Starter cells were enumerated on LM17 agar (Merck, Darmstadt, Germany) after incubation at 30°C for 3 days. Total NSLAB (lactobacilli) were enumerated on Lactobacillus selective (LBS) agar (BD, Oxford, UK) after 5 days incubation at 30°C. Coliforms and enterococci were enumerated on violet red bile agar (BD) and Kanamycin aesculin azide agar (Merck) after 24 h incubation at 30°C.
To confirm that the majority of NSLAB belonged to the inoculated adjuncts, Pulsed-Field Gel Electrophoresis (PFGE) was performed as described by Stefanovic et al. (2017a (link)). Isolates from Month 3 samples were analyzed, and the PFGE patterns were compared with the patterns of the three adjuncts (Figure 2B).

A culture of L. lactis DPC3147 was maintained on LM17 agar (Merck KGaA, Darmstadt, Germany) at 30°C. A single colony of DPC3147 was inoculated into 10 ml LM17 broth, and incubated aerobically at 30°C under static conditions overnight. One milliliter of the overnight culture was sub-cultured into 100 ml LM17 broth, and incubated aerobically at 30°C overnight. This overnight culture was centrifuged at 8000 × g for 15 min at 4°C and the cell pellet was then washed twice with ice cold water for injection (WFI) (BioSciences Ltd., Dun Laoghaire, Dublin, Ireland), and concentrated ten-fold by suspending the cells in 10 ml WFI for downstream viability assays. A schematic of the preparation of the DPC3147 live bio-therapeutic formulation is included in Figure 1A.