Tributyrin agar

The salt tolerance of CNS strains was determined by growth on TSA supplemented with NaCl [final concentration 19%–24% (w/v)] with incubation at 37°C for 4 days. Protease activity was determined on TSA containing 2% skim milk (w/v). Lipase activity was determined on tributyrin agar (Sigma-Aldrich, USA) containing 1% tributyrin (v/v). Enzyme activity was determined from the clear zone developed after incubation at 37°C for 4 days. The effect of NaCl concentration on protease and lipase activities was determined by addition of NaCl to the activity test media. The test strains were incubated in TSB to optical density 0.5 at 600 nm and 1 μl of this suspension was inoculated onto each plate. Experiments were performed at least three times on separate days.

The pH values were measured on the mini-cheeses that were mixed with a spatula. Proteolytic activity was determined on calcium caseinate agar modified according to Frazier and Rupp (Merck, Darmstadt, Germany) supplemented with 1% (w/v) skim milk powder (BD Difco^TM). Lipolytic activity was determined on tributyrin agar (Sigma-Aldrich) supplemented with 1% (w/v) tributyrin (Sigma-Aldrich). Ten microliters of cell suspensions at 10⁶ CFU/ml in physiological water (NaCl 9 g/l) were spot-inoculated onto the plates, which were then incubated at 15 or 25°C for 21 days. Proteolytic and lipolytic activities were evaluated by the formation of a clear zone around the spots.

Acid production was determined on TSA containing 0.5% (w/v) glucose and 0.7% (w/v) CaCO₃. Protease activity was determined on TSA containing 0.5% (w/v) glucose and 2% (w/v) skim milk. Lipase activity was tested on tributyrin agar (Sigma-Aldrich, USA) containing 1% (v/v) tributyrin and 0.5% (w/v) glucose. The tributyrin-supplemented medium was emulsified by sonication before autoclaving. To check enzymatic activity, filter paper discs were placed on each substrate-supplemented agar medium surface and 10 μl of Leu. lactis cultured on MRS broth was dropped onto these filter paper discs. Substrate-supplemented agar plates were then incubated at 30°C for 18 h. The relative size of the zone of clearing around the filter paper disc was used as an indicator of enzymatic activity. The effect of NaCl on protease activity was determined by adding NaCl to each medium up to a final concentration of 6% (w/v). All experiments were conducted at least two times on separate days.

To evaluate the proteolytic activity of M. caseolyticus subsp. caseolyticus strains, reconstituted skim milk (RSM) Agar was prepared from skim milk powder (Kerry ingredients, Cheshire, United Kingdom) at 10% (w/v) and Agar (Agar; Sigma-Aldrich, Wicklow, Ireland) at 1.5% (w/v). The inoculated plates were then incubated for 24 h at 37°C. To determine lipolytic activity, tributyrin Agar (Sigma-Aldrich) was prepared according to the manufacturer’s instructions, with the modification prescribed by Bertuzzi (2017) . Y. lipolytica DPC6266 was used as a positive control. The plates were incubated for 48 h at 37°C. A positive result for protease and lipase activity was scored on the basis of the presence of halo of clearing around the growth of the organism. The test assay was performed in triplicate.

The salt tolerance of E. faecium strains was determined by examining growth on TSA supplemented with NaCl at a final concentration of 0.5–9% (w/v). Growth on 0.5%, 3%, 6%, and 9% NaCl was observed after 1, 2, 3, and 4 days of incubation. The protease and lipase activities were determined on TSA containing 2% (w/v) skim milk and tributyrin-agar (Sigma-Aldrich, Burlington, MA, USA) containing 1% (v/v) tributyrin, respectively. The effect of NaCl was determined by adding NaCl to the appropriate medium. The enzyme activities were determined by clear halo formation around colonies.

Acid production was determined on TSA supplemented with 0.7% (w/v) CaCO₃. Protease and lipolytic activities were determined on TSA containing 2% (w/v) skim milk, and tributyrin agar (Sigma-Aldrich, St. Louis, MO, USA) containing 1% (v/v) tributyrin, respectively. The enzymatic activity and acid production ability were determined by clear halo formation around the colony on each appropriate agar medium after incubation at 37 °C for 18 h. The effect of NaCl on each activity was determined by the addition of NaCl to each medium up to a final concentration of 6% (w/v).