Optimisation of Culture Conditions for Proteolytic Activity in Supernatants of Two Infant-Derived Lactobacilli

We examined cultivation-dependent proteolytic responses in clarified supernatants of two infant-derived lactobacilli (TRM58163 and TRM59525) using single-factor screening, a 12-run Plackett–Burman design, a five-point one-dimensional bracketing scan, and Box–Behnken response-surface modelling. The original optimisation stage yielded relative, assay-dependent casein–Folin responses on an unvalidated nominal scale, without subtraction of sterile-medium background; these values are not absolute enzyme activities. The practical selected media contained 32/31 g/L yeast extract/glucose at nominal pre-sterilisation pH 6.0 for TRM58163 and 30/35 g/L at pH 6.3 for TRM59525. In independently cultivated, matrix-matched confirmation (three batches, three bottles per group per batch), medium-background-corrected apparent casein–Folin responses were 7.99 ± 0.26 versus 22.21 ± 0.88 U/mL for TRM58163 and 7.27 ± 0.21 versus 23.61 ± 0.74 U/mL for TRM59525 (basal versus selected medium); mixed-model selected-to-basal ratios were 2.78 and 3.25, respectively. An independent azocasein readout supported the direction of change. The results demonstrate enhanced crude-supernatant assay response under the selected cultivation conditions, not increased proteinase synthesis per cell or functionality in food or feed matrices.