Comparative Screening of Probiotic-Derived Formulations for Inhibition of Biofilm Formation by Milking Equipment Isolates and Reference Strains

Biofilms in dairy production lines, including milking equipment, can contribute to persistent microbial contamination. This exploratory study screened 32 formulations derived from Lactobacillus acidophilus, Lactobacillus helveticus, Lacticaseibacillus rhamnosus, and Saccharomyces cerevisiae to identify candidates for further biofilm-control studies. Nine bacterial targets, including isolates from milking equipment and reference strains, were tested individually. The attached biomass was measured using crystal-violet staining, with De Man–Rogosa–Sharpe (MRS) controls adjusted to pH 3.8 and 6.8. Planktonic growth was assessed after exposure to native-pH cell-free supernatants (CFSs). The screening comprised one independent experiment with three technical replicates per formulation target combination. All four native-pH CFSs inhibited attached-biomass formation by the six strong biofilm-forming targets, with displayed inhibition values of 71–100% and no or low formulation-only staining. The three lactobacillus-derived CFSs also generally reduced planktonic turbidity to near-background levels, whereas the effect of S. cerevisiae CFS on planktonic growth varied among targets. L. acidophilus pH-adjusted CFS and S. cerevisiae heat-inactivated whole-culture preparation also retained broad antibiofilm activity. Meanwhile, several other cell-containing or pH-adjusted formulations showed target-dependent effects or produced substantial surface-associated material. Native-pH CFSs were the most consistent candidates for independent confirmation and further testing on equipment-relevant surfaces.