Robust Biofilm-Forming Bacillus Isolates from the Dairy Environment Demonstrate an Enhanced Resistance to Cleaning-in-Place Procedures

One of the main strategies for maintaining the optimal hygiene level in dairy processing facilities is regular cleaning and disinfection, which is incorporated in the cleaning-in-place (CIP) regimes. However, a frail point of the CIP procedures is their variable efficiency in eliminating biofilm bacteria. In the present study, we evaluated the susceptibility of strong biofilm-forming dairy Bacillus isolates to industrial cleaning procedures using two differently designed model systems. According to our results, the dairy-associated Bacillus isolates demonstrate a higher resistance to CIP procedures, compared to the non-dairy strain of B. subtilis. Notably, the tested dairy isolates are highly persistent to different parameters of the CIP operations, including the turbulent flow of liquid (up to 1 log), as well as the cleaning and disinfecting effects of commercial detergents (up to 2.3 log). Moreover, our observations indicate an enhanced resistance of poly-γ-glutamic acid (PGA)-overproducing B. subtilis, which produces high amounts of proteinaceous extracellular matrix, to the CIP procedures (about 0.7 log, compared to the wild-type non-dairy strain of B. subtilis). We therefore suggest that the enhanced resistance to the CIP procedures by the dairy Bacillus isolates can be attributed to robust biofilm formation. In addition, this study underlines the importance of evaluating the efficiency of commercial cleaning agents in relation to strong biofilm-forming bacteria, which are relevant to industrial conditions. Consequently, we believe that the findings of this study can facilitate the assessment and refining of the industrial CIP procedures.