A systematic characterization of the distribution, biofilm-forming potential and the resistance of the biofilms to the CIP processes of the bacteria in a milk powder processing factory

Abstract

Owing to the resistance to conventional cleaning and sanitizing agents, biofilms formed on surfaces of dairy processing equipment pose a hazard to the dairy industry. The objective of this study was to evaluate the diversity of the microflora attached to various surfaces of the processing lines of a milk powder processing factory based on 16S rRNA gene sequence analysis. The production of biofilms by the bacterial strains was evaluated on polystyrene (PS) and stainless-steel (SS) surfaces by crystal violet staining method. Especially, effects of temperatures (e.g., 37 °C and 55 °C) and growth mediums (e.g., nutrient broth, NB; tryptic soy broth, TSB) on the production of biofilms by these strains on PS surfaces were explored. Besides, the tolerance of the biofilms of the strains to CIP processes (1.5% v/v HNO₃ solution or 2.0% w/v NaOH solution at 80 °C) were analyzed. Forty-five isolates from eleven interior surfaces of the facilities were identified and the distribution of strains had high species diversity, which indicated that multiple spoilage and pathogenic microorganisms remained in milk powder processing lines after CIP processes. Bacteria showed higher biofilm-forming abilities on SS compared to PS surfaces under the same condition. Moreover, effects of the incubation temperature and growth medium on biofilm formation varied between genera, species, and strains. One strain of each species with biofilm-forming abilities on SS surfaces was selected to check the resistance of their biofilms formed on SS coupons to the CIP processes. Biofilms of all of the nine strains were highly or moderately alkali and acid resistant, posing a threat to the milk powder production. These results suggested that organisms within the biofilm might withstand temperature and pH changes better than planktonic organisms. More research is needed to investigate different species isolated from different facilities of the processing lines and to improve the key controlling points in the CIP processes.

Keywords: Acid tolerance; Alkali tolerance; Bacillus spp.; Bacterial biofilms; Milk powder processing facility.