Characterisation and spoilage potential of Bacillus subtilis complex group isolated from milk processing environment

Abstract

Thermophilic Bacillus and their spores are important in dairy processing due to their ubiquity, resistance to high temperature, chemical inactivation and their biofilm forming potential. The presence of such microbial contaminants results in shelf-life reduction of processed foods serving as a potential hotspot of outbreaks and spoilage of post-processed milk. Therefore, this study seeks to characterise Bacillus subtilis and Bacillus velezensis from raw, pasteurised and packaged extended shelf-life (ESL) milk samples, determine their biofilm forming and spoilage potential as well as the effect of ultraviolet C (UVC) in the inactivation of their spores with the intent of mitigating their deleterious presence in the dairy processing plant. The isolates were identified B. subtilis and B. velezensis with the potential of forming weak, moderate and strong biofilms with B. velezensis strain LPL-K103 (B44) with the most robust biofilm. All the isolates are novel sequence types (STs) using their multi-locus sequence type profile with the closest STs are 96 for B. velezensis and 128 for B. subtilis mostly isolated from the soil. The heat resistance profile indicated all 12 isolates are psychrotolerant as well as thermophilic with temperature ranges of 6 °C to 55 °C, 6 °C to 60 °C and 15 °C to 60 °C. All isolates can produce both lipolytic and proteolytic enzymes both in their planktonic and biofilm states. The maximum lethality rate after UVC exposure is 6.5 for B. subtilis strain SRCM103689 (B47) and highest percentage hydrophobicity was 54.9 % from the sample B. velezensis strain LPL-K103 (B44). Flow cytometry analysis of UVC treated spore suspensions showed a divergence into subpopulations unaccounted for by plate counting on growth media which are: inactivated, live spores, dormant, sub-lethally injured and an unknown subpopulation. The Raman spectroscopy identified B. subtilis CECT 4002 (B4002) spores as the isolate possessing the highest concentration of Ca-DPA. The spoilage potential of the isolates was determined by quantifying the concentrations of proteolytic and lipolytic enzymes produced by the biofilm and planktonic cells by using azocasein and p-nitrophenol palmitate (p-NPP) assays. In the planktonic cells, B48 has the highest proteolysis with 1033.6 ρL/CFU while B50 has highest lipolysis of 34.5 ρL/CFU. For the biofilms, B168 has the highest proteolysis and lipolysis per cell with a mean 3706 ρL and 179.9 ρL. The result of this study indicated that the spoilage potential (proteolysis and lipolysis) both of biofilms and planktonic culture are strain-dependent and that there seems to be a relationship between the strength or complexity of the biofilms and spoilage potential of the isolates. The study presents the significance of thermophilic B. subtilis B. velezensis and possible reason for their perpetuation in the dairy processing plant. The result linked the isolates to the raw milk used in the production of ESL milk-fed into the downstream processing line suggesting the survivability of the isolates by adaptation to the processing condition either as spores or as a community as in biofilms. The quality of the raw milk is thereby compromised which in turn affect the shelf-life of the final product. The result highlighted the effect of UVC in the inactivation of the spores and spore surface hydrophobicity are heterogenous with some strain-to-strain variations at molecular level among the organisms used.