Molecular characterization and biofilm formation potential of Bacillus subtilis and Bacillus velezensis in extended shelf life milk processing line

摘要

This study aims to characterize Bacillus subtiliscomplex group from raw, pasteurized, and packagedextended shelf-life (ESL) milk samples, to determinetheir biofilm potential and source-track the microbialcontaminants to control their presence during processing.Isolates were characterized using multi-locus sequencetyping (MLST) with 7 housekeeping genes. Theprimers used were designed from the coding regionswith the highest number of polymorphic sites. The heatresistance profile indicated that all 12 isolates are psychrotolerantas well as thermophilic, with temperatureranges of 6°C to 55°C (B43, B44, B52, B54, B55, B56,B57), 6°C to 60°C (B46, B47, B48), and 15°C to 60°C(B49, B50). A general linear model 2-way repeatedmeasureANOVA of the biofilm-forming potential of theisolates shows a statistically significant difference acrossthe time of incubation (6, 12, 18, and 24 h). All isolatesexcept 2 formed moderate to strong biofilms, with B44having the most robust biofilm formation (3.14 ± 0.60).Scanning electron and confocal microscopy images revealthe strain specificity of the biofilm structure. TheMLST analysis identified all isolates as belonging toeither B. subtilis or Bacillus velezensis. All the isolateswere novel sequence types (ST) when compared withthe PubMLST database butshowed relatedness to isolates in the raw milk that wasprocessed. The closest ST are 96 for B. velezensis and128 for B. subtilis, mostly isolated from soil. This studypresents the significance of biofilms of thermophilic B.Subtilis and B. velezensis and their possible perpetuationin the dairy processing plant. The informationprovided is a call for an innovative food contact surfaceor any other intervention that can minimize or preventmicrobial adhesion in the processing plant, to preventnegative effects in ESL milk.