Application of fluorescent probes to determine localised salt concentrations within cheese matrices and their influence on metabolic activity of entrapped bacterial cells

Abstract Text:

The influence of salt on microbial growth and activity in cheese has received much prior attention. However, the way in which salt within cheese matrices affects bacterial physiology to control cell growth is not fully elucidated. Application of advanced microscopy techniques to determine salt concentration at a localised level is of interest to understand interactions between cheese matrix physico-chemistry and microbial activity prior to, during and post-brining of cheese. The objective of this study was to determine the presence of and effects of localised salt gradients associated with brine salting on microflora physiology and metabolic activity of the individual starters, S. thermophilus and L. helveticus used in cheese manufacture. Cheeses were manufactured in 3 replicate trials, brined for 66 hrs and sampled at the high-salt outside and low-salt inside layers prior to, during and post-brining. Localised salt concentrations were determined using CoroNa™ Green Sodium Indicator by Confocal Laser Scanning Microscopy. The average salt content in the outside layer post brining was ~3.8%. The response of cytoplasmic membrane integrity of bacterial cells and levels of free reactive oxygen species to salt concentrations were assessed using fluorescent probes combined with Flow Cytometry. There were greater levels of membrane damage and oxidative stress observed in L. helveticus compared to S. thermophilus at all times. Confocal imaging clearly identified localised variations in salt concentrations and illustrated the penetration distance of the brine solution into the matrix during and post-brining. Overall, this study showed a differing impact of varying salt levels on cheese starter physiology and metabolic activity in vivo, dependant on starter type and confirmed that the methodologies used have the potential to identify ripening hotspots at a localised level.  It opens up further opportunities to apply fluorescent probes to gain a deeper understanding of the influence of cheese matrix physicochemistry on the metabolic activity of entrapped bacteria and thus to control cheese manufacture processes to achieve greater consistency in ripened cheese quality.

Keywords: Cheese, Salt Gradients, Metabolic Activity