Changes in Sodium, Calcium, and Magnesium Ion Concentrations That Inhibit Geobacillus Biofilms Have No Effect on Anoxybacillus flavithermus Biofilms

Abstract

cThis study investigated the effects of varied sodium, calcium, and magnesium concentrations in specialty milk formulations on biofilm formation by Geobacillus spp. and Anoxybacillus flavithermus. The numbers of attached viable cells (log CFU per square centimeter) after 6 to 18 h of biofilm formation by three dairy-derived strains of Geobacillus and three dairy-derived strains of A. flavithermus were compared in two commercial milk formulations. Milk formulation B had relatively high sodium and low calcium and magnesium concentrations compared with those of milk formulation A, but the two formulations had comparable fat, protein, and lactose concentrations. Biofilm formation by the three Geobacillus isolates was up to 4 log CFU cm ؊2 lower in milk formulation B than in milk formulation A after 6 to 18 h, and the difference was often significant (P < 0.05). However, no significant differences (P < 0.05) were found when biofilm formations by the three A. flavithermus isolates were compared in milk formulations A and B. Supplementation of milk formulation A with 100 mM NaCl significantly decreased (P < 0.05) Geobacillus biofilm formation after 6 to 10 h. Furthermore, supplementation of milk formulation B with 2 mM CaCl 2 or 2 mM MgCl 2 significantly increased (P < 0.05) Geobacillus biofilm formation after 10 to 18 h. It was concluded that relatively high free Na ؉ and low free Ca 2؉ and Mg 2؉ concentrations in milk formulations are collectively required to inhibit biofilm formation by Geobacillus spp., whereas biofilm formation by A. flavithermus is not impacted by typical cation concentration differences of milk formulations.T hermophilic bacilli belonging to the Geobacillus spp. and Anoxybacillus flavithermus groups are the predominant spoilage bacteria that may contaminate milk during its manufacture into milk powder (1, 2). The number of thermophilic bacilli in milk powder is of major importance because it is a measure of its quality and determines its market selling price (1, 2). Geobacillus spp. and A. flavithermus grow as biofilms on product contact surfaces in regions of milk powder manufacturing plants, such as in plate heat exchangers and evaporators, that are held at high temperatures (up to 70°C) (1, 2). It is postulated that these biofilms act as a reservoir of cells that slough off and disperse into milk as it transits through the plant (1, 2). The majority of thermophilic bacilli that appear in milk powder originate from biofilms on product contact surfaces (2).The concentrations and ratios of free cations in the aqueous phase that immerses a biofilm can influence biofilms in many ways. Electrostatic interactions of free cations with bacterial polymers in a biofilm matrix can influence the structural integrity and cohesion of a biofilm (3, 4). In addition, bacteria respond to fluctuations in free cation concentrations by adapting their physiology, which may influence biofilm formation. For example, Kara et al. (5) showed that increasing Na ϩ concentrations increased the proportion of negatively char...

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