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

Abstract

This 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 CaCl2 or 2 mM MgCl2 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.

FIG 1

Biofilm formation, after 6 to 18 h of incubation at 55°C, by viable Geobacillus sp. F75 cells on stainless steel coupons fully submerged in milk formulation A (a), milk formulation A supplemented with 50 mM NaCl (b), milk formulation A supplemented with 100 mM NaCl (c), milk formulation B (d), milk formulation B supplemented with 2 mM CaCl₂ (e), and milk formulation B supplemented with 2 mM MgCl₂ (f). Experiments were repeated as triplicates, and error bars represent ±1 standard deviation. An asterisk indicates a significant difference (P ≤ 0.05) between cation-supplemented and unsupplemented milk formulations for the respective milk formulation and time point.

FIG 2

Biofilm formation, after 6 to 18 h of incubation at 55°C, by viable Geobacillus sp. TRa cells on stainless steel coupons fully submerged in milk formulation A (a), milk formulation A supplemented with 50 mM NaCl (b), milk formulation A supplemented with 100 mM NaCl (c), milk formulation B (d), milk formulation B supplemented with 2 mM CaCl₂ (e), and milk formulation B supplemented with 2 mM MgCl₂ (f). Experiments were repeated as triplicates, and error bars represent ±1 standard deviation. An asterisk indicates a significant difference (P ≤ 0.05) between cation-supplemented and unsupplemented milk formulations for the respective milk formulation and time point.

FIG 3

Biofilm formation, after 6 to 18 h of incubation at 55°C, by viable Geobacillus sp. 183 cells on stainless steel coupons fully submerged in milk formulation A (a), milk formulation A supplemented with 50 mM NaCl (b), milk formulation A supplemented with 100 mM NaCl (c), milk formulation B (d), milk formulation B supplemented with 2 mM CaCl₂ (e), and milk formulation B supplemented with 2 mM MgCl₂ (f). Experiments were repeated as triplicates, and error bars represent ±1 standard deviation. An asterisk indicates a significant difference (P ≤ 0.05) between cation-supplemented and unsupplemented milk formulations for the respective milk formulation and time point.

FIG 4

Biofilm formation, after 6 to 18 h of incubation at 55°C, by viable Anoxybacillus flavithermus E16 (a and b), A. flavithermus TRb (c and d), and A. flavithermus 136 (e and f) cells on stainless steel coupons fully submerged in milk formulation A (a, c, and e) and milk formulation B (b, d, and f). Experiments were repeated as triplicates, and error bars represent ±1 standard deviation.