The effect of calcium and milk formulations on biofilm formation of Geobacillus stearothermophilus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology, the School of Food and Advanced Technology, Massey University, Palmerston North, New Zealand
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Date
2021
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Massey University
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Abstract
G. stearothermophilus contaminates milk powder products from bacteria spores released from biofilms on product contact surfaces in dairy manufacturing plants. The dairy industry has observed that calcium-reduced milk protein powder is associated with reduced spore contamination of thermophilic bacteria during milk protein powder manufacture. Calcium, as a major component of milk minerals, was previously found to affect G. stearothermophilus biofilms grown on stainless steel exposed to milk formulations. Additionally, G. stearothermophilus cells cultured with additional calcium showed an increased attachment to stainless steel. The current study investigated the effect of calcium and milk formulations on cell attachment, biofilm formation, spore production and spore heat resistance of G. stearothermophilus to pinpoint the potential factors contributing to the reduced thermophile contamination in the calcium-reduced milk protein powder.
The effect of calcium on biofilm formation of G. stearothermophilus dairy isolates A1, P3, and one reference strain 7953 in modified TSB media was studied to gain more insights into the role of calcium in biofilm formation of G. stearothermophilus. The presence of calcium increased biofilm cell numbers of strain A1, but reduced biofilm cell numbers of the reference strain and showed minimal effect on strain P3. Extracellular polymeric substances (EPS) quantity, in particular extracellular protein, varied between strains. Unlike the consistent biofilm promotive effect of calcium in milk formulations found in a previous study, the current findings suggest that a strain-to-strain difference exists in biofilm formation of G. stearothermophilus species in the presence of calcium.
Calcium plays an important role in cell attachment by changing cell surface properties, cell physiology and metabolism, modifying cell surface structure and bridging between bacteria and substrata. In the dairy industry, milk formulations with different cation profiles may affect the cell attachment of a common contaminant G. stearothermophilus. The current study investigated the effect of calcium on cell attachment of G. stearothermophilus strains A1, P3 and 7953 to stainless steel and polystyrene substrata and characterized the cell surface charge, hydrophobicity and cell surface polymers. In addition, cell attachment in milk formulations on stainless steel was characterized. The presence of calcium increased the cell attachment of dairy isolates on polystyrene but not the reference strain, while calcium did not affect cell attachment of all strains on stainless steel. The presence of calcium changed the amount of cell surface polymers produced but not hydrophobicity. Although calcium affected the zeta potential, this did not correlate with the trend in cell attachment. It is assumed that the cell attachment of G. stearothermophilus is affected by the substrata, strain specific cell surface polymers, as well as calcium induced changes in cell surface polymers. Milk formulations (MF1 and MF2) with different cation profiles showed little effect on cell attachment of G. stearothermophilus on stainless steel, indicating a minimal effect of cell attachment to contamination levels of different cation-modified milk protein powder products in dairy manufacturing plant.
The effect of total calcium concentration, total sodium concentration and bacteria growth history were investigated on biofilm formation of G. stearothermophilus in MF1 and MF2. The numbers of culturable biofilm cells of G. stearothermophilus strain A1, P3 and 7953 were compared in MF1 and MF2 over 18 h. MF1 and MF2 have similar protein, lactose and fat content except for cation profiles, where MF2 has relatively high sodium and low calcium concentrations. Biofilm formation of all three strains was lower in MF2 than in MF1, but the inhibition of MF2 was conditional and was highly dependent on the growth history of bacterial inocula. The inhibition of MF2 on dairy isolates A1 and P3 were further investigated by cation supplementation. Supplementation of MF1 with sodium decreased biofilm formation at 18 h for A1 and P3. Supplementation of MF2 with either 2 or 26 mM calcium increased biofilm formation of A1 at 18 h, and P3 at 10 h. High sodium and low calcium concentrations in the milk formulation seem to be required to inhibit biofilm formation of G. stearothermophilus. However, it is not known if the inhibitory effect of MF2 was due to the direct effect of cations on biofilms or the change of milk protein structure on biofilms due to calcium removal.
Cations such as calcium and sodium were shown to affect sporulation and spore heat resistance of G. stearothermophilus. MF1 and 2 have distinctive cation profiles and therefore the sporulation and spore heat resistance of G. stearothermophilus A1, P3 and 7953 in milk
formulations were investigated. MF2 effectively reduced the total biofilm spore numbers of A1 and P3 over the 18 h culture period compared to MF1, but the effect was not observed in 7953. The sporulation percentage of A1 was higher in MF1 than MF2 at 14 and 18 h, and a similar effect was observed for P3 at 10 and 14 h. Supplementation of calcium to MF2 increased the sporulation percentage of A1 at 14 and 18 h, as well as P3 at 14 h. Supplementation of sodium to MF1 decreased the sporulation percentage of A1 at 18 h. No significant difference in spore heat resistance of A1, P3 and 7953 was observed between spores produced from MF1 and MF2.
Overall, the reduced biofilm formation and sporulation percentage of G. stearothermophilus in MF2 provided evidence to the reduced thermophile contamination in the calcium-reduced milk protein powder.
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Figure 2-2 is re-used under a CC BY license.
Keywords
Dried milk, Thermophilic bacteria, Biofilms, Calcium, Milk, Composition