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    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
    (Massey University, 2021) Wang, Tianyang
    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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    Characterisation of dairy strains of Geobacillus stearothermophilus and a genomics insight into its growth and survival during dairy manufacture : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2016) Burgess, Sara
    The thermophilic bacilli, such as G. stearothermophilus, are an important group of contaminants in the dairy industry. Although these bacilli are generally not pathogenic, their presence in dairy products is an indicator of poor hygiene and high numbers are unacceptable to customers. In addition, their growth may result in milk product defects caused by the production of acids or enzymes, potentially leding to off-flavours. These bacteria are able to grow in sections of dairy manufacturing plants where temperatures reach 40 – 65 °C. Furthermore, because they are spore formers, they are difficult to eliminate. In addition, they exhibit a fast growth rate and tend to readily form biofilms. Many strategies have been tested to prevent the formation of thermophilic bacilli biofilms in dairy manufacture, but with limited success. This is, in part, because little is known about the diversity of strains found in dairy manufacture, the structure of thermophilic bacilli biofilms and how these bacteria have adapted to grow in a dairy environment. In Chapters 2 and 3, phenotypic approaches were taken to understand the diversity of strains within a manufacturing plant. Specifically in Chapter 2, strains of the most dominant thermphilic bacilli, G. stearothermophilus, were isolated from the surface of various locations within the evaporator section and ten strains were evaluated for different phenotypic characteristics. Biochemical profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fatty profiling demonstrated that the population was diverse. In Chapter 3, it was shown that the same ten strains varied in their ability to form biofilms and produce spores. Three strains of G. stearothermophilus, A1, P3 and D1, were selected for further analysis. SEM demonstrated that there were differences in biofilm morphologies between the three strains, particularly D1 versus the other two strains, A1 and P3. In Chapters 4, 5 and 6 a comparative genomics approach was taken to determine how these bacteria are able to grow and survive within a dairy manufacturing environment, as well as how they differ from other strains of Geobacillus. In Chapter 4 draft genome sequences were generated for three strains of G.stearothermophilus. Identification of a putative lactose operon in the three dairy strains provided evidence of dairy adaptation. In Chapter 5 a phylogenomics approach was taken to resolve relationships within the Geobacillus genus and to identify differences within the G. stearothermophilus group itself. Finally in Chapter 6 comparison with the model organism B. subtilis, gave a genomics insight into the potential mechanisms of sporulation for Geobacillus spp.
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    The growth of thermophilic bacteria in a milk powder plant and the formation of spores in biofilms of the dairy thermophile Anoxybacillus flavithermus : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Microbiology at Massey University
    (Massey University, 2005) Scott, Sara
    Dairy production makes up 20% of New Zealand's export earnings, with whole milk powder being the number one dairy export. However, contamination of milk powder by spores of thermophilic bacteria is an ongoing problem in the manufacture of milk powder. These spores survive manufacturing processes, contaminate final product and have potential to spoil foods manufactured with milk powder. The main thermophilic organisms that cause concern in the New Zealand dairy industry are Geobacillus spp. and Anoxybacillus flavithermus. The vegetative forms of these organisms are able to grow within biofilms, but there is very little information as to the origin of their spores found in the product and the conditions under which they are produced. We have now monitored the dynamics and location of spore formation in the industrial and controlled laboratory settings. A survey was undertaken at the Pahiatua milk powder manufacturing plant to determine the origin and rate of spore formation. The predominant sites of spore formation were the plate heat exchanger and evaporator. Spores began to develop approximately 11 h into an 18 h manufacturing run. The spores were identified as Anoxybacillus flavithermus and Geobacillus species. To examine the dependence of spore formation on the development of the A. flavithermus biofilm under controlled laboratory conditions, a continuous flow reactor was used. The release of spores and vegetative cells into the milk was measured using change in impedance. Impedance change confirmed the presence of both vegetative cells and spores on stainless steel sample tubes. At the end of an 8.5 h run at 55°C, using the continuous flow reactor, the total number of thermophilic bacteria released into the milk reached up to 106 cells mL -1. At least 10 % of cells attached to the stainless steel surface were spores. These results indicate that spores form readily in biofilms of A. flavithermus believed to colonise the surface of the manufacturing plant. When the temperature of the continuous flow reactor was decreased to 48°C no spores were detected within the biofilm. The results from this study have provided key information about where thermophilic spores form in a milk powder manufacturing plant and how biofilms of one of the typical thermophilic bacteria, Anoxybacillus flavithermus, develop. This knowledge will help the dairy industry to design strategies to prevent spore formation.
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    Effect of cations on biofilm formation by Geobacillus species and Anoxybacillus flavithermus dairy isolates : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2013) Somerton, Benjamin Thomas
    The concentration of free cations is one factor that may influence biofilm formation and consequent contamination of milk formulations by Geobacillus spp. and Anoxybacillus flavithermus during the manufacture of milk powders. Culture optical densities were measured to show that Ca2+ and Mg2+ predominantly increased the planktonic growth of Geobacillus spp. and A. flavithermus cultures. Culture cell numbers were enumerated, and a protein quantification assay was used to indicate that increases in optical density elicited by Ca2+ and Mg2+ supplementation was due to increased production of bacterial surface protein rather than an increase in cell numbers. High individual concentrations of Na+, K+ or Ca2+ (63 – 250 mM) inhibited the planktonic growth of Geobacillus spp., and Mg2+ protected Geobacillus spp. from high, inhibitory concentrations of Na+, K+ or Ca2+. The number of viable cells attached to stainless steel coupons was enumerated to show that cation concentrations or the monovalent to divalent cation ratio (2:1 compared to 10:1) did not influence the transition of bacteria from a planktonic to surface-attached form, or the subsequent formation of an established biofilm. However, preconditioning of the bacteria with cations increased their subsequent attachment. It was proposed that the transition of bacteria from a planktonic to surface-attached form is primarily mediated by the expression of bacterial surface proteins, as induced by cation preconditioning. The number of attached Geobacillus spp. was up to 4 log CFU cm-2 lower, for up to 18 h of biofilm formation, in a milk formulation that had a high monovalent to divalent cation ratio (greater than 10:1) relative to a milk formulation that had a monovalent to divalent cation ratio that resembled that found in unprocessed milk. Supplementation of a milk formulation that had a high monovalent to divalent cation ratio with Ca2+ or Mg2+ fully alleviated the inhibitory effect of the milk formulation on biofilm formation by Geobacillus spp. It was concluded that there is potential for the total thermophile count in milk powders that have high monovalent to divalent cation ratios to be markedly reduced. This would increase the quality and selling price of the milk powders.
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    The adhesion force study of dairy thermophile Anoxybacillus flavithermus CM with atomic force microscopy : a thesis presented in partial fulfilment of the requirements for the degree of Master of Engineering in Chemical & Nanotechnology at Massey University, Manawatu, New Zealand
    (Massey University, 2014) Mohd Saidi, Mohd Salihin
    Anoxybacillus flavithermus is a common species of thermophilic bacteria discovered in most milk powder manufacturing plants through out New Zealand. The contamination of it’s spores into the finished milk powder is an on-going problem as these spores are able to survive the sterilization process. Cheating death, A. flavithermus spores were then believed to attached on the stainless steel surface piping of the production line and germinate into a mature bacteria. A single surviving spore could grow to produce more spores that eventually dislodged from the colony and deposited together with the packaged milk powder. Over the storage time, the contaminated product will gives an off flavor as it deteriorates from bacterial action within. Currently, the applied cleaning method is by rinsing the target section with 1% sodium hydroxide & acid solutions before being flushed out to remove any microorganisms attached on the interior surfaces. However, it is not very effective in removing spores and there is very little information on the value of the spore’s adhesion force on a stainless steel surface. With that in mind, the aim of this study is to determine a proper adhesion force value between a dairy strain spore, A.flavithermus CM and stainless steel surface using the Atomic Force Microscopy (AFM) system. Meanwhile, Geobacillus strearothermophilus ATCC 2641 which is also a thermophilic organism was used over the study for comparison purpose. To measure the adhesion force under an Atomic Force Microscopy (AFM), the crude suspension was first purified using two-phase separation method. Polyethylene glycol (PEG) and phosphate buffer were used as the phase separation chemicals while 0.1% polysorbate 20 was added to the freshly purified spores’ suspension to aid the imaging sequence under the AFM. All AFM imaging and force measurements were done in air and conducted using the silicon type CSG 11/Au cantilever. The crucial Force-Volume imaging was done on a 32x32 grid scan size (1024 samples) on a scan rate of 0.5 Hz. It was calculated that a single A. flavithermus CM spore has an adhesive force value of 16.8 µN when attached on a stainless steel surface. It has a stronger localize adhesive value of 3.9 nN than a G.stearothermophilus ATCC 2641 spore with just 3.6 nN. However, G.stearothermophilus ATCC 2641 has a larger adhesive force of 21.1 µN on a stainless steel surface due to it’s larger spore size. It was also found that spore’s hydrophobicity does not dictates the magnitude of it’s adhesion on any surface. The results from this study have provide the dairy industry an extra sight on the quantitative value of the adhesion force of thermophilic spores, particularly A.flavithermus CM. This will help the dairy industry to design strategies in preventing spores from adhering to its production lines.