Formation and control of biofilms of thermo-resistant streptococci on stainless steel : a thesis presented in partial fulfilment of the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
The aim of this study was to develop improved methods of controlling biofilms of thermo-resistant streptococci in dairy manufacturing plant. A method to rapidly and accurately detect viable cells of thermo-resistant streptococci on stainless steel surfaces involving the use of the Malthus microbiological growth analyser was developed. A modified Robbins device was designed and installed in a dairy manufacturing plant to monitor biofilm development and obtain isolates for study. These studies confirmed that routine cleaning programmes were not eliminating biofilms of thermo-resistant streptococci from the stainless steel surface. The isolates obtained were identified using biochemical and molecular techniques. As well as the expected Streptococcus thermophilus, a new species, S. waiu representing 24% of the isolates was also described. Molecular techniques (polymerase chain reaction and fluorescent in situ hybridisation) were developed to rapidly identify the bacteria. The cell surface hydrophobicity of all isolates was determined, with those obtained from dairy manufacture being highly hydrophobic compared with mixed hydrophobicity in the general population. There was no correlation between many factors often associated with adhesion (such as hydrophobicity, polysaccharide production, surface charge) and the rate of cell adhesion. However, treatment of the bacteria with proteolytic agents reduced the number of all isolates adhering to stainless steel by approximately 100-fold. A 55 kDa protein with an N-terminal sequence matching that of β-lactoglobulin was identified as being associated with adhesion, through comparisons between cell proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis before and after treatment with proteolytic agents. Further evidence of the involvement of this protein in adhesion was the reduction in adhesion following treatment of the cells with specific antiserum to the 55 kDa "adhesion protein". The presence of the protein on the surface of the cells was demonstrated by immunolabelling. A continuous flow laboratory reactor was developed to generate biofilms of thermo-resistant streptococci on stainless steel surfaces in the presence of skim milk. Trials using biofilms developed in laboratory reactors and on the surface of coupons in pilot plants, indicated that chemicals routinely used in dairy manufacturing plants were inadequate to remove or inactivate thermo-resisant streptococci. Proteolytic enzyme treatments removed more bacteria from the surface than any other treatment, reducing the total number of cells by at least 100-fold. This was confirmed in a pilot-scale trial using a commercial proteolytic-enzyme-based cleaner. In addition, no viable cells were detected following treatment with this cleaner. Proteolytic enzyme cleaners may be more effective than the caustic and acid cleaners for the routine cleaning of biofilms of thermo-resistant streptococci from dairy manufacturing plants.