Atividade proteolítica de Pseudomonas fluorescens em biofilmes e detecção das células por anti-soro policlonal

• Autor(a) principal: Machado, Ana Diolina Soares
• Data de Publicação: 2006
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1574
• topic: Pseudomonas fluorescens; Biofilmes; Proteólise em leite; Pseudomonas fluorescens; Biofilms; Proteolytic activity of milk; CNPQ::CIENCIAS AGRARIAS::CIENCIA E TECNOLOGIA DE ALIMENTOS::CIENCIA DE ALIMENTOS

The purposes of this work were to investigate the formation of biofilms from Pseudomonas fluorescens strains isolated from cold raw milk and proteolytic activity in these biofilms. Viability to detect these cells in milk was also investigated through immunological methods. Potential development of biofilms in AISI 304 stainless steel surface, finishing #4 was evaluated under temperatures of 7 ºC and 22 °C. The number of cells from a mixed culture composed of seven P. fluorescens strains adhered to stainless steel plates increased proportionally to the increase of planktonic cell population reaching about 108 UFC.mL-1 after 72 hours of incubation in 12% reconstituted skimmed milk (RSM) at 22 °C. Proteolytic activity verified in RSM supernatant 12% in the presence of stainless steel with adhered cells was higher than in the absence, which suggests that adhered cells, or in biofilms may secrete enzymes to the external medium. The evaluation of cell growth from pure P. fluoresnces cultures 041 and 097 in tryptone, yeast extract, phosphate (TYEP) medium and in RSM 1%, or 12% has indicated that TYEP medium stimulated growth, but not proteolytic activity at 7 ºC and 22 °C. It was verified that adhesion capacity with the development of biofilms is different among strains from the same species. Adhesion capacity with the development of biofilms and proteolytic activity are independent properties in the bacteria tested. P. fluorescens 041 presented low adhesion capacity to stainless steel plates at 7°C and high proteolytic activity whereas the number of P. fluorescens 097 adhered cells was about109 UFC.mL-1 at 7 ºC and 22°C. Proteolytic activity, however, was lower. Biofilm investigation witth scanning electron microscope made clear that P. fluorescens 097 has formed multilayered cell agglomerates on the surface of stainless steel plates. Polyclonal antisera produced in nine rabbits against mixtures of P. fluorescens strains presented different sensivtivity levels, with the capacity to detect cells in concentrations which vary from 103 UFC.mL-1 to 108 UFC.mL-1. Produced antisera have not presented cross-reaction with other contaminating bacterial species from cold raw milk, with the exception of 2A and 3A antisera which reacted with Aeromonas. P. fluorescens cells were detected at RSM 12% in 103 UFC mL-1 and 105 UFC.mL-1 concentrations when drop agglutination and imunodotblot techniques were applied. These results indicate the potential of these procedures to detect these damaging bacteria at a concentration level which does not affect the quality of the milk.