Implantação de íons de prata em aço inoxidável e infecção fágica para o controle de adesão e formação de biofilmes bacterianos na indústria de alimentos

• Ano de defesa: 2012
• Autor(a) principal: Novello, Junia Cápua de Lima
• Orientador(a): Andrade, Nélio José de
• Banca de defesa: Pinto, Cláudia Lúcia de Oliveira , Carvalho, Antônio Fernandes de , Azeredo, Joana Cecília Valente de Rodrigues
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Ciência e Tecnologia de Alimentos
• Departamento: Ciência de Alimentos; Tecnologia de Alimentos; Engenharia de Alimentos
• País: BR
• Palavras-chave em Português: Adesão bacteriana; Biofilme; Rugosidade; Tensão interfacial; Implantação de íons
• Palavras-chave em Inglês: Bacterial adhesion, biofilm; Roughness; interfacial tension; Ion Implantation
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::CIENCIA E TECNOLOGIA DE ALIMENTOS::CIENCIA DE ALIMENTOS
• Link de acesso: http://locus.ufv.br/handle/123456789/466

The bacterial adhesion and biofilm formation on food contact surfaces can lead to the increased in corrosion of equipment surfaces and pipes blockage. Moreover, it can also cause foodborne illness, which results in economic losses, and, especially, in public health problems. The aims of this work were to evaluate the adhesion of Listeria monocytogenes CECT 4031T on the austenitic stainless steel (AISI 304) and ferritic (AISI 430) surfaces implanted with Ag+1 ions; study the biofilm formation of L. monocytogenes (strains 747 and 994) in single and mixed culture with cells of Pseudomonas fluorescens ATCC 27663 on austenitic stainless steel surfaces, AISI 304, finishing #4, in the biofilm and; study the biocontrol of L. monocytogenes 747 in mixed culture with P. fluorescens ATCC 27663 by infection with bacteriophage &#981;IBB PF7A. Ag+1 ions were implanted in austenitic stainless steel, AISI 304, and ferritic surfaces, AISI 430, finishing #2B, 4, 6 and 8, with the energy of 200 keV, and austenitic stainless steel, AISI 304, finish #2B, with the energy of 70 keV. The depth of Ag+1 ions, chemical composition, hydrophobicity (qualitative and quantitative), and roughness for stainless steel surfaces were evaluated by Rutherford backscattering spectrometry (RBS) spectroscopy, X-ray photoelectron (XPS), contact angle and atomic force microscopy, respectively. Qualitatively and quantitatively hydrophobicity for bacterial strains, L. monocytogenes and P. fluorescens, were also evaluated. The technique of fluorescence in situ hybridization of peptide nucleic acids (PNA Fish) was used to study the biofilm formation ability of L. monocytogenes 747 in single or mixed culture with P. fluorescens, before or after infection with bacteriophage- &#981;IBBPF7A. The results showed that Ag+1 ions implanted with energy of 200 keV presented an average depth of 25 nm and, when implanted with energy of 70 keV, these ions were found shallower, with an average depth of 15 nm. Additionally, the XPS analyzes confirmed the presence of silver in the implanted surfaces. There was significant difference (p < 0.05) between the roughness surfaces of stainless steel (Ra and Rz). Ferritic stainless steel, AISI 430, finishing # 4, with Ag+1 ions showed highest roughness and ferritic stainless steel, AISI 430, finishing #8, with and without Ag+1 lowest. After implantation of Ag+1, it was possible to observe a significant reduction on values of the contact angles with water (&#952;w) (p < 0.05) and alterations on the free energy of interaction between identical surfaces immersed in water (&#916;G TOT SWS). Ferritic stainless steel, AISI 430, finishing #8, without Ag+1 ions implanted, showed highest values of hydrophobicity (- 64.67 mJ.m-2). The number of L. monocytogenes CECT 4031T cells adhered proved to be materials dependents (p < 0.05), with lowest values for stainless steel, AISI 304, finishing #2B, with silver implanted at 70 keV (2.63 ± 0.32 log CFU.cm-2). Furthermore, there was no correlation between the number of adhered cells of L. monocytogenes CECT 4031T and the variation of the total free energy of interaction (&#916;G). Important is to emphasize that no direct relationship was observed between the number of adhered cells and the roughness of the surfaces of stainless steel. L. monocytogenes and P. fluorescens cells shown to be hydrophilic, and the greatest variation in total free energy of interaction was observed for P. fluorescens (- 27.91 mJ.m-2). L. monocytogenes 747 and 994, in single culture, were able to adhere at 4 °C and formed biofilms at 25 °C and 37 ºC in stainless steel AISI 304, finishing #4. Moreover, there was significant difference (p < 0.05) in the number of viable cells in single biofilms of P. fluorescens under the tested temperatures measured with scores of 7,26 log CFU.cm-2 ,at 25 ºC, and 5,26 log CFU.cm-2, at 37 ºC. The number of viable bacteria cells measured in planktonic forms or single biofimes decreased (p < 0.05) after contact with the &#981;IBB-PF7A bacteriophage, reaching 5.5 log reduction, after 6 h, and 2.03 log reduction, after 24 h. In mixed biofilms with L. monocytogenes 747, it was also observed a reduction in the number of P. fluorescens viable cells (p < 0.05), after 6 h with phage contact, reaching 5.6 log reduction. However, after 24 h, the reduction in the number of P. fluorescens cells was not significant (p > 0.05) (2.19 log reduction). From this time, there was an increase of population, which can be attributed to bacterial resistance to phage, possibly initiated during planktonic cells lifestyle. Epifluorescence microscopy images allow us to confirm that both L. monocytogenes 747 and P. fluorescens were able to form biofilms on glass surfaces in single and mixed culture at 25 °C. It was also observed that the structure of biofilm of P. fluorescens and L. monocytogenes 747 consists of a monolayer of cells with a mixture of both microorganisms. Important is to highlight that the Ag+1 ion implantation is a causative agent able for reducting the number of adhered cells of L. monocytogenes to stainless steel. In the initial time, phage infection was able to cause a reduction on the number of adhered cells of L. monocytogenes and P. fluorescens in biofilms. So, this work presents itself as an important step in researching new technologies that can be employed to reduce or prevent bacterial adhesion and biofilms formation.