Navegando por Autor "Machado, Solimar Gonçalves"
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Item The biodiversity of the microbiota producing heat-resistant enzymes responsible for spoilage in processed bovine Milk and dairy products(Frontiers in Microbiology, 2017-03-01) Machado, Solimar Gonçalves; Baglinière, François; Marchand, Sophie; Coillie, Els Van; Vanetti, Maria Cristina Dantas; Block, Jan De; Heyndrickx, MarcRaw bovine milk is highly nutritious as well as pH-neutral, providing the ideal conditions for microbial growth. The microbiota of raw milk is diverse and originates from several sources of contamination including the external udder surface, milking equipment, air, water, feed, grass, feces, and soil. Many bacterial and fungal species can be found in raw milk. The autochthonous microbiota of raw milk immediately after milking generally comprises lactic acid bacteria such as Lactococcus, Lactobacillus, Streptococcus, and Leuconostoc species, which are technologically important for the dairy industry, although they do occasionally cause spoilage of dairy products. Differences in milking practices and storage conditions on each continent, country and region result in variable microbial population structures in raw milk. Raw milk is usually stored at cold temperatures, e.g., about 4°C before processing to reduce the growth of most bacteria. However, psychrotrophic bacteria can proliferate and contribute to spoilage of ultra-high temperature (UHT) treated and sterilized milk and other dairy products with a long shelf life due to their ability to produce extracellular heat resistant enzymes such as peptidases and lipases. Worldwide, species of Pseudomonas, with the ability to produce these spoilage enzymes, are the most common contaminants isolated from cold raw milk although other genera such as Serratia are also reported as important milk spoilers, while for others more research is needed on the heat resistance of the spoilage enzymes produced. The residual activity of extracellular enzymes after high heat treatment may lead to technological problems (off flavors, physico-chemical instability) during the shelf life of milk and dairy products. This review covers the contamination patterns of cold raw milk in several parts of the world, the growth potential of psychrotrophic bacteria, their ability to produce extracellular heat-resistant enzymes and the consequences for dairy products with a long shelf life. This problem is of increasing importance because of the large worldwide trade in fluid milk and milk powder.Item Detecção de Pseudomonas fluorescens em leite cru pela reação em cadeia da polimerase(Universidade Federal de Viçosa, 2011-07-15) Machado, Solimar Gonçalves; Bazzolli, Denise Mara Soares; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4761710D6; Araujo, Elza Fernandes de; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783675E2; Vanetti, Maria Cristina Dantas; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783874H3; http://lattes.cnpq.br/8037317132459423; Pinto, Cláudia Lúcia de Oliveira; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4783521J6; Martins, Maurilio Lopes; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794520A8; Silveira, Wendel Batista da; http://lattes.cnpq.br/7361036485940798Entre os micro-organismos psicrotróficos que são capazes de crescer durante a refrigeração, alguns produzem proteases termorresistentes relacionadas com problemas tecnológicos nos derivados lácteos. Visto que a demanda por métodos que permitem alta taxa de processamento exigida em indústrias de alimentos para controle da qualidade de leite cru é crescente, a avaliação de métodos mais sensíveis e mais rápidos para detecção de psicrotróficos, especialmente Pseudomonas fluorescens, espécie predominante, é de grande importância. Com a finalidade de estabelecer um protocolo para detecção de P. fluorescens pela reação em cadeia da polimerase (PCR), foram avaliados cinco métodos de extração de DNA total do leite, dois genes alvos na PCR e o limite de detecção dos métodos para análise de leite cru e leite esterilizado e inoculado com P. fluorescens. O kit comercial e o método de filtração modificado foram os métodos mais adequados para extração de DNA total de amostras de leite, por isso, foram utilizados nas análises seguintes para detecção dos genes alvos. No entanto, a utilização do método de filtração modificado para análise de leite inoculado com P. fluorescens e leite cru resultou em um menor limite de detecção quando comparado com o kit comercial. Por meio da amplificação do gene alvo rDNA 16S, foi possível observar um amplificado de 850 pb quando a população do leite inoculado com P. fluorescens era da ordem de 102 UFC/ml. Em amostras de leite cru, foi possível detectar o produto quando a contagem de Pseudomonas era da ordem de 106 UFC/ml e a população de psicrotróficos estava entre 107 e 109 UFC/ml. A análise do grau de proteólise das amostras de leite cru por eletroforese em gel de poliacrilamida demonstrou que a relação entre a população de psicrotróficos e a degradação das proteínas do leite depende de outros fatores além da contagem de psicrotróficos.Item Identificação de bactérias psicrotróficas proteolíticas isoladas de leite cru refrigerado e caracterização do seu potencial deteriorador(Revista do Instituto Laticínios Cândido Tostes, 2015-05-25) Machado, Solimar Gonçalves; Pinto, Cláudia Lúcia de Oliveira; Martins, Maurilio Lopes; Vanetti, Maria Cristina DantasOs objetivos deste estudo foram identificar bactérias psicrotróficas proteolíticas isoladas de leite cru refrigerado, avaliar o seu poder deteriorador e a sua capacidade de adesão em superfícies de aço inoxidável sob temperaturas de refrigeração. Dentre as bactérias gram-positivas, 23,81% e 33,33% dos isolados pertenciam aos gêneros Bacillus e Paenibacillus respectivamente. No grupo de bactérias gram-negativas fermentadoras de glicose foram identificados os gêneros Cedecea, Enterobacter, Hafnia, Klebsiella, Pantoea, Providencia, Rhanella e Serratia e dentre as bactérias gram-negativas não-fermentadoras, os gêneros Acinetobacter,Aeromonas, Burkholderia, Chryseobacteriun (Flavobacterium), Chryseomonas, Moraxella e Pseudomonas. O gênero Pseudomonas foi predominante entre as bactérias identificadas e representou 33,98% do total de isolados. Dentre as bactérias fermentadoras de glicose 14,6%, 15,3% e 20,8% dos isolados apresentaram predominantemente atividade proteolítica a 6,5 ºC, 21 ºC e 35 ºC, respectivamente, enquanto as bactérias gram-negativas não-fermentadoras apresentaram 25% e 23,6% dos isolados com atividades associadas de proteases, lipases e lecitinases a 6,5 ºC e 21 ºC, respectivamente, o que indicou maior potencial deteriorador. Em espécies de bactérias gram-negativas e gram-positivas, constatou-se a produção de enzimas termorresistentes e adesão à superfície de aço inoxidável a 6,5 ºC. Os resultados deste estudo reforçaram a importância da implementação das boas práticas de produção e de fabricação na cadeia produtiva do leite para fins de prevenção de sua deterioração e minimização de perdas de qualidade e econômicas.Item Performance of two alternative methods for Listeria detection throughout Serro Minas cheese ripening(Brazilian Journal of Microbiology, 2016-04-22) Mata, Gardênia Márcia Silva Campos; Martins, Evandro; Machado, Solimar Gonçalves; Vanetti, Maria Cristina Dantas; Pinto, Maximiliano Soares; Carvalho, Antônio Fernandes deThe ability of pathogens to survive cheese ripening is a food-security concern. Therefore, this study aimed to evaluate the performance of two alternative methods of analysis of Listeria during the ripening of artisanal Minas cheese. These methods were tested and compared with the conventional method: Lateral Flow System™, in cheeses produced on laboratory scale using raw milk collected from different farms and inoculated with Listeria innocua; and VIDAS®-LMO, in cheese samples collected from different manufacturers in Serro, Minas Gerais, Brazil. These samples were also characterized in terms of lactic acid bacteria, coliforms and physical–chemical analysis. In the inoculated samples, L. innocua was detected by Lateral Flow System™ method with 33% false-negative and 68% accuracy results. L. innocua was only detected in the inoculated samples by the conventional method at 60-days of cheese ripening. L. monocytogenes was not detected by the conventional and the VIDAS®-LMO methods in cheese samples collected from different manufacturers, which impairs evaluating the performance of this alternative method. We concluded that the conventional method provided a better recovery of L. innocua throughout cheese ripening, being able to detect L. innocua at 60-day, aging period which is required by the current legislation.Item Proteolytic potential of pseudomonas fluorescens isolated from refrigerated raw milk(Revista Brasileira de Agropecuária Sustentável, 2014-12-22) Machado, Solimar Gonçalves; Pinto, Cláudia Lúcia Oliveira; Cardoso, Rodrigo Rezende; Alves, Rita Maria; Vanetti, Maria Cristina DantasThe growth rate and the proteolytic activity of Pseudomonas fluorescens strains 07A and 041, isolated from cow’s milk, were evaluated at 2, 4, 7 and 10ºC. P. fluorescens promoted protein degradation during storage of milk samples as observed by Proteolytic activity measurement, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and heat stability of milk. Casein hydrolysis resulted in loss of thermal stability of milk and in formation of fragments of low and medium molecular mass. Temperatures up to 10°C did notItem Sedimentação, atividade proteolítica e proteólise de leite UHT integral durante o armazenamento(Revista do Instituto Laticínios Cândido Tostes, 2017-05-17) Pinto, Cláudia Lúcia de Oliveira; Machado, Solimar Gonçalves; Cardoso, Rodrigo Rezende; Vanetti, Maria Cristina DantasA desestabilização do leite UHT durante a estocagem pode ser atribuída, entre outros fatores, à ação de proteases endógenas e, ou bacterianas e constitui um dos principais entraves na cadeia produtiva desse produto. Objetivou-se avaliar a qualidade microbiológica do leite cru refrigerado e seu efeito sobre a formação de sedimentos, grau de proteólise e atividade proteolítica de leite UHT integral durante o período de armazenamento. As médias das contagens de bactérias mesófilas, psicrotróficas, psicrotróficas proteolíticas e Pseudomonas spp. das amostras do leite cru refrigerado foram, respectivamente, de 5,5 x 106 UFC/mL, 3,0 x 106 UFC/mL, 8,0 x 105 UFC/mL e 1,1 x 106 UFC/mL. O tratamento UHT reduziu 93,2% da atividade proteolítica em relação ao leite cru. Ao longo do período de estocagem do leite UHT integral ocorreu o aumento da massa de sedimentos, do grau de proteólise e da atividade proteolítica. Todas as amostras de leite UHT apresentaram baixa contagem de mesófilos e permaneceram termicamente estáveis e sem sinais de gelificação. Considerando a atividade residual de proteases no leite UHT e a sua relação com a formação de sedimentos, atividade proteolítica e proteólise, o emprego de leite com baixas contagens de bactérias psicrotróficas e de bactérias psicrotróficas proteolíticas deve ser considerado para fins de prevenção de defeitos tecnológicos como sedimentação e gelificação, os quais reduzem, consideravelmente, a aceitação do produto pelo consumidor além de ocasionarem prejuízos à indústria.Item Serratia and Pseudomonas as important heat-resistant protease producers in cold raw milk(Universidade Federal de Viçosa, 2015-07-17) Machado, Solimar Gonçalves; Vanetti, Maria Cristina Dantas; http://lattes.cnpq.br/8037317132459423The storage of raw milk at cold temperatures does not prevent growth of psychrotrophic bacteria, which can produce heat-resistant proteases that compromise the quality and reduce the shelf life of dairy products. To minimize the technological problems resulting from proteolytic activity, these enzymes should be detected and quantified in raw milk before processing by a rapid method. However, there is no efficient method for this purpose. This work aimed to identify the predominant psychrotrophic species with spoilage potential in Brazilian raw milk, to identify and characterize the heat-resistant proteases produced by this microbiota and to developed a rapid method to detect these proteases in raw milk samples. The cold storage conditions were simulated and the psychrotrophic proteolytic bacteria isolated. The heat-resistant protease-producing bacteria were typing by Rep-PCR and clustered. Biochemical tests, 16S rDNA and rpoB gene sequencing were used for identifying one representative isolate from each cluster. The heat-resistant protease produced by Serratia liquefaciens was characterized. The encoding gene was identified after mass spectrometry analysis of tryptic peptides from the heat-resistant protease. The biotinylated casein was coated on microtiter plates and used as substrate to quantify proteolytic activity in solution and milk samples. Highly proteolytic strains were identified and characterized. The isolates were separated into eight different clusters and four solitary fingerprints. The most proteolytic isolates belonged to Serratia liquefaciens and Pseudomonas species. The S. liquefaciens isolates may produce Ser2, which is a a metalloprotease resistant to the heat treatment of 95 °C for 8.45 min. This metalloprotease showed a molecular weight of approximately, 52 kDa and a heat- resistance similar to AprX from Pseudomonas spp. Although nucleotide sequences of ser2 gene were conserved among S. liquefaciens isolates, the spoilage potential among them was heterogeneous indicating differences in enzyme expression levels or post-transcriptional modifications. The developed immunoassay was efficient for quantification of trypsin, papain, pepsin, thermolysin and protease from bovine x pancreas activity. However, further research should be performed to minimize the influence of milk components on the developed assay for detecting proteases in milk samples. This work highlighted the poor conditions of hygiene in milk farms and the high spoilage potential of the microbiota found in raw milk samples besides the development of a promising assay for detection and quantification of proteolytic activity useful for dairy industry.Item Treatment of soy milk with Debaryomyces hansenii cells immobilised in alginate(Food Chemistry, 2009-05-15) Souza Júnior, Waldeck Campanha de; Rezende, Sebastião Tavares de; Viana, Pollyanna Amaral; Falkoski, Daniel Luciano; Reis, Angélica Pataro; Machado, Solimar Gonçalves; Barros, Everaldo Gonçalves de; Guimarães, Valéria MontezeWhole cells of Debaryomyces hansenii UFV-1 were permeabilised with ethanol and immobilised in calcium alginate hydrogel. The optimum pH and temperature for α-galactosidase activities of permeabilised free (PFC) and permeabilised immobilised cells (PIC) were 4.5 and 60 °C; and 4.0 and 70 °C, respectively. PIC α-galactosidase was more stable than that of PFC. The incubation of PIC at 60 and 70 °C promoted an increase in α-galactosidase activity. The α-galactosidase activity was maintained when PIC was used in three repeated batches. The Km values for PIC and PFC α-galactosidases, with ρNPαGal, were 0.82 mM and 0.30 mM, respectively. Soy milk treatment with PIC for 6 h at 60 °C promoted 100% hydrolysis of raffinose oligosaccharides.Item α-Galactosidases production by Debaryomyces hansenii UFV-1(Food Science and Biotechnology, 2011-06-30) Viana, Pollyanna Amaral; Rezende, Sebastião Tavares de; Passos, Flávia Maria Lopes; Machado, Solimar Gonçalves; Maitan, Gabriela Picollo; Coelho, Vinicio Tadeu da Silva; Guimarães, Valéria MontezeExtracellular and intracellular α-galactosidases were produced by yeast Debaryomyces hansenii UFV-1 grown on different media with several carbon sources. D. hansenii grown in YP-medium (1% yeast extract and 2% peptone) presented maximum cell mass (8.45 mg/mL) after 36 h of cultivation, with lactose as carbon source, followed by sucrose, glucose, raffinose, and galactose. Higher extracellular and intracellular α-galactosidases activities were observed at 48 h of D. hansenii cultivation in YPmedium containing galactose (0.97 and 5.27 U/mL) and lactose (1.28 and 4.88 U/mL), supporting the evidence for the model of induction for the yeast GAL/MEL regulon, such as described in Sacharomyces cereviseae.