Navegando por Autor "Miranda, Rodrigo Otávio"
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Item Desenvolvimento do meio de cultura Rafinose-Propionato Mupirocina de Lítio, seletivo para bifidobactérias, e avaliação de sua associação com o PetrifilmTM Aerobic Count(Universidade Federal de Viçosa, 2013-03-22) Miranda, Rodrigo Otávio; Nero, Luís Augusto; http://lattes.cnpq.br/7496821877571187Os leites fermentados são ideais carreadores de micro-organismos probióticos devido a seu histórico de consumo e fabricação. A aplicação de bifidobactérias em produtos lácteos probióticos é geralmente associada a outras bactérias fermentadoras, com objetivos de garantir a qualidade tecnológica e sensorial do produto. A microbiota mista dos leites fermentados deve ser analisada diferencialmente com uso de meios seletivos para garantir a viabilidade e concentração de células. Metodologias alternativas rápidas para enumeração de micro-organismos, como o PetrifilmTM, são vantajosas para a indústria de alimentos, porém requerem prévia padronização. O objetivo desse trabalho foi adaptar meios seletivos para bifidobactérias com plaqueamento em PetrifilmTM AC. Cepas de bifidobactérias, lactobacilos e estreptococos foram pesquisadas em relação ao perfil de fermentação de carboidratos, redução e inibição por cloreto de 2,3,5- trifeniltetrazólio (TTC) e desenvolvimento em meios seletivos para bifidobactérias. Adicionalmente, o meio de cultura Rafinose-Propionato-MUP (RP-MUP) com plaqueamento convencional e em PetrifilmTM AC com diferentes tempos de incubação foi comparado com a metodologia da ISO29981 com meio TOS-MUP em leites fermentados produzidos. A rafinose foi o carboidrato que apresentou maior especificidade de fermentação por bifidobactérias, sendo considerada a fonte de carbono a ser utilizada no desenvolvimento do meio de cultura alternativo RP-MUP. Todas as cepas de Bifidobacterium spp. conseguiram reduzir adequadamente o TTC, e não foram inibidas nas concentrações 25, 50 e 100 mg/L. TOS-MUP e RP-MUP foram os meios de cultura que apresentaram melhor seletividade para bifidobactérias, quando comparadas aos meios MRS-ABC e MRS-NNLP. A associação do RP-MUP a placas PetrifilmTM AC permitiu a enumeração seletiva de bifidobactérias em leites fermentados, com equivalência de resultados com o RP-MUP e TOS-MUP utilizados pela metodologia convencional de plaqueamento (ANOVA, p < 0.05), e altos índices de correlação (r = 0.99, p < 0.05), inclusive em um tempo inferior de incubação (48 h). O protocolo alternativo proposto para enumeração de bifidobactérias em leites fermentados apresentou desempenho adequado, e representa uma vantagem para utilização pelas indústrias de alimentos no controle de qualidade de produtos probióticos com esses micro-organismos.Item Development of a selective culture medium for bifidobacteria, Raffinose-Propionate Lithium Mupirocin (RP-MUP) and assessment of its usage with PetrifilmÔ Aerobic Count plates(Food Microbiology, 2013-11-26) Miranda, Rodrigo Otávio; Carvalho, Antonio Fernandes de; Nero, Luís AugustoThis study aimed to develop a selective culture media to enumerate bifidobacteria in fermented milk and to assess this medium when used with PetrifilmÔ AC plates. For this purpose, Bifidobacterium spp., Lactobacillus spp. and Streptococcus thermophilus strains were tested to verify their fermentation patterns for different carbohydrates. All bifidobacteria strains were able to use raffinose. Based on these characteristic, a selective culture medium was proposed (Raffinose-Propionate Lithium Mupirocin, RP-MUP), used with PetrifilmÔ AC plates, and was used to enumerate bifidobacteria in fermented milk. RP-MUP performance was assessed by comparing the results with this medium to reference protocols and culture media for bifidobacteria enumeration. RP-MUP, whether used or not with PetrifilmÔ AC, presented similar performance to TOS-MUP (ISO 29981), with no significant differences between the mean bifi dobacteria counts (p < 0.05) and with high correlation indices (r 1⁄4 0.99, p < 0.05). As an advantage, reliable results were obtained after just 48 h of incubation when RP-MUP was used with PetrifilmÔ AC, instead of the 72 h described in the ISO 29981 protocol.Item Development of a selective culture medium for bifidobacteria, Raffinose-Propionate Lithium Mupirocin (RP-MUP) and assessment of its usage with Petrifilm™ Aerobic Count plates(Food Microbiology, 2014-05) Miranda, Rodrigo Otávio; Carvalho, Antonio Fernandes de; Nero, Luís AugustoThis study aimed to develop a selective culture media to enumerate bifidobacteria in fermented milk and to assess this medium when used with Petrifilm™ AC plates. For this purpose, Bifidobacterium spp., Lactobacillus spp. and Streptococcus thermophilus strains were tested to verify their fermentation patterns for different carbohydrates. All bifidobacteria strains were able to use raffinose. Based on these characteristic, a selective culture medium was proposed (Raffinose-Propionate Lithium Mupirocin, RP-MUP), used with Petrifilm™ AC plates, and was used to enumerate bifidobacteria in fermented milk. RP-MUP performance was assessed by comparing the results with this medium to reference protocols and culture media for bifidobacteria enumeration. RP-MUP, whether used or not with Petrifilm™ AC, presented similar performance to TOS-MUP (ISO 29981), with no significant differences between the mean bifidobacteria counts (p < 0.05) and with high correlation indices (r = 0.99, p < 0.05). As an advantage, reliable results were obtained after just 48 h of incubation when RP-MUP was used with Petrifilm™ AC, instead of the 72 h described in the ISO 29981 protocol.Item Enumeration of bifidobacteria using Petrifilm™ AC in pure cultures and in a fermented milk manufactured with a commercial culture of Streptococcus thermophilus(Food Microbiology, 2011-07-21) Miranda, Rodrigo Otávio; Gama Neto, Gabriel; Freitas, Rosangela de; Carvalho, Antônio Fernandes de; Nero, Luís AugustoBifidobacteria are probiotic microorganisms that are widely used in the food industry. With the aim of using of Petrifilm™ Aerobic Count (AC) plates associated with selective culture media, aliquots of sterile skim milk were inoculated separately with four commercial cultures of bifidobacteria. These cultures were plated by both the conventional method and Petrifilm™AC, using the culture media NNLP and ABC. The cultures were incubated under anaerobiosis at 37 °C for 24, 48 and 72 h. No significant differences (p > 0.05) were observed between the obtained counts at 48 and 72 h. Bifidobacteria counts in ABC were usually higher than in NNLP, independent of the plating method. Subsequently, fermented milk was prepared with a Streptococcus thermophilus strain, and aliquots were inoculated with the same bifidobacteria. Then, the fermented milks were submitted to microbiological analysis for bifidobacteria enumeration using the same culture media and methodologies previously described, incubated under anaerobiosis at 37 °C for 48 h. Again, bifidobacteria counts in ABC were higher than in NNLP, with significant differences for some cultures (p < 0.05). The counts obtained by both methodologies presented significant correlations (p < 0.05). The results indicate the viability of Petrifilm™AC as an alternative method for bifidobacteria enumeration when associated to specific culture media, specially the ABC.Item Enumeration of bifidobacteria using PetrifilmTM AC in pure cultures and in a fermented milk manufactured with a commercial culture of Streptococcus thermophilus(Food Microbiology, 2011-07-21) Miranda, Rodrigo Otávio; Freitas, Rosangela de; Carvalho, Antônio Fernandes de; Nero, Luís Augusto; Gama Neto, GabrielBifidobacteria are probiotic microorganisms that are widely used in the food industry. With the aim of using of PetrifilmÔ Aerobic Count (AC) plates associated with selective culture media, aliquots of sterile skim milk were inoculated separately with four commercial cultures of bifidobacteria. These cultures were plated by both the conventional method and PetrifilmÔAC, using the culture media NNLP and ABC. The cultures were incubated under anaerobiosis at 37 C for 24, 48 and 72 h. No significant differences (p > 0.05) were observed between the obtained counts at 48 and 72 h. Bifidobacteria counts in ABC were usually higher than in NNLP, independent of the plating method. Subsequently, fermented milk was prepared with a Streptococcus thermophilus strain, and aliquots were inoculated with the same bifidobacteria. Then, the fermented milks were submitted to microbiological analysis for bifidobacteria enumeration using the same culture media and methodologies previously described, incubated under anaerobiosis at 37 C for 48 h. Again, bifidobacteria counts in ABC were higher than in NNLP, with significant differences for some cultures (p < 0.05). The counts obtained by both methodologies presented significant correlations (p < 0.05). The results indicate the viability of PetrifilmÔAC as an alternative method for bifidobacteria enumeration when associated to specific culture media, specially the ABC.Item Expressão de genes associados a condições de estresse por Listeria monocytogenes em interação com Lactococcus lactis produtor de nisina(Universidade Federal de Viçosa, 2017-06-26) Miranda, Rodrigo Otávio; Nero, Luís Augusto; http://lattes.cnpq.br/7496821877571187A utilização de cepas fermentadoras de Lactococcus lactis subsp. lactis produtoras de nisina em alimentos fermentados tem vantagem para a indústria pois permite um controle adicional de contaminantes, como o patógeno de origem alimentar Listeria monocytogenes. No entanto, as interações microbianas devem ser avaliadas para garantir a produção da bacteriocina no alimento e o efeito na população do patógeno. L. monocytogenes tem a capacidade de resistir a diversas condições de estresse encontradas no alimento e durante o processamento, expressando diferentes genes como o fator siga alternativo (sigB), a enzima glutamato descarboxilase (gadD), a chaperona GroEL e o transportador de glicina betaína (gbu). A exposição a uma condição de estresse em nível subletal é capaz de conferir uma maior resistência a L. monocytogenes de sobreviver a outras situações. Nesse contexto, o objetivo deste trabalho foi avaliar a expressão de genes de estresse de L. monocytogenes em interação com L. lactis subsp. lactis produtor de nisina em meio de cultura e leite. A produção de nisina em caldo BHI e leite foi avaliada por sua detecção no sobrenadante do meio de crescimento e pela expressão do gene nisK. A expressão dos genes de estresse de L. monocytogenes sigB, gadD2, groEL e gbu foi avaliada relativamente a cultura pura e na interação com L. lactis subsp. lactis. A expressão relativa dos genes de estresse de L. monocytogenes foi variável. No entanto, a expressão dos genes sigB, groEL e gbu foi inferior no tempo de 24 h durante a interação, em relação a cultura pura, o que pode indicar uma menor capacidade de sobrevivência aos estresses quando a bactéria se encontra em interação com L. lactis subsp. lactis produtor de nisina.Item Expression of genes associated with stress conditions by Listeria monocytogenes in interaction with nisin producer Lactococcus lactis(Food Research International, 2017-12-13) Miranda, Rodrigo Otávio; Campos-Galvão, Maria Emilene Martino; Nero, Luís AugustoThe use of nisin producers in foods is considered a mitigation strategy to control foodborne pathogens growth, such as Listeria monocytogenes, due to the production of this bacteriocin in situ. However, when the bacteriocin does not reach an adequate concentration, the target bacteria can develop a cross-response to different stress conditions in food, such as acid, thermal and osmotic. This study aimed to evaluate the interaction of a nisin-producing strain of Lactococcus lactis DY-13 and L. monocytogenes in BHI and skim milk, and its influence on general (sigB), acid (gadD2), thermal (groEL) and osmotic (gbu) stress-related genes of the pathogen. L. monocytogenes populations decreased approximately 2 log in BHI and 1 log in milk after 24 h in co-culture with the nisin producer L. lactis, coherent with the increasing expression of nisK. Expression of stress-related genes by L. monocytogenes presented lower oscillation in BHI than in milk, indicating its better ability to survive in milk, despite the higher nisin production. Stress-related genes presented a varied expression by L. monocytogenes in the tested conditions: sigB expression remained stable or reduced over time; gadD2 presented high expression in milk; groEL presented low expression in BHI when compared to milk, trending to decrease overtime; gbu expression in milk after 24 h was lower than in BHI. The presented study demonstrated the growth of a nisin producer L. lactis can affect the expression of stress-related genes by L. monocytogenes, and understating these mechanisms is crucial to enhance the conservation methods employed in foods.Item Virulence, antibiotic resistance and biogenic amines of bacteriocinogenic lactococci and enterococci isolated from goat milk(International Journal of Food Microbiology, 2014-06-12) Perin, Luana Martins; Miranda, Rodrigo Otávio; Todorov, Svetoslav Dimitrov; Franco, Bernadette Dora Gombossy de Melo; Nero, Luís AugustoThe present study aimed to investigate the virulence, antibiotic resistance and biogenic amine production in bacteriocinogenic lactococci and enterococci isolated from goat milk in order to evaluate their safety. Twenty-nine bacteriocinogenic lactic acid bacteria (LAB: 11 Lactococcus spp., and 18 Enterococcus spp.) isolated from raw goat milk were selected and subjected to PCR to identify gelE, cylA, hyl, asa1, esp, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc genes. The expression of virulence factors (gelatinase, hemolysis, lipase, DNAse, tyramine, histamine, putrescine) in different incubation temperatures was assessed by phenotypic methods, as well as the resistance to vancomycin, gentamicin, chloramphenicol, ampicillin and rifampicin (using Etest®). The tested isolates presented distinct combinations of virulence related genes, but not necessarily the expression of such factors. The relevance of identifying virulence-related genes in bacteriocinogenic LAB was highlighted, demanding for care in their usage as starter cultures or biopreservatives due to the possibility of horizontal gene transfer to other bacteria in food systems.