Comparison of phenotypic and molecular tests to identify lactic acid bacteria.

Twenty-nine lactic acid bacteria (LAB) isolates were submitted for identification using Biolog, API50CHL, 16S rDNA sequencing, and species-specific PCR reactions. The identification results were compared, and it was concluded that a polyphasic approach is necessary for proper LAB identification, being the molecular analyzes the most reliable.

Enumeration of coagulase and thermonuclease-positive Staphylococcus spp. in raw milk and fresh soft cheese: an evaluation of Baird-Parker agar, Rabbit Plasma Fibrinogen agar and the Petrifilm Staph Express count system.

Staphylococcus spp. are microorganisms that are naturally present in milk and dairy products and are often associated with food-borne diseases outbreaks due to the ability of some strains to produce thermostable enterotoxins. This ability is usually associated with coagulase and thermonuclease production, characteristics that are considered in the microbiological analyses for the control of such microorganisms. The objective of this study was to evaluate the culture media and the methodologies used for the enumeration of coagulase and thermonuclease-positive Staphylococcus spp. in raw milk and fresh soft cheese. Samples of artificially contaminated milk (with coagulase-positive Staphylococcus reference strains) and samples of naturally contaminated raw milk and cheese were submitted for enumeration in Baird-Parker agar (BP), Rabbit Plasma Fibrinogen agar (RPFA) and in the Petrifilm Staph Express count system (STX). No significant differences (P > 0.05) were observed between the mean counts obtained in all of the evaluated culture media. RPFA and STX had good correlation indices between the total and typical colony counts as well as with coagulase and the thermonuclease-positive colony counts. Thus, there is a better association between coagulase and thermonuclease production to typical colony morphology developed on these culture media, leading to more accurate and reliable results than with BP, which demonstrated lower correlation indices between these counts.

Microbiological quality and safety of raw milk and soft cheese and detection of autochthonous lactic acid bacteria with antagonistic activity against Listeria monocytogenes, Salmonella Spp., and Staphylococcus aureus.

This study aimed to characterize the microbiological quality and safety of raw milk and soft cheese, verifying possible associations between microbial populations, and the detection of lactic acid bacteria (LAB) with antagonistic activity against foodborne pathogens. Raw milk (n = 36) and soft cheese (n = 18) samples were collected and submitted for the analysis of mesophilic aerobes, total coliforms, Escherichia coli, LAB, coagulase-positive Staphylococcus (CPS), Listeria monocytogenes, and Salmonella spp. In all, 389 LAB isolates were randomly selected and submitted for antagonistic tests against L. monocytogenes, St. aureus, Salmonella Typhimurium, and Lactobacillus sakei. The samples presented high counts of mesophilic aerobes, total coliforms, and LAB, and also high and significant correlation indices between these populations. Low levels of CPS and E. coli were observed, as well as an absence of Salmonella spp. and L. monocytogenes. A substantial portion of the analyzed samples presented LAB cultures with antagonistic activity, but not against Salmonella Typhimurium. The obtained results indicate the antimicrobial potential of the autochthonous microbiota of raw milk and soft cheese. Despite the spoilage potential, the LAB present in the studied food products can be isolated and properly characterized as antagonistic cultures, to be used in bioconservation studies for pathogen control in foods.

Foodborne pathogens and microbiological characteristics of raw milk soft cheese produced and on retail sale in Brazil.

The consumption of raw milk soft cheeses (RMSC), which are typically manufactured in small dairy farms under unsatisfactory hygiene conditions, is common in Brazil. Due to these production characteristics, this type of cheese is a potential carrier of pathogenic microorganisms, such as Listeria monocytogenes, Salmonella, and enterotoxin-producing Staphylococcus spp. Considering these characteristics, in this work, we aimed to detect the presence of these pathogenic microorganisms in RMC and to evaluate their microbiological quality. Fifty-five samples of this product were collected from different noninspected commercial establishments and submitted to the enumeration of mesophilic aerobes (MA), total coliforms (TC), Escherichia coli, and coagulase-positive staphylococci (CPS), and detection of L. monocytogenes and Salmonella spp. All analyzed samples were negative for Salmonella spp. and L. monocytogenes. All samples presented counts of MA higher than 10(6) colony forming units/g (CFU/g; range, 3.0x10(6) to 4.0x10(9)). TC were present at levels between 1.0x10(3) and 1.8x10(8) CFU/g, and E. coli between 1.0x10(2) and 3.5x10(6) CFU/g. CPS were detected in 17 (30.9%) samples at levels higher than 10(4) CFU/g. These results confirm the poor microbiological quality of raw milk used in the manufacturing of RMC samples, and also the inadequate production conditions. Therefore, the evaluation of microbiological safety and quality of these products must be constantly reported to alert the official agencies about the significance of proper inspection.

Lantibiotics biosynthesis genes and bacteriocinogenic activity of Lactobacillus spp. isolated from raw milk and cheese.

Lactobacillus species are usually used as starters for the production of fermented products, and some strains are capable of producing antimicrobial substances, such as bacteriocins. Because these characteristics are highly desirable, research are continually being performed for novel Lactobacillus strains with bacteriocinogenic potential for use by food industries. The aim of this study was to characterise the bacteriocinogenic potential and activity of Lactobacillus isolates. From a lactic acid bacteria culture collection obtained from raw milk and cheese, 27 isolates were identified by 16S rDNA as Lactobacillus spp. and selected for the detection of lantibiotics biosynthesis genes, bacteriocin production, antimicrobial spectra, and ideal incubation conditions for bacteriocin production. Based on the obtained results, 21 isolates presented at least one of the three lantibiotics biosynthesis genes (lanB, lanC or lamM), and 23 isolates also produced antimicrobial substances with sensitivity to at least one proteinase, indicating their bacteriocinogenic activity. In general, the isolates had broad inhibitory activity, mainly against Listeria spp. and Staphylococcus spp. strains, and the best antimicrobial performance of the isolates occurred when they were cultivated at 25 °C for 24 or 48 h or at 35 °C for 12 h. The present study identified the bacteriocinogenic potential of Lactobacillus isolates obtained from raw milk and cheese, suggesting their potential use as biopreservatives in foods.

Práticas de produção aplicadas no controle de contaminação microbiana na produção de leite cru / Producing practices applied on the control of microbiological contamination in raw milk production

A qualidade do leite cru é influenciada diretamente pelas condições de higiene na ordenha earmazenamento, bem como sanidade dos animais. Isso determina que a adoção de Boas Práticas de Produção éfundamental para obtenção de produtos final com baixas contagens microbianas, característica indicativa de boa qualidade. Visando avaliar a eficácia de práticas higiênicas durante o procedimento de ordenha em uma propriedade rural, pontos específicos foram selecionados e amostrados em duas etapas: uma fase inicial, com as práticas do produtor, e uma faseseguinte, em que práticas higiênicas específicas foram adotadas (soluções de cloro para pré-dipping, a 750ppm, e higiene de utensílios, a 300 ppm, além de descarte de primeiros jatos de leite). As amostras foram coletadas em 3 repetições em cada etapa e submetidas a contagens de aeróbios mesófilos (Petrifilm™ AC, 35ºC por 48h), psicrotróficos (superfície de ágar padrão de contagem, 7ºC por 10 dias), coliformes totais e Escherichia coli (Petrifilm™ EC, 35ºC por 48h). Osresultados finais obtidos foram comparados em cada etapa. Na etapa inicial os grupos identificados como principais contaminantes da linha de ordenha foram os aeróbios mesófilos e psicrotróficos, presentes em altos níveis nos tetos e leite dos animais. As práticas adotadas geraram redução da contaminação dos microrganismos indicadores pesquisados em vários pontos da linha de ordenha, indicando a eficácia das soluções de cloro para higienização de tetos dos animais eutensílios de ordenha.

The quality of raw milk is directly influenced by hygiene conditions during milking and storage, and also by animal sanity. Considering these characteristics, Good Producing Practices (GPP) must be adoptedsystematically in milk production in order to obtain final products with low microbial counts, what is indicative of quality. A dairy farm was selected to evaluate the efficiency of some GPP during milking process, when milk and surface samples were collected from specific milking steps in two stages: a first one, when hygienic procedures from the farmers were conducted, and a following one, when specific hygienic procedures were adopted (chlorine solutions for pre-dipping, at 750ppm, and utensils cleaning, at 300ppm, and discard of the forestrip milk). The samples were collected in threerepetitions in each phase and submitted to microbiological analysis of mesophilic aerobes (Petrifilm™ AC, 35ºC for 48h),psychrotrophics (surface of plate count agar, 7ºC for 10 days), total coliforms e Escherichia coli (Petrifilm™ EC, 35ºC for48h). The final results were then compared in each phase. At the initial phase, mesophilic aerobes and psychrotrophics were identified as the main contaminants of milking procedure by their high levels in the teats and milk. The adopted procedures generated reduction of microbial contamination in several sampling points, indicating the efficacy of chlorine solutions for hygiene of teats and milking utensils.

Glucuronidase activity of Escherichia coli isolated from chicken carcasses

To identify Escherichia coli through the production of â-D-glucuronidase (GUD), 622 suspect cultures were isolated from chicken carcasses and plated in PetrifilmTM EC. Of these cultures, only 44 (7.1 percent) failed to produce GUD. This result indicates the usefulness of GUD production for estimating E. coli populations in chicken.