a b s t r a c tThe objective of this study was to evaluate culture media and methodologies for isolation and detection of lactic acid bacteria (LAB) capable to produce bacteriocin-like substances. Samples of milk and cheese were pour plated on de Mann-Rogosa-Sharpe agar (MRS) and Kang-Fung-Sol agar (KFS) (both at 35 C/48 h, under anaerobiosis), from which 389 and 256 LAB cultures were selected. The antagonistic activity of them was evaluated using the spot-on-the-lawn and two culture media: brain-heart infusion agar with catalase (BHI þ C) and M17 (both at 35 C/24 h). The proteinaceous nature of the antagonistic cultures was verified using: spot-on-the-lawn (MRS, 25 C/24 h, under anaerobiosis) and well-diffusion (cultures amplified on modified MRS broth at 25 C/24 h, and then neutralized using NaOH). Listeria monocytogenes ATCC 7644 was used as indicator. A larger number of antagonist cultures were isolated from MRS (83 by M17 and 65 by BHI þ C) in comparison to KFS (24 by M17 and 15 by BHI þ C). The spot-onthe-lawn identified a higher frequency of LAB capable of producing bacteriocin-like substances. MRS was considered to be the best culture media for the isolation of LAB capable to produce bacteriocin-like substances, activity that was better identified using the spot-on-the-lawn methodology.

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.

Lactic acid bacteria (LAB) are currently used by food industries because of their ability to produce metabolites with antimicrobial activity against gram-positive pathogens and spoilage microorganisms. The objectives of this study were to identify naturally occurring bacteriocinogenic or bacteriocinogenic-like LAB in raw milk and soft cheese and to detect the presence of nisin-coding genes in cultures identified as Lactococcus lactis. Lactic acid bacteria cultures were isolated from 389 raw milk and soft cheese samples and were later characterized for the production of antimicrobial substances against Listeria monocytogenes. Of these, 58 (14.9%) LAB cultures were identified as antagonistic; the nature of this antagonistic activity was then characterized via enzymatic tests to confirm the proteinaceous nature of the antimicrobial substances. In addition, 20 of these antagonistic cultures were selected and submitted to genetic sequencing; they were identified as Lactobacillus plantarum (n=2) and Lactococcus lactis ssp. lactis (n=18). Nisin genes were identified by polymerase chain reaction in 7 of these cultures. The identified bacteriocinogenic and bacteriocinogenic-like cultures were highly variable concerning the production and activity of antimicrobial substances, even when they were genetically similar. The obtained results indicated the need for molecular and phenotypic methodologies to properly characterize bacteriocinogenic LAB, as well as the potential use of these cultures as tools to provide food safety.

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Em 2005 o Brasil produziu 23.320.000 toneladas de leite, o que posicionou o país como sétimo maior produtor mundial e gerou um lucro de 12.572 milhões de reais (EMBRAPA GADO DE LEITE, 2007). Esses valores expressivos mostram a relevância da atividade leiteira para a economia brasileira, tanto no cenário interno como no externo. Como consequência, a produção leiteira no Brasil vem crescendo nos últimos anos em conjunto com a exportação, revelando seu grande potencial de desenvolvimento e consolidação como produto de importância na economia nacional (EMBRAPA GADO DE LEITE, 2007).As características da produção leiteira no Brasil são os principais fatores que impedem um desenvolvimento mais acelerado dessa atividade. De forma geral, a maior parte dos produtores pode ser classificada como pequenos ou médios, com produção diária de 50 a 100 L e de caráter familiar (EMBRAPA GADO DE LEITE, 2007;BRITO et al., 2004). Como consequência ocorre pouco investimento na atividade, resultando em problemas em toda a cadeia produtiva, como baixa tecnificação, falta de controle sanitário dos animais e condições higiênicas inadequadas durante a ordenha, conservação e transporte (SANTOS;FONSECA, 2007;VALEEVA et al., 2005). Os reflexos dessas deficiências da produção leiteira são observados na baixa produtividade do rebanho nacional (apenas 1.219 L/vaca/ano em 2005, EMBRAPA GADO DE LEITE, 2007) e na baixa qualidade do leite produzido.A baixa qualidade do leite cru é notoriamente conhecida em todo o território nacional, e como consequência resulta em produtos beneficiados de qualidade insatisfatória (ARCURI et al., 2006;ROCHA et al., 2006;NERO et al., 2005;NERO et al., 2004; GUIMARÃES, 2002;SILVA et al., 2001;BELOTI et al., 1999;SILVEIRA et al., 1989). O principal parâmetro utilizado para se verificar a qualidade desse produto é o seu perfil microbiológico, determinado principalmente pela forma de obtenção, armazenamento e transporte. Grupos específicos de microrganismos são pesquisados para esse fim, como os aeróbios mesófilos, coliformes e psicrotróficos (CHAMBERS, 2002; GUIMARÃES, 2002). A presença de altos níveis de contaminação microbiana em leite e em seus derivados compromete a durabilidade desses produtos, já que AbstractMilk production has been considered increasingly important in the Brazilian economy despite being characterized as typical practice of small producers with low productivity. Nevertheless, little financial investment has been made resulting in low quality milk production with high microbiological counts. The objective of this work was to establish a relation between hygienic practices of milking and production and the microbiological quality of milk. A questionnaire focusing on the characteristics of production, milking, and sanitary conditions was administered in 60 milk farms in the city of Viçosa, MG -Brazil, from which milk samples were collected for determining the counts of mesophilic aerobes (MA) and psychrotrophics (PSI). The majority of the producers was characterized as small with less than 15 animals ...

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.

This study aimed to compare Petrifilm Aerobic Count (AC) plates and the conventional pour plate methodology using de Mann-Rogosa-Sharpe (MRS), Kang-Fung (KF) and Kang-Fung-Sol (KFS) culture media for screening and enumeration of lactic acid bacteria (LAB) in milk. Suspensions of 10 LAB species in reconstituted powder skim milk and 30 raw milk samples, without experimental inoculation, were tested. For selective enumeration, all samples were previously diluted in MRS, KF and KFS broths and then plated in Petrifilm AC and conventional pour plate methodology, using the same culture media with added agar. All plates were incubated at 37 degrees C for 48 h in anaerobic conditions. Differences in the counts were observed only for raw milk samples using KFS in conventional methodology, when compared with the counts obtained from MRS and KF (P0.05). The results showed excellent correlation indexes between both methodologies using the three culture media for LAB suspensions (r=0.97 for MRS, KF and KFS). For raw milk samples, the correlation indexes were excellent (r=0.97, for MRS) and good (r=0.84 for KF, and r=0.82 for KFS), showing some interference in Petrifilm AC when supplements were added, especially lactic acid. These results indicate the possibility of using Petrifilm AC plates for enumeration of LAB in milk, even with the use of selective supplements.

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.