Listeria monocytogenes inhibition by lactic acid bacteria and coliforms in Brazilian fresh white cheese.

INTRODUCTION: Minas fresh cheese (MFC), a Brazilian white cheese, is one of the most popular cheeses nationwide. Studies have shown that Listeria monocytogenes occurrence in this product is generally low, while high populations of coliforms can be found. This study aimed to evaluate the influence of coliforms in the behavior of L. monocytogenes in MFC. METHODS: Pasteurized milk was inoculated with L. monocytogenes and coliforms, and the acidification was made by lactic acid or by the addition of a starter culture. The cheeses of each production were divided into 3 groups and stored at 5 ºC, 12 ºC and cycles of 5 ºC followed by 25 ºC. In predetermined days, samples were taken and L. monocytogenes, coliforms and lactic acid bacteria populations were evaluated, besides the pH, water activity (aw), titratable acidity and NaCl concentration. RESULTS: The inhibition of L. monocytogenes in the presence of coliforms was observed (p < 0.05), except for those samples prepared with lactic acid and stored at temperature cycles. The values of pH and aw were not sufficiently low to cause inhibition; however, titratable acidity was higher in cheeses containing coliforms. In vitro tests containing lactic acid and L. monocytogenes showed that the bacterium is sensitive to concentration of lactic acid ≥ 0.3%, indicating that lactic acid produced by coliforms strongly influences the population of L. monocytogenes. CONCLUSIONS: Thus, it can be concluded that coliforms negatively impact populations of L. monocytogenes in MFC. We strongly recommend that producers of MFC adopt good hygiene practices to not only avoid contamination with L. monocytogenes, but also coliforms.

Viability of probiotic Lactobacillus casei in yoghurt: defining the best processing step to its addition.

Probiotics are live microorganisms capable of producing beneficial effects on its host when consumed in adequate amounts. To exert these effects, foods must contain probiotic microorganisms in populations above 10(6) CFU/g or mL throughout its shelf life. One of the strategies to ensure high population of probiotics in fermented milk is to add them during or after the fermentation process separately from the starter cultures. The objective of this study was to investigate the behavior of the probiotic microorganism Lactobacillus casei added to yoghurt in different stages of production. Yoghurts with L. casei were produced at different stages: before addition of starter (Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus), added together with this culture and at the end of fermentation. Yoghurt without probiotic added was produced as a control. The products were stored at 4 degrees C and analyzed after 1, 7, 14 and 21 days of storage. In these periods, the populations ofprobiotic and starter cultures were enumerated and the parameters pH and acidity were analyzed. The results were evaluated using analysis of variance and Tukey's test, both at 5% significance level. L. casei remained viable in populations of more than 10(8) CFU / g during 21 days of storage, which is suitable to define the formulations as probiotics. When the different stages of the addition of probiotics in yoghurts were evaluated there was no statistical difference between the formulations (p < 0.05) for populations of L. casei except for the first day of storage.

Viability of probiotic Lactobacillus casei in yoghurt: defining the best processing step to its addition / Viabilidade de Lactobacillus casei probiótico em iogurte natural: definição da melhor etapa de processamento para sua adição

Probiotics are live microorganisms capable of producing beneficial effects on its host when consumed in adequate amounts. To exert these effects, foods must contain probiotic microorganisms in populations above 106 CFU / g or mL throughout its shelf life. One of the strategies to ensure high population of probiotics in fermented milk is to add them during or after the fermentation process separately from the starter cultures. The objective of this study was to investigate the behavior of the probiotic microorganism Lactobacillus casei added to yoghurt in different stages of production. Yoghurts with L. casei were produced at different stages: before addition of starter (Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus), added together with this culture and at the end of fermentation. Yoghurt without probiotic added was produced as a control. The products were stored at 4 °C and analyzed after 1, 7, 14 and 21 days of storage. In these periods, the populations of probiotic and starter cultures were enumerated and the parameters pH and acidity were analyzed. The results were evaluated using analysis of variance and Tukey's test, both at 5% significance level. L. casei remained viable in populations of more than 108 CFU / g during 21 days of storage, which is suitable to define the formulations as probiotics. When the different stages of the addition of probiotics in yoghurts were evaluated there was no statistical difference between the formulations (p<0.05) for populations of L. casei except for the first day of storage.

Probióticos são microrganismos capazes de produzir efeitos benéficos sobre seu hospedeiro, quando consumidos vivos e em quantidades adequadas. Para que exerçam esses efeitos, os alimentos probióticos devem conter tais microrganismos em populações acima de 106 UFC/g ou mL, durante toda sua validade. Uma das estratégias para garantir a alta população dos probióticos em leites fermentados é adicioná-los durante ou após o processo de fermentação separadamente das culturas starter. Assim, o objetivo deste trabalho foi verificar o comportamento do microrganismo probiótico Lactobacillus casei adicionado em iogurte natural, em diferentes etapas do processo de produção. Foram produzidos iogurtes com adição de L. casei em diferentes etapas: antes da suplementação com o starter (Streptococcus salivarius subsp. thermophilus e Lactobacillus delbrueckii subsp. bulgaricus), juntamente com essa cultura e após o término da fermentação. Além disso, um iogurte controle (sem probiótico) foi produzido. Os produtos foram estocados a 4°C e analisados após 1, 7, 14 e 21 dias de armazenamento. Nesses períodos, foram enumeradas as populações das culturas probiótica e starter e avaliados os parâmetros físico-químicos (pH e acidez). Os resultados obtidos foram avaliados através de análise de variância e teste de Tukey, ambos ao nível de 5% de significância.L. casei mantevese viável e em populações superiores a 108 UFC/g durante os 21 dias de armazenamento, suficientes para que as formulações fossem definidas como probióticas. Quando as diferentes etapas de adição do probiótico aos iogurtes foram avaliadas, com exceção do 1º dia, não houve diferença estatística entre as formulações (p<0,05) para as populações de L. casei.

Antimicrobial action of lactoferrin on Staphylococcus aureus inoculated in Minas frescal cheese

The effect of the addition of lactoferrin on Staphylococcus aureus to Minas frescal cheese was evaluated. Three cheeses were made: control (S. aureus), Lf-2% (2% lactoferrin + S. aureus) and Lf-4% (4% lactoferrin + S. aureus). Cheeses were packaged and S. aureus populations were determined on days 1, 8, 15, 22 and 29 of storage at 6°C. The experiment was repeated three times. S. aureus population increased 1.3 logarithmic cycles in the control cheese during storage, while it decreased to numbers below the detection limit in cheeses containing lactoferrin, over the same period. Moreover, antimicrobial effect showed to be dose-dependent.

Ação antimicrobiana de lactoferrina sobre Staphylococcus aureus inoculado em queijo Minas frescal. O efeito da adição de lactoferrina sobre a viabilidade de Staphylococcus aureus inoculado em queijo Minas frescal foi avaliado. Três queijos foram produzidos: controle (S. aureus), Lf-2% (2% lactoferrina + S. aureus) e Lf-4% (4% lactoferrina + S. aureus). Os queijos foram armazenados e as populações de S. aureus foram determinadas durante o armazenamento refrigerado a 6°C nos dias 1, 8, 15, 22 e 29. Os experimentos foram realizados em triplicata. As populações de S. aureus aumentaram 1,3 ciclos logarítmicos nos queijos controle durante o armazenamento, enquanto que essas populações foram reduzidas abaixo do limite de detecção nos queijos contendo lactoferrina no mesmo período. Além disso, observou-se que o efeito antimicrobiano foi dose-dependente.

Antimicrobial action of lactoferrin on Staphylococcus aureus inoculated in Minas frescal cheese.

The effect of the addition of lactoferrin on Staphylococcus aureus to Minas frescal cheese was evaluated. Three cheeses were made: control (S. aureus), Lf-2% (2% lactoferrin + S. aureus) and Lf-4% (4% lactoferrin + S. aureus). Cheeses were packaged and S. aureus populations were determined on days 1, 8, 15, 22 and 29 of storage at 6 degrees C. The experiment was repeated three times. S. aureus population increased 1.3 logarithmic cycles in the control cheese during storage, while it decreased to numbers below the detection limit in cheeses containing lactoferrin, over the same period. Moreover, antimicrobial effect showed to be dose-dependent.

[Physico-chemical and microbiological evaluation of UHT milk commercialized in three Mercosul countries (Brazil, Argentina and Paraguay)]. / Avalição físico-química e microbiológica do leite UHT comercializado em três países do Mercosul (Brasil, Argentina e Paraguai).

With the aim to evaluate the physico-chemical and microbiological quality of UHT milk commercialized in three countries of Mercosul, samples of four different brands were acquired in each city (Foz do Iguaçu-Brazil, Puerto Iguazú-Argentina and Ciudad del Este-Paraguay) and submitted to the following analysis: fat content, titratable acidity, milk ethanol stability (with the following ethanol concentrations: 68, 72, 76 and 80%), total dry extract and no fat dry extract, pH, density and freezing point. Counts of mesophilic and psychrotrophic microorganisms were already done. In the physico-chemical evaluation of UHT milk, a significant number of samples were in disagree with the established patterns for fat content, no fat dry extract, density and freezing point. Except one brand from Brazil, milk samples showed stability to 68% ethanol. pH averages of Brazilian milk were in agree with the patterns and highest values were observed in samples acquired on Paraguay. Observing the microbiological analysis, 37.5%, 62.5% and 12.5% of samples acquired from Brazil, Argentina and Paraguay, respectively, showed counts above the established patterns for mesophilic microorganisms. Counts of psychrotrophic microorganisms were in disagree with the established patterns in 50%, 50% and 100% of samples from Brazil, Argentina and Paraguay, respectively.

Enterobacter sakazakii in dried infant formulas and milk kitchens of maternity wards in São Paulo, Brazil.

This study was the first conducted in Brazil to evaluate the presence of Enterobacter sakazakii in milk-based powdered infant formula manufactured for infants 0 to 6 months of age and to examine the conditions of formula preparation and service in three hospitals in São Paulo State, Brazil. Samples of dried and rehydrated infant formula, environments of milk kitchens, water, bottles and nipples, utensils, and hands of personnel were analyzed, and E. sakazakii and Enterobacteriaceae populations were determined. All samples of powdered infant formula purchased at retail contained E. sakazakii at <0.3 [corrected] most probable number (MPN)/100 g. In hospital samples, E. sakazakii was found in one unopened formula can (0.3 MPN/100 g) and in the residue from one nursing bottle from hospital A. All other cans of formula from the same lot bought at a retail store contained E. sakazakii at <0.3 [corrected] MPN/100 g. The pathogen also was found in one cleaning sponge from hospital B. Enterobacteriaceae populations ranged from 10(1) to 10(5) CFU/g in cleaning aids and <5 CFU/g in all formula types (dry or rehydrated), except for the sample that contained E. sakazakii, which also was contaminated with Enterobacteriaceae at 5 CFU/g. E. sakazakii isolates were not genetically related. In an experiment in which rehydrated formula was used as the growth medium, the temperature was that of the neonatal intensive care unit (25 degrees C), and the incubation time was the average time that formula is left at room temperature while feeding the babies (up to 4 h), a 2-log increase in levels of E. sakazakii was found in the formula. Visual inspection of the facilities revealed that the hygienic conditions in the milk kitchens needed improvement. The length of time that formula is left at room temperature in the different hospitals while the babies in the neonatal intensive care unit are being fed (up to 4 h) may allow for the multiplication of E. sakazakii and thus may lead to an increased health risk for infants.

Enterotoxigenic and genetic profiles of Bacillus cereus strains of food origin in Brazil.

In Brazil, the incidence of Bacillus cereus outbreaks is unknown, and there is little information about B. cereus occurrence in food. In addition, data on toxin production and genetic characterization of the B. cereus isolates cannot be found. This pathogen causes two distinct types of toxin-mediated foodborne illnesses known as diarrheal and emetic syndromes. Diarrheal syndrome has been linked to three different enterotoxins: two protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE); and an enterotoxic protein, cytotoxin K (cytK). Emetic syndrome is related to cereulide, a toxin encoded by the ces gene. In this study, NHE and HBL production capacities of 155 strains of B. cereus isolated from Brazilian food products were evaluated with an immunoassay. Strains were also tested for the presence of the genes of the HBL and NHE complexes, cytK, cytK-1, cytK-2, and ces, using PCR. HBL was detected in 105 (67.7%) strains and NHE in 154 (99.4%) strains. All the strains harbored at least one gene of the NHE complex, while 96.1% of them were positive for at least one of those of the HBL complex. Genes cytK1 and ces were not detected. All strains showed toxigenic capacity and could represent a risk for consumers if good practices are not followed. This is the first report on toxigenic and genetic profiles of B. cereus strains isolated in Brazil.

Influência dos coliformes no comportamento de Listeria monocytogenes em queijo Minas frescal / Influence of coliformS in the behavior of Listeria monovytogenes in fresh white Minas cheese

Os queijos macios são veículos conhecidos de surtos de listeriose. Os chamados 'queso blanco' ou 'Latin-style fresh cheese' representam um grupo heterogêneo de queijos macios brancos e não maturados produzidos e consumidos em diferentes países da América Latina. O queijo Minas Frescal é o representante brasileiro deste grupo e pode ser produzido utilizando-se diferentes tecnologias. Vários estudos têm mostrado que a ocorrência de Listeria monocytogenes (Lm) em queijo Minas Frescal é muito variável, enquanto altas populações de coliformes fecais (>104UFC/g) são muito comuns. O objetivo deste trabalho foi avaliar a influência dos coliformes no comportamento de Lm em queijo Minas Frescal produzido por acidificação direta do leite com ácido lático e por adição de cultura lática. Leite pasteurizado foi contaminado com Lm (106UFC/g e 1UFC/g) e coliformes (107UFC/g) e os queijos foram preparados seguindo-se os procedimentos de fabricação comerciais. Queijos preparados com leite contaminado somente com Lm (106CFU/g e 1CFU/g) ou coliformes (107UFC/g) foram utilizados como controle. As produções foram repetidas 3 vezes. Os queijos embalados em sacos de polietileno foram divididos em três grupos, sendo cada um estocado em uma das seguintes condições: 20 dias a 5oC; 20 dias a 12oC (temperatura de abuso); 8 dias a 5oC/16h seguido por 25oC/8h (para simular condições em feiras-livres). A cada 5 dias para os queijos armazenados a 5oC e 12oC, e a cada 2 dias para o outro grupo, duplicata de amostras foram retiradas e as populações de Lm, coliformes e bactérias láticas foram determinadas utilizando-se procedimentos padrões. pH e atividade de água também foram mensurados. A inibição de Lm foi observada nos queijos em que os coliformes estavam presentes (pelo menos 1 log de diferença em cada temperatura, com exceção dos queijos preparados com ácido lático e estocados com alternância de temperaturas). Os valores de pH e aw não foram suficientemente baixos para causarem essa inibição, entretanto, a acidez titulável foi maior em queijo contendo coliformes. Testes em ágar tripticase de soja (TSA) contendo diferentes concentrações de ácido lático e contaminados com Lm (106UFC/g e 1UFC/g) mostraram que, quando altas concentrações de ácido lático foram utilizadas (0,3% ou mais), a população de Lm não aumentou, indicando que o ácido lático produzido pelos coliformes pode ser um fator importante no controle de Lm em queijo Minas Frescal. É importante ressaltar que não é nossa intenção utilizar coliformes para inibir Lm nesse tipo de queijo

Soft cheeses are a well known cause of listeriosis outbreaks. The so called 'queso blanco' or 'Latin-style fresh cheese' represents a heterogeneous group of white, unripened soft cheese produced and consumed in different Latin America countries. Minas Frescal cheese is the Brazilian representative of this group and it can be produced by different technologies. Several studies have shown that Listeria monocytogenes (Lm) occurrence in Minas cheese is highly variable, while high population of fecal coliforms (>104CFU/g) is very common. The objective of this study was to evaluate the influence of coliforms in the behavior of Lm in Minas Frescal cheeses produced by direct acidification of the milk with lactic acid and by the addition of a lactic culture. Pasteurized milk was spiked with Lm (106CFU/g and 1CFU/g) and coliforms (107CFU/g) and the cheeses were lab prepared following regular commercial procedures. Cheeses prepared with milk spiked only with Lm (106CFU/g and 1CFU/g) or coliforms (107CFU/g) were used as controls. The production had been repeated 3 times. The cheeses packed in polyethylene bags were divided in 3 groups and each group was stored in one of the following conditions: up to 20 days at 5oC; up to 20 days at 12oC (abuse temperature); up to 8 days at 5oC/16h followed by 25oC/8h (to simulate open market conditions). Every 5 days for cheeses stored at 5oC and 12oC, and every 2 days for the other group, duplicate samples were taken and Lm, coliforms and lactic acid bacteria population were determined using standard procedures. pH and water activity were also measured. An inhibition of Lm was observed in the cheeses when in the presence of coliforms (at least 1 log difference at any temperature, except cheeses prepared with lactic acid and stored with temperature alternation). The values of pH and aW had not been sufficiently low to cause this inhibition, however, the titratable acidity was higher in cheeses containing coliforms. Tests in tryptone soy agar containing different concentrations of lactic acid and spiked with Lm (106CFU/g and 1CFU/g) showed that when higher concentrations of lactic acid were used (0.3 % or more), the population of Lm did not increase, indicating that the lactic acid produced by coliforms may be an important factor in controlling Lm in Minas Frescal cheese. It is important to ressaltar that it is not our intention to use coliforms to inhibit Lm in this type of cheese