A New Bacteriocin from Latilactobacillus sakei: In vitro and In situ Application.

AIMS AND BACKGROUND: Natural preservatives are a viable alternative to replace chemical preservatives that have potential toxicity and carcinogenic effects. OBJECTIVE: To prove the effectiveness in increasing the microbiological stability of Minas Frescal cheese with the addition of a bacteriocin obtained from Latilactobacillus sakei as a natural preservative. METHODS: A new broad-spectrum bacteriocin was evaluated for its functional activity in vitro and in situ when applied in the formulation of Minas Frescal cheese. A commercial bacteriocin was used as a positive control. RESULTS: The inhibitory action of the bacteriocin studied was confirmed, with a reduction of 42.86% in the count of coagulase-positive Staphylococcus in relation to the negative control, at the end of the 30 days of study. For the group of thermotolerant coliforms, the bacteriocin studied showed greater efficiency than the commercial preservative. In vitro analyzes showed the inhibitory action of bacteriocin, above 87% inhibition against S. aureus, E. coli and Salmonela enteritidis, and approximately 90% against Listeria monocytogenes. CONCLUSION: It was concluded that the bacteriocin produced by the Latilactobacillus sakei strain has great potential for application in foods such as Minas Frescal cheese.

Licury Cake in Diets for Lactating Goats: Qualitative Aspects of Milk and Cheese.

The study was carried out to evaluate the effects of licury cake (LC) inclusion in diets for lactating goats on milk chemical composition and fatty acid profile, and physicochemical composition and sensory attributes of Minas Frescal cheese. Twelve lactating goats were used (eight Saanen and four Anglo Nubian goats, with 35 ± 5 days in lactation and average body weight of 37.93 ± 9.22 kg), distributed in a triplicate 4 × 4 Latin Square design. The increasing levels of LC studied were: 0, 67, 133 and 200 g/kg of dry matter (DM). The analyses were adjusted using orthogonal polynomial contrasts, in which the probability level of 5% was considered. For sensory attributes, principal component analysis was performed. The LC inclusion promoted the reduction in moisture (p = 0.013) and mineral matter (p < 0.001) content in cheese. The ether extract content in cheese showed a quadratic effect (p = 0.021). Lauric acid showed a linear (p = 0.002) increasing effect, and myristic (p < 0.001) and rumenic (p = 0.018) acids showed quadratic effects. The sensory attributes analysis in cheese indicated that the inclusion of 133 and 200 g/kg of LC improve its texture. Flavor was improved with the inclusion of 67 and 133 g/kg. The inclusion of up to 133 g/kg of LC in the diet of lactating goats is recommended because it promotes improvements in flavor and texture of Minas Frescal cheese.

Possibilities for using ohmic heating in Minas Frescal cheese production.

The use of ohmic heating (OH, 4, 8, or 12 V/cm, 72-75 °C/15 s) for pasteurization of milk intended for the manufacture of Minas Frescal cheese was investigated. The cheeses were characterized for the gross composition, bioactive compounds (antioxidant, antihypertensive and antidiabetic activities), fatty acid profile, rheological parameters, volatiles profile, and sensory acceptance. OH decreased the elasticity, hardness, and firmness while improved the sensory acceptance of Minas Frescal cheese. In addition, higher antioxidant, antihypertensive and antidiabetic activities values as well as higher concentrations of short, medium and long-chain fatty acids were observed. Overall, OH can be considered a suitable technology to be used in milk for the Minas Frescal cheese processing.

Ohmic heating does not influence the biochemical properties of Minas Frescal cheese but decreases uric acid levels in healthy Wistar rats.

The objective of this study was to evaluate the effects of the consumption of Minas Frescal cheese (2 g for 15 d) produced with pasteurized milk or milk subjected to ohmic heating on the health variables (biochemical, hematological, and uric acid levels) of rats, as well as on the identity variables and proteolysis indices of cheese. Three groups of rats (n = 6 per group) were studied, as follows: control group, without cheese consumption (CON), rats fed Minas Frescal cheese produced with milk pasteurized by conventional technology (PC, 72 to 75°C for 15 s), and rats fed Minas Frescal cheese produced with milk subjected to ohmic heating (OHC, 8 V/cm, 72 to 75°C for 15 s). Ohmic heating did not alter the macronutrient levels (moisture, fat, protein) of the cheeses, but an increase in the proteolysis index was observed. For both cheeses, no significant differences were observed for feed intake (139 to 143 g/d), water consumption (196 to 206 mL/d), or final animal weight (236 to 252 g), and no changes were detected in hematological variables (counts of red blood cells, hemoglobin, leukocytes, and platelets) or levels of potassium, calcium, magnesium, urea, creatinine, total cholesterol, high-density lipoprotein cholesterol, blood glucose, and bilirubin (total, direct, and indirect) of rats. In addition, both cheeses led to a reduction of serum triglycerides and an increase in monocyte, eosinophil, and basophil levels. However, consumption of OHC resulted in decreased sodium, uric acid, and lymphocyte levels. The present results suggest that ohmic heating in the processing of Minas Frescal cheese has positive effects on the health of healthy rats.

Pseudomonas spp. and other psychrotrophic microorganisms in inspected and non-inspected Brazilian Minas Frescal cheese: proteolytic, lipolytic and AprX production potential / Pseudomonas spp. e outros micro-organismos psicrotróficos em queijos Minas Frescal inspecionados e não inspecionados: potencial proteolítico, lipolítico e produção de AprX

The most consumed cheese in Brazil, Minas Frescal cheese (MFC) is highly susceptible to microbial contamination and clandestine production and commercialization can pose a risk to consumer health. The storage of this fresh product under refrigeration, although more appropriate, may favor the growth of spoilage psychrotrophic bacteria. The objective of this study was to quantify and compare Pseudomonas spp. and other psychrotrophic bacteria in inspected and non-inspected MFC samples, evaluate their lipolytic and proteolytic activities and their metalloprotease production potentials. Twenty MFC samples were evaluated: 10 inspected and 10 non-inspected. Counts of psychrotrophic bacteria and Pseudomonas spp., evaluation of the proteolytic and lipolytic potential of the isolates, and identification of potential producers of alkaline metalloprotease (AprX) were assessed. The mean total psychrotrophic counts were 1.07 (±2.18) × 109CFU/g in the inspected samples and 4.5 (±5.86) × 108CFU/g in the non-inspected, with no significant difference (p=0.37). The average score of Pseudomonas spp. was 6.86 (±18.6) × 105 and 2.08 (±3.65) × 106 CFU/g for the inspected and non-inspected MFC samples, respectively, with no significant difference (p=0.1). Pseudomonas spp. represented 0.06% and 0.004% of psychrotrophic bacteria found in inspected and non-inspected MFC samples, respectively. Collectively, 694 psychrotrophic strains and 47Pseudomonas spp. were isolated, of which 59.9% and 68.1% were simultaneously proteolytic and lipolytic, respectively. Of the 470 psychrotrophs isolated from inspected and 224 from non-inspected cheese samples, 5.74% and 2.23% contained aprX, respectively, while 100 and 86.96% of the Pseudomonas spp. isolates in inspected and non-inspected cheese samples contained the gene. The production potential of AprX did not, however, determine the proteolytic activity on plates of these isolates under the conditions evaluated in this study...(AU)

O mais consumido no Brasil, o queijo Minas Frescal (QMF) é altamente suscetível à contaminação microbiana e a produção e comercialização clandestina podem representar um risco para a saúde do consumidor. O armazenamento deste produto fresco sob refrigeração, embora mais apropriado, pode favorecer a multiplicação de bactérias psicrotróficas deteriorantes. O objetivo deste estudo foi quantificar e comparar Pseudomonas spp. e outras bactérias psicrotróficas em amostras de QMF inspecionadas e não inspecionadas, avaliar o potencial lipolítico, proteolítico e de produção de metaloprotease alcalina. Vinte amostras de QMF foram avaliadas: 10 inspecionadas e 10 não inspecionadas. Foram avaliadas as contagens de bactérias psicrotróficas e Pseudomonas spp., o potencial proteolítico e lipolítico dos isolados e a identificação de potenciais produtores de metaloprotease alcalina (AprX). A média total das contagens de bactérias psicrotróficas foi de 1,07 (±2,18) × 109UFC/g nas amostras inspecionadas e 4,5 (±5,86) × 108UFC/g nas não inspecionadas, sem diferença significativa (p=0,37). A média de Pseudomonasspp. foi de 6,86 (±18,6) × 105 e 2,08 (±3,65) × 106UFC/g para as amostras QMF inspecionadas e não-inspecionadas, respectivamente, sem diferença significativa (p=0,1). Pseudomonas spp. representaram 0,06% e 0,004% de bactérias psicrotróficas encontradas em amostras QMF inspecionadas e não-inspecionadas, respectivamente. Das amostras inspecionadas e não inspecionadas, foram isoladas 694 colônias psicrotróficas e 47 Pseudomonasspp., dos quais 59,9% e 68,1% foram simultaneamente proteolíticos e lipolíticos, respectivamente. Dos 470 isolados de psicrotróficos das amostras de queijo inspecionados e dos 224 isolados das não inspecionadas, 5,74% e 2,23% continham o gene aprX, respectivamente, enquanto 100 e 86,96% das Pseudomonasspp. isoladas em amostras de queijo inspecionadas e não inspecionadas continham o potencial de expressão de AprX...(AU)

Pseudomonas spp. and other psychrotrophic microorganisms in inspected and non-inspected Brazilian Minas Frescal cheese: proteolytic, lipolytic and AprX production potential / Pseudomonas spp. e outros micro-organismos psicrotróficos em queijos Minas Frescal inspecionados e não inspecionados: potencial proteolítico, lipolítico e produção de AprX

The most consumed cheese in Brazil, Minas Frescal cheese (MFC) is highly susceptible to microbial contamination and clandestine production and commercialization can pose a risk to consumer health. The storage of this fresh product under refrigeration, although more appropriate, may favor the growth of spoilage psychrotrophic bacteria. The objective of this study was to quantify and compare Pseudomonas spp. and other psychrotrophic bacteria in inspected and non-inspected MFC samples, evaluate their lipolytic and proteolytic activities and their metalloprotease production potentials. Twenty MFC samples were evaluated: 10 inspected and 10 non-inspected. Counts of psychrotrophic bacteria and Pseudomonas spp., evaluation of the proteolytic and lipolytic potential of the isolates, and identification of potential producers of alkaline metalloprotease (AprX) were assessed. The mean total psychrotrophic counts were 1.07 (±2.18) × 109CFU/g in the inspected samples and 4.5 (±5.86) × 108CFU/g in the non-inspected, with no significant difference (p=0.37). The average score of Pseudomonas spp. was 6.86 (±18.6) × 105 and 2.08 (±3.65) × 106 CFU/g for the inspected and non-inspected MFC samples, respectively, with no significant difference (p=0.1). Pseudomonas spp. represented 0.06% and 0.004% of psychrotrophic bacteria found in inspected and non-inspected MFC samples, respectively. Collectively, 694 psychrotrophic strains and 47Pseudomonas spp. were isolated, of which 59.9% and 68.1% were simultaneously proteolytic and lipolytic, respectively. Of the 470 psychrotrophs isolated from inspected and 224 from non-inspected cheese samples, 5.74% and 2.23% contained aprX, respectively, while 100 and 86.96% of the Pseudomonas spp. isolates in inspected and non-inspected cheese samples contained the gene. The production potential of AprX did not, however, determine the proteolytic activity on plates of these isolates under the conditions evaluated in this study. Of total, 65.63% of the psychrotrophs that contained aprX gene were confirmed as Pseudomonas spp., using genus-specific PCR. Phylogenetic analysis of the 16S rRNA gene of the other psychrotrophs that were potential producers of AprX identified them as Serratia spp. (n=7), Raoultella ornithinolytica (n=1), and Acinetobacter schindleri (n=1) in the inspected samples and Psychrobacter sanguinis (n=1) and Leuconostoc mesenteroides (n=1) in the non-inspected samples. The production conditions of Brazilian MFC of these samples, while meeting the legal determinations, are not sufficient to control Pseudomonas and other spoilage-related psychrotrophs. Thus, stricter hygienic measures are required during the formal production of this type of cheese.(AU)

O mais consumido no Brasil, o queijo Minas Frescal (QMF) é altamente suscetível à contaminação microbiana e a produção e comercialização clandestina podem representar um risco para a saúde do consumidor. O armazenamento deste produto fresco sob refrigeração, embora mais apropriado, pode favorecer a multiplicação de bactérias psicrotróficas deteriorantes. O objetivo deste estudo foi quantificar e comparar Pseudomonas spp. e outras bactérias psicrotróficas em amostras de QMF inspecionadas e não inspecionadas, avaliar o potencial lipolítico, proteolítico e de produção de metaloprotease alcalina. Vinte amostras de QMF foram avaliadas: 10 inspecionadas e 10 não inspecionadas. Foram avaliadas as contagens de bactérias psicrotróficas e Pseudomonas spp., o potencial proteolítico e lipolítico dos isolados e a identificação de potenciais produtores de metaloprotease alcalina (AprX). A média total das contagens de bactérias psicrotróficas foi de 1,07 (±2,18) × 109UFC/g nas amostras inspecionadas e 4,5 (±5,86) × 108UFC/g nas não inspecionadas, sem diferença significativa (p=0,37). A média de Pseudomonasspp. foi de 6,86 (±18,6) × 105 e 2,08 (±3,65) × 106UFC/g para as amostras QMF inspecionadas e não-inspecionadas, respectivamente, sem diferença significativa (p=0,1). Pseudomonas spp. representaram 0,06% e 0,004% de bactérias psicrotróficas encontradas em amostras QMF inspecionadas e não-inspecionadas, respectivamente. Das amostras inspecionadas e não inspecionadas, foram isoladas 694 colônias psicrotróficas e 47 Pseudomonasspp., dos quais 59,9% e 68,1% foram simultaneamente proteolíticos e lipolíticos, respectivamente. Dos 470 isolados de psicrotróficos das amostras de queijo inspecionados e dos 224 isolados das não inspecionadas, 5,74% e 2,23% continham o gene aprX, respectivamente, enquanto 100 e 86,96% das Pseudomonasspp. isoladas em amostras de queijo inspecionadas e não inspecionadas continham o potencial de expressão de AprX. Esse potencial, no entanto, não determinou a atividade proteolítica em placas desses isolados nas condições avaliadas neste estudo. Do total, 65,63% dos psicrotróficos que continham o gene aprX foram confirmados como Pseudomonasspp., utilizando PCR gênero-específico. A análise filogenética do gene 16S rRNA dos outros psicrotróficos que foram produtores potenciais de AprX os identificou como Serratia spp. (n=7), Raoultella ornithinolytica (n=1) e Acinetobacter schindleri (n=1) nas amostras inspecionadas e Psychrobacter sanguinis (n=1) e Leuconostoc mesenteroides (n=1) nas amostras não inspecionadas. As condições de produção do QMF dessas amostras, atendendo às determinações legais, não são suficientes para controlar a Pseudomonas e outros psicrotróficos relacionados à deterioração. Assim, medidas higiênicas mais rígidas são necessárias durante a produção formal deste tipo de queijo.(AU)

Study of the effectiveness of staphylococcins in biopreservation of Minas fresh (Frescal) cheese with a reduced sodium content.

Reducing salt content in foods such as cheeses, while limiting the growth of spoilage microorganisms and foodborne pathogens, is a difficult challenge. One method that may prove useful is use of staphylococcins, which are bacteriocins produced by staphylococci. Therefore, staphylococcin antimicrobial activity against six strains of S. aureus isolated from cheese was tested aiming at their industrial application in biopreservation of Minas fresh (Frescal) cheese with reduced sodium content. Three staphylococcins were selected for these tests: Pep 5, aureocin A53 and lysostaphin. All three staphylococcins proved to be bacteriolytic against all six strains of S. aureus. The antimicrobial activity of the partially purified staphylococcins was subsequently investigated against strains S. aureus Q1 and QJ3 in cheese matrices (6.0 log CFU/g) with different NaCl contents (control, a 25% reduction, and a 50% reduction), kept under refrigeration at 4 °C, for 21 days. Both strains were shown to be of concern for food industry as they carry the SEA, SEB and SEH enterotoxin genes, and are resistant to ß-lactam drugs and moderate biofilm formers when grown in TSB. When used singly, Pep5, aureocin A53 and lysostaphin reduced approximately 95%, 99% and 99.99% of the viable cell counts, respectively, irrespective of the sodium content of the cheese matrix. The combined action of aureocin A53 and Pep5 resulted in an additional and significant reduction (p < 0.05) of ~1.0 log CFU/g when compared with the reduction caused by the use of either one singly. The combined action of lysostaphin and aureocin A53 or lysostaphin and Pep5 resulted in a reduction similar to or slightly smaller (p > 0.05) than that observed when lysostaphin was employed singly. Lysostaphin also proved to reduce the number of the staphylococcal viable cells to a level (~ 2.0 log CFU/g) at which enterotoxin production should not reach a sufficient quantity to cause food poisoning. Therefore, lysostaphin may have a practical application in the food industry to control staphylococcal contamination of Minas fresh cheese with a sodium content reduced up to 50%, providing consumers with more safe options to reduce their intake of sodium.

Pseudomonas spp. and other psychrotrophic microorganisms in inspected and non-inspected Brazilian Minas Frescal cheese: proteolytic, lipolytic and AprX production potential

ABSTRACT: The most consumed cheese in Brazil, Minas Frescal cheese (MFC) is highly susceptible to microbial contamination and clandestine production and commercialization can pose a risk to consumer health. The storage of this fresh product under refrigeration, although more appropriate, may favor the growth of spoilage psychrotrophic bacteria. The objective of this study was to quantify and compare Pseudomonas spp. and other psychrotrophic bacteria in inspected and non-inspected MFC samples, evaluate their lipolytic and proteolytic activities and their metalloprotease production potentials. Twenty MFC samples were evaluated: 10 inspected and 10 non-inspected. Counts of psychrotrophic bacteria and Pseudomonas spp., evaluation of the proteolytic and lipolytic potential of the isolates, and identification of potential producers of alkaline metalloprotease (AprX) were assessed. The mean total psychrotrophic counts were 1.07 (±2.18) × 109CFU/g in the inspected samples and 4.5 (±5.86) × 108CFU/g in the non-inspected, with no significant difference (p=0.37). The average score of Pseudomonas spp. was 6.86 (±18.6) × 105 and 2.08 (±3.65) × 106 CFU/g for the inspected and non-inspected MFC samples, respectively, with no significant difference (p=0.1). Pseudomonas spp. represented 0.06% and 0.004% of psychrotrophic bacteria found in inspected and non-inspected MFC samples, respectively. Collectively, 694 psychrotrophic strains and 47Pseudomonas spp. were isolated, of which 59.9% and 68.1% were simultaneously proteolytic and lipolytic, respectively. Of the 470 psychrotrophs isolated from inspected and 224 from non-inspected cheese samples, 5.74% and 2.23% contained aprX, respectively, while 100 and 86.96% of the Pseudomonas spp. isolates in inspected and non-inspected cheese samples contained the gene. The production potential of AprX did not, however, determine the proteolytic activity on plates of these isolates under the conditions evaluated in this study. Of total, 65.63% of the psychrotrophs that contained aprX gene were confirmed as Pseudomonas spp., using genus-specific PCR. Phylogenetic analysis of the 16S rRNA gene of the other psychrotrophs that were potential producers of AprX identified them as Serratia spp. (n=7), Raoultella ornithinolytica (n=1), and Acinetobacter schindleri (n=1) in the inspected samples and Psychrobacter sanguinis (n=1) and Leuconostoc mesenteroides (n=1) in the non-inspected samples. The production conditions of Brazilian MFC of these samples, while meeting the legal determinations, are not sufficient to control Pseudomonas and other spoilage-related psychrotrophs. Thus, stricter hygienic measures are required during the formal production of this type of cheese.

RESUMO: O mais consumido no Brasil, o queijo Minas Frescal (QMF) é altamente suscetível à contaminação microbiana e a produção e comercialização clandestina podem representar um risco para a saúde do consumidor. O armazenamento deste produto fresco sob refrigeração, embora mais apropriado, pode favorecer a multiplicação de bactérias psicrotróficas deteriorantes. O objetivo deste estudo foi quantificar e comparar Pseudomonas spp. e outras bactérias psicrotróficas em amostras de QMF inspecionadas e não inspecionadas, avaliar o potencial lipolítico, proteolítico e de produção de metaloprotease alcalina. Vinte amostras de QMF foram avaliadas: 10 inspecionadas e 10 não inspecionadas. Foram avaliadas as contagens de bactérias psicrotróficas e Pseudomonas spp., o potencial proteolítico e lipolítico dos isolados e a identificação de potenciais produtores de metaloprotease alcalina (AprX). A média total das contagens de bactérias psicrotróficas foi de 1,07 (±2,18) × 109UFC/g nas amostras inspecionadas e 4,5 (±5,86) × 108UFC/g nas não inspecionadas, sem diferença significativa (p=0,37). A média de Pseudomonasspp. foi de 6,86 (±18,6) × 105 e 2,08 (±3,65) × 106UFC/g para as amostras QMF inspecionadas e não-inspecionadas, respectivamente, sem diferença significativa (p=0,1). Pseudomonas spp. representaram 0,06% e 0,004% de bactérias psicrotróficas encontradas em amostras QMF inspecionadas e não-inspecionadas, respectivamente. Das amostras inspecionadas e não inspecionadas, foram isoladas 694 colônias psicrotróficas e 47 Pseudomonasspp., dos quais 59,9% e 68,1% foram simultaneamente proteolíticos e lipolíticos, respectivamente. Dos 470 isolados de psicrotróficos das amostras de queijo inspecionados e dos 224 isolados das não inspecionadas, 5,74% e 2,23% continham o gene aprX, respectivamente, enquanto 100 e 86,96% das Pseudomonasspp. isoladas em amostras de queijo inspecionadas e não inspecionadas continham o potencial de expressão de AprX. Esse potencial, no entanto, não determinou a atividade proteolítica em placas desses isolados nas condições avaliadas neste estudo. Do total, 65,63% dos psicrotróficos que continham o gene aprX foram confirmados como Pseudomonasspp., utilizando PCR gênero-específico. A análise filogenética do gene 16S rRNA dos outros psicrotróficos que foram produtores potenciais de AprX os identificou como Serratia spp. (n=7), Raoultella ornithinolytica (n=1) e Acinetobacter schindleri (n=1) nas amostras inspecionadas e Psychrobacter sanguinis (n=1) e Leuconostoc mesenteroides (n=1) nas amostras não inspecionadas. As condições de produção do QMF dessas amostras, atendendo às determinações legais, não são suficientes para controlar a Pseudomonas e outros psicrotróficos relacionados à deterioração. Assim, medidas higiênicas mais rígidas são necessárias durante a produção formal deste tipo de queijo.

Análise microbiológica de queijos tipo minas frescal oriundos de diferentes formas de produção / Microbiological analysis of minas frescal cheese from different forms of production

Introdução: o queijo tipo Minas Frescal, obtido através da coagulação do leite, é amplamente consumido no Brasil, e apresenta um alto teor de umidade, sendo um ótimo meio para crescimento microbiano. Por isso, para sua produção, é necessária a pasteurização do leite cru, além de boas técnicas e práticas de fabricação seguindo as normas sanitárias, evitando, assim, contaminações graves por micro-organismos causadores de doenças transmitidas por alimentos (DTAs). Objetivo: o presente trabalho teve como objetivo avaliar a qualidade microbiológica de 3 amostras de queijo Minas Frescal oriundas de diferentes processos produtivos. Materiais e métodos: os micro-organismos pesquisados foram coliformes totais e termotolerantes, S. aureus e Salmonella spp. As origens das amostras foram: industrializada, produzida em açougue de bairro em Bauru e oriunda de produção caseira/artesanal. Resultados: das 3 amostras analisadas, os resultados foram: a artesanal e a de açougue apresentaram crescimento de coliformes totais e termotolerantes acima do permitido; as 3 amostras apresentaram crescimento de colônias atípicas incontáveis nas análises de S. aureus, impossibilitando a contagem dos micro-organismos em questão; nenhuma delas apresentou crescimento de Salmonella spp. Conclusão: considerando a qualidade microbiológica, foi possível concluir que somente o queijo Minas Frescal industrializado está dentro dos padrões determinados pela ANVISA, enquanto as outras amostras apresentaram resultados insatisfatórios, o que pode comprometer a saúde do consumidor.

Introduction: Minas Frescal cheese, which is obtained through milk coagulation, is widely consumed in Brazil, and presents a high moisture content, being a great way for microbial growth. That's why, for its production, it's necessary raw milk's pasteurization, besides good manufacturing techniques and practices following sanitary standards, thus avoiding serious contaminations caused by foodborne illness. Objective: the present study had as objective to evaluate the microbiologic quality of 3 Minas Frescal cheese samples coming from different manufacturing processes. Materials and methods: the microorganisms searched were total and thermotolerants coliforms, S. aureus and Salmonella spp. The origins of the samples were: industry, butchery and homemade/craft production. Results: from the 3 analyzed samples, the results were: the craft production and butchery ones presented above the allowed growth of total and thermotolerants coliforms; the 3 samples showed uncountable atypical colonies growth in the analysis of S. aureus preventing the count of the microorganisms in question; and none of them presented Salmonella spp's growth. Conclusion: thus, considering microbiologic quality, it was possible to conclude that only the industrialized Minas Frescal chesse is within standards given by ANVISA, while the other samples presented unsatisfactory results, which can harm the consumer's health.

Antibiotic Resistance Genetic Markers and Integrons in White Soft Cheese: Aspects of Clinical Resistome and Potentiality of Horizontal Gene Transfer.

Antibiotic resistance poses an important threat to global public health and has become a challenge to modern medicine. The occurrence of antibiotic-resistant bacteria in a broad range of foods has led to a growing concern about the impact that food may have as a reservoir of antibiotic resistance genes. Considering Minas Frescal Cheese (MFC)-a typical Brazilian white soft cheese-and its economic and cultural values, in this study, medically relevant antimicrobial-resistance genetic markers (AR genes) were screened, and the occurrence of integrons were evaluated in manufactured MFC using culture-independent approaches. Through a fingerprinting analysis, the tested MFCs were brand-clustered, indicating reproducibility along the production chain. A common core of resistance markers in all brands evaluated and related antimicrobials such as ß-lactams, tetracyclines, quinolones, and sulfonamide was detected. Several other markers, including efflux pumps and aminoglycosides-resistance were distributed among brands. Class 1 and 2 integrons were observed, respectively, in 77% and 97% of the samples. The presence of AR genes is of special interest due to their clinical relevance. Taken together, the data may suggest that the production chain of MFC might contribute to the spread of putative drug-resistant bacteria, which could greatly impact human health. Furthermore, detection of class 1 and class 2 integrons in MFC has led to discussions about resistance gene spread in this traditional cheese, providing evidence of potential horizontal transfer of AR genes to human gut microbiota.

Reformulating Minas Frescal cheese using consumers' perceptions: Insights from intensity scales and check-all-that-apply questionnaires.

We evaluated the performance of check-all-that-apply (CATA) questions and intensity scales to describe Minas Frescal cheese and its reformulation based on consumers' perceptions. Ten commercial samples with different formulations (full-fat, low-fat, or low-lactose) were evaluated by 200 consumers divided equally into 2 groups: 1 evaluated samples and described their ideal cheese using intensity scales and 1 did the same using CATA questions. Both methodologies provided similar information about the sensory characteristics of the Minas Frescal cheeses, the description of the ideal product, and directions for product reformulation. The ideal Minas Frescal cheese was characterized by high moisture, intense white color, homogeneous mass, typical Minas Frescal cheese aroma and flavor, softness, and juiciness. For the intensity scales, the recommendation was to increase the typical aroma and flavor, salty taste, and juiciness, and to decrease the bitter flavor; for the CATA questions, only increasing the typical Minas Frescal cheese flavor was important for all classes of cheeses. Even for a heterogeneous product with no defined manufacturing protocol, both methodologies presented satisfactory results that should be considered for use by cheese producers and the dairy industry.

Viabilidade de Escherichia coli O153:H25, O113:H21 e O111: H8 (STEC não-O157) produtoras de toxina Shiga em queijo minas frescal / Viability of Escherichia coli O153: H25, O113:H21 e O111:H8 (STEC non-O157) Shiga toxin-producing in minas frescal cheese

A existência de um reservatório animal é de grande importância na transmissão de Escherichia coli, produtora de toxina shiga (STEC) aos humanos. Epidemiologicamente, o sorotipo O157:H7 tem sido o mais envolvido em surtos de doença humana causada por STEC, porém surtos envolvendo STEC não pertencentes ao sorogrupo O157 (STEC não-O157) têm sido descritos. Inúmeros trabalhos constatam uma elevada ocorrência destes microrganismos em fezes de bovinos no Brasil, entretanto, pouco se sabe sobre a transmissão destes aos produtos de origem animal em nosso país. Neste trabalho, foi avaliada a viabilidade de E.coli O153:H25; O113:H21 e O111:H8 em Queijo Minas Frescal (QMF), produzido com inóculos de STEC não O157: H7 e armazenados a 8ºC. Realizaram-se contagens de E. coli e psicrotróficos totais após o processamento do queijo e com intervalos de sete e quinze dias. Foi observado aumento nas contagens de E. coli STEC não O157: H7 e psicrotróficos totais logo após o processamento do QMF, bem como durante o armazenamento a 8ºC, temperatura máxima recomendada pela legislação brasileira. Demonstra-se que, caso haja contaminação da matéria-prima com STEC não O157: H7 (deste estudo), o processamento do QMF não elimina os microrganismos e a temperatura máxima recomendada pela legislação não inibe a multiplicação bacteriana, mantendo-se o risco à população. Reforça-se, portanto, a atenção à qualidade da matéria-prima, das ferramentas de qualidade no campo e na indústria de alimentos para garantir a inocuidade do produto final.(AU)

The existence of an animal reservoir is of great importance in the transmission of shiga toxin-producing E. coli (STEC) to humans. Epidemiologically serotype O157:H7 has been the most involved in human disease outbreaks caused by STEC, however STEC not belonging to serogroup O157 (STEC non-O157) have been described in outbreaks. Studies have revealed a risk in occurrence of these organisms in feces of cattle in Brazil, but little is known about the transmission to animal's products origin in our country. This study evaluated the viability of E. coli O153:H25, O113:H21 and O111:H8 in Minas Frescal Cheese (MFC), produced with non- STEC O157:H7 and stored at 8ºC. Counts of E. coli and total psychrotrophic were performed after processing and at intervals seven and fifteen days. There were an increase in the counts of E. coli and total psychrotrophic just after MFC processing as well as during storage at 8°C. The results demonstrated that, if the raw material (milk) is contaminated with STEC non O157:H7 (this study), the MFC processing does not eliminate the microorganisms and the maximum temperature recommended does not eliminate bacterial growth, keeping the risk to the population. The results reinforces, the attention to the quality of the raw material, the quality tools in the field and in the food industry to ensure the safety of final products.(AU)

Viabilidade de Escherichia coli O153:H25, O113:H21 e O111:H8 (STEC não-O157) produtoras de toxina Shiga em queijo minas frescal / Viability of Escherichia coli O153:H25, O113:H21 e O111:H8 (STEC non-O157) Shiga toxin-producing in minas frescal cheese

A existência de um reservatório animal é de grande importância na transmissão de Escherichia coli, produtora de toxina shiga (STEC) aos humanos. Epidemiologicamente, o sorotipo O157:H7 tem sido o mais envolvido em surtos de doença humana causada por STEC, porém surtos envolvendo STEC não pertencentes ao sorogrupo O157 (STEC não-O157) têm sido descritos. Inúmeros trabalhos constatam uma elevada ocorrência destes microrganismos em fezes de bovinos no Brasil, entretanto, pouco se sabe sobre a transmissão destes aos produtos de origem animal em nosso país. Neste trabalho, foi avaliada a viabilidade de E.coli O153:H25; O113:H21 e O111:H8 em Queijo Minas Frescal (QMF), produzido com inóculos de STEC não O157: H7 e armazenados a 8ºC. Realizaram-se contagens de E. coli e psicrotróficos totais após o processamento do queijo e com intervalos de sete e quinze dias. Foi observado aumento nas contagens de E. coli STEC não O157: H7 e psicrotróficos totais logo após o processamento do QMF, bem como durante o armazenamento a 8ºC, temperatura máxima recomendada pela legislação brasileira. Demonstra-se que, caso haja contaminação da matéria-prima com STEC não O157: H7 (deste estudo), o processamento do QMF não elimina os microrganismos e a temperatura máxima recomendada pela legislação não inibe a multiplicação bacteriana, mantendo-se o risco à população. Reforça-se, portanto, a atenção à qualidade da matéria-prima, das ferramentas de qualidade no campo e na indústria de alimentos para garantir a inocuidade do produto final.

The existence of an animal reservoir is of great importance in the transmission of shiga toxin-producing E. coli (STEC) to humans. Epidemiologically serotype O157:H7 has been the most involved in human disease outbreaks caused by STEC, however STEC not belonging to serogroup O157 (STEC non-O157) have been described in outbreaks. Studies have revealed a risk in occurrence of these organisms in feces of cattle in Brazil, but little is known about the transmission to animal's products origin in our country. This study evaluated the viability of E. coli O153:H25, O113:H21 and O111:H8 in Minas Frescal Cheese (MFC), produced with non- STEC O157:H7 and stored at 8ºC. Counts of E. coli and total psychrotrophic were performed after processing and at intervals seven and fifteen days. There were an increase in the counts of E. coli and total psychrotrophic just after MFC processing as well as during storage at 8°C. The results demonstrated that, if the raw material (milk) is contaminated with STEC non O157:H7 (this study), the MFC processing does not eliminate the microorganisms and the maximum temperature recommended does not eliminate bacterial growth, keeping the risk to the population. The results reinforces, the attention to the quality of the raw material, the quality tools in the field and in the food industry to ensure the safety of final products.

PCR and ELISA (VIDAS ECO O157(®)) Escherichia coli O157:H7 identification in Minas Frescal cheese commercialized in Goiânia, GO.

Escherichia coli O157:H7 has been incriminated in food poisoning outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome in many countries. Considering the high susceptibility of Minas Frescal cheese to contamination by E. coli O157:H7, the aim of this study was to determine the occurrence of this pathogen through PCR (Polymerase Chain Reaction) and ELISA (VIDAS ECO O157(®), bioMérieux, Lyon, France) test. Thirty cheese samples manufactured by artisan farmhouse producers were collected from open-air markets in Goiânia and thirty from industries under Federal Inspection located in Goiás State which trade their products in supermarkets in Goiânia. E. coli O157:H7 was detected in 6.67% samples collected in open air markets using ELISA, and 23,33% with PCR. The pathogen was not detected in samples from industries under Federal Inspection.

PCR and ELISA (VIDAS ECO O157(R)) Escherichia coli O157: H7 identification in Minas Frescal cheese commercialized in Goiânia, GO

Escherichia coli O157:H7 has been incriminated in food poisoning outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome in many countries. Considering the high susceptibility of Minas Frescal cheese to contamination by E. coli O157:H7, the aim of this study was to determine the occurrence of this pathogen through PCR (Polymerase Chain Reaction) and ELISA (VIDAS ECO O157®, bioMérieux, Lyon, France) test. Thirty cheese samples manufactured by artisan farmhouse producers were collected from open-air markets in Goiânia and thirty from industries under Federal Inspection located in Goiás State which trade their products in supermarkets in Goiânia. E. coli O157:H7 was detected in 6.67% samples collected in open air markets using ELISA, and 23,33% with PCR. The pathogen was not detected in samples from industries under Federal Inspection.(AU)

PCR and ELISA (VIDAS ECO O157®) Escherichia coli O157:H7 identification in Minas Frescal cheese commercialized in Goiânia, GO

Escherichia coli O157:H7 has been incriminated in food poisoning outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome in many countries. Considering the high susceptibility of Minas Frescal cheese to contamination by E. coli O157:H7, the aim of this study was to determine the occurrence of this pathogen through PCR (Polymerase Chain Reaction) and ELISA (VIDAS ECO O157®, bioMérieux, Lyon, France) test. Thirty cheese samples manufactured by artisan farmhouse producers were collected from open-air markets in Goiânia and thirty from industries under Federal Inspection located in Goiás State which trade their products in supermarkets in Goiânia. E. coli O157:H7 was detected in 6.67% samples collected in open air markets using ELISA, and 23,33% with PCR. The pathogen was not detected in samples from industries under Federal Inspection.

Perfil de resistência de aeromonas spp. Isolada no fluxograma de produção do queijo minas frescal industrial e artesanal / Resistance profile of aeromonas spp. Isolated in dairy products industry

Bactérias do gênero Aeromonas podem ser isoladas de amostras clínicas, alimentares e ambientais. Para os derivados lácteos a contaminação decorre da presença do agente na matéria prima utilizada para produção, ou devido a condições higiênicas insatisfatórias durante o processamento. Considerando a importância da Aeromonas spp. como patógeno emergente de doenças de origem alimentar, o uso indiscriminado de antimicrobianos nas patologias humanas, além do papel fundamental que o leite e seus derivados representam na alimentação humana, o presente estudo teve por objetivo verificar o comportamento dos isolados frente à ação de antimicrobianos de uso comum. Foram pesquisados produtos e pontos do fluxograma do processamento de derivados lácteos, que poderiam veicular o gênero Aeromonas e permitir sua ocorrência nos produtos finais, ou atuar como disseminadores do micro-organismo nas diferentes etapas do processamento do queijo Minas Frescal e do queijo Colonial. Os resultados demonstram altos níveis de resistência: 33,6% dos 98 isolados de Aeromonas spp. foram resistentes a todos os 15 antimicrobianos testados, correspondendo a 7,5% dos isolados do queijo Colonial e 51,7% do queijo Minas Frescal, ocorrendo, portanto, maior resistência entre as amostras coletadas na indústria. 100% dos isolados apresentaram resistência múltipla, resultado que revela um perfil preocupante para saúde pública.(AU)

Bacteria of the genus Aeromonas can be isolated from clinical, food and environmental samples. For dairy products contamination can be caused by the presence of the agent in the feedstock used for production or due to unsatisfactory hygienic conditions during processing. The present study aimed to assess Aeromonas isolates behavior in the presence of common antimicrobials, considering the importance of Aeromonas spp. as an emerging pathogen of foodborne diseases, the indiscriminate use of antimicrobials to treat human pathologies, in addition to the role that milk and its derivatives play in human feeding habits. The processing steps during dairy products manufacturing were studied looking for vulnerable points or products where Aeromonas could be present and, subsequently, either pass it to the final products or disseminate the micro-organism in different processing stages of Minas and Colonial cheeses. The results show high levels of resistance: 33.6% of 98 isolates of Aeromonas spp. were resistant to all 15 antibiotics tested, corresponding to 7.5% of the isolates from Colonial cheese and 51.7% from Minas cheese, thus showing greater resistance among the samples collected in the industry. Multiple antimicrobial resistance was verified in 100% of the isolates, which reveals a profile which is alarming for public health.(AU)

Perfil de resistência de aeromonas spp. Isolada no fluxograma de produção do queijo minas frescal industrial e artesanal / Resistance profile of aeromonas spp. Isolated in dairy products industry

Bactérias do gênero Aeromonas podem ser isoladas de amostras clínicas, alimentares e ambientais. Para os derivados lácteos a contaminação decorre da presença do agente na matéria prima utilizada para produção, ou devido a condições higiênicas insatisfatórias durante o processamento. Considerando a importância da Aeromonas spp. como patógeno emergente de doenças de origem alimentar, o uso indiscriminado de antimicrobianos nas patologias humanas, além do papel fundamental que o leite e seus derivados representam na alimentação humana, o presente estudo teve por objetivo verificar o comportamento dos isolados frente à ação de antimicrobianos de uso comum. Foram pesquisados produtos e pontos do fluxograma do processamento de derivados lácteos, que poderiam veicular o gênero Aeromonas e permitir sua ocorrência nos produtos finais, ou atuar como disseminadores do micro-organismo nas diferentes etapas do processamento do queijo Minas Frescal e do queijo Colonial. Os resultados demonstram altos níveis de resistência: 33,6% dos 98 isolados de Aeromonas spp. foram resistentes a todos os 15 antimicrobianos testados, correspondendo a 7,5% dos isolados do queijo Colonial e 51,7% do queijo Minas Frescal, ocorrendo, portanto, maior resistência entre as amostras coletadas na indústria. 100% dos isolados apresentaram resistência múltipla, resultado que revela um perfil preocupante para saúde pública.

Bacteria of the genus Aeromonas can be isolated from clinical, food and environmental samples. For dairy products contamination can be caused by the presence of the agent in the feedstock used for production or due to unsatisfactory hygienic conditions during processing. The present study aimed to assess Aeromonas isolates behavior in the presence of common antimicrobials, considering the importance of Aeromonas spp. as an emerging pathogen of foodborne diseases, the indiscriminate use of antimicrobials to treat human pathologies, in addition to the role that milk and its derivatives play in human feeding habits. The processing steps during dairy products manufacturing were studied looking for vulnerable points or products where Aeromonas could be present and, subsequently, either pass it to the final products or disseminate the micro-organism in different processing stages of Minas and Colonial cheeses. The results show high levels of resistance: 33.6% of 98 isolates of Aeromonas spp. were resistant to all 15 antibiotics tested, corresponding to 7.5% of the isolates from Colonial cheese and 51.7% from Minas cheese, thus showing greater resistance among the samples collected in the industry. Multiple antimicrobial resistance was verified in 100% of the isolates, which reveals a profile which is alarming for public health.

Genetic heterogeneity of Escherichia coli strains isolated from raw milk, Minas Frescal cheese, and food handlers / Heterogeneidade genética de cepas de Escherichia coli isoladas de leite cru, queijo Minas Frescal e manipuladores

From February 2004 to March 2005, 140 samples of food handlers - hands and nostrils - (92), raw milk (24), and minas frescal cheese (24) were analyzed for the presence of Escherichia coli in a dairy processing plant of Goiás State. Forty-seven E. coli strains were obtained and compared by DNA macrorestriction patterns obtained from pulsed-field gel electrophoresis following XbaI restriction in order to investigate the possible sources of cheese contaminations. Based on PFGE genotyping, one strain isolated from food the hands of a handler and five strains isolated from raw milk were identical or closely related to six strains from cheese suggesting, in these cases, the probable source of E. coli contamination in cheeses. No strain isolated from the nostrils was related to those found in cheeses or milk strains. The results showed high diversity among the strains, demonstrating a lack of predominance of an endemic clone in the dairy plant. This paper highlights the usefulness of PFGE as an epidemiological tool for determining the source of E. coli contamination in the food industry.(AU)

Durante um ano, de fevereiro de 2004 a março de 2005, 140 amostras retiradas das mãos e das narinas de manipuladores de alimentos (92), do leite cru (24) e do queijo-de-minas frescal (24) foram analisadas para a presença de Escherichia coli, em um laticínio do Estado de Goiás. As 47 cepas obtidas foram comparadas por macrorrestrição do DNA com enzima XbaI, seguida de eletroforese em gel em campo pulsado (PFGE), a fim de investigar as possíveis fontes de contaminação do queijo. Baseado na genotipagem pelo PFGE, uma cepa obtida do leite cru e cinco cepas obtidas dos manipuladores mostraram similaridade maior que 80 por cento com seis cepas isoladas do queijo, denotando forte correlação genética entre elas e sugerindo, nestes casos, a fonte provável de contaminação do produto final. Nenhuma cepa isolada do nariz foi relacionada às isoladas do queijo ou do leite. Os resultados mostraram grande diversidade entre as cepas, demonstrando ausência de um clone endêmico no laticínio avaliado. Este estudo destaca a utilidade do PFGE como uma ferramenta importante em investigações epidemiológicas e na determinação de possíveis fontes de contaminação por E. coli na indústria de alimentos.(AU)

Genetic heterogeneity of Escherichia coli strains isolated from raw milk, Minas Frescal cheese, and food handlers / Heterogeneidade genética de cepas de Escherichia coli isoladas de leite cru, queijo Minas Frescal e manipuladores

From February 2004 to March 2005, 140 samples of food handlers - hands and nostrils - (92), raw milk (24), and minas frescal cheese (24) were analyzed for the presence of Escherichia coli in a dairy processing plant of Goiás State. Forty-seven E. coli strains were obtained and compared by DNA macrorestriction patterns obtained from pulsed-field gel electrophoresis following XbaI restriction in order to investigate the possible sources of cheese contaminations. Based on PFGE genotyping, one strain isolated from food the hands of a handler and five strains isolated from raw milk were identical or closely related to six strains from cheese suggesting, in these cases, the probable source of E. coli contamination in cheeses. No strain isolated from the nostrils was related to those found in cheeses or milk strains. The results showed high diversity among the strains, demonstrating a lack of predominance of an endemic clone in the dairy plant. This paper highlights the usefulness of PFGE as an epidemiological tool for determining the source of E. coli contamination in the food industry.

Durante um ano, de fevereiro de 2004 a março de 2005, 140 amostras retiradas das mãos e das narinas de manipuladores de alimentos (92), do leite cru (24) e do queijo-de-minas frescal (24) foram analisadas para a presença de Escherichia coli, em um laticínio do Estado de Goiás. As 47 cepas obtidas foram comparadas por macrorrestrição do DNA com enzima XbaI, seguida de eletroforese em gel em campo pulsado (PFGE), a fim de investigar as possíveis fontes de contaminação do queijo. Baseado na genotipagem pelo PFGE, uma cepa obtida do leite cru e cinco cepas obtidas dos manipuladores mostraram similaridade maior que 80 por cento com seis cepas isoladas do queijo, denotando forte correlação genética entre elas e sugerindo, nestes casos, a fonte provável de contaminação do produto final. Nenhuma cepa isolada do nariz foi relacionada às isoladas do queijo ou do leite. Os resultados mostraram grande diversidade entre as cepas, demonstrando ausência de um clone endêmico no laticínio avaliado. Este estudo destaca a utilidade do PFGE como uma ferramenta importante em investigações epidemiológicas e na determinação de possíveis fontes de contaminação por E. coli na indústria de alimentos.