CARNOBACTERIUM MALTAROMATICUM AS BIOPROTECTIVE CULTURE AGAINST SPOILAGE BACTERIA IN MEAT PRODUCTS

O consumo médio de produtos cárneos e carne está aumentando gradativamente ao longo dos anos. Em 2019, A produção mundial de carne é de 335,2 milhões de toneladas (peso equivalente em carcaça). A produção de carne bovina e suína representam 72,2 e 110,5 milhões de toneladas, respectivamente. A carne é um alimento rico em nutrientes, que em conjunto com pH moderado e alta atividade de água, favorece o crescimento microbiano. A indústria alimentícia e vendedores estimam que 40% da carne produzida é perdida por causa da deterioração microbiana, acarretando impactos econômicos, sociais e ambientais. Portanto, métodos de preservação da carne e prolongamento da sua vida de prateleira são de suma importância para a indústria e consumidores. A técnica de biopreservação consiste no uso de uma bactéria artificialmente ou naturalmente presente no alimento, inoculada em concentrações suficientes para inibir o crescimento de outras bactérias patogênicas e deteriorantes. Essa técnica é uma opção atrativa para preservar os produtos cárneos em razão dos mínimos impactos nos parâmetros nutricionais e sensoriais do produto. Bactérias ácido lácticas (LAB lactic acid bacteria) são fortemente empregadas com essa finalidade. A Carnobacterium maltaromaticum apresenta características desejáveis de uma cultura bioprotetora. O gênero Carnobacterium é capaz de crescer em condições ambientais adversas como baixas temperatura usadas na refrigeração de alimentos, altas concentrações de sal e moderada variação de pH. Bactérias desse gênero são naturalmente encontradas em vários alimentos e ambientes. Alguns representantes, como C. maltaromaticum e Carnobacterium divergens, apresentam espectro inibitório de bactérias deteriorantes e patogênicas. Dessa forma, este estudo objetivou avaliar o potencial efeito inibitório de C. maltaromaticum previamente isolada de bife australiano (longissimus dorsi) armazenado a vácuo com 140 d de vida de prateleira a -1 °C Três cepas de C. maltaromaticum (CM_B824, CM_B827 e CM_B829) e o pool dessas cepas (CM) foram testadas contra Pseudomonas fluorescens ATCC®1355TM (PF), pool de três cepas de Brochothrix thermosphacta (BT- ATCC®11509TM, s109 e s153) e Listeria monocytogenes ATCC®19117 (LM) in vitro. As bactérias foram crescidas em caldo BHI por 24 h a 25 °C (deteriorantes) e a 37 °C (patógeno), dos quais 7 log UFC/mL de Carnobacteria e 3 log UFC/mL de bactéria alvo foram crescidos em co-cultura em BHI por 72 h a 15 °C em condições aeróbia e anaeróbia. Todos os tratamentos contendo Carnobacteria tiveram um efeito inibitório elevado contra BT e LM, com diferentes desempenhos de acordo com a atmosfera. A inibição da Listeria corrobora com estudos anteriores feitos com as mesmas cepas de Carnobacteria em outras temperaturas. O crescimento da PF foi inibido com maior eficácia em anaerobiose. A Carnobacteria atingiu altas concentrações independente da presença do competidor nas condições testadas. O efeito bioprotetor do pool de Carnobacteria foi testado em duas matrizes alimentares (carne moída crua e presunto cozido fatiado) contra P. fluorescens ATCC®1355TM e B. thermosphacta (ATCC®11509TM, s109 e s153). A carne moída foi inoculada com as concentrações de 6,76 (CM), 3,49 (BT) e 3,48 (PF) log UFC/mL e o presunto com 6,37 (CM), 3,99 (BT) e 3,79 (PF) UFC/m. A carne moída foi armazenado em atmosfera modificada com elevada concentração de oxigênio (MAP - 66% O2, 4% N2 e 30% CO2) por 7 d (inicialmente a 4 °C por 3 d e depois a 8°C por 4 d); e o presunto foi armazenado em MAP com baixa concentração de oxigênio (70% N2 e 30% CO2) por 28 d (inicialmente a 4 °C por 10 d e depois a 8°C por 18 d). As análises microbiológicas e físico químicas (pH, cor instrumental e composição gasosa) foram desenvolvidas para 0, 3 e 7 d (carne moída) e 0, 5, 10, 19 e 28 d (presunto) de armazenamento. Considerando a carne moída, o pool de Carnobacteria reduziu a contagem da população de BT e PF inoculadas e Brochothrix spp. e Pseudomonas spp. naturalmente presentes no alimento. A contagem final de CM não foi afetada pela BT. Tratamentos com CM tiveram leve caimento de pH e elevada concentração de CO2. As características de qualidade da carne moída praticamente não foram afetadas. Considerando o presunto, ambas bactérias deteriorantes foram inibidas. O crescimento de CM não foi afetado por nenhuma das bactérias deteriorantes. A presença da CM reduziu fracamente o pH e elevou a concentração de CO2. CM manteve as características de cor instrumental preservadas e abaixo da rejeição teórica do produto pelo consumidor. Os resultados se justificam nas habilidades da Carnobacteria: inibir bactérias deteriorantes; crescer e manter uma população elevada e estável até o final da vida de prateleira; causar impactos mínimos nas características físico-químicas dos produtos cárneos. Dessa forma, o uso da C. maltaromaticum pode ser considerado como uma alternativa natural para a bioproteção de produtos cárneos.

The consumption of meat and meat products has increased over the years. In 2019, the world meat production was 335.2 million tons (carcass weight equivalent), and beef and pork production represented 72.2 and 110.5 million tons, respectively. Meat contains a high amount of nutrients, which along with the moderate pH and the high-water content favors microbial growth. Food industry and retailers estimate that 40% of the meat produced is lost due to microbial spoilage, leading to economic, social and environmental impacts. Therefore, methods to preserve meat and extend the product shelf life play a crucial role for both consumers and industry. Biopreservation consists of using a microorganism naturally present or artificially inoculated on the food in concentrations high enough to inhibit the growth of other spoilage and pathogenic microorganisms. This preservation method is an interesting alternative to preserve meat products due to the minimal impacts on the nutritional and sensorial parameters of the product. Lactic acid bacteria are strongly used for this method. Carnobacterium maltaromaticum shows desirable characteristics as a bioprotective culture. The genus Carnobacterium can grow under adverse environmental conditions, such as low temperature, high salt concentration and moderate pH range. Bacteria from this genus are naturally found in several foods and environments. Some representatives, C. maltaromaticum and Carnobacterium divergens have inhibitory effects against spoilage and pathogenic microorganisms. Thus, this study aimed to evaluate the potential inhibitory effect of C. maltaromaticum previously isolated from vacuum packaged Australian beef (longissimus dorsi) with 140 d of shelf life at -1 °C. Three strains of C. maltaromaticum (CM_B824, CM_B827 and CM_B829) and the pool of these strains (CM) were tested against Pseudomonas fluorescens ATCC®1355TM (PF), a pool of three strains of Brochothrix thermosphacta (ATCC®11509TM, s109 and s153) (BT) and Listeria monocytogenes ATCC®19117 (LM) in vitro. The bacteria were grown in BHI broth for 24 h at 25 °C (spoilers) and at 37 °C (pathogen), from which 7 log CFU/mL of Carnobacteria and 3 log CFU/mL of target bacteria were cocultured in BHI broth for 72 h at 15 °C in aerobic and anaerobic conditions. All treatments containing Carnobacteria showed a high inhibitory effect against BT and LM, the performance varied according to the atmosphere. The inhibition of Listeria corroborates with previous studies conducted with the same Carnobacteria strains in other temperatures. The PF growth was more efficiently inhibited in anaerobiosis. Carnobacteria reached high concentrations independently of the presence of the competitor under the conditions tested. Moreover, the bioprotective effect of the pool of Carnobacteria was tested in two food matrices (raw ground beef and sliced cooked ham) against P. fluorescens ATCC®1355TM and B. thermosphacta (ATCC®11509TM, s109 and s153). The ground beef was inoculated with the concentrations of 6.76 (CM), 3.49 (BT) and 3.48 (PF) log CFU/mL and the sliced cooked ham with 6.37 (CM), 3.99 (BT) and 3.79 (PF). The ground beef was stored under modified atmosphere with high oxygen content (MAP - 66% O2, 4% N2 and 30% CO2) for 7 d (initially at 4 °C for 3 d and after at 8°C for 4 d); and the sliced cooked ham was stored under MAP with low oxygen content (70% N2 and 30% CO2) for 28 d (initially at 4 °C for 10 d and after at 8°C for 18 d). The microbiological and physicochemical analyses (pH, instrumental colors and headspace gas composition) were performed at the 0, 3 and 7 d (ground beef) and at 0, 5, 10, 19 and 28 d (ham) of storage. Results for ground beef: the pool of Carnobacteria significantly reduced the counts of BT and PF inoculated populations and indigenous Brochothrix spp. and Pseudomonas spp. The final counts of CM were not influenced by BT presence. CM treatments showed slight pH values decrease and increase in CO2 package concentration. The quality characteristics of the ground beef were practically not affected by Carnobacteria. Results for sliced cooked ham: both spoilage bacteria were inhibited. The CM growth was not influenced by the spoilage bacteria. The presence of CM weakly reduced the pH values and increased the CO2 concentration in package. CM maintained the color of the product and color differences during storage were below theoretical decline of the product by the costumer. The results corroborate with the abilities of Carnobacteria to inhibit spoilage bacteria; to grow and maintain its population at a high number until the end of storage and has low impact on the physical-chemical characteristics of the meat products. Thus, the use of C. maltaromaticum can be considered a natural alternative to the bioprotection of meat products.

Bio-preservation of ground beef meat by Enterococcus faecalis CECT7121

Meat and particularly ground beef is frequently associated with Food Poisoning episodes and breeches in Food Safety. The main goal of this research was to evaluate the bactericide effect of the probiotic Enterococcus faecalis CECT7121, against different pathogens as: Escherichia coli O157:H7, Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes, inoculated in ground beef meat. Three studies were performed to evaluate the inhibition of E. faecalis CECT7121 on ground beef meat samples inoculated with pathogens: Study I: Samples (100 g meat) were inoculated with pathogens (10³ CFU/g)) and E. faecalis CECT7121 (10(4) CFU/g) simultaneously. Study II: Samples were inoculated with E. faecalis CECT7121 24 h before the pathogens. Study III: E. faecalis CECT7121were inoculated 24 h after pathogens. The viable counts were performed at 0, 24, 48 and 72 h post-inoculation. The simultaneous inoculation of E. faecalis CECT7121 with E. coli O157:H7 strains resulted in the absence of viable counts of bacteria at 72 h post-treatment. However, when the probiotic was added 24 h before and 24 h after the pathogen E. coli O157:H7, viable cells were not detected at 24 h and 48 h post-treatment, respectively. Consistently, neither S. aureus nor Cl. perfringens viable bacteria were detected at 48 h in whole assays when inoculated with E. faecalis CECT7121. The same trend than described before was obtained after applying the 3 models assayed for L. monocytogenes. The current assays demonstrated the bactericide activity of E. faecalis CECT7121 strain on bacterial pathogens in ground beef meat.(AU)

Molecular characterization of Listeria monocytogenes isolated from animal products in a city of Northern Brazil

Listeria monocytogenes, a foodborne pathogen causes listeriosis, a fatal disease in about 30% of cases that affects mainly immunocompromised persons. The aim of this research was to characterize L. monocytogenes pulsed-field gel electrophoresis (PFGE) types isolated from meat products collected at public markets in Araguaina city, TO. Sixty samples of raw ground beef and frescal sausage were analyzed during the second half of 2008. Five out of 30 samples (16.7%) of raw ground beef tested positive for L. monocytogenes, three of which were classified as serotype 1/2b and two as serotype 4b. Among the 30 samples of sausage collected, two strains of L. monocytogenes were isolated (6.7%), one of them belonging to serotype 1/2a and the other belonging to serotype 1/2b. The restriction enzymes used were ApaI and SmaI. Similarities among the strains were determined by Dice coefficient. The macro restriction profile obtained by using SmaI enzyme allowed the distribution of seven strains in two clusters, two pulsotypes and two subtypes. The result indicates that L. monocytogenes isolates, belonging to serotype 4b, 1/2a and 1/2b, are strongly correlated within the same serotype group, and in some cases among different serotypes, suggesting that they have a common source.

Listeria monocytogenes é um patógeno de origem alimentar que causa a listeriose, doença fatal em aproximadamente 30% dos casos, e que afeta principalmente pessoas imunocomprometidas. O presente trabalho teve como objetivo analisar os perfis PFGE de cepas de L. monocytogenes isoladas de produtos de origem animal, obtidos em mercados públicos da cidade de Araguaína, TO. Foram analisadas 60 amostras de carne moída crua e de linguiça frescal, no segundo semestre de 2008. Cinco (16,7%) das 30 amostras de carne moída crua foram positivas ao patógeno, sendo que três pertenciam ao sorotipo 1/2b e duas ao sorotipo 4b. Das 30 amostras de linguiça mista frescal, duas (6,7%) foram positivas para L. monocytogenes, sendo uma do sorotipo 1/2a e outra do 1/2b. Foram utilizadas as enzimas de restrição ApaI e SmaI. A similaridade entre eles foi determinada pelo coeficiente de Dice. A análise do perfil de macrorestrição com a enzima SmaI permitiu a distribuição dos sete isolados em dois clusters, dois pulsotipos e dois subtipos. Os resultados permitiram concluir que os isolados de L. monocytogenes sorotipos 4b, 1/2a e 1/2b foram fortemente correlacionados dentro dos mesmos sorotipos e em alguns casos entre diferentes sorotipos, sugerindo uma fonte comum.

Molecular characterization of Listeria monocytogenes isolated from animal products in a city of Northern Brazil

Listeria monocytogenes, a foodborne pathogen causes listeriosis, a fatal disease in about 30% of cases that affects mainly immunocompromised persons. The aim of this research was to characterize L. monocytogenes pulsed-field gel electrophoresis (PFGE) types isolated from meat products collected at public markets in Araguaina city, TO. Sixty samples of raw ground beef and frescal sausage were analyzed during the second half of 2008. Five out of 30 samples (16.7%) of raw ground beef tested positive for L. monocytogenes, three of which were classified as serotype 1/2b and two as serotype 4b. Among the 30 samples of sausage collected, two strains of L. monocytogenes were isolated (6.7%), one of them belonging to serotype 1/2a and the other belonging to serotype 1/2b. The restriction enzymes used were ApaI and SmaI. Similarities among the strains were determined by Dice coefficient. The macro restriction profile obtained by using SmaI enzyme allowed the distribution of seven strains in two clusters, two pulsotypes and two subtypes. The result indicates that L. monocytogenes isolates, belonging to serotype 4b, 1/2a and 1/2b, are strongly correlated within the same serotype group, and in some cases among different serotypes, suggesting that they have a common source.

Listeria monocytogenes é um patógeno de origem alimentar que causa a listeriose, doença fatal em aproximadamente 30% dos casos, e que afeta principalmente pessoas imunocomprometidas. O presente trabalho teve como objetivo analisar os perfis PFGE de cepas de L. monocytogenes isoladas de produtos de origem animal, obtidos em mercados públicos da cidade de Araguaína, TO. Foram analisadas 60 amostras de carne moída crua e de linguiça frescal, no segundo semestre de 2008. Cinco (16,7%) das 30 amostras de carne moída crua foram positivas ao patógeno, sendo que três pertenciam ao sorotipo 1/2b e duas ao sorotipo 4b. Das 30 amostras de linguiça mista frescal, duas (6,7%) foram positivas para L. monocytogenes, sendo uma do sorotipo 1/2a e outra do 1/2b. Foram utilizadas as enzimas de restrição ApaI e SmaI. A similaridade entre eles foi determinada pelo coeficiente de Dice. A análise do perfil de macrorestrição com a enzima SmaI permitiu a distribuição dos sete isolados em dois clusters, dois pulsotipos e dois subtipos. Os resultados permitiram concluir que os isolados de L. monocytogenes sorotipos 4b, 1/2a e 1/2b foram fortemente correlacionados dentro dos mesmos sorotipos e em alguns casos entre diferentes sorotipos, sugerindo uma fonte comum.

Presença de sulfito de sódio e sua influência nas características físico-químicas e microbiológicas de carnes bovinas moídas resfriadas / Sodium sulfite presence and its influence on physicochemical and microbiological characteristics of refrigerated ground beef

Background: Ground meat stands among the most consumed meat products, being its safety easily damaged by increasing the surface area of contact after grinding it, what facilitates the growth of pathogenic and deteriorating micro-organisms. In order to decrease the lost because of deterioration, trade has been utilizing fraudulent devices as the addition of intentional preservative such as sodium sulfite. This research aimed to identify the presence of sodium sulfite in cooled ground beef commercialized in business establishments located in Porto Alegre city, state of Rio Grande do Sul, Brazil. In addition, it addressed the evaluation of the influence of using this addictive in cooled ground meat front its physic-chemical and microbiological characteristics and residual capacity in the first 48 hours. Materials, Methods & Results: A total of 55 samples of cooled ground beef, from non-noble cuts, acquired in business establishments located in Porto Alegre city, were collected. Thus, microbiological analyses for total and thermotolerant coliphorms, Clostridium Sulfite reducers, Staphylococcus coagulase-positive, Salmonella sp. and a qualitative test, for verifying the presence of sodium sulfite, were carried out. Subsequently, half of the negative samples (24) in the sulfite test were separated, and 0.04 g of this addictive for each 50 g of ground beef (0.08%) was, then, added. In these samples, microbiological and color analyses, and the confirmation of the presence of sodium sulfite, at times 0, 24 and 48, after the simulation of fraud of the samples, were realized. Moreover, for the verification of safety, microbiological analyses for Salmonella sp., Staphylococcus coagulase-positive, Clostridium Sulfite reducers, total and thermo tolerant coliphorms were carried out. Data were analyzed by Analysis of Variance (ANOVA), with pairwise comparisons done by the Tukey test, for a level of significance of 5% (P < 0.05). Thereby, from the total of 55 (100%) analyzed samples of cooled ground beef, two (3.63%) showed the presence of sodium sulfite addictive, and the performed microbiological analyses indicated the presence of Salmonella sp. in eight (14.54%) samples. The values of L*, observed in the samples added with sulfite, maintained constant at times (zero, 24 and 48) compared with the control samples (without addition of sulfite) (42.92; 40.35 and 41.69, vs 44.67; 37.65 and 32.38, respectively), which demonstrated lost of luminosity in the course of time. Also, the values of a* and b* demonstrated stable at times (zero, 24 and 48) in the samples added with addictive (5.72; 6.89; 5.47 and 9.73; 10.69; 9.56, respectively), although this has not occurred with the control samples that presented a gradual lost of these tonalities (11.48; 7.21; 5.91 and 16.11; 12.88; 7.84, respectively) (P < 0.05). Discussion: The results indicated that two (3.63%) samples showed the presence of sodium sulfite, being in disagreement with the Ordinance n. 1004 that forbids the addition of addictive a in natura meat. The presence of Salmonella sp. in samples of ground meat contradicts the Resolution RDC n. 12 that advocates its absence in 25 g of meat products. Furthermore, the conducted analyses of color proved that the addition of addictive in meat maintains its fresh color and appearance, beyond inhibiting reactions of enzymatic and non-enzymatic darkness during processing and storage. By considering the obtained results, it may be concluded that despite of a low incidence of fraud for finding sodium sulfite, this practice exists in business establishments located in Porto Alegre city, state of Rio Grande do Sul, Brazil, even though it can be concealed for the lack of continuous studies and legal analytical inspections, endangering sanitary-hygienic conditions of the products.

Presença de sulfito de sódio e sua influência nas características físico-químicas e microbiológicas de carnes bovinas moídas resfriadas / Presença de sulfito de sódio e sua influência nas características físico-químicas e microbiológicas de carnes bovinas moídas resfriadas

Background: Ground meat stands among the most consumed meat products, being its safety easily damaged by increasing the surface area of contact after grinding it, what facilitates the growth of pathogenic and deteriorating micro-organisms. In order to decrease the lost because of deterioration, trade has been utilizing fraudulent devices as the addition of intentional preservative such as sodium sulfite. This research aimed to identify the presence of sodium sulfite in cooled ground beef commercialized in business establishments located in Porto Alegre city, state of Rio Grande do Sul, Brazil. In addition, it addressed the evaluation of the influence of using this addictive in cooled ground meat front its physic-chemical and microbiological characteristics and residual capacity in the first 48 hours.Materials, Methods & Results: A total of 55 samples of cooled ground beef, from non-noble cuts, acquired in business establishments located in Porto Alegre city, were collected. Thus, microbiological analyses for total and thermotolerant coliphorms, Clostridium Sulfite reducers, Staphylococcus coagulase-positive, Salmonella sp. and a qualitative test, for verifying the presence of sodium sulfite, were carried out. Subsequently, half of the negative samples (24) in the sulfite test were separated, and 0.04 g of this addictive for each 50 g of ground beef (0.08%) was, then, added.

Background: Ground meat stands among the most consumed meat products, being its safety easily damaged by increasing the surface area of contact after grinding it, what facilitates the growth of pathogenic and deteriorating micro-organisms. In order to decrease the lost because of deterioration, trade has been utilizing fraudulent devices as the addition of intentional preservative such as sodium sulfite. This research aimed to identify the presence of sodium sulfite in cooled ground beef commercialized in business establishments located in Porto Alegre city, state of Rio Grande do Sul, Brazil. In addition, it addressed the evaluation of the influence of using this addictive in cooled ground meat front its physic-chemical and microbiological characteristics and residual capacity in the first 48 hours.Materials, Methods & Results: A total of 55 samples of cooled ground beef, from non-noble cuts, acquired in business establishments located in Porto Alegre city, were collected. Thus, microbiological analyses for total and thermotolerant coliphorms, Clostridium Sulfite reducers, Staphylococcus coagulase-positive, Salmonella sp. and a qualitative test, for verifying the presence of sodium sulfite, were carried out. Subsequently, half of the negative samples (24) in the sulfite test were separated, and 0.04 g of this addictive for each 50 g of ground beef (0.08%) was, then, added.

Microrganismos patogênicos e indicadores de condições higiênico-sanitária em carne moída comercializada na cidade de Barra do Garças, MT / Pathogenic microorganisms and hygienic and sanitary conditions indicatorsin ground beef sold in Barra do Garças, MT

Este estudo objetivou averiguar a qualidade da carne moída comercializada em 10 estabelecimentos, açougues e supermercados, na cidade de Barra do Garças-MT, quanto a presença de Salmonella spp., e ocorrência de bactérias aeróbias mesófilas, coliformes totais, Escherichia coli, Staphylococcus aureus e fungos (bolores e leveduras). Para tanto, coletou-se três amostras por estabelecimento perfazendo um total de 30, que foram analisadas pelos métodos: tradicional para determinação da presença de Samonellaspp. e pelo método rápido, com placas Petrifilm TM 3M, para os demais microrganismos pesquisados. Destas 30 amostras, cinco (17%) foram positivas para a presença de Salmonellaspp., A contagem de S. aureus em 20% das amostras variaram de 2,7 x 102 UFC/g a 9,3 x 102 UFC/g.E. colifoi verificada em contagens de 1,0 x 101 UFC/g até 6,5 x 102 UFC/g. Encontrou-se Coliformes totais em 13,3% das amostras, com contagens entre 1,2 x 103 UFC/g a 4,4 x 104 UFC/g. As bactérias aeróbias mesófilas nas amostras variaram de 1,3 x 103 UFC/g a 2,7 x 105 UFC/g, e observou-se contagens de bolores e leveduras de 1,1 x 102 UFC/g a 4,4 x 104 UFC/g. A presença de cepas de Salmonellaspp. e S. aureus nas amostras de carne moída demonstram falha higiênica na manipulação desta, e representando risco a saúde dos consumidores.

This study aimed to investigate ground beef sold in 10 establishments, like as butcher shops and supermarkets, in the town of Barra do Garças-MT, for presence of Salmonella spp.,. and occurrence of mesophilic aerobic bacteria, coliforms, Escherichia coli and Staphylococcus aureus. For this, three samples were collected in the places of sale for a total of 30 samples which were analyzed by the methods: traditional (Salmonellaspp.) and fast using Petrifilm TM 3M plates (others microorganisms). Of these 30 samples, five (17%) were positive for the presence of Salmonellaspp., and 20% for S. aureuscounts ranging from 2,7 x 102 CFU/g to 9,3 x 102 CFU/g, and 100% with 1,0 x 101 CFU/g to 6,5 x 102 CFU/g in E. coli. Total coliforms were found in 13, 3% of the samples with counts between 1,2 x 103 CFU/gto 4,4 x 104 CFU/g, and yeast and mold counts between 1,1 x 102 CFU/g to 4,4 x 104 CFU/g and mesophilic aerobic bacteria between 1,3 x 103 to 2,7 x 104 CFU/g. The presence of Salmonellaspp. strains and S. aureusin ground beef samples showed fault in hygienic handling of these establishments, and still represent a health risk for consumers.

PREVALENCE OF SHIGA TOXIN-PRODUCING Escherichia coli IN SOUTHERN BRAZIL ISOLATED FROM GROUND BEEF AND RAW MILK / PREVALÊNCIA DE Escherichia coli PRODUTORA DE SHIGA TOXINAS ISOLADA DE CARNE MOÍDA E LEITE CRU NO SUL DO BRASIL

The aim of this work was to investigate the occurrence of shiga toxin-producing Escherichia coli (STEC) in ground beef and raw milk in Southern Brazil, and to study the fate of STEC isolates from cattle faeces in ground beef and milk, and its resistance in acid and alcoholic media. Among 464 E. coli isolated from ground beef and raw milk there were no stx1 and stx2 genes. The population of STEC isolates from cattle faeces was quite stable when inoculated in ground beef and increased in inoculated milk along the 120 hours of storage at 8oC.  These STEC isolates were inactivated when exposed to pH 2.5 and 3.0, but they were viable after eight hours at pH 4.0. The STEC isolates did not survive 48 hours in medium containing 12% ethanol. At 6% ethanol, STEC O174:H21, O163:H19 and O112:H2 have shown an increase in population and STEC O91:H21 and O22:H8 did not resist beyond 24 and 48 hours of incubation, respectively. The low prevalence of STEC in foods together with the attributes of the STEC found in Brazilian cattle could be among the reasons for the low prevalence of foodborne diseases caused by STEC in Brazil.KEY WORDS: Acid, ethanol, ground beef, milk, Shiga toxin-producing Escherichia coli.

O trabalho teve como objetivo determinar a ocorrência de Escherichia coli produtora de toxina Shiga (STEC) em carne moída e leite cru no sul do Brasil e estudar o comportamento de STEC isoladas de fezes de bovinos de corte e leite, verificando sua resistência em meios ácido e alcoólico. Não foram identificados genes stx1 e stx2 nas 464 E. coli isoladas de carne moída e leite cru. STEC isoladas de fezes de bovinos mantiveram populações estáveis e apresentaram crescimento, respectivamente, em carne moída e em leite experimentalmente contaminados, durante 120 horas a 8ºC. Esses isolados foram inativados quando expostos a pH 2,5 e 3,0, mas permaneceram viáveis após oito horas em pH 4,0. Os isolados de STEC não sobreviveram 48 horas em meio contendo 12% de etanol. Em 6% de etanol, STEC O174:H21, O163:H19 e O112:H2 apresentaram crescimento, ao passo que STEC O91:H21 e O22:H8 não resistiram além de 24 e 48 horas de incubação, respectivamente. A baixa prevalência de STEC em alimentos e as características das cepas encontradas em bovinos podem estar relacionadas com a baixa prevalência de enfermidades de origem alimentar causadas por STEC no Brasil.PALAVRAS-CHAVES: Ácido, carne moída, etanol, leite, Escherichia coli produtora de toxina Shiga.

Qualidade higiênico-sanitária de carne bovina moída e de embutidos frescais comercializados no sul do rio grande do sul, brasil

ABSTRACT Meat is an excellent medium for the growth of microorganisms and it can be responsible for the transmission of pathogenic bacteria to human. The aim of this work was to analyze the sanitary-hygienic quality of ground beef and fresh sausages commercialized in southern Brazil and to evaluate the resistance profile of Salmonella isolates to antimicrobial agents. The methods used in the thermotolerant coliforms counting and in the Salmonella isolation were according to Instrução Normativa nº 62/2003 from Brazils Ministério da Agricultura, Pecuária e Abastecimento. The Salmonella isolates were tested in regard to susceptibility to ampicilin (10 mcg), norfloxacin (10 mcg), tetracycline (30 mcg), nalidixic acid (30 mcg) and chloramphenicol (30 mcg) antimicrobials. From 67 samples analized, 12 were not within the limits established by the current legislation to thermotolerant coliforms and Salmonella. Salmonella Typhimurium was isolated from 1 (4.2%) sample of ground beef. Salmonella serotypes Typhimurium and Infantis were isolated from 2 (9.5%) samples of pork meat sausages, and serotypes Typhimurium, Infantis, Derby from 3 (13.6%) samples of poultry sausages. The 6 (100%) isolates were sensitive to norfloxacina and ampicilina. Three (50%) isolates were sensitive to tetracycline, 3 (50%) to chloramphenicol and 2 (33.3%) to nalidixic acid. The results highlight the importance of meat products from swine, cattle and chicken as sources of Salmonella contamination.

RESUMO A carne constitui excelente meio para a multiplicação de microrganismos, podendo ser responsável pela transmissão de doenças para o homem através de bactérias patogênicas. O trabalho teve por objetivo analisar a qualidade higiênico-sanitária da carne bovina moída e de embutidos frescais comercializados no sul do Brasil e avaliar o perfil de resistência dos isolados de Salmonella a agentes antimicrobianos. Os métodos utilizados na contagem de coliformes termotolerantes e no isolamento de Salmonella foram realizados de acordo com a Instrução Normativa nº 62/2003 do Ministério da Agricultura, Pecuária e Abastecimento. Os isolados de Salmonella foram testados quanto à sensibilidade aos antimicrobianos ampicilina (10 mcg), norfloxacina (10 mcg), tetraciclina (30 mcg), ácido nalidíxico (30 mcg) e cloranfenicol (30 mcg). De 67 amostras analisadas, doze não atenderam aos limites estabelecidos pela legislação vigente para coliformes termotolerantes e Salmonella. Salmonella Typhimurium foi isolada de uma (4,2%) amostra de carne moída. Salmonella sorotipos Typhimurium e Infantis foram isoladas de duas (9,5%) amostras de lingüiça suína e sorotipos Typhimurium, Infantis e Derby de três (13,6%) amostras de lingüiça de frango. Os seis (100%) isolados foram sensíveis à norfloxacina e à ampicilina. Três (50%) isolados foram sensíveis à tetraciclina, três (50%) ao cloranfenicol e dois (33,3%) ao ácido nalidíxico. Os resultados obtidos ressaltam a importância dos produtos cárneos de origem suína, bovina e avícola como fonte de contaminação por Salmonella.

Carne moída homogeneizada: especificações para sua produção e qualidade microbiológica / Homogenized ground beef: specifications for its production and microbiological quality

A carne moída é um produto que todo consumidor conhece, porém existe outra opção, a carne moída homogeneizada, que apesar de estar no mercado há mais de 20 anos, não é ainda conhecida, tanto na forma da sua obtenção, quanto sobre sua qualidade. Este trabalho teve como objetivo descrever como é a produção deste produto, bem como avalia a qualidade através de indicadores microbiológicos. A metodologia aplicada foi a colheita de 325 amostras de carne moída homogeneizada, em oitenta e três lojas, de uma rede de supermercados, escolhidas aleatoriamente, em nove estados da federação, no período de novembro de 2009 a abril de 2011. Os resultados foram avaliados segundo a RDC 12 da ANVISA verificando se as amostras estavam ou não dentro do padrão legal.(AU)

The ground beef is a product thatevery consumer knows, but there isanother option, the homogenizedground beef, which despite being inthe market for over 20 years, is notyet known, both in the form of obtainingit. as about its quality. Thisstudy had as targets to describehow to produce this type of productas well as assess the microbiologicalquality by means of indicators.The utilized methodology was thecollection of 325 samples of homogenizedground beef in 83 randomlyselected stores of a supermarketchain located in nine states of theBrazilian federation, held from November2009 to April 2011. The resultswere evaluated according tothe RDC ANVISA 12 to verify whetherthe analyzed samples were inthe legal standards.(AU)

Presence of extraintestinal pathogenic Escherichia coli in butcheries in Taquaritinga, SP, Brazil

The study was conducted in twenty-three butcheries in the city of Taquaritinga, State of São Paulo, Brazil, surveyed during a 10 months period. Among two hundred and eighty-seven Escherichia coli strains isolated from samples of ground beef, meat-grinding-machines and the hands of manipulators, five were recognized as extraintestinal pathogenic E. coli (ExPEC), showing virulence factors (P and S fimbriae, hemolysin and aerobactin) and presenting multidrug resistance. Retail-sold food may constitute an important vehicle for the dissemination of ExPEC in communities, giving rise to reasons for concern.

O trabalho foi desenvolvido em 23 açougues em Taquaritinga, Estado de São Paulo, Brasil, durante um período de 10 meses. De duzentas e oitenta e sete cepas de E.coli isoladas de carne moída, moedor de carne e mãos de manipuladores de carne, cinco foram caracterizadas como E. coli patogênica extraintestinal (ExPEC) apresentando fatores de virulência (fimbria P e S, hemolisina e aerobactina), assim como multiresistencia a drogas antimicrobianas. Retalhos de carne podem ser um veiculo importante para a disseminação de ExPEC, o que representa um motivo de preocupação.

Prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) strains in ground beef in São Paulo, Brazil

This study aims to assess the prevalence of Shiga toxin-producing Escherichia coli (STEC) in ground beef collected in two cities located in the State of São Paulo, Brazil. A total of 250 samples of raw ground beef were collected in local grocery stores during the period of March to December 2002 in the cities of Ribeirão Preto (114 samples) and Campinas (136 samples), São Paulo State, Brazil. The samples were processed according to standard methods. The resulting 591 E.coli colonies were screened for STEC by hybridization assays using the specific DNA probes, stx1,stx2 and eae. Further characterization of STEC isolates included the search for the ehxA sequence, detection of enterohemolysin and expression of Shiga toxin using the Vero cell assay. STEC isolates belonging to serotypes O93:H19, ONT:HNT, ONT:H7, and O174:HNT we recovered from four samples (3.5%) collected in Ribeirão Preto. All samples from Campinas were negative for STEC. Three of the strains carried stx2 and ehxA sequences while one harbored stx1,stx2 and ehxA sequences. Considering that among foods of animal origin, ground beef is an important vehicle for STEC transmission, these data emphasize the need of a closer surveillance of these microorganisms. They can survive in unfavorable conditions specially when the products are refrigerated or frozen for long periods of time and can be the cause of outbreaks affecting a great number of consumers.

O objetivo deste estudo foi verificar a ocorrência de Escherichia coli produtora de toxina Shiga (STEC) em amostras de carne moída crua comercializadas em duas cidades do Estado de São Paulo, Brasil. Um total de 250 amostras de carne moída crua foi coletado de açougues locais durante o período de Março a Dezembro de 2002, nas cidades de Ribeirão Preto (114 amostras) e de Campinas (136 amostras), Estado de São Paulo, Brasil. As amostras foram processadas de acordo com os métodos de referência. Um total de 591 colônias de E.coli foi submetido à técnica de hibridização de colônias usando sondas específicas de DNA para a detecção das seqüências stx1,stx2 e eae. Caracterizações adicionais das cepas STEC incluíram a pesquisa da seqüência ehxA, a detecção da enterohemolisina e a pesquisa da expressão de toxina Shiga utilizando testes com células Vero. Em quatro amostras (3,5%) coletadas em Ribeirão Preto, foram encontradas cepas STEC, mas todas aquelas da região de Campinas foram negativas. As cepas de STEC pertenciam aos sorotipos O93:H19, ONT:HNT, ONT:H7, e O174:HNT. Três cepas tinham o perfil stx2 ehxA e uma era portadora das seqüências stx1,stx2 e ehxA. Considerando que entre os alimentos de origem animal, a carne moída ainda representa um importante veículo de transmissão de STEC, estes dados alertam para a necessidade de uma vigilância da presença destes microrganismos capazes de sobreviver em condições desfavoráveis, especialmente quando os produtos são refrigerados ou congelados por longos períodos, podendo ser causas de importantes surtos afetando grande número de consumidores.

Isolation of Shiga toxigenic Escherichia coli from butcheries in Taquaritinga city, State of São Paulo, Brazil

Shiga toxigenic Escherichia coli (STEC) has been implicated as the cause of several human diseases. Samples (ground beef, grinding-machines and human hands) from 23 butcheries were assayed for E. coli using standard microbiological methods, and 287 isolates were submitted to polymerase chain reaction for the detection of stx 1, stx 2 and eae genes. Four STEC isolates were recovered, two from ground beef and two from grinding-machines; all harbored the stx 2 gene and were negative for the eae gene. All E. coli isolates including the four STEC were screened for antibiotic resistance. High levels of resistance against several antimicrobial agents were detected; those most commonly observed were to tetracycline (76.6%), amoxicillin (64.1%) and cephalothin (58.8%). Such high levels of antimicrobial resistance highlight the need for a more rational use of these agents in cattle.

Escherichia coli Shiga toxigênica (STEC) tem sido responsabilizada como o agente etiológico de diversas doenças nos seres humanos. Neste estudo foram analisadas amostras provenientes de 23 açougues (carne moída, moedor de carne e mãos de manipuladores) visando o isolamento de cepas de E. coli utilizando os métodos microbiológicos tradicionais. Um total de 287 cepas de E. coli, isoladas destas amostras, foram submetidas a reação em cadeia da polimerase para a detecção dos genes stx 1, stx 2 e eae. Foram identificadas 4 cepas STEC, 2 provenientes de carne moída e 2 provenientes do moedor de carne, todas as cepas apresentando o gene stx 2 e negativas para a presença do gene eae. Todas as cepas de E. coli, incluindo as 4 cepas STEC, foram examinadas para verificar a resistência antimicrobiana. Foram detectados altos níveis de resistência a diferentes agentes antimicrobianos, sendo as resistências mais elevadas para a tetraciclina (76,6%), amoxicilina (64,1%) e cefalotina (58,8%). Estes altos índices de resistência ressaltam a necessidade de uma utilização mais racional destes agentes no gado bovino.

PREVALENCE OF SHIGA TOXIN-PRODUCING Escherichia coli IN SOUTHERN BRAZIL ISOLATED FROM GROUND BEEF AND RAW MILK / PREVALÊNCIA DE Escherichia coli PRODUTORA DE SHIGA TOXINAS ISOLADA DE CARNE MOÍDA E LEITE CRU NO SUL DO BRASIL

The aim of this work was to investigate the occurrence of shiga toxin-producing Escherichia coli (STEC) in ground beef and raw milk in Southern Brazil, and to study the fate of STEC isolates from cattle faeces in ground beef and milk, and its resistance in acid and alcoholic media. Among 464 E. coli isolated from ground beef and raw milk there were no stx1 and stx2 genes. The population of STEC isolates from cattle faeces was quite stable when inoculated in ground beef and increased in inoculated milk along the 120 hours of storage at 8oC.  These STEC isolates were inactivated when exposed to pH 2.5 and 3.0, but they were viable after eight hours at pH 4.0. The STEC isolates did not survive 48 hours in medium containing 12% ethanol. At 6% ethanol, STEC O174:H21, O163:H19 and O112:H2 have shown an increase in population and STEC O91:H21 and O22:H8 did not resist beyond 24 and 48 hours of incubation, respectively. The low prevalence of STEC in foods together with the attributes of the STEC found in Brazilian cattle could be among the reasons for the low prevalence of foodborne diseases caused by STEC in Brazil.KEY WORDS: Acid, ethanol, ground beef, milk, Shiga toxin-producing Escherichia coli.

O trabalho teve como objetivo determinar a ocorrência de Escherichia coli produtora de toxina Shiga (STEC) em carne moída e leite cru no sul do Brasil e estudar o comportamento de STEC isoladas de fezes de bovinos de corte e leite, verificando sua resistência em meios ácido e alcoólico. Não foram identificados genes stx1 e stx2 nas 464 E. coli isoladas de carne moída e leite cru. STEC isoladas de fezes de bovinos mantiveram populações estáveis e apresentaram crescimento, respectivamente, em carne moída e em leite experimentalmente contaminados, durante 120 horas a 8ºC. Esses isolados foram inativados quando expostos a pH 2,5 e 3,0, mas permaneceram viáveis após oito horas em pH 4,0. Os isolados de STEC não sobreviveram 48 horas em meio contendo 12% de etanol. Em 6% de etanol, STEC O174:H21, O163:H19 e O112:H2 apresentaram crescimento, ao passo que STEC O91:H21 e O22:H8 não resistiram além de 24 e 48 horas de incubação, respectivamente. A baixa prevalência de STEC em alimentos e as características das cepas encontradas em bovinos podem estar relacionadas com a baixa prevalência de enfermidades de origem alimentar causadas por STEC no Brasil.PALAVRAS-CHAVES: Ácido, carne moída, etanol, leite, Escherichia coli produtora de toxina Shiga.

Avaliação microbiológica da carne bovina moída comercializada no Município de Jaboticabal, SP / Microbiological evaluation of ground beef sold in Jaboticabal, SP

Com o objetivo de avaliar as condições higiênico-sanitárias e a evolução dos microrganismos presentes na carne bovina moída ao longo do período de sua comercialização, foram analisadas 40 amostras, adquiridas em dez estabelecimentos comerciais, incluindo açougues e supermercados de diferentes pontos da cidade de Jaboticabal-SP. Foram feitas a pesquisa de Salmonella sp e quantificação de bactérias mesófilas e psicrotróficas viáveis. Os resultados evidenciaram que a higiene inadequada e más condições de conservação dos produtos contribuíram para o aumento das populações de microrganismos ao longo do período de sua comercialização, indicando um produto de qualidade microbiológica comprometida.(AU)

Grourad beef samples collected at the butches in Jaboticabal, SP, Brazil: They were submitted to microbiological fourty samples proceding of different commercial dot. They were quantified the microorganisms mesophilic, psicotrophic and research Salmonella sp. The found results evidencing that the analyzed samples present were out of the microbiological standards permited indicating a bad hygienic- sanitary quality of product. (AU)

Presença de sulfito de sódio e sua influência nas características físico-químicas e microbiológicas de carnes bovinas moídas resfriadas / Presença de sulfito de sódio e sua influência nas características físico-químicas e microbiológicas de carnes bovinas moídas resfriadas

Background: Ground meat stands among the most consumed meat products, being its safety easily damaged by increasing the surface area of contact after grinding it, what facilitates the growth of pathogenic and deteriorating micro-organisms. In order to decrease the lost because of deterioration, trade has been utilizing fraudulent devices as the addition of intentional preservative such as sodium sulfite. This research aimed to identify the presence of sodium sulfite in cooled ground beef commercialized in business establishments located in Porto Alegre city, state of Rio Grande do Sul, Brazil. In addition, it addressed the evaluation of the influence of using this addictive in cooled ground meat front its physic-chemical and microbiological characteristics and residual capacity in the first 48 hours.Materials, Methods & Results: A total of 55 samples of cooled ground beef, from non-noble cuts, acquired in business establishments located in Porto Alegre city, were collected. Thus, microbiological analyses for total and thermotolerant coliphorms, Clostridium Sulfite reducers, Staphylococcus coagulase-positive, Salmonella sp. and a qualitative test, for verifying the presence of sodium sulfite, were carried out. Subsequently, half of the negative samples (24) in the sulfite test were separated, and 0.04 g of this addictive for each 50 g of ground beef (0.08%) was, then, added.

Background: Ground meat stands among the most consumed meat products, being its safety easily damaged by increasing the surface area of contact after grinding it, what facilitates the growth of pathogenic and deteriorating micro-organisms. In order to decrease the lost because of deterioration, trade has been utilizing fraudulent devices as the addition of intentional preservative such as sodium sulfite. This research aimed to identify the presence of sodium sulfite in cooled ground beef commercialized in business establishments located in Porto Alegre city, state of Rio Grande do Sul, Brazil. In addition, it addressed the evaluation of the influence of using this addictive in cooled ground meat front its physic-chemical and microbiological characteristics and residual capacity in the first 48 hours.Materials, Methods & Results: A total of 55 samples of cooled ground beef, from non-noble cuts, acquired in business establishments located in Porto Alegre city, were collected. Thus, microbiological analyses for total and thermotolerant coliphorms, Clostridium Sulfite reducers, Staphylococcus coagulase-positive, Salmonella sp. and a qualitative test, for verifying the presence of sodium sulfite, were carried out. Subsequently, half of the negative samples (24) in the sulfite test were separated, and 0.04 g of this addictive for each 50 g of ground beef (0.08%) was, then, added.