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Orientador : Antonio de Melo Serrano Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: A presente pesquisa teve por objetivo verificar a eficiência de alguns meios para isolamento de Listeria a partir de alimentos, assim como avaliar a contaminação de alguns produtos alimentícios por este grupo de bactérias. Numa primeira fase, avaliamos a capacidade seletiva dos meios ágar McBride modificado ("MLA"), ágar Vogel-Johnson modificado ("MVJ") e ágar cloreto de lítio feniletanol moxalactam ("LPM") para amostras de leite e de carne bovina moída artificialmente contaminadas. Para as amostras de leite, os 3 meios mostraram-se semelhantes, mas para as amostras de carne moída os meios "LPM" e "MVJ" foram mais seletivos, permitindo o isolamento de colônias típicas de Listeria de modo mais eficiente, após incubação por 48 horas. Na segunda fase examinamos 140 amostras de alimentos colhidas no varejo em Campinas, SP. Bactérias do gênero Listeria estiveram presentes em 67 amostras (48%) sendo a maior incidência para carnes e derivados (95% de amostras positivas). Os leites e queijos apresentaram 12,5% de amostras positivas, sendo a totalidade dos leites pasteurizados, tipos B e C, negativa para o gênero. Os produtos cárneos examinados (carne bovina moída, salsicha e lingüiça, 20 amostras de cada produto) apresentaram 71,7% das amostras positivas para Listeria monocytogenes; 80,0% para L. innocua; 1,7% para L. seeligeri; 3,3% para L. welshimeri e 1,7% para L. murrayi. Já a partir dos produtos lácteos (leite cru tipo B e queijo minas frescal, 20 amostras de cada produto) foi possível observar a presença de L. monocytogenes em 5,0% das amostras, L. innocua em 20,0% e tanto L. seeligei quanto L. welshimeri em 2,5%. Abstract: The purpose of this research was to evaluate some plating media and a methodology to recover Listeria spp from food, as well as to evaluate the problem of food contamination by this genus of bacteria. In the first step we evaluated the selectivity of 3 media: modified McBride Listeria agar (MLA), modified Vogel-Johnson agar (MVJ) and lithium chloride-phenylethanol-moxalactam (LPM). Pasteurized milk and raw minced meat artificially innoculated were used. All 3 media worked equally well for the milk samples but MVJ and LPM were the best for the meat samples. The best incubation time was 48 hours. The second step was the evaluation of 140 samples obtained from retail stores in Campinas, SP. Listeria spp was detected in 67 (48%) samples. From 60 meat products samples 95% showed Listeria spp as well as 12,5% of the milk and milk products. Listeria spp was not detected in all pasteurized milk samples. In meat and meat products (20 samples per product) Listeria monocytogenes could be islolated from 71,7%; Listeria innocua from 80,0%; L. welshimeri from 3,3% and L. seeligeri and L. murrayi from 1,7% . In raw milk and cheese samples (20 samples per product) Listeria monocytogenes was detected in 5,0%; L. innocua in 20,0% and L. seeligeri & L. welshimeri in 2,5%. Mestrado Mestre em Tecnologia de Alimentos

Bacteriocins bacST202Ch and bacST216Ch, produced by Lactobacillus plantarum strains isolated from Beloura and Chouriço, respectively, inhibited the growth of a number of Gram-positive and Gram-negative meat spoilage bacteria. According to trycine-SDS-PAGE, bacST202Ch and bacST216Ch are approximately 3.5 and 10.0 kDa in size, respectively. Maximal activity of bacST202Ch (25,600 AU/ml) was recorded after 27 h of and bacST216Ch (102,400 AU/ml) after 22 h of growth. The mode of activity, as determined against Enterococcus faecium HKLHS, is bactericidal. Both peptides adsorb to the surface of the producer cells, but at very low concentrations. Both peptides remained active after 120 min at 100 degrees C and after 2 h of incubation at pH 2.0-12.0. Treatment for 120 min at 121 degrees C did not affect bacST216Ch activity. Activity of bacST202Ch and bacST216Ch was not affected by 1% Triton X-100, Tween 80, Tween 20, SDS, NaCl, urea and EDTA. Bacteriocin ST216Ch was deactivated in the presence of 1% Triton X-114. The nucleotide sequence of a 1044 bp DNA fragment amplified from L. plantarum ST202Ch is identical to the structural gene encoding pediocin PA-1, suggesting that the two bacteriocins are identical. Based on the broad spectrum of antibacterial activity, strains ST202Ch and ST216Ch may be used as starter cultures in the fermentation of meat products.

Eggs and their products have been implicated in food-borne disease outbreaks due to contamination with SalmonellaEnteritidis. Irradiation, as a food preservation technology, could minimize such contamination. The aim of this study was to determine the effects of irradiation in powdered egg yolk, egg white, and whole egg samples spiked with Salmonella Enteritidis. The powdered egg samples were exposed to doses of 0.5, 1.0, 1.0, 2.0, 2.5, 3.0, 3.5, or 4.0 kGy irradiation. Odors and tastes of raw and cooked non-inoculated, irradiated samples, and non-irradiated samples, were analyzed by a trained panel. A dose of 2.0 kGy resulted in slight to moderate changes in powdered egg yolk samples, compared to non-irradiated controls, while minimal differences were observed in powdered egg white samples. The dose of 3.5 kGy, when applied to samples of powdered egg white, resulted in taste and odor slightly stronger than the non-irradiated control. The maximum irradiation threshold before changes in flavor and odor occurred was 2.28 kGy, for powdered yolk and whole eggs, and 2.58 kGy, for powdered egg white. Those thresholds were enough to reduce S.Enteritidis to safe levels. RESUMOOs ovos e os seus produtos derivados têm sido implicados em doenças de origem alimentar devido à contaminação com SalmonellaEnteritidis. A irradiação é um método tecnológico de preservação de alimentos que pode minimizar esse tipo de contaminação. O objetivo do presente estudo foi determinar os efeitos da irradiação nas amostras de gema de ovo em pó, clara de ovo em pó e ovo integral em pó contaminadas artificialmente com Salmonella Enteritidis. As amostras de ovo em pó foram expostas a doses de 0,5, 1,0, 1,5, 2,0, 2,5, 3,0, 3,5 ou 4,0 kGy de irradiação. As amostras não contaminadas irradiadas e não irradiadas foram avaliadas por um painel de especialistas que avaliaram odor e sabor cru e cozido não inoculados, amostras irradiadas e não irradiadas. A dose de 2,0 kGy resultou em alterações leves a moderadas nas amostras de gema de ovo em pó em comparação aos controles não-irradiados, enquanto diferenças mínimas foram observados em amostras de ovo em pó. A dose de 3,5 kGy quando aplicada às amostras de clara de ovo em pó resultou em sabor e odor mais fortes do que no controle não irradiado. O limite máximo da dose de radiação sem causar alteração significativa no sabor e odor foi 2,28 kGy para gema em pó e ovos inteiros e 2,58 kGy para clara em pó e suficiente para reduzir S. Enteritidis para níveis seguros.

Listeria monocytogenes is a pathogen capable of adhering to many surfaces and forming biofilms, which may explain its persistence in food processing environments. This study aimed to genetically characterise L. monocytogenes isolates obtained from bovine carcasses and beef processing facilities and to evaluate their adhesion abilities. DNA from 29 L. monocytogenes isolates was subjected to enzymatic restriction digestion (AscI and ApaI), and two clusters were identified for serotypes 4b and 1/2a, with similarities of 48% and 68%, respectively. The adhesion ability of the isolates was tested considering: inoculum concentration, culture media, carbohydrate source, NaCl concentration, incubation temperature, and pH. Each isolate was tested at 108 CFU mL− 1 and classified according to its adhesion ability as weak (8 isolates), moderate (17) or strong (4). The isolates showed higher adhesion capability in non-diluted culture media, media at pH 7.0, incubation at 25 °C and 37 °C, and media with NaCl at 5% and 7%. No relevant differences were observed for adhesion ability with respect to the carbohydrate source. The results indicated a wide diversity of PFGE profiles of persistent L. monocytogenes isolates, without relation to their adhesion characteristics. Also, it was observed that stressing conditions did not enhance the adhesion profile of the isolates.

This study evaluated the growth of naturally occurring L. monocytogenes in sliced, vacuum-packed mortadella samples during storage at 5ºC until the expiration date. Tukey's test indicated that counts of L. monocytogenes on 0, 10, 20, 30 and 40 days of storage were significantly different (p

This study was carried out to evaluate the shelf life of minimally processed (MP) watercress exposed to gamma radiation with doses of 1.0; 3.0; 4.0 kGy. Packaged irradiated and non-irradiated MP watercress was subject to sensory analysis. A panel consisting of 25-30 non-trained members, aged 20-55 years, was used. Sensory evaluation was carried out on days 0, 2, 5, 7, 9 and 12 after treatment with packages maintained at 7 °C. The members of the panel rated each sample for overall liking on a hybrid 10 hedonic scale (0 = extremely dislike; 10 = extremely like). Microbiological analyses were also conducted. Compared to the non-irradiated sample, 1 kGy irradiated watercress increased its shelf life by one day (16 days). Shelf lives of samples exposed to higher doses were reduced to 9 days (3 kGy) and 6 days (4 kGy) due to changes in appearance. The microbiological quality was good throughout the experiment. Este estudo foi desenvolvido para avaliar a vida de prateleira de agrião minimamente processado exposto a doses de 1,0; 3,0 e 4,0 kGy. Amostras de agrião não irradiadas e irradiadas foram submetidas à análise sensorial. Um painel composto por 25-30 membros não treinados e idade entre 20 e 55 anos foi utilizado. A avaliação sensorial foi realizada nos tempos 0, 2, 5, 7, 9 e 12 após o tratamento com radiação, utilizando embalagens mantidas sob refrigeração (7 °C). Os provadores avaliaram cada amostra utilizando a escala hedônica híbrida de 10 cm (0 = desgostei muitíssimo; 10 = gostei muitíssimo). Em paralelo foram realizadas análises microbiológicas. A amostra de agrião irradiada com 1 kGy apresentou vida de prateleira de 16 dias, um dia a mais que a da amostra testemunha. Por outro lado, amostras expostas a doses de 3 e 4 kGy apresentaram vida de prateleira menor, com 9 dias e 6 dias, respectivamente. A qualidade microbiológica do produto permaneceu adequada durante o experimento.

Several alternative analytical methods are currently available for the rapid microbiological testing of food. Due to their many advantages, particularly their convenience of use, the popularity of ready-to-use systems for the enumeration of hygiene indicator microorganisms is increasing. However, the ability of these systems to enumerate stressed microorganisms, such as those that may be found growing in acidic foods, is unknown. Therefore, the aim of this study was to evaluate the performance of Petrifilm™ and SimPlate™ plates for the enumeration of total aerobes and fungi (yeasts and molds) in acidic fruit juices, using non-pasteurized orange juice as an experimental model. The samples were analyzed before and after neutralization of pH, and the results were compared with those obtained using conventional procedures, i.e. pour-plates containing Standard Methods Agar, acidified potato dextrose agar, or dichloran-glycerol agar. The results obtained with Petrifilm and SimPlate for counts of mesophilic aerobes as well as for yeast and mold correlated well with those obtained using conventional procedures. Although no statistically significant differences were observed between counts of non-neutralized and neutralized samples (α ≤0.05), better correlation indexes were observed in the neutralized samples. Both Petrifilm and SimPlate proved to be good alternative methods for testing the microbiological quality of acidic fruit juices.

RESUMO. Pouco se sabe, no Brasil e na América do Sul, sobre a ocorrência de Listeria monocytogenes em embutidos cárneos fermentados, fatiados, embalados a vácuo. Por suas características de produção e armazenamento, estes produtos são potenciais veiculadores deste patógeno ao ser humano, principalmente imunodeprimidos, idosos e grávidas, levando a disfunções gastrintestinais e neurológicas. Neste trabalho, foram analisadas 45 amostras de diferentes tipos e marcas de salames fatiados embalados a vácuo, refrigerados, provenientes de diferentes pontos do comércio varejista da mais populosa cidade da América do Sul, São Paulo - Brasil. Do total de amostras analisadas, 3 (6,7%) apresentaram populações de L. monocytogenes de 9,2 NMP/g, sendo duas pertencentes ao sorotipo 1/2a e uma ao 1/2b, os mais frequentemente encontrados também em outros países. Esta contaminação demonstra a necessidade de um rastreamento maior e em nível nacional da presença desta bactéria em produtos pronto para consumo. Isto pode, no futuro, auxiliar os órgãos governamentais de fiscalização a adotarem níveis máximos permitidos de Listeria monocytogenes nestes alimentos e as indústrias e comércio a implementarem medidas de controle higiênico-sanitário como GMP e sistemas como HACCP, garantindo, assim, um produto seguro para a saúde do consumidor SUMMARY. Occurrence of Listeria monocytogenes in pre-sliced vacuum-packaged salami in São Paulo - Brazil. There is scarce information in Brazil and other South American countries about the occurrence of Listeria monocytogenes in food, mainly refrigerated ready-to-eat products. The consumption of sliced vacuum-packaged meat products has increased in the last few years. Nevertheless, a complete assessment of the risk associated with L. monocytogenes in these products is still necessary. Because of the production and storage characteristics of these products, they can be considered potential vehicles for L. monocytogenes to humans, mainly immunocompromised, elderly, and pregnant women. The objectives of this study was to evaluate the population of L. monocytogenes in salami, a ready-to-eat meat product with extended shelf life, acquired in retail stores in São Paulo - Brazil. The three-tube most probable number technique was used and the methodology was that from Health Canada. Strains were biochemically identified and serotyped. Among the 45 samples, 3 (6,7%) harboured 9,2 MPN/g of L. monocytogenes and the others < 0,3 MPN/g. All the strains belonged to serotypes 1/2a and 1/2b, the most frequent serotypes found in food everywhere. Even being low, the population of L. monocytogenes found in this product could be a cause of concern to public health authorities as it can pose a threat to population at risk. This contamination highlights the importance of implementing systems like HACCP to assure safe products to consumers.

Avaliou-se um novo procedimento para detecção rápida de Salmonella em alimentos, baseado na combinação entre SPRINT®, MSRV® e Salmonella Latex Test® . SPRINT® é um sistema para reduzir as etapas de pré-enriquecimento e enriquecimento seletivo para 24 h. MSRV® é um meio seletivo semi-sólido para detecção de salmonelas móveis. Salmonella Latex Test® é um teste rápido de aglutinação de látex. A combinação dos três sistemas permite que a detecção de Salmonella em alimentos possa ser feita em apenas 48 h. O procedimento foi avaliado em alimentos infantis prontos para consumo, experimentalmente contaminados com Salmonella exclusivamente e com uma mistura de Salmonella e várias espécies de Enterobacteriaceae e também em cem amostras de lingüiças de porco e de frango sem adição artificial de microrganismos. O método convencional de cultura foi empregado como método de referência. A avaliação em alimentos infantis indicou que o procedimento proposto apresentava boa sensibilidade (80%) e especificidade (100%), sem reação cruzada com outras Enterobacteriaceae. Entretanto, quando aplicado a lingüiças, seu desempenho não foi adequado: os valores de x² (5,062, α >; 0,05) e do índice de concordância de Kappa (0,171, p=0,089) indicaram que as diferenças entre os resultados obtidos pelos dois métodos foram estatisticamente significantes e a correlação entre eles foi baixa. A new procedure for rapid detection of Salmonella in foods, based on the combination of SPRINT TM, MSRV TM and Salmonella Latex TestTM, was evaluated. SPRINT TM is a system to reduce the preenrichment and selective enrichment steps to 24 hours. MSRV TM is a semi-solid selective media for detection of motile Salmonella. Salmonella Latex TestTM is a rapid latex agglutination test for Salmonella. Using the three systems in combination, the total time for detection of Salmonella in a food sample is 48h. Evaluations were performed in artificially contaminated ready-to-eat baby-foods and raw Brazilian sausages (lingüiça) containing no added microorganisms. The BAM conventional culture procedure was used as reference method. The study with baby foods indicated that the new procedure had good sensitivity (89%) and specificity (100%), without cross-reactions with Enterobacteriaceae. However, when applied to naturally contaminated foods, the performance was poor: chi square (x² = 5.062, α>; 0. 05) and Kappa-Cohen agreement (K = 0.171, p=0.089) indexes indicated that the differences between results given by the two procedures were significant and the correlation between them was low.

This study reports results on the biochemical characteristics of 274 Staphylococcus aureus strains isolated from mastitic milk, hands of milkmen and milking machines. Features included colony morphology on Baird Parker agar (BPA), catalase, coagulase and thermonuclease tests. API-Staph (bioMérieux, France) was used as reference identification system. 39.4% of the colonies on BPA identified as S.aureus were atypical. They were more frequent in milk samples (94.4%). All S.aureus strains were coagulase positive but intensity of the test varied according to the source: environmental strains were 3+ or 4+, while 17.1% of strains isolated from milk presented only 1+ or 2+ results. The majority of atypical S.aureus strains presented a 4+ coagulase result. 2.2% of S.aureus strains were thermonuclease negative. Sixteen thermonuclease positive and coagulase positive strains were identified as S.hyicus. Esse trabalho relata as características bioquímicas de 274 cepas de Staphylococcus aureus isoladas de leite mastítico, de mãos de ordenhadores e de ordenhadeiras mecânicas. Foram avaliadas as características morfológicas em agar Baird Parker (BPA) e os resultados para os testes de catalase, coagulase e termonuclease. API-Staph foi empregado como sistema de referência para identificação. 39,4% das colônias em BPA identificadas como S.aureus apresentaram morfologia atípica, sendo mais freqüentes em leite (94,4%). Todas as cepas identificadas como S.aureus foram coagulase positivas mas a intensidade da reação variou de acordo com a fonte: cepas isoladas de amostras ambientais foram 3+ ou 4+, enquanto 17,1% daquelas isoladas de leite foram apenas 1+ ou 2+. A maioria das cepas de S aureus com morfologia atípica em agar Baird-Parker apresentaram coagulase 4+. 2,2% das cepas de S.aureus foram termonuclease negativas. 16 cepas termonuclease positivas e coagulase positivas foram identificadas como S.hyicus.