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Marc Heyndrickx - One of the best experts on this subject based on the ideXlab platform.
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emended descriptions of Bacillus Sporothermodurans and Bacillus oleronius with the inclusion of dairy farm isolates of both species
International Journal of Systematic and Evolutionary Microbiology, 2012Co-Authors: Marc Heyndrickx, Patsy Scheldeman, An Coorevits, Liesbeth Lebbe, Peter Schumann, Marina Rodriguezdiaz, Gillian Forsyth, Anna E Dinsdale, Jeroen HeyrmanAbstract:Bacillus Sporothermodurans is an industrially important micro-organism because of its ability to produce endospores which resist ultra-high temperature (UHT) and industrial sterilization processes. It was described by Pettersson et al. (1996) [Pettersson, B., Lembke, F., Hammer, P., Stackebrandt, E. & Priest, F. G. (1996). Int J Syst Bacteriol 46, 759-764] based on seven genetically homogeneous isolates all from UHT milk. Bacillus oleronius, the closest phylogenetic neighbour of B. Sporothermodurans, was described by Kuhnigk et al. (1995) [Kuhnigk, T., Borst, E.-M., Breunig, A., Konig, H., Collins, M. D., Hutson, R. A. & Kampfer, P. (1995). Can J Microbiol 41, 699-706] based on a single strain, isolated from the hindgut of the termite Reticulitermes santonensis. A polyphasic study of a heterogeneous collection of B. Sporothermodurans and B. oleronius strains isolated from various sources and geographical origins led to an emended description of both species. Additional data presented are the results of fatty acid, quinone and/or cell wall (polar lipid) analyses. DNA-DNA hybridization confirmed 3 subgroups of strains obtained after SDS-PAGE analysis of cellular proteins as B. Sporothermodurans. One named B. Sporothermodurans strain (R-7489) was reclassified as a Bacillus fordii strain. The phenotypic profiles of both species were rather heterogeneous, sometimes different from the original descriptions and did not differ in a large number of characteristics, although B. oleronius generally gave stronger reactions in its positive tests than did B. Sporothermodurans; the variable and weak reactions for both organisms with some substrates blurred the distinction between the two. However, differences in polar lipid, SDS-PAGE and menaquinone profiles clearly allow distinction between the two species.
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Bacillus Sporothermodurans and other highly heat resistant spore formers in milk
Journal of Applied Microbiology, 2006Co-Authors: Patsy Scheldeman, Lieve Herman, Simon J Foster, Marc HeyndrickxAbstract:A recent example of a micro-organism causing undesired growth in consumer milk is Bacillus Sporothermodurans producing highly heat-resistant spores (HRS) which may survive ultra-high temperature (UHT) treatment or industrial sterilization. Molecular typing showed a heterogeneous group of farm isolates (non-HRS strains), but a clonal group of UHT isolates from diverse European countries and other continents (HRS-clone) suggesting a common source. During a survey of Belgian dairy farms for the presence of potentially highly heat-resistant spore formers, high numbers of these spores were detected in filter cloth, green crop and fodder samples. The strain collection showed a high taxonomic diversity with 18 potentially new species and with Bacillus licheniformis and GeoBacillus pallidus as predominating species overall. Seventeen B. Sporothermodurans isolates were identified, mainly originating from feed concentrate. Heat resistance studies showed the UHT resistance of B. Sporothermodurans spores present in industrially contaminated UHT milk, but a lower heat resistance of laboratory-grown strains (HRS and non-HRS). Hydrogen peroxide, used as sanitizer in the dairy industry, was found to induce higher heat resistance of laboratory-grown B. Sporothermodurans strains to a certain level. This indicates that sublethal stress conditions may affect the heat resistance. By transmission electron microscopy, structural differences at the spore level were found between HRS and non-HRS strains. The data indicate that the attainment of extreme heat resistance is rather multifactorial.
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Incidence and Diversity of Potentially Highly Heat-Resistant Spores Isolated at Dairy Farms
Applied and environmental microbiology, 2005Co-Authors: Patsy Scheldeman, Annelies Pil, Lieve Herman, Paul De Vos, Marc HeyndrickxAbstract:The presence of highly heat-resistant spores of Bacillus Sporothermodurans in ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100°C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera. Bacillus licheniformis and Bacillus pallidus were the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.
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genetic heterogeneity in Bacillus Sporothermodurans as demonstrated by ribotyping and repetitive extragenic palindromic pcr fingerprinting
Applied and Environmental Microbiology, 2002Co-Authors: Olivier Guillaumegentil, Patsy Scheldeman, Lieve Herman, Joey D Marugg, Han Joosten, Marc HeyndrickxAbstract:Thirty-eight strains of Bacillus Sporothermodurans isolated from ultra-high-temperature (UHT)-treated milk or sterilized milk (UHT isolates) and from animal feed or raw milk (farm isolates) were characterized by automated ribotyping and by repetitive extragenic palindromic (REP)-PCR fingerprinting. By investigating the genetic relationships among isolates from these various sources, the relative importance of different contamination sources could be evaluated. The results of the separate clustering analyses of the PvuII and EcoRI ribopatterns and the REP-PCR patterns were largely consistent with each other and revealed the existence of two main clusters; there was one homogeneous group containing all (REP-PCR) or most (ribotyping) of the UHT isolates, and there was a second more diverse group comprising the farm isolates. A combined three-dimensional analysis of all data showed that three German UHT isolates did not belong to the compact group containing the majority of the UHT isolates. These results demonstrate that B. Sporothermodurans is more heterogeneous than previously assumed and that most of the UHT isolates form a genetically distinct subgroup and are capable of producing highly heat-resistant spores. The close genetic relationship of these UHT isolates suggests a clonal origin of a few predominant strains of B. Sporothermodurans that can be found in UHT-treated or sterilized milk products.
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polymerase chain reaction identification of Bacillus Sporothermodurans from dairy sources
Journal of Applied Microbiology, 2002Co-Authors: Patsy Scheldeman, Lieve Herman, Johan Goris, P De Vos, Marc HeyndrickxAbstract:Aims: A new polymerase chain reaction (PCR) method for the identification of Bacillus Sporothermodurans strains from sterilized or ultrahigh temperature-treated milk and milk products and from other non-milk sources and environments, including the dairy farm. Methods and Results: Two strains from raw milk and feed concentrate could be allocated to B. Sporothermodurans based on 16S rDNA sequencing and DNA-DNA hybridization results. Two specific PCR primers were derived from the 16S rRNA gene of B. Sporothermodurans. Conclusions: The PCR identification method was validated using a collection of B. Sporothermodurans strains from different sources and on a large collection of dairy and non-dairy Bacillus spp. and other relevant taxa. Significance and Impact of the Study: This PCR method was used as a screening method for strains with very heat-resistant endospores, isolated at the dairy farm level after heat treatment for 30 min at 100°C. Seventeen strains isolated at the dairy farm were identified as B. Sporothermodurans. They originated mainly from feed concentrate and also from soy, pulp and silage. The PCR identification method described here can, therefore, contribute to a better understanding of the route by which B. Sporothermodurans contaminates raw and/or heat-treated milk.
Patricia Blumer Zacarchenco - One of the best experts on this subject based on the ideXlab platform.
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Aperfeiçoamento da metodologia para contagem de Bacillus Sporothermodurans e sua ocorrencia em leite UAT/UHT
2017Co-Authors: Patricia Blumer ZacarchencoAbstract:Resumo: Nos últimos anos, em nível internacional, o microrganismo Bacillus Sporothermodurans (BSP) vem sendo isolado a partir de leite processado pelo sistema UAT/UHT (Ultra Alta TemperaturalUltra High Temperature). Também no Brasil, mais recentemente, o microrganismo foi isolado a partir de amostras de leite UAT produzido em unidades industriais do Estado de São Paulo e Paraná. As amostras contaminadas apresentaram contagens em torno de 105UFC/ml sem evidências maiores de alterações organolépticas. No entanto, nestas condições, o leite UAT fica fora dos padrões exigidos pela legislação para o produto quanto a contagem de microrganismos aeróbios mesófilos, que é de 102UFC/ml(portaria SVSIMS n12451/97). Na primeira parte desta pesquisa, procurou-se avaliar e otimizar a metodologia para detecção de BSP, usando-se como referência a cepa DSMZ 10599, da Coleção Alemã de Microorganismos e Culturas Celulares (Deutsche Sammlug von Mikroorganismen und Zellkulturen). Em uma primeira etapa, avaliou-se qualitativa e quantitativamente o desempenho do ágar Infusão de Cérebro e Coração (BHI), ágar Padrão de Contagem (PCA) e o ágar Nutriente (NA) na contagem de BSP. Além disso, comparou-se as técnicas de inoculação em profundidade (pour p/ate) e espalhamento superficial (spread plate). Os melhores resultados foram obtidos pelo uso do ágar BHI (colônias mais diferenciadas e com melhor crescimento) e o uso da técnica de espalhamento superficial (maior uniformidade e crescimento das colônias). Numa segunda etapa formulou-se o meio diferencial (ágar BHI-E), com base na adição de 1g/I de esculina e 0,5gl1 de citrato férrico amoniacal, permitindo, assim, a melhor caracterização das colônias de BSP pelo seu enegrecimento e formação de halo escuro pela hidrólise da esculina. Numa terceira etapa, comprovou-se que a adição destes componentes não ocasionou quaisquer efeitos inibitórios comparativamente ao ágar BHI. Finalmente, numa última etapa, avaliou-se a eficiência da ágar BHI-E na diferenciação de BSP frente a outros Bacillus spp de ocorrência comum em produtos lácteos, caso de Bacillus cereus, Bacillus subtilis e Bacillus firmus. Na segunda parte da pesquisa, analisaram-se 100 amostras de leite UAT de 3 regiões do Brasil (Centro-Oeste, Sudeste e Sul) utilizando o ágar BHI-E desenvolvido na primeira fase. Verificou-se que 45% das amostras estavam fora dos padrões da Portaria SVS/MS no 451/97 e que 55% delas apresentavam esporos mesófilos termo-resistentes. Isolaram-se 300 colônias que hidrolisaram esculina e tinham aspecto semelhante às colônias do Bacillus Sporothermodurans em ágar BHI-E. A maior parte dos isolados apresentou morfologia típica e produzia oxidase, sendo que poucos não fermentaram a glicose. Vinte e quatro cepas, da segunda etapa da pesquisa, tiveram o seu DNA cromossômico avaliado pelo método RAPD ("random amplified polymorphic DNA"). Os perfis de DNA obtidos permitiram dividí-Ias em 3 grupos que apresentaram perfiJde DNA muito semelhantes ao do BSP Abstrat: Bacillus Sporothermodurans - BSP - has been isolated worldwide in milk processed by the ultra high temperature system (UHT-milk). More recently BSP has also been isolated in Brazil, partieularly in industrial samples processed in São Paulo and Paraná states. Ali the samples showed total counts around 105cfu/ml without any organoleptie ehanges. However based on Brazilian microbiological standards (Portaria SVS/MS nQ451/97) fixing a limit of 1& cfu/ml, the samples were eonsidered inadequate, being rejected. The objective of the first part of this research was the evaluation and improvement of the methodology for Bsp detection, employing as reference the strain DSMZ 10599 from Germany Colection of Microorganisms and Cells Cultures (Deutsche Sammlug von Mikroorganismen und Zellkulturen). In a first step, the performanee of Brain Heart Infusion agar (BHI agar), Total Plate Count Agar (PCA) e Nutrient agar (NA) were comparatively evaluated, considering tooa the inoculation methods (pour plate compared to spread plate). The results showed the best performance of BHI agar with more uniform and better growth of Bsp, the same being valid eoncerning the use of spread plate compared to the pour plate method. In a seeond step, it was formulated a differential medium named BHI-E agar, based on addition of 19/1 of esculin and 0,5gl1 of ferrie ammonium eitrate; with this modification, BSP showed typical black colonies with a black halo due to esculin hydrolisis. In a third step, it was concluded that BHI-E agar did not show any inhibitory effect when compared to BHI agar. Finally, in a last step, BHI-E agar was evaluated in relation to the other Bacillus spp commonly found em milk products, particularly Bacillus cereus, Bacillus subtilis and Bacillus firmus. In the second part of the research work, a total of 100 comercial samples of UHT milk produced in 3 different Brazilian regions (Center-east, southern and southeast) were analysed using BHI-E agar previously developed and tested. The results showed that 45% of the examined samples did not attend the Brazilian standards (Portarja SVS/MS nQ451197)and 55% were contamined with mesophilic thermoresistant bacterial species. A total of 300 esculin positive cultures were isolated with morphological and cultural aspects similar to BSP besides being oxidase positive but generally showing a slow glucose fermenting activity. Twenty four strains isolated at the second part of this research were sub-tiped by molecular typing (RAPO). These 24 strains could be separated in 2 groups according to their ONA pattern. The profiles of 3 groups were very similar to that ONA pattern of Bacillus Sporothermodurans (BSP
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ocorrencia de Bacillus Sporothermodurans em leite uat uht brasileiro e a influencia do tratamento termico
Food Science and Technology International, 2000Co-Authors: Patricia Blumer Zacarchenco, Mauro Faber De Freitas Leitao, Maria Tereza Destro, Cristiano AndrighetoAbstract:O Bacillus Sporothermodurans (BSP) tem sido isolado em todo o mundo, principalmente nos paises europeus, como um contaminante usual do leite processado pelo sistema UAT/UHT. Embora o BSP nao seja patogenico, o leite contaminado fica fora dos padroes da legislacao, que fixa o limite de contagem de microrganismos aerobios mesofilos em 100ufc/ml. Tal bacilo foi pesquisado em amostras de leite UAT produzido nas regioes Sul, Sudeste e Centro-Oeste do Brasil, processado pelos sistemas de aquecimento indireto (trocadores a placas ou tubulares) e direto (injecao ou infusao de vapor). Foi analisado um total de 100 amostras, avaliadas quantitativamente pela tecnica de espalhamento superficial no meio diferencial agar infusao de cerebro e coracao - esculina (agar BHI-E). Na analise qualitativa as amostras foram submetidas a choque termico (115oC/7min), incubadas e plaqueadas no referido agar. Os resultados revelaram um total de 45% de amostras com contagens acima do limite toleravel (100ufc/ml), oscilando entre 2x104 e 9,5x105ufc/ml. No entanto, em funcao do tipo de sistema de aquecimento, as variacoes foram muito acentuadas, sendo que na avaliacao quantitativa os indices de rejeicao foram de 71,4% nas amostras aquecidas indiretamente e naquelas processadas diretamente. Isto se deve, provavelmente, as condicoes mais drasticas de tratamento termico utilizada pelos laticinios com sistema direto de aquecimento. Na analise qualitativa, os indices de rejeicao foram de 71,7 e 22,2%, respectivamente. Trezentas culturas foram isoladas das amostras contaminadas, revelando caracteristicas culturais, morfologicas e bioquimicas similares as de BSP, exceto pela capacidade de fermentacao lenta da glicose evidenciada pela maioria delas. Deste total, 24 cepas tiveram seu DNA cromossomico avaliado pelo metodo PCR-RAPD, sendo que os perfis obtidos foram muito similares ao de BSP referencia (DSMZ 10599) confirmando tratar-se de Bacillus Sporothermodurans.
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occurrence of Bacillus Sporothermodurans and the influence of the thermal processing procedure on its presence in brazilian uht milk
Food Science and Technology International, 2000Co-Authors: Patricia Blumer Zacarchenco, Mauro Faber De Freitas Leitao, Maria Tereza Destro, Cristiano AndrighetoAbstract:Bacillus Sporothermodurans (BSP) has been isolated worldwide, but mainly in European contries as an usual contaminant of milk by the UHT system. Even being non pathogenic, generally its presence results in product rejection based on international microbiological standards limiting the counts to a maximum of 100 cfu/ml. In this research work, the occurrence of BSP was evaluated in UHT milk samples produced in different regions of Brazil (Central, South and Southeast) and processed by the indirect thermal treatment system (plate or tube exchangers) or by the direct system (steam infusion or steam injection). A total of 100 commercial samples were analysed both quantitatively by spread plate technique using Brain Heart Infusion Esculin Agar (BHI-E agar) and qualitatively after thermal shock treatment (115°C/7min), followed by incubation and plating in that medium. The results showed a total of 45% of the examined samples with counts above 100 cfu/ml, varying from 2 .104 and 9,5 .105 cfu/ml. However, 71.4% of the samples processed by indirect system were above limits, while none of the samples heat treated by the direct system, that allows a more drastic temperature condition, were rejected. A similar picture was observed in the qualitative evaluation, with contamination levels of 71,7 and 22,2% for the indirect and direct system, respectively. A total of 300 cultures were isolated from contaminated samples, showing cultural, morphological and biochemical patterns similar to BSP, except a late glucose fermentation capacity showed by most of the isolated strains. When 24 representative strains were submitted to PCR-RAPD evaluation and compared to the reference BSP strain (DSMZ 10599), they were confirmed as Bacillus Sporothermodurans based on their DNA pattern.
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Aperfeiçoamento da metodologia para contagem de Bacillus Sporothermodurans e sua ocorrencia em leite UAT/UHT
Universidade Estadual de Campinas . Faculdade de Engenharia de Alimentos, 2000Co-Authors: Patricia Blumer ZacarchencoAbstract:Nos últimos anos, em nível internacional, o microrganismo Bacillus Sporothermodurans (BSP) vem sendo isolado a partir de leite processado pelo sistema UAT/UHT (Ultra Alta TemperaturalUltra High Temperature). Também no Brasil, mais recentemente, o microrganismo foi isolado a partir de amostras de leite UAT produzido em unidades industriais do Estado de São Paulo e Paraná. As amostras contaminadas apresentaram contagens em torno de 105UFC/ml sem evidências maiores de alterações organolépticas. No entanto, nestas condições, o leite UAT fica fora dos padrões exigidos pela legislação para o produto quanto a contagem de microrganismos aeróbios mesófilos, que é de 102UFC/ml(portaria SVSIMS n12451/97). Na primeira parte desta pesquisa, procurou-se avaliar e otimizar a metodologia para detecção de BSP, usando-se como referência a cepa DSMZ 10599, da Coleção Alemã de Microorganismos e Culturas Celulares (Deutsche Sammlug von Mikroorganismen und Zellkulturen). Em uma primeira etapa, avaliou-se qualitativa e quantitativamente o desempenho do ágar Infusão de Cérebro e Coração (BHI), ágar Padrão de Contagem (PCA) e o ágar Nutriente (NA) na contagem de BSP. Além disso, comparou-se as técnicas de inoculação em profundidade (pour p/ate) e espalhamento superficial (spread plate). Os melhores resultados foram obtidos pelo uso do ágar BHI (colônias mais diferenciadas e com melhor crescimento) e o uso da técnica de espalhamento superficial (maior uniformidade e crescimento das colônias). Numa segunda etapa formulou-se o meio diferencial (ágar BHI-E), com base na adição de 1g/I de esculina e 0,5gl1 de citrato férrico amoniacal, permitindo, assim, a melhor caracterização das colônias de BSP pelo seu enegrecimento e formação de halo escuro pela hidrólise da esculina. Numa terceira etapa, comprovou-se que a adição destes componentes não ocasionou quaisquer efeitos inibitórios comparativamente ao ágar BHI. Finalmente, numa última etapa, avaliou-se a eficiência da ágar BHI-E na diferenciação de BSP frente a outros Bacillus spp de ocorrência comum em produtos lácteos, caso de Bacillus cereus, Bacillus subtilis e Bacillus firmus. Na segunda parte da pesquisa, analisaram-se 100 amostras de leite UAT de 3 regiões do Brasil (Centro-Oeste, Sudeste e Sul) utilizando o ágar BHI-E desenvolvido na primeira fase. Verificou-se que 45% das amostras estavam fora dos padrões da Portaria SVS/MS no 451/97 e que 55% delas apresentavam esporos mesófilos termo-resistentes. Isolaram-se 300 colônias que hidrolisaram esculina e tinham aspecto semelhante às colônias do Bacillus Sporothermodurans em ágar BHI-E. A maior parte dos isolados apresentou morfologia típica e produzia oxidase, sendo que poucos não fermentaram a glicose. Vinte e quatro cepas, da segunda etapa da pesquisa, tiveram o seu DNA cromossômico avaliado pelo método RAPD ("random amplified polymorphic DNA"). Os perfis de DNA obtidos permitiram dividí-Ias em 3 grupos que apresentaram perfiJde DNA muito semelhantes ao do BSP Abstrat: Bacillus Sporothermodurans - BSP - has been isolated worldwide in milk processed by the ultra high temperature system (UHT-milk). More recently BSP has also been isolated in Brazil, partieularly in industrial samples processed in São Paulo and Paraná states. Ali the samples showed total counts around 105cfu/ml without any organoleptie ehanges. However based on Brazilian microbiological standards (Portaria SVS/MS nQ451/97) fixing a limit of 1& cfu/ml, the samples were eonsidered inadequate, being rejected. The objective of the first part of this research was the evaluation and improvement of the methodology for Bsp detection, employing as reference the strain DSMZ 10599 from Germany Colection of Microorganisms and Cells Cultures (Deutsche Sammlug von Mikroorganismen und Zellkulturen). In a first step, the performanee of Brain Heart Infusion agar (BHI agar), Total Plate Count Agar (PCA) e Nutrient agar (NA) were comparatively evaluated, considering tooa the inoculation methods (pour plate compared to spread plate). The results showed the best performance of BHI agar with more uniform and better growth of Bsp, the same being valid eoncerning the use of spread plate compared to the pour plate method. In a seeond step, it was formulated a differential medium named BHI-E agar, based on addition of 19/1 of esculin and 0,5gl1 of ferrie ammonium eitrate; with this modification, BSP showed typical black colonies with a black halo due to esculin hydrolisis. In a third step, it was concluded that BHI-E agar did not show any inhibitory effect when compared to BHI agar. Finally, in a last step, BHI-E agar was evaluated in relation to the other Bacillus spp commonly found em milk products, particularly Bacillus cereus, Bacillus subtilis and Bacillus firmus. In the second part of the research work, a total of 100 comercial samples of UHT milk produced in 3 different Brazilian regions (Center-east, southern and southeast) were analysed using BHI-E agar previously developed and tested. The results showed that 45% of the examined samples did not attend the Brazilian standards (Portarja SVS/MS nQ451197)and 55% were contamined with mesophilic thermoresistant bacterial species. A total of 300 esculin positive cultures were isolated with morphological and cultural aspects similar to BSP besides being oxidase positive but generally showing a slow glucose fermenting activity. Twenty four strains isolated at the second part of this research were sub-tiped by molecular typing (RAPO). These 24 strains could be separated in 2 groups according to their ONA pattern. The profiles of 3 groups were very similar to that ONA pattern of Bacillus Sporothermodurans (BSP
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Ocorrência de Bacillus Sporothermodurans em leite UAT/UHT brasileiro e a influência do tratamento térmico Occurrence of Bacillus Sporothermodurans and the influence of the thermal processing procedure on its presence in brazilian UHT milk
Sociedade Brasileira de Ciência e Tecnologia de Alimentos, 2000Co-Authors: Patricia Blumer Zacarchenco, Mauro Faber De Freitas Leitao, Maria Tereza Destro, Cristiano AndrighetoAbstract:O Bacillus Sporothermodurans (BSP) tem sido isolado em todo o mundo, principalmente nos países europeus, como um contaminante usual do leite processado pelo sistema UAT/UHT. Embora o BSP não seja patogênico, o leite contaminado fica fora dos padrões da legislação, que fixa o limite de contagem de microrganismos aeróbios mesófilos em 100ufc/ml. Tal bacilo foi pesquisado em amostras de leite UAT produzido nas regiões Sul, Sudeste e Centro-Oeste do Brasil, processado pelos sistemas de aquecimento indireto (trocadores a placas ou tubulares) e direto (injeção ou infusão de vapor). Foi analisado um total de 100 amostras, avaliadas quantitativamente pela técnica de espalhamento superficial no meio diferencial ágar infusão de cérebro e coração - esculina (ágar BHI-E). Na análise qualitativa as amostras foram submetidas a choque térmico (115ºC/7min), incubadas e plaqueadas no referido ágar. Os resultados revelaram um total de 45% de amostras com contagens acima do limite tolerável (100ufc/ml), oscilando entre 2x10(4) e 9,5x10(5)ufc/ml. No entanto, em função do tipo de sistema de aquecimento, as variações foram muito acentuadas, sendo que na avaliação quantitativa os índices de rejeição foram de 71,4% nas amostras aquecidas indiretamente e naquelas processadas diretamente. Isto se deve, provavelmente, às condições mais drásticas de tratamento térmico utilizada pelos laticínios com sistema direto de aquecimento. Na análise qualitativa, os índices de rejeição foram de 71,7 e 22,2%, respectivamente. Trezentas culturas foram isoladas das amostras contaminadas, revelando características culturais, morfológicas e bioquímicas similares as de BSP, exceto pela capacidade de fermentação lenta da glicose evidenciada pela maioria delas. Deste total, 24 cepas tiveram seu DNA cromossômico avaliado pelo método PCR-RAPD, sendo que os perfis obtidos foram muito similares ao de BSP referência (DSMZ 10599) confirmando tratar-se de Bacillus Sporothermodurans.Bacillus Sporothermodurans (BSP) has been isolated worldwide, but mainly in European contries as an usual contaminant of milk by the UHT system. Even being non pathogenic, generally its presence results in product rejection based on international microbiological standards limiting the counts to a maximum of 100 cfu/ml. In this research work, the occurrence of BSP was evaluated in UHT milk samples produced in different regions of Brazil (Central, South and Southeast) and processed by the indirect thermal treatment system (plate or tube exchangers) or by the direct system (steam infusion or steam injection). A total of 100 commercial samples were analysed both quantitatively by spread plate technique using Brain Heart Infusion Esculin Agar (BHI-E agar) and qualitatively after thermal shock treatment (115°C/7min), followed by incubation and plating in that medium. The results showed a total of 45% of the examined samples with counts above 100 cfu/ml, varying from 2 .10(4) and 9,5 .10(5) cfu/ml. However, 71.4% of the samples processed by indirect system were above limits, while none of the samples heat treated by the direct system, that allows a more drastic temperature condition, were rejected. A similar picture was observed in the qualitative evaluation, with contamination levels of 71,7 and 22,2% for the indirect and direct system, respectively. A total of 300 cultures were isolated from contaminated samples, showing cultural, morphological and biochemical patterns similar to BSP, except a late glucose fermentation capacity showed by most of the isolated strains. When 24 representative strains were submitted to PCR-RAPD evaluation and compared to the reference BSP strain (DSMZ 10599), they were confirmed as Bacillus Sporothermodurans based on their DNA pattern
Lieve Herman - One of the best experts on this subject based on the ideXlab platform.
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Bacillus Sporothermodurans and other highly heat resistant spore formers in milk
Journal of Applied Microbiology, 2006Co-Authors: Patsy Scheldeman, Lieve Herman, Simon J Foster, Marc HeyndrickxAbstract:A recent example of a micro-organism causing undesired growth in consumer milk is Bacillus Sporothermodurans producing highly heat-resistant spores (HRS) which may survive ultra-high temperature (UHT) treatment or industrial sterilization. Molecular typing showed a heterogeneous group of farm isolates (non-HRS strains), but a clonal group of UHT isolates from diverse European countries and other continents (HRS-clone) suggesting a common source. During a survey of Belgian dairy farms for the presence of potentially highly heat-resistant spore formers, high numbers of these spores were detected in filter cloth, green crop and fodder samples. The strain collection showed a high taxonomic diversity with 18 potentially new species and with Bacillus licheniformis and GeoBacillus pallidus as predominating species overall. Seventeen B. Sporothermodurans isolates were identified, mainly originating from feed concentrate. Heat resistance studies showed the UHT resistance of B. Sporothermodurans spores present in industrially contaminated UHT milk, but a lower heat resistance of laboratory-grown strains (HRS and non-HRS). Hydrogen peroxide, used as sanitizer in the dairy industry, was found to induce higher heat resistance of laboratory-grown B. Sporothermodurans strains to a certain level. This indicates that sublethal stress conditions may affect the heat resistance. By transmission electron microscopy, structural differences at the spore level were found between HRS and non-HRS strains. The data indicate that the attainment of extreme heat resistance is rather multifactorial.
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Incidence and Diversity of Potentially Highly Heat-Resistant Spores Isolated at Dairy Farms
Applied and environmental microbiology, 2005Co-Authors: Patsy Scheldeman, Annelies Pil, Lieve Herman, Paul De Vos, Marc HeyndrickxAbstract:The presence of highly heat-resistant spores of Bacillus Sporothermodurans in ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100°C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera. Bacillus licheniformis and Bacillus pallidus were the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems.
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genetic heterogeneity in Bacillus Sporothermodurans as demonstrated by ribotyping and repetitive extragenic palindromic pcr fingerprinting
Applied and Environmental Microbiology, 2002Co-Authors: Olivier Guillaumegentil, Patsy Scheldeman, Lieve Herman, Joey D Marugg, Han Joosten, Marc HeyndrickxAbstract:Thirty-eight strains of Bacillus Sporothermodurans isolated from ultra-high-temperature (UHT)-treated milk or sterilized milk (UHT isolates) and from animal feed or raw milk (farm isolates) were characterized by automated ribotyping and by repetitive extragenic palindromic (REP)-PCR fingerprinting. By investigating the genetic relationships among isolates from these various sources, the relative importance of different contamination sources could be evaluated. The results of the separate clustering analyses of the PvuII and EcoRI ribopatterns and the REP-PCR patterns were largely consistent with each other and revealed the existence of two main clusters; there was one homogeneous group containing all (REP-PCR) or most (ribotyping) of the UHT isolates, and there was a second more diverse group comprising the farm isolates. A combined three-dimensional analysis of all data showed that three German UHT isolates did not belong to the compact group containing the majority of the UHT isolates. These results demonstrate that B. Sporothermodurans is more heterogeneous than previously assumed and that most of the UHT isolates form a genetically distinct subgroup and are capable of producing highly heat-resistant spores. The close genetic relationship of these UHT isolates suggests a clonal origin of a few predominant strains of B. Sporothermodurans that can be found in UHT-treated or sterilized milk products.
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polymerase chain reaction identification of Bacillus Sporothermodurans from dairy sources
Journal of Applied Microbiology, 2002Co-Authors: Patsy Scheldeman, Lieve Herman, Johan Goris, P De Vos, Marc HeyndrickxAbstract:Aims: A new polymerase chain reaction (PCR) method for the identification of Bacillus Sporothermodurans strains from sterilized or ultrahigh temperature-treated milk and milk products and from other non-milk sources and environments, including the dairy farm. Methods and Results: Two strains from raw milk and feed concentrate could be allocated to B. Sporothermodurans based on 16S rDNA sequencing and DNA-DNA hybridization results. Two specific PCR primers were derived from the 16S rRNA gene of B. Sporothermodurans. Conclusions: The PCR identification method was validated using a collection of B. Sporothermodurans strains from different sources and on a large collection of dairy and non-dairy Bacillus spp. and other relevant taxa. Significance and Impact of the Study: This PCR method was used as a screening method for strains with very heat-resistant endospores, isolated at the dairy farm level after heat treatment for 30 min at 100°C. Seventeen strains isolated at the dairy farm were identified as B. Sporothermodurans. They originated mainly from feed concentrate and also from soy, pulp and silage. The PCR identification method described here can, therefore, contribute to a better understanding of the route by which B. Sporothermodurans contaminates raw and/or heat-treated milk.
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the presence of intragenically located rep like elements in Bacillus Sporothermodurans is sufficient for rep pcr typing
Research in Microbiology, 2000Co-Authors: Lieve Herman, Marc HeyndrickxAbstract:The technique of repetitive extragenic palindromic polymerase chain reaction (REP-PCR) enables the identification and discrimination of clonally related Bacillus Sporothermodurans isolates from ultra-high temperature and sterilized milk. The aim of this study was to investigate the genetic basis for the generation of these highly informative REP-PCR patterns. The major 947-bp REP-PCR fragment of B. Sporothermodurans was cloned, together with its 5' and 3' flanking sequences. Only partial homology with the REP consensus sequence was established at the borders of the REP-PCR fragment. Moreover, these border sequences were located within two distinct open reading frames, with great homology to the uvrA and uvrB genes of Escherichia coli. The presence of these REP-like elements in B. Sporothermodurans was thus sufficient for high resolution REP-PCR typing of this Gram-positive organism. In some cases (and especially with Gram-positives), REP-PCR could thus be considered more as an arbitrary primed PCR, albeit at a somewhat higher annealing temperature and using conserved primers. The random priming effect at the less stringent annealing conditions of REP-PCR was also demonstrated for enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) on another Gram-positive organism, Listeria monocytogenes.
Chedia Aouadhi - One of the best experts on this subject based on the ideXlab platform.
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effect of sporulation conditions on the resistance of Bacillus Sporothermodurans spores to nisin and heat
Food Microbiology, 2016Co-Authors: Chedia Aouadhi, Slah Mejri, Zeineb Rouissi, Souhir Kmiha, Abderrazak MaaroufiAbstract:Bacillus Sporothermodurans cells were grown in various environmental conditions to study the effects of temperature, pH, and water activity of the sporulation medium on spore inactivation by nisin and heat treatment. The obtained results showed that the modification of the sporulation conditions affected the spore properties. In fact, the resistance of B. Sporothermodurans spores to heat treatment combined with nisin (treatment at 95 °C for 12 min in the presence of 125 UI/ml) increases with increasing of pH level (7–9) and decreases as the acidity of the medium increases. While the sensitivity of B. Sporothermodurans spores depends on the sporulation temperature. It increases with increasing the temperature from 4 to 37 °C, but starting with 42 °C, the sensitivity decreases. In the presence of NaCl as depressor, the resistance of B. Sporothermodurans spores was higher compared with standard conditions (pH 7, 37 °C without NaCl or glycerol) with an inactivation rate does not exceed 2.79 log. This is the first study which contributed to understand the relationship between the sporulation conditions (pH, temperature and water activity) and the resistance of B. Sporothermodurans spores to nisin and heat treatment. It shows that the sporulation conditions, particularly sporulation temperature, must be carefully taken into account during settling sterilization processes applied in the dairy industry.
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germination et inactivation des spores de Bacillus Sporothermodurans
2015Co-Authors: Chedia AouadhiAbstract:Dans le cadre de trouver des alternatives a la methode thermiques pour detruire les spores dans le lait, nous avons etudie la germination et l’inactivation,par des methodes non-thermiques,de spores tres thermoresistantes de Bacillus Sporothermodurans. Tout d'abord, nous avons reussi a mettre au point un nouveau milieu de culture specifique a la germination de cette bacterie.En effet,une germination optimale, provoquee par les elements nutritifs, a ete obtenue apres incubation des spores, pendant 60 min a 35°C, en presence du 9mM de D-glucose et 60mM de L-alanine. En outre, une germination optimale provoquee par la pression hydrostatique a ete obtenue apres un traitement de 163MPa pendant 25 min a 20°C et apres une incubation post-pressurisation de 90 min dans l’eau distillee et un traitement de 147MPa pendant 20 min a 20°C et apres une incubation post-pressurisation de 55 min dans le lait. En ce qui concerne l’inactivation, une reduction de 5log du nombre initial des spores a ete obtenue dans les conditions de 477 MPa/48°C pendant 26 min dans l'eau distillee ou 495 MPa/49°C pendant 30 min dans le lait ou 472 MPa/53°C pendant 5 min en presence de la nisine (121 UI/ml).
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Inhibitory effects of nisin and potassium sorbate alone or in combination on vegetative cells growth and spore germination of Bacillus Sporothermodurans in milk.
Food Microbiology, 2015Co-Authors: Chedia Aouadhi, Slah Mejri, Abderrazak MaaroufiAbstract:The inhibitory activities of nisin or/and potassium sorbate on spores and vegetative cells of Bacillus Sporothermodurans LTIS27, which are known to be a contaminant of dairy products and to be extremely heat-resistant, were investigated. First, the tested concentrations of nisin or potassium sorbate inhibited vegetative cell growth; with the minimum inhibitory concentrations were 5 × 10(3) IU/ml and 2% (w/v), respectively. Then, the behaviour of vegetative cells and spores in presence of sub-lethal concentrations of nisin (50 UI/ml) or/and potassium sorbate (0.2%), in milk at 37 °C for 5 days, were evaluated. In the absence of inhibitors, strain grew and sporulated at the end of the exponential phase. Nisin (50 UI/ml) was able to inhibit spore outgrowth but didn't affect their germination. It induced an immediate and transitory reduction (1.6log(10) after 1 h and 2.8log(10) after 6 h of incubation) of vegetative cell growth which reappeared between 10 h and 24 h. Potassium sorbate (0.2%) had a durable bacteriostatic effect (1.1log(10) after 6 h), on vegetative cells, followed by a slower regrowth. It was able to inhibit both germination and outgrowth of spores. Association of nisin and potassium sorbate, at sub-lethal concentrations, showed a synergistic effect and resulted in a total inhibition of cells growth after 5 days. The results illustrate the efficacy of nisin and potassium sorbate in combination, and the commercial potential of applying such treatment to decontaminate any product that has a problem with persistence of bacterial spores.
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Inactivation of Bacillus Sporothermodurans spores by nisin and temperature studied by design of experiments in water and milk.
Food Microbiology, 2014Co-Authors: Chedia Aouadhi, Slah Mejri, Zeineb Rouissi, Abderrazak MaaroufiAbstract:Spores of Bacillus Sporothermodurans are known to be a contaminant of dairy products and to be extremely heat-resistant. A central composite experimental design with three factors using response surface methodology was used to evaluate the effect of nisin (50-150 UI/mL), temperature (80-100 °C), and temperature-holding time (10-20 min) on the inactivation of B. Sporothermodurans LTIS27 spores in distilled water, in skim milk and in chocolate milk. The experimental values were shown to be significantly in good agreement with the values predicted by the quadratic equation since the adjusted determination coefficients (Radj(2)) were around 0.97. By analyzing the response surfaces plots, the inactivation was shown to be higher in distilled water than in skim milk under all the conditions tested. Five-log cycle reductions of B. Sporothermodurans spores were obtained after a treatment at 95 °C for 12 min in presence of 125 UI of nisin/mL in distilled water or at 100 °C for 13 min in presence of 134 UI of nisin/mL in skim milk or at 100 °C for 15 min in presence of 135 UI of nisin/mL in chocolate milk. This study showed the efficiency of nisin (15-184 UI/mL) in combination with temperature (73-106 °C) to inactivate spores of B. Sporothermodurans in milk.
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Optimization of nutrient-induced germination of Bacillus Sporothermodurans spores using response surface methodology.
Food Microbiology, 2013Co-Authors: Chedia Aouadhi, Abderrazak Maaroufi, Hélène Simonin, Slah MejriAbstract:Spores of Bacillus Sporothermodurans are known to be contaminant of dairy products and to be extremely heat-resistant. The induction of endospore germination before a heat treatment could be an efficient method to inactivate these bacteria and ensure milk stability. In this study, the nutrient-induced germination of B. Sporothermodurans LTIS27 spores was studied. Testing the effect of 23 nutrient elements to trigger an important germination rate of B. Sporothermodurans spores, only D-glucose, L-alanine, and inosine were considered as strong independent germinants. Both inosine and L-alanine play major roles as co-germinants with several other amino acids. A central composite experimental design with three factors (L-alanine, D-glucose, and temperature) using response surface methodology was used to optimize the nutrient-induced germination. The optimal rate of nutrient-induced germination (100%) of B. Sporothermodurans spores was obtained after incubation of spore for 60 min at 35 °C in presence of 9 and 60 mM of D-glucose and L-alanine, respectively. The results in this study can help to predict the effect of environmental factors and nutrients on spore germination, which will be beneficial for screening of B. Sporothermodurans in milk after induction their germination. Moreover, the chosen method of optimization of the nutrient-induced germination was efficient in finding the optimum values of three factors.
Zacarchenco, Patricia Blumer - One of the best experts on this subject based on the ideXlab platform.
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Aperfeiçoamento da metodologia para contagem de Bacillus Sporothermodurans e sua ocorrencia em leite UAT/UHT
[s.n.], 2018Co-Authors: Zacarchenco, Patricia BlumerAbstract:Orientador: Mauro Faber de Freitas LeitãoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: Nos últimos anos, em nível internacional, o microrganismo Bacillus Sporothermodurans (BSP) vem sendo isolado a partir de leite processado pelo sistema UAT/UHT (Ultra Alta TemperaturalUltra High Temperature). Também no Brasil, mais recentemente, o microrganismo foi isolado a partir de amostras de leite UAT produzido em unidades industriais do Estado de São Paulo e Paraná. As amostras contaminadas apresentaram contagens em torno de 105UFC/ml sem evidências maiores de alterações organolépticas. No entanto, nestas condições, o leite UAT fica fora dos padrões exigidos pela legislação para o produto quanto a contagem de microrganismos aeróbios mesófilos, que é de 102UFC/ml(portaria SVSIMS n12451/97). Na primeira parte desta pesquisa, procurou-se avaliar e otimizar a metodologia para detecção de BSP, usando-se como referência a cepa DSMZ 10599, da Coleção Alemã de Microorganismos e Culturas Celulares (Deutsche Sammlug von Mikroorganismen und Zellkulturen). Em uma primeira etapa, avaliou-se qualitativa e quantitativamente o desempenho do ágar Infusão de Cérebro e Coração (BHI), ágar Padrão de Contagem (PCA) e o ágar Nutriente (NA) na contagem de BSP. Além disso, comparou-se as técnicas de inoculação em profundidade (pour p/ate) e espalhamento superficial (spread plate). Os melhores resultados foram obtidos pelo uso do ágar BHI (colônias mais diferenciadas e com melhor crescimento) e o uso da técnica de espalhamento superficial (maior uniformidade e crescimento das colônias). Numa segunda etapa formulou-se o meio diferencial (ágar BHI-E), com base na adição de 1g/I de esculina e 0,5gl1 de citrato férrico amoniacal, permitindo, assim, a melhor caracterização das colônias de BSP pelo seu enegrecimento e formação de halo escuro pela hidrólise da esculina. Numa terceira etapa, comprovou-se que a adição destes componentes não ocasionou quaisquer efeitos inibitórios comparativamente ao ágar BHI. Finalmente, numa última etapa, avaliou-se a eficiência da ágar BHI-E na diferenciação de BSP frente a outros Bacillus spp de ocorrência comum em produtos lácteos, caso de Bacillus cereus, Bacillus subtilis e Bacillus firmus. Na segunda parte da pesquisa, analisaram-se 100 amostras de leite UAT de 3 regiões do Brasil (Centro-Oeste, Sudeste e Sul) utilizando o ágar BHI-E desenvolvido na primeira fase. Verificou-se que 45% das amostras estavam fora dos padrões da Portaria SVS/MS no 451/97 e que 55% delas apresentavam esporos mesófilos termo-resistentes. Isolaram-se 300 colônias que hidrolisaram esculina e tinham aspecto semelhante às colônias do Bacillus Sporothermodurans em ágar BHI-E. A maior parte dos isolados apresentou morfologia típica e produzia oxidase, sendo que poucos não fermentaram a glicose. Vinte e quatro cepas, da segunda etapa da pesquisa, tiveram o seu DNA cromossômico avaliado pelo método RAPD ("random amplified polymorphic DNA"). Os perfis de DNA obtidos permitiram dividí-Ias em 3 grupos que apresentaram perfiJde DNA muito semelhantes ao do BSP.Abstrat: Bacillus Sporothermodurans - BSP - has been isolated worldwide in milk processed by the ultra high temperature system (UHT-milk). More recently BSP has also been isolated in Brazil, partieularly in industrial samples processed in São Paulo and Paraná states. Ali the samples showed total counts around 105cfu/ml without any organoleptie ehanges. However based on Brazilian microbiological standards (Portaria SVS/MS nQ451/97) fixing a limit of 1& cfu/ml, the samples were eonsidered inadequate, being rejected. The objective of the first part of this research was the evaluation and improvement of the methodology for Bsp detection, employing as reference the strain DSMZ 10599 from Germany Colection of Microorganisms and Cells Cultures (Deutsche Sammlug von Mikroorganismen und Zellkulturen). In a first step, the performanee of Brain Heart Infusion agar (BHI agar), Total Plate Count Agar (PCA) e Nutrient agar (NA) were comparatively evaluated, considering tooa the inoculation methods (pour plate compared to spread plate). The results showed the best performance of BHI agar with more uniform and better growth of Bsp, the same being valid eoncerning the use of spread plate compared to the pour plate method. In a seeond step, it was formulated a differential medium named BHI-E agar, based on addition of 19/1 of esculin and 0,5gl1 of ferrie ammonium eitrate; with this modification, BSP showed typical black colonies with a black halo due to esculin hydrolisis. In a third step, it was concluded that BHI-E agar did not show any inhibitory effect when compared to BHI agar. Finally, in a last step, BHI-E agar was evaluated in relation to the other Bacillus spp commonly found em milk products, particularly Bacillus cereus, Bacillus subtilis and Bacillus firmus. In the second part of the research work, a total of 100 comercial samples of UHT milk produced in 3 different Brazilian regions (Center-east, southern and southeast) were analysed using BHI-E agar previously developed and tested. The results showed that 45% of the examined samples did not attend the Brazilian standards (Portarja SVS/MS nQ451197)and 55% were contamined with mesophilic thermoresistant bacterial species. A total of 300 esculin positive cultures were isolated with morphological and cultural aspects similar to BSP besides being oxidase positive but generally showing a slow glucose fermenting activity. Twenty four strains isolated at the second part of this research were sub-tiped by molecular typing (RAPO). These 24 strains could be separated in 2 groups according to their ONA pattern. The profiles of 3 groups were very similar to that ONA pattern of Bacillus Sporothermodurans (BSP).MestradoMestre em Tecnologia de Alimento
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Occurrence of Bacillus Sporothermodurans and the influence of the thermal processing procedure on its presence in brazilian UHT milk
Sociedade Brasileira de Ciência e Tecnologia de Alimentos, 2015Co-Authors: Zacarchenco, Patricia Blumer, LeitÃo Mauro Faber De Freitas, Destro Maria Tereza, Andrigheto CristianoAbstract:Bacillus Sporothermodurans (BSP) has been isolated worldwide, but mainly in European contries as an usual contaminant of milk by the UHT system. Even being non pathogenic, generally its presence results in product rejection based on international microbiological standards limiting the counts to a maximum of 100 cfu/ml. In this research work, the occurrence of BSP was evaluated in UHT milk samples produced in different regions of Brazil (Central, South and Southeast) and processed by the indirect thermal treatment system (plate or tube exchangers) or by the direct system (steam infusion or steam injection). A total of 100 commercial samples were analysed both quantitatively by spread plate technique using Brain Heart Infusion Esculin Agar (BHI-E agar) and qualitatively after thermal shock treatment (115°C/7min), followed by incubation and plating in that medium. The results showed a total of 45% of the examined samples with counts above 100 cfu/ml, varying from 2 .10(4) and 9,5 .10(5) cfu/ml. However, 71.4% of the samples processed by indirect system were above limits, while none of the samples heat treated by the direct system, that allows a more drastic temperature condition, were rejected. A similar picture was observed in the qualitative evaluation, with contamination levels of 71,7 and 22,2% for the indirect and direct system, respectively. A total of 300 cultures were isolated from contaminated samples, showing cultural, morphological and biochemical patterns similar to BSP, except a late glucose fermentation capacity showed by most of the isolated strains. When 24 representative strains were submitted to PCR-RAPD evaluation and compared to the reference BSP strain (DSMZ 10599), they were confirmed as Bacillus Sporothermodurans based on their DNA pattern.O Bacillus Sporothermodurans (BSP) tem sido isolado em todo o mundo, principalmente nos países europeus, como um contaminante usual do leite processado pelo sistema UAT/UHT. Embora o BSP não seja patogênico, o leite contaminado fica fora dos padrões da legislação, que fixa o limite de contagem de microrganismos aeróbios mesófilos em 100ufc/ml. Tal bacilo foi pesquisado em amostras de leite UAT produzido nas regiões Sul, Sudeste e Centro-Oeste do Brasil, processado pelos sistemas de aquecimento indireto (trocadores a placas ou tubulares) e direto (injeção ou infusão de vapor). Foi analisado um total de 100 amostras, avaliadas quantitativamente pela técnica de espalhamento superficial no meio diferencial ágar infusão de cérebro e coração - esculina (ágar BHI-E). Na análise qualitativa as amostras foram submetidas a choque térmico (115ºC/7min), incubadas e plaqueadas no referido ágar. Os resultados revelaram um total de 45% de amostras com contagens acima do limite tolerável (100ufc/ml), oscilando entre 2x10(4) e 9,5x10(5)ufc/ml. No entanto, em função do tipo de sistema de aquecimento, as variações foram muito acentuadas, sendo que na avaliação quantitativa os índices de rejeição foram de 71,4% nas amostras aquecidas indiretamente e naquelas processadas diretamente. Isto se deve, provavelmente, às condições mais drásticas de tratamento térmico utilizada pelos laticínios com sistema direto de aquecimento. Na análise qualitativa, os índices de rejeição foram de 71,7 e 22,2%, respectivamente. Trezentas culturas foram isoladas das amostras contaminadas, revelando características culturais, morfológicas e bioquímicas similares as de BSP, exceto pela capacidade de fermentação lenta da glicose evidenciada pela maioria delas. Deste total, 24 cepas tiveram seu DNA cromossômico avaliado pelo método PCR-RAPD, sendo que os perfis obtidos foram muito similares ao de BSP referência (DSMZ 10599) confirmando tratar-se de Bacillus Sporothermodurans
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Ocorrência de Bacillus Sporothermodurans em leite UAT/UHT brasileiro e a influência do tratamento térmico
Sociedade Brasileira de Ciência e Tecnologia de Alimentos, 2000Co-Authors: Zacarchenco, Patricia Blumer, LeitÃo Mauro Faber De Freitas, Destro Maria Tereza, Andrigheto CristianoAbstract:O Bacillus Sporothermodurans (BSP) tem sido isolado em todo o mundo, principalmente nos países europeus, como um contaminante usual do leite processado pelo sistema UAT/UHT. Embora o BSP não seja patogênico, o leite contaminado fica fora dos padrões da legislação, que fixa o limite de contagem de microrganismos aeróbios mesófilos em 100ufc/ml. Tal bacilo foi pesquisado em amostras de leite UAT produzido nas regiões Sul, Sudeste e Centro-Oeste do Brasil, processado pelos sistemas de aquecimento indireto (trocadores a placas ou tubulares) e direto (injeção ou infusão de vapor). Foi analisado um total de 100 amostras, avaliadas quantitativamente pela técnica de espalhamento superficial no meio diferencial ágar infusão de cérebro e coração - esculina (ágar BHI-E). Na análise qualitativa as amostras foram submetidas a choque térmico (115ºC/7min), incubadas e plaqueadas no referido ágar. Os resultados revelaram um total de 45% de amostras com contagens acima do limite tolerável (100ufc/ml), oscilando entre 2x10(4) e 9,5x10(5)ufc/ml. No entanto, em função do tipo de sistema de aquecimento, as variações foram muito acentuadas, sendo que na avaliação quantitativa os índices de rejeição foram de 71,4% nas amostras aquecidas indiretamente e naquelas processadas diretamente. Isto se deve, provavelmente, às condições mais drásticas de tratamento térmico utilizada pelos laticínios com sistema direto de aquecimento. Na análise qualitativa, os índices de rejeição foram de 71,7 e 22,2%, respectivamente. Trezentas culturas foram isoladas das amostras contaminadas, revelando características culturais, morfológicas e bioquímicas similares as de BSP, exceto pela capacidade de fermentação lenta da glicose evidenciada pela maioria delas. Deste total, 24 cepas tiveram seu DNA cromossômico avaliado pelo método PCR-RAPD, sendo que os perfis obtidos foram muito similares ao de BSP referência (DSMZ 10599) confirmando tratar-se de Bacillus Sporothermodurans
