Potencial deteriorante da microbiota mesófila, psicrotrófica, termodúrica e esporulada do leite cru / Deteriorating potential of the mesophilic microbiota, psychrotrophic, termoduric and spore raw milk

Considerando os diversos problemas tecnológicos e econômicos que os microrganismos deteriorantes acarretam aos laticínios, o objetivo deste trabalho foi quantificar aeróbios mesófilos, psicrotróficos, termodúricos e esporulados no leite cru produzido na região Norte do Paraná e verificar a capacidade proteolítica e lipolítica desses microrganismos. Foram analisadas 20 amostras de leite cru e determinadas contagens de aeróbios mesófilos, psicrotróficos, termodúricos mesófilos, termodúricos psicrotróficos e esporos aeróbios e o perfil proteolítico e lipolítico de cada grupo. A contagem média de aeróbios mesófilos foi de 1,7x106UFC/mL acima do limite estabelecido pela Instrução Normativa 76 (3,0x105UFC/mL); 55% das amostras apresentaram contagens dentro dos padrões. As contagens de psicrotróficos variaram de 1,0x102 a 9,9x106UFC/mL e em 30% das amostras esse valor foi superior ao de mesófilos. A média de termodúricos mesófilos foi de 2,4x104UFC/mL, de termodúricos psicrotróficos 1,7x102UFC/mL e de esporos aeróbios foi de 5,0x101UFC/mL. Quanto ao perfil deteriorante, todos os grupos apresentaram atividade lipolítica e proteolítica elevadas, com predomínio de bactérias lipolíticas. Os valores encontrados indicam que o manejo higiênico-sanitário inadequado durante a ordenha e o resfriamento prolongado favorece a proliferação da microbiota deteriorante, que pode promover alterações significativas na qualidade e no rendimento do leite e seus derivados, mesmo após tratamento térmico.

Considering that technological problems and economic losses are basically caused by spoilage microorganisms on dairy industry, therefore the aim of this study was to quantify the mesophilic aerobic, psychrotrophic, thermoduric and aerobic spore-forming in raw milk produced in northern Paraná and also verify the proteolytic and lipolytic capacity of these microorganisms. Twenty samples of raw milk were analyzed and were determined mesophilic aerobic, psychrotrophic, thermoduric mesophilic, psychrotrophic thermoduric and aerobicspore-forming counts and proteolytic and lipolytic capacity from each group. The average counts of aerobic mesophilic were 1,79x106CFU/mL, above the limit established to Normative Instruction 76 (3,0x105CFU/mL); 55% samples had scores within the standards. Psychrotrophic counts ranged from 1,0x102 to 9,9x106CFU/mL and in 30% samples this count was higher than mesophilic. The average mesophilic thermoduric was 2,4x104CFU/mL, psychrotrophic thermoduric was 1,7x102CFU/mL and aerobic spore-forming was 5,0x101CFU/mL. As for the spoilage profile, all groups showed higher lipase and proteolytic activity, with a predominance of lipolytic bacteria. The values found indicates the inappropriate hygienic-sanitary management during milking and maintaining molk refrigerated for long periods until pasteurize, it can favorsthe proliferation of spoilage microbiota, which can promote significant changes in the quality and yield of milk and dairy products, even after the heat treatment.

Potencial deteriorante da microbiota mesófila, psicrotrófica, termodúrica e esporulada do leite cru / Deteriorating potential of the mesophilic microbiota, psychrotrophic, termoduric and spore raw milk

Considerando os diversos problemas tecnológicos e econômicos que os microrganismos deteriorantes acarretam aos laticínios, o objetivo deste trabalho foi quantificar aeróbios mesófilos, psicrotróficos, termodúricos e esporulados no leite cru produzido na região Norte do Paraná e verificar a capacidade proteolítica e lipolítica desses microrganismos. Foram analisadas 20 amostras de leite cru e determinadas contagens de aeróbios mesófilos, psicrotróficos, termodúricos mesófilos, termodúricos psicrotróficos e esporos aeróbios e o perfil proteolítico e lipolítico de cada grupo. A contagem média de aeróbios mesófilos foi de 1,7x106UFC/mL acima do limite estabelecido pela Instrução Normativa 76 (3,0x105UFC/mL); 55% das amostras apresentaram contagens dentro dos padrões. As contagens de psicrotróficos variaram de 1,0x102 a 9,9x106UFC/mL e em 30% das amostras esse valor foi superior ao de mesófilos. A média de termodúricos mesófilos foi de 2,4x104UFC/mL, de termodúricos psicrotróficos 1,7x102UFC/mL e de esporos aeróbios foi de 5,0x101UFC/mL. Quanto ao perfil deteriorante, todos os grupos apresentaram atividade lipolítica e proteolítica elevadas, com predomínio de bactérias lipolíticas. Os valores encontrados indicam que o manejo higiênico-sanitário inadequado durante a ordenha e o resfriamento prolongado favorece a proliferação da microbiota deteriorante, que pode promover alterações significativas na qualidade e no rendimento do leite e seus derivados, mesmo após tratamento térmico.(AU)

Considering that technological problems and economic losses are basically caused by spoilage microorganisms on dairy industry, therefore the aim of this study was to quantify the mesophilic aerobic, psychrotrophic, thermoduric and aerobic spore-forming in raw milk produced in northern Paraná and also verify the proteolytic and lipolytic capacity of these microorganisms. Twenty samples of raw milk were analyzed and were determined mesophilic aerobic, psychrotrophic, thermoduric mesophilic, psychrotrophic thermoduric and aerobicspore-forming counts and proteolytic and lipolytic capacity from each group. The average counts of aerobic mesophilic were 1,79x106CFU/mL, above the limit established to Normative Instruction 76 (3,0x105CFU/mL); 55% samples had scores within the standards. Psychrotrophic counts ranged from 1,0x102 to 9,9x106CFU/mL and in 30% samples this count was higher than mesophilic. The average mesophilic thermoduric was 2,4x104CFU/mL, psychrotrophic thermoduric was 1,7x102CFU/mL and aerobic spore-forming was 5,0x101CFU/mL. As for the spoilage profile, all groups showed higher lipase and proteolytic activity, with a predominance of lipolytic bacteria. The values found indicates the inappropriate hygienic-sanitary management during milking and maintaining molk refrigerated for long periods until pasteurize, it can favorsthe proliferation of spoilage microbiota, which can promote significant changes in the quality and yield of milk and dairy products, even after the heat treatment.(AU)

Influence of the microbiological quality of raw milk on the shelf life of pasteurized milk / Influência da qualidade microbiológica do leite cru na vida útil do leite pasteurizado

The dairy industry strives to produce high quality products with high nutritional value as well as to meet the legal standards for longer shelf life. However, these goals are made unfeasible by the poor quality of raw milk produced in some regions of Brazil. Others Brazilian dairy regions, however, already succeed in producing milk with low microbial counts, such as the municipality of Castro, Paraná state, designated as the ‘Brazilian dairy capital. In order to evaluate the effect of raw milk quality on microbial counts during the shelf life of pasteurized milk, samples were collected from two dairy regions of Paraná: the northern and Castro region, characterized by milk production with high and low microbiological counts, respectively. Samples were experimentally pasteurized and the total microorganism counts were analyzed for 18 days at 7°C, using the Brazilian standard microbiological count limit for pasteurized milk (8 x 104 CFU/mL) as the end of the shelf life. Low microbiological counts in raw milk (Castro) resulted in significantly lower counts shortly after pasteurization and over the entire shelf life, meeting the pasteurized milk standard for 18 days. The temporal evolution in the counts over 18 days for the milks of high and low microbiological count was similar; however, the disparity between the absolute counts between the regions was significant (p < 0.05). Of the milk samples from northern Paraná, four (44.4%) already had counts higher than that of the legislative limit for pasteurized milk immediately after pasteurization. The others (five) reached the maximum microbiological count limit for pasteurized milk on the 6th day after pasteurization. In contrast, the milk from the Castro region remained below the limit throughout the analysis period...(AU)

Produtos de alta qualidade, com elevado valor nutricional e longa vida útil, são objetivos almejados pela indústria de laticínios, dificultados pela má qualidade microbiológica do leite cru produzido no Brasil. No entanto, algumas regiões já produzem leite com baixas contagens microbianas, como a capital brasileira do leite, Castro, no Paraná. Com a intenção de avaliar o efeito da qualidade do leite cru nas contagens microbianas durante o período de vida útil de leite pasteurizado, amostras de leite cru foram coletadas de duas regiões do Paraná: a região norte e a região de Castro, caracterizadas pela produção de leite com contagens microbiológicas altas e baixas, respectivamente. As amostras foram experimentalmente pasteurizadas e analisadas as contagens totais de micro-organismos pelo período de 18 dias a 7ºC, utilizando o padrão determinado na IN62/2011 para o fim da vida de útil (máx. 8 x 104 UFC/mL). Baixas contagens microbiológicas no leite cru resultaram em contagens significativamente menores logo após a pasteurização e ao longo da vida útil, atendendo ao padrão de qualidade para leite pasteurizado por 18 dias. As evoluções nas contagens durante o período de análise para os leites de alta e baixa contagem microbiológica foram proporcionais, no entanto, a disparidade entre as contagens absolutas entre as regiões foi significativa. O leite do norte do Paraná atingiu o limite máximo para o leite pasteurizado no 6°dia após a pasteurização, enquanto o leite da região de Castro manteve-se abaixo da contagem limite durante todo o período de análise. Dessa forma, é possível afirmar que as contagens de microrganismos e a vida útil do leite pasteurizado está diretamente relacionada às contagens apresentadas pelo leite antes da pasteurização. Assim, para prolongar a vida útil do leite pasteurizado, deve-se diminuir as contagens do leite cru e, como se sabe, as boas práticas de higiene na produção são essenciais para atingir esse objetivo.(AU)

Influence of the microbiological quality of raw milk on the shelf life of pasteurized milk / Influência da qualidade microbiológica do leite cru na vida útil do leite pasteurizado

The dairy industry strives to produce high quality products with high nutritional value as well as to meet the legal standards for longer shelf life. However, these goals are made unfeasible by the poor quality of raw milk produced in some regions of Brazil. Others Brazilian dairy regions, however, already succeed in producing milk with low microbial counts, such as the municipality of Castro, Paraná state, designated as the ‘Brazilian dairy capital’. In order to evaluate the effect of raw milk quality on microbial counts during the shelf life of pasteurized milk, samples were collected from two dairy regions of Paraná: the northern and Castro region, characterized by milk production with high and low microbiological counts, respectively. Samples were experimentally pasteurized and the total microorganism counts were analyzed for 18 days at 7°C, using the Brazilian standard microbiological count limit for pasteurized milk (8 x 104 CFU/mL) as the end of the shelf life. Low microbiological counts in raw milk (Castro) resulted in significantly lower counts shortly after pasteurization and over the entire shelf life, meeting the pasteurized milk standard for 18 days. The temporal evolution in the counts over 18 days for the milks of high and low microbiological count was similar; however, the disparity between the absolute counts between the regions was significant (p < 0.05). Of the milk samples from northern Paraná, four (44.4%) already had counts higher than that of the legislative limit for pasteurized milk immediately after pasteurization. The others (five) reached the maximum microbiological count limit for pasteurized milk on the 6th day after pasteurization. In contrast, the milk from the Castro region remained below the limit throughout the analysis period...

Produtos de alta qualidade, com elevado valor nutricional e longa vida útil, são objetivos almejados pela indústria de laticínios, dificultados pela má qualidade microbiológica do leite cru produzido no Brasil. No entanto, algumas regiões já produzem leite com baixas contagens microbianas, como a capital brasileira do leite, Castro, no Paraná. Com a intenção de avaliar o efeito da qualidade do leite cru nas contagens microbianas durante o período de vida útil de leite pasteurizado, amostras de leite cru foram coletadas de duas regiões do Paraná: a região norte e a região de Castro, caracterizadas pela produção de leite com contagens microbiológicas altas e baixas, respectivamente. As amostras foram experimentalmente pasteurizadas e analisadas as contagens totais de micro-organismos pelo período de 18 dias a 7ºC, utilizando o padrão determinado na IN62/2011 para o fim da vida de útil (máx. 8 x 104 UFC/mL). Baixas contagens microbiológicas no leite cru resultaram em contagens significativamente menores logo após a pasteurização e ao longo da vida útil, atendendo ao padrão de qualidade para leite pasteurizado por 18 dias. As evoluções nas contagens durante o período de análise para os leites de alta e baixa contagem microbiológica foram proporcionais, no entanto, a disparidade entre as contagens absolutas entre as regiões foi significativa. O leite do norte do Paraná atingiu o limite máximo para o leite pasteurizado no 6°dia após a pasteurização, enquanto o leite da região de Castro manteve-se abaixo da contagem limite durante todo o período de análise. Dessa forma, é possível afirmar que as contagens de microrganismos e a vida útil do leite pasteurizado está diretamente relacionada às contagens apresentadas pelo leite antes da pasteurização. Assim, para prolongar a vida útil do leite pasteurizado, deve-se diminuir as contagens do leite cru e, como se sabe, as boas práticas de higiene na produção são essenciais para atingir esse objetivo.

Spoilage potential of spore-forming bacteria from refrigerated raw milk / Potencial deteriorante de bactérias formadoras de esporos do leite cru refrigerado

Aerobic bacterial spores are an important group of microorganisms in raw milk. These microbes are thermoduric, whereas the vegetative forms are thermophilic, thermoduric and psychrotrophic and reduce the shelf life of pasteurized milk. In Brazil, there are a lack of studies on the load of aerobic spores in raw milk; thus, little is known about the spoilage activity of these organisms. The aim the present study was to quantify the aerobic spores in Brazilian refrigerated raw milk of dairy region of Castro, Paraná state, assess the potential proteolytic and/or lipolytic isolates and identify the microorganisms derived from the germination. Twenty milk samples were evaluated, and the aerobic spore count was performed after plating the samples following heat treatment at 80ºC for 12 min. The activity proteolytic and lipolytic isolates were evaluated through subculture on milk agar and tributyrin agar, respectively, and these microorganisms were identified using partial 16S rRNA gene sequences that were compared through GenBank. The aerobic spore counts ranged from 1 to 3.7 log CFU.mL-¹, with a mean of 1.75 (± 0.59) log CFU.mL-¹. After spore germination, 137 aerobic bacterial isolates were obtained, 40 of which (29.2%) showed milk spoilage activity. Among these, 31 isolates (77.5%) were proteolytic and lipolytic, seven isolates (17.5%) were exclusively lipolytic and two isolates (5%) were only proteolytic. Based on the 16S rRNA gene analysis, Bacillus licheniformis (55%), Bacillus spp. (27.5%), Paenibacillus spp. (7.5%), Bacillus pumilus (5%), Bacillus circulans (2.5%) and Brevibacillus spp. (2.5%) were identified. Studies of Brazilian raw milk microbiota have not yet described B. circulans which are frequently detected in milk from other countries. Among the 22 B. licheniformis isolates, 21 microbes (95.5%) showed proteolytic and lipolytic activity, and one isolate (4.5%) exhibited only proteolytic activity. The two B. pumilus isolates were proteolytic...(AU)

Esporos de bactérias aeróbias são um importante grupo de micro-organismos no leite cru. Esses micro-organismos são termodúricos, e suas formas vegetativas são termofílicas, termodúricas e psicrotróficas e reduzem a vida útil do leite pasteurizado. No Brasil, não há estudos sobre os esporos aeróbios no leite cru, assim, pouco se sabe sobre o potencial deteriorante dessa microbiota no leite cru brasileiro. O objetivo do presente trabalho foi quantificar esporos aeróbios no leite cru refrigerado brasileiro, avaliar o seu potencial proteolítico e/ou lipolítico e identificar esses micro-organismos originários da germinação dos esporos. Foram avaliadas 20 amostras de leite cru refrigerado, nas quais foi realizada a contagem de esporos aeróbios após o tratamento térmico de 80ºC por 12 min. A atividade proteolítica e/ou lipolítica dos isolados foi avaliada após o repique das colônias em ágar leite e tributirina, respectivamente, e a identificação desses micro-organismos deteriorantes foi realizada pelo sequenciamento parcial do gene 16S rRNA e comparação com as sequencias depositadas no GenBank. As contagens de esporos aeróbios variaram de 1 a 3.7 log CFU.mL-¹, com média de 1.75 (± 0.59) log UFC.mL-¹. Após a germinação dos esporos, foram obtidas 137 colônias, das quais 40 (29.2%) apresentaram algum tipo de atividade deteriorante: 31 (77.5%) isolados foram proteolíticos e lipolíticos, sete (17.5%) foram exclusivamente lipolíticos e dois (5%) foram apenas proteolíticos. Baseado nas sequencias do gene 16S rRNA, foram identificados Bacillus licheniformis (55%), Bacillus spp. (27.5%), Paenibacillus spp. (7.5%), Bacillus pumilus (5%), Bacillus circulans (2.5%) e Brevibacillus spp. (2.5%) entre os esporulados deteriorantes. Estudos preliminaries sobre a microbiota do leite brasileiro ainda não haviam descrito B. circulans e Paenibacillus, gêneros frequentemente descritos no leite de outros países. Entre os 22 B. licheniformis...(AU)

Draft genome sequence of pectic polysaccharide-degrading moderate thermophilic bacterium Geobacillus thermodenitrificans DSM 101594

Geobacillus thermodenitrificans DSM 101594 was isolated as a producer of extracellular thermostable pectic polysaccharide degrading enzymes. The completely sequenced genome was 3.6 Mb in length with GC content of 48.86%. A number of genes encoding enzymatic active against the high molecular weight polysaccharides of potential biotechnological importance were identified in the genome.(AU)

Thermoduric psychrotrophic proteolytic microbiota from refrigerated raw milk / Microbiota termodúrica psicrotrófica proteolítica do leite cru refrigerado

Thermoduric microorganisms may withstand high temperatures during the pasteurization of milk. Therefore, the microbiota in pasteurized milk consist of thermoduric microbes and directly influence the shelf-life of the milk. The aim of this study was to identify thermoduric psychrotrophic proteolytic microbiota in refrigerated raw milk. Twenty samples, previously heat-treated, were streaked and incubated at 7°C for 10 days. The strains isolated were streaked on milk agar to assess proteolytic activity and were initially analyzed morphologically by light microscopy and then by molecular techniques to identify the species. In 40% of the samples analyzed was observed only one bacterial growth and others 10 thermoduric psychrotrophic fungi. All isolates were proteolytic. The sequencing of 16S rRNA gene identified the bacterial strain as Bacillus pumilus and analysis of the ITS1-5.8S-ITS2 region of fungal isolates revealed the Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), and the 3 remaining strains were identified as Geotrichum candidum (30%). This is a fist description of B. pumilus in Brazilian raw milk. Considering the spoilage potential of all isolates and of the fungi present in raw milk and their survival in pasteurized milk, it is extremely important to carry out further studies to your resistant to heat, their impact on the shelf-life of pasteurized milk, ultra-high temperature (UHT) milk and dairy products.(AU)

Os micro-organismos termodúricos são aqueles capazes de resistir ao processo de pasteurização do leite. Assim, a microbiota do leite pasteurizado é constituída por esses micro-organismos que podem influenciar diretamente na vida sua vida útil. O objetivo do presente trabalho foi identificar a microbiota termodurica psicrotrófica proteolítica do leite cru refrigerado. Foram avaliadas 20 amostras de leite cru refrigerado, plaqueadas e incubadas a 7°C por 10 dias. As cepas isolados foram repicadas em ágar leite para verificação da atividade proteolítica e inicialmente avaliadas microscopicamente e posteriormente identificadas por sequenciamento de DNA. Foi observado apenas um crescimento bacteriano e outros 10 isolados fúngicos termodúricos psicrotróficos em 40% das amostras avaliadas. Todos os isolados foram proteolíticos. O sequenciamento parcial do gene 16S rRNA da cepa bacteriana permitiu a identificação de Bacillus pumilus e a análise da região ITS1-5.8S-ITS2 dos isolados fúngicos os identificou como Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), e Geotrichum candidum (30%). Não foram encontrados estudos anteriores sobre a presença de B. pumilus no leite cru brasileiro. Considerando o potencial deteriorante de todos os isolados e presença de fungos termodúricos psicrotróficos no leite cru, é importante a continuidade trabalhos que avaliem sua resistência térmica e seu impacto na vida útil do leite pasteurizado, longa vida e derivados.(AU)

Análise microbiológica e físico-química de material proteico comestível derivado do processamento da gordura oriunda do abate de bovinos / Microbiological and Physical-Chemical Analysis of Edible Protein Obtained as a By-Product of Fat Rendering in Cattle Slaughter

Background: The use of all by-products of bovine slaughter is of high economic importance for the industries of products of animal origin. Among these products, fat has an important role, once fat rendering may generate several different products, such as protein material that may be used in the manufacture of meat products. However, in spite of the importance that the use of all by-products has for the economic balance of the industry, there are no reports on their use in Brazil, or studies that supply data on microbiological and physical-chemical local standards for this protein. Thus, the objective of this study was to evaluate microbiological and physical-chemical characteristics of protein material obtained from fat rendering, as well as to provide support for companies to use fat rendering to generate protein material, adding value to industrialized meat products. Materials, Methods & Results: The experimental production of edible protein obtained of fat rendering was conducted in slaughterhouse with supervision of the Brazilian Ministry of Agriculture, Livestock and Food Supply. Protein material was obtained in a continuous, humid heat system at high temperatures. Fat scraps containing protein were ground and cooked at high temperature (85°C), and placed in a three phase decanter centrifuge. After centrifugation, protein material was ground again and packed. Samples were collected from 15 batches of protein material, and the following microbiological analyses were carried out: counts of aerobic mesophilic and psychrotrophic microorganisms, coliforms at 35ºC, Escherichia coli, sulfite-reducing Clostridium, and Staphylococcus aureus, besides presence or absence of Salmonella and Listeria monocytogens. The following physical-chemical analyses were also carried out: protein, total lipid, moisture, ash, carbohydrate, and energy content. Mean counts of mesophiles, psychrotrophs, and coliforms at 35ºC were 4.17; 3.69 and 1.87 (log CFU/g), respectively. Levels of protein, total lipids, moisture, ashes and carbohydrates were 27.50; 7.83; 63.88%; 0.24%; and 0.55%, respectively, and energy content was 182.63 kcal/100g. Discussion: Results of microbiological analyses demonstrated that, although low, the final product showed to be contaminated. Contamination that occurred during the second grinding procedure may be an explanation for these bacterial counts. Also, the temperature used for fat fusion was not enough to eliminate thermoduric microorganisms. However, even with the presence of indicator microorganisms in the samples, none was contaminated by E. coli, sulfite-reducing Clostridium, S. aureus, Salmonella or L. monocytogenes. Physical-chemical analyses showed that the product had adequate nutritional quality. Based on these results, it was possible to conclude that protein material obtained in fat rendering showed characteristics that enable the use of this product as raw material for processed meat products. Besides, the present study was the first one to present scientific results in relation to edible by-products obtained in fat rendering, supplying important information for slaughterhouses and meat-processing plants. The study also produced relevant data on the innocuousness of the product, which may be used to guide decision-making of health inspectors.

MICRO-ORGANISMOS DETERIORANTES DO LEITE: ATIVIDADE PROTEOLÍTICA E LIPOLÍTICA DE BACTÉRIAS PSICROTRÓFICAS E TERMODÚRICAS MESÓFILAS

A presença de enzimas microbianas deteriorantes termoestáveis proteolíticas e lipolíticas pode comprometer a qualidade e vida útil do leite processado e derivados, mesmo que o leite cru apresente baixas contagens totais de micro-organismos. Além disso, as formas microbianas vegetativas ou esporuladas podem resistir à pasteurização e também atuarem como deteriorantes do leite pasteurizado. Como passo preliminar para redução dos micro-organismos deteriorantes no leite cru refrigerado, aumento da vida útil e do potencial tecnológico do leite pasteurizado, o presente trabalho teve como objetivo identificar a diversidade genética de micro-organismos psicrotróficos e termodúricos com potencial deteriorante proteolítico e/ou lipolítico no leite cru refrigerado de alta qualidade microbiológica. Foram avaliadas 20 amostras de leite cru refrigerado produzido na bacia leiteira da região de Castro, Paraná, nas quais foram utilizados métodos oficiais para a contagem e isolamento de psicrotróficos e termodúricos e técnicas biomoleculares de agrupamento genético hierárquico (Restriction Fragment Length Polymorphism) e caracterização (sequenciamento parcial do gene 16S rRNA e análise filogenética) dos isolados. As contagens médias de psicrotróficos e termodúricos foram 1,1 x 104 e 3,2 x 102 UFC/mL, respectivamente. Entre os psicrotróficos, 47,8% do total de isolados (n = 295) apresentaram atividade deteriorante, dos quais 30,5% foram proteolíticos e lipolíticos simultaneamente, 31,9% exclusivamente proteolíticos e 37,6% lipolíticos. Pelos dendrogramas foram determinados 36 clusters de psicrotróficos proteolíticos e 47 de lipolíticos com 70% de similaridade hierárquica, cuja identificação filogenética determinou o predomínio das espécies com os mesmos perfis polimórficos de Lactococcus lactis (24,5%), Enterobacter kobei (15,4%), Aerococcus urinaeequi (8,4%) e Acinetobacter lwoffii (6,3%) entre os psicrotróficos deteriorantes. Serratia ureilytica, Enterobacter kobei, Pseudomonas spp. e Yersinia enterocolitica foram os psicrotróficos identificados como potencialmente produtores de metaloprotease alcalina (aprX). Entre os termodúricos, 42,6% do total de isolados (n = 310) foram deteriorantes, dos quais 32,6% apresentaram atividade proteolítica e lipolítica simultaneamente, 31% exclusivamente proteolítica e 36,4% apenas lipolítica. Esses isolados proteolíticos e lipolíticos foram agrupados em 41 e 38 clusters, respectivamente, considerando 50% de similaridade. A análise filogenética das sequências parciais do gene 16S rRNA revelou que predominam entre os termodúricos deteriorantes as espécies Bacillus licheniformis (34,1%), Brachybacterium nesterenkovii (14,4%), Enterococcus faecalis (9,1%) e Streptococcus agalactiae (8,3%). Foi observado que 50% das cepas de termodúricos pertencem aos gêneros Bacillus e Paenibacillus, frequentemente descritos como esporulados que resistem a tratamentos térmicos mais intensos que a pasteurização e que comprometem a vida útil do leite pasteurizado dos Estados Unidos. Os resultados do presente trabalho demonstram que a composição da microbiota é tão importante quanto a sua quantificação. Apesar de baixas contagens, psicrotróficos e termodúricos podem comprometer a qualidade do leite e derivados pela sua atividade deteriorante, sendo um fator limitante na manutenção da qualidade do leite fluido e derivados. Portanto, é necessário desenvolver ferramentas sensíveis e eficientes para monitorar e controlar a presença desses micro-organismos no leite cru.

The presence of heat-stable spoilage enzymes can compromise the milk and dairy products quality and shelf life, even if raw milk with low total microorganisms counts. In addition, the vegetative or sporulated microbial forms may resist pasteurization and also deteriorate pasteurized milk. The present work aimed to identify the genetic diversity of psychrotrophic and thermoduric microorganisms with proteolytic and/or lipolytic potential in high microbiological quality refrigerated raw milk. Twenty samples of refrigerated raw milk produced in the dairy region of Castro, Paraná State, Brazil, were evaluated per official methods for the counting and isolation of psychrotrophic and thermoduric. Biomolecular techniques of hierarchical genetic grouping (Restriction Fragment Length Polymorphism) and identification by partial sequencing of the 16S rRNA gene and phylogenetic analysis. The mean of psychrotrophic and thermoduric counts were 1.1 x 104 and 3.2 x 102 CFU/mL, respectively. Among the psychrotrophs, 47.8% of the total isolates (n = 295) presented spoilage potential, of which 30.5% were proteolytic and lipolytic simultaneously, 31.9% exclusively proteolytic and 37.6% lipolytic. The dendrograms determined 36 clusters of proteolytic and 47 lipolytic psychrotrophs with 70% genetic hierarchical similarity, whose phylogenetic identification shows the predominance of the species with the same polymorphic profiles of Lactococcus lactis (24.5%), Enterobacter kobei (15.4%), Aerococcus urinaeequi (8.4%) and Acinetobacter lwoffii (6.3%) among the deteriorating psychrotrophs. Serratia ureilytica, Enterobacter kobei, Pseudomonas spp. and Yersinia enterocolitica were the psychrotrophs identified as potentially producing of alkaline metalloprotease (aprX gene positive). Among the thermoduric, 42.6% of the total isolates (n = 310) shows spoilage potential, of which 32.6% presented proteolytic and lipolytic activity simultaneously, 31% were exclusively proteolytic and 36.4% only lipolytic. These proteolytic and lipolytic isolates were grouped in 41 and 38 clusters, respectively, considering 50% of genetic similarity. Phylogenetic analysis of partial sequences of 16S rRNA gene revealed that Bacillus licheniformis (34.1%), Brachybacterium nesterenkovii (14.4%), Enterococcus faecalis (9.1%) and Streptococcus agalactiae (8.3%). It was observed that 50% of the thermoduric strains belong to the genus Bacillus and Paenibacillus, often described as sporulated that resist heat treatments that are more intense at pasteurization and that compromise the shelf life of the pasteurized milk in the United States. The results of the present study demonstrate that the microbiota composition of raw milk as so important that your quantification. Although low counts, psychrotrophic and thermoduric can compromise the milk quality and dairy produtcs by their deteriorating activity, being a limiting factor for the maintenance of the quality. Therefore, it is necessary to development of a sensitive and efficient tools to monitor and control the presence of these microorganisms in raw milk.

Caracterização bioquímica de bactérias psicrotróficas e produção de enzimas termorresistentes em leite cru

Objetivou-se com o presente trabalho isolar e caracterizar bioquimicamente a microbiota psicrotrófica, bem como avaliar seu poder deteriorador no leite cru refrigerado, armazenado em tanques de expansão, individuais e coletivos, de propriedades das regiões Agreste e Sertão do Estado de Alagoas. Foram analisadas amostras de leite coletadas em 23 tanques individuais e 13 coletivos, onde se verificou a presença de bactérias psicrotróficas nos dois tipos de tanques de refrigeração, sendo um total de 105 isolados bacterianos. Foi detectada a predominância de bactérias psicrotróficas gram-negativas 78,09% (82/105), dos gêneros Pseudomonas, Enterobacter, Aeromonas, Burkholderia, Acinetobacter e Shigella, enquanto que as bactérias gram-positivas foram da ordem de 21,90% (23/105) dos gêneros Streptococcus, Corynebacterium, Enterococcus, Micrococcus e Bacillus. Pseudomonas foi o gênero predominante com 43,81% (46/82). A grande maioria dos isolados bacterianos demonstrou alto poder de produção de enzimas extracelulares protease, lipase e/ou lecitinase, evidenciando assim potencial deteriorador do leite e derivados. Estatisticamente, não foi significativa a diferença de produção enzimática pelos isolados entre tanques individuais e coletivos, não havendo dependência entre o número de isolados bacterianos produtores de enzimas e o tipo de tanque.

The goal of this study was the isolation and biochemical characterization of psychotrophic microbiota, evaluating its deteriorating potential on refrigerated raw milk stored in both individual and collective expansion tanks located in farms from Agreste and Sertão regions of Alagoas State. Milk samples from 23 (individual) and 13 (collective) tanks were analyzed, and the presence of psychotropic bacteria was diagnosed in both types of refrigeration tanks, totalizing 105 bacterial isolates. There was a predominance of gram-negative psychotropic bacteria 78,09% (82/105) from genus Pseudomonas, Enterobacter, Aeromonas, Burkholderia, Acinetobacter e Shigella, whereas gram-positive bacteria represented 21,90% (23/105) of total isolates, from genus Streptococcus, Corynebacterium, Enterococcus, Micrococcus and Bacillus. In general, Pseudomonas was the most prevalent genus isolated 43,81% (46/82). The majority of bacterial isolates had high extracellular enzymes production protease, lipase and/or lecithinase, which evidenced elevate deteriorating potential for milk and its derivatives. There was no significant difference on enzymatic production among isolates or between individual and collective tanks, as well as no dependency between total number of enzymatic producer bacterial isolates and tank type. Keywords: Gram-negative, Proteases, Lipases, Pseudomonas, Thermoduric.

ISOLAMENTO E IDENTIFICAÇÃO DA MICROBIOTA ESPORULADA E FÚNGICA ASSOCIADA À DETERIORAÇÃO DO LEITE

A produção de leite vem aumentado no estado do Paraná e no Brasil, porém, a sua qualidade ainda é um problema. A baixa qualidade do leite cru refrigerado brasileiro faz com que a vida útil do leite pasteurizado não alcance seis dias. O aumento da vida útil do leite pasteurizado está diretamente relacionado à redução das contagens de micro-organismos deteriorantes proteolíticos e lipolíticos, sobretudo os termodúricos e esporulados no leite cru, que constituirão a microbiota remanescente no leite pasteurizado. Não há dados no Brasil sobre a composição da microbiota esporulada e fúngica termodúrica presente no leite, dificultando o seu controle. O presente trabalho teve por objetivo identificar os micro-organismos formadores de esporos e a microbiota fúngica do leite cru refrigerado com atividade proteolítica e/ou lipolítica por técnicas moleculares. Foram avaliadas 20 amostras de leite cru refrigerado produzido na bacia leiteira de Castro, região central do estado do Paraná, nas quais foram realizadas a contagem de esporos aeróbios, verificação da atividade proteolítica e lipolítica dos isolados, amplificação parcial do gene 16S rRNA, sequenciamento e identificação por similaridade usando a ferramenta BLAST para comparação com as sequencias depositadas no GenBank do National Center for Biotechnology Information (NCBI). Para o isolamento dos fungos, as amostras de leite cru foram submetidas à pasteurização laboratorial, semeadas e incubadas em temperaturas apropriadas para a multiplicação de bolores e leveduras termodúricos mesófilos e psicrotróficos. Também foi avaliado o potencial proteolítico dos isolados obtidos, que foram identificados por microscopia óptica e confirmados por sequenciamento da região ITS do DNA. As contagens de esporos aeróbios variaram de 1 a 3,7 log UFC.mL-1, com média de 1,75 (±0,59) log UFC.mL-1. Foram obtidos 137 isolados puros de bactérias a partir da germinação dos esporos, dos quais 40 (29,2%) apresentaram atividade deteriorante do leite. Dessas, 31 (77,5%) foram proteolíticas e lipolíticas, 7 (17,5%) foram exclusivamente lipolíticas e 2 (5%) foram apenas proteolíticas. As 40 cepas de esporos aeróbios proteolíticos e/ou lipolíticos submetidas ao sequenciamento do 16S rRNA, foram identificadas como: Bacillus licheniformis (55%), Bacillus sp. (27,5%), Paenibacillus sp. (7,5%), Bacillus pumilus (5%), Bacillus circulans (2,5%) e Brevibacillus sp. (2,5%). Estudos anteriores sobre a composição da microbiota do leite cru refrigerado brasileiro não relatam a presença de micro-organismos do gênero Paenibacillus, apesar de ser um grupo de micro-organismos frequentemente descrito nos trabalhos desenvolvidos em outros países. Dos 22 isolados de B. licheniformis, 21 (95,5%) apresentaram atividade proteolítica e lipolítica e 1 (4,5%) foi apenas proteolítico. As duas cepas de B. pumilus foram proteolíticas e lipolíticas, enquanto a cepa de B. circulans foi apenas lipolítica. Em relação aos 11 isolados de Bacillus sp., oito (72,7%) foram proteolíticos e lipolíticos, um (9,1%) foi proteolítico e outros dois (18,2%) lipolíticos. Os três isolados de Paenibacillus sp. foram apenas lipolíticos, assim como a cepa de Brevibacillus sp. Com relação aos fungos, foram obtidos 11 isolados, um mesófilo e outros 10 psicrotróficos. Todos os isolados foram proteolíticos. O sequenciamento do DNA fúngico amplificado confirmou a identificação microscópica, sendo a cepa termodúrica mesófila identificada como espécies do complexo Fusarium chlamydosporum (9,09%), e os psicrotróficos como Cladosporium cladosporoides (54,54%), Curvularia geniculatus (9,09%) e os outros 3 isolados foram identificados como Geotrichum candidum (27,27%). A presença de fungos termodúricos mesófilos e psicrotróficos proteolíticos no leite cru pode resultar em redução da vida útil do leite pasteurizado, bem como em risco à saúde pela presença de cepas patogências e produtoras de micotoxinas. A redução dos micro-organismos termodúricos, proteolíticos e/ou lipolíticos no leite cru, diminui a quantidade de micro-organismos e enzimas deteriorantes termoestáveis no leite pasteurizado, aumentando sua vida útil.

Milk production has increased in the Paraná state and Brazil, but its quality is still a important problem. The low quality of brazilian refrigerated raw milk makes the shef-life of pasteurized milk not reach 6 days. Increased shelf-life of pasteurized milk is directly related to the reduction of microorganisms counts proteolytic and lipolytic rough, especially thermoduric and spores in raw milk, which constitute the remaining microbiota in pasteurized milk. There is no data in Brazil about the composition of the spore-forming and thermoduric fungal microbiota in milk, hindering the control. The aim this study was to identify the microorganisms spore-forming and mycoflora of refrigerated raw milk with proteolytic and/or lipolytic activity by molecular biological techniques. Were evaluated 20 samples of refrigerated raw milk produced in the dairy region Castro, Paraná, Brazil, in which were performed aerobic spore count, verification of proteolytic and lipolytic activity of the isolated, partial amplification of the 16S rRNA gene, identification by sequencing data and comparison with the sequences deposited in GenBank of National Center for Biotechnology Information (NCBI). For the isolation of thermoduric fungi, the raw milk samples were subjected to laboratory pasteurization, plated and incubated at appropriate temperatures for multiplication of molds and yeasts mesophilic and psychrotrophic. We also assessed the proteolytic potential of these isolates that were identified by optical microscopy and confirmed by sequencing data the ITS region of DNA. Aerobic spore counts ranged from 1 to 3.7 log CFU.mL-1, with a mean of 1.75 (± 0.59) log CFU.mL-1. One hundred thirty-seven bacterial isolates were obtained from germination of spores, of which 40 (29.2%) showed deteriorating activity of milk. Of these, 31 (77.5%) were proteolytic and lipolytic, 7 (17.5%) were exclusively lipolytic and 2 (5%) were only proteolytic. The 40 strains of aerobic spore-forming proteolytic and/or lipolytic sequecing to 16S rRNA were identified as Bacillus licheniformis (55%), Bacillus sp. (27.5%), Paenibacillus sp. (7.5%), Bacillus pumilus (5%), Bacillus circulans (2.5%) and Brevibacillus sp. (2.5%). Previous studies about the microbiota of brazilian refrigerated raw milk not report the presence of Paenibacillus genus, although a group of microrganisms frequently described the work undertaken in other countries. Of the 22 isolates of B. licheniformis, 21 (95.5%) showed proteolytic and lipolytic and 1 (4.5%) was only proteolytic. The 2 strains of B. pumilus are proteolytic and lipolytic while the strain of B. circulans was only lipolytic. Of the 11 isolates of Bacillus sp., 8 (72.7%) were proteolytic and lipolytic, 1 (9.1%) was proteolitic and other 2 (18.2%) were lipolytic only. The 3 isolates of Paenibacillus sp. were just lipolytic as well as the strain of Brevibacillus sp.. For fungi were obtained 11 strains, 1 mesophilic and 10 psychrotrophic. All isolates were proteolytic. The sequencing data of the amplified DNA confirmed fungal microscopic identification, and the strain identified as thermoduric mesophilic species complex Fusarium chlamydosporum (9.09%) and the psychrotrophic as Cladosporium cladosporoides (54.54%), Curvularia geniculatus (9.09%) and the other 3 isolates were identified as Geotrichum candidum (27.27%). The presence of mesophilic and psychrotrophic thermoduric proteolytic fungi in raw milk may result in shortening the shelf-life of pasteurized milk, as well as health risk by the presence of patogenic strains and mycotoxin production. The reduction of microorganisms thermoduric, proteolytic and/or lipolytic on raw milk decreases the amount of spoilage microorganisms and thermostable enzymes in pasteurized milk, increasing its shelf-life.

Thermoduric psychrotrophic proteolytic microbiota from refrigerated raw milk / Microbiota termodúrica psicrotrófica proteolítica do leite cru refrigerado

Thermoduric microorganisms may withstand high temperatures during the pasteurization of milk. Therefore, the microbiota in pasteurized milk consist of thermoduric microbes and directly influence the shelf-life of the milk. The aim of this study was to identify thermoduric psychrotrophic proteolytic microbiota in refrigerated raw milk. Twenty samples, previously heat-treated, were streaked and incubated at 7°C for 10 days. The strains isolated were streaked on milk agar to assess proteolytic activity and were initially analyzed morphologically by light microscopy and then by molecular techniques to identify the species. In 40% of the samples analyzed was observed only one bacterial growth and others 10 thermoduric psychrotrophic fungi. All isolates were proteolytic. The sequencing of 16S rRNA gene identified the bacterial strain as Bacillus pumilus and analysis of the ITS1-5.8S-ITS2 region of fungal isolates revealed the Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), and the 3 remaining strains were identified as Geotrichum candidum (30%). This is a fist description of B. pumilus in Brazilian raw milk. Considering the spoilage potential of all isolates and of the fungi present in raw milk and their survival in pasteurized milk, it is extremely important to carry out further studies to your resistant to heat, their impact on the shelf-life of pasteurized milk, ultra-high temperature (UHT) milk and dairy products.

Os micro-organismos termodúricos são aqueles capazes de resistir ao processo de pasteurização do leite. Assim, a microbiota do leite pasteurizado é constituída por esses micro-organismos que podem influenciar diretamente na vida sua vida útil. O objetivo do presente trabalho foi identificar a microbiota termodurica psicrotrófica proteolítica do leite cru refrigerado. Foram avaliadas 20 amostras de leite cru refrigerado, plaqueadas e incubadas a 7°C por 10 dias. As cepas isolados foram repicadas em ágar leite para verificação da atividade proteolítica e inicialmente avaliadas microscopicamente e posteriormente identificadas por sequenciamento de DNA. Foi observado apenas um crescimento bacteriano e outros 10 isolados fúngicos termodúricos psicrotróficos em 40% das amostras avaliadas. Todos os isolados foram proteolíticos. O sequenciamento parcial do gene 16S rRNA da cepa bacteriana permitiu a identificação de Bacillus pumilus e a análise da região ITS1-5.8S-ITS2 dos isolados fúngicos os identificou como Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), e Geotrichum candidum (30%). Não foram encontrados estudos anteriores sobre a presença de B. pumilus no leite cru brasileiro. Considerando o potencial deteriorante de todos os isolados e presença de fungos termodúricos psicrotróficos no leite cru, é importante a continuidade trabalhos que avaliem sua resistência térmica e seu impacto na vida útil do leite pasteurizado, longa vida e derivados.

Thermoduric psychrotrophic proteolytic microbiota from refrigerated raw milk / Microbiota termodúrica psicrotrófica proteolítica do leite cru refrigerado

Thermoduric microorganisms may withstand high temperatures during the pasteurization of milk. Therefore, the microbiota in pasteurized milk consist of thermoduric microbes and directly influence the shelf-life of the milk. The aim of this study was to identify thermoduric psychrotrophic proteolytic microbiota in refrigerated raw milk. Twenty samples, previously heat-treated, were streaked and incubated at 7C for 10 days. The strains isolated were streaked on milk agar to assess proteolytic activity and were initially analyzed morphologically by light microscopy and then by molecular techniques to identify the species. In 40% of the samples analyzed was observed only one bacterial growth and others 10 thermoduric psychrotrophic fungi. All isolates were proteolytic. The sequencing of 16S rRNA gene identified the bacterial strain as Bacillus pumilus and analysis of the ITS1-5.8S-ITS2 region of fungal isolates revealed the Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), and the 3 remaining strains were identified as Geotrichum candidum (30%). This is a fist description of B. pumilus in Brazilian raw milk. Considering the spoilage potential of all isolates and of the fungi present in raw milk and their survival in pasteurized milk, it is extremely important to carry out further studies to your resistant to heat, their impact on the shelf-life of pasteurized milk,

Os micro-organismos termodúricos são aqueles capazes de resistir ao processo de pasteurização do leite. Assim, a microbiota do leite pasteurizado é constituída por esses micro-organismos que podem influenciar diretamente na vida sua vida útil. O objetivo do presente trabalho foi identificar a microbiota termodurica psicrotrófica proteolítica do leite cru refrigerado. Foram avaliadas 20 amostras de leite cru refrigerado, plaqueadas e incubadas a 7C por 10 dias. As cepas isolados foram repicadas em ágar leite para verificação da atividade proteolítica e inicialmente avaliadas microscopicamente e posteriormente identificadas por sequenciamento de DNA. Foi observado apenas um crescimento bacteriano e outros 10 isolados fúngicos termodúricos psicrotróficos em 40% das amostras avaliadas. Todos os isolados foram proteolíticos. O sequenciamento parcial do gene 16S rRNA da cepa bacteriana permitiu a identificação de Bacillus pumilus e a análise da região ITS1-5.8S-ITS2 dos isolados fúngicos os identificou como Cladosporium cladosporioides (60%), Curvularia geniculatus (10%), e Geotrichum candidum (30%). Não foram encontrados estudos anteriores sobre a presença de B. pumilus no leite cru brasileiro. Considerando o potencial deteriorante de todos os isolados e presença de fungos termodúricos psicrotróficos no leite cru, é importante a continuidade trabalhos que avaliem sua resistência té

Spoilage potential of spore-forming bacteria from refrigerated raw milk / Potencial deteriorante de bactérias formadoras de esporos do leite cru refrigerado

Aerobic bacterial spores are an important group of microorganisms in raw milk. These microbes are thermoduric, whereas the vegetative forms are thermophilic, thermoduric and psychrotrophic and reduce the shelf life of pasteurized milk. In Brazil, there are a lack of studies on the load of aerobic spores in raw milk; thus, little is known about the spoilage activity of these organisms. The aim the present study was to quantify the aerobic spores in Brazilian refrigerated raw milk of dairy region of Castro, Paraná state, assess the potential proteolytic and/or lipolytic isolates and identify the microorganisms derived from the germination. Twenty milk samples were evaluated, and the aerobic spore count was performed after plating the samples following heat treatment at 80ºC for 12 min. The activity proteolytic and lipolytic isolates were evaluated through subculture on milk agar and tributyrin agar, respectively, and these microorganisms were identified using partial 16S rRNA gene sequences that were compared through GenBank. The aerobic spore counts ranged from 1 to 3.7 log CFU.mL-¹, with a mean of 1.75 (± 0.59) log CFU.mL-¹. After spore germination, 137 aerobic bacterial isolates were obtained, 40 of which (29.2%) showed milk spoilage activity. Among these, 31 isolates (77.5%) were proteolytic and lipolytic, seven isolates (17.5%) were exclusively lipolytic and two isolates (5%) were only proteolytic. Based on the 16S rRNA gene analysis, Bacillus licheniformis (55%), Bacillus spp. (27.5%), Paenibacillus spp. (7.5%), Bacillus pumilus (5%), Bacillus circulans (2.5%) and Brevibacillus spp. (2.5%) were identified. Studies of Brazilian raw milk microbiota have not yet described B. circulans which are frequently detected in milk from other countries. Among the 22 B. licheniformis isolates, 21 microbes (95.5%) showed proteolytic and lipolytic activity, and one isolate (4.5%) exhibited only proteolytic activity. The two B. pumilus isolates were proteolytic...

Esporos de bactérias aeróbias são um importante grupo de micro-organismos no leite cru. Esses micro-organismos são termodúricos, e suas formas vegetativas são termofílicas, termodúricas e psicrotróficas e reduzem a vida útil do leite pasteurizado. No Brasil, não há estudos sobre os esporos aeróbios no leite cru, assim, pouco se sabe sobre o potencial deteriorante dessa microbiota no leite cru brasileiro. O objetivo do presente trabalho foi quantificar esporos aeróbios no leite cru refrigerado brasileiro, avaliar o seu potencial proteolítico e/ou lipolítico e identificar esses micro-organismos originários da germinação dos esporos. Foram avaliadas 20 amostras de leite cru refrigerado, nas quais foi realizada a contagem de esporos aeróbios após o tratamento térmico de 80ºC por 12 min. A atividade proteolítica e/ou lipolítica dos isolados foi avaliada após o repique das colônias em ágar leite e tributirina, respectivamente, e a identificação desses micro-organismos deteriorantes foi realizada pelo sequenciamento parcial do gene 16S rRNA e comparação com as sequencias depositadas no GenBank. As contagens de esporos aeróbios variaram de 1 a 3.7 log CFU.mL-¹, com média de 1.75 (± 0.59) log UFC.mL-¹. Após a germinação dos esporos, foram obtidas 137 colônias, das quais 40 (29.2%) apresentaram algum tipo de atividade deteriorante: 31 (77.5%) isolados foram proteolíticos e lipolíticos, sete (17.5%) foram exclusivamente lipolíticos e dois (5%) foram apenas proteolíticos. Baseado nas sequencias do gene 16S rRNA, foram identificados Bacillus licheniformis (55%), Bacillus spp. (27.5%), Paenibacillus spp. (7.5%), Bacillus pumilus (5%), Bacillus circulans (2.5%) e Brevibacillus spp. (2.5%) entre os esporulados deteriorantes. Estudos preliminaries sobre a microbiota do leite brasileiro ainda não haviam descrito B. circulans e Paenibacillus, gêneros frequentemente descritos no leite de outros países. Entre os 22 B. licheniformis...