Effect of bactofugation of raw milk on total bacterial and aerobic spore counts and on microbial diversity of sporeformers / Efeito da bactofugação do leite cru nas contagens bacteriana total, esporos aeróbios e na diversidade microbiana de formadores de esporos

This study describes the effect of bactofugation (10,000 × g in a continuous flow of 10,000 L/h) of three batches of raw milk on total bacterial count (TBC) and aerobic spore count, and it also shows the effect on the microbial diversity of spore-forming bacteria by analysing their genetic variability through molecular approaches. Given that milk must be preheated to approximately 55 °C before being bactofuged, for comparison, the three batches were evaluated at different stages as refrigerated raw, preheated, and bactofuged milk. For preheated milk, it was found that bactofugation caused a significant reduction (p < 0.05) of 99.52% (from 4.5 × 106 to 2.1 × 104 CFU/mL) in the mean TBC and of 95% (from 333 to 17 CFU/mL) in the aerobic mesophilic spore count. Due to the effect of bactofugation on preheated milk, a reduction of 82% was observed in both TBC and aerobic spore count. With respect to diversity, 107 isolates from raw milk, prior to bactofugation, and 16 isolates from bactofuged milk were recovered and grouped into 40 and 8 clusters, respectively. The predominant species detected in raw and preheated milk were Bacillus toyonensis (63% - 20 clusters) and Lysinibacillus fusiformis (15% - 8 clusters). Proportionally, B. toyonensis (69% - 6 clusters) and L. fusiformis (25% - 1 cluster) were predominant in bactofuged milk. B. pumilus, L. varians, B. flexus, B. invictae, and B. megaterium, bacteria with a known milk spoilage potential, were isolated from milk prior to bactofugation, and they reduced to undetectable levels in bactofuged milk. Bactofugation of milk, therefore, reduces the TBC and aerobic spore count, with a significant effect in reducing the microbial diversity of spore-forming bacteria, proportional to their incidence in raw milk. Therefore, bactofugation can be an alternative to increase the shelf life and technological potential of milk.(AU)

Esse estudo descreve o efeito da bactofugação (10,000 × g em fluxo contínuo de 10,000 L/h) de três lotes de leite cru nas contagens bacterianas totais (CBT) e de esporos aeróbios, verificando também o efeito sobre a diversidade microbiana dos formadores de esporos por abordagem molecular de variabilidade genética. Como o leite a ser bactofugado deve ser pré-aquecido (≈55ºC), os três lotes foram avaliados enquanto cru refrigerado, pré-aquecido e bactofugado. Em associação com o pré-aquecimento, foi verificado que a bactofugação promoveu a redução significativa (p < 0.05) de 99,52% (4,5 x 106 para 2,1 x 104 UFC/mL) na contagem bacteriana total e 95% (333 para 17 UFC/mL) dos esporos aeróbios mesófilos. Considerando o efeito isolado da bactofugação sobre o leite já pré-aquecido, foi observada a redução de 82% tanto para CBT quanto para formadores de esporos. Em relação à diversidade, foram recuperados 107 isolados do leite anterior à bactofugação e 16 isolados do leite bactofugado, agrupados em 40 e 8 clusters, respectivamente. Foi observado predomínio das espécies Bacillus toyonensis (63% - 20 clusters) e Lysinibacillus fusiformis (15% - 8 clusters) no leite cru e pré-aquecido e, proporcionalmente, B. toyonensis (69% - 6 clusters) e L. fusiformis (25% - 1 cluster) no leite bactofugado. Bacillus pumilus, Lysinibacillus varians, B. flexus, B. invictae, e B. megaterium, isolados do leite antes da bactofugação e com potencial deteriorante conhecido, foram reduzidos a níveis indetectáveis no leite bactofugado. A bactofugação do leite, portanto, reduz a CBT e as contagens de esporos aeróbios, também com efeito significativo na redução da diversidade de formadores de esporos, proporcionalmente, conforme a sua incidência no leite cru, tendo potencial para ser utilizado como alternativa para aumento da vida útil e potencial tecnológico do leite.(AU)

Inoculation of arbuscular mycorrhizal fungi as a strategy to improve annatto (Bixa orellana L. ) growth

This study aimed to assess the occurrence of arbuscular mycorrhizal fungi (AMF) in annatto (Bixa orellana L.) cultivars and their response to AMF inoculation using biometric parameters. The occurrence surveys were conducted in annatto fields in three municipalities from Pernambuco Forest Zone: Lagoa de Itaenga, Gloria de Goitá, and Vitoria de Santo Antão, and in four cultivars (Red Piave, Green Piave, Red Peruvian Paulista, and Green Peruvian Paulista). In a greenhouse, biometric parameters of annatto seedlings of Red Piave, Red Peruvian Paulista, Embrapa-36, and Embrapa-37 cultivars inoculated with AMF isolated from annatto fields. The Red Piave cultivar exhibited greater root colonization than the Green Peruvian Paulista in the Lagoa de Itaenga and Vitoria de Santo Antão municipalities. The cultivar Red Piave showed a more beneficial association with AMF in plants and soil than cultivar Green Peruvian Paulista did, in both Lagoa de Itaenga and Vitoria de Santo Antão. AMF inoculation was effective in promoting the growth of annatto plants, particularly those inoculants with S. heterogama and C. etunicatum.

Inoculation of arbuscular mycorrhizal fungi as a strategy to improve annatto (Bixa orellana L. ) growth

This study aimed to assess the occurrence of arbuscular mycorrhizal fungi (AMF) in annatto (Bixa orellana L.) cultivars and their response to AMF inoculation using biometric parameters. The occurrence surveys were conducted in annatto fields in three municipalities from Pernambuco Forest Zone: Lagoa de Itaenga, Gloria de Goitá, and Vitoria de Santo Antão, and in four cultivars (Red Piave, Green Piave, Red Peruvian Paulista, and Green Peruvian Paulista). In a greenhouse, biometric parameters of annatto seedlings of Red Piave, Red Peruvian Paulista, Embrapa-36, and Embrapa-37 cultivars inoculated with AMF isolated from annatto fields. The Red Piave cultivar exhibited greater root colonization than the Green Peruvian Paulista in the Lagoa de Itaenga and Vitoria de Santo Antão municipalities. The cultivar Red Piave showed a more beneficial association with AMF in plants and soil than cultivar Green Peruvian Paulista did, in both Lagoa de Itaenga and Vitoria de Santo Antão. AMF inoculation was effective in promoting the growth of annatto plants, particularly those inoculants with S. heterogama and C. etunicatum.(AU)

Enterococcus Spp. Survival Through the Use of Standard Protocol for Clostridium Sp. Isolation

The genera Clostridium and Enterococcus are very different from each other, both morphologically and physiologically. Due to the high resistance by the sporulation capacity of Clostridium species, the thermal shock is a characteristic tool used for the isolation and identification of these microorganisms, this way, it would eliminate any other bacteria that did not present spores. The objective of this work is to show that Enterococcus sp. resist the temperature treatment and grow in culture media used for the isolation of Clostridium sp. For this, the present study initially attempted to identify reducing sulfite clostridia in poultry products, through the use of specific culture media and heat shock treatment. However, the PCR did not detect the presence of Clostridium sp. Then, sequencing of the 16S rDNA region was performed, which showed that the reducing sulfite colonies that were being isolated were, actually, Enterococcus spp. With this, some tests were carried out using different temperature and time combinations in the thermal shock, as well as the use of five different selective and differential culture media, in an attempt to eliminate any contaminants, but all without success, because these bacteria resisted to all modification. Therefore, the standard protocol for the isolation of bacteria of the genus Clostridium does not eliminate Enterococcus, which can lead to failures in the quantification and qualification of sulfite reducing microorganisms, a fact that can significantly affect food safety and animal health.(AU)

Enterococcus Spp. Survival Through the Use of Standard Protocol for Clostridium Sp. Isolation

The genera Clostridium and Enterococcus are very different from each other, both morphologically and physiologically. Due to the high resistance by the sporulation capacity of Clostridium species, the thermal shock is a characteristic tool used for the isolation and identification of these microorganisms, this way, it would eliminate any other bacteria that did not present spores. The objective of this work is to show that Enterococcus sp. resist the temperature treatment and grow in culture media used for the isolation of Clostridium sp. For this, the present study initially attempted to identify reducing sulfite clostridia in poultry products, through the use of specific culture media and heat shock treatment. However, the PCR did not detect the presence of Clostridium sp. Then, sequencing of the 16S rDNA region was performed, which showed that the reducing sulfite colonies that were being isolated were, actually, Enterococcus spp. With this, some tests were carried out using different temperature and time combinations in the thermal shock, as well as the use of five different selective and differential culture media, in an attempt to eliminate any contaminants, but all without success, because these bacteria resisted to all modification. Therefore, the standard protocol for the isolation of bacteria of the genus Clostridium does not eliminate Enterococcus, which can lead to failures in the quantification and qualification of sulfite reducing microorganisms, a fact that can significantly affect food safety and animal health.

Contribution of environmental factors in the formation of biofilms by Alicyclobacillus acidoterrestris on surfaces of the orange juice industry / Contribuição de fatores ambientais na formação de biofilmes por Alicyclobacillus acidoterrestris em superfícies da indústria de suco de laranja

The objective of this study was to evaluate the effect of the initial microbial load, temperature and contact time on the biofilm formation of Alicyclobacillus acidoterrestris on stainless steel and natural food-grade rubber using orange juice as culture medium. The low initial load of A. acidoterrestris (2 log CFU/mL) led to biofilm formation on the stainless steel surface after 48 h of contact at 28 ºC and after 24 h at 45 ºC, and on natural food-grade rubber surface after 48 h of contact at both temperatures. The high initial microbial load (5 log CFU/mL) led to biofilm formation on stainless steel after 4 h of contact at 28 °C and 45 °C, while biofilm was formed on natural food-grade rubber after 8 h of contact at 28 °C and 4 h at 45 °C. The microbial load also affected the presence of spores in biofilm, which was observed on both surfaces only at high initial loads of A. acidoterrestris.(AU)

O objetivo deste estudo foi avaliar o efeito da carga microbiana inicial, temperatura e tempo de contato na formação de biofilme de Alicyclobacillus acidoterrestris em aço inoxidável e borracha natural de qualidade alimentar utilizando suco de laranja como meio de cultura. A baixa carga inicial de A. acidoterrestris (2 log UFC/mL) levou à formação de biofilme na superfície do aço inoxidável após 48 h de contato a 28 ºC e após 24 h a 45 ºC, e na superfície natural de borracha de qualidade alimentar após 48 h de contato nas duas temperaturas. A alta carga microbiana inicial (5 log UFC/mL) levou à formação de biofilme em aço inoxidável após 4 h de contato a 28 °C e 45 °C, enquanto o biofilme foi formado em borracha natural de qualidade alimentar após 8 h de contato a 28 °C e 4 h a 45 °C. A carga microbiana também afetou a presença de esporos no biofilme, o que foi observado em ambas as superfícies apenas com altas cargas iniciais de A. acidoterrestris.(AU)

Intercropping Acacia mangium stimulates AMF colonization and soil phosphatase activity in Eucalyptus grandis

Arbuscular mycorrhizal fungi (AMF) are very important to plant nutrition, mostly in terms of acquisition of P and micronutrients. While Acacia mangium is closely associated with AMF throughout the whole cycle, Eucalyptus grandis presents this symbiosis primarily at the seedling stage. The aim of this study was to evaluate the dynamics of AMF in these two tree species in both pure and mixed plantations during the first 20 months after planting. We evaluated the abundance, richness and diversity of AMF spores, the rate of AMF mycorrhizal root colonization, enzymatic activity and soil and litter C, N and P. There was an increase in AMF root colonization of E. grandis when intercropped with A. mangium as well as an increase in the activity of acid and alkaline phosphatase in the presence of leguminous trees. AMF colonization and phosphatase activities were both involved in improvements in P cycling and P nutrition in soil. In addition, P cycling was favored in the intercropped plantation, which showed negative correlation with litter C/N and C/P ratios and positive correlation with soil acid phosphatase activity and soil N and P concentrations. Intercropping A. mangium and E. grandis maximized AMF root colonization of E. grandis and phosphatase activity in the soil, both of which accelerate P cycling and forest performance.

Intercropping Acacia mangium stimulates AMF colonization and soil phosphatase activity in Eucalyptus grandis

Arbuscular mycorrhizal fungi (AMF) are very important to plant nutrition, mostly in terms of acquisition of P and micronutrients. While Acacia mangium is closely associated with AMF throughout the whole cycle, Eucalyptus grandis presents this symbiosis primarily at the seedling stage. The aim of this study was to evaluate the dynamics of AMF in these two tree species in both pure and mixed plantations during the first 20 months after planting. We evaluated the abundance, richness and diversity of AMF spores, the rate of AMF mycorrhizal root colonization, enzymatic activity and soil and litter C, N and P. There was an increase in AMF root colonization of E. grandis when intercropped with A. mangium as well as an increase in the activity of acid and alkaline phosphatase in the presence of leguminous trees. AMF colonization and phosphatase activities were both involved in improvements in P cycling and P nutrition in soil. In addition, P cycling was favored in the intercropped plantation, which showed negative correlation with litter C/N and C/P ratios and positive correlation with soil acid phosphatase activity and soil N and P concentrations. Intercropping A. mangium and E. grandis maximized AMF root colonization of E. grandis and phosphatase activity in the soil, both of which accelerate P cycling and forest performance.(AU)

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)

Bacteria from the Bacillus cereus group as contaminants in requeijão curd cheeses and especialidade láctea tipo requeijão / Bactérias do grupo Bacillus cereus como contaminantes em queijos fundidos requeijão e especialidade láctea tipo requeijão

This study aimed to evaluate the occurrence of Bacillus cereus group in requeijões and especialidades lácteas tipo requeijão (regular and light) and to verify if there is differences in relation to this occurrence among different categories of these products. A set of 14 (35%) lots was contaminated with this bacterial group from the 40 lots with low counts (maximum 3.1 × 10 CFU/g), and no significant difference regarding counts or presence/absence were observed among the categories of the products. It can be concluded that contamination by B. cereus group in these products is unable to consist in risk to consumers, regarding adequate refrigeration during selling. This study was the first one to report this bacteria group for these dairy products and highlights the needs of further investigations to evaluate the impact of its spoilage during shelf life.(AU)

Este estudo objetivou avaliar a ocorrência de bactérias do grupo de Bacillus cereus em requeijões e especialidades lácteas (normais e light) e verificar se há diferença em relação a essa ocorrência entre as diferentes categorias desses produtos. Entre os 40 lotes avaliados, 14 (35%) estavam contaminados por bactérias desse grupo, com baixas contagens (máxima de 3,1 × 10 UFC/g) e sem diferenças significativas no que tange às contagens ou à presença/ausência entre as diversas categorias. Conclui-se que as bactérias do grupo de B. cereus em requeijões e especialidades lácteas são incapazes de consistir em risco aos consumidores desses produtos, desde que as condições de refrigeração sejam adequadas durante armazenamento e venda. O presente trabalho é o primeiro a relatar a presença de tal grupo de micro-organismos nesses tipos de produtos, porém, são necessários novos estudos para avaliar o impacto de sua ação deteriorante sobre o período em que passa na prateleira.(AU)

Bacteria from the Bacillus cereus group as contaminants in requeijão curd cheeses and especialidade láctea tipo requeijão / Bactérias do grupo Bacillus cereus como contaminantes em queijos fundidos requeijão e especialidade láctea tipo requeijão

This study aimed to evaluate the occurrence of Bacillus cereus group in requeijões and especialidades lácteas tipo requeijão (regular and light) and to verify if there is differences in relation to this occurrence among different categories of these products. A set of 14 (35%) lots was contaminated with this bacterial group from the 40 lots with low counts (maximum 3.1 × 10 CFU/g), and no significant difference regarding counts or presence/absence were observed among the categories of the products. It can be concluded that contamination by B. cereus group in these products is unable to consist in risk to consumers, regarding adequate refrigeration during selling. This study was the first one to report this bacteria group for these dairy products and highlights the needs of further investigations to evaluate the impact of its spoilage during shelf life.(AU)

Este estudo objetivou avaliar a ocorrência de bactérias do grupo de Bacillus cereus em requeijões e especialidades lácteas (normais e light) e verificar se há diferença em relação a essa ocorrência entre as diferentes categorias desses produtos. Entre os 40 lotes avaliados, 14 (35%) estavam contaminados por bactérias desse grupo, com baixas contagens (máxima de 3,1 × 10 UFC/g) e sem diferenças significativas no que tange às contagens ou à presença/ausência entre as diversas categorias. Conclui-se que as bactérias do grupo de B. cereus em requeijões e especialidades lácteas são incapazes de consistir em risco aos consumidores desses produtos, desde que as condições de refrigeração sejam adequadas durante armazenamento e venda. O presente trabalho é o primeiro a relatar a presença de tal grupo de micro-organismos nesses tipos de produtos, porém, são necessários novos estudos para avaliar o impacto de sua ação deteriorante sobre o período em que passa na prateleira.(AU)

Spoilage potential of Paenibacillussp. in Brazilian raw milk / Potencial deteriorante de Paenibacillus sp. no leite cru refrigerado brasileiro

Bacterial spores are widespread in the environment and can contaminate milk. Spores are resistant to thermal conditions and your germination reduces milk shelf-life because the aerobic bacteria that are sporulated produce proteases and lipases. The aim of this study was identify Paenibacillus sp., the spoilage microbiota, arising from the germination of spores in raw milk and your spoilage potential. Twenty different milk samples were treated at 80°C/12min and plated to isolate spore-forming bacteria. These strains were picked in milk agar and tributyrin agar for verification of their potential proteolytic and lipolytic activities, respectively. Amplification and sequencing of the 16S rRNA gene of the strains for identification by similarity to the DNA sequences deposited in GenBank was performed. One hundred and thirty-seven isolates were obtained, of which 40 (29.2%) showed spoilage activity for milk. Of these, three (7.5%) were identified as strains of Paenibacillus sp., and all were lipolytic. Paenibacillus sp. have been identified as primarily responsible for the spoilage of pasteurized milk with a long shelf-life in other countries. To increase the shelf-life of Brazilian pasteurized milk, it is important to identify the sporulated microbes to determine their origin and to control the contamination of milk by vegetative forms such as spores.(AU)

Os esporos de bactérias estão amplamente difundidos no ambiente e podem contaminar o leite. Os esporos conferem resistência aos processos térmicos e sua germinação pode reduzir a vida útil do leite pasteurizado, uma vez que bactérias esporuladas aeróbias são muito frequentemente produtoras de proteases e lipases. O objetivo do presente trabalho foi identificar Paenibacillus sp. na microbiota deteriorante, oriunda da germinação de esporos no leite cru, e avaliar o seu potencial deteriorante. Vinte amostras de leite de propriedades distintas foram tratadas a 80ºC/12min e semeadas para isolamento de bactérias formadoras de esporos. Essas cepas foram repicadas em ágar leite e tributirina para verificação do seu potencial proteolítico e lipolítico, respectivamente. Foi realizada a amplificação e sequenciamento do gene 16S do rRNA para identificação das cepas por similaridade com as sequências de DNA depositadas no GenBank. Foram obtidos 137 isolados, dos quais 40 (29,2%) apresentaram atividade deteriorante do leite. Dessas, 3 (7,5%) cepas foram identificadas como Paenibacillus sp., todas lipolíticas. Paenibacillus sp. são considerados os principais responsáveis pela deterioração do leite pasteurizado de longa vida útil em outros países. Para o aumento da vida útil do leite pasteurizado brasileiro, é importante conhecer a microbiota esporulada, de forma a determinar sua origem e poder controlar a contaminação do leite tanto pelas formas vegetativas como por seus esporos.(AU)

Spoilage potential of Paenibacillussp. in Brazilian raw milk / Potencial deteriorante de Paenibacillus sp. no leite cru refrigerado brasileiro

Bacterial spores are widespread in the environment and can contaminate milk. Spores are resistant to thermal conditions and your germination reduces milk shelf-life because the aerobic bacteria that are sporulated produce proteases and lipases. The aim of this study was identify Paenibacillus sp., the spoilage microbiota, arising from the germination of spores in raw milk and your spoilage potential. Twenty different milk samples were treated at 80°C/12min and plated to isolate spore-forming bacteria. These strains were picked in milk agar and tributyrin agar for verification of their potential proteolytic and lipolytic activities, respectively. Amplification and sequencing of the 16S rRNA gene of the strains for identification by similarity to the DNA sequences deposited in GenBank was performed. One hundred and thirty-seven isolates were obtained, of which 40 (29.2%) showed spoilage activity for milk. Of these, three (7.5%) were identified as strains of Paenibacillus sp., and all were lipolytic. Paenibacillus sp. have been identified as primarily responsible for the spoilage of pasteurized milk with a long shelf-life in other countries. To increase the shelf-life of Brazilian pasteurized milk, it is important to identify the sporulated microbes to determine their origin and to control the contamination of milk by vegetative forms such as spores.

Os esporos de bactérias estão amplamente difundidos no ambiente e podem contaminar o leite. Os esporos conferem resistência aos processos térmicos e sua germinação pode reduzir a vida útil do leite pasteurizado, uma vez que bactérias esporuladas aeróbias são muito frequentemente produtoras de proteases e lipases. O objetivo do presente trabalho foi identificar Paenibacillus sp. na microbiota deteriorante, oriunda da germinação de esporos no leite cru, e avaliar o seu potencial deteriorante. Vinte amostras de leite de propriedades distintas foram tratadas a 80ºC/12min e semeadas para isolamento de bactérias formadoras de esporos. Essas cepas foram repicadas em ágar leite e tributirina para verificação do seu potencial proteolítico e lipolítico, respectivamente. Foi realizada a amplificação e sequenciamento do gene 16S do rRNA para identificação das cepas por similaridade com as sequências de DNA depositadas no GenBank. Foram obtidos 137 isolados, dos quais 40 (29,2%) apresentaram atividade deteriorante do leite. Dessas, 3 (7,5%) cepas foram identificadas como Paenibacillus sp., todas lipolíticas. Paenibacillus sp. são considerados os principais responsáveis pela deterioração do leite pasteurizado de longa vida útil em outros países. Para o aumento da vida útil do leite pasteurizado brasileiro, é importante conhecer a microbiota esporulada, de forma a determinar sua origem e poder controlar a contaminação do leite tanto pelas formas vegetativas como por seus esporos.

Efficiency of boiling and four other methods for genomic DNA extraction of deteriorating spore-forming bacteria from milk / Eficiência da fervura e outros quatro métodos para extração do DNA genômico de bactérias esporuladas deteriorantes do leite

The spore-forming microbiota is mainly responsible for the deterioration of pasteurized milk with long shelf life in the United States. The identification of these microorganisms, using molecular tools, is of particular importance for the maintenance of the quality of milk. However, these molecular techniques are not only costly but also labor-intensive and time-consuming. The aim of this study was to compare the efficiency of boiling in conjunction with four other methods for the genomic DNA extraction of sporulated bacteria with proteolytic and lipolytic potential isolated from raw milk in the states of Paraná and Maranhão, Brazil. Protocols based on cellular lysis by enzymatic digestion, phenolic extraction, microwave-heating, as well as the use of guanidine isothiocyanate were used. This study proposes a method involving simple boiling for the extraction of genomic DNA from these microorganisms. Variations in the quality and yield of the extracted DNA among these methods were observed. However, both the cell lysis protocol by enzymatic digestion (commercial kit) and the simple boiling method proposed in this study yielded sufficient DNA for successfully carrying out the Polymerase Chain Reaction (PCR) of the rpoB and 16S rRNA genes for all 11 strains of microorganisms tested. Other protocols failed to yield sufficient quantity and quality of DNA from all microorganisms tested, since only a few strains have showed positive results by PCR, thereby hindering the search for new microorganisms. Thus, the simple boiling method for DNA extraction from sporulated bacteria in spoiled milk showed the same efficacy as that of the commercial kit. Moreover, the method is inexpensive, easy to perform, and much less time-consuming.

A microbiota esporulada é a principal responsável pela deterioração do leite pasteurizado de longa vida útil nos Estados Unidos. A identificação destes micro-organismos é de especial importância para a qualidade do leite e ferramentas moleculares são fundamentais nesse processo. No entanto, exigem a execução de etapas onerosas e laboriosas que podem inviabilizar algumas pesquisas. O objetivo do presente trabalho foi comparar a eficiência do método de extração de DNA por fervura com outros quatro métodos para extração de DNA genômico de bactérias esporuladas com potencial proteolítico e lipolítico isoladas do leite cru dos estados do Paraná e Maranhão, Brasil. Foram utilizados protocolos que se baseavam na lise celular por digestão enzimática, agitação com fenol, aquecimento em micro-ondas, tiocianato de guanidina e este trabalho propõe um método por fervura simples para o estudo desses micro-organismos. Observaram-se variações nos métodos de quantificação do DNA extraído e baixo coeficiente de correlação de Person entre esses métodos. No entanto, observou-se que tanto no protocolo de lise celular por digestão enzimática (kit comercial) quanto na fervura simples proposta pelo presente estudo, houve êxito na realização da Reação em Cadeia da Polimerase (PCR) para pesquisa dos genes rpoB e 16S rRNA para todas as 11 cepas de micro-organismos testadas. Os outros protocolos não apresentaram sucesso na extração de DNA para a totalidade da microbiota avaliada, já que somente algumas amostras tiveram êxito nas reações de PCR, fato que os inviabiliza para a pesquisa desses micro-organismos. Dessa forma, o método de fervura simples das suspensões de bactérias esporuladas deteriorantes do leite demonstrou a mesma eficiência do kit comercial para a extração do DNA desses micro-organismos, sendo um método de baixo custo e de fácil e rápida execução.

Efficiency of boiling and four other methods for genomic DNA extraction of deteriorating spore-forming bacteria from milk / Eficiência da fervura e outros quatro métodos para extração do DNA genômico de bactérias esporuladas deteriorantes do leite

The spore-forming microbiota is mainly responsible for the deterioration of pasteurized milk with long shelf life in the United States. The identification of these microorganisms, using molecular tools, is of particular importance for the maintenance of the quality of milk. However, these molecular techniques are not only costly but also labor-intensive and time-consuming. The aim of this study was to compare the efficiency of boiling in conjunction with four other methods for the genomic DNA extraction of sporulated bacteria with proteolytic and lipolytic potential isolated from raw milk in the states of Paraná and Maranhão, Brazil. Protocols based on cellular lysis by enzymatic digestion, phenolic extraction, microwave-heating, as well as the use of guanidine isothiocyanate were used. This study proposes a method involving simple boiling for the extraction of genomic DNA from these microorganisms. Variations in the quality and yield of the extracted DNA among these methods were observed. However, both the cell lysis protocol by enzymatic digestion (commercial kit) and the simple boiling method proposed in this study yielded sufficient DNA for successfully carrying out the Polymerase Chain Reaction (PCR) of the rpoB and 16S rRNA genes for all 11 strains of microorganisms tested. Other protocols failed to yield sufficient quantity and quality of DNA from all microorganisms tested, since only a few strains have showed positive results by PCR, thereby hindering the search for new microorganisms. Thus, the simple boiling method for DNA extraction from sporulated bacteria in spoiled milk showed the same efficacy as that of the commercial kit. Moreover, the method is inexpensive, easy to perform, and much less time-consuming.(AU)

A microbiota esporulada é a principal responsável pela deterioração do leite pasteurizado de longa vida útil nos Estados Unidos. A identificação destes micro-organismos é de especial importância para a qualidade do leite e ferramentas moleculares são fundamentais nesse processo. No entanto, exigem a execução de etapas onerosas e laboriosas que podem inviabilizar algumas pesquisas. O objetivo do presente trabalho foi comparar a eficiência do método de extração de DNA por fervura com outros quatro métodos para extração de DNA genômico de bactérias esporuladas com potencial proteolítico e lipolítico isoladas do leite cru dos estados do Paraná e Maranhão, Brasil. Foram utilizados protocolos que se baseavam na lise celular por digestão enzimática, agitação com fenol, aquecimento em micro-ondas, tiocianato de guanidina e este trabalho propõe um método por fervura simples para o estudo desses micro-organismos. Observaram-se variações nos métodos de quantificação do DNA extraído e baixo coeficiente de correlação de Person entre esses métodos. No entanto, observou-se que tanto no protocolo de lise celular por digestão enzimática (kit comercial) quanto na fervura simples proposta pelo presente estudo, houve êxito na realização da Reação em Cadeia da Polimerase (PCR) para pesquisa dos genes rpoB e 16S rRNA para todas as 11 cepas de micro-organismos testadas. Os outros protocolos não apresentaram sucesso na extração de DNA para a totalidade da microbiota avaliada, já que somente algumas amostras tiveram êxito nas reações de PCR, fato que os inviabiliza para a pesquisa desses micro-organismos. Dessa forma, o método de fervura simples das suspensões de bactérias esporuladas deteriorantes do leite demonstrou a mesma eficiência do kit comercial para a extração do DNA desses micro-organismos, sendo um método de baixo custo e de fácil e rápida execução.(AU)

Effects of temperature and light regime on Corynespora cassiicola and of temperature and of leaf wetness period in development of target spot in barbados cherry / Efeitos da temperatura e regime de luz sobre Corynespora cassiicola e da temperatura e período de molhamento foliar no desenvolvimento da mancha-alvo em acerola

The objective of this study were: (i) to assess, in vitro, the effect of temperature on mycelial growth (10-35o C) and spores germination (10-45o C); (ii) to evaluate, in vitro¸ the effect of the light regime (continuous light, 12 h photoperiod and continuous darkness) on mycelial growth, sporulation and viability of the spores of C. cassiicola; and (iii) to assess, in vivo, the effect of temperature (20-30o C) and the duration of leaf wetness (4-48 h) in the development of target spot in leaves of barbados cherry. The estimated maximum temperatures for mycelia growth and spore germination were 30 and 29o C, respectively. The light regime did not affect the mycelial growth and spores germination. However, continuous light regime favored the sporulation, and the absence of light during spore production decreased the spore viability. The target spot lesions area in barbados cherry leaves increased with the increment in the temperature up to 30o C. Twelve hours of leaf wetness were necessary for the infection and lesion development of target spot.

O presente estudo teve como objetivos: (i) determinar, in vitro, o efeito da temperatura sobre o crescimento micelial (10 a 35o C) e a germinação de conídios (10 a 45o C) de Corynespora cassiicola; (ii) avaliar, in vitro, o efeito do regime de luz (luz contínua, fotoperíodo de 12 h e escuro) sobre o crescimento micelial, a esporulação e a viabilidade dos esporos de C. cassiicola; e (iii) verificar, in vivo, a influência da temperatura (20 a 30o C) e da duração do período de molhamento foliar (4 a 48 h) no desenvolvimento da mancha-alvo em folhas de acerola. As temperaturas ótimas, estimadas a partir das equações, para o crescimento micelial e a germinação de esporos foram de 30 e 29o C, respectivamente. O regime de luz não afetou o crescimento micelial e a germinação dos esporos, entretanto, luz contínua favoreceu a esporulação, e a ausência de luz durante a produção dos esporos diminuiu sua viabilidade. Houve um incremento na área das lesões da mancha-alvo em folhas de acerola conforme a temperatura aumentou até 30o C. Foram necessárias 12 h de molhamento foliar para que a infecção e as lesões ocorressem.

Efeitos da temperatura e regime de luz sobre Corynespora cassiicola e da temperatura e período de molhamento foliar no desenvolvimento da mancha-alvo em acerola

ABSTRACT The objective of this study were: (i) to assess, in vitro , the effect of temperature on mycelial growth (10-35oC) and spores germination (10-45oC); (ii) to evaluate, in vitro¸the effect of the light regime (continuous light, 12 h photoperiod and continuous darkness) on mycelial growth, sporulation and viability of the spores of C. cassiicola ;and (iii) to assess, in vivo,the effect of temperature (20-30oC) and the duration of leaf wetness (4-48 h) in the development of target spot in leaves of barbados cherry. The estimated maximum temperatures for mycelia growth and spore germination were 30 and 29oC, respectively. The light regime did not affect the mycelial growth and spores germination. However, continuous light regime favored the sporulation, and the absence of light during spore production decreased the spore viability. The target spot lesions area in barbados cherry leaves increased with the increment in the temperature up to 30oC. Twelve hours of leaf wetness were necessary for the infection and lesion development of target spot.

RESUMO O presente estudo teve como objetivos: (i) determinar, in vitro, o efeito da temperatura sobre o crescimento micelial (10 a 35oC) e a germinação de conídios (10 a 45oC) de Corynespora cassiicola ; (ii) avaliar, in vitro , o efeito do regime de luz (luz contínua, fotoperíodo de 12 h e escuro) sobre o crescimento micelial, a esporulação e a viabilidade dos esporos de C. cassiicola ; e (iii) verificar, in vivo , a influência da temperatura (20 a 30oC) e da duração do período de molhamento foliar (4 a 48 h) no desenvolvimento da mancha-alvo em folhas de acerola. As temperaturas ótimas, estimadas a partir das equações, para o crescimento micelial e a germinação de esporos foram de 30 e 29oC, respectivamente. O regime de luz não afetou o crescimento micelial e a germinação dos esporos, entretanto, luz contínua favoreceu a esporulação, e a ausência de luz durante a produção dos esporos diminuiu sua viabilidade. Houve um incremento na área das lesões da mancha-alvo em folhas de acerola conforme a temperatura aumentou até 30oC. Foram necessárias 12 h de molhamento foliar para que a infecção e as lesões ocorressem.

Efeitos da temperatura e regime de luz sobre Corynespora cassiicola e da temperatura e período de molhamento foliar no desenvolvimento da mancha-alvo em acerola / Effects of temperature and light regime on Corynespora cassiicola and of temperature and of leaf wetness period in development of target spot in barbados cherry

O presente estudo teve como objetivos: (i) determinar, in vitro, o efeito da temperatura sobre o crescimento micelial (10 a 35oC) e a germinação de conídios (10 a 45oC) de Corynespora cassiicola ; (ii) avaliar, in vitro , o efeito do regime de luz (luz contínua, fotoperíodo de 12 h e escuro) sobre o crescimento micelial, a esporulação e a viabilidade dos esporos de C. cassiicola ; e (iii) verificar, in vivo , a influência da temperatura (20 a 30oC) e da duração do período de molhamento foliar (4 a 48 h) no desenvolvimento da mancha-alvo em folhas de acerola. As temperaturas ótimas, estimadas a partir das equações, para o crescimento micelial e a germinação de esporos foram de 30 e 29oC, respectivamente. O regime de luz não afetou o crescimento micelial e a germinação dos esporos, entretanto, luz contínua favoreceu a esporulação, e a ausência de luz durante a produção dos esporos diminuiu sua viabilidade. Houve um incremento na área das lesões da mancha-alvo em folhas de acerola conforme a temperatura aumentou até 30oC. Foram necessárias 12 h de molhamento foliar para que a infecção e as lesões ocorressem.(AU)

The objective of this study were: (i) to assess, in vitro , the effect of temperature on mycelial growth (10-35oC) and spores germination (10-45oC); (ii) to evaluate, in vitro¸the effect of the light regime (continuous light, 12 h photoperiod and continuous darkness) on mycelial growth, sporulation and viability of the spores of C. cassiicola ;and (iii) to assess, in vivo,the effect of temperature (20-30oC) and the duration of leaf wetness (4-48 h) in the development of target spot in leaves of barbados cherry. The estimated maximum temperatures for mycelia growth and spore germination were 30 and 29oC, respectively. The light regime did not affect the mycelial growth and spores germination. However, continuous light regime favored the sporulation, and the absence of light during spore production decreased the spore viability. The target spot lesions area in barbados cherry leaves increased with the increment in the temperature up to 30oC. Twelve hours of leaf wetness were necessary for the infection and lesion development of target spot.(AU)

Effects of temperature and light regime on Corynespora cassiicola and of temperature and of leaf wetness period in development of target spot in barbados cherry / Efeitos da temperatura e regime de luz sobre Corynespora cassiicola e da temperatura e período de molhamento foliar no desenvolvimento da mancha-alvo em acerola

The objective of this study were: (i) to assess, in vitro, the effect of temperature on mycelial growth (10-35o C) and spores germination (10-45o C); (ii) to evaluate, in vitro¸ the effect of the light regime (continuous light, 12 h photoperiod and continuous darkness) on mycelial growth, sporulation and viability of the spores of C. cassiicola; and (iii) to assess, in vivo, the effect of temperature (20-30o C) and the duration of leaf wetness (4-48 h) in the development of target spot in leaves of barbados cherry. The estimated maximum temperatures for mycelia growth and spore germination were 30 and 29o C, respectively. The light regime did not affect the mycelial growth and spores germination. However, continuous light regime favored the sporulation, and the absence of light during spore production decreased the spore viability. The target spot lesions area in barbados cherry leaves increased with the increment in the temperature up to 30o C. Twelve hours of leaf wetness were necessary for the infection and lesion development of target spot.(AU)

O presente estudo teve como objetivos: (i) determinar, in vitro, o efeito da temperatura sobre o crescimento micelial (10 a 35o C) e a germinação de conídios (10 a 45o C) de Corynespora cassiicola; (ii) avaliar, in vitro, o efeito do regime de luz (luz contínua, fotoperíodo de 12 h e escuro) sobre o crescimento micelial, a esporulação e a viabilidade dos esporos de C. cassiicola; e (iii) verificar, in vivo, a influência da temperatura (20 a 30o C) e da duração do período de molhamento foliar (4 a 48 h) no desenvolvimento da mancha-alvo em folhas de acerola. As temperaturas ótimas, estimadas a partir das equações, para o crescimento micelial e a germinação de esporos foram de 30 e 29o C, respectivamente. O regime de luz não afetou o crescimento micelial e a germinação dos esporos, entretanto, luz contínua favoreceu a esporulação, e a ausência de luz durante a produção dos esporos diminuiu sua viabilidade. Houve um incremento na área das lesões da mancha-alvo em folhas de acerola conforme a temperatura aumentou até 30o C. Foram necessárias 12 h de molhamento foliar para que a infecção e as lesões ocorressem.(AU)

Efeitos da temperatura e regime de luz sobre Corynespora cassiicola e da temperatura e período de molhamento foliar no desenvolvimento da mancha-alvo em acerola

ABSTRACT The objective of this study were: (i) to assess, in vitro , the effect of temperature on mycelial growth (10-35oC) and spores germination (10-45oC); (ii) to evaluate, in vitro¸the effect of the light regime (continuous light, 12 h photoperiod and continuous darkness) on mycelial growth, sporulation and viability of the spores of C. cassiicola ;and (iii) to assess, in vivo,the effect of temperature (20-30oC) and the duration of leaf wetness (4-48 h) in the development of target spot in leaves of barbados cherry. The estimated maximum temperatures for mycelia growth and spore germination were 30 and 29oC, respectively. The light regime did not affect the mycelial growth and spores germination. However, continuous light regime favored the sporulation, and the absence of light during spore production decreased the spore viability. The target spot lesions area in barbados cherry leaves increased with the increment in the temperature up to 30oC. Twelve hours of leaf wetness were necessary for the infection and lesion development of target spot.

RESUMO O presente estudo teve como objetivos: (i) determinar, in vitro, o efeito da temperatura sobre o crescimento micelial (10 a 35oC) e a germinação de conídios (10 a 45oC) de Corynespora cassiicola ; (ii) avaliar, in vitro , o efeito do regime de luz (luz contínua, fotoperíodo de 12 h e escuro) sobre o crescimento micelial, a esporulação e a viabilidade dos esporos de C. cassiicola ; e (iii) verificar, in vivo , a influência da temperatura (20 a 30oC) e da duração do período de molhamento foliar (4 a 48 h) no desenvolvimento da mancha-alvo em folhas de acerola. As temperaturas ótimas, estimadas a partir das equações, para o crescimento micelial e a germinação de esporos foram de 30 e 29oC, respectivamente. O regime de luz não afetou o crescimento micelial e a germinação dos esporos, entretanto, luz contínua favoreceu a esporulação, e a ausência de luz durante a produção dos esporos diminuiu sua viabilidade. Houve um incremento na área das lesões da mancha-alvo em folhas de acerola conforme a temperatura aumentou até 30oC. Foram necessárias 12 h de molhamento foliar para que a infecção e as lesões ocorressem.