Refrigerated raw milk may contain psychrotrophic microorganisms that produce thermoresistant exoproteases and lipases, which may compromise the quality of processed fluid milk and dairy products during storage. The aim of this work was to quantify and identify the deteriorating psychrotrophic microbiota in Brazilian refrigerated raw milk using genetic diversity analysis. The mean psychrotrophic count was 1.1 × 10 cfu/mL. Of the total isolates, 47.8 and 29.8% showed deteriorating activity at 35°C within 48 h and 7°C within 10 d, respectively. Among the proteolytic species, more isolated by this study were Lactococcus lactis (27.3%), Enterobacter kobei (14.8%), Serratia ureilytica (8%), Aerococcus urinaeequi (6.8%), and Bacillus licheniformis (6.8%). Observed among lipolytics were E. kobei (17.7%), L. lactis (15.6%), A. urinaeequi (12.5%), and Acinetobacter lwoffii (9.4%). The isolates S. ureilytica, E. kobei, Pseudomonas spp., and Yersinia enterocolitica potentially produced alkaline metalloprotease (aprX). Despite the low counts, a considerable portion of the psychrotrophic microbiota presented spoilage potential, which reaffirms the need for rigor in the control of contamination and the importance of rapid processing as factors that maintain the quality of milk and dairy products.

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. Key words: Bacillus. Paenibacillus. PCR. Spores. ResumoA 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 microondas, 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 Cad...

RESUMOO queijo devido à sua rica composição de nutrientes oferece condições para a multiplicação de microrganismos. A presença de coliformes totais e termotolerantes indica contaminação de origem ambiental e fecal, respectivamente, o que pode caracterizar, dependendo da contagem, baixa qualidade microbiológica e condições higiênico-sanitárias insatisfatórias durante o processo de produção do queijo, além da possibilidade da presença de enteropatógenos. O objetivo do presente trabalho foi avaliar a condição higiênica da produção de queijo tipo mussarela e Minas frescal, produzidos na região Norte do Paraná. Foram analisadas 50 amostras de queijo, sendo 14 de mussarela e 36 de Minas frescal, no período de junho de 2011 a junho de 2016. As condições sanitárias de produção foram avaliadas através da contagem de coliformes totais e termotolerantes conforme a metodologia preconizada pela legislação brasileira. Para o queijo mussarela, foi observado que todas as amostras apresentavam contagens de acordo com os padrões preconizados para coliformes totais e termotolerantes. No entanto, foi observado que 55,6% das amostras de queijo Minas frescal estavam em desacordo com os padrões estabelecidos pela legislação para coliformes totais, assim como 27,8% para termotolerantes. Estes resultados indicam que parte considerável dos queijos tipo Minas frescal apresentam condições higiênicas insatisfatórias durante as etapas de produção e risco à saúde dos consumidores, fazendo-se necessárias a adoção de boas práticas de fabricação, aplicando medidas corretivas e de monitoramento que permitam a redução dessa

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. Thus, it can be stated that the microbiological quality of raw milk is directly related to the initial count of microorganisms after pasteurization, and that pasteurized milk produced from raw milk with low microbiological counts complies with the Brazilian legislation for 18 days following thermal processing.

Contamination of water by microcystins is a global problem. These potent hepatotoxins demand constant monitoring and control methods in potable water. Promising approaches to reduce contamination risks have focused on natural microcystin biodegradation led by enzymes encoded by the mlrABCD genes. The first enzyme of this system (mlrA) linearizes microcystin structure, reducing toxicity and stability. Heterologous expression of mlrA in different microorganisms may enhance its production and activity, promote additional knowledge on the enzyme, and support feasible applications. In this context, we intended to express the mlrA gene from Sphingosinicella microcystinivorans B9 in an industrial Saccharomyces cerevisiae strain as an innovative biological alternative to degrade microcystins. The mlrA gene was codon-optimized for expression in yeast, and either expressed from a plasmid or through chromosomal integration at the URA3 locus. Recombinant and wild yeasts were cultivated in medium contaminated with microcystins, and the toxin content was analyzed during growth. Whereas no difference in microcystins content was observed in cultivation with the chromosomally integrated strain, the yeast strain hosting the mlrA expression plasmid reduced 83% of toxins within 120 h of cultivation. Our results show microcystinase A expressed by industrial yeast strains as a viable option for practical applications in water treatment.

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