Lipolytic and proteolytic activity of Pseudomonas spp. isolated during milking and storage of refrigerated raw milk.

UNLABELLED: The aim of this study was to verify the presence of lipolytic and proteolytic Pseudomonas spp. during milking and storage of refrigerated raw milk. We also intended to compare samples collected during rainy and dry seasons, from farms with manual and mechanical milking systems. For this, samples of milkers' hands, cows' teats, water, expansion tanks, equipment, and utensils used during milking were analyzed regarding Pseudomonas spp. COUNT: Positive samples were tested for the production of lipolytic and proteolytic enzymes. Microorganisms of the genus Pseudomonas were isolated from all sampling points. A higher isolation rate of the bacterium was found in the rainy season except for 6 sampling points, with all of these associated with mechanical milking systems. Pseudomonas spp. exhibiting lipolytic activity were found to be predominant during the dry season, since no activity was detected during the rainy season in 26 of the 29 sampling sites. The highest number of lipolytic Pseudomonas isolates was obtained from water. Presence of lipase-producing Pseudomonas spp. was verified in 7 and 36% of the samples collected from farms with manual and mechanical milking, respectively. When analyzing raw milk collected from expansion tanks immediately (0 h) and 24h after milking, we observed that for dairy properties with manual milking process, 10% of the Pseudomonas isolates were positive for lipolytic activity. The percentage increased to 12% 48h after milking. Mean averages were 32, 33, and 39% immediately after, 24 and 48h after milking, respectively, for farms with mechanical milking. All sampling points showed the presence of proteolytic strains of Pseudomonas. The highest proteolytic activity was found during the rainy season, except for the samples collected from milkers' hands before milking, buckets, and teat cup inner surfaces after milking and from the water in dairy farms with mechanical milking system. Of these samples, 72, 56, and 50%, respectively, were positive for proteolysis during the dry season. For the water samples, a statistical difference was observed between mechanical (50%) and manual (7%) milking systems in the percentage of proteolytic activity. No production of proteolytic enzyme was detected in the samples from milkers' hands taken after milking and no statistically significant difference was found among manual (19.91%) and mechanical (47.85%) milking. During the rainy months, no proteolysis was detected in the samples taken from cows' teats after the predipping. It is evident, therefore, that preventive measures capable of minimizing the contamination with Pseudomonas spp. during milking and storage of refrigerated raw milk are needed, regardless of season.

Detection of Bacillus cereus isolated during ultra high temperature milk production flowchart through random amplified polymorphic DNA polymerase chain reaction / Detecção de Bacillus cereus isolado durante o fluxograma de produção do leite tratado por ultra alta temperatura através do DNA polimórfico amplificado ao acaso através da reação em cadeia pela polimerase

ABSTRACT: The present study focused on isolation Bacillus cereus during the UHT milk production and shelf life, to assess the enterotoxigenic production capacity of isolates and to evaluate the use of the RAPD-PCR technique to verify whether Bacillus cereus isolated at different phases of UHT milk processing belongs to the same strain. For this, six groups of milk samples composed of raw, pasteurized and UHT milk were collected from a processing plant. The results revealed that bacteria belonging to the Bacillus cereus group were isolated from 51.6%, 81.6% and from 13.8% of raw, pasteurized and UHT milk samples, respectively. About 50.0% of isolates from raw milk, 19.2% isolates from pasteurized milk and 70.7% isolates from UHT milk were capable of producing enterotoxins. It was confirmed the genetic similarity among Bacillus cereus isolates from raw, pasteurized and UHT milk, therefore demonstrating that the microorganism is able to withstand UHT treatment. These results should serve as a warning to health authorities, given that 13.8% of samples were not in accordance with standards established by the Department of Health for containing a potentially pathogen agent, therefore indicating that contamination of milk by sporulating bacteria should be avoided.

RESUMO: Objetivou-se realizar o isolamento de bactérias do grupo do Bacillus cereus e verificação da semelhança genética entre elas, através da PCR-RAPD, durante a produção e vida de prateleira do leite UAT, bem como verificar sua capacidade enterotoxigência. Para isso, foram colhidas em uma usina de beneficiamento seis grupos de amostras de leite compostos cada um por amostras de leite cru, pasteurizado e UAT. Os resultados obtidos evidenciaram que bactérias do grupo do Bacillus cereus foram isoladas de 51,6% das amostras de leite cru, de 81,6% das de leite pasteurizado e de 13,8% das de leite UAT. Demonstraram-se capazes de produzir enterotoxinas 50,0% dos isolados de leite cru, 19,2% de leite pasteurizado e 70,7% de leite UAT. Também se pode constatar a semelhança genética entre as cepas de Bacillus cereus isoladas do leite cru, pasteurizado e UAT, evidenciando assim que o micro-organismo é capaz de resistir ao tratamento por UAT. Tais resultados devem servir de alerta às autoridades sanitárias, tendo em vista que 13,8% deles estavam em desacordo com o estabelecido pelo Ministério da Saúde, por serem capazes de veicular um agente potencialmente patogênico, demonstrando assim que a contaminação do leite por bactérias esporuladas deve ser motivo de preocupação.