Pseudomonas fluorescens and Pseudomonas putida from refrigerated raw milk: genetic diversity and lipoproteolytic activity.

In this research communication the genetic diversity of Pseudomonas fluorescens (n = 67) and Pseudomonas putida (n = 44) isolated from refrigerated raw milk from bulk tank trucks were verified. The relationship between the genetic profile of the isolates and their lipoproteolytic potential was evaluated using skim milk agar and tributyrin agar (21°C/72 h). The lipoproteolytic potential (low or high), evaluated by the diameter of the halos (cm), was correlated with the number of milk producing properties that contributed to each sample (one sample = one bulk tank truck; 8-80 producers/sample) and the distance between the dairy properties and the processing plant (21-370 km). P. fluorescens was confirmed in all samples, while P. putida in 60% samples. For both species, two clusters (I and II) were observed, and the first one showed lower genotypic diversity and the presence of isolates with 100% similarity. P. fluorescens isolates presenting at least 70% similarity were 83.9% in Cluster I (n = 31) and 44.4% in Cluster II. In both clusters (I and II) observed in the P. fluorescens dendrogram, the occurrence of high proteolytic and lipolytic potential were equivalent. The higher the number of farms per milk sample, the greater the lipoproteolytic intensity of P. fluorescens isolates. In relation to P. putida isolates, 74% presented at least 50% similarity in Cluster I (n = 27) and only 35% in Cluster II (n = 17). The occurrence of high proteolysis linked to P. putida was proportional between both Clusters, but the occurrence of high lipolysis was greater in Cluster II. No significant association was detected between P. putida isolates and the variables studied. The results indicate the circulation of P. putida and P. fluorescens with 100% similarity in different milk producing regions. The level of genetic diversity was related only to the lipolytic capacity of P. putida.

Efeito da bactofugação nas contagens e diversidade microbiana de psicrotróficos do leite cru / Effect of bactofugation of raw milk on counts and microbial diversity of psychrotrophs

Esse estudo teve por objetivo verificar o efeito da bactofugação do leite na quantidade e diversidade de psicrotróficos. Como o leite cru era pré-aquecido (≈55ºC) para ser bactofugado, foram coletadas amostras de três lotes de leite cru refrigerado, pré-aquecido e bactofugado a 10.000g. O pré-aquecimento foi suficiente para eliminação de 99,99% dos psicrotróficos do leite cru. A bactofugação reduziu 89,66% das contagens de psicrotróficos do leite pré-aquecido. Lysinibacillus fusiformis predominou (45, 7%) no leite cru, pré -aquecido (37,5%) e bactofugado (60%). Bacillus invictae (20%), Enterococcus faecalis (10%) e Kurthia gibsonii (10%) também foram remanescentes à bactofugação, que aparentemente não tem efeito sobre uma população específica de micro-organismos, mas reduz proporcionalmente toda a microbiota psicrotrófica do leite cru.

Lipolytic capacity of Pseudomonas spp. isolated from refrigerated raw milk / Capacidade lipolítica de Pseudomonas spp. isoladas de leite cru refrigerado

The psychrotrophic bacteria growth in refrigerated milk is responsible for the production of heat resistant enzymes, including those which have lipolytic capacity. Lipases can cause rancidity and off-flavor in pasteurized and UHT milk, milk powder and cheese. Pseudomonas spp. is the major genera responsible by lipases synthesis, mainly Pseudomonas fluorescens. The lipolytic capacity of Pseudomonas spp. (1182 strains) and P. fluorescens (158 strains) isolated from cooling tank and bulk milk transportation were tested. Pseudomonas spp. strains were isolated by Pseudomonas Agar Base, adding the supplement cephalotin, fusidic acid, cetrimide- CFC (30º C/48 h) and Pseudomonas Cetrimide Agar with 10% of glicerol (21º C/ 48 h) to determine P. fluorescens. The strains lipolytic capacity were tested with Tributyrin Agar (21 oC/72 h). Lipolytic indexes of Pseudomonas spp. were 51.4% and 67.2% in cooling tank and bulk milk transportation (p< 0.05), respectively. P. fluorescens lipolytic capacity indexes were 26.4% from strains isolated from cooling tank and 41% (p < 0.05) from strains isolated from bulk milk transportation. High lipolytic indexes had been found in the strains isolated from refrigerated raw milk sent to manufacturing. Refrigeration temperature and storage time may contribute to the difference observed between two points of collection evaluated in this study.

A multiplicação de bactérias psicrotróficas em leite refrigerado pode promover a síntese de enzimas resistentes ao calor, entre elas aquelas que tem capacidade lipolítica. As lipases podem promover rancidez, e defeitos de sabor em leite pasteurizado, UHT, leite em pós e queijos. Pseudomonas spp. é o principal gênero responsável pela síntese desta enzimas, principalmente a espécie Pseudomonas fluorescens. Avaliou-se a capacidade lipolítica de 1182 cepas de Pseudomonas spp. e 158 cepas de P. fluorescens isoladas de amostras de leite de tanques de resfriamento (TR) e caminhão tanque (CT) enviado ao beneficiamento. Foi utilizado o Agar base Pseudomonas com suplemento cephalotin, fusidic acid, cetrimide - CFC (30º C/48 h ) para isolamento das pseudomonas e Pseudomonas Cetrimide Agar com 10% de glicerol (21º C/ 48 h) para a espécie P. fluorescens. Para avaliação da capacidade lipolítica as cepas fora semeadas em Ágar Tributirina (21 ºC/72 h). O índice de lipólise das cepas de Pseudomonas spp. provenientes do leite do tanque de resfriamento e do caminhão tanque (p<0,05) foram de 51,4% e 62,7%, respectivamente. Foram também observados índices de capacidade lipolítica de P. fluorescens de 26,4% e 41% (p<0,05) para TR e CT, respectivamente. Altos índices de cepas lipolíticas foram encontrados nas cepas isoladas do leite cru refrigerado enviado ao beneficiamento. Temperatura de refrigeração e tempo de estocagem podem ter contribuído para a diferença observada na população lipolítica dos dois pontos de coleta avaliados.

Pseudomonas spp. and P. fluorescens: population in refrigerated raw milk / Pseudomonas spp. e P. fluorescens: população em leite cru refrigerado

ABSTRACT: Raw milk samples were collected from cooling tanks (after they cooled for 48 h) in five dairy farms and the corresponding bulk tank (bulk milk transportation, BMT) when they arrived to the industry. Routine physical chemical analyzes and quantification of psychrotrophic ( Pseudomonas spp. and P. fluorescens ) and aerobic mesophilic (AM) populations were performed. Only relative density and titratable acidity values for samples of milk from three farms were in agreement to the quality parameters required by law. In the BMT, only the protein content has not reached the minimum value established by law, and counting was performed for AM (>105 colony forming units (CFU) mL-1) and psychrotrophic bacteria (2.8x106CFU mL-1). Pseudomonas spp. counting corresponded to 17.9% of the psychrotrophic population, and P. fluorescens was 3.4% of Pseudomonas spp . count. In milk samples from dairy farms, counts were variable for AM (3.4x105 to 3.7 x107CFU mL-1), psychrotrophic (4.0x104 to 3.1x106CFU mL-1), Pseudomonas spp. (2.3x104 to 1.8x105CFU mL-1), and P. fluorescens (62 to 8.4x103CFU mL-1). For the populations studied, no statistical difference (P>0.05) was observed between counts reported in milk samples collected in dairy farms (cooling tanks) and BMT. Therefore, the genera Pseudomonas spp. and P. fluorescens were not the most frequent psychrotrophic bacteria in this studied milk transportation line.

RESUMO: Amostras de leite foram coletadas de tanques de refrigeração (após 48h de refrigeração) em cinco fazendas de produção e do respectivo caminhão tanque (leite de conjunto, LC) ao chegar à indústria beneficiadora. Análises físico químicas de rotina e quantificação das populações de bactérias psicrotróficas ( Pseudomonas spp. e P. fluorescens ) e aeróbios mesófilos (AM) foram realizadas. Apenas os valores de densidade relativa e acidez titulável de amostras de leite de três fazendas estavam de acordo com a legislação. No LC, apenas o teor de proteína não atingiu o valor mínimo estabelecido por lei, e a contagem foi realizada para AM (>105 unidades formadoras de colônias (UFC) mL-1) e psicrotróficos (2,8x106UFC mL-1). A contagem de Pseudomonas spp. correspondeu a 17,9% da população de psicrotróficos, e 3,4% da contagem de Pseudomonas spp. eram da espécie P. fluorescens . Nas amostras de leite das fazendas de produtoras, as contagens foram variáveis para AM (3,4x105a 3,7x107UFC mL-1), psicrotróficos (4,0x104 a 3,1x106UFC mL-1), Pseudomonas spp. (2,3x104 a 1,8x105UFC mL-1) e P. fluorescens (62 a 8,4x103UFC mL-1). Para as populações estudadas, nenhuma diferença estatística (P>0,05) foi observada entre as contagens encontradas nas amostras de leite coletadas nas fazendas (tanques de resfriamento) e do LC. Portanto, o gênero Pseudomonas spp. e a espécie P. fluorescens não foram os psicrotróficos mais frequentes nesta linha de transporte de leite estudada.