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

ABSTRACT

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...

RESUMO

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...