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Spoilage potential of spore-forming bacteria from refrigerated raw milk

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 80°C 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 and lipolytic, whereas the B. circulans isolate was only lipolytic. Among the 11 Bacillus spp. isolates, eight isolates (72.7%) were proteolytic and lipolytic, one isolate (9.1%) was proteolytic and the other two isolates (18.2%) were lipolytic. The three Paenibacillus spp. and Brevibacillus spp. isolates were primarily lipolytic. Therefore, to extend the shelf life of pasteurized milk, preventive measures must be adopted to reduce contamination with spores because one-third of these microorganisms exhibited proteolytic and/or lipolytic activity.