Enzymes from isolates of Pseudomonas fluorescens involved in food spoilage

Aims: Psychrotrophic Gram-negative bacteria, such as Pseudomonas species, pose a significant spoilage problem in refrigerated meat and dairy products due to secretion of hydrolytic enzymes, especially lipases and proteases. This study characterized the enzymes produced by strains of Pseudomonas fluorescens isolated from pasteurized milk. Methods and Results: Thirty-seven isolates of Ps. fluorescens from skimmed, semiskimmed and whole milk were all shown to be proteolytic and lipolytic on casein and tributyrin agar, respectively. The highest level of protease production by one isolate, SMD 31, from skimmed milk was in minimal salts medium containing 1 mmol l(-1) calcium chloride at 20degreesC. The proteases belonged to the class of metallo-proteases, as there was no residual activity with 10 mmol l(-1) EDTA. They were heat stable and retained activity even after treatment at 121degreesC for 20 min. One protease of 45-48 kDa was detected in unconcentrated supernatant fluid samples but, in three isolates from different milk sources, five proteases with molecular masses between 28 and 48 kDa were detected on a 12% zymogram casein gel following ultrafiltration. Attempts to purify the lipases proved unsuccessful. Conclusions: The characteristics of the major protease of 45-48 kDa correspond to those of proteases described for other Pseudomonas species isolated from a range of environments. However, the smaller proteases have not been described previously. Significance and Impact of the Study: In the absence of ultrafiltration the presence of the minor protease species may be missed and they may act as contaminants of the major protease in unpurified or semipurified samples.