Abstract: Pseudomonas frequently occur in refrigerated raw milk as the predominant bacteria,which secret heat-resistant enzymes that can cause milk spoilage.A total of 102 strains of Pseudomonas isolated from raw milk were identified by rpoB gene sequencing at the species level,and azocasein was used to quantify the proteolytic activity and heat resistance of their extracellular peptidases under two sets of cultivation conditions (30 ℃ for 2 days or 6 ℃ for 8 days).Pseudomonas fluorescens was the most dominant Pseudomonas species in the samples,followed by Pseudomonasfragi and Pseudomonas sp1.A significant variation in the proteolytic activity of different Pseudomonas species was observed.Most isolates of Pseudomonasfluorescens (23/41) showed proteolytic activity after cultivation at two temperatures (30 or 6 ℃),while most isolates of Pseudomonas sp1 did not.In addition,all isolates of Pseudomonasfragi (13/13) showed proteolytic activity only after cultivation at 6 ℃.Temperature stability of their extracellular peptidases was determined.After cultivation at 6 ℃,P.fluorescens M02 had the highest peptidase activity (11.78 ΔA/(h·mL)).The peptidase showed maximum activity at pH 6.5 and 40 ℃.The addition of 103 CFU/mL P.fluorescens M02 resulted in destabilization of skim milk during 5 days of culture at 6 ℃.Monitoring the predominant psychrotrophic bacteria at species level will be useful in designing effective monitoring methods and tools to improve the quality and safety of raw milk or dairy products.

Key words: psychrotrophic bacteria; heat resistance; peptidase; Pseudomonas

摘要： 在冷藏的原料乳中,假单胞菌经常出现并成为主导菌群,其分泌的耐热酶会使原料乳变质。对从原料乳中分离的102株假单胞菌经rpoB基因测序进行菌种分析,用偶氮酪蛋白测试所有假单胞菌在2种培养条件(30℃培养2d或6℃培养8d)下胞外肽酶的蛋白水解活性和耐热性。结果表明:荧光假单胞菌(Pseudomonas fluorescens)是假单胞菌中最主要的菌种,其次是莓实假单胞菌(Pseudomonas fragi)和假单胞菌sp1 (Pseudomonas sp1)。不同假单胞菌菌种之间的产肽酶能力存在明显差异。大多数荧光假单胞菌(23/41)在不同温度(30℃和6℃)条件下培养后能产肽酶,而大多数的假单胞菌sp1不产肽酶,所有分离的莓实假单胞菌(13/13)只在6℃条件下产肽酶,大多数的假单胞菌所产肽酶都有热稳定性。在6℃培养条件下,荧光假单胞菌菌株M02有最高肽酶活力(11.78ΔA/ (h·mL)),其胞外肽酶最佳催化pH值为6.5,最佳催化温度为40℃。当M02的接种浓度为103 CFU/mL时,6℃培养5d就能使脱脂乳溶液产生沉淀。对耐冷假单胞菌的种类和产肽酶能力进行分析和考察有助于提高原料乳或乳制品的质量和安全性。

关键词: 耐冷菌; 耐热性; 肽酶; 假单胞菌