变质原料乳中假单胞菌的分离鉴定及产蛋白酶特性研究

目的]为了解导致原料乳变质的微生物种类及其产蛋白酶活力。方法]对广东省恒远市某规模化牧场提供的蛋白水解、脂肪上浮的变质原料乳稀释，涂布在MPC琼脂平板上。将平板于6.5 ℃恒温培养箱培养10 d，挑取形态不同的单菌落在LB液体培养基复壮增菌，提取其DNA，利用16S rDNA方法对分离的菌株进行鉴定，并对该菌株的蛋白酶活力及高温钝化后酶残留率进行测定。结果]MPC琼脂板菌落形态单一，原料乳中分离鉴定出的两株菌均为霍氏假单胞菌，该菌株在28 ℃下对乳平板的水解圈为（15.35±0.56）mm，经135 ℃处理5 s，其蛋白酶残留率为43.63%。结论]变质原料乳中分离到两株霍氏假单胞菌，并且该菌可产生热不敏感的蛋白酶，该酶的存在会造成原料乳中蛋白质水解，导致UHT乳在货架期内出现蛋白水解、胀包等质量问题。

Isolation and Identification of Pseudomonas From Spoiled Raw Milk and Determination of Protease Activity

Objective] To identify the microbial species and protease-producing activity that cause the deterioration of raw milk. Method] The proteolyzed and fat-floating metamorphic raw materials provided by a large-scale ranch in Hengyuan City，Guangdong Province，were diluted and coated on MPC agar plates. The plates were incubated in a 6.5 ℃ thermostatic incubator for ten days， and the monobacterial colonies with different morphologies were picked up and reinvigorated in LB liquid medium， their DNA was extracted，and the isolated strains were identified by the 16S rDNA method. The protease activity and enzyme residue rate of this strain were also determined. Result] MPC agar plate colony form is single，and the two strains isolated and identified in the raw milk are Pseudomonas rhodesiae. The hydrolysis circle of the milk plate at 28 ℃ was （15.35±0.56）mm，and the protease residue rate was 43.63% after 5 s treatment at 135 ℃.Conclusion] Two strains of Pseudomonas rhodesiae were isolated from spoiled raw milk，and the bacterium produces heat-insensitive proteases. The presence of this enzyme will cause protein hydrolysis in raw milk，resulting in quality problems such as proteolysis and swelling of UHT milk during the shelf life.