Identification Of The Sources Of Psychrophile In Raw Milk And Establishment Of Rapid Detection Method

Refrigerated transportation is the key measure to ensure the quality of raw milk,but psychrophile can continue to grow and become the main microorganisms in raw milk under low temperature conditions.The quality of UHT milk in the shelf life were damaged by residual heat-resistant proteases heated 5 s at 135℃from the psychrophile bacteria.In this study,the diversity and the sources of psychrophile in raw milk were analyzed by using pure culture technology combined with metagenomic shotgun sequencing technology.The key pollution strains in raw milk were determined according to the protease activity and enzyme residue rate of psychrophile.And a rapid detection system for Loop-mediated isothermal amplification combined with Lateral flow dipstick（LAMP-LFD）was established and optimized for these strains.It aims to quickly detect psychrophile that affect the quality of UHT milk in raw milk,so as to ensure the quality of raw milk.The specific results of this study are as follows:（1）A total of 64 samples were collected from Yinchuan City,Ningxia Hui Autonomous Region,Qiqihar City,Heilongjiang Province,Hengshui City,Hebei Province,and Qingyuan City,Guangdong Province,and 330 strains of psychrophile were isolated,including 76species from 21 genera.Isolated strains include Pseudomonas,Psychrobacter,Acinetobacter,Serratia,Glutamicibacter,Exiguobacterium,etc.Among them,Pseudomonas accounted for57.27%of the total isolated strains,including 33 Pseudomonas such as P.azotoformans,P.lactis,P.paralactis,P.gessardii,and P.weihenstephanensis.In addition,it was found that the species and abundance of psychrophile were obviously different in seasons and different regions,but the structure of psychrophile in the region or the season did not show a strong correlation.The diversity of psychrophile were abundant in the cow barn bedding,the first three handfuls of milk and TMR feed,but almost no psychrophile were isolated in drinking water and ensilage feed.（2）The diversity,species and abundance of Pseudomonas in raw milk samples and environmental samples were further studied by metagenomic shotgun sequencing technology.The data show that the phylum level in raw milk is mainly Proteobacteria,Firmicutes,and Actinobacteria.99.63%of Pseudomonas in normal milk and 96.39%milk container can be traced to cow barn bedding,indicating that the cow barn bedding is a potential source of various types of Pseudomonas..（3）At 28℃,61.38%of Pseudomonas strains could produce protease,and up to 92.24%of Pseudomonas strains carried aprX gene.After being treated at 135℃for 5 s,the protease residue rate of 21.55%was more than 60%.Among them,the protease activity of 1 strain of P.fluorescens exceeded 40 U/m L,and the residual rate of protease of 5 strains of P.azotoformans exceeded 80%.（4）The detection method of LAMP-LFD was established by using P.azotoformans CAAS 2022A16-3 and P.fluorescensis CAAS 2022L6-1 of as the detection strains and aprX as the detection genes.Under the optimal primer group,LAMP amplification was best when the volume of Bst DNA polymerase was 0.8μL,the concentrations of Mg²⁺and d NTPs were8 m M and 1.2 m M,and the reaction temperature and time were 63°C and 40 min,respectively.The limit of detection of LAMP amplification products on Lateral flow dipstick for Pseudomonas in raw milk was as low as 4.02×10¹CFU/m L.The detection method is simple to operate,has high specificity,strong sensitivity and short detection time,which can meet the rapid detection of Pseudomonas protease-producing in raw milk.The results of this study provide basic theoretical knowledge for the scientific prevention and control of psychrophiles in pasture,and provide a basis for the graded use of raw milk.