Abstract: 【Objective】 To investigate the changes of protein concentration in UHT milk at different storage temperatures when UHT milk was exposed to different orders of cold-philic bacteria. 【Methods】 (1) Mixed culture method was used to dilute the raw milk from the original liquid to a 10^-5 dilution gradient, and each gradient was parallel to each other.They were cultured at 6.5℃ for 10 days to isolate and purify and retain a single colony.(2) Genomic DNA extraction and PCR reaction were used to identify the dominant cold-philic bacteria.(3) Milk plate visual identification method was used to screen the strain with the strongest protease production ability.(4) BCA protein concentration determination method was applied, UHT milk was diluted 8 times, 12 times, 20 times, 24 times and 28 times dilution concentration in phosphate buffer, and the linear dilution ratio of UHT milk in protein concentration determination experiment was determined.(5) UHT milk was simultaneously inoculated with 10³, 10⁴ and 10⁵ CFU/mL dominant thermophilic bacteria.The three experimental groups were stored at 4, 7 and 21℃ for 7 days, respectively.Protein concentration was detected every 24hBCA. 【Results】 (1) A total of 212 strains of thermophilic bacteria were screened out from 33 fresh milk samples.(2) Through PCR identification, pseudomonas was the dominant cold-philic bacteria in fresh milk, and the isolation rate was 5.19%; (3) Through the milk plate visual identification method, pseudomonas strain 99 showed the strongest protease production ability, and the diameter of the transparent ring was 0.3mm; (3) The linear coefficient of protein quantification in UHT milk at 28 times diluted concentration was R²=0.998, and the linear relationship was the best.(4) At 4℃, there was no significant change in the protein concentration of the three experimental groups supplemented with 10³ CFU/mL, 10⁴ CFU/mL and 10⁵ CFU/mL pseudomonas (P > 0.05); At 7℃, the protein concentration of the inoculated bacteria in the three groups was significantly different from that in the control group (P<0.05); At 21℃, except for 10⁴ CFU/mL group, the protein concentration decreased significantly (P<0.05), the protein concentration in 10³ CFU/mL and 10⁵ CFU/mL groups decreased significantly (P<0.01). 【Conclusion】 The content of cryophilic bacteria in fresh milk, storage temperature and storage time all affect the protein concentration of UHT milk.The higher the temperature is, the higher the content of cryophilic bacteria is, and the more obvious the degree of protein hydrolysis is, thus affecting the quality of UHT milk.