Raw skim milk was incubated at 7 C for 15 days after inoculation with six psychrotrophic bacterial cultures previously isolated from raw milk. Effects of the microbial activities on proteins of milk were evaluated by polyacrylamide gel electrophoresis. Results showed that all psychrotrophs hydrolyzed milk proteins. The K- and β -caseins were most susceptible to proteolysis while the a -casein was less affected. Most of the isolates required extended incubation periods for hydrolysis of the whey proteins. Commercially sterilized milk samples inoculated with pure cultures developed bitterness after 4 days of storage at 7 C when the psychrotrophic count was 2.5 × 10 6 /ml. The addition of 9.8 enzyme units to UHT milk caused a bitter flavor within 28 days at 7 C and in less than 3 days at room temperature. The presence of only 2 units resulted in bitterness in less than 7 days at room temperature.
Twelve gram-negative psychrotrophic bacteria producing heat-resistant proteases that hydrolyzed casein were isolated from refrigerated raw milk. All were pseudomonads and the enzymes of the six most proteolytic cultures were examined further. The proteases were partially purified, and gel electrophoresis indicated that only a single enzyme was present in the preparation. The molecular weight of most of the proteases was approximately 45,000. All six enzymes retained some activity after being heated at 121 C for 10 min and casein was hydrolyzed at pH levels found in normal milk and many cultured dairy products. Although proteolysis was highest at about 40 C, considerable activity was evident at refrigeration temperatures.