Heat-Resistant Psychrotrophic Bacteria Isolated from Pasteurized Milk

Abstract

Psychrotrophic bacteria were isolated from 227 pasteurized milk samples which had a shelf life in excess of 20 days at 7.2 C. Of 700 cultures isolated, 135 were resistant to heating at 72 C for 16 sec and were able to re-establish growth at 7.2 C. Thirty-five cultures, representing 15 different types were subjected to detailed examination to determine their actions on refrigerated milk, growth temperatures, thermal resistance at various temperatures, and their identities. The spore-forming genus Bacillus occured most frequently. The non-sporing types were assigned to the genera Arthrobacter, Microbacterium, Streptococcus, and Corynebacterium. More than a decade ago psychrotrophs in milk were called psychrophiles and were reported to be destroyed by proper pasteurization (2,3,38,41,50,53). Some organisms were observed to survive laboratory pasteurization and grow at 10 C, but failed to grow at 7.2 C (4,28). Olson et al. (37) and Davis (JJ) claimed that a critical difference in growth response of microorganisms existed between 7.2 C and 10 C and that use of grov.1h at 10 C as a criterion for grouping would lead to the inclusion of "thermodurics" in the psychrophilic count. Boyd et al. (9) made the same observation in comparing 5 and 10 C incubation temperatures. Those researchers who did isolate bacteria that could survive pasteurization of milk and grow at 7.2 C or below found them to be rather uncommon. Mikolajcik and Burgwald (33) found only 8 of 150 thermoduric isolates could grow at 7.2 C and Boyd et al. (9) reported that only 17.9o/o of thermoduric isolates they obtained grew at 5 C. An earlier study by Stark and Scheib (47) revealed 4 of 486 isolates from butter were thermoduric and could grow at sc. Over the past decade changes in production methods, farm bulk tank storage of milk, long distance hauling of milk, silo storage of raw milk at the processing plant, and the trend in the dairy industry toward higher pasteurization temperatures have caused a definite change in the microbial flora of milk. This change is 1 Journal Paper No. J 3157 qf' the Oklahoma Agricultural Experiment Station, evident in both raw and pasteurized milk and may be the result of disruption of the ecosystem of milk as it passed on from the cow to the consumer (46). The trend toward higher pasteurization temperatures and longer refrigerated storage of milk before comsumption have brought to attention selected flora that may be described as "thermoduric psychrotrophs". This paper presents a study of identification, pasteurization resistance, grov.1h characteristics and possible commercial impact of several such organisms isolated from a large number of processed milk samples. MATERIALS AND METHODS Samples During an 18-month period samples of pasteurized milk at various stages of processing were subjected to a shelf life test at 7.2 C. Two hundred and twenty seven samples showing no organoleptic signs of spoilage at 20 days were retained until off flavors developed, while samples spoiling before 20 days were discarded. Cultrue isolation Thermoduric psychrotrophs were isolated from the spoiled pasteurized milk samples by inoculating a loopful of each sample into a 16-mm screw capped test tube containing 10 ml of sterile litmus milk and subjecting the inoculated milk to laboratory pasteurization at 71.7 C for 16 sec. Tubes were cooled and held at 7.2 C for 14 days, streaked on Plate count age (Difco) and incubated at approximately 25 C until colonies developed. Two colonies of each predominant type were picked into litmus milk. After incubation at approximately 25 C for 5 to 7 days, one tube of each type of reaction was saved for further study. The isolated cultures were grouped according to staining characteristics, colony morphology, cell morphology and reaction in litmus milk. From each group, depending on the number of isolates involved, one to five cultures were selected for futher study. Thennal resistance All cultures were subjected to laboratory pasteurization at 71.7 C for 16 sec at least four times, and some as many as nine times, followed by incubation at 7.2 C for 14 days. In separate trials. cultures from each group were heated at 71.7 C for 20,30,40, and SO sec. In additional trials cultures were heated for 16 see at 74.4, 77.2, and 80 C. A low temperature exposure of 60.7 C for 30 min and a high extreme of93.3 C for 5 min were also used. Trials involving temperatures of 71.7 C or higher were accomplished by placing the test tube support and test tubes, containing one loopful of culture in 5 ml of previously sterilized litmus milk, in a container with 12 mm of water, a depth not sufficient to touch the bottoms of the tubes. The container was covered and a thermometer inserted through the lid into a control tube. The water was heated and the generated steam served to heat the tubes. When the thermometer reached a