Browsing by Author "Rouvinen, Vilma-Riitta"

Psychrotrophs, which are mostly gram negative bacteria, can produce heat stable proteases and lipases. Even though these bacteria cannot survive pasteurization of milk, the enzymes can. This can cause problems for the dairy industry. Psychrotrophic milk isolates also have multidrug-resistant traits for antimicrobials and may act as reservoir for resistance genes. The aims of the study were to test denaturing gradient gel electrophoresis (DGGE) for PCR products obtained from organic and conventional raw milk bacteria, to optimize DNA-extraction protocols and PCR-conditions for the raw milk samples and study the bacterial population changes during the cold storage. The aim was also to study the antimicrobial susceptibility of the bacterial isolates. The DNA was extracted from the raw milk samples, when received and after 4 days cold storage, using commercial kits. Nested-PCR was performed and samples were analysed using DGGE. Susceptibility to antimicrobials was determined by growing bacteria on plates that contained two different concentrations of five antibiotics. From the five antibiotics two were used as a combination. The composition of the bacterial population changed during the cold (4ºC) storage: the difference in DGGE profiles was clear between 0 and 4 days cold storage. Fingerprint profile analysis showed that irrespective of the origin of the raw milk, the sample profiles were clustered according to the sampling date (day 0 or day 4). There was no clear difference between DGGE-profiles from conventional and organic milk. Proportion of psychrotrophic bacteria increased and antimicrobial resistance seemed to be more prevailing in conventional than in organic raw milk. Antimicrobial resistance decreased after four days storage at 4ºC, in most of the cases. We showed that the PCR-DGGE-method is an efficient tool to analyse the changes in bacterial populations in raw milk and that cold storage has an evident effect in population composition.