Browsing by Subject "raakamaito"

The cold storage, utilized to increase the shelf life of raw milk, favors the growth of psychrotrophic bacteria. Psychrotrophs are considered as problematic because they produce heat-stable spoilage enzymes. In addition, some pathogenic bacteria are capable of growing at cold storage temperatures and it has been observed in previous studies that the psychrotrophs in raw milk can be resistant to several antibiotics. In the literature review section of this study, the microbial composition of raw milk and the effect of cold storage, nitrogen gas (N2) treatment and activation of the lactoperoxidase system (LPS) on the raw milk bacteria was reviewed; moreover, the evolution, spread and mechanisms of bacterial antibiotic resistance and the antibiotic resistance on dairy farms and of raw milk bacteria was reviewed. Also the methods to study raw milk bacteria and their antibiotic resistance were documented. The aim of the experimental part was to study the effects of cold storage, N2 gas treatment and LPS treatment on the levels of antibiotic resistant bacteria in raw milk. Untreated, N2-treated and LPS-treated raw milk samples were stored at 6 °C and the counts of bacteria resistant to gentamicin, ceftatzidime, levofloxacin and trimethoprim-sulfamethoxazole were determined in the beginning of the experiments and after three and seven days of cold storage. The antibiotic resistance and spoilage features of bacterial isolates selected from different plates were compared. In addition, DNA fingerprints of selected isolates were obtained by rep-PCR method and for a couple of isolates 16S rRNA gene partial sequencing was performed. The presence of certain antibiotic resistance genes for the isolates partially identified was investigated using a PCR-based method. The length of cold storage affected the proportions of antibiotic resistant bacteria in raw milk. The proportions of antibiotic resistant bacteria in the control milk sample were generally, with the exception of TS-resistant bacteria in some cases, at their lowest after seven days of cold storage when the milk was, though, microbiologically unacceptable. There was a lot of variation in the prevalence of bacterial antibiotic resistance between the milk samples treated differently. The proportions of resistant bacteria were on average clearly higher in LPS samples than in control samples, whereas the effect of the N2 treatment on the proportions of resistant bacteria varied between the experiments. The bacterial isolates resistant to all the considered antibiotics produced less frequently protease and phospholipase than the isolates resistant to fewer antibiotics. The isolates from LPS samples were more frequently resistant to all the antibiotics studied and produced less frequently protease and phospholipase than the isolates from control and N2-treated samples. On the basis of the rep-PCR fingerprints, there were both similar and different strains among the bacterial isolates. Based on the 16S rRNA partial gene sequence, two isolates were identified as Pseudomonas spp. and two isolates as Stenotrophomonas spp. The targeted antibiotic resistance genes were not detected among the considered isolates. Possible reasons can be variations in the gene sequences, suboptimal PCR conditions or that the isolates lacked the genes studied. A PCR product was obtained for one Pseudomonas isolate using mexA-F/R primers, but according to the database the gene sequence did not show homology with antibiotic resistance genes. Further analyses would be required to confirm if the gene studied is linked to antibiotic resistance.

Propionibacterium freudenreichii is a known as secondary ripening culture that occurs spontaneously in small amounts in raw milk. Contamination of raw milk with P. freudenreichii and other microbes can occur during milking, transporting and processing, either from the air or through surfaces. Microbes that spontaneously end up in raw milk survive on heat treatments that are milder than pasteurization, which makes the microbiome of raw milk cheeses more diverse than cheeses made from pasteurized milk, where a higher heating temperature inactivates enzymes and kills microbes. During the ripening of the cheese, a lot of changes occur in the microbiome. The aim of this master thesis was to discover the presence and number of lactic acid bacteria and propionic bacteria in raw milk cheeses by culturing samples in different culture media, and to identify P. freudenreichii by using a polymerase chain reaction (PCR) method and P. freudenreichii -specific primers. In addition, selected P. freudenreichii strains were tested for their ability to produce vitamin B12 by liquid chromatography and the effects of different NaCl concentrations on the growth of P. freudenreichii isolates were investigated. There were differences in the number bacterial colony forming units of the cheeses. Of the selected catalase-positive isolates resembling P. freudenreichii isolates morphologically, 65% were identified as P. freudenreichii strains by PCR. Most strains of P. freudenreichii were identified from isolates of Vorarlberger Bergkäs and Liechtensteiner cheese samples (92–94%). The least strains of P. freudenreichii were identified from isolates of Tete de Moine cheese samples (25%). Possibly Vorarlberger Bergkäs and Liechtensteiner cheeses had more P. freudenreichii bacteria compared to other propionic bacteria with similary colony morphology, and Tete de Moine had less of P. freudenreichii bacteria than other propionic bacteria. Based on growth experiments, increasing the NaCl content on agar plates slowed the growth of P. freudenreichii strains and impaired the color of the colonies. Based on the vitamin assays, all 14 strains of P. freudenreichii tested produced active vitamin B12 in humans, but there were differences in the amounts of vitamins produced per strain. Strain-specific differences were also observed in the ratios of vitamin forms produced (vitamin B12 / pseudovitamin).