Microbial Diversity in Raw and Pasteurized Milk with Terminal Restriction Fragment Length Polymorphism (T-RFLP)

Abstract In this thesis, a molecular PCR-based method was used to study the bacterial diversity in milk. The aim was to compare the microbiota of traditionally pasteurized milk with milk treated with a novel pasteurization technique, using terminal restriction fragment length polymorphism (T-RFLP). A second aim was to analyze the microbial composition of cheese produced from the two milk variants. Results of this molecular approach were compared with outcomes from traditional culturing on non-selective media, followed by 16S rRNA sequencing in order to evaluate the usefulness of the methods. Overall, the results demonstrated that T-RFLP is a powerful tool for analyzing microbiota in foods. In conclusion, both pasteurization techniques proved to be effective in reducing the number of bacteria. The initial hypothesis, that the two pasteurization techniques affect different parts of the microbiota of the raw milk, was confirmed. As expected, the molecular approach of DNA extraction direct from the milk detected a more differentiated microflora, compared to DNA extracted from cultivated bacteria from the milk. The results also indicated that the molecular approach was more reproducible between the sampling occasions. The cultivation and sequencing showed that the microbiota mainly consisted of Firmicutes and Actinobacteria, as well as Bacteriodetes. Popular science summary: Microbial Diversity in Raw and Pasteurized milk In this thesis, a molecular PCR-based method was used to study the bacterial diversity in milk. The aim was to compare the microbiota of traditionally pasteurized milk with milk treated with a novel pasteurization technique, using terminal restriction fragment length polymorphism (T-RFLP). A second aim was to analyze the microbial composition of cheese produced from the two milk variants. Results of this molecular approach were compared with outcomes from traditional culturing on non-selective media, followed by 16S rRNA sequencing in order to evaluate the usefulness of the methods. Much of the flavor and aroma of milk and cheese originates in its microbial content. Pasteurization is used to minimize the pathogens and extend the shelf-life for food. It decreases the number of microorganisms in the product by heating, followed by an immediate cooling. The treatment does not sterilize the product, but reduces the number of viable microorganisms. The awareness and control of the bacterial diversity in the milk is therefore a very important aspect in production of cheese, dairy product and other food products. Traditional methods to assess community diversity often include culturing on plates. In addition to being labor-intensive, it has proven to give a deceptive reflection of the original community structure. Molecular biology methods are based on variations in the DNA sequences between bacteria species. T-RFLP visualizes differences in the prokaryotic 16S rRNA. The analysis typically involve four steps: DNA isolation and purification, PCR amplification and restriction enzyme digestion, separation of digested products via capillary gel electrophoresis and finally analysis and clustering of data to generate a fragment profile for each sample. Identity of the terminal restriction fragments can be obtained by creation of a clone library. Effect of pasteurization The two pasteurization techniques affect different parts of the microbiota of the raw milk. Traditional pasteurization showed the greatest decrease in number of bacterial species. The cheese microbiota was considerably less diverse. As expected, the number of bacterial species found from the non-cultured population was greater than from the cultivatable population. The results also indicated that the molecular approach was more reproducible between the sampling occasions. A majority of the identified bacteria belonged to the Firmicutes phylum and were lactic acid bacteria (LAB). In conclusion, both pasteurization techniques proved to be effective in reducing the number of bacteria. The results confirmed the advantages of using molecular opposed to culture-based approaches to characterize microbiota. The study also verified the applicability of T-RFLP for analyzing microbiota in foods. Advisor: Klara Bath Degree Project 30 credits in Cell and Molecular Biology 2012 Department of Biology, Lund University