DNA based analyses of microbiota in fat fraction of raw milk
The microbiota of raw milk is complex and the analysis methods have during the years developed from culturing to molecular based methods, allowing more precise descriptions of the composition. This project included both cultivation and molecular methods to compile the distri-bution of microorganisms...
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| Formato: | H2 |
| Lenguaje: | Inglés sueco |
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SLU/Department of Molecular Sciences
2017
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| Materias: |
| _version_ | 1855572131636576256 |
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| author | Blänning, Caroline |
| author_browse | Blänning, Caroline |
| author_facet | Blänning, Caroline |
| author_sort | Blänning, Caroline |
| collection | Epsilon Archive for Student Projects |
| description | The microbiota of raw milk is complex and the analysis methods have during the years developed from culturing to molecular based methods, allowing more precise descriptions of the composition. This project included both cultivation and molecular methods to compile the distri-bution of microorganisms in the whole milk, since the most common way to day to investigate milk microbiota is based on defatted milk. The goal was to explore how big part of the microbiota that is connect-ed to the milk fat fraction and if the already existing method for DNA based analyses of milk microbiota is suitable also for detection of mi-crobiota in whole milk. In this project, raw milk samples were spiked with Lactobacillus reuteri DSM, Lactobacillus reuteri PTA, Escherichia coli and Staphylococcus aureus. The samples were centrifuged and the fractions (pellet, supernatant and fat) were cultured to identify the bac-terial distribution. The molecular method consisted of DNA extractions, Terminal Restriction Fragment Length Polymorphism (T-RFLP) and quantitative PCR (qPCR) also based on whole milk, spiked with the same bacteria as during the cultivation part. PowerFood Microbial DNA Isolation kit (MoBio Laboratories Inc.) was used for four different ex-traction methods. For comparing the DNA extraction methods and bac-terial community, the T-RFLP was used. Additionally, qPCR was per-formed for total and specific targeting of bacteria and quantification. The results from the cultivation part showed that the highest amounts of colony forming units (CFU) originated from the fat fraction. The mo-lecular methods confirmed the pattern as the T-RFLP analyses from the whole milk samples showed more traces of bacteria. The qPCR showed that the whole milk samples contained more DNA than the skim milk samples. In conclusion, the PowerFood kit without additions of solvents can be used for DNA preparation followed by PCR and/or T-RFLP/sequencing when analyzing the microbiota in whole milk. |
| format | H2 |
| id | RepoSLU12791 |
| institution | Swedish University of Agricultural Sciences |
| language | Inglés swe |
| publishDate | 2017 |
| publishDateSort | 2017 |
| publisher | SLU/Department of Molecular Sciences |
| publisherStr | SLU/Department of Molecular Sciences |
| record_format | eprints |
| spelling | RepoSLU127912017-10-24T06:43:44Z DNA based analyses of microbiota in fat fraction of raw milk DNA-baserad analys av mikrobiota i råmjölkens fettfraktion Blänning, Caroline raw milk microbiota whole milk DNA T-RFLP PCR The microbiota of raw milk is complex and the analysis methods have during the years developed from culturing to molecular based methods, allowing more precise descriptions of the composition. This project included both cultivation and molecular methods to compile the distri-bution of microorganisms in the whole milk, since the most common way to day to investigate milk microbiota is based on defatted milk. The goal was to explore how big part of the microbiota that is connect-ed to the milk fat fraction and if the already existing method for DNA based analyses of milk microbiota is suitable also for detection of mi-crobiota in whole milk. In this project, raw milk samples were spiked with Lactobacillus reuteri DSM, Lactobacillus reuteri PTA, Escherichia coli and Staphylococcus aureus. The samples were centrifuged and the fractions (pellet, supernatant and fat) were cultured to identify the bac-terial distribution. The molecular method consisted of DNA extractions, Terminal Restriction Fragment Length Polymorphism (T-RFLP) and quantitative PCR (qPCR) also based on whole milk, spiked with the same bacteria as during the cultivation part. PowerFood Microbial DNA Isolation kit (MoBio Laboratories Inc.) was used for four different ex-traction methods. For comparing the DNA extraction methods and bac-terial community, the T-RFLP was used. Additionally, qPCR was per-formed for total and specific targeting of bacteria and quantification. The results from the cultivation part showed that the highest amounts of colony forming units (CFU) originated from the fat fraction. The mo-lecular methods confirmed the pattern as the T-RFLP analyses from the whole milk samples showed more traces of bacteria. The qPCR showed that the whole milk samples contained more DNA than the skim milk samples. In conclusion, the PowerFood kit without additions of solvents can be used for DNA preparation followed by PCR and/or T-RFLP/sequencing when analyzing the microbiota in whole milk. Mjölkråvarans mikrobiota är komplex och genom åren har analysmetoderna utvecklats från odling på agarplattor till molekylära metoder, vilket resulte-rat i en tydligare bild av de förekommande bakterierna. Detta projekt har inkluderat både odling och molekylära metoder för att jämföra distribution-en av bakterier i helmjölk, eftersom dagens analyser är baserade på avfettad mjölk. Syftet med detta projekt är att undersöka hur stor del av mikrobiotan som binder till fettet och om befintliga metoder för DNA-analyser av mjölk även går att applicera för analyser av mjölkfettets mikrobiota. Första delen av projektet innebar att undersöka hur bakterierna fördelades i pellet-, supernatant- eller fettfraktionerna efter centrifugering. Detta gjordes genom att inokulera mjölkprover separat med Lactobacillus reuteri DSM, Lacto-bacillus reuteri PTA, Escherichia coli and Stafylococcus aureus och däref-ter gjordes spädningar som odlades på selektiva agarplattor. Nästa del best-od av DNA-extrahering med hjälp av PowerFood Microbial DNA Isolation kit (MoBio Laboratories Inc.) med några avvikelser from tillverkarens pro-tokoll, vilket resulterade i fyra metoder. Tre av dessa användes för vidare analys med metoden Terminal Restriction Fragment Length Polymorphism (T-RFLP) för att jämföra mikrobiotan i proverna och om det förekom fler typer av bakterier i helmjölkproverna. För undersökning av de specifika bakterierna och kvantifiering av dem utfördes även kvantitativ PCR (qPCR). Odlingen på agarplattorna resulterade i att flest kolonibildande enheter (CFU) bildades från fettfraktionerna, vilket betyder att mjölkfettet innehål-ler en stor del bakterier som annars kan förbises vid analyser då avfettad mjölk används. För T-RFLP analysen visade det sig att det inte var någon större skillnad i extraheringsmetoderna, men vid analys med metoder base-rade på helmjölk tenderade proverna att innehålla högre antal bakterier. Som slutsats kan det konstateras att PowerFood kittet går att använda utan att ursprungsprotokollet modifieras för DNA-isolering, PCR och T-RFLP/sekvensering för att analysera bakterier även i helmjölk. SLU/Department of Molecular Sciences 2017 H2 eng swe https://stud.epsilon.slu.se/12791/ |
| spellingShingle | raw milk microbiota whole milk DNA T-RFLP PCR Blänning, Caroline DNA based analyses of microbiota in fat fraction of raw milk |
| title | DNA based analyses of microbiota in fat fraction of raw milk |
| title_full | DNA based analyses of microbiota in fat fraction of raw milk |
| title_fullStr | DNA based analyses of microbiota in fat fraction of raw milk |
| title_full_unstemmed | DNA based analyses of microbiota in fat fraction of raw milk |
| title_short | DNA based analyses of microbiota in fat fraction of raw milk |
| title_sort | dna based analyses of microbiota in fat fraction of raw milk |
| topic | raw milk microbiota whole milk DNA T-RFLP PCR |