Visualization of fluorescent Lactobacillus reuteri in milk using epifluorescence microscopy : a model study to investigate the distribution and affinity of L. reuteri in bovine milk fat fraction
Abstract: Microbiota composition and content in raw milk is diverse and crucial for the quality of milk as a raw material and subsequent processing. Investigation and identification of microbiota in raw milk is mainly performed based on molecular-based techniques, which are rapid and accurate. At present, defatted milk is used to isolate milk microbiota. A recent study (unpublished) showed that a significant part of milk microbiota is associated with milk fat fraction. Therefore, present study endeavour to continue and confirm the association of microbiota, visually using epifluorescence microscope. A model study was designed to investigate the distribution of recombinant Lactobacillus reuteri in bovine milk varied with three levels of fat content (~0.1%, 1.5% and 3.0%). Two fluorescently active recombinant bacterial strains of L. reuteri (6475 mCherry and R2LC mCherry) and their wild-type (fluorescently inactive) were cultured in milk. Dilution series for each strains were made by respective cultures and bacteria cells were counted by suitable dilutions to make stock solutions. Milk samples were spiked using stock solutions of bacteria and observed through a fluorescence microscope using UV light. The recombinant bacteria were observed with red light fluorescence filter (Cy3 red filter) and the epifluorescent images captured by a high-definition colour camera (Nikon DS-Fi2) which was attached to the fluorescence microscope. The results from the fluorescent labelled microscopic images showed that L. reuteri 6475 mCherry and L. reuteri R2LC mCherry strains were associated with milk fat and they were attached to milk fat globules without being free in milk serum. In conclusion, Lactobacillus reuteri showed a great-er affinity to milk fat fraction in bovine milk than serum fraction. Thus, the results confirmed the previ-ous study and whole milk based microbiota investigation in raw milk should be encouraged as the defat-ted milk based techniques have the tendency to underestimate the microbiota in raw milk.
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