Two techniques for investigation of proteins and short-chain fatty acids of Lactobacillus plantarum in presence of galactooligosaccharides
Sammanfattning: Abstract The long-term aim of this project was to develop synbiotic drinks presenting food supplements with beneficial effects on human health. A synbiotic consists of a combination of probiotic bacteria and one or more prebiotics. For the part of the project that was covered by this Master thesis, a probiotic strain, Lactobacillus plantarum F44 and a prebiotic galactooligosaccharide (GOS) were tested in vitro. The probiotic L. plantarum F44 was selected due to previous systematic studies of the strain characteristics. The selected probiotic strain was cultured with GOS, but also grown without GOS (control), to compare growth efficiency, gene expression, and fermentation products. By monitoring the cell density, it was shown that GOS in a moderate concentration (2 mg/mL) increased the bacterial growth rate. Bacterial proteins play a major role in interaction with the cells in the human gastrointestinal tract. Investigation of these proteins can help understand and predict how probiotics affect the human host. Methods for protein extraction of the L. plantarum F44 showed that the strain had a high resistance to osmotic stress but not to lysozyme treatment. Two-dimensional protein maps were obtained of the extracted proteins. However, no distinct differences were noted of the protein patterns of bacteria grown in the presence and absence of GOS. A prospect for the future is to analyse whether there are differences when using other prebiotics, such as pectin and resistant starches, and with other probiotic strains. The concentrations of short-chain fatty acids (SCFAs) in the culture, mainly butyric and propionic acids, produced as a result of bacterial fermentation of the GOS, were determined by high performance liquid chromatography (HPLC). The conditions for the chromatographic separation were carefully evaluated and clear chromatograms of the standard solutions of butyric and propionic acids established. However, a different sample preparation will be required for accurate measurement of the SCFAs produced by the bacteria. The attempted liquid/liquid back extraction was not sufficient since too many impurities remained in the sample, interfering with the chromatography. Further purification steps are needed.
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