A new liquid chromatography method for the simultaneous and sensitive quantification of lactose and lactulose in milk

The disaccharide lactulose is known to have a large influence on human digestion, the effects of which range from prebiotic to laxative action. Lactulose can be derived from lactose by either alkaline isomerization or enzymatic transgalactosylation of fructose. The aim of this study was to establish a new analytical method for the quantification of both disaccharides lactose and lactulose using high performance liquid chromatography (HPLC) and evaporative light scattering detection (ELSD). The new method needs to provide good chromatographic separation of lactose and lactulose while being fast and reliable. Therefore, four analytical methods using different HPLC columns were developed and compared. A HPLC column with an amino-bonded polymeric matrix yielded better results compared to amino-bonded silica-phase resin or cation-exchange resin. The new method had detection limits for lactose and lactulose of 3.8 and 2.5 mg·L−1 and was successfully applied to the quantification of lactulose and lactose in commercial milk products

Mutational analysis of the Rhizobium lupini H13-3 and Sinorhizobium meliloti flagellin genes: importance of flagellin A for flagellar filament structure and transcriptional regulation

Complex flagellar filaments are unusual in their fine structure composed of flagellin dimers, in their right-handed helicity, and in their rigidity, which prevents a switch of handedness. The complex filaments of Rhizobium lupini H13-3 and those of Sinorhizobium meliloti are composed of three and four flagellin (Fla) subunits, respectively. The Fla-encoding genes, named flaA through flaD, are separately transcribed from sigma(28)-specific promoters. Mutational analysis of the fla genes revealed that, in both species, FlaA is the principal flagellin and that FlaB, FlaC, and FlaD are secondary. FlaA and at least one secondary Fla protein are required for assembling a functional flagellar filament. Western analysis revealed a ratio close to 1 of FlaA to the secondary Fla proteins (= FlaX) present in wild-type extracts, suggesting that the complex filament is assembled from FlaA-FlaX heterodimers. Whenever a given mutant combination of Fla prevented the assemblage of an intact filament, the biosynthesis of flagellin decreased dramatically. As shown in S. meliloti by reporter gene analysis, it is the transcription of flaA, but not of flaB, flaC, or flaD, that was down-regulated by such abortive combinations of Fla proteins. This autoregulation of flaA is unusual. We propose that any combination of Fla subunits incapable of assembling an intact filament jams the flagellar export channel and thus prevents the escape of an (as yet unidentified) anti-sigma(28) factor that antagonizes the sigma(28)-dependent transcription of flaA