Physiology of exopolysaccharide biosynthesis by Lactococcus lactis

Several lactic acid bacteria (LAB) produce exopolysaccharides (EPS). EPSs produced by LAB are a potential source of natural additives and because LAB are food grade organisms, these EPSs can also be produced in situ . The amount of EPS in milk fermented with strain NIZO B40, which produces an anionic EPS composed of glucose, rhamnose, galactose and phosphate, is very low. This relatively low concentration could be increased by optimising the culture conditions and medium composition. Using pH-controlled fermentations and a chemically defined medium, the total EPS production was highest at pH 5.8 and 25 °C. Glucose was demonstrated to be the most efficient sugar source for EPS production by L. lactis NIZO B40. With fructose as the sugar source only a minor amount of EPS was produced. The intracellular levels of sugar nucleotides, the EPS precursors, were much lower in fructose- than in glucose-grown cultures. The activity of the enzymes involved in the biosynthesis of the sugar nucleotides were however unaffected by the source of sugar but the activity of fructose-1,6-bisphosphatase (FBPase) was very low. FBPase catalyses the conversion of fructose-1,6-diphosphate into fructose-6-phosphate, an essential step for the biosynthesis of sugar nucleotides from fructose but not from glucose. Overexpression of the fbp gene resulted in increased EPS synthesis on fructose.Most culture conditions influenced growth as well as EPS formation and EPS synthesis itself was also influenced by the growth rate. EPS production by strain NIZO B40 starts at the exponential growth phase but continues during the stationary phase in batch cultures, indicating that EPS biosynthesis and growth are not strictly coupled. Indeed we found that non-growing cultures were still able to produce EPS, making it possible to study the influence of different culture conditions on EPS biosynthesis independent of growth.The amounts of EPS produced by L. lactis NIZO B40 and NIZO B891 were comparable under glucose and leucine limitation. The efficiency of EPS production, the quantity of EPS produced per quantity of glucose consumed, was however much higher under conditions of glucose limitation. The production of phosphorylated B40 EPS and of unphosphorylated B891 EPS was strongly reduced under conditions of phosphate limitation. The sugar composition of both B40 and B891 EPS and the phosphate content of B40 EPS were unaffected by the type of limitation but surprisingly, glucose limitation resulted in the production of EPSs with strongly reduced molecular masses.Anionic B40 EPS in suspension and a cell-associated layer of this EPS protected the bacteria against toxic copper ions and nisin, probably due to charge interactions. Furthermore, cell-associated EPS resulted in a decrease in the sensitivity of the bacteria to bacteriophages and lysozyme, most likely by masking the targets for the phages and the enzyme. The protection of EPS against nisin and bacteriophages could be a competitive advantage in mixed strain dairy starter cultures. Unfortunately, the EPS yields were not increased in the presence of copper, bacteriophages, nisin or lysozyme.