Influence of carbohydrates on proteolytic activity of Lactococcus lactis strains

Lactococcus lactis is a Gram-positive lactic acid bacterium that is commonly used in traditional food industry such as cheese, fermented milk products and butter production. In addition, it is extensively used in modern biotechnological applications. Recently,many studies have been done on physiology and genetics of this bacterium, therefore a wide variety of genetic tools have been developed. Nowadays several genomes of L. lactis strains are completely sequenced. One of important properties of lactic acid bacterium is their proteolytic activity. The strains with a high level of proteolytic activity are used in cheese production, but their usage in fermented milk products production can lead to flaw initiation. Several carbohydrates, for example, prebiotic lactulose can influence starter microflora properties. In this research work the influence of lactulose, lactose and glucose on proteolytic activity of 2 strains L. lactis pNZ45subC and NZ48CE was studied. First, the gene expression was induced by the addition of nisin to the final concentration of 0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, 2 ng/ml, 5 ng/ml, 10 ng/ml and 20 ng/ml. The induced and noninduced cells were harvested after 20 h, and the proteolitic activity assay was performed. The highest proteolitic activity was observed after induction of 1 ng/ml and 5 ng/ml, in noninduced strains proteolytic activity was close to zero. To verify the optimal condition for the protein overexpression, a range of the nisin and the NZ9700 supernatant concentration was tested. All inducted cultures showed higher enzymatic activity than the non-inducted culture. The highest activity around 120 U/mL was obtained when 10 000-20 000 diluted supernatantfrom L. lactis NZ9700 was used. These results are comparable with the results received for 1-5 ng/mL nisin induction. In further experiments the supernatant of the NZ9700 strain was used. The highest proteolytic activity for both strains was observed during the growth on M17 medium supplemented with 10% milk and 0.5-1.0% of glucose. Growth of bacteria only in M17 did not lead to high enzymatic activity of the culture under induction, what might suggest that the secreted protease is not stable under these conditions. Lactose and lactulose presence probably block nisin action and the level of proteolytic activity was close to zero. It means that adding lactose and lactulose to fermented milk products will not lead to activation of L. lactis pNZ45subC and NZ48CE proteolytic activity and will not cause flaw initiation.

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