Disabling lactose metabolism in proteinase-positive Lactococcus lactis to modulate proteolysis in cheese

Excessive bitterness is a well-recognized flavor defect in cheese which is caused by hydrophobic peptides that primarily accumulate due to the action of rennet and the cell-envelope bound protease (CEP) of the starter. Here, we present a new method to limit CEP-related proteolysis by preventing starter bacteria from metabolizing lactose. First, we generated a lactose-negative (lac − ) variant of a CEP positive (Prt + ) Lactococcus lactis LAC1-L. Surprisingly, the growth of LAC1-L could be partially restored when co-cultured with L. lactis strain EC02, which lacks a CEP but can metabolize lactose. EC02 shows strong autolysis, releasing intracellular peptidases under starvation conditions. During co-cultivation, LAC1-L accounted for only 8% of the population, regardless of the strain ratio at the time of inoculation. In contrast, when EC02 was cocultured with the lactose-fermenting, protease-positive strain RD01, RD01 grew better and ended up comprising more than 20% of the population. When cheeses were prepared with the LAC1-L/EC02 culture, LAC1-L remained at a low level throughout ripening, while free amino acids (FAAs) accumulated rapidly, peaking after 2 weeks. The increase in FAAs confirmed that EC02, through the release of peptidases, could efficiently degrade peptides released by the rennet and the CEP of LAC1-L, and more efficiently than in cheeses prepared with RD01/EC02, which is aligned with previous findings that the CEP hampers autolysis by degrading the major autolysin AcmA. Thus, by rationally designing the starter culture composition, this strategy can modulate proteolysis, accelerate cheese ripening, and offer a promising approach for more efficient and sustainable cheese production.