Nutty-flavour compound 3-methylbutanal production in milk by <i>Lactococcus cremoris</i> : biosynthesis pathway and role of pyruvate dehydrogenase

This research paper investigated the hypothesis that the high production of 3-methylbutanal by lactic acid bacteria is achieved not only through a direct metabolic pathway but also via an indirect pathway. 3-methylbutanal is a flavour compound that contributes to the sensory profile of various cheeses, often described as nutty, malty, or chocolate-like depending on its concentration and the cheese matrix. Although it is desired in certain cheese varieties, its acceptability varies among different cheese types. Among the various compounds responsible for this nutty scent, 3-methylbutanal plays a pivotal role. Certain strains of lactic acid bacteria are known to produce 3-methylbutanal from leucine, via both direct and indirect pathways. While it has commonly been assumed that the direct pathway is the primary mechanism, this study reports the discovery of <i>Lactococcus cremoris</i> AJ01, which produces 3-methylbutanal predominantly through an indirect pathway. We first investigated the effects of milk fermentation conditions on 3-methylbutanal and acid production by strain AJ01. The effect of leucine supplementation on 3-methylbutanal production was then examined. Finally, we explored the metabolic pathway responsible for 3-methylbutanal synthesis in this strain, determined the nucleotide sequence of the pyruvate dehydrogenase gene involved in the indirect pathway, and evaluated its enzyme activity to assess its contribution to production. When cultured in milk at 20 °C, strain AJ01 produced titratable acidity at >0.6% and 3-methylbutanal at >6 mg/kg within 18 h. Adding leucine to the milk enhanced 3-methylbutanal production, while excessive leucine supplementation inhibited it. Strain AJ01 exhibited activity of pyruvate dehydrogenase, while no activity of α-keto acid decarboxylase, the direct-pathway enzyme, was detected, suggesting that it produces 3-methylbutanal via the indirect pathway. These findings may contribute to the effective management and enhancement of flavour profiles in fermented dairy products, particularly cheeses.