Formate producing capacity provided by reducing ability of Streptococcus thermophilus NADH oxidase determines yogurt acidification rate

Yogurt is made by fermenting milk with 2 lactic acid bacteria, Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus (S. thermophilus). To comprehensively understand the protocooperation mechanism between S. thermophilus and L. bulgaricus in yogurt fermentation, we examined 24 combinations of cocultures comprising 7 fast- or slow- acidifying S. thermophilus strains with 6 fast- or slow- acidifying L. bulgaricus strains. Furthermore, 3 NADH oxidase deficient mutants (Δnox) and one pyruvate formate-lyase deficient mutant (ΔpflB) of S. thermophilus were used to evaluate the factor that determines the acidification rate of S. thermophilus. The results revealed that the acidification rate of S. thermophilus monoculture determined the yogurt fermentation rates, despite the co-existence of L. bulgaricus, whose acidification rate was either fast or slow. Significant correlation was found between the acidification rate of S. thermophilus monoculture and the amount of formate production. Result using ΔpflB showed that the formate was indispensable for the acidification of S. thermophilus. Moreover, results of the Δnox experiments revealed that formate production required Nox activity, which not only regulated dissolved oxygen (DO), but also the redox potential. NADH oxidase provided the large decrease in redox potential required by pyruvate formate lyase to produce formate. A highly significant correlation was found between formate accumulation and NADH oxidase activity in S. thermophilus. In conclusion, the formate production ability provided by the action of NADH oxidase activity determines the acidification rate of S. thermophilus, and consequently regulates yogurt coculture fermentation.