Temporal microbial succession informs the reformulation of a defined four-member starter for fermented milk.

Temporal microbial succession influences fermented-milk quality, but its use in multi-strain starter formulation remains limited. A defined consortium comprising Streptococcus thermophilus TCX4, Lactococcus lactis HYE8, Lactobacillus kefiri BCK1, and Lacticaseibacillus rhamnosus ENY6 was monitored during 0-14 h of fermentation using PacBio full-length 16S rRNA gene sequencing. Based on the combined community-composition and product-quality profiles, the 8 h and 10 h profiles were converted into candidate inoculum formulations and compared with the initial formulation and a commercial reference starter. The 10 h-derived formulation achieved the shortest coagulation time of 5.0 h and showed the highest water-holding capacity and sensory score, together with favorable texture and rheological properties. These results indicate that temporal community-composition profiles can provide biologically informed starting points for screening and validating defined multi-strain fermented-milk starters.