Microbial functional differentiation and flavor formation across layer exchange in cereal vinegar fermentation: Multi-omics insights and targeted bioaugmentation.

Spatial heterogeneity is common in cereal vinegar fermentation, but microbial and metabolic changes across the layer-exchange process remain unclear. We combined metagenomics and metatranscriptomics with physicochemical, organic-acid, and volatile profiling to investigate layer-associated changes during Tianjin Duliu mature vinegar (TDMV) fermentation and guide bioaugmentation. Before exchange, the frequently turned upper layer consumed ethanol at 1.14 times the lower-layer rate, rapidly accumulated acetic acid, and was enriched in Acetobacter and Komagataeibacter. The lower layer retained more ethanol and lactic acid, was enriched in lactic acid bacteria and yeasts, and contained more ethyl esters. After upward transfer, the original lower-layer Cupei rapidly accumulated acetic acid as several ethyl esters decreased; after downward transfer, the original upper-layer Cupei acidified more slowly while phenethyl alcohol, phenethyl acetate, ethyl lactate, and 2,3-butanediol increased. Genus-level transcript profiles showed that Acetobacter-affiliated transcripts related to acetaldehyde oxidation and the acetolactate/acetoin branch were more pronounced in upper-positioned Cupei, whereas LAB-affiliated lactate-forming transcripts were prominent in the lower layer. At the 100-kg scale, a targeted two-strain treatment using Lactobacillus helveticus and Acetobacter pasteurianus increased substrate conversion efficiency from 47.35 ± 1.36% to 51.19 ± 0.32% and final total acid by approximately 9.7% in both portions. In a → A, day-17 acetic acid was 43.8% higher than in the control, but the difference was not significant at the endpoint. The bioaugmentation samples showed changes in aroma-related volatile composition. These findings support complementary microbial functional differentiation across Cupei positions and inform position-targeted regulation of TDMV fermentation.