Optimization of fermentation conditions for 3‑methylthiopropanol production by Saccharomycopsis fibuligera in a tobacco-based medium and its impact on tobacco quality.

In this study, we investigated the potential of the Baijiu-derived yeast Saccharomycopsis fibuligera (S. fibuligera) Y1402 to synthesize the key flavor compound 3-methylthiopropanol (3-Met) using a tobacco-based substrate. The culture medium composition and fermentation conditions were systematically optimized using single-factor experiments, Plackett-Burman design, and response surface methodology. The results showed that under the following conditions: glucose concentration of 60 g/L, inoculum size of 0.09%, tobacco leaf size of 40-60 mesh, tobacco addition of 3%, shaking speed of 225 rpm, initial pH of 5.0, yeast extract concentration of 0.4 g/L, liquid volume of 26.52 mL/250 mL, L-methionine concentration of 6.95 g/L, and fermentation time of 47.7 h, the yield of 3-Met reached 3.99 g/L, which was in excellent agreement with the model-predicted value (4.00 g/L). Sensory evaluation indicated that when the L-methionine addition was 2.0 g/L, the aroma quality, smoothness, delicacy, and sweetness of the fermented tobacco shreds were significantly improved, whereas the off-flavor intensity was markedly reduced, resulting in substantially enhanced sensory quality of the tobacco shreds. This study is the first to report efficient 3-Met synthesis by S. fibuligera in a tobacco matrix, providing a novel strategy for the microbe-directed modulation of tobacco flavor.