Bioaugmentation of Bacillus amyloliquefaciens-Bacillus kochii co-cultivation to improve sensory quality of flue-cured tobacco.

Flue-cured tobacco (FCT) with irritating and undesirable flavor must be aged. However, the spontaneous aging usually takes a very long time for the low efficiency. Bioaugmentation with functional strains is a promising method to reduce aging time and improve sensory quality. To eliminate the adverse effect of excessive starch or protein content on the FCT quality, we used the flow cytometry to sort Bacillus amyloliquefaciens LB with high alpha-amylase and Bacillus kochii SC with high neutral protease from the FCT microflora. The mono, co-culture of strains was performed the solid-state fermentation with FCT. Bacillus amyloliquefaciens monoculture for 2 days and Bacillus kochii monoculture for 2.5 days achieved the optimum quality. B. amyloliquefaciens-B. kochii co-culture at a ratio of 3:1 for 2 days of fermentation showed a more comprehensive quality enhancement and higher functional enzyme activity than mono-cultivation. Through OPLS-DA model (orthogonal partial least-squares-discriminant analyzes), there were 38 differential compounds between bioaugmentation samples. In co-cultivation, most of Maillard reaction products and terpenoid metabolites were at a higher level than other samples, which promoted an increase in aroma, softness and a decrease in irritation. This result validated the hypothesis of quality improvement via the co-culture. In our study, we presented a promising bioaugmentation technique for changing the sensory attributes of FCT in a short aging time.