Screening of Aroma-Enhancing Strains for Low-Grade Tobacco Leaves,Optimization of Fermentation Process and Characterization of Volatile Metabolites

Fu Jincun; Wei Zimeng; Zong Dongyue; Wang Fushen; Qu Lili; Li Jiaxin; Fang Yaning; Liu Yuanshang; Xu Chunping

doi:10.15933/j.cnki.1004-3268.2026.07.017

Abstract：To screen a strain of aroma-enhancing bacteria that can be used for low-grade tobacco leaves,strains were isolated from tobacco leaves,Damask roses,Oolong tea leaves,and Osmanthus fragrans,and re-screened by sensory scores.After single factor optimization of fermentation conditions,headspace solid-phase microextraction-gas chromatography-mass spectrometry(HS-SPME-GC-MS)and multivariate statistical methods were further used to analyze the changes of volatile metabolites and significant differential metabolic pathways when the strain was applied to the liquid fermentation of low-grade tobacco leaves.The results showed that 119 strains were isolated from tobacco leaves,Damask roses,tea leaves,and Osmanthus fragrans,of which 9 strains produced obvious aroma.Among them,MG-1(the first strain isolated from the Damask rose sample collected in Dali City,Yunnan Province)achieved the highest total sensory score for cigarette sensory evaluation(54.00 points),which was 8.30%higher than that of the control group without bacterial inoculation(49.86 points).It was identified as Enterobacter asburiae by molecular analysis.The optimal liquid fermentation conditions of the strain were determined as follows:inoculation amount 3%,fermentation time 12 h,liquid volume 100 mL,shaking speed 180 r/min.A total of 1 583 volatile metabolites were identified by HS-SPME-GC-MS.Multivariate statistical analysis showed that 197 of them were differential metabolites(132 up-regulated and 65 down-regulated),with an up-regulation ratio of 67.0%.The differential metabolites with variable importance in projection(VIP)values in the top 40 and fold change(FC)≥2 or FC≤0.5 were further screened.After fermentation,the contents of aroma components such as 6-methyl-5-hepten-2-ol(citrus aroma),myrcene(citrus aroma),dimethyl succinate(fruit aroma),phenylacetaldehyde(almond aroma),phenylethyl alcohol(rose aroma)and safranal(spicy and floral aroma)were significantly up-regulated,while the contents of irritant substances such as allyl isothiocyanate were significantly down-regulated.Further KEGG pathway analysis showed that the differential metabolites were mainly enriched in six pathways:phenylalanine metabolism,biosynthesis of various plant secondary metabolites,microbial metabolism in diverse environments,degradation of aromatic compounds,metabolic pathway and biosynthesis of secondary metabolites.Among them,biosynthesis of various plant secondary metabolites and biosynthesis of secondary metabolites were significantly up-regulated,while metabolic pathway,degradation of aromatic compounds and microbial metabolism in diverse environments were significantly down-regulated.In summary,the liquid fermentation of Enterobacter asburiae MG-1 can effectively improve the aroma quality of the extract of low-grade tobacco leaves,and has a clear potential for aroma enhancement,which provides a theoretical basis for the improvement of the quality and efficiency of low-grade tobacco leaves.