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

doi:10.15933/j.cnki.1004-3268.2026.07.017

Abstract

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.

摘要： 为筛选一株可用于低次烟叶的增香菌，首先分别从烟叶、大马士革玫瑰、乌龙茶叶、桂花中分离、初筛菌株，然后通过感官得分进行复筛。采用单因素优化发酵条件后，进一步采用顶空-固相微萃取-气相色谱-质谱联用技术（HS-SPME-GC-MS）与多元统计学方法，分析所筛菌株应用于低次烟叶液态发酵时挥发性代谢物的变化及差异显著代谢途径。结果表明，从烟叶、玫瑰、茶叶、桂花中共分离到119株菌株，有9株产生较为明显的香气。其中，MG-1（从云南省大理市大马士革玫瑰样品中分离得到的第1株菌）的卷烟感官评吸总分最高（54.00分），较不接菌的对照组（49.86分）提升8.30%，该菌株经分子鉴定为阿氏肠杆菌（Enterobacter asburiae）。该菌株的最优液态发酵条件为接种量3%、发酵时间12 h、装液量100 mL、摇床转速180 r/min。利用HS-SPME-GC-MS共鉴定出1 583种挥发性代谢物，多元统计分析显示，其中197种为差异代谢物（上调132种，下调65种），上调比例为67.0%。进一步筛选变量投影重要性（VIP）值前40且倍数变化（FC）≥2或FC≤0.5的差异代谢物，其中发酵后6-甲基-5-庚烯-2-醇（柑橘清香）、月桂烯（柑橘清香）、琥珀酸二甲酯（果香）、苯乙醛（杏仁香）、苯乙醇（玫瑰香）、藏红花醛（辛香、花香）等香气成分含量均显著上调，而异硫氰酸烯丙酯等刺激性化合物含量显著下调。进一步的KEGG通路分析显示，差异代谢物主要富集到苯丙氨酸代谢、植物次生代谢产物生物合成、多样化环境中微生物代谢、芳香族化合物降解、代谢途径以及次生代谢物生物合成等6个通路。其中，植物次生代谢产物生物合成和次生代谢物生物合成通路显著上调，而代谢途径、芳香族化合物降解及多样化环境中微生物代谢相关通路显著下调。综上，阿氏肠杆菌MG-1液态发酵能有效提升低次烟叶浸提液香气品质，具有明确的增香应用潜力，为低次烟叶的提质增效提供了理论依据。

关键词: 低次烟叶, 阿氏肠杆菌, 液态发酵, 非靶向代谢组学, 烟叶增香

CLC Number:

S572
TS426

Fu Jincun, Wei Zimeng, Zong Dongyue, Wang Fushen, Qu Lili, Li Jiaxin, Fang Yaning, Liu Yuanshang, Xu Chunping. Screening of Aroma⁃Enhancing Strains for Low⁃Grade Tobacco Leaves，Optimization of Fermentation Process and Characterization of Volatile Metabolites[J]. Journal of Henan Agricultural Sciences, 2026, 55(7): 167-180.

付金存, 魏子梦, 宗东岳, 王富申, 曲利利, 李嘉欣, 方亚宁, 刘远上, 许春平. 低次烟叶增香菌株的筛选、发酵工艺优化及挥发性代谢物分析[J]. 河南农业科学, 2026, 55(7): 167-180.

Figures/Tables 15

References

［1］刘远上，吴攀，赵怡凡，等. 菌酶协同改善低次烟叶品质的工艺优化及对香气成分的影响［J］.河南农业科学，2025，54（10）：159⁃170.
Liu Y S，Wu P，Zhao Y F，et al. Process optimization of microbe⁃enzyme synergy fermentation of low⁃grade tobacco leaves and impact on aroma components［J］.Journal of Henan Agricultural Sciences，2025，54（10）：159⁃170.
［2］罗海涛，王硕赢，齐雅婷，等. 不同酶处理对低次烟叶提取物提质效果的影响［J］.河南农业科学，2025，54（3）：170⁃180.
Luo H T，Wang S Y，Qi Y T，et al. Effect of different enzymes on the quality improvement effect of discarded tobacco extract［J］. Journal of Henan Agricultural Sciences，2025，54（3）：170⁃180.
［3］Guo S D，Li Y S，Zhu B B，et al. Introducing CCD1 into isolated Rhodotorula strain enhances flavor production and improves cigar fermentation［J］.Frontiers in Bioengineering and Biotechnology，2024，12：1510075.
［4］Isleten Hosoglu M. Aroma characterization of five microalgae species using solid⁃phase microextraction and gas chromatography⁃mass spectrometry/olfactometry［J］.Food Chemistry，2018，240：1210⁃1218.
［5］Zhang J，He Y G，Yin L G，et al. Isolation of aroma⁃producing Wickerhamomyces anomalus yeast and analysis of its typical flavoring metabolites［J］.Foods，2023，12（15）：2934.
［6］赵佳，邓明蕊，范文荣，等. 黄花菜梨汁发酵过程中理化性质与风味物质变化分析［J］. 中国酿造，2024，43（11）：99⁃104.
Zhao J，Deng M R，Fan W R，et al.Analysis on the changes of physicochemical properties and flavor substances during the fermentation of Hemerocallis citrina and pear juice［J］. China Brewing，2024，43（11）：99⁃104.
［7］Pei Q，Jiang X，Li Z Q，et al. Study on quality enhancement during cigar tobacco fermentation by Staphylococcus nepalensis： insights into microbial community，volatile substances and sensory evaluation［J］.Frontiers in Microbiology，2025，16：1526178.
［8］Navarro L，Gil Cortiella M，Gutiérrez⁃Moraga A，et al.Antarctic soil yeasts with fermentative capacity and potential for the wine industry［J］. Foods，2023，12（24）：4496.
［9］王慧慧，王超凡，林晓阳，等. 山药酒精饮料工艺优化及其挥发性成分分析［J］. 粮食与油脂，2024，37（5）：120⁃124.
Wang H H，Wang C F，Lin X Y，et al.Process optimization of yam alcoholic beverage and its volatile components analysis［J］. Cereals & Oils，2024，37（5）：120⁃124.
［10］李晋华，晏慧君，杨锦红，等. 香水月季复合群（Rosa odorata complex）花香成分分析［J］. 西南农业学报，2018，31（3）：587⁃591.
Li J H，Yan H J，Yang J H，et al. Analysis of volatile components from Rosa odorata complex by SPME⁃GC/MS［J］.Southwest China Journal of Agricultural Sciences，2018，31（3）：587⁃591.

［11］雷高明. 几种鲜花芳香水化学成分分析及吸附分离研究［D］. 杭州：浙江大学，2016.

Lei G M. Analysis and adsorptive isolation of chemical components from hydrolates of fresh flowersfrom several species［D］. Hangzhou：Zhejiang University，2016.

［12］Ning Y，Zhang L Y，Mai J，et al. Tobacco microbial screening and application in improving the quality of tobacco in different physical states［J］.Bioresources and Bioprocessing，2023，10（1）：32.

［13］Zheng T F，Zhang Q Y，Wu Q Y，et al. Effects of inoculation with Acinetobacter on fermentation of cigar tobacco leaves［J］. Frontiers in Microbiology，2022，13：911791.

［14］叶建斌，齐晓娜，杨宗灿，等. 肠杆菌发酵对烟草浸膏化学成分及卷烟品质的影响［J］. 食品与机械，2021，37（5）：33⁃38.

Ye J B，Qi X N，Yang Z C，et al. Effects of Enterobacter fermentation on chemical components of tobacco extract and cigarette quality［J］. Food & Machinery，2021，37（5）：33⁃38.

［15］Yang Y，Peng Q R，Ou M Y，et al. Research progress in tobacco fermentation［J］. Journal of Biosciences and Medicines，2018，6（6）：105⁃114.

［16］YC/T 497—2014 卷烟中式卷烟风格感官评价方法［S］.

YC/T 497—2014 Cigarette：sensory evaluation method for Chinese⁃style cigarette style［S］.

［17］张诺妮，谢云波，李斌，等. 烟草马铃薯Y病毒拮抗细菌ZF-10的筛选鉴定及发酵条件优化［J］. 烟草科技，2024，57（5）：64⁃74.

Zhang N N，Xie Y B，Li B，et al. Screening，identification and fermentation condition optimization of an antagonistic biocontrol bacterium ZF⁃10 for tobacco potato virus Y［J］. Tobacco Science &Technology，2024，57（5）：64⁃74.

［18］ YC/T 159—2019 烟草及烟草制品水溶性糖的测定连续流动法［S］.

YC/T 159—2019 Tobacco and tobacco products：determination of water⁃soluble sugars：continuous flow method［S］.

［19］YC/T 468—2021 烟草及烟草制品总植物碱的测定连续流动（硫氰酸钾）法［S］.

YC/T 468—2021 Tobacco and tobacco products：determination of total alkaloids： continuous flow（potassium thiocyanate）method［S］.

［20］YC/T 162—2011 烟草及烟草制品氯的测定连续流动法［S］.

YC/T 162—2011 Tobacco and tobacco products：determination of chlorine：continuous flow method［S］.

［21］YC/T 217—2007 烟草及烟草制品钾的测定连续流动法［S］.

YC/T 217—2007 Tobacco and tobacco products：determination of potassium：continuous flow method ［S］.

［22］YC/T 161—2002 烟草及烟草制品总氮的测定连续流动法［S］.

YC/T 161—2002 Tobacco and tobacco products：determination of total nitrogen：continuous flow method［S］.

［23］YC/T 448—2012 烟草及烟草制品游离氨基酸的测定离子色谱-积分脉冲安培法［S］.

YC/T 448—2012 Tobacco and tobacco products：determination of free amino acids：ion chromatography with integrated pulsed amperometric detection［S］.

［24］吴晓茜，钟秋平，张云竹. 山兰米醋发酵过程中理化指标及挥发性风味物质的变化规律［J］. 食品与发酵工业，2024，50（5）：108⁃115.
Wu X Q，Zhong Q P，Zhang Y Z. Changes of physicochemical parameters and volatile flavor substances in the fermentation process of Shanlan rice vinegar［J］. Food and Fermentation Industries，2024，50 （5）：108⁃115.

［25］吴婷，邓秀娟，李沅达，等. 云茶香1号不同萎凋工艺白茶的化学品质研究［J］. 食品安全质量检测学报，2021，12（24）：9530⁃9538.
Wu T，Deng X J，Li Y D，et al. Study on the chemical quality of white tea with different withering processes of Camellia sinensis vs Yunchaxiang 1［J］. Journal of Food Safety and Quality，2021，12（24）：9530⁃9538.

［26］蒙小玉，冯建文，黄欣艳，等.“糖心”苹果香气物质生物合成的转录代谢联合分析［J］. 核农学报，2025，39（4）：696⁃706.
Meng X Y，Feng J W，Huang X Y，et al. Combined transcriptional and metabolic analysis of aroma substances in watercore apples［J］. Journal of Nuclear Agricultural Sciences，2025，39（4）：696⁃706.

［27］周奎，王润东，宫梦学，等. 冷鲜波动温度对生食三文鱼肌肉代谢物的影响［J］. 食品科学，2024，45（22）：229⁃238.
Zhou K，Wang R D，Gong M X，et al. Effect of temperature fluctuations during chilled storage on the metabolite composition of salmon consumed raw［J］.Food Science，2024，45（22）：229⁃238.

［28］郑杰，王平平，郭春生，等. 初烤阶段酶制剂不同喷施方式下烟叶中挥发性代谢物的差异分析［J］. 山东农业科学，2024，56（4）：56⁃65.
Zheng J，Wang P P，Guo C S，et al. Differential analysis of volatile metabolites in flue⁃cured tobacco leaves under different spraying methods of exogenous enzyme at initial curing stage［J］. Shandong Agricultural Sciences，2024，56（4）：56⁃65.

［29］Liu J B，Jiang J，Bai Y J，et al. Mimicking a new 2⁃phenylethanol production pathway from Proteus mirabilis JN458 in Escherichia coli［J］. Journal of Agricultural and Food Chemistry，2018，66（13）：3498⁃3504.

［30］Bukhari S I，Manzoor M，Dhar M K. A comprehensive review of the pharmacological potential of Crocus sativus and its bioactive apocarotenoids［J］.Biomedicine & Pharmacotherapy，2018，98：733⁃745.

［31］Wang X，Wang J J，Zhang X Y，et al. Efficient myrcene production using linalool dehydratase isomerase and rational biochemical process in Escherichia coli［J］.Journal of Biotechnology，2023，371：33⁃40.

［32］罗佳兴，金桂勇，艾丹，等. 水热反应釜法提取蓝莓风味物质工艺优化及其成分分析［J］. 粮食与油脂，2025，38（3）：117⁃121.
Luo J X，Jin G Y，Ai D，et al. Optimization of extraction process of blueberry flavor substances by hydrothermal reactor method and its component analysis［J］. Cereals & Oils，2025，38（3）：117⁃121.

［33］张斌，宋笑龙，张警予，等. 信阳市家庭农场烤烟适宜种植规模研究［J］. 种业导刊，2025（6）：60⁃63.
Zhang B，Song X L，Zhang J Y，et al. Study on the suitable planting scale of flue⁃cured tobacco for family farms in Xinyang City［J］. Journal of Seed Industry Guide，2025（6）：60⁃63.