富集功能菌株的烟叶培养基筛选及其对烤烟提质研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-1252

摘要/Abstract

摘要：

目的探究采用烟叶源液体培养基进行靶向富集，对功能菌株发酵液代谢特性及烤烟提质效果的影响。方法从醇化烟叶中筛选得到两株功能菌株，经生理生化与16S rDNA鉴定为砖红微杆菌M2和枯草芽胞杆菌M24。分别在LB液体培养基和烟叶液体培养基中对菌株进行富集，利用非靶向代谢组学分析发酵液代谢物差异，并系统评估了经发酵液喷施处理后烟叶的化学成分、大分子物质含量及感官品质变化。结果菌株M2与M24均能产淀粉酶、蛋白酶和纤维素酶，且M2能产半纤维素酶。非靶向代谢组学分析表明，不同培养基富集后的发酵液代谢物组成差异显著，烟叶培养基组中绿原酸、右旋奎宁酸等多酚类物质含量显著高于LB培养基组。喷施处理后，烟叶培养基富集所得的发酵液（T2、T4）对烟叶提质效果更优，与LB培养基组相比，其处理的烟叶纤维素含量降幅达5.32%-14.63%，总糖与还原糖含量显著提升，糖碱比提高，总氮与氮碱比降低，感官评吸中杂气与刺激性明显减轻，香气质、香气量与甜度提升显著。结论烟叶培养基通过优化功能菌株的代谢特性，促使发酵液富集更多有益于烟叶品质的多酚类物质，从而更有效实现烤烟的增香降杂。本研究为微生物发酵技术在烟草加工中定向应用提供了新的理论依据。

关键词: 功能微生物, 烟叶培养基, 酶活性, 化学品质, 感官品质

Abstract:

Objective To investigate the effects of targeted enrichment using a tobacco leaf-derived liquid medium on the metabolic characteristics of functional strain fermentation broths and their impact on improving the quality of flue-cured tobacco. Method Two functional strains were isolated from aged tobacco leaves and identified as Microbacterium lateritum M2 and Bacillus subtilis M24 based on physiological, biochemical, and 16S rDNA sequence analyses. The strains were enriched in LB liquid medium and tobacco leaf liquid medium, respectively. Non-targeted metabolomics was employed to analyze differences in metabolite composition in the fermentation broths. Systematic evaluations were conducted on changes in chemical components, macromolecular substance content, and sensory quality of tobacco leaves after spraying treatment with the fermentation broths. Result Both strains M2 and M24 were capable of producing amylase, protease, and cellulase, with strain M2 additionally producing hemicellulase. Non-targeted metabolomics analysis revealed significant differences in the metabolite profiles of fermentation broths enriched in different media. The content of polyphenols, such as chlorogenic acid and quinic acid, was significantly higher in the tobacco leaf medium group compared to the LB medium group. After spraying treatment, the fermentation broths obtained from tobacco leaf medium enrichment (T2, T4) demonstrated superior tobacco quality improvement effects. Compared to the LB medium group, tobacco leaves treated with these broths showed a reduction in cellulose content by 5.32%–14.63%, a significant increase in total sugar and reducing sugar content, an improved sugar-to-alkaloid ratio, a decrease in total nitrogen and nitrogen-to-alkaloid ratio, and notable enhancements in sensory quality, including reduced off-flavors and irritancy, as well as improved aroma quality, aroma quantity, and sweetness. Conclusion Tobacco leaf medium optimizes the metabolic characteristics of functional strains, promoting the enrichment of more polyphenols beneficial for tobacco quality in the fermentation broth, thereby more effectively enhancing aroma and reducing harshness in flue-cured tobacco. This study provides a new theoretical basis for the targeted application of microbial fermentation technology in tobacco processing.

Key words: functional microorganisms, tobacco medium, enzyme activity, chemical quality, sensory quality

胡逸超, 秦天, 孙建生, 苏赞, 周奕, 刘金仓, 刘政钦, 张同琢, 关铭鑫, 马婷婷, 黎娟. 富集功能菌株的烟叶培养基筛选及其对烤烟提质研究[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1252.

HU Yi-chao, QIN Tian, SUN Jian-sheng, SU Zan, ZHOU Yi, LIU Jin-cang, LIU Zheng-qin, ZHANG Tong-zhuo, GUAN Ming-xin, MA Ting-ting, LI Juan. Screening of Tobacco Leaf Medium for Enriching Functional Microorganisms and Its Effect on Improving Flue-cured Tobacco Quality[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1252.

图/表 10

图1 M2及M24菌株菌落形态及电镜扫描图

Fig. 1 Morphology and electron microscope scans of M2 and M24 strains

图2 M2及M24菌株分子生物学鉴定系统发育树A：M2菌株；B：M24菌株；括号中数值为GeneBank登录号，分支处数字为自展值，标尺为核苷酸替换率

Fig. 2 Phylogenetic tree depicting the molecular identification of strains M2 and M24A:M2 strain;B:M24 strain. The values in parentheses are GenBank accession numbers. Numbers at the nodes are bootstrap values, and the scale bar indicates the nucleotide substitution rate

图3 M2及M24菌株产酶能力A：淀粉酶；B：蛋白酶；C：纤维素酶；D：半纤维素酶

Fig. 3 Enzyme production capacity of strain M2 and M24A: Amylase; B: Protease; C: Cellulase; D: Hemicellulase

表1 M2及M24菌株生理生化功能鉴定

Table 1 Physiological and biochemical characterization of M2 and M24 strains

编号

葡萄糖发酵试验

Glucose fermentation test

甲基红试验

Methyl red

伏普试验

Voges-Proskauer

吲哚

试验Indole

硫化氢试验Hydrogen sulfide

明胶液化试验

Gelatin liquefaction

柠檬酸盐利用试验

Citrate utilization

M24

图4 菌株生长曲线A：菌株M2生长曲线；B：菌株M24生长曲线

Fig. 4 Growth curves of the strainsA: Growth curve of strain M2. B: Growth curve of strain M24

图5 菌株最适生长条件A-D：分别为M2菌株的最适碳源种类、最适碳源浓度、最适氮源种类、最适氮源浓度。E-F：分别为M24菌株的最适碳源种类、最适碳源浓度、最适氮源种类、最适氮源浓度

Fig. 5 Optimal growth conditions for strains M2 and M24A-D: The optimal carbon source type, optimal carbon source concentration, optimal nitrogen source type, and optimal nitrogen source concentration for strain M2, respectively. E-H: Represent the optimal carbon source type, optimal carbon source concentration, optimal nitrogen source type, and optimal nitrogen source concentration for strain M24, respectively

图6 不同培养基富集菌株发酵液代谢物组成A：不同培养基富集菌株发酵液代谢物聚类热图；B：M24菌株发酵液主要多酚类化合物含量；C：M2菌株发酵液主要多酚类化合物含量，占比经log2处理。Tobacco：烟叶培养基；LB：LB培养基

Fig. 6 Metabolite composition of fermentation broth from enriched strains on different mediaA: Cluster heatmap of metabolites in fermentation broth of enriched strains on different media. B: Contents of major polyphenolic compounds in fermentation broth of M24 strain. C: Contents of major polyphenolic compounds in fermentation broth of M2 strain, with proportions log2-transformed. Tobacco: Tobacco leaf culture medium; LB: LB culture medium

表2 菌株发酵液处理烟叶后烟叶化学成分含量

Table 2 Chemical composition of tobacco leaves after bacterial treatment with fermentation broth

处理Treatment	总氮 Nitrogen （%）	全钾 Potassium （%）	氯离子 Chloride ion （%）	总糖 Total sugar （%）	还原糖 Reduced sugar （%）	烟碱Nicotine （%）	糖碱比 Sugar-nicotine ratio	氮碱比Nitrogen-nicotine ratio	钾氯比Potassium-chloride ratio
CK	1.96±0.03a	2.94±0.07a	0.42±0.07a	32.60±1.59ab	22.37±0.79c	2.74±0.02a	11.88±0.55b	0.71±0.01bc	6.99±0.42a
T1	1.97±0.11a	2.96±0.12a	0.42±0.12a	30.61±0.97b	23.65±1.25ab	2.62±0.07a	11.69±0.23b	0.75±0.01a	7.32±1.73a
T2	1.88±0.08cd	2.91±0.15a	0.39±0.15a	34.13±0.47a	24.66±1.04a	2.62±0.16a	13.04±0.43a	0.72±0.02bc	7.50±1.07a
T3	1.92±0.05bc	3.03±0.26a	0.35±0.26a	31.75±1.00ab	23.23±0.67ab	2.62±0.04a	12.11±0.32b	0.73±0.03ab	8.86±2.00a
T4	1.87±0.08d	3.03±0.09a	0.43±0.09a	32.44±2.04ab	25.19±0.86a	2.69±0.12a	12.04±0.57b	0.69±0.02c	7.13±1.47a

表3 菌株发酵液处理烟叶后烟叶大分子物质含量

Table 3 Contents of macromolecular substances in tobacco leaves after bacterial treatment with fermentation broth

处理Treatment	蛋白质Protein （%）	淀粉Starch （%）	纤维素Cellulose （%）
CK	12.22±0.20a	6.47±0.21a	13.30±0.98a
T1	11.97±0.30ab	6.20±0.54ab	13.17±1.70a
T2	11.77±0.19b	5.75±0.09b	12.47±1.27bc
T3	11.86±0.24ab	6.45±0.41a	13.47±1.86a
T4	11.73±0.10b	5.99±0.18ab	11.50±0.75c

表4 发酵液喷施处理后烟叶感官品质得分

Table 4 Sensory quality scores of tobacco leaves after treatment with fermentation broth spray

处理 Treatment	评吸单项得分 Score of single evaluation （0-9.0）								综合得分Total score
处理 Treatment	香气质Aroma quality	香气量Aroma quantity	刺激性Irritation	杂气Offensive flavor	余味 Aftertaste	甜度Sweetness	燃烧性Burning property	灰色 Ash color	综合得分Total score
CK	7	6.3	6.5	6.5	6	6.3	3	4	45.6
T1	7.3	6.5	6.5	6.3	6	6.3	3	4	45.9
T2	7.5	6.8	6.8	7	6.5	7	3	4	48.6
T3	7	6.5	6.3	6.3	6.4	6.5	3	4	46.0
T4	7.5	6.7	7	6.8	6.5	6.8	3	4	48.3

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