夏冬两季发酵雪茄烟叶的代谢组差异分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-1101

摘要/Abstract

摘要：

【目的】 解析夏冬季节发酵的雪茄烟叶代谢物差异。【方法】 采用超高效液相色谱串联质谱（UPLC-MS/MS）技术对发酵前、冬季发酵、夏季发酵的雪茄烟叶样品进行广泛靶向代谢物检测。【结果】 发酵期间（历时34 d）夏季、冬季发酵房平均温度分别是27.2℃、14.7℃，二者相差12.5℃，平均湿度分别是77.6%、66.7%，二者相差10.9%，夏季更符合烟叶发酵相对高温、高湿的环境要求；雪茄烟叶样品检测得到905种代谢物包含10类：生物碱、酚酸、黄酮、脂质、氨基酸、有机酸、萜类、糖类、核苷酸、木质素，其中生物碱、有机酸、脂质含量比例居前三，分别是24.5%、23.7%、15.0%。烟叶发酵后，有机酸、脂质、氨基酸、酚酸的含量都显著提升，生物碱、萜类、黄酮、糖类、木质素的含量有不同程度的减少。夏季发酵的烟叶生物碱、萜类代谢转化更剧烈，有机酸、氨基酸、黄酮、脂质、糖类含量较高，化学成分更协调，香气质量更丰富，烟气柔顺、余味甜润，感官品质更优。【结论】 鄂西南地区夏季自然高温高湿环境，更顺应雪茄烟农业发酵理想状态，将当年生产的雪茄烟叶在冬季自然醇化后推迟到次年夏季发酵，或可作为探索推动雪茄烟发酵工序提质增效的参考方案。

关键词: 雪茄烟叶, 发酵, 代谢组学, 感官质量, 差异分析

Abstract:

【Objective】 The differences of metabolites in cigar tobacco fermented in summer and winter were analyzed. 【Method】 Widely targeted metabolomics detection（UPLC-ESI-MS/MS）was used in combination with multivariate statistical analysis（PCA and OPLS-DA）. 【Result】 During the fermentation period（lasting for 34 d）, the average temperature of the fermentation room in summer and winter were 27.2℃ and 14.7℃, respectively, with a difference of 12.5℃, and the average humidity was 77.6% and 66.7%, respectively, with a difference of 10.9%, thus summer was more in line with the environmental requirements of relatively high temperature and high humidity for tobacco fermentation. A total of 905 metabolites were detected in cigar leaf samples, including alkaloids, phenolic acids, flavonoids, lipids, amino acids, organic acids, terpenes, sugars, nucleotides, and lignin. The top three metabolites were alkaloids, organic acids, and lipids, accounting for 24.5%, 23.7%, and 15.0%, respectively. After the fermentation, the contents of organic acids, lipids, amino acids and phenolic acids significantly increased, and the contents of alkaloids, terpenes, flavonoids, sugars and lignin decreased to varying degrees. In summer fermented tobacco leaves, the metabolic transformation of alkaloids and terpenoids was more intense, the contents of organic acids, amino acids, flavonoids, lipids and sugars were higher, the chemical composition was more coordinated, the aroma quality was richer, the flue gas was soft, the aftertaste was sweet, and the sensory quality was better.【Conclusion】 The natural high temperature and high humidity environment in summer in southwest Hubei is more suitable for the ideal state of cigar tobacco agricultural fermentation. The cigar leaves produced in the current year should be naturally aged in winter and then fermented in the next summer, which may be used as a reference scheme to explore and promote the quality and efficiency of the fermentation process of cigar.

Key words: cigar tobacco, fermentation, metabolism, sensory quality, difference analysis

虞昕磊, 何结望, 林国平, 李金海, 王大爱, 袁跃斌, 刘圣高, 李志豪, 陶德欣. 夏冬两季发酵雪茄烟叶的代谢组差异分析[J]. 生物技术通报, 2024, 40(6): 260-270.

YU Xin-lei, HE Jie-wang, LIN Guo-ping, LI Jin-hai, WANG Da-ai, YUAN Yue-bin, LIU Sheng-gao, LI Zhi-hao, TAO De-xin. Metabolome Difference Analysis of Fermented Cigar Tobacco Leaves in Summer and Winter[J]. Biotechnology Bulletin, 2024, 40(6): 260-270.

图/表 7

图1 夏冬两季雪茄烟发酵环境条件对比

Fig. 1 Comparison of tobacco fermentation environment in summer and winter

图2 夏冬两季雪茄烟发酵垛芯温度对比

Fig. 2 Comparison of tobacco stacks core temperature in summer and winter fermentation

图3 夏冬两季发酵雪茄烟叶的代谢谱全貌解析 A：代谢物含量组成；B：聚类分析；C：主成分分析得分；D：主成分分析散点（X：第一主成分，Y：第二主成分）。SF：夏季发酵烟叶；CK：发酵前烟叶；WF：冬季发酵烟叶；QC：质控样本，下同

Fig. 3 Overview of global metabolic profiles in fermented cigar tobacco samples of summer and winter A: Metabolites content categories. B: Clustering analysis. C: Principal component analysis score. D: Principal component analysis scatter（X: First principal component;Y: Second principal component）. SF: Tobacco fermentation in summer. CK：Tobacco before fermentation. WF: Tobacco fermentation in winter. QC: Quality control sample. The same below

图4 夏冬两季雪茄烟发酵叶中代谢物离子丰度对比不同小写字母表示3个样本数据在基于Tukey HSD的多重比较在P< 0.05水平存在显著差异

Fig. 4 Comparison of metabolite ion intensity in the fermented cigar tobacco leaves of summer and winter Different lowercase letters indicate Tukey HSD multiple comparison exists significant differences at the P< 0.05 level

图5 夏冬两季雪茄烟发酵叶差异生物碱聚类热图

Fig. 5 Clustering heat map of differential alkaloids in the fermented cigar leaves of summer and winter

表1 差异生物碱的关联代谢通路

Table 1 Metabolic pathways associated with differential alkaloids

编号 No.	代谢通路 Metabolic pathway	关联代谢物 Associated metabolites	通路ID Pathway ID
1	代谢途径 Metabolic pathways	胍丁胺、N-甲基-5-羟基色胺、顺式-5'-（2-氧代丙基）-烟碱、2-苯乙胺、去甲基烟碱、苯基乙醇胺、烟胺、3-吲哚乙腈	ko01100
2	甘氨酸、丝氨酸和苏氨酸代谢 Glycine, serine and threonine metabolism	乙酸胍	ko00260
3	半胱氨酸和甲硫氨酸代谢 Cysteine and methionine metabolism	烟胺	ko00270
4	精氨酸和脯氨酸代谢Arginine and proline metabolism	胍丁胺、N-乙酰腐胺、阿魏酰腐胺	ko00330
5	酪氨酸代谢Tyrosine metabolism	L-酪胺	ko00350
6	苯丙氨酸代谢 Phenylalanine metabolism	2-苯乙胺	ko00360
7	色氨酸代谢 Tryptophan metabolism	N-甲基-5-羟基色胺、甲氧基吲哚乙酸、3-吲哚乙腈	ko00380
8	核黄素代谢Riboflavin metabolism	7,8-二甲基咯嗪	ko00740
9	烟酸和烟酰胺代谢Nicotinate and nicotinamide metabolism	1,4-二氢-1-甲基-4-氧代-3-吡啶甲酰胺、烟酸甲酯	ko00760
10	异喹啉生物碱生物合成Isoquinoline alkaloid biosynthesis	L-酪胺	ko00950
11	莨菪烷、哌啶和吡啶生物碱生物合成 Tropane, piperidine and pyridine alkaloid biosynthesis	去甲基烟碱	ko00960
12	植物次生代谢产物的生物合成 Biosynthesis of various plant secondary metabolites	烟胺、3-吲哚乙腈、阿魏酰腐胺、1-甲基胍、对香豆酰酪胺	ko00999

表2 夏冬两季雪茄烟发酵叶的感官评价

Table 2 Sensory quality of fermented cigar tobacco leaves from summer and winter

样品 Sample	香气质 Aroma quality	香气量 Aroma quantity	杂气 Disorder	刺激性 Thrill	余味 Aftertaste	甜度 Sweetness	燃烧性 Inflammability	灰色 Grayness	合计 Total
发酵初始样	12	12	6	7.5	8	4	8	7.5	65
冬季发酵叶	13	12	7	8	9	6.5	8.5	8	72
夏季发酵叶	15	13	7.5	9	10	7	8.5	8	78

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