Article(id=1217471082697773202, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1217471079325549522, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250472, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1750089600000, receivedDateStr=2025-06-17, revisedDate=null, revisedDateStr=null, acceptedDate=1755187200000, acceptedDateStr=2025-08-15, onlineDate=1768197325633, onlineDateStr=2026-01-12, pubDate=1767456000000, pubDateStr=2026-01-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1768197325633, onlineIssueDateStr=2026-01-12, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1768197325633, creator=13701087609, updateTime=1768197325633, updator=13701087609, issue=Issue{id=1217471079325549522, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='1', pageStart='1', pageEnd='475', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1768197324830, creator=13701087609, updateTime=1768198886678, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217477630291530315, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1217471079325549522, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217477630291530316, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1217471079325549522, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=187, endPage=212, ext={EN=ArticleExt(id=1217471082932654229, articleId=1217471082697773202, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Characteristics of microbial communities in aged tobacco leaves from diverse production areas in Yunnan and their correlations with chemical components, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

[Objective] This study examined the characteristics of the microbial communities and their associations with chemical components in aged tobacco leaves from different production areas in Yunnan Province, aiming to provide a theoretical foundation for the targeted exploitation of tobacco microbial resources and quality improvement of tobacco. [Methods] A systematic approach integrating metagenomic sequencing, GC/LC-MS, and Spearman’s correlation analysis was employed to investigate the microbial communities, chemical components, and their correlations of aged tobacco leaf samples collected from Dali, Wenshan, Honghe, Luoping, Pu’er, Zhaotong, and Lincang. [Results] The dominant bacterial phylum was Pseudomonadota in aged tobacco leaf samples. The dominant bacterial genera varied among different production areas. Specifically, Salinivibrio was identified as the core genus in the samples from Wenshan, while Methylobacterium and Sphingomonas exhibited significant enrichment in the samples from Luoping. For fungal communities, Ascomycota and Mucoromycota were the predominant phyla, and dominant fungal genera showed negligible variations. Alpha diversity analysis revealed the highest microbial richness and diversity in the samples from Lincang and the lowest in the samples from Wenshan. KEGG analysis revealed that metabolic pathways exhibited high relative abundance across samples from all regions, whereas environmental information processing pathways were more abundant in the samples from Wenshan. CAZy analysis showed that genes annotated as glycoside hydrolases (GHs) and glycosyl transferases (GTs) were the most prevalent, with both generally exhibiting lower abundance in the samples from Wenshan. Chemical profiling revealed that reducing sugar, total sugar and other conventional components were more concentrated in the samples from Dali, whereas total sugar-to-nicotine ratio and potassium-to-chloride ratio were elevated in the samples from Pu’er. Additionally, neutral aroma compounds, including benzyl alcohol, had significantly higher levels in the samples from Wenshan, while polyphenols such as anthocyanins exhibited markedly higher concentrations in the samples from Luoping. Correlation analysis further disclosed that Microbacterium had a significantly positive correlation with total nitrogen, while Aureobasidium showed a significantly negative correlation with total nitrogen. Leucosporidium exhibited significantly positive correlations with potassium ions and potassium-to-chloride ratio. Salinivibrio had significantly positive correlations with multiple neutral aroma components. In contrast, Methylobacterium and Friedmanniomyces showed significantly negative correlations with the majority of neutral aroma components. Additionally, Methylobacterium, Sphingomonas, Friedmanniomyces, and Metschnikowia had significantly positive correlations with multiple polyphenols. [Conclusion] Microbial communities and chemical components of aged tobacco leaves exhibited marked differences across different production areas in Yunnan. Notably, dominant genera are strongly correlated with aroma compounds, which lays a theoretical basis for targeted screening of functional microbes and the development of biotechnologies to improve tobacco quality.

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*E-mail:
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【目的】 探究云南不同产区醇化烟叶的微生物群落特征及其与化学成分的关系,为烟草微生物资源的定向挖掘与烟草品质改良提供理论依据和支持。 【方法】 综合运用宏基因组学、气相色谱/液相色谱-质谱联用技术及斯皮尔曼相关性分析等方法,系统分析大理、文山、红河、罗平、普洱、昭通和临沧共7个云南产区醇化烟叶的微生物群落、化学成分以及两者间的相关性。 【结果】 在各产区醇化烟叶中,细菌以假单胞菌门(Pseudomonadota)为优势菌门,优势细菌属呈现明显的地域差异。其中,文山产区以盐水弧菌属(Salinivibrio)为核心菌属,罗平产区以甲基杆菌属(Methylobacterium)和鞘氨醇单胞菌属(Sphingomonas)为典型代表;真菌以子囊菌门(Ascomycota)和毛霉门(Mucoromycota)为主,优势真菌属无地域差异。临沧产区的微生物丰富度和多样性最高,文山产区最低。KEGG注释显示,新陈代谢通路在各产区均具有较高丰度,环境信息处理通路则在文山产区呈现较高丰度。CAZy结果显示,被注释的糖苷水解酶(glycoside hydrolases, GHs)和糖基转移酶(glycosyl transferases, GTs)基因数目最多,且二者在文山产区的丰度较低。化学分析结果显示,大理产区烟叶的还原糖、总糖等常规成分含量较高,普洱产区的糖碱比和钾氯比均较高;文山产区含有较高含量的苯甲醇等多种中性致香成分,罗平产区则含有较高含量的花青素等多酚物质。相关性分析表明,微杆菌属(Microbacterium)与总氮呈显著正相关,短柄霉属(Aureobasidium)与总氮呈显著负相关;白冬孢酵母属(Leucosporidium)与钾离子和钾氯比呈显著正相关;盐水弧菌属(Salinivibrio)与多种中性致香成分呈显著正相关,甲基杆菌属(Methylobacterium)和弗莱德门氏菌属(Friedmanniomyces)则与多数中性致香成分呈显著负相关;甲基杆菌属(Methylobacterium)、鞘氨醇单胞菌属(Sphingomonas)、弗莱德门氏菌属(Friedmanniomyces)和梅奇酵母属(Metschnikowia)与大部分多酚物质呈显著正相关。 【结论】 云南不同产区醇化烟叶的微生物群落及化学成分均存在显著的地域差异,且醇化烟叶中的优势菌属与香气化合物存在显著关联,这为功能微生物的定向筛选和烟叶提质生物技术开发提供了理论支撑。

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作者贡献声明

刘蓉:数据收集与分析、论文撰写和修改;唐游:样本收集、论文讨论及提供技术支持;吕祥敏:化学检测;王鹏:常规化学成分分析;唐杰:检测方法建立;牛国清:论文框架设计与审阅;马明:论文构思、框架设计和审阅。

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A: Domain level; B and C: Bacterial phylum and genus levels, respectively; D and E: Fungal phylum and genus levels, respectively. The top 10 taxa in relative abundance at phylum and genus levels were color-coded, with the remaining taxa grouped as others., figureFileSmall=X+KM4ZzvjtWtj+4UpWfKrQ==, figureFileBig=dkg/8ozuYOomGzBtVcLDKA==, tableContent=null), ArticleFig(id=1226557141549498435, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=图1, caption=云南不同产区醇化烟叶微生物群落的界、门及属水平分布特征。A:界水平;B、C:细菌的门和属水平;D、E:真菌的门和属水平。门和属水平相对丰度前10个类群着色,其余统一为others。, figureFileSmall=X+KM4ZzvjtWtj+4UpWfKrQ==, figureFileBig=dkg/8ozuYOomGzBtVcLDKA==, tableContent=null), ArticleFig(id=1226557141700493390, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Figure 2, caption=Clustered heatmap illustrating the relative abundances of the top 35 species at genus level for bacterial communities (A) and fungal communities (B), respectively., figureFileSmall=vIc6okO8t6+9laRyXPUjtw==, figureFileBig=qqEbz/5EL2m2F0Zc2Wy2Bw==, tableContent=null), ArticleFig(id=1226557141830516818, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=图2, caption=细菌(A)和真菌(B)在属水平排名前35的物种相对丰度聚类热图, figureFileSmall=vIc6okO8t6+9laRyXPUjtw==, figureFileBig=qqEbz/5EL2m2F0Zc2Wy2Bw==, tableContent=null), ArticleFig(id=1226557141943763032, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Figure 3, caption=Petal plot of bacterial (A) and fungal (B) genus-level community composition in aged tobacco leaves from different production areas., figureFileSmall=Sl7ig7R9j0n9OS76j84iHA==, figureFileBig=iW5e1839Nkxkt6P+CRaA7w==, tableContent=null), ArticleFig(id=1226557142031843423, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=图3, caption=不同产区醇化烟叶细菌(A)与真菌(B)属水平的物种花瓣图, figureFileSmall=Sl7ig7R9j0n9OS76j84iHA==, figureFileBig=iW5e1839Nkxkt6P+CRaA7w==, tableContent=null), ArticleFig(id=1226557142136701029, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Figure 4, caption=Characterization of KEGG-annotated genes (A) and functional annotation relative abundances (B) in microbial communities of aged tobacco leaves across diverse production areas., figureFileSmall=fkeQGg0Q4DxqRm4C+nGm4Q==, figureFileBig=M4Z39kSiBMmOhGpm/T5L2A==, tableContent=null), ArticleFig(id=1226557142279307371, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=图4, caption=不同产区醇化烟叶的KEGG数据库注释功能基因数目(A)及其相对丰度分析(B), figureFileSmall=fkeQGg0Q4DxqRm4C+nGm4Q==, figureFileBig=M4Z39kSiBMmOhGpm/T5L2A==, tableContent=null), ArticleFig(id=1226557142405136501, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Figure 5, caption=Characterization of eggNOG-annotated genes (A) and functional annotation relative abundance of microbial communities (B) in aged tobacco leaves from different production areas., figureFileSmall=2YRa8pnw3C3vnisZu1ujeA==, figureFileBig=ajcvMwUZPXmSEgB7hrm0lg==, tableContent=null), ArticleFig(id=1226557142539354232, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=图5, caption=不同产区醇化烟叶的eggNOG数据库注释功能基因数目(A)及其相对丰度分析(B), figureFileSmall=2YRa8pnw3C3vnisZu1ujeA==, figureFileBig=ajcvMwUZPXmSEgB7hrm0lg==, tableContent=null), ArticleFig(id=1226557143978000515, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Figure 6, caption=Characterization of CAZy-annotated genes (A) and functional annotation relative abundance of microbial communities (B) in aged tobacco leaves from different production areas., figureFileSmall=4I8472Thxu0SyR12WUfK7g==, figureFileBig=qSdIqcagOYSgowE6WrwUNw==, tableContent=null), ArticleFig(id=1226557144133189773, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=图6, caption=不同产区醇化烟叶的CAZy数据库注释功能基因数目(A)及其相对丰度分析(B), figureFileSmall=4I8472Thxu0SyR12WUfK7g==, figureFileBig=qSdIqcagOYSgowE6WrwUNw==, tableContent=null), ArticleFig(id=1226557144246435985, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Figure 7, caption=Analysis of conventional chemical components in aged tobacco leaves from different production areas. A: Chloride ion; B: Reducing sugar; C: Total sugar; D: Nicotine; E: Potassium ion; F: Total nitrogen; G: Chloride ion/nicotine; H: Total sugar/nicotine; I: Potassium ion/chloride ion. Different lowercase letters indicate that there are statistically significant differences in the same conventional chemical component among different producing areas (P<0.05)., figureFileSmall=iBGnkESCVaB8cF9DBCkVjA==, figureFileBig=S9520iDxYbh13Ep1XA0nxQ==, tableContent=null), ArticleFig(id=1226557144430985369, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=图7, caption=不同产区醇化烟叶的常规化学成分分析。A:氯离子;B:还原糖;C:总糖;D:烟碱;E:钾离子;F:总氮;G:氯碱比;H:糖碱比;I:钾氯比。不同小写字母表示不同产区间同一常规化学成分的差异有统计学意义(P<0.05)。, figureFileSmall=iBGnkESCVaB8cF9DBCkVjA==, figureFileBig=S9520iDxYbh13Ep1XA0nxQ==, tableContent=null), ArticleFig(id=1226557144653283487, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Figure 8, caption=Correlation between microbial flora and conventional chemical components in aged tobacco leaves. A: Bacteria; B: Fungi. The color intensity represents the correlation coefficient value, and the color type denotes positive correlation (red) or negative correlation (blue). * indicates statistical significance at P<0.05, ** indicates statistical significance at P<0.01. The same below., figureFileSmall=AnAL2bpCETIN+jVCMRkTCQ==, figureFileBig=6vJfVbgZ4mw1703THZDLaw==, tableContent=null), ArticleFig(id=1226557144816861350, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=图8, caption=醇化烟叶微生物菌群与常规化学成分的相关性分析。A:细菌;B:真菌。颜色深浅代表相关系数值,颜色类型代表正相关(红色)或负相关(蓝色),*表示在P<0.05时具有统计学意义,**表示在P<0.01时具有统计学意义。下同。, figureFileSmall=AnAL2bpCETIN+jVCMRkTCQ==, figureFileBig=6vJfVbgZ4mw1703THZDLaw==, tableContent=null), ArticleFig(id=1226557144951079087, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Figure 9, caption=Correlation between core microbial flora and neutral aroma components in aged tobacco leaves., figureFileSmall=9g5/RY36ZA+QQd62SgL9ig==, figureFileBig=bZR+IRQ4RzZ6VfpbceAJFA==, tableContent=null), ArticleFig(id=1226557145089491128, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=图9, caption=醇化烟叶核心菌群与中性致香成分的相关性分析, figureFileSmall=9g5/RY36ZA+QQd62SgL9ig==, figureFileBig=bZR+IRQ4RzZ6VfpbceAJFA==, tableContent=null), ArticleFig(id=1226557145257263293, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Figure 10, caption=Correlation between core microbial flora and polyphenolic components in aged tobacco leaves., figureFileSmall=cyf9y8sg9pCdSgQLKmmb8w==, figureFileBig=xIRzlQ6gKlLipiviDEpEEQ==, tableContent=null), ArticleFig(id=1226557145425035460, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=图10, caption=醇化烟叶核心菌群与多酚成分的相关性分析, figureFileSmall=cyf9y8sg9pCdSgQLKmmb8w==, figureFileBig=xIRzlQ6gKlLipiviDEpEEQ==, tableContent=null), ArticleFig(id=1226557145555058889, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Table 1, caption=

Overview of aged tobacco leaves from different production areas in Yunnan

, figureFileSmall=null, figureFileBig=null, tableContent=

产地

Production areas

YearVarietyGradeWarehouse
大理Dali2022‘Honghuadajinyuan’C2FCore raw material
文山Wenshan2022‘Yunyan 116’C2F+C3FCore raw material
红河Honghe2022‘K326’C2F+C3FTobacco leaf No. 4
罗平Luoping2022‘K326’C3FCore raw material
普洱Pu’er2022‘Yunyan 116’C3FCore raw material
昭通Zhaotong2022‘K326’C2FCore raw material
临沧K Lincang K2022‘K326’C3FCore raw material
临沧Y Lincang Y2022‘Yunyan 87’C3FCore raw material
), ArticleFig(id=1226557145685082321, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=表1, caption=

云南不同产区醇化烟叶样品概况

, figureFileSmall=null, figureFileBig=null, tableContent=

产地

Production areas

YearVarietyGradeWarehouse
大理Dali2022‘Honghuadajinyuan’C2FCore raw material
文山Wenshan2022‘Yunyan 116’C2F+C3FCore raw material
红河Honghe2022‘K326’C2F+C3FTobacco leaf No. 4
罗平Luoping2022‘K326’C3FCore raw material
普洱Pu’er2022‘Yunyan 116’C3FCore raw material
昭通Zhaotong2022‘K326’C2FCore raw material
临沧K Lincang K2022‘K326’C3FCore raw material
临沧Y Lincang Y2022‘Yunyan 87’C3FCore raw material
), ArticleFig(id=1226557145848660186, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Table 2, caption=

Alpha diversity indexes of microbial communities in aged tobacco leaves from different production areas

, figureFileSmall=null, figureFileBig=null, tableContent=
Tobacco-growing areasACE indexChao1 indexShannon indexSimpson indexGoods coverage
Dali7 178.7877 272.4606.110.930.999 91
Wenshan4 060.7774 091.8625.540.920.999 94
Honghe7 235.8697 218.7306.800.950.999 96
Luoping4 086.2944 133.1055.510.910.999 96
Pu’er9 029.8689 109.4047.030.950.999 92
Zhaotong5 976.6506 043.0175.690.910.999 92
Lincang K9 793.5759 850.7747.790.970.999 94
Lincang Y8 803.7518 899.1276.910.950.999 92
), ArticleFig(id=1226557145961906397, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=表2, caption=

不同产区醇化烟叶微生物群落的α多样性指数

, figureFileSmall=null, figureFileBig=null, tableContent=
Tobacco-growing areasACE indexChao1 indexShannon indexSimpson indexGoods coverage
Dali7 178.7877 272.4606.110.930.999 91
Wenshan4 060.7774 091.8625.540.920.999 94
Honghe7 235.8697 218.7306.800.950.999 96
Luoping4 086.2944 133.1055.510.910.999 96
Pu’er9 029.8689 109.4047.030.950.999 92
Zhaotong5 976.6506 043.0175.690.910.999 92
Lincang K9 793.5759 850.7747.790.970.999 94
Lincang Y8 803.7518 899.1276.910.950.999 92
), ArticleFig(id=1226557146087735528, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Table 3, caption=

Concentrations of 21 neutral aroma components in aged tobacco leaves from different production areas

, figureFileSmall=null, figureFileBig=null, tableContent=

化学物质名称

Compound names

DaliWenshanHongheLuopingPu’erZhaotongLincang KLincang Y
糠醛Furfural102.52±0.15a47.47±0.43c73.79±0.86b15.76±0.10f17.27±0.09e23.17±0.12d8.54±0.12h11.86±0.09g
糠醇Furfuryl alcohol16.58±0.03b24.02±0.24a15.74±0.36c5.99±0.11e5.37±0.08e13.67±0.21d2.35±0.03f2.08±0.03f
苯甲醇Benzyl alcohol1.01±0.17h26.69±0.12a1.58±0.21g5.57±0.15d7.62±0.17c14.00±0.20b4.33±0.15e3.33±0.21f
苯乙醛Phenylacetaldehyde4.78±0.29e12.75±0.39b11.22±1.07c9.00±0.28d4.55±0.37e15.58±0.34a3.45±0.39e4.21±0.41e
芳樟醇Linalool2.59±0.08e7.56±0.21b2.55±0.20e9.16±0.24a8.76±0.10a6.02±0.08d7.62±0.14b6.72±0.25c

氧化异佛尔酮

Isophorone oxide

0.65±0.02a0.46±0.02b0.69±0.03a0.32±0.03c0.21±0.02d0.21±0.03d0.22±0.01d0.25±0.03cd
苯乙醇Phenethyl alcohol4.72±0.48cd14.06±0.44a5.58±0.20c3.91±0.40cd4.58±0.37d12.39±0.47b3.13±0.25de2.40±0.31e
异佛尔酮Isophorone8.65±0.83f63.42±0.82a11.80±0.74e63.49±0.80a53.59±0.79b34.40±0.83d45.77±0.75c55.04±0.77b
茄酮Solanesone1 598.82±68.21b1 630.89±106.47b2 001.41±125.75a468.80±95.18d792.70±89.63c1 807.66±118.53ab427.12±72.35d406.42±80.71d
β-大马酮β-amascenone1.01±0.00c2.56±0.03a0.80±0.04d0.82±0.02d1.07±0.02c2.14±0.03b0.79±0.01d0.59±0.09e
α-大马酮α-damascenone0.81±0.01cd2.20±0.02a0.89±0.03b0.94±0.02b0.83±0.02c2.16±0.02a0.77±0.01d0.70±0.01h

香叶基丙酮

Geranyl acetone

4.45±0.23b4.15±0.38bc4.46±0.41b3.19±0.037c2.33±0.34c8.03±0.39a3.16±0.29c2.59±0.32c
β-紫罗兰酮β-ionone0.72±0.05d1.00±0.05b0.74±0.02d0.90±0.04bc0.86±0.03c1.31±0.05a0.60±0.03e0.66±0.03de

二氢猕猴桃内酯

Dihydroactinidiolide

3.45±0.05a2.92±0.07b2.86±0.08b1.46±0.07cd1.55±0.08c2.99±0.06b1.39±0.06cd1.28±0.06d

巨豆三烯酮

Megastigmatrienone

105.04±4.79e416.02±4.67a147.92±2.25c165.06±4.36b131.23±4.02d407.20±3.69a111.67±2.81e107.41±3.21e
雪松醇Cedrol0.09±0.00bc0.04±0.01c0.14±0.01ab0.15±0.00ab0.09±0.00bc0.04±0.01c0.12±0.00b0.19±0.01a
红没药醇Bisabolol0.10±0.01f0.47±0.01a0.12±0.01f0.25±0.01d0.28±0.01c0.27±0.01cd0.22±0.01e0.32±0.01b

苯甲酸苯甲酯

Benzyl benzoate

1.80±0.00a0.97±0.00c1.81±0.05a0.52±0.02de0.60±0.03d1.12±0.04b0.42±0.01f0.51±0.03e
新植二烯Neophytadiene104.38±0.18a28.51±2.13d69.93±4.36b24.71±3.01d44.72±4.11c23.99±3.56d26.42±3.82d43.26±2.58c

金合欢基丙酮

Farnesyl acetone

2.33±0.09c5.17±0.11a2.52±0.11c1.51±0.10d1.34±0.11de2.87±0.10b1.15±0.10e1.51±0.10d

十六酸甲酯

Methyl hexadecanoate

4.94±0.13f10.52±0.11d5.18±0.05f9.51±0.10e20.27±0.09a10.56±0.12d15.29±0.06b13.25±0.08c
), ArticleFig(id=1226557146242924782, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=表3, caption=

不同产区醇化烟叶中21种中性致香成分含量的测定结果

, figureFileSmall=null, figureFileBig=null, tableContent=

化学物质名称

Compound names

DaliWenshanHongheLuopingPu’erZhaotongLincang KLincang Y
糠醛Furfural102.52±0.15a47.47±0.43c73.79±0.86b15.76±0.10f17.27±0.09e23.17±0.12d8.54±0.12h11.86±0.09g
糠醇Furfuryl alcohol16.58±0.03b24.02±0.24a15.74±0.36c5.99±0.11e5.37±0.08e13.67±0.21d2.35±0.03f2.08±0.03f
苯甲醇Benzyl alcohol1.01±0.17h26.69±0.12a1.58±0.21g5.57±0.15d7.62±0.17c14.00±0.20b4.33±0.15e3.33±0.21f
苯乙醛Phenylacetaldehyde4.78±0.29e12.75±0.39b11.22±1.07c9.00±0.28d4.55±0.37e15.58±0.34a3.45±0.39e4.21±0.41e
芳樟醇Linalool2.59±0.08e7.56±0.21b2.55±0.20e9.16±0.24a8.76±0.10a6.02±0.08d7.62±0.14b6.72±0.25c

氧化异佛尔酮

Isophorone oxide

0.65±0.02a0.46±0.02b0.69±0.03a0.32±0.03c0.21±0.02d0.21±0.03d0.22±0.01d0.25±0.03cd
苯乙醇Phenethyl alcohol4.72±0.48cd14.06±0.44a5.58±0.20c3.91±0.40cd4.58±0.37d12.39±0.47b3.13±0.25de2.40±0.31e
异佛尔酮Isophorone8.65±0.83f63.42±0.82a11.80±0.74e63.49±0.80a53.59±0.79b34.40±0.83d45.77±0.75c55.04±0.77b
茄酮Solanesone1 598.82±68.21b1 630.89±106.47b2 001.41±125.75a468.80±95.18d792.70±89.63c1 807.66±118.53ab427.12±72.35d406.42±80.71d
β-大马酮β-amascenone1.01±0.00c2.56±0.03a0.80±0.04d0.82±0.02d1.07±0.02c2.14±0.03b0.79±0.01d0.59±0.09e
α-大马酮α-damascenone0.81±0.01cd2.20±0.02a0.89±0.03b0.94±0.02b0.83±0.02c2.16±0.02a0.77±0.01d0.70±0.01h

香叶基丙酮

Geranyl acetone

4.45±0.23b4.15±0.38bc4.46±0.41b3.19±0.037c2.33±0.34c8.03±0.39a3.16±0.29c2.59±0.32c
β-紫罗兰酮β-ionone0.72±0.05d1.00±0.05b0.74±0.02d0.90±0.04bc0.86±0.03c1.31±0.05a0.60±0.03e0.66±0.03de

二氢猕猴桃内酯

Dihydroactinidiolide

3.45±0.05a2.92±0.07b2.86±0.08b1.46±0.07cd1.55±0.08c2.99±0.06b1.39±0.06cd1.28±0.06d

巨豆三烯酮

Megastigmatrienone

105.04±4.79e416.02±4.67a147.92±2.25c165.06±4.36b131.23±4.02d407.20±3.69a111.67±2.81e107.41±3.21e
雪松醇Cedrol0.09±0.00bc0.04±0.01c0.14±0.01ab0.15±0.00ab0.09±0.00bc0.04±0.01c0.12±0.00b0.19±0.01a
红没药醇Bisabolol0.10±0.01f0.47±0.01a0.12±0.01f0.25±0.01d0.28±0.01c0.27±0.01cd0.22±0.01e0.32±0.01b

苯甲酸苯甲酯

Benzyl benzoate

1.80±0.00a0.97±0.00c1.81±0.05a0.52±0.02de0.60±0.03d1.12±0.04b0.42±0.01f0.51±0.03e
新植二烯Neophytadiene104.38±0.18a28.51±2.13d69.93±4.36b24.71±3.01d44.72±4.11c23.99±3.56d26.42±3.82d43.26±2.58c

金合欢基丙酮

Farnesyl acetone

2.33±0.09c5.17±0.11a2.52±0.11c1.51±0.10d1.34±0.11de2.87±0.10b1.15±0.10e1.51±0.10d

十六酸甲酯

Methyl hexadecanoate

4.94±0.13f10.52±0.11d5.18±0.05f9.51±0.10e20.27±0.09a10.56±0.12d15.29±0.06b13.25±0.08c
), ArticleFig(id=1226557146385531125, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=EN, label=Table 4, caption=

Concentrations of 20 polyphenolic components in aged tobacco leaves from different production areas

, figureFileSmall=null, figureFileBig=null, tableContent=

化学物质名称

Compound names

DaliWenshanHongheLuopingPu’erZhaotongLincang KLincang Y
奎宁酸Quinic acid2 529.72±23.51f3 059.02±27.90d2 385.77±35.29g2 891.83±25.18e5 512.80±30.46a2 490.10±3.11f4 204.28±33.88bb3 601.82±28.54c
新绿原酸Nclrgn acid2 650.02±41.38d2 474.26±12.10e2 455.16±1.22e3 151.08±15.73a2 897.49±25.67b3 088.25±33.13a2 810.11±29.81c3 157.77±18.46a

绿原酸

Chlorogenic acid

17 587.42±390.60a12 345.51±56.39e12 446.63±19.38e14 000.43±45.28c12 305.89±98.76e14 818.52±21.57b13 420.40±89.42d14 881.11±52.11b
东莨菪苷Scopolin162.93±15.63f657.82±14.32d500.13±4.56e748.50±5.22c816.54±8.45b110.15±6.89g685.41±12.79d1 130.31±10.37a
隐绿原酸Cclrgn acid2 864.60±125.94c2 875.43±78.63c2 779.37±48.60c3 292.24±95.49b3 034.41±52.18c3 442.57±63.59ab3 156.01±88.35bc3 585.66±91.73a
咖啡酸Caffeic acid480.90±9.83c384.47±7.25e329.48±5.98f407.07±8.64d1 124.47±6.84a221.76±6.32g667.26±7.93b472.96±9.11c
七叶亭Esculetin24.49±1.89d28.20±1.41cd43.60±1.13a26.20±1.57cd25.40±1.69d29.03±1.39cd35.98±1.76b30.28±1.26c

3-O-阿魏酰奎宁酸

3-O-feruloylquinic acid

45.69±2.04g138.14±1.51e40.03±0.75h202.15±1.83a125.85±0.93f159.24±1.34b147.23±0.88c145.51±1.18c
花青素Anthocyanidin5.47±0.23e8.16±0.09d4.19±0.01g23.94±0.15a5.49±0.03e23.36±0.21b5.60±0.11e16.00±0.06c
芦丁Rutin7 651.41±5.92e8 225.22±61.79c9 032.14±67.03a9 155.89±59.34a8 224.05±63.22c8 012.67±65.78d7 708.60±58.62e8 623.70±66.41b
异槲皮苷Isoquercetin61.34±0.57f69.29±0.13d73.20±0.07b71.53±0.29c71.90±0.56c73.68±0.50b68.00±0.36e80.97±0.41a

山奈酚-3-O-芸香糖苷

Kaem-3-O-rut

334.45±2.26f352.08±1.87e312.84±0.45g643.07±2.02a498.18±0.64c402.73±1.24d335.02±0.98f559.24±1.57b
水仙苷Isor-3-O-rut24.81±0.28g53.16±0.17e26.83±0.14f65.64±0.27b68.07±0.19a63.94±0.22c56.44±0.24d66.19±0.25b
阿魏酸Ferulic acid21.52±0.05b13.63±0.07e11.24±0.15f24.25±0.10a19.84±0.12c13.85±0.10e18.28±0.13d18.55±0.06d
莨菪亭Scopoletin325.75±13.75e423.89±11.47d549.42±6.57c526.33±9.61c586.51±7.38b169.60±10.23f610.22±8.76ab624.10±12.15a
二氢槲皮素Taxifolin0.24±0.01e2.62±0.02c0.43±0.02d2.89±0.07a2.72±0.03bc2.84±0.06ab2.74±0.04b2.78±0.05ab

紫云英苷

Kaem-3-O-glu

6.93±0.05d7.50±0.18d7.91±0.01cd8.43±0.09c9.28±0.10b8.09±0.23cd8.27±0.43c9.91±0.27a

3,5-di-O-咖啡酰奎宁酸

3,5-二-O-caffeoylquinic acid

82.18±0.19c71.67±0.15d73.57±1.77d105.69±1.53a84.40±0.45c63.16±0.78f66.81±1.02e90.80±0.97b
槲皮素Quercetin1.63±0.06e7.71±0.29c4.04±1.21d4.19±0.03d7.43±0.75c3.76±1.12d12.69±0.90a10.27±0.12b
异鼠李素Isorhamnetin0.23±0.01c0.56±0.09ab0.23±0.04c0.42±0.00b0.59±0.00a0.52±0.05ab0.57±0.07a0.56±0.02ab
), ArticleFig(id=1226557146482000125, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1217471082697773202, language=CN, label=表4, caption=

不同产区醇化烟叶中20种多酚成分含量的检测结果

, figureFileSmall=null, figureFileBig=null, tableContent=

化学物质名称

Compound names

DaliWenshanHongheLuopingPu’erZhaotongLincang KLincang Y
奎宁酸Quinic acid2 529.72±23.51f3 059.02±27.90d2 385.77±35.29g2 891.83±25.18e5 512.80±30.46a2 490.10±3.11f4 204.28±33.88bb3 601.82±28.54c
新绿原酸Nclrgn acid2 650.02±41.38d2 474.26±12.10e2 455.16±1.22e3 151.08±15.73a2 897.49±25.67b3 088.25±33.13a2 810.11±29.81c3 157.77±18.46a

绿原酸

Chlorogenic acid

17 587.42±390.60a12 345.51±56.39e12 446.63±19.38e14 000.43±45.28c12 305.89±98.76e14 818.52±21.57b13 420.40±89.42d14 881.11±52.11b
东莨菪苷Scopolin162.93±15.63f657.82±14.32d500.13±4.56e748.50±5.22c816.54±8.45b110.15±6.89g685.41±12.79d1 130.31±10.37a
隐绿原酸Cclrgn acid2 864.60±125.94c2 875.43±78.63c2 779.37±48.60c3 292.24±95.49b3 034.41±52.18c3 442.57±63.59ab3 156.01±88.35bc3 585.66±91.73a
咖啡酸Caffeic acid480.90±9.83c384.47±7.25e329.48±5.98f407.07±8.64d1 124.47±6.84a221.76±6.32g667.26±7.93b472.96±9.11c
七叶亭Esculetin24.49±1.89d28.20±1.41cd43.60±1.13a26.20±1.57cd25.40±1.69d29.03±1.39cd35.98±1.76b30.28±1.26c

3-O-阿魏酰奎宁酸

3-O-feruloylquinic acid

45.69±2.04g138.14±1.51e40.03±0.75h202.15±1.83a125.85±0.93f159.24±1.34b147.23±0.88c145.51±1.18c
花青素Anthocyanidin5.47±0.23e8.16±0.09d4.19±0.01g23.94±0.15a5.49±0.03e23.36±0.21b5.60±0.11e16.00±0.06c
芦丁Rutin7 651.41±5.92e8 225.22±61.79c9 032.14±67.03a9 155.89±59.34a8 224.05±63.22c8 012.67±65.78d7 708.60±58.62e8 623.70±66.41b
异槲皮苷Isoquercetin61.34±0.57f69.29±0.13d73.20±0.07b71.53±0.29c71.90±0.56c73.68±0.50b68.00±0.36e80.97±0.41a

山奈酚-3-O-芸香糖苷

Kaem-3-O-rut

334.45±2.26f352.08±1.87e312.84±0.45g643.07±2.02a498.18±0.64c402.73±1.24d335.02±0.98f559.24±1.57b
水仙苷Isor-3-O-rut24.81±0.28g53.16±0.17e26.83±0.14f65.64±0.27b68.07±0.19a63.94±0.22c56.44±0.24d66.19±0.25b
阿魏酸Ferulic acid21.52±0.05b13.63±0.07e11.24±0.15f24.25±0.10a19.84±0.12c13.85±0.10e18.28±0.13d18.55±0.06d
莨菪亭Scopoletin325.75±13.75e423.89±11.47d549.42±6.57c526.33±9.61c586.51±7.38b169.60±10.23f610.22±8.76ab624.10±12.15a
二氢槲皮素Taxifolin0.24±0.01e2.62±0.02c0.43±0.02d2.89±0.07a2.72±0.03bc2.84±0.06ab2.74±0.04b2.78±0.05ab

紫云英苷

Kaem-3-O-glu

6.93±0.05d7.50±0.18d7.91±0.01cd8.43±0.09c9.28±0.10b8.09±0.23cd8.27±0.43c9.91±0.27a

3,5-di-O-咖啡酰奎宁酸

3,5-二-O-caffeoylquinic acid

82.18±0.19c71.67±0.15d73.57±1.77d105.69±1.53a84.40±0.45c63.16±0.78f66.81±1.02e90.80±0.97b
槲皮素Quercetin1.63±0.06e7.71±0.29c4.04±1.21d4.19±0.03d7.43±0.75c3.76±1.12d12.69±0.90a10.27±0.12b
异鼠李素Isorhamnetin0.23±0.01c0.56±0.09ab0.23±0.04c0.42±0.00b0.59±0.00a0.52±0.05ab0.57±0.07a0.56±0.02ab
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云南不同产区醇化烟叶微生物群落特征及其与化学成分的关联性分析
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刘蓉 1 , 唐游 1 , 吕祥敏 1 , 王鹏 1 , 唐杰 1 , 牛国清 2 , 马明 1, *
微生物学报 | 研究报告 2026,66(1): 187-212
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微生物学报 | 研究报告 2026, 66(1): 187-212
云南不同产区醇化烟叶微生物群落特征及其与化学成分的关联性分析
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刘蓉1, 唐游1, 吕祥敏1, 王鹏1, 唐杰1, 牛国清2, 马明1, *
作者信息
  • 1.重庆中烟工业有限责任公司技术中心,重庆
  • 2.西南大学 农学与生物科技学院,重庆
Characteristics of microbial communities in aged tobacco leaves from diverse production areas in Yunnan and their correlations with chemical components
Rong LIU1, You TANG1, Xiangmin LÜ1, Peng WANG1, Jie TANG1, Guoqing NIU2, Ming MA1, *
Affiliations
  • 1.Technical Center of China Tobacco Chongqing Industrial Co. , Ltd. , Chongqing, China
  • 2.College of Agronomy and Biotechnology, Southwest University, Chongqing, China
出版时间: 2026-01-04 doi: 10.13343/j.cnki.wsxb.20250472
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【目的】 探究云南不同产区醇化烟叶的微生物群落特征及其与化学成分的关系,为烟草微生物资源的定向挖掘与烟草品质改良提供理论依据和支持。 【方法】 综合运用宏基因组学、气相色谱/液相色谱-质谱联用技术及斯皮尔曼相关性分析等方法,系统分析大理、文山、红河、罗平、普洱、昭通和临沧共7个云南产区醇化烟叶的微生物群落、化学成分以及两者间的相关性。 【结果】 在各产区醇化烟叶中,细菌以假单胞菌门(Pseudomonadota)为优势菌门,优势细菌属呈现明显的地域差异。其中,文山产区以盐水弧菌属(Salinivibrio)为核心菌属,罗平产区以甲基杆菌属(Methylobacterium)和鞘氨醇单胞菌属(Sphingomonas)为典型代表;真菌以子囊菌门(Ascomycota)和毛霉门(Mucoromycota)为主,优势真菌属无地域差异。临沧产区的微生物丰富度和多样性最高,文山产区最低。KEGG注释显示,新陈代谢通路在各产区均具有较高丰度,环境信息处理通路则在文山产区呈现较高丰度。CAZy结果显示,被注释的糖苷水解酶(glycoside hydrolases, GHs)和糖基转移酶(glycosyl transferases, GTs)基因数目最多,且二者在文山产区的丰度较低。化学分析结果显示,大理产区烟叶的还原糖、总糖等常规成分含量较高,普洱产区的糖碱比和钾氯比均较高;文山产区含有较高含量的苯甲醇等多种中性致香成分,罗平产区则含有较高含量的花青素等多酚物质。相关性分析表明,微杆菌属(Microbacterium)与总氮呈显著正相关,短柄霉属(Aureobasidium)与总氮呈显著负相关;白冬孢酵母属(Leucosporidium)与钾离子和钾氯比呈显著正相关;盐水弧菌属(Salinivibrio)与多种中性致香成分呈显著正相关,甲基杆菌属(Methylobacterium)和弗莱德门氏菌属(Friedmanniomyces)则与多数中性致香成分呈显著负相关;甲基杆菌属(Methylobacterium)、鞘氨醇单胞菌属(Sphingomonas)、弗莱德门氏菌属(Friedmanniomyces)和梅奇酵母属(Metschnikowia)与大部分多酚物质呈显著正相关。 【结论】 云南不同产区醇化烟叶的微生物群落及化学成分均存在显著的地域差异,且醇化烟叶中的优势菌属与香气化合物存在显著关联,这为功能微生物的定向筛选和烟叶提质生物技术开发提供了理论支撑。

云南产区  /  醇化烟叶  /  宏基因组分析  /  微生物多样性  /  化学成分  /  相关性分析

[Objective] This study examined the characteristics of the microbial communities and their associations with chemical components in aged tobacco leaves from different production areas in Yunnan Province, aiming to provide a theoretical foundation for the targeted exploitation of tobacco microbial resources and quality improvement of tobacco. [Methods] A systematic approach integrating metagenomic sequencing, GC/LC-MS, and Spearman’s correlation analysis was employed to investigate the microbial communities, chemical components, and their correlations of aged tobacco leaf samples collected from Dali, Wenshan, Honghe, Luoping, Pu’er, Zhaotong, and Lincang. [Results] The dominant bacterial phylum was Pseudomonadota in aged tobacco leaf samples. The dominant bacterial genera varied among different production areas. Specifically, Salinivibrio was identified as the core genus in the samples from Wenshan, while Methylobacterium and Sphingomonas exhibited significant enrichment in the samples from Luoping. For fungal communities, Ascomycota and Mucoromycota were the predominant phyla, and dominant fungal genera showed negligible variations. Alpha diversity analysis revealed the highest microbial richness and diversity in the samples from Lincang and the lowest in the samples from Wenshan. KEGG analysis revealed that metabolic pathways exhibited high relative abundance across samples from all regions, whereas environmental information processing pathways were more abundant in the samples from Wenshan. CAZy analysis showed that genes annotated as glycoside hydrolases (GHs) and glycosyl transferases (GTs) were the most prevalent, with both generally exhibiting lower abundance in the samples from Wenshan. Chemical profiling revealed that reducing sugar, total sugar and other conventional components were more concentrated in the samples from Dali, whereas total sugar-to-nicotine ratio and potassium-to-chloride ratio were elevated in the samples from Pu’er. Additionally, neutral aroma compounds, including benzyl alcohol, had significantly higher levels in the samples from Wenshan, while polyphenols such as anthocyanins exhibited markedly higher concentrations in the samples from Luoping. Correlation analysis further disclosed that Microbacterium had a significantly positive correlation with total nitrogen, while Aureobasidium showed a significantly negative correlation with total nitrogen. Leucosporidium exhibited significantly positive correlations with potassium ions and potassium-to-chloride ratio. Salinivibrio had significantly positive correlations with multiple neutral aroma components. In contrast, Methylobacterium and Friedmanniomyces showed significantly negative correlations with the majority of neutral aroma components. Additionally, Methylobacterium, Sphingomonas, Friedmanniomyces, and Metschnikowia had significantly positive correlations with multiple polyphenols. [Conclusion] Microbial communities and chemical components of aged tobacco leaves exhibited marked differences across different production areas in Yunnan. Notably, dominant genera are strongly correlated with aroma compounds, which lays a theoretical basis for targeted screening of functional microbes and the development of biotechnologies to improve tobacco quality.

production areas in Yunnan  /  aged tobacco leaves  /  metagenomic sequencing  /  microbial diversity  /  chemical component  /  correlation analysis
刘蓉, 唐游, 吕祥敏, 王鹏, 唐杰, 牛国清, 马明. 云南不同产区醇化烟叶微生物群落特征及其与化学成分的关联性分析. 微生物学报, 2026 , 66 (1) : 187 -212 . DOI: 10.13343/j.cnki.wsxb.20250472
Rong LIU, You TANG, Xiangmin LÜ, Peng WANG, Jie TANG, Guoqing NIU, Ming MA. Characteristics of microbial communities in aged tobacco leaves from diverse production areas in Yunnan and their correlations with chemical components[J]. Acta Microbiologica Sinica, 2026 , 66 (1) : 187 -212 . DOI: 10.13343/j.cnki.wsxb.20250472
烟叶醇化是烟草品质提升的关键工艺环节,通过微生物活动、酶促反应及化学变化的协同作用,可促进纤维素、蛋白等大分子的降解与转化,生成香味成分,进而改善烟叶品质[1]。近年来,随着宏基因组学和高通量测序技术的发展,烟叶表面微生物群落的结构特征、功能潜力以及有益菌株筛选研究成为热点[2-5]。黄申等[6]通过比较3种不同香型再造烟叶的微生物群落发现其菌群结构存在显著差异,其中清香型烟叶的细菌多样性水平最高,且芽孢杆菌属(Bacillus)为优势细菌属。前期研究发现泛菌属(Pantoea)、不动杆菌属(Acinetobacter)、假单胞菌属(Pseudomonas)和芽孢杆菌属(Bacillus)共同构成了不同醇化卷烟烟叶的主要核心菌属;鞘氨醇单胞菌属(Sphingomonas)和芽孢杆菌属(Bacillus)等在醇化过程中可降解淀粉、蛋白质和果胶等大分子物质,促进糖类和中性致香物质等的释放,从而对烟叶风味的形成产生重要影响[7-8]。此外,已有研究表明不同产地醇化烟叶微生物群落的地域性差异是产区烟叶风格特色形成的重要驱动因素[9-11]
在农产品和食品风味塑造中,微生物菌群通过代谢活动参与酯类和酚类等致香前体物质的降解或合成,赋予产品独特的香型风格与品质属性。微生物与化学成分间的相关性研究是深入解析“微生物-化学成分”互作机制的关键切入点。例如,Li等[12]通过相关性分析发现罗河杆菌属(Rhodanobacter)和出芽单胞菌属(Gemmatimonas)与苯丙酮、2,3-丁二酮和香叶基丙酮等风味化合物呈显著正相关,从而揭示了这些菌属在雪茄烟叶风味形成中的潜在促进作用。刘晓敏等[13]通过探究不同香型醇化烟叶微生物与常规化学成分的关系,从云南玉溪清香型烟叶中筛选到具有降解烟碱能力的假单胞菌属(Pseudomonas),其菌群丰度与烟叶清甜香物质积累呈显著正相关。在发酵食品方面,Gao等[14]指出虾酱中优势的四联球菌属(Tetragenococcus)与多种游离氨基酸正相关,葡萄球菌属(Staphylococcus)与5′-核苷酸呈正相关,二者构成影响非挥发性风味成分的核心菌属。Yang等[15]通过相关性分析筛选出鞘氨醇单胞菌属(Sphingomonas)、不动杆菌属(Acinetobacter)和盐单胞菌属(Halomonas)等陈香型铁观音的核心功能微生物,并证实这些菌属具有通过积累代谢前体促进陈香风格形成的潜力。
现有研究多聚焦于大产区或不同香型烟叶的微生物群落组成特征,却未关注到同一香型不同产区的微生物群落与多维度化学成分在香气风格细分及品质特色塑造中的关键作用,更缺乏对“微生物-化学品质”之间的潜在关系及核心菌属的代谢贡献的系统探究。因此,本研究以云南清香型典型烤烟产区2年醇化期的烟叶为研究对象,利用宏基因组学、色谱-质谱联用以及斯皮尔曼相关性分析等方法系统解析不同产区醇化烟叶微生物群落构成,深入探寻其与烟叶化学品质间的内在关联,以期为特定功能菌株的挖掘及烟叶提质生物技术的开发奠定研究基础,助力特色产区烟叶品质的高质量发展。
烟叶醇化是指经复烤的片烟装箱后,在温度20-30 ℃、湿度60%-70%、含水率10%-13%的仓库条件下,经过1-3年储存所发生的缓慢发酵过程。在此过程中,需定期翻垛以保持堆垛烟包醇化的质量与均一性。为探究云南同一香型下不同产区醇化烟叶的微生物群落与化学成分特征及两者的内在联系,供试样品于2024年8月采集自重庆中烟工业有限责任公司醇化仓库,均为经过2年自然醇化的清香型烤烟,样本涵盖7个典型产区、4个不同品种,其中临沧产区采集了2个品种,共计8个样品(表1)。每个样品均从烟箱上、中、下三部分分别采样后混匀,总质量约1 kg,于-80 ℃超低温条件下保存备用。
将采集的醇化烟叶用无菌剪刀剪切为小于0.3 cm×0.3 cm的碎片后混匀,采用十六烷基三甲基溴化铵法(cetyltrimethylammonium bromide, CTAB)提取样本基因组DNA[16]。通过1%琼脂糖凝胶电泳方法检测DNA提取质量,再用无菌蒸馏水稀释至1 ng/μL。取1 μg样本基因组DNA,用超声波破碎仪(Covaris公司)随机打断为约350 bp的片段,经末端修复、加A尾、测序接头、纯化和PCR扩增等步骤进行文库构建。构建好的文库先使用AATI检测文库片段的完整性及插入片段大小,符合预期后,再采用qPCR方法对文库的有效浓度进行准确定量(文库有效浓度>3 nmol/L)。库检合格后,依据有效浓度和目标下机数据量的需求对不同文库进行pooling操作,再使用Illumina NovaSeq 6000平台上机测序,以上实验委托北京诺禾致源科技股份有限公司完成。
利用fastp (https://github.com/OpenGene/fastp)和Bowtie 2软件对NovaSeq测序平台产生的原始数据(raw data)进行预处理,获得用于后续分析的有效数据(clean data)。通过MEGAHIT软件对clean data进行组装并从N连接处打断,得到不含N的scaftigs。随后经MetaGeneMark (http://topaz.gatech.edu/GeneMark)的ORF预测过滤掉100 nt以下信息,并利用CD-HIT软件去冗余后得到非冗余的初始gene catalogue。使用Bowtie 2将各样品的clean data比对初始gene catalogue,统计基因在各样品中比对上的reads数目,过滤掉reads数目≤2的基因,最终得到unigenes。
采用Diamond软件将unigenes与NCBI非冗余蛋白质数据库(http://www.ncbi.nlm.nih.gov)中抽提出的Mircro_NR进行同源对比,并结合LCA算法和Krona分析完成群落中物种注释、物种相对丰度量化以及基于丰度差异的样本聚类热图分析。基于物种注释Absolute表,计算有效数据覆盖率(goods coverage)指数反映样本文库覆盖率;计算Shannon指数、Simpson指数分析样本中微生物群落多样性;计算Chao1指数、ACE指数分析样本中微生物丰富度。使用Diamond软件将unigenes与功能数据库(KEGG、eggNOG和CAZy)进行比对用于基因功能预测及其相对丰度分析。利用联川生物平台(http://www.omicstudio.cn)中云工具的斯皮尔曼(Spearman)相关性算法研究微生物与化学成分的关联性。
将醇化烟叶在25 ℃恒温平衡12 h后打成烟末,用于测定烟叶常规化学成分。其测定采用以下标准方法:YC/T 159—2019《烟草及烟草制品 水溶性糖的测定 连续流动法》、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》、YC/T 162—2011《烟草及烟草制品 氯的测定 连续流动法》、YC/T 217—2007《烟草及烟草制品 钾的测定 连续流动法》、YC/T 161—2002《烟草及烟草制品 总氮的测定 连续流动法》、YC/T 31—1996《烟草及烟草制品试样的制备和水分测定 烘箱法》[17-22]
将醇化烟叶在25 ℃恒温平衡12 h后打成烟末备用。参照现有方法进行中性致香成分的样品前处理及气相色谱-质谱联用仪(gas chromatography-mass spectrometry, GC-MS)检测[23]。称取约2 g (精确至0.01 g)烟末样品置于50 mL具盖离心管中,加入10 mL水,振荡直至样品充分浸润后静置10 min。随后移取10 mL乙腈和0.2 mL乙酸苯乙酯(上海希格玛高技术有限公司)内标工作液至该离心管,2 000 r/min振荡1 min,冷冻10 min。加入高色素样品萃取试剂包(河南嵩检标物科技有限公司)后,立即2 000 r/min振荡2 min防止无水硫酸镁结块,再以4 000 r/min离心10 min。移取1 mL上清液于1.5 mL离心管,加入香味成分净化包(河南嵩检标物科技有限公司),2 000 r/min振荡2 min后,6 000 r/min离心2 min,收集上清液待测。GC-MS检测条件:使用气相色谱-质谱仪(Agilent公司),色谱柱为DB-5MS (60 m×0.25 mm×0.25 μm)毛细管色谱柱,载气为氦气,流速1.0 mL/min (恒流);进样量为1.0 μL,分流比为10:1,进样口温度为250 ℃。升温程序:初始温度50 ℃保持2 min,以8 ℃/min升至280 ℃并保持25 min;传输线温度为280 ℃,离子源温度为230 ℃,四极杆温度为150 ℃;EI电离能量70 eV;溶剂延迟时间5.7 min;全扫描质量数范围为33-400 amu。全扫描后定性分析采用NIST标准谱库,定量分析采用选择离子检测模式(selected ion monitoring, SIM)。
醇化烟叶中多酚成分的样品前处理及高效液相色谱-质谱联用仪(high performance liquid chromatography-mass spectrometry, HPLC-MS)检测方法如下:称取0.5 g烟末样品,加入20 mL 50%甲醇-水溶液和200 µL内标溶液(上海安谱实验科技股份有限公司),以1 500 r/min涡旋萃取30 min,过0.22 μm滤膜后待测[24]。HPLC-MS检测条件:使用超高效液相色谱-三重四级杆串联质谱仪(Agilent公司),色谱柱为ZORBAX SB-C18 RRHD (2.1 mm×150 mm, 1.8 µm);流动相A为含0.1%甲酸的乙腈,流动相B为0.1%甲酸水溶液;进样量2 μL,流速0.2 mL/min,柱温:30 ℃。梯度洗脱流程:0-1 min,8% A;1-15 min,8%-45% A;15-18 min,45%-92% A;18-19 min,92%-8% A;19-22 min,8% A。质谱工作条件:离子源为电喷雾离子源Jet Stream ESI;干燥气温度325 ℃,流量6 L/min;雾化器压力35 psi;鞘气温度300 ℃,流量10 L/min;毛细管电压3 500 V;四级杆温度100 ℃;扫描方式为动态多反应监视(dynamic multiple reaction monitoring, dynamic MRM)。
基于醇化烟叶的宏基因组学数据,对界、门及属水平的微生物组成与相对丰度进行了分析。结果显示,在界水平,醇化烟叶中的微生物包括细菌(bacteria)、真菌(fungi)、古菌(archaea)、病毒(viruses)和其他类群(others)。各产区样品中,细菌均为绝对优势菌群,其相对丰度范围为31.80%-59.28%;真菌次之,相对丰度在0.71%-5.86%之间(图1A)。在门水平和属水平,细菌和真菌群落结构呈现出不同的特征。在细菌方面,优势菌门构成基本一致,主要包括假单胞菌门(Pseudomonadota,相对丰度为69.69%-89.22%)、放线菌门(Actinomycetota, 1.14%-6.79%)、拟杆菌门(Bacteroidota, 0.11%-2.99%)和芽孢杆菌门(Bacillota, 0.46%-2.99%) (图1B)。优势细菌属的组成呈现明显的产区差异:在大理、红河、普洱、昭通和临沧产区的6个样品中假单胞菌属(Pseudomonas, 12.19%-25.23%)和鞘氨醇单胞菌属(Sphingomonas, 7.29%-14.52%)均为核心菌属,而第三优势菌属则有所不同,大理、红河、普洱和昭通产区以泛菌属(Pantoea, 5.00%-11.46%)为主,临沧产区的‘K326’和‘云烟87’品种分别为不动杆菌属(Acinetobacter, 6.84%)和甲基杆菌属(Methylobacterium, 6.60%) (图1C);文山产区的特征菌属为盐水弧菌属(Salinivibrio, 12.60%)和不动杆菌属(Acinetobacter, 2.70%);罗平产区则以鞘氨醇单胞菌属(Sphingomonas, 25.82%)和甲基杆菌属(Methylobacterium, 26.50%)为优势菌属。与细菌属水平的产区差异不同,真菌在门及属水平上均表现出较高的相似性。在门水平上,子囊菌门(Ascomycota,相对丰度21.50%-46.40%)为绝对优势类群,其次是毛霉门(Mucoromycota, 5.50%-21.90%)和担子菌门(Basidiomycota, 6.20%-17.00%) (图1D)。在属水平上,弗莱德门氏菌属(Friedmanniomyces, 4.18%-28.92%)、根霉属(Rhizopus, 3.51%-18.27%)、硬皮地星属(Astraeus, 5.39%-10.81%)和短柄霉属(Aureobasidium, 2.66%-7.60%)为各产区共有的优势菌属(图1E)。上述结果表明,不同产区细菌属水平的群落结构地域差异显著,这种细菌群落结构的地域特异性或可作为产区识别的微生物标志物,为云南烟叶产区的精准筛选提供科学依据。
云南产区8个样本中,属水平排名前35的物种相对丰度差异及其聚类情况如图2所示。整体而言,细菌群落在产区间的相似性远低于真菌群落,且临沧产区2个样本的聚类分支距离较远。在细菌菌群聚类结构中,普洱、昭通和临沧Y样本形成紧密的聚类分支,其余产区样本则分布于较远的聚类分支,呈现出明显的地域差异性特征。物种丰度分布显示:黄单胞菌属(Xanthomonas)、丛毛单胞菌属(Comamonas)和新鞘氨醇菌属(Novosphingobium)在临沧K样本中呈现较高分布;微杆菌属(Microbacterium)和甲基杆菌属(Methylobacterium)在罗平样本中呈现高丰度聚集;埃希氏菌属(Escherichia)、居海事城球杆菌属(Phocaeicola)和巨单胞菌属(Megamonas)在大理样本中丰度较高;盐水弧菌属(Salinivibrio)和肠球菌属(Enterococcus)、假杜擀氏菌属(Pseudoduganella)和西地西菌属(Cedecea)、芽孢杆菌属(Bacillus)和罗尔斯通氏菌属(Ralstonia)、土地芽孢杆菌属(Terribacillus)以及红球菌属(Rhodococcus)分别在文山、红河、普洱、昭通和临沧Y样本中呈现较高丰度。
对于真菌群落聚类而言,罗平和文山产区样本在聚类树中形成了距离较远的独立分支,其余产区样本则聚集于相近分支,反映出共有菌属的广泛分布及较高的群落组成相似性。然而,真菌核心菌属的丰度热图仍呈现出较明显的地域差异:罗平产区中合头霉属(Syncephalis)、粪壳菌属(Sordaria)、弗莱德门氏菌属(Friedmanniomyces)和梅奇酵母属(Metschnikowia)等丰度较高;曲霉属(Aspergillus)和内养孢囊菌属(Entrophospora)在文山产区呈现较高丰度;红菇属(Russula)、Knufia、青霉属(Penicillium)、根囊菌属(Rhizophagus)、链格孢属(Alternaria)和白冬孢酵母属(Leucosporidium)则分别在临沧K、红河、大理、昭通、普洱和临沧Y样本中呈现高富集特征,与聚类分析结果相呼应。
为明确不同产区醇化烟叶微生物群落的组成差异,采用花瓣图对细菌和真菌在属水平的物种组成进行了分析(图3)。结果显示:在细菌方面共有属鉴定出301个,特有属以临沧K最为丰富,文山产区最低,其余样本按特有属数量递减依次为普洱、临沧Y、红河、大理、昭通和罗平。真菌群落则以共有属为主,各产区醇化烟叶中共有真菌属高达386个,仅有临沧K、普洱和红河3个样本存在少量特有真菌属,分别为4、2、2个。
基于α多样性指数对不同产区醇化烟叶微生物群落生态特征的解析结果显示(表2):各产区样品的测序覆盖度指数均达到0.999 00以上,表明该测序数据完整度高,能够真实反映微生物群落结构。此外,临沧K样本的丰富度指数(ACE和Chao1)与多样性指数(Shannon和Simpson)均为最高,表明该产区K326醇化烟叶的物种丰富度及多样性较高;文山产区样品各项指数均最低,说明其微生物群落结构相对单一。进一步比较分析发现,各产区醇化烟叶微生物的丰富度和多样性指数呈现一致的递减趋势,依次为临沧K、普洱、临沧Y、红河、大理、昭通、罗平和文山,这表明微生物丰富度较高的产区其物种分布均匀性更佳。临沧产区特有的菌属优势与其显著的多样性特征相互印证,揭示了地理环境和烟叶品种对醇化烟叶微生物的双重塑造作用。
基于微生物群落结构解析结果,综合运用KEGG (https://www.genome.jp/kegg)、eggNOG (http://eggnogdb.embl.de)及CAZy (https://www.cazy.org)三大功能数据库开展功能注释及相对丰度分析,系统探寻不同产区醇化烟叶微生物群落的代谢功能潜力。
基于Diamoda软件,将496 735条unigenes序列与KEGG数据库进行比对注释,对微生物群落的功能进行预测[25-28]。KEGG结果显示(图4A),醇化烟叶的微生物群落中注释得到的总功能基因包含6个一级通路,分别为:细胞过程(cellular processes)、环境信息处理(environmental information processing)、遗传信息处理(genetic information processing)、人类疾病(human diseases)、新陈代谢(metabolism)和生物体系统(organismal systems)。在已注释的总基因中,新陈代谢通路基因和环境信息处理通路基因占比合计超过50%。在新陈代谢通路中,碳水化合物代谢(carbohydrate metabolism)基因数量为53 978个,氨基酸代谢(amino acid metabolism)基因数量为48 693个,表明微生物群落中二者的功能基因储备更为丰富,无疑为烟叶醇化阶段风味物质的形成创造了有利条件。对于环境信息处理通路,膜转运(membrane transport)基因有37 308个,信号转导(signal transduction)基因34 370个,二者的高丰度富集在保障烟叶醇化中风味前体高效转化、调控菌群协同以促进风味物质定向积累,以及感知烟叶基质及仓储环境变化、调整代谢策略维持活性等方面具有巨大潜能。不同产区一级通路相对丰度分析结果显示(图4B),新陈代谢和环境信息处理两大通路在所有样品中均有较高的相对丰度。其中,文山产区以环境信息处理通路为主,相对丰度高达32%;其他产区样品则以新陈代谢通路为主导,其相对丰度介于17%-19%之间。
通过eggNOG数据库的功能注释分析共鉴定出1 215 318个基因(图5A)。经过筛选去除未知功能(S单元)后,成功定位到23个一级功能单元。其中,注释功能基因数量居前6的功能单元依次为:氨基酸转运和代谢(E单元)、无机离子转运和代谢(P单元)、细胞壁/膜/包膜生物发生(M单元)、碳水化合物转运和代谢(G单元)、转录(K单元)以及信号转导机制(T单元)。eggNOG一级功能单元相对丰度前10的分析结果显示(图5B),除功能未知的S单元外,其余9个功能单元在各样本间的相对丰度较为接近,且氨基酸转运和代谢功能单元的相对丰度在各样本中始终最高。
凭借CAZy数据库功能注释,各产区醇化烟叶中共鉴定出85 713个碳水化合物活性酶(CAZyme)编码基因,分属6个一级功能分类(图6A),分别是糖苷水解酶(glycoside hydrolases, GHs)、糖基转移酶(glycosyl transferases, GTs)、碳水化合物结合模块(carbohydrate binding modules, CBMs)、碳水化合物酯酶(carbohydrate esterases, CEs)、辅助氧化还原酶(auxiliary activities, AAs)和多糖裂解酶(polysaccharide lyases, PLs)。其中,GHs和GTs两大家族被注释到的基因数量最多,分别占已注释基因总数的41.61%和39.42%,累计占比高达81.03%。GHs家族能够水解糖苷键,促使单糖或寡糖生成,在寡糖、芳香基糖苷的合成以及氨基酸和多样的糖基化进程中承担关键作用;GTs则主要负责催化糖分子与其他生物分子的共价连接,因而高丰度的GHs和GTs可为醇化过程中风味物质合成提供关键前体。功能相对丰度分析结果显示(图6B),所有产区样品中GHs和GTs是碳水化合物活性酶的主要组成。除文山产区的碳水化合物活性酶相对丰度偏低外,其余产区样品的一级功能单元相对丰度则呈现出较高的一致性。
醇化烟叶的常规化学成分及其比值是决定烟叶品质的重要因素,直接影响卷烟的感官品质、香气特征与燃烧性能。基于此,对不同产区醇化烟叶的常规化学成分进行了测定,结果如图7所示。不同产区烟叶的常规化学成分呈现显著差异,大理产区的氯离子(chloride ion, Cl-)、还原糖(reducing sugar, RS)、总糖(total sugar, TS)及氯碱比(chloride ion/nicotine, Cl-/NIC)含量显著高于其他产区,而钾离子(potassium ion, K+)和钾氯比(potassium ion/chloride ion, K+/Cl-)含量最低;红河产区的烟碱(nicotine, NIC)含量明显高于其他产区;临沧Y样品的钾离子(potassium ion, K+)含量最高,与其他产区存在显著差异;罗平和昭通产区的总氮(total nitrogen, TN)含量比其他产区高,但两者间无显著差异;普洱产区的糖碱比(total sugar/nicotine, TS/NIC)和钾氯比(potassium ion/chloride ion, K+/Cl-)显著高于其他产区,氯离子含量则在所有产区中最低。
中性致香物质是烟草品质的核心要素,其种类、含量及相互作用直接决定了烟草的香气风格。为多维度解析云南不同产区醇化烟叶的品质特征,对醇化烟叶中的21种中性致香成分进行了定量分析。结果显示(表3):文山产区的苯甲醇、苯乙醇、β-大马酮、巨豆三烯酮和金合欢基丙酮等花香型成分含量显著高于其他产区;大理产区的糠醛、二氢猕猴桃内酯和新植二烯等烘烤香成分含量明显高于其他产区;昭通产区高含量的香叶基丙酮和β-紫罗兰酮等脂香成分与其他产区存在显著差异;红河产区的氧化异佛尔酮、茄酮和苯甲酸苯甲酯含量最高,与其他产区差异性显著;普洱产区的十六酸甲酯含量显著高于其他产区;临沧产区整体中性致香成分含量较其他产区偏低。这种不同产区中性致香物质差异性分布特征表明产区种植环境可能在烟叶香气风格形成过程中发挥了重要作用。
多酚类物质是烟草中重要的次生代谢产物,它们不仅影响烟草的香气和风味,还与烟草的燃烧特性与加工性能密切相关。为从多维度解析不同产区烟叶的品质特征,对醇化烟叶中的多酚类物质进行了定量分析。结果显示(表4):绿原酸、新绿原酸、隐绿原酸及芸香苷4种核心成分含量之和占多酚类物质总量的74.7%-88.4%。大理产区的绿原酸含量最高;红河产区的七叶亭含量高于其他产区;罗平产区的3-O-阿魏酰奎宁酸、花青素、山柰酚-3-O-芸香糖苷、阿魏酸和二氢槲皮素含量显著高于其他产区;普洱产区的奎宁酸、咖啡酸和水仙苷含量明显更高;临沧K的槲皮素含量显著高于其他产区;临沧Y的东莨菪苷、异槲皮素苷和莨菪亭含量显著高于其他产区。在不同产区的醇化烟叶中,稳定且占比高的核心多酚组分构成了云南清甜香型烟叶的物质基础,差异化分布的非核心多酚则形成了各产区独特的化学指纹图谱。
微生物是调控烟叶品质的核心因素,为明确醇化过程中烟叶微生物与化学成分的潜在关联,选取了相对丰度前10且兼具生物大分子降解功能及产区优势特征的细菌属和真菌属进行相关性分析,结果如图8所示。细菌方面:仅微杆菌属(Microbacterium)与总氮呈显著正相关,其他细菌属与常规化学成分相关性不显著(图8A);真菌方面:短柄霉属(Aureobasidium)与总氮呈显著负相关;白冬孢酵母属(Leucosporidium)与钾离子及钾氯比呈显著正相关(图8B)。
为解析醇化烟叶微生物关键菌属对中性致香物质的影响,对两者的关联性进行分析。结果显示(图9A),在细菌关键菌属中。甲基杆菌属(Methylobacterium)、鞘氨醇单胞菌属(Sphingomonas)、不动杆菌属(Acinetobacter)、黄单胞菌属(Xanthomonas)均与多种中性致香成分呈负相关,其中甲基杆菌属与二氢猕猴桃内酯、糠醛、糠醇、苯乙醇、金合欢基丙酮和香叶基丙酮呈显著性负相关;鞘氨醇单胞菌属与糠醇和氧化异佛尔酮的负相关性显著。盐水弧菌属(Salinivibrio)、微杆菌属(Microbacterium)、泛菌属(Pantoea)则与绝大部分中性致香成分呈正相关,尤其是盐水弧菌属与苯甲醇、α-大马酮、β-紫罗兰酮和巨豆三烯酮呈现显著性正相关。此外,假单胞菌属(Pseudomonas)与香叶基丙酮呈显著正相关,与红没药醇、十六酸甲酯呈显著负相关;马赛菌属(Massilia)与芳樟醇、红没药醇和十六酸甲酯呈显著负相关。在真菌关键菌属中(图9B),弗莱德门氏菌属(Friedmanniomyces)、梅奇酵母属(Metschnikowia)、短柄霉属(Aureobasidium)、根霉属(Rhizopus)则与多数中性致香成分呈负相关,其中弗莱德门氏菌属与氧化异佛尔酮、糠醇和金合欢丙酮呈显著负相关;短柄霉属与红没药醇的负相关性显著,根霉属与苯甲醇呈显著的负相关性。青霉属(Penicillium)与十六酸甲酯呈显著负相关性。该关联性分析揭示了与中性致香成分显著相关的核心菌属,为阐明微生物与烟草品质的互作机制和产香菌属定向筛选提供了有益参考。
烟叶中的多酚物质对醇化烟叶独特香味的形成有较大贡献,而关键微生物菌群在多酚化合物的转化与降解过程中起着关键作用。为此,本研究深入剖析了细菌/真菌群落在属水平的相对丰度与多酚化合物含量之间的潜在关系,如图10所示。在细菌关键菌属中,假单胞菌属(Pseudomonas)、马赛菌属(Massilia)、泛菌属(Pantoea)以及不动杆菌属(Acinetobacter)与10余种多酚呈现负相关,其中马赛菌属与花青素、山奈酚-3-O-芸香糖苷的负相关性显著;寡养单胞菌属(Stenotrophomonas)、黄单胞菌属(Xanthomonas)均与芦丁呈显著负相关。甲基杆菌属(Methylobacterium)、鞘氨醇单胞菌属(Sphingomonas)则与15种以上多酚呈较强正相关,具体表现为甲基杆菌属与新绿原酸、二氢槲皮素和紫云英苷的正相关性显著;鞘氨醇单胞菌属与隐绿原酸、新绿原酸、3-O-阿魏酰奎宁酸、二氢槲皮素、花青素和山奈酚-3-O-芸香糖苷的正相关性显著。
在真菌关键菌属中,短柄霉属(Aureobasidium)、根霉属(Rhizopus)、Knufia、硬皮地星属(Astraeus)、白僵菌属(Beauveria)及青霉属(Penicillium)与多数多酚成分呈负相关,其中短柄霉属与花青素、Knufia与芦丁分别呈显著负相关。白冬孢酵母属(Leucosporidium)、弗莱德门氏菌属(Friedmanniomyces)及梅奇酵母属(Metschnikowia)则与多种多酚呈正相关,具体表现为:白冬孢酵母属与异槲皮苷呈显著正相关;弗莱德门氏菌属与3-O-阿魏酰奎宁酸、二氢槲皮素、隐绿原酸、新绿原酸、紫云英苷呈现显著的正相关性;梅奇酵母属与阿魏酸和3,5-二-O-咖啡酰奎宁酸呈显著正相关。多项研究表明,上述提及的根霉属等真菌属与多酚成分的负相关性,与它们分泌的阿魏酸酯酶、漆酶、过氧化物酶等的直接降解能力密切相关[29-32]。综上所述,烟叶关键菌群具有通过特定代谢酶调控多酚转化与降解的潜力,这为定向调控烟叶风味品质提供了重要依据。
通过宏基因组技术分析不同产区醇化烟叶的微生物群落组成及潜在功能,结果显示:在细菌群落中假单胞菌门是各产区的优势门,其相对丰度介于69.69%-89.22%之间,这表明假单胞菌门在烟叶醇化过程中发挥着关键作用[33-34]。进一步分析优势细菌属发现,优势细菌属呈现出明显的产区特异性,如盐水弧菌属在文山产区丰度较高,甲基杆菌属和鞘氨醇单胞菌属则在罗平产区高度富集。在真菌群落中子囊菌门和毛霉门是主要优势类群,且各产区的优势真菌属组成较为一致,这可能与真菌代谢的普适性有关[13]。KEGG注释的新陈代谢通路在各产区的相对丰度较高,环境信息处理通路则在文山产区高度富集。CAZy分析结果显示,糖苷水解酶和糖基转移酶占碳水化合物活性酶总基因数量的81.03%,且二者在除文山外的其余产区相对丰度均较高,这表明多糖等大分子的降解与转化是烟叶醇化过程中的核心要素[8,35],值得特别关注的是GHs家族。李石头等[8]研究发现烟叶醇化特定阶段富集的糖苷酶和淀粉酶,能高效催化纤维素、淀粉等大分子水解为葡萄糖等还原糖。王文婷等[35]指出,降解淀粉、纤维素和果胶的GHs家族在发酵中后期丰度显著升高,且多数糖苷水解酶与中性致香成分呈正相关,如GH1家族寡糖水解酶相对丰度与茄酮、巨豆三烯酮等呈显著正相关。后续研究将动态监测烟叶醇化过程中优势菌属与GHs等关键活性酶的表达谱,围绕这些优势菌群以及具有多糖降解活性酶的有益菌属系统开展菌株分离、鉴定与开发利用工作,进一步丰富烟草功能菌株资源,拓展其在烟叶提质生物技术开发和工业烟叶原料品质改良中的应用。
在烟草品质形成过程中,微生物与化学成分的相互作用是关键因素。之前的研究证实假单胞菌能以烟碱作为底物,在发酵中降解烟碱以实现自身大量繁殖,这与本文假单胞菌属与烟碱正相关的结果高度契合[36-38]。王祯等[34]发现假单胞菌属能分泌蛋白酶与纤维素酶,促进醇化烟叶中蛋白质等大分子降解转化,并且绿原酸等多酚含量显著提升。该发现合理解释了其与总氮负相关、与绿原酸正相关的内在联系,为筛选有益微生物提供了重要参考。本研究结果还显示盐水弧菌属和曲霉属与多种花香型物质呈较强正相关,说明二者可能是产区特征香气形成的关键驱动因素[39-40]。烟草微生物领域虽未报道过盐水弧菌属,但其作为豆酱等发酵食品的重要嗜盐菌,已证实对风味物质生成至关重要,如与曲霉菌属协同分解蛋白质等大分子物质,促进中性风味成分积累[41-42]。这些研究佐证了盐水弧菌属和曲霉属在烟叶特征香气形成中的潜在功能,为定向筛选产香有益菌株提供了科学依据。关键菌属与多酚的相关性分析显示,甲基杆菌属、鞘氨醇单胞菌属、弗莱德门氏菌属以及梅奇酵母属与新绿原酸、花青素等多酚存在显著正相关,这些核心菌属或许在多酚代谢的转化过程中发挥着正向调控作用。以梅奇酵母属为例,它们分泌的β-葡萄糖苷酶、纤维素酶等胞外水解酶可高效降解植物细胞壁多糖及糖苷,释放出多酚等香气前体物质,揭示了该类菌属在香味物质调控中的应用潜力[43]
通过解析云南不同产区醇化烟叶微生物群落特征及其与化学成分的潜在联系,不仅揭示了各产区微生物群落组成及其化学成分的地域特性,更基于相关性分析筛选出了与烟草风味成分密切关联的重要微生物菌属。这些研究发现为阐明烟草农产品风味形成的微生物驱动机制提供了关键靶点,而功能菌株的定向筛选有望为精准调控发酵工艺与品质改良奠定技术支持[44-47]。然而,当前所发现的核心菌属功能基因及代谢通路与烟草风味物质生成的相互作用机制仍缺乏系统性解析。未来研究将综合利用微生物组、代谢组和转录组等多组学技术,结合无菌发酵验证体系系统解析烟叶醇化过程中微生物群落的动态演替规律,重点探究萜烯类、苯丙氨酸代谢产物等中性致香成分以及多酚类物质与关键微生物菌群的互作网络。通过上述研究为定向筛选具有产香能力的功能菌株,实现烟叶提质生物技术开发与烟叶工业原料改良提供依据和支持。
本文利用宏基因组学、色谱-质谱联用以及斯皮尔曼相关性分析等方法对云南不同产区醇化烟叶的微生物群落及其化学成分进行深入剖析,并对其核心微生物菌属与化学成分的相关性进行研究,得出以下重要结论:微生物群落组成方面,各产区醇化烟叶的优势细菌门均为假单胞菌门,但优势细菌属存在显著的产区差异。例如,文山产区以盐水弧菌属为特征菌属,罗平产区以甲基杆菌属为优势菌属,其余产区多以假单胞菌属为主。各产区的优势真菌门均为子囊菌门,优势真菌属地域差异较小。多样性分析结果显示,临沧产区微生物丰富度和多样性最高,文山产区最低。地理环境与烟叶品种对微生物群落具有双重塑造作用。功能预测研究发现,新陈代谢通路在各产区丰度均较高,而环境信息处理通路在文山产区丰度较高;糖苷水解酶和糖基转移酶基因在碳水化合物活性酶基因数中占比最高,且二者仅在文山产区的丰度较低。化学成分检测结果表明,大理产区的还原糖、总糖和氯离子含量较高,而普洱产区的糖碱比和钾氯比均较高;文山产区的多种中性香型成分显著高于其余产区;所有产区的绿原酸及其异构体和芸香苷4种核心多酚构成稳定且占比高,多种非核心多酚含量在罗平产区烟叶中较高。相关性研究表明:微杆菌属与总氮呈显著正相关,短柄霉属与总氮呈显著负相关;白冬孢酵母属与钾离子、钾氯比呈现显著正相关;盐水弧菌属与多种中性致香物质呈显著性正相关,甲基杆菌属和弗莱德门氏菌属则与多数中性致香成分呈显著负相关;甲基杆菌属、鞘氨醇单胞菌属、弗莱德门氏菌属及梅奇酵母属与新绿原酸、花青素等多酚存在显著性正相关。本研究系统阐述了云南不同产区醇化烟叶微生物群落与化学成分的差异性特征及两者间潜在互作关系,为烟草微生物资源挖掘、功能菌株定向筛选及烟叶品质提升提供了科学依据。
  • 重庆中烟工业有限责任公司科技项目(HX20230207)
  • 中国烟草总公司重点研发项目(110202202023)
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2026年第66卷第1期
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doi: 10.13343/j.cnki.wsxb.20250472
  • 接收时间:2025-06-17
  • 首发时间:2026-01-12
  • 出版时间:2026-01-04
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  • 收稿日期:2025-06-17
  • 录用日期:2025-08-15
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Technology Project of China Tobacco Chongqing Industrial Co., Ltd(HX20230207)
重庆中烟工业有限责任公司科技项目(HX20230207)
Key Research and Development Project of China National Tobacco Corporation(110202202023)
中国烟草总公司重点研发项目(110202202023)
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    1.重庆中烟工业有限责任公司技术中心,重庆
    2.西南大学 农学与生物科技学院,重庆

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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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