抑制雪茄烟叶TSNAs生成的弯曲芽胞杆菌Ni3菌株的分离筛选及其关键基因挖掘  

Isolation,screening and key gene discovery of a Bacillus flexus strain Ni3 to inhibit formation of TSNAs in cigar tobacco

作　　者：叶长文[1] 董欣雨 崔中月 黄阔 刘丹丹 魏雪团[2] 刘萌萌 李栋[1] YE Changwen;DONG Xinyu;CUI Zhongyue;HUANG Kuo;LIU Dandan;WEI Xuetuan;LIU Mengmeng;LI Dong(Zhengzhou Tobacco Research Institute of CNTC,Zhengzhou 450001,China;Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]中国烟草总公司郑州烟草研究院,郑州高新技术产业开发区450001 [2]华中农业大学,武汉市洪山区430070

出　　处：《烟草科技》2024年第8期32-41,共10页Tobacco Science & Technology

基　　金：湖北省烟草公司科技项目“雪茄烟叶降TSNAs特色菌种资源精准筛选和应用技术研究”[027Y2021-015];中国烟草总公司重大科技项目“手工雪茄烟加工过程中霉变生防控制技术研究与应用”[11202201034(XJ-05)]。

摘　　要：为了明确微生物抑制烟草特有亚硝胺(TSNAs)形成的机制,以TSNAs的关键前体物烟碱为底物,经定向筛选从雪茄烟叶中分离出1株耐高温且高效降解烟碱的弯曲芽胞杆菌(Bacillus flexus)Ni3。通过摇瓶发酵和仿真农业堆积发酵试验评估了菌株Ni3对雪茄烟叶中TSNAs的抑制能力,并通过全基因组测序分析初步挖掘出菌株Ni3中降解TSNAs前体物的关键基因。结果表明:①菌株Ni3在初始质量浓度为1.5 g/L的烟碱发酵培养基中72 h时烟碱的降解率为56%;在4种TSNAs成分初始质量浓度为10 mg/L的TSNAs发酵培养基中,NNN、NNK、NAT和NAB的降解率分别为23%、64%、26%和27%;②将Ni3菌悬液应用于雪茄烟叶CX-7发酵过程中,在发酵第9天时NNN、NNK、NAT和NAB的积累量分别减少41%、53%、53%和53%,总TSNAs减少46%;③全基因组测序分析表明,菌株Ni3中有7个基因可能参与调控TSNAs前体物的代谢。因此,弯曲芽胞杆菌Ni3在抑制雪茄烟叶发酵过程中TSNAs形成方面具有较大的应用潜力。To elucidate the mechanism of microbial inhibition on the formation of tobacco-specific nitrosamines(TSNAs),Bacillus flexus Ni3,a high-temperature-resistant strain capable of degrading nicotine with high efficiency,was isolated from cigar tobacco using the major precursors of TSNAs as the substrate.The inhibitory ability of strain Ni3 on TSNAs in cigar tobacco leaves was evaluated through shake flask culture experiments and simulated agricultural stack fermentation.The key genes involved in the degradation of TSNAs in strain Ni3 were identified by whole genome sequencing.The results showed that:1)The nicotine degradation rate of strain Ni3 was 56%within 72 h in the nicotine fermentation medium with an initial nicotine mass concentration of 1.5 g/L.When the initial concentration of TSNAs was 10 mg/L,the degradation rates of NNN,NNK,NAT and NAB were 23%,64%,26%,and 27%,respectively.2)Compared with the control group,the accumulation amounts of NNN,NNK,NAT and NAB on the 9th day of fermentation with Ni3 bacterial suspension decreased by 41%,53%,53%and 53%,respectively,and the total TSNAs decreased by 46%.3)The whole genome sequencing analysis showed that there were 7 genes of strain Ni3 that might be involved in the degradation of TSNAs precursors.Therefore,B.flexus Ni3 has greater application potential in inhibiting the formation of TSNAs during the fermentation of cigar tobacco leaves.

关 键 词：雪茄烟 烟草特有亚硝胺(TSNAs) 弯曲芽胞杆菌Ni3 烟碱 全基因组测序 

分 类 号：TS414[农业科学—烟草工业]