一株源于醇化烟叶表面高效降解TSNA 菌株AS97 的分离筛选、鉴定及应用

Identification and primary application of TSNA degrading bacterial strain AS97 isolated from aging tobacco leaves

摘要:

摘要:【目的】烟草特有亚硝胺(Tobacco-specific nitrosamines，简称TSNA)是烟叶中的主要致癌物质。本研究从筛选建立的特有菌库中发现了1 株可有效降解TSNA 的菌株AS97，并对其进行了鉴定及初步应用研究。【方法】采用富集驯化及选择培养基筛选得到硝酸盐与亚硝酸盐转化能力最强的菌株AS97;根据菌株的形态特征、生理生化特性及16S rRNA 基因序列分析对其进行鉴定;并将AS97 自制发酵液喷施于烟丝表面，确定适宜接种量和发酵条件，采用LC-MS /MS(液相色谱串联质谱)方法检测TSNA 中四种主要成分的含量。【结果】菌株AS97 源于云南玉溪烤烟样品表面，经分析确定其为荧光假单胞菌(Pseudomonas fluorescens，GenBank 登录号:JF449445)。将AS97 自制发酵液以5% 的接种量喷施于烟丝，30℃(相对湿度是60%)条件下发酵10 d 检测烟叶中硝酸盐、亚硝酸盐、TSNA、4-(N-甲基-亚硝基) -1-(3-吡啶基)-1-丁酮(NNK)、N-亚硝基去甲基烟碱(NNN)、N-亚硝基新烟草碱(NAT) 及N-亚硝基假木贼碱(NAB) 的转化率分别达到68. 77%、45. 57%、45. 47%、59. 08%、38. 79%、21. 41% 及11. 76%。相关性分析结果表明烤烟中硝酸盐、亚硝酸盐与TSNA 的含量均呈极显著相关(P＞0.01)，进一步证实硝酸盐与亚硝酸盐是TSNA 的主要前体物质。【结论】醇化烟叶表面的荧光假单胞菌AS97 能够显著降低TSNA 的含量。本文首次报道了源于醇化烤烟表面对TSNA 有良好转化能力的Pseudomonas fluorescens。可将其开发成新型微生物制剂，应用于低害卷烟制品的生 产实践中。

Abstract:

Abstract:［Objective］ The purpose of this work was to screen strains having tobacco-specific nitrosamines (TSNA) deteriorating activity，isolated from the inner and superficial of tobacco plants． Then strain AS97 was isolated and identified for further application.[Methods] Strain AS97，with the highest conversion ability against both nitrate and nitrite，was screened by enrichment and selective medium． The strain was identified by rphological，physio-biochemical characteristics and 16S rRNA gene sequence analysis．The concentration of 4-(methylnitrosamino)-1-(3-pyridy)-1-butanone (NNK)，N-nitrosonicotine (NNN)，N-nitrosoanatabine (NAT) and N-nitrosoanabasine (NAB) were determined by LC-MS/MS．The fermentation broth of strain AS97 was spraied on the leaves of tobacco to define inoculum concentration and fermentation condition. [Results] AS97 was identified as Pseudomonas fluorescens (Genbank accessionnumber:JF 449445)．Under the optimal growth conditions with inoculum concentration of 5%，at 30℃ for 10 d，AS97 had high biological activity against NNK and NNN with a degradation rate of 59. 08% and 38.79%，respectively．The correlation analysis displayed a pronounced correlation(p ＞0.01) among the concentration of nitrate，nitrite and TSNA．Furthermore，the results also exhibited that nitrate and nitrite were antecedent substance of TSNA. [Conclusion] These results indicated that Pseudomonas fluorescens AS97 could be a promising microorganism in the practice of non-harmful cigarette production．