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【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。
【Objective】 Tobacco-specific nitrosamines (TSNAs) are the main carcinogens in tobacco leaves. In this study, we found 1 strain TS97 that can effectively degrade TSNA from the endemic bacteria screened and identified and preliminary application research. 【Method】 The strain AS97 with the highest nitrate and nitrite conversion ability was screened by enrichment, acclimation and selection medium. The strain AS97 was identified according to its morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. AS97 Homemade fermentation broth was sprayed on the surface of tobacco to determine the appropriate inoculum size and fermentation conditions. The contents of four main components in TSNA were detected by LC-MS / MS (liquid chromatography-tandem mass spectrometry). 【Result】 Strain AS97 originated from the surface of Yuxi flue-cured tobacco samples from Yunnan. The strain was identified as Pseudomonas fluorescens (GenBank accession number: JF449445). The AS97 homemade fermentation broth was sprayed on tobacco at 5% inoculum for 10 d to detect nitrate, nitrite, TSNA, 4- (N-methyl- Nitroso) -1- (3-pyridyl) -1-butanone (NNK), N- nitrosomylnicotine (NNN) The conversion of nitrosamylamine (NAB) reached 68.77%, 45.57%, 45.47%, 59.08%, 38.79%, 21.41% and 11.76%, respectively. Correlation analysis showed that the content of nitrite, nitrite and TSNA in flue-cured tobacco showed a highly significant correlation (P> 0.01), which further confirmed that nitrate and nitrite were the main precursors of TSNA. 【Conclusion】 Pseudomonas fluorescens AS97 on alcoholic tobacco leaves can significantly reduce the content of TSNA. This paper reports for the first time the Pseudomonas fluorescens derived from the surface of alcoholic flue-cured tobacco that has a good ability to transform TSNA. Can be developed into a new type of microbial agents, used in the production of low-harm cigarette products in practice.