微生物发酵口含烟原料提质降害的研究

【作者】 杨静；

【导师】 何腊平；

【作者基本信息】 贵州大学 ， 食品加工与安全， 2021， 硕士

【摘要】 随着社会经济的发展,人们逐渐意识到吸烟的危害并开始尝试选择新型无烟气烟草制品。口含烟为新型无烟气烟草制品之一,目前在国际控烟环境下,各大烟草公司争相进行探索研究。由于口含烟吸食方式异于传统烟草制品,传统烟叶直接入口会产生浓烈辛辣味和刺激性而不适于直接制作口含烟,且烟叶中含有烟碱、特有亚硝胺等有害物质,威胁人体健康。因此,有必要采用合适的工艺手段以提高口含烟的烟草原料适用性和质量。本文旨在利用外源微生物对口含烟烟草原料进行发酵的方法来改善烟草原料的品质,降低其烟碱、特有亚硝胺含量,并利用复合菌进行口含烟烟草原料发酵实验研究。研究结果如下:(1)使用连续流动分析仪对贵州黔东南州生产的云烟87不同等级部位烟叶进行基本化学成分分析,发现中上部叶作为口含烟烟草原料比较适宜。解淀粉芽孢杆菌(GUHP86)和枯草芽孢杆菌(B01)发酵口含烟烟草原料可增强感官特性,缩短发酵时间。从嗅香、满足感、口感特征、异味、口腔余香五个方面对发酵烟草原料进行综合评价,并结合发酵的结团情况,对烟草原料发酵工艺进行优化,GUHP86发酵烟草原料的最优工艺为:接菌量为20%,有糖盐溶液20%,发酵温度37°C,发酵时间为15 d。B01发酵烟草原料的最优工艺为:接菌量为30%,有糖盐溶液25%,发酵温度37°C,发酵时间为15 d。(2)利用GUHP86和B01在最优化工艺条件下发酵,通过SDE提取,K-D法浓缩,气相色谱-三重四级杆串联质谱仪(GC-MS-MS)测定两种微生物发酵前后的致香物质变化,比较发酵前后致香物质的变化。结果发现经过GUHP86、B01发酵后口含烟烟草原料致香物质种类及相对含量均发生变化,并产生一系列具有独特香味的物质。两种微生物发酵都具有增香作用,GUHP86产生的致香物质比B01种类多,含量高。GUHP86发酵后增加9种致香物质,主要是醛、酮、酯类,并减少了7种容易产生刺激性的烯酸、烷类物质。B01发酵后增加6种醇类和酮类物质,减少5种物质。发酵后得到整体更为谐调、香味复杂浓烈、刺激性小、滋味丰富、风味更佳的口含烟烟草原料。(3)利用GUHP86和B01在最优化工艺条件下发酵,通过连续流动分析仪、液相色谱-三重四级杆串联质谱仪(LC-MS-MS)测定发酵前后烟碱和特有亚硝胺N-亚硝基去甲烟碱(NNN)、4-甲基亚硝胺基-1-3-吡啶基-1-丁酮(NNK)含量变化,结果发现GUHP86、B01发酵均具有降低烟碱、NNN和NNK的作用,降解效果最佳时间均为发酵后10-15 d。GUHP86对不同部位烟叶烟碱降解率存在差异,上部最高50.78%,中部次之40.89%,下部最低19.46%;B01降解效果次于GUHP86,上部最高44.36%,中部降解率38.43%,下部降解率15%。GUHP86对NNN的降解率下部叶28.15%,中部叶56.73%,上部叶64.39%;对NNK的降解率,下部叶33.33%%,中部叶59.28%,上部叶63.73%。B01对NNN的降解率下部叶53.23%,中部叶54.86%,上部叶55.60%;对NNK的降解率下部叶41.67%%,中部叶55.43%,上部叶65.68%。(4)利用GUHP86与B01对口含烟烟草原料进行混菌发酵,烟碱降解率最高47.37%,优于单菌发酵,提高了感官特性。以发酵后烟碱含量及感官评价为指标,通过监测发酵过程中烟草原料中烟碱含量的变化,采用感官评分确定混菌发酵的条件。经过混菌发酵后,口含烟烟草原料中的烟碱含量在不同的菌种比、接菌量、有糖盐溶液添加量条件下均发生明显变化,在GUHP86和B01比例为3:1,接种量为30%,有糖盐溶液添加量为20%的条件下,37°C发酵15 d,口含烟烟草原料中烟碱含量达到最低值1.51%,发酵后口含烟烟草原料感官评分达到最高94。更多还原

【Abstract】 With the development of economy,the harm of smoking was gradually realized by the public and the new smoke-free tobacco products would like to be introdued.Snuff cigarette,one of the new substitutes for cigarette,has been developed by many cigarette companies under the international environment for control of tobacco.Comparing to the combustion way of traditional cigarettes,the Snuff cigarette would be put into mouth directly.Therefore the traditional tobacco leaves which will produce a greater pungent taste and irritation is not suitable for direct production of Snuff cigarette.Moreover,tobacco contains nicotine,unique nitrosamines and other harmful substances,which pose a threat to human health.As a result,it is necessary to adopt appropriate technology to improve the suitability and quality of tobacco raw materials.This dissertation aims to improve the quality of tobacco raw materials and reduce the content of nicotine and specific nitrosamine by the fermentation of tobacco raw materials with foreign microorganisms.The experimental study on the fermentation of the prepared tobacco raw materials with compound bacteria was also carried out.The results are shown as follows:(1)The basic chemical components of tobacco(cv.Yunyan 87)leaves of different grades produced in Qiandongnan Prefecture of Guizhou Province were analyzed by continuous flow analyzer.It was found that the middle and upper leaves were suitable for Snuff cigarette.Bacillus amyloliticus(GUHP86)and(B01)had the ability to enhance the sensory properties of Snuff in a shortened fermentation time.For sensory properties from nose,satisfaction,taste characteristics,bad breath,mouth mouth,and agglomeration of tobacco during fermentation,it can be known that the optimal conditions for GUHP86 fermentation were: the inoculation quantity of 20%,sugar salt solution of 20%,fermentation temperature 37°C,fermentation time of 15 days.The optimal fermentation conditions B01 to ferment the for tobacco raw material was: inoculation amount of 30%,sugar and salt solution of 25%,fermentation temperature at 37°C,and fermentation time of 15 d.(2)The tobacco leaves were fermented by GUHP86 and B01 under the optimized conditions,extracted by SDE,concentrated by K-D method,and analyzed by gas chromatography triple quadrupole tandem mass spectrometry(GC-MS-MS).The changes of aroma components before and after fermentation were determined,and the changes of aroma components before and after fermentation were compared.The results showed that the types and relative contents of the aromatic substances were changed after the fermentation by these strains.In addition,some substances with unique fragrance were produced after fermentation.The fermentation have the function of flavor enhancement,GUHP86 produced more aroma compounds than B01.After GUHP86 fermentation,9 kinds of aroma substances were added,mainly aldehydes,ketones and esters,and 7 kinds of irritating enoids and alkanes were reduced.After B01 fermentation,six alcohols and ketones were added and five substances were decreased.The fermentation could help to obtain more harmonious,complex and strong aroma,less irritation,rich taste and better flavor tobacco raw materials.(3)The tobacco leaves were fermented by GUHP86 and B01 under the optimized conditions.The contents of nicotine and two specific nitrosamines,nitrosamine n-nitrosodimethylnicotine(NNN)and 4-methylnitrosamine-1-3-pyridyl-1-butanone(NNK),before and after fermentation were determined by gas continuous flow analyzer and liquid chromatography tandem mass spectrometry(LC-MS-MS).The result showed that both of GUHP86 and B01 could reduce nicotine and nitrosamines,and the best fermentation time for two microorganisms was10-15 d.GUHP86 showed different ability to degraded the amount of nicotine from different part of tobacco,which is in the order of 50.78% for the upper part of tobacco,40.89% for the central part of tobacco,and 19.46% for the the lower part of tobacco.Comparing to GUHP86,B01 showed a lower ability to degrade the amount of nicotine,which is in the order of 44.36% for the upper part of tobacco,38.43% for the central part of tobacco,and 15% for the the lower part of tobacco.The degradation rates of NNN by GUHP86 were 28.15%,56.73% and 64.39% for the lower leaves,middle leaves and upper leaves of tobacco,respectively.The degradation rate of NNK was 33.33%% in the lower leaf,59.28% in the middle leaf and 63.73% in the upper leaf.The degradation rate of NNN by B01 was 53.23% in the lower leaf,54.86% in the middle leaf and 55.60% in the upper leaf,and the degradation rate of NNK was41.67% in lower leaves,55.43% in middle leaves and 65.68% in upper leaves.(4)The co-fermentation by GUHP86 and B01 could degrade 47.37% of nicotine in tobacco and improve its sensory properties,which were higher than that from single fermentation.Taking the nicotine content and sensory properties as evaluated base,the change of nicotine content in tobacco raw materials during fermentation was monitored,and the sensory score was used to determine the fermentation conditions of co-fermentation.It can be found that the nicotine content in tobacco raw material can be significantly changed under different strain ratio,inoculation quantity,sugar,salt solution.The optimized conditions for co-fermentation was starch GUHP86 and B01 ratio of 3:1,inoculation amount of30%,sugar salt solution of 20%,fermentation time of 15 d,and temperature of 37°C.Under the optimized conditions,the nicotine content in tobacco raw material was 1.51%,which can endow the mouth smoke tobacco with 94 sensory score.更多还原