微生物发酵降解造纸法烟草薄片有害前体物的研究
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摘要
造纸法烟草薄片是以烟梗和烟末等烟草加工废弃物为原料的一种资源再生利用产品，具有焦油含量低，有害成分释放量小等特点。但是由于烟梗中以纤维素和果胶为主的细胞壁物质含量较多，会导致造纸法烟草薄片有害成分的释放量增加。因此，降解造纸法烟草薄片原料中纤维素和果胶等大分子物质的含量对实现调控造纸法烟草薄片烟气焦油有害成分组成和含量至关重要。
本课题根据造纸法烟草薄片化学组成的特性，从造纸法烟草薄片原料中分离筛选出能够降解纤维素、果胶、蛋白质和淀粉等大分子物质的微生物菌株，并利用这些微生物进行发酵处理，达到调控造纸法烟草薄片和烟草原料烟气焦油组成的目的。同时，采用热重红外联用技术和热重质谱联用技术研究菌株发酵对造纸法烟草薄片热裂解过程中主要有害成分和致香成分释放轨迹的影响作用机理。
（1）以降解纤维素、果胶、蛋白质及淀粉能力为评价指标，采用平板分离技术从造纸法烟草薄片原料中分离出优势菌群，并对优势菌群进行筛选、纯化及种群鉴定研究。研究结果显示，菌株GX5的果胶酶活性最高，达295.79U/mL，其蛋白酶活性也较高；GX6的淀粉酶活性最强，达到31.74U/mL；菌株GX8的纤维素酶活性最高，达到435.52U/mL。三种优势菌株的种群鉴定结果为：GX5为枯草芽孢杆菌（Bacillus subtilis），GX6为蜡状芽孢杆菌（Bacillus cereus）GX8为短短芽孢杆菌（Brevibacillus brevis）。通过发酵工艺优化及正交实验设计最终确定较佳的发酵培养基成分及最佳发酵工艺为：玉米粉2%、黄豆粉3.0%、淀粉2.0%、桔皮粉1.5%、培养24h、培养温度30℃、起始pH值为7.0，整个液体培养的固含量为7.5%；发酵时，三种高酶活菌先单独发酵12h后再按照GX5：GX6：GX8为1:2:2的进行混合，混合均匀后继续发酵24h。
（2）研究了各高酶活菌株发酵对造纸法烟草薄片原料和造纸法烟草薄片化学组成的影响；同时，采用小型热裂解装置进行热裂解实验模拟烟草燃吸过程，采用气相色谱质谱联用分析（GC/MS）对热裂解焦油组成特性进行表征，分析各菌株发酵处理后热裂解焦油中有害成分的变化情况。实验结果表明，菌株发酵处理会导致造纸法烟草薄片中纤维素、果胶、蛋白质及淀粉等大分子物质含量降低，总糖和还原糖的含量增加。从调控原料化学组成来看，采用对造纸法烟草薄片基片浆料进行菌株发酵处理优于直接处理烟草原料。混合菌株发酵在抑制焦油中杂化化合物和PAHs的生成，降解焦油中烟碱含量以及促进焦油中酮类物质的生成方面的能力明显优于其他菌株。菌株GX8和混合菌株发酵抑制烟草原料和造纸法烟草薄片热裂解烟气中CO生成的效果均优于菌株GX5和菌株GX6，烟气中CO的生成主要与原料中纤维素含量有关。
（3）以还原糖、挥发性组分以及热裂解焦油中致香成分的含量为评价指标，研究各高酶活菌株及高酶活混合发酵液对造纸法烟草提取物的发酵效果。研究发现，菌株发酵处理促使蛋白质组分热裂解过程向更有利于形成2,6-二甲基吡嗪、大马酮和新植二烯等致香成分的化学反应进行。混合菌株发酵处理在改善烟梗提取物挥发性成分及增加烟气焦油中香气化合物含量方面都优于单一菌株。不同发酵时间对烟梗提取物热裂解焦油中吡嗪类、吡啶类和吡咯类化合物产生显著影响。烟梗提取物热裂解焦油的组成特性不仅与其烟梗提取物中的挥发性组分有关，更受热裂解升温过程中发生的美拉德反应影响。
（4）采用热重红外联用技术（TG-FTIR）和热重质谱联用技术（TG-MS）对造纸法烟草薄片热裂解焦油中主要有害成分和致香成分的释放轨迹进行研究。研究发现，混合菌株发酵处理会导致造纸法烟草薄片和烟梗提取物热稳定性减弱，还会导致造纸法烟草薄片热裂解过程中酮类和酚类化合物在300℃左右出现新的释放峰，使酚类化合物释放温度向低温区移动，避免高温区芳烃类化合物的释放，从而减少了烟气中有害成分的形成。此外混合菌株发酵处理不仅会导致烟草薄片热裂解过程糠醛、茚和萘的释放量减少，同时会导致其释放温度区间更加集中。经过混合菌株发酵处理后，糠醛的释放温度区间主要集中在400至650o℃；茚和萘的释放温度区间集中在500至800℃；这些有害成分释放温度的集中有助于调控烟气中这些物质的形成。苯酚的释放温度区间主要集中在400至600℃。混合菌株发酵处理对烟梗提取物热裂解过程中吡咯和3-乙烯基吡啶的释放温度区间没有影响作用，但是会导致3-乙烯基吡啶的释放量的减小。同时，混合菌株发酵处理不仅会导致2,6-二甲基吡嗪释放温度区间的增大，而且会导致其释放量的增加，对增加卷烟烟气香味起到重要作用。
The paper-process reconstituted tobacco, using tobacco wastes as raw materials, can beregards as a resource recycling products. It has the following and main characteristics: lowyields of tars and small releasing amounts of harmful compounds. The high content ofhazardous compound precursors, including cellulose, pectin andproteins in tobacco stem willlead to increasing the evolution of harmful compounds.Therefore, it was important todegradethe hazardous compound precursors of paper-process reconstituted tobacco to regulate thecomponent and yield of harmful compounds.
According to the chemical composition characteristics of the paper of paper-processreconstituted tobacco, thisstudy focused on the separation of strains from the raw material,which has high capacity of degrading cellulose, pectin, protein and starch, then using thesemicrobial strains fermentation regulating the components of tar from paper-processreconstituted tobacco and raw materials. Meanwhile, the influences of fermentation processon the evolution mechanism of main harmful and chiefly fragrant compounds were studied byTG-FTIR and TG-MS.
Based on the degradation capacity of cellulose, pectin, protein and starch, thepredominant bacterium were separated by theplate separation technology, and thenpurification and population identification.The research results show that the strain GX5hashighest pectinase activity, the value up to295.79U/mL, protease activity of the strainGX5was also higher; the strain GX6has highest amylase activity, the value up to31.74U/mL;the strain GX8has highest celluloseactivity, the value up to435.52U/mL. The threepredominant strains were identified as Bacillus subtilis (GX5), Bacillus cereus (GX6) andBrevibacillus brevis (GX8), respectively.Ingredients and the best fermentation process:2%ofthe corn flour,3.0%of soybean meal,2.0%of starch,1.5%of the orange peel powder,cultured24h, incubation temperature30°C, and initial pH value was7.0, the solids content of7.5%. The optimal proportion of GX5: GX6: GX8was1:2:2identified by the orthogonalexperimental design.
Study on the effect of high enzyme activity strains on the chemical composition ofpaper-process reconstituted tobacco and raw materials. Meanwhile, thermal pyrolysis wasused to simulatethe reaction of tobacco burning suction process, the evolution tars during thisreaction were analyzed by GC/MS. Experimental results show that the fermentation processwill reduce the content of cellulose, pectin, protein and starch, however, total increase thecontent of total sugar and reducing sugar.From the regulation of the chemical composition of raw materials, the fermentation of reconstituted tobacco pulp is superior to the fermentation ofraw material. Mixed strains fermentation inhibit the formation of hybrid generationcompounds and PAHs, promote the generation of ketones, and the effect capacity of mixedstrains fermentation were better than the other single strain. The inhibition capacity of COduring the strain GX8and the mixed strain fermentation process are better than the strainsGX5and GX6. The formation of CO is directly related to the cellulose content.
The effect of high enzyme activity strains and mixed strains fermentation on thecomponent and yield of reducing sugar, volatile components, and pyrolysis tars were studied.This study found that the fermentation process promote the formation of2,6-dimethylpyrazine, Malaysiaone and neophytadiene. The improve effect of volatile components andfragrant compounds during the mixed strains fermentation process are superior to single strainfermentation process.The fermentation time has significant effect on the releasing of pyrazine,pyridine and pyrrole.The composition characteristics of pyrolysis tar werenot only affectsbythe volatile components of tobacco stem extracts, but also affect by the Maillard reaction inthe heating process.
The releasing laws of harmful and fragrant compounds from paper-process reconstitutedtobacco pyrolysis were studied by TG-FTIR and TG-MS. This study found that mixedfermentation process will lead to the thermal stability of reconstituted tobacco and tobaccostem extract reduce. Anew release peak of phenols appearsaround300℃during mixed strainfermentation process. Meanwhile, the mixed strain fermentation lead to the releasetemperature of ketones and phenols shift to low temperature zone, avoiding the formation ofaromaticsin high temperature zone, finally, reducing the formation of harmfulcomponents.Mixed strain fermentation will not only result in the reduction of furfural, indeneand naphthalene releasing, but also lead to the releasing temperature more focused. Aftermixed strain fermentation process, furfural release temperature range mainly in the400to650℃; the releasing temperature range of indene and naphthalene is concentrated in the500to800℃. The release temperature range of Phenols is mainly concentrated in the400to600℃. Mixed strain fermentation has nosignificant effect on the releasing temperature ofpyrrole and3–vinylpyridine, but will reduce the formation of3–vinylpyridine. Meanwhile,the releasing temperature range of2,6-dimethyl pyrazine will be extended, and the releasing amount of2,6-dimethyl pyrazine will be increased during the mixed strain fermentationprocess.
本课题根据造纸法烟草薄片化学组成的特性，从造纸法烟草薄片原料中分离筛选出能够降解纤维素、果胶、蛋白质和淀粉等大分子物质的微生物菌株，并利用这些微生物进行发酵处理，达到调控造纸法烟草薄片和烟草原料烟气焦油组成的目的。同时，采用热重红外联用技术和热重质谱联用技术研究菌株发酵对造纸法烟草薄片热裂解过程中主要有害成分和致香成分释放轨迹的影响作用机理。
（1）以降解纤维素、果胶、蛋白质及淀粉能力为评价指标，采用平板分离技术从造纸法烟草薄片原料中分离出优势菌群，并对优势菌群进行筛选、纯化及种群鉴定研究。研究结果显示，菌株GX5的果胶酶活性最高，达295.79U/mL，其蛋白酶活性也较高；GX6的淀粉酶活性最强，达到31.74U/mL；菌株GX8的纤维素酶活性最高，达到435.52U/mL。三种优势菌株的种群鉴定结果为：GX5为枯草芽孢杆菌（Bacillus subtilis），GX6为蜡状芽孢杆菌（Bacillus cereus）GX8为短短芽孢杆菌（Brevibacillus brevis）。通过发酵工艺优化及正交实验设计最终确定较佳的发酵培养基成分及最佳发酵工艺为：玉米粉2%、黄豆粉3.0%、淀粉2.0%、桔皮粉1.5%、培养24h、培养温度30℃、起始pH值为7.0，整个液体培养的固含量为7.5%；发酵时，三种高酶活菌先单独发酵12h后再按照GX5：GX6：GX8为1:2:2的进行混合，混合均匀后继续发酵24h。
（2）研究了各高酶活菌株发酵对造纸法烟草薄片原料和造纸法烟草薄片化学组成的影响；同时，采用小型热裂解装置进行热裂解实验模拟烟草燃吸过程，采用气相色谱质谱联用分析（GC/MS）对热裂解焦油组成特性进行表征，分析各菌株发酵处理后热裂解焦油中有害成分的变化情况。实验结果表明，菌株发酵处理会导致造纸法烟草薄片中纤维素、果胶、蛋白质及淀粉等大分子物质含量降低，总糖和还原糖的含量增加。从调控原料化学组成来看，采用对造纸法烟草薄片基片浆料进行菌株发酵处理优于直接处理烟草原料。混合菌株发酵在抑制焦油中杂化化合物和PAHs的生成，降解焦油中烟碱含量以及促进焦油中酮类物质的生成方面的能力明显优于其他菌株。菌株GX8和混合菌株发酵抑制烟草原料和造纸法烟草薄片热裂解烟气中CO生成的效果均优于菌株GX5和菌株GX6，烟气中CO的生成主要与原料中纤维素含量有关。
（3）以还原糖、挥发性组分以及热裂解焦油中致香成分的含量为评价指标，研究各高酶活菌株及高酶活混合发酵液对造纸法烟草提取物的发酵效果。研究发现，菌株发酵处理促使蛋白质组分热裂解过程向更有利于形成2,6-二甲基吡嗪、大马酮和新植二烯等致香成分的化学反应进行。混合菌株发酵处理在改善烟梗提取物挥发性成分及增加烟气焦油中香气化合物含量方面都优于单一菌株。不同发酵时间对烟梗提取物热裂解焦油中吡嗪类、吡啶类和吡咯类化合物产生显著影响。烟梗提取物热裂解焦油的组成特性不仅与其烟梗提取物中的挥发性组分有关，更受热裂解升温过程中发生的美拉德反应影响。
（4）采用热重红外联用技术（TG-FTIR）和热重质谱联用技术（TG-MS）对造纸法烟草薄片热裂解焦油中主要有害成分和致香成分的释放轨迹进行研究。研究发现，混合菌株发酵处理会导致造纸法烟草薄片和烟梗提取物热稳定性减弱，还会导致造纸法烟草薄片热裂解过程中酮类和酚类化合物在300℃左右出现新的释放峰，使酚类化合物释放温度向低温区移动，避免高温区芳烃类化合物的释放，从而减少了烟气中有害成分的形成。此外混合菌株发酵处理不仅会导致烟草薄片热裂解过程糠醛、茚和萘的释放量减少，同时会导致其释放温度区间更加集中。经过混合菌株发酵处理后，糠醛的释放温度区间主要集中在400至650o℃；茚和萘的释放温度区间集中在500至800℃；这些有害成分释放温度的集中有助于调控烟气中这些物质的形成。苯酚的释放温度区间主要集中在400至600℃。混合菌株发酵处理对烟梗提取物热裂解过程中吡咯和3-乙烯基吡啶的释放温度区间没有影响作用，但是会导致3-乙烯基吡啶的释放量的减小。同时，混合菌株发酵处理不仅会导致2,6-二甲基吡嗪释放温度区间的增大，而且会导致其释放量的增加，对增加卷烟烟气香味起到重要作用。
The paper-process reconstituted tobacco, using tobacco wastes as raw materials, can beregards as a resource recycling products. It has the following and main characteristics: lowyields of tars and small releasing amounts of harmful compounds. The high content ofhazardous compound precursors, including cellulose, pectin andproteins in tobacco stem willlead to increasing the evolution of harmful compounds.Therefore, it was important todegradethe hazardous compound precursors of paper-process reconstituted tobacco to regulate thecomponent and yield of harmful compounds.
According to the chemical composition characteristics of the paper of paper-processreconstituted tobacco, thisstudy focused on the separation of strains from the raw material,which has high capacity of degrading cellulose, pectin, protein and starch, then using thesemicrobial strains fermentation regulating the components of tar from paper-processreconstituted tobacco and raw materials. Meanwhile, the influences of fermentation processon the evolution mechanism of main harmful and chiefly fragrant compounds were studied byTG-FTIR and TG-MS.
Based on the degradation capacity of cellulose, pectin, protein and starch, thepredominant bacterium were separated by theplate separation technology, and thenpurification and population identification.The research results show that the strain GX5hashighest pectinase activity, the value up to295.79U/mL, protease activity of the strainGX5was also higher; the strain GX6has highest amylase activity, the value up to31.74U/mL;the strain GX8has highest celluloseactivity, the value up to435.52U/mL. The threepredominant strains were identified as Bacillus subtilis (GX5), Bacillus cereus (GX6) andBrevibacillus brevis (GX8), respectively.Ingredients and the best fermentation process:2%ofthe corn flour,3.0%of soybean meal,2.0%of starch,1.5%of the orange peel powder,cultured24h, incubation temperature30°C, and initial pH value was7.0, the solids content of7.5%. The optimal proportion of GX5: GX6: GX8was1:2:2identified by the orthogonalexperimental design.
Study on the effect of high enzyme activity strains on the chemical composition ofpaper-process reconstituted tobacco and raw materials. Meanwhile, thermal pyrolysis wasused to simulatethe reaction of tobacco burning suction process, the evolution tars during thisreaction were analyzed by GC/MS. Experimental results show that the fermentation processwill reduce the content of cellulose, pectin, protein and starch, however, total increase thecontent of total sugar and reducing sugar.From the regulation of the chemical composition of raw materials, the fermentation of reconstituted tobacco pulp is superior to the fermentation ofraw material. Mixed strains fermentation inhibit the formation of hybrid generationcompounds and PAHs, promote the generation of ketones, and the effect capacity of mixedstrains fermentation were better than the other single strain. The inhibition capacity of COduring the strain GX8and the mixed strain fermentation process are better than the strainsGX5and GX6. The formation of CO is directly related to the cellulose content.
The effect of high enzyme activity strains and mixed strains fermentation on thecomponent and yield of reducing sugar, volatile components, and pyrolysis tars were studied.This study found that the fermentation process promote the formation of2,6-dimethylpyrazine, Malaysiaone and neophytadiene. The improve effect of volatile components andfragrant compounds during the mixed strains fermentation process are superior to single strainfermentation process.The fermentation time has significant effect on the releasing of pyrazine,pyridine and pyrrole.The composition characteristics of pyrolysis tar werenot only affectsbythe volatile components of tobacco stem extracts, but also affect by the Maillard reaction inthe heating process.
The releasing laws of harmful and fragrant compounds from paper-process reconstitutedtobacco pyrolysis were studied by TG-FTIR and TG-MS. This study found that mixedfermentation process will lead to the thermal stability of reconstituted tobacco and tobaccostem extract reduce. Anew release peak of phenols appearsaround300℃during mixed strainfermentation process. Meanwhile, the mixed strain fermentation lead to the releasetemperature of ketones and phenols shift to low temperature zone, avoiding the formation ofaromaticsin high temperature zone, finally, reducing the formation of harmfulcomponents.Mixed strain fermentation will not only result in the reduction of furfural, indeneand naphthalene releasing, but also lead to the releasing temperature more focused. Aftermixed strain fermentation process, furfural release temperature range mainly in the400to650℃; the releasing temperature range of indene and naphthalene is concentrated in the500to800℃. The release temperature range of Phenols is mainly concentrated in the400to600℃. Mixed strain fermentation has nosignificant effect on the releasing temperature ofpyrrole and3–vinylpyridine, but will reduce the formation of3–vinylpyridine. Meanwhile,the releasing temperature range of2,6-dimethyl pyrazine will be extended, and the releasing amount of2,6-dimethyl pyrazine will be increased during the mixed strain fermentationprocess.
引文
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