利用酶法和外加香料法对下部烟叶的增香提质研究
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摘要
烟草，作为特殊的嗜好性产品，是我国重要的经济作物之一。在农田采收、烟叶贮藏、卷烟加工过程中，会产生大量的低等级烟叶如下部烟等，此类低等级烟叶通常品质差，利用率低，造成了极大的资源浪费。同时，由于中式卷烟品种结构的调整，中上等烟叶用量早已不足，若能将下部低等烟叶经处理转化成制造高档低焦油混合型卷烟的补充原料，对于提高我国下部低等烟叶利用价值，提高经济效益及环境效益都具有重要现实意义。本课题针对我国下部等低等级烟叶吸食品质差，可用性差，生产利用率低的问题，对其进行了提质增香的改良研究，旨在改良其内在品质及感官特性，提高其加工品质及可用性。主要研究内容及结果如下：
目前我国上、下部烟叶的品质较差，相对于品质较好的中部烟叶，利用率较低，但造成这种结果的不同部位烟叶之间内在化学成分的具体差异原因尚不明确。因此首先针对我国不同部位烟叶之间缺乏化学成分差异性分析的缺陷，考察了上、中、下不同部位烟叶之间基本化学成分、大分子物质之间的差异，并通过主成分分析法比较了不同部位烟叶中性香味成分的差异，结果表明：上部烟叶水溶性总糖及还原糖含量低，烟碱含量高，因此烟叶成分协调性差，刺激性强；而中部烟叶各指标基本适中，是不同部位烟叶中品质最好的，烟叶整体感官品质优良；下部烟叶烟碱含量低，因此烟气劲头小，浓度低。细胞壁物质含量高，中性香味成分含量低，香气质量差。中性香味成分主成分分析结果表明：上部烟叶与中部烟叶之间的差异主要与类胡萝卜素降解产物相关，而下部烟叶与中部烟叶之间的差异与西柏烷类降解产物、萜类降解产物、美拉德反应产物和苯丙氨酸降解产物均有关。
选择细胞壁物质含量高、具有代表性的下部低等烟叶进行低等烟的提质研究。首先通过细胞壁酶的作用对烟叶本身进行提质，主要采用了以多糖酶和蛋白酶复配进行非溶液酶解烟叶细胞壁物质和酶解法提取烟叶浸膏并反加香于下部低等烟叶两种方法。通过将多糖酶、中性蛋白酶、淀粉酶、果胶酶单独及复配喷施于下部低等级烟叶样品（亳州X3L）进行了酶降解烟叶细胞壁物质的研究。以烟叶细胞壁物质降解效果为指标，确定了多糖酶、蛋白酶作用效果适宜。通过单因素及正交试验确定了多糖酶和蛋白酶复配降解亳州X3L细胞壁物质的最佳工艺条件：多糖酶用量490U/g，蛋白酶用量208U/g，酶解时间4h，在此条件下烟叶细胞壁物质含量可降解至25.74%。并通过扫描电镜观察、GC-MS检测、TG-DSC热重分析考察了酶处理后烟叶的表面结构、燃烧性能及感官性质的变化，最后考察了复配酶处理对于烟叶及烟气成分的影响。结果表明：在多糖酶和蛋白酶的共同作用下，亳州X3L烟叶的细胞壁结构发生了一定程度的破坏，有利于烟叶中的香气成分转移至烟气中；复配酶处理增加了烟叶中美拉德反应产物类香味成分及中性香味成分总量的含量；复配酶处理仅略微减缓了烟草多糖类物质的热解和碳化过程，基本不改变烟叶的燃烧性质。经复配酶处理后的烟叶水溶性糖含量和成分协调性均得到了提高。
由于果胶酶喷施于烟叶造成烟叶结构较大程度的破坏，因此利用了果胶酶解法提取烟草浸膏并用于亳州X3L加香，结果表明当果胶酶用量为64U/g，酶解时间2h时得到的烟叶浸膏，用于为低等级烟草增香时，感官评价得分最高。果胶酶处理制备的烟叶浸膏为亳州X3L加香适宜添加量烟丝质量的30～40g/g，添加此烟草浸膏后的烟叶和烟气中性香味成分的含量均得到提高。
针对下部低等烟叶萜类降解产物等中性香味成分含量低的问题，对其进行了外加富含萜类的中草药（艾叶）提取物的增香研究。分别选择以艾叶精油、艾叶浸膏两种添加形式实现为低等烟叶的针对性补香。首先通过同时蒸馏萃取（SDE）、固相微萃取（SPME）与全二维气相色谱飞行时间质谱技术（GC×GC-TOFMS）联用分析了艾叶中的挥发性成分，结果表明艾叶中的挥发性成分主要为单萜(C10H16)和倍半萜(C15H24）类及其衍生物。接下来以艾叶精油形式进行增香实验。艾叶精油烟气转移实验证实，艾叶中的萜类物质可以增加烟气中香味成分的含量。针对艾叶精油在用于低等级烟草增香时易挥发、释放不均匀的问题，采用了微乳化技术对艾叶油进行了包埋。分别考察了不同表面活性剂、助表面活性剂及Km值对艾叶油微乳区域的影响，确定了以乙醇为助表面活性剂、吐温80为表面活性剂，Km=3：1时，能够与艾叶油形成载油量较高、加水稀释稳定的的微乳体系；并考察了此微乳体系的性质：以（吐温80+乙醇）(km=3)：艾叶油=7：3的微乳体系在水分含量超过66%时，微乳结构为水包油型；此微乳体系在47°C发生相转变；不同水分含量的微乳贮藏2个月，微乳体系粒径及PDI均保持稳定。将制备的艾叶油微乳加入烟丝中，分析了艾叶精油及微乳在主流烟气中的逐口抽吸转移规律，结果表明艾叶精油经包埋后再添加至烟草中，可使得烟丝60天时持留率由＜60%提高至＞80%，并促使主流烟气抽吸口数之间艾叶油分布趋于均匀化，在一定程度上起到了缓释加香的作用。
针对直接添加艾叶精油易挥发，烟丝持留率低的问题，设计了以艾叶浸膏形式为亳州X3L增香的实验。结果表明添加艾叶浸膏与直接添加艾叶精油相比，烟丝持留率得到了提高，但低于添加艾叶精油微乳的样品。艾叶浸膏直接裂解与添加至烟丝后的裂解反应证实，艾叶浸膏中的挥发性成分主要在300℃以下转移，300～600℃下部分转移，600～900℃发生裂解反应；艾叶浸膏中的挥发性成分可以在烟草燃烧条件下转移至烟气中，为亳州X3L低等烟叶增加萜类香味成分的含量，少量增加烟气总粒相物、焦油含量，对烟气其它常规成分影响不大。当艾叶浸膏在烟丝中的添加量为30g/g时，香气协调性好。
Tobacco, as special hobby product, is an important economic crop in China. During theprocessing of harvesting, storage, manufacturing process, large amounts of low-grade tobaccoare produced. The abandoned low grade tobacco with poor quality and low utilization causedgreat waste of resources, as well as serious environmental pollution. Meanwhile, the provisionof high grade tobacco was inadequate, since the restructuring policy of Chinese cigarettesblends. It will be of great importance for the industry and environment if the low-gradetobacco could be improved as supplement raw materials for low-tar cigarettes manufacturing.In this resrerch, the low-grade tobacco of poor flavoring quality, poor usability, and lowutilization were improved by different means, with the aims of improving the intrinsic qualityand organoleptic characteristics of low-grade tobacco. The main research contents and resultsare as follows.
At first, the differences of basic chemical compositions and macromolecule substancesbetween the upper, middle and lower leaves were compared, due to the the lack of appropriatephysical and chemical indicators support defects in currenent Chinese flue-cured tobaccoquality evaluation system. Thereafter, Principal Component Analysis (PCA) was applied toinvestigate the differences of neutral voltiles in tobacco flavor between different positions oftobacco stalk. The results obtained showed that: the upper leaves had low contents of totalsugar and reducing sugar, high levels of nicotine, which led to poor components coordinationand strong irritant feeling; All of the chemical composition index examined in the middleleaves were in an moderate range, which led to middle leaves with the best sensory qualitybetween different positions of tobacco; the lower leaves had low contents of nicotine andneutral volatiles but high cell wall material contents, which led to the low mainstream smokevolume and poor aroma quality. PCA results showed that: the main differences between upperleaves and middle leaves were the content of carotenoids degradation products, while thedifference between the middle and lower leaves were releted to the cembratriendid alkyldegradation products, terpenes degradation products, Maillard reaction products andphenylalanine degradation products.
In order to decrease the negative effects of high content of tobacco cell wall substancesto the tobacco aroma, polysaccharide enzyme (Viscozyme), neutral protease (Protease),amylase (BAN), pectinase (Pectine) in non-solution system were applied to degrade the cellwall substances of lower position tobacco. Cell wall degradation ratio and sensory scoreswere used as indicators to determine the efficiency of enzymtic degradtion. The resultsindicated that Viscozyme and Protease showed the most satisfied profits. The technologyparameters of degradation with Viscozyme and Protease were optimized by using theorthogonal experiment design combined with practical process. The optimized process was asfollows: Viscozyme of490U/g, Protease of208U/g, Reaction time of4h; under thiscondition tobacco cell wall substance content was decreased to25.74%. Meanwhile, theeffects of enzymatic treatment on tobacco surface structure, the combustion property and the content of neutral volatiles were also investigated by scanning electron microscopy (SEM),GC-MS detection and TG-DSC thermal analysis, respectively. The results showed that: thetobacco surface became smoother and the contents of neutral volatiles were raised afterenzymolysis; the combustion prosperity was rarely influenced by enzymatic process.
As Pectinase caused serious damage to the low-grade tobacco structure, the pectinasesolution was consequently used to extract the flavor components of low-grade tobacco forflavoring. When the Pectinase dosage was64U/g and the reaction time was2h, tobaccoextract obtained and used as low-grade tobacco flavoring showed good sensory properties.The cased cigarettes provided coordinate aroma when the addition amount of tobacco extractwas in the dosage of30～40g/g.
In order to increase the TDPs content of lower leaves, the Artemisia argyi leaves rich interpenoids was applied. Simultaneous distillation extraction (SDE), solid phase microextraction (SPME) and comprehensive two-dimensional gas chromatography time of flightmass Spectrometry (GC×GC-TOFMS) were used to determine the volatiles of the Artemisiaargyi leaves. The results showed that the major volatiles of Artemisia argyi leaves weremonoterpenes (C10H16), sesquiterpenes (C15H24) and their oxygend derivatives. Smoketransfer experiments indicated that terpenes can transfer into mainstream smoke and increasethe content of neutral volatiles in tobacco smoke. Because of high volatility of essential oil,the added Artemisia argyi essential oils would lost during storage and uneven release inmainstream smoke. Therefore, the microemulsion of Artemisia argyi essential oil was appliedto solve this problem. The effects of different surfactants, cosurfacant and Km values to themicroemulsion area were examined. The results indicated that ethanol as co-surfactant, Tween80as surfactant and Km=3:1were the optimal choices for Artemisia argyi essential oilmicroemulsion system, which had high loading capacity and good water dilution. Themicroemulsion system was O/W type when the moisture content was over66%, and thephase transition temperature was47°C. The microemulsion particle size and PDI remainedstable under two months storage. The Artemisia argyi essential oil microemulsion was addedto low grade tobacco leaves and the mainstream somke puff-by-puff transfer rules werechecked. The results showed that Artemisia argyi essential oil added in embedding type couldraise the60-day essential oil holding rate in tobacco leaves from <60%up to>80%. Also,Artemisia argyi essential oil microemulsion promoted the volatiles, in a certain extent,distributed more evenly among puffs in mainstream smoke.
Artemisia argyi ethanol extract was also applied for lower leaves casing. The aromaretention rates and prolysis properties of Artemisia argyi ethanol extract were determined.The results indicated that retention rate of volatiles in tobacco experiments was higher inArtemisia argyi extract than that in Artemisia argyi essential oil, but lower than that inArtemisia argyi essential oil microemulsion. The pyrolysis products at different temperatures(300℃,600℃,900℃) were identified. The results showed that most of the volatiles directlytransferred via distilling under300℃. Part of the neutral volatiles transferred to the smoke atthe range of300～600℃and most of the volatiles pyrolysed at the range of600～900℃.The terpenoids in tobacco smoke can be raised under the combustion situation. The contents ofTPN, tar were also increased slightly, while the CO, nicotine were not influenced. Sensoryevaluation results showed that the cased cigarette provided coordinate aroma when theaddition amount of Artemisia argyi extract was30g/g.
目前我国上、下部烟叶的品质较差，相对于品质较好的中部烟叶，利用率较低，但造成这种结果的不同部位烟叶之间内在化学成分的具体差异原因尚不明确。因此首先针对我国不同部位烟叶之间缺乏化学成分差异性分析的缺陷，考察了上、中、下不同部位烟叶之间基本化学成分、大分子物质之间的差异，并通过主成分分析法比较了不同部位烟叶中性香味成分的差异，结果表明：上部烟叶水溶性总糖及还原糖含量低，烟碱含量高，因此烟叶成分协调性差，刺激性强；而中部烟叶各指标基本适中，是不同部位烟叶中品质最好的，烟叶整体感官品质优良；下部烟叶烟碱含量低，因此烟气劲头小，浓度低。细胞壁物质含量高，中性香味成分含量低，香气质量差。中性香味成分主成分分析结果表明：上部烟叶与中部烟叶之间的差异主要与类胡萝卜素降解产物相关，而下部烟叶与中部烟叶之间的差异与西柏烷类降解产物、萜类降解产物、美拉德反应产物和苯丙氨酸降解产物均有关。
选择细胞壁物质含量高、具有代表性的下部低等烟叶进行低等烟的提质研究。首先通过细胞壁酶的作用对烟叶本身进行提质，主要采用了以多糖酶和蛋白酶复配进行非溶液酶解烟叶细胞壁物质和酶解法提取烟叶浸膏并反加香于下部低等烟叶两种方法。通过将多糖酶、中性蛋白酶、淀粉酶、果胶酶单独及复配喷施于下部低等级烟叶样品（亳州X3L）进行了酶降解烟叶细胞壁物质的研究。以烟叶细胞壁物质降解效果为指标，确定了多糖酶、蛋白酶作用效果适宜。通过单因素及正交试验确定了多糖酶和蛋白酶复配降解亳州X3L细胞壁物质的最佳工艺条件：多糖酶用量490U/g，蛋白酶用量208U/g，酶解时间4h，在此条件下烟叶细胞壁物质含量可降解至25.74%。并通过扫描电镜观察、GC-MS检测、TG-DSC热重分析考察了酶处理后烟叶的表面结构、燃烧性能及感官性质的变化，最后考察了复配酶处理对于烟叶及烟气成分的影响。结果表明：在多糖酶和蛋白酶的共同作用下，亳州X3L烟叶的细胞壁结构发生了一定程度的破坏，有利于烟叶中的香气成分转移至烟气中；复配酶处理增加了烟叶中美拉德反应产物类香味成分及中性香味成分总量的含量；复配酶处理仅略微减缓了烟草多糖类物质的热解和碳化过程，基本不改变烟叶的燃烧性质。经复配酶处理后的烟叶水溶性糖含量和成分协调性均得到了提高。
由于果胶酶喷施于烟叶造成烟叶结构较大程度的破坏，因此利用了果胶酶解法提取烟草浸膏并用于亳州X3L加香，结果表明当果胶酶用量为64U/g，酶解时间2h时得到的烟叶浸膏，用于为低等级烟草增香时，感官评价得分最高。果胶酶处理制备的烟叶浸膏为亳州X3L加香适宜添加量烟丝质量的30～40g/g，添加此烟草浸膏后的烟叶和烟气中性香味成分的含量均得到提高。
针对下部低等烟叶萜类降解产物等中性香味成分含量低的问题，对其进行了外加富含萜类的中草药（艾叶）提取物的增香研究。分别选择以艾叶精油、艾叶浸膏两种添加形式实现为低等烟叶的针对性补香。首先通过同时蒸馏萃取（SDE）、固相微萃取（SPME）与全二维气相色谱飞行时间质谱技术（GC×GC-TOFMS）联用分析了艾叶中的挥发性成分，结果表明艾叶中的挥发性成分主要为单萜(C10H16)和倍半萜(C15H24）类及其衍生物。接下来以艾叶精油形式进行增香实验。艾叶精油烟气转移实验证实，艾叶中的萜类物质可以增加烟气中香味成分的含量。针对艾叶精油在用于低等级烟草增香时易挥发、释放不均匀的问题，采用了微乳化技术对艾叶油进行了包埋。分别考察了不同表面活性剂、助表面活性剂及Km值对艾叶油微乳区域的影响，确定了以乙醇为助表面活性剂、吐温80为表面活性剂，Km=3：1时，能够与艾叶油形成载油量较高、加水稀释稳定的的微乳体系；并考察了此微乳体系的性质：以（吐温80+乙醇）(km=3)：艾叶油=7：3的微乳体系在水分含量超过66%时，微乳结构为水包油型；此微乳体系在47°C发生相转变；不同水分含量的微乳贮藏2个月，微乳体系粒径及PDI均保持稳定。将制备的艾叶油微乳加入烟丝中，分析了艾叶精油及微乳在主流烟气中的逐口抽吸转移规律，结果表明艾叶精油经包埋后再添加至烟草中，可使得烟丝60天时持留率由＜60%提高至＞80%，并促使主流烟气抽吸口数之间艾叶油分布趋于均匀化，在一定程度上起到了缓释加香的作用。
针对直接添加艾叶精油易挥发，烟丝持留率低的问题，设计了以艾叶浸膏形式为亳州X3L增香的实验。结果表明添加艾叶浸膏与直接添加艾叶精油相比，烟丝持留率得到了提高，但低于添加艾叶精油微乳的样品。艾叶浸膏直接裂解与添加至烟丝后的裂解反应证实，艾叶浸膏中的挥发性成分主要在300℃以下转移，300～600℃下部分转移，600～900℃发生裂解反应；艾叶浸膏中的挥发性成分可以在烟草燃烧条件下转移至烟气中，为亳州X3L低等烟叶增加萜类香味成分的含量，少量增加烟气总粒相物、焦油含量，对烟气其它常规成分影响不大。当艾叶浸膏在烟丝中的添加量为30g/g时，香气协调性好。
Tobacco, as special hobby product, is an important economic crop in China. During theprocessing of harvesting, storage, manufacturing process, large amounts of low-grade tobaccoare produced. The abandoned low grade tobacco with poor quality and low utilization causedgreat waste of resources, as well as serious environmental pollution. Meanwhile, the provisionof high grade tobacco was inadequate, since the restructuring policy of Chinese cigarettesblends. It will be of great importance for the industry and environment if the low-gradetobacco could be improved as supplement raw materials for low-tar cigarettes manufacturing.In this resrerch, the low-grade tobacco of poor flavoring quality, poor usability, and lowutilization were improved by different means, with the aims of improving the intrinsic qualityand organoleptic characteristics of low-grade tobacco. The main research contents and resultsare as follows.
At first, the differences of basic chemical compositions and macromolecule substancesbetween the upper, middle and lower leaves were compared, due to the the lack of appropriatephysical and chemical indicators support defects in currenent Chinese flue-cured tobaccoquality evaluation system. Thereafter, Principal Component Analysis (PCA) was applied toinvestigate the differences of neutral voltiles in tobacco flavor between different positions oftobacco stalk. The results obtained showed that: the upper leaves had low contents of totalsugar and reducing sugar, high levels of nicotine, which led to poor components coordinationand strong irritant feeling; All of the chemical composition index examined in the middleleaves were in an moderate range, which led to middle leaves with the best sensory qualitybetween different positions of tobacco; the lower leaves had low contents of nicotine andneutral volatiles but high cell wall material contents, which led to the low mainstream smokevolume and poor aroma quality. PCA results showed that: the main differences between upperleaves and middle leaves were the content of carotenoids degradation products, while thedifference between the middle and lower leaves were releted to the cembratriendid alkyldegradation products, terpenes degradation products, Maillard reaction products andphenylalanine degradation products.
In order to decrease the negative effects of high content of tobacco cell wall substancesto the tobacco aroma, polysaccharide enzyme (Viscozyme), neutral protease (Protease),amylase (BAN), pectinase (Pectine) in non-solution system were applied to degrade the cellwall substances of lower position tobacco. Cell wall degradation ratio and sensory scoreswere used as indicators to determine the efficiency of enzymtic degradtion. The resultsindicated that Viscozyme and Protease showed the most satisfied profits. The technologyparameters of degradation with Viscozyme and Protease were optimized by using theorthogonal experiment design combined with practical process. The optimized process was asfollows: Viscozyme of490U/g, Protease of208U/g, Reaction time of4h; under thiscondition tobacco cell wall substance content was decreased to25.74%. Meanwhile, theeffects of enzymatic treatment on tobacco surface structure, the combustion property and the content of neutral volatiles were also investigated by scanning electron microscopy (SEM),GC-MS detection and TG-DSC thermal analysis, respectively. The results showed that: thetobacco surface became smoother and the contents of neutral volatiles were raised afterenzymolysis; the combustion prosperity was rarely influenced by enzymatic process.
As Pectinase caused serious damage to the low-grade tobacco structure, the pectinasesolution was consequently used to extract the flavor components of low-grade tobacco forflavoring. When the Pectinase dosage was64U/g and the reaction time was2h, tobaccoextract obtained and used as low-grade tobacco flavoring showed good sensory properties.The cased cigarettes provided coordinate aroma when the addition amount of tobacco extractwas in the dosage of30～40g/g.
In order to increase the TDPs content of lower leaves, the Artemisia argyi leaves rich interpenoids was applied. Simultaneous distillation extraction (SDE), solid phase microextraction (SPME) and comprehensive two-dimensional gas chromatography time of flightmass Spectrometry (GC×GC-TOFMS) were used to determine the volatiles of the Artemisiaargyi leaves. The results showed that the major volatiles of Artemisia argyi leaves weremonoterpenes (C10H16), sesquiterpenes (C15H24) and their oxygend derivatives. Smoketransfer experiments indicated that terpenes can transfer into mainstream smoke and increasethe content of neutral volatiles in tobacco smoke. Because of high volatility of essential oil,the added Artemisia argyi essential oils would lost during storage and uneven release inmainstream smoke. Therefore, the microemulsion of Artemisia argyi essential oil was appliedto solve this problem. The effects of different surfactants, cosurfacant and Km values to themicroemulsion area were examined. The results indicated that ethanol as co-surfactant, Tween80as surfactant and Km=3:1were the optimal choices for Artemisia argyi essential oilmicroemulsion system, which had high loading capacity and good water dilution. Themicroemulsion system was O/W type when the moisture content was over66%, and thephase transition temperature was47°C. The microemulsion particle size and PDI remainedstable under two months storage. The Artemisia argyi essential oil microemulsion was addedto low grade tobacco leaves and the mainstream somke puff-by-puff transfer rules werechecked. The results showed that Artemisia argyi essential oil added in embedding type couldraise the60-day essential oil holding rate in tobacco leaves from <60%up to>80%. Also,Artemisia argyi essential oil microemulsion promoted the volatiles, in a certain extent,distributed more evenly among puffs in mainstream smoke.
Artemisia argyi ethanol extract was also applied for lower leaves casing. The aromaretention rates and prolysis properties of Artemisia argyi ethanol extract were determined.The results indicated that retention rate of volatiles in tobacco experiments was higher inArtemisia argyi extract than that in Artemisia argyi essential oil, but lower than that inArtemisia argyi essential oil microemulsion. The pyrolysis products at different temperatures(300℃,600℃,900℃) were identified. The results showed that most of the volatiles directlytransferred via distilling under300℃. Part of the neutral volatiles transferred to the smoke atthe range of300～600℃and most of the volatiles pyrolysed at the range of600～900℃.The terpenoids in tobacco smoke can be raised under the combustion situation. The contents ofTPN, tar were also increased slightly, while the CO, nicotine were not influenced. Sensoryevaluation results showed that the cased cigarette provided coordinate aroma when theaddition amount of Artemisia argyi extract was30g/g.
引文
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