【作者】 刘萍；

【导师】 张广民； 张修国；

【作者基本信息】 山东农业大学 ， 植物病理学， 2003， 硕士

【摘要】 本研究从云南、山东等主要产烟区采集烟田罹病烟叶、仓库储存(陈化)烟叶及土壤标样，从中分离纯化获得细菌菌株。分别在烟叶储藏期、大田期及烘烤期喷施细菌菌剂进行诱香试验，依据评价烟叶品质优劣的综合技术指标，筛选诱香效果较好的细菌菌株；并对其进行诱香机理的酶学、生物学及生理生化特性的研究，探讨菌株的发酵工艺参数。结果概括如下：1．从采集标样内共分离获得249个细菌菌株，经储藏期烟叶处理后，测定烟叶内化学物质(包括可溶性总糖、烟碱、总氮及类胡萝卜素)含量变化，根据评价烟叶品质优劣的综合技术指标及感官评吸结果表明，菌株B07、B21和B45处理的烟叶主要成分适中，糖氮比、糖碱比和氮碱比趋于协调。该三个菌株处理过的烟叶，香气质平均值皆比对照提高3.54%，香气量的平均值分别比对照提高3.13%、2.50%和3.13%，余味比对照更舒适，杂气和刺激性皆下降，评吸总分值分别比对照提高2.96%、2.02%和3.36%。2．大田生长期烟叶诱香试验结果表明，菌株B03、B05、B07和B09可提高烟叶外观质量，与对照相比，处理烟叶内化学成分变化明显，其含量和比值更接近优质烤烟的标准值。烘烤期烟叶增质增香试验结果表明，菌株B16、B35、B38和B28处理的烟叶与对照相比，化学成分含量适中，烟碱含量下降，香气指标糖/氮、糖/碱和氮/碱均比对照有所改善。3．针对诱香效果较好的菌株B07、B21、B45生物学及生理生化特性进行了研究，阐明适宜诱香菌株生长的温度、pH值、碳源、氮源等，为各菌株适宜发酵工艺参数提供依据。菌株B07细菌杆状，产芽孢，周鞭；产淀粉水解酶和过氧化氢酶，水解明胶，吲哚试验阳性，甲基红试验阴性；适宜温度25～40℃；适宜pH值8.0；适宜渗透压0.5%；适宜碳源葡萄糖和蔗糖，适宜氮源蛋白胨。菌株B21细菌杆状，极鞭，不产芽孢；产过氧化氢酶，淀粉水解试验、明胶液化试验、甲基红试验、吲哚试验皆为阴性；适宜温度25～30℃；适宜pH值6～8；适宜渗透压1%；适宜碳源葡萄糖和蔗糖，适宜氮源蛋白胨和硫酸氨。菌株B45细菌杆状，产芽孢，周鞭；产淀粉水解酶及过氧化氢酶，水<WP=11>解明胶，吲哚试验阴性，甲基红试验阳性；适宜温度25～36℃；最适宜pH值8.0；适宜渗透压1%；适宜碳源葡萄糖和蔗糖，适宜氮源牛肉浸膏。4．针对三个诱香效果较好的细菌菌株进行诱香机理的酶学研究，测定了烟叶内与香气化学物质有关的活性酶，主要包括α-淀粉酶、纤维素酶、蛋白酶、多酚氧化酶和过氧化物酶。试验结果表明：诱香细菌处理的烟叶，上述各种酶活性累加值明显比对照处理增加。烟叶内纤维素酶活性的动态变化无规律，过氧化物酶活性基本呈下降趋势，α-淀粉酶、蛋白酶和多酚氧化酶总体呈现前期活性较高，而后期下降的明显趋势。三个诱香细菌中，菌株B45处理烟叶内各种酶的活性高于B07和B21处理，该结果与感官评吸得分呈正相关。上述三个诱香效果显著的细菌菌株，经准中试和中试试验验证后，具一定的应用开发价值。更多还原

【Abstract】 This research content is to improve tobacco quality by bacterial strain. The bacteria were used during the growing，curing and fermenting. The synergistic bacteria would screened through the assessment with sense organ by smoking and analysis of chemical ingredient，such as soluble sugar，total nitrogen，protein，caritenoid and nicotine. Study on the mechanism of the bacterial fermentation，biological，physiological and biochemical characters，discussing fermentation parameters. The main achievements or conclusions and results are acquired as follow:1. More 240 strains were isolated from specimens of leafage and soil，which were collected from Yunnan and Shandong province. The analysis of the chemical ingredient of flue-cured tobacco leaves and the panel of test indicated that 3 strains，B07，B21 and B45 can improve tobacco quality. Aroma quality increased 3.54%，aroma quantity increased 3.13%，2.50% and 3.13%，respectively. Offensive and irritancy were reduced.2. The analysis of the chemical ingredient of tobacco leaves with strains demonstrated that 4 strains of 10 strains made in growing period，B03，B09，B07 and B05 that improve aspect quality of flue-cured tobacco leaves，and 4 strains of 28 stains were made during curing period，all could make chemical substance changed largely，and more harmonies than that of the control.3. In order to exert the role of bacterial strains in improving tobacco qulity greatly，and offer proper technical parameter，biological，physiological and biochemical characters of the strains B07，B21 and B45 were studied. The<WP=13>result indicated that strains B07 and B45 belong to bacillus，peripheral flagella，Gram-positive，no capsule，salt toleration. Gelatin liquefaction test，amyloclastic test and hydrogen peroxide test were all positive reaction. As for the strain B07，the range of optimal temperature is 25～40℃，the range of optimal pH-value is 8.0，the optimum aton is glucose and saccharose，and the optimum nitrogen source is peptone. It can not utilize sorbose. As for the strain B45，the range of optimal temperature is 25～36℃，optimum pH-value is 8.0，the optimum aton is glucose and saccharose，and the optimum nitrogen source is beef extract. It can not utilize sorbose and rhamnose. As for B21，thallus is rod，polar flagellum，Gram-negative，no capsule. Amyloclastic and hydrogen peroxide test were positive reaction. Gelatin liquefaction test，indole test and methyl red test were all negative reaction. The range of optimal temperature is 25～30℃，the range of optimal pH-value is 6.0～8.0，the optimum aton is glucose and saccharose，the optimum nitrogen source is peptone. It can not utilize sorbose.4. Study of the changes of five enzyme activities(including α-amylase，cellulase，proteinase，polyphenol oxidase and peroxidase) in flue-cured tobacco leaves during bacterial fermentation. The result showed that the enzyme activities of bacterial fementation were better than that of the control，and the assessment value have closely relationship with enzyme activities. The changes of cellulase activity were not consistent during bacterial fermentation. The changes of peroxidase activity was continuously decreased. For α-amylase，proteinase and polyphenol oxidase which were higher than that of the control，the enzyme activities ascended firstly，but respectively descended on the 3th or 9th day，更多还原