【作者】 郑小嘎；

【导师】 张天宇； 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】 本论文分离筛选了对烟叶具诱香增质的真菌菌株，筛选出4株诱香增质效果较好的菌株，并对这4株的生物学及菌株诱香增质的酶学机理进行了研究。从云南、山东（沂水、临朐、诸城三地）等地采集的烟田罹病烟叶、仓库贮存（陈化）烟叶及烟田土壤标样上共分离到250个真菌菌株，经过发酵处理烟叶后，对叶内化学成分（可溶性糖、烟碱、总氮及类胡萝卜素等）的含量及其变化进行测定，依据评价烟叶品质优劣的技术指标，初步筛选出7个具有诱香效果的真菌菌株。对筛选出的7个菌株处理贮藏期烟叶，经化学测定和专家评吸，进一步筛选出4个诱香效果较好的菌株BF06 、BF07、 BF63和BF05。化学测定结果表明，菌株处理过的烟叶内化学成分间更趋于协调，两次专家评吸结果显示，香气质平均分别比对照提高6.06%、 5.41%、5.04%和2.75%，香气量平均分别比对照提高1.24% 、3.6%、2.24%和1.93%，余味更加舒适，杂气和刺激性减小，两次平均得分分别比对照高出4.34%、4.01%、3.47% 和1.74%。从上述数据看出，该4个真菌菌株的诱香效果较好且较稳定。大田期和烘烤期烟叶经真菌菌剂处理后，烟叶内总糖、总氮、蛋白质及烟碱含量测定结果表明，部分菌株使烟叶内化学成分趋于协调，且更接近于优质烤烟的技术指标。在诱香烟叶化学成分测定及专家评吸的基础上，对4个菌株生物学特性进行了研究，探索诱香菌株生长所需的适宜温度、pH值、培养基、碳源、氮源及光照条件等，作为制定各菌株适宜发酵工艺参数和条件的依据。菌株BF05适宜生长温度为25℃～30℃，最适温度为27.5℃；适宜pH值为6～11，最适pH值为8；在供选的4种培养基中， PDA为最适培养基；在供试6种碳源中，以果糖为最适碳源；在供试的7种氮源中，以硝酸钾为最适氮源；在12h/12h光暗交替光照条件下该菌生长最佳。菌株BF06适宜生长温度为25℃～30℃，最适温度为27.5℃；<WP=10>适宜pH值为7～10，最适pH值为8；在供试4种培养基中，以PDA为最适培养基；在供试6种碳源中，以麦芽糖为最适碳源；在供试7种氮源中，以硝酸钾为最适氮源；在黑暗24h/紫光12h/完全黑暗光照条件下该菌生长最好。菌株BF07适宜生长温度为25℃～35℃，最适温度为35℃；适宜pH值为5～8，最适pH值为6；在供选的4种培养基中，以在PDA培养基上生长势最优；在供试6种碳源中，以果糖为最适碳源；在供试7种氮源中，以在硫酸铵为最适氮源；该菌在完全黑暗和12h/12h光暗交替光照条件下生长最佳。菌株BF63适宜生长温度为25℃～30℃，最适温度为30℃；适宜pH值为5～7，最适pH值为7；在4种供试培养基中，以在L-B培养基上生长势最优；在6种供试碳源中，以果糖为最适碳源；在7种供试氮源中，以硝酸钾为最适氮源；在完全黑暗光照条件下该菌生长势最好。本研究还就上述菌株的诱香酶学机理进行探索，对菌剂处理后的烟叶内与香气化学物质有关的活性酶α-淀粉酶、蛋白酶、多酚氧化酶和过氧化氢酶的活性进行了测定。结果表明，经4个菌株BF05、BF06、BF07及BF63处理烟叶30天，烟叶内α-淀粉酶活性、蛋白酶活性和多酚氧化酶活性的累加值均高于对照，菌株BF05和BF06的过氧化物酶活性的累加值也高于对照。菌株诱香处理后烟叶内化学成分的变化与诱香烟叶内上述几种酶的活性基本上呈正相关。同时也说明，供试菌株对烟叶内多种与香气有关的酶活性具有激活作用，表明科学有效地喷施一种诱香微生物制剂可同时诱导几种酶的活性，促使烟叶内多种与烟叶品质改善有关的物质进行转化与合成，这种措施比单纯施用外源单一酶的效果将会更好。更多还原

【Abstract】 The main purpose of this study is to find fungus strains that are capable of inducing aroma and improving tobacco quality. 4 strains were screened. Biological characteristics and the enzyme mechanism of them were determined.More than 250 isolates were isolated from samples of leafage and soil collected from Yunnan and Shandong provinces. Based on analyzing of chemical, such as soluble sugar, total nitrogen, protein and carotenoid, and referring to the quality standard of tobacco, 7 fungus strains were screened.The results of determination of main chemical contents and the panel test showed that, 4 strains, BF05, BF06, BF07 and BF63 were capable of improving tobacco quality. Aroma quality increased 6.06%, 5.41%, 5.04%, 2.75%, respectively, aroma quantity increased 1.24%, 3.60%, 2.24%, and 1.93% respectively, and offensive and irritancy were reduced. The chemical analysis showed that several main chemical contents of the tobacco leaves treated with fungus strains during growing season and curing period changed remarkably, and showed more harmonious than the control.For the sake of bring the capacity of the fungus strains in improving the aroma quality of tobacco into full play, and to offer proper technical parameter, biological characteristics of 4 strains were determined. As for BF05, the range of temperature of hyphae growth is 25℃-30℃, the optimum is 27.5℃. The range of pH adapted is 6 -11, the optimum pH is 8. PDA is the optimum media. The optimum carbon source is D-fructose and the optimum nitrogen source is potassium nitrate. The natural light (12h/12h) is the optimum light.As for BF06, the range of temperature of hyphae growth is 25℃-30℃, the optimum is 27.5℃. The range of pH adapted is 7-10, and the optimum pH is 8. PDA is the optimum media. The optimum carbon source is maltose and the optimum nitrogen source is potassium nitrate. The optimum light is black light (24h)/purple light (12h)/black light (12h).<WP=12>As for BF07, the range of temperature of hyphae growth is 25℃-35℃, the optimum is 35℃. The range of pH adapted is 5-8, and the optimum pH is 6. PDA is the optimum media. The optimum carbon source is D-fructose and the optimum nitrogen source is vitriol ammonium. The optimum light is natural light (12h/12h) and black light.As for BF63, the range of temperature of hyphae growth is 25℃-30℃, the optimum is 30℃. The range of pH adapted is 5-7, and the optimum pH is 7. LB is the best media. The optimum carbon source is D-fructose and the optimum nitrogen source is potassium nitrate. The optimum light is black light.Changes of enzyme activities (including α-amylase, proteinase, polyphenol oxidase and peroxidase) in treated tobacco leaves were determined. Results showed that the total values of each of enzyme activities was higher than the control, and total values of peroxidase of BF05 and BF06 were higher than the control too, although enzyme activities were different in different time. And changes of chemical substance have closely relativity with enzyme activities.更多还原