【作者】 牛燕丽；

【导师】 宫长荣；

【作者基本信息】 河南农业大学 ， 食品科学， 2005， 硕士

【摘要】 为探索烟叶发酵过程中添加生物酶制剂以降解烟叶中淀粉和蛋白质的可行性,本文进行了大量正交设计试验,并利用生物技术的有效手段筛选菌株用于发酵烟叶（丝）,试验结果如下: 1、为了有效转化烟叶中残留的淀粉,试验选用α-淀粉酶和葡萄糖淀粉酶两种淀粉酶,由正交试验结果分析可知:两种酶对B2F等级烟叶淀粉降解率分别为21.88%和24.17%;分别作用于C3F等级烟叶后平均淀粉降解率为33.23%和34.68%,影响两种酶作用的主要发酵条件不同;葡萄糖淀粉酶对X2L等级烟叶淀粉降解率较强,平均淀粉降解率达33.83%。两种淀粉酶同时用于烟叶淀粉的降解,仍通过正交试验得到针对B2F、C3F、X2L三个等级烟叶的相对最佳的酶作用条件为:对于B2F,α-淀粉酶和葡萄糖淀粉酶比例1:1,加酶量为4×,在温度45℃和相对湿度70%下作用30×;对于C3F,α-淀粉酶和葡萄糖淀粉酶比例1:5,加酶量8×,在温度50℃和60%相对湿度下作用30×;对于X2L,α-淀粉酶和葡萄糖淀粉酶比例1:5,加酶量8×,在温度35℃和60%相对湿度下作用30×。 2、试验选用中性蛋白酶用于发酵烟叶,B2F、C3F、X2L三个等级烟叶中蛋白质的平均降解率分别为5.20%、4.71%和9.28%。但先在各等级相对最佳条件下施加α-淀粉酶、葡萄糖淀粉酶后再进行该试验,三种酶分别添加在发酵过程后,其针对的淀粉和蛋白质有所降解,B2F、C3F、X2L的淀粉降解率分别为26.77%、29.54%和35.00%,蛋白质降解率分别为13.36%、3.10%和11.79%。B2F、C3F、X2L三个等级烟叶中所测得的氨基酸含量均有显著提高,其增加率分别为7.11%、11.45%和9.52%。三个等级烟叶的还原糖含量、总氮和烟碱含量变化不明显。 3、试验筛选出高产用于分解烟叶淀粉的α-淀粉酶的新菌株G₁。该菌在以烟叶内含物质为主要营养源配制的培养基,并在其中添加玉米粉等物质补充营养,调pH7.0左右,以12%的培养液浓度在250ml三角瓶装量25ml的条件下发酵,选择发酵温度37℃,摇床转速220 r/m,2%接种量,发酵时间48h。在此条件组合下制得的发酵液,离心后测定其中各种酶的活力,由结果可知,驯化后的菌株G₁在烟草内含物质为营养来源的生长环境中仍保留了较高的α-淀粉酶活力,达到187.36 U/ml,并且有糖化酶、蛋白酶等产出。从电泳扫描图谱和酶谱检测,可以明显看出驯化后菌株G₁与出发菌株C₁发酵液蛋白条带数量和位置的区别。 将新菌株制得的发酵液以1:10的比例加入已平衡水分的烟丝中,混合均匀后放入恒温恒湿培养箱。在45℃、70%相对湿度的条件下发酵烟丝,由试验结果可知,加入发酵液发酵烟丝后,蛋白质和烟碱含量能明显的降低,淀粉也有所降解。发酵24h后蛋白质降解率达到20.13%,之后有所上升,可能由于酶液部分残留在烟叶表面;烟碱的降解在72h达到最大,降解率达到18.70%:淀粉含量在48h降解了6.38%达到最大,之后趋于稳定;发酵后烟丝中氨基酸、还原糖含量明显提高,发酵24h后,氨基酸提高了14.76%;72h后还原糖含量更多还原

【Abstract】 To explore the feasibility of adding enzyme to hydrolyze starch and protein of flue-cured tobacco leaves during fermentation, this paper had a lot of orthogonal matrix experiments and used the effectual means of biotechnology screened a bacterial strain to ferment tobacco leaves （cut tobacco）.There were conclusion as below:1 In order to hydrolyze the starch of tobacco leaves, this paper selected a-amylase and glucose-amylase, the result showed that adding two kinds of amylase respectively also could decrease the starch content of B2F leaves by 21.88% and 24.17% on average; adding a-amylase and glucose-amylase respectively could decrease the starch content of C3F leaves by 33.23% and 34.68% on average, but the main influential factors were not identical; adding glucose-amylase could effectively hydrolyze the starch of X2L leaves, the average decreasing rate was 33.83%. Mixed two kinds of amylase, added it to reduce the starch content of tobacco leaves, B2F, C3F, X2L, and analyzed the results by means of orthogonal matrix. The preferable treatment conditions were as follows: For B2F ,4x of mixture of a-amylase and glucose-amylase （1:1,w/w）,temperature 45℃ and relative humidity 70% for 30×; For C3F ,8× of mixture of a-amylase and glucose-amylase （1:5,w/w）,temperature 50℃ and relative humidity 60% for 30×; For X2L ,8× of mixture of a-amylase and glucose-amylase （1:5,w/w）,temperature 35℃ and relative humidity 60% for 30×.2 This experiment selected neutral protease to hydrolyze the protein of tobacco leaves ,and it could decrease the protein content of B2F, C3F and X2L by 5.20% ,4.71% and 9.28% respectively. But we added the mixture of a-amylase and glucose-amylase at that preferable treatment conditions first , and then added the neutral protease . The starch content of B2F, C3F and X2L were decreased respectively by 26.77%, 29.54%, 35.00%; The protein content were decreased respectively by 13.36%, 3.10%, and 11.79%; The amino acid content of three grade were increased remarkablely , the rates of increase were 7.11%,11.45%,9.52%; The changes of RS, TN and nicotine were not obvious.3 This experiment screened a new strain named G₁ , it producted a-amylase which could hydrolyze the starch of tobacco leaves . The optimal medium consisted the contents of tobacco leaves and other material such as cornmeal, the initial pH of the更多还原