【作者】 王晓云；

【导师】 陈红菊； 季相山；

【作者基本信息】 山东农业大学 ， 养殖（专业学位）， 2015， 硕士

【摘要】 枯草芽孢杆菌是一种益生菌,不仅存在于土壤、空气、水中,有些还存在于人与动物肠道内。枯草芽孢杆菌具有很强的抗性,在恶劣环境下可以通过内生孢子来保存生命,同时该细菌具有强大的产酶特性,主要分泌胞外蛋白酶、淀粉酶、纤维素酶等酶类。在水产养殖的应用中,枯草芽孢杆菌能够分泌的各种酶类来降解鱼类养殖池塘中的残留饵料与动物自身分泌物,同时还可以提高水产动物机体消化酶活性,其被广泛用来制备净水、肥水的微生态制剂。但在实际应用中,枯草芽孢杆菌因温度等环境因素的影响,达不到应有的酶活性标准。因此,筛选高酶活性的枯草芽孢杆菌和提高已有枯草芽孢杆菌的产酶活性变得十分有意义。为了获得高产蛋白酶活性的枯草芽孢杆菌,本文研究了高产蛋白酶枯草芽孢杆菌的筛选鉴定和通过紫外诱变提高枯草芽孢杆菌产蛋白酶活力。该研究的结果分为以下两个部分:1、高温环境下高产蛋白酶枯草芽孢杆菌的筛选与鉴定。本文从温泉养殖池塘水样中获得了8株菌株,将其分别命名为BY1到BY8。通过对该菌株菌落形态、菌体革兰氏染色观察分析,同时使用MEGA5.1软件建立8株芽孢杆菌的进化树并进行同源性分析,结果显示在除BY6外所有菌株为枯草芽孢杆菌,菌株BY6为坚强芽孢杆菌。同时对通过BIOLOG系统对菌株进行生化鉴定,进一步确定了所得结果。利用透明圈法和福林酚法测定菌株的产蛋白酶活力并与分离的商业菌株作比较。结果分别为:在37℃培养条件下,菌株BY7具有较高产酶活力。BY7的透明圈的直径与菌落直径比值为5.4,明显高于其他菌。商业菌株BS2的产蛋白酶活力为16.61U/m L,菌株BY7的蛋白酶活力为43.28U/m L;BY7的产蛋白酶活力远远高于商业菌株BS2。将BY7应用于罗非鱼养殖,以致病菌无乳链球菌为对照,检测BY7的安全性。试验结果显示灌服枯草芽孢杆菌和生理盐水的死亡率均为0,而灌服无乳链球菌的罗非鱼死亡率根据浓度由低到高分别为10%、30%、30%、30%。因此可以确定枯草芽孢杆菌BY7是安全菌株。2、枯草芽孢杆菌BC2的紫外诱变选育对试验室保藏菌株BC2进行紫外诱变,诱变后以酪素培养基为定性培养基,测定60~100个单克隆的透明圈大小,并对前10%的菌株进行-80℃保存。每次诱变完后选择最大透明圈的菌株继续诱变。连续诱变6次后,利用福林酚法测定诱变后突变菌株的产蛋白酶活力,结果显示,培养96h时,出发菌株BC2的产蛋白酶活性为27.68U/m L,而突变菌株B38的活性经过诱变后达到了86.82U/mL,提高了3.14倍,差异极显著(P<0.01)。为了检测紫外诱变对菌株其他产酶活性的影响,本文利用DNS法测定突变菌株B38和出发菌株BC2的产纤维素酶活性,结果显示,培养48h时,B38和BC2的产纤维素活性到达最高。BC2的纤维素酶活力为0.85U/m L,B38的纤维素酶活力为0.97U/m L,两者的产酶活性没有显著差异。测定突变菌株B38遗传稳定性试验结果显示:该菌株在传代10次后,菌株的产蛋白酶活力为77.01U/m L,纤维素酶活力为0.913U/m L,分别是诱变菌株产蛋白酶活力的88.70%和94.6%。这说明了该突变菌株具有较稳定的产蛋白酶遗传性。总而言之,本研究从温泉养殖池塘中筛选到了一株37℃培养温度下高产蛋白酶的枯草芽孢杆菌;同时利用紫外诱变处理的方法提高了已有枯草芽孢杆菌BC2在25℃条件下的产蛋白酶活力。该研究为水产微生态制剂的制备提供了两株可选择的枯草芽孢杆菌。更多还原

【Abstract】 Bacillus subtilis is one probiotic. It is not only present in the soil, air, water, and some present in the human and animal intestinal tract. B. subtilis has a strong resistance. In the harsh environment it can save lives by endogenous spores. At the same time it also has a powerful strain of enzyme production characteristics, primarily secreted extracellular protease, amylase, cellulase enzymes. In aquaculture enzymes from Bacillus subtilis can reduce fodder residue and decompose animal excreta, in addition can also promote digestion and absorption of aquatic animals. It is widely used for the preparation of probiotics. However, in practice, due to the effect of Bacillus subtilis temperature, can not achieve the desired enzyme standard.So screening of high activity of Bacillus subtilis and improving Bacillus subtilis’ production activity becomes very meaningful.In order to obtain a strain of Bacillus subtilis that produced prolific protease.This article study the screening and identification of Bacillus subtilis protease yield and improving Bacillus subtilis produced protease activity by UV mutagenesis.The main results of this study are as follows:1. screening and identification of Bacillus subtilis protease yield under high temperature environment.We obtained 8 strains from the pond water, which were named BY1 to BY8. According to the chracteristics of morphology, cultivation, physology and 16 SrDNA squence, they were identified as Bacillus sp. We used MEGA5.1 software to build the evolutionary tree of eight Bacillus and analyze their homology. The results showed that the strains in addition BY6 are in the same branch and have 99% homology with Bacillus subtilis. The strain of BY6 is e in the same branch and has 99% homology with B. lentus. What’s more, We further determine the outcome by Biolog biochemical identification.The producing protease strains and a commercial strain were measured using Casein transparent circle and Folin-Ciocalteu method.The results shows that strain BY7 has the largest transparent circle(5.4)under the culture temperature 37 ℃, significantly higher than other bacteria; Commercial strains BS2 produced protease activity is 16.61 U/m L,and strain BY7 protease activity is 43.28 U/mL. Its production protease activity is much higher than commercial strains BS2The strain BY7 is applied to tilapia aquaculture, with streptococcus pneumoniae as the control group. The lethal rates of perfusing BY7 and NS are 0%,but according to the concentration of streptococcus pneumoniae from low to high respectively is 10% 30% 30% and30%. So we can determine that the bacillus subtilis strain BY7 is safety.2. the UV mutation of Bacillus subtilisHaving an ultraviolet mutagenesis to strain BC2 of our laboratory, and after screened by casein medium, we will get 60-100 monoclonal srains. The strains producing a transparent circle were identified, and the transparent circle diameter/colony diameter was calculated. The ratio of top ten percent of colony the transparent circle diameter/colony diameter will be stored at-80℃, and choose ratio of biggest of the colony mutagenized again. We got B38 strain after six mutation screening. We determine the protease-producing activity between B38 strain and BC2 strain. Research shows that after cultured for 96 hours, the BC2 strain protease-producing activity is 27.68U/mL after the mutafacient protease-producing activity is 86.82U/mL, increased 3.14 fold.In order to detect ultraviolet mutagenesis effects on other enzyme-producing activity of strains. We determine the cellulase-producing activity between B38 mutant strain and B32 original strain by DNS method. Research shows that after cultured for 48 hours, the BC2 strain cellulase-producing activity is 0.85U/m L after the mutafacient cellulase-producing activity is 0.97U/m L. There were no significant difference between BC2 and B38.This study analyzes the genetic stability of the mutant strain B38. The results showed that after ten times of subculture the strain producing strains of protease activity was77.01 U /m L and cellulase activity was 0.913U/mL. They maintained 88.70% and 94.6% respectively. This shows that BY7 has genetic stability.In summary, In this study a high protease activity bacillus subtilis was screened from hot spring breeding pond under cultural temperature 37℃. And by using the method of UV mutagenic treatment improved the existing strains BC2 producing protease activity under under cultural temperature 25℃. The study on provides two strains of bacillus subtilis for Microecologics.更多还原