【作者】 马颖辉；

【导师】 曹龙奎；

【作者基本信息】 黑龙江八一农垦大学 ， 农产品加工及贮藏工程， 2012， 硕士

【摘要】 益生素又称微生态制剂或活菌制剂，作为一种无残留、无毒副作用、无抗药性的绿色添加剂，在畜牧业中的应用越广泛。目前常用的微生态制剂主要有乳酸菌、芽孢杆菌、酵母菌、光合细菌等四大类。其中饲用芽孢杆菌由于具有抗逆性强、耐高温、易贮存等独特的特性成为研究热点。本试验的目的是通过原生质体融合技术，得到一株降解淀粉和蛋白质能力较强的菌株。试验从样品中分离筛选出一株高产淀粉酶和蛋白酶菌株，通过诱变育种的方法得到五株高产酶菌株，并进行原生质体融合，研究原生质体方法的适宜融合条件，通过原生质体融合来提高枯草芽孢杆菌的产淀粉酶和蛋白酶能力，为枯草芽孢杆菌微生态制剂的研究提供了理论依据。1、本文首先对高产淀粉酶及蛋白酶功能饲用菌的选育进行了研究。从土壤和新鲜猪粪样品中分离得到45株产降解淀粉酶和蛋白酶的菌株，通过淀粉水解培养基和酪蛋白水解培养基初筛得到10株菌株，对这10株菌株进行淀粉酶及蛋白酶活性测定，筛选出1株淀粉酶和蛋白酶高产菌株JASSB2，淀粉和蛋白酶活力分别为1.846U/mL和9.848U/mL，对出发菌株的生长曲线和产酶曲线进行测定，确定出发菌株的对数生长期为8-12h，12h后进入稳定期；酶活力升高时间为8-14h。2、高产淀粉酶及蛋白酶功能饲用菌诱变育种的研究。通过紫外（UV），硫酸二乙酯（DES）单一诱变剂处理后测定突变菌株酶活性，筛选出高产酶突变菌株，即UJ-3、UJ-5、DJ-1和DJ-2，测定其淀粉酶和蛋白酶活性分别为4.457U/mL和11.509U/mL、5.092U/mL和11.728U/mL、4.513U/mL和12.306U/mL、5.124U/mL和13.031U/mL。单一诱变处理的最佳条件分别为紫外照射160s；DES浓度为1.5mg/mL，处理40min。采用紫外-硫酸二乙酯复合诱变剂，以JASSB2枯草芽孢杆菌为出发菌株，在单因素试验的基础上，采用二次回归正交旋转组合试验设计，优化复合诱变JASSB2枯草芽孢杆菌的诱变条件，并进一步对JASSB2枯草芽孢杆菌进行诱变处理，测定复合诱变所得突变菌株酶活性，实验结果表明：在紫外照射时间160s，硫酸二乙酯浓度1.5mg/mL，处理时间40min条件下，筛选得到突变菌株U-DJ4，其淀粉酶和蛋白酶活性分别5.280U/mL和13.324U/mL。3、基因组改组技术选育高产淀粉酶及蛋白酶菌株的研究。实验结果表明，原生质体制备、灭活及融合最佳条件为：处理时间为15min，溶菌酶浓度为1.0mg/mL，37℃酶解15h；原生质体紫外灭活15min；热灭活为60℃下处理90min；原生质体融合条件为PEG6000浓度40%，融合温度34℃，融合时间40min，此时融合率可达5.6×10^-5。以诱变所得菌株为出发菌，经过两轮改组获得改组菌F299，其淀粉酶活性及蛋白酶活性分别为6.732U/mL和18.842U/mL。更多还原

【Abstract】 Probiotics, also known as Living bacterium agent or Microbial ecological agent, as a kind of noresidue, non-toxic side effects, no drug-resistant to green additive, it is more widely used in animalhusbandry. Commonly used four main categories probiotics are lactic acid bacteria, Bacillus, yeast,photosynthetic bacteria. Which forage Bacillus become a hot topic because of strong resistance, hightemperature resistant, easy storage and other unique features. The purpose of this test is to get a strongstrain of the degradation of starch and protein by protoplast fusion technique.Test separate a high yieldof amylase and protease strains from the sample, get five high-yield enzyme through the mutationbreeding methods, and protoplast fusion, and use it to improve the yield of bacillus subtilis amylase andprotease ability, provides a theoretical basis for Bacillus subtilis probiotics study.1. This study first to high-yield amylase and protease feeding function bacteria breeding theresearch.45degradation with high amylase and protease activity were isolated from soil and fresh pigmanure samples. Through the hydrolysis of starch Medium and casein hydrolysis medium to getscreening screened10strains and measure the amylase and protease activity of these strains. Screeningout one high amylase and protease strain of JAPPS2. Starch and protease activity respectively is1.846IU/mL和9.848IU/mL. Determination the growth curve and enzyme curve of the starting strain.Determine the logarithmic phase of the starting strain is8-12h, after12h into stable period, enzymeactivity elevate at8-14h.2. The high yield of amylase and protease forage bacteria mutation breeding the research. Used byultraviolet radiation, Ethyl sulfate as single mutagen treated, determinate the enzyme activity of incomemutant strains. Filter out the high-yield enzyme mutants strains, these are UJ-3、UJ-5、DJ-1and DJ-2.Determination of amylase and protease activity respectively are4.457U/mLand1.509U/mL、5.092U/mLand11.728U/mL、4.513U/mLand12.306U/mL、5.124U/mLand13.031U/mL.The best conditions forsingle mutagenic treatment were ultraviolet irradiation160s; the concentration of DES is1.5mg/mL,processing40min. Using UV–DES compound mutagens, JASSB2Bacillus subtilis as original strain,on the basis of the single-factor test, used by the quadratic regression orthogonal rotation combinationtrial design. Optimize the mutagenesis conditions of composite mutagenesis JASSB2Bacillus subtilisand mutagenic treatment the JASSB2Bacillus subtilis under the optimized mutagenic conditions,determination the mutant enzyme activity of combined mutagenesis. The experimental results showthat under the conditions of ultraviolet irradiation160s; the concentration of DES is1.5mg/mL,processing40min can screening the mutant strains of U-DJ4,the amylase and protease activityrespectively is5.280U/mL和13.324U/mL3. Genome shuffling breeding high amylase and protease strains the research. The experimentalresults show that: the best conditions for Protoplast formation, inactivated, and fusion is processing15min, Lysozyme concentration is1.0mg/mL,37℃enzymolysis15h; Protoplast uv-inactivated is15min, heat-inactivated is60℃processing90min; Protoplast fusion conditions are the PEG6000concentration is40%, fusion temperature is34℃, fusion time is40min, at this point fusion rate can up to5.6×10^-5. To use mutagenesis proceeds strain as Original strain, After two rounds of shuffling get thereorganization bacteria of F299, It amylase and protease activity respectively is6.732U/mL and18.842U/mL。更多还原