枯草芽孢杆菌脉冲强光诱变及其发酵性能研究
发布时间：2018-04-18 13:36
本文选题：脉冲强光 + 枯草芽孢杆菌 ； 参考：《沈阳农业大学》2017年硕士论文
【摘要】：枯草芽孢杆菌是当今工业生产中重要的食品级益生菌,诱变选育高耐受性和发酵性能良好的菌株,不仅能提高菌株在生产加工中受不良环境影响的耐受能力,而且提高了其产酶水平和抑菌效果,在食品酶制剂的应用和食品防腐等领域发挥了重要作用。脉冲强光诱变技术是以脉冲强光为独特的诱变光源,利用其瞬时、高强度照射的光能影响微生物细胞,改变遗传物质结构,使蛋白质合成发生变化,从而引起微生物性状的改变。目前国内外利用脉冲强光技术诱变枯草芽孢杆菌的研究报道极少,因此,本研究采用脉冲强光技术诱变处理枯草芽孢杆菌,筛选出性状优良的菌株,并初步分析其诱变机理,探究脉冲强光诱变枯草芽孢杆菌的可行性。试验结果如下:1.以脉冲电压、脉冲次数、照射距离为影响因素,菌株致死率为指标,通过响应面法优化并确定脉冲强光致死菌株的最佳条件是:脉冲电压2450 V、脉冲次数65次、照射距离5 cm。保持最佳脉冲电压和最佳照射距离不变,在脉冲次数为8、14、21、27、34、40和47次条件下,探究不同脉冲次数对菌株致死率的影响,确定出脉冲次数为40次,筛选出八株抗性菌株B1～B8。2.以原始菌株B0和脉冲强光筛选出的B1～B8八株抗性菌株进行了高温耐受性、强酸耐受性和高浓度胆盐耐受性试验,最终筛选得出兼具三类高耐受性的优越抗性变异菌株 B3、B4、B7。3.对比分析了原始菌株和变异菌株的发酵性能,结果表明变异菌株的发酵性能良好,产酶遗传稳定性和抗菌活性均明显高于原始菌株。(1)采用平板透明圈法和Yoo改良法,分别测定菌株水解淀粉能力和发酵产α-淀粉酶活力。结果表明,变异菌株B3和B7水解淀粉能力比原始菌株B0更强;B3和B7的α-淀粉酶活分别为(114.06±0.43)U/mL 和(120.89±0.32)U/mL,是 B0 酶活(68.3±0.14)U/mL的1.67倍和1.77倍,差异极显著(P0.01);B3和B7分别传代培养五次,产酶遗传稳定性良好。(2)参照菲利普的方法和福林-酚法,分别测定菌株降解蛋白能力和发酵产蛋白酶活力。结果表明,变异菌株B3和B7降解蛋白能力比原始菌株B0更强;B3和B7的蛋白酶活分别为(88.3±0.35)U/mL 和(96.79±0.26)U/mL,是B0 酶活(56.6±0.21)U/mL的1.56倍和1.71倍,差异极显著(P0.01);B3和B7分别传代培养五次,产酶遗传稳定性良好。(3)采用平板透明圈法和橄榄油乳化法,分别测定菌株分解脂肪能力和发酵产脂肪酶活力。结果表明,变异菌株B3和B7分解脂肪能力比原始菌株BO更强;B3和B7的脂肪酶活分别为(16.21 土0.27)U/mL和(17.18±0.36)U/mL,是B0酶活(12.1±0.15)U/mL的1.34倍和1.42倍,差异显著(P0.05);B3和B7分别传代培养五次,产酶遗传稳定性良好。(4)测定菌株发酵产α-乙酰乳酸脱羧酶活力,结果表明,变异菌株B3和B7的α-乙酰乳酸脱羧酶活分别为(0.64±0.12)U/mL和(0.71±0.17)U/mL,分别是原始菌株B0酶活(0.45±0.23)U/mL的1.42倍和1.58倍,差异显著(P0.05);B3和B7分别传代培养五次,产酶遗传稳定性良好。(5)采用牛津杯法测定枯草芽孢杆菌的抗菌活性,并计算对金黄色葡萄球菌、大肠杆菌、沙门氏菌这三种不同菌的抗菌率。结果表明,变异菌株B3和B7对三种致病菌的抑菌效果最佳,均同属等级一;原始菌株B0对大肠杆菌和沙门氏菌的抑菌效果稍差,属于等级二;B3、B7对三种致病菌的抗菌率均高于B0,当菌悬液浓度为103个/mL和104个/mL时,差异显著(P0.05);变异菌株B3、B7的抗菌活性良好。4.对经脉冲强光诱变处理后的变异菌株B3、B7和原始菌株B0进行菌体SDS-PAGE电泳试验,结果表明,枯草芽孢杆菌经脉冲强光诱变处理前后,菌体受到影响,菌株蛋白在电泳道上不同分子量处对应呈现出来的条带的有无和明暗深浅的不同,说明脉冲强光引起了菌株蛋白质表达的改变。
[Abstract]:Bacillus subtilis is a food grade important probiotics in today's industrial production, breeding of high tolerance and good fermentation performance can not only improve the strain, strain due to adverse environmental impact in the production and processing of tolerance, but also increase the level of enzyme production and antibacterial effect, play an important role in food enzyme preparation and application the preservation of food and other fields. In light pulse mutagenesis technique of pulsed light source for the mutation unique, using the instantaneous, high strength light irradiation effect of microbial cells, change the structure of the genetic material, the changes of protein synthesis, causing the microbial properties change. At present domestic and foreign research reports using pulsed light mutagenesis of Bacillus subtilis rarely, therefore, this study uses pulsed light treatment of Bacillus subtilis, select the excellent character of strain, and its preliminary analysis Mutation mechanism, probe into the feasibility of pulsed light mutagenesis of Bacillus subtilis. The experimental results are as follows: 1. pulse voltage, pulse frequency, irradiation distance factor, strain death rate as the index, through the optimization of response surface method and determine the optimum conditions of light pulse lethal strain is: 2450 V pulse voltage, pulse frequency of 65, irradiation a distance of 5 cm. pulse voltage to maintain the best and the best irradiation distance unchanged, in the pulse number for 8,14,21,27,34,40 and the 47 conditions, explore the different effect on the rate of pulse number of lethal strain, determined the pulse number is 40 times, screened eight strains of resistant strains of B1 ~ B8.2. to the original strain B0 and pulsed light screened B1 ~ eight strains of B8 resistant strains of high temperature tolerance, acid tolerance and high concentration of bile salt tolerance test, finally obtained has superior resistance mutation strain B3, three kinds of high tolerance to B4, B7.3. Comparative analysis of the fermentation performance of the original strain and mutant strain. The results showed that the fermentation performance of strains of good enzyme producing genetic stability and antibacterial activity were significantly higher than that of the original strain. (1) by plate culture method and Yoo method, the determination of strains hydrolyzed starch fermentation capacity and amylase activity respectively. The results showed that B3 and B7 strains, starch hydrolysis ability stronger than the original strain B0; B3 and B7 of the alpha amylase activity were (114.06 + 0.43) U/mL and (120.89 + 0.32) U/mL, B0 enzyme activity (68.3 + 0.14) U/mL 1.67 times and 1.77 times the difference was significant (P0.01); B3 and B7 were subcultured five times, enzyme producing good genetic stability. (2) according to the method and Folin Philip - phenol method, determination of protein degradation ability and protease activity of fermentation respectively. The results showed that the strains of B3 and B7 protein degradation ability than the original strain B0 is more B3 and B7; 鐨勮泲鐧介叾娲诲垎鍒负(88.3卤0.35)U/mL 鍜，
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