本文選題：脈沖強(qiáng)光 + 枯草芽孢桿菌�。� 參考：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：枯草芽孢桿菌是當(dāng)今工業(yè)生產(chǎn)中重要的食品級(jí)益生菌,誘變選育高耐受性和發(fā)酵性能良好的菌株,不僅能提高菌株在生產(chǎn)加工中受不良環(huán)境影響的耐受能力,而且提高了其產(chǎn)酶水平和抑菌效果,在食品酶制劑的應(yīng)用和食品防腐等領(lǐng)域發(fā)揮了重要作用。脈沖強(qiáng)光誘變技術(shù)是以脈沖強(qiáng)光為獨(dú)特的誘變光源,利用其瞬時(shí)、高強(qiáng)度照射的光能影響微生物細(xì)胞,改變遺傳物質(zhì)結(jié)構(gòu),使蛋白質(zhì)合成發(fā)生變化,從而引起微生物性狀的改變。目前國(guó)內(nèi)外利用脈沖強(qiáng)光技術(shù)誘變枯草芽孢桿菌的研究報(bào)道極少,因此,本研究采用脈沖強(qiáng)光技術(shù)誘變處理枯草芽孢桿菌,篩選出性狀優(yōu)良的菌株,并初步分析其誘變機(jī)理,探究脈沖強(qiáng)光誘變枯草芽孢桿菌的可行性。試驗(yàn)結(jié)果如下:1.以脈沖電壓、脈沖次數(shù)、照射距離為影響因素,菌株致死率為指標(biāo),通過(guò)響應(yīng)面法優(yōu)化并確定脈沖強(qiáng)光致死菌株的最佳條件是:脈沖電壓2450 V、脈沖次數(shù)65次、照射距離5 cm。保持最佳脈沖電壓和最佳照射距離不變,在脈沖次數(shù)為8、14、21、27、34、40和47次條件下,探究不同脈沖次數(shù)對(duì)菌株致死率的影響,確定出脈沖次數(shù)為40次,篩選出八株抗性菌株B1～B8。2.以原始菌株B0和脈沖強(qiáng)光篩選出的B1～B8八株抗性菌株進(jìn)行了高溫耐受性、強(qiáng)酸耐受性和高濃度膽鹽耐受性試驗(yàn),最終篩選得出兼具三類高耐受性的優(yōu)越抗性變異菌株 B3、B4、B7。3.對(duì)比分析了原始菌株和變異菌株的發(fā)酵性能,結(jié)果表明變異菌株的發(fā)酵性能良好,產(chǎn)酶遺傳穩(wěn)定性和抗菌活性均明顯高于原始菌株。(1)采用平板透明圈法和Yoo改良法,分別測(cè)定菌株水解淀粉能力和發(fā)酵產(chǎn)α-淀粉酶活力。結(jié)果表明,變異菌株B3和B7水解淀粉能力比原始菌株B0更強(qiáng);B3和B7的α-淀粉酶活分別為(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,差異極顯著(P0.01);B3和B7分別傳代培養(yǎng)五次,產(chǎn)酶遺傳穩(wěn)定性良好。(2)參照菲利普的方法和福林-酚法,分別測(cè)定菌株降解蛋白能力和發(fā)酵產(chǎn)蛋白酶活力。結(jié)果表明,變異菌株B3和B7降解蛋白能力比原始菌株B0更強(qiáng);B3和B7的蛋白酶活分別為(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,差異極顯著(P0.01);B3和B7分別傳代培養(yǎng)五次,產(chǎn)酶遺傳穩(wěn)定性良好。(3)采用平板透明圈法和橄欖油乳化法,分別測(cè)定菌株分解脂肪能力和發(fā)酵產(chǎn)脂肪酶活力。結(jié)果表明,變異菌株B3和B7分解脂肪能力比原始菌株BO更強(qiáng);B3和B7的脂肪酶活分別為(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,差異顯著(P0.05);B3和B7分別傳代培養(yǎng)五次,產(chǎn)酶遺傳穩(wěn)定性良好。(4)測(cè)定菌株發(fā)酵產(chǎn)α-乙酰乳酸脫羧酶活力,結(jié)果表明,變異菌株B3和B7的α-乙酰乳酸脫羧酶活分別為(0.64±0.12)U/mL和(0.71±0.17)U/mL,分別是原始菌株B0酶活(0.45±0.23)U/mL的1.42倍和1.58倍,差異顯著(P0.05);B3和B7分別傳代培養(yǎng)五次,產(chǎn)酶遺傳穩(wěn)定性良好。(5)采用牛津杯法測(cè)定枯草芽孢桿菌的抗菌活性,并計(jì)算對(duì)金黃色葡萄球菌、大腸桿菌、沙門氏菌這三種不同菌的抗菌率。結(jié)果表明,變異菌株B3和B7對(duì)三種致病菌的抑菌效果最佳,均同屬等級(jí)一;原始菌株B0對(duì)大腸桿菌和沙門氏菌的抑菌效果稍差,屬于等級(jí)二;B3、B7對(duì)三種致病菌的抗菌率均高于B0,當(dāng)菌懸液濃度為103個(gè)/mL和104個(gè)/mL時(shí),差異顯著(P0.05);變異菌株B3、B7的抗菌活性良好。4.對(duì)經(jīng)脈沖強(qiáng)光誘變處理后的變異菌株B3、B7和原始菌株B0進(jìn)行菌體SDS-PAGE電泳試驗(yàn),結(jié)果表明,枯草芽孢桿菌經(jīng)脈沖強(qiáng)光誘變處理前后,菌體受到影響,菌株蛋白在電泳道上不同分子量處對(duì)應(yīng)呈現(xiàn)出來(lái)的條帶的有無(wú)和明暗深淺的不同,說(shuō)明脈沖強(qiáng)光引起了菌株蛋白質(zhì)表達(dá)的改變。
[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; 鐨勮泲鐧介叾媧誨垎鍒負(fù)(88.3鹵0.35)U/mL 鍜，

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