高产胞外多糖乳酸乳杆菌的选育及其发酵条件的优化
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摘要
乳酸菌(lactic acid bacteria, LAB)胞外多糖(exopolysaccharide, EPS)是乳酸菌在生长代谢过程中分泌到细胞壁外常渗于培养基的一类糖类化合物,除了对细菌自身具有生物学意义,赋予发酵乳制品特殊的质构和风味,而广泛用于各种食品的增稠、稳定、乳化、胶凝及持水,起到安全食品添加剂的作用。大量研究表明,LAB在抗变异原性、防癌抗癌和增强机体免疫力方面有着不可低估的作用,而它营养保健特性的发挥主要是通过其代谢过程中形成的胞外多糖物质来完成的。本实验旨在对于乳酸菌的特定菌株产的胞外多糖进行一定深入的研究,提高菌株胞外多糖的产率,得到胞外多糖高产菌株及优化其发酵条件,满足工业化生产要求。
研究结果如下：
1、本课题是从市售酸奶中分离纯化得到菌株,经生理生化鉴定,均成阴性反应,结合伯杰明手册可知其与乳杆菌属相符,再经糖发酵实验,16S rDNA检测鉴定得到保加利亚乳杆菌、乳酸乳杆菌。
2、初步探索了野生型乳杆菌的生长特性,接着对菌株分别采用物理、化学、生物学方法进行诱变选育。紫外诱变筛选出2株高产胞外多糖的正突变株,对应的多糖产量为2.85g/L,3.15g/L；亚硝基胍(NTG)筛选出4株高产胞外多糖的正突变株,对应的多糖产量为2.413g/L,2.841g/L,2.743g/L,2.694g/L；菌株UV-NTG复合诱变,筛选出3株高产胞外多糖的正突变株,对应的多糖产量为5.219g/L,4.926g/L,4.805g/L；将UV和NTG筛选到的6株高产菌作为基因组改组的出发菌株,经第一轮改组得到4株高产菌株F1,产量最多可达6.827g/L；经过筛选获得4株第二轮改组菌株F2,产量最多可达9.38g/L。
3、探索了乳杆菌产胞外多糖的发酵特性,从6种适合乳酸菌生长的培养基中确定Elliker作为最适发酵培养基；采用N=16的Plackett-Burman试验设计,对Elliker培养基的成分和菌株生长条件等各变量进行考察,筛选出对乳酸乳杆菌产胞外多糖影响显著的因子：[酵母粉](P<0.001)、初始pH(P=0.0360)、发酵时间(P=0.0047)；然后采用中心组合设计试验,响应面分析得到确定最佳发酵条件为：[酵母粉]=17.35g/L,初始pH=5.85,发酵时间为23.18h。在预测发酵条件进行发酵实验,得到实际产量为3.910g/L。
4、初步探索了不同酵母粉浓度(0g/L、10g/L、20g/L)对野生型乳酸乳杆菌发酵过程生长速率和EPS产率的影响,都显示出高浓度的初始酵母粉浓度对菌株生长及代谢产物EPS有一定抑制作用；经过基因组改组后改组菌株的生物量(OD值)和EPS产量明显高于原始菌株和传统诱变菌株；经第二轮改组([酵母粉]30g/L)后的菌株,在酵母粉浓度15g/L时,生物量OD值约为原始菌株的2倍,筛选到的F2-1菌株EPS浓度最高达10.021g/L,提高了534.64%。结果表明基因组改组技术能够快速提高乳酸菌耐酵母粉和产EPS能力两个表型。
Lactic acid bacterium exopolysaccharides is produced with Lactic acid bacteria growing and penetrates into culture medium. Exopolysaecharides by lactic acid bacteria plays an important role in the theological properties of dairy products, as stabilizers and thickeners. Important features of the starter are rapid acidification, microbial preservation of the milk, formation of specific flavours, texturing capacities and health benefits. In recent years, There has been a upsurge in interest in immunostimulating activity, enhance lymphocyte mitogenicity and macrophage cytostaticity. The following are main results:
1. Two strains were obtained by sceening, isolate and identify Lactic acid bacterium from yoghurt from market. Based on results of the characteristics of morphology, physiology, and biochemistry, sugar fermentation, they are identified by API bacterial identifiation system as Lactobacillus lactis and Lactobacillus bulgaricus.
2. After the characteristic study of strain L. lactis, we studied physical, chemic, biologic mutantation to enhance the yield of exopolysaccarides. Two mutant strains were obtained by ultraviolet irradiation, the high yield of which was 2.85g/L,3.15 g/L. Four mutant strains were obtained by NTG mutagenesis, the high yield of which was 2.413g/L,2.841 g/L,2.743g/L,2.694g/L. Three mutant strains were obtained by ultraviolet irradiation and NTG mutagenesis, the high yield of which was 5.219g/L, 4.926g/L,4.805g/L. The mutant strains with high yield, which were obtained by ultraviolet irradiation and NTG mutagenesis, as a parent library in recursive fusion. Through two rounds of genome shuffling, a mutant strains with highest yield were obtained, the yield of which was 9.38g/L,
3. After the characteristic study of strain L. lactis in the fermentation, the results showed that Elliker was the optimal medium. Three statistically significant ingredients, yeast extract, the initial pH and fermentation time, which were determined by Plackett-Burman design. Based on the results of the first phase, the five-level three-factor central composite design was employed to optimize the above critical factors. It showes that the maxmium yield of exopolysaccharid was 3.910g/L, with yeast extract 17.35g/L, the initial pH5.85, fermentation time 23.18h.
4. After the characteristic study of strain L. lactis in deffierent yeast extract concentration medium, the results indicated high yeast extract The high yield mutant strains with improved yeast extract tolerance, which were obtained by ultraviolet irradiation and NTG mutagenesis, as a parent library in recursive fusion. The fusants were screened under YE plates. Through two rounds of genome shuffling, the mutants obtained the compatibility of two optimal phenotypes of yeast extract tolerance and EPS enhancement, the highest yield was 10.021g/L. The results indicated application of the genome shuffling in improving the yeast extract tolerance of L. lactis and enhance EPS production.
研究结果如下：
1、本课题是从市售酸奶中分离纯化得到菌株,经生理生化鉴定,均成阴性反应,结合伯杰明手册可知其与乳杆菌属相符,再经糖发酵实验,16S rDNA检测鉴定得到保加利亚乳杆菌、乳酸乳杆菌。
2、初步探索了野生型乳杆菌的生长特性,接着对菌株分别采用物理、化学、生物学方法进行诱变选育。紫外诱变筛选出2株高产胞外多糖的正突变株,对应的多糖产量为2.85g/L,3.15g/L；亚硝基胍(NTG)筛选出4株高产胞外多糖的正突变株,对应的多糖产量为2.413g/L,2.841g/L,2.743g/L,2.694g/L；菌株UV-NTG复合诱变,筛选出3株高产胞外多糖的正突变株,对应的多糖产量为5.219g/L,4.926g/L,4.805g/L；将UV和NTG筛选到的6株高产菌作为基因组改组的出发菌株,经第一轮改组得到4株高产菌株F1,产量最多可达6.827g/L；经过筛选获得4株第二轮改组菌株F2,产量最多可达9.38g/L。
3、探索了乳杆菌产胞外多糖的发酵特性,从6种适合乳酸菌生长的培养基中确定Elliker作为最适发酵培养基；采用N=16的Plackett-Burman试验设计,对Elliker培养基的成分和菌株生长条件等各变量进行考察,筛选出对乳酸乳杆菌产胞外多糖影响显著的因子：[酵母粉](P<0.001)、初始pH(P=0.0360)、发酵时间(P=0.0047)；然后采用中心组合设计试验,响应面分析得到确定最佳发酵条件为：[酵母粉]=17.35g/L,初始pH=5.85,发酵时间为23.18h。在预测发酵条件进行发酵实验,得到实际产量为3.910g/L。
4、初步探索了不同酵母粉浓度(0g/L、10g/L、20g/L)对野生型乳酸乳杆菌发酵过程生长速率和EPS产率的影响,都显示出高浓度的初始酵母粉浓度对菌株生长及代谢产物EPS有一定抑制作用；经过基因组改组后改组菌株的生物量(OD值)和EPS产量明显高于原始菌株和传统诱变菌株；经第二轮改组([酵母粉]30g/L)后的菌株,在酵母粉浓度15g/L时,生物量OD值约为原始菌株的2倍,筛选到的F2-1菌株EPS浓度最高达10.021g/L,提高了534.64%。结果表明基因组改组技术能够快速提高乳酸菌耐酵母粉和产EPS能力两个表型。
Lactic acid bacterium exopolysaccharides is produced with Lactic acid bacteria growing and penetrates into culture medium. Exopolysaecharides by lactic acid bacteria plays an important role in the theological properties of dairy products, as stabilizers and thickeners. Important features of the starter are rapid acidification, microbial preservation of the milk, formation of specific flavours, texturing capacities and health benefits. In recent years, There has been a upsurge in interest in immunostimulating activity, enhance lymphocyte mitogenicity and macrophage cytostaticity. The following are main results:
1. Two strains were obtained by sceening, isolate and identify Lactic acid bacterium from yoghurt from market. Based on results of the characteristics of morphology, physiology, and biochemistry, sugar fermentation, they are identified by API bacterial identifiation system as Lactobacillus lactis and Lactobacillus bulgaricus.
2. After the characteristic study of strain L. lactis, we studied physical, chemic, biologic mutantation to enhance the yield of exopolysaccarides. Two mutant strains were obtained by ultraviolet irradiation, the high yield of which was 2.85g/L,3.15 g/L. Four mutant strains were obtained by NTG mutagenesis, the high yield of which was 2.413g/L,2.841 g/L,2.743g/L,2.694g/L. Three mutant strains were obtained by ultraviolet irradiation and NTG mutagenesis, the high yield of which was 5.219g/L, 4.926g/L,4.805g/L. The mutant strains with high yield, which were obtained by ultraviolet irradiation and NTG mutagenesis, as a parent library in recursive fusion. Through two rounds of genome shuffling, a mutant strains with highest yield were obtained, the yield of which was 9.38g/L,
3. After the characteristic study of strain L. lactis in the fermentation, the results showed that Elliker was the optimal medium. Three statistically significant ingredients, yeast extract, the initial pH and fermentation time, which were determined by Plackett-Burman design. Based on the results of the first phase, the five-level three-factor central composite design was employed to optimize the above critical factors. It showes that the maxmium yield of exopolysaccharid was 3.910g/L, with yeast extract 17.35g/L, the initial pH5.85, fermentation time 23.18h.
4. After the characteristic study of strain L. lactis in deffierent yeast extract concentration medium, the results indicated high yeast extract The high yield mutant strains with improved yeast extract tolerance, which were obtained by ultraviolet irradiation and NTG mutagenesis, as a parent library in recursive fusion. The fusants were screened under YE plates. Through two rounds of genome shuffling, the mutants obtained the compatibility of two optimal phenotypes of yeast extract tolerance and EPS enhancement, the highest yield was 10.021g/L. The results indicated application of the genome shuffling in improving the yeast extract tolerance of L. lactis and enhance EPS production.
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