CN116064360A - 一种提高菌株冷冻干燥存活率的方法 - Google Patents
一种提高菌株冷冻干燥存活率的方法 Download PDFInfo
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Abstract
本发明涉及一种提高菌株冷冻干燥存活率的方法,通过在植物乳杆菌胞内构建出海藻糖的生成途径,证明胞内海藻糖能提高菌株冻干存活率,且使海藻糖实现其最大化的保护效果,避免菌株差异性。与现有技术相比,本发明能高效提高冷冻干燥后菌株的存活率,还能初步判断海藻糖对菌株发挥保护效果的位点,具有较好的可靠性。
Description
技术领域
本发明涉及生物技术领域,尤其是涉及一种提高菌株冷冻干燥存活率的方法。
背景技术
益生菌,尤其是包括植物乳杆菌在内的乳酸菌,以其生长速度快、价格低廉,而且具有营养功能、肠道保护功能、免疫功能以及降血糖血脂功能等方面的特性,日益受到人们的重视,相关研究也越来越广泛且深入,相关应用越来越广泛,包括医疗和食品等行业,有着广阔的发展前景。随着它们在工业上广泛使用的同时,在运输以及储存过程中,如何保持较高的活菌数量和活力面临着巨大的挑战。
冷冻干燥凭借其在益生菌保存和运输方面的优势起到了很大的作用,该方法能在运输以及储存过程中,保持较高的活菌数量和活力。而为提高细胞对冷冻干燥的抗性,通常会通过使用保护剂以减轻冷冻干燥带来的损伤,并且通过优化外源所添加保护剂来降低此过程中造成的损伤,但保护剂的效果存在明显的菌株差异性,给定一个保护剂如何实现其最大化的保护效果是目前尚未解决的问题。
海藻糖在逆环境中具有保护效果,让菌株在逆环境中损伤降至最低,众多研究证明外源海藻糖对植物乳杆菌[1]、嗜酸乳杆菌[2]、干酪乳杆菌[3]在处于冷冻干燥或者干燥储存过程都具有保护效果;其中与山梨醇保护效果不相同的是,添加海藻糖能将植物乳杆菌冻干存活率提升到40%的水平[4],但同样存在菌株差异性。已有研究证明海藻糖水平与细胞对抗所处逆环境的应激能力相关,如在酸胁迫作用下,菌株胞内的海藻糖含量会上升[5],因此许多文献通过增加胞内海藻糖含量的方法来增强菌株对抗逆环境的耐受力[6][7]。
[1]Wang G,Luo L,Dong C,et al.Polysaccharides can improve the survivalof Lactiplantibacillus plantarum subjected to freeze-drying[J].Journal ofDairy Science,2021,104(3):2606-14.
[2]Shu G,Wang Z,Chen L,et al.Characterization of freeze-driedLactobacillus acidophilus in goat milk powder and tablet:Optimization of thecomposite cryoprotectants and evaluation of storage stability at differenttemperature[J].LWT-Food Science and Technology,2018,90:70-6.
[3]Dimitrellou D,Kandylis P,Kourkoutas Y.Effect of cooling rate,freeze-drying,and storage on survival of free and immobilized Lactobacilluscasei ATCC 393[J].LWT-Food Science and Technology,2016,69:468-73.
[4]Wang G Q,Pu J,Yu X Q,et al.Influence of freezing temperaturebefore freeze-drying on the viability of various Lactobacillus plantarumstrains[J].Journal of Dairy Science,2020,103(4):3066-75.
[5]Cardoso F S,Gaspar P,Hugenholtz J,et al.Enhancement of trehaloseproduction in dairy propionibacteria through manipulation of environmentalconditions[J].International Journal of Food Microbiology,2004,91(2):195-204.
[6]Purvis J E,Yomano L P,Ingram L O.Enhanced trehalose productionimproves growth of Escherichia coli under osmotic stress[J].Applied andEnvironmental Microbiology,2005,71(7):3761-9.
[7]Termont S,Vandenbroucke K,Iserentant D,et al.Intracellularaccumulation of trehalose protects Lactococcus lactis from freeze-dryingdamage and bile toxicity and increases gastric acid resistance[J].Applied andEnvironmental Microbiology,2006,72(12):7694-700.
发明内容
本发明的目的就是为了克服上述现有技术存在的缺陷而提供一种提高菌株冷冻干燥存活率的方法,通过在植物乳杆菌胞内构建出海藻糖的生成途径,证明胞内海藻糖能提高菌株冻干存活率,且使海藻糖实现其最大化的保护效果,避免菌株差异性。
本发明的目的可以通过以下技术方案来实现:
一种提高菌株冷冻干燥存活率的方法,将otsA基因和otsB基因(简称otsBA)导入植物乳杆菌中,实现植物乳杆菌异源合成海藻糖,从而提高植物乳杆菌冻干存活率;
所述otsA基因的核苷酸序列如SEQ ID NO.7所示;
所述otsB基因的核苷酸序列如SEQ ID NO.8所示。
进一步地,所述otsA基因和otsB基因来源于大肠杆菌DH5ɑ。
进一步地,所述植物乳杆菌为植物乳杆菌AR113。
进一步地,利用植物乳杆菌中的高拷贝质粒过表达otsA基因和otsB基因,编码合成海藻糖的相关酶。
上述更进一步地,所述高拷贝质粒为pIB184。
上述更进一步地,所述酶包括海藻糖-6-磷酸合成酶和海藻糖-6-磷酸酶。
上述更进一步地,所述合成海藻糖的具体过程为:葡萄糖通过海藻糖-6-磷酸合成酶催化合成海藻糖-6-磷酸,再通过海藻糖-6-磷酸酶催化海藻糖-6-磷酸去磷化合成海藻糖。
上述更进一步地,所述过表达otsA基因和otsB基因的植物乳杆菌正常生长。
进一步地,将otsA基因和otsB基因导入植物乳杆菌后,得到重组植物乳杆菌,在重组植物乳杆菌外部添加保护剂,进一步提高植物乳杆菌冻干存活率。
进一步地,海藻糖提高植物乳杆菌冻干存活率的表现为:植物乳杆菌细胞结构完整,植物乳杆菌细胞与PBS均匀结合。
本发明的原理如下:
益生菌作为发酵剂在工业上的应用依赖于其技术性能、浓度和保鲜技术,在长期贮藏过程中保持较高的生存力、酸化活性和酶活性是必需的。迄今为止,人们已经探索了各种封装策略和干燥技术,以保护生产过程和储存期间的细菌活力,在各种干燥技术中,冷冻干燥技术是工业生产中保持细菌稳定性最常用、最有效的方法。
海藻糖在大肠杆菌中存在一种代谢途径:首先由海藻糖-6-磷酸合成酶和海藻糖-6-磷酸磷酸酶催化合成,分别是由otsA和otsB编码;otsA和otsB形成的操纵子分别在菌株处于高渗透压和进入静止阶段时进行诱导,胞内所积累的海藻糖被细胞质内的海藻糖酶treF分解成葡萄糖,确保海藻糖的连续利用。大肠杆菌中的otsBA操纵子大小为2200bp,包含两个基因:otsB和otsA;1-801bp为otsB基因,编码海藻糖-6-磷酸酶(EC3.1.3.12)催化反应海藻糖-6-磷酸水解为海藻糖;776-220bp为otsA基因,编码海藻糖-6-磷酸合成酶(EC2.4.1.15),催化反应葡萄糖-6-磷酸合成海藻糖-6-磷酸。
在植物乳杆菌中代谢途径中,缺乏与海藻糖相关的合成途径otsA和otsB,且植物乳杆菌中未发现存在海藻糖所谓合成途径的证明,因此尝试通过在植物乳杆菌中表达otsA和otsB来实现胞内海藻糖的产生。植物乳杆菌中存在这两种酶的反应底物,因此在植物乳杆菌中异源表达来自大肠杆菌中的otsBA对合成胞内海藻糖的可能性最大。实现在植物乳杆菌中产生海藻糖的目的,通过胞内产生海藻糖的方法提供菌株的冻干存活率。
目前有些多物理方法可以将小分子保护剂有效地传递到细胞中,常见的一些传递方法有热击、渗透、电击、液相内吞和磷脂相变等;液相内吞所需要的加载时间较长且效率较低,渗透所需条件和时间较长。其中电穿孔的方式用于加载小分子进入细胞有巨大的应用潜力,PEF电场已证明是一种可用于加载保护剂进入菌株中的方法,因此通过PEF的方法加载已被证明在胞内保护效果更好的小分子保护剂海藻糖进入胞内提高菌株的冷冻干燥存活率。
脉冲电场(PEF)可应用于微生物灭活、稳定生物活性化合物和营养物质、减少食品中的污染、以及从生物体中提取营养成分等,PEF处理过程中会对细胞膜产生对应的影响,导致细胞膜中孔隙的形成,根据所应的PEF和密封的条件,孔隙分为可逆和不可逆的。使用PEF系统加载海藻糖保护剂,增加胞内保护剂含量达到增加菌株冷冻干燥存活率目的。
与现有技术相比,本发明的优点如下:
1.本发明能高效提高冷冻干燥后菌株的存活率,还能初步判断海藻糖对菌株发挥保护效果的位点,具有较好的可靠性;
2.本发明提供了一种存在于胞内的保护剂,对菌株处于冷冻干燥过程产生更好的保护效果;
3.本发明从菌株本身内部角度进行更加深入的研究,并分析保护剂的相应生物合成特性以及相关应用的差异性,达到减少菌株在冻干过程中受的损伤这一目的。
附图说明
图1为过表达质粒构建流程;
图2为PEF处理流程图;
图3为大肠杆菌胞内海藻糖的代谢工程系统,其中,图画中浅灰色部分代表植物乳杆菌中所缺的代谢途径;
图4为构建过表达菌株的电泳图和生长情况;
图5为重组菌株的目的蛋白的表达;
图6为重组菌株经过冷冻干燥处理后的存活率;
图7为AR113和AR113-otsBA经过冷冻干燥后的菌株形态以及受损状态,其中,a为AR113菌株在SEM电镜200μm的菌株形态;a为AR113菌株在SEM电镜200μm的菌株形态;b为AR113菌株在SEM电镜500μm的菌株形态;c为AR113菌株在SEM电镜100μm的菌株形态;d为AR113-otsBA菌株在SEM电镜200μm的菌株形态;e为AR113-otsBA菌株在SEM电镜500μm的菌株形态;f为AR113-otsBA菌株在SEM电镜100μm的菌株形态;
图8为不同植物乳杆菌经过PEF处理后的冷冻干燥存活率,其中,a为PBS介质PEF处理(对照组:胞内不含海藻糖),b为海藻糖介质PEF处理(胞内含海藻糖);
图9为不同益生菌经过PEF处理后的冷冻干燥存活率,其中,a为PBS介质PEF处理(对照组:胞内不含海藻糖),b为海藻糖介质PEF处理(胞内含海藻糖)。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明。本实施例以本发明技术方案为前提进行实施,给出了详细的实施方式和具体的操作过程,但本发明的保护范围不限于下述的实施例。
以下各实施例中,所需材料如下所示:
MRS培养基(1L):蛋白胨10.0g,酵母粉5.0g,牛肉浸粉10.0g,柠檬酸氢二铵2.0g,葡萄糖20.0g,吐温-80 1.0mL,乙酸钠5.0g,磷酸氢二钾2.0g,硫酸镁0.58g,硫酸锰0.25g,pH为6.2~6.6,用于乳杆菌的活化、培养和活菌计数,115℃灭菌20min;
LB培养基(1L):胰蛋白胨10.0g,酵母浸出粉5.0g,NaCl 10.0g,121℃灭菌15min;
试剂:Trizol、无缝克隆酶、PBS缓冲液、海藻糖。
菌株及质粒来源如表1所示:
表1菌株及其特性
植物乳杆菌AR113保藏信息如下:植物乳杆菌(Lactobacillus plantarum)AR113保藏编号为CGMCC No.13909,保藏单位为中国普通微生物保藏中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏日期为2017年03月22日,在CN111304134A专利中公开过。
嗜热链球菌S-3保藏信息如下:嗜热链球菌(Streptococcus thermophilus)保藏编号为CGMCC No.12098,保藏单位为中国普通微生物菌种保藏管理中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏日期为2016年1月22日,在CN108220201A专利中公开过。
植物乳杆菌AR307的保藏信息如下:植物乳杆菌(Lactobacillusplantarum)保藏编号为CGMCC No.10773,保藏单位为中国普通微生物菌种保藏管理中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏日期为2015年4月29日,在CN105400725A专利中公开过。
植物乳杆菌AR495的保藏信息如下:植物乳杆菌(Lactobacillusplantarum)保藏编号为CGMCC No.14004,保藏单位为中国普通微生物菌种保藏管理中心,保藏地址为北京市朝阳区北辰西路1号院3号,保藏日期为2017年04月07日,在CN114196600A专利中公开过。
植物乳杆菌WCFS1、乳酸乳球菌NZ9000来自市售。
植物乳杆菌AR514为发明人在实验室自行筛选得到。
以下各实施例中,所需质粒如下所示:
pIB184质粒来源于文章:
Shuttle expression plasmids for genetic studies in Streptococcusmutans.
Biswas I,Jha JK,Fromm N.Microbiology.2008Aug;154(Pt 8):2275-82.doi:10.1099/mic.0.2008/019265-0.
以下各实施例中,所需仪器如下所示:
PCR仪、ChemiDoc XRS+凝胶成像仪、厌氧培养箱、真空冷冻干燥机、MicroPulse电转化仪、SEM电镜。
以下实施例中,核苷酸序列如下所示:
引物otsBA-F的核苷酸序列,序列如SEQ ID NO.1所示:
5’-TGGATCCCCGCGGTACCCGGGAATTCTTATCCTCGCGATTTAC-3’;
引物otsBA-R的核苷酸序列,序列如SEQ ID NO.2所示:
5’-AATCACTTAAGCTTATCGATAGATCTATGACGACAGATTGGTT-3’;
pIB184质粒的核苷酸序列,序列如SEQ ID NO.3所示:
aactttgcaatttgttctagtgtttttatggttggatctgattttcctgattctattcgtgaataatttgatctactcatttctaattcttggggtaccgccagcatttcggaaaaaaaccacgctaaggattttttctataaaaagagccgttatattaagaataaaacggctcttttatacgtaaaggacgtaaattcatttgcccagtgtcatgtaatccttcaaatttgtattctccaagaaaattgatatgttcccatcctaacggccacgcatatggcattaaatcttctctaaattctcctcttgcttttaattcttctacggctttttccatatatacagtgttccacacacttatagcgttaataattatgtttagtgcactagctctttgtaactggtcttggagagcacgttctctaaattctccacgttgtccaaaaaatatagttctagctaatgcattgattgcttctcctttatttaaacctttttgaacccgtctccttacggctttattagatatgtaatccagcgtaaagagggttttctcgattcgtcccatttctccaagtgctgttgcgagtttattttgtcttgcatatgatccgagcttccccatgataagagcgctagggacctctttagctccttggaagctgtcagtagtatacctaataatttatctacattccctttagtaacgtgtaactttccaaatttacaaaagcgactcatagaattatttcctcccgttaaataatagataactattaaaaatagacaatacttgctcataagtaacggtacttaaattgtttactttggcgtgtttcattgcttgatgaaactgatttttagtaaacagttgacgatattctcgattgacccattttgaaacaaagtacgtatatagcttccaatatttatctggaacatctgtggtatggcgggtaagttttattaagacactgtttacttttggtttaggatgaaagcattccgctggcagcttaagcaattgctgaatcgagacttgagtgtgcaagagcaaccctagtgttcggtgaatatccaaggtacgcttgtagaatccttcttcaacaatcagatagatgtcagacgcatggctttcaaaaaccacttttttaataatttgtgtgcttaaatggtaaggaatactcccaacaattttatacctctgtttgttagggaattgaaactgtagaatatcttggtgaattaaagtgacacgagtattcagttttaatttttctgacgataagttgaatagatgactgtctaattcaatagacgttacctgtttacttattttagccagtttcgtcgttaaatgccctttacctgttccaatttcgtaaacggtatcggtttcttttaaattcaattgttttattatttggttgagtactttttcactcgttaaaaagttttgagaatattttatatttttgttcatgtaatcactccttcttaattacaaatttttagcatctaatttaacttcaattcctattatacaaaattttaagatactgcactatcaacacactcttaagtttgcttctaagtcttatttccataacttcttttacgtttccgccattctttgctgtttcgatttttatgatatggtgcaagtcagcacgaacacgaaccgtcttatctcccattatatctttttttgcactgattggtgtatcatttcgtttttcttttgcgcttcttgataaaagggatagtaattcattcctggttgcaaattttgaaaaccgctacggatcacatctttttctaaactattgatccatagtcttttatacgttttatctttagaaaaggcatttgctttatgaatgatcgaccaggcaatgttttcgccttctctgtcgctatctgttgcgacaatgattgtatttgcttgttttaaaagttcagcaacaattttaaactgcttttttttatctgttgccacttcaaaatcgtatcgattctagatttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgtctagaatcgataagcttggctgcaggtcgataaggctattggtgtttatggctctcttggtcgtcagactgatgggccccgaaaagccctgacaacccttgttcctaaaaaggaataagcgttcggtcagtaaataatagaaataaaaaatcagacctaagactgatgacaaaaagagaaaattttgataaaatagtcttagaattaaattaaaaagggaggccaaatataatgaaaaatatgaatgacaatgatgttggatccccgcggtacccgggaattctagagctcgagatctatcgataagcttaagtgattagtcaaagaatggtgatgacaattgtaaattctatttaatcactttgactagcaaatactaacaacaagacacacacaccaaaaatcaaaaattcactacttttagttaaaaaccacgtaaccacaagaactaatccaatccatgtaatcgggttcttcaaatatttctccaagattttcctcctctaatatgctcaacttaaatgacctattcaataaatctattatgctgctaaatagtttataggacaaataagtatactctaatgacctataaaagatagaaaattaaaaaatcaagtgttcgcttcgctctcactgcccctcgacgttttagtagcctttccctcacttcgttcagtccaagccaactaaaagttttcgggctactctctccttctccccctaataattaattaaaatcttactctgtatatttctgctaatcattcgctaaacagcaaagaaaaaacaaacacgtatcatagatataaatgtaatggcatagtgcgggttttattttcagcctgtatcatagctaaacaaatcgagttgtgtgtccgttttagggcgttctgctagcttgtttaaagtctcttgaatgaatgtatgctctaagtcaaaagaatttgtcagcgcctttatatagctttctttttcttctttttttactttaatgatcgatagcaacaatgatttaacactagcaagttgaatgccaccatttcttcctggtttaatcttaaagaaaatttcctgattcgccttcagtaccttcagcaatttatctaatgtccgttcaggaatgcctagcacttctctaatctcttttttggtcgtcactaaataaggcttgtatacatcgcttttttcgctaatataagccattaaatcttctttccattctgacaaatgaacacgttgacgttcgcttctttttttcttgaatttaaaccacccttgacggacaaataaatctttactggttaaatcacttgatacccaagctttgcaaagaatggtaatgtattccctattagccccttgatagttttctgaataggcacttctaacaattttgattacttctttttcttctaagggttgatctaatcgattattaaactcaaacatattatattcgcacgtttcgattgaatagcctgaactaaagtaggctaaagagagggtaaacatgacgttattacgccctattaaacccttttctcctgaaaatttcgtttcgtgcaataagagattaaaccagggttcatctacttgttttttgccttctgtaccgcttaaaaccgttagacttgaacgagtaaagcccttattatctgtttgtttgaaagaccaatcttgccattctttgaaagaataacggtaattaggatcaaaaaattctacattgtccgttcttggtatgcgagcaataccaaaatgattacacgttagatcaactggcaaagactttccaaaatattctcggatattttgcgaaattattttggctgctttgacagatttaaattctgattttgaagtcacatagactggcgtttctaaaacaaaatatgcttgataacctttatcagatttgataatcatagtaggcataaaacctaaatcaatagcggttgttaaaatatcgcttgctgaaatagtttcttttgccgtgtgaatatcaaaatcaataaagaaggtattgatttgtcttaaattgttttcagaatgtcctttcgtgtatgaacggttttcgtctgcatacgttccataacgataaacgttgggtgtccaatgtgtaaatgtatcttgattttcttgaatcgcttcctcggaagtcagaacaacaccacgaccgccaatcatgcttgattttgagcgatacgcaaaaatagcccctttgcttttacctggcttggtagtgattgagcgaattttactatttttaaatttgtactttaacaagccgtcatgaagcacagtttctacaacaaaagggatattcattcagctgttctcctttcctataaatcctataaaataggttgtttaattaacttggtttgctttttcattcaactgtttcaatattgcatgttttgaaaaagatttttttcctttataagtcaatttttttccactaatcgaataaattattttgttattttctattaacttatatatataatcttccccctccgaagaaaaatacttatctgattttgtttctaagtagatatttctcttttctaactctttcttaaacgtttctagtgtatagatatttgctaattttcttatctccaataaactattttttatataagttttacattcatcatgattcatacaaactccaccttctataaatgaatacaaaaaaagcaatcaaacgatttccgattgattgcttaacaattcttaaattcagtagcttagatacttgaaaactctctgatttccctatataatgatagtacggttatataccgtcttcaaacaaagttaattaaataacttcttacgagggaagagttcatctgactaactgataagcgttggtttggcaatcttatcgggctatgcatttataaaatgtcgtcaaacattttataaatgtgtcatggctcttttttcgtttctattcagttcgttgtttcgttatatctagtataccgcttttaaaaaaaaataagcaacgatttcgtgcattattcacacgaagtcattgcttttttcttcttccatttctaaatccaatgttacttgttctgattctgtttctggctctggttctgttggctcatttgggattaaatccactactagcgttgagttagtt;
引物pIB184-yz-F的核苷酸序列,序列如SEQ ID NO.4所示:
5’-GATGGGCCCCGAAAAGC-3’;
引物pIB184-yz-R的核苷酸序列,序列如SEQ ID NO.5所示:
5’-GAGAAATATTTGAAGAACCCGATTACATGG-3’;
质粒pIB184-otsBA的核苷酸序列,序列如SEQ ID NO.6所示:
aactttgcaatttgttctagtgtttttatggttggatctgattttcctgattctattcgtgaataatttgatctactcatttctaattcttggggtaccgccagcatttcggaaaaaaaccacgctaaggattttttctataaaaagagccgttatattaagaataaaacggctcttttatacgtaaaggacgtaaattcatttgcccagtgtcatgtaatccttcaaatttgtattctccaagaaaattgatatgttcccatcctaacggccacgcatatggcattaaatcttctctaaattctcctcttgcttttaattcttctacggctttttccatatatacagtgttccacacacttatagcgttaataattatgtttagtgcactagctctttgtaactggtcttggagagcacgttctctaaattctccacgttgtccaaaaaatatagttctagctaatgcattgattgcttctcctttatttaaacctttttgaacccgtctccttacggctttattagatatgtaatccagcgtaaagagggttttctcgattcgtcccatttctccaagtgctgttgcgagtttattttgtcttgcatatgatccgagcttccccatgataagagcgctagggacctctttagctccttggaagctgtcagtagtatacctaataatttatctacattccctttagtaacgtgtaactttccaaatttacaaaagcgactcatagaattatttcctcccgttaaataatagataactattaaaaatagacaatacttgctcataagtaacggtacttaaattgtttactttggcgtgtttcattgcttgatgaaactgatttttagtaaacagttgacgatattctcgattgacccattttgaaacaaagtacgtatatagcttccaatatttatctggaacatctgtggtatggcgggtaagttttattaagacactgtttacttttggtttaggatgaaagcattccgctggcagcttaagcaattgctgaatcgagacttgagtgtgcaagagcaaccctagtgttcggtgaatatccaaggtacgcttgtagaatccttcttcaacaatcagatagatgtcagacgcatggctttcaaaaaccacttttttaataatttgtgtgcttaaatggtaaggaatactcccaacaattttatacctctgtttgttagggaattgaaactgtagaatatcttggtgaattaaagtgacacgagtattcagttttaatttttctgacgataagttgaatagatgactgtctaattcaatagacgttacctgtttacttattttagccagtttcgtcgttaaatgccctttacctgttccaatttcgtaaacggtatcggtttcttttaaattcaattgttttattatttggttgagtactttttcactcgttaaaaagttttgagaatattttatatttttgttcatgtaatcactccttcttaattacaaatttttagcatctaatttaacttcaattcctattatacaaaattttaagatactgcactatcaacacactcttaagtttgcttctaagtcttatttccataacttcttttacgtttccgccattctttgctgtttcgatttttatgatatggtgcaagtcagcacgaacacgaaccgtcttatctcccattatatctttttttgcactgattggtgtatcatttcgtttttcttttgcgcttcttgataaaagggatagtaattcattcctggttgcaaattttgaaaaccgctacggatcacatctttttctaaactattgatccatagtcttttatacgttttatctttagaaaaggcatttgctttatgaatgatcgaccaggcaatgttttcgccttctctgtcgctatctgttgcgacaatgattgtatttgcttgttttaaaagttcagcaacaattttaaactgcttttttttatctgttgccacttcaaaatcgtatcgattctagatttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgtctagaatcgataagcttggctgcaggtcgataaggctattggtgtttatggctctcttggtcgtcagactgatgggccccgaaaagccctgacaacccttgttcctaaaaaggaataagcgttcggtcagtaaataatagaaataaaaaatcagacctaagactgatgacaaaaagagaaaattttgataaaatagtcttagaattaaattaaaaagggaggccaaatataatgaaaaatatgaatgacaatgatgttggatccatgacagaaccgttaaccgaaacccctgaactatccgcgaaatatgcctggttttttgatcttgatggaacgctggcggaaatcaaaccgcatcccgatcaggtcgtcgtgcctgacaatattctgcaaggactacagctactggcaaccgcaagtgatggtgcattggcattgatatcagggcgctcaatggtggagcttgacgcactggcaaaaccttatcgcttcccgttagcgggcgtgcatggggcggagcgccgtgacatcaatggtaaaacacatatcgttcatctgccggatgcgattgcgcgtgatattagcgtgcaactgcatacagtcatcgctcagtatcccggcgcggagctggaggcgaaagggatggcttttgcgctgcattatcgtcgggctccgcagcatgaagacgcattaatgacattagcgcaacgtattactcagatctggccacaaatggcgttacagcagggaaagtgtgttgtcgagatcaaaccgagaggtaccagtaaaggtgaggcaattgcagcttttatgcaggaagctccctttatcgggcgaacgcccgtatttctgggcgatgatttaaccgatgaatctggcttcgcagtcgttaaccgactgggcggaatgtcagtaaaaattggcacaggtgcaactcaggcatcatggcgactggcgggtgtgccggatgtctggagctggcttgaaatgataaccaccgcattacaacaaaaaagagaaaataacaggagtgatgactatgagtcgtttagtcgtagtatctaaccggattgcaccaccagacgagcacgccgccagtgccggtggccttgccgttggcatactgggggcactgaaagccgcaggcggactgtggtttggctggagtggtgaaacagggaatgaggatcagccgctaaaaaaggtgaaaaaaggtaacattacgtgggcctcttttaacctcagcgaacaggaccttgacgaatactacaaccaattctccaatgccgttctctggcccgcttttcattatcggctcgatctggtgcaatttcagcgtcctgcctgggacggctatctacgcgtaaatgcgttgctggcagataaattactgccgctgttgcaagacgatgacattatctggatccacgattatcacctgttgccatttgcgcatgaattacgcaaacggggagtgaataatcgcattggtttctttctgcatattcctttcccgacaccggaaatcttcaacgcgctgccgacatatgacaccttgcttgaacagctttgtgattatgatttgctgggtttccagacagaaaacgatcgtctggcgttcctggattgtctttctaacctgacccgcgtcacgacacgtagcgcaaaaagccatacagcctggggcaaagcatttcgaacagaagtctacccgatcggcattgaaccgaaagaaatagccaaacaggctgccgggccactgccgccaaaactggcgcaacttaaagcggaactgaaaaacgtacaaaatatcttttctgtcgaacggctggattattccaaaggtttgccagagcgttttctcgcctatgaagcgttgctggaaaaatatccgcagcatcatggtaaaattcgttatacccagattgcaccaacgtcgcgtggtgatgtgcaagcctatcaggatattcgtcatcagctcgaaaatgaagctggacgaattaatggtaaatacgggcaattaggctggacgccgctttattatttgaatcagcattttgaccgtaaattactgatgaaaatattccgctactctgacgtgggcttagtgacgccactgcgtgacgggatgaacctggtagcaaaagagtatgttgctgctcaggacccagccaatccgggcgttcttgttctttcgcaatttgcgggagcggcaaacgagttaacgtcggcgttaattgttaacccctacgatcgtgacgaagttgcagctgcgctggatcgtgcattgactatgtcgctggcggaacgtatttcccgtcatgcagaaatgctggacgttatcgtgaaaaacgatattaaccactggcaggagtgcttcattagcgacctaaagcagatagttccgcgaagcgcggaaagccagcagcgcgataaagttgctacctttccaaagcttgcgtagatctatcgataagcttaagtgattagtcaaagaatggtgatgacaattgtaaattctatttaatcactttgactagcaaatactaacaacaagacacacacaccaaaaatcaaaaattcactacttttagttaaaaaccacgtaaccacaagaactaatccaatccatgtaatcgggttcttcaaatatttctccaagattttcctcctctaatatgctcaacttaaatgacctattcaataaatctattatgctgctaaatagtttataggacaaataagtatactctaatgacctataaaagatagaaaattaaaaaatcaagtgttcgcttcgctctcactgcccctcgacgttttagtagcctttccctcacttcgttcagtccaagccaactaaaagttttcgggctactctctccttctccccctaataattaattaaaatcttactctgtatatttctgctaatcattcgctaaacagcaaagaaaaaacaaacacgtatcatagatataaatgtaatggcatagtgcgggttttattttcagcctgtatcatagctaaacaaatcgagttgtgtgtccgttttagggcgttctgctagcttgtttaaagtctcttgaatgaatgtatgctctaagtcaaaagaatttgtcagcgcctttatatagctttctttttcttctttttttactttaatgatcgatagcaacaatgatttaacactagcaagttgaatgccaccatttcttcctggtttaatcttaaagaaaatttcctgattcgccttcagtaccttcagcaatttatctaatgtccgttcaggaatgcctagcacttctctaatctcttttttggtcgtcactaaataaggcttgtatacatcgcttttttcgctaatataagccattaaatcttctttccattctgacaaatgaacacgttgacgttcgcttctttttttcttgaatttaaaccacccttgacggacaaataaatctttactggttaaatcacttgatacccaagctttgcaaagaatggtaatgtattccctattagccccttgatagttttctgaataggcacttctaacaattttgattacttctttttcttctaagggttgatctaatcgattattaaactcaaacatattatattcgcacgtttcgattgaatagcctgaactaaagtaggctaaagagagggtaaacatgacgttattacgccctattaaacccttttctcctgaaaatttcgtttcgtgcaataagagattaaaccagggttcatctacttgttttttgccttctgtaccgcttaaaaccgttagacttgaacgagtaaagcccttattatctgtttgtttgaaagaccaatcttgccattctttgaaagaataacggtaattaggatcaaaaaattctacattgtccgttcttggtatgcgagcaataccaaaatgattacacgttagatcaactggcaaagactttccaaaatattctcggatattttgcgaaattattttggctgctttgacagatttaaattctgattttgaagtcacatagactggcgtttctaaaacaaaatatgcttgataacctttatcagatttgataatcatagtaggcataaaacctaaatcaatagcggttgttaaaatatcgcttgctgaaatagtttcttttgccgtgtgaatatcaaaatcaataaagaaggtattgatttgtcttaaattgttttcagaatgtcctttcgtgtatgaacggttttcgtctgcatacgttccataacgataaacgttgggtgtccaatgtgtaaatgtatcttgattttcttgaatcgcttcctcggaagtcagaacaacaccacgaccgccaatcatgcttgattttgagcgatacgcaaaaatagcccctttgcttttacctggcttggtagtgattgagcgaattttactatttttaaatttgtactttaacaagccgtcatgaagcacagtttctacaacaaaagggatattcattcagctgttctcctttcctataaatcctataaaataggttgtttaattaacttggtttgctttttcattcaactgtttcaatattgcatgttttgaaaaagatttttttcctttataagtcaatttttttccactaatcgaataaattattttgttattttctattaacttatatatataatcttccccctccgaagaaaaatacttatctgattttgtttctaagtagatatttctcttttctaactctttcttaaacgtttctagtgtatagatatttgctaattttcttatctccaataaactattttttatataagttttacattcatcatgattcatacaaactccaccttctataaatgaatacaaaaaaagcaatcaaacgatttccgattgattgcttaacaattcttaaattcagtagcttagatacttgaaaactctctgatttccctatataatgatagtacggttatataccgtcttcaaacaaagttaattaaataacttcttacgagggaagagttcatctgactaactgataagcgttggtttggcaatcttatcgggctatgcatttataaaatgtcgtcaaacattttataaatgtgtcatggctcttttttcgtttctattcagttcgttgtttcgttatatctagtataccgcttttaaaaaaaaataagcaacgatttcgtgcattattcacacgaagtcattgcttttttcttcttccatttctaaatccaatgttacttgttctgattctgtttctggctctggttctgttggctcatttgggattaaatccactactagcgttgagttagtt;
otsA基因的核苷酸序列,序列如SEQ ID NO.7所示:
atcgcttcccgttagcgggcgtgcatggggcggagcgccgtgacatcaatggtaaaacacatatcgttcatctgccggatgcgattgcgcgtgatattagcgtgcaactgcatacagtcatcgctcagtatcccggcgcggagctggaggcgaaagggatggcttttgcgctgcattatcgtcgggctccgcagcatgaagacgcattaatgacattagcgcaacgtattactcagatctggccacaaatggcgttacagcagggaaagtgtgttgtcgagatcaaaccgagaggtaccagtaaaggtgaggcaattgcagcttttatgcaggaagctccctttatcgggcgaacgcccgtatttctgggcgatgatttaaccgatgaatctggcttcgcagtcgttaaccgactgggcggaatgtcagtaaaaattggcacaggtgcaactcaggcatcatggcgactggcgggtgtgccggatgtctggagctggcttgaaatgataaccaccgcattacaacaaaaaagagaaaataacaggagtgatgacta;
otsB基因的核苷酸序列,序列如SEQ ID NO.8所示:
atgacagaaccgttaaccgaaacccctgaactatccgcgaaatatgcctggttttttgatcttgatggaacgctggcggaaatcaaaccgcatcccgatcaggtcgtcgtgcctgacaatattctgcaaggactacagctactggcaaccgcaagtgatggtgcattggcattgatatcagggcgctcaatggtggagcttgacgcactggcaaaaccttatcgcttcccgttagcgggcgtgcatggggcggagcgccgtgacatcaatggtaaaacacatatcgttcatctgccggatgcgattgcgcgtgatattagcgtgcaactgcatacagtcatcgctcagtatcccggcgcggagctggaggcgaaagggatggcttttgcgctgcattatcgtcgggctccgcagcatgaagacgcattaatgacattagcgcaacgtattactcagatctggccacaaatggcgttacagcagggaaagtgtgttgtcgagatcaaaccgagaggtaccagtaaaggtgaggcaattgcagcttttatgcaggaagctccctttatcgggcgaacgcccgtatttctgggcgatgatttaaccgatgaatctggcttcgcagtcgttaaccgactgggcggaatgtcagtaaaaattggcacaggtgcaactcaggcatcatggcgactggcgggtgtgccggatgtctggagctggcttgaaatgataaccaccgcattacaacaaaaaagagaaaataacaggagtgatgactatgagtcgtttagtcgtagtatctaa。
其余如无特别说明的原料或处理技术,则表明其均为本领域的常规市售原料或常规处理技术。
实施例1
1.1过表达质粒的构建
otsBA异源表达质粒的构建:以大肠杆菌DH5ɑ为模板使用引物otsBA-F和otsBA-R扩增出otsBA的片段,片段大小为2200bp左右;将pIB184质粒使用限制性内切酶ScaII和XhoI进行双酶切37℃3h,纯化回收后的载体与DNA片段进行无缝克隆。将无缝克隆体系在37℃条件下反应30min,然后转入E.coil Top10感受态热击42℃90s,涂布在含有红霉素终浓度为400ng/mL的平板上,孵育24h后,挑选单个菌株用验证引物pIB184-yz-F和pIB184-yz-R进行验证。目的条带在2300bp左右,测序成功后获得重组质粒pIB184-otsBA,该质粒电子克隆流程见图1。
1.2重组菌株的构建
将重组质粒pIB184-otsBA电转入AR113感受态:转化具体步骤:将0.1~1μg质粒转入感受态中,冰上静置10min,转入提前紫大风杀菌40min预冷后的2mm电转杯(Bio-rad)中,以2.5kV,400Ω,25μF的条件进行电转,立即加入900μL复苏液,于37℃静置复苏3h,然后将菌株在4500rpm条件下离心3min,去除700μL上清液后,涂布于对应抗性的平板上,37℃厌氧培养24~48h;挑取Emr抗性平板筛选出的阳性克隆子,用验证引物pIB184-yz-F和pIB184-yz-R进行PCR验证,获得正确的过表达菌株命名为L.plantarum AR113-otsBA。
1.3PEF加载海藻糖进入胞内
将过夜培养OD600为1左右的菌液离心出每个样品2mL的菌泥,将菌泥用生理盐水清洗两次后重悬于10%海藻糖溶液中(对照组重悬于PBS),整个实验流程见图2。将电转杯置于超净台内紫外大风吹30min后,分别在电转杯中加入400μL样品,使用Gene-Pulser Xcell设备进行电击,设定参数为1500V电压、两个0.1ms脉冲持续时间、方波脉冲、脉冲间隔时间:5s、在室温(25±1℃)进行PEF处理。处理结束后将样品置于37℃培养箱内重新密封30min。处理前和结束处理后的样品分别取样进行计数。
1.6菌株冷冻干燥存活率的测定
将处理后的菌泥重悬于1mL海藻糖保护剂(对照组为PBS)中,然后转移到西林瓶中,并将其放置于已设置好程序的冷冻干燥机中进行冷冻干燥,冷冻干燥程序设置如下:首先样品在-40℃下预冻3h,再以1℃/min的速率升温至-30℃干燥800min,然后以1℃/min的速率将温度提高到25℃进行二次干燥2h。其中冷阱温度为-80℃左右,真空度20Pa。将冷冻干燥结束后的菌粉用梯度稀释计数法进行计数,吸取10μL稀释液进行平板计数,平板置于37℃培养36~48h。冷冻干燥存活率为:冻干后的活菌数/冻干前的活菌数。
实施例2结果
2.1胞内产海藻糖植物乳杆菌的构建
图3代表海藻糖在大肠杆菌中的代谢途径。植物乳杆菌中缺乏合成海藻糖的途径,通过在植物乳杆菌中重组海藻糖合成相关酶系,达到体内合成海藻糖的目的。利用植物乳杆菌中的高拷贝质粒pIB184对来源于大肠杆菌DH5α中otsBA操纵子(分别包含otsA和otsB两个的基因)分别编码合成海藻糖的相关酶:海藻糖-6-磷酸合成酶和海藻糖-6-磷酸酶进行过表达。
利用植物乳杆菌中的高拷贝质粒pIB184对来源于大肠杆菌DH5α中otsBA操纵子(分别包含otsA和otsB两个的基因)分别编码合成海藻糖的相关酶:海藻糖-6-磷酸合成酶和海藻糖-6-磷酸酶进行过表达。过表达质粒的构建:以DH5α基因组DNA为模板,用引物otsBA-F和otsBA-R进行PCR扩增出目的片段为2200bp左右与酶切后的载体相连,构建出目的质粒pIB184-otsBA转入植物乳杆菌AR113感受态中,用验证引物扩增出2200bp左右大小的条带(图4a),将菌株命名为AR113-otsBA。异源表达菌株的生长趋势与野生型菌株一致,都在3-4h左右进入对数生长期,虽表中的野生型的最大生长速率略高于重组菌株,但重组菌株的迟滞期时间较短,且最大生物量与野生型几乎无显著的差异性(图4b)。表明过表达来自大肠控制海藻糖产生相关酶系的基因对于菌株的正常生长无显著的影响。
2.2重组菌株中目的蛋白表达以及胞内海藻糖含量的测定
在重组菌株AR113-otsBA中海藻糖的生物合成始于葡萄糖,然后通过海藻糖-6-磷酸合成酶催化海藻糖-6-磷酸的合成,最后通过海藻糖-6-磷酸酶催化海藻糖-6-磷酸去磷化生成海藻糖。对两种酶的表达进行测定,验证otsA和otsB基因是否能在植物乳杆菌中成功表达。
表2异源表达菌株中胞内海藻糖的含量
注:所得海藻糖标曲的R2为0.997;ND表达未达到该方法的检出限。
将重组菌株AR113-otsBA和对照组野生型AR113的菌株破碎取上清进行SDS-PAGE检测。结果见图5,AR113-otsBA菌株在50kDa处存在一条与对照组差异明显的条带,与otsA的理论大小53.7kDa相符合,该基因在植物乳杆菌中得到了很好的表达。而在25kDa处的上面存在一条接近otsB理论大小29.1kDa的条带,但是与对照组的灰度差异不太明显,原因可能有菌株本身表达出来与目的蛋白大小相接近的蛋白,导致目的蛋白条带被掩盖,以至于不易观察出otsB表达蛋白的水平。
为了探究在菌株中表达海藻糖合成相关酶系对胞内海藻糖产量的影响,通过HPLC方法对培养后的重组菌株进行破碎上清海藻糖含量的检测。结果如表2所示,通过本检测方法在野生型菌株中未检出海藻糖,但是在重组菌株AR113-otsBA中检测到32.72mg/L的海藻糖。该结果说明通过在植物乳杆菌过表达来自大肠杆菌的otsBA操纵子,成功地将植物乳杆菌中所缺乏的海藻糖合成相关酶系进行表达,从而在菌株内部产生合成海藻糖途径,最终重组菌株中检测出了海藻糖的含量。
2.3重组菌株冷冻干燥存活率的分析
通过上述结果表明重组菌株内部已有海藻糖的产生,为了探究胞内海藻糖含量的增加对菌株冷冻干燥的影响,将重组菌株AR113-otsBA和野生型AR113在添加PBS和海藻糖的情况下进行冷冻干燥处理,测定其不同情况下的存活率。由图6所示:AR113-otsBA在外部不添加保护剂的情况下,冻干存活率能达到48.75%,相比于对照组,提升了34%左右;说明胞内海藻糖的存在会显著提升菌株的冷冻干燥存活率,菌株因为胞内海藻糖的增加而获得了更强的抗冻性。其中当海藻糖作为外源保护剂添加时,野生型菌株的存活率为55.21%,而重组菌株在该条件下的存活率提升了43%左右。因此在胞内海藻糖增加的情况下,菌株冻干存活率得到显著的提升。
综上所述,通过基因工程技术将大肠杆菌中海藻糖的产生途径异源表达在植物乳杆菌中,使得重组菌株能在胞内合成海藻糖,提高了重组菌株的抗冻性,证明了胞内保护剂的增加对菌株冷冻干燥过程的保护效果。
2.4异源表达菌株冷冻干燥后的形态
不同菌株在冷冻干燥过程中添加外源保护剂后对菌株形态的影响,结果见图7;在大多数干燥配方中,细胞不可见,因为它们被包裹在无定形基质中,AR113-otsBA菌株在海藻糖的条件下的细胞形态也没有看见;分组1)、2)分别是野生型菌株AR113和AR113-otsBA菌株在PBS条件下冷冻干燥后的菌株图像结果,结果显示AR113-otsBA菌株中的细胞相互紧密结合,在野生型中细胞与PBS紧密的结合,随着倍数越小,视野越广,可以看到细胞结构的完整性、细胞与PBS的均匀结合,可以看出AR113-otsBA菌株中的细胞比野生型中相互结合更紧密,且细胞间的松散结合增加了细胞表面的水分吸收,这是细胞活力丧失的主要原因,野生型的细胞间结合较为松散,该现象与存活率结果相对应,AR113-otsBA抗冻性优于野生型,胞内保护剂的增加提高菌株的抗冻性。
2.5PEF加载海藻糖进入胞内提高植物乳杆菌冷冻干燥存活率
应用PEF技术加载海藻糖进入四株植物乳杆菌(AR495、AR514、AR307和WCFS1)中,通过增加胞内海藻糖的方式提高菌株的冷冻干燥存活率。与对照组相比,所有菌株通过PEF加载海藻糖的处理后的冻干存活率均出现不同程度的提升,提升倍数分别为:AR495:4.8、AR514:15.3、AR307:3.2和WCFS1:8.75。因此PEF加载海藻糖进入胞内的方法可以提高这4株植物乳杆菌的抗冻性。而在正常条件下添加海藻糖后AR307的冷冻干燥存活率为15%,WCFS1也仅为60%,PEF处理后菌株的冻干存活率也高于正常条件下添加保护剂的情况。利用PEF加载保护剂进入胞内有效地提高植物乳杆菌的冷冻干燥存活率。
2.6 PEF加载海藻糖进入胞内提高益生菌冷冻干燥存活率
PEF加载海藻糖进入胞内对不同益生菌(植物乳杆菌AR113、嗜热乳杆菌S-3和乳酸乳球菌NZ9000)冷冻干燥存活率的影响:加载海藻糖介质PEF处理后的存活率见图9b,三株菌的冻干存活率均得到提升;AR113冻干存活率从24.1%提升到56.6%、S-3的冻干存活率从10.7%提升到41.2%、NZ9000存活率相比于对照组提升了2.25倍,存活率为64.28%。因此经过海藻糖介质PEF处理后,菌株的冻干存活率基本得到提高,其中处理后所有菌株的存活率均处于40%以上,且在海藻糖介质PEF处理后外源添加海藻糖冻干基质的条件下,所有菌株冻干存活率均高于50%。通过PEF加载海藻糖进入胞内的方法有效地提高益生菌的抗冻性。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种提高菌株冷冻干燥存活率的方法,其特征在于,将otsA基因和otsB基因导入植物乳杆菌中,实现植物乳杆菌异源合成海藻糖,从而提高植物乳杆菌冻干存活率;
所述otsA基因的核苷酸序列如SEQ ID NO.7所示;
所述otsB基因的核苷酸序列如SEQ ID NO.8所示。
2.根据权利要求1所述的一种提高菌株冷冻干燥存活率的方法,其特征在于,所述otsA基因和otsB基因来源于大肠杆菌DH5ɑ。
3.根据权利要求1所述的一种提高菌株冷冻干燥存活率的方法,其特征在于,所述植物乳杆菌为植物乳杆菌AR113。
4.根据权利要求1所述的一种提高菌株冷冻干燥存活率的方法,其特征在于,利用植物乳杆菌中的高拷贝质粒过表达otsA基因和otsB基因,编码合成海藻糖的相关酶。
5.根据权利要求4所述的一种提高菌株冷冻干燥存活率的方法,其特征在于,所述高拷贝质粒为pIB184。
6.根据权利要求4所述的一种提高菌株冷冻干燥存活率的方法,其特征在于,所述酶包括海藻糖-6-磷酸合成酶和海藻糖-6-磷酸酶。
7.根据权利要求6所述的一种提高菌株冷冻干燥存活率的方法,其特征在于,所述合成海藻糖的具体过程为:葡萄糖通过海藻糖-6-磷酸合成酶催化合成海藻糖-6-磷酸,再通过海藻糖-6-磷酸酶催化海藻糖-6-磷酸去磷化合成海藻糖。
8.根据权利要求4所述的一种提高菌株冷冻干燥存活率的方法,其特征在于,所述过表达otsA基因和otsB基因的植物乳杆菌正常生长。
9.根据权利要求1所述的一种提高菌株冷冻干燥存活率的方法,其特征在于,将otsA基因和otsB基因导入植物乳杆菌后,得到重组植物乳杆菌,在重组植物乳杆菌外部添加保护剂,进一步提高植物乳杆菌冻干存活率。
10.根据权利要求1所述的一种提高菌株冷冻干燥存活率的方法,其特征在于,海藻糖提高植物乳杆菌冻干存活率的表现为:植物乳杆菌细胞结构完整,植物乳杆菌细胞与PBS均匀结合。
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