CN114711434B - 一种人乳铁蛋白突变体及其联合乳杆菌在制备食品或保健品中的应用 - Google Patents
一种人乳铁蛋白突变体及其联合乳杆菌在制备食品或保健品中的应用 Download PDFInfo
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- CN114711434B CN114711434B CN202210377984.XA CN202210377984A CN114711434B CN 114711434 B CN114711434 B CN 114711434B CN 202210377984 A CN202210377984 A CN 202210377984A CN 114711434 B CN114711434 B CN 114711434B
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Abstract
本发明涉及一种人乳铁蛋白突变体及其联合乳杆菌在制备食品或保健品中的应用。具体涉及一种人乳铁蛋白突变体,能够显著促进双歧杆菌、乳杆菌、乳球菌等益生菌增殖,对沙门氏菌、金黄色葡萄球菌、绿脓杆菌等有害细菌具有明显抑制作用;该人乳铁蛋白突变体可与罗伊乳杆菌配合使用,显著提高机体免疫球蛋白水平,降低氧自由基含量,防止氧化应激对人体造成损伤;所述人乳铁蛋白突变体及其组合物可口服使用,为保健食品和保健品的开发提供了新途径。
Description
技术领域
本发明属于生物技术领域,具体涉及一种人乳铁蛋白突变体及其联合乳杆菌在制备食品或保健品中的应用。
背景技术
免疫力是人体自身的防御机制,是人体识别和消灭外来侵入的任何异物(如病毒、细菌等)、处理衰老、损伤、死亡、变性的自身细胞以及识别和处理体内突变细胞和病毒感染细胞的能力。免疫力低下者更容易被感染或患病,常常变现为体质虚弱、营养不良、精神萎靡、疲乏无力、食欲降低、睡眠障碍等状态,严重者还会影响身体和智力发育,诱发腹泻、感冒、哮喘、肿瘤等严重疾病。提高机体免疫力是增强体质,改善机体免疫环境的重要方面,也是食品研究、保健品开发的重要研究方向,其中具有免疫调节功能的蛋白质和益生菌的应用受到研究人员的高度重视。
乳铁蛋白(Lactoferrin,LF),又称乳转铁蛋白(Lactotransferrin,LTF),分子量约为80kDa,属于铁结合蛋白家族成员,是一类非血红素铁结合的糖基化蛋白,在其氨基酸结构呈现“二枚银杏叶型”,分别在其分子的N端和C端形成2个环状结构(loops),每叶在内部缝隙处都有一个铁结合位点。乳铁蛋白于20世纪处被首次从牛乳中分离出来,因其晶体呈红色,能够高亲和性可逆地与铁结合,因此被称为乳铁蛋白。乳铁蛋白在母乳中的含量非常丰富,据报道可高达7g/L,是母乳中含量最丰富的蛋白之一。需要说明的是,乳铁蛋白不仅仅存在于动物乳汁中,而是广泛存在于多种组织及其分泌液中,包括但不限于动物乳汁、血液、泪液、粘液等,其具有丰富多样的生物学功能,包括抗菌、抗病毒、抗氧化、参与免疫调节、促进铁元素吸收等等;此外,乳铁蛋白为天然乳汁中的主要成分之一,对于胃肠消化***的适应性较强,通常的服用过程中可采用口服方式,如Heidi Chan等(A randomized,double-blind,placebo-controlled trial to determine the efficacy and safety oflactoferrin with vitamin E and zinc as an oral therapy for mild to moderateacne vulgaris,International Journal of Dermatology,2017,56(6):686-690)、Mahmoud A El-Hawy等(Comparing oral iron bisglycinate chelate,lactoferrin,lactoferrin with iron and iron polymaltose complex in the treatment ofchildren with iron deficiency anemia,Clin Nutr ESPEN,2021,46:367-371)、阿拉腾珠拉等(裂壶藻和乳铁蛋白对大肠杆菌K99攻毒哺乳犊牛腹泻、生长性能、粪便评分及血清抗氧化指标的影响,动物营养学报,2020,32(9):4166-4176)均公开了使用口服方式研究乳铁蛋白的生理功能,这也为基于乳铁蛋白的食品或保健品的开发提供了方便,避免了一般免疫调节蛋白易被消化道破坏而难以直接开发制作食品或保健品的困境。然而,天然的牛乳或人乳中的乳铁蛋白在免疫调节方面的活性仍有待提高,尤其是与益生菌协同作用方面有待于改善和加强。
益生菌是通过定殖在人体内,改变宿主某一部位菌群组成的一类对宿主有益的活性微生物,通过调节宿主黏膜与***免疫功能或通过调节肠道内菌群平衡,促进营养吸收保持肠道健康,产生有利于健康作用的单微生物或组成明确的混合微生物,其生理功能相当丰富,包括促进营养物质的消化吸收、提高机体免疫力、维持肠道菌群结构平衡和防止消化道炎症、对抗氧化应激等等。从益生菌的种类来看,人体中的益生菌主要包括酵母菌、益生芽孢菌、丁酸梭菌、乳杆菌、双歧杆菌、放线菌等,其中乳杆菌为兼性厌氧、耐酸、发酵终产物以乳酸为主的革兰阳性菌,它广泛存在于人体的口腔、胃肠道等部位,参与人体多项生理活动,目前发现的乳杆菌种类较多,据报道嗜酸乳杆菌、短乳杆菌、干酪乳杆菌、保加利亚乳杆菌、约氏乳杆菌、副干酪乳杆菌、罗伊乳杆菌、鼠李糖乳杆菌和唾液乳杆菌等均可作为益生菌使用。乳杆菌的作用也十分广泛,包括抑制致病菌生长、改善肠道屏障功能、调节免疫***等,但是在某些个别报道中,乳杆菌似乎也可诱导某些疾病,如鼠李糖乳杆菌可导致败血症、心内膜炎等疾病,因此在食品和保健品开发领域如何选用适当的乳杆菌,以及如何改善其促进免疫力提高能力成为研究热点和现实需求之一。
本发明基于上述现有技术中存在的问题,提供了一种具有全新氨基酸结构的人乳铁蛋白突变体,与天然乳铁蛋白相比,可以显著提高乳杆菌的增殖能力,抑制致病菌生长;并且适合与乳杆菌构成复合制剂,大幅度提升机体免疫能力,帮助机体消除氧自由基过量导致的不良后果,为相关食品和保健品的开发提供了新的研究思路和开发途径。
发明内容
本发明的主要目的是提供一种提高机体免疫力的方式,具体提供了一种人乳铁蛋白突变体,可增强对乳杆菌的促增殖能力;该乳铁蛋白与乳杆菌构成组合物,口服使用后可大幅度提高机体免疫能力,对抗氧自由基能力也有所提高,可全面提升机体的防御机能,为现代食品和保健品的开发提供了新手段。
本发明的详细技术方案如下:
提供了一种人乳铁蛋白突变体,其氨基酸序列如SEQ ID NO:1所示。
所述人乳铁蛋白突变体,以人源乳铁蛋白为基础,利用生物信息学手段和已有人乳铁蛋白结构解析数据,设计相应的定点突变,经过分子水平、细胞水平和动物水平实验验证筛选而得,相对于野生型蛋白,在促进乳杆菌增殖(尤其是罗伊乳杆菌增殖)、提高机体免疫力和清除机体内氧自由基方面均展现出显著技术优势。
提供了一种编码所述人乳铁蛋白突变体基因,
进一步的,所述基因的核苷酸序列如SEQID NO:2所示。
提供了一种编码所述人乳铁蛋白突变体基因的重组载体。
提供了一种含有上述载体的宿主细胞。
进一步的,所述宿主细胞为大肠杆菌、酵母菌或昆虫细胞
提供了一种组合物,包括本发明中所述的人乳铁蛋白突变体和益生菌,所述益生菌选自双歧杆菌、乳杆菌和/或乳酸菌中的至少一种。
进一步的,所述益生菌选自罗伊乳杆菌、鼠李糖乳杆菌和/或植物乳杆菌中的至少一种。
进一步的,所述益生菌选自罗伊乳杆菌。
据报道,乳铁蛋白可促进双歧杆菌、乳球菌、乳杆菌等益生菌生长,而对于包括沙门氏菌、金黄色葡萄球菌、绿脓杆菌在内的大多数有害细菌却展现出较强的抑制作用,因而其在肠道菌群调解方面具有一定的选择性。即使是对于益生菌类,乳铁蛋白对于不同种类的益生菌也展现了不同程度的促进作用,因此在本发明中经过严格实验验证和筛选,最终确定所述人乳铁蛋白突变体与罗伊乳杆菌的组合在改善机体免疫力方面最为有效,有望在相关食品或保健品开发中发挥更大作用。
提供了一种所述人乳铁蛋白突变体、基因、重组载体、宿主细胞或组合物在制备食品中的应用。
提供了一种所述人乳铁蛋白突变体、基因、重组载体、宿主细胞或组合物在制备保健品中的应用。
与现有技术相比,本发明的有益效果为:
本发明提供了一种新型人乳铁蛋白突变体,具有全新的氨基酸结构,能够显著促进双歧杆菌、乳杆菌、乳球菌等益生菌增殖,对沙门氏菌、金黄色葡萄球菌、绿脓杆菌等有害细菌具有明显抑制作用,而且该蛋白保留了人乳铁蛋白可口服使用的特性,为保健食品和保健品的开发提供了便利;所述人乳铁蛋白突变体与罗伊乳杆菌构成组合物,能够大幅度提升机体免疫能力,快速清除体内过量的氧自由基,维持机体内环境平衡。
附图说明
图1人乳铁蛋白突变体对益生菌的生长促进作用;
图2人乳铁蛋白突变体对有害菌的生长抑制作用;
图3乳铁蛋白与罗伊乳杆菌组合物对免疫球蛋白的影响;
图4乳铁蛋白与罗伊乳杆菌组合物对ROS的影响;
具体实施方式
以下非限制性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何形式限制本发明。凡基于本发明上述内容所实现的技术均应当属于本申请要求保护的范围之中。
以下实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂生物材料、检测试剂盒,如无特殊说明,均可从商业途径获得。
实施例1人乳铁蛋白突变体的确定和制备
所述人乳铁蛋白突变体,以人源乳铁蛋白为基础,利用生物信息学手段和已有人乳铁蛋白结构解析数据,设计相应的定点突变,经过分子水平和细胞水平实验验证筛选而得,经鉴定所述人乳铁蛋白突变体的氨基酸序列如SEQ ID No.1所示,其核苷酸序列如SEQID No.2所示。
以大肠杆菌为宿主菌制备本发明中所述的人乳铁蛋白突变体。首先设计上下游PCR引物扩增人乳铁蛋白突变体(以下简称mu-hLF)基因,并在上下游引物中分别引入EcoRI和XhoI酶切位点;其次用EcoRI和XhoI双酶切表达载体pET28a和mu-hLF基因片段,酶切连接后得到重组表达载体pET28a-mu-hLF,然后将该重组表达载体电转化导入宿主菌E.coliBL21(DE3)中;在5L生物反应器中培养上述宿主菌,达到对数生长状态后,流加输入诱导剂IPTG,诱导表达生产目标蛋白,诱导表达16-24h后,4℃、8000rpm离心收集菌体,超声破碎后通过亲和层析色谱柱分离获得重组人乳铁蛋白突变体蛋白,经高效液相色谱鉴定所述蛋白纯度达98%以上,满足后续实验要求。
实施例2重组人乳铁蛋白突变体对不同微生物生长的影响
2.1重组人乳铁蛋白突变体促进益生菌生长
取冻存的双歧杆菌、乳杆菌(罗伊乳杆菌、鼠李糖乳杆菌和植物乳杆菌)、乳球菌菌株,上述菌株为本实验室保存,复苏后接种与无菌LB培养基中活化培养24小时;离心收集菌体,使用无菌LB培养基调整菌体浓度至1×105CFU/mL,分别取上述5种益生菌1mL接种于20mL无菌LB培养基中;将各个益生菌分为三组,分别加入50mg/mL的mu-hLF蛋白、50mg/mL天然人乳铁蛋白(以下简称hLF)和等体积的无菌水(作为空白对照),于摇床上37℃培养24后,使用分光光度计测量OD600数值。
结果如图1所示,天然和突变的人乳铁蛋白均能够促进双歧杆菌、乳杆菌和乳球菌生长,但是本发明中所提供的mu-hLF在促进乳杆菌生长方面具有明显优势,并且在不同的乳杆菌种属之间也具有差异,mu-hLF可显著促进罗伊乳杆菌生长,对于植物乳杆菌相比于hLF仅具有微弱优势(无显著差异),在鼠李糖乳杆菌方面促增殖能力却弱于hLF;但在双歧杆菌菌和乳球菌中,虽然mu-hLF也有明显的生长促进作用,但是相比于天然hLF未见显著差异。
2.2重组人乳铁蛋白突变体抑制有害菌生长
取沙门氏菌、金黄色葡萄球菌、绿脓杆菌、产气荚膜梭菌菌株(上述菌株由本实验室保存),验证人乳铁蛋白对其抑制作用,具体实验步骤同2.1节。结果图2所示,天然人乳铁蛋白和突变修饰的人乳铁蛋白具有展现出了对多四种有害微生物的抑制作用,其中在沙门氏菌和产气荚膜梭菌中,mu-hLF的抑制作用似乎强于天然人乳铁蛋白,但在其他两种有害细菌中二者取未见显著差异。
实施例3重组人乳铁蛋白与罗伊乳杆菌组合物的制备
本实施例中选择在前期实验中促增殖作用最强的罗伊乳杆菌,与人乳铁蛋白突变体制成组合物,在该组合中,一方面,人乳铁蛋白突变体显著促进罗伊乳杆菌的增殖和生长,以便在体内发挥更大免疫调节作用;另一方面,所述人乳铁蛋白突变体在促进乳杆菌增殖方面展现了一定的选择性,可避免诸如鼠李糖乳杆菌、植物乳杆菌等其他类型乳杆菌导致的潜在致病风险,提高组合物的使用安全性。具体制备过程如下:
首先制备mu-hLF蛋白和hLF蛋白,具体步骤参考实施例1,将获得的蛋白冷冻干燥,获得蛋白质粉;其次制备罗伊乳杆菌菌粉,将罗伊乳杆菌接种于无菌LB培养基中活化培养约24h,将活化后的菌体接种于5L生物反应器中,37℃下进行培养,每小时检测pH和OD值,待其生长状态进入稳定期后结束发酵,低温离心、收集菌体,磷酸盐缓冲液(PBS)洗涤后,加冻干保护剂(脱脂奶粉)后进行乳化,完成后进行真空冷冻干燥,得到罗伊乳杆菌菌粉;最后制备人乳铁蛋白与罗伊乳杆菌组合物,将上述两种组分按照1:10比例混合,分别获得mu-hLF-Lr组合物和hLF-Lr组合物。
实施例4重组人乳铁蛋白与罗伊乳杆菌组合物的体内实验
为验证本发明中所提供的mu-hLF蛋白与益生菌联合改善机体免疫机能的效果,本实施例中采用大鼠模型,口服人乳铁蛋白与罗伊乳杆菌组合物后,考察其对于免疫能力的影响。
4.1 实验动物处理
选取健康SD大鼠,SPF级,雌雄各半,体重180±20g,随机分组,每组10只,共3组,包括生理盐水组、mu-hLF-Lr组、hLF-Lr组,其中mu-hLF-Lr组每日口服100mg/kg的mu-hLF-Lr组合物,hLF-Lr组每日口服100mg/kg的hLF-Lr组合物,生理盐水组每日口服1mL无菌生理盐水,共口服30天后进行检测。
4.2机体免疫能力检测
免疫球蛋白是具有抗体活性的,与抗体分子相似的球蛋白,它是机体免疫的重要组成部分,可帮助机体对抗多种有害物质的侵袭,包括细菌、病毒、有害异物等等,免疫球蛋白分为五类,即免疫球蛋白G(IgG)、免疫球蛋白A(IgA)、免疫球蛋白M(IgM)、免疫球蛋白D(IgD)和免疫球蛋白E(IgE),其中IgG、IgA、IgM在血清中的含量较多,其生理活性也最为重要,因此本实施例中通过检测上述三种免疫球蛋白的含量来考察对于机体免疫能力的影响。
大鼠尾静脉取血,将全血以3000rpm的速率离心10min。吸取上清液,采用IgA、IgG、IgM免疫球蛋白试剂盒(购自南京建成生物公司)检测血液中IgA、IgG、IgM含量,结果如图3所示,乳铁蛋白和罗伊乳杆菌构成的组合物对于上述三种免疫球蛋白浓度均有提升作用,但是在IgG方面,mu-hLF-Lr组合物相比于hLF-Lr组合物并没有展现出更多优势,血液中的IgG水平没有显著变化;在IgA、IgM方面,使用人乳铁蛋白突变体与罗伊乳杆菌组合,则能够显著增加相应的免疫球蛋白水平。
众所周知,IgA是血清中的含量仅次于IgG,占血清免疫球蛋白的10~20%,多存在于黏膜组织,如消化道、呼吸道、泌尿生殖***等,也具有强大的抗菌、抗病毒和免疫调节功能。IgM是人体循环***中的最大抗体,因其分子量极大而在抗原凝集反应中非常有效,初次遭遇外来抗原后,IgM为最早回应出现的循环性抗体,因此是清除抗原异物的主要效应抗体。本申请中所提供的mu-hLF-Lr组合物能够显著提升IgA、IgM水平,说明其能够有效激活和促进机体的自身免疫水平,具有特异性抵御外源有害物质的潜力。
4.3抗氧化能力检测
活性氧(reactive oxygen species,ROS)是体内一类氧的单电子还原产物,是线粒体由状态Ⅲ向状态Ⅳ转换中高氧的环境和高还原态的呼吸链使大量电子漏出并还原氧分子而形成。活性氧是机体内正常代谢的产物,具有积极作用,如辅助吞噬细胞发挥杀伤作用,但是当其含量升高,造成活性氧的产生和清除失衡,则会引起活性氧对人体中的核苷酸、蛋白质、细胞膜造成广泛的损伤,从而引发多种疾病。
本实施例中为研究所述组合物对ROS的作用效果,使用试剂盒(购自南京建成生物公司)检测血清中活性氧含量。结果如图4所示,mu-hLF-Lr组合物可显著降低血清中ROS含量,其作用效果强于hLF-Lr组合物,表明这种联合使用可产生有效的协同作用,清除体内过量的氧自由基,保护机体免疫氧化应激损伤。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
序列表
<110> 广州泰术生物科技有限公司
<120> 一种人乳铁蛋白突变体及其联合乳杆菌在制备食品或保健品中的应用
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ctgtgcgcgc tgtgcattgg cgatgaacag ggcgaaaaca aatgcgtgcc gaacagcaac 1620
gaacgctatt atggctatac cggcgcgttt cgctgcctgg cggaaaacgc gggcgatgtg 1680
gcgtttgtga aagatgtgac cgtgctgcag aacaccgatg gcaacaacaa cgatgcgtgg 1740
gcgaaagatc tgatgctggc ggattttgcg ctgctgtgcc tgccgggcaa aaccaccccg 1800
gtgaccgaag cgcgcagctg ccatctggcg atggcgccga accatgcggt ggtgagccgc 1860
atggataaag tggaacgcct gaaacaggtg ctgctgcatc agcaggcgaa atttggccgc 1920
aacggcagcg attgcccgga taaattttgc ctgtttcaga gcgaaaccaa aaacctgctg 1980
tttaacgata acaccgaatg cctggcgcgc ctgcatggca aaaccaccta tgaaaaatat 2040
ctgggcccgc agtatgtggc gggcattacc aacctgaaaa aatgcagcac cagcccgctg 2100
ctggaagcgt gcgaatttct gcgcaaa 2127
Claims (10)
1.一种人乳铁蛋白突变体,其特征在于,其氨基酸序列如SEQ ID No.1所示。
2.一种编码权利要求1所述人乳铁蛋白突变体的基因,其核苷酸序列如SEQ ID No.2所示。
3.一种含有权利要求2所述的基因的重组载体。
4.一种含有权利要求3所述的重组载体的宿主细胞。
5.根据权利要求4所述的宿主细胞,其特征在于所述宿主细胞为大肠杆菌、酵母菌或昆虫细胞。
6.一种组合物,包括权利要求1中所述的人乳铁蛋白突变体和益生菌,所述益生菌选自双歧杆菌和/或乳杆菌。
7.根据权利要求6所述的组合物,其特征在于益生菌选自罗伊乳杆菌、鼠李糖乳杆菌和/或植物乳杆菌中的至少一种。
8.根据权利要求7所述的组合物,其特征在于益生菌为罗伊乳杆菌。
9.一种如权利要求1中所述的人乳铁蛋白突变体或权利要求2所述的基因或权利要求3所述的重组载体或权利要求4-5所述的宿主细胞或权利要求6-8中所述组合物在制备食品中的应用。
10.一种如权利要求1中所述的人乳铁蛋白突变体或权利要求2所述的基因或权利要求3所述的重组载体或权利要求4-5所述的宿主细胞或权利要求6-8中所述组合物在制备保健品中的应用。
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