CN115109130A - 一种细菌素Thurisin A2及其应用 - Google Patents
一种细菌素Thurisin A2及其应用 Download PDFInfo
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Abstract
本发明属于微生物应用领域，具体涉及一种细菌素Thurisin A2及其应用。所述细菌素Thurisin A2具有如SEQ ID NO.1所示的氨基酸序列，可以采用多肽固相合成法或液相多肽合成法合成，也可以将所述细菌素Thurisin A2的编码基因在工程菌中表达得到。其结构简单，安全低毒，合成较容易，能高效杀灭金黄色葡萄球菌、蜡样芽胞杆菌、产气荚膜梭菌、单核细胞增生李斯特菌，且对真核动物红细胞无溶血活性，具备生物安全性，可用于制备食品防腐剂、饲料添加剂、抗菌药物等，具有广阔的应用前景。
Description
技术领域
本发明属于微生物应用领域，具体涉及一种细菌素Thurisin A2及其应用。
背景技术
食品腐败变质引起的食源性疾病严重威胁人类健康、阻碍经济和社会的可 持续发展。2015年世卫组织发布的《全球食源性疾病负担的估算报告》中指出 每年全世界约6亿人因食用受污染的食品而患病，并有42万人死亡(包括5 岁以下儿童12.5万人)。2018年世界银行《关于食源性疾病经济负担的报告》 中指出，低收入和中等收入国家与食源性疾病相关的生产力和医疗费用损失达 1100亿美元。
在诸多影响食品安全的因素中微生物污染高居首位。食品加工业通常使用 食品防腐剂来抑制或杀灭食品中的微生物、防止食品的腐败变质、延长食品的 保质期。食品防腐剂按来源的不同分为化学食品防腐剂和天然食品防腐剂。传 统的化学防腐剂虽然有较好的防腐效果，但使用不当会有一定副效应，长期过 量摄入会对人体健康造成一定损害。天然食品防腐剂不同于化学食品防腐剂， 它是由生物体分泌或体内存在的、具有抗菌作用的物质，经人工提取或加工后 制成的一类食品防腐剂。天然食品防腐剂是天然物质，有的本身就是食品的组 分，具有无毒、无残留、高效、不影响食品口感等特点，是一类有重要发展前 景的食品防腐剂。在消费者日益追求天然、绿色、健康食品趋势下，从动植物、 微生物中寻找安全、无毒、高效的天然防腐剂是食品科学发展的主要研究方向， 也是提升食品产业效益和安全性的重要措施，具有重要的研究和应用价值。
细菌素(Bacteriocins)是细菌在生长过程中通过核糖体合成途径产生的一 类具有抗菌活性的多肽类或蛋白类物质。细菌素本身具有很多优良属性：体内、 体外的高效抗菌活性；对人体或动物无毒，且易被动物消化道中的一些蛋白酶 所降解而无残留；独特的抗菌谱，有很好的选择性杀菌效果；易于生物改造， 满足人们不同的需求；具有热稳定性及耐酸、碱能力，便于贮藏；本身无味， 不会影响食品、饲料的口感、味道；使用后可降低食品的杀菌温度、减少热处 理时间，因此能改进食品的营养价值、风味、颜色等。因此，细菌素在食品添 加剂、饲料添加剂、药物等方面有着广阔的开发、应用前景。
本发明要解决的技术问题是：发掘属性优良的新细菌素。
发明内容
本发明的目的之一是提供一种细菌素Thurisin A2，其具有如SEQ ID NO.1 所示的氨基酸序列。本发明的细菌素Thurisin A2可以采用多肽固相合成法或液 相多肽合成法合成，也可以将所述细菌素Thurisin A2的编码基因在工程菌中表 达得到。
本发明的目的之二是提供所述细菌素Thurisin A2在制备抗菌和/或防腐产 品中的应用。
进一步的，所述抗菌和/或防腐产品抑制金黄色葡萄球菌、蜡样芽胞杆菌、 产气荚膜梭菌和单核细胞增生李斯特菌中的任一种或多种。
本发明的目的之三是提供一种抗菌和/或防腐产品，包括所述的细菌素 ThurisinA2。
本发明的目的之四是提供所述细菌素Thurisin A2在制备治疗和/或预防细 菌感染性疾病的药物中的应用。
进一步的，所述细菌金黄色葡萄球菌、蜡样芽胞杆菌、产气荚膜梭菌和单 核细胞增生李斯特菌中的任一种或多种。
本发明的目的之五是提供一种治疗和/或预防细菌感染性疾病的药物，包括 所述的细菌素Thurisin A2。
本发明具有如下有益效果：
本发明提供的细菌素Thurisin A2结构简单，安全低毒，合成较容易，能高 效杀灭蜡样芽胞杆菌、单核细胞增生李斯特菌、金黄色葡萄球菌、产气荚膜梭 菌，且对真核动物红细胞无溶血活性，具备生物安全性。本发明的细菌素 Thurisin A2可用于制备食品防腐剂、饲料添加剂、抗菌药物等，具有广阔的应 用前景。
附图说明
图1为实施例3中细菌素Thurisin A2的溶血活性、细胞毒性测试结果。
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，但不应理解为本发明 的限制。如未特殊说明，下述实施例中所用的技术手段为本领域技术人员所熟 知的常规手段，下述实施例中所用的材料、试剂等，如无特殊说明，均可从商 业途径得到。
实施例1：细菌素Thurisin A2的获得
本发明提出的细菌素Thurisin A2来源于我们构建的苏云金芽胞杆菌XIN- LX43(Bacillus thuringensis XIN-LX43)多肽序列数据库，该菌已于2021年6月 15日保藏于中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO：M 2021719；保藏地址：中国武汉武汉大学。该细菌素Thurisin A2的氨基酸序列(SEQ ID NO.1所示)如下： MVAFLRIVGQLGAKAASWAWANKGRVLGWIRDGMAIEWIINKINDMVS。通 过在线BlastP比对分析(Protein BLAST:search protein databases using a protein query(nih.gov))，该细菌素Thurisin A2与已鉴定、报道的细菌素的氨基酸序列 均无同源性，说明它是一种新颖细菌素，属发明人首次研究、鉴定、报道。根据 所示的氨基酸序列，经强耀生物科技有限公司(上海)合成得到对应的抗菌多肽 其纯度>95％。
实施例2：细菌素Thurisin A2对多种食源性病原菌抗菌活性的测定
以生理盐水为稀释液，通过二倍稀释法配置系列梯度的细菌素溶液(0.5， 1，2，4，8,16,32,64,128μM)。利用琼脂扩散法检测不同浓度下的Thurisin A2 对多种食源性病原菌的最低抑菌浓度(MIC)。在未凝固的琼脂培养基中加入适 量的指示菌(细菌个数约为5×105cfu/mL)，混匀，倒平板。选取蜡样芽胞杆菌 CMCC 63301、蜡样芽胞杆菌CMCC 63303、产气荚膜梭菌ATCC 13124、单 核细胞增生李斯特菌ATCC 19111、单核细胞增生李斯特菌ATCC19115、金黄 色葡萄球菌ATCC 6538、金黄色葡萄球菌ATCC 43300为指示菌。待其凝固后 用孔径为6mm的打孔器打孔。每孔中加入约50μL样品后将平板先放置于4℃， 2h左右，让待测样品充分扩散，而后放置于30℃下培养12h，观察抑菌效果 (观察有无透明的抑菌圈出现)。经测定Thurisin A2对蜡样芽胞杆菌 CMCC 63301、蜡样芽胞杆菌CMCC 63303、产气荚膜梭菌ATCC 13124、单 核细胞增生李斯特菌ATCC 19111、单核细胞增生李斯特菌ATCC 19115、金黄 色葡萄球菌ATCC 6538、金黄色葡萄球菌ATCC 43300的最小抑菌浓度分别为 0.5μM，0.5μM，2μM，1μM，0.5μM，0.5μM，2μM(表1)。
表1 Thurisin A2对4类食源性病原菌最低抑菌浓度的测定
实施例3：细菌素Thurisin A2溶血活性的测定
采集健康人新鲜血液3mL(添加抗凝血剂)，3000g离心5min，弃血浆收 集红细胞。将收集的红细胞用生理盐水洗涤三遍，并按2％(V/V)的比例重 悬血细胞。在无菌的96孔细胞培养板中加入100μL红细胞重悬液，再分别加 入不同浓度的细菌素溶液，每个细菌素浓度设置3个平行。以0.1％Tritonx-100 作为完全溶血的阳性对照，以生理盐组作为不溶血的阴性对照。37℃孵育2h， 3000g离心10min。将离心后的上清转移到新的无菌96孔细胞培养板中，并 利用酶标仪检测405nm处的光吸收值，计算细菌素的溶血活性。
细菌素Thurisin A2的溶血活性如图1所示，该细菌素在高浓度64μM的浓 度下仅表现出1.1％的溶血活性，说明其溶血活性非常低，具备生物安全性。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基 本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要 求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发 明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及 其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
序列表
<110> 淮北师范大学
<120> 一种细菌素Thurisin A2及其应用
<160> 1
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 人工序列
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Met Val Ala Phe Leu Arg Ile Val Gly Gln Leu Gly Ala Lys Ala Ala
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Ser Trp Ala Trp Ala Asn Lys Gly Arg Val Leu Gly Trp Ile Arg Asp
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Gly Met Ala Ile Glu Trp Ile Ile Asn Lys Ile Asn Asp Met Val Ser
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Claims (7)
1.一种细菌素Thurisin A2，其特征在于，具有如SEQ ID NO.1所示的氨基酸序列。
2.权利要求1所述细菌素Thurisin A2在制备抗菌和/或防腐产品中的应用。
3.根据权利要求2所述的应用，其特征在于，所述抗菌和/或防腐产品抑制金黄色葡萄球菌、蜡样芽胞杆菌、产气荚膜梭菌和单核细胞增生李斯特菌中的任一种或多种。
4.一种抗菌和/或防腐产品，其特征在于，包括权利要求1所述的细菌素Thurisin A2。
5.权利要求1所述细菌素Thurisin A2在制备治疗和/或预防细菌感染性疾病的药物中的应用。
6.根据权利要求5所述的应用，其特征在于，所述细菌为金黄色葡萄球菌、蜡样芽胞杆菌、产气荚膜梭菌和单核细胞增生李斯特菌中的任一种或多种。
7.一种治疗和/或预防细菌感染性疾病的药物，其特征在于，包括权利要求1所述的细菌素Thurisin A2。
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