CN116041453B - 一种抗多种食源性病原菌的无前导肽细菌素subticin A1及应用 - Google Patents
一种抗多种食源性病原菌的无前导肽细菌素subticin A1及应用 Download PDFInfo
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Abstract
本发明公开了一种抗多种食源性病原菌的无前导肽细菌素subticin A1及应用,属于医药食品应用领域。上述无前导肽细菌素subticin A1的氨基酸序列为:MIAFLRIVAQLGARAARWAWANKDRVLGWIRDGMAIDWIINKINDMVS,氨基酸序列中第一个氨基酸为甲酰甲硫氨酸;该细菌素subticin A1通过芽孢杆菌Bacillus subtilis发酵产物获取,或者通过化学合成方式获取。经过实验验证,该细菌素subticin A1pH稳定性高、安全低毒,能高效杀灭肉毒梭菌、蜡样芽胞杆菌等多种食源性病原微生物,可应用到食品、饲料、抗菌药物等不同领域。
Description
技术领域
本发明涉及医药食品应用领域,特别是涉及一种抗多种食源性病原菌的无前导肽细菌素subticin A1及应用。
背景技术
食品质量和安全一直是消费者和食品加工业最关心的问题,由食源性病原菌引起的食品质量安全问题已严重威胁人们的健康。在2016年世界卫生组织发布的报告中,全球每年有近1/10人口因食物污染而患病,其中约42万人死亡。肉毒梭菌(C.botulinum)是一种能形成网球拍状芽胞的革兰氏阳性芽胞杆菌,有较强抗逆性,生长速度快,在厌氧条件可产生毒性极强的肉毒毒素,毒素包括A、B、E等8个类型,我国多由A型肉毒毒素引起中毒。蜡样芽胞杆菌、单增李斯特菌是常见的食源性病原菌,广泛存在于水、土壤、植物中,可污染牛奶、肉制品、蔬菜等食品,在加工运输和冷藏条件下均可生长。蜡样芽胞杆菌可导致呕吐或腹泻等疾病,单增李斯特菌可引起脑膜炎、胃肠炎等疾病,严重威胁人类生命和健康。目前一般采用高温灭菌或添加化学防腐剂如含氯消毒剂、甲基内酰脲类化合物等进行处理,但高温会破坏食品颜色、风味、口味、质地,降低某些有效成分的含量及生物可利用性,化学防腐剂的使用会对人体健康造成危害,还污染环境。
细菌素是细菌在代谢过程中由核糖体合成的、对产生菌具有自身免疫性的一类具有抗菌活性的多肽类物质,根据其结构的不同,可分为翻译后可修饰细菌素(Ⅰ类细菌素)和翻译后未修饰细菌素(Ⅱ类细菌素)两大类。细菌素在生物合成、作用模式、抗性机制及抗菌活性方面与抗生素存在明显不同,易在人类肠道系统降解,具有安全、无毒、无残留、对热稳定,作用于细胞膜而不与抗生素产生交叉抗性,能有效抑制或杀灭食源性病原菌等很多优点,而被作为生物食品防腐剂应用于食品防腐领域。早在1969年,联合国粮食及农业组织、世界卫生组织(FAL/WHO)食品添加剂联合专家委员会就批准来源于乳酸链球菌的Ⅰ类细菌素nisin作为食品防腐剂使用。然而,由于多数细菌素不易规模化生产、成本较高等缺点大大限制了其商业开发价值。迄今为止,目前被授权作为食品防腐剂的细菌素仅有Nisin、pediocinPA-1两种,其中Nisin已广泛使用多年,其仅在pH<7的条件下有抑菌活性,pediocinPA-1主要对单增李斯特菌有抑菌活性。而无前导肽细菌素是细菌素家族中一类由核糖体合成的、不进行任何翻译后修饰的、N端没有前导肽序列的Ⅱ类细菌素。该类细菌素因其在生物合成过程中不形成前导肽序在翻译后不经任何修饰即转变为有活性的成熟肽,这种简单的遗传结构使其在其它细菌或真核细胞中更易表达,更适合通过生物工程进行规模化生产,具有巨大的商业应用潜能。因此,开发能有效抑制食源性病原菌、耐受较高pH值、易于规模化生产的无前导肽细菌素,将为由食源性病原菌引起的食品腐败以及所导致的相关疾病提供有效防治和治疗,也为细菌素类食品防腐剂的开发提供新的途径。
发明内容
本发明的目的是提供一种抗多种食源性病原菌的无前导肽细菌素subticin A1及应用,以解决上述现有技术存在的问题,该无前导肽细菌素subticin A1结构简单,合成容易,安全低毒,能高效杀灭肉毒梭菌等多种食源性病原微生物,可应用到食品、饲料、抗菌药物等不同领域。
为实现上述目的,本发明提供了如下方案:
本发明提供一种无前导肽细菌素subticin A1,其氨基酸序列为:MIAFLRIVAQLGARAARWAWANKDRVLGWIRDGMAIDWIINKINDMVS,氨基酸序列中第一个氨基酸为甲酰甲硫氨酸。
本发明还提供所述的无前导肽细菌素subticin A1的制备方法,所述细菌素通过枯草芽孢杆菌Bacillus subtilis发酵产物获取,或者通过化学合成方式获取。
优选的是,所述枯草芽孢杆菌(Bacillus subtilis)的保藏编号为CGMCCNo.25552;保藏时间为2022年8月19日;保藏单位为中国微生物菌种保藏管理委员会普通微生物中心(CGMCC);保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所。
优选的是,所述芽胞杆菌的发酵条件为:将所述LB芽胞杆菌接种LB液体培养基,37℃,220rpm振摇培养8h,获取发酵产物。
本发明还提供所述的无前导肽细菌素subticin A1在抑菌方面的应用,所述菌包括食源性病原菌肉毒梭菌(C.botulinum)、蜡样芽胞杆菌(Bacillus cereus)、单增李斯特菌(Listeria monocytogenes)和金黄色葡萄球菌(Staphylococcus aureus)。
本发明还提供所述的无前导肽细菌素subticin A1在制备食品防腐剂或保鲜剂方面的应用。
优选的是,所述无前导肽细菌素subticin A1应用于制备由食源性病原菌肉毒梭菌(C.botulinum)、蜡样芽胞杆菌(Bacillus cereus)、单增李斯特菌(Listeriamonocytogenes)、金黄色葡萄球菌(Staphylococcus aureus)引起的食品变质腐败的食品防腐剂中。
本发明还提供所述的无前导肽细菌素subticin A1在在制备抗菌药物中的应用,其特征在于,所述抗菌药物包括抗食源性病原菌肉毒梭菌(C.botulinum)、蜡样芽胞杆菌(Bacillus cereus)、单增李斯特菌(Listeria monocytogenes)以及金黄色葡萄球菌(Staphylococcus aureus)的药物。更优选的,所述抗菌药物包括抗食源性病原菌肉毒梭菌(C.botulinum)、蜡样芽胞杆菌(Bacillus cereus)、单增李斯特菌(Listeriamonocytogenes)以及金黄色葡萄球菌(Staphylococcus aureus)引起的人类或者动物腹泻的药物。
本发明还提供一种产品,含有所述的无前导肽细菌素subticin A1。
优选的是,所述产品包括食品防腐剂、食品保鲜剂和抗菌药物。
本发明公开了以下技术效果:
本发明公开了通过生物合成或者化学合成的方式获取一种新型无前导肽细菌素subticin A1,该细菌素subticin A1有较好的pH稳定性,可耐受pH的范围为2-11;并且结构简单,合成容易,安全低毒,能高效杀灭肉毒梭菌等多种食源性病原微生物,可用于抗菌药物、饲料、食品等领域,具有广阔的应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为细菌素subticin A1的一级质谱图;图中M.W.表示分子量;
图2为细菌素subticin A1的氨基酸序列结构及二级质谱图;“*”表示该氨基酸被甲酰化形成甲酰甲硫氨酸,小写字母表示碎片离子;
图3为细菌素subticin A1的高效液相色谱(HPLC)分离纯化结果;
图4为不同浓度细菌素subticin A1对pH的稳定性,指示菌为Bacillus cereusATCC 14579;
图5为不同浓度subticin A1的细胞毒性;由不同的小写字母表示显著性差异,P<0.05;
图6为细菌素subticin A1在牛乳保鲜中的应用效果;A:指示菌为Bacilluscereus ATCC 14579,B:指示菌为Listeria monocytogenes LM201。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
以下实施例中所使用的枯草芽孢杆菌(Bacillus subtilis)ZXF04,是发明人从自然界的土壤中分离得到的一株菌,已于2022年8月19日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC);保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所;保藏编号为CGMCC No.25552。该菌株的分离鉴定方法如下:
1、土壤采集自河南省开封市兰考县某耕地,风干粉碎过40目筛,取1g置于装100mL无菌水的三角瓶中振摇20min,80℃水浴15min,然后采用涂布法涂布于LB固体平板,30℃培养15h,挑取单菌落。
2、培养特征
菌落形态:菌落灰白,粗制不透明,表明有褶皱,边缘不整齐(LB平板)。
菌体细胞:LB液体培养基中37℃,220rpm培养20h后,显微镜观察有明显的芽胞产生。
在LB液体培养基中培养特征:接种后37℃、220rpm振荡培养,8h开始产生抗菌物质,其发酵上清液对蜡样芽胞杆菌、单增李斯特菌、猪链球菌等病原菌有抗菌活性。
3、基因鉴定
以该菌株的基因组为模板,采用鉴定细菌通用引物27F和1541R进行PCR扩增,27F:5′-AGAGTTTGATCCTGGCTCAG-3′,1541R:5′-AAGGAGGTGATCCAGCCGCA-3′;扩增体系及扩增程序见表1:
表1:PCR扩增体系及扩增程序
PCR扩增产物送生物测序公司进行测序,结果显示,该菌株的16srDNA序列(SEQ IDNO:2)为:
CGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACA ATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAA。
序列比对显示,该菌株16srDNA序列与枯草芽孢杆菌模式菌株Bacillus subtilisNCIB 3610(T)(NCBI登录号为ABQL01000001)的16srDNA序列一致性为99.86%。根据其菌落形态、细胞形态及16srDNA序列比对情况,确认该菌株为枯草芽孢杆菌。
实施例1细菌素subticin A1的合成和鉴定
细菌素subticin A1可以采用枯草芽孢杆菌(Bacillus subtilis)ZXF04进行生物合成,然后经高效液相色谱(HPLC)分离纯化获得,也可根据其氨基酸序列采用化学合成法直接合成。
(1)生物合成法
将菌株Bacillus subtilis ZXF04在LB培养基37℃,220rpm振摇培养8h,取发酵上清液经离子交换树脂Amberlite XAD7HP交换,分别用蒸馏水、30%、80%乙醇(pH=2)洗脱,活性组分经低温旋蒸浓缩后,高效液相色谱(HPLC)TC-C18色谱柱进一步分离,色谱条件为流动相乙腈,上样量10uL,流速1mL/min,梯度洗脱程序为乙腈浓度由10%增加到80%洗脱55min,检测波长210nm,收集保留时间49min的馏分进行旋转蒸发,冷冻干燥,可获得纯度大于95%的subticin A1,见图3。
对所得subticin A1经高分辨率液质联用仪Agilent Technologies 6540UDHAccurate-Mass Q-TOF LC/MS。质谱检测条件为毛细管电压:3,500V,喷雾压力:35lb/in2,Q-TOF扫描范围:500-2,000m/z,干燥气流速:9liters/min,温度:300°C,数据采集速率:1spectrum/s。
一级质谱分析如图1所示,分子量为5552.0154Da,二级质谱分析如图2所示,细菌素subticin A1氨基酸序列(SEQ ID NO:1)为:MIAFLRIVAQLGARAARWAWANKDRVLGWIRDGMAIDWIINKINDMVS,其中第一个氨基酸甲酰化为甲酰甲硫氨酸。BlastP分析显示:subticin A1的氨基酸序列与目前已报道并鉴定的细菌素的氨基酸序列均不相同,说明细菌素subticinA1是一种新型的无前导肽细菌素,为发明人首次研究、鉴定并报道。
(2)细菌素subticin A1也可根据其氨基酸序列经生物科技公司化学合成获得,纯度大于98%。
实施例2细菌素subticin A1对肉毒梭菌等多种食源性病原微生物的最小抑菌浓度(MIC)测定
用生理盐水将色谱纯制品subticin A1配制成60μM的溶液,然后进行2倍系列稀释,以肉毒梭菌或其他食源性病原微生物为指示菌(如表1所示),采用打孔法测定其抑菌活性,最小抑菌浓度MIC是指在加有subticin A1的孔周围有明显抑菌圈显示的subticin A1的最小浓度(μM)。细菌素subticin A1对肉毒梭菌及其他食源性病原菌的最小抑菌浓度(MIC)如下表所示。
表1细菌素Subticin A1对多种食源性病原菌的最小抑菌浓度
注ATCC表示American Type Culture Collection,其它指示菌来源于实验室保藏。
实施例3细菌素subticin A1对pH的耐受性
将浓度分别为1.88μM、3.75μM、7.5μM、15μM的细菌素Subticin A1溶液的pH用0.1M的NaOH或HCl调节为2、3、4、5、6、7、8、9、10、11、12后,37℃水浴1h,然后将其pH调至6,以Bacillus cereus ATCC 14579为指示菌采用打孔法测定其抑菌活性。
结果如图4所示:不同浓度的细菌素subticin A1均在pH2-11范围内对指示菌有抗菌活性,说明细菌素subticin A1对pH有较好的稳定性。
实施例4细菌素subticin A1的细胞毒性的测定
将Hela细胞加放有DMEM培养液的96孔板中,使每个孔中细胞数量约为103个,然后放置在37℃、5%的CO2培养箱中培养24h,用含浓度分别为3.75、7.5、15、30、60μM的新鲜DMEM培养液替换96孔板中的细胞培养液,37℃培养24h,同时将未添加细菌素的培养液和添加1% Triton X-100的培养液分别作为阴性对照和阳性对照,在孔中加入5mg/mL的MTT[3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐]溶液,在37℃条件下孵育3h后除去培养基,然后加入50uL二甲基亚砜(DMS),采用酶标仪检测590nm下各孔的吸光值(OD值),并通过下面公式计算细胞活性。
细胞活性(%)={100-[(Abs590阴性对照样品)–(Abs590细菌素处理样品)]}/[(Abs590阴性对照样品)–(Abs590 Triton X-100处理样品]×100。
结果如图5所示:Subticin A1在60μM的浓度下与阳性对照有极显著差异(P≤0.01),说明Subticin A1对Hela细胞无细胞毒性,毒副作用非常低,是安全的。
实施例5细菌素subticin A1在牛奶保鲜中的应用
将subticin A1分别加入到接种有蜡样芽胞杆菌或单增李斯特菌的灭菌牛奶中(菌浓为105CFU/mL,subticin A1浓度为分别为8×MIC、16×MIC),以未加subticin A1的样品作对照,在25℃条件下放置7d,观察结果。
结果如图6所示;与对照相比,当浓度为16×MIC时,subticin A1可在1天后完全杀灭牛奶中的蜡样芽胞杆菌和单增李斯特菌,因此,细菌素subticin A1可用于由蜡样芽胞杆菌、单增李斯特菌等病原微生物引起的食品防腐或保鲜中。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (4)
1.一种无前导肽细菌素subticin A1的制备方法,其特征在于,所述无前导肽细菌素subticin A1通过枯草芽孢杆菌(Bacillus subtilis)发酵产物获取;
所述枯草芽孢杆菌的保藏编号为CGMCC No.25552;
所述枯草芽孢杆菌的发酵条件为:将所述枯草芽孢杆菌接种LB液体培养基,37℃,220rpm振摇培养8h,获取发酵产物;
取所述发酵产物的上清液经离子交换树脂交换,分别用蒸馏水、30%、80%乙醇洗脱,活性组分经低温旋蒸浓缩后,高效液相色谱进一步分离,色谱条件为流动相乙腈,上样量10μL,流速1mL/min,梯度洗脱程序为乙腈浓度由10%增加到80%洗脱 55min,检测波长210nm,收集保留时间49min的馏分进行旋转蒸发,冷冻干燥,获得所述无前导肽细菌素subticin A1;
所述无前导肽细菌素subticin A1的氨基酸序列为:MIAFLRIVAQLGARAARWAWANKDRVLGWIRDGMAIDWIINKINDMVS,氨基酸序列中第一个氨基酸甲酰化为甲酰甲硫氨酸。
2.一种无前导肽细菌素subticin A1在制备抑菌剂方面的应用,其特征在于,所述抑菌剂为抑制食源性病原菌肉毒梭菌(C.botulinum)、蜡样芽胞杆菌(Bacillus cereus)、单增李斯特菌(Listeria monocytogenes)和金黄色葡萄球菌(Staphylococcus aureus)的菌剂;
所述无前导肽细菌素subticin A1的氨基酸序列为:MIAFLRIVAQLGARAARWAWANKDRVLGWIRDGMAIDWIINKINDMVS,氨基酸序列中第一个氨基酸甲酰化为甲酰甲硫氨酸。
3.一种无前导肽细菌素subticin A1在制备食品防腐剂方面的应用,其特征在于,所述无前导肽细菌素subticin A1应用于制备由食源性病原菌肉毒梭菌(C.botulinum)、蜡样芽胞杆菌(Bacillus cereus)、单增李斯特菌(Listeria monocytogenes)及金黄色葡萄球菌(Staphylococcus aureus)引起的食品变质腐败的食品防腐剂中;
所述无前导肽细菌素subticin A1的氨基酸序列为:MIAFLRIVAQLGARAARWAWANKDRVLGWIRDGMAIDWIINKINDMVS,氨基酸序列中第一个氨基酸甲酰化为甲酰甲硫氨酸。
4.一种无前导肽细菌素subticin A1在制备抗菌药物中的应用,其特征在于,所述抗菌药物为抗食源性病原菌肉毒梭菌(C.botulinum)、蜡样芽胞杆菌(Bacillus cereus)、单增李斯特菌(Listeria monocytogenes)以及金黄色葡萄球菌(Staphylococcus aureus)的药物;
所述无前导肽细菌素subticin A1的氨基酸序列为:MIAFLRIVAQLGARAARWAWANKDRVLGWIRDGMAIDWIINKINDMVS,氨基酸序列中第一个氨基酸甲酰化为甲酰甲硫氨酸。
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