CN107021996B - 一种短肽、其应用以及由其得到的抗菌组合物 - Google Patents
一种短肽、其应用以及由其得到的抗菌组合物 Download PDFInfo
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Abstract
本发明提供了一种短肽、其应用以及由其得到的抗菌组合物，属于生物技术领域，该短肽能够有效应用于抑制黄曲霉的生长和黄曲霉毒素的生物合成中。该短肽具有如下通式结构：
其中，X代表天冬酰胺或鸟氨酸，当n＝0时，m为2‑9的正整数；当n＝1时，m为1‑8的正整数。本发明能够应用于黄曲霉生长的抑制中。
Description
技术领域
本发明属于生物技术领域，尤其涉及一种短肽、其应用以及由其得到的抗菌组合物。
背景技术
黄曲霉毒素主要由黄曲霉和寄生曲霉产生，具有强的毒性、致畸性和致癌性，农作物如花生、玉米、大米、棉籽、坚果等容易被黄曲霉毒素污染。黄曲霉毒素B1是已知毒性最强的天然致癌物，其主要引发肝癌。
现在常用物理、化学、生物法去除原料中的黄曲霉毒素。物理法如加热、吸附、提取、伽马射线辐照、紫外线照射等。化学法如用臭氧、氨水、氯气、过氧化氢、甲醛处理等。生物法如用微生物发酵原料去毒，用植物提取物抑制毒素产生等。然而，每种方法都有限制，或因原料的营养受到损失，或因需要昂贵的设备等。例如，黄曲霉毒素结构非常稳定，270℃分解，如用加热处理是一般温度越高、加热时间越长，黄曲霉毒素去除率越高，但很明显，加热法容易使物料的营养损失，且需要耐高压的设备。因此，目前急需提供一种有效的方法来抑制黄曲霉毒素的生物合成。
发明内容
本发明提供了一种短肽、其应用以及由其得到的抗菌组合物，该短肽能够有效应用于黄曲霉的生长和黄曲霉毒素的生物合成中。
为了达到上述目的，本发明的一方面提供了一种短肽，所述短肽具有如下通式结构：
其中，X代表天冬酰胺或鸟氨酸，当n＝0时，m为2-9的正整数；当n＝1时，m为1-8的正整数。
作为优选技术方案，当n＝0时，所述短肽的氨基酸序列选自序列表中SEQ IDNO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8中的任意一种。
作为优选技术方案，当X代表天冬酰胺，且n＝1时，所述短肽的氨基酸序列选自序列表中SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13、SEQ IDNO.14、SEQ ID NO.15、SEQ ID NO.16中的任意一种。
作为优选技术方案，当X代表鸟氨酸，且n＝1时，所述短肽的氨基酸序列选自序列表中SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19、SEQ ID NO.20、SEQ ID NO.21、SEQ IDNO.22、SEQ ID NO.23、SEQ ID NO.24中的任意一种。
本发明的另一方面提供了一种如上述任一项技术方案所述的短肽在固体培养基上抑制黄曲霉生长中的应用。
本发明的再一方面提供了一种如上述任一项技术方案所述的短肽在农作物储存中抑制黄曲霉的生长和黄曲霉毒素的生物合成中的应用。
本发明的又一方面提供了一种抗菌组合物，所述抗菌组合物包括至少一种治疗或预防有效量的如上述任一项技术方案所述的短肽和至少一种的药学上可接受的载体。
与现有技术相比，本发明的优点和积极效果在于：
1、本发明所提供的短肽为从巨大芽孢杆菌发酵液中首次分离得到的，具有很好的黄曲霉毒素的抑制作用。
2、本发明所提供的短肽不仅在固体培养基上对黄曲霉的生长具有显著的抑制作用，而且在农作物储存中也对黄曲霉的生长和黄曲霉毒素的生物合成具有显著的抑制作用，其中，部分短肽在2mg/mL的浓度时即可显著抑制黄曲霉的生长。
附图说明
图1为本发明实施例1中所提供的经Sephadex G-25层析分离得到的巨大芽孢杆菌发酵液的组分纯化图，包括A1和A2两个组分；
图2为本发明实施例1所提供的A2组分经反相高效液相色谱的分离纯化图，包括B1-B9共9个组分；
图3为本发明实施例1所提供的巨大芽孢杆菌中各短肽的液质联用色谱图，其中，图(a)为经反相高效液相色谱分离得到的B6组分的液质图；(b)为m/z 942.9606的短肽的质谱图；(c)为m/z 938.0045的短肽的质谱图。
图4为本发明实施例2所提供的24种短肽在固体培养基上的抑菌效果图；
图5为本发明实施例3所提供的不同构型的Asp-Orn、Asp-Asn和Asp-Asp-Asn在农作物储存中的抑菌效果图；
图6为本发明实施例3所提供的扫描电镜下观察得到的L-Asp-L-Orn对于黄曲霉菌菌丝形态的影响；
图7为本发明实施例3所提供的透射电镜下观察得到的L-Asp-L-Orn对于黄曲霉菌菌丝形态的影响。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
本发明实施例提供了一种短肽，该短肽从巨大芽孢杆菌发酵液中首次分离，具体如下:
所利用的微生物：
黄曲霉A.flavus NRRL 3357：培养于PDA培养基中(200g土豆浸提物，20g葡萄糖，20g琼脂，1L水)。
巨大芽孢杆菌Bacillus megaterium(CGMCC7086，保存于中国微生物菌种保藏管理委员会普通微生物中心)：培养于牛肉膏蛋白胨培养基中(牛肉膏3g，蛋白胨10g，氯化钠5g，1L水)。
纯化和鉴定:
培养12升巨大芽孢杆菌(160rpm，37℃，3天)，发酵液经8,000r/min离心20分钟。将离心后的上清液经80％硫酸铵沉淀，沉淀冷冻干燥后，经Sephadex G-25层析，流速为5mL/min，检测波长为220nm。如图1所示，沉淀中共有两个组分，实验表明A2组分具有抑制黄曲霉的作用，收集A2组分进一步研究。
将A2组分进一步经反相高效液相色谱(RP-HPLC)(Agilent,Santa Clara,CA,USA)纯化，所采用的色谱柱为C18柱(25×250mm)，流动相为含0.1％三氟乙酸(TFA)的乙腈。如图2所示，共收集9个组分，实验表明B6组分具有抑制黄曲霉的作用，收集B6组分进一步研究。
将B6组分进一步经液质联用色谱(LC-MS/MS)鉴定，所采用的仪器为AccurateMass quadruple time-of-flight(Q-TOF)Liquid Chromatography(Triple TOF 5006,ABSCIEX,USA)。如图3可知，鉴定出3种短肽，分别为Asp-peptide、Asn-peptide和Orn-peptide，这3种短肽分别包含了多种含2-7个Asp(天冬氨酸残基)的短肽。实验表明，Asp-peptide、Asn-peptide和Orn-peptide对黄曲霉具有抑制作用。
结合谱图分析可知，所述短肽具有如下通式结构：
其中，X代表天冬酰胺或鸟氨酸，当n＝0时，m为2-9的正整数；当n＝1时，m为1-8的正整数。
根据上述条件进行分类，所得到的短肽在不同情况下均为一系列短肽，具体如下：
1)当n＝0时，所得到的短肽如下：
2)当X代表天冬酰胺，且n＝1时，所得到的短肽如下：
| 序列号 | 短肽 |
| SEQ ID NO.9 | Asp-Asn |
| SEQ ID NO.10 | Asp-Asp-Asn |
| SEQ ID NO.11 | Asp-Asp-Asp-Asn |
| SEQ ID NO.12 | Asp-Asp-Asp-Asp-Asn |
| SEQ ID NO.13 | Asp-Asp-Asp-Asp-Asp-Asn |
| SEQ ID NO.14 | Asp-Asp-Asp-Asp-Asp-Asp-Asn |
| SEQ ID NO.15 | Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asn |
| SEQ ID NO.16 | Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asn |
3)当X代表鸟氨酸，且n＝1时，所得到的短肽如下：
| 序列号 | 短肽 |
| SEQ ID NO.17 | Asp-Orn |
| SEQ ID NO.18 | Asp-Asp-Orn |
| SEQ ID NO.19 | Asp-Asp-Asp-Orn |
| SEQ ID NO.20 | Asp-Asp-Asp-Asp-Orn |
| SEQ ID NO.21 | Asp-Asp-Asp-Asp-Asp-Orn |
| SEQ ID NO.22 | Asp-Asp-Asp-Asp-Asp-Asp-Orn |
| SEQ ID NO.23 | Asp-Asp-Asp-Asp-Asp-Asp-Asp-Orn |
| SEQ ID NO.24 | Asp-Asp-Asp-Asp-Asp-Asp-Asp-Asp-Orn |
以上需要说明的是，在3)中所示的序列SEQ ID NO.17-SEQ ID NO.24中，由于鸟氨酸本身并没有与之相对应的密码子，其均是由精氨酸通过精氨酸分解酶分解得到的，因此，在对应的氨基酸序列中，鸟氨酸对应的密码子均表述为精氨酸所对应的密码子，该表述方法在实际科研工作中是可行的，这主要是因为在实际科研工作中，为了得到鸟氨酸，本领域技术人员也是通过对精氨酸进行处理得到。
可以理解的是，在上述组分分离鉴定中，从B6组分中还鉴定得到了：
CA肽(Asp-Asp-Asp-Asp-Asp-Asp-Asp-2-羟基-辛酸-)；和
His肽(Asp-Asp-Asp-Asp-Asp-Asp-Asp-His-，其为由组氨酸上的α-氨基基团和天冬氨酸上的α-羰基基团形成的环)。但由于上述两种肽对黄曲霉的抑菌效果不显著，因此不作保护，但不排除上述两种肽对于其他菌的抑制效果。
实施例2
固体培养基上的抑菌试验
对上述所得到的24种短肽(均为L型)分别进行黄曲霉的抑菌效果试验，分别命名为：a,b,c,d,e,f,g,h,i,j,k,l,m,n,o,p,q,r,s,t,u,v,w,x，并以实验室消毒剂-75％酒精以及强杀菌剂-1％高锰酸钾作为对比进行抑菌效果的评价(重复试验3次)。其中，试验方法具体为：
配制固体PDA培养基，将PDA培养基倾注在96孔板中，使其凝固；分别在PDA培养基上涂布4微升106个/毫升的黄曲霉孢子，然后分别取10微升16毫克/毫升的各种短肽、75％酒精、1％高锰酸钾涂布于96孔板上各加样孔，28℃培养3天，观察抑菌结果。
如图4所示，所得到的24种短肽对黄曲霉均有一定抑制作用，且抑制黄曲霉的效果普遍好于75％酒精，例如a、b、d、g、h、k、m、q、t、u等短肽；某些短肽的抑制黄曲霉的效果甚至可以和1％高锰酸钾相媲美，例如b、g、h、k等短肽。由此可推断，所得到的这24种结构相似的短肽可能对其他真菌，如青霉、酵母、脉胞菌等也有一定抑制作用。
实施例3
在农作物储存中的抑菌试验
本实施例中的农作物可为花生、玉米、大米、棉籽、坚果等，其中，以花生储存为例，具体如下：
首先需要说明的是，由于24种短肽通过实施例2已证明具有良好的抑菌效果，因此，本实施例仅以Asp-Orn、Asp-Asn和Asp-Asp-Asn为例进行测试，同时，为了判断每种肽的构型对于抑菌效果的影响，还分别以上述3种肽为例进行了不同构型的合成。将所得到的不同构型的短肽配成不同浓度的溶液，喷洒到花生上，28℃培养7天，观察黄曲霉的生长状况(作为对照比较：每个花生上同时接种105个黄曲霉孢子)。
由图5可知，与对照相比，抑制作用最强的是L-Asp-L-Orn,L-Asp-L-Asn和L-Asp-L-Asp-L-Asn，且相对于D型的短肽，L型的短肽效果更加显著，在达到2mg/mL时即可更加显著抑制黄曲霉在花生上的生长以及显著抑制黄曲霉毒素在花生上的生成。以L-Asp-L-Orn为例，如图6-7所示，显示了其对于黄曲霉菌菌丝形态的影响，A、B为对照组菌丝，C、D为短肽处理组菌丝，其中，在A、B组中，菌丝的细胞形态饱满，各细胞器清晰完整，而C、D组中，菌丝的细胞形态发生变化，液泡变大，细胞器变型。由此可说明，本实施例所提供的短肽可有效抑制黄曲霉在花生上的生长以及黄曲霉毒素在花生上的生成。
实施例4
基于实施例2-3，本施例还提供了一种抗菌组合物，所述抗菌组合物包括如上述实施例所述的至少一种治疗或预防有效量的如SEQ ID NO:1-SEQ ID NO.24所示氨基酸序列的短肽和至少一种的药学上可接受的载体。由于上述实施例所提供的24种短肽可有效抑制黄曲霉在花生上的生长以及黄曲霉毒素在花生上的生成，因此，配合至少一种的药学上可接受的载体组成的抗菌组合物也可有效应用在黄曲霉的抑制中，尤其是农作物储存中的黄曲霉以及黄曲霉毒素的生长。
Claims (6)
1.一种短肽，其特征在于，所述短肽为如下通式结构：
其中，X代表天冬酰胺，n＝1，m为2-6的正整数；
所述短肽的氨基酸序列选自序列表中SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.13、SEQ ID NO.14中的任意一种。
2.一种如权利要求1所述的短肽在固体培养基上抑制黄曲霉生长中的应用。
3.一种结构为
其中X代表天冬酰胺，n＝1，m＝1，且氨基酸序列为SEQ ID NO.9的短肽在固体培养基上抑制黄曲霉生长中的应用。
4.一种如权利要求1所述的短肽在农作物储存中抑制黄曲霉的生长和黄曲霉毒素的生物合成中的应用。
5.一种结构为
其中X代表天冬酰胺，n＝1，m＝1，且氨基酸序列为SEQ ID NO.9的短肽在农作物储存中抑制黄曲霉的生长和黄曲霉毒素的生物合成中的应用。
6.一种抗菌组合物，其特征在于，所述抗菌组合物包括至少一种有效量的如权利要求1所述的短肽和至少一种的药学上可接受的载体。
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