CN113912676A - 一种酒糟黄鱼抗菌肽fah34及其应用 - Google Patents
一种酒糟黄鱼抗菌肽fah34及其应用 Download PDFInfo
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Abstract
本发明公开了一种酒糟黄鱼抗菌肽FAH34,其氨基酸序列为KTISFILK,抗菌肽的分子量为949道尔顿。本发明抗菌肽具有强烈的抑制副溶血性弧菌生长的作用,且在经过高温处理后依旧具有良好的抑菌活性,可作为制备预防或抑制副溶血性弧菌感染的药物。本发明为后续进一步研究酒糟黄鱼抗菌肽作为食品防腐剂和防止鱼病的饲料添加剂开发奠定了基础。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种酒糟黄鱼抗菌肽FAH34及其应用。
背景技术
食源致病微生物是食品、食用农产品产供过程的关键潜在的生物危害因素,严重威胁食品、食用农产品质量安全,给人类和动物健康造成严重危害。据世界卫生组织(WHO)统计,全球每年约有15亿人感染食源性疾病,其中70%为致病性微生物污染所造成的。其中常见的致病微生物有:沙门氏菌(禽畜肉、蛋类)、弯曲菌(禽肉)、金黄色葡萄球菌(畜禽肉、乳品)、副溶血性弧菌(水产品)、蜡样芽孢杆菌(剩饭)、肉毒梭菌(发酵制品、肉制品)、单核细胞增生李斯特菌(乳制品、即食食品)以及致病性大肠杆菌(畜禽肉)等。而沙门氏菌以及副溶血性弧菌是沿海地区两种最常见的食源性致病菌,也是引起沿海地区人民食物中毒的首要食源性致病菌。
副溶血性弧菌广泛存在于海洋环境中,是引起食物中毒爆发的主要病原菌之一,也是一种人和海洋生物共感染的病原菌。既可引起人食物中毒、肠胃炎等,危害人体健康,又可引起水产品养殖病,造成经济损失。传统抗生素在致病微生物防控中发挥着极重要的作用,但在近年来,由于抗生素的滥用所带来的副作用及病菌抗药性问题也愈发凸显,严重影响了人类健康及生态安全。抗菌肽作为广泛存在于自然界生物中的一类多肽物质,对细菌具有良好的抑制作用,可作为传统抗生素和饲料添加剂的代替品。大部分抗菌肽具有热稳定性,在l00℃下加热10~15min仍能保持其活性,超过100℃仍能保持活性的抗菌肽则比较罕见。
因此,提供一种能有效抑制副溶血性弧菌感染,同时能保持良好的热稳定性的抗菌肽是本领域亟待解决的技术问题。
发明内容
本发明的目的在于提供一种酒糟黄鱼抗菌肽及其应用,通过酒糟黄鱼抗菌肽FAH34对副溶血性弧菌抑菌活性以及热稳定性研究,为寻找新的食品防腐剂和预防鱼病水产饲料添加剂奠定基础。
为了解决上述目的,本发明采用的技术方案如下:
本发明解决其技术问题所采用的技术方案之一是:提供了一种酒糟黄鱼蛋白抗菌肽FAH34,其氨基酸序列KTISFILK,如SEQ ID NO:1所示。所示抗菌肽FAH34的分子量为949道尔顿,所带电荷为+2,总疏水性比为50%。
抗菌肽FAH34是从如下原理对细菌造成破坏:一方面,抗菌肽肽FAH34具有的正电荷可与细菌细胞膜发生作用,增加细菌细胞膜成孔活性。另一方面,破坏细胞膜的同时改变细菌细胞膜的通透性,使细胞内物质外渗从而导致细菌死亡。
本发明解决其技术问题所采用的技术方案之二是:提供了一种酒糟黄鱼抗菌肽FAH34在制备抗菌药物中的应用,该抗菌药物用于抑制和/或杀灭副溶血性弧菌。
本发明解决其技术问题所采用的技术方案之三是:提供了一种抗菌药物,其有效成分包括酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1。
进一步地,所述抗菌药物用于抑制和/或杀灭副溶血性弧菌。
本发明解决其技术问题所采用的技术方案之四是:提供了一种水产饲料添加剂,其有效成分包括酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1。
进一步地,所述水产饲料添加剂用于抑制和/或杀灭副溶血性弧菌。
本发明解决其技术问题所采用的技术方案之五是:提供了一种食品防腐剂,其有效成分包括酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1。
进一步地,所述食品防腐剂用于抑制和/或杀灭副溶血性弧菌。
本发明的抗菌肽可以采用本领域技术人员已知的方法合成,例如固相合成,并采用本领域技术人员已知的方法进行纯化,例如高效液相色谱法。
实施本发明,具有如下有益效果:
本发明以酒糟黄鱼为研究对象,通过LCMS质谱结果对所的肽段进行生物信息学预测,发现一个全新氨基酸序列的多肽FAH34。研究多肽FAH34对副溶血性弧菌的抑菌活性;并以副溶血性弧菌为例利用透射电镜观察FAH34对其破坏程度;最后研究了121℃以及80℃两种高温处理后该多肽的抑菌活性,验证其耐热性。实验结果表明,该肽对副溶血性弧菌具有强烈的抑制作用,且在经过高温处理后依旧具有良好的抑菌活性。它的抑菌机理是首先吸附在细菌表面,然后破坏细菌的细胞膜,并抑制膜的生成使细胞内物质外渗,从而达到使细菌失活的作用。良好的热稳定性使其在食品防腐剂的应用方面具备一定的潜力。
附图说明
图1为抗菌肽FAH34的质谱图。
图2为抗菌肽FAH34的结构示意图。
图3为抗菌肽FAH34对副溶血性弧菌最低抑制浓度(MIC)测定对照图。其中,
A:抗菌肽浓度0μg/mL;
B:抗菌肽浓度3.90625μg/mL;
C:抗菌肽浓度7.8125μg/mL;
D:抗菌肽浓度15.625μg/mL;
E:抗菌肽浓度31.25μg/mL;
F:抗菌肽浓度62.5μg/mL。
图4为抗菌肽FAH34时间杀伤曲线图。
(▲)空白对照;(■)MIC;(●)2MIC;
图5为副溶血性弧菌透射电镜图.
(A)空白对照组;(B)抗菌肽FAH34处理的副溶血性弧菌;
图6为80℃加热30min后抗菌肽FAH34对副溶血性弧菌最低抑制浓度(MIC)测定对照图。其中,
A:抗菌肽浓度0μg/mL;
B:抗菌肽浓度3.90625μg/mL;
C:抗菌肽浓度7.8125μg/mL;
D:抗菌肽浓度15.625μg/mL;
E:抗菌肽浓度31.25μg/mL;
F:抗菌肽浓度62.5μg/mL。
图7为121℃加热15min后抗菌肽FAH34对副溶血性弧菌最低抑制浓度(MIC)测定对照图。其中,
A:抗菌肽浓度0μg/mL;
B:抗菌肽浓度3.90625μg/mL;
C:抗菌肽浓度7.8125μg/mL;
D:抗菌肽浓度15.625μg/mL;
E:抗菌肽浓度31.25μg/mL;
F:抗菌肽浓度62.5μg/mL。
图8为80℃加热30min以及121℃加热15min后抗菌肽FAH34时间杀伤曲线图。
具体实施方式
为了更好地理解本发明,下面结合实施例和附图对本发明做进一步的详细说明,但本领域技术人员了解,下述实施例不是对本发明保护范围的限制,任何在本发明基础上做出的改变和变化,都在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
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:酒糟黄鱼的液相色谱/质谱联用技术(LCMS)
称取100~200g酒糟黄鱼样品鱼肉于烧杯中,加入适量超纯水用搅拌机破碎3~5min,然后将破碎的组织于100℃水浴锅煮2~3h。在4℃条件下用11000g离心力离心20min制备样品。再将离心好的上清液过0-3000目筛后于冰箱隔夜冷冻后进行冻干。再将冻干后的样品进行LCMS实验。
色谱条件:进样量:5.0μl
色谱柱:C18分析色谱柱,长度25cm,内径75μm.
流动相:A:0.1%甲醇水溶液
B:乙腈
结合搜库软件:MAXQUANTv1.6.5.0、数据库:uniprot大黄鱼蛋白库对所得肽段质谱图进行区分鉴定,得到85种肽段。抗菌肽FAH34质谱结果如图1所示。
实施例2:酒糟黄鱼中抗菌肽的筛选
通过LCMS结果所得85种氨基酸序列,利用抗菌肽预测在线服务器APD3、CAMP对酒糟黄鱼蛋白序列中可能存在的抗菌序列进行预测,并对可能具有抗菌序列的电荷、疏水性进行分析,最终筛选出氨基酸序列KTISFILK化学合成(由北京中科亚光生物科技有限公司合成),并进行抑菌活性验证。筛选结果如下表所示。
表1酒糟黄鱼LCMS结果潜在抗菌肽的预测
从表1可以看出,序列KTISFILK具有典型抗菌肽的特征,即氨基酸数量(<50)、总正电荷(+2到+9)和疏水氨基酸百分比(>30%)。合成符合特征的2个肽(KTISFILK、AALDLLVPMKKNSNTAGGK),分别对副溶血性弧菌进行抑菌实验,结果显示肽序列KTISFILK的抑菌作用最强。
利用在线服务器I-TASSER对肽序列KTISFILK的结构进行预测,同时利用软件PyMOL观察其二级结构。结果如图2所示。
实施例3:最低抑制浓度(MIC)测定
将副溶血性弧菌在37℃培养12h至对数生长期,在0.01MpH7.2磷酸盐缓冲液中稀释至106-7CFU/mL。将肽溶于磷酸盐缓冲中,37℃等体积与菌混合处理2h。最低抑菌浓度(MIC)是指在37℃孵育过夜后,从微量滴定板上看不到细菌生长的抗菌肽最低浓度。如图3所示,FAH34对副溶血性弧的最低抑菌浓度(MIC)为3.90625μg/mL。
实施例4:时间杀伤曲线TIMEKILL测定
将副溶血性弧菌在37℃培养12h至对数生长期,在0.01MpH7.2磷酸盐缓冲液中稀释至103-4CFU/mL。取MIC和2×MIC浓度肽于37℃等体积与菌混合后分别进行孵育,每隔30分钟取样涂平板,37℃培养过夜后记录菌落总数。由结果可知,抗菌肽对副溶血性弧菌均在2.5h开始有明显效果;随后继续呈下降趋势。在抗菌肽作用下,细菌数量减少的更快。表明抗菌肽FAH34对副溶血性弧菌随着作用时间增加有明显的抑制效果(图4)。
实施例5:透射电镜分析
以106-7CFU/mL的细菌在37℃下用2×MIC的FAH34处理2h,然后在2700g下离心10min,用磷酸盐缓冲液(pH7.2)洗涤两次。用1%的锇酸固定后,用95%乙醇脱水,然后丙酮处理20min。样品在70℃下烘烤24h,在铜网格上制备70-90nm的薄片,然后用柠檬酸铅和乙酸铀酯染色。用H-7650透射电子显微镜观察和捕获超微结构。
如图5A所示,对于未经处理的细菌,细菌细胞的胞内组织和结构完整性良好。然而,如图5B所示,经过肽FAH34处理后,可以看到细菌细胞膜结构开始随着细胞空泡化而模糊,细胞的形状变得不规则,细胞膜完全塌陷,细胞质内容物流出。透射电子显微镜结果表明,FAH34肽对副溶血性弧菌的细胞膜和内部结构具有破坏作用。
实施例6:高温处理后抗菌肽FAH34的抑菌活性测定
分别对肽FAH34在121℃加热15min以及80℃加热30min,处理完成后对其最低抑菌浓度(MIC)以及时间杀灭动力学曲线TIMEKILL进行测定。测定方法同例3、例4中的测定方法。结果表明,在经过121℃加热15min以及80℃加热30min两种高温热处理后,肽FAH34保持了其抑菌活性,最低抑制浓度(MIC)和时间杀伤曲线(TIMEKILL)均未发生明显变化。如图6、7和8所示,经过热处理后的抗菌肽FAH34的最低抑菌浓度(MIC)仍为3.90625μg/mL,时间杀伤曲线(TIMEKILL)表明其在2.5h后有明显的杀菌效果。说明抗菌肽FAH34具有较好的热稳定性,可被用作热加工中的食品防腐剂以及水产饲料添加剂,在食品工业中具有良好的潜在应用前景。
综上,本发明提供了一种全新的抗菌肽FAH34,抗菌肽FAH34对副溶血弧菌的最低抑菌浓度(MIC)为3.90625μg/mL,对副溶血弧菌具有强烈的抑制作用,且具有较好的热稳定性。本发明抗菌肽FAH34是通过吸附在细菌表面,破坏细菌的细胞膜,并抑制膜的生成使细胞内物质外渗的原理而达到使细菌失活的作用。
虽然以上描述了本发明的具体实施方式,但是熟悉本技术领域的技术人员应当理解,我们所描述的具体的实施例只是说明性的,而不是用于对本发明的范围的限定,熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化,都应当涵盖在本发明的权利要求所保护的范围内。
序列表
<110> 集美大学
<120> 一种酒糟黄鱼抗菌肽FAH34及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Pseudosciaena crocea
<400> 1
Lys Thr Ile Ser Phe Ile Leu Lys
1 5
Claims (9)
1.一种酒糟黄鱼抗菌肽FAH34,其氨基酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的酒糟黄鱼抗菌肽FAH34,其特征在于:所述抗菌肽的分子量为949道尔顿。
3.如权利要求1~2中任一项所述的酒糟黄鱼抗菌肽FAH34在制备抗菌药物中的应用,其特征在于:所述抗菌药物用于抑制和/或杀灭副溶血性弧菌。
4.一种抗菌药物,其特征在于:其有效成分包括酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1,所述抗菌药物用于抑制和/或杀灭副溶血性弧菌。
5.如权利要求4所述的抗菌药物,其特征在于:其有效成分为酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1,所述抗菌药物用于抑制和/或杀灭副溶血性弧菌。
6.一种水产饲料添加剂,其特征在于:其有效成分包括酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1,所述水产饲料添加剂用于抑制和/或杀灭副溶血性弧菌。
7.如权利要求6所述的水产饲料添加剂,其特征在于:其有效成分为酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1,所述水产饲料添加剂用于抑制和/或杀灭副溶血性弧菌。
8.一种食品防腐剂,其特征在于:其有效成分包括酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1,所述食品防腐剂用于抑制和/或杀灭副溶血性弧菌。
9.如权利要求8所述的食品防腐剂,其特征在于:其有效成分为酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1,所述食品防腐剂用于抑制和/或杀灭副溶血性弧菌。
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