CN113024663A - 一种泥蚶血红蛋白α螺旋抗菌肽及其应用 - Google Patents
一种泥蚶血红蛋白α螺旋抗菌肽及其应用 Download PDFInfo
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Abstract
本发明涉及一种抗菌肽,尤其涉及一种泥蚶血红蛋白α螺旋抗菌肽及其应用。该抗菌肽包括选自SEQ ID NO:1的氨基酸序列或由其组成,具有有效地抑菌作用,并通过低强度超声加工增效其对细菌的抑菌活性。低强度超声辅助处理的TGH2对细菌具有强烈的抑制作用,同时抗菌肽TGH2的α螺旋结构保持稳定。本发明为低强度超声的杀菌方法和TGH2作为食品防腐剂提供了实验依据。
Description
技术领域
本发明涉及一种抗菌肽,尤其涉及一种泥蚶血红蛋白α螺旋抗菌肽及其应用。
背景技术
致病微生物引起的食源性疾病是消费者、行业和监管机构面临的主要问题。食源性致病菌在食品原料、加工和贮存过程中对人体健康构成威胁。
超声波是一种频率超过20kHz的声波,作为一种具有良好方向性、穿透性和反射率的振动能量形式,成为食品工业的前沿抗菌技术。一般认为超声抑菌机制分为声空化、声穿孔和声化学三种机制,声空化和声化学机制在中高强度超声下才能实现。据报道中高强度超声波可以抑制杏仁奶中的大肠杆菌O157:H7和单核细胞增生李斯特菌,并延长冷藏条件下的保质期,同样,对莴苣上这两种细菌的具有相同的抗菌活性。而低强度超声条件下不能灭活病原微生物。
抗菌肽(AMPs)主要是由不同物种产生的阳离子、两亲性肽,用于抵抗入侵的病原微生物,具有高效、低毒、无耐药性等特点。与传统的抗生素不同,大多数抗菌肽通过破坏细菌细胞膜发挥作用,但其α螺旋结构在中高强度超声条件下不稳定,易被破坏而失去抑菌作用。
发明内容
本发明要解决的技术问题,在于提供一种泥蚶血红蛋白α螺旋抗菌肽及其应用,在一定超声条件下,该抗菌肽的抑菌效果得到了很大的提高。
本发明是这样实现的:
本发明首先提供了一种泥蚶血红蛋白α螺旋抗菌肽,包括选自SEQ ID NO:1的氨基酸序列或由其组成。
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。
还提供了所述泥蚶血红蛋白α螺旋抗菌肽的编码基因,以及含有所述编码基因的表达盒、重组菌、重组载体和转基因细胞系。
进一步地,一种抑菌剂,其包括至少一种下列各项:所述的泥蚶血红蛋白α螺旋抗菌肽;或所述的编码基因。
进一步地,一种食品防腐剂,其包括至少一种下列各项:所述的泥蚶血红蛋白α螺旋抗菌肽;或所述的编码基因。
本发明最后提供了所述泥蚶血红蛋白α螺旋抗菌肽与超声协同增效抑菌的方法,经过反复多次实验,在超声频率为40MHz,功率为0.5W/cm2,超声时间1h的条件下,抗菌肽TGH2的对大肠杆菌的抑菌效果最好。
超声结合α螺旋抗菌肽可以从至少如下两方面对细菌造成破坏:一方面,大大增加大肠杆菌细胞膜的通透性,导致细菌死亡;另一方面,抗菌肽TGH2的α螺旋结构未被破坏,使得抗菌肽进入细菌细胞后具有抑菌活性。
本发明具有如下优点:本发明以α螺旋抗菌肽TGH2为研究对象,通过低强度超声加工增效其对细菌的抑菌活性;测定细菌细胞膜通透性的变化,并利用透射电镜观察超声结合抗菌肽TGH2对其破坏程度;最后对它的α螺旋结构稳定性进行评估。实验结果表明,低强度超声辅助处理的TGH2对细菌具有强烈的抑制作用。它的抑菌机理是首先增加细菌细胞膜的通透性,然后抗菌肽快速透过细胞膜进入细菌体内,破坏细菌内部结构,同时细菌内大量内溶物流出,导致细菌死亡,同时抗菌肽TGH2的α螺旋结构保持稳定。本发明为低强度超声的杀菌方法和TGH2作为食品防腐剂提供了实验依据。
附图说明
下面参照附图结合实施例对本发明作进一步的说明。
图1为本发明所选用菌种大肠杆菌生长曲线。(■)为细菌正常条件下的生长曲线,(●)为细菌在单独超声处理下的生长曲线。
图2为超声处理对大肠杆菌作用对照图,其中,A:未经超声处理的大肠杆菌;B:低强度超声处理后的大肠杆菌。
图3为超声处理对金黄色葡萄球菌作用对照图,其中,A:未经超声处理的金黄色葡萄球菌;B:低强度超声处理后的金黄色葡萄球菌。
图4为本发明抗菌肽TGH2单独处理及超声辅助抗菌肽TGH2对大肠杆菌最低抑制浓度(MIC)测定测定对照图,其中,A:孵育条件,抗菌肽浓度500μg/mL;B:超声条件,抗菌肽浓度500mg/mL;C:孵育条件,抗菌肽浓度125μg/mL;D:超声条件,抗菌肽浓度125μg/mL;E:孵育条件,抗菌肽浓度62.5μg/mL;F:超声条件,抗菌肽浓度62.5μg/mL;G:孵育条件,抗菌肽浓度31.25μg/mL;H:超声条件,抗菌肽浓度31.25μg/mL。
图5为本发明抗菌肽TGH2对大肠杆菌的时间杀伤测定曲线;(■)为超声结合抗菌肽TGH2处理,(●)为抗菌肽TGH2单独处理。
图6为本发明抗菌肽TGH2作用大肠杆菌的透射电镜观察图,其中,A:空白对照组;B:抗菌肽TGH2处理的大肠杆菌;C:低强度超声辅助抗菌肽TGH2处理的大肠杆菌。
图7为本发明低强度超声辅助抗菌肽TGH2对大肠杆菌膜通透性变化折线图。
图8为本发明低强度超声辅助抗菌肽TGH2对大肠杆菌电导率变化折线图。
图9为本发明低强度超声辅助抗菌肽TGH2二级结构变化图。
具体实施方式
实施例1:超声条件的优化
将稀释好的菌液与肽混合后置于超声水浴中,温度37℃。以超声频率、超声功率和超声时间为参数,测定不同条件下抗菌肽TGH2对大肠杆菌的抑菌活性及其它抑菌参数。根据超声的声穿孔机制需要,超声功率应尽量控制在5.0W/cm2以下,这样抗菌肽TGH2的α螺旋结构才可能保持稳定。
实施例2:抗菌肽的筛选
首先登录NCBI(美国国立生物技术信息中心)网站,查找泥蚶血红蛋白序列,然后在利用抗菌肽预测在线服务器AntiBP预测血红蛋白序列可能存在的抗菌区域,并对抗菌序列的电荷、疏水性和可靠性进行分析。最终筛选出氨基酸序列AEFLREKLGDKCTDR,命名为TGH2。泥蚶的血红蛋白序列由152个氨基酸组成,TGH2序列位于序号119-136。利用在线服务器APD3对TGH2序列疏水性计算,其总疏水性比为33%。最后验证TGH2的抑菌活性。
实施例3:大肠杆菌生长曲线
将20uL的活化菌株接种到20mL营养肉汤液体培养基中,达到102-4CFU/mL浓度,然后在0.5W/cm2下超声处理1h,取20ul(0,1,2,3,4,5,6,7,8小时)样品进行平板计数,观察生长曲线。如图1所示,经低强度超声处理后的大肠杆菌未有明显损伤,生长趋势与培养条件下基本一致。
实施例4:最低抑制浓度(MIC)测定
将大肠杆菌在37℃培养12h至对数生长期,在0.01M pH 7.2磷酸盐缓冲液中稀释至106-7CFU/mL。将肽TGH2溶于磷酸盐缓冲中,37℃等体积与菌混合后分别进行孵育、0.5W/cm2超声处理1h。MIC是指在37℃孵育过夜后,看不到细菌生长的抗菌肽最低浓度。如图4所示,抗菌肽TGH2对大肠杆菌的MIC为125μg/mL、低强度超声辅助抗菌肽TGH2对大肠杆菌的MIC为31.25μg/mL。抗菌肽TGH2对副溶血弧菌及溶藻弧菌的MIC为125μg/mL。同时由泥蚶血红蛋白合成的其他肽段B7(EMVSGKKKNGVVLMI)、B8(MVSGKKKNGVVLMIK)对金黄色葡萄球菌及大肠杆菌作用较弱,MIC为500μg/mL。
结合图1、图2及图3可知,低强度超声处理未能有效杀灭损伤大肠杆菌及金黄色葡萄球菌,经低强度处理后的大肠杆菌、金黄色葡萄球菌能够正常生长、繁殖。
实施例4:时间杀灭动力学(Time-kill kinetics)
将大肠杆菌在37℃培养12h至对数生长期,在0.01M pH 7.2磷酸盐缓冲液中稀释至106-7CFU/mL。取1×MIC浓度肽37℃等体积与菌混合后分别进行孵育、0.5W/cm2超声处理。每隔0.5h取样涂平板,37℃培养过夜后记录菌落总数。由结果可知,低强度超声辅助处理抗菌肽TGH2明显优于抗菌肽TGH2单独处理(图5)。
实施例5:透射电镜分析
以106-7CFU/mL的细菌在37℃下用2×MIC的抗菌肽TGH2处理2h,然后在2700g下离心10min,用磷酸盐缓冲液(pH 7.2)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。
用透射电镜观察了抗菌肽TGH2和超声波对大肠杆菌超微结构的影响。对照样品显示组织分布均匀,无渗漏,细胞膜和细胞壁光滑(图6A)。然而,用肽TGH2处理后,细胞膜和细胞壁出现一些膜模糊和不规则,虽然它在细胞质中呈现均匀的电子密度(图6B)。TGH2协同超声处理后,大肠杆菌细胞内溶质渗漏,空泡化完全(图6C)。有趣的是,细菌的细胞膜和细胞壁连续而光滑,这进一步证实了低强度超声辅助肽TGH2通过声穿孔的机制进行杀菌。
实施例6:TGH2对细菌细胞膜通透性的影响
为了研究肽TGH2对通透性影响,将大肠杆菌置于低浓度的TGH2下培养,观察其对细菌细胞膜通透性的影响。具体操作如下:通过离心收集大肠杆菌细胞,再重悬于以乳糖为唯一碳源的M9培养基中,37℃摇床培养至OD600<0.4,然后与不同浓度的等体积TGH2相应稀释液混合。将混合物加入96孔平底板中,在37℃下分别进行孵育、0.5W/cm2超声处理1h,然后加入0.5mg/mL的ONPG混匀后进行摇床培养观察并测定其在(0-8h)的OD420的变化。细胞通透性与TGH2浓度呈正相关。但当TGH2肽浓度低于1/2×MIC时,对细胞膜通透性影响不大,低强度辅助超声TGH2能够有效增加细胞膜通透性,如图7所示。
生物膜的形成代表了一种受保护的生长方式,为细菌细胞能够在恶劣的环境中生存提供保护,这被认为是食品加工行业的主要健康风险,而膜通透性的增加有助于抗菌物质更容易进入细菌内部,因此,低强度辅助超声TGH2能明显增加大肠杆菌膜通透性是该肽的一个很好的特征,表明它可以被用作防腐剂。
实施例7:电导率测定
将大肠杆菌在37℃培养12h至对数生长期,在0.01M pH 7.2磷酸盐缓冲液中稀释至106-7CFU/mL。取MIC浓度肽37℃等体积与菌混合后分别进行孵育、0.5W/cm2超声处理。每隔30分钟取样测定电导率。结果表明低强度超声辅助TGH2抗菌肽可以有效提高大肠杆菌的电导率,从而加速细菌的裂解死亡,提高抑菌效率。如图8所示。
实施例8:圆二色谱测定低强度超声处理对肽TGH2二级结构的影响
用Jasco810光谱偏振仪(Jasco,东京)CD在25℃下以100nm/min的扫描速度测定了肽的平均残基摩尔椭圆度。将肽TGH2溶于25mM十二烷基硫酸钠(SDS)中,最终浓度为0.20mg/mL,然后将不同超声条件下的肽TGH2溶液加到1mm石英比色皿中,用两次扫描从190-2nm扫描其光谱。
肽TGH2具有α螺旋结构,一般认为具有一定疏水性的α螺旋抗菌肽可能通过环孔形模型、地毯模型或桶壁模型破坏细菌的细胞膜。透射电镜结果显示,肽TGH2在细胞膜上形成小孔,导致内溶物流出,推断其抑菌机制可能是环孔形模型或桶壁模型机制,即抗菌肽先接触细胞膜,进而在膜上形成小孔,导致内溶物流出,细菌死亡,超声的辅助作用加速了这一机制的形成。
如图9所示,与对照相比,0.5w超声1小时不会对抗菌肽结构产生影响,而超声时间增长及强度增大都会使抗菌肽α-螺旋下降,影响抗菌效果,如0.5w超声2小时或1.0w超声1小时。
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> 一种泥蚶血红蛋白α螺旋抗菌肽及其应用
<130> P68277
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Ala Glu Phe Leu Arg Glu Lys Leu Gly Asp Lys Cys Thr Asp Arg
1 5 10 15
Claims (9)
1.一种泥蚶血红蛋白α螺旋抗菌肽,其特征在于:包括选自SEQ ID NO:1的氨基酸序列或由其组成。
2.一种如权利要求1所述泥蚶血红蛋白α螺旋抗菌肽的编码基因。
3.含有如权利要求2所述编码基因的表达盒。
4.含有如权利要求2所述编码基因的重组菌。
5.含有权利要求2所述编码基因的重组载体。
6.含有如权利要求2所述编码基因的转基因细胞系。
7.一种抑菌剂,其包括至少一种下列各项:根据权利要求1所述的泥蚶血红蛋白α螺旋抗菌肽;或根据权利要求2所述的编码基因。
8.一种食品防腐剂,其包括至少一种下列各项:根据权利要求1所述的泥蚶血红蛋白α螺旋抗菌肽;或根据权利要求2所述的编码基因。
9.如权利要求1所述泥蚶血红蛋白α螺旋抗菌肽与超声协同增效抑菌的方法,其特征在于:超声条件为:超声频率40MHz,功率为0.5W/cm2,超声时间1h。
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