单增李斯特菌鸡卵黄抗体的抑菌性能及其机理研究
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摘要
单增李斯特菌是水产食品中最为常见的致病菌之一,其生长的温度范围广,在4℃的普通冷藏条件下能够缓慢生长繁殖,在动物养殖及鲜活产品储运过程中往往难以有效控制其污染和安全危害。卵黄抗体(IgY)作为禽类蛋黄中存在的唯一一种免疫球蛋白,不仅能与其抗原菌特异性的结合,而且结合后还能够抑制抗原菌的生长,是一种潜在的食品微生物抑菌剂。
本研究以单增李斯特菌作为菌体抗原免疫蛋鸡制备IgY,并针对高纯度特异性IgY的制备纯化及其生物活性展开研究探讨,研究其对于水产品样本中单增李斯特菌的免疫亲和能力以及生长抑制效果,并对IgY的抑菌机理进行探讨,初步验证其应用于水产品加工储藏和分析检测的可行性,同时为水产品中其他致病菌和腐败菌的有效控制提供技术参考,论文的主要研究成果如下：
以单增李斯特菌为抗原免疫产蛋母鸡,比较不同方法对于鸡卵黄中单增李斯特菌IgY的分离效果,研究IgY在鸡蛋中产生的免疫应答过程,分析确定所得IgY的主要理化性质。结果表明,采用硫酸铵盐析法可以取得较好的提取纯化效果,提取液中的IgY效价高且蛋白组分较为单一,IgY具有较好的理化稳定性,在pH 3～9范围内的抗体活性能够保持在70%以上,经60℃、4 h处理后免疫活性仍然良好。
IgY对单增李斯特菌的生长有显著的抑制作用,在液体培养基中添加IgY,可有效抑制单增李斯特菌的生长,而且抗体浓度越大,抑菌效果越明显,培养基中添加200 mg/mL的IgY,37℃下孵育8小时,单增李斯特菌细胞数量减少90%,4℃下孵育120小时,单增李斯特菌细胞数量也可减少50%；固体培养基经200 mg/mL的1gY浸泡30分钟后,可使单增李斯特菌细胞数量减少80%以上；采用200 mg/mL的1gY处理过的新鲜三文鱼和烟熏三文鱼,真空包装后在6±1℃条件下储存15天,单增李斯特菌的细胞数量较空白对照可以降低两个对数单位,证实了1gY作为食品中致病菌抑制剂的可行性。
通过对单增李斯特菌菌体细胞的预处理,建立了两种免疫亲和纯化方法对特异性IgY进行分离纯化,并对纯化后抗体的性能进行了研究。结果表明,两种方法均成功对特异性IgY和非特异性IgY进行了有效分离；相同浓度的IgY,纯化后抗体效价较纯化前提高了大约4,倍；在以新鲜牛奶为样本的抑菌实验中,50mg/mL的纯化后IgY的抑菌性能要比纯化前200 mg/mL IgY的抑菌性能高,纯化后的抑菌效率较纯化前提高4倍以上。
对IgY的抑菌机理进行研究探讨,透射电镜观察发现,单增李斯特菌经200mg/mL IgY处理6小时后,菌体的细胞壁变薄,细胞的完整性被损坏,说明IgY通过对菌体细胞壁的破坏发挥抑菌性能；对IgY与单增李斯特菌相互作用后的反应液组分进行高效液相分离收集和核磁质谱结构分析,结果表明反应液中含有磷壁酸的结构组成单体,从分子水平上初步明确了IgY与菌体细胞的作用位点是细胞壁中的磷壁酸,为今后IgY抑菌效率的提高、IgY增效因子的研究以及各种增效因子的协同作用提供了理论支持。
Listeria monocytogenes is a common pathogen in seafood. Because it can grow at a wide range of temperature and survive even at 4℃,it is difficult to effectively control the pollution and resulted hazards of Listeria monocytogenes in fresh products during their storage and transportation. Egg yolk antibody (IgY), a unique immunoglobulin in egg yolk, has a potent inhibitory activity to its antigen bacteria, and could be used as a potential antimicrobial agent to food micro-organisms. In this study, IgY antibodies against Listeria monocytogens were isolated, purified, characterized and validated to be used as an antimicrobial agent in sea food samples. The main results were summarized as follows:
Four different methods were evaluated for extraction and purification of IgY, and the best outcoming was observed with ammonium sulfate precipitation, where the titer of anti-Listeria monocytogens antibodies was the highest and the SDS-PAGE analysis showed comparatively high purity of protein contents. The content of IgY in egg yolk reach to a high level after three times of booster immunization,-and-maintained stable for six weeks. The prepared IgY demonstrated excellent pH and thermal stabilities: more than 70% bioactivity could be retained in the range of pH 3-9, and only 33% decrease in the titer after 4-h incubation at 60℃.
The inhibitory effect of the specific IgY on the growth of Listeria monocytogenes was tested in liquid medium, solid medium and food samples. After 8-h incubation with the specific IgY at 37℃,the growth of Listeria monocytogenes significantly decreased compared with the control ones, and positive correlation between inhibitory efficiency and IgY concentration is observed.Similar growth inhibitory effects were observed with incubation at 4℃for 120 h.The IgY also inhibited the growth of Listeria monocytogenes when coated onto solid medium, fresh or smoked salmon samples. More than 80% of Listeria monocytogenes were inhibited for growth when 200 mg/mL of the IgY was incubated with the solid medium for 30 min at room temperature. Compared with those of blanks, the amounts of Listeria monocytogenes in fresh or smoked salmon samples were reduced more than 2 log cycles after 15 days of storage at 6±1℃in the presence of 200 mg/mL of the specific IgY.
Two different affinity chromatography methods were developed for the separation of the specific IgY from non-specific ones. After purification by either of the two methods, the titer of the IgY could increase more than 4 times, and the antibacterial efficiency could also improve more than 4 times when tested with milk stored at 6±1℃for 10 days as sample.
The results of transmission electron microscopy showed that the binding of the specific IgY to Listeria Monocytogens resulted in significant destruction of cell wall and structural alterations on the bacterial surface. The products of the IgY and Listeria monocytogenes were collected and analysed by NMR and GC-MS.The result showed that the structure of those products was similar with that of teichoic acid, which is an important component of cell wall.The above data illustrated that the antibacterial mechanism of the IgY might be the destruction of teichoic acid.
本研究以单增李斯特菌作为菌体抗原免疫蛋鸡制备IgY,并针对高纯度特异性IgY的制备纯化及其生物活性展开研究探讨,研究其对于水产品样本中单增李斯特菌的免疫亲和能力以及生长抑制效果,并对IgY的抑菌机理进行探讨,初步验证其应用于水产品加工储藏和分析检测的可行性,同时为水产品中其他致病菌和腐败菌的有效控制提供技术参考,论文的主要研究成果如下：
以单增李斯特菌为抗原免疫产蛋母鸡,比较不同方法对于鸡卵黄中单增李斯特菌IgY的分离效果,研究IgY在鸡蛋中产生的免疫应答过程,分析确定所得IgY的主要理化性质。结果表明,采用硫酸铵盐析法可以取得较好的提取纯化效果,提取液中的IgY效价高且蛋白组分较为单一,IgY具有较好的理化稳定性,在pH 3～9范围内的抗体活性能够保持在70%以上,经60℃、4 h处理后免疫活性仍然良好。
IgY对单增李斯特菌的生长有显著的抑制作用,在液体培养基中添加IgY,可有效抑制单增李斯特菌的生长,而且抗体浓度越大,抑菌效果越明显,培养基中添加200 mg/mL的IgY,37℃下孵育8小时,单增李斯特菌细胞数量减少90%,4℃下孵育120小时,单增李斯特菌细胞数量也可减少50%；固体培养基经200 mg/mL的1gY浸泡30分钟后,可使单增李斯特菌细胞数量减少80%以上；采用200 mg/mL的1gY处理过的新鲜三文鱼和烟熏三文鱼,真空包装后在6±1℃条件下储存15天,单增李斯特菌的细胞数量较空白对照可以降低两个对数单位,证实了1gY作为食品中致病菌抑制剂的可行性。
通过对单增李斯特菌菌体细胞的预处理,建立了两种免疫亲和纯化方法对特异性IgY进行分离纯化,并对纯化后抗体的性能进行了研究。结果表明,两种方法均成功对特异性IgY和非特异性IgY进行了有效分离；相同浓度的IgY,纯化后抗体效价较纯化前提高了大约4,倍；在以新鲜牛奶为样本的抑菌实验中,50mg/mL的纯化后IgY的抑菌性能要比纯化前200 mg/mL IgY的抑菌性能高,纯化后的抑菌效率较纯化前提高4倍以上。
对IgY的抑菌机理进行研究探讨,透射电镜观察发现,单增李斯特菌经200mg/mL IgY处理6小时后,菌体的细胞壁变薄,细胞的完整性被损坏,说明IgY通过对菌体细胞壁的破坏发挥抑菌性能；对IgY与单增李斯特菌相互作用后的反应液组分进行高效液相分离收集和核磁质谱结构分析,结果表明反应液中含有磷壁酸的结构组成单体,从分子水平上初步明确了IgY与菌体细胞的作用位点是细胞壁中的磷壁酸,为今后IgY抑菌效率的提高、IgY增效因子的研究以及各种增效因子的协同作用提供了理论支持。
Listeria monocytogenes is a common pathogen in seafood. Because it can grow at a wide range of temperature and survive even at 4℃,it is difficult to effectively control the pollution and resulted hazards of Listeria monocytogenes in fresh products during their storage and transportation. Egg yolk antibody (IgY), a unique immunoglobulin in egg yolk, has a potent inhibitory activity to its antigen bacteria, and could be used as a potential antimicrobial agent to food micro-organisms. In this study, IgY antibodies against Listeria monocytogens were isolated, purified, characterized and validated to be used as an antimicrobial agent in sea food samples. The main results were summarized as follows:
Four different methods were evaluated for extraction and purification of IgY, and the best outcoming was observed with ammonium sulfate precipitation, where the titer of anti-Listeria monocytogens antibodies was the highest and the SDS-PAGE analysis showed comparatively high purity of protein contents. The content of IgY in egg yolk reach to a high level after three times of booster immunization,-and-maintained stable for six weeks. The prepared IgY demonstrated excellent pH and thermal stabilities: more than 70% bioactivity could be retained in the range of pH 3-9, and only 33% decrease in the titer after 4-h incubation at 60℃.
The inhibitory effect of the specific IgY on the growth of Listeria monocytogenes was tested in liquid medium, solid medium and food samples. After 8-h incubation with the specific IgY at 37℃,the growth of Listeria monocytogenes significantly decreased compared with the control ones, and positive correlation between inhibitory efficiency and IgY concentration is observed.Similar growth inhibitory effects were observed with incubation at 4℃for 120 h.The IgY also inhibited the growth of Listeria monocytogenes when coated onto solid medium, fresh or smoked salmon samples. More than 80% of Listeria monocytogenes were inhibited for growth when 200 mg/mL of the IgY was incubated with the solid medium for 30 min at room temperature. Compared with those of blanks, the amounts of Listeria monocytogenes in fresh or smoked salmon samples were reduced more than 2 log cycles after 15 days of storage at 6±1℃in the presence of 200 mg/mL of the specific IgY.
Two different affinity chromatography methods were developed for the separation of the specific IgY from non-specific ones. After purification by either of the two methods, the titer of the IgY could increase more than 4 times, and the antibacterial efficiency could also improve more than 4 times when tested with milk stored at 6±1℃for 10 days as sample.
The results of transmission electron microscopy showed that the binding of the specific IgY to Listeria Monocytogens resulted in significant destruction of cell wall and structural alterations on the bacterial surface. The products of the IgY and Listeria monocytogenes were collected and analysed by NMR and GC-MS.The result showed that the structure of those products was similar with that of teichoic acid, which is an important component of cell wall.The above data illustrated that the antibacterial mechanism of the IgY might be the destruction of teichoic acid.
引文
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