CN112521443B - 一种三七花蛋白的制备方法及应用 - Google Patents
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Abstract
本发明公开了一种三七花蛋白的制备方法及应用,具体涉及一种采用缓冲盐提取、DEAE离子交换柱层析、凝胶柱层析及透析纯化制备三七花蛋白的方法;本发明方法制备的三七花蛋白PNP1的分子量为35~40kDa,该蛋白对尖孢镰刀菌、腐皮镰刀菌均具有较好的抑制作用;本发明的制备方法操作简单,以三七花为原料,得到的三七花蛋白具有显著的抗菌活性,拓展其在植物蛋白工程领域的应用,为制备新型抗菌食药提供新途径,具有广阔的应用前景。
Description
技术领域
本发明涉及一种具有抗菌活性的三七花蛋白的制备方法及应用,属于生物医药领域。
背景技术
三七花又名田七花,为五加科人参属植物生长2年以上的三七尚未开放花序的干燥品。三七花作为三七的副产物,未能得到有效的开发与利用。在我国民间三七花有作为茶饮料使用的习惯。三七花既是名贵中药药品,又是优良食品。三七花性甘凉,具有生津止渴、降压、平肝明目的功效。现代药理研究表明三七花具有镇静、抗炎、护肝、活血化瘀、降血脂、抗癌等作用,但其抗菌作用未见报道。关于三七花中活性成分的研究主要集中在皂苷和挥发油方面,对于三七中蛋白类成分的研究涉及较少。据报道,三七花中粗蛋白含量丰富,为20%以上。但目前三七花中蛋白的分离纯化和功能研究未见报道。
抗菌蛋白肽作为一类防御性多肽活性物质,可有效地抑制病原体生长,广泛分布于病毒、细菌以及各种动植物体内。抗菌蛋白具有抑菌、不易产生耐药性等作用,食用安全性非常高。
发明内容
针对现有技术的不足,本发明提供了一种抗菌三七花蛋白的制备方法及其在制备抗菌食品及药物中的应用;本发明从三七花中提取分离出抗菌蛋白,制备方法简便,适合于工业生产,并可将其应用于医药和健康食品的开发。
为实现以上目的,本发明采用的技术方案如下:
(1)将三七花鲜品用粉碎机磨碎,加入粉碎物质量5~10倍的pH=7~8、浓度0.01mol/L的 Tris-HCl缓冲液充分浸泡24h,使得三七花各部位细胞溶胀破裂,浸提液用绢布过滤,滤渣二次加入Tris-HCl缓冲液充分浸泡,过滤后合并滤液,将滤液离心取上清,即为三七花总蛋白提取液;
(2)将三七花总蛋白提取液装入截留分子量为10kDa的透析袋中,于4℃透析12~24h,透析除盐及小分子化合物,收集内滤液,冻干后即为三七花总蛋白粉;
(3)将三七花总蛋白粉用0.01mol/L、pH=7~8的Tris-HCl缓冲液溶解后,用DEAE离子交换柱层析对其进行分离纯化,采用NaCl溶液梯度洗脱的方法进行洗脱,收集0.1~0.2mol/L NaCl溶液的洗脱液,洗脱液采用截留分子量为10kDa透析袋于4℃透析12~24 h,收集内滤液,冻干后备用;
(4)将步骤(3)冻干品用0.01 mol/L、pH=7~8的Tris-HCl缓冲液溶解,采用Sephadex G-50 凝胶柱分离,采用NaCl溶液进行梯度洗脱,洗脱流速0.2mL/min,采用紫外分光光度仪在280nm下检测,收集第一个吸收峰的洗脱液,再采用截留分子量为10kDa透析袋于4℃透析12~24 h,收集内滤液,冻干后得到三七花蛋白PNP1,分子量范围为35~40 kDa。
对上述方法制得的三七花蛋白PNP1做抗菌活性检测,实验结果显示,本发明方法制得的三七花蛋白具有良好的抗真菌活性,可以将三七花蛋白PNP1应用在制备抗菌食品或抗菌药物中。
本发明的显著优点在于:
1、本发明制备获得的三七花蛋白溶于水,纯度高,且制备方法简单;
2、本发明制备的三七花蛋白对尖孢镰刀菌、腐皮镰刀菌具有较好的抑制作用;
3、本发明制备方法简单,适于工业化生产和市场推广应用。
附图说明
图1为实施例1中制备的三七花蛋白PNP1的SDS-PAGE电泳图。
具体实施方式
下面通过附图和实施例对本发明方法作进一步详细说明,但本发明保护范围不局限于所述内容。
实施例1:三七花蛋白的制备方法如下:
1、三七花总蛋白的提取:
取三七花鲜品50g,用粉碎机磨碎,加入500mL pH=7.4、0.01mol/L的 Tris-HCl缓冲液充分浸泡24h,浸提液用绢布过滤,滤渣二次加入500mL Tris-Hcl缓冲液充分浸泡,过滤后合并滤液,滤液于4000r/min离心20min,取上清,得到三七花总蛋白提取液1000mL;将三七花总蛋白提取液1000mL装入截留分子量为10kDa透析袋中,4℃透析12h,除去盐及小分子化合物,收集内滤液,冻干后得到三七花总蛋白10g;
2、三七花蛋白PNP1的分离纯化
取三七花总蛋白粉10g,用0.01mol/L、pH=7.4的Tris-HCl缓冲液溶解后,用DEAE离子交换层析对其进行分离纯化,采用0.05~0.5mol/L NaCl溶液进行梯度洗脱(按0.05mol/L、0.1 mol/L、0.2 mol/L、0.3 mol/L、0.4 mol/L、0.5 mol/L进行梯度洗脱),收集0.1~0.2mol/L NaCl溶液洗脱的洗脱液,洗脱液采用截留分子量为10kDa透析袋于4℃透析12h,收集内滤液,冻干后得到固体样品200mg;
取冻干品200mg用0.01mol/L、pH=7.4的Tris-HCl缓冲液溶解,采用Sephadex G-50凝胶柱分离,采用0.1~1mol/L NaCl溶液进行洗脱(0.1mol/L、0.3 mol/L、0.5 mol/L、0.7mol/L、0.9mol/L、1mol/L),洗脱流速0.2mL/min,采用紫外分光光度仪在280nm下检测,收集第一个吸收峰的洗脱液,再采用截留分子量为10kDa透析袋于4℃透析24h,收集内滤液,冻干后得到三七花蛋白PNP1 25mg,其分子量为37kDa,SDS-PAGE蛋白电泳图见图1。
实施例2:三七花蛋白的制备方法如下:
1、三七花总蛋白的提取:
取三七花鲜品50g,用粉碎机磨碎,加入400mL pH=7.4、0.01mol/L的 Tris-HCl缓冲液充分浸泡24h,浸提液用绢布过滤,滤渣二次加入400mL Tris-HCl缓冲液充分浸泡,过滤后合并滤液,滤液于4000r/min离心20min,取上清,得到三七花总蛋白提取液800mL;将三七花总蛋白提取液800mL装入截留分子量为10kDa透析袋中,4℃透析15h,除去盐及小分子化合物,收集内滤液,冻干后得到三七花总蛋白9g;
2、三七花蛋白PNP1的分离纯化
取三七花总蛋白粉9g,用0.01 mol/L、pH=7.4的Tris-HCl缓冲液溶解后,用DEAE离子交换层析对其进行分离纯化,采用0.05~0.5mol/L NaCl溶液进行梯度洗脱(按0.05mol/L、0.1 mol/L、0.2 mol/L、0.3 mol/L、0.4 mol/L、0.5 mol/L进行梯度洗脱),收集0.1~0.2mol/L NaCl溶液洗脱的洗脱液,洗脱液采用截留分子量为10kDa透析袋于4℃透析15h,收集内滤液,冻干后得到固体样品170mg;
取冻干品170mg用0.01mol/L、pH=7.4的Tris-HCl缓冲液溶解,采用Sephadex G-50凝胶柱分离,采用0.1~1mol/L NaCl溶液进行洗脱(0.1mol/L、0.3 mol/L、0.5 mol/L、0.7mol/L、0.9mol/L、1mol/L),洗脱流速0.2mL/min,采用紫外分光光度仪在280nm下检测,收集第一个吸收峰的洗脱液,再采用截留分子量为10kDa透析袋于4℃透析15h,收集内滤液,冻干后得到三七花蛋白PNP1 20mg,其分子量为37kDa。
实施例3:三七花蛋白PNP1的抗菌活性
本实施例研究了上述实施例制得三七花蛋白PNP1对尖孢镰刀菌(Fusarium oxysporum)、腐皮镰刀菌(Fusarium solani )的抑制作用,结果见表1;结果显示,三七花蛋白PNP1能够抑制尖孢镰刀菌、腐皮镰刀菌菌丝的生长,抑制效果明显,其半最大效应浓度EC50分为0.23mg/mL、0.36mg/mL,说明三七花蛋白具有良好的抗菌活性;
表1 三七花蛋白对尖孢镰刀菌、腐皮镰刀菌的抑制作用
。
Claims (1)
1.一种三七花蛋白PNP1在制备抗菌药物中的应用,抗菌菌种为尖孢镰刀菌、腐皮镰刀菌;
所述三七花蛋白PNP1的制备方法如下:
(1)在粉碎的三七花鲜品中加入其质量5~10倍的pH=7.4、浓度0.01mol/L的Tris-HCl缓冲液充分浸泡24h,使得三七花各部位细胞溶胀破裂,浸提液过滤后,滤渣二次加入Tris-HCl缓冲液充分浸泡过滤,合并滤液,滤液离心取上清,即得三七花总蛋白提取液;
(2)将三七花总蛋白提取液装入截留分子量为10 kDa的透析袋中,于4℃透析12~24 h,收集内滤液,冻干后即为三七花总蛋白粉;
(3)将三七花总蛋白粉用0.01 mol/L、pH=7.4的Tris-HCl缓冲液溶解后,用DEAE离子交换柱层析对其进行分离纯化,采用NaCl溶液梯度洗脱,收集0.1~0.2 mol/L NaCl溶液洗脱的洗脱液,洗脱液采用截留分子量为10kDa透析袋于4℃透析12~24h,收集内滤液,冻干后备用;
所述NaCl溶液梯度浓度为0.05mol/L、0.1 mol/L、0.2 mol/L、0.3 mol/L、0.4 mol/L、0.5 mol/L;
(4)将步骤(3)冻干品用0.01mol/L、pH=7.4的Tris-HCl缓冲液溶解,采用Sephadex G-50 凝胶柱分离,采用NaCl溶液进行梯度洗脱,洗脱流速0.2mL/min,采用紫外分光光度仪在280nm下检测,收集第一个吸收峰的洗脱液,再采用截留分子量为10kDa透析袋于4℃透析12~24 h,收集内滤液,冻干后得到三七花蛋白PNP1;
所述NaCl溶液梯度浓度为0.1mol/L、0.3 mol/L、0.5 mol/L、0.7 mol/L、0.9mol/L、1mol/L。
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