CN117264018B - 一种鸽蛋清中功能肽的制备及其应用 - Google Patents
一种鸽蛋清中功能肽的制备及其应用 Download PDFInfo
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- CN117264018B CN117264018B CN202311253068.6A CN202311253068A CN117264018B CN 117264018 B CN117264018 B CN 117264018B CN 202311253068 A CN202311253068 A CN 202311253068A CN 117264018 B CN117264018 B CN 117264018B
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Classifications
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Abstract
本发明公开了一种鸽蛋清中功能肽的制备及其应用,属于生物技术领域。本发明从鸽蛋清中制备得到了氨基酸序列如KEWTSPNVM的肽,该肽具有优异的抗氧化活性,同时具有抑制结肠癌细胞扩增、促进上皮细胞跨膜转运、抑制ACE的能力,可以用在化妆品、保健品、医药制品领域。同时,本发明利用鸽蛋清制备肽的方法,工艺简单、温和、成本低廉、环境友好且对人体无害。
Description
技术领域
本发明涉及一种鸽蛋清中功能肽的制备及其应用,属于生物技术领域。
背景技术
鸽蛋(Pigeon egg),据《本草纲目》中记载,具有调精益气、生津止渴、滋阴补阳的功效。鸽蛋清是鸽蛋的蛋白成分,富含多种矿物质,还包含高生物价的蛋白质,属于理想蛋白。前期研究发现,鸽蛋清中蛋白质氨基酸组成和糖基化位点与鸡蛋、鹅蛋、鹌鹑蛋等来源蛋白均有较大区别,结构差别与其生理功能密切相关。
目前对于鸽蛋清中提取蛋白的现有技术主要局限于对鸽蛋清中卵白蛋白。例如,一种鸽蛋清卵白蛋白的提取方法(CN112028990B),优化从鸽蛋清中提取卵白蛋白的工艺,对鸽蛋清卵白蛋白的提取率达到107.44mg/g;鸽蛋清中卵白蛋白的提取及其在伤口修复、抗衰老方面的应用(CN116421708A)公开了从鸽蛋清中提取的卵白蛋白的相关应用。
上述现有技术对于鸽蛋清中的开发均只停留于大分子的卵白蛋白,忽视了鸽蛋清中有效的活性小分子肽。现有技术对鸽蛋清功能肽的挖掘较少,小分子肽的制备技术更复杂,其分离难度更高,难以定位和纯化;而现有研究对小分子肽的研究也停留于混合肽,难以定位具体发挥活性作用的小分子肽。因此,挖掘鸽蛋清中功能肽的制备及其应用对于鸽蛋产品开发具有重要意义,充分挖掘鸽蛋清中这些有潜力的活性小分子是当前亟需解决的问题。
发明内容
功能肽作为一种具有特定生理活性的肽类化合物,具有抗氧化、抗肿瘤、调节免疫功能以及降血压等多种功能活性,鸽蛋清经酶水解可获得具有多种生理功能的低分子活性肽,是制备功能肽的良好原料。
本发明提供了一种肽(KEWTSPNVM)的制备方法及肽的应用。本发明提供的肽具有较强的抗氧化性,具有能够抑制结肠癌细胞扩增、促进上皮细胞跨膜转运、降低血压等多种功能。此外,本发明的肽可以从鸽蛋清中分离得到,制备方法简单、成本低廉。
本发明的第一个目的是提供一种肽,所述肽的氨基酸序列为KEWTSPNVM(Lys-Glu-Trp-Thr-Ser-Pro-Asn-Val-Met,如SEQ ID NO:1所示)。
本发明的第二个目的是提供一种核酸分子,所述核酸分子可以编码本发明所述的肽。
本发明的第三个目的是提供一种肽的衍生物,所述肽衍生物含有本发明所述肽的氨基酸序列。
本发明的第四个目的是提供一种生物材料,所述生物材料含有编码本发明所述肽的氨基酸序列或者本发明所述核酸分子的核苷酸序列,包括重组DNA、表达盒、转座子、质粒载体,病毒载体,工程菌或转基因细胞系。
本发明的第五个目的是提供一种本发明所述肽的制备方法,所述方法可以是通过鸽蛋清制备、通过基因工程菌合成,或通过人工合成。
在一种实施方式,所述肽的制备方法从鸽蛋清中制备,包括如下步骤:
(1)从新鲜鸽蛋中分离的鸽蛋清,采用滤网过滤以去除鸽蛋清中悬浮物和杂质,加入纯净水稀释至一定浓度;
(2)对步骤(1)稀释后的蛋清液进行热处理,经热变性后冷却至室温;
(3)调节步骤(2)溶液的pH至5.0-7.0,在水浴摇床中预热,加入蛋白酶水浴振荡数小时,加热灭酶后冷却至室温;
(4)再次步骤(3)中溶液的pH至2.0-4.0,加入蛋白酶进行二次酶解水浴振荡数小时后加热灭酶,冷却后调节pH至6.0-8.0后离心;
(5)将步骤(4)中所得上清液进行超滤分离,留存滤过液;
(6)将(5)中所得滤过液经离子交换柱分离,干燥,得到鸽蛋清功能肽粉末。
其中,在上述鸽蛋清肽的制备方法中,步骤(2)所述的热变性条件为,75-90℃变性15-25min。
其中,在上述鸽蛋清肽的制备方法中,步骤(3)所述的蛋白酶浓度为底物质量分数的3%-7%,其水浴振荡温度为40-60℃,加热灭酶温度为85-100℃,时间为5-15min。
其中,在上述鸽蛋清肽的制备方法中,步骤(4)所述的蛋白酶浓度为底物质量分数的1%-3%,其水浴振荡温度为40-60℃,加热灭酶温度为85-100℃,时间为10-20min。
其中,在上述鸽蛋清肽的制备方法中,步骤(5)所述的超滤分离涉及的膜,分子量为3000-10000Da。
其中,在上述鸽蛋清肽的制备方法中,步骤(6)中所述的干燥方式为喷雾干燥,使用条件为80℃-120℃喷雾干燥,进料流量为8-15mL/min,进料温度20-40℃,出口温度60-80℃。
本发明的第六个目的是提供一种制备具有抗氧化功能的组合物、或抑制结肠癌细胞扩增的药物、或促进上皮细胞的跨膜转运的药物、或抑制ACE的药物中的方法,所述方法是利用本发明所述肽或者核酸分子或者肽的衍生物或者生物材料。
本发明的第七个目的是提供一种制品,所述制品含有本发明所述的肽或者所述的肽的衍生物。
在一种实施方式中,所述制品为具有抗氧化功能的化妆品或保健品。
在一种实施方式中,所述化妆品为霜剂、乳剂、喷雾剂、洗剂、粉剂、膏剂或面膜。
在一种实施方式中,保健品为粉剂、口服液、丸剂等形式。
在一种实施方式中,所述制品为具有抑制结肠癌细胞扩增功能的药物。
在一种实施方式中,所述制品为促进上皮细胞的跨膜转运的药物。
在一种实施方式中,所述制品为抑制ACE的药物。
在一种实施方式中,所述制品中含有药物辅料。
在一种实施方式中,所述药用辅料包含赋形剂以及附加剂。
在一种实施方式中,所述药用辅料包含抗黏合剂、渗透促进剂、缓冲剂、增塑剂、表面活性剂、消泡剂、增稠剂、包合剂、吸收剂、保湿剂、溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、pH值调节剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、整合剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、发泡剂、助悬剂、包衣材料、芳香剂、稀释剂、絮凝剂与反絮凝剂、助滤剂以及释放阻滞剂。
在一种实施方式中,所述附加剂包含微晶纤维素、羟丙基甲基纤维素以及精制卵磷脂。
在一种实施方式中,所述药品的剂型包含颗粒剂、胶囊剂、片剂、丸剂或口服液。
在一种实施方式中,所述制品以本发明的肽或者肽的衍生物为主要活性成分。
有益效果:
本发明的纯九肽KEWTSPNVM(以下简称为肽)同时具备多项功能,优秀的抗氧化活性、抑制结肠癌细胞增殖、促进上皮细胞跨膜转运、抑制血管紧张素转化酶活性,具体如下:
(1)本发明的肽,氨基酸序列为KEWTSPNVM,具有优秀的抗氧化活性,对DPPH和ABTS显示出良好的抗氧化能力,在1mg/mL时对DPPH自由基清除率达到45.89±1.89%;对ABTS的清除率达到36.76±1.83%;本发明的九肽能够有效减轻氧化应激、清除自由基,预防细胞损伤和老化,可应用于化妆品及保健品行业,作为天然抗氧化剂使用;
(2)本发明的肽具有抑制结肠癌细胞扩增的能力,在肽的添加量为20μg/L的情况下,与不添加相比,Caco-2细胞的平均迁移率降低23.9%,这显示出本发明的肽潜在的抗肿瘤活性,是开发抗肿瘤药物医药配方中潜在的肿瘤抑制剂;
(3)本发明的肽具有促进上皮细胞跨膜转运的特性,可改善细胞膜通透性和物质运输过程,增强上皮细胞的功能,250μg/mL的九肽在60分钟内跨膜运输量达到46.9%,表明本发明的肽可应用于药物递送系统的开发,作为跨膜转运增强剂,以提高药物在细胞内的吸收和传递效率;
(4)本发明的肽抑制血管紧张素转化酶(Angiotensin-Converting Enzyme,ACE)抑制作用,在1mg/mL时对ACE抑制率到达51.32%;本发明得肽能够调节血管紧张素转换酶的活性,有助于降低血压水平,可作为作为天然的、无副作用的高血压治疗方法,血压调节剂或辅助治疗药物使用;
本发明还提供了一种简便可行的利用鸽蛋清制备肽的方法,工艺简单、温和,成本低廉,扩大了鸽蛋的应用范围,并提升了其商业价值;同时,该制备方法未添加任何有机溶剂,对环境友好且对人体无害。
附图说明
图1为实施例1中鸽蛋清一次水解及二次水解产物组成的液相图;
图2为实施例1中经3000Da超滤后组分的液相图;
图3为实施例1纯九肽的液相图;
图4为鸽蛋清功能肽的分子模型;
图5为实施例2中各组分对DPPH自由基清除率的影响;
图6为实施例2中各组分对ABTS总抗氧化性的影响;
图7为实施例3肽对结肠癌细胞扩增的抑制作用;
图8为实施例5肽的ACE抑制效果。
具体实施方式
以下实施例中,若无特殊说明,所用的方法均为常规方法,所用试剂均可通过商业途径购买。
实施例1:纯九肽KEWTSPNVM的制备
1、从鸽蛋清中分离制备纯九肽KEWTSPNVM
(1)一次酶解鸽蛋清
从新鲜鸽蛋中分离的鸽蛋清,采用滤网过滤以去除鸽蛋清中悬浮物和杂质,加入纯净水与鸽蛋清1:1比例混合,搅匀后放置在85℃中热变性20min,待冷却至室温,调节溶液pH至6.5,放置在水浴摇床中预热5min,加入鸽蛋清质量分数6%的风味蛋白酶,在120r/min、55℃的水浴摇床中反应4h,95℃灭酶10min在6000r/min下离心5min,得到一次酶解液,将一次酶解液喷雾干燥最终获得鸽蛋清混合肽粉末,利用高效液相色谱和离子交换柱分析产物组成,结果如图1所示。
(2)二次酶解鸽蛋清
取步骤(1)制备得到的一次酶解液,调节一次酶解液pH至2.0,加入鸽蛋清质量分数2%的胃蛋白酶,在120r/min、40℃的水浴摇床中反应2h,100℃灭酶15min,待冷却后调节溶液pH至7.0,在6000r/min下离心5min,得到二次酶解液,将二次酶解液喷雾干燥最终获得鸽蛋清混合肽粉末,利用高效液相色谱和离子交换柱分析产物组成,结果如图1所示,可以发现经过二次酶解的鸽蛋清其分子量较一次酶解的更小。
(3)超滤和离子交换柱分离,喷雾干燥得到鸽蛋清肽粉末
取步骤(2)制备得到的二次酶解液,采用截留分子量为3000Da超滤管将上清液超滤处理,使用液相色谱检测,结果如图2所示,滤过液采用C18离子交换柱进行分离,将上述液体分别喷雾干燥,最终获得鸽蛋清肽粉末。
(4)液相色谱-质谱联用纯化并鉴定
采用液相色谱-质谱联用仪纯化并鉴定步骤(3)制备得到鸽蛋清肽粉末,所得肽的氨基酸序列通过MaxQuant进行数据搜索对比,鉴定出该肽为九肽,氨基酸序列为KEWTSPNVM。
因此,采用如步骤(1)~(4)所示的方法,可以从鸽蛋清中分离制备纯九肽KEWTSPNVM。
2、采用化学合成法制备纯九肽KEWTSPNVM
采用化学合成法纯九肽KEWTSPNVM,合成的肽段经液相检测为单一峰,结果如图3所示,且出峰时间与步骤(3)中分离的肽段一致,该九肽的分子模型如图4所示。
实施例2:纯九肽KEWTSPNVM在制备抗氧化产品中的应用
(1)纯九肽KEWTSPNVM对DPPH自由基清除率
采用购自南京建成生物工程研究所的DPPH试剂盒,将二次酶解未经纯化的混合肽、纯九肽配置成不同浓度(1mg/mL、2mg/mL、5mg/mL),Vc作为阳性对照,按照试剂盒说明书,在517nm处测定各样品的吸光度,利用公式DPPH清除率%=[1-(A样品-A对照)/A空白]×100%,计算鸽蛋清肽的DPPH清除率。
结果如图5所示,混合肽(1mg/mL)的DPPH清除率为28.55±1.13%;纯九肽(1mg/mL)对DPPH自由基清除率达到45.89±1.89%,纯九肽较混合肽提高了71.24%。
(2)纯九肽KEWTSPNVM对ABTS清除率
采用购自南京建成生物工程研究所的ABTS试剂盒,将混合肽、纯九肽配置成不同浓度(1mg/mL、2mg/mL、5mg/mL),Vc作为阳性对照,按照试剂盒说明书,在734nm处测定各样品的吸光度。利用公式ABTS清除率%=[1-(A测定-A对照)/A空白)]×100%,计算鸽蛋清肽的ABTS清除率。
结果如图6所示,混合肽(1mg/mL)的ABTS清除率为18.79±0.94%;纯九肽(1mg/mL)对ABTS的清除率达到36.76±1.83%,纯九肽较混合肽提高了98.6%。
结合结果可以发现纯九肽KEWTSPNVM显示出较好的抗氧化能力。因此,本发明的纯九肽KEWTSPNVM可以用于制备抗氧化的化妆品或保健品。具体的,化妆品可以霜剂、乳剂、喷雾剂、洗剂、粉剂、膏剂、面膜等形式;保健品可以配制为粉剂、口服液、丸剂等形式。
实施例3:纯九肽KEWTSPNVM应用于抑制结肠癌细胞扩增
取处于对数生长期的Caco-2细胞,经处理调整其浓度为1×105cell/mL,每孔加入2mL细胞悬液,带细胞铺满单层约80%,设置空白组、低剂量组和高剂量组进行划痕,蛋白多肽剂量分别为0μg/mL、20μg/mL、100μg/mL,时间点为0h、24h、48h,通过痕距计算平均迁移率,在添加20μg/L纯九肽培养48h时,较不添加纯九肽,Caco-2细胞的平均迁移率降低23.9%,结果如图7和表1所示,九肽的添加会明显抑制Caco-2细胞的扩增和迁徙。
表1肽对结肠癌细胞扩增的抑制作用
基于本实施例的效果,本发明的纯九肽KEWTSPNVM可以用于制备抑制结肠癌细胞增殖的药物。
实施例4:纯九肽KEWTSPNVM应用于促进上皮细胞跨膜转运
将Caco-2细胞(浓度为1×105cell/mL)接种至24孔板,待其贴壁之后,基底侧(BL)加入600μL的完全培养基,刷状缘侧(AP)加入100μL细胞悬液,置于5% CO2,95% O2,37℃,相对湿度95%的CO2培养箱中培养。
接种后隔天更换一次AP侧和BP侧完全培养基,一周后每天更换AP侧和BL侧的完全培养基直到21天。取培养至21天的细胞,弃去培养基,用PBS清洗3次,最后一次将PBS加入小室,孵育30min后弃去。AP侧向BL侧转运,在AP侧加入100μL样品作为供给池,BL侧加入600μLPBS作为接收池;BL侧向AP侧转运,在BL侧加入600μL样品作为供给池,AP侧加入600μL PBS作为接收池。设置样品浓度为250μg/mL,时间点为0.5h、1h、2h,吸取跨膜转运液,离心过膜后进行液相结合Inertsil WP300 C18色谱柱检测,结果如表2所示,在60分钟内,九肽的跨膜运输量达到46.9%。
表2跨膜转运物质浓度
基于本实施例的效果,本发明的纯九肽KEWTSPNVM可以用于制备促进上皮细胞的跨膜转运的药物。
实施例5:纯九肽KEWTSPNVM应用于抑制血管紧张素转化酶(ACE)
设置空白组、对照组以及样品组(浓度设置为1mg/mL、2mg/mL、4mg/mL),取5μL鸽蛋清肽溶液,加入15μL 1mg/mL HHL溶液,混匀后在37℃预热5min,再加入5μL ACE溶液,混匀后置于37℃水浴30min,随后加入50μL 1mol/L HCI溶液停止反应,加入0.5mL乙酸乙酯漩涡萃取1min,离心后取上清液烘干,加入1mL RO水,在228nm处测定吸光度,利用公式ACE抑制率=[(A对照-A样品)/(A对照-A空白)]×100%,计算鸽蛋清肽的ACE抑制率。
结果如图8所示,纯九肽KEWTSPNVM(1mg/mL)对ACE抑制率到达51.32%,且随着浓度的增加,抑制率增加。基于本实施例的效果,本发明的纯九肽KEWTSPNVM可以用于制备ACE抑制剂,用于治疗治疗高血压、心力衰竭和肾脏疾病。
实施例6:纯九肽KEWTSPNVM药物的制备
基于纯九肽KEWTSPNVM得较好抗氧化作用、抑制结肠癌细胞增殖,促进上皮细胞跨膜转运以及抑制血管紧张素转化酶得作用,本发明的纯九肽KEWTSPNVM可以用于制备预防或者辅助治疗或者治疗相关疾病的药物,药物可以为片剂、颗粒剂、糖制剂、液体剂。
药用辅料包含抗黏合剂、渗透促进剂、缓冲剂、增塑剂、表面活性剂、消泡剂、增稠剂、包合剂、吸收剂、保湿剂、溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、pH值调节剂、黏合剂、崩解剂、填充剂、润滑剂、润湿剂、整合剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、发泡剂、助悬剂、包衣材料、芳香剂、稀释剂、絮凝剂与反絮凝剂、助滤剂以及释放阻滞剂。
可选地,药物具体剂型制备方法如下:
片剂的制备:100mg纯九肽KEWTSPNVM与100mg玉米淀粉、100mg乳糖以及2mg硬脂酸镁混合,其混合物根据传统方法压制成片剂。
胶囊剂的制备:将500mg纯九肽KEWTSPNVM装入软明胶胶囊中,即制得胶囊产品。
糖制剂的制备:将1g纯九肽KEWTSPNVM与10g异构糖、5g甘露醇和适量的纯化水混合,根据传统方法将该混合物制备成100ml糖制剂。
液体剂的制备:将200mg纯九肽KEWTSPNVM加热并分散在200mg的含聚氧乙烯氢化蓖麻油的生理盐水中,即制成含提取物浓度0.1%的注射液。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (11)
1.一种功能肽,其特征在于,所述肽的氨基酸序列为KEWTSPNVM(Lys-Glu-Trp-Thr-Ser-Pro-Asn-Val-Met)。
2.核酸分子,其特征在于,所述核酸分子用于编码权利要求1所述的肽。
3.生物材料,其特征在于,所述生物材料含有权利要求2所述的核酸分子,所述生物材料为表达盒、转座子、质粒载体、病毒载体、工程菌或转基因细胞系。
4.一种权利要求1所述的肽的制备方法,其特征在于,所述方法包括通过鸽蛋清制备或通过基因工程菌合成或通过人工合成。
5.根据权利要求4所述的制备方法,其特征在于,功能肽从鸽蛋清中制备,制备方法包括如下步骤:
(1)从鸽蛋中分离的鸽蛋清,采用滤网过滤以去除鸽蛋清中悬浮物和杂质,加入水稀释至一定浓度;
(2)对步骤(1)中稀释后的蛋清液进行热处理,经热变性后冷却;
(3)调节步骤(2)中溶液的pH至5.0-7.0,在水浴摇床中预热,加入风味蛋白酶水浴振荡数小时,加热灭酶后冷却;
(4)再次调节步骤(3)中溶液的pH至2.0-4.0,加入胃蛋白酶进行二次酶解水浴振荡数小时后加热灭酶,冷却后调节pH至6.0-8.0后离心;
(5)将步骤(4)中所得上清液进行超滤分离,留存滤过液;
(6)将(5)中所得滤过液经离子交换柱分离,干燥,得到鸽蛋清功能肽粉末。
6.权利要求1所述的肽或者权利要求2所述的核酸分子或者权利要求3所述的生物材料在制备具有抗氧化功能的化妆品或保健品中的应用。
7.权利要求1所述的肽或者权利要求2所述的核酸分子或者权利要求3所述的生物材料在制备治疗高血压或心力衰竭的药物中的应用。
8.一种制品,其特征在于,所述制品含有权利要求1所述的肽;所述制品为化妆品或保健品。
9.根据权利要求8所述的制品,所述制品以权利要求1所述的肽为主要活性成分。
10.一种制品,其特征在于,所述制品含有权利要求1所述的肽;所述制品为治疗高血压或心力衰竭的药物。
11.根据权利要求10所述的制品,所述制品以权利要求1所述的肽为主要活性成分。
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