CN115505025A - 一种基于老年人静态消化模型的乳清蛋白源抗氧化肽的筛选 - Google Patents
一种基于老年人静态消化模型的乳清蛋白源抗氧化肽的筛选 Download PDFInfo
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Abstract
本发明公开了一种基于老年人静态消化模型的乳清蛋白源抗氧化肽的筛选,通过体外模拟老年人消化、LC‑MS/MS肽谱鉴定及分子对接技术,筛选出最具抗氧化潜力的乳清蛋白源肽段DDQNPHSSNICNISCDK。该多肽的获得来源于老年人静态消化模型下的乳清蛋白的消化、消化前后抗氧化活性的测定、多肽序列鉴定及分子对接筛选。乳清蛋白经过老年人肠消化后,可以达到较高的抗氧化活性,因此对老年人肠消化进行后续的肽段筛选。通过本发明的筛选方法,可以方便快速的得到适合老年人胃肠道的抗氧化肽,并且为乳制品行业针对老年人乳品研发提供了新思路。
Description
技术领域
本发明属于乳产品功能应用技术领域,具体涉及一种基于老年人静态消化模型的乳清蛋白源抗氧化肽的筛选。
背景技术
近年来,越来越多的研究不仅关注食物本身的相应功能性,更多关注的是其在人体或动物体内的功能性作用,但人体或动物体试验的成本较高、资源有限且在伦理学方面存在争议。在人体消化过程中,摄入的食物会通过消化酶和胃肠道蠕动提供的机械运动被分解为生物大分子或更小的食糜碎片,用于人体生长发育所需,因此需要对食物营养成分消化过程的变化进行探究,以了解食物在人体胃肠消化中的功能性变化。然而随着年龄的增长,老年人的消化酶活性和肠道蠕动的速率会显著下降,因此本发明在参考INFOGEST中建立的体外模拟消化国际共识的基础上,结合国内外文献中所使用的老年人静态消化模型,建立了一种符合老年人特殊消化模式的消化模型。
乳清分离蛋白(Whey Protein Isolates,WPI)是一种由干酪生产的副产物乳清通过特殊的交叉流过滤生产工艺,以及专用的分馏技术分离,从而得到的蛋白含量在90%以上的乳清蛋白产品,相较乳清浓缩蛋白,它的蛋白含量更高,且几乎不含乳糖和乳脂肪,由于乳清蛋白包含多种功能性蛋白成分,如β-乳球蛋白、α-乳白蛋白、牛血清白蛋白和免疫球蛋白,因此目前它被广泛用于特医食品方向的研究,然而大多数关于生物活性肽的筛选和制备的研究往往以其体外理化测定指标为依据,但其在体内胃肠消化过程中的功能性及变化少有研究。
目前比较认可的抗氧化肽的作用机制有以下三种:(1)抗氧化肽的氨基酸作为氢供体或电子供体消除自由基;(2)通过螯合金属离子抑制脂质过氧化;(3)调节生物体内相关抗氧化酶的活性。Keap1-Nrf2-ARE信号通路是参与调节细胞内氧化还原反应的重要途径,当机体内细胞处于氧化应激条件下,外来的抗氧化剂会干扰Keap1-Nrf2相互作用,从而最终激活ARE序列调控细胞内一系列具有抗氧化功能的酶(如过氧化氢酶、超氧化物歧化酶和谷胱甘肽过氧化物酶等)的基因表达,从而促进机体的抗氧化能力。
发明内容
本发明所要解决的技术问题,提供一种基于老年人静态消化模型的乳清蛋白源抗氧化肽的筛选方法。
为解决上述技术问题,本发明所采用的技术方案如下:
一种基于老年人静态消化模型的乳清蛋白源抗氧化肽的筛选方法,包括以下步骤:
(1)乳清蛋白溶液配制:将乳清分离蛋白配制成10mg/mL的蛋白液,在3000r/min转速中离心10min后,取上清液保存,进一步去除乳脂肪。
(2)体外模拟老年人消化:由于乳清蛋白溶液属于液体蛋白,因此不需要进行模拟口腔消化。先进行模拟胃消化,将20mL乳清蛋白溶液与16mL模拟胃液(SGF)混合均匀,加入5.6mg胃蛋白酶(10000U/mg)使溶液最终酶活达到1400U/mL,再加入40μL 0.3M CaCl2溶液,再缓慢滴入1M HCl溶液且计下盐酸加入量,最终滴定pH=3.0,最后补足蒸馏水使溶液体积到40mL,将混合后的胃消化液在37℃,转速100rpm振荡消化2h;再进行模拟肠消化,取20mL胃消化食糜与16mL模拟肠液(SIF)混合均匀,加入16mg胰蛋白酶(250U/mg)使溶液最终酶活达到100U/mL,加入0.163g牛胆盐使溶液最终胆盐浓度达到10mM,加入40μL 0.3M CaCl2溶液,再缓慢滴入1M NaOH溶液且记下NaOH加入量,最终滴定pH=6.5,最后补足蒸馏水使溶液体积到40mL,将混合后的肠消化液在37℃,转速100rpm振荡消化2h。将制备好的胃肠消化液在6000r/min转速下离心5分钟,离心后的消化液均在-80℃中贮存。
(3)抗氧化活性测定:进行体外抗氧化活性检测,包括ABTS、DPPH及FRAP法进行测定。
(4)乳清蛋白肽序列鉴定:通过Triple TOF 5600+液质联用系统对乳清蛋白模拟老年人肠道消化产物进行肽谱鉴定。
(5)抗氧化肽的筛选:通过Peptide Ranker进行生物活性肽打分,再通过分子对接技术将符合打分要求的乳清蛋白肽与Keap1分子进行对接,筛选出结合能最强的乳清蛋白肽。
优选的,步骤(1)的操作,需用磁力搅拌器搅拌3小时以上使乳清蛋白充分溶解。
优选的,步骤(2)的操作,模拟胃肠消化后的乳清蛋白溶液需及时放在冰盒上或放入冰箱中,以抑制胃蛋白酶和胰蛋白酶进一步消化。
优选的,步骤(3)的操作,测定抗氧化活性时需要将消化前的WPI、胃消化物及肠消化调整到相同的蛋白浓度进行测定。
优选的,步骤(4)的操作,将质谱下机后的原始MS/MS文件使用ProteinPilot中的Paragon算法对uniprot数据库进行搜索。参数设定如下:仪器为TripleTOF5600,用碘乙酰胺修饰半胱氨酸;选择生物修饰作为ID focus。对鉴定的蛋白结果,选定一定的过滤标准,肽段unused score>1.3(可信度95%以上)认为是可信肽段,保留包含至少1条unique肽段的蛋白质。
优选的,步骤(5)的操作,用Peptide Ranker>0.5作为筛选条件,使用DiscoveryStudio V2019软件对鉴定出的乳清蛋白肽进行分子对接。
优选的,步骤(5)的操作,Keap1分子来源于RCSB数据库(PDB:2FLU),并需在分子对接前对原PDB文件进行删除水分子、氢原子及原有受体所携带的配体,并对蛋白进行加氢处理等前处理。
本发明公开了一种基于老年人静态消化模型的乳清蛋白源抗氧化肽的筛选方法,本发明的所述的抗氧化肽主要经历乳清蛋白溶液配制和体外模拟老年人消化,对消化前后的蛋白溶液进行抗氧化活性测定,消化产物中乳清蛋白肽的鉴定及具有抗氧化潜力的肽的分子对接筛选。经过体外和细胞抗氧化活性实验证明,消化过程中肠消化产物的抗氧化活性最强,因此鉴定肠消化物中的肽的组成。通过肽谱鉴定出216条肽段,符合PeptideRanker>0.5的肽段有21条,将21条肽段与Keap1分子对接后,分子对接结果得到乳清蛋白肽-DDQNPHSSNICNISCDK的“-CDOCKER Energey”值最高,说明此肽段与Keap1分子结合能力最强,最具有抗氧化活性潜力。
附图说明
图1为WPI消化前、胃消化及肠消化后的ABTS(a)、DPPH(b)及FRAP(c);
图2为多肽DDQNPHSSNICNISCDK与Keap1活性位点相互作用的3D(上)和2D(下)模式图。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例以及实验例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料;所用的设备,如无特殊说明,均为常规实验设备。
实施例1:
本发明基于老年人静态消化模型的乳清蛋白源抗氧化肽的筛选包括消化前后抗氧化活性的测定,抗氧化肽序列的鉴定。
具体步骤包括:
(1)抗氧化活性测定:三种方法测定结果如图1所示。结果显示,通过老年人胃肠消化后的抗氧化活性显著增加,因此本发明选定WPI肠消化物进行肽谱测定,筛选最具有抗氧化潜力的多肽片段。
(2)LC-MS/MS多肽序列鉴定:将WPI肠消化物多肽样品蛋白浓度稀释至1μg/μL。设定上样量5μL,采集60min的扫描模式。扫描样品中质荷比在350-1200的肽段。质谱数据采集使用的是Triple TOF 5600+液质联用系统(ABSCIEX,USA)。将多肽样品溶解于2%乙腈/0.1%甲酸中,并使用与Eksigent nanoLC系统(ABSCIEX,USA)偶联的Triple TOF 5600plus质谱仪进行分析。将多肽溶液加到C18捕获柱(3μm,350μm×0.5mm,AB Sciex,USA)上,并以60min时间梯度,300nL/min的流速在C18分析柱(3μm,75μm×150mm,Welch Materials,Inc)上进行梯度洗脱。两个流动相是缓冲液A(2%乙腈/0.1%甲酸/98%H2O)和缓冲液B(98%乙腈/0.1%甲酸/2% H2O)。对于IDA(信息依赖性采集),以250ms的离子累积时间进行一级质谱图的扫描,并以50ms的离子累积时间采集30个前体离子的二级质谱图。在350-1200m/z的范围内采集MS1光谱,并且在100-1500m/z的范围内采集MS2光谱。将前体离子动态排除时间设置为15s。
表1 WPI肠消化物肽谱
实施例2:
基于分子对接技术的WPI抗氧化活性肽的筛选:
首先根据Peptide Ranker将上述通过LC-MS鉴定的肽段进行初步筛选,筛选出生物活性预测分数>0.5的肽段,并将其与Keap1分子进行分子对接,“-CDOCKER Energey”数值越大表明对接效果越好,可能具有的抗氧化潜力越高。对接结果如表2所示。
表2潜在的抗氧化肽与Keap1的分子对接评分
将“-CDOCKER Energey”数值最高的肽段-DDQNPHSSNICNISCDK进行分子对接相互作用分析,结果如图2所示。Keap1的分子突变实验证明,ETGE基序是与Keap1的Kelch结构域结合时关键基序。研究表明,接触ETGE基序相互作用的残基分别是三个Arg残基Arg380、Arg415和Arg483,四个Ser363、Ser508、Ser555和Ser602的Ser残基,以及Kelch结构域中Tyr334、Asn382和Gln530的残基,此外Asn387、His436、Tyr525、Tyr572和Phe577的残基在Keap1-Nrf2蛋白质-蛋白质相互作用中也起着关键作用。通过2D分子对接作用图分析可知,多肽DDQNPHSSNICNISCDK具有显著影响关键残基的能力,因此可被认为是一种潜在的抗氧化活性肽。
以上实施例仅是本发明的优选实施方式,本发明的保护范围并不仅局限于上述实施例。凡属于本发明思路下的技术方案均属于本发明的保护范围。应该指出,对于本技术领域的普通技术人员来说,在脱离本发明原理的前提下的改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种基于老年人静态消化模型的乳清蛋白源抗氧化肽的筛选方法,其特征在于,包括以下步骤:
(1)多肽序列鉴定:通过Triple TOF 5600+液质联用系统对乳清蛋白模拟老年人肠道消化产物进行肽谱鉴定;
(2)抗氧化肽的筛选:通过Peptide Ranker进行生物活性肽打分,再通过分子对接技术将符合打分要求的乳清蛋白肽与Keap1分子进行对接,通过“-CDOCKER Energey”得分筛选出结合能最强的乳清蛋白肽。
2.根据权利要求1所述的制备方法,其特征在于,具体包括以下步骤:
(1)体外模拟老年人消化:由于乳清蛋白属于液体蛋白,因此不需要进行口腔消化。按照文献方法依次进行模拟胃消化和肠消化。胃肠消化液按照国际共识方法进行配制;
(2)抗氧化活性测定:进行体外抗氧化活性检测,包括ABTS、DPPH及FRAP法进行测定;
(3)肠消化物序列鉴定:将乳清蛋白肠消化物进行LC-MS/MS测定,分析其多肽组成;
(4)抗氧化肽筛选:将鉴定出的多肽片段先筛选出Peptide Ranker>0.5的潜在生物活性肽,然后将其通过Discovery Studio V2019软件进行分子对接,筛选最具潜力的抗氧化肽。
3.根据权利要求2所述的制备方法,其特征在于,模拟胃肠消化后的乳清蛋白溶液需及时放在冰盒上或放入冰箱中,以抑制胃蛋白酶和胰蛋白酶进一步消化。
4.根据权利要求2所述的制备方法,其特征在于,测定抗氧化活性时需要将消化前的WPI、胃消化物及肠消化调整到相同的蛋白浓度进行测定。
5.根据权利要求1所述的制备方法,其特征在于,对鉴定的蛋白结果,选定一定的过滤标准,肽段unused score>1.3(可信度95%以上)认为是可信肽段,保留包含至少1条unique肽段的蛋白质。
6.根据权利要求1所述的制备方法,其特征在于,Keap1分子来源于RCSB数据库(PDB:2FLU)。
7.根据权利要求6所述的制备方法,其特征在于,需在分子对接前对原PDB文件进行删除水分子、氢原子及原有受体所携带的配体,并对蛋白进行加氢处理等前处理。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040259161A1 (en) * | 2003-03-12 | 2004-12-23 | Fredrik Nilsson | Screening assay |
CA2611416A1 (en) * | 2005-06-08 | 2006-12-14 | Isidra Recio Sanchez | Bioactive peptides identified in enzymatic hydrolyzates of milk caseins and method for the production thereof |
WO2008057434A2 (en) * | 2006-11-06 | 2008-05-15 | Merck & Co., Inc. | Method for identifying modulators of the nrf2-keap1-are pathway |
CN107964040A (zh) * | 2017-11-13 | 2018-04-27 | 江苏大学 | 乳球蛋白活性肽的超声辅助模拟消化方法及功能食品应用 |
-
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- 2022-11-10 CN CN202211404356.2A patent/CN115505025A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040259161A1 (en) * | 2003-03-12 | 2004-12-23 | Fredrik Nilsson | Screening assay |
CA2611416A1 (en) * | 2005-06-08 | 2006-12-14 | Isidra Recio Sanchez | Bioactive peptides identified in enzymatic hydrolyzates of milk caseins and method for the production thereof |
WO2008057434A2 (en) * | 2006-11-06 | 2008-05-15 | Merck & Co., Inc. | Method for identifying modulators of the nrf2-keap1-are pathway |
CN107964040A (zh) * | 2017-11-13 | 2018-04-27 | 江苏大学 | 乳球蛋白活性肽的超声辅助模拟消化方法及功能食品应用 |
Non-Patent Citations (3)
Title |
---|
张勇慧: "微生物天然药物化学研究", 华中科技大学出版社, pages: 84 - 85 * |
李相怡;方曦;任皓威;程琳;张拓;付国红;刘宁;: "基质辅助激光解吸电离飞行时间串联质谱法分析人乳β-酪蛋白新生儿体外消化模型多肽组", 分析化学, no. 05, pages 631 - 636 * |
李良煜: "基于Keap1-Nrf2通路的蛋清源抗氧化小肽筛选及其作用机制研究", 中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑, no. 1, pages 024 - 451 * |
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