CN115260295A - 一种具有抗氧化功能的生物活性肽tdefqlhtnvndgtefggsiyqk - Google Patents
一种具有抗氧化功能的生物活性肽tdefqlhtnvndgtefggsiyqk Download PDFInfo
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Abstract
本发明公开了一种具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK,其氨基酸序列为:Thr‑Asp‑Glu‑Phe‑Gln‑Leu‑His‑Thr‑Asn‑Val‑Asn‑Asp‑Gly‑Thr‑Glu‑Phe‑Gly‑Gly‑Ser‑Ile‑Tyr‑Gln‑Lys。本发明的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK能够耐受胃肠道消化酶的消化,并可以被肠上皮细胞完整吸收,不会被肠上皮细胞的酶类降解。并且通过细胞实验,验证了TDEFQLHTNVNDGTEFGGSIYQK具有良好的抗氧化功能。该生物活性肽对开发具有抗氧化功能的食品、保健品和药物具有十分重要的意义。
Description
技术领域
本发明涉及功能性食品技术领域,具体涉及一种具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK。
背景技术
抗氧化肽是指具有清除自由基、抑制脂质过氧化、螯合过渡金属离子和降低细胞自氧化速率,并且能够有效捕获并中和自由基的一类活性物质。研究表明,肉与肉制品是抗氧化肽的良好来源。其中发酵肉制品,如发酵香肠和火腿,在经过长期发酵成熟过程中,蛋白质在各种内源酶以及微生物的作用下发生强烈降解,生成了丰富的多肽、小肽类物质。因此传统发酵肉制品是抗氧化肽的良好来源。
抗氧化肽经口服在机体发挥抗氧化功能之前,要克服胃酸、小肠碱性环境及胃蛋白酶、胰酶、肽酶等一系列酶的作用,也要克服小肠的吸收屏障进入机体,是发挥其生理功能的重要前提条件。但是关于肉制品来源的天然的、耐消化、可通过小肠黏膜上皮具有高生物利用度的抗氧化肽国内外还未见报道。
发明内容
本发明以云南的宣威火腿为原料,通过体外消化模型和小肠上皮细胞吸收模型筛选得到了具有抗氧化性、耐消化、可完整通过小肠黏膜上皮的抗氧化肽。
本发明所采用的技术方案是一种具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK,其氨基酸序列为:Thr-Asp-Glu-Phe-Gln-Leu-His-Thr-Asn-Val-Asn-Asp-Gly-Thr-Glu-Phe-Gly-Gly-Ser-Ile-Tyr-Gln-Lys。
一种具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK的制备方法,通过固相合成法制备。
一种具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK的应用,所述具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK在制备具有抗氧化功能的食品、保健品或药物中的应用。
一种抗氧化功能产品,包括具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK,所述的抗氧化功能产品包括抗氧化食品或抗氧化保健品或抗氧化药物。
本发明的有益效果是:(1)以宣威火腿为原料,经过模拟体外消化、Caco-2细胞模型筛选获得既耐消化,又可完整通过小肠黏膜上皮生物活性肽TDEFQLHTNVNDGTEFGGSIYQK。(2)生物活性肽TDEFQLHTNVNDGTEFGGSIYQK对氧化损伤的HepG2细胞具有较好的保护作用,效果好于谷胱甘肽(GSH)。
附图说明
图1:F4组分肽溶液总离子流图。
图2:粗肽中F4组分中能通过小肠黏膜上皮的肽溶液总离子流图。
图3:合成肽段TK-23的HPLC液相色谱保留时间。
图4:合成肽段TK-23的二级质谱图。
图5:肽段TK-23对HepG2细胞CAT活率的影响情况。
图6:肽段TK-23对HepG2细胞SOD活率的影响情况。
图7:肽段TK-23对HepG2细胞GSH-Px活率的影响情况。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。通常在此处附图中描述和示出的本发明实施例的组件能够以各种不同的配置来布置和设计。
本发明的目的在于提供一种耐消化、可完整通过小肠黏膜上皮的抗氧化肽,以解决生物活性肽产品的生物利用度问题。其获得是将宣威火腿经体外模拟消化得到粗肽液后,通过Caco-2细胞模型对既耐消化又可被小肠细胞吸收利用的肽段进行筛选,然后通过多凝胶过滤色谱和液相色谱得到,也可通过固相合成的方法得到,优选固相合成法。
(1)活性肽的制备、分离纯化及鉴定
取5-15g宣威火腿肱二头肌部分于100-500mL离心管中,分别加入15-30mL模拟唾液电解质、10-30mL模拟胃液电解质、15-30mL模拟肠液电解质,恒温摇床中以100-200rpm转速匀速振荡反应2-4小时以模拟人体小肠消化。得到模拟消化液后,将样品于90-100℃水浴锅中水浴加热10-20min。离心、真空冷冻干燥,即得到体外模拟消化液粗肽粉。
宣威火腿体外模拟消化产物经3-5kDa超滤膜超滤后,凝胶过滤层析得到F1、F2、F3和F4四个组分。选取分子量最小的F4组分进行Caco-2细胞转运实验,在Caco-2细胞模型的上侧和下侧分别添加1.5-3mL含粗肽样品的平衡盐缓冲液,将培养板放入二氧化碳细胞培养箱吸收转运2-3h,然后收集两侧溶液进行LC-MS/MS分析。确定F4组分中被Caco-2细胞完整吸收的肽段包括:
Thr-Asp-Glu-Phe-Gln-Leu-His-Thr-Asn-Val-Asn-Asp-Gly-Thr-Glu-Phe-Gly-Gly-Ser-Ile-Tyr-Gln-Lys(TDEFQLHTNVNDGTEFGGSIYQK)。
(2)活性肽具有抗氧化作用
对可完整通过Caco-2细胞的肽段TDEFQLHTNVNDGTEFGGSIYQK应用固相合成法进行合成,合成的肽段经体外实验表明具有较好的抗氧化活性,并且Fe2+螯合能力显著高于GSH。同时HepG2氧化损伤细胞实验结果表明,肽段TDEFQLHTNVNDGTEFGGSIYQK可以调节抗氧化酶活性,对HepG2细胞的氧化应激具有保护作用。
实施示例1:活性肽的制备、分离纯化及鉴定。
(1)取5g宣威火腿肱二头肌部分于100mL离心管中,分别加入15mL模拟唾液电解质与75μL二水氯化钙溶液,将离心管置于烧杯中冰浴。使用匀浆机均质以模拟人体口腔咀嚼。
(2)分别在均质好的肉糜样品中加入10mL模拟胃液电解质,将样品的pH值调节至2.5,再补充适量模拟胃液电解质至30mL,按照酶:样品=1:20(w/w)加入0.24g胃蛋白酶粉末,将恒温摇床培养箱设置参数为温度37℃,转速为100rpm,反应时间为2小时,匀速振荡以模拟人体胃部消化。
(3)取上述胃食糜样品,加入24mL模拟肠液电解质,将样品的pH值调节至7.0,再补充适量模拟肠液电解质至60mL,加入0.25g牛胆盐及按照酶:样品=1:50(w/w)加入0.1g胰蛋白酶粉末,置于37℃恒温摇床中以100rpm转速匀速振荡反应2小时以模拟人体小肠消化,得到模拟消化液。
(4)将经过体外模拟消化的消化液样品于95℃水浴锅中水浴加热10min后。于4℃、12000×g条件下离心20min。收集离心管中的上层清液进行抽滤后,再进行真空冷冻干燥,即得到体外模拟消化液粗肽粉。
(5)用超滤离心管(超滤膜孔径为3kDa)对粗肽粉进行超滤,取冻干后小于3kDa的肽粉复溶,用层析柱(固定相:葡聚糖凝胶G-15,流动相:去离子水,流速:1mL/min)对肽粉进行分离纯化;检测器的检测波长为280nm。待样品峰出现后,选用蛋白收集器每管收集5mL洗脱液,将属于同一峰段的洗脱液混合成同一样品,根据分子量得到抗氧化能力不同的四个多肽组分F1、F2、F3和F4,其中F4组分的分子量最小,抗氧化能力最强。
(6)先将Caco-2细胞培养于25cm2细胞培养瓶中,培养液为含有16%胎牛血清(FBS,v/v),1%非必需氨基酸(NEAA,v/v),100U/mL青霉素和0.1mg/mL链霉素的高糖培养基(DMEM)。培养条件为37℃、含5%CO2,相对湿度为90%。细胞培养21d。
(7)细胞转运实验。用PBS轻轻地清洗Caco-2细胞3次,在Caco-2细胞模型的上侧(AP)和下侧(BL)分别添加1.5mL和3mL 37℃预热后的平衡盐缓冲液,37℃平衡30min。然后移除AP侧平衡盐缓冲液,添加含肽样品F4的平衡盐缓冲液溶液,将Transwell培养板放入二氧化碳细胞培养箱吸收转运2h,然后收集两侧溶液进行液质联用(LC-MS/MS)分析。
(8)液质联用测定F4组成多肽序列的条件为。液相条件:流动相A:0.1%甲酸水溶液;流动相B:0.1%甲酸溶于80%的乙腈。洗脱程序:0~95min,流动相B 3%;95~105min,流动相B 32%;105~120min,流动相B 100%。流速:200nL/min;进样量:3μL。质谱条件:MS扫描范围(m/z):355-1700,分辨率:70000,最大离子注入时间:100ms;MS/MS扫描范围(m/z):200-2000,离子注入时间:75ms,分辨率:35000;碎裂模式:高能碰撞;碎裂能量:27。结果见附表1。
(9)得到F4组分中被Caco-2细胞完整吸收的肽段有3个,肽段长度分别为19、20和23个氨基酸。其中由23个氨基酸组成的肽段TDEFQLHTNVNDGTEFGGSIYQK体外抗氧化效果最好。
实施示例2:生物活性肽TDEFQLHTNVNDGTEFGGSIYQK(TK-23)具有良好的抗氧化作用
(1)采用固相合成法合成活性肽TDEFQLHTNVNDGTEFGGSIYQK,纯度为99%。
(2)HepG2细胞培养。先将HepG2细胞培养液为含有10%胎牛血清(FBS,v/v),100U/mL青霉素和0.1mg/mL链霉素的高糖培养基(DMEM)。培养条件为37℃,含5%CO2,相对湿度为90%。每隔2d更换一次培养基,当HepG2细胞长满培养瓶培养面积的80%左右时,用含0.25%EDTA的胰酶消化细胞,进行传代培养。
(3)实验设计及造模方法。实验室分为空白组、模型组(氧化损伤模型)、GSH组和活性肽组(设置4个梯度0.50、0.75、1.00和1.25mg/mL)。HepG2氧化损伤细胞造模条件H2O2浓度分别为100、200、300、400、500、600、700、800、1000μmol/L,分别培养2h、4h、6h后采用四甲基偶氮唑盐比色法(MTT法)检测细胞活力,并计算HepG2细胞存活率。空白组加入100μL的培养基。将存活率在50%左右的H2O2浓度作为细胞的造模浓度。
(4)取1×105细胞/mL的HepG2细胞接种于六孔板中,每孔接种2.5mL细胞,培养至细胞铺满80%聚合度后,用不同浓度梯度的活性肽和GSH孵育细胞24h,之后弃去培养基,每孔加入含有800μmol/L的过氧化氢(H2O2),孵育2h。用细胞裂解液裂解HepG2细胞,离心后取上清,用牛血清白蛋白(BCA)试剂盒测定蛋白浓度,用细胞超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)及过氧化氢酶(CAT)试剂盒对细胞中抗氧化酶活力进行测定。
(5)结果分析。肽段TK-23对HepG2细胞抗氧化酶活性影响附图5-7所示。加入TK-23后,细胞的CAT、SOD和GSH-Px活性显著增强。由CAT的活性可知,在0.50mg/mL GSH孵育条件下,CAT活性与空白细胞组相比没有显著差异(P>0.05)。0.50、0.75mg/mL TK-23孵育HepG2细胞后的CAT活性显著高于氧化损伤模型组和GSH组(*P<0.05,**P<0.01)。由SOD活性可知,在0.50mg/mL GSH孵育条件下,SOD活性显著高于空白细胞和氧化损伤模型组(##P<0.01),说明GSH可以降低HepG2细胞的氧化应激水平。而0.50、0.75、1.00、1.25mg/mL的T-23孵育HepG2细胞后的SOD活性显著高于GSH组(**P<0.01)。说明T-23对HepG2细胞中抗氧化酶的调节作用优于GSH。由GSH-Px活性可知,在0.50mg/mL GSH孵育条件下,SOD活性与空白细胞组相比没有显著差异(P>0.05)。0.75mg/mL的TK-23孵育HepG2细胞后的GSH-Px活性显著高于氧化损伤组和GSH组(**P<0.01)。综合以上结果可知,TK-23进入细胞后可调节HepG2细胞内抗氧化酶活性,进而调节细胞的氧化应激水平,从而起到防止细胞受到氧化损伤的作用。
表1,粗肽F4组分的肽段序列及能通过Caco-2细胞的F4组分的肽段。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 东莞理工学院
<120> 一种具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK
<130> 1835
<141> 2022-06-14
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> PRT
<213> Artificial Sequence
<400> 1
Thr Asp Glu Phe Gln Leu His Thr Asn Val Asn Asp Gly Thr Glu Phe
1 5 10 15
Gly Gly Ser Ile Tyr Gln Lys
20
Claims (4)
1.一种具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK,其特征在于:其氨基酸序列为:Thr-Asp-Glu-Phe-Gln-Leu-His-Thr-Asn-Val-Asn-Asp-Gly-Thr-Glu-Phe-Gly-Gly-Ser-Ile-Tyr-Gln-Lys。
2.如权利要求1所述的具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK的制备方法,其特征在于:通过固相合成法制备。
3.如权利要求1所述的具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK的应用,其特征在于:所述的具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK在制备具有抗氧化功能的食品、保健品或药物中的应用。
4.一种抗氧化功能产品,其特征在于,包括如权利要求1所述具有抗氧化功能的生物活性肽TDEFQLHTNVNDGTEFGGSIYQK,所述的抗氧化功能产品包括抗氧化食品或抗氧化保健品或抗氧化药物。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007134328A2 (en) * | 2006-05-15 | 2007-11-22 | Van Andel Research Institute | Geldanamycins and their quinone moieties inhibit cancer by acting on mitochondrial voltage-dependent anion channel (vdac) protein |
CA2875418A1 (en) * | 2013-12-17 | 2015-06-17 | Agada Biosciences, LLC | Method and agents to quantify proteins from tissues |
CN112625112A (zh) * | 2020-12-16 | 2021-04-09 | 熊猫乳品集团股份有限公司 | 一种生物活性多肽paapaqlpkki及其制备方法和应用 |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007134328A2 (en) * | 2006-05-15 | 2007-11-22 | Van Andel Research Institute | Geldanamycins and their quinone moieties inhibit cancer by acting on mitochondrial voltage-dependent anion channel (vdac) protein |
CA2875418A1 (en) * | 2013-12-17 | 2015-06-17 | Agada Biosciences, LLC | Method and agents to quantify proteins from tissues |
CN112625112A (zh) * | 2020-12-16 | 2021-04-09 | 熊猫乳品集团股份有限公司 | 一种生物活性多肽paapaqlpkki及其制备方法和应用 |
Non-Patent Citations (1)
Title |
---|
YAO-YAO HU ET AL: "Transepithelial transport and cytoprotection of novel antioxidant peptides isolated from simulated gastrointestinal digestion of Xuanwei ham", FOOD FUNCT, vol. 2023, no. 14, 6 March 2023 (2023-03-06), pages 3552 - 3563 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116589534A (zh) * | 2023-07-11 | 2023-08-15 | 中国农业大学 | 青稞来源的抗氧化肽及其制备方法和应用 |
CN116589534B (zh) * | 2023-07-11 | 2023-10-31 | 中国农业大学 | 青稞来源的抗氧化肽及其制备方法和应用 |
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