CN107964040A - 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 - Google Patents
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 Download PDFInfo
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Abstract
本发明公开了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,属于乳制品精深加工及功能食品制备技术领域。本发明先采用超声预处理β‑乳球蛋白,其次蛋白酶进行酶解制备β‑乳球蛋白抗炎多肽,再通过模拟胃肠道消化追踪β‑乳球蛋白抗炎多肽的活性,然后Caco‑2细胞模拟小肠上皮细胞吸收后,表征出了经过胃肠道消化后,被Caco‑2细胞模拟小肠内壁吸收的高抗炎活性的β‑乳球蛋白抗炎多肽。本发明首次鉴定出了2种经过胃肠道消化后,被Caco‑2细胞模拟小肠内壁吸收具有很强的抗炎活性β‑乳球蛋白功能多肽;本发明首次报道了β‑乳球蛋白水解产物对血管内皮细胞具有良好的抗炎活性。
Description
技术领域
本发明提供了一种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,属于乳制品精深加工及功能食品制备技术领域,可作为功能性用于制备功能性食品或药物等。
背景技术
慢性炎症参与人体多种疾病的发生发展,是多种疾病的致病基础。衬于心、血管和淋巴管内表面的单层扁平上皮细胞即血管内皮细胞在血管生物学中起着至关重要的作用,如调节血管紧张度、止血、嗜中性粒细胞募集、激素运输和液体过滤等。血管炎症是导致内皮功能失调的关键性因素之一,与多种疾病密切相关,包括动脉粥样硬化、糖尿病、冠状动脉疾病、高血压和高胆固醇血症。目前,临床上针对炎症的治疗药物主要包括甾体类和非甾体类两种。然而这些药物不仅对人体健康产生一定影响,还需要较大的经济投入。鉴于对长时间使用这些药物的副作用的担忧,以食源性功能食品作为抗炎药物的替代品成为了研究热点,前景广阔。
β-乳球蛋白(占乳清蛋白的68%)是除α-乳白蛋白之外的牛乳乳清蛋白的两种主要蛋白之一,它所具有的结构和功能性质日益引起人们的重视。β-乳球蛋白的分子量为18277~18363,分子为一条多肽链,由162个氨基酸残基组成,主要以二聚体形式存在,由非共价键连接的两个单体亚基组成。每个单体含有2个二硫键Cys66-Cys160和Cysl06-Cys199,还有一个Cys121为游离巯基。近年来人们对活性肽研究的范围逐渐扩大,陆续发现了一些源于β-乳球蛋白、有着较强生物活性的肽段,这些肽具有较强的抗高血压的活性、ACE抑制活性、抑菌活性、降低血清胆固醇水平和显著的镇静、止痛、安神的作用。MurakamiM等发现一种来自β-乳球蛋白具有较强的ACE抑制活性的四肽(ALPM)。Pellegrini A等(2001)通过蛋白酶对β-乳球蛋白的水解产生了4个具有较强抑菌活性的肽段:VAGTWY f(15-20)、AASDISLLDAQSAPLR f(25-40)、IPAVFK f(78-83)、VLVLDTDYK f(92-100)。但是关于β-乳球蛋白多肽对血管内皮细胞的抗炎活性以及其相关的抗炎多肽的制备方法至今无人报道。
现阶段关于β-乳球蛋白功能活性肽的制备方法的研究,主要集中在蛋白酶的筛选、酶解工艺的优化和高活性肽的分离、纯化和鉴定等方法。然而上述研究忽略了口服生物活性肽必须经过胃肠道消化,小肠内皮细胞吸收后,以活性肽形式达到目标器官,才能发挥生理活性作用。胃肠道中含有大量的胃蛋白酶,胰酶,对生物活性肽在吸收之前进行二次酶解;小肠内皮细胞分泌产生细胞酶对途经多肽进一步的分解;与此同时,小肠内皮细胞对多肽的吸收具有多种途经,如转运蛋白介导、跨细胞被动扩散以及细胞旁路转运。把体内胃肠道消化吸收考虑进生物活性肽的制备方法已成必然。专利“从皱纹盘鲍鲍鱼内脏中分离的抗炎肽及其用途”(201510594885.7),虽然模拟胃肠道消化方法制备抗炎肽,却忽略了小肠上皮细胞对其选择性吸收的影响。综上所述,目前关于β-乳球蛋白制备功能多肽的方法都忽略了胃肠道的消化吸收,并不能真实的模拟生物活性肽在胃肠道消化以及小肠内皮细胞对多肽的吸收。
发明内容
鉴于上述不足之处,本发明先采用超声预处理β-乳球蛋白,其次蛋白酶进行酶解制备β-乳球蛋白抗炎多肽,再通过模拟胃肠道消化追踪β-乳球蛋白抗炎多肽的活性,然后Caco-2细胞模拟小肠上皮细胞吸收后,表征出了经过胃肠道消化后,被Caco-2细胞模拟小肠内壁吸收的高抗炎活性的β-乳球蛋白抗炎多肽。
本发明的目的是首次利用β-乳球蛋白水解产物模拟人体胃肠道消化及Caco-2细胞吸收制备得到两种β-乳球蛋白抗炎多肽。
其氨基酸序列为:
Phe-Tyr-Gln-Ala
Leu-Gln-Tyr
本发明的另一个目的是提供了利用超声预处理β-乳球蛋白,蛋白酶酶解β-乳球蛋白制备功能多肽,模拟人体胃肠道消化及Caco-2细胞吸收制备得到β-乳球蛋白抗炎多肽的方法,按照下述步骤进行:
(1)β-乳球蛋白的提取
将乳清粉用蒸馏水充分搅拌溶解配成质量浓度为7%的蛋白溶液,然后加入NaCl使其最终的质量浓度为7%,用HCl调节pH至2,用高速离心机在5000r/min下离心20min,收集上清液;用分子截量为14000Da的透析袋取一定量的上清液,放于30倍体积的蒸馏水中静置20h,收集透析袋中的溶液;得到β-乳球蛋白溶液;
(2)超声预处理β-乳球蛋白:将底物浓度为1~4g/L的β-乳球蛋白溶液加入超声袋中,密封,然后将样品超声袋直接置于超声反应釜中,进行超声波处理;
(3)β-乳球蛋白酶解液制备:调节上述蛋白溶液pH 7.5~8.0,加入蛋白酶,酶与底物的比例为1:20~50(w/w),均匀混合,酶解温度50~70℃,酶解时间2~4h;酶解结束后调节混合液pH为7.0,并在沸水浴中灭酶10min,离心获得上清液,脱盐、浓缩、冷冻干燥成粉末。
(4)模拟胃肠道消化:配置人工胃液。在37℃下,将将步骤(3)制备得到的β-乳球蛋白水解物与胃液以1:20~50混合,在震荡频率为120~180rpm条件下模拟胃消化2~4h。用NaOH调节pH为6.8,以1:100(w/v)加入胰酶至混合液,继续酶解4~6h;沸水浴灭酶10min,离心取上清液,脱盐、浓缩、冷冻干燥成粉末。
(5)Caco-2模拟小肠上皮细胞吸收:构建Caco-2细胞转运模型,将步骤(4)制备得到的β-乳球蛋白多肽消化产物配置成20mg/ml,加入到Caco-2细胞的绒毛面AP侧(apical,肠腔侧),0.5~4h后,收集基底面BL侧(basolateral,肠内壁侧)转运的β-乳球蛋白肽,脱盐、冷冻干燥。
(6)采用UPLC-MC鉴定及分析多肽序列:对步骤(5)经过Caco-2细胞吸收的β-乳球蛋白多肽进行鉴定及多肽序列分析,筛选出离子强度强,小于1000的多肽序列;
(7)多肽的合成以及活性验证:对步骤(6)筛选出来的多肽进行合成,并验证其抗炎活性,最终验证得到两种β-乳球蛋白抗炎活性多肽,其氨基酸序列为:
Phe-Tyr-Gln-Ala
Leu-Gln-Tyr。
其中步骤(2)所述的超声波处理工艺条件如下:超声处理,超声处理时间10-30min,间歇比10s/3s,温度25℃;超声波处理频率及频率组合为:单频:20kHz、28kHz、40kHz;双频:20/28kHz、20/40kHz、28/40kHz;三频:20/28/40kHz。
其中步骤(3)所述的蛋白酶为碱性蛋白酶、木瓜蛋白酶、中性蛋白酶或嗜热蛋白酶;优选嗜热蛋白酶。
其中步骤(6)中的β-乳球蛋白抗炎多肽优选Leu-Gln-Tyr。
上述β-乳球蛋白活性肽作为保健食品应用,经过公知方法的制造成胶囊或者片剂,作为抗炎的保健食品或者功能食品。
本发明的优势在于:
(1)本发明首次鉴定出了2种经过胃肠道消化后,被Caco-2细胞模拟小肠内壁吸收具有很强的抗炎活性β-乳球蛋白功能多肽;
(2)本发明首次报道了一种新颖的用于分离鉴定出可以经过胃肠道消化、吸收的抗炎肽的方法;
(3)本发明首次研究了β-乳球蛋白多肽消化产物在Caco-2模拟小肠内壁吸收系统的吸收率;
(4)本发明首次报道了β-乳球蛋白水解产物对血管内皮细胞具有良好的抗炎活性。
附图说明
图1为本发明的技术路线。
具体实施方式
一、实验方法:
1、水解度和蛋白转化率的测定
使用2,4,6-三硝基苯磺酸(TNBS)法测定β-乳球蛋白的水解度。
水解度的测定采用pH-stat方法,水解度(DH)即蛋白质在酶解的过程中,断裂的肽键数占总蛋白质肽键数的百分比。
式中:V—NaOH消耗量(mL);N—NaOH的摩尔浓度(mol/L);α—β-乳球蛋白中α-NH2的平均解离度,试验条件下为0.99;M—水解蛋白的质量(g);htot—单位质量蛋白质中肽键的数量(mmol/g),对于不同的蛋白质htot为不同值,取经验值β-乳球蛋白的htot=7.35mmol/g。
通过凯氏定氮法测定β-乳球蛋白和其水解产物的总氮量,计算β-乳球蛋白蛋白的转化率。蛋白转化率(%)=水解物含氮量/底物蛋白氮含量*100%
2、细胞培养:
人结肠腺癌细胞系Caco-2(HTB-37TM)和内皮细胞系EA.hy926(CRL-2922TM)细胞购自美国菌种保藏中心,细胞用含10%胎牛血清的、1%非必需氨基酸、1%抗生素和2.5%HEPES缓冲液的DMEM培养液,在37℃,5%CO2的培养箱中培养。每周更换3次培养液。使用0.25%胰蛋白酶-EDTA处理将细胞传代培养。
3、细胞毒性实验:
使用Alamar Blue试剂检测β-乳球蛋白多肽对细胞的毒性。将Caco-2细胞以1×104个细胞/孔的密度接种在96孔板中24小时,细胞用不同浓度(10~50mg/ml)的β-乳球蛋白多肽再处理24小时。处理24小时后,弃去培养液,加入含有10%Alamar Blue试剂的培养液,并在37℃再培养4小时。在激发波长560nm和发射波长590nm下测量荧光强度。细胞活力表示为相比于未处理细胞的百分比。
4、抗炎活性实验:
通过蛋白印记法分析血管内皮细胞即EA.hy926细胞炎症因子细胞间黏附分子(ICAM-1)和血管细胞黏附分子(VCAM-1)的表达水平来检测抗炎效果。代数<12的EA.hy926细胞转接到48孔板中,密度达到80-90%后,用2.5mg/ml的水解产物和消化物,0.2mM和3.0mM的合成肽处理18h。然后加入TNF-α(10ng/mL)刺激细胞6h产生炎症。
移去细胞培养液,使用含有50μM二硫苏糖醇(还原剂)和0.2%Triton X-100煮沸的Laemmle缓冲液裂解细胞。然后将细胞裂解物在9%十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)上跑电泳。将细胞蛋白转移到硝酸纤维素膜上,并用ICAM-1/VCAM-1抗体进行免疫印迹。用α-tubulin标准化ICAM-1和VCAM-1的含量。tubulin内参抗体的用量为0.4μg/mL,其他的用量为0.1μg/mL。蛋白条带用Licor Odyssey BioImager扫描,并通过使用ImageStudio Lite 5.2(Licor Biosciences,Lincoln,NB,USA)的光密度测定法进行定量。所有数据表示为相应阳性对照(单独用TNF-α处理的细胞)的百分比。
5、Caco-2模拟小肠内皮吸收率的测定:
收集Caco-2细胞的AP和BL表面的多肽,通过UPLC测定多肽的吸收率,反相C18柱(100mm×2.1mm id,1.7μm,Waters,Milford,MA,USA)上样15μL,流动相A(含1%TFA的纯水),溶剂B(含1%TFA的乙腈)。洗脱条件:100-75%A,25min;75-50%A,25-35min;流速为0.3ml/min;检测波长为220nm。多肽的吸收率表示为相应时间点(0.5h、1h、2h和4h)对应的BL收集多肽的吸收峰占加入到AP表面的多肽(0h)的吸收峰的积分面积比。
6、UPLC-MS分析多肽的序列:
液相色谱分析柱为nanoACQUITY BEH130C18(75μm x 150mm,1.7μm),流动相A为含0.1%甲酸的乙腈,流动相B含0.1%甲酸的水洗脱梯度为:1-6%B,0-2min;6-25%B,2-25min;25-45%B,25-40min;45-75%B 40-45min;75-95%B,45-50min;95%B,50-55min。质谱采用电喷雾正离子模式采集数据,毛细管电压为3.5kV,离子源温度为100℃,扫描范围m/z为200-1000。用Mass Lynx软件(Micromass U.K.Ltd)分析肽的氨基酸序列。PeaksViewer4.5(Bioinformatics Solutions Inc.,Waterloo,ON,Canada)与手动测序结合来处理MS/MS数据。通过Genscript Corp(Piscataway,NJ)合成鉴定的肽序列(>98%纯度)并用于抗炎测定。
本发明β-乳球蛋白的提取的方法如下:
在100mL蒸馏水中加入7g乳清粉充分搅拌,溶解后加入7%的NaCl,用HCL调节pH至2,用高速离心机在5000r/min下离心20min,收集上清液。用分子截量为14000Da的透析袋取一定量的上清液,放于30倍体积的蒸馏水中静置20h,收集透析袋中的溶液。
实施例1
将底物浓度为1g/L的β-乳球蛋白溶液200mL加入超声袋中,密封,然后将样品超声袋直接置于超声反应釜中,单频(40kHz)超声处理。超声处理时间30min,间歇比10s/3s,温度25℃。
β-乳球蛋白酶解液制备:调节上述蛋白溶液pH 8,加入碱性蛋白酶,酶与底物的比例为1:20(w/w),均匀混合,酶解温度50℃,酶解时间2h。酶解结束后调节混合液pH为7.0,并在沸水浴中灭酶10min混合液在沸水浴中灭酶10min,离心获得上清液,脱盐、浓缩、冷冻干燥成粉末。测β-乳球蛋白的水解度,蛋白转化率,以及其水解产物对EA.hy926细胞的抗炎活性。
模拟胃肠道消化:根据美国药典(USP30-NF25)配置人工胃液。在37℃下,将β-乳球蛋白水解物与胃液以1:20混合,在震荡频率为120rpm条件下模拟胃消化4h。用NaOH调节pH为6.8,以1:100(w/v)加入胰酶至混合液,继续酶解6h。沸水浴灭酶10min,离心取上清液,脱盐、浓缩、冷冻干燥成粉末。测4h和10h后的消化产物对EA.hy926细胞的抗炎活性。
相比于传统酶解,单频超声预处理后,β-乳球蛋白的水解度由10.32%提高到了13.70%,蛋白转化率由30.27%提高到了35.17%(表1)。碱性蛋白酶酶解β-乳球蛋白所得β-乳球蛋白酶解液对血管内皮细胞具有良好的抗炎效果,对血管内皮细胞VCAM-1、ICAM-1炎症因子表达产生了显著影响,相比于对照组的,其表达量分别为42.3%、62.7%;模拟胃消化后,其酶解液的消化产物对血管内皮细胞仍然具有良好的抗炎效果,VCAM-1、ICAM-1炎症因子表达量相比于对照组分别为48.2%、55.3%;模拟肠消化后,最终消化产物对血管内皮细胞依然保持良好的抗炎效果,VCAM-1、ICAM-1炎症因子表达量相比于对照组为分别为50.7%、63.2%(表2)。结果表明了,模拟消化对β-乳球蛋白酶解液的抗炎活性影响不大。
实施例2
将底物浓度为4g/L的β-乳球蛋白溶液200mL加入超声袋中,密封,然后将样品超声袋直接置于超声反应釜中,双频(20/28kHz)超声处理。超声处理时间20min,间歇比10s/3s,温度25℃。
β-乳球蛋白酶解液制备:调节上述蛋白溶液pH 7.5,加入中性蛋白酶,酶与底物的比例为1:30(w/w),均匀混合,酶解温度55℃,酶解时间4h。酶解结束后调节混合液pH为7.0,并在沸水浴中灭酶10min,离心获得上清液,脱盐、浓缩、冷冻干燥成粉末。测β-乳球蛋白的水解度,蛋白转化率,以及其水解产物对EA.hy926细胞的抗炎活性。
模拟胃肠道消化:根据美国药典(USP30-NF25)配置人工胃液。在37℃下,将β-乳球蛋白水解物与胃液以1:30混合,在震荡频率为150rpm条件下模拟胃消化3h。用NaOH调节pH为6.8,以1:100(w/v)加入胰酶至混合液,继续酶解4h。沸水浴灭酶10min,离心取上清液,脱盐、浓缩、冷冻干燥成粉末。测4h和10h后的消化产物对EA.hy926细胞的抗炎活性。
相比于传统酶解,双频超声预处理后β-乳球蛋白的水解度由6.19%提高到了9.53%,蛋白转化率由15.11%提高到了22.34%(表1)。碱性蛋白酶酶解β-乳球蛋白所得β-乳球蛋白酶解液对血管内皮细胞具有良好的抗炎效果,对血管内皮细胞VCAM-1、ICAM-1炎症因子表达产生了显著影响,相比于对照组的,其表达量分别为63.2%、52.3%;模拟胃消化后,其酶解液的消化产物对血管内皮细胞仍然具有良好的抗炎效果,VCAM-1、ICAM-1炎症因子表达量相比于对照组分别为50.3%、47.3%;模拟肠消化后,最终消化产物对血管内皮细胞依然保持良好的抗炎效果,VCAM-1、ICAM-1炎症因子表达量相比于对照组为分别为53.4%、53.8%(表2)。结果表明了,模拟消化对β-乳球蛋白酶解液的抗炎活性影响不大。
实施例3
将底物浓度为4g/L的β-乳球蛋白溶液200mL加入超声袋中,密封,然后将样品超声袋直接置于超声反应釜中,三频(20/28/40kHz)超声处理。超声处理时间10min,间歇比10s/3s,温度25℃。
β-乳球蛋白酶解液制备:调节上述蛋白溶液pH 8,加入嗜热蛋白酶,酶与底物的比例为1:50(w/w),均匀混合,酶解温度70℃,酶解时间2h。酶解结束后调节混合液pH为7.0,并在沸水浴中灭酶10min,离心获得上清液,脱盐、浓缩、冷冻干燥成粉末,测其水解度,蛋白转化率,以及对EA.hy926细胞的抗炎活性。
模拟胃肠道消化:根据美国药典(USP30-NF25)配置人工胃液。在37℃下,将β-乳球蛋白水解物与胃液以1:50混合,在震荡频率为180rpm条件下模拟胃消化2h。用NaOH调节pH为6.8,以1:100(w/v)加入胰酶至混合液,继续酶解4h。沸水浴灭酶10min,离心取上清液,脱盐、浓缩、冷冻干燥成粉末。测4h和10后的消化产物对EA.hy926细胞的抗炎活性。
相比于传统酶解,三频超声预处理后,β-乳球蛋白的水解度由13.20%提高到了21.41%,蛋白转化率由36.90%提高到了41.02%(表1)。
表1.超声预处理对不同酶酶解β-乳球蛋白的水解度、蛋白转化率的影响
碱性蛋白酶酶解β-乳球蛋白所得β-乳球蛋白酶解液对血管内皮细胞具有良好的抗炎效果,对血管内皮细胞VCAM-1、ICAM-1炎症因子表达产生了显著影响,相比于对照组的,其表达量分别为48.9%、36.5%;模拟胃消化后,其酶解液的消化产物对血管内皮细胞仍然具有良好的抗炎效果,VCAM-1、ICAM-1炎症因子表达量相比于对照组分别为43.3%、30.1%;模拟肠消化后,最终消化产物对血管内皮细胞依然保持良好的抗炎效果,VCAM-1、ICAM-1炎症因子表达量相比于对照组为分别为49.1%、36.2%(表2)。结果表明了,模拟消化对β-乳球蛋白酶解液的抗炎活性影响不大。
表2.不同β-乳球蛋白水解产物在模拟胃肠道消化前、消化中、消化结束后对TNF-α诱导EA.hy926细胞的炎症因子ICAM-1,VCAM-1表达
实施例4选择实施例3的消化产物多肽进行Caco-2模拟小肠内皮细胞吸收:
检测β-乳球蛋白多肽对Caco-2的细胞毒性。进行Caco-2模拟小肠内皮细胞吸收模型的建立:Caco-2细胞以2×105cells/mL接种于12孔Transwell板的滤膜上,隔天换一次培养液,21天后测定Caco-2细胞模型的评价指标:上皮细胞电阻,碱性磷酸酶活力,荧光素钠渗漏实验。实验开始前用HBSS缓冲液清洗Caco-2细胞,在绒毛面AP侧加入由HBSS缓冲液配制的β-乳球蛋白多肽0.5ml,浓度为20mg/mL,在BL侧加入HBSS缓冲液1.5mL,置于37℃,5%CO2培养箱中转运4h,在0.5h、1h、2h从BL侧吸取0.2ml转运的β-乳球蛋白多肽检测β-乳球蛋白多肽的Caco-2细胞的吸收率。在Caco-2模拟小肠内皮细胞吸收4h的时候收集所有的AP侧(apical,肠腔侧)和BL侧(basolateral,肠内壁侧)的多肽分别测定其对EA.hy926的抗炎活性;并且采用UPLC-MC分析肽序:对β-乳球蛋白多肽消化产物以及经过Caco-2细胞吸收的多肽进行多肽序列分析,筛选出离子强度强的小肽;并且对这些多肽进行合成,验证合成的多肽的抗炎活性。
表3.β-乳球蛋白多肽(嗜热蛋白酶酶解)消化产物对Caco-2细胞的活力影响
多肽浓度(mg/ml) | Caco-2细胞活力 | |
对照 | - | 100 |
组1 | 5 | 125.8±4.7 |
组2 | 10 | 124.3±9.0 |
组3 | 20 | 110.2±10.8 |
组4 | 50 | 106.5±8.0 |
表3显示加入β-乳球蛋白多肽后,Caco-2细胞的活力不仅没有下降,反而有所上升,该结果表明了β-乳球蛋白多肽对Caco-2细胞没有任何毒性;β-乳球蛋白水解产物的模拟消化后所得多肽经过Caco-2细胞的吸收,随着转运时间的延长,其多肽的转运率也随着增加,在4h结束转运的时候,其转运率达到了2.67%,表明了Caco-2细胞对多肽具有选择性的吸收(表4);β-乳球蛋白多肽经过Caco-2模拟小肠内皮细胞吸收后,其BL侧的多肽对血管内皮细胞的抗炎效果大幅度提高,其VCAM-1、ICAM-1炎症因子表达量相比于对照组分别为22.1%、16.9%,和AP侧的多肽相比,其表达量分别降低了17.0%、19.3%(表2)。
表4.β-乳球蛋白多肽(嗜热蛋白酶酶解)消化产物对在Caco-2模拟小肠吸收的0.5h,1.0h,2.0h和4.0h相对应的多肽穿透率
穿透时间(h) | 穿透率(%) |
0.5 | 0.22±0.01 |
1 | 0.71±0.02 |
2 | 1.35±0.01 |
4 | 2.67±0.03 |
上述结果表明了β-乳球蛋白多肽经过Caco-2模拟小肠内皮细胞吸收后,其抗炎活性大幅度提高,间接表明了吸收的多肽具有很好的抗炎活性。对β-乳球蛋白多肽消化产物以及经过Caco-2细胞吸收的多肽的结构进行鉴定、分析,筛选出离子强度强的小肽进行人工合成,检测其抗炎活性。经过活性筛选,最后获得两种高抗炎活性的β-乳球蛋白肽,其结构为Phe-Tyr-Gln-Ala(FYQA)、Leu-Gln-Tyr(LQY)。两种多肽都对血管内皮细胞具有极好的抗炎活性,在低浓度100μM的时候,其对VCAM-1炎症因子的表达量相比于对照组分别为41.3%、33.7%;其对ICAM-1炎症因子的表达量相比于对照组分别为56.6%、48.2%(表5)。
表5.不同β-乳球蛋白水解产物在模拟胃肠道消化前、消化中、消化结束后对TNF-α诱导EA.hy926细胞的炎症因子ICAM-1,VCAM-1表达
Claims (9)
1.β-乳球蛋白活性多肽,其特征在于其氨基酸序列为:
Phe-Tyr-Gln-Ala
Leu-Gln-Tyr。
2.根据权利要求1所述的β-乳球蛋白活性多肽,其特征在于其氨基酸序列为:
Leu-Gln-Tyr。
3.根据权利要求1或2所述的β-乳球蛋白活性多肽的制备方法,其特征在于按照下述步骤进行:
(1)β-乳球蛋白的提取
将乳清粉用蒸馏水充分搅拌溶解配成质量浓度为7%的蛋白溶液,然后加入NaCl使其最终的质量浓度为7%,用HCl调节pH至2,用高速离心机在5000r/min下离心20min,收集上清液;用分子截量为14000Da的透析袋取一定量的上清液,放于30倍体积的蒸馏水中静置20h,收集透析袋中的溶液;得到β-乳球蛋白溶液;
(2)超声预处理β-乳球蛋白:将底物浓度为1~4g/L的β-乳球蛋白溶液加入超声袋中,密封,然后将样品超声袋直接置于超声反应釜中,进行超声波处理;
(3)β-乳球蛋白酶解液制备:调节上述蛋白溶液pH 7.5~8.0,加入蛋白酶,酶与底物的比例为1:20~50(w/w),均匀混合,酶解温度50~70℃,酶解时间2~4h;酶解结束后调节混合液pH为7.0,并在沸水浴中灭酶10min,离心获得上清液,脱盐、浓缩、冷冻干燥成粉末;
(4)模拟胃肠道消化:配置人工胃液;在37℃下,将将步骤(3)制备得到的β-乳球蛋白水解物与胃液以1:20~50混合,在震荡频率为120~180rpm条件下模拟胃消化2~4h;用NaOH调节pH为6.8,以1:100(w/v)加入胰酶至混合液,继续酶解4~6h;沸水浴灭酶10min,离心取上清液,脱盐、浓缩、冷冻干燥成粉末;
(5)Caco-2模拟小肠上皮细胞吸收:构建Caco-2细胞转运模型,将步骤(4)制备得到的β-乳球蛋白多肽消化产物配置成20mg/ml,加入到Caco-2细胞的绒毛面AP侧(apical,肠腔侧),0.5~4h后,收集基底面BL侧(basolateral,肠内壁侧)转运的β-乳球蛋白肽,脱盐、冷冻干燥;
(6)采用UPLC-MC鉴定及分析多肽序列::对步骤(5)经过Caco-2细胞吸收的β-乳球蛋白多肽进行鉴定及多肽序列分析,筛选出离子强度强,小于1000的多肽序列;
(7)多肽的合成以及活性验证:对步骤(6)筛选出来的多肽进行合成,并验证其抗炎活性,最终验证得到两种β-乳球蛋白抗炎活性多肽,其氨基酸序列为:
Phe-Tyr-Gln-Ala
Leu-Gln-Tyr。
4.根据权利要求3所述的β-乳球蛋白活性多肽的制备方法,其特征在于其中步骤(2)所述的超声波处理工艺条件如下:超声处理,超声处理时间10-30min,间歇比10s/3s,温度25℃;超声波处理频率及频率组合为:单频:20kHz、28kHz、40kHz;双频:20/28kHz、20/40kHz、28/40kHz;三频:20/28/40kHz。
5.根据权利要求4所述的β-乳球蛋白活性多肽的制备方法,其特征在于超声波处理频率及频率组合为三频:20/28/40kHz。
6.根据权利要求3所述的β-乳球蛋白活性多肽的制备方法,其特征在于其中步骤(3)所述的蛋白酶为碱性蛋白酶、木瓜蛋白酶、中性蛋白酶或嗜热蛋白酶。
7.根据权利要求6所述的β-乳球蛋白活性多肽的制备方法,其特征在于其中步骤(3)所述的蛋白酶为嗜热蛋白酶。
8.根据权利要求3所述的β-乳球蛋白活性多肽的制备方法,其特征在于其中步骤(6)中的β-乳球蛋白活性多肽的氨基酸序列为Leu-Gln-Tyr。
9.根据权利要求1或2所述的β-乳球蛋白活性多肽的作为保健食品应用,经过公知方法的制造成胶囊或者片剂,作为抗炎的保健食品或者功能食品。
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