CN115093456A - 一种具有抗氧化活性的杏仁多肽及其提取方法、应用 - Google Patents
一种具有抗氧化活性的杏仁多肽及其提取方法、应用 Download PDFInfo
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Abstract
本发明属于多肽提取技术领域,具体涉及一种具有抗氧化活性的杏仁多肽及其提取方法、应用。所述杏仁多肽的序列为:肽段1如SEQ ID NO.1所示:TEDDWRWH;肽段2如SEQ ID NO.2所示:WYDNEWGYR;肽段3如SEQ ID NO.3所示:AEDHEWWWR。本发明分离得到的肽段,分子量分布在814.46~1648.70 Da之间,通过受体‑配体相互作用发现杏仁肽可能通过竞争性结合keap1和抑制keap1‑Nrf2相互作用来释放Nrf2因子,抗氧化活性高。
Description
技术领域
本发明属于多肽提取技术领域,具体涉及一种具有抗氧化活性的杏仁多肽及其提取方法、应用。
背景技术
杏仁是我国传统药食两用资源,含有丰富的蛋白质,是制备生物活性肽的良好原料。细胞内积累过多的自由基和活性氧会产生氧化应激,进而导致生物体受损。尽管内源性抗氧化防御系统起主要作用,但外源性抗氧化剂,尤其是食源性天然抗氧化剂,对于维持人体细胞的氧化还原稳态非常重要。
随着仁用杏产量的不断提高,国内对杏仁的研究日益看中,但是国内研究主要集中于杏仁油以及苦杏仁苷上,对生产过程中的副产物脱脂杏仁饼粕利用较少,大量的杏仁饼粕被做成饲料,杏仁蛋白的利用以及精深加工没有得到充分发挥。对于其中具有抗氧化活性多肽的研究较少。
发明内容
针对现有技术中存在的问题,本发明提供了一种具有抗氧化活性的杏仁多肽。
本发明还提供了一种具有抗氧化活性的杏仁多肽的提取方法。
本发明的另一目的为提供了一种杏仁多肽在抗氧化活性产品中的应用。
本发明为了实现上述目的所采用的技术方案为:
本发明提供了一种具有抗氧化活性的杏仁多肽,所述杏仁多肽的序列为:
肽段1如SEQ ID NO.1所示:TEDDWRWH;
肽段2如SEQ ID NO.2所示:WYDNEWGYR;
肽段3如SEQ ID NO.3所示:AEDHEWWWR。
本发明还提供了一种杏仁多肽的提取方法,包括以下步骤:
(1)称取脱脂杏仁粉置于烧杯中,加入去离子水,调pH值至8,超声提取,然后将提取液离心,留上清液;将所得的上清液调节pH值至4.5,静置,再次离心,收集沉淀后水洗,将沉淀置于真空冷冻干燥机中冻干获得杏仁蛋白;
(2)称取杏仁蛋白溶于去离子水配置成蛋白质溶液,水浴预热,之后加入蛋白酶,酶解,过程中保持蛋白液pH不变,;酶解完成后,终止反应,然后离心,取上清液,真空冷冻干燥得杏仁多肽粉,记为APHs;
(3)将APHs依次经过截留分子量为3 kDa,10 kDa的切向错流膜分离,得到分子量<3 kDa、3 kDa~10 kDa、>10 kDa 三个组分,收集<3 kDa、3 kDa~10 kDa、>10 kDa三个组分并冻干,分别记作APHs-1、APHs-2、APHs-3;
(4)使用Sephadex G-25作为柱层析填料对APHs-1进行分离,在220 nm波长下,按出峰顺序命名为APHs-1-a、APHs-1-b、APHs-1-c;洗脱多次分别收集三个组份的洗脱液,真空冷冻干燥后备用;
(5)对APHs-1-c样品采用0.15%乙酸复溶;取多肽萃取液,0.15%乙酸清洗2次,所得滤液使用C18 StageTip脱盐,真空冷冻干燥;干燥后肽段用0.1% TFA复溶,260 nm测定肽段浓度,进行LC-MS/MS分析及定性分析,根据-CDOCKER-Energy值筛选出杏仁多肽。
进一步的,步骤(1)中,所述脱脂杏仁粉和去离子水的液料比为25-35:1;所述超声提取的条件为:功率200-300W,时间20-40min;所述离心为7000 r/min条件下离心20 min;所述再次离心为7000 r/min条件下离心15min。
进一步的,步骤(2)中,所述蛋白质溶液的质量分数为2%;所述水浴预热至70℃;所述蛋白酶为中性蛋白酶,复合蛋白酶,碱性蛋白酶,风味蛋白酶和木瓜蛋白酶中的一种或几种;所述酶的加入量为4-6%;所述酶解的温度为45-55℃、pH为6.0-10.0、酶解时间为6h;所述终止反应为100℃下加热15min;所述离心为7000 r/min条件下离心20 min。
本发明所使用的LC-MS/MS分析具体条件为:使用EasynLC1200色谱系统进行色谱分离;缓冲液:A液为0.1%甲酸,B液为0.1%甲酸、80%乙腈;样品进样到100 µm×20 mm×5 µm,C18的Trap Column后经过75 µm×150 mm×3 µm,C18的色谱分析柱进行梯度分离,流速为300 nl/min;液相分离梯度如下:0-2 min,2%~5%B液;2-44 min,5%~28% B液;44-51 min,28%~40% B液;51-53 min,40%~100% B液;53-60 min,100% B液。
上述质谱分析的检测模式:正离子,母离子扫描范围:350-1800 m/z,一级质谱分辨率:60,000 @m/z 200,AGC target:3e6,一级Maximum IT:50 ms;肽段二级质谱分析按照下列方法采集:每次全扫描后触发采集20个最高强度母离子的二级质谱图谱,二级质谱分辨率:15,000 @ m/z 200,AGC目标:1e5,最大 IT: 50 ms,二级质谱解离模式: HCD,隔离窗口:1.6 m/z,NCE: 28。
本发明还提供了一种杏仁多肽在制备抗氧化产品中的应用。
本发明通过分子对接预测杏仁肽与Keap1的结合能力。首先从RCSB蛋白质数据库中获得Keap1(kelch-like ECH-associated protein 1)的X射线晶体结构,将其定义为受体。通过Discovery Studio软件对受体靶标去除水分子、添加氢原子后,在“Receptor-Ligand Interactions”模块中定义其活性中心(坐标:x=7.449330,y=8.441914,z=1.674659;半径:9.22 Å)。所选杏仁肽的结构由Discovery Studio 2014客户端软件绘制,其能量通过CHARMm力场最小化。将这些肽定义为配体。使用-CDOCKER-协议进行对接。-CDOCKER-能量高的Keap1-多肽复合物被认为是最稳定的构象。此外,通过受体-配体相互作用图,获得杏仁肽与Keap1的相互作用机制。
本发明的有益效果为:
(1)本发明采用超声辅助法提取杏仁蛋白,提高了蛋白提取率,通过各步骤协同作用下,本发明杏仁蛋白提取率达(61.57±0.364)%;
(2)本发明提取的杏仁蛋白溶解性、持水性、持油性、起泡性、泡沫稳定性、乳化性、乳化稳定性等理化性能优异,能够作为食品添加剂进行使用。
(3)本发明分离得到的杏仁蛋白水解物APHs-1-c对TBHP引起的HepG2细胞氧化损伤具有显著的保护作用,同时对于细胞内ROS具有非常强的清除作用。APHs-1-c还可以通过激活Keap1-Nrf2/ARE通路保护HepG2细胞免受TBHP引起的氧化应激损伤;
(4)本发明分离得到的肽段,分子量分布在814.46~1648.70 Da之间,通过受体-配体相互作用发现杏仁肽可能通过竞争性结合 keap1和抑制 keap1-Nrf2相互作用来释放Nrf2因子,抗氧化活性高。
附图说明
图1为实施例1中液料比对杏仁蛋白提取率的影响图。
图2为实施例1中温度对杏仁蛋白提取率的影响图。
图3为实施例1中时间对杏仁蛋白提取率的影响图。
图4为实施例1中超声功率对杏仁蛋白提取率的影响图。
图5为实施例2中不同蛋白酶酶解对杏仁蛋白水解度的影响图。
图6为实施例2中不同蛋白酶酶解对杏仁蛋白水解产物DPPH自由基清除率的影响图。
图7为实施例2中酶用量对蛋白水解度和DPPH自由基清除能力的影响图。
图8为实施例2中酶解时间对蛋白水解度和DPPH自由基清除能力的影响图。
图9为实施例3中超滤后各组分ABTS自由基清除率图。
图10为实施例3中 APHs-1的洗脱曲线图。
图11为实施例3中APHs-1-c对氧化损伤HepG2细胞存活率的影响图。
图12为实施例3中APHs-1-c对HepG2细胞内ROS清除能力的影响图。
图13 为实施例3中APHs-1-c对HeapG2细胞中Keap1、Nrf2和HO-1表达的影响图。
图14为实施例4中分离得到的APHs-1-c质谱基峰图。
图15为肽段TEDDWRWH -Keap1相互作用图。
图16为肽段 WYDNEWGYR -Keap1相互作用图。
图17为肽段 AEDHEWWWR-Keap1相互作用图。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步的解释和说明。
本发明所使用的杏仁粉为珍珠油杏的脱脂杏仁粉,杏仁品种为珍珠油杏,由山东省农业科学院果树研究所牛庆霖副研究员鉴定。杏仁粉碎后过40目筛,用正己烷在常温下将杏仁粉搅拌浸提脱脂。脱脂后的杏仁粉于通风橱内晾干,-20℃保存备用。
其基本成分如表1所示。
表1
实施例1 珍珠油杏杏仁蛋白的提取
称取脱脂后的杏仁粉100 g置于烧杯中,加入300g去离子水,用1 mol/L NaOH溶液调pH值,设置超声功率、温度及超声时间;将提取液7000 r/min离心20 min,留上清液。将所得的上清液用1 mol/L HCL调节pH值至4.5,静置30 min后,7000 r/min离心15 min,收集沉淀后水洗3次,将沉淀置于真空冷冻干燥机中冻干获得杏仁蛋白,-20℃保存备用。经凯氏定氮法检测,杏仁蛋白粉的纯度为86.5%。
随着液料比的增加,超声条件:250w、45℃条件下提取30min;杏仁蛋白提取率呈现先增加后平缓的趋势,由图1可见,当液料比为30:1时,杏仁蛋白提取率达到最大(53.84%),随着液料比的进一步增大,蛋白提取率趋于平缓,表明此条件下杏仁蛋白的溶出量已达到最大值。此外,随着料液比的进一步加大,一些非蛋白物质成分也逐渐增加,反应体系也逐渐变大,不利于后续蛋白的沉淀与收集。
随着温度的升高(其他条件同上),杏仁蛋白的提取率呈现先增加后降低的趋势。由图2可见,当温度为45℃时,杏仁蛋白提取率达到最大,为55.17%。当温度大于45℃时,杏仁蛋白提取率明显降低,可能是由于温度过高导致蛋白质发生变性等不可逆的化学变化,导致蛋白质不易溶出。
随着提取时间的增加(其他条件同上),杏仁蛋白提取率逐渐升高。由图3所示,10min到30 min之间,随着时间的增加,杏仁蛋白提取率提高较为迅速。30 min之后,蛋白提取率虽然仍在提高,但接近平缓,这是由于随着时间的增加,杏仁粉逐步充分溶胀,蛋白溶出达到饱和。
随着超声功率(其他条件同上)的增加,杏仁蛋白提取率先升高后降低。由图4所示,当超声功率达到250 W时,杏仁蛋白提取率达到最大值。继续增加超声功率,杏仁蛋白提取率逐渐降低,可能是由于较强的超声功率产生过高的热效应,使部分蛋白质变性导致难以溶出。
实施例2 杏仁蛋白的酶解工艺
称取一定量的杏仁蛋白溶于去离子水配置成质量分数为2%的蛋白质溶液,70℃水浴预热15 min,之后加入适量蛋白酶,在55℃的酶解温度,pH=8条件下酶解一定的时间,过程中需持续滴加1 mol/L NaOH保持蛋白液pH不变,并记录NaOH溶液的消耗量,用于计算杏仁蛋白的水解度。酶解完成后100℃加热15min终止反应,然后后7000 r/min离心20 min取上清液,真空冷冻干燥得杏仁多肽粉,记为APHs,-20℃保存备用。
5种蛋白酶酶解杏仁蛋白,酶解2.5h,加酶量为6%,水解度随着时间变化的曲线如图 5所示。5种蛋白酶水解反应都符合典型的酶促进程曲线,前1h内,5种蛋白酶水解杏仁蛋白过程较为迅速。1h后只有碱性蛋白酶对杏仁蛋白的水解度仍具有较为明显的促进作用,其 余4种蛋白酶的作用逐渐变缓。
5种蛋白酶酶解生成的杏仁蛋白酶解产物对DPPH自由基清除率如图6所示。5种蛋白酶酶解得到的多肽的抗氧化能力不尽相同,其中碱性蛋白酶酶解产物抗氧化能力最强,DPPH自由基清除率达到49.87%。5种蛋白酶酶解产物对DPPH自由基清除清理清理强弱关系为:碱性蛋白酶>木瓜蛋白酶>复合蛋白酶>风味蛋白酶>中性蛋白酶。
如图7所示,选择碱性蛋白酶进行酶解,酶解2.5h,加酶量为6%,随着酶用量的增加,反应体系的水解度逐渐增大,随着酶用量的增加,蛋白质与酶的结合几率增大,促进水解度逐渐增加。随着酶用量的增加,酶解产物的DPPH自由基清除活性先呈现不断上升的趋势,当酶用量为5%时,DPPH自由基清除活性达到最大值(48.67%)。
如图8所示,选用碱性蛋白酶,酶解2.5h,加酶量为6%,随着酶解时间的增加,蛋白的水解度呈现逐渐上升的趋势。而酶解产物对DPPH自由基清除能力呈现先上升后下降的趋势,在2.5 h时清除率达到最大值47.86%。
实施例3 APHs的超滤分离
(1)将APHs依次经过截留分子量为3,10的切向错流膜分离,得到分子量<3 kDa、3kDa~10 kDa、>10 kDa 三个组分。收集<3 kDa、3 kDa~10 kDa、>10 kDa三个组分并冻干,记作APHs-1、APHs-2、APHs-3,-20℃保存备用。测定各超滤组分体外抗氧化能力进行比较。
(2)超滤后各组分ABTS自由基清除率如图9。由图9可知,在0.25~4.00 mg/mL浓度范围内,各组均表现出较强的ABTS自由基清除能力,且具有剂量依赖性。其中APHs-1在所有浓度都表现出较强的ABTS自由基清除活性,其IC50达到0.42 mg/mL。
(3)使用Sephadex G-25作为柱层析填料对APHs-1进行下一步分离,洗脱曲线见图10。在220 nm波长下检测到三个较为明显的洗脱峰,按出峰顺序命名为APHs-1-a、APHs-1-b、APHs-1-c。洗脱多次分别收集三个组份的洗脱液,真空冷冻干燥后备用。
具体步骤为:
(a)葡聚糖凝胶的预处理
称取葡聚糖凝胶G-25大约50 g,用去离子水洗去悬浮的破碎颗粒,于500 mL无水乙醇中浸泡2 h,用去离子水充分洗涤,之后在沸水浴中加热搅拌6 h,使其充分溶胀,冷却后装柱。
(b)装柱
选取长度100 cm,直径1.6 cm的层析柱,垂直固定于铁架台上,在层析柱中倒入1/3柱体积的去离子水,将溶胀好的葡聚糖凝胶G-25自柱子顶端边搅拌边缓慢倒入柱子中,使凝胶在柱中自然沉降,同时打开柱子底端,使柱中去离子水经细管缓慢流出。装好的葡聚糖凝胶层析柱必须无分层无气泡,凝胶上层有5 cm水柱,之后用相当于柱体积3倍的去离子水对凝胶层析柱进行平衡。
(c)加样及洗脱
将凝胶上层水柱流干或吸干,自柱子顶端加入3 mL 50 mg/mL样品液,打开柱子底端,待样品液与凝胶柱最高点重合时,向柱中加入3 cm水柱。之后用流速为1.5 mL/min的去离子水对样品进行洗脱。
(d)样品收集
在220 nm紫外波长下对洗脱液进行检测,洗脱液用自动部分收集器进行收集,每4min收集一管,根据吸收值曲线中分离峰出现的时间合并同一分离峰所对应的洗脱液,冷冻干燥后测定各洗脱组分体外抗氧化能力进行比较。
(4)APHs-1-c对HepG2细胞氧化损伤的保护作用
建立TBHP诱导HepG2细胞氧化损伤模型。不同浓度APHs-1-c对HepG2细胞保护作用如图11所示。由图中可知,加TBHP的损伤组细胞存活率为51.46%,约为空白组细胞存活率的一半,说明建模成功。在实验组中,APHs-1-c对TBHP诱导的HepG2细胞氧化损伤保护作用呈剂量依赖性,APHs-1-c浓度为800 μm/mL时,具有最大细胞存活率为89.61%。结果表明,APHs-1-c可以显著保护HepG2细胞免受TBHP引起的氧化损伤。
(5)APHs-1-c对HepG2细胞内ROS清除作用
利用DCFH-DA荧光探针,使用流式细胞仪测定细胞内活性氧的变化情况进行检测,结果如图12所示。HepG2细胞经TBHP诱导氧化损伤后,荧光强度明显上升,约为对照组的6倍,说明TBHP诱导HepG2细胞氧化应激导致细胞内大量累计活性氧。而经杏仁肽APHs-1-c预处理后,HepG2细胞内荧光强度随杏仁肽浓度的增大而明显降低,并具有明显的剂量依赖性,说明杏仁肽APHs-1-c能够有效地清除TBHP诱导HepG2细胞氧化应激产生的细胞内ROS。
(6)APHs-1-c对HepG2细胞内Keap1、Nrf2和HO-1表达的影响
如图13所示,与空白组相比,在TBHP作用下,Keap1蛋白表达水平略微上调,Nrf2和HO-1的蛋白表达水平下降,说明HeapG2细胞处于氧化应激状态。与损伤组相比,APHs-1-c给药组的Keap1蛋白的表达水平显著下调,Nrf2和HO-1的表达水平显著上调,并表现出剂量依赖性。试验结果表明,APHs-1-c可以通过激活Keap1-Nrf2/ARE通路来保护HepG2细胞免受TBHP引起的氧化应激损伤。
实施例4 定性分析
样品粉末取适量用0.15%乙酸复溶。取多肽萃取液,0.15%乙酸清洗2次,所得滤液使用C18 StageTip脱盐,真空冷冻干燥。干燥后肽段用0.1% TFA复溶,260 nm测定肽段浓度,以备LC-MS/MS分析。
(1)LC-MS/MS分析
对APHs-1-c进行质谱分析和搜库定性分析,APHs-1-c质谱Basepeak结果见图14。
取适量样品使用EasynLC1200色谱系统进行色谱分离。缓冲液:A液为0.1%甲酸,B液为0.1%甲酸、80%乙腈。色谱柱以100%的A液平衡。样品进样到Trap Column(100 µm×20mm×5 µm,C18)后经过色谱分析柱(75 µm×150 mm×3 µm,C18)进行梯度分离,流速为300nl/min。液相分离梯度如下:0-2 min,2%~5%B液;2-44 min,5%~28% B液;44-51 min,28%~40% B液;51-53 min,40%~100% B液;53-60 min,100% B液。
肽段分离后用Q-Exactive HF-X质谱仪进行DDA(数据依赖采集)质谱分析。分析时长为60 min,检测模式:正离子,母离子扫描范围:350-1800 m/z,一级质谱分辨率:60,000@m/z 200,AGC target:3e6,一级Maximum IT:50 ms。肽段二级质谱分析按照下列方法采集:每次全扫描后触发采集20个最高强度母离子的二级质谱图谱,二级质谱分辨率:15,000@ m/z 200,AGC target:1e5,二级Maximum IT: 50 ms,MS2 Activation Type: HCD,Isolation window:1.6 m/z,Normalized collisionenergy: 28。
(2)数据库检索
本发明采用的质谱数据库检索软件为PFind;使用如下蛋白质数据库:来源于Uniprot Protein Database,物种为杏仁,共53251条蛋白序列,下载于2021年11月22日。PFind分析参数设置如表2所示。
表2 PFind分析参数设置
共鉴定到40种杏仁活性肽,检测到的肽段氨基酸数目在7~13个之间,分子量分布在814.46~1648.70 Da之间。将keap1作为受体蛋白,根据-CDOCKER-Energy值筛选潜在抗氧化效果好的活性肽,结果如表3所示。40个杏仁肽中,有24个活性肽可与靶蛋白keap1发生不同程度的结合,-CDOCKER-Energy值最高的三个肽依次为TEDDWRWH(148.72 kcal/mol)、WYDNEWGYR(145.31 kcal/mol)、AEDHEWWWR(143.88 kcal/mol)。
表3APHs-1-c氨基酸序列鉴定结果
No. | Sequence | MH<sup>+</sup>(Da) | -CDOCKER-Energy(kcal/mol) |
1 | GPVWRSL | 814.456961 | 62.1695 |
2 | FYGPGGPY | 857.382798 | 67.5204 |
3 | APTKIWR | 871.514807 | 65.8605 |
4 | RWDGVPF | 876.436226 | 86.0308 |
5 | KFPIMPF | 879.47967 | 63.8257 |
6 | RWEGVPF | 890.451875 | 88.6921 |
7 | KIPDWFL | 918.508326 | 103.6570 |
8 | HKDPIFW | 942.483174 | 97.3578 |
9 | EPWWPKM | 973.459995 | 60.5327 |
10 | DWYKGPTL | 979.488317 | / |
11 | LRLPLWPS | 981.587967 | / |
12 | GKPIYHFM | 992.502191 | 64.8751 |
13 | WHDWDKL | 999.468251 | 117.5860 |
14 | GNEWKKPF | 1005.515198 | 111.3570 |
15 | KNSWGEDW | 1021.437344 | 132.5330 |
16 | KIPDWFLN | 1032.551248 | 105.4000 |
17 | RSTNLDWY | 1054.495189 | 101.5060 |
18 | FFDTPRTW | 1069.510112 | 97.7187 |
19 | NYDFRNPF | 1072.484626 | 118.8290 |
20 | FHLDRPMY | 1078.513815 | / |
21 | DWYKGPTLL | 1092.572375 | / |
22 | FQPFPRPPH | 1122.584276 | 53.0171 |
23 | TEDDWRWH | 1144.4806 | 148.7220 |
24 | YLEDFYRF | 1152.53599 | / |
25 | WYDNEWGYS | 1219.469034 | / |
26 | ILDEWKRLY | 1235.678228 | / |
27 | WDQPIRLPGY | 1244.642178 | 116.5020 |
28 | RWWNEINDL | 1245.601043 | / |
29 | MTEDDWRWH | 1275.521079 | 139.8610 |
30 | WYDNEWGYR | 1288.538111 | 145.3050 |
31 | AEDHEWWWR | 1314.564993 | 143.8810 |
32 | YDNDFGWGRPI | 1339.60652 | 139.6860 |
33 | YRHPWEDVLY | 1377.658554 | / |
34 | GRWEKPGHSPLF | 1410.727631 | / |
35 | HAHNPVDWYPW | 1421.638487 | / |
36 | APYDPDWYYIR | 1458.668782 | / |
37 | YRHPWEDVLYT | 1478.706227 | / |
38 | NWDDMEKIWHH | 1510.653145 | / |
39 | LAPYDPDWYYIR | 1571.75284 | / |
40 | VSWYDNEWGYSSR | 1648.702593 | / |
分子对接用于表明活性肽和keap1的确切结合位点。三种杏仁肽与keap1结合的对接结果如图15~17所示。与其他两种肽相比,TEDDWRWH含有更多的活性基团。从图15可发现TEDDWRWH可以与His254、Arg15、Tyr251和Asn61残基形成7个常规氢键,与Ser234、Arg15、Ser281、Gly43残基形成4个碳氢键,与Arg94、Arg59和Tyr13残基形成π-阳离子相互作用,与Pro63残基产生π-烷基相互作用。TEDDWRWH与Keap1的一些残基形成范德华相互作用,包括Phe14、Thr255、Ser42和Gly282等。上述结果表明,TEDDWRWH有可能与Keap1竞争性结合释放Nrf2,并通过激活Keap1-Nrf2/ARE途径在体内显示抗氧化活性。
Claims (7)
1.一种具有抗氧化活性的杏仁多肽,其特征在于,所述杏仁多肽的序列为:
肽段1如SEQ ID NO.1所示:TEDDWRWH;
肽段2如SEQ ID NO.2所示:WYDNEWGYR;
肽段3如SEQ ID NO.3所示:AEDHEWWWR。
2.一种如权利要求1所述的杏仁多肽的提取方法,其特征在于,包括以下步骤:
(1)称取脱脂杏仁粉置于烧杯中,加入去离子水,调pH值至8,超声提取,然后将提取液离心,留上清液;将所得的上清液调节pH值至4.5,静置,再次离心,收集沉淀后水洗,将沉淀置于真空冷冻干燥机中冻干获得杏仁蛋白;
(2)称取杏仁蛋白溶于去离子水配置成蛋白质溶液,水浴预热,之后加入蛋白酶,酶解,过程中保持蛋白液pH不变,;酶解完成后,终止反应,然后离心,取上清液,真空冷冻干燥得杏仁多肽粉,记为APHs;
(3)将APHs依次经过截留分子量为3 kDa,10 kDa的切向错流膜分离,得到分子量<3kDa、3 kDa~10 kDa、>10 kDa 三个组分,收集<3 kDa、3 kDa~10 kDa、>10 kDa三个组分并冻干,分别记作APHs-1、APHs-2、APHs-3;
(4)使用Sephadex G-25作为柱层析填料对APHs-1进行分离,在220 nm波长下,按出峰顺序命名为APHs-1-a、APHs-1-b、APHs-1-c;洗脱多次分别收集三个组份的洗脱液,真空冷冻干燥后备用;
(5)对APHs-1-c样品采用0.15%乙酸复溶;取多肽萃取液,0.15%乙酸清洗2次,所得滤液使用C18 StageTip脱盐,真空冷冻干燥;干燥后肽段用0.1% TFA复溶,260 nm测定肽段浓度,进行LC-MS/MS分析及定性分析,根据-CDOCKER-Energy值筛选出杏仁多肽。
3.根据权利要求2所述的提取方法,其特征在于,步骤(1)中,所述脱脂杏仁粉和去离子水的液料比为25-35:1;所述超声提取的条件为:功率200-300W,时间20-40min;所述离心为7000 r/min条件下离心20 min;所述再次离心为7000 r/min条件下离心15min。
4.根据权利要求2或3所述的提取方法,其特征在于,步骤(2)中,所述蛋白质溶液的质量分数为2%;所述水浴预热至70℃;所述蛋白酶为中性蛋白酶,复合蛋白酶,碱性蛋白酶,风味蛋白酶和木瓜蛋白酶中的一种或几种;所述酶的加入量为4-6%;所述酶解的温度为45-55℃、pH为6.0-10.0、酶解时间为6h;所述终止反应为100℃下加热15min;所述离心为7000 r/min条件下离心20 min。
5.根据权利要求2所述的提取方法,其特征在于,所述LC-MS/MS分析具体条件为:使用EasynLC1200色谱系统进行色谱分离;缓冲液:A液为0.1%甲酸,B液为0.1%甲酸、80%乙腈;样品进样到100 µm×20 mm×5 µm,C18的Trap Column后经过75 µm×150 mm×3 µm,C18的色谱分析柱进行梯度分离,流速为300 nl/min;液相分离梯度如下:0-2 min,2%~5%B液;2-44min,5%~28% B液;44-51 min,28%~40% B液;51-53 min,40%~100% B液;53-60 min,100% B液。
6.根据权利要求5所述的提取方法,其特征在于,所述质谱分析的检测模式:正离子,母离子扫描范围:350-1800 m/z,一级质谱分辨率:60,000 @m/z 200,AGC target:3e6,一级Maximum IT:50 ms;肽段二级质谱分析按照下列方法采集:每次全扫描后触发采集20个最高强度母离子的二级质谱图谱,二级质谱分辨率:15,000 @ m/z 200,AGC目标:1e5,最大IT: 50 ms,二级质谱解离模式: HCD,隔离窗口:1.6 m/z,NCE: 28。
7.一种如权利要求1所述的杏仁多肽在制备抗氧化产品中的应用。
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