CN113480608B - 一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法 - Google Patents
一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法 Download PDFInfo
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Abstract
一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法,氨基酸序列为:Lys‑Arg‑Gln‑Lys‑Tyr‑Asp。本发明所涉及的活性肽同时兼具改善肠道菌群和减少肝脏脂质堆积,缓解肝压力功能,具有结构简单、安全、活性强等特点,发挥出营养和保健的作用,有望为开发无毒副作用的缓解酒精性肝损伤新食品提供有效成分,具有广泛的应用前景。
Description
技术领域
本发明属于功能性多肽制备技术领域,涉及一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法。
背景技术
酒精性肝病(ALD)是一种由长期大量饮酒引起的破坏性肝脏疾病。其病理特点主要是肝内脂肪堆积,导致脂肪肝、肝纤维化和硬化。ALD是肝损伤疾病相关发病率和死亡率提高的主要原因,占全球因疾病死亡总人数的5.9%。虽然近年来研究重点开始聚焦于慢性酒精性肝损伤,但是ALD的发病机制尚未明确,可能的机制包括肠道稳态失衡,活性氧介导的氧化应激损伤,肝细胞脂质代谢紊乱和由病原体分子相关的脂多糖介导的肝细胞炎症。其中,调节肠道稳态和修复氧化应激损伤在治疗ALD的潜在靶点中发挥了重要作用。
在许多国家,肉制品中的干腌火腿都很受欢迎。在干腌火腿漫长的成熟过程中,火腿中多达10%的蛋白质被水解成多肽,这些多肽可以对人体产生特殊的生物学效应。例如从西班牙帕尔玛火腿中分离得到的多肽具有抗高血压、抗2-型糖尿病、抗氧化、抗炎和抗菌的活性。先前已有研究对中国的浙江金华火腿(JHP)中大量天然抗氧化肽进行了筛选和鉴定。然而,目前JHP的研究局限于抗氧化剂的试管实验,对JHP中是否存在缓解ALD的多肽尚未见报道。虽然有证据表明,增加合成抗氧化剂(美他多辛)、抗生素(阿莫西林克拉维酸、利福昔明、环丙沙星)和免疫抑制剂(利隆纳普)的应用可抑制ALD的发病过程,但是在治疗酒精性肝损伤方面生物活性肽相比抗生素更稳定、更安全也更易吸收。外源生物活性肽如蘑菇多糖肽、云芝多糖肽、灵芝多糖肽、海带多糖肽、玉米肽等均可减轻肝脏损伤。其中以干腌火腿肌球蛋白为主要源的多肽与缓解酒精性肝损伤关系紧密,在肝病保护中的应用具有良好前景。
发明内容
本发明的目的在于提供一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法。
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种可缓解酒精性肝损伤的干腌火腿源多肽,氨基酸序列为:Lys-Arg-Gln-Lys-Tyr-Asp。
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种可缓解酒精性肝损伤的干腌火腿源多肽的制备方法,包括下列步骤:
步骤1:将干腌火腿的股二头肌与磷酸盐缓冲液混合,然后于冰浴条件下用匀浆机进行均质,接着离心取上清,得到干腌火腿多肽提取物;
步骤2:对干腌火腿多肽提取物进行超滤,再用凝胶柱分离,洗脱条件为:洗脱液为0.01mol/L的HCl,恒温分离,流速0.8mL/min,用280nm紫外检测器测定馏分,用自动馏分收集器收集每管馏分;馏分在真空冷冻干燥机中干燥;
步骤3:采用液相分离将步骤2得到的缓解酒精性肝损伤最有效的组分进一步分离,获得最具缓解酒精性肝损伤活性的多肽;
步骤4:采用质谱对液相分离得到的最具缓解酒精性肝损伤活性的多肽进行分析,鉴定活性肽的结构,得到一种保护肝活性的肽序列为:Lys-Arg-Gln-Lys-Tyr-Asp。
优选的技术方案为:步骤3包括将冻干后的样品溶于1mL蒸馏水,注入HPLC系统,采用BEH C18色谱柱进行梯度洗脱,流速为0.3 mL/min,洗脱液A为0.1%甲酸,洗脱液B为100%乙腈;流速梯度为:0-10 min,100% A,10-22 min,30-80% B;22-23 min,100% A;肽峰在280nm紫外波长下检测;将多肽对应的峰分为七个部分,冷冻干燥;收集七个干腌火腿小肽分离组分,干燥后测定各个小肽的缓解酒精性肝损伤的活性。
优选的技术方案为:鉴定方法包括:采用Acquity高效液相色谱系统,采用反相BEHC18色谱柱对最具缓解酒精性肝损伤活性的多肽峰进行分离;梯度洗脱:流速0.3mL/min,洗脱液a为0.1%甲酸,洗脱液b为100% CAN;流动梯度为:0-10 min,100%a,10-22min,30-80%b;22-23 min,100% a;柱温保持在25℃;流量直接进入MS/MS系统进行多反应测量;前体离子记录的质量范围为m/z=200-4000;使用Mass Lynx V4.1操作仪器,分析质谱图信息。
由于上述技术方案运用,本发明与现有技术相比具有的优点是:
1、本发明的干腌火腿源的缓解酒精性肝损伤肽具有潜在的医学价值。
2、本发明所涉及的活性肽同时兼具改善肠道菌群和减少肝脏脂质堆积,缓解肝压力功能,具有结构简单、安全、活性强等特点,发挥出营养和保健的作用,有望为开发无毒副作用的缓解酒精性肝损伤新食品提供有效成分,具有广泛的应用前景。
附图说明
图1为排阻色谱法获得的肽对雄性小鼠酒精性肝损伤(ALD)的影响。
图2为空白组(CTRL)、酒精组(EtOH)、酒精+联苯双酯组(EtOH)、酒精+肽组(EtOH+JHP)喂食35天对小鼠肠道内酶表达的影响。
图3A为六肽Leu-Pro-Gly-Val-Leu-Pro-Val-Ala(KRQKYD)的HPLC图。
图3B为六肽Leu-Pro-Gly-Val-Leu-Pro-Val-Ala(KRQKYD)的一级质谱(MS)图。
图3C为六肽Leu-Pro-Gly-Val-Leu-Pro-Val-Ala(KRQKYD)的二级质谱(MS/MS)图。
图4为喂食对照饮食或含乙醇饮食(含或不含JHP)对小鼠肠道菌群的影响。
图5 为KRQKYD对小鼠肠道紧密连接的影响。
图6 为KRQKYD对小鼠肝脏氧化应激反应的影响。
图7为本发明的技术路线图。
具体实施方式
以下由特定的具体实施例说明本发明的实施方式,熟悉此技术的人士可由本说明书所揭露的内容轻易地了解本发明的其他优点及功效。
请参阅图1-7。须知,本说明书所附图式所绘示的结构、比例、大小等,均仅用以配合说明书所揭示的内容,以供熟悉此技术的人士了解与阅读,并非用以限定本发明可实施的限定条件,故不具技术上的实质意义,任何结构的修饰、比例关系的改变或大小的调整。提供以下实施例以便更好地理解本发明,而非限制本发明。以下实施例中的实验方法如无特殊说明,均为常规方法。下述实施例中所用的实验材料如无特殊说明,均为常规生化试剂商店购买所得。
实施例1:一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法
本发明的可缓解酒精性肝损伤的干腌火腿源多肽,其氨基酸序列为下:
六肽:Lys-Arg-Gln-Lys-Tyr-Asp;下文中,该六肽也可以简写为:KRQKYD。
本发明的火腿源缓解酒精性肝损伤六肽序列,包括所述活性六肽序列为核心。
本发明的干腌火腿源缓解酒精性肝损伤肽的制备方法,包括以下步骤:
(1)以金华火腿/宣威火腿/如皋火腿的股二头肌为原料进行均质
将20g股二头肌与80mL磷酸盐缓冲液(0.2mmol/L,pH7.2)混合,于冰浴下使用数显匀浆机均质;均质4次;每次10s;22000rpm。
(2)离心
对步骤(1)得到的浆料进行离心,取上清液。离心时离心机设置转速12000g;时间20min;温度4℃。
(3)对多肽提取物进行超滤和凝胶过滤
对干腌火腿多肽提取物进行超滤,然后用凝胶柱分离,洗脱条件为:洗脱液为0.01mol/L的HCl,恒温分离,流速0.8 mL/min。用280nm紫外检测器(AmershamBiosciences)测定馏分,用自动馏分收集器收集每管馏分。馏分在真空冷冻干燥机中干燥以供进一步分析。
超滤的工艺参数为:P/N: S02-E003-05-N,介质/额定值:mPES/3KDa,表面积:790cm2。
真空冷冻干燥的工艺条件为:-40℃~-50℃,24h。
(4)干腌火腿粗肽提取物的反相高效液相色谱(RP-HPLC)分离纯化
将步骤(3)得到的缓解酒精性肝损伤最有效的组分进一步分离。步骤(4)具体为:将冻干后的样品溶于1mL蒸馏水,注入HPLC系统,采用BEH C18色谱柱(1.7μm,2.1 × 100mm,Waters Inc.,Milford,MA,USA)进行梯度洗脱,流速为0.3 mL/min,洗脱液A为0.1%甲酸,洗脱液B为100%乙腈。流速梯度为:0-10 min,100% A;10-22 min,30-80% B;22-23 min,100% A。肽峰在280 nm紫外波长下检测。将多肽对应的峰分为七个部分,冷冻干燥;收集七个干腌火腿小肽分离组分,干燥测定各个小肽缓解酒精性肝损伤的活性。
(5)缓解酒精性肝损伤活性肽结构鉴定
采用质谱对液相分离得到的最具缓解酒精性肝损伤的活性峰进行分析,鉴定活性肽的结构,得到一种保护肝活性的肽序列:KRQKYD;步骤(5)具体技术方案为:采用Acquity(Waters Inc.)高效液相色谱系统,采用反相BEH C18色谱柱(1.7μm,2.1 × 100 mm,Waters Inc.)对具有缓解酒精性肝损伤最有效的峰进行分离。梯度洗脱:流速0.3 mL/min,洗脱液a为0.1%甲酸,洗脱液b为100%ACN。流动梯度为:0-10min,100% a;10-22min,30-80%b;22-23min,100% a。柱温保持在25℃。流量直接进入MS/MS系统进行多反应测量。前体离子记录的质量范围为m/z=200-4000。使用Mass Lynx V4.1操作仪器,分析质谱图信息。
(6)人工合成KRQKYD并验证其抗炎活性。
步骤(6)中人工合成KRQKYD方法如下:
1、准确称量0.2mmol树脂(2-氯三酰氯树脂),分别加入3ml二甲基甲酰胺(DMF)、二氯甲烷(DCM)溶胀树脂30min;
2、按缩合剂(HCTU): 氨基酸:碱(DIEA):树脂= 4:4:8:1置入10ml EP管中备用;
3、连接第一氨基酸(不加HCTU),将氨基酸溶于3mL DMF中,加入132μl DIEA,摇匀3min,倒入合成管,将合成管置于45℃空气浴摇床中8h;
4、树脂清洗:先用DMF洗3次,再用DCM洗3次,最后用DMF洗3次;
5、使用5%甲醇溶液封口:合成管中放入5ml 1%甲醇,摇匀30min;
6、清洗树脂:重复步骤4的内容;
7、去除氨基保护基团(Fmoc):加入20%哌啶试剂摇匀5min;
8、连接氨基酸:将称好的氨基酸与缩合后的混合物溶解在3mL DMF中,加入132μLDIEA,摇匀3min,倒入合成管,摇匀30min,重复步骤6、7、8依次插入所需氨基酸,使肽链延伸;
9、清洗树脂:重复步骤4、的内容;
10、沥干树脂,用准备好的切割液(TFA:苯酚:水:TIPS=88:5:5:2)切割肽段;
11、将溶液压出,用氮气浓缩至5ml;
12、加入35mL冰乙醚沉淀肽,3500g离心15min。沉淀的肽为目标肽。采用BEH C18色谱柱(1.7 μm,2.1 × 100 mm,Waters Inc., Milford,MA,USA),采用反相高效液相色谱(RP-HPLC)对合成的多肽进行纯化。流动相为0.1%甲酸(溶剂A)和100%乙腈(溶剂B),流动相为:0-10 min,100%溶剂A;10-22 min,30-80%溶剂B;22-23 min,100%溶剂A,流速0.3ml/min。分离过程在280 nm紫外波长下进行检测。用液相色谱-质谱联用法对合成的多肽进行鉴定。
本发明的干腌火腿源的缓解酒精性肝损伤的肽同时具有改善肠道菌群和降低肝细胞氧化应激损伤的功能,非常适合用于制备缓解肠道和肝脏不适的食品以及修复酒精性肝损伤的药品。
干腌火腿源缓解酒精性肝损伤肽(JHP)对雄性小鼠酒精性肝损伤(ALD)的影响;JHP是一种分子量范围广泛的多肽的复杂混合物。确定36个月的干腌火腿中的功能性成分对ALD预防作用。
组分A由于分子量最大首先被洗脱出来,而组分I分子量最小,最后被洗脱出来。收集所有馏分,冷冻干燥。用36个月的干腌火腿中分离的不同组分喂养194只小鼠,测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)和丙二醛(MDA)(图1的B-D)。G组ALT (128.89 U/L)、AST (294.39 U/L)、MDA (80.47 mmol/mL)水平均低于其他各组。因此,JHP-G具有最大的缓解酒精性肝损伤特性。为了进一步分析,JHP-G在真空冷冻干燥机中干燥。
雄性小鼠中分别喂食对照饮食或含乙醇饮食(含或不含JHP)35天血清中谷丙转氨酶ALT(B)、谷草转氨酶AST(C)和丙二醛MDA(D)浓度的影响;
采用反相高效液相色谱法进一步纯化G组分,该组分存在分子间极性差异。从G-1到G-7共分离出7个馏分(图2的A)。将分离的馏分冻干,测定其对ALD的活性(图2的B-D)。G-6部位具有最大的缓解损伤作用。
采用反相高效液相色谱质谱联用(LC-MS/MS)对G-6进行氨基酸测序。发现是来自于Lys-Arg-Gln-Lys-Tyr-Asp(KRQKYD)。
对合成的干腌火腿源缓解酒精性肝损伤肽进行结构鉴定(图3);
采用Acquity(Waters Inc.)高效液相色谱系统,采用反相BEH C18色谱柱(1.7μm,2.1 × 100 mm,Waters Inc.)对具有缓解酒精性肝损伤最有效的峰进行分离。
梯度洗脱:流速0.3 mL/min,洗脱液a为0.1%甲酸,洗脱液b为100% ACN。流动梯度为:0 -10 min,100% a;10-22min,30-80% b;22-23 min,100% a。柱温保持在25℃。流量直接进入MS/MS系统进行多反应测量。
前体离子记录的质量范围为m/z=200-4000。使用Mass Lynx V4.1操作仪器,分析质谱图信息。
KRQKYD通过抑制氧化应激对小鼠酒精性肝损伤的影响;
为了确定KRQKYD是否影响酒精处理小鼠肝脏氧化应激损伤,需要检测肝脏中活性氧(ROS)、超氧化物歧化酶(SOD)和谷胱甘肽氧化酶(GSH-Px)的水平以及CYP2E1、Nrf2和HO-1基因的表达。
如图4的A所示,酒精处理(EtOH)组的活性氧水平(161.21 U/mg prot)高于空白组(94.66 U/mg prot),说明酒精处理小鼠产生了更多的活性氧。KRQKYD处理组活性氧水平为71.19 U/mg prot,低于对照组。这表明KRQKYD预处理显著降低了EtOH组活性氧水平。饮酒可显著降低肝脏中GSH-Px和SOD的水平,如图4的B所示。
KRQKYD预处理显著改善了EtOH组GSH301 Px和SOD活性的下降。结果表明,KRQKYD可以通过抑制活性氧的产生,提高GSH-Px和SOD活性来减少酒精诱导的肝脏氧化应激损伤;KRQKYD对ALD的保护作用可能是通过下调CYP2E1的表达降低氧化应激,并通过激活Nrf2/HO319通路增强氧化防御系统。
KRQKYD对酒精处理小鼠(EtOH组)GM谱的影响;
KRQKYD对酒精处理小鼠GM谱的影响通过16S rRNA高通量测序分析结肠微生物区系组成。共在32个样本(每组n=8)中检测到4201828个细菌16S rRNA,每个样本有131307个,共检测到749个不同的操作分类单元。
EtOH组的Chao1和Shannon指数明显高于空白组,KRQKYD组,和EtOH+ KRQKYD 组,这些结果表明酒精摄入显著提高了变形杆菌的比例,而KRQKYD预处理显著降低了酒精处理小鼠肠道中变形杆菌的比例。
在门水平上,GM主要由疣微菌门、拟杆菌门、放线菌门、厚壁菌门和变形菌门组成(图5的A)。EtOH组疣微菌门、放线菌门和脱硫弧菌门的比例明显减少,而KRQKYD预处理后的呈进行性增加。酒精摄入显著提高了变形杆菌的比例,而KRQKYD预处理显著降低了酒精处理小鼠的变形杆菌比例。
在属水平上, EtOH处理组比空白组大肠杆菌和肠球菌的丰度高,而艾克曼菌、杜博氏菌和脱硫弧菌的丰度低(图5的B)。KRQKYD预处理后,艾克曼菌、杜博氏菌和脱硫弧菌的相对丰度增加,大肠杆菌、志贺氏菌和肠球菌的相对丰度降低。这表明KRQKYD对肝脏的保护作用可能与调节酒精诱导小鼠的细菌组成有关。
KRQKYD可提高对紧密蛋白的表达继而影响肠道稳态;
首先进行阿利新蓝染色以确定KRQKYD是否影响酒精处理小鼠的肠道内稳态根据结肠的组织病理学分析(图6的A)。用Western blot法检测Reg3g和Reg3b的表达,进一步探讨KRQKYD对肠道平衡的保护作用。
如图6的B-D所示,与空白组相比,酒精处理(Etoh处理)小鼠Reg3b和Reg3g蛋白表达水平略低。然而,用KRQKYD预处理后,在酒精处理过的小鼠中,这些蛋白的含量越来越高。采用RT-qPCR检测结肠中密封蛋白、封闭蛋白-1和紧密连接-1的mRNA表达水平。图6的A显示,酒精处理后小鼠密封蛋白、封闭蛋白-1和紧密连接-1的mRNA表达水平明显降低,而经KRQKYD预处理的小鼠则相反,mRNA表达水平明显升高。证实出调控机制包括了封闭蛋白-1、密封蛋白和紧密连接-1,使用免疫组织化学和Western -blot技术评估了上游信号通路蛋白的变化。如图6的B-Etoh处理小鼠的密封蛋白、封闭蛋白-1和紧密连接-1蛋白表达水平明显降低。
KRQKYD预处理可逐步提高酒精处理小鼠中这些蛋白的水平。KRQKYD促进紧密连接的成分,增强了屏障功能,促进了肠道完整性。结果表明,KRQKYD提高了抗菌肽和紧密蛋白的表达,改善了小鼠的肠道平衡。
图1 凝胶排阻色谱法获得的肽对雄性小鼠ALD的影响。(A)干腌火腿中多肽的SephadexTM 10/300 GL尺寸排斥色谱分析;(B-D):JHP-(A-I)对雄性小鼠血清中谷丙转氨酶ALT;(B)、谷草转氨酶AST;(C)和丙二醛MDA (D)浓度的影响。
图2空白组(CTRL)、酒精组(EtOH)、酒精+联苯双酯组(EtOH)、酒精+肽组(EtOH+JHP)喂食35天对小鼠肠道内酶表达的影响(A)反相高效液相色谱法分离G组分;(B-D): G-(1-7)对雄性小鼠血清中谷丙转氨酶ALT(B)、谷草转氨酶AST;(C)和丙二醛MDA(D)浓度的影响。
图3 A-图3C分别为六肽Leu-Pro-Gly-Val-Leu-Pro-Val-Ala(KRQKYD)的MS结构鉴定图。图3 AMS/MS谱图中G-6组分的总颗粒;图3B峰在9.74 min时的质谱(G-6);图3C用MS/MS谱鉴定纯化的多肽的分子量和氨基酸序列。
图4 KRQKYD对小鼠酒精性肝脏氧化应激的影响。(A)肝脏活性氧水平;(B)肝脏抗氧化酶活性水平;(C) HO-1、Nrf2和CYP2E1基因在肝脏中的表达水平;(D-E)肝组织HO-1、Nrf2、CYP2E1蛋白表达水平。
图5喂食对照饮食或含乙醇饮食(含或不含JHP) 35天对小鼠肠道菌群的影响。(A)肠道细菌门水平的相对丰度;(B)肠道细菌属水平的相对丰度。
图6 KRQKYD对小鼠肠道屏障稳态的影响。(A)定量RT-PCR检测紧密连接-1(ZO-1)、封闭蛋白-1(Claudin-1)、密封蛋白(Occludin) mRNA表达水平;(B-C)western blot检测ZO-1、Claudin-1、Occludin表达水平。
实施例2:一种可缓解酒精性肝损伤的干腌火腿源多肽及其制备方法
一种可缓解酒精性肝损伤的干腌火腿源多肽,氨基酸序列为:Lys-Arg-Gln-Lys-Tyr-Asp。
为实现上述目的及其他相关目的,本发明提供的技术方案是:一种可缓解酒精性肝损伤的干腌火腿源多肽的制备方法,包括下列步骤:
步骤1:将干腌火腿的股二头肌与磷酸盐缓冲液混合,然后于冰浴条件下用匀浆机进行均质,接着离心取上清,得到干腌火腿多肽提取物;
步骤2:对干腌火腿多肽提取物进行超滤,再用凝胶柱分离,洗脱条件为:洗脱液为0.01mol/L的HCl,恒温分离,流速0.8mL/min,用280nm紫外检测器测定馏分,用自动馏分收集器收集每管馏分;馏分在真空冷冻干燥机中干燥;
步骤3:采用液相分离将步骤2得到的缓解酒精性肝损伤最有效的组分进一步分离,获得最具缓解酒精性肝损伤活性的多肽;
步骤4:采用质谱对液相分离得到的最具缓解酒精性肝损伤活性的多肽进行分析,鉴定活性肽的结构,得到一种保护肝活性的肽序列为:Lys-Arg-Gln-Lys-Tyr-Asp。
步骤3包括将冻干后的样品溶于1mL蒸馏水,注入HPLC系统,采用BEH C18色谱柱进行梯度洗脱,流速为0.3 mL/min,洗脱液A为0.1%甲酸,洗脱液B为100%乙腈;流速梯度为:0-10 min,100% A,10-22 min,30-80% B;22-23 min,100% A;肽峰在280 nm紫外波长下检测;将多肽对应的峰分为七个部分,冷冻干燥;收集七个干腌火腿小肽分离组分,干燥后测定各个小肽的缓解酒精性肝损伤的活性。
鉴定方法包括:采用Acquity高效液相色谱系统,采用反相BEH C18色谱柱对最具缓解酒精性肝损伤活性的多肽峰进行分离;梯度洗脱:流速0.3mL/min,洗脱液a为0.1%甲酸,洗脱液b为100% CAN;流动梯度为:0-10 min,100%a,10-22min,30-80% b;22-23 min,100% a;柱温保持在25℃;流量直接进入MS/MS系统进行多反应测量;前体离子记录的质量范围为m/z=200-4000;使用Mass Lynx V4.1操作仪器,分析质谱图信息。
该六肽同时具有增加肠道中脱硫菌门相对丰度(由5.2±0.04%升至7.1±0.02%)和降低后壁菌门相对丰度(由66.5±0.02%降至57.3±0.01%)等功能,相较于酒精受损肠道,可以增加肠上皮细胞的紧密连接,提高密封蛋白(上调18.2±0.2%)、封闭蛋白-1(上调15.8±0.3%)和紧密连接-1(上调22.5±0.3%)蛋白的表达,降低肝脏炎症级联反应并可用于功能性食品或药品的制备。本发明中干腌火腿源的缓解酒精性肝损伤肽上调了Nrf2/HO-1抗氧化防御系统的表达,降低了肝细胞氧化应激损伤,具有抑制活性氧的生成的功能,从而非常适合作为主要功效配料用于制备缓解酒精性肝损伤的食品或药品。
以上所述者仅为用以解释本发明之较佳实施例,并非企图具以对本发明做任何形式上之限制,是以,凡有在相同之发明精神下所作有关本发明之任何修饰或变更,皆仍应包括在本发明意图保护之范畴。
Claims (3)
1.一种可缓解酒精性肝损伤的干腌火腿源多肽的制备方法,其特征在于:包括下列步骤:
步骤1:将干腌火腿的股二头肌与磷酸盐缓冲液混合,然后于冰浴条件下用匀浆机进行均质,接着离心取上清,得到干腌火腿多肽提取物;
步骤2:对干腌火腿多肽提取物进行超滤,再用凝胶柱分离,洗脱条件为:洗脱液为0.01mol/L的HCl,恒温分离,流速0.8mL/min,用280nm紫外检测器测定馏分,用自动馏分收集器收集每管馏分;馏分在真空冷冻干燥机中干燥;
步骤3:采用液相分离将步骤2得到的缓解酒精性肝损伤最有效的组分进一步分离,获得最具缓解酒精性肝损伤活性的多肽;
步骤4:采用质谱对液相分离得到的最具缓解酒精性肝损伤活性的多肽进行分析,鉴定活性肽的结构,得到一种保护肝活性的肽序列为:Lys-Arg-Gln-Lys-Tyr-Asp。
2.根据权利要求1所述的可缓解酒精性肝损伤的干腌火腿源多肽的制备方法,其特征在于:步骤3包括将冻干后的样品溶于1mL蒸馏水,注入HPLC系统,采用BEH C18色谱柱进行梯度洗脱,流速为0.3 mL/min,洗脱液A为0.1%甲酸,洗脱液B为100%乙腈;流速梯度为:0-10min,100% A,10-22 min,30-80% B;22-23 min,100% A;肽峰在280 nm紫外波长下检测;将多肽对应的峰分为七个部分,冷冻干燥;收集七个干腌火腿小肽分离组分,干燥后测定各个小肽的缓解酒精性肝损伤的活性。
3.根据权利要求1所述的可缓解酒精性肝损伤的干腌火腿源多肽的制备方法,其特征在于:鉴定方法包括:采用Acquity高效液相色谱系统,采用反相BEH C18色谱柱对最具缓解酒精性肝损伤活性的多肽峰进行分离;梯度洗脱:流速0.3mL/min,洗脱液a为0.1%甲酸,洗脱液b为100% CAN;流动梯度为:0-10 min,100%a,10-22min,30-80% b;22-23 min,100% a;柱温保持在25℃;流量直接进入MS/MS系统进行多反应测量;前体离子记录的质量范围为m/z=200-4000;使用Mass Lynx V4.1操作仪器,分析质谱图信息。
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