CN109517035B - 一种西兰花蛋白来源的ace抑制肽、ace抑制肽酶切代谢产物及其制备方法和应用 - Google Patents
一种西兰花蛋白来源的ace抑制肽、ace抑制肽酶切代谢产物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种西兰花蛋白来源的ACE抑制肽、其酶切代谢产物及其制备方法和应用,特点是该ACE抑制肽序列如SEQ ID NO1所示,其酶切代谢产物序列如SEQ ID NO2‑5所示,其制备方法如下:(1)以西兰花新鲜茎叶为原料进行压榨加热后,沉淀物经离心干燥得西兰花蛋白粗提物;(2)西兰花茎叶中多肽提取物经酶法制备得到西兰花多肽提取物;(3)西兰花多肽提取物经超滤和凝胶过滤收集ACE抑制活性最强的组分;(4)西兰花多肽提取物高效液相色谱分离纯化收集ACE抑制活性最强的组分得ACE抑制肽,通过模拟胃肠道消化得到肽代谢产物,可用于制备辅助降血压食品或药品,优点是结构简单、安全、活性强。
Description
技术领域
本发明涉及食品医药技术领域,尤其是涉及一种西兰花蛋白来源的ACE抑制肽、ACE抑制肽酶切代谢产物及其制备方法和应用。
背景技术
西兰花是一种季节性较强的蔬菜作物,其花球采收时间集中在每年的11月至次年3月之间,采收花球后的植株茎叶作为废弃物堆放田间地头,严重污染西兰花主产区环境。据测算,西兰花茎叶一般亩产为2.5~3吨,仅以浙江省临海市上盘镇 7万亩西兰花种植基地计算,其总量可达17.5~21.0万吨。过分集中堆积的西兰花茎叶严重影响土壤耕作层生态环境的良性循环,过量还田复耕后的西兰花茎叶腐烂后产生的氮、磷元素造成水体富营养化,导致养殖鱼类及其他水生物大量死亡。在西兰花集散加工过程中产生的大量叶片和切削花球时产生的边角料腐烂后产生大量的污水和氨、硫化氢等恶臭气体,严重影响周边居民身体健康和生活质量。因此,西兰花茎叶等废弃物的治理对于保障西兰花产业可持续发展意义重大。
西兰花中的多肽对心血管疾病有一定的帮助。随着世界的发展和社会的进步,人们的生活水平不断提高;大众的生活方式和饮食结构都发生了巨大变化,现代文明病也接踵而来。现代文明病主要包括肥胖症、糖尿病、高血压、抑郁症等。在这些常见病中,高血压是严重危害人类健康的慢性病之一,也是世界范围内的普遍病症,其发生的原因,目前尚不清楚;同时,高血压是心血管疾病发展过程中主要的可控危险因子,每降低5mmHg收缩压,患心血管疾病的风险就降低16%;持续升高的血压极可能增加中风、心脏病发作和肾衰竭的风险,每年,全世界有大约30%或更多的成年人受高血压疾病的困扰,并有数百万人因高血压及其并发症而死亡。高血压已成为危及人类身体健康的主要疾病之一,据世界卫生组织预测,到2020年,非传染性疾病将占死亡原因的79%,而其中高血压等心血管疾病将会占到首位。作为现代文明病标志之一的高血压,通常被人们称为“无预兆”的疾病。那是由于高血压引起的后果总要等到对机体产生明显的损害之后才被发觉,这时往往已经太迟了。因此,高血压疾病的预防和治疗工作尤为重要。高血压发病期间,患者需要经常服用一些辅助降压药物,但这些降压药会产生许多副作用,尤其对肾功能影响很大。所以,如果能在日常饮食中食用一些含有抗高血压成分的食品,那么不仅可以有效控制高血压的发病频率,而且对身体无任何副作用。
生物活性肽是具有生理调节功能的肽类总称,一般而言,这类肽大多相对分子质量较小,在人体内的消化吸收较蛋白质容易。这些小肽不仅能提供人体生长发育所需要的营养,还能调节人体生理机能,起到预防、甚至治疗疾病的作用。食源性生物活性肽的种类较多,包括血管紧张素转化酶(angiotension converting enzyme,ACE)抑制肽、免疫调节肽、抗氧化肽、抗菌肽、抗血栓肽、阿片样活性肽、促钙吸收肽。其中ACE抑制肽与高血压疾病的预防和治疗关系紧密,已引起各国科学家与政府的高度关注。目前,国内外较少研究关于从西兰花茎叶中提取有效的多肽提取物用于高血压疾病的预防和治疗。
发明内容
本发明所要解决的技术问题是提供一种结构简单、安全、活性强的西兰花蛋白来源的ACE抑制肽、ACE抑制肽酶切代谢产物及其制备方法和应用。
本发明解决上述技术问题所采用的技术方案为:
1、一种西兰花蛋白来源的ACE抑制肽,所述的ACE抑制肽氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys。
2、一种西兰花蛋白来源的ACE抑制肽酶切代谢产物,所述的ACE抑制肽酶切代谢产物为上述ACE抑制肽经胃蛋白酶和胰蛋白酶消化酶解所得,其氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu(LL-7),Leu-Val-Leu-Pro-Gly-Glu(LE-6),Leu-Ala-Lys(LK-3),Ala-Lys(AK-2)。
3、上述西兰花蛋白来源的ACE抑制肽以及ACE抑制肽酶切代谢产物的制备方法,步骤如下:
(1)以西兰花新鲜茎叶为原料进行压榨,将所得汁液加热到85-95℃,保温8-12min,沉淀物经离心,干燥,即得西兰花蛋白粗提物;
(2)西兰花茎叶中多肽提取物的酶法制备
以西兰花蛋白粗提物为原料,按料液质量比1:15-25加入蒸馏水,在温度55-65℃、pH8.0条件下,加入西兰花蛋白粗提物质量1.5-2.5%的Aclase碱性蛋白酶,酶解3h后,灭酶,冷却,并经离心,旋转蒸发,浓缩后冷冻干燥,得到西兰花多肽提取物;
(3)西兰花多肽提取物超滤和凝胶过滤
将西兰花多肽提取物采用Valflow 50超滤,然后用G-15凝胶柱分离,洗脱条件为:洗脱液为去离子水,流速13-17mL/h,上样量90-110mg,收集三个西兰花小肽分离组分,经过滤,干燥测定ACE抑制活性;
(4)西兰花多肽提取物的高效液相色谱分离纯化
将凝胶过滤后得到的ACE抑制活性最强的组分,进一步进行液相分离,色谱条件:色谱柱CAPCELL PAK C18 AQ S-5,柱温30℃,流动相A:水+0.2%甲酸,流动相B:乙腈,洗脱方式:梯度洗脱,0-30min流动相A:100%-92.5%,流动相B:0-7.5%;30-33min流动相A:92.5%-15%,流动相B:7.5%-85%;33-43min流动相A:15%,流动相B:85%;43-45min流动相A:15%-100%,流动相B 85%-0%;45-55min流动相A:100%,流动相B 0%,流速1.0 ml/min,检测波长:280nm,进样体积100uL,收集四个西兰花小肽分离组分,干燥测定ACE抑制活性;
(5)ACE抑制肽的结构鉴定
采用质谱对液相分离得到的ACE抑制活性最强的峰进行分析,鉴定ACE抑制肽的结构,获得其氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys;
(6)ACE抑制肽胃肠道酶切代谢后肽段鉴定
将步骤(5)得到的ACE抑制肽依次用胃蛋白酶和胰蛋白酶酶切消化后产生的新的ACE抑制肽,鉴定ACE抑制肽酶切代谢产物,获得其氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu,Leu-Val-Leu-Pro-Gly-Glu,Leu-Ala-Lys和Ala-Lys。
步骤(6)中所述的胃蛋白酶与所述的ACE抑制肽的质量比为 1∶100,所述的胰蛋白酶与所述的ACE抑制肽的质量比为1∶50。
上述西兰花蛋白来源的ACE抑制肽以及ACE抑制肽酶切代谢产物在制备辅助降血压食品或降压药品方面的应用。
与现有技术相比,本发明的优点在于:本发明一种西兰花茎叶来源的ACE抑制肽、ACE抑制肽酶切代谢产物及其制备方法和应用。该西兰花茎叶来源的血管紧张素转换酶抑制肽是以西兰花新鲜茎叶为原料分离纯化鉴定得到,ACE抑制肽的氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys,该肽可有效抑制ACE活性,IC50值为184 uM,酶切代谢产物的序列为Leu-Val-Leu-Pro-Gly-Glu-Leu,Leu-Val-Leu-Pro-Gly-Glu,Leu-Ala-Lys,Ala-Lys,IC50值分别为100 uM,13.5 uM,50uM,80 uM。本发明所涉及的ACE抑制肽具有结构简单、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降压功能新药物提供有效成分,具有广泛的应用前景。
附图说明
图1为西兰花多肽分子量分布图;
图2为西兰花多肽G-15凝胶柱组分分离图;
图3为西兰花多肽高效液相色谱(HPLC)组分分离图;
图4为本发明ACE抑制肽Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys (LK-9) 的MS/MS结构鉴定图;
图5为Leu-Val-Leu-Pro-Gly-Glu-Leu (LL-7) 的MS/MS结构鉴定图;
图6为Leu-Val-Leu-Pro-Gly-Glu(LE-6) 的MS/MS结构鉴定图;
图7为Leu-Ala-Lys(LK-3) 结构鉴定图;
图8为Ala-Lys(AK-2)的MS/MS结构鉴定图。
具体实施方式
以下结合附图实施例对本发明作进一步详细描述。
实施例1
一种西兰花蛋白来源的ACE抑制肽,该ACE抑制肽氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys,其酶切代谢产物的氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu(LL-7),Leu-Val-Leu-Pro-Gly-Glu(LE-6),Leu-Ala-Lys(LK-3),Ala-Lys(AK-2)。
实施例2
上述实施例1中西兰花蛋白来源的ACE抑制肽的制备方法,包括以下步骤:
1、西兰花茎叶中蛋白的提取
以西兰花新鲜茎叶为原料进行压榨,将所得汁液加热到85-95℃,保温8-12min,沉淀物经离心,干燥,即得西兰花蛋白粗提物;
2、西兰花茎叶中多肽提取物的酶法制备及多肽含量测定
以西兰花蛋白粗提物为原料,按料液质量比1:15-25加入蒸馏水,在温度55-65℃、pH8.0条件下,加入西兰花蛋白粗提物质量1.5-2.5%的Aclase碱性蛋白酶,酶解3h后,灭酶,冷却,并经离心,旋转蒸发,浓缩后冷冻干燥,得到西兰花多肽提取物备用,西兰花多肽含量测定采用Folllin酚法;采用凝胶过滤色谱法测定西兰花多肽提取物的相对分子质量分布,结果如图1所示。由图1可知,西兰花多肽提取物中的多肽分子质量主要分布在1000Da以下;
3、西兰花多肽提取物的初步纯化实验
西兰花多肽提取物采用Valflow 50超滤,然后用G-15凝胶柱分离,洗脱条件为:洗脱液为去离子水,流速13-17mL/h,上样量90-110mg,重复收集三个西兰花小肽分离组分Ⅰ、Ⅱ和Ⅲ(如图2所示),经过滤,蒸干,并多次收集干燥备用。
表1 西兰花多肽G-15凝胶柱组分ACE抑制率
4、分离组分的高效液相色谱(HPLC)分离纯化
将凝胶过滤后得到的ACE抑制活性最强的组分,进一步进行液相分离,色谱条件:色谱柱CAPCELL PAK C18 AQ S-5,柱温30℃,流动相A:水+0.2%甲酸,流动相B:乙腈,洗脱方式:梯度洗脱,0-30min流动相A:100%-92.5%,流动相B:0-7.5%;30-33min流动相A:92.5%-15%,流动相B:7.5%-85%;33-43min流动相A:15%,流动相B:85%;43-45min流动相A:15%-100%,流动相B 85%-0%;45-55min流动相A:100%,流动相B 0%。流速1.0 ml/min,检测波长:280nm,进样体积100uL,组分收集后真空冻干备用。西兰花多肽高效液相色谱(HPLC)组分分离图如图3所示,共有Fraction_1、Fraction_2、Fraction_3和Fraction_4四个组分。
表2 西兰花多肽高效液相色谱(HPLC)组分ACE抑制率
其中血管紧张素转化酶抑制活性(ACEI)检测方法如下:反应体系:在离心管中加入80uL 5mmol/L HHL(马尿酰-组氨酰-亮氨酸)(溶于HEPES缓冲液,PH8.3)和30uL不同浓度的样品溶液(溶于双蒸水),混合后置于37℃水浴5min,再加入40uL 0.025U/mL ACE(溶于HEPES缓冲液,PH8.3),37℃孵育1h,然后加入150uL 1M盐酸终止反应。空白组在加入ACE的同时加入盐酸,对照组使用30ul双蒸水代替样品溶液,卡托普利(10ng/mL)作为阳性对照。反应完成后用RP-HPLC检测样品中马尿酸(HA)的含量,通过与马尿酸标准品峰面积比较计算得出检测样品中马尿酸含量。色谱条件:色谱柱(CAPCELL PAK C18 AQ S-5, 4.6*150mm),柱温30℃,流动相A:水+0.2%甲酸,流动相C:乙腈,流动相比例A:C=85%:15%,流速1.5 ml/min,检测波长:228nm,进样体积100uL,分析时间8min。
抑制率I% = ([HA]b − [HA]s)/ ([HA]b − [HA]c) × 100%,其中[HA]b表示对照组的马尿酸峰面积,[HA]s表示样品的马尿酸峰面积,[HA]c表示的空白组的马尿酸峰面积。
5、ACE抑制活性最强组分的肽的结构鉴定
样品取适量肽段使用纳升流速Easy nLC 1200色谱系统(Thermo Scientific)进行色谱分离。缓冲液:A液为0.1%甲酸水溶液,B液为0.1%甲酸、乙腈和水混合溶液(其中乙腈为85%)。色谱柱以95%的A液平衡。样品进样到Trap Column(100μm*20mm, 5μm,C18,Dr.Maisch GmbH)后经过色谱分析柱(75μm*150mm, 3μm,C18,Dr. Maisch GmbH)进行梯度分离,流速为300 nl/min。液相分离梯度如下:0分钟---2分钟,B液线性梯度从5% 到8% ;2分钟---42分钟,B液线性梯度从8% 到23%;42分钟---50分钟,B液线性梯度从23%到40%;50分钟---52分钟,B液线性梯度从40%到100%;52分钟----60分钟,B液维持在100%。肽段分离后用Q-Exactive Plus质谱仪(Thermo Scientific)进行DDA(数据依赖采集)质谱分析。分析时长为120min,检测模式:正离子,母离子扫描范围:350-1800m/z,一级质谱分辨率:70,000@m/z 200,AGC target:1e6,一级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(full scan)后触发采集15个最高强度母离子的二级质谱图谱(MS2 scan),二级质谱分辨率:17,500 @ m/z 200,AGC target: 1e5,二级Maximum IT:100 ms,MS2Activation Type: HCD,Isolation window:1.6 Th,Normalized collision energy:27(结果如图4所示)。ACE抑制肽的氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys,该肽可有效抑制ACE活性,IC50值为184 uM。
表 3 Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys (LK-9)西兰花蛋白归属
由上表可知,Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys (LVLPGELAK)所属蛋白为西兰花组蛋白H2B,位置从130-138,是西兰花组蛋白H2B的特异性肽段。
6、ACE抑制肽胃肠道酶切对ACE抑制活性的影响及酶切代谢后肽段鉴定
ACE抑制肽的体外消化稳定性测定,用胃蛋白酶和胰蛋白酶模拟胃肠道消化,将胃蛋白酶溶解在 6 M HCl(pH 2.0)中,之后添加到1mg/ml ACE抑制肽溶液中(pH 2.0),胃蛋白酶和ACE抑制肽的质量比为 1∶100。在 37 ℃ 消化2 h后用1M NaHCO3 将pH调节至 7.2使酶灭活。随后添加胰蛋白酶,胰蛋白酶和ACE抑制肽的质量比为 1∶50。在 37 ℃消化 2 h后加热至 95 ℃保持 10 min 使 酶灭活。将其冻干后,用去离子水使其溶解后测定 ACE抑制活性并进行质谱分析。
Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys肽段具有以下特点,肽段由9个氨基酸组成,肽段中富含亮氨酸(Leu),缬氨酸(Val),脯氨酸(Pro),甘氨酸(Gly),谷氨酸(Glu),丙氨酸(Ala),赖氨酸(Lys)等。疏水氨基酸含量较高,达到 55.6%,在体外经胃蛋白酶和胰蛋白酶消化两小时后,非常稳定,稳定性为82.9%,如下表4所示。
表4 Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys (LK-9)体外经胃蛋白酶和胰蛋白酶消化稳定性结果
由上表可知,LK-9体外经胃蛋白酶和胰蛋白酶消化后,稳定性为82.9%,说明IK-7在胃蛋白酶和胰蛋白酶作用下,不大容易被酶切破坏,具有广泛的应用前景。对酶切后的溶液进行ACE抑制活性测定,酶切后的溶液的抑制活性较酶切前提高了将近2倍,
表 5 Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys (LK-9)酶切前后ACE抑制活性的变化
对酶切后产生的肽段进行了质谱鉴定序列,结果如图5、图6、图7和图8所示,新产生的肽段序列为Leu-Val-Leu-Pro-Gly-Glu-Leu(LL-7),Leu-Val-Leu-Pro-Gly-Glu(LE-6),Leu-Ala-Lys(LK-3),Ala-Lys(AK-2)。理论上,在血液及细胞中蛋白酶作用下会形成多种疏水性含量高的小肽,可能发挥更强的ACE抑制作用,在食品和药品中具有较大的应用意义。
本发明所述抑制肽为西兰花茎叶蛋白来源的具有高ACE抑制活性肽,此肽是西兰花茎叶蛋白经食品级酶在温和条件下水解后获得的,其安全性高,并且能够大量制备,因此,有望为开发新降血压药物提供有效成分。
上述说明并非对本发明的限制,本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内,做出的变化、改型、添加或替换,也应属于本发明的保护范围。
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<110> 宁波大学
<120> 一种西兰花蛋白来源的ACE抑制肽、ACE抑制肽酶切代谢产物及其制备方法和应用
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 9
<212> PRT
<213> LK-9
<400> 1
Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys 9
<210> 2
<211> 7
<212> PRT
<213> LL-7
<400> 2
Leu-Val-Leu-Pro-Gly-Glu-Leu 7
<210> 3
<211> 6
<212> PRT
<213> LE-6
<400> 3
Leu-Val-Leu-Pro-Gly-Glu 6
<210> 4
<211> 3
<212> PRT
<213> LK-3
<400> 4
Leu-Ala-Lys 3
<210> 5
<211> 2
<212> PRT
<213> AK-2
<400> 5
Ala-Lys 2
Claims (4)
1.一种西兰花蛋白来源的ACE抑制肽酶切代谢产物,其特征在于:所述的ACE抑制肽酶切代谢产物为ACE抑制肽经胃蛋白酶和胰蛋白酶消化酶解所得,其氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu,Leu-Val-Leu-Pro-Gly-Glu,Leu-Ala-Lys和Ala-Lys,所述的ACE抑制肽氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys。
2.一种权利要求1所述的西兰花蛋白来源的ACE抑制肽酶切代谢产物的制备方法,其特征在于步骤如下:
(1)以西兰花新鲜茎叶为原料进行压榨,将所得汁液加热到85-95℃,保温8-12min,沉淀物经离心,干燥,即得西兰花蛋白粗提物;
(2)西兰花茎叶中多肽提取物的酶法制备
以西兰花蛋白粗提物为原料,按料液质量比1:15-25加入蒸馏水,在温度55-65℃、pH8.0条件下,加入西兰花蛋白粗提物质量1.5-2.5%的Aclase碱性蛋白酶,酶解3h后,灭酶,冷却,并经离心,旋转蒸发,浓缩后冷冻干燥,得到西兰花多肽提取物;
(3)西兰花多肽提取物超滤和凝胶过滤
将西兰花多肽提取物采用Valflow 50超滤,然后用G-15凝胶柱分离,洗脱条件为:洗脱液为去离子水,流速13-17mL/h,上样量90-110mg,收集三个西兰花小肽分离组分,经过滤,干燥测定ACE抑制活性;
(4)西兰花多肽提取物的高效液相色谱分离纯化
将凝胶过滤后得到的ACE抑制活性最强的组分,进一步进行液相分离,色谱条件:色谱柱CAPCELL PAK C18 AQ S-5,柱温30℃,流动相A:水+0.2%甲酸,流动相B:乙腈,洗脱方式:梯度洗脱,0-30min流动相A:100%-92.5%,流动相B:0-7.5%;30-33min流动相A:92.5%-15%,流动相B:7.5%-85%;33-43min流动相A:15%,流动相B:85%;43-45min流动相A:15%-100%,流动相B 85%-0%;45-55min流动相A:100%,流动相B 0%,流速1.0 ml/min,检测波长:280nm,进样体积100uL,收集四个西兰花小肽分离组分,干燥测定ACE抑制活性;
(5)ACE抑制肽的结构鉴定
采用质谱对液相分离得到的ACE抑制活性最强的峰进行分析,鉴定ACE抑制肽的结构,获得其氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu-Ala-Lys;
(6)ACE抑制肽胃肠道酶切代谢后肽段鉴定
将步骤(5)得到的ACE抑制肽依次用胃蛋白酶和胰蛋白酶酶切消化得到ACE抑制肽酶切代谢产物,鉴定ACE抑制肽酶切代谢产物获得其氨基酸序列为Leu-Val-Leu-Pro-Gly-Glu-Leu,Leu-Val-Leu-Pro-Gly-Glu,Leu-Ala-Lys和Ala-Lys。
3.根据权利要求2所述的西兰花蛋白来源的ACE抑制肽酶切代谢产物的制备方法,其特征在于:步骤(6)中所述的胃蛋白酶与所述的ACE抑制肽的质量为 1∶100,所述的胰蛋白酶与所述的ACE抑制肽的质量比为1∶50。
4.根据权利要求1所述的西兰花蛋白来源的ACE抑制肽酶切代谢产物在制备辅助降血压食品或降压药品方面的应用。
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