CN110269129A - 一种鸭肉源ace抑制肽及其制备方法 - Google Patents
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Abstract
本发明公开了一种鸭肉源ACE抑制肽及其制备方法。选用碱性蛋白酶酶解鸭肉蛋白制备ACE抑制肽提取物,并采用超滤、凝胶过滤色谱、反相高效液相色谱对鸭肉源ACE抑制肽进行分离纯化,并通过Nano‑LC‑ESI‑LTQ‑Orbitrap MS/MS液质联用仪进行氨基酸序列鉴定,共鉴定出7条肽段,分别为KLSH,FVAG,LHALLLL,TLVP,KALVG,AYDT,VLGV,其中肽段TLVP表现出最强的ACE抑制活性,可用于高血压治疗相关的食品、药品中。
Description
技术领域
本发明属于食品领域,具体涉及一种鸭肉源ACE抑制肽及其制备方法。
背景技术
高血压是一种以动脉血压持续升高为主要特征的慢性心血管疾病,血压过高甚至能够引起肾、心脏及脑等器官的器质性病变。统计数据表明,2008年全球高血压患病人数高达10亿人次,如不采取有效的预防及治疗措施,2025年全球将有15.6亿人次罹患这一疾病。血管紧张素转化酶(ACE)是一种分布在机体血液和组织中的外肽酶,它可以通过肾素-血管紧张素系统(RAS)引起血压升高,因此ACE抑制肽被视为能够有效缓解高血压的肽类物质。目前临床上治疗高血压的药物以合成药物为主,主要有卡托普利、赖诺普利、培哚普利和福辛普利等,这些药物虽具有良好的临床降压效果,但若长期服用会产生一些严重的副作用,如咳嗽、皮疹、高钾血症和肾功能减退等。食源性ACE抑制肽的降压作用虽不及合成类药物,但其性质比较温和,且其只对高血压患者有降压作用,对血压正常者无影响。因此,食源性ACE抑制肽可作为食品辅料添加于功能性食品中,以此改善人体健康。
我国是鸭养殖大国,出栏量和消费量居世界第一。联合国粮食及农业组织的统计数据表明,2017年我国肉鸭出栏量约34.7亿只,约占世界总出栏量的74.2%。鸭肉营养价值高,富含蛋白质和各种人体所必需的微量元素,但我国鸭肉深加工能力不足,高值化利用率低,资源未得到充分利用。ACE抑制肽因具有成本低、易吸收、稳定性强和不易致敏等特点,现已成为科学领域的研究热点。目前关于动物蛋白源ACE抑制肽的研究主要以畜肉和鱼肉为主,而有关禽肉特别是鸭肉中ACE抑制肽的研究还鲜见报道。
发明内容
发明目的:针对现有技术的不足,本发明的目的是提供一种鸭肉源ACE抑制肽,具有成本低、易吸收、稳定性强、不易致敏和性质温和等特点,可作为食源性ACE抑制肽以食品辅料添加于功能性食品中;本发明的另一目的是提供上述鸭肉源ACE抑制肽的制备方法,选用碱性蛋白酶酶解鸭肉蛋白制备ACE抑制肽,条件温和,水解程度易控制,可通过选用特定的蛋白酶水解蛋白质产生具有某种特定功能的生物活性肽。
技术方案:为了实现上述发明目的,本发明采用的技术方案为:
一种鸭肉源ACE抑制肽,至少具有以下氨基酸序列的短肽中的一种,所述的氨基酸序列分别为KLSH,FVAG,LHALLLL,TLVP,KALVG,AYDT,VLGV。
所述ACE抑制肽氨基酸序列为TLVP。
鸭肉源ACE抑制肽的制备方法,其特征在于,包括以下步骤:
1)将冷冻的鸭胸肉放于4℃冰箱中过夜解冻,去除肌膜等结缔组织后绞碎,按照肉水比称取肉样,匀浆,置于水浴箱中水浴15min,酶解pH值为6~10,加酶量为1000~5000U/g,酶解温度为55~75℃,酶解时间为1~5h,样品取出后,将酶解液的pH调至7,置于沸水浴中灭酶10min,冷却至室温后在转速为10000g、温度4℃下离心10min后取上清液进行冻干,得到鸭肉源ACE抑制肽提取物;
2)鸭肉源ACE抑制肽提取物经超滤、凝胶过滤色谱、反相高效液相色谱逐级分离纯化得到ACE抑制肽。
步骤1)中肉水质量体积比为1∶3g·mL-1。
步骤1)中酶的种类为:碱性蛋白酶,复合蛋白酶,胃蛋白酶。
步骤1)中酶的种类为:碱性蛋白酶。
步骤1)中酶解pH值为9,加酶量为2000U/g,酶解温度为65℃,酶解时间为4h。
步骤2)中超滤分离纯化中超滤膜的截留分子量小于3kDa。
步骤2)中制得的ACE抑制肽经液质联用鉴定得到的肽段分子量为386.5Da~792.0Da。
鸭肉源ACE抑制肽在辅助降血压的食品和药品中的应用。
有益效果:与现有技术相比,本发明选用碱性蛋白酶酶解鸭肉蛋白制备ACE抑制肽,通过测定酶解条件对碱性蛋白酶酶解效果的影响,得到的最优酶解工艺为:酶解时间4h,加酶量2000U/g,pH9,酶解温度65℃。采用超滤、凝胶过滤色谱、反相高效液相色谱对鸭肉源ACE抑制肽进行分离纯化,并通过Nano-LC-ESI-LTQ-Orbitrap MS/MS液质联用仪进行氨基酸序列鉴定,共鉴定出7条肽段,分别为KLSH,FVAG,LHALLLL,TLVP,KALVG,AYDT,VLGV,其中肽段TLVP表现出最强的ACE抑制活性,食源性ACE抑制肽性质比较温和,且其只对高血压患者有降压作用,对血压正常者无影响。因此,食源性ACE抑制肽可作为食品辅料添加于功能性食品中,以此改善人体健康。
附图说明
图1是酶种类对鸭肉蛋白水解度的影响图;
图2是酶种类对鸭肉蛋白酶解产物ACE抑制活性的影响图;
图3是不同酶解时间对水解度和ACE抑制活性的影响图;
图4是不同加酶量对水解度和ACE抑制活性的影响图;
图5是不同pH对水解度和ACE抑制活性的影响图;
图6是不同酶解温度对水解度和ACE抑制活性的影响图;
图7是膜超滤法分离纯化鸭肉酶解产物后各组分的ACE抑制活性图;图中,a-c不同字母表示组间差异显著(P<0.05);
图8是凝胶过滤色谱分离纯化鸭肉源ACE抑制肽图;
图9是经凝胶过滤色谱后各组分的ACE抑制活性图;图中,a-b不同字母表示组间差异显著(P<0.05);
图10是反相高效液相色谱分离纯化鸭肉源ACE抑制肽图;
图11是经反相高效液相色谱后各组分的ACE抑制活性图;图中,a-f不同字母表示组间差异显著(P<0.05)。
具体实施方式
下面结合具体实施例进一步说明本发明,但这些实例并不用来限制本发明。下述实施例中所使用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材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例所使用的材料和试剂如下:
冷冻樱桃谷鸭胸肉购自江苏益客食品有限公司;Alcalase 2.4L(碱性蛋酶,2.4AU-A/g),购自诺维信(中国)生物技术有限公司;血管紧张素转化酶(ACE,来源于兔肺)、马尿酰-组氨酰-亮氨酸(HHL),购自美国Sigma-Aldrich公司;BCA试剂盒、甲酸(色谱纯)、三乙胺(色谱纯),购自南京瑞翼特生物科技有限公司;乙腈(色谱纯),购自南京晚晴化玻仪器有限公司;其他试剂均为分析纯。
实施例1
将冷冻的鸭胸肉放于4℃冰箱中过夜解冻,去除肌膜等结缔组织后绞碎,称取100g绞碎的肉样于烧杯中,加入300mL蒸馏水,匀浆(8000r/min,3次,每次20s)。将匀浆液的pH调至各蛋白酶的最适pH值,然后置于水浴箱中水浴15min,加入3000U/g的酶,恒温搅拌酶解。酶解过程中,分别于30min、1h、2h、3h、4h、5h、6h、7h、8h、9h、10h时进行取样。样品取出后,将酶解液的pH调至7,置于沸水浴中灭酶10min,冷却至室温后在转速10000g、温度4℃下离心10min后取上清液进行水解度及ACE抑制活性的测定。以鸭肉蛋白为酶解底物,选择碱性蛋白酶、复合蛋白酶和胃蛋白酶为水解蛋白酶。各蛋白酶的最适反应条件如下:Alcalase2.4L(碱性蛋白酶,pH8,温度60℃),Protamex(复合蛋白酶,pH6,温度50℃),Pepsin(胃蛋白酶,pH2,温度37℃)。
1)酶种类对蛋白质水解度的影响
鸭肉蛋白经三种不同的蛋白酶酶解后,其水解度变化趋势如图1所示。从图中可以看出,碱性蛋白酶在0-5h的酶解过程中,水解度随着酶解时间的增加而快速增加,而从5h到10h,蛋白质水解度的增加幅度逐渐变缓;复合蛋白酶在酶解的0-7h期间,水解度随着时间的延长而不断增加,随后呈下降趋势;此外,与碱性蛋白酶和复合蛋白酶相比,胃蛋白酶在整个酶解过程中的水解度最低。
2)酶种类对ACE抑制活性的影响
由图2可知,三种蛋白酶酶解产物的ACE抑制活性之间存在显著差异(P<0.05)。在酶解初期的前30min内,三种酶解产物的ACE抑制率迅速上升,其可能的原因是酶解初期底物蛋白充足,在蛋白酶的作用下原本隐藏在蛋白质内部的氨基酸残基暴露出来,从而造成酶解产物中的ACE抑制肽含量迅速上升。在酶解的0-10h内,碱性蛋白酶酶解产物的ACE抑制活性随着酶解时间的延长先上升后下降,在4h时达到最大值60.78±1.12%;在酶解的0-7h内,复合蛋白酶酶解产物的ACE抑制活性波动性较强,7h后ACE抑制肽的活性呈下降趋势。可能是复合蛋白酶兼具内切酶和外切酶的活性,无特异性的酶切位点,它可以产生具有ACE抑制活性的多肽,也可以将已有的活性肽水解;在整个胃蛋白酶酶解过程中,肽的ACE抑制活性始终最低,其可能的原因是鸭肉蛋白不是胃蛋白酶的适宜底物。
综合考虑水解度和ACE抑制活性两个指标,碱性蛋白酶为酶解鸭肉蛋白制备ACE抑制肽的最适蛋白酶。
实施例2
将冷冻的鸭胸肉放于4℃冰箱中过夜解冻,去除肌膜等结缔组织后绞碎,按照肉水比为1∶3称取肉样,匀浆(8000r/min,3次,每次20s)。将匀浆液的pH调至9,然后置于水浴箱中水浴15min,加入2000U/g的酶,65℃恒温搅拌酶解。样品取出后,将酶解液的pH调至7,置于沸水浴中灭酶10min,冷却至室温后在转速为10000g、温度4℃下离心10min后取上清液进行冻干,将冻干粉置于-20℃。酶解时间分别设为1、2、3、4、5h,离心取上清液,测定其水解度和ACE抑制活性。
如图3所示,酶解时间对水解度和ACE抑制率有显著影响(P<0.05)。在酶解的0-5h内,水解度随着酶解时间的延长而增加;在酶解的前4h,酶解产物的ACE抑制活性随着酶解时间的延长而增加,在4h时达到最大值63.78±0.59%(蛋白浓度3mg/mL,下同),4h后酶解产物的ACE抑制活性开始下降。这一结果表明水解度和ACE抑制率的变化趋势并不是完全一致的,随着酶解时间的增加,ACE抑制肽中的活性基团可能会被过度水解,从而造成活性下降。因此,选择4h为最佳酶解时间。
实施例3
将冷冻的鸭胸肉放于4℃冰箱中过夜解冻,去除肌膜等结缔组织后绞碎,按照肉水质量体积比为1∶3g·mL-1称取肉样,匀浆(8000r/min,3次,每次20s)。将匀浆液的pH调至9,然后置于水浴箱中水浴15min,加入酶,65℃恒温搅拌酶解4h。样品取出后,将酶解液的pH调至7,置于沸水浴中灭酶10min,冷却至室温后在转速为10000g、温度4℃下离心10min后取上清液进行冻干,将冻干粉置于-20℃。加酶量分别设为1000、2000、3000、4000、5000U/g,离心取上清液,测定其水解度和ACE抑制活性。
如图4所示,加酶量对水解度和ACE抑制率有显著影响(P<0.05)。随着加酶量的增加(1000-2000U/g),水解度和ACE抑制率都显著增加,ACE抑制率在2000U/g时达到最大值62.89±0.47%。继续增加加酶量,水解度稍微上升(P<0.05),但酶解产物的ACE抑制活性有下降趋势。出现这一现象的原因可能是在一定的加酶量范围内,随着加酶量的增加会增大底物与酶结合的可能性,但继续增加加酶量,底物蛋白产生饱和现象,会使原已生成的ACE抑制肽被水解,从而造成活性下降。因此,选择2000U/g为最适加酶量。
实施例4
将冷冻的鸭胸肉放于4℃冰箱中过夜解冻,去除肌膜等结缔组织后绞碎,按照肉水质量体积比为1∶3g·mL-1称取肉样,匀浆(8000r/min,3次,每次20s)。调节匀浆液的pH值,然后置于水浴箱中水浴15min,加入2000U/g的酶,65℃恒温搅拌酶解4h。样品取出后,将酶解液的pH调至7,置于沸水浴中灭酶10min,冷却至室温后在转速为10000g、温度4℃下离心10min后取上清液进行冻干,将冻干粉置于-20℃。匀浆液的pH分别设为6、7、8、9、10,离心取上清液,测定其水解度和ACE抑制活性。
如图5所示,pH对水解度和ACE抑制率有显著影响(P<0.05)。pH在6-9范围内变动时,随着pH的增加,水解度和ACE抑制活性都显著上升,在pH为9时,二者均达到最大值,分别为17.82±0.46%、63.73±0.65%。随着pH的继续增大,水解度和ACE抑制率均显著下降。出现这一现象的原因可能是pH可以影响酶和底物分子的解离状态,在最适的pH下,底物蛋白和酶才能发生最有效地结合。因此,确定最佳酶解pH为9。
实施例5
将冷冻的鸭胸肉放于4℃冰箱中过夜解冻,去除肌膜等结缔组织后绞碎,按照肉水质量体积比为1∶3g·mL-1称取肉样,匀浆(8000r/min,3次,每次20s)。将匀浆液的pH调至9,然后置于水浴箱中水浴15min,加入酶,恒温搅拌酶解4h。样品取出后,将酶解液的pH调至7,置于沸水浴中灭酶10min,冷却至室温后在转速为10000g、温度4℃下离心10min后取上清液进行冻干,将冻干粉置于-20℃。酶解温度分别设为55、60、65、70、75℃,离心取上清液,测定其水解度和ACE抑制活性。
如图6所示,酶解温度对水解度和ACE抑制率有显著影响(P<0.05)。在温度为55-70℃内,随着酶解温度的增加,水解度逐渐上升,温度继续增加,则水解度开始下降;温度在55-65℃范围内,随着温度的升高,酶解产物的ACE抑制率逐渐上升,在温度为65℃时,达到最大值66.32±0.4%。此后随着温度的继续升高,ACE抑制率显著下降。出现这一现象的原因可能是温度过高或过低,会导致酶的活性发生变化,甚至会使酶失活。因此综合考虑水解度和ACE抑制率两个指标,确定65℃为最适酶解温度。
实施例6
将冷冻的鸭胸肉放于4℃冰箱中过夜解冻,去除肌膜等结缔组织后绞碎,按照肉水比为1∶3称取肉样,匀浆(8000r/min,3次,每次20s)。将匀浆液的pH调至9,然后置于水浴箱中水浴15min,加入2000U/g的酶,65℃恒温搅拌酶解4h。样品取出后,将酶解液的pH调至7,置于沸水浴中灭酶10min,冷却至室温后在转速为10000g、温度4℃下离心10min后取上清液进行冻干,将冻干粉置于-20℃。
实施例7
1膜超滤法分离鸭肉蛋白酶解产物
称取2g冻干粉溶解于100mL蒸馏水中,然后采用截留分子量(MWCO)分别为3kDa和10kDa的超滤膜对鸭肉蛋白酶解产物进行逐级分离,收集所得组分MWCO-1(>10kDa)、MWCO-2(3-10kDa)和MWCO-3(<3kDa),将各组分冷冻干燥后测其ACE抑制率。
如图7所示,鸭肉酶解产物用超滤膜分离后得到三个组分,分别为MWCO-1(>10kDa)、MWCO-2(3-10kDa)和MWCO-3(<3kDa),其ACE抑制率分别为30.38±0.83%、47.45±0.74%和55.15±0.36%(蛋白浓度为1.0mg/mL)。
2凝胶过滤色谱分离鸭肉源ACE抑制肽
选用填充料为葡聚糖凝胶Sephadex G-25的凝胶色谱柱(5×60cm),用超纯水平衡色谱柱。将超滤获得的ACE抑制活性较强的冻干组分溶解于超纯水中,配制成质量浓度为20mg/mL的溶液,涡旋混匀,经0.45μm水系滤膜过滤后进样,上样量为2mL。在洗脱过程中,超纯水为洗脱剂,洗脱流速为2mL/min,检测波长为280nm。分别收集各个经洗脱分离后的峰组分,冷冻干燥后进行ACE抑制活性的测定。
如图8和图9所示,通过凝胶过滤色谱后,MWCO-3组分被分离成两个主要组分,分别为组分A和组分B。两个组分的ACE抑制活性之间存在显著差异(P<0.05),组分A和B的ACE抑制率分别为33.21±1.47%和62.16±0.52%(蛋白浓度为1.0mg/mL)。该结果表明分子量较小的肽组分,其ACE抑制活性较强。因此,选择组分B进行下一步的反相高效液相色谱。
3反相高效液相色谱(RP-HPLC)分离鸭肉源ACE抑制肽
将通过凝胶过滤色谱获得的ACE抑制活性最强的组分溶解于超纯水中,配制成质量浓度为20mg/mL的溶液,涡旋混匀,经0.45μm水系滤膜过滤后用RP-HPLC进行分离。选用XBridge Peptide BEH C18柱,柱流速为0.8mL/min,流动相为:流动相A-含0.1%甲酸的超纯水溶液,流动相B-含0.1%甲酸的乙腈溶液。进样前,先使用2%的流动相B溶液进行冲洗,100μL样品进样后,流动相梯度洗脱程序如下:0-25min,2-20%B;25-30min,20-2%B。整个洗脱程序在280nm波长下进行,柱温25℃。分别收集各个经洗脱分离后的峰组分,冷冻干燥后进行ACE抑制活性的测定。
由图10可知,凝胶过滤组分B经过反相高效液相色谱后,被分离成B1-B7等7个组分。从图11中可以看出,各组分之间的ACE抑制能力存在显著差异(P<0.05)。组分B6的ACE抑制率最高,为68.31±2.79%(蛋白浓度为0.35mg/mL),组分B2的ACE抑制率次之,为55.79+2.1%,组分B1和B4的ACE抑制率最低。由于组分B6表现出最强的ACE抑制活性,因此选择组分B6进行下一步的质谱鉴定。
4Nano-LC-ESI-MS/MS分离鉴定
通过Nano-LC-ESI-LTQ-Orbitrap MS/MS液质联用设备鉴定鸭肉源ACE抑制肽的氨基酸序列。高效液相色谱条件:Thermo C18色谱柱(100×2.1cm,3μm,);流动相A:含有0.1%甲酸的超纯水溶液,流动相B:20%超纯水,80%乙腈,0.1%甲酸;梯度洗脱程序:0-1.5min(97%A,3%B),1.5-19min(97%-65%A,3%-35%B),19-26min(97%-5%A,35%-95%B),26-30min(5%-97%A,95%-3%B)。流速为0.2mL/min,上样量20μL。
质谱条件:样品经过HPLC后,在纳升电喷雾离子源作用下电离,然后进入质谱仪。电离模式为电喷雾电离,电离电压2.2kV,毛细管温度保持在200℃。一级质谱扫描通过Orbitrap进行,其扫描范围在100-1800m/z,分辨率为60000。二级质谱通过数据依赖性扫描模式进行,Orbitrap分辨率在7500,碰撞能量设为40%,活化q值为0.25,活化时间30ms。选取6个一级质谱中的最高峰进行碎片扫描,所得结果用Peaks软件进行从头测序(De Novo)分析,并通过鸭属(Anas)蛋白数据库进行多肽序列的溯源。
在氨基酸序列鉴定过程中,精确质量数扫描可以确定离子所带电荷数,通过从二级质谱中获得的碎片离子可进行氨基酸序列的识别,质谱结果通过Peaks软件分析。利用DeNovo从头测序共发现9条可信度在75%以上的肽段,通过Uniprot蛋白数据库进行溯源,发现有7条肽段与鸭属(Anas)蛋白库完全匹配。如表1所示(a-f同列不同字母表示差异显著(P<0.05),n=3),这些肽段由4-7个氨基酸残基组成,分子量在386.5Da-792.0Da之间。将这7条肽段进行人工合成,并对其ACE抑制活性进行验证,结果表明肽段TLVP表现出最强的ACE抑制活性(75.34±1.34%,0.35mg/mL)。
表1反相高效液相色谱组分B6经液质联用鉴定得到的肽段
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<110> 南京黄教授食品科技有限公司
<120> 一种鸭肉源ACE抑制肽及其制备方法
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Claims (10)
1.一种鸭肉源ACE抑制肽,其特征在于,所述ACE抑制肽至少具有以下氨基酸序列的短肽中的一种,所述的氨基酸序列分别为KLSH,FVAG,LHALLLL,TLVP,KALVG,AYDT,VLGV。
2.一种鸭肉源ACE抑制肽,其特征在于,所述ACE抑制肽氨基酸序列为TLVP。
3.一种如权利要求1所述的鸭肉源ACE抑制肽的制备方法,其特征在于,包括以下步骤:
1)将冷冻的鸭胸肉放于4℃冰箱中过夜解冻,去除肌膜等结缔组织后绞碎,按照肉水比称取肉样,匀浆,置于水浴箱中水浴15min,酶解pH值为6~10,加酶量为1000~5000U/g,酶解温度为55~75℃,酶解时间为1~5h,样品取出后,将酶解液的pH调至7,置于沸水浴中灭酶10min,冷却至室温后在转速为10000g、温度4℃下离心10min后取上清液进行冻干,得到鸭肉源ACE抑制肽提取物;
2)鸭肉源ACE抑制肽提取物经超滤、凝胶过滤色谱、反相高效液相色谱逐级分离纯化得到ACE抑制肽。
4.根据权利要求3所述的鸭肉源ACE抑制肽的制备方法,其特征在于,步骤1)中肉水质量体积比为1∶3g·mL-1。
5.根据权利要求3所述的鸭肉源ACE抑制肽的制备方法,其特征在于,步骤1)中酶的种类为:碱性蛋白酶,复合蛋白酶,胃蛋白酶。
6.根据权利要求3所述的鸭肉源ACE抑制肽的制备方法,其特征在于,步骤1)中酶的种类为:碱性蛋白酶。
7.根据权利要求3所述的鸭肉源ACE抑制肽的制备方法,其特征在于,步骤1)中酶解pH值为9,加酶量为2000U/g,酶解温度为65℃,酶解时间为4h。
8.根据权利要求3所述的鸭肉源ACE抑制肽的制备方法,其特征在于,步骤2)中超滤分离纯化中超滤膜的截留分子量小于3kDa。
9.根据权利要求3所述的鸭肉源ACE抑制肽的制备方法,其特征在于,步骤2)中制得的ACE抑制肽经液质联用鉴定得到的肽段分子量为386.5Da~792.0Da。
10.权利要求1所述的鸭肉源ACE抑制肽在辅助降血压的食品和药品中的应用。
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