CN112442104A - 发酵法制备枸杞活性肽及方法和应用与抑制剂、功能食品 - Google Patents
发酵法制备枸杞活性肽及方法和应用与抑制剂、功能食品 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A61P9/00—Drugs for disorders of the cardiovascular system
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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Abstract
本发明涉及一种发酵法制备枸杞活性肽的方法,本发明利用微生物发酵枸杞蛋白质制备枸杞活性肽,经本发明技术制备的枸杞活性肽具有以下特点:经过微生物的蛋白质酶体系作用,枸杞蛋白中的大分子量蛋白质被降解为小分子肽,不仅避免了枸杞蛋白引起过敏等不良反应,又使肽的活性位点暴露而表现出血管紧张素转换酶(angiotensin‑converting enzyme,ACE)和二肽基肽酶(dipeptidyl peptidase IV,DPP‑IV)抑制活性,枸杞活性肽可用于药品、功能性食品、食品添加剂等领域,具有广阔应用前景。
Description
技术领域
本发明涉及一种发酵法制备枸杞活性肽的方法,本发明利用微生物发酵枸杞蛋白质制备枸杞活性肽。
背景技术
生物活性肽是一类由2~20一个氨基酸组成的、相对分子质量小于6kDa的对生物机体的生命活动有益或是具有生理作用的肤类化合物的总称,具有非常重要的科学意义。
天然产物仍然是生物活性肽的主要来源,因为植物、动物和乳制品都含有大量的蛋白质。有些肽在亲本蛋白中不活跃,可以通过酶解或微生物发酵释放。枯草芽孢杆菌因其强大的蛋白水解活性而被广泛应用于微生物发酵。与未发酵蛋白相比,发酵产物具有更好的溶解性和稳定性。
枸杞是茄科植物,枸杞果实枸杞子是我国传统的药食同源药材,《中药大辞典》记载枸杞子具有滋补肝肾、益精明目的功效,枸杞子含有枸杞多糖、黄酮、多酚、类胡萝卜素等多种活性物质,具有抗氧化、降血脂、抗衰老、保肝等生物活性。干枸杞中枸杞蛋白质含量为14.67%,含有18种氨基酸,其中8种为人体必需氨基酸,是良好的生物活性肽来源。
血管紧张素转换酶(angiotensin-converting enzyme,ACE)作为一种多功能酶,广泛存在于人体中,通过作用于体内的肾素-血管紧张素系统和激肽释放酶-激肽系统起到调节血压的功能。因此ACE抑制剂作为一种降血压药物被广泛利用。二肽基肽酶(dipeptidyl peptidase IV,DPP-IV)是一种细胞表面的丝氨酸蛋白酶,可以灭活多种生物活性肽,包括胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)。DPP-IV抑制剂通过使DPP-IV失活,提高体内GLP-1水平,产生调节血糖的作用。但是由于西药类合成药物会造成肝肾负担,伴随极大地副作用,所以具有生物功能的活性肽在治疗和/或预防高血压、高血糖的药物及保健食品具有良好的发展前景。
发明内容
本发明涉及一种发酵法制备枸杞活性肽的方法,本发明利用微生物发酵枸杞蛋白质制备枸杞活性肽,活性肽表现出血管紧张素转换酶和二肽基肽酶抑制活性。
为实现上述目的,本发明采用的技术方案如下:
提取枸杞蛋白,经过微生物发酵枸杞蛋白制备枸杞活性肽。
(1)枸杞蛋白提取物的制备
以干枸杞为原料,称取枸杞并加入其8~20倍(枸杞g/mL水)去离子水浸泡2~8小时后粉碎,超声提取20~60min,于3000~8000rpm离心10~20min,沉淀用于提取枸杞蛋白;
沉淀冷冻干燥后再次粉碎得到枸杞粉;枸杞粉中加入其5~20倍(枸杞粉g/mL提取液)体积的有机溶剂提取液,于温度20~80℃、转速80~200rpm搅拌提取0.5~2小时,去除液体,保留固体,重复提取过程2~4次,滤饼冷冻干燥后为枸杞蛋白;
(2)枸杞活性肽的制备
将枸杞蛋白加入其5~20倍(枸杞蛋白g/mL水)去离子水,用1~5M的氢氧化钠溶液调节pH至7.2~7.5,接种微生物菌种,发酵液菌含量为1*106~2*108CFU/mL,于35~50℃发酵24~72小时;发酵结束后在9000~15000rpm离心5~20min,取上清液冷冻干燥,得到发酵粗提取物;
发酵粗提取物中加入其2~10倍体积(发酵粗提取物g/mL盐酸)的0.01~0.1M的盐酸,在0~8℃匀浆5~20min后,于5000~15000rpm离心10~30min,取上清液加入1~5倍体积的无水乙醇,0~8℃静置24~48小时,于5000~15000rpm离心10~30min,取上清冷冻干燥,得到枸杞多肽。
枸杞蛋白提取物制备过程中所用有机溶剂为正己烷、乙酸乙酯、正丁醇、乙醇、甲醇中的一种或者两种溶剂以1:1~1:5的体积比混合。
发酵所用的微生物菌种为枯草芽孢杆菌、乳酸杆菌、嗜热链球菌中的一种或者两种以上的菌种。
本发明具有如下优点:
1.反应条件温和,本发明提取枸杞蛋白,使用微生物发酵,反应条件温和,保留了枸杞有活性组分。
2.去除了可能引起过敏的大分子蛋白质,增加了新活性。通过微生物发酵技术可将易引起过敏的大分子蛋白质去除。发酵技术可将大分子蛋白质降解为小分子物质,消除了枸杞的致敏原,扩大了其适用人群。发酵枸杞蛋白质可制备成为具有ACE抑制活性、DPP-IV抑制活性的生物活性肽。
3.本发明作为降血压、降血脂药物或保健食品研发的潜在来源具有广泛的应用前景。
具体实施方式
实施例1
(1)枸杞蛋白提取物的制备
以10g干枸杞为原料,称取枸杞并加入100mL去离子水浸泡2小时后粉碎,超声提取60min,于5000rpm离心10min,9.6g沉淀用于提取枸杞蛋白。
沉淀冷冻干燥后再次粉碎得到枸杞粉,枸杞粉中加入192mL的有机溶剂提取液,正己烷:乙醇=2.6:1(v/v),于温度50℃、转速150rpm搅拌提取1小时,过滤,滤饼再用与上述相同的有机溶剂提取液提取一次,提取两次后过滤,滤饼冷冻干燥后为7.5g枸杞蛋白。
(2)枸杞活性肽的制备
将枸杞蛋白加入112mL离子水,用1M的氢氧化钠溶液调节pH至7.5,接种微生物菌种(枯草芽孢杆菌),菌含量为2*108CFU/mL,于37℃发酵48小时;发酵结束后在13000rpm离心15min,取上清液冷冻干燥,得到1g发酵粗提取物。
发酵粗提取物中加入5mL的0.01M的盐酸,在2℃匀浆8min后,于12000rpm离心20min,取上清液加入其3倍体积的无水乙醇,4℃静置24小时,于12000rpm离心20min,取上清冷冻干燥,得到0.383g枸杞多肽。
实施例2
提取枸杞蛋白,经过微生物发酵枸杞蛋白制备枸杞活性肽。
(1)枸杞蛋白提取物的制备
以20g干枸杞为原料,称取枸杞并加入40mL去离子水浸泡2小时后粉碎,超声提取20min,于7000rpm离心10min,得到19.1g沉淀用于提取枸杞蛋白。
沉淀冷冻干燥后再次粉碎得到枸杞粉,枸杞粉中加入97.5mL的有机溶剂提取液,乙酸乙酯:甲醇=1:1(v/v),于温度20℃、转速80rpm搅拌提取0.5小时,提取一次后过滤,滤饼冷冻干燥后为15g枸杞蛋白。
(2)枸杞活性肽的制备
将枸杞蛋白加入75mL去离子水,用1M的氢氧化钠溶液调节pH至7.5,接种乳酸杆菌,菌含量为1*106CFU/mL,于35℃发酵24小时;发酵结束后在12000rpm离心5min,取上清液冷冻干燥,得到2.09g发酵粗提取物。
发酵粗提取物中加入14.63mL的0.01M的盐酸,在2℃匀浆5min后,于5000rpm离心10min,取上清液加入1倍体积的无水乙醇,6℃静置24小时,于5000rpm离心10min,取上清冷冻干燥,得到0.766g枸杞多肽。
实施例3
提取枸杞蛋白,经过微生物发酵枸杞蛋白制备枸杞活性肽。
(1)枸杞蛋白提取物的制备
以30g干枸杞为原料,称取枸杞并加入120mL去离子水浸泡2小时后粉碎,超声提取40min,于5000rpm离心15min,28.7mL沉淀用于提取枸杞蛋白。
沉淀冷冻干燥后再次粉碎得到枸杞粉,枸杞粉中加入143.5mL的有机溶剂提取液,乙酸乙酯:甲醇=1:4(v/v),于温度20℃、转速80rpm搅拌提取0.5小时,过滤,滤饼再用与上述相同的有机溶剂提取液提取一次,提取两次后过滤,滤饼冷冻干燥后为22.67g枸杞蛋白。
(2)枸杞活性肽的制备
将枸杞蛋白加入113.35mL去离子水,用1M的氢氧化钠溶液调节pH至7.5,接种嗜热链球菌,菌含量为6*106CFU/mL,于35℃发酵24小时;发酵结束后在12000rpm离心30min,取上清液冷冻干燥,得到3.14g发酵粗提取物。
发酵粗提取物中加入31.4mL的0.01M的盐酸,在8℃匀浆5min后,于5000rpm离心28min,取上清液加入1倍体积的无水乙醇,1℃静置24小时,于5000rpm离心10min,取上清冷冻干燥,得到1.15g枸杞多肽。
(3)枸杞活性肽的鉴定
将枸杞多肽用LTQ Orbitrap Velos进行质谱分析:将枸杞多肽除盐,商品化C18SPE预处理柱,经2mL乙腈活化,再用2mL的0.1%(v/v)TFA溶液洗去乙腈。样品用50%(v/v)TFA溶液调节p H值至2后过柱。经2mL的0.1%(v/v)TFA液脱盐后,用1mL 80%(v/v)乙腈/0.1%(v/v)TFA溶液分3次洗脱,洗脱液冷冻干燥后于-20℃保存,用于质谱分析。将冻干样品加适量0.1%(v/v)甲酸复溶,配制成0.5μg/μL的溶液,使用线性离子阱静电场轨道阱组合质谱仪(LTQ Orbitrap Velos)对样品进行质谱分析,设置离子传输毛细管的温度250℃,电喷雾电压2.2kV,归一化碰撞能量35.0%。均使用数据依赖模式(data-dependent mode)对MS和MS/MS进行图谱采集。质谱扫描条件设定为:从每次m/z=400~2000的全扫描中选择10个最高丰度离子峰进行MS/MS扫描。样品平行分析三次,取三次分析鉴定到的共同肽段进行统计。将采集的*.raw文件数据用Thermo Proteome Discoverer Daemon(v1.4)转换成*.mgf格式,再用Mascot(version 2.3.0,Matrix Science,London,UK)谱图在茄科植物蛋白库solanaceae.fasta(https://www.uniprot.org/)中进行检索。三次分析共鉴定到来源于14个不同蛋白的75条共同肽段,41.33%的肽段来自蛋白Fibrillin,肽段中氨基酸数量在8~27之间,计算分子质量在920.4491~2742.4664(Da),表一为部分枸杞多肽的质谱鉴定结果。
表一枸杞多肽的质谱鉴定结果
实施例4枸杞多肽的ACE抑制活性检测
N-(3-(2-呋喃酰)丙烯酰-苯氨酰-谷氨酰-谷氨酸(FAPGG,λmax=340nm,ε=2270M-1cm-1,分子量399.40)可以被ACE酶解成N-[3-(呋喃)丙稀醇酰]-2-苯丙氨酸(FAP,λmax=340nm,ε=1512M-1cm-1)和双甘氨肽(GG,在340nm处无吸收),因此可以作为ACE的模拟底物。1mM FAPGG完全转化成FAP和GG的吸光值为0.758,因此可根据340nm吸光度的变化值计算抑制率。
反应体系
(1)缓冲溶液:0.1M PBS缓冲液(pH=8.2,含300mM NaCl)
(2)底物溶液:使用上述缓冲液配制浓度为1.6mM的FAPGG溶液。
(3)酶溶液:使用上述缓冲溶液将ACE配制成0.2U/mL的溶液。
(4)样品溶液:按照实验需要将枸杞多肽用上述缓冲液配制成15.0,10.0,5.0mg/mL浓度的溶液。
实验在96孔板中进行。按照表二依次加入样品溶液、ACE溶液和底物溶液,混合均匀,立即用酶标仪于340nm波长下测定吸光度,记为OD0,37℃温育30min后再次于340nm波长下测定吸光度,记为OD1。测定各个样品的孔的吸光度,令ΔOD=OD0-OD1。每个样品平行测定3次。
表二ACE抑制活性加样方法
“-”代表加入该列等体积的PBS缓冲液
ACE抑制率计算公式:
I=[(ΔODControl-ΔODSample)/(ΔODControl-ΔODblank)]*100%
ΔODcontrol表示反应中样品溶液被等量的缓冲液取代后吸光度的变化;ΔODSample表示反应中样品溶液的吸光度变化;而ΔODblank表示反应中样品溶液、酶溶液被等量的缓冲液取代后吸光度的变化
表三ACE抑制活性实验的吸光度
表四枸杞多肽对ACE的抑制活性
按上述方法对不同浓度枸杞多肽进行ACE抑制活性检测。表三为不同浓度枸杞多肽对于ACE抑制活性实验的吸光度,表四为不同浓度的枸杞多肽对ACE的抑制活性,由此计算枸杞多肽半抑制浓度(IC50)为7.13±0.29mg/mL。
Claims (9)
1.微生物发酵法制备枸杞活性肽的方法,其特征在于:提取枸杞中的蛋白质,制备枸杞蛋白提取物,利用微生物发酵枸杞蛋白提取物制备枸杞活性肽;
具体过程为:
(1)枸杞蛋白提取物的制备
以干枸杞为原料,称取枸杞并加入其8~20倍(枸杞g/mL水)去离子水浸泡2~8小时后粉碎,超声提取20~60min,于3000~8000rpm离心10~20min,沉淀用于提取枸杞蛋白;
沉淀冷冻干燥后再次粉碎得到枸杞粉;枸杞粉中加入其5~20倍(枸杞粉g/mL提取液)体积的有机溶剂提取液,于温度20~80℃、转速80~200rpm搅拌提取0.5~2小时,去除液体,保留固体,重复提取过程2~4次,滤饼冷冻干燥后为枸杞蛋白;
(2)枸杞活性肽的制备
将枸杞蛋白加入其5~20倍(枸杞蛋白g/mL水)去离子水,用1~5M的氢氧化钠溶液调节pH至7.2~7.5,接种微生物菌种,发酵液菌含量为1*106~2*108CFU/mL,于35~50℃发酵24~72小时;发酵结束后在9000~15000rpm离心5~20min,取上清液冷冻干燥,得到发酵粗提取物;
发酵粗提取物中加入其2~10倍体积(发酵粗提取物g/mL盐酸)的0.01~0.1M的盐酸,在0~8℃匀浆5~20min后,于5000~15000rpm离心10~30min,取上清液加入1~5倍体积的无水乙醇,0~8℃静置24~48小时,于5000~15000rpm离心10~30min,取上清冷冻干燥,得到枸杞多肽。
2.按照权利要求1所述的方法,其特征在于:
枸杞蛋白提取物制备过程中所用有机溶剂为正己烷、乙酸乙酯、正丁醇、乙醇、甲醇中的一种或者两种以上;
当采用两种溶剂时,以1:1~1:5的体积比混合。
3.按照权利要求1所述的方法,其特征在于:
发酵所用的微生物菌种为枯草芽孢杆菌、乳酸杆菌、嗜热链球菌中的一种或者两种以上的菌种。
4.一种权利要求1、2或3所述方法制备获得的枸杞活性肽。
5.一种抑制剂,其为血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制剂和/或二肽基肽酶(dipeptidyl peptidase IV,DPP-IV)抑制剂,其以权利要求4所述的枸杞活性肽为活性成份。
6.按照权利要求5所述抑制剂,其特征在于:
抑制剂中还可加入药物学可接受的载体制备成任何剂型形式,例如口服剂型:粉剂、片剂、胶囊剂、软胶囊剂、水性药物、糖浆剂、酊剂、丸剂、散剂、小包、或颗粒剂。
7.一种功能食品,权利要求4所述的枸杞活性肽加入到食品中,制备成具有血管紧张素转换酶抑制活性和/或二肽基肽酶抑制活性的功能食品。
8.一种权利要求4所述枸杞活性肽的应用,其特征在于:
所述枸杞活性肽作为血管紧张素转化酶(angiotensin-converting enzyme,ACE)抑制剂、和/或二肽基肽酶(dipeptidyl peptidase IV,DPP-IV)抑制剂的活性成份,用于制备药品或功能食品。
9.按照权利要求8所述枸杞活性肽的应用,其特征在于:
可加入药物学可接受的载体或食品中制备成任何剂型形式,例如口服剂型:粉剂、片剂、胶囊剂、软胶囊剂、水性药物、糖浆剂、酊剂、丸剂、散剂、小包、或颗粒剂。
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