CN107459555A - 寡肽、浓厚味赋予剂、调味料及其制备方法与应用 - Google Patents
寡肽、浓厚味赋予剂、调味料及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了寡肽、浓厚味赋予剂、调味料及其制备方法与应用。本发明的寡肽γ‑Glu‑γ‑Glu‑γ‑Glu‑Abu具有良好的稳定性，在60‑100mg/kg的浓度下可显著提高食品特别是调味品的厚味。具体制备步骤为：将谷氨酰胺与2‑氨基丁酸混合后溶于水中，添加溶液质量0.001‑0.4%（w/w）的谷氨酰胺酶或γ‑谷氨酰胺转肽酶，用10 mol/l的氢氧化钠溶液调节溶液的pH至7.0‑10.0，在25‑50℃反应3‑12小时，后用4 mol/L 盐酸回调pH 至 7~8之间，90℃灭酶10 min。
Description
技术领域
本发明涉及具有呈味效果的肽及其制备方法，具体涉及具有厚味的γ-谷氨酰肽及其制备方法。
背景技术
γ-谷氨酰肽广泛存与多种动植物和发酵食品中，是一种安全天然、安全的食品成分。常见的谷胱甘肽其一级结构是γ-Glu-Cys-Gly，是一种典型的γ-谷氨酰肽。现代研究表明γ-谷氨酰肽与生物体的代谢活动和生理功能有关，此外大部分γ-谷氨酰肽具有镇定减压、舒缓疲劳、抗癌解毒、络合重金属离子、维持细胞金属离子平衡等生理功能。
近年来，人们发现γ-谷氨酰肽还具有较强的呈味特性。2002年日本味之素株式会社公开了通过探索具有CaSR激动剂活性的多种多样的化合物，发现具有更优异的浓味赋予作用，特别是具有前味型的浓味赋予作用，且具有优异的稳定性，可以简便而低成本地生产的能够赋予浓味的物质，提供包含该物质的浓味赋予剂，以及将该物质与其他具有CaSR激动剂活性的物质组合使用而成的复合浓味赋予剂。包含γ-Glu-Abu(L-γ-谷氨酰-L-2-2-氨基丁酸)的浓味赋予剂，以及该物质与其他具有CaSR激动剂活性的物质组合使用而成的复合浓味赋予剂(专利申请号201080024054.1)。
在低浓度条件下的大部分γ-谷氨酰肽具有轻微涩味，随着浓度的提高，γ-谷氨酰肽开始呈现基本味道。例如γ-Glu-Glu在浓度为10mmol/L时有鲜味，γ-Glu-tyr的苦味阈值是其涩味阈值的两倍。在鲜味溶液中，γ-谷氨酰肽具有厚味，阈值也在明显下降。随着人们对呈味肽的进一步了解，学者发现γ-谷氨酰肽具有厚味物质的特征：1.在水溶液中基本没有味道或只有轻微的味道(涩味或酸味)；2.能够与体系中的其他呈味物质发生协同增效作用，添加到含有鲜味物质的溶液体系中，不会改变溶液的基本味觉强度，但是能协调各种味道，显著增加浓厚感(kokumi)；3.与纯水溶液相比，在鲜味溶液中阈值明显下降。
表1已报道γ-谷氨酰肽呈味特性和阈值一览表
迄今为止，尚未见γ-Glu-γ-Glu-γ-Glu-Abu具有浓厚味的报道。
发明内容
本发明的目的在于克服现有技术存在的上述不足，提供一种利用谷氨酰胺酶合成γ-谷氨酰肽的制备方法。
本发明的目的通过以下技术方案实现。
一种寡肽，其氨基酸序列为γ-Glu-γ-Glu-γ-Glu-Abu。
本发明提供的浓厚味赋予剂，由所述寡肽和如下一种以上的肽组合而成：γ-Glu-X-Gly(X表示氨基酸或氨基酸衍生物)、γ-Glu-Val-Y(Y表示氨基酸或氨基酸衍生物)、γ-Glu-Abu、γ-Glu-Ala、γ-Glu-Gly、γ-Glu-Cys、γ-Glu-Met、γ-Glu-Thr、γ-Glu-Val、γ-Glu-Orn、Asp-Gly、Cys-Gly、Cys-Met、Glu-Cys、Gly-Cys、Leu-Asp、D-Cys、γ-Glu-Met(O)、γ-Glu-γ-Glu-Val、γ-Glu-Val-NH2、γ-Glu-Val-ol、γ-Glu-Ser、γ-Glu-Tau、γ-Glu-Cys(S-Me)(O)、γ-Glu-Leu、γ-Glu-Ile、γ-Glu-t-Leu和γ-Glu-Cys(S-Me)。
一种含有所述寡肽的调味料，其含有60重量mg/kg以上的γ-Glu-γ-Glu-γ-Glu-Abu。
一种含有所述寡肽的调味料，含有60重量mg/kg以上的
γ-Glu-γ-Glu-γ-Glu-Abu。
一种含有所述寡肽的调味料，含有500重量mg/kg以上的
γ-Glu-γ-Glu-γ-Glu-Abu。
所述寡肽的制备方法，具体是：谷氨酰胺与游离2-氨基丁酸，摩尔比为1-2:1，溶于水中得溶液，调节反应pH为7.0-10.0，添加谷氨酰胺酶或γ-谷氨酰胺转肽酶0.02-1％(w/w)，在25-50℃反应3-12小时，后用4mol/L盐酸回调pH至7～8之间，90℃灭酶10min，得到富含γ-Glu-γ-Glu-Abu的反应液。采用XSelect HSS T3 5μm 4.6x250mm分析柱，流动相A液为0.1％(V/V)甲酸-水溶液；流动相B液为0.1％(V/V)甲酸-乙腈溶液，柱温为40℃，流速为1mL/min，反应液的进样量10μL，采用高效液相色谱对反应液进行梯度洗脱，第2个峰即为寡肽γ-Glu-γ-Glu-Abu。
进一步地，所述溶液中谷氨酰胺和游离2-氨基丁酸的浓度在400-1000mmol/L。
本发明与现有技术相比，具有如下优点：
1)本发明首次公开了γ-Glu-γ-Glu-γ-Glu-Abu具有厚味特性。
2)首次公开了利用谷氨酰胺酶或γ-谷氨酰胺转肽酶催化制备γ-Glu-γ-Glu-γ-Glu-Abu。
附图说明
图1为2-氨基丁酸与谷氨酰胺混合物添加谷氨酰胺酶对呈味特性的差异对比图；
图2为实例1反应后混合物的液相色谱图；
图3为γ-Glu-γ-Glu-γ-Glu-Abu的呈味特性分析；
图4为γ-Glu-γ-Glu-γ-Glu-Abu的二级质谱图；
图5a、图5b分别是γ-Glu-γ-Glu-Abu对酱油和鸡汤的呈味增效作用。
具体实施方式
以下结合附图和实例对本发明的具体是实施作进一步举例，但本发明的实施和保护不限于此。
实施例1
将400mM谷氨酰胺与400mM2-氨基丁酸溶于100g水中，调节溶液的pH值至10.0，加入0.4g谷氨酰胺酶(日本天野株式会社，活力为100GTU/g)，在25℃分别反应12h，后用4mol/L盐酸回调pH至7.0后，90℃灭酶10min，对产物呈味特性进行感官评价，其反应前后呈味特性如图1。
由图1可见，反应结束后，反应液的厚味、鲜味和咸味都显著提高了，甜味显著降低。谷氨酰胺呈甜味，反应后溶液中谷氨酰胺含量降低，导致反应液的甜味减弱。
HPLC液相对反应产物进行定量呈味分析：分析柱：XSelect HSS T3 5μm 4.6x250mm；流动相：A液：0.1％(V/V)甲酸-水溶液；B液：0.1％(V/V)甲酸-乙腈溶液；柱温：40℃，流速：1mL/min。进样量10μL。其液相色谱图见图2，流动相进行梯度变化洗脱如表2所示。
表2
| Time/min | 0 | 5 | 10 | 15 | 20 |
| A/％ | 90 | 80 | 20 | 90 | 90 |
图2中，与标准品进行对照，经验证，出峰时间为5.577对应的峰3别为γ-Glu-γ-Glu-γ-Glu-Abu，对峰3进行收集，冷冻干燥得到γ-Glu-γ-Glu-γ-Glu-Abu的单体。称取0.01gγ-Glu-γ-Glu-γ-Glu-Abu，以及0.5g食盐和0.3g味精，溶解于100g水中，并和空白样(0.5g食盐和0.3g味精溶于100g水中)进行感官评价。感官评价实验由30-41岁有感官评价经验的7位男性和9位女性组成，感官评价结果见图3。
由图3可见，添加0.01g的γ-Glu-γ-Glu-γ-Glu-Abu的样品与对照相比，溶液的鲜味和咸味明显提高。
UPLC-MS/MS对反应产物进行定性分析：包括：An Agilent 1290 series UPLCsystem(Agilent Technologies)用于各γ-Glu-peptides,带有an elec-trosprayionization(ESI)的质谱***(Q-TOF MS/MS，Bruker Daltonics)进行定性分析。色谱柱为：Agilent ZORBAX RRHD SB-C18(2.1x50mm,1.8μm)。进样量为5μL。液相条件为：溶液A为0.1％甲酸-水溶液，和溶液B为0.1％甲酸-乙腈溶液，梯度洗脱程序为：0–10％B,0-5.0min；10-15％B,5.0-10.0min；然后100％B,10.0-12.0min。MS条件：Ionization(离子化)：正离子模式；Drying gas(干燥气体)：350℃下10L/min；Nebulizer(喷雾器压力)：25psig；Fragmentor(毛细管电压)：30V。对峰3进行UPLC-MS/MS分析，其检测结果如图4。
由图4可知，其一级结构为γ-Glu-γ-Glu-γ-Glu-Abu。
实施例2
将鸡胸肉放入15倍重量清水中，煮沸后保温60min，冷冻祛除汤面油脂，过滤得鸡汤。加入鸡汤0.5％(％为占鸡汤质量百分比，下同)的食盐,添加0.01％γ-Glu-γ-Glu-γ-Glu-Abu于鸡汤中，评价其呈味特性变化。感官评价实验由30-41岁有感官评价经验的7位男性和9位女性组成，感官评价结果表明，添加0.01％的γ-Glu-γ-Glu-γ-Glu-γ-Abu的鸡汤的鸡汤的感觉、鲜味、甜味和厚味均有明显增加，见图5b。
酱油为市售海天金标酱油，分别添加0.01％(质量百分比，下同)γ-Glu-γ-Glu-γ-Glu-Abu于酱油中。感官评价实验由30-41岁有感官评价经验的7位男性和9位女性组成，感官评价结果表明，添加0.01％的γ-Glu-γ-Glu-γ-Glu-γ-Abu的酱油的咸味有所降低，但鲜味、甜味和厚味增加明显，见图5a。
实施例3
将解淀粉芽孢杆菌(Bacillus amyloliquefaciens)SWJS22(中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCCNo.8425)以1％的接种量接种于发酵培养基(发酵培养基的组成为：4％豆粕、2％麸皮、2％玉米粉)中，37～39℃发酵培养30h。将发酵液于4℃，10000r/min条件下高速离心10min，过滤得上清液，即谷氨酰胺酶粗酶液。
将600mM谷氨酰胺和300mM 2-氨基丁酸溶于100g水中，调节溶液的pH值至7.0，加入1g粗酶液，在50℃分别反应3h后，用4mol/L盐酸回调pH至7.0后，90℃灭酶10min，得到富含γ-Glu-γ-Glu-γ-Glu-Abu的反应液。采用XSelect HSS T3 5μm 4.6x250mm分析柱，流动相A液为0.1％(V/V)甲酸-水溶液；流动相B液为0.1％(V/V)甲酸-乙腈溶液，柱温为40℃，流速为1mL/min，反应液的进样量10μL，采用高效液相色谱对反应液进行梯度洗脱，第3个峰即为寡肽γ-Glu-γ-Glu-γ-Glu-Abu。
实施例4
将枯草芽胞杆菌ATCC 6633以1％的接种量接种于发酵培养基(发酵培养基的组成为：4％豆粕、2％麸皮、2％玉米粉)中，37～39℃发酵培养48h。将发酵液于4℃，10000r/min条件下高速离心10min，过滤得上清液，即γ-谷氨酰胺转肽酶粗酶液。
将600mM谷氨酰胺和400mM2-氨基丁酸溶于100g水中，分别调节溶液的pH值至8.0，加入1gγ-谷氨酰胺转肽酶粗酶液，在40℃分别反应6h后，用4mol/L盐酸回调pH至7.0后，90℃灭酶10min，得到富含γ-Glu-γ-Glu-γ-Glu-Abu的反应液。采用XSelect HSS T3 5μm4.6x250mm分析柱，流动相A液为0.1％(V/V)甲酸-水溶液；流动相B液为0.1％(V/V)甲酸-乙腈溶液，柱温为40℃，流速为1mL/min，反应液的进样量10μL，采用高效液相色谱对反应液进行梯度洗脱，第3个峰即为寡肽γ-Glu-γ-Glu-γ-Glu-Abu。
表3
a:酸味(mg/L),b:厚味.溶液pH值调到6.5
表3显示γ-谷氨酰肽在不同溶液中的刺激呈味阈值，％为占水的质量百分比。由表3可见，γ-Glu-γ-Glu-γ-Glu-Abu和乳酸有较强的呈味相互作用，添加味精和乳酸可明显降低γ-Glu-γ-Glu-γ-Glu-Abu的刺激呈味阈值。
Claims (8)
1.一种寡肽，其特征在于其氨基酸序列为γ-Glu-γ-Glu-γ-Glu-Abu。
2.浓厚味赋予剂，其特征在于由权利要求1所述寡肽和如下一种以上的肽组合而成：γ-Glu-X-Gly、γ-Glu-Val-Y、γ-Glu-Abu、γ-Glu-Ala、γ-Glu-Gly、γ-Glu-Cys、γ-Glu-Met、γ-Glu-Thr、γ-Glu-Val、γ-Glu-Orn、Asp-Gly、Cys-Gly、Cys-Met、Glu-Cys、Gly-Cys、Leu-Asp、D-Cys、γ-Glu-Met(O)、γ-Glu-γ-Glu-Val、γ-Glu-Val-NH2、γ-Glu-Val-ol、γ-Glu-Ser、γ-Glu-Tau、γ-Glu-Cys(S-Me)(O)、γ-Glu-Leu、γ-Glu-Ile、γ-Glu-t-Leu和γ-Glu-Cys(S-Me)； X、Y 表示氨基酸或氨基酸衍生物。
3.一种含有权利要求1所述寡肽的调味料，其特征在于含有60重量mg/kg以上的γ-Glu-γ-Glu-γ-Glu-Abu。
4.一种含有权利要求2所述浓厚味赋予剂的调味料，其特征在于含有60重量mg/kg以上的γ-Glu-γ-Glu-γ-Glu-Abu。
5.根据权利要求3或4所述的调味料，其特征在于含有500重量mg/kg以上的γ-Glu-γ-Glu-γ-Glu-Abu。
6.权利要求1所述寡肽的制备方法，其特征在于谷氨酰胺与游离2-氨基丁酸，摩尔比为1-2:1，溶于水中得溶液，调节反应pH为7.0-10.0，添加谷氨酰胺酶或γ-谷氨酰胺转肽酶0.02-1%（w/w），在25-50℃反应3-12小时，后用4 mol/L 盐酸回调pH至7~8之间，90℃灭酶10min，得到寡肽含γ-Glu-γ-Glu-γ-Glu-Abu反应液。
7.根据权利要求6所述的制备方法，其特征在于：采用XSelect HSS T3 5μm 4.6x250mm分析柱，流动相A 液为0.1％（V/V） 甲酸-水溶液；流动相B 液为0.1％(V/V) 甲酸-乙腈溶液，柱温为40℃，流速为1 mL/min，反应液的进样量 10 μL，采用高效液相色谱对反应液进行梯度洗脱，第3个峰即为寡肽γ-Glu-γ-Glu-γ-Glu-Abu。
8.根据权利要求6所述的制备方法，其特征在于所述溶液中谷氨酰胺和游离2-氨基丁酸的浓度在400-1000mmol/L。
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