CN108715597A - 甜味肽γ-L-Glu-D-Val及其制备方法与应用 - Google Patents
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Abstract
本发明公开了甜味肽γ‑L‑Glu‑D‑Val及其制备方法与应用。具体制备步骤为：将L‑谷氨酰胺与D‑缬氨酸混合后溶于水中，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8.0‑10.0，在25‑50℃反应3‑12小时，后用4 mol/L盐酸回调pH至6~7之间，90℃灭酶10 min。本发明制得这种γ‑谷氨酰胺肽（γ‑L‑Glu‑D‑Val）具有良好的稳定性，具有天然的类似蔗糖的甜味，应用于食品中能增加或提高食品的甜味。
Description
技术领域
本发明涉及具有呈味效果的肽及其制备方法，具体涉及甜味肽γ-L-Glu-D-Val及其制备方法与应用。
背景技术
呈味肽是重要的滋味物质和风味前体物质之一，按其呈味特性的不同可分为甜味肽、苦味肽、酸味肽、咸味肽和鲜味肽。陆续从奶酪、酒、肉等多种食物中分离鉴定得到确定序列呈苦、鲜、酸、咸、甜等味的呈味肽。自从发现L-天冬氨酞-L-苯丙氨酸甲酯的甜味以来,已有大量的天冬氨酞二肽、三肽、四肽和五肽类似物被合成以用于扩大甜味肽的种类。
γ-谷氨酰肽广泛存在于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。其中最为典型的是谷胱甘肽（γ-Glu-Cys-Gly，GSH）。GSH已经是总所周知的具有多种生理功能的小分子肽，但对于它的呈味特性提及甚少。事实上，目前对于此类γ-谷氨酰肽的研究主要主要针对的是L型氨基酸，对于D型氨基酸的研究甚少。迄今为止，尚未见γ-L-Glu-D-Val具有天然的类似蔗糖的甜味的报道。
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，提供甜味肽γ-L-Glu-D-Val及其制备方法与应用。
本发明的目的通过以下技术方案实现。
一种甜味肽γ-L-Glu-D-Val，其氨基酸序列为γ-L-Glu-D-Val。
制备所述甜味肽的方法是，将谷氨酰胺与游离缬氨酸按摩尔比为1-2:1，溶于水中得溶液，调节反应pH为8.0-10.0，添加占溶液质量0.02-1%（w/w）的谷氨酰胺酶或γ-谷氨酰胺转肽酶，在25-50℃反应3-12小时，后用盐酸回调pH至6~7之间，灭酶，得到富含二肽γ-L-Glu-D-Val的反应液。
进一步地，溶液中谷氨酰胺和游离缬氨酸的浓度在400-1000mmol/L。
进一步优化地，所述方法利用高相液相色谱分离：采用XSelect HSS T3 5μm 4.6× 250mm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富含二肽γ-L-Glu-D-Val的反应液进行梯度洗脱，出峰时间为4.267min的峰即为γ-L-Glu-D-Val。
本发明提供的一种甜味肽γ-L-Glu-D-Val的应用，具体是在食品中添加500 mg/kg以上的γ-L-Glu-D-Val，以增加或提高甜味。
进一步优化地，所述食品中含有1500 mg/kg以上的γ-L-Glu-D-Val。进一步地，溶液中谷氨酰胺和游离缬氨酸的浓度在400-1000mmol/L。
进一步地，本发明关键参数还有：所述盐酸浓度为4 mol/L 盐酸，所述灭酶为90℃灭酶10min，只有在这样的参数下才能取得说明书实施例所述的效果。
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-L-Glu-D-Val具有甜味特性，这种γ-谷氨酰胺肽（γ-L-Glu-D-Val）具有良好的稳定性，具有天然的类似蔗糖的甜味。
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-L-Glu-D-Val。
附图说明
图1为缬氨酸与谷氨酰胺混合物添加酶否呈味特性的差异对比图；
图2为反应后混合物的液相色谱图；
图3为γ-L-Glu-D-Val的呈味特性分析图；
图4为γ-L-Glu-D-Val的二级质谱图；
图5a~图5b分别为二肽对黑咖啡和红茶的呈味改善效果对比图。
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300mM谷氨酰胺与300mM缬氨酸溶于100g水中，用10 mol/l的氢氧化钠溶液调节溶液的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37℃分别反应12h，后用4 mol/L 盐酸回调pH 至 7.0后，90℃灭酶10 min，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，反应液的甜味、鲜味都显著提高了。说明反应液物质发生了改变，产生了具有甜味的物质。为了进一步确定物质的改变，将反应液用于高效液相色谱法确定产物。
HPLC液相对反应产物进行定量分析：分析柱：XSelect HSS T3 5μm 4.6x 250mm；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：1 mL/min。进样量 10 μL。其液相色谱图见图4。流动相进行梯度变化如表1所示。
表1
| Time/min | 0 | 5 | 10 | 15 | 20 |
| A/% | 90 | 85 | 20 | 90 | 90 |
图2中,出峰时间为4.267对应的峰4为γ-L-Glu-D-Val，对峰4进行收集，冷冻干燥得到γ-L-Glu-D-Val，并进行感官评价。称取0.01gγ-L-Glu-D-Val，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0.01gγ-L-Glu-D-Val的样品与对照相比，显著地赋予空白溶液甜味特性。
利用UPLC-MS/MS对产物进行结构分析：包括：An Agilent 1290 series UPLCsystem (Agilent Technologies) 用于分离各 γ-Glu-peptides, 带有an elec-trospray ionization (ESI)的质谱系统（Q-TOF MS/MS，Bruker Daltonics) 进行定性分析。色谱柱为：Agilent ZORBAX RRHD SB-C18 (2.1 x 50mm , 1.8μm)。进样量为5 μL。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 -5.0 min; 10 -15% B, 5.0 -10.0 min; 然后100% B, 10.0 -12.0 min。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。采用UPLC-MS/MS对峰6，其检测结果如图4。由图4可知，图中碎片离子主要为谷氨酸和缬氨酸残基。利用从头测序方法确定目标产物一级结构为γ-L-Glu-D-Val。
实施例2
将0.01g纯化过的γ-L-Glu-D-Val加入含有适量无糖奶粉的黑咖啡溶液或者红茶中，对产品进行感官评价。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5 a 和图5b。由图5a可知，将此甜味肽添加至含有奶粉的黑咖啡中，对照空白组而言，溶液的甜味明显增加，苦味、酸味明显降低，说明了此甜味肽产生了类似于蔗糖等咖啡伴侣的作用，赋予了黑咖啡甜味。如图5b，对于红茶而言，添加甜味肽后出现出现了类似奶茶的口感，甜味、丝滑感增加。由此两种应用可以判断，此甜味肽可用于取代蔗糖。
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CGMCC No.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缬氨酸溶于100g水中，分别调节溶液的pH值至9.0，加入1g粗酶液，在40℃分别反应12h后，用4 mol/L 盐酸回调pH 至 7.0后，90℃灭酶10 min，得到γ-L-Glu-D-Val的反应液。采用XSelect HSS T3 5μm 4.6x 250mm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，第四个峰即为甜味肽γ-L-Glu-D-Val。
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400mM缬氨酸溶于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4 mol/L 盐酸回调pH 至 7.0后，90℃灭酶10 min，得到含有γ-L-Glu-D-Val的反应液。采用XSelect HSS T3 5μm 4.6x 250mm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，第四个峰即为γ-L-Glu-D-Val。
Claims (7)
1.一种甜味肽γ-L-Glu-D-Val，其特征在于氨基酸序列为γ-L-Glu-D-Val。
2.制备权利要求1所述一种甜味肽γ-L-Glu-D-Val的方法，其特征在于，将谷氨酰胺与游离缬氨酸按摩尔比为1-2:1，溶于水中得溶液，调节反应pH为8.0-10.0，添加质量为溶液质量0.02-1%（w/w）的谷氨酰胺酶或γ-谷氨酰胺转肽酶，在25-50℃反应3-12小时，后用盐酸回调pH至6~7之间，灭酶，得到富含二肽γ-L-Glu-D-Val的反应液。
3.根据权利要求2所述的方法，其特征在于，所述盐酸浓度为4 mol/L 盐酸，所述灭酶为90℃灭酶10min。
4.根据权利要求2所述的方法，其特征在于溶液中谷氨酰胺和游离缬氨酸的浓度在400-1000mmol/L。
5.根据权利要求2或3所述的方法，其特征在于利用高相液相色谱分离：采用XSelectHSS T3 5μm 4.6× 250mm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富含二肽γ-L-Glu-D-Val的反应液进行梯度洗脱，出峰时间为4.267min的峰即为甜味肽γ-L-Glu-D-Val。
6.权利要求1所述一种甜味肽γ-L-Glu-D-Val的应用，其特征在于在食品中添加500mg/kg以上的γ-L-Glu-D-Val，以增加或提高甜味。
7.根据权利要求6所述的应用，其特征在于食品中含有1500 mg/kg以上的γ-L-Glu-D-Val。
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