CN116751246A - 一种鲜味寡肽及其制备方法 - Google Patents
一种鲜味寡肽及其制备方法 Download PDFInfo
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- CN116751246A CN116751246A CN202310603685.8A CN202310603685A CN116751246A CN 116751246 A CN116751246 A CN 116751246A CN 202310603685 A CN202310603685 A CN 202310603685A CN 116751246 A CN116751246 A CN 116751246A
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- umami
- halophilus
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Classifications
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- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
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- C—CHEMISTRY; METALLURGY
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- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
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Abstract
本发明涉及功能微生物筛选与食品生物技术领域,具体提供了一种鲜味寡肽及其制备方法与应用。所述鲜味寡肽是从嗜盐四链球菌(Tetragenococcus halophilus)发酵液中分离提取得到的,具有浓郁的鲜味,口味延伸感强,且具有鲜味增强效果,可广泛应用于食品和调味品领域。
Description
技术领域
本发明涉及功能微生物筛选与食品生物技术领域,具体是涉及一种新型鲜味寡肽及其制备方法与应用。
背景技术
鲜味肽是由食物中提取或经氨基酸合成得到的有鲜味特性的小分子肽,其分子质量通常为150至3000Da。作为近年来提出的一种新型鲜味物质,鲜味肽具有良好的呈鲜效果、加工特性、以及营养价值,成为近年来食品鲜味科学的研究热点和鲜味剂开发的重点方向,引起人们的广泛关注。
鲜味肽可用于补充和增强食物的整体味道,使其更加和谐,柔软和浓郁;也能够在不影响食品的酸、甜、苦或咸等其他味感的基础上,通过协同作用或美拉德反应,进一步补充或增强食品的总体味感,使其风味更加协调、柔和、浓郁,在带来愉悦的味觉感受的同时,还能提供肽及氨基酸类营养成分。与此同时,鲜味肽还可以通过肽与肽、肽与核苷酸、肽与阳离子这三种方式与其他鲜味物质发生协同作用,进而达到增鲜的目的。除此之外,Kim发现鲜味肽除了本身的呈鲜、增鲜效果,还可以掩盖和减弱苦味,改善食品的风味,同时具有良好的加工特性及热稳定性。
现代营养学研究发现,膳食蛋白经消化道各种酶作用后,相比于游离氨基酸,寡肽消化更快、吸收更多。近年来,国内外学者在许多动物源、植物源等加工或未加工食品中都发现含有鲜味肽。2018年Yu等人从蚕蛹蛋白水解物中提取得到具有鲜味、甜味、涩味的寡肽VPY、AAPY、GFP、TAY。2019年,Zhang等人从花生分离蛋白水解物中提取得到DQR、NNP、EGF、EDG等具有轻微鲜味的寡肽。2021年,Shen等人从猪骨蛋白水解物中提取得到鲜味寡肽SY,PN,GS,LP、APHR。
采用传统方法从肉类、谷类或真菌类中提取的鲜味肽往往呈味不足,而且生产成本高,不能满足食品加工领域的广泛需求。与传统鲜味肽提取方法相比,从发酵食品中筛选得到高产鲜味肽的益生菌,再从其发酵液中提取鲜味肽的方法,可以进一步富集鲜味,得到呈鲜含量更高、增鲜效果更好的鲜味肽;与此同时,也更加安全、绿色、健康。因此,筛选新型的鲜味寡肽及其生产菌株,提高鲜味寡肽的品质和产量,具有重要的意义。
发明内容
本发明为解决现有技术问题,提供了一种新型鲜味寡肽及其制备方法与应用。所述鲜味寡肽是从嗜盐四链球菌(Tetragenococcus halophilus)发酵液中分离提取得到的,具有浓郁的鲜味,口味延伸感强,且具有鲜味增强效果,可广泛应用于食品和调味品领域。
本发明一方面提供了一种新型鲜味寡肽,所述鲜味寡肽的氨基酸序列为DFE(Asp-Phe-Glu)、LAGE(Leu-Ala-Gly-Glu)或QLQ(Gln-Leu-Gln)中的任意一种。
本发明一方面提供了上述鲜味寡肽在制备调味品、发酵食品或保健品中的应用。
所述调味品为味精、鸡精、鸡粉、酱油、汤块、耗油、酱、腐乳中的任意一种。
本发明还提供了一种鲜味剂,包含上述鲜味寡肽。
本发明还提供了一种鲜味剂的制备方法,包括如下步骤:
(1)将嗜盐四链球菌进行发酵培养,得发酵液;
(2)将嗜盐四链球菌发酵液进行离心,得上清液;
(3)将上清液进行冷冻干燥,即得鲜味剂。
本发明还提供了一种鲜味剂的制备方法,包括如下步骤:
(1)将嗜盐四链球菌进行发酵培养,得发酵液;
(2)将嗜盐四链球菌的发酵液经40%、60%、80%(v/v)乙醇分级沉淀;
(3)将80%(v/v)乙醇沉淀物进行冷冻干燥,即得鲜味剂。
所述嗜盐四联球菌为嗜盐四联球菌SNTH-1(Tetragenococcus halophilus SNTH-1),已于2021年08月20日保藏至中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCCNo.23165。
进一步优选的,所述鲜味剂的制备方法包括以下步骤:
(1)嗜盐四链球菌的发酵培养:
①菌株活化:
按1%(v/v)接种量将嗜盐四链球菌菌株接种于50mL MRS培养基中,调整pH值为8.3,在37℃条件下摇瓶培养40h,待菌株生长至对数生长期末期时,取0.5mL发酵菌液重复接种于50mL MRS培养基中,得到活化的嗜盐四联球菌;
②种子液培养:
将活化后的嗜盐四联球菌菌液接种到5L发酵罐,内有3L富集培养基,在温度37℃,转速200rpm,空气流量比1:0.5,罐压0.1kg/cm2的条件下培养24h,得到种子液;
③发酵罐培养:
将种子液转入到50L的发酵罐中,内有30L大豆蛋白基质培养基,在温度37℃,转速200rpm,空气流量比1:0.55,罐压0.12kg/cm2的条件下发酵42h,得到富含鲜味肽的发酵液;
(2)乙醇分级:
①取嗜盐四联球菌的发酵液,浓缩后加入食用级无水乙醇,配成乙醇终浓度为40%的溶液,常温搅拌30min,8000g离心30min,得沉淀物1和上清液1;
②将上清液1与食用级无水乙醇混合配成乙醇终浓度为60%的溶液,常温搅拌30min,8000g离心15min,得沉淀物2和上清液2;
③将上清液2与食用级无水乙醇混合配成乙醇终浓度为80%的溶液,常温搅拌30min,8000g离心15min,得沉淀物3和上清液3;
(3)将80%乙醇沉淀物3进行冷冻干燥,即得鲜味剂。
所述的富集培养基中各组分及其含量分别为:蛋白胨10g/L、无水乙酸钠3g/L、磷酸氢二钾2g/L、七水合硫酸镁0.575g/L、一水合硫酸锰0.25g/L、葡萄糖20g/L、柠檬酸三钠2.42g/L、酵母浸粉4g/L、牛肉浸膏8g/L、吐温80 1g/L;氯化钠150g/L、那他霉素2g/L、结晶紫500μL/L。
所述的大豆蛋白基质培养基中各组分及其含量分别为:大豆蛋白胨20g/L、无水乙酸钠3g/L、磷酸氢二钾2g/L、七水合硫酸镁0.575g/L、一水合硫酸锰0.25g/L、葡萄糖20g/L、柠檬酸三钠2.42g/L、吐温80 1g/L、氯化钠100g/L。
本发明的有益效果
申请人从东北自然发酵的豆酱中筛选出一株高产鲜味肽的菌株嗜盐四联球菌SNTH-1,其发酵上清液中鲜味多肽的含量高达25.35mg/mL-26.62mg/mL;电子舌测定鲜味值高达16.22,呈鲜味效果非常好。
本发明提供的三条生物源鲜味寡肽DFE、LAGE和QLQ,是从嗜盐四联球菌SNTH-1发酵液中分离提取到的,其呈鲜阈值分别为0.4517mmol/L、0.2893mmol/L和0.2256mmol/L。DFE的鲜味和酸味较为突出,甜味也较强,具有一定的咸味,苦味最弱,其中鲜味味感评分为6.54,超过标准品,鲜味十分显著;LAGE的鲜味和甜味较为突出,其中鲜味值为5.68弱于DFE,但甜味更高,同时有一定酸味,苦味和咸味稍弱;QLQ的鲜味评分为3.67,苦味较为突出,酸味、甜味和咸味均较低。
所述鲜味寡肽DFE为嗜盐四联球菌SNTH-1特产鲜味肽,鲜味最显著,有一定的厚味,并且具有鲜味增强效果,增鲜阈值达0.3839mmol/L,能明显减少模拟肉汤和酱油的苦味,增加其鲜味和咸味,可广泛添加在食品和复合调味品中,显著提升其风味,增强口味的厚重感。
所述鲜味寡肽DFE由其CDP-甘油磷酸转移酶[EC:2.7.8.45]调控生成;LAGE和QLQ是由CRISPR相关解旋酶Cas3[EC:3.1.-.-3.6.4.-]调控生成。鲜味寡肽DFE基于分子模拟可以与鲜味受体T1R1/T1R3对接,主要结合位点为位于T1R3亚基上的氨基酸残基SER256。
此外,本发明还提供了利用嗜盐四联球菌SNTH-发酵生产得到的鲜味剂。所述鲜味剂中多肽含量高达31.83mg/g-32.25mg/g,鲜味值高达16.22,呈鲜味效果非常好。所述鲜味剂中总氨基酸、游离氨基酸和肽基氨基酸含量分别为208.28mg/g、104.25mg/g和104.03mg/g,其中鲜味氨基酸含量达93.81mg/g,占全部总氨基酸的40%,游离氨基酸中鲜味氨基酸的含量达41.74mg/g,占全部游离氨基酸的45%。所述鲜味剂中富含鲜味寡肽DFE、LAGE和QLQ和其他小分子鲜味肽类物质,具有呈味能力强、吸收速度快和营养价值高等特点。
本发明提供的鲜味寡肽以及鲜味剂均可广泛应用于味精、鸡精、鸡粉、酱油、汤块、耗油、酱、腐乳等调味品的生产,还可添加于发酵食品或保健品中,用于改善食品或保健品的口味应用。
附图说明:
图1为嗜盐四联球菌SNTH-1发酵液不同乙醇分级提取组分的感官滋味轮廓图;
图2为嗜盐四联球菌SNTH-1发酵液80%乙醇提取组分的葡聚糖G-15凝胶色谱图;
图3为凝胶柱层析后不同鲜味组分的感官滋味轮廓图;
图4为凝胶层析后鲜味组分(G-A-2)的反相高效液相色谱图;
图5为UPLC-ESI-MS/MS二级质谱图,其中(A)DFE,(B)LAGE,(C)QLQ;
图6为合成肽的呈味特性分析,其中(A)感官滋味轮廓图,(B)电子舌滋味轮廓图;
图7为鲜味受体T1R1/T1R3的同源模拟受体模型;
图8为鲜味寡肽DFE与鲜味受体T1R1/T1R3的分子对接结果图;
图9为鲜味寡肽DFE对肉汤和酱油的感官滋味轮廓图,其中(A)模拟肉汤,(B)酱油。
具体实施方式
下面结合具体实施例对本发明做详细的描述。
本发明实施例中使用的嗜盐四联球菌SNTH-1(Tetragenococcus halophilusSNTH-1)筛选自辽阳市小屯镇上平洲村自然发酵半年的豆酱中,其16s rDNA序列为SEQ IDNO:1,已于2021年08月20日保藏至中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCCNo.23165。
嗜盐四联球菌SNTH-1能够高产鲜味多肽,在50L发酵罐中发酵42h后,其发酵上清液中鲜味多肽的含量高达25.35mg/mL-26.62mg/mL,电子舌测定鲜味值高达16.22,呈鲜味效果非常好。
嗜盐四联球菌SNTH-1还能高产蛋白酶、淀粉酶和γ-谷氨酰胺转肽酶,其发酵上清液中蛋白酶活力为85.42U/mL-85.48U/mL,淀粉酶活力为57.76±0.04U/mL;其细胞裂解液中γ-谷氨酰胺转肽酶活力6.44±0.01U/mL。
嗜盐四联球菌SNTH-1可广泛应用于鲜味肽的生产,具有生产周期短、产量高、安全无毒副作用的优点。该菌株还可用于制备发酵食品,有助于改善发酵食品的风味,提高发酵食品的安全性,具有广阔的应用前景。
本发明实施例使用的培养基及其配方如下:
MRS培养基(g/L):蛋白胨10g、乙酸钠(无水)3g、磷酸氢二钾2g、七水合硫酸镁0.575g、一水合硫酸锰0.25g、葡萄糖20g、柠檬酸三钠2.42g、酵母浸粉4g、牛肉浸膏8g、吐温80 1g;
富集培养基(g/L):MRS培养基中添加氯化钠150g、那他霉素2g、结晶紫500μL、调整pH值为7;
固体分离培养基(g/L):MRS培养基中添加氯化钠150g、那他霉素2g、碳酸钙10g、琼脂20g,调整pH值为7;
大豆蛋白基质培养基(g/L):大豆蛋白胨20g、乙酸钠(无水)3g、磷酸氢二钾2g、七水合硫酸镁0.575g、一水合硫酸锰0.25g、葡萄糖20g、柠檬酸三钠2.42g、吐温80 1g、氯化钠100g,调整pH值为7。
下面结合具体实施例,对本发明作进一步阐述。
实施例1嗜盐四联球菌SNTH-1在发酵生产鲜味肽中的应用
1、菌株活化:
从-80℃超低温冰箱中取已保藏的嗜盐四联球菌SNTH-1(Tetragenococcushalophilus SNTH-1)菌株1份,待化冰后按1%(v/v)接种量将其接种于50mL MRS培养基中,调整pH值为8.3,在37℃条件下摇瓶培养40h,待菌株生长至对数生长期末期时,取0.5mL发酵菌液重复接种于50mL MRS培养基中,得到已经活化的嗜盐四联球菌SNTH-1菌株;
2、种子液培养:
将上述活化后的菌液接种到5L发酵罐,内有3L富集培养基,在温度37℃,转速200rpm,空气流量比1:0.5,罐压0.1kg/cm2的条件下培养24h,得到种子液;
3、发酵罐培养:
将生长旺盛的种子液转入到50L的发酵罐中,内有30L大豆蛋白基质培养基,在温度37℃,转速200rpm,空气流量比1:0.55,罐压0.12kg/cm2的条件下发酵42h,得到嗜盐四联球菌SNTH-1的发酵液。
将发酵液在4℃条件下8000rpm离心20min,取上清液,参照下述方法分别进行多肽含量测定和电子舌呈味分析测定。
结果显示:所述嗜盐四联球菌SNTH-1发酵上清液中鲜味多肽的含量高达25.35mg/mL-26.62mg/mL;电子舌测定鲜味值高达16.22,呈鲜味效果非常好。
所述嗜盐四联球菌SNTH-1发酵上清液经过冷冻干燥后,可直接作为鲜味剂应用于调味品或食品中,其中鲜味多肽含量达到31.83mg/g-32.25mg/g。
产多肽能力测定方法:
将1mL稀释的发酵上清液添加到4mL双缩脲试剂中。充分摇动混合物,并在室温(24℃-26℃)下放置30min,然后在540nm处测定吸光度值。将0、2、4、8、10、12、16和20mg/mL的Gly-Gly-Tyr-Arg四肽标准溶液设置为标准曲线,以空白培养基作空白对照。试验一式三份进行,结果以每毫升发酵上清液中的肽毫克数报告。计算公式如下:
多肽含量(mg/mL)=N*(A-0.0006)/0.0141。
电子舌(SA402B电子舌)呈味分析方法:
该电子舌配备了5个测试传感器和2个参比传感器,其中AAE、CT0、CA0、AE1和C00测试传感器分别用于测试鲜味、咸味、酸味、涩味还有苦味。
从测试传感器中取出Ag/AgCl电极后,加入内部溶液后重新组装传感器,将其放入参比溶液中活化24h,备用。参比传感器的活化:从参比传感器中取出电极后加入内部溶液,重新组装传感器,并置于3.33M的KCl溶液中活化24h,备用。
电子舌系统自检完成后,将样品均匀的倒在两个电子舌专用烧杯中,AAE、CT0、CA0、AE1和C00等6个传感器先在阴离子或阳离子溶液中浸泡,以便清洗传感器,而后在参比溶液1和2中依次清洗,接着在参比溶液3中清洗得到参比溶液电势Vr,然后在样品杯中浸泡得到样品电势Vs,通过Vs-Vr的电势差可对鲜味、酸味、咸味、苦味、涩味等基本值进行评价;参比溶液4和5中分别清洗后,于溶液6中浸泡测试回味,检测到电势Vr,通过Vr’-Vr的电势差可检测样品鲜味、苦味或涩味的回味。每个样品重复检测4次,为减少系统误差,原始数据选取后3次测量的数据,参比溶液1-6成分完全相同。
实施例2嗜盐四联球菌SNTH-1产鲜味肽的提取与纯化
1、乙醇分级:
(1)取200mL实施例1所述嗜盐四联球菌SNTH-1的发酵液,浓缩至100mL,然后将浓缩后的发酵液加入67mL食用级无水乙醇后常温搅拌30min并离心(8000g,30min),得沉淀物E1和上清液1;
(2)取沉淀物E1冻干,即得40%乙醇提取的组分;将上清液1与80mL乙醇混合配成乙醇终浓度为60%的溶液,常温搅拌30min并离心(8000g,15min),得沉淀物E2和上清液2;
(3)取沉淀物E2冻干,即得60%乙醇提取的组分;将上清液2加入200mL乙醇混合配成乙醇终浓度为80%的溶液,常温搅拌30min并离心(8000g,15min),得到沉淀物E3和上清液3;
(4)取沉淀物E3冻干,即得80%乙醇提取的组分;将上清液3冻干,收集得上清液组分。
取上述各级乙醇提取组分的感官滋味轮廓图结果如图1所示。
从图1可知,随着乙醇浓度的提升,粗肽中的鲜味和甜味先升高后下降,苦味、酸味和咸味则始终升高。当乙醇浓度升至80%时,鲜味最为明显,其鲜味值达到7.2,超过了原液鲜味值。从而说明,80%乙醇有效提取了该菌发酵液粗肽中的鲜味组分。
进一步,将80%乙醇提取组分进行氨基酸组成分析,方法如下:
氨基酸含量测定方法:采用日立L-8800型全自动氨基酸分析仪测定。游离氨基酸:取适量冻干组分溶解于超纯水中,振荡混合均匀后,取定容后的样液1mL,按样品:磺基水杨酸(15%)=1:1加入磺基水杨酸,混合均匀,置于4℃冰箱中冷藏静置30min,并4℃下5000g离心5min,将上述上清液经活性炭柱脱色后,过0.22μm滤膜后于氨基酸分析仪进行检测。
总氨基酸测定方法:取适量冻干组分溶解于超纯水中,TCA溶液提取离心后,取2mL上清液于蒸发皿中蒸干,加入2mL 6M盐酸溶液,抽真空后置于110℃的环境中水解24h,过滤后蒸干,加入2mL 0.02M盐酸溶液溶解,充分溶解后过滤,通过活性炭柱脱色,再经0.22μm滤膜过滤后待测。
具体结果见表1。
表1嗜盐四联球菌SNTH-1发酵液80%乙醇提取组分的氨基酸组成
从表1的数据可知,80%乙醇提取组分的总氨基酸中鲜味氨基酸含量达93.81mg/g,占全部总氨基酸的40%,说明嗜盐四联球菌SNTH-1发酵液提取粗肽中很有可能具有鲜味肽。游离氨基酸中鲜味氨基酸的含量达41.74mg/g,占全部游离氨基酸的45%。发酵液中鲜味氨基酸比例越高,其鲜味强度越高。因此,80%乙醇提取组分的呈鲜效果是最优的,可以安全高效的富集鲜味肽。
申请人选取80%乙醇提取组分进行下一步的分离纯化。
2、Sephadex G-15过滤层析:
将上述获得的嗜盐四联球菌SNTH-1发酵液80%乙醇提取组分(沉淀物E3)用超纯水复溶,过0.22μm滤膜后于4℃保存待用。
将处理好的葡聚糖凝胶Sephadex G-15一次性水平填装于1.6cm×50cm的层析柱中,取1mL样品上柱,用超纯水以1.0mL/min的流速进行洗脱,在220nm下监测紫外吸光值,多次平行进样,按峰面积和保留时间的不同分别对每个组分分离峰进行收集,冷冻干燥后保存于-80℃冰箱备用。Sephadex G-15凝胶柱洗脱图谱如图2所示。
从图2可知,发酵液80%乙醇提取所得的鲜味组分经层析后出现2个峰,依次命名为G-A-1和G-A-2。峰面积与对应鲜味组分的含量成正比。与G-A-1相比,G-A-2是鲜味组分中面积比例最大的组分,说明其含量相对较高。
取Sephadex G-15凝胶层析分离得到的G-A-1和G-A-2组分分别进行感官滋味分析和氨基酸组成分析,结果如图3和表2所示。
表2G-A-1和G-A-2的氨基酸组成分析
从表2的数据可知,G-A-1和G-A-2层析组分的肽基氨基酸含量分别为36.13mg/g、57.48mg/g,分别占总氨基酸的44%、73%。肽基氨基酸中鲜味氨基酸的含量分别为11.19mg/g、29mg/g,分别占肽基全部氨基酸的31%、50%。从而说明,经过凝胶过滤层析纯化的鲜味组分,尤其是G-A-2组分,其总氨基酸和肽基氨基酸含量仍然较高,同时对应的鲜味氨基酸占比较大,因此鲜味肽类物质可能对不同层析组分的呈鲜味特性起着主要作用,此结果与上述讨论相符。
从图3可知,80%乙醇提取的粗肽经过凝胶柱层析后,在感官滋味轮廓图中,G-A-1和G-A-2组分的鲜味较为突出,甜味也较丰富,呈苦味咸味不明显,但有一定酸味产生。其中,G-A-2组分的鲜味、甜味和酸味要高于G-A-1组分,其中鲜味更为显著,咸味和苦味则低于G-A-1组分。因此,层析组分中G-A-2的鲜味值最高,具有更好的呈味效果,申请人选取G-A-2组分继续做下一步的分离纯化研究。
3、RP-HPLC纯化:
采用半制备柱XBridgeTMBEH130 pre C18(直径为10μm,尺寸为10×150mm)对G-A-2组分作进一步分离。
将G-A-2组分冻干后用超纯水复溶,配成20mg/mL的溶液,经0.22μm水相膜过滤后通过反相高效液相色谱纯化;流动相速度为3.00mL/min,流动相为:A:0.1%三氟乙酸水溶液;B:0.1%三氟乙酸乙腈溶液。最适洗脱的程序为:0-10min,5% B;10-15min,5%-10%B;15-20min,10%-30%B;20-25min,30%-50%B。洗脱液在波长218nm下进行检测(检测器为Waters 2487),采集均具有有效的保留时间、峰形相对独立的各组分冻干后,进行后续鲜味肽结构的鉴定。G-A-2组分的RP-HPLC分离结果如图4所示。
从图4可知,采用RP-HPLC进行纯化具有较好的分离效果,根据峰组分的峰高、峰面积以及出峰时间,对主要的峰组分进行大批量收集以作结构鉴定。鲜味组分G-A-2经过RP-HPLC色谱分离纯化后,产生了四个峰组分G-A-2-I、G-A-2-II、G-A-2-III和G-A-2-IV,因G-A-2-II所占峰高较高、峰面积较大,所以收集G-A-2-II组分进行序列鉴定。
实施例3嗜盐四联球菌SNTH-1产鲜味肽的鉴定合成与评价
1、UPLC-ESI-MS/MS序列分析鉴定:
取实施例2中RP-HPLC分离得到的鲜味组分G-A-2-II进行氨基酸序列分析。
具体方法如下:
液相所用的A液为0.1%甲酸水溶液,B液为0.1%甲酸乙腈水溶液(乙腈为84%)。液相色谱柱(0.15mm*150mm,RP-C18,Column Technology Inc.)以95%的A液进行平衡,样品由自动进样器上样到Zorbax 300SB-C18 peptide traps(Agilent Technologies,Wilmington,DE),再经过液相色谱柱分离,相关液相梯度设置如下:0-50min,B液线性梯度从4%-50%;50-54min,B液线性梯度从50%-100%;54-60min,B液维持在100%。产物经毛细管高效液相色谱分离后用Q Exactive质谱仪(Thermo Fisher)进行质谱分析,分析时长:60min。离子源:ESI。检测方式:正离子。多肽和多肽的碎片的质量电荷比按照下列方法采集:每次全扫描(fullscan)后采集10个碎片图谱(MS2 scan)。质谱测试原始文件(RawFile)使用软件MaxQuant1.5.5.1检索相应的嗜盐四联球菌数据库(UniProt_Tetragenococcus_halophilus_15322),进而得到目标多肽鉴定及定量分析结果,具体结果见表3。UPLC-ESI-MS/MS二级质谱图如图5所示。
表3G-A-2-II氨基酸序列分析
从表3和图5的数据可知,G-A-2-II组分中鉴定出的潜在鲜味肽多以小分子肽为主,三肽更多,评分均大于20,氨基酸序列准确度较高。从而说明,经乙醇分级、Sephadex G-15凝胶过滤层析和RP-HPLC分离纯化后,鲜味小分子肽得到了充分的富集。
G-A-2-II组分中鉴定出的三条肽段,其序列分别为DFE(Asp-Phe-Glu)、LAGE(Leu-Ala-Gly-Glu)和QLQ(Gln-Leu-Gln),分子量分别为409.14851Da、388.1958Da和387.21178Da,肽段注释到的对应蛋白为A0A3G5FJU2和A0A6I5YLN3,而后通过GenBank查询嗜盐四联球菌物种数据库,成功注释到了嗜盐四联球菌相对应的基因,分别是C7H83_09190和cas3。
2、肽的固相合成:
委托上海厚基生物科技有限公司,采用固相合成法分别合成氨基酸序列为DFE、LAGE和QLQ的三种寡肽,合成肽纯度均超过99%。
3、感官评价:
将合成寡肽分别配制为浓度为1mg/mL溶液50mL。以0.22um滤膜过滤后的滤液作为评定样液。感官评定小组由10名试验室成员组成(5男5女,年龄在25-35岁之间),小组成员均经过感官培训。评分从0分到10分,0分代表没有味道,10分表示具有显著的味道。甜味、苦味、酸味、咸味、鲜味评价标准品分别为蔗糖(1%)、L-异亮氨酸(0.25%)、柠檬酸(0.08%)、氯化钠(0.35%)、味精(0.35%),在此浓度下的标准品被认定为5分。三种合成寡肽的呈味特性分析感官滋味轮廓图如图6(A)所示。
从图6(A)可知,合成后的3条肽段在0.2mg/mL浓度下的感官评价和滋味轮廓基本一致。DFE的5种基本味感中鲜味和酸味较为突出,甜味也较强,具有一定的咸味,苦味最弱,与凝胶层析组分滋味轮廓相近,其中鲜味味感评分为6.54,超过标准品,鲜味十分显著。LAGE的鲜味和甜味较为突出,其中鲜味值为5.68弱于DFE,但甜味更高,同时有一定酸味,苦味和咸味稍弱。QLQ鲜味评分最低,为3.67,苦味较为突出,评分为2.9分,高于另外两种寡肽,酸味、甜味和咸味均较低。其中DFE是嗜盐四联球菌SNTH-1特异性产出的鲜味寡肽。
4、电子舌呈味分析测定:
该电子舌配备了6个测试传感器和2个参比传感器,其中AAE、CT0、CA0、AE1、C00和GL1测试传感器分别用于测试鲜味、咸味、酸味、涩味、苦味还有甜味。对传感器进行活化,电子舌系统自检完成后,将合成肽配置为浓度为0.2mg/mL的溶液,倒入电子舌专用烧杯中,每个样品重复检测4-5次,为减少系统误差,原始数据选取后3次测量的数据。合成肽的电子舌呈味特性分析轮廓图如图6(B)所示。
从图6(B)可知,与乙醇分级提取的鲜味组分、凝胶层析纯化的鲜味组分相比,合成寡肽的鲜味无显著性差异,咸味和苦味均有所降低,这是因为乙醇分级提取组分和凝胶层析纯化组分均为混合物,含有带咸味的无机盐离子和苦味物质,使得其咸味和苦味较高。
5、描述性鉴评试验:
感官评定小组由10名试验室成员组成(5男5女,年龄在25-35岁之间)。
将合成寡肽分别配制成浓度为1mg/mL的母液(通过加入微量氢氧化钠溶液调节溶剂pH至6),采用滋味稀释分析法(TDA),以1:1体积比例逐步稀释,各个分部的逐步稀释的溶液按照浓度增加的顺序呈给经过训练的评员,每个稀释水平溶液采用三角检验(TriangleTest,1个样品实验组,2个空白对照组)。当某个稀释水平的溶液与2个空白(纯净水)之间的滋味差异刚好能被识别出来(或者样品与两个空白刚好分不清),所识别的合成肽的最低浓度和第二最低浓度取平均值作为其识别阈值,每个评定组的阈值采用各个评员评定结果的平均值,并要求品评人员对所品尝到的滋味进行描述。三种合成寡肽的滋味描述和呈鲜阀值如表4所示。
表4合成肽的滋味描述和呈鲜阀值
从表4的数据可知,本发明合成的三种鲜味寡肽的呈鲜阈值分别为0.4517mg/mL、0.2893mg/mL和0.2256mg/mL,其中DFE的鲜味最显著,而且可产生一定令人愉悦的感觉,QLQ的鲜味较弱,同时带有一定酸味。
实施例4鲜味寡肽DFE与鲜味受体T1R1/T1R3的分子对接
鲜味味觉受体是一种C类G蛋白偶联受体(GPCR),对L-谷氨酸和一定程度上的L-天冬氨酸产生反应。受体属于T1R家族,包括T1R1和T1R3。鲜味寡肽DFE的蛋白受体为异二聚体T1R1/T1R3。
1、以mGluR1的配体结合区(封闭-开放状态)为模板(PDB编码1EWK)进行同源性建模,同源性分别为23.2%和23.0%。采用Modeller 10.1建立同源性模型,通过PDF总能量和DOP评分评分选择模型。进一步使用PDBsum在线服务(http://www.ebi.AC.uk/Thornton-SRV/databases/pdb sum/generate.html)进行模型评估,并构建一个Ramachandran图来检查初始模型的结构稳定性。结果如图7所示。
2、选择与Chimera v1.13相关的Autodock Vina套装v4.0进行分子对接分析。鲜味肽的三维结构是在Chem3D中构建和优化的。配置文件加载了网格生成参数(T1R1:中心X=-2.3,Y=1.6,Z=7.3尺寸X=18.5,Y=20.0,Z=20.9T1R3:中心X=4.8,Y=4.7,Z=14.3尺寸X=20.1,Y=18.8,Z=16.8),耗尽值=24,能量范围在=4,模式数=5在配置文件中设置,运行多次迭代并获得一致的结果。通过Discovery Studio visualizer(美国加利福尼亚州圣地亚哥市Accelrys Inc.)对停靠得分最高的停靠姿势进行可视化分析,以获得2D和3D交互模式的形态。结果如图7所示。
3、鲜味寡肽DFE与T1R3亚基的结合主要是通过与受体氨基酸残基之间产生的静电相互作用、氢键作用、疏水相互作用和范德华力。鲜味寡肽DFE与鲜味受体T1R1/T1R3的对接能量如表5所示,鲜味寡肽DFE与鲜味受体T1R1/T1R3的结合位点如图8所示。
由图8可知,鲜味寡肽DFE与T1R3亚基的结合位点主要为T1R3亚基上的氨基酸残基SER256。
表5鲜味寡肽DFE与鲜味受体T1R1/T1R3的对接能量
从表5的数据可知,鲜味寡肽DFE单独与T1R1,T1R3结合时,与右侧T1R3的结合力更强,所需对接总能量更低。
实施例5鲜味寡肽DFE增鲜实验
为了解合成寡肽DFE的鲜味增强效果,以0.5g/L的味精为原料,制备合成寡肽DFE在不同浓度(0、0.25、0.5、1和2g/L)下的鲜味评价溶液。这些样品的评分从1分到9分,其中1分即味精溶液(即不含DFE)。将0.5、1、1.5g/L味精溶液的鲜味强度分别定义为1、5、9分。
结果显示,与对照组相比,本发明所述鲜味寡肽DFE的增鲜阈值为0.3839mmol/L,滋味描述为鲜味和厚味突出,取得了意料不到的效果。
实施例6鲜味寡肽DFE对肉汤/酱油的增鲜效果
为了解合成寡肽DFE在复杂食品体系(模拟肉汤/酱油)中的鲜味增强效果,将合成寡肽DFE用模拟肉汤(2mg/mL味精、2mg/mL氯化钠、0.4mg/mL蔗糖)稀释至0.5mg/mL,并用微量氢氧化钠溶液(2.0M)调整pH值为6.5;将市售的酱油稀释30倍,将合成寡肽DFE用稀释后的酱油溶液稀释至0.5mg/mL,并用微量氢氧化钠溶液(2.0M)调整pH值为6。以未加DFE的模拟肉汤和酱油为对照,小组成员(5男5女,年龄在25-35岁之间)用10分制分别评估合成寡肽DFE对肉汤和酱油的增鲜效果。结果如图9所示。
从图9可知,本发明所述鲜味寡肽DFE能明显减少模拟肉汤和酱油的苦味,增加鲜味和咸味。取得了意料不到的技术效果。
本发明提供的鲜味寡肽增鲜效果突出,可广泛应用于味精、鸡精、鸡粉、酱油、汤块、耗油、酱、腐乳等调味品的生产,还可添加于发酵食品或保健品中,用于改善食品或保健品的口味应用。
Claims (10)
1.一种鲜味寡肽,其特征在于,所述鲜味寡肽的氨基酸序列为LAGE(Leu-Ala-Gly-Glu)。
2.权利要求1所述的鲜味寡肽在制备调味品、发酵食品或保健品中的应用。
3.如权利要求2所述的应用,其特征在于,所述的调味品为味精、鸡精、鸡粉、酱油、汤块、耗油、酱、腐乳中的任意一种。
4.一种鲜味剂,其特征在于,所述的鲜味剂包含权利要求1所述的鲜味寡肽。
5.权利要求4所述的鲜味剂的制备方法,其特征在于,所述方法包括如下步骤:
(1)将嗜盐四链球菌进行发酵培养,得发酵液;
(2)将嗜盐四链球菌发酵液进行离心,得上清液;
(3)将上清液进行冷冻干燥,即得鲜味剂。
6.权利要求4所述的鲜味剂的制备方法,其特征在于,所述方法包括如下步骤:
(1)将嗜盐四链球菌进行发酵培养,得发酵液;
(2)将嗜盐四链球菌的发酵液经40%、60%、80%(v/v)乙醇分级沉淀;
(3)将80%(v/v)乙醇沉淀物进行冷冻干燥,即得鲜味剂。
7.如权利要求5或6所述的制备方法,其特征在于,所述方法中嗜盐四联球菌为嗜盐四联球菌SNTH-1(Tetragenococcus halophilus SNTH-1),已于2021年08月20日保藏至中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCC No.23165。
8.如权利要求7所述的制备方法,其特征在于,所述方法包括以下步骤:
(1)嗜盐四链球菌的发酵培养:
①菌株活化:
按1%(v/v)接种量将嗜盐四链球菌菌株接种于50mL MRS培养基中,调整pH值为8.3,在37℃条件下摇瓶培养40h,待菌株生长至对数生长期末期时,取0.5mL发酵菌液重复接种于50mL MRS培养基中,得到活化的嗜盐四联球菌;
②种子液培养:
将活化后的嗜盐四联球菌菌液接种到5L发酵罐,内有3L富集培养基,在温度37℃,转速200rpm,空气流量比1:0.5,罐压0.1kg/cm2的条件下培养24h,得到种子液;
③发酵罐培养:
将种子液转入到50L的发酵罐中,内有30L大豆蛋白基质培养基,在温度37℃,转速200rpm,空气流量比1:0.55,罐压0.12kg/cm2的条件下发酵42h,得到富含鲜味肽的发酵液;
(2)乙醇分级:
①取嗜盐四联球菌的发酵液,浓缩后加入食用级无水乙醇,配成乙醇终浓度为40%的溶液,常温搅拌30min,8000g离心30min,得沉淀物1和上清液1;
②将上清液1与食用级无水乙醇混合配成乙醇终浓度为60%的溶液,常温搅拌30min,8000g离心15min,得沉淀物2和上清液2;
③将上清液2与食用级无水乙醇混合配成乙醇终浓度为80%的溶液,常温搅拌30min,8000g离心15min,得沉淀物3和上清液3;
(3)将80%乙醇沉淀物3进行冷冻干燥,即得鲜味剂。
9.如权利要求8所述的制备方法,其特征在于,所述的富集培养基中各组分及其含量分别为:蛋白胨10g/L、无水乙酸钠3g/L、磷酸氢二钾2g/L、七水合硫酸镁0.575g/L、一水合硫酸锰0.25g/L、葡萄糖20g/L、柠檬酸三钠2.42g/L、酵母浸粉4g/L、牛肉浸膏8g/L、吐温801g/L;氯化钠150g/L、那他霉素2g/L、结晶紫500μL/L。
10.如权利要求8或9所述的制备方法,其特征在于,所述的大豆蛋白基质培养基中各组分及其含量分别为:大豆蛋白胨20g/L、无水乙酸钠3g/L、磷酸氢二钾2g/L、七水合硫酸镁0.575g/L、一水合硫酸锰0.25g/L、葡萄糖20g/L、柠檬酸三钠2.42g/L、吐温80 1g/L、氯化钠100g/L。
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