CN116375799B - 来源于臭鳜鱼的鲜味肽 - Google Patents
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Abstract
本发明公开了来源于臭鳜鱼的鲜味肽,涉及食品生物技术领域;所述鲜味肽的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。本发明从自然发酵生产的臭鳜鱼产品中筛选得到了两种具有明显鲜味的肽,这两种肽与鲜味识别受体T1R1/T1R3的对接能量分别为‑98.25kcal/mol和‑96.30kcal/mol,鲜味为谷氨酸钠的2倍,与已报道的鲜味肽相比具有明显的优势,可用于制备具有明显鲜味的调味包、调味料等调味品,具有广阔的市场应用前景。
Description
技术领域
本发明涉及食品生物技术领域,特别是涉及来源于臭鳜鱼的鲜味肽。
背景技术
食品增鲜剂是一类改善和增强食品风味的物质,不改变原有食品中的酸味、甜味、苦味和咸味,从而增强食品的可口性。增鲜剂中的鲜味主要是由游离氨基酸、风味核苷酸、有机酸类和鲜味肽等鲜味化合物提供的,包括谷氨酸,丙氨酸,肌苷酸二钠和谷氨酸钠(味精)等,另外水解动植物蛋白是新型食品增鲜剂,用以制备大量适口性优良的鲜味肽,鲜味肽大多是具有低分子量的活性肽,在发酵食品的鲜味上起到重要作用。谷氨酸和天冬氨酸的存在以及它们在鲜味肽的序列在鲜味肽的呈味中发挥重要作用,而且具有高比例的谷氨酸和天冬氨酸的鲜味肽呈现出更明显的鲜味。鲜味肽是具有鲜味特征的小分子肽,通常来讲,鲜味肽含有谷氨酸和天冬氨酸并且分子质量小于3000Da。
食物源的鲜味肽具有良好的呈鲜效果,可用于制备具有明显鲜味的调味包、调味料等调味食品。鲜味肽中谷氨酸残基和天冬氨酸残基对于其鲜味的提升具有重要作用。传统的鲜味肽筛选方法是利用商品化蛋白酶水解食物蛋白,然后利用分离纯化方式获得肽,通过质谱分析获得抑制肽的结构。这种筛选方法的通量低、效率低、操作复杂、耗时耗力,分离纯化获得的鲜味肽的纯度较低。而且由于商品化蛋白酶水解位点固定,水解得到的多肽片段的多样性降低,大大降低了获得新型鲜味肽的可能性。臭鳜鱼是一种以鳜鱼为原料制作的传统水产发酵食品。臭鳜鱼中的微生物菌群结构复杂,且能够分泌多种多样的蛋白酶,因此会产生更为丰富多样的肽序列。而且,鳜鱼蛋白中富含谷氨酸和天冬氨酸,为鲜味肽的制备提供了可能。
发明内容
本发明的目的是提供来源于臭鳜鱼的鲜味肽,以解决上述现有技术存在的问题,本发明提供的臭鳜鱼源鲜味肽具有明显鲜味,与鲜味识别受体T1R1/T1R3的对接能量可分别达到-98.25kcal/mol和-96.30kcal/mol。
为实现上述目的,本发明提供了如下方案:
本发明提供来源于臭鳜鱼的鲜味肽,所述鲜味肽的氨基酸序列如SEQ ID NO.1或SEQ IDNO.2所示。
本发明还提供上述的鲜味肽的制备方法,包括以下步骤:
(1)鳜鱼经自然发酵得到臭鳜鱼;
(2)利用盐酸提取法,从所述臭鳜鱼中提取得到多肽提取物;
(3)所述多肽提取物经脱盐处理后,利用超高效液相四级杆高分辨串联质谱鉴定得到所述鲜味肽。
进一步地,步骤(1)的具体操作包括:将鳜鱼去鳞、去鳃、去内脏,清洗后置于发酵容器中,加入盐水,自然发酵,得到所述臭鳜鱼。
进一步地,所述盐水的浓度为6wt%。
进一步地,所述自然发酵的温度为18℃,时间为8d。
进一步地,所述盐酸提取法所使用的盐酸溶液的浓度为0.01mol/L。
进一步地,所述盐酸提取法的盐酸浸提时间为40min。
本发明还提供上述的鲜味肽在提高食品鲜味中的应用。
本发明还提供上述的鲜味肽在制备调味品中的应用。
本发明还提供一种调味品,所述调味品的鲜味成分包括上述的鲜味肽。
本发明公开了以下技术效果:
本发明从自然发酵生产的臭鳜鱼产品中筛选得到了两种具有明显鲜味的肽Asn-Trp-Asp-Asp-Met-Glu-Lys和Trp-Phe-Lys-Asp-Glu-Glu-Phe,这两种肽与鲜味识别受体T1R1/T1R3的对接能量分别为-98.25kcal/mol和-96.30kcal/mol,鲜味为谷氨酸钠的2倍,与已报道的鲜味肽相比具有明显的优势,可用于制备具有明显鲜味的调味包、调味料等调味品,具有广阔的市场应用前景。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为Asn-Trp-Asp-Asp-Met-Glu-Lys与鲜味识别受体T1R1/T1R3的分子对接图;其中,A为三维结合图,B为局部放大图,C为平面相互作用示意图;
图2为Trp-Phe-Lys-Asp-Glu-Glu-Phe与鲜味识别受体T1R1/T1R3的分子对接图;其中,A为三维结合图,B为局部放大图,C为平面相互作用示意图;
图3为Asn-Trp-Asp-Asp-Met-Glu-Lys和Trp-Phe-Lys-Asp-Glu-Glu-Phe的滋味轮廓图。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
实施例1
(1)臭鳜鱼所用原料的处理:选择大小为500g的鳜鱼(Sinipercachuatsi),经去鳞、去鳃、去内脏及清洗,然后整齐码放于发酵桶。
(2)臭鳜鱼的自然发酵生产:加入含盐量为6wt%的食盐水,发酵温度为18℃,发酵8d后获得臭鳜鱼产品。
(3)多肽的提取:臭鳜鱼产品经过去皮后,将鱼肉绞碎后用0.01mol/L的稀盐酸浸40min,离心获得多肽提取物。
(4)鲜味肽的筛选:多肽提取物经过脱盐柱处理后,借助超高效液相四级杆高分辨串联质谱进行多肽组分析,通过与鳜鱼蛋白质进行比对,获得全部多肽的序列。
实施例2
利用谷氨酸和天冬氨酸占肽序列的比例初步筛选鲜味肽。借助ChemDraw软件对筛选到的鲜味肽进行三维结构构建,并使用DS优化分子结构,设置成最低能量的稳定结构。并将其作为受体蛋白,使用SwissModel对鲜味识别受体T1R1/T1R3进行三维结构模拟构建,并将其中打分结果最高的三维晶体结构作为受体,通过DS软件进行分子对接筛选能与鲜味识别受体T1R1/T1R3位点紧密结合的肽。以对接能量为筛选指标,得到两条最具潜在鲜味的肽Asn-Trp-Asp-Asp-Met-Glu-Lys(SEQ ID NO.1)和Trp-Phe-Lys-Asp-Glu-Glu-Phe(SEQ IDNO.2)。这两条肽的丰度和分子对接结果如表1和图1-2所示。Asn-Trp-Asp-Asp-Met-Glu-Lys和Trp-Phe-Lys-Asp-Glu-Glu-Phe与鲜味识别受体T1R1/T1R3的对接能量分别为-98.25kcal/mol和-96.30kcal/mol,丰度分别达到了3.30×108和1.56×108。
实施例3
将鲜味肽Asn-Trp-Asp-Asp-Met-Glu-Lys和Trp-Phe-Lys-Asp-Glu-Glu-Phe与此鳜鱼蛋白质数据进行比对,得到其前体蛋白质和酶切位点。借助在线网站ToxinPred分析风味肽序列的毒性,使用ExPASy用于分析风味肽序列的等电点和相对分子质量,使用PepDraw预测风味肽序列的电荷和疏水性,使用ProtParam分析风味肽序列的稳定性。这两种肽的物理化学特性如表2所示。Asn-Trp-Asp-Asp-Met-Glu-Lys来源于鳜鱼的肌动蛋白,酶切位点是107-113,等电点为pH4.03,分子量为937.08Da,带电荷为-2,具有较高的疏水性和稳定性。Trp-Phe-Lys-Asp-Glu-Glu-Phe来源于鳜鱼的核苷二磷酸激酶,酶切位点是152-158,等电点为pH4.14,分子量为1000.16Da,带电荷为-2,具有较高的疏水性和稳定性。
实施例4
通过电子舌对Asn-Trp-Asp-Asp-Met-Glu-Lys和Trp-Phe-Lys-Asp-Glu-Glu-Phe进行味觉评价,用10mMKCl将Asn-Trp-Asp-Asp-Met-Glu-Lys和Trp-Phe-Lys-Asp-Glu-Glu-Phe稀释至多肽浓度为1mg/mL,其中KCl溶液作为支撑电解质,使传感器输出更稳定。将配制溶液置于电子舌专用烧杯中,于室温条件下进行数据采集。设置采集条件为:每个样本进行4次采集,每次采集时间为120s,每次采集结束后以味觉标准溶液进行两步清洗,待测样品和清洗液按照交替的序列进行分析检测。以1mg/mL谷氨酸钠为对照溶液。
这两种肽的滋味轮廓图如图3所示。这两种肽均具有较强的鲜味,Asn-Trp-Asp-Asp-Met-Glu-Lys和Trp-Phe-Lys-Asp-Glu-Glu-Phe的鲜味达到对照物谷氨酸钠的2倍。这两种肽可用于制备具有明显鲜味的调味包、调味料等调味品,具有广阔的市场应用前景。
表1
表2
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (4)
1.来源于臭鳜鱼的鲜味肽,其特征在于,所述鲜味肽的氨基酸序列如SEQ ID NO.2所示。
2.如权利要求1所述的鲜味肽在提高食品鲜味中的应用。
3.如权利要求1所述的鲜味肽在制备调味品中的应用。
4.一种调味品,其特征在于,所述调味品的鲜味成分包括权利要求1所述的鲜味肽。
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