WO2020232975A1 - 从牡蛎酶解液中分离出的一种呈味肽及其制备方法与应用 - Google Patents

从牡蛎酶解液中分离出的一种呈味肽及其制备方法与应用 Download PDF

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WO2020232975A1
WO2020232975A1 PCT/CN2019/113799 CN2019113799W WO2020232975A1 WO 2020232975 A1 WO2020232975 A1 WO 2020232975A1 CN 2019113799 W CN2019113799 W CN 2019113799W WO 2020232975 A1 WO2020232975 A1 WO 2020232975A1
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oyster
ethanol
taste
supernatant
preparation
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French (fr)
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崔春
舒丹阳
张典
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华南理工大学
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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
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/50—Molluscs
    • 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
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/65—Addition of, or treatment with, microorganisms or enzymes
    • 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
    • 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

Definitions

  • the invention relates to the field of taste-presenting peptides, in particular to a taste-presenting peptide separated from an oyster enzymatic hydrolysate, and a preparation method and application thereof.
  • Oysters are the largest farmed shellfish in the world and the largest economic shellfish produced in China. Oysters are delicious, rich in a variety of physiologically active ingredients, and have important potential value in food and medicine.
  • the Ministry of Health of my country has listed oysters as one of the first batch of raw materials that can be used as both food and medicine.
  • the products developed on the market using oysters as raw materials mainly include functional health products, seafood condiments and medicines.
  • Enzyme technology is a new type of marine raw material processing technology, which has attracted attention because of its mild reaction conditions, easy control of the process, and good retention of nutrients in food raw materials.
  • the current enzyme technology has been widely used in the development of various oyster products, and its applications mainly include three aspects: enzyme modification to improve protein function, enzymatic hydrolysis to extract nutrients in protein, and enzymatic hydrolysis to produce flavor substances.
  • enzymatic modification can change the structure of oyster protein, thereby improving its emulsification, solubility and foaming properties.
  • Using enzymatic technology to hydrolyze oyster protein can make it hydrolyze to produce a variety of small molecule peptides and free amino acids, thereby giving the oyster enzymatic hydrolysis product rich nutrition and full flavor.
  • the choice of protease is very important. Different proteases have their own unique modes of action, so they will also produce different enzymatic hydrolysis effects.
  • the commonly used proteases for enzymatic hydrolysis of oysters include Protemax complex enzyme, trypsin, alkaline protease, neutral protease, and flavor protease.
  • the enzymatic hydrolysate prepared by Protemax complex enzyme has better flavor and color; pancreatin is a mixed enzyme preparation, composed of trypsin, pancreatic amylase, pancrelipase, aminopeptidase and other single enzymes, with hydrolysis efficiency
  • alkaline protease has high hydrolysis efficiency and poor specificity, and has a good hydrolysis effect on aromatic amino acids, hydrophobic amino acids and basic amino acids; neutral protease is an endonuclease It has the advantages of high hydrolysis efficiency and good flavor of the hydrolysate. It is widely used in functional food and condiment industries; flavor protease can hydrolyze protein to produce free amino acids and oligopeptides with good flavor, so flavor protease is widely used Among the new condiment
  • Taste peptides are a type of small molecule peptides that can affect or improve the flavor of food.
  • the main methods of obtaining taste peptides are enzymatic hydrolysis and biotechnology synthesis.
  • Taste peptides are rich in types, and their taste covers the five basic flavors of sour, sweet, bitter, salty and fresh. Therefore, taste peptides can be added to foods instead of traditional sucrose, salt, sodium glutamate and other seasonings.
  • the food grade ethanol separation method is non-toxic, harmless, high-throughput, and fast in separation. The advantages have been widely used.
  • the purpose of the present invention is to separate and identify a new type of taste peptide from the enzymatic hydrolysate of oysters hydrolyzed by complex enzymes, using fresh oyster meat as raw materials, using complex enzymatic hydrolysis technology to obtain enzymatic hydrolysates, using ethanol fractionation and UPLC -MS/MS separation and identification of taste peptides, the taste peptides are sour, rich in umami taste, without obvious fishy bitterness, and seafood condiments can be developed by industrial synthesis of the taste peptides.
  • a taste peptide the amino acid sequence of the taste peptide is Thr-Gly-Ser-Ser-Pro-Ala-Gly-Glu.
  • the method for separating the taste peptide from the oyster hydrolysate includes the following steps:
  • Pretreatment Take oyster meat as raw material, wash and drain, beat into meat emulsion, and add water to prepare oyster homogenate;
  • Enzymatic hydrolysis adding compound enzymes to the oyster homogenate for enzymatic hydrolysis, after the reaction is completed, the enzymes are inactivated and centrifuged, and the resulting filtrate is the oyster hydrolysate;
  • Ethanol fractionation to extract the peptides in the oyster hydrolysate add ethanol to the oyster hydrolysate, stir evenly, centrifuge to obtain the precipitate (I) and the supernatant (I), and then add the supernatant (I) Add ethanol, stir and centrifuge to obtain precipitate (II) and supernatant (II); then add ethanol to the supernatant (II), stir and centrifuge to obtain the supernatant (III), and then supernatant (III) Remove excess ethanol.
  • the material-to-liquid ratio of the oyster homogenate in step (1) is 1g:1ml.
  • the complex enzymes in step (2) are Pancreatin (pancreatin), Papain (papain), Acid Protease (acid protease), Ns 37071 (alkaline protease), Protamex (complex hydrolase), Flavorzyme 500MG (flavor protease) ) And Alcalase2.4L (alkaline protease).
  • the compound enzyme is a mixed enzyme of Flavorzyme 500MG and Pancreatin.
  • the mass ratio of Flavorzyme 500MG and Pancreatin in the mixed enzyme is 1:3.
  • the added amount of the compound enzyme in step (2) is 0.1-0.5 wt% of the oyster meat, more preferably 0.2 wt%.
  • the temperature of the enzymatic hydrolysis in step (2) is 45-55°C, more preferably 50°C; the time of enzymatic hydrolysis is 4-8h, more preferably 7h.
  • the enzyme inactivation time in step (2) is 10-20 min.
  • step (3) 3-5 times the volume of the oyster hydrolysate is added with ethanol; the supernatant (I) is added with ethanol so that the final concentration of ethanol in the system is 70-90 vol%; Ethanol is added to the clear solution (II) so that the final concentration of ethanol in the system is 95 vol%.
  • step (3) 4 times the volume of ethanol is added to the oyster hydrolysate; the supernatant (I) is added with ethanol so that the final concentration of ethanol in the system is 90 vol%; the supernatant ( II) Add ethanol to make the final concentration of ethanol in the system 95 vol%.
  • the present invention has the following advantages:
  • the present invention uses a complex enzyme to carry out enzymatic hydrolysis from multiple sites to increase the degree of hydrolysis of oyster protein and at the same time increase the yield of polypeptides;
  • the taste-presenting peptide of the present invention is an 8-peptide with a molecular weight of less than 800 Da, which is easily absorbed by the human body and improves the nutritional value.
  • reaction conditions of the present invention are mild, the reaction time is short, the enzyme used in the reaction is low in price, high in quality and small in dosage, which effectively saves costs.
  • Figure 1 is a flow chart of ethanol fractionation extraction
  • Figure 2 Sensory evaluation diagram of different enzymolysis conditions.
  • Fig. 3a, Fig. 3b, Fig. 3c, Fig. 3d are diagrams showing the effect of different concentrations of oyster enzymatic hydrolysate on food.
  • Figure 4 is the secondary mass spectrum of the taste peptide of UPLC-MS/MS.
  • E-1 0.1wt% mixed enzyme, 50°C enzymatic hydrolysis for 7h
  • E-2 0.3wt% mixed enzyme, 50°C enzymatic hydrolysis for 7h
  • E-3 0.5wt% mixed enzyme, Enzymatic hydrolysis at 50°C for 7 hours
  • E-4 0.3wt% mixed enzymes, enzymatic hydrolysis at 45°C for 7 hours
  • E-5 0.3wt% mixed enzymes, enzymatic hydrolysis at 55°C for 7 hours;
  • the sensory evaluation team in this study selected five men and five women (aged between 24 and 30) without dysgeusia, and controlled the temperature of the evaluation room at room temperature (25 ⁇ 2°C). According to the characteristics of the experiment, the group members were trained with the reference solution.
  • the reference solution is as follows: Umami (MSG solution, 16mmol/L, 8mmol/L, 4mmol/L); sweet (sucrose solution, 50mmol/L, 30mmol/L, 10mmol/L); sour (citric acid solution, 8mmol) /L, 4mmol/L, 2mmol/L); bitter taste (L-isoleucine solution, 40mmol/L, 20mmol/L, 10mmol/L); thick taste is more complex, this study uses white water boiled mother Chicken broth prepared from chicken was used as a reference solution (chicken broth cooked for 6 hours, chicken broth cooked for 4 hours, chicken broth cooked for 2 hours).
  • the flavor scores of the three concentrations of the reference solution are respectively positioned 10 points, 5 points, and 1 point according to the strength.
  • Each flavor score refers to the score of the reference liquid that is similar to its flavor, and the final result is the average of the scores of each group member.
  • the sensory evaluation results of the oyster hydrolysate are shown in Figure 1. It can be seen from Figure 1 that the sensory score of the oyster hydrolysate E-2 is: umami taste 6.67, sour taste 1.96, sweet taste 3.75, thick taste 4.18, and bitter taste 1.65. Its umami taste and sweet taste are higher than other enzymatic hydrolysates, the thick taste is moderately scored, and the sour taste and bitter taste are lower. This shows that the amount of enzyme added is 0.3wt%, and the oyster hydrolysate obtained by enzymatic hydrolysis at 50°C for 7 hours at natural pH is rich in flavour, full taste and longer aftertaste, moderate sweetness, no obvious sourness and bitterness, and the overall flavor is relatively good it is good.
  • Figure 3a shows: salt and monosodium glutamate mixed solution
  • Figure 3b shows: salt, monosodium glutamate and I+G
  • Figure 3c shows: model chicken soup
  • Figure 3d shows: model beef soup, and use sodium hydroxide solution and formic acid solution to adjust the pH value of each solution prepared to 6.5.
  • the prepared solutions were presented to the sensory evaluation panel in the order of the concentration of the enzymatic hydrolysate products from small to large, and their taste enhancement effects were evaluated.
  • the “5-point” intensity scale (0, undetectable; 5, strong detectable) is used to evaluate the sensory characteristics of each solution.
  • the target sensory characteristics include umami and thick taste (the taste is more complicated, and the assessor will respond after tasting the food for 10 seconds. It is evaluated) and persistence (the sensory effect that can be maintained continuously or the taste-enhancing effect, the assessor evaluates the food after 25 seconds).
  • Pretreatment Take oyster meat as raw material, wash and drain, beat into meat emulsion, and add water to prepare an oyster homogenate with a material-to-liquid ratio of 1g:1ml;
  • Ultra-high performance liquid chromatography tandem mass spectrometry and de novo sequencing were used to separate and identify the peptides in the ethanol extracted components.
  • the samples were first separated by ultra-high performance liquid chromatography, and then electrospray dissociation (ESI) into the high-resolution mass spectrometer After being bombarded by the secondary mass spectrum, fragment ions are broken into fragments, which are separated according to the different mass-to-charge ratios to form a secondary mass spectrum (see Figure 2).
  • ESI electrospray dissociation
  • fragment ions are broken into fragments, which are separated according to the different mass-to-charge ratios to form a secondary mass spectrum (see Figure 2).
  • Peaks software and database search a new peptide chain was matched from the ethanol component, and its molecular weight was less than 800Da.
  • the identified peptide chain was Thr-Gly-Ser-Ser-Pro-Ala-Gly-Glu. Can be artificially synthesized.
  • the sensory standard is a 0.35% monosodium glutamate and salt mixed solution, and the score is 5 points.
  • Add the synthetic peptide to the standard solution adjust the peptide concentration to 2mg/mL, present each solution prepared to the sensory evaluation team (10: 5 males, 5 females, trained sensory assessors), and then It is evaluated as flavor enhancement.
  • the sensory characteristics of each solution are evaluated.
  • the target sensory characteristics include umami taste, thick taste (the taste is more complicated, and the assessor evaluates the food 10 seconds after tasting the food) and persistence (the sensory effect that can be maintained continuously or the taste is enhanced, The rater evaluates the food 25 seconds after tasting it). Evaluate its umami taste, salty taste and its flavor enhancement.
  • Enhancement effect of synthetic peptide-table salt the sensory standard is 0.35% salt solution, and the score is 5 points.
  • the synthetic peptide was added to the standard solution, the concentration of the peptide was adjusted to 2 mg/mL, and the salty taste and its flavor enhancement were evaluated.

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Abstract

从牡蛎酶解液中分离出的一种呈味肽及其制备方法与应用。该方法包括以下步骤：以牡蛎肉为原料，洗净后打成肉糜，加水制备成匀浆；向匀浆液中加入复合酶酶解，反应结束后，灭酶、离心，所得滤液为酶解液；乙醇分级提取酶解液中的肽；UPLC-MS/MS分离鉴定该呈味肽，氨基酸序列为Thr-Gly-Ser-Ser-Pro- Ala-Gly-Glu。还包括所述呈味肽在食品中的应用。

Description

从牡蛎酶解液中分离出的一种呈味肽及其制备方法与应用 技术领域
本发明涉及呈味肽领域，具体涉及从牡蛎酶解液中分离出的一种呈味肽及其制备方法与应用。
背景技术
牡蛎是世界上最大的养殖贝类，同时也是中国产量最大的经济贝类。牡蛎味道鲜美、富含多种生理活性成分，在食品及药用方面均存在重要的潜在价值，我国***已将牡蛎列为首批既是食品又可作为药材的原料之一。目前市场上以牡蛎为原料开发的产品主要有功能性保健品、海鲜调味品以及药品等。我国的牡蛎加工类产品相对较少，大多为传统意义上的牡蛎产品，如干制品、罐头等，当下如何利用发酵技术、生物酶解技术等现代食品工艺高新技术对牡蛎肉进行精深加工，从而充分利用牡蛎资源已成为研究的热点。
酶技术是一种新型的海洋原料加工技术，因其反应条件温和，过程易于控制，且可以较好地保留食品原料的营养物质而受到关注。当下酶技术已经广泛应用于各类牡蛎产品的开发中，其应用主要包括酶法改性提升蛋白质功能、酶解提取蛋白中的营养成分、以及酶解产生风味物质这三个方面。
其中酶法改性可以改变牡蛎蛋白的结构，从而提高其乳化性、溶解性和起泡性等功能特性。采用酶技术酶解牡蛎蛋白，可以使其水解产生多种小分子肽和游离氨基酸，从而赋予牡蛎酶解产物丰富的营养和饱满的滋味。采用酶技术酶解牡蛎蛋白时，蛋白酶的选择十分关键，不同的蛋白酶有其独特的作用方式，因此也会产生不同的酶解效果。目前酶解牡蛎常用的蛋白酶有Protemax复合酶、胰蛋白酶、碱性蛋白酶、中性蛋白酶、风味蛋白酶等。采用Protemax复合酶制备的酶解液的风味与色泽都更好；胰酶是一种混合酶制剂，由胰蛋白酶、胰淀粉酶、胰脂肪酶、氨肽酶等单酶共同组成，具有水解效率高、专一性强的优点；碱性蛋白酶水解效率高、专一性较差，对芳香族氨基酸、疏水性氨基酸和碱性氨 基酸有较好的水解效果；中性蛋白酶是一种内切酶，具有水解效率高、水解产物风味好的优点，被广泛应用于功能性食品和调味品工业中；风味蛋白酶可以使蛋白水解产生具有良好风味的游离氨基酸和寡肽，因此风味蛋白酶被广泛应用于新型调味品中。
呈味肽是一类小分子肽，可以影响或改善食品的风味，当下获取呈味肽的主要方式有酶法水解和生物技术合成。呈味肽种类丰富，其呈味涵盖了酸、甜、苦、咸、鲜五种基本味，因此呈味肽可以代替传统的蔗糖、食盐、谷氨酸钠等调味料添加到食品中。近些年，关于从食物中分离并鉴定呈味肽的研究有很多，在诸多呈味肽分离纯化的手段中，食用级乙醇分离法因具有无毒无害、高通量、分离速度快的优点而受到了广泛的应用。在呈味肽结构鉴定方面，超高效液相色谱-串联质谱(UPLC-MS/MS)技术和从头测序(De Novo sequencing)法的精确度和灵敏度都大大超过常规的检测方法，能够快速准确地鉴别和测定多肽。
发明概述
技术问题
问题的解决方案
技术解决方案
本发明的目的是从复合酶酶解牡蛎的酶解液中分离鉴定出一种新型呈味肽，利用新鲜的牡蛎肉为原料，采用复合酶解技术得到酶解液，利用乙醇分级法和UPLC-MS/MS分离鉴定呈味肽，该呈味肽酸味、鲜味浓郁，无明显的腥苦味，可通过工业合成该呈味肽来开发海鲜调味品。
本发明的目的通过以下技术方案实现。
一种呈味肽，该呈味肽的氨基酸序列为Thr-Gly-Ser-Ser-Pro-Ala-Gly-Glu。
从牡蛎酶解液中分离出所述的一种呈味肽的方法，包括以下步骤：
(1)预处理：以牡蛎肉为原料，清洗沥干，打成肉糜，加水制备成牡蛎匀浆；
(2)酶解：向牡蛎匀浆中加入复合酶进行酶解反应，反应结束后灭酶、离心，所得滤液为牡蛎酶解液；
(3)乙醇分级提取牡蛎酶解液中的肽：向牡蛎酶解液中加入乙醇，搅拌均匀 ，离心后得沉淀物(I)和上清液(I)，然后向上清液(I)中加入乙醇，搅拌均匀后离心分离得沉淀物(II)和上清液(II)；再向上清液(II)中加入乙醇，搅拌均匀后离心得到上清液(III)，再将上清液(III)去除多余乙醇。
优选的，步骤(1)所述牡蛎匀浆的料液比为1g∶1ml。
优选的，步骤(2)所述复合酶为Pancreatin(胰酶)、Papain(木瓜蛋白酶)、Acid Protease(酸性蛋白酶)、Ns 37071(碱性蛋白酶)、Protamex(复合水解酶)、Flavorzyme500MG(风味蛋白酶)和Alcalase2.4L(碱性蛋白酶)中的两种以上。
进一步优选的，所述复合酶为Flavorzyme500MG和Pancreatin的混合酶。
更优选的，所述混合酶中Flavorzyme500MG和Pancreatin的质量比为1∶3。
优选的，步骤(2)所述复合酶的添加量为牡蛎肉的0.1-0.5wt％，进一步优选为0.2wt％。
优选的，步骤(2)所述酶解的温度为45-55℃，进一步优选为50℃；酶解的时间为4-8h，进一步优选为7h。
优选的，步骤(2)所述灭酶的时间为10-20min。
优选的，步骤(3)中，向牡蛎酶解液中加入牡蛎酶解液3-5倍体积的乙醇；向上清液(I)中加入乙醇使得体系中乙醇最终浓度为70-90vol％；向上清液(II)中加入乙醇使得体系中乙醇最终浓度为95vol％。
进一步优选的，步骤(3)中，向牡蛎酶解液中加入牡蛎酶解液4倍体积的乙醇；向上清液(I)中加入乙醇使得体系中乙醇最终浓度为90vol％；向上清液(II)中加入乙醇使得体系中乙醇最终浓度为95vol％。
以上所述的呈味肽在食品中的应用。
发明的有益效果
有益效果
与现有技术相比，本发明具有如下优点：
(1)本发明采用复合酶从多个位点进行酶解，提高牡蛎蛋白的水解度，同时提高了多肽的得率；
(2)本发明的呈味肽是分子量小于800Da的8肽，易于被人体吸收，提高了营 养价值。
(3)本发明反应条件温和，反应时间短，反应中采用的酶价格低廉、品质高且用量少，有效的节约了成本。
(4)本发明分离鉴定出的呈味肽与其他调味品组合能显著增强调味品的鲜味，削弱腥苦味。
对附图的简要说明
附图说明
图1为乙醇分级提取流程图；
图2不同酶解条件的感官评价图。
图3a、图3b、图3c、图3d为不同浓度的牡蛎酶解产物对食品的呈味增效作用图。
图4为UPLC-MS/MS的呈味肽的二级质谱图。
发明实施例
本发明的实施方式
以下结合实例与图对本发明的具体实施做进一步的说明，但本发明的实施方式不限于此。
实施例1
筛选滋味最优的牡蛎酶解液
1、以牡蛎肉为原料，清洗沥干，打成肉糜，加水制备成料液比为1g∶1ml的牡蛎匀浆；
2、向牡蛎匀浆中加入牡蛎肉0.1-0.5wt％的Flavorzyme500MG和Pancreatin的混合酶(Flavorzyme500MG和Pancreatin的质量比为1∶3)，在自然pH值下，45-55℃酶解7h，反应终止后，将酶解液置于沸水中进行灭酶处理10min，酶解液离心、过滤，所得滤液为酶解产物，按酶添加量分为E-1，E-2，E-3，E-4，E-5，其中E-1：0.1wt％混合酶，50℃酶解7h；E-2：0.3wt％混合酶，50℃酶解7h；E-3：0.5wt％混合酶，50℃酶解7h；E-4：0.3wt％混合酶，45℃酶解7h；E-5：0.3wt％混合酶，55℃酶解7h；
3、酶解液的感官评定
本研究的感官评定小组选择没有味觉障碍的男性和女性各五名(年龄在24～30之间)，将品评房间的温度控制在室温(25±2℃)。针对实验的特点，用参比溶液对小组成员进行训练。参比溶液如下：鲜味(MSG溶液，16mmol/L，8mmol/L，4mmol/L)；甜味(蔗糖溶液，50mmol/L，30mmol/L，10mmol/L)；酸味(柠檬酸溶液，8mmol/L，4mmol/L，2mmol/L)；苦味(L-异亮氨酸溶液，40mmol/L，20mmol/L，10mmol/L)；厚味的口感较为复杂，本研究采用白水熬煮的老母鸡制得的鸡汤作参比溶液(熬煮6h的鸡汤，熬煮4小时的鸡汤，熬煮2小时的鸡汤)。其中参比液的3种浓度的风味分值按强弱分别定位10分，5分，1分。各风味分值参考与其风味相近的参比液分值，最终结果取各小组成员评分的平均值。
牡蛎酶解液的感官评定结果如图1所示。由图1可知，牡蛎酶解液E-2的感观评分为：鲜味6.67，酸味1.96，甜味3.75，厚味4.18，苦味1.65。其鲜味、甜味较其他酶解液的评分较高，厚味的评分适中，酸味和苦味的评分较低。这表明酶添加量为0.3wt％，自然pH下，50℃酶解7h所得的牡蛎酶解液鲜味浓郁，口感饱满且余味较长，甜度适中，无明显的酸味和苦味，整体风味较好。
实施例2
滋味最优酶解液呈味增效评价
1、以牡蛎肉为原料，清洗沥干，打成肉糜，加水制备成料液比为1g∶1ml的牡蛎匀浆；
2、向牡蛎匀浆中加入牡蛎肉0.3wt％的Flavorzyme500MG和Pancreatin的混合酶(Flavorzyme500MG和Pancreatin的质量比为1∶3)，在自然pH值下，50℃酶解7h，反应终止后，将酶解液置于沸水中进行灭酶处理10min，酶解液离心、过滤，所得滤液为酶解产物。
3、呈味增效评定
分别加入鸡汤溶液、牛肉汤溶液、味精与盐水的混合溶液以及味精、盐水与I+G的混合溶液中，其中图3a表示：食盐与味精混合溶液；图3b表示：食盐、味精与I+G的混合溶液；图3c表示：模型鸡汤；图3d表示：模型牛肉汤，并采用氢氧化钠溶液和甲酸溶液将所配制的各溶液的pH值调整为6.5。将所配制的各溶液按 其酶解产物浓度从小到大的顺序呈现给感官评定小组，并对其呈味增效进行评价。采用“5点”强度量表(0，不可检测；5，强可检测)评估各溶液的感官特征，目标感官特征包括鲜味、厚味(口感较为复杂，评定人员在品尝食物10秒后对其进行评估)和持续性(可以持续维持的感官效果或呈味增效，评定人员在品尝食物25秒后对其进行评估)。
由图3a、图3b、图3c、图3d可知，牡蛎酶解液对四种溶液的鲜味、厚味和持续性都有不同程度的增强效果，且添加量为0.1％酶解液对四种溶液的呈味增效值均显著高于添加量为0.05％酶解液。牡蛎酶解液对食盐与味精混合溶液的持续性和鲜味的增强效果较好，其呈味增效值依次为：持续性＞鲜味＞厚味，呈味增效值的范围分别为0.52～0.81、0.58～0.71和0.18～0.24；对盐水、味精与I+G混合溶液的持续性增强效果较好，其呈味增值依次为：持续性＞鲜味＞厚味，呈味增效值的范围分别为0.71～1.2、0.23～0.42和0.32～0.51；对鸡汤的鲜味和厚味的增强效果较好，其呈味增效值依次为：鲜味＞厚味＞持续性，呈味增效值的范围分别为0.69～0.84、0.61～0.82和0.21～0.39；对牛肉汤的鲜味和厚味的增强效果较好，其呈味增效值依次为厚味＞鲜味＞持续性，呈味增效值的范围分别为0.73～0.9、0.41～0.66和0.19～0.38。由此可见，牡蛎酶解液可以有效地提升食品的风味，其对不同食品的呈味影响存在一定的差异。
实施例3
1.预处理：以牡蛎肉为原料，清洗沥干，打成肉糜，加水制备成料液比为1g∶1ml的牡蛎匀浆；
2、向牡蛎匀浆中加入牡蛎肉0.39wt％的Flavorzyme500MG和Pancreatin的混合酶(Flavorzyme500MG和Pancreatin的质量比为1∶3)，在自然pH(6.8)值下，50℃酶解7h，反应终止后，将酶解液置于沸水中进行灭酶处理10min，酶解液离心、过滤，所得滤液为酶解产物。
3.乙醇分级提取牡蛎酶解液中的肽：
取牡蛎酶解液20mL加入80mL无水乙醇后在25℃下搅拌30min，离心(8000r/min，4℃，20min)得沉淀物(1)和上清液(1)，向上清液(1)中加入无水乙醇使得体系中乙醇最终浓度为90vol％，在上述同等条件下搅拌、离心分离得 沉淀物(2)和上清液(2)；再向上清液(2)中加入200mL无水乙醇使得体系中乙醇最终浓度为95vol％，以上述同等条件搅拌、离心得到上清液(3)，上清液(3)去除多余乙醇后复溶于去离子水，得待测组分。具体操作流程见图1所示。
4.UPLC-MS/MS分离鉴定组分中新型呈味肽
采用超高效液相色谱串联质谱法和从头测序法对乙醇提取组分中的多肽进行分离鉴定，样品首先经超高效液相色谱分离，然后经电喷雾离解(ESI)后进入高分辨率质谱仪中，经二级质谱轰击后断裂成碎片离子，根据质荷比的不同而分离开来，形成二级质谱图(见图2)。通过Peaks软件和数据库搜索从乙醇组分中匹配出1条新型肽链，其分子量小于800Da，所鉴定出的肽链为Thr-Gly-Ser-Ser-Pro-Ala-Gly-Glu，该肽链可人工合成。
5.利用人工感官评定方法测定短肽的呈味特性
(1)合成肽-食盐与味精混合物的增强效应
感官标准品为0.35％味精与食盐混合溶液，评分为5分。将合成多肽添加至标准品溶液中，调节多肽浓度为2mg/mL，将所配制的各溶液呈现给感官评定小组(10名：5男，5女，经过培训的感官评定员)，并对其呈味增效进行评价。评估各溶液的感官特征，目标感官特征包括鲜味、厚味(口感较为复杂，评定人员在品尝食物10秒后对其进行评估)和持续性(可以持续维持的感官效果或呈味增效，评定人员在品尝食物25秒后对其进行评估)。评价其鲜味、咸味及其呈味增效。
(2)合成肽-食盐的增强效应：感官标准品为0.35％食盐溶液，评分为5分。将合成肽添加至标准品溶液中，调节多肽浓度为2mg/mL，评价其咸味及其呈味增效。
表1
Figure PCTCN2019113799-appb-000001
从表1可以看出，合成肽在食盐溶液中的鲜味有所增强，酸味减弱，表示食盐溶液对呈味肽的鲜味有一定的提升效果；在食盐与味精混合溶液中呈现一定的鲜味增强效果。

Claims (10)

  1. 一种呈味肽，其特征在于，该呈味肽的氨基酸序列为Thr-Gly-Ser-Ser-Pro-Ala-Gly-Glu。
  2. 从牡蛎酶解液中分离出权利要求1所述的一种呈味肽的方法，其特征在于，包括以下步骤：
    (1)预处理：以牡蛎肉为原料，清洗沥干，打成肉糜，加水制备成牡蛎匀浆；
    (2)酶解：向牡蛎匀浆中加入复合酶进行酶解反应，反应结束后灭酶、离心，所得滤液为牡蛎酶解液；
    (3)乙醇分级提取牡蛎酶解液中的肽：向牡蛎酶解液中加入乙醇，搅拌均匀，离心后得沉淀物(I)和上清液(I)，然后向上清液(I)中加入乙醇，搅拌均匀后离心分离得沉淀物(II)和上清液(II)；再向上清液(II)中加入乙醇，搅拌均匀后离心得到上清液(III)，再将上清液(III)去除多余乙醇。
  3. 根据权利要求2所述的制备方法，其特征在于，步骤(2)所述复合酶为Pancreatin、Papain、Acid Protease、Ns 37071、Protamex、Flavorzyme500MG和Alcalase2.4L中的两种以上。
  4. 根据权利要求3所述的制备方法，其特征在于，所述复合酶为Flavo rzyme500MG和Pancreatin的混合酶。
  5. 根据权利要求4所述的制备方法，其特征在于，所述混合酶中Flavo rzyme500MG和Pancreatin的质量比为1∶3。
  6. 根据权利要求2所述的制备方法，其特征在于，步骤(2)所述复合酶的添加量为牡蛎肉的0.1-0.5wt％。
  7. 根据权利要求2所述的制备方法，其特征在于，步骤(2)所述酶解的温度为45-55℃，时间为4-8h。
  8. 根据权利要求7所述的制备方法，其特征在于，步骤(2)所述酶解的温度为50℃，时间为7h。
  9. 根据权利要求2所述的制备方法，其特征在于，步骤(3)中，向牡蛎酶解液中加入牡蛎酶解液3-5倍体积的乙醇；向上清液(I)中加入乙醇使得体系中乙醇最终浓度为70-90vol％；向上清液(II)中加入乙醇使得体系中乙醇最终浓度为95vol％。
  10. 权利要求1所述的呈味肽在食品中的应用。
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