WO2020232975A1 - Flavor peptide isolated from oyster enzymatic hydrolysate, preparation method therefor and use thereof - Google Patents

Abstract

A flavor peptide isolated from an oyster enzymatic hydrolysate, a preparation method therefor and use thereof. Said method comprises the following steps: taking oyster meat as a raw material, washing and crushing same to form meat paste, and adding water to prepare a homogenate; adding compound enzymes to the homogenate solution for enzyme digestion, and performing enzyme deactivation and centrifugation after the reaction is finished, the obtained filtrate being an enzymatic hydrolysate; performing ethanol fractional extraction of peptides in the enzymatic hydrolysate; and using UPLC-MS/MS to separate and identify the flavor peptide, the amino acid sequence being Thr-Gly-Ser-Ser-Pro- Ala-Gly-Glu. Also included is use of the flavor peptide in foodstuffs.

Description

A taste peptide isolated from oyster enzymatic hydrolysate and its preparation method and application Technical field

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.

Background technique

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. At present, the products developed on the market using oysters as raw materials mainly include functional health products, seafood condiments and medicines. There are relatively few oyster processed products in my country, most of which are traditional oyster products, such as dried products, canned food, etc. How to use fermentation technology, biological enzymatic hydrolysis technology and other modern food technology to carry out intensive processing of oyster meat. Making full use of oyster resources has become a research focus.

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.

Among them, 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. When using enzymatic technology to hydrolyze oyster protein, 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. At present, 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 The advantages of high and specificity; 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 condiments.

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. In recent years, there have been many studies on the separation and identification of taste peptides from food. Among many methods for the separation and purification of taste peptides, the food grade ethanol separation method is non-toxic, harmless, high-throughput, and fast in separation. The advantages have been widely used. In terms of structural identification of taste peptides, the accuracy and sensitivity of ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology and de novo sequencing (De Novo sequencing) greatly exceed those of conventional detection methods, and can quickly and accurately Identify and measure peptides.

Summary of the invention

technical problem

The solution to the problem

Technical solutions

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.

The purpose of the present invention is achieved through the following technical solutions.

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:

(1) Pretreatment: Take oyster meat as raw material, wash and drain, beat into meat emulsion, and add water to prepare oyster homogenate;

(2) 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;

(3) 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.

Preferably, the material-to-liquid ratio of the oyster homogenate in step (1) is 1g:1ml.

Preferably, 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).

Further preferably, the compound enzyme is a mixed enzyme of Flavorzyme 500MG and Pancreatin.

More preferably, the mass ratio of Flavorzyme 500MG and Pancreatin in the mixed enzyme is 1:3.

Preferably, 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%.

Preferably, 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.

Preferably, the enzyme inactivation time in step (2) is 10-20 min.

Preferably, in 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%.

Further preferably, in 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%.

Application of the above-mentioned taste peptide in food.

The beneficial effects of the invention

Beneficial effect

Compared with the prior art, the present invention has the following advantages:

(1) 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;

(2) 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.

(3) The 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.

(4) The combination of the taste peptide isolated and identified in the present invention and other condiments can significantly enhance the umami taste of the condiments and weaken the bitterness.

Brief description of the drawings

Description of the drawings

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.

Invention embodiment

Embodiments of the invention

The specific implementation of the present invention will be further described below in conjunction with examples and figures, but the implementation of the present invention is not limited thereto.

Example 1

Screen the best taste of oyster hydrolysate

1. Take oyster meat as raw material, clean and drain, beat into meat emulsion, and add water to prepare an oyster homogenate with a material-to-liquid ratio of 1g:1ml;

2. Add 0.1-0.5wt% oyster meat Flavorzyme500MG and Pancreatin mixed enzyme (the mass ratio of Flavorzyme500MG and Pancreatin is 1:3) to the oyster homogenate, and enzymatically hydrolyze at 45-55℃ for 7h under natural pH value, and react After termination, place the enzymatic hydrolysate in boiling water for enzymatic inactivation for 10 minutes, centrifuge and filter the enzymatic hydrolysate. The filtrate obtained is the product of enzymatic hydrolysis, which is divided into E-1, E-2, E-3, E according to the amount of enzyme added. -4, E-5, where E-1: 0.1wt% mixed enzyme, 50℃ enzymatic hydrolysis for 7h; E-2: 0.3wt% mixed enzyme, 50℃ 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;

3. Sensory evaluation of enzyme hydrolysate

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). Among them, 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.

Example 2

Evaluation of the best taste of the enzyme hydrolysis

1. Take oyster meat as raw material, clean and drain, beat into meat emulsion, and add water to prepare an oyster homogenate with a material-to-liquid ratio of 1g:1ml;

2. Add oyster meat 0.3wt% Flavorzyme500MG and Pancreatin mixed enzyme (the mass ratio of Flavorzyme500MG and Pancreatin is 1:3) to the oyster homogenate, and enzymatically hydrolyze at 50℃ for 7h at natural pH value. After the reaction is terminated, The enzymolysis solution is placed in boiling water for enzymatic inactivation treatment for 10 minutes, and the enzymolysis solution is centrifuged and filtered, and the obtained filtrate is the enzymatic hydrolysis product.

3. Appraisal of taste enhancement

Add chicken broth solution, beef broth solution, mixed solution of monosodium glutamate and brine, and mixed solution of monosodium glutamate, brine and I+G, respectively. 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).

From Figure 3a, Figure 3b, Figure 3c, Figure 3d, it can be seen that the oyster enzymatic hydrolysate has different degrees of enhancement effects on the umami taste, thick taste and persistence of the four solutions, and the addition of 0.1% enzymatic hydrolysate has different effects on the four solutions. The taste enhancement value of the two solutions were significantly higher than the 0.05% enzymatic hydrolysis solution. The oyster enzymatic hydrolysate has a better effect on the persistence and umami taste of the mixed solution of salt and monosodium glutamate. The order of its taste synergistic value is: persistence> umami taste> thick taste, and the range of taste synergistic value is 0.52. ～0.81, 0.58～0.71 and 0.18～0.24; the effect of continuous enhancement of salt water, monosodium glutamate and I+G mixed solution is better, and the increase in taste is in the order of persistence> umami> thick taste, showing a taste enhancement value The ranges of are 0.71～1.2, 0.23～0.42 and 0.32～0.51 respectively; the enhancement effect on the umami and thick taste of chicken soup is better, and its taste enhancement value is in order: umami> thick taste> persistent, present taste The range of synergistic value is 0.69～0.84, 0.61～0.82 and 0.21～0.39, respectively; the enhancement effect on the umami and thick taste of beef broth is better, and the order of its taste synergistic value is thick taste> umami taste> persistence The range of taste-enhancing value is 0.73-0.9, 0.41-0.66 and 0.19-0.38, respectively. It can be seen that the oyster enzymatic hydrolysate can effectively enhance the flavor of food, and its influence on the taste of different foods has certain differences.

Example 3

1. 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;

2. Add oyster meat 0.39wt% Flavorzyme500MG and Pancreatin mixed enzyme (the mass ratio of Flavorzyme500MG and Pancreatin is 1:3) to the oyster homogenate, and enzymatically hydrolyze at 50℃ for 7h at natural pH (6.8), and the reaction is terminated Afterwards, the enzymatic hydrolysate is placed in boiling water for enzymatic inactivation treatment for 10 minutes, the enzymatic hydrolysate is centrifuged and filtered, and the resulting filtrate is the enzymatic hydrolysate product.

3. Ethanol fractionation to extract peptides in oyster hydrolysate:

Take 20mL of oyster enzymatic hydrolysate and add 80mL of absolute ethanol, stir at 25℃ for 30min, centrifuge (8000r/min, 4℃, 20min) to obtain precipitate (1) and supernatant (1), supernatant (1) Add absolute ethanol to make the final concentration of ethanol in the system 90 vol%, stir and centrifuge under the same conditions above to obtain precipitate (2) and supernatant (2); then add 200 mL of anhydrous to supernatant (2) Ethanol makes the final concentration of ethanol in the system 95 vol%, stirring and centrifuging under the same conditions as above to obtain the supernatant (3), the supernatant (3) is reconstituted in deionized water after removing excess ethanol to obtain the components to be tested. The specific operation process is shown in Figure 1.

4. UPLC-MS/MS separation and identification of new taste peptides

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). Through 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.

5. Using artificial sensory evaluation methods to determine the taste characteristics of short peptides

(1) Enhancement effect of synthetic peptide-table salt and MSG mixture

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.

(2) 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.

Table 1

It can be seen from Table 1 that the umami taste of the synthetic peptides in the salt solution is enhanced, and the acidity is weakened, indicating that the salt solution has a certain effect on improving the umami taste of the taste peptide; in the mixed solution of salt and monosodium glutamate, there is a certain amount of freshness. Taste enhancing effect.

Claims (10)

1. A taste peptide characterized in that the amino acid sequence of the taste peptide is Thr-Gly-Ser-Ser-Pro-Ala-Gly-Glu.
2. The method for separating the taste peptide according to claim 1 from the oyster enzymatic hydrolysate is characterized by comprising the following steps:

   (1) Pretreatment: Take oyster meat as raw material, wash and drain, beat into meat emulsion, and add water to prepare oyster homogenate;

   (2) 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;

   (3) 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.
3. The preparation method according to claim 2, wherein the complex enzyme in step (2) is two or more of Pancreatin, Papain, Acid Protease, Ns 37071, Protamex, Flavorzyme 500MG and Alcalase 2.4L.
4. The preparation method according to claim 3, wherein the compound enzyme is a mixed enzyme of Flavorzyme 500MG and Pancreatin.
5. The preparation method according to claim 4, wherein the mass ratio of Flavorzyme 500MG and Pancreatin in the mixed enzyme is 1:3.
6. The preparation method according to claim 2, wherein the addition amount of the compound enzyme in step (2) is 0.1-0.5 wt% of oyster meat.
7. The preparation method according to claim 2, wherein the temperature of the enzymatic hydrolysis in step (2) is 45-55°C, and the time is 4-8h.
8. The preparation method according to claim 7, characterized in that the temperature of the enzymatic hydrolysis in step (2) is 50°C and the time is 7h.
9. The preparation method according to claim 2, wherein in step (3), 3-5 times the volume of the oyster hydrolysate is added to the oyster hydrolysate; ethanol is added to the supernatant (I) to make the system The final concentration of middle ethanol is 70-90vol%; adding ethanol to the supernatant (II) makes the final concentration of ethanol in the system 95vol%.
10. The use of the taste peptide of claim 1 in food.

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