CN108893515B

CN108893515B - High F value oligopeptide and preparation method thereof

Info

Publication number: CN108893515B
Application number: CN201810804406.3A
Authority: CN
Inventors: 田亚平; 吴警涛; 李婷婷; 周楠迪
Original assignee: Jiangnan University
Current assignee: Hubei Yuelai Bioengineering Co.,Ltd.
Priority date: 2018-07-20
Filing date: 2018-07-20
Publication date: 2020-07-07
Anticipated expiration: 2038-07-20
Also published as: CN108893515A; WO2020014997A1

Abstract

The invention relates to a preparation method of high-F-value oligopeptides, which comprises the following steps of adding alkaline protease into a yeast protein solution, carrying out enzymolysis at 45-55 ℃ under the condition that the pH value is 7.0-9.0 to obtain yeast extract, wherein the adding amount of the alkaline protease is 400-600U/g of yeast protein, concentrating the yeast extract to remove molecules with the molecular weight of below 500Da in the yeast extract to obtain concentrated solution, adding α -chymotrypsin into the concentrated solution, carrying out enzymolysis at 35-45 ℃ for 2-8 h under the condition that the pH value is 6.5-8.5, adding carboxypeptidase A into an enzymolysis product after enzyme deactivation, carrying out enzymolysis at 35-45 ℃ for 4-8 h under the condition that the pH value is 6.5-7.5, and carrying out impurity removal by using activated carbon adsorption after enzyme deactivation to obtain the high-F-value oligopeptides.

Description

High F value oligopeptide and preparation method thereof

Technical Field

The invention relates to the technical field of enzyme preparations, in particular to a high F value oligopeptide and a preparation method thereof.

Background

The high F value oligopeptide is a mixed small peptide mixture consisting of 2-9 amino acid residues, the F value refers to the molar ratio of branched chain amino acids (leucine, isoleucine and valine, BCAA for short) to aromatic amino acids (phenylalanine and tyrosine, AAA for short) in the mixture, and is named for commemorating the 'pseudo neurotransmitter hypothesis' proposed by Fischer in the last 70 th century of Germany famous scholars. The high F value oligopeptide should have an F value greater than 20. Oligopeptides are protein precursors composed of 2-9 amino acids, or protein degradation products composed of 2-9 amino acids from protein degradation, which can also be called small peptides, short peptides, etc. In actual production, the high F value oligopeptide can be widely applied to the aspects of medicines for treating liver diseases, liver protection foods, protein nutrition foods for surgical patients, intestinal nutrients for digestive enzyme deficiency patients, food nutrition enhancers for high-intensity workers and the like. Compared with a branched chain amino acid formula, the high F oligopeptide mixture has more practical effect, has high attention to unique physiological functions of people, and has good development prospect.

Yeast is a microbial industrial product that contributes most to humans. The method has been handed over with yeast since the beginning of the history of human brewing, vinegar making and sauce making, and can even trace back to the ancient Babylon times thousands of years ago and the trends of China. The protein content of the yeast is between 45 and 55 percent, and compared with common-grade yeast, the active dry yeast has the water content of 4 to 6 percent, small particles and high fermentation speed. The yeast protein contains rich essential amino acids, wherein the branched chain amino acids account for 18.96 percent, the aromatic amino acids account for 7.45 percent, the initial F value is 3.41, the amino acid content is higher than that of common animal and plant protein powder such as soybean protein, rice protein and the like, and the amino acid-containing yeast protein is a raw material which is very suitable for producing high-F-value oligopeptides.

At present, high F value products sold at home and abroad are all compounded by amino acid, the product types are single, and no high F value oligopeptide product is sold. Modern nutritional studies have shown that: compared with free amino acids, the oligopeptide can be used as a nutrient substance, has the advantages of low osmotic pressure, low energy consumption and the like, and is easier to be absorbed and utilized by organisms.

The preparation process of the high F value oligopeptide is complicated due to strict preparation requirements. At present, according to the current research situation at home and abroad, zein, soy protein, lactalbumin, casein and the like are mainly used as raw materials, and most of the used hydrolases are actin, pronase, pepsin and papain, so that the development of a new preparation way is very important. The key point in increasing the F value during purification is the removal of aromatic amino acids. For free aromatic amino acids released in the enzymolysis process, the aromatic amino acids need to be effectively removed for dearomatization and high F value. At present, the dearomatization method mainly comprises an ion exchange method, a membrane separation method, a gel chromatography method, an affinity chromatography method and an active carbon chromatography method. According to the research reports at home and abroad, Sephadex G-15 or Bio-Gel P-2 Gel chromatography is generally used for separation and purification, but the large-scale production is difficult because the sample loading amount is small and the process is not easy to amplify.

The active dry yeast is easy to transport and store, and the protein content is rich, so that the application potential of yeast cells is explored, and the development of the yeast high-F-value oligopeptide which is simple and convenient in method, low in cost and easy to absorb has certain market value.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide the high-F-value oligopeptide and the preparation method thereof.

In one aspect, the present invention provides a method for preparing a high F value oligopeptide, comprising the following steps:

(1) adding alkaline protease into a yeast protein aqueous solution, and carrying out enzymolysis at 45-55 ℃ under the condition that the pH value is 7.0-9.0 to obtain a yeast extract, wherein the adding amount of the alkaline protease is 400-600U/g yeast protein;

(2) concentrating the yeast extract to remove molecules with molecular weight of less than 500Da in the yeast extract to obtain a concentrated solution;

(3) adding α -chymotrypsin into the concentrated solution, carrying out enzymolysis for 2-8 h at 35-45 ℃ under the condition that the pH value is 6.5-8.5, carrying out enzyme deactivation and centrifugation, taking supernatant, adding carboxypeptidase A into an enzymolysis product, carrying out enzymolysis for 4-8 h at 35-45 ℃ under the condition that the pH value is 6.5-7.5, carrying out enzyme deactivation and centrifugation, taking supernatant, and then carrying out adsorption impurity removal by using activated carbon to obtain the high-F-value oligopeptide.

Extraction refers to a process of extracting and separating a desired substance from a solid or a liquid mixture by a solvent according to its characteristics.

Further, in the step (1), the concentration of the yeast protein solution is 160-250 g/L.

Further, in the step (1), the enzymolysis time is 8-12 h.

Further, in step (1), the yeast protein solution is obtained by adding active dry yeast to water, followed by ultrasonication. The ultrasonic crushing power is 300-450W, preferably 400W, and the crushing time is 10-25 min, preferably 20 min.

Further, in the step (2), nanofiltration is used for concentration to remove molecules with molecular weight of less than 500Da, such as free amino acids and/or salt ions, in the yeast extract.

Furthermore, the nanofiltration method is carried out under 2-4 MPa.

Further, in the step (2), the pH value of the yeast extract before concentration is controlled to be 6.5-7.5, and the ratio of the volume of the liquid after concentration to the volume of the liquid before concentration is 1: 1.5-3.

Further, before the step (2), the method also comprises the steps of inactivating enzyme, centrifuging and taking supernatant. The enzyme deactivation condition is boiling water bath enzyme deactivation for 15 min. The centrifugal speed is preferably 13000r/min, and the centrifugal time is preferably 15 min.

Further, in the step (3), α -chymotrypsin is added in an amount of 4000-6000U/g yeast protein.

Further, in the step (3), the addition amount of the carboxypeptidase A is 2-6U/mL of yeast protein.

Further, in the step (3), the solid-to-liquid ratio of the activated carbon to the enzymolysis liquid is 1: 10-20. Most of aromatic amino acids are removed by activated carbon adsorption, and branched-chain amino acids are retained.

Further, in the step (3), the adsorption temperature is 30-40 ℃, and the adsorption is carried out under the condition that the pH value is 2.0-3.0.

Further, in the step (3), the adsorption time is 2-5 h.

Further, in the step (3), the activated carbon is washed with dilute hydrochloric acid to remove ash in advance before use, so as to change the surface group property of the activated carbon.

Further, in the step (3), the adsorbed hydrolysate is subjected to freeze drying to obtain high F value oligopeptide powder.

The active dry yeast is a living cell, the extraction efficiency is low and the time consumption is long by using α -chymotrypsin and carboxypeptidase A, so that the yeast protein is extracted by using nonspecific alkaline protease to decompose into small-fragment polypeptide, then the large part of aromatic amino acid can be liberated by α -chymotrypsin (mainly cutting the carboxyl end of aromatic amino acid residue as aromatic amino acid) and carboxypeptidase A (mainly cutting the carboxyl end of the first amino acid residue as aromatic amino acid) for directional enzymolysis, and then the high-F-value mixed oligopeptide liquid can be obtained by activated carbon adsorption.

On the other hand, the invention also claims a high F value oligopeptide prepared by the method, wherein the F value is 30-40.

Furthermore, the high F value oligopeptide from the yeast protein is formed by mixing polypeptide molecules, the molecular weight of the oligopeptide is mainly concentrated below 1500Da and accounts for 99.82 percent of the total content. Wherein the oligopeptide with the molecular weight of 180-1500 Da accounts for 66.42% of the total content, the oligopeptide with the molecular weight range mainly comprises 3-6 amino acid residues, and the molecular weight requirement of the high-F-value oligopeptide is met; the molecular weight is less than 180Da and accounts for 32.86%, and the molecules in the molecular weight range comprise dipeptide and/or free amino acid.

Further, the high F value oligopeptide from yeast protein of the present invention has a free amino acid content of 14.67% in the mixture of oligopeptide and free amino acid; in the free amino acids, the content of aromatic amino acids was 3.6%, and the content of branched chain amino acids was 55.1%. The yeast protein hydrolysate contains not only various amino acids, but also 35.48% essential amino acids and 64.52% nonessential amino acids, and among many amino acids, the total content (mass fraction) of branched-chain amino acids valine (Val), isoleucine (Ile) and leucine (Leu) is 21.73%, and the total content (mass fraction) of aromatic amino acids tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr) is 0.75%.

By the scheme, the invention at least has the following advantages:

the invention takes the active dry yeast as the protein raw material, utilizes the characteristics of high protein content, high initial F value, wide source, low price, environmental protection and the like, utilizes the enzyme engineering technology and the modern separation and purification technology, has sufficient raw material source and controllable enzymolysis process, can produce a pure natural and easily absorbed high F value bioactive oligopeptide mixture through one-step separation, provides a basis for the reasonable application and development of yeast protein resources, and provides an important application way for further improving the added value of the yeast protein resources.

The invention selects specific protease to replace protease with wide enzyme cutting sites for directional hydrolysis, improves the F value and the practical value of the raw materials, provides a method for remarkably improving the F value of yeast extract by specific internal and external protease collaborative directional hydrolysis and one-step purification, and provides a new idea for solving the problem of the lack of high F value oligopeptide products at present.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.

Drawings

FIG. 1 is a schematic view of the process flow of the present invention for the enzymatic hydrolysis of yeast proteins by multi-enzymes;

FIG. 2 shows the results of DH test on the protein yield and degree of hydrolysis of dry yeast proteins with different enzymatic hydrolysis activities;

FIG. 3 shows the results of tests on the influence of different concentration factors on the total free amino acid removal rate and the protein recovery rate under nanofiltration conditions;

FIG. 4 shows the results of measuring the release rate of aromatic amino acid Phe (low phenylalanine Phe is an example of aromatic amino acid) in α -chymotrypsin-digested yeast extract under the conditions of different temperatures, times, pH values and enzyme addition amounts;

FIG. 5 shows the results of the ratio measurement of the absorbance 220/280 after the activated carbon adsorbs the enzymatic hydrolysate under different conditions of time, pH, feed-to-liquid ratio, and temperature;

FIG. 6 shows the results of testing the free amino acid content of yeast extract, α -chymotrypsin and carboxypeptidase A after reaction and the total amino acid content of hydrolyzed oligopeptides after activated carbon adsorption;

FIG. 7 is a molecular weight distribution diagram of oligopeptide lyophilized powder obtained after yeast extract and bi-enzyme directional enzymolysis.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

In the following examples of the present invention, the test methods used include the following:

and (3) enzyme activity determination: reference is made to GB/T23527-2009.

Degree of Hydrolysis (DH): ratio of free amino nitrogen content to total protein nitrogen content. Wherein, the content of free amino nitrogen is determined by adopting a formaldehyde titration method, and the content of total protein nitrogen is determined by adopting a Kjeldahl nitrogen determination method.

Formaldehyde titration method: reference is made to GB 5009.235-2016.

Polypeptide distribution determination: centrifuging the solution at 12000r/min for 10min, collecting supernatant, and determining polypeptide molecular weight distribution by HPLC method.

And (3) amino acid content determination:

(1) free amino acids: and (3) adding an equal volume of 10% TCA (trichloroacetic acid) solution into the centrifuged supernatant, standing for 3h, centrifuging for 30min at the rotating speed of 15000rpm, filtering 2mL of the supernatant again through a 0.22-micron organic phase membrane, putting 400 mu L of the supernatant into a liquid phase sample bottle, and determining the content of the free amino acid by an HPLC method.

(2) Hydrolyzing amino acid: taking a hydrolysis tube, measuring 1mL of a sample (liquid), adding 1mL of concentrated hydrochloric acid, adding 6mL of 6mol/L HCl, then introducing nitrogen for 3min, screwing the hydrolysis tube after the completion, and putting the tube in an oven at 120 ℃ for hydrolysis for 22 h. After 22h, transferring all samples in the hydrolysis tube into a volumetric flask, adding 4.8mL of 10mol/L NaOH for neutralization, then fixing the volume to 25mL by using distilled water, placing the mixture in an oscillator for uniform mixing, filtering the mixture by using double-layer filter paper, taking 1mL of filtrate into a 1.5mL centrifuge tube, centrifuging the filtrate for 30min at the rotating speed of 15000rpm, taking 400 mu L of supernatant into a liquid phase sample bottle, and determining the total amino acid content in the solution by adopting an HPLC method; the solid was tested as a liquid except that about 100.00mg of the solid was added to 8mL of 6mol/L HCl.

F value: the ratio of moles of branched chain amino acids to moles of aromatic amino acids. The calculation formula is as follows:

n represents a mole number.

EXAMPLE 1 preparation of an aqueous Yeast protein solution

Adding distilled water into active dry yeast to prepare a yeast protein solution with a plurality of groups of certain solid content, wherein the concentration can be selected from 160-210 g/L, and carrying out ultrasonic crushing on the solution. The ultrasonic power is 300W, 350W, 400W or 450W, the ultrasonic time is 20min, and the time interval is 2s/2 s.

Taking soluble protein in the crushed supernatant as an index, increasing the crushing degree of the yeast and then tending to be gentle along with the enhancement of the ultrasonic power, and when the power reaches 400W, the change degree of the protein yield tends to be stable, so that the ultrasonic power of 400W is selected for ultrasonic crushing in consideration of the energy consumption problem.

Then selecting the ultrasonic power as 400W, preparing a plurality of groups of yeast protein solutions according to the method, and respectively carrying out ultrasonic treatment for 10min, 15min, 20min and 25min under the condition of 400W.

By taking soluble protein in the crushed supernatant as an index, the crushing degree of the yeast tends to be gentle after being increased along with the increase of the ultrasonic time, the protein yield is not changed greatly after the time reaches 20min, and the yeast protein solution can be crushed under the optimal conditions that the ultrasonic power is 400W and the ultrasonic time is 20min in consideration of energy consumption.

EXAMPLE 2 preparation of Yeast extract

Preparing yeast protein solution according to the same procedure as in example 1, subjecting the yeast protein solution to ultrasonic treatment at 400W for 20min, performing enzymolysis with neutral protease, pepsin, alkaline protease and trypsin, respectively, setting at suitable temperature and pH, reacting for 10h, and adding 500U g^-1。

The enzymolysis conditions are as follows:

neutral protease: at 40 to 50 ℃, pH 6.0 to 7.0.

Pepsin: at 35-45 ℃ and pH 1.5-2.5.

Alkaline protease: at 45-55 ℃, pH is 7.0-9.0.

Trypsin: at 35-45 deg.C and pH 7.5-8.5.

As shown in FIG. 2, it is clear from FIG. 2 that the degree of hydrolysis by alkaline protease is highest in yeast protein, and that the hydrolysis effect by pepsin is lowest in neutral protease. Meanwhile, the protein recovery rate is positively correlated with the hydrolysis degree. And then comparing the absorbance ratios of the four enzymes hydrolyzed and adsorbed by activated carbon at the wavelength of 220/280nm to find that the removal rates of the aromatic amino acids of the products after enzymatic hydrolysis of the enzymes are not greatly different. In conclusion, the alkaline protease selected by the invention is the best active enzyme for extracting the dry yeast.

EXAMPLE 3 nanofiltration concentration of Yeast extract

On the basis of example 2 (alkaline protease is used as the enzyme for extracting active dry yeast), yeast extract is obtained, then enzyme is inactivated in boiling water bath for 15min, and supernatant is obtained after centrifugation for 15min at 13000 r/min. And then, carrying out nanofiltration concentration on the supernatant, wherein the nanofiltration condition is to adopt a PES roll-type membrane with the molecular weight cutoff of 500Da, the operating pressure is 2-4 MPa, the initial pH of the yeast extract is 6.5-7.5, and the concentration times (the ratio of the volume of the cut-off liquid to the volume of the stock solution is 1/1.5-1/3) are 1.0, 1.5, 2.0, 2.5 and 3.0 times (the concentration time of 3.0 refers to the volume ratio of the cut-off liquid to the stock solution is 1/3, and other concentration times are similar to the meanings in the above step).

As shown in FIG. 3, the salt ion removal rate and the free amino acid removal rate increased with the increase of the concentration factor, but the excessive concentration factor caused a part of the polypeptide to be lost, resulting in a decrease in the protein recovery rate, so that the concentration factor of 2.5 was the optimal nanofiltration concentration factor.

Example 4 two-step Directional enzymatic hydrolysis

On the basis of example 3 (concentration multiple of 2.5 times), α -chymotrypsin is added into the nanofiltration yeast extract with the dosage of 2000-8000 U.g^-1(5000U·g^-1、5500U·g^-1、6000U·g^-1、6500U·g^-1、7000U·g^-1And the enzymolysis time is 1-8 h (1h, 2h, 4h, 6h and 8h), the enzymolysis pH is 6.5-9.0 (pH is 7.0, 7.5, 8.0, 8.5 and 9.0), and the enzymolysis temperature is 34-46 ℃ (34 ℃, 37 ℃, 40 ℃, 43 ℃ and 46 ℃). Adding carboxypeptidase A with the dosage of 2 U.mL^-1The enzymolysis time is 4h, the enzymolysis pH is 7.5, the enzymolysis temperature is 40 ℃, and the enzymolysis effect is inspected according to the index of the free rate of aromatic amino acid.

The results are shown in FIG. 4, FIGS. 4a-d are the results of testing the liberation rate of aromatic amino acids of α -chymotrypsin under the conditions of different pH, different temperature, different enzyme adding amount and different enzymolysis time, in addition, the orthogonal experiment obtains more ideal action conditions, and the action strength of single factor on the enzymolysis experiment is analyzed, the results are shown in Table 1, the results show that the optimum action condition of α -chymotrypsin is the addition amount of 5500 U.g^-1The enzymolysis time is 4h, the enzymolysis pH is 8, the enzymolysis temperature is 40 ℃,and the order of the strong and weak influence of the single factor on the enzymolysis experiment is as follows: time of day>pH>Temperature of>The amount of enzyme added.

TABLE 1 α chymotrypsin enzymatic hydrolysis orthogonal experiments and results

Example 5 two-step Directional enzymolysis

Based on example 4 (α -chymotrypsin addition amount is 5500U g)^-1The enzymolysis time is 4h, the enzymolysis pH is 8, the enzymolysis temperature is 40 ℃), the α -chymotrypsin is inactivated in boiling water bath for 15min after the enzymolysis is finished, the cooled solution is centrifuged at 13000r/min for 15min, and then carboxypeptidase A is added into the enzymolysis solution with the dosage of 2-6 U.mL^-1The enzymolysis time is 2-6 h, the enzymolysis pH is 7.0-8.0, the enzymolysis temperature is 35-45 ℃, and the enzymolysis effect is inspected according to the index of the free rate of aromatic amino acid.

As shown in Table 2, the optimum conditions for carboxypeptidase A were 4 U.mL^-1The enzymolysis time is 4h, the enzymolysis pH is 7.5, the enzymolysis temperature is 40 ℃, and the order of the strong and weak influence of single factors on the enzymolysis experiment is as follows: enzyme addition amount>Temperature of>pH>Time.

TABLE 2 carboxypeptidase A enzyme hydrolysis orthogonal experiment and results

Example 6 activated carbon adsorption

The amount of α -chymotrypsin added was 5500U g based on example 5^-1The enzymolysis time is 4h, the enzymolysis pH is 8, and the enzymolysis temperature is 40 ℃; carboxypeptidase A was added in an amount of 4 U.mL^-1The enzymolysis time is 4h, the enzymolysis pH is 7.5, and the enzymolysis temperature is 40 ℃. Boiling the product after the two steps of enzymolysis for 15min to inactivate enzyme, cooling, centrifuging at 13000r/min for 15min, adding activated carbon treated by dilute hydrochloric acid, and passing through a reactor at 1 mol.L^-1NaOH and 1 mol. L^-1Adjusting pH of the product after enzymolysis to 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 with HCl, and adding the powder at a ratio of 1:10Adsorbing with activated carbon powder on a 35 deg.C shaker for 2 hr, centrifuging at 4 deg.C and rotation speed of 10000rpm/min for 15 min. The absorbance of the supernatant after adsorption was measured at 220nm and 280nm, and the adsorption effect of activated carbon was compared by absorbance 220/280.

As shown in FIG. 5b, the absorbance 220/280 was the largest at pH2.5, and when the pH was lower, the isoelectric point of the amino acid was exceeded, the amino acid was likely to precipitate, the intermolecular force was decreased, and the adsorption by activated carbon was not likely to occur. When the pH is high, the group modification on the surface of the activated carbon is insufficient, and aromatic amino acids cannot be effectively bound. Therefore, pH2.5 is the optimum pH for adsorption on activated carbon.

The results of activated carbon adsorption treatment under the above-mentioned optimum pH adsorption conditions were shown in FIGS. 5a, 5c and 5d, with different adsorption times (1h, 3h, 4h and 5h), different feed-to-liquid ratios (1:5, 1:15, 1:20 and 1:25) and different temperatures (25 ℃, 30 ℃, 40 ℃ and 45 ℃). As can be seen from the figure, the value of 220/280 gradually increases with the increase of the adsorption time and becomes stable after 2h, the value of 220/280 of activated carbon gradually decreases with the increase of the feed-liquid ratio, and the value of 220/280 is higher between 30 ℃ and 40 ℃, and the temperature in the range is the optimal adsorption temperature. Example 7 polypeptide distribution assay and amino acid composition analysis

And (4) carrying out operation according to the same steps as 6, and carrying out freeze drying on the enzymatic hydrolysate adsorbed by the activated carbon to obtain oligopeptide freeze-dried powder.

The amino acid composition analysis of the lyophilized powder after dearomatization by activated carbon adsorption was performed by HPLC, and the same test was performed on the initial yeast extract, with the results shown in fig. 6. Calculating according to a formula of the F value to obtain 35.47F value of the freeze-dried powder, wherein the F value is increased by 10.4 times compared with the initial F value of the raw materials; in the mixture of oligopeptide and free amino acid, the content of free amino acid is 14.67%; in the free amino acids, the content of aromatic amino acids was 3.6%, and the content of branched chain amino acids was 55.1%. These data illustrate that: the F value of the freeze-dried powder after dearomatization treatment meets the requirement of high F value oligopeptide, and free aromatic amino acid is basically adsorbed and removed, so that the expected experiment purpose is achieved.

The molecular weight distribution of the polypeptide was analyzed by HPLC on the lyophilized powder after dearomatization by activated carbon adsorption, and the same test was performed on the initial yeast extract and the product without carboxypeptidase A for enzymatic hydrolysis, the results are shown in FIG. 7. FIG. 7 shows that the molecular weight of the lyophilized powder is mainly concentrated below 1500Da, accounting for 99.82% of the total content. Wherein the oligopeptide with the molecular weight of 180-1500 Da accounts for 66.42% of the total content, the oligopeptide with the molecular weight range mainly comprises 3-6 amino acid residues, and the molecular weight requirement of the high-F-value oligopeptide is met; the molecular weight is less than 180Da and accounts for 32.86%, and the content analysis of the combined amino acid shows that: this molecular weight range may be a dipeptide or free amino acid, with the dipeptide being the major portion, about 24%, and the remaining 8% of free amino acids having a very low aromatic amino acid content, with branched chain amino acids and other amino acids as the major components.

According to the invention, α -chymotrypsin and carboxypeptidase A directional hydrolysis yeast extract are selected and purified by one step (activated carbon adsorption), the F value is increased by 10.4 times, the F value is 3.41 to 35.47 of the raw material, the requirements of the F value and the molecular weight of the high F value oligopeptide are met, multiple verification experiments have stable repeatability, and the expected effect is achieved.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. The high-F-value oligopeptide is characterized by having an F value of 30-40, wherein the high-F-value oligopeptide comprises 66.42% of oligopeptides with a molecular weight of 180-1500 Da and 32.86% of oligopeptides with a molecular weight of less than 180Da, and the preparation method of the high-F-value oligopeptide comprises the following steps:

(1) adding alkaline protease into a yeast protein aqueous solution, wherein the concentration of the yeast protein aqueous solution is 160-250 g/L, and carrying out enzymolysis at 45-55 ℃ under the condition that the pH value is 7.0-9.0 to obtain a yeast extract, wherein the addition amount of the alkaline protease is 400-600U/g of yeast protein;

(2) concentrating the yeast extract to remove molecules with molecular weight of less than 500Da in the yeast extract to obtain a concentrated solution; the pH value of the yeast extract before concentration is controlled to be 6.5-7.5, and the ratio of the volume of the liquid after concentration to the volume of the liquid before concentration is 1: 1.5-3;

(3) adding α -chymotrypsin into the concentrated solution, wherein the adding amount of the α -chymotrypsin is 4000-6000U/g yeast protein, carrying out enzymolysis for 2-8 h at 35-45 ℃ under the condition that the pH value is 6.5-8.5, adding carboxypeptidase A into an enzymolysis product after enzyme deactivation, wherein the adding amount of the carboxypeptidase A is 2-6U/mL yeast protein, carrying out enzymolysis for 4-8 h at 35-45 ℃ under the condition that the pH value is 6.5-7.5, adsorbing and removing impurities by using activated carbon after enzyme deactivation, wherein the solid-liquid ratio of the activated carbon to enzymolysis liquid is 1: 10-20, the adsorption temperature is 30-40 ℃, and adsorbing is carried out under the condition that the pH value is 2.0-3.0, so as to obtain the high-F-value oligopeptide.

2. The high F value oligopeptide according to claim 1, wherein: in the step (2), nanofiltration is adopted for concentration so as to remove molecules with the molecular weight of less than 500Da in the yeast extract.

3. The high F value oligopeptide according to claim 1, wherein: before the step (2), the method also comprises the steps of inactivating enzyme, centrifuging and taking supernatant.