WO2020014997A1

WO2020014997A1 - High f-ratio oligopeptide and preparation method therefor

Info

Publication number: WO2020014997A1
Application number: PCT/CN2018/096955
Authority: WO
Inventors: 田亚平; 吴警涛; 李婷婷; 周楠迪
Original assignee: 江南大学
Priority date: 2018-07-20
Filing date: 2018-07-25
Publication date: 2020-01-23
Also published as: CN108893515A; CN108893515B

Abstract

Provided is a preparation method for a high-F ratio oligopeptide, comprising the following steps: adding an alkaline protease to a yeast protein solution, and performing enzymolysis under conditions of pH 7.0-9.0 at 45 to 55 °C to obtain a yeast extract; concentrating the yeast extract to remove molecules having a molecular weight of 500 Da or less to obtain a concentrate; adding alpha-chymotrypsin to the concentrate, and performing enzymolysis under the conditions of pH 6.5-8.5 at 35 °C to 45 °C for 2 to 8 h; and inactivating enzymes, adding carboxypeptidase A to the enzymatic hydrolysate, performing enzymolysis under conditions of pH 6.5-7.5 at 35 °C to 45 °C for 4 to 8h, inactivating said enzyme, and using activated carbon to adsorb and remove impurities, so as to obtain the high F-ratio oligopeptide.

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 technique

High F value oligopeptides are a mixture of small peptides composed of 2 to 9 amino acid residues. F value refers to the branched chain amino acids (leucine, isoleucine and valine, BCAA for short) and aromatics in the mixture. The molar ratio of family amino acids (phenylalanine, tyrosine, AAA for short) is named in honor of the "pseudo-neurotransmitter hypothesis" proposed by the famous German scholar Fischer in the 1970s. The high F value oligopeptide should have an F value greater than 20. An oligopeptide is a protein precursor composed of a combination of 2 to 9 amino acids, or a protein degradation product composed of 2 to 9 amino acids that is degraded by a protein. It can also be referred to as a small peptide or short peptide. In actual production, high-F-number oligopeptides can be widely used in the treatment of liver diseases, liver protection foods, protein nutrition foods for surgical patients, intestinal nutrients for patients with digestive enzyme deficiency, and food nutrition fortified laborers. Agents and other aspects. The high-F oligopeptide mixture has more practical effects than the branched-chain amino acid formula, and its unique physiological function has been highly concerned by people and has good development prospects.

Yeast is the microbial industrial product that contributes the most to humans. Since the beginning of the history of human brewing, vinegar, and sauce making, we have dealt with yeast, even dating back to the Babylonian era thousands of years ago and before the Zhou Dynasty in China. The protein content of yeast is between 45% and 55%. Compared with ordinary yeast, the active dry yeast has a water content of 4% to 6%, and the particles are small and the fermentation speed is fast. Yeast protein is rich in amino acids, with branched chain amino acids accounting for 18.96% and aromatic amino acids accounting for 7.45%. Its initial F value is 3.41, which exceeds common animal and plant protein powders such as soybean protein, rice protein, etc., and is a very suitable method for producing high F. Raw material for oligopeptides.

At present, the high-F-value products sold at home and abroad are made of amino acid compounds, and the product types are relatively single. No high-F-value oligopeptide products have yet been sold. And modern nutrition research shows that compared with free amino acids, oligopeptides can not only be used as nutrients, but also have the advantages of low osmotic pressure and low energy consumption, and are more easily absorbed and utilized by the body.

High F-number oligopeptides are complicated due to their strict requirements for preparation. At present, according to research status at home and abroad, zein, soy protein, whey protein, casein, etc. are mainly used as raw materials, and the hydrolytic enzymes used are mostly actin, streptomycin, pepsin, and papain, so new preparations have been opened up. The approach is extremely important. In the purification process, the key point to increase the F value is the removal of aromatic amino acids. For the free aromatic amino acids released in the enzymolysis process, it is necessary to effectively remove the aromatic amino acids for dearomatization and high F value. At present, the methods of dearomatization mainly include ion exchange method, membrane separation method, gel chromatography method, affinity chromatography method, and activated carbon chromatography method. According to research reports at home and abroad, Sephadex G-15 or Bio-Gel P-2 gel chromatography is generally used for separation and purification, but because of the small amount of sample and difficult to scale up the process, it has brought difficulties to large-scale production.

Active dry yeast transportation is easy to save and has rich protein content, so exploring the richer application potential of yeast cells, and developing a yeast high-F-number oligopeptide with simple method, low cost, and easy absorption has certain market value.

Summary of the invention

In order to solve the above technical problems, the object of the present invention is to provide a high F value oligopeptide and a preparation method thereof. The method of the present invention is simple, low cost, sufficient source of raw materials, controllable enzymolysis process, and is prepared to be pure natural and easy to absorb. High F-value bioactive oligopeptides.

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

(1) Adding alkaline protease to the yeast protein aqueous solution, and performing enzymolysis at 45-55 ° C under the condition of pH 7.0-9.0 to obtain a yeast extract, wherein the amount of the alkaline protease to be added is 400 ~ 600U / g yeast protein;

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

(3) adding α-chymotrypsin to the concentrated solution, and enzymolyzing at 35 ° C to 45 ° C for 2 to 8 hours under the conditions of pH 6.5 to 8.5; inactivating the enzyme, centrifuging and removing the supernatant to the enzyme Carboxypeptidase A is added to the hydrolysis product, and the enzyme is hydrolyzed at 35 ° C to 45 ° C for 4 to 8 hours at a pH of 6.5 to 7.5. After the enzyme is inactivated and centrifuged, the supernatant is removed by adsorption with activated carbon to obtain the high F value oligopeptide.

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

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

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

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

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

Further, the nanofiltration method is performed at 2 to 4 MPa.

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

Further, before step (2), the method further comprises the steps of inactivating the enzyme and taking a supernatant after centrifugation. Enzyme inactivation conditions were 15min in a boiling water bath. The centrifugation speed is preferably 13000 r / min, and the centrifugation time is preferably 15 min.

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

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

Further, in step (3), the solid-liquid ratio of the activated carbon to the enzymatic hydrolysis liquid is 1: 10-20. Adsorption through activated carbon to remove most of the aromatic amino acids and retain branched chain amino acids.

Further, in step (3), the adsorption temperature is 30 to 40 ° C., and the adsorption is performed at a pH of 2.0 to 3.0.

Further, in step (3), the adsorption time is 2 to 5 hours.

Further, in step (3), the activated carbon is washed with dilute hydrochloric acid to remove ash before use to change the properties of the surface groups of the activated carbon.

Further, in step (3), the hydrolyzed solution after adsorption is freeze-dried to obtain a high-F-number oligopeptide powder.

Active dry yeast is a kind of living cell. The simple extraction with α-chymotrypsin and carboxypeptidase A is relatively inefficient and time-consuming. Therefore, the yeast protein was first extracted with non-specific alkaline protease to make it Peptide decomposed into small fragments, then oriented by α-chymotrypsin (the main amino acid cleavage site of the aromatic amino acid carboxyl terminus) and carboxypeptidase A (the first amino acid residue of the carboxyl terminus is aromatic amino acid) Enzymolysis can release most of the aromatic amino acids. Thereby, a high-F-value mixed oligopeptide solution can be obtained through activated carbon adsorption.

In another aspect, the present invention also claims a high F-number oligopeptide prepared by using the above method, the F-number of which is 30-40.

Further, the high-F-number oligopeptide derived from yeast protein of the present invention is made by mixing peptide molecules, and its molecular weight is mainly concentrated below 1500 Da, accounting for 99.82% of the total content. Among them, oligopeptides with a molecular weight of 180 to 1500 Da account for 66.42% of the total content. The oligopeptides in this molecular weight range are mainly composed of 3 to 6 amino acid residues, which meet the molecular weight requirements of high F-number oligopeptides; 32.86 are less than 180 Da %, Molecules in this molecular weight range include dipeptides and / or free amino acids.

Further, the high F value oligopeptide derived from yeast protein of the present invention, in the mixture of oligopeptide and free amino acid, the free amino acid content accounts for 14.67%; among the free amino acids, the aromatic amino acid content accounts for 3.6%, and the branched chain amino acid The content accounts for 55.1%. There are not only many types of amino acids in the yeast proteolytic solution, but also essential amino acids account for 35.48% of the total amino acids and non-essential amino acids account for 64.52%. Among the many amino acids, the branched chain amino acids valine (Val) and isoleucine (Ile ) And leucine (Leu) total content (mass fraction) is 21.73%, while the aromatic amino acids tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr) total content (mass fraction) It is 0.75%.

With the above solution, the present invention has at least the following advantages:

The present invention uses active dry yeast as a protein raw material, takes advantage of its high protein content, high initial F value, wide source and low price, and is environmentally friendly. Using enzyme engineering technology and modern separation and purification technology, the raw material source is sufficient and the enzymatic hydrolysis process can be controlled. After one-step separation, a pure natural, easily absorbed, high-F-value biologically active oligopeptide mixture can be produced, which provides a basis for the rational use and development of yeast protein resources and an important application approach for further improving its added value.

The invention selects a specific protease to replace a protease with a wide range of cleavage sites for targeted hydrolysis, improves the F value and practical value of the raw material, and provides a specific endo- and exo-protease cooperative targeted hydrolysis and one-step purification to significantly increase the F value of yeast extract The method provides a new idea for solving the problem of lack of high-F-number oligopeptide products.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and can be implemented according to the contents of the description, the following describes in detail the preferred embodiments of the present invention and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a process for the enzymatic hydrolysis of yeast protein by multiple enzymes according to the present invention; FIG.

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

Figure 3 shows the test results of the effects of different concentration multiples on the total free amino acid removal rate and protein recovery rate under nanofiltration conditions;

Fig. 4 shows the aromatic amino acid Phe of α-chymotrypsin-digested yeast extract under different conditions of temperature, time, pH, and amount of enzyme. Amino acid) free test results;

Figure 5 is the test result of the ratio of absorbance 220/280 after the activated carbon adsorbs the enzymolysis solution under different time, pH, material-liquid ratio, and temperature conditions;

FIG. 6 is a test result of the free amino acid content of the yeast extract, α-chymotrypsin, and carboxypeptidase A after the reaction and the total amino acid content of the hydrolyzed oligopeptide after activated carbon adsorption;

FIG. 7 is a molecular weight distribution diagram of the oligopeptide lyophilized powder obtained after the yeast extract and the dual-enzyme directed enzymatic hydrolysis.

detailed description

The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following examples are used to illustrate the present invention, but not to limit the scope of the present invention.

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

Enzyme activity measurement: refer to GB / T23527-2009.

Degree of hydrolysis (DH): ratio of free amino nitrogen content to total protein nitrogen content. Among them, the free amino nitrogen content was measured by formaldehyde titration method, and the total protein nitrogen content was measured by Kjeldahl method.

Formaldehyde titration method: refer to GB 5009.235-2016.

Peptide distribution determination: The solution was centrifuged at 12000 r / min for 10 minutes, the supernatant was taken, and the molecular weight distribution of the peptide was determined by HPLC.

Determination of amino acid content:

(1) Free amino acid: take the supernatant after centrifugation, add an equal volume of 10% TCA (trichloroacetic acid) solution, leave it for 3h, centrifuge at 15000rpm for 30min, and then take 2mL of the supernatant for 0.22μm The organic phase membrane was filtered again, and then 400 μL of the supernatant was taken in a liquid sample bottle, and the free amino acid content was determined by HPLC method.

(2) Hydrolysis of amino acid: Take a hydrolysis tube, measure 1mL of sample (liquid), add 1mL of concentrated hydrochloric acid, then add 6mL of 6mol / L HCl, and then fill with nitrogen for 3min. After that, tighten the hydrolysis tube and put it in an oven at 120 ℃ for hydrolysis 22h. After 22h, transfer all the samples in the hydrolysis tube to a volumetric flask, add 4.8mL of 10mol / L NaOH for neutralization, then make up to 25mL with distilled water, mix in a shaker and filter through double-layer filter paper, take 1mL The filtrate was centrifuged in a 1.5 mL centrifuge tube at 15000 rpm for 30 minutes, and then 400 μL of the supernatant was taken in a liquid sample bottle, and the total amino acid content in the solution was determined by HPLC. The solid test method is the same as that of the liquid, except that the solid Take about 100.00mg and add 8mL of 6mol / L HCl.

F value: The molar ratio of branched chain amino acids to aromatic amino acids. Calculated as follows:

n represents the number of moles.

Example 1 Preparation of aqueous yeast protein solution

Distilled water is added to the active dry yeast to prepare a yeast protein solution with multiple groups of a certain solid content. The concentration can be selected from 160 to 210 g / L, and the solution is sonicated. The ultrasonic power is 300W, 350W, 400W or 450W, the ultrasonic time is 20min, and the time interval is 2s / 2s.

Taking the soluble protein in the supernatant after the disruption as an indicator, with the increase of the ultrasonic power, the degree of yeast disruption first increased and then flattened. When the power reached 400W, the degree of protein yield change became stable, so the issue of energy consumption was considered. , Select the ultrasonic power of 400W for ultrasonic crushing.

Then select the ultrasonic power of 400W, configure multiple groups of yeast protein solution according to the above method, and sonicate at 400W for 10min, 15min, 20min and 25min, respectively.

Taking the soluble protein in the supernatant after crushing as an indicator, with the increase of ultrasound time, the degree of yeast crushing first increased and then became gentle. When the time reached 20 minutes, the protein yield began to change little. Considering the energy consumption, The ultrasonic power was 400 W and the ultrasonic time was 20 min.

Example 2 Preparation of Yeast Extraction Solution

Follow the same steps as in Example 1 to configure the yeast protein solution, sonicate the yeast protein solution at 400 W for 20 min, and then use neutral protease, pepsin, alkaline protease, and trypsin to digest them. pH, reaction time was 10h, and the amount of enzyme added was 500U · g ^-1 .

The enzymatic conditions are as follows:

Neutral protease: 40-50 ° C, pH = 6.0-7.0.

Pepsin: 35-45 ° C, pH = 1.5-2.5.

Alkaline protease: 45-55 ° C, pH = 7.0-9.0.

Trypsin: 35-45 ° C, pH = 7.5-8.5.

The results are shown in FIG. 2. As can be seen from FIG. 2, the degree of hydrolysis of the yeast protein by alkaline protease was the highest, followed by the neutral protease, and the hydrolysis effect of pepsin was the lowest. At the same time, protein recovery was positively correlated with the degree of hydrolysis. After comparing the absorbance ratios at the 220 / 280nm wavelengths after the four enzymes were hydrolyzed and adsorbed by activated carbon, it was found that the removal rates of aromatic amino acids of the products after enzymatic hydrolysis were not much different. In summary, the alkaline protease used in the present invention is the best active enzyme for extracting 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), a yeast extract is obtained, and then the enzyme is inactivated in a boiling water bath for 15 minutes, and the supernatant is taken after centrifugation at 13000 r / min for 15 minutes. Then take the supernatant for nanofiltration and concentration operation. The nanofiltration conditions are to use a PES roll membrane with a cut-off molecular weight of 500 Da, the operating pressure is 2 to 4 MPa, the initial pH of the yeast extract is 6.5 to 7.5, and the concentration factor (the volume of the interception liquid The ratio to the volume of the original solution 1 / 1.5 ～ 1/3) is 1.0, 1.5, 2.0, 2.5, 3.0 times (concentration 3.0 times means that the volume ratio of the retentate to the original solution is 1/3, and other concentration times are similar to the meaning here ).

The results are shown in Figure 3. With the increase of the concentration factor, the salt ion removal rate and free amino acid removal rate increased, but the excessive concentration factor caused some peptides to be lost, resulting in a decrease in protein recovery rate. Therefore, the concentration factor was 2.5 times. For best nanofiltration concentration.

Example 4 Two-step directed enzymolysis

Based on Example 3 (concentration factor is 2.5 times), α-chymotrypsin was added to the yeast extract after nanofiltration, and the amount was 2000-8000U · g ^-1 (5000U · g ^-1 , 5500U · G ^-1 , 6000U · g ^-1 , 6500U · g ^-1 , 7000U · g ^-1 ,), enzymolysis time is 1 ～ 8h (1h, 2h, 4h, 6h, 8h), enzymolysis pH is 6.5 ～ 9.0 (pH = 7.0, 7.5, 8.0, 8.5, 9.0), and the enzymatic hydrolysis temperature was 34-46 ° C (34 ° C, 37 ° C, 40 ° C, 43 ° C, 46 ° C). Then, carboxypeptidase A was added in an amount of 2U · mL ^-1 , the enzymolysis time was 4h, the enzymolysis pH was 7.5, the enzymolysis temperature was 40 ° C., and the effect of enzymatic hydrolysis was examined by the index of aromatic amino acid free ratio.

The results are shown in Fig. 4. Figs. 4a-d are the test results of the free rate of aromatic amino acids under different pH, different temperature, different amount of enzyme, and different hydrolysis time. The cross test obtained more ideal conditions, and analyzed the effect of the single factor on the enzymolysis experiment. The results are shown in Table 1. The results showed that the optimal conditions for α-chymotrypsin were the addition of 5500 U · g ^-1 , the enzymolysis time was 4 hours, the enzymolysis pH was 8 and the enzymolysis temperature was 40 ° C., and the single factor had a strong effect on the enzymolysis experiment. The order of weak effects is: time>pH>temperature> enzyme addition.

Table 1 Orthogonal experiment and results of α-chymotrypsin hydrolysis

Example 5 Two-step directional enzymolysis

Based on Example 4 (α-chymotrypsin addition amount 5500U · g ^-1 , enzymolysis time 4h, enzymolysis pH 8 and enzymolysis temperature 40 ° C), the action of α-chymotrypsin was completed The enzyme was inactivated by a boiling water bath for 15 minutes, and then cooled at 13,000 r / min for 15 minutes after cooling. Then add carboxypeptidase A to the enzymatic hydrolysis solution in an amount of 2 to 6 U · mL ^-1 , the enzymatic hydrolysis time is 2 to 6 hours, the enzymatic hydrolysis pH is 7.0 to 8.0, the enzymatic hydrolysis temperature is 35 to 45 ° C., and the aromatic Amino acid free rate index to investigate the effect of enzymatic hydrolysis.

The results are shown in Table 2. The optimal conditions for carboxypeptidase A are the addition amount of 4U · mL ^-1 , the enzymolysis time is 4h, the enzymolysis pH is 7.5, and the enzymolysis temperature is 40 ° C. The order of the effect of the strength of the effect is: the amount of enzyme added>temperature>pH> time.

Table 2 Orthogonal experiments and results of carboxypeptidase A digestion

Example 6 Activated carbon adsorption

Based on Example 5, α-chymotrypsin was added in an amount of 5500U · g ^-1 , enzymolysis time was 4h, enzymolysis pH was 8 and enzymolysis temperature was 40 ° C; carboxypeptidase A was added in 4U · mL ^-1 , enzymolysis time is 4h, enzymolysis pH is 7.5, and enzymolysis temperature is 40 ° C. The product after two-step enzymolysis was boiled to destroy the enzyme for 15min. After cooling, it was centrifuged at 13000r / min for 15min. The activated carbon treated with dilute hydrochloric acid was added. The product was digested with 1mol·L ^-1 NaOH and 1mol·L ^-1 HCL. The pH was adjusted to 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and powdered activated carbon was added according to the material-liquid ratio of 1:10, followed by adsorption on a 35 ° C shaker for 2h. After the adsorption was completed, it was placed at 4 ° C and rotated at 10,000 rpm / min to centrifuge. 15min. The absorbance of the supernatant after the adsorption was measured at 220 nm and 280 nm, and the adsorption effect of the activated carbon was compared by the absorbance of 220/280.

The results are shown in Figure 5b. At pH 2.5, the value of the absorbance 220/280 is the largest. When the pH is lower, the isoelectric point of the amino acid is exceeded, the amino acid is liable to settle, the intermolecular force is weakened, and it is not easy to be adsorbed by activated carbon. When the pH is high, the groups on the surface of the activated carbon are insufficiently modified to effectively bind aromatic amino acids. So pH 2.5 is the optimal pH for activated carbon adsorption.

Under the above optimal pH adsorption conditions, adjust different adsorption times (1h, 3h, 4h, 5h), different material-liquid ratios (1: 5, 1:15, 1:20, 1:25), and different temperatures (25 ° C, 30 ° C, 40 ° C, 45 ° C), activated carbon adsorption treatments were performed under the above conditions, and the results are shown in Figs. 5a, 5c, and 5d. It can be seen from the figure that with the increase of the adsorption time, the value of 220/280 gradually increases, and then stabilizes after 2 h. With the increase of the material-liquid ratio, the value of 220/280 gradually decreases, at 30 Between -40 ℃, the value of 220/280 is high, and the temperature in this range is the optimal adsorption temperature. Example 7 Determination of Peptide Distribution and Analysis of Amino Acid Composition

The operation was performed in the same manner as in step 6. The enzymolysis solution after activated carbon adsorption was freeze-dried to obtain an oligopeptide lyophilized powder.

Amino acid composition analysis was performed on the lyophilized powder after desorption of activated carbon by HPLC, and the same test was performed on the initial yeast extract. The results are shown in FIG. 6. According to the formula of the F value, the F value of the lyophilized powder is 35.47. This F value is 10.4 times higher than the starting F value of the raw material. In the mixture of oligopeptides and free amino acids, the free amino acid content accounts for 14.67%; among the free amino acids, the aromatic amino acid content is 3.6%, and the branched chain amino acid content is 55.1%. These data show that the F value of the lyophilized powder after dearomatization treatment meets the requirements of high F value oligopeptides, and the free aromatic amino acids are basically removed by adsorption, which achieves the intended experimental purpose.

The HPLC method was used to analyze the molecular weight distribution of the lyophilized powder after adsorption and dearomatization by activated carbon. At the same time, the same test was performed on the initial yeast extract and the product hydrolyzed without adding carboxypeptidase A. The results are shown in FIG. 7. Figure 7 shows that the molecular weight of the lyophilized powder is mainly concentrated below 1500 Da, accounting for 99.82% of the total content. Among them, oligopeptides with a molecular weight of 180 to 1500 Da account for 66.42% of the total content. The oligopeptides in this molecular weight range are mainly composed of 3 to 6 amino acid residues, which meet the molecular weight requirements of high F-number oligopeptides; 32.86 are less than 180 Da %, Combined with the analysis of amino acid content, it can be known that the molecular weight range may be dipeptide or free amino acid, and the dipeptide is the main part, accounting for about 24%. Among the remaining 8% of free amino acid, the aromatic amino acid content is very small, and the branched chain amino acid is And other amino acids are the main ingredients.

In the present invention, α-chymotrypsin and carboxypeptidase A are used to selectively hydrolyze yeast extract and one-step purification (activated carbon adsorption), the F value is increased by 10.4 times, and the F value is from 3.41 to 35.47 of the raw material, which meets the high F value oligopeptide F value and molecular weight requirements, and repeated verification experiments have stable repeatability and achieve the expected results: while selecting new raw materials for preparing high F value oligopeptides, it also provides an efficient and stable preparation of high F value The oligopeptide method helps to solve the current problem of extreme shortage of high F-number oligopeptide products.

The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the technical principles of the present invention. And modifications, these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

A method for preparing a high F-number oligopeptide, which comprises the following steps:

(1) Adding alkaline protease to the yeast protein aqueous solution, and performing enzymolysis at 45-55 ° C under the condition of pH 7.0-9.0 to obtain a yeast extract, wherein the amount of the alkaline protease to be added is 400 ~ 600U / g yeast protein;

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

(3) adding α-chymotrypsin to the concentrated solution, and enzymolyzing at 35 ° C to 45 ° C for 2 to 8 hours under the conditions of pH 6.5 to 8.5; adding carboxypeptide to the hydrolysis product after the enzyme is eliminated Enzyme A is hydrolyzed at 35 ° C to 45 ° C for 4 to 8 hours under the conditions of pH 6.5 to 7.5, and the impurities are removed by adsorption with activated carbon to obtain the high F value oligopeptide.
The method according to claim 1, wherein in step (1), the concentration of the yeast protein solution is 160-250 g / L.
The method according to claim 1, wherein in step (2), nanofiltration is used to concentrate to remove molecules with a molecular weight of 500 Da or less in the yeast extract.
The method according to claim 1, characterized in that in step (2), the pH value of the yeast extract before concentration is controlled to 6.5 to 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 to 3.
The method according to claim 1, characterized in that before step (2), the method further comprises the steps of inactivating the enzyme and taking the supernatant after centrifugation.
The method according to claim 1, characterized in that in step (3), the added amount of α-chymotrypsin is 4000-6000 U / g yeast protein.
The method according to claim 1, characterized in that: in step (3), the amount of carboxypeptidase A added is 2 to 6 U / mL yeast protein.
The method according to claim 1, wherein in step (3), the solid-liquid ratio of the activated carbon to the enzymatic hydrolysis liquid is 1: 10-20.
The method according to claim 1, characterized in that in step (3), the adsorption temperature is 30 to 40 ° C, and the adsorption is performed at a pH of 2.0 to 3.0.
A high F value oligopeptide prepared by the method according to any one of claims 1 to 9, wherein the F value is 30 to 40.