1. A method for preparing high F value oligopeptide by microbial fermentation is characterized in that a fermentation product of bacillus natto and flavourzyme are used for carrying out enzymolysis on a raw material containing corn gluten meal, and the product obtained after the enzymolysis is fermented by aspergillus oryzae to produce the high F value oligopeptide.

2. The method of claim 1, wherein the Aspergillus oryzae is Aspergillus oryzae CICC 40214.

3. The method according to claim 1 or 2, wherein the fermentation product of the bacillus natto is a nattokinase obtained by the bacillus natto under the condition that the temperature is 35-39 ℃ and the pH is 8.5-9.5.

4. The method according to claim 1 or 3, wherein the corn gluten is obtained by enzymatic hydrolysis of corn gluten with alpha-amylase, the corn gluten is then enzymatically hydrolyzed with the obtained nattokinase, and the product obtained by the enzymatic hydrolysis is used as the fermentation medium of Aspergillus oryzae.

5. The method according to claim 4, wherein the nattokinase comprises alkaline protease, and is added in an amount of 3500-4500U per gram of zein; and (3) carrying out enzymolysis for 4 hours at the temperature of 45-55 ℃ and under the condition of pH 9.0, adding 1.0-2.0% of sucrose and 0.1-0.2% of sodium nitrate after the enzymolysis is finished, and inactivating the mixture to obtain the fermentation culture medium of aspergillus oryzae.

6. The method as claimed in claim 5, wherein Aspergillus oryzae is inoculated to the fermentation medium in an amount of 5-7%, and the mixture is fermented at 25-34 ℃ for 60-76 h at a pH of 60-7.0 to obtain a fermentation broth.

7. The method of claim 6, wherein the fermentation broth is subjected to adsorptive dearomatization using activated carbon.

8. The method of claim 7, wherein the modified activated carbon is added according to the proportion of 1 (10-15), the mixture is adsorbed for 2 hours at the temperature of 20-25 ℃ and under the condition of pH 2.0-0, and finally the adsorption solution is filtered to obtain the adsorption solution containing the high F value oligopeptide.

9. A high F value oligopeptide prepared by the method of any one of claims 1 to 8.

10. Use of the high F value according to claim 9 for the preparation of food, pharmaceutical, health care products.

Background

The high F value oligopeptide is a mixed oligopeptide (peptide chain consisting of 2-9 amino acids) with the molecular weight of 200-1000 Da and the BCAA/AAA (branched chain amino acid/aromatic amino acid) molar ratio of more than 20. BCAA refers to three amino acids of valine (Val), leucine (Leu) and isoleucine (Ile); AAA refers to tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe). The high F value oligopeptide has wide application value in the functional food and medicine industries as an oligopeptide containing high proportion of branched chain amino acids and low aromatic amino acids. Modern nutritional studies show that oligopeptides are more easily absorbed and utilized by organisms than amino acids, and some oligopeptides can be directly absorbed as active substances and further participate in physiological metabolism regulation. According to research, partial dipeptide and tripeptide can be directly transported into cells by oligopeptide transporter, and a small part of oligopeptide can be completely absorbed into systemic circulation through a paracellular route and a carrier transport system. The functional food containing high F value oligopeptide has effects of relieving fatigue, relieving hangover, resisting oxidation, and improving protein nutrition status of patients

The corn yellow meal (CGM) is a main byproduct of wet-process production of corn starch, the corn is the largest crop per unit yield in China according to the data of the national statistical bureau, the yield in 2020 is 26,067 ten thousand tons, and the yield of the corn is still increased year by year. According to the statistics and reports of the Chinese starch industry Association, the corn starch yield is 3,097.4 ten thousand tons in 2019, the yield is increased by 10 percent on a same scale, and the corn starch industry has a trend of rising year by year. The annual yield of CGM is increased, but the CGM is lack of two essential amino acids (lysine and tryptophan) in human bodies, so the CGM has low application value in the food industry. The subsequent utilization of the feed mainly lies in feed processing at present, and the added value is low. With the rapid development of national economy to high-quality development, the deep utilization of CGM is more urgent. According to the characteristics of high protein per se, the CGM deep processing and utilization are mainly directed to extracting corn protein and producing and preparing corn peptide at present, and if the nutritive value of the corn peptide can be improved, the corn peptide has important significance for the utilization and sustainable development of corn.

Disclosure of Invention

Aiming at the existing problems, the invention provides a method for preparing high F value oligopeptide, which comprises the steps of hydrolyzing a raw material of corn gluten with a bacillus natto fermentation product, fermenting the product obtained after enzymolysis by using aspergillus oryzae, realizing utilization of the corn gluten, and successfully preparing the high F value oligopeptide.

The invention provides a method for preparing high F value oligopeptide by microbial fermentation, which is characterized in that a fermentation product of bacillus natto and flavourzyme are used for carrying out enzymolysis on a raw material containing corn gluten meal, and the product obtained after the enzymolysis is fermented by aspergillus oryzae to produce the high F value oligopeptide.

In one embodiment, the Aspergillus oryzae is Aspergillus oryzae CICC40214, and is purchased from China center for Industrial culture Collection of microorganisms.

In one embodiment, the bacillus natto is obtained by mutagenesis of bacillus natto CICC10023, and is disclosed in Zhang Yangyang et al, mutagenesis screening of bacillus natto with high alkaline protease yield (disclosed in 2021, 4 months), the applicant promises to release the strain to the public in a legal way within 20 years from the filing date.

In one embodiment, the fermentation product of Bacillus natto comprises an alkaline protease and a neutral protease, the alkaline protease is not less than 50U/mL, and the neutral protease is not less than 100U/mL.

In one embodiment, the alkaline protease is 50-60U/mL and the neutral protease is 100-130U/mL.

In one embodiment, the OD is₆₀₀Inoculating 7-8 bacillus natto into a fermentation medium in an amount of 7mL/100mL for fermentation.

In one embodiment, the fermentation product of the bacillus natto is natto protease obtained by bacillus natto at the temperature of 35-39 ℃ and the pH of 8.5-9.5.

In one embodiment, the fermentation product of bacillus natto is a nattokinase obtained from bacillus natto at a temperature of 37 ℃ and a pH of 9.5.

In one embodiment, the fermentation medium comprises: 8-10 g/L of glucose, 15-20 g/L of fish meal peptone, 1-2 g/L of calcium chloride and 1-2 g/L of magnesium sulfate.

In one embodiment, the corn gluten is obtained by enzymolysis of corn gluten powder with alpha-amylase, and the corn gluten is obtained by enzymolysis of the fermentation product of bacillus natto, and the product obtained by enzymolysis is used as the fermentation medium of aspergillus oryzae.

In one embodiment, the raw material containing the corn gluten meal is pretreated by: adding corn gluten meal into PBS buffer solution with pH of 5.0-6.0 at a solid-to-liquid ratio of 1: 5-1: 12, adding 150-200U/g of medium-temperature alpha-amylase, performing heat preservation and enzymolysis at 55-60 ℃ for 4-6 h, deactivating enzyme at 100 ℃ for 10min after enzymolysis, adding sodium sulfite reducing agent with final concentration of 0.5-0.7 mg/mL, heating at 115-125 ℃ for 1.5-2.5 h, washing, drying and crushing to obtain the pretreated zein.

In one embodiment, the fermentation product of the bacillus natto contains alkaline protease, and the bacillus natto fermentation product is added with the bacillus natto protease according to the addition amount of 3500-4500U per gram of corn protein; carrying out enzymolysis for 4 hours at the temperature of 45-55 ℃ and under the condition of pH value of 9.0-9.5, adding 1.0-2.0% of cane sugar and 0.1-0.2% of sodium nitrate after enzymolysis, and inactivating to obtain the fermentation culture medium of aspergillus oryzae.

In one embodiment, the enzymatic hydrolysis is carried out at 55 ℃ for 4 hours at pH 9.0, followed by addition of 1.5% (1.5g/100mL) sucrose and 0.15% (0.15g/100mL) sodium nitrate.

In one embodiment, the natto protease is added in an amount of 4000U per gram of zein.

In one embodiment, Aspergillus oryzae is inoculated to the fermentation medium in an amount of 5-7%, and the mixture is fermented at pH 6.0-7.0 and 25-34 ℃ for 60-76 h to obtain a fermentation liquid.

In one embodiment, the composition will contain 1.0X 10 per ml⁸Inoculating the Aspergillus oryzae spore liquid of each spore into the prepared Aspergillus oryzae fermentation culture medium according to the amount of 6mL/100mL, and fermenting at the pH of 6.0-7.0 and the temperature of 25-34 ℃ for 60-76 h to obtain fermentation liquid.

In one embodiment, the fermentation broth is subjected to adsorptive dearomatization using activated carbon.

In one embodiment, modified activated carbon is added at a carbon-to-liquid ratio of 1 (10-15) (1 g of activated carbon is added to every 10-15 ml of fermentation liquid), the mixture is adsorbed for 2 hours at 20-25 ℃ and under the condition of pH 2.0-3.0, and finally the adsorption liquid is filtered to obtain the adsorption liquid containing the high-F-value oligopeptide.

In one embodiment, the modified activated carbon is added at a carbon to liquid ratio of 1: 10.

The invention provides a high F value oligopeptide prepared by the method.

The invention provides application of the high F value in preparing foods, medicines and health-care products.

Has the advantages that: the method comprises the steps of preparing corn protein by using corn gluten as a raw material, preparing a culture medium for aspergillus oryzae fermentation by utilizing a fermentation product of bacillus natto to carry out enzymolysis on the corn protein, fermenting aspergillus oryzae in the culture medium, adsorbing and dearomatizing fermentation liquor obtained after fermentation by activated carbon to obtain oligopeptides, wherein the oligopeptide content less than 1000Da is more than 85 percent, the F value is more than 22, and the oligopeptide content is 9.43 g.L-¹The oligopeptide accounts for 86 percent. The zeaxanthin powder is fully utilized, and the high-F-value oligopeptide is prepared, so that the preparation method has a wide application prospect.

Drawings

FIG. 1 shows the hydrolysis effect of nattokinase at different temperatures.

Detailed Description

The corn gluten meal mainly comprises the following components: 60% of protein, 17% of starch, 2-5% of crude fat and 1% of cellulose.

Aspergillus oryzae CICC40214 is purchased from China center for culture Collection of Industrial microorganisms.

The Bacillus natto used in the examples is obtained by mutagenesis of Bacillus natto CICC10023, and is disclosed in Zhang Zheng Yang, et al, mutagenesis and screening of Bacillus natto with high yield of alkaline protease (disclosed in 4 months of 2021).

Protease activity determination: reference is made to the standard GB/T23527-2009.

And (3) measuring the content of the peptide: measurement by biuret method.

Preparation of biuret reagent

a) NaOH solution: 6g of NaOH is weighed by a beaker, 25mL of distilled water is weighed and added into the beaker to dissolve the NaOH, and the concentration is prepared to be 6mol/L for standby.

b) Biuret reagent: weighing 0.75g copper sulfate (CuSO)₄·5H₂O) was dissolved in 500mL of freshly prepared distilled water, and 2.25g of sodium potassium tartrate (KNaC) was added₄H₄O₆·4H₂O for bonding Cu²⁺For preventing CuO from precipitating under alkaline conditions) and 1.5g of KI (for preventing alkaline copper tartrate from auto-reducing and for preventing Cu from being added₂Isolation of O), until complete dissolution, 25mL of 6g/L NaOH solution was added with stirring and the volume was adjusted to 250mL with water, and the mixture was placed in a plastic bottle and tightly capped for storage.

Taking ten 10mL volumetric flasks, using 5% TCA to prepare 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8mg/mL Gly-Gly-Tyr-Arg tetrapeptide standard solutions in sequence, then respectively taking 6.0mL standard solutions, adding 4.0mL biuret reagent, mixing uniformly, standing for 10min, centrifuging at 2000r/min for 10min, taking the supernatant, and determining the OD value at 540nm (taking the first tube as a blank control). A standard curve was prepared with the peptide concentration as abscissa X (mg/mL) and the OD value as ordinate Y.

The method comprises the following steps: taking 2.5mL of sample solution, adding 2.5mL of 10% (W/V) trichloroacetic acid (TCA) aqueous solution, uniformly mixing on a vortex mixer, standing for 10min, centrifuging for 15min at 4000r/min, transferring all supernate into a 50mL volumetric flask, fixing the volume to the scale with 5% TCA, and uniformly shaking. Then 6.0mL of the solution is put into another test tube, 4.0mL of biuret reagent (sample solution: biuret reagent is 3:2, V/V) is added, the mixture is uniformly mixed on a vortex mixer, the mixture is kept stand for 10min, the mixture is centrifuged at 2000r/min for 10min, the supernatant is taken and the OD value is measured at 540nm, the polypeptide concentration C (mg/mL) in the sample solution is obtained by contrasting a standard curve, and the polypeptide content in the sample can be further obtained.

F value determination of the enzymolysis liquid: and (3) carrying out detection analysis by using a high performance liquid chromatography OPA on-line derivation method.

And (3) detecting the molecular weight distribution: and detecting and analyzing the distribution of the molecular weight of the sample by using high performance liquid chromatography.

Protein conversion rate:

in the formula: the protein content was 83.1%.

Example 1

(1) Adding corn gluten meal into PBS buffer solution with pH 5.2 according to a solid-to-liquid ratio of 1:10, adding 165U/mL medium-temperature alpha-amylase, carrying out heat preservation and enzymolysis for 4h at 57 ℃, carrying out water bath at 100 ℃ to inactivate enzyme for 10min after the enzymolysis is finished, adding a sodium sulfite reducing agent with the final concentration of 0.7mg/mL, placing at 120 ℃ for heating treatment for 2.0h, and finally washing, drying and crushing to obtain the pretreated zein;

(2) using Bacillus natto as protease fermentation strain, and fermenting with OD₆₀₀Inoculating 7 percent (7mL/100mL) of Bacillus natto into a fermentation medium under the fermentation conditions of 37 ℃ of temperature, 9.5 of pH and 180-220 r/min of shaking table rotation speed, wherein the fermentation medium is 10g/L of glucose, 20g/L of fish meal peptone, 1g/L of calcium chloride and 1g/L of magnesium sulfate, and taking out after fermentation for 44 hours;

(3) centrifuging the fermentation liquor obtained in the step (2) for 20min at 8000r/min, and removing thallus and impurities to obtain natto protease;

(4) measuring the enzyme activity of the natto protease in the step (3), wherein the alkaline protease is 50-60U/mL, and the neutral protease is 100-130U/mL;

(5) carrying out enzymolysis on the corn protein obtained in the step (1) by using the natto protease in the step (4), wherein the addition amount of the corn protein is 5% (m/v), the addition amount of the enzyme is 200U/mL, carrying out enzymolysis for 4 hours at 55 ℃ and under the condition of pH 9.0, adding 1.5% (1.5g/100mL) of sucrose and 0.15% (0.15g/100mL) of sodium nitrate after enzymolysis, and carrying out damp-heat inactivation to obtain an aspergillus oryzae fermentation medium;

(6) preparing an aspergillus oryzae seed solution: firstly, Aspergillus oryzae CICC40214 is activated, the activated strain is inoculated to a slant culture medium, cultured at 28 ℃ until spores grow, hyphae are washed into a triangular flask with preset glass beads by sterile physiological saline water, and filtered by gauze after full shaking, and the filtrate is the spore liquid of Aspergillus oryzae. Diluting spore liquid with normal saline by appropriate times, and counting with blood count plateNumber, counted at OD₆₀₀0.148 hours the number of spores was about 1X 10⁸one/mL.

Inoculating Aspergillus oryzae, fermenting, and collecting spore liquid (1.0 × 10 per ml)⁸Spores) of the strain was inoculated into the Aspergillus oryzae fermentation medium prepared in step (5) in an amount of 6mL/100mL, the liquid loading was 70/250mL, the initial pH of the fermentation was 6.5, the fermentation temperature was 28 ℃, and the fermentation time was 68 hours. And filtering to remove thalli after fermentation is finished to obtain fermentation liquor.

(7) And (4) detecting the peptide content and the molecular weight in the step (6), wherein the oligopeptide content is 19.8g/L, and the oligopeptide accounts for 84.6%.

(8) And (3) adsorbing and dearomatizing the fermentation liquor obtained in the step (6) by using activated carbon, wherein the ratio of the activated carbon to the fermentation liquor in the adsorption process is 1: adding modified activated carbon into the fermentation liquor with the carbon-liquid ratio of 10 (1 g of activated carbon is added into every 10ml of fermentation liquor), adsorbing for 2 hours at the temperature of 20-25 ℃ and under the condition of pH 2.0-3.0, and finally filtering to obtain the adsorption liquid.

(9) Detecting the molecular weight distribution and the F value of the adsorption solution in the step (8), wherein the oligopeptide less than 1000Da is more than 85 percent, the F value is more than 22, and the oligopeptide content is 9.43 g.L-¹The oligopeptide accounts for 86 percent.

Example 2

In the same way as in example 1, the enzymolysis temperature in step (5) is adjusted to 45 ℃ and 50 ℃, and the result is shown in fig. 1, and the hydrolysis degree of enzymolysis is 13-15% in 4-6 h at 45-50 ℃.

Example 3

In the same manner as in example 1, the amounts of sucrose to be added in step (5) were 0, 0.5%, 1.0%, and 2.0%. And (3) detecting the content of oligopeptides and oligopeptides after the fermentation is finished, wherein the content of oligopeptides is respectively 15.44, 15.81, 16.19 and 15.94g/L, and the ratio of oligopeptides is respectively 63.25, 75.86, 79.85 and 75.23%.

Example 4

In the same manner as in example 1, the inoculation amount in step (6) was changed to 5% and 7%, the contents of oligopeptides were 16.55 and 16.44g/L, respectively, and the ratios of oligopeptides were 83.1 and 80.2%, respectively.

Example 5

The specific implementation way is the same as that of the example 1, the fermentation time in the step (6) is changed to 60 hours, 64 hours, 72 hours and 76 hours, the content of oligopeptides is respectively 15.33, 16.12, 15.88 and 14.91g/L, and the ratio of oligopeptides is 72.61, 82.13, 82 and 78.56 percent.

Example 6

The specific implementation manner is the same as that of example 1, except that the fermentation temperature in step (6) is changed to 25 ℃, 31 ℃ and 34 ℃, the contents of oligopeptides are respectively 15.33, 17.68 and 16.35g/L, and the ratio of oligopeptides is respectively 72.61, 83.97 and 82%.

Example 7 preparation of high F-value oligopeptide in fermenter

The amplification experiment is carried out in a 5L fermentation tank, and the fermentation conditions of the bacillus natto are as follows: liquid loading amount is 2.5L, culture medium is the same as that of shake flask fermentation, and ventilation amount is set to 200 L.h^-1The dissolved oxygen is set as a fixed value of 60 percent, and the rotating speed is related to the dissolved oxygen and ranges from 20 to 999 r.min^-1The activity of the alkaline protease after 44h fermentation is 132 U.mL^-1The CGM is added in a fermentation tank according to the activity of alkaline protease for enzymolysis (4000U/g of maize yellow powder), 2L of enzymolysis liquid is taken after the enzymolysis is finished, 1.5% (w/v) of cane sugar and 0.15% (w/v) of sodium nitrate are added, and the aspergillus oryzae is inoculated for fermentation after the protease is inactivated. The initial fermentation conditions are the same as those of shake flask fermentation, the dissolved oxygen is set to be 60%, the rotation speed is related to the dissolved oxygen, and the range is 20-999 r.min^-1. Fermenting Aspergillus oryzae in 5L fermenter for 66 hr to obtain oligopeptide content of 22.36 g.L^-1The oligopeptide content is 84.2%.

Comparative example 1

The specific implementation manner is the same as that of example 1, except that the enzymolysis temperature in the step (5) is adjusted to 35 ℃ and 40 ℃, and as a result, as shown in fig. 1, the hydrolysis degree reaches 9-13% after enzymolysis for 4-6 h at 35-40 ℃.

Comparative example 2

The specific embodiment is the same as example 1 except that the moist heat sterilization process is eliminated in the step (5). And (4) directly carrying out the step (6), and detecting the peptide content and the molecular weight in the step (6), wherein the oligopeptide content is 13.5g/L, and the oligopeptide content is 64.3%.

Comparative example 3

The specific embodiment is the same as example 1 except that the amount of sucrose to be added in step (5) is 2.5%. And (3) detecting the content of the oligopeptides and the oligopeptides after the fermentation is finished, wherein the content of the oligopeptides is 14.34g/L, and the ratio of the oligopeptides is 73.33%.

Comparative example 4

In the same manner as in example 1, the inoculation amount in step (6) was changed to 5% and 7%, the contents of oligopeptides were 16.55 and 16.44g/L, respectively, and the ratios of oligopeptides were 83.1 and 80.2%, respectively.

Comparative example 5

In the same way as in example 1, the fermentation time in step (6) was changed to 56 hours, the oligopeptide content was 13.48g/L, and the oligopeptide content was 63.25%.

Comparative example 6

The specific implementation manner is the same as that of example 1, except that the fermentation temperature in step (6) is changed to 22 ℃, the oligopeptide content is 13.50g/L, and the oligopeptide content is 64.21% respectively.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.