CN114854576A

CN114854576A - Preparation system and method of protein polypeptide with antioxidant activity

Info

Publication number: CN114854576A
Application number: CN202210747369.3A
Authority: CN
Inventors: 薛刚
Original assignee: Beijing Fangfei Story Technology Development Co ltd
Current assignee: Beijing Fangfei Story Technology Development Co ltd
Priority date: 2022-06-28
Filing date: 2022-06-28
Publication date: 2022-08-05

Abstract

The invention provides a system and a method for preparing protein polypeptide with antioxidant activity, which belong to the field of antioxidant polypeptide preparation, wherein straw liquid is stored in a straw pulp tank, the straw liquid is conveyed to a high-temperature container through a conveyor, pretreated protein pulp is input into the high-temperature container, the straw liquid is mixed, the mixed pulp is subjected to steam decomposition treatment in the high-temperature container, the high-temperature container is connected with a mixing container, a cooling medium in a cooling medium tank is added into the mixing container through a conduit and is used for cooling the mixed pulp output by the high-temperature container in the mixing container, the pH value of the mixed pulp is increased through mixing alkaline substances, a main enzyme hydrolysis tank is used for incomplete enzyme hydrolysis, the pulp output by the mixing container is converted into liquefied material, one part of the liquefied material is led into a heat exchanger, the heat exchanger reduces the temperature of the liquefied material and generates the cooling medium, another portion of the liquefied material is passed to a secondary enzymatic hydrolysis tank where complete enzymatic hydrolysis takes place.

Description

Preparation system and method of protein polypeptide with antioxidant activity

Technical Field

The invention relates to preparation of protein polypeptide, in particular to a system and a method for preparing protein polypeptide with antioxidant activity, and belongs to the field of preparation of antioxidant polypeptide.

Background

The modification of proteins has not been widely used in food processing, but its importance is increasingly recognized by the public. Protein modification is important for food, pharmaceutical and industrial applications because it can alter the microstructure and physicochemical properties of biopolymers to alter the functional properties of proteins to achieve industry standard desired functional properties, including enhancement of food texture, flavor, color, solubility, foaming, whipping, and digestibility, among others. In general, depending on the requirements of the application, the modification of food proteins is effected principally by crosslinking or hydrolysis. Protein cross-links are cross-links between polypeptide chains in a protein to form covalent bonds or protein-protein cross-links. Hydrolysis of proteins is a reaction in which a protein molecule is cleaved to form a polypeptide or amino acid catalyzed by an acid, base, or protease. At present, the method for modifying food protein mainly comprises physical modification, enzymatic modification, chemical modification or combination of the first three modifications.

Conventional physical modification methods such as extrusion, mechanical treatment, heating, etc. have been extensively studied. At present, the technology of ultrasound, ultrahigh pressure, irradiation, high hydrostatic pressure and the like is applied to the physical modification technology of protein at home and abroad, and certain achievements are obtained. For example, the ultra-high pressure has no effect on the primary structure of the protein, but has a certain effect on the secondary structure and the tertiary structure thereof, thereby causing changes in functional properties of the protein, such as gel properties. Enzymatic modification is a method for releasing bioactive peptides by carrying out enzymolysis on proteins by utilizing different specificities of enzymes, and is used for improving the functional characteristics and the nutritional characteristics of the proteins. For the enzymatic modification, it is hot to obtain polypeptides with different physiological activities, such as angiotensin inhibitory peptide, antioxidant peptide, antibacterial peptide, etc. from various proteins of different sources by enzymatic hydrolysis. The chemical modification method comprises acid, alkali and salt modification, phthalylation modification, phosphorylation modification, saccharification modification and the like.

The enzyme method is mainly to utilize the catalytic action of enzyme to hydrolyze the protein under the condition of proper enzyme activity, thereby breaking some chemical bonds, changing the structure of the protein and finally changing the functional characteristics of the protein. When the enzymolysis modification is researched at home and abroad, the enzyme preparation mainly comprises bromelain, papain and other plant-derived proteases; animal-derived proteases such as trypsin and pepsin; protease derived from microorganisms such as thermolysin and alkaline protease. The specific property of the enzyme determines the hydrolysis of specific belly bonds or specific groups of the protein, and different protease has different enzyme cutting sites on the protein, for example, the enzyme cutting sites of trypsin mainly comprise lysine residues and arginine residues; the enzyme cutting site of the pepsin is mainly amino acid connected with hydrophobic amino acid and comprises phenylalanine, methionine, leucine and tryptophan; the enzyme cutting sites of the alkaline protease mainly comprise alanine, leucine, valine, tyrosine and phenylalanine. The degree of enzymatic modification can be expressed by the Degree of Hydrolysis (DH) of the protein, i.e., the proportion of peptide bonds cleaved by hydrolysis of the protein molecule to the total peptide bonds in the protein molecule, and is generally measured by the formaldehyde titration method or the trinitrobenzene sulfonic acid (TNBS) method.

The use of antioxidants protects the human body from oxidative stress and prevents food ingredients from deteriorating due to the negative effects of donating electrons to ROS and neutralizing ROS. Although many synthetic antioxidants successfully scavenge ROS, their use is often limited due to their toxic side effects. Therefore, it is necessary to develop a new antioxidant having higher activity. The structure of the protein is changed by the hydrolysis of the protease, the active functional group of the hydrophobic region is exposed, and the free amino acid is increased along with the cleavage of peptide bonds, so that protons or electron sources can be provided, a higher redox potential is kept, the protein has the capability of scavenging active free radicals (such as ROS), reducing power, the capability of scavenging peroxynitrite and inhibiting the occurrence of lipid peroxidation, and the antioxidant activity of the enzymolysis modified product is improved.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a system for preparing a protein polypeptide having antioxidant activity, comprising: the straw pulp tank, the high-temperature container, the mixing container, the cooling medium tank, the main enzyme hydrolysis tank, the secondary enzyme hydrolysis tank and the heat exchanger;

straw liquid with 40 wt% of straw solid is stored in the straw pulp tank and is transmitted to the high-temperature container through a conveyor,

inputting the pretreated protein pulp into the high-temperature container, mixing the straw liquid, performing steam decomposition treatment on the mixed pulp in the high-temperature container,

the high-temperature container is connected with the mixing container, the cooling medium in the cooling medium tank is added into the mixing container through a conduit and is used for cooling the mixed pulp output by the high-temperature container in the mixing container and increasing the pH value of the mixed pulp through mixing alkaline substances,

the main enzymatic hydrolysis tank is used for carrying out incomplete enzymatic hydrolysis, converting the pulp output by the mixing container into liquefied material with low consistency, introducing a part of the liquefied material into the heat exchanger, reducing the temperature of the liquefied material to 50-30 ℃ by the heat exchanger and generating the cooling medium,

another portion of the liquefied material flows into the secondary enzymatic hydrolysis tank, which is used to perform complete enzymatic hydrolysis.

And the ultrafiltration system is connected with the secondary enzyme hydrolysis tank and is used for intercepting polypeptide macromolecules and colloids with large relative molecular mass to form concentrated solution.

Further, in the steam decomposition treatment process, the mixed pulp is decomposed at a temperature higher than 100 ℃ in an acid environment, and the gas in a high-temperature container is 133m ³ The rate of/h is discharged.

Further, the ultrafiltration system firstly carries out ultrafiltration on the polypeptide liquid by a membrane component with the molecular weight cut-off of 10KDa to obtain a filtrate and a concentrated solution; and then the filtrate is ultrafiltered by a 3KDa membrane component to obtain 3-10KDa ultrafiltrate and filtrate.

The invention also provides a preparation method of the protein polypeptide with antioxidant activity, which is realized by adopting the preparation system and comprises the following steps:

mixing the straw liquid with 40 wt% of solid with the pretreated protein slurry to form mixed slurry,

the mixed pulp is subjected to steam decomposition treatment at a temperature higher than 100 ℃ in an acidic environment,

cooling the mixed pulp after the steam decomposition treatment, increasing the pH value of the mixed pulp through mixing alkaline substances,

incomplete enzymatic hydrolysis is carried out by a primary enzymatic hydrolysis reaction, a part of the liquefied material produced in the incomplete enzymatic hydrolysis process is introduced into a heat exchanger to produce a cooling medium, and the other part of the liquefied material is subjected to a secondary enzymatic hydrolysis reaction, thereby realizing complete enzymatic hydrolysis.

Further, under the driving force of static pressure difference of 0.1-1MPa, firstly, performing ultrafiltration on the polypeptide liquid by using a membrane component with molecular weight cutoff of 10KDa to obtain filtrate and concentrated solution; and then the filtrate is ultrafiltered by a 3KDa membrane component to obtain 3-10KDa ultrafiltrate and filtrate, and polypeptide macromolecules and colloid with large relative molecular mass are intercepted.

Further, performing suction filtration and vacuum rotary evaporation on the ultrafiltrate respectively to obtain concentrated solution, placing the concentrated solution in a culture dish, sealing the concentrated solution by using a preservative film, placing the culture dish in a refrigerator at the temperature of 18 ℃ below zero for pre-freezing for 24 hours, and then performing vacuum freeze drying to obtain protein polypeptide powder.

Further, the primary enzymatic hydrolysis reaction is 0.5 to 6.0 hours, the secondary enzymatic hydrolysis reaction is operated in a continuous or batch mode, and the secondary enzymatic hydrolysis reaction is carried out for 21 to 69 hours, thereby achieving complete enzymatic hydrolysis.

Further, the total nitrogen mass M1 of the protein slurry was measured, the mass M2 of the amino nitrogen in the enzymatic hydrolysate was measured, and the degree of hydrolysis was calculated according to the following formula:

compared with the prior art, the invention has the following beneficial technical effects:

1. compared with the existing preparation system, the preparation system of the protein polypeptide with the antioxidant activity, disclosed by the invention, is provided with a main enzyme hydrolysis tank and a secondary enzyme hydrolysis tank; the main enzyme hydrolysis tank is used for carrying out incomplete enzyme hydrolysis, the secondary enzyme hydrolysis tank is used for carrying out complete enzyme hydrolysis, and the catalytic action of the two enzymes is utilized to carry out full hydrolysis on the protein under the condition of proper enzyme activity, so that some chemical bonds are broken, the structure of the protein is changed, and finally the functional characteristics of the protein are changed.

2. Compared with the existing preparation system, the preparation system of the protein polypeptide with the antioxidant activity has an ultrafiltration system, and can firstly carry out ultrafiltration on polypeptide liquid by using a membrane component with the molecular weight cutoff of 10KDa, then carry out ultrafiltration on filtered liquid by using a membrane component with 3KDa, finally obtain 3-10KDa ultrafiltrate and filtrate, and intercept polypeptide macromolecules and colloid with large relative molecular mass.

3. The invention utilizes the correlation between the hydrolysis degree and the oxidation resistance and the correlation between the hydrolysis degree of the enzyme and the chelating force of ferrous ions, and adds the ferrous ions to promote the hydrolysis degree of the enzyme through an ion adding system.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive labor.

FIG. 1 is a schematic diagram showing the overall structure of a system for preparing a protein polypeptide having antioxidant activity according to the present invention.

FIG. 2 is a graph of a spectral scan of two protein polypeptide solutions of different concentrations according to the present invention.

FIG. 3 is a graph showing the change of the final pH after saccharification reaction of the protein polypeptide of the present invention and glucose at different temperatures and ratios.

FIG. 4 is a flow chart of a method for preparing the protein polypeptide having antioxidant activity of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

In the drawings of the embodiments of the present invention, in order to better and more clearly describe the working principle of each element in the system, the connection relationship of each part in the apparatus is shown, only the relative position relationship between each element is clearly distinguished, and the restriction on the signal transmission direction, the connection sequence, and the size, the dimension, and the shape of each part structure in the element or structure cannot be formed.

Prior to the preparation of the protein polypeptide, the material is first pretreated, in this example, fish skin is used as the preparation raw material.

Thawing and cleaning fish skin, adding 0.05mol/L NaOH solution according to the ratio of 1: 10(g/mL), stirring and soaking at room temperature for 30min, washing with running water to be neutral, then adding 0.05mol/L HCl solution according to the ratio of 1: 10(g/mL), stirring and soaking at room temperature for 10min, washing with running water to be neutral, draining, and pulping the fish skin to form slurry.

FIG. 1 is a schematic diagram showing the overall structure of a system for producing a protein polypeptide having antioxidant activity according to the present invention.

Inputting the pretreated pulp into a high-temperature container, connecting the upper end of the high-temperature container with a straw pulp tank through a conveyor, conveying the straw liquid to the conveyor, wherein the straw liquid in the straw pulp tank is at a temperature of about 100 ℃, is a solid-liquid mixture with 40 weight percent of solids and has a pH of 4, and during the conveying process of the conveyor, the temperature of the pulp is reduced and the pH is increased.

The pulp mixed with the straw liquid is subjected to steam decomposition treatment in a high-temperature container. During the steam decomposition treatment, the pulp is decomposed at a temperature higher than 100 deg.C in an acidic environment, and in order to ensure the air pressure balance in the high-temperature container, the gas discharged from the high-temperature container is 133m ³ The rate of/h is discharged.

The straw can be, but is not limited to, hardwood, softwood, bagasse, energy cane, corn stover, corn cobs, corn fiber, straw from rice, wheat, rye, and other crop or forestry residues.

The high temperature vessel is connected to a mixing vessel which is used to cool the pretreated slurry and increase the PH of the slurry by mixing with alkaline substances such as ammonia, lime or other earth metal based hydroxides, etc.

In the mixing vessel, the cooling medium in the cooling medium tank is added to the pulp in the mixing vessel via a conduit. The cooling medium is the liquefied material discharged from the main enzymatic hydrolysis tank. The liquefied material is pumped through a heat exchanger using a pump, which can reduce the temperature of the liquefied material to about 50 ℃ to 30 ℃ and produce a cooling medium.

The main enzymatic hydrolysis tank is a conical vessel that converts the pulp output from the mixing vessel into a liquid having a relatively low consistency during enzymatic hydrolysis in the main enzymatic hydrolysis tank.

The cooled and PH controlled pulp is maintained in the main enzymatic hydrolysis tank for a relatively short holding time, about 0.5 to 6.0 hours. In the main enzymatic hydrolysis tank, where the straw in the cooled and PH-adjusted pulp is converted from the solid fraction into sugar and entrained product liquefied material, the retention time required for the reaction is about 6 hours, but in which the enzymatic hydrolysis of the pulp is not completely completed, i.e. no sufficient enzymatically hydrolyzed material is produced.

During enzymatic hydrolysis in the main enzymatic hydrolysis tank, the pulp reduces the apparent viscosity of hydrolysis as the long chain polymers of the pulp are broken down into short molecules. Since the liquefied material after hydrolysis is substantially liquid, a part of the liquefied material is introduced into the heat exchanger, and the other part of the liquefied material flows into the secondary enzymatic hydrolysis tank through the conduit.

The secondary enzymatic hydrolysis tank is a cylindrical vessel that operates in either a continuous or batch mode. The time to complete enzymatic hydrolysis in the secondary enzymatic hydrolysis tank may be relatively long, for example, may be about 21 to 69 hours or more, to achieve complete enzymatic hydrolysis.

The enzymatic hydrolysis reaction mainly utilizes the catalytic action of enzyme to hydrolyze protein under the condition of proper enzyme activity, thereby breaking some chemical bonds, changing the structure of the protein and finally changing the functional characteristics of the protein.

In this embodiment, neutral protease, alkaline protease, trypsin and papain can be selected to perform single-enzyme enzymolysis on the pulp, and hydrolysis is performed under the conditions of the optimal PH value and the optimal enzymolysis temperature of each protease according to the instructions of the use of the enzyme preparation.

As the hydrolysis reaction proceeds, the degree of hydrolysis gradually increases and the substrate protein is gradually hydrolyzed into free amino acids. The degree of enzymatic hydrolysis modification can be expressed by the Degree of Hydrolysis (DH) of the protein, i.e., the proportion of peptide bonds cleaved by hydrolysis of the protein molecule to the total peptide bonds in the protein molecule, and is generally measured by the formaldehyde titration method or the trinitrobenzene sulfonic acid (TNBS) method.

There is a correlation between the degree of hydrolysis and the resistance to oxidation, and there is a very significant correlation between the degree of hydrolysis of the enzyme and the chelating power of ferrous ions.

Thus, in a preferred embodiment, the degree of hydrolysis of the enzyme may be promoted by the addition of ferrous ions via an ion addition system.

The total nitrogen mass M1(g) of the protein slurry is determined by using a Kjeldahl method, the mass M2(g) of amino nitrogen in the enzymolysis liquid is determined by an automatic potentiometric titration method, and the hydrolysis degree is calculated according to the following formula:

Fe ²⁺ the binding rate is measured by a phenazine colorimetry, a 5mg/mL sample solution is prepared by using a sodium acetate buffer solution (0.2mol/L, PH5.0),mixing 700 μ L sample solution and 100 μ L ferrous sulfate solution (0.2mmol/L) in 96-well plate, keeping at 37 deg.C for 2h, adding 200 μ L phenanthroline solution (5mmol/L), standing at room temperature for 10min, reading absorbance at 562nm wavelength with enzyme labeling instrument, using tryptic zymolyte of casein and glutathione as positive control, and Fe ²⁺ The binding rate was calculated as follows:

in the formula: a. the ₀ Is the absorbance of the added equal volume of water; a. the ₁ Is the absorbance after addition of the sample.

Containing Fe ²⁺ The correlation between the reducing power of the enzymolysis product of the protein polypeptide solution and the chelating power of ferrous ions is very obvious; the reducing power of the enzymolysis product has very obvious correlation with the capacity of eliminating DPPH free radicals.

Accurately suck 1.00mL of Fe-containing solution ²⁺ Mixing the protein polypeptide sample solution (5mg/mL) with 1.00mL DPPH-ethanol solution (0.15mmol/L, dissolved in 95% ethanol solution), reacting at room temperature in dark for 30min, and measuring the absorbance A of the solution at 517nm _s The blank group was prepared by adding 1mL of a 95% ethanol solution instead of the DPPH solution, mixing, and measuring the absorbance A at a wavelength of 517nm _b The control group was prepared by adding 1mL of a DPPH solution and 1mL of a 95% ethanol solution, and measuring the absorbance A at a wavelength of 517nm _c Taking glutathione as a positive control, the DPPH free radical clearance rate is calculated according to the following formula:

after the protein is modified by enzymolysis, a large amount of peptides or amino acids with similar relative molecular mass, hydrophilicity and hydrophobicity are generated, and the enzymolysis product is a mixture of the protein, the peptides and the amino acids, which brings great challenges to the separation and purification of the protein, the peptides and the amino acids. Thus, at the end of the preparation system, the product released from the secondary enzymatic hydrolysis tank is a polypeptide liquor that is sufficiently hydrolyzed by the enzymes and delivered to the ultrafiltration system.

The ultrafiltration system is driven by static pressure difference of 0.1-1MPa, the filtering particle size is between microfiltration and reverse osmosis, about 5-10nm, and polypeptide macromolecules and colloids with large relative molecular mass are intercepted to form concentrated solution, so that the purposes of separating, purifying and concentrating the solution are achieved.

In a preferred embodiment, the ultrafiltration system first ultrafilters the polypeptide liquid with a membrane module with a molecular weight cut-off of 10KDa (pressure < o.1mpa, room temperature) to obtain a filtrate and a concentrate. Ultrafiltering the filtrate with 3KDa (pressure <0.1MPa, room temperature) membrane module to obtain 3-10KDa ultrafiltrate and filtrate.

And (3) respectively carrying out suction filtration and vacuum rotary evaporation on the ultrafiltrate to obtain a concentrated solution, placing the concentrated solution in a culture dish, sealing the concentrated solution by using a preservative film, pre-freezing the concentrated solution in a refrigerator at the temperature of-18 ℃ for 24 hours, punching holes on the preservative film for ventilation, and then carrying out vacuum freeze drying to obtain the protein polypeptide powder.

In another embodiment of the invention, after the protein polypeptide powder is obtained, the protein polypeptide microporous carrier microcapsule can be prepared by adopting a coagulation bath preparation system.

Weighing 2.52g of sodium alginate, dissolving in 100mL of distilled water, uniformly dissolving by using a magnetic stirrer, slowly adding 1g of Tween 80 into the solution, and continuously stirring to completely disperse and dissolve the Tween 80 and the sodium alginate. 2.2176g of protein polypeptide powder is weighed according to the proportion of 0.88:1 of the core wall ratio, 20mL of distilled water is used for dissolving, meanwhile, corn micropores which are equal to the polypeptide are added as core material carriers and dissolved in the protein polypeptide liquid, and the mixture is stirred to be fully dissolved so as to enable the micropore carriers to carry the polypeptide. Adding the mixture into the wall material solution, and magnetically stirring until the mixture is completely dissolved. Preparing a calcium chloride solution with the concentration of 2.48 percent as a curing agent, preparing microcapsules by using a probe with the particle size of 808m by using a microcapsule granulator, dripping the microcapsules into the curing solution for curing for 0.5 hour, repeatedly washing the curing solution on the surfaces of the microcapsule particles by using distilled water, separating the microcapsule particles, flatly spreading the microcapsule particles in a culture dish, freezing the microcapsule particles in a refrigerator at the temperature of-80 ℃ for 8 hours, then carrying out vacuum freeze drying for 24 hours, and storing the microcapsule particles in a dryer.

Weighing freeze-dried microcapsule O.lg, sufficiently shaking and washing with cold distilled water to ensure that polypeptide adhered to the surface is dissolved in washing liquid, fixing the volume of the washing liquid to 5mL, filtering with a 0.45-micrometer filter membrane, measuring an absorption peak at 248nm, and calculating the content in a standard curve, wherein the peptide in the washing liquid is the content of the peptide on the surface of the microcapsule. Weighing freeze-dried microcapsule O.lg, fully grinding to break the wall of the microcapsule O.lg, adding a little distilled water, shaking to fully dissolve the polypeptide and fixing the volume to 5mL, filtering by using a 0.45-micrometer filter membrane, measuring an absorption peak at 248nm, corresponding to a standard curve, and calculating the content, wherein the polypeptide in the dissolved solution is the content of the total polypeptide of the microcapsule.

The embedding rate calculation formula is as follows:

the encapsulation efficiency (%) × (1-microcapsule surface peptide content/microcapsule total polypeptide content) × 100%.

The protein polypeptide liquid with two different concentrations is subjected to spectral scanning, the protein polypeptide liquid has a maximum absorption peak at 248nm, and the absorption peak trends of the two concentrations show high consistency, as shown in figure 2.

In another embodiment of the invention, a saccharification system is adopted, and after protein polypeptide powder is obtained, glucose and the protein polypeptide powder are subjected to saccharification reaction to prepare a product with stronger antioxidant activity.

Dissolving the freeze-dried protein polypeptide in distilled water to enable the final mass concentration to be 6mg/mL, adding glucose with different masses to enable the mass ratio of the polypeptide to the glucose to be 2:1, 1:1, 1:2 and 1:4 respectively, adjusting the pH to 7.5 by using 1M NaOH and HCl, and freeze-drying for 24 hours. Weighing a certain amount of freeze-dried powder, carrying out glycosylation reaction in a dryer filled with saturated potassium iodide solution (relative humidity is 65%), reacting at 50 ℃ and 60 ℃ for 24 hours, and taking out to obtain the protein polypeptide-glucose complex.

In this example, a continuous plug flow reactor was used instead of a high temperature vessel, which allowed rapid heating and acidification of the slurry, control of the reaction time by selecting the correct flow rate and length of the continuous plug flow reactor, and rapid quenching of the pretreated slurry by flash cooling. Short reaction times and rapid cooling allow the degree of polymerization of the straw material to be reduced, but do not allow these straw chains to recombine. Structural changes are not limited to a reduction in the degree of polymerization, but also include significant removal of half of the straw washed out during pretreatment, and changes in straw lignin binding. All these effects together result in a pretreated pulp with a significantly increased enzymatic hydrolysis rate and thus a higher glucose yield.

Once the slurry is at the desired reaction temperature, a concentrated stream of acid, typically sulfuric acid or any mineral acid, is continuously injected into the hot slurry so that it is rapidly mixed into the slurry, resulting in a final acid concentration in the aqueous phase of 0% to 1%, preferably suitable for slurries in the range of 5-10% solids.

Protein polypeptide-glucose complex samples were diluted to appropriate concentrations and their absorbance values were measured at 294nm and 420nm, respectively, using an ultraviolet spectrophotometer. The light absorption value under 294nm is an intermediate product with ultraviolet absorption in the protein polypeptide-glucose saccharification reaction, and the light absorption value under 420nm is the browning degree of the saccharification reaction.

As shown in FIG. 3, the change of the final pH after saccharification reaction of protein polypeptide and glucose at different temperatures and ratios is shown schematically. In the figure, the abscissa represents the mass ratio of protein polypeptide to glucose, and the ordinate represents the pH.

The invention also provides a preparation method of the protein polypeptide with antioxidant activity, which is realized by adopting the preparation system, as shown in figure 4, and comprises the following steps:

mixing the straw liquid with 40 wt% straw solid with the pretreated protein slurry to form mixed slurry,

cooling the mixed pulp after the steam decomposition treatment, increasing the pH value of the mixed pulp by mixing alkaline substances,

Preferably, under the driving force of static pressure difference of 0.1-1MPa, firstly, the polypeptide liquid is ultrafiltered by a membrane component with molecular weight cut-off of 10KDa to obtain filtrate and concentrated solution; and then carrying out ultrafiltration on the filtrate by using a 3KDa membrane component to obtain 3-10KDa ultrafiltrate and filtrate, and intercepting polypeptide macromolecules and colloids with large relative molecular mass.

Preferably, the ultrafiltrate is respectively filtered and vacuum rotary evaporated to obtain a hyperconcentration solution, the hyperconcentration solution is placed in a culture dish and sealed by a preservative film, the culture dish is placed in a refrigerator at the temperature of 18 ℃ below zero for pre-freezing for 24 hours, and then the hyperconcentration solution is subjected to vacuum freeze drying to obtain protein polypeptide powder.

In the above embodiments, the implementation may be wholly or partially realized by software, hardware, firmware, or any combination thereof. When implemented in software, may be implemented in whole or in part in the form of a computer program product. The computer program product includes one or more computer instructions. The procedures or functions described in accordance with the embodiments of the application are all or partially generated when the computer program instructions are loaded and executed on a computer. The computer may be a general purpose computer, a special purpose computer, a network of computers, or other programmable device. The computer instructions may be stored in or transmitted over a computer-readable storage medium. The computer-readable storage medium can be any available medium that can be accessed by a computer or a data storage device, such as a server, a data center, etc., that incorporates one or more of the available media. The usable medium may be a magnetic medium (e.g., floppy disk, hard disk, magnetic tape), an optical medium (e.g., DVD), or a semiconductor medium (e.g., Solid State Disk (SSD)), among others.

While the invention has been described with reference to specific embodiments, the scope of the invention is not limited thereto, and those skilled in the art can easily conceive various equivalent modifications or substitutions within the technical scope of the invention. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. A system for producing a protein polypeptide having antioxidant activity, comprising: the straw pulp tank, the high-temperature container, the mixing container, the cooling medium tank, the main enzyme hydrolysis tank, the secondary enzyme hydrolysis tank and the heat exchanger;

straw liquid containing 40 wt% of straw solids is stored in the straw pulp tank and is transmitted to the high-temperature container through a conveyor,

2. The system for preparing a polypeptide of claim 1, further comprising an ultrafiltration system connected to the secondary enzymatic hydrolysis tank for entrapping polypeptide macromolecules and colloids having a relatively large molecular mass to form a concentrate.

3. The system for preparing as claimed in claim 1, wherein the mixed slurry is decomposed at a temperature higher than 100 ℃ in an acidic environment during the steam decomposition treatment, and the gas in the high temperature vessel is 133m ³ The rate of/h is discharged.

4. The preparation system of claim 2, wherein the ultrafiltration system first ultrafilters the polypeptide solution with a membrane module having a molecular weight cut-off of 10KDa to obtain a filtrate and a concentrate; and ultrafiltering the filtrate with 3KDa membrane module to obtain 3-10KDa ultrafiltrate and filtrate.

5. A method for producing a protein polypeptide having antioxidant activity, which is carried out by the production system according to any one of claims 1 to 4, comprising:

mixing straw liquid containing 40 wt% of straw solids with the pretreated protein slurry to form mixed slurry,

6. The preparation method according to claim 5, characterized in that, under the driving force of static pressure difference of 0.1-1MPa, the polypeptide liquid is firstly ultrafiltered by a membrane component with molecular weight cut-off of 10KDa to obtain filtrate and concentrated solution; and ultrafiltering the filtrate with 3KDa membrane module to obtain 3-10KDa ultrafiltrate and filtrate, and intercepting polypeptide macromolecules and colloid with large relative molecular mass.

7. The preparation method according to claim 6, wherein the ultrafiltrates are respectively subjected to suction filtration and vacuum rotary evaporation, the obtained hyperconcentration solution is placed in a culture dish, sealed by a preservative film and pre-frozen in a refrigerator at-18 ℃ for 24h, and then subjected to vacuum freeze drying to obtain the protein polypeptide powder.

8. The method according to claim 5, wherein the mixed slurry is decomposed at a temperature higher than 100 ℃ in an acidic environment during the steam decomposition treatment, and the gas in the high-temperature vessel is 133m ³ The rate of/h is discharged.

9. The process according to claim 5, wherein the primary enzymatic hydrolysis is carried out for 0.5 to 6.0 hours, the secondary enzymatic hydrolysis is carried out in a continuous or batch mode, and the secondary enzymatic hydrolysis is carried out for 21 to 69 hours, thereby achieving complete enzymatic hydrolysis.

10. The method according to claim 5, wherein the mass M1 of total nitrogen in the protein slurry is measured, the mass M2 of amino nitrogen in the enzymatic hydrolysate is measured, and the degree of hydrolysis is calculated as follows: