A method for preparing short peptides from gluten by double enzyme complex enzymolysis

Abstract

The invention relates to the technical field of methods for preparing gluten short peptides, and in particular to a method for preparing gluten short peptides by double-enzyme composite enzymolysis, comprising: first preparing immobilized pullulanase and a hydrogel material for embedding alpha-amylase; subjecting gluten to ball milling and sieving to obtain gluten fine powder; mixing the gluten fine powder with water, and heating to obtain a mixed slurry; adding a buffer solution to the slurry and adjusting the pH value, and then adding the previously prepared immobilized pullulanase to perform a first step of enzymolysis; then adding the previously prepared alpha-amylase embedding hydrogel material, and performing a second step of enzymolysis under acidic conditions and in the presence of acetylacetone; and finally obtaining the gluten short peptides through enzyme inactivation, centrifugation and drying.

Description

Technical Field

[0001] The invention relates to the technical field of methods for preparing gluten short peptides, and in particular to a method for preparing gluten short peptides by double-enzyme composite enzymolysis. Background Art

[0002] Wheat is the most widely planted grain in the world, and its total output is second only to rice and slightly higher than corn. In my country, the annual output of wheat is about 100 million tons, ranking first among all grains. Gluten, also known as active gluten powder and wheat gluten protein, is a kind of light yellow powdery natural grain protein extracted from wheat through deep processing. It has wide application value in noodle products, meat products, aquatic products and beverage industries. According to statistics, about 500,000 tons of gluten are produced in the world every year. Gluten contains more than 70% gluten protein and is a high-quality and low-cost plant protein resource. As a by-product of wheat starch processing, gluten contains a large amount of protein, 5%-10% starch and a small amount of crude fiber. These components wrap the gluten protein molecules, prevent the contact between gluten protein and proteolytic enzymes, and limit the enzymatic hydrolysis of gluten protein. At present, most of the gluten enzymatic hydrolysis technologies only use proteases to hydrolyze proteins. Therefore, there is an urgent need for a new method for hydrolyzing gluten to obtain short peptides without using proteases. Summary of the invention

[0003] The present invention provides a method for preparing short gluten peptides by double-enzyme composite enzymolysis, so as to solve the defects pointed out in the background technology.

[0004] According to a first aspect of an embodiment of the present invention, a method for preparing short gluten peptides by double enzyme complex enzymolysis comprises the following steps:

[0005] Step 1: ball-milling gluten powder to obtain a crude gluten powder, and sieving the crude gluten powder to obtain gluten powder;

[0006] Step 2: mixing the gluten powder with water, and then heating at 75° C.-90° C. for 1.5-4 hours to obtain a first mixed slurry;

[0007] Step 3: adding acetic acid-sodium acetate buffer to the first mixed slurry, and adjusting the pH value to 5.0-6.0 to obtain a second mixed slurry;

[0008] Step 4: lowering the temperature of the second mixed slurry to 50° C.-65° C., and then adding immobilized pullulanase for enzymolysis to obtain a first enzymolysis solution; during the enzymolysis process of step 4, keeping the pH value and temperature unchanged;

[0009] Step 5: preparing a hydrogel material for embedding α-amylase;

[0010] Step 6: adding an acidic solution and acetylacetone to the first enzymatic hydrolyzate, adjusting the pH value to 3.0-3.5, then adding a hydrogel material encapsulating α-amylase, and performing enzymolysis at 40° C.-50° C. to obtain a second enzymatic hydrolyzate; keeping the pH value and temperature unchanged during the enzymatic hydrolysis process in step 6;

[0011] Step 7: The second enzymatic hydrolysate is subjected to inactivation of enzymes, centrifugation and drying to obtain the gluten short peptides.

[0012] In one aspect of the embodiment of the present invention, in step 1, the ball milling process includes: adding the gluten powder to anhydrous ethanol for ball milling, the mass ratio of the gluten powder to anhydrous ethanol is 1: (3.5-5), 5-10 10 mm agate balls and 25-40 6 mm agate balls are used as grinding balls, and the ball mill parameters are set to a rotation speed of 200-250 rpm and a ball milling time of 30-60 min.

[0013] In one aspect of an embodiment of the present invention, specifically, in step 1, the ball milling process includes: adding the gluten powder to anhydrous ethanol for ball milling, the mass ratio of the gluten powder to anhydrous ethanol is 1:4, 8 10 mm agate balls and 30 6 mm agate balls are used as grinding balls, and the ball mill parameters are set to a rotation speed of 250 rpm and a ball milling time of 45 min.

[0014] In one aspect of the embodiment of the present invention, in step 1, the mesh size of the sieving is 150-200 meshes.

[0015] In one aspect of the embodiment of the present invention, specifically, in step 1, the mesh size of the sieving is 150-200 meshes.

[0016] In one aspect of the embodiments of the present invention, in step 2, the mass ratio of the gluten powder to water is 1:(10-15).

[0017] In one aspect of the embodiments of the present invention, specifically, in step 2, the mass ratio of the gluten powder to water is 1:12.

[0018] In one aspect of the embodiments of the present invention, in step 4, the enzymatic hydrolysis time is 1.5-3 h.

[0019] In one aspect of the embodiment of the present invention, specifically, the enzymatic hydrolysis time is 2 hours.

[0020] In one aspect of the embodiments of the present invention, in step 6, the acidic solution is dilute hydrochloric acid or glacial acetic acid.

[0021] In one aspect of the embodiment of the present invention, in step 6, the enzymatic hydrolysis time is 1-2 hours.

[0022] In one aspect of the embodiments of the present invention, specifically, in step 6, the enzymatic hydrolysis time is 1.5 h.

[0023] In one aspect of the embodiments of the present invention, the immobilized pullulanase is prepared by the following steps:

[0024] Step 1-a: adding pullulanase and magnetic Fe3O4 nanoparticles to PBS buffer solution, respectively, shaking for 1-3 hours, and then mixing the two, combining at 4°C for 1.5-3 hours to obtain a third mixed solution;

[0025] Step 2-a: adding the first cross-linking agent to the third mixed solution, shaking at 20°C-35°C for 8-12 hours, and then standing for 8-12 hours, using a magnet to adsorb magnetic Fe3O4 nanoparticles to obtain the immobilized pullulanase.

[0026] In one aspect of the embodiment of the present invention, the pullulanase used in step 1-a is obtained after purification, but is not limited thereto.

[0027] In one aspect of the embodiment of the present invention, in the third mixed solution, the mass percentage of magnetic Fe3O4 nanoparticles is 2.5-5%.

[0028] In one aspect of an embodiment of the present invention, specifically, in the third mixed solution, the mass percentage of the magnetic Fe3O4 nanoparticles is 3.5%.

[0029] In one aspect of the embodiment of the present invention, in step 4, the amount of pullulanase used is 1000-2000 U / g raw material.

[0030] In one aspect of the embodiment of the present invention, in step 6, the dosage of α-amylase is 600-1500 U / g raw material.

[0031] In one aspect of an embodiment of the present invention, in step 2-a, after using a magnet to adsorb the magnetic Fe3O4 nanoparticles, a drying process is performed to obtain the immobilized pullulanase.

[0032] In one aspect of the embodiments of the present invention, the first cross-linking agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0033] In one aspect of the embodiment of the present invention, in the third mixed solution, the mass percentage of the first cross-linking agent is 1%-1.5%.

[0034] In one aspect of the embodiments of the present invention, the immobilized pullulanase is prepared by the following steps:

[0035] Step 1-a: Pullulanase and magnetic Fe3O4 nanoparticles were added to the same volume of PBS buffer solution, respectively, and shaken for 1.5 hours, and then the two were mixed and combined at 4°C for 2 hours to obtain a third mixed solution;

[0036] Step 2-a: Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the third mixed solution, shake at 25°C for 10 hours, then let it stand for 8 hours, use a magnet to adsorb magnetic Fe3O4 nanoparticles, and obtain the immobilized pullulanase after drying.

[0037] In one aspect of the embodiments of the present invention, the hydrogel material encapsulating α-amylase is prepared by the following steps:

[0038] Step 1-c: adding polyethylene glycol diacrylate and polyethylene glycol methacrylate to water, heating to 45° C.-60° C. and stirring, and then adding α-amylase solution dropwise to obtain a fourth mixed solution;

[0039] Step 2-c: lowering the temperature of the fourth mixed solution to room temperature, then adding a photoinitiator, and shaking for 2-4 hours to obtain a fifth mixed solution;

[0040] Step 3-c: irradiating the fifth mixed solution under a 365 nm ultraviolet lamp for 1-2 hours to obtain a hydrogel;

[0041] Step 4-c: The hydrogel is rinsed with a PBS buffer solution, and then a second cross-linking agent is added, and a cross-linking reaction is performed for 30-60 minutes to obtain a crude hydrogel material encapsulating α-amylase.

[0042] In one aspect of the embodiments of the present invention, the second cross-linking agent is glutaraldehyde.

[0043] In one aspect of the embodiment of the present invention, the mass of the second cross-linking agent is 1.5%-2.5% of the mass of the hydrogel obtained in step 3-c.

[0044] In one aspect of the embodiment of the present invention, the mass ratio of the polyethylene glycol diacrylate to the polyethylene glycol methacrylate is 1:(0.5-1.5). Preferably, the mass ratio of the polyethylene glycol diacrylate to the polyethylene glycol methacrylate is 1:(0.75-0.85).

[0045] In one aspect of the embodiment of the present invention, the sum of the masses of the polyethylene glycol diacrylate and the polyethylene glycol methacrylate in the fourth mixed solution accounts for 8%-20%, but is not limited thereto.

[0046] In one aspect of the embodiment of the present invention, the photoinitiator is 2-hydroxy-2-methylpropiophenone (HMPP), ethyl 2,4,6-(trimethylbenzoyl)phenylphosphinate (TPOL) or methyl benzoylformate (MBF). Preferably, the photoinitiator is 2-hydroxy-2-methylpropiophenone.

[0047] In one aspect of the embodiment of the present invention, in the fifth mixed solution, the mass percentage of the photoinitiator is 0.75%-1.2%.

[0048] In one aspect of the embodiment of the present invention, after step 4-c, step 5-c is further included:

[0049] Step 5-c: The crude product of the hydrogel material encapsulating α-amylase is crushed and passed through a 80-120 mesh sieve to obtain the hydrogel material encapsulating α-amylase.

[0050] In one aspect of the embodiments of the present invention, the hydrogel material encapsulating α-amylase is prepared by the following steps:

[0051] Step 1-c: adding polyethylene glycol diacrylate and polyethylene glycol methacrylate to water, heating to 55° C. and stirring, and then adding α-amylase solution dropwise to obtain a fourth mixed solution; wherein the mass ratio of polyethylene glycol diacrylate to polyethylene glycol methacrylate is 1:(0.75-0.85);

[0052] Step 2-c: lowering the temperature of the fourth mixed solution to room temperature, then adding 2-hydroxy-2-methylpropiophenone as a photoinitiator, and shaking for 2.5 hours to obtain a fifth mixed solution; wherein the mass percentage of the photoinitiator is 0.75%-1.2%;

[0053] Step 3-c: irradiating the fifth mixed solution under a 365 nm ultraviolet lamp for 1.5 h to obtain a hydrogel;

[0054] Step 4-c: Rinse the hydrogel with PBS buffer solution, then add glutaraldehyde as a cross-linking agent, and after 45 minutes of cross-linking reaction, obtain the crude hydrogel material encapsulating α-amylase; wherein the mass of glutaraldehyde is 1.5%-2.5% of the mass of the hydrogel obtained in step 3-c.

[0055] Step 5-c: The crude product of the hydrogel material encapsulating α-amylase is crushed and passed through a 100-mesh sieve to obtain the hydrogel material encapsulating α-amylase.

[0056] In one aspect of the embodiments of the present invention, the enzyme inactivation process comprises: heating the second enzymatic hydrolysate to 90° C.-95° C. and maintaining the temperature for 15-30 minutes.

[0057] In one aspect of the embodiments of the present invention, the centrifugation process includes: centrifuging the enzyme-inactivated hydrolyzate at 3500-4500 rpm for 15-30 min.

[0058] In one aspect of the embodiments of the present invention, the drying process is spray drying.

[0059] According to a second aspect of an embodiment of the present invention, a gluten short peptide is provided, and the gluten short peptide is prepared by the aforementioned method.

[0060] In one aspect of the embodiments of the present invention, the content of the short gluten peptides with a molecular weight less than 1000 Da is 89%-98%.

[0061] The technical solution provided by the embodiments of the present invention may have the following beneficial effects:

[0062] It can be seen from the above embodiments that, by the method provided by the present invention, a gluten short peptide with a molecular weight concentrated below 1000Da is prepared without using protease or cellulase; the present invention first enzymatically hydrolyzes the amylopectin of gluten by pullulanase, then destroys the wheat crude fiber by acetylacetone, and prepares a hydrogel-protected α-amylase, and the α-amylase has acid and oxidation resistance due to the protection of the hydrogel, and can further enzymatically hydrolyze gluten in the presence of acetylacetone and an acidic solution, so that a gluten short peptide with a molecular weight concentrated below 1000Da can be obtained.

[0063] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. The relevant embodiments described herein are illustrative and are used to provide a basic understanding of the present invention. The embodiments of the present invention should not be interpreted as limiting the present invention.

[0065] For simplicity, only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.

[0066] In this article, the terms "comprises", "comprising" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, an element defined by the sentence "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device that includes the element.

[0067] In the description herein, unless otherwise specified, “above” and “below” include the number.

[0068] Unless otherwise specified, the terms used in the present invention have the commonly known meanings generally understood by those skilled in the art. Unless otherwise specified, the numerical values ​​of the various parameters mentioned in the present invention can be measured by various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the embodiments of the present invention).

[0069] The term "about" is used to describe and illustrate small changes. When used in conjunction with an event or situation, the term may refer to an example in which the event or situation occurs precisely and an example in which the event or situation occurs very approximately. For example, when used in conjunction with a numerical value, the term may refer to a range of variation less than or equal to ±10% of the numerical value, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. In addition, amounts, ratios, and other numerical values ​​are sometimes presented in this article in a range format. It should be understood that such a range format is for convenience and simplicity, and should be flexibly understood to include not only numerical values ​​explicitly designated as range limits, but also all individual 10 numerical values ​​or subranges encompassed within the range, as if each numerical value and subrange were explicitly designated.

[0070] A list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms may mean any combination of the listed items. For example, if items A and B are listed, the phrase "at least one of A and B" means only A; only B; or A and B. In another example, if items A, B, and C are listed, the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A may contain a single component or multiple components. Item B may contain a single component or multiple components. Item C may contain a single component or multiple components.

[0071] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0072] Embodiment 1

[0073] A. Preparation of purified pullulanase and immobilized pullulanase:

[0074] Reagents and materials: PulASK expression strain Escherichia coli BL21 (DE3): purchased from Novagen; PuLASK gene (NCBI accession number EPZ37738): synthesized by BGI and codon optimized for Escherichia coli expression. Nde I / Xho I restriction site to construct expression plasmid pET-21PulASK; pullulan: purchased from Sigma; peptone and yeast extract: purchased from OXOID; other reagents are domestic analytical grade. LB medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, sterilized at 121℃ for 15 min.

[0075] The constructed expression plasmid pET-21PulASK was transferred into BL21 (DE3), transferred to LB medium containing Amp (100μg / mL), cultured at 37℃, 200 r / min until the OD 600nm value was 0.6-0.8, and IPTG with a final concentration of 0.2mmol / L was added, and the expression was induced at 18℃, 200 r / min for 16h. The induced cultured bacteria were collected by centrifugation, resuspended in lysis buffer (20 mmol / LPBS, 150 mmol / L NaCl, 10 mmol / L imidazole, pH 7.4), broken by high-pressure homogenizer, centrifuged at 10000r / min, 4℃ for 60 min, and the supernatant was collected for use. Since pET-21a (+) carries a histidine tag, BeyoGold TM His-tag filler can be used to purify the target protein. First, filter the supernatant with a 0.45 μm filter membrane, then use the equilibrated filler to bind at 4°C for 1 hour, then wash with equilibration buffer (20 mmol / L PBS, 150 mmol / L NaCl, 20mmol / L imidazole, pH 7.4) for 5 times the column volume, and finally elute the target protein with elution buffer (20 mmol / L PBS, 150 mmol / LNaCl, 500 mmol / L imidazole, pH 7.4). After SDS-PAGE detection, the target protein was desalted and concentrated using a 50 K ultrafiltration concentrator for later use.

[0076] A certain amount of purified Pulask was added to a buffer solution (20 mmol / L PBS, 150 mmol / L NaCl, pH 6.0), and the magnetic material Fe3O4 was added to the buffer solution (20 mmol / L PBS, 150 mmol / L NaCl, pH 6.0), and the two solutions were shaken for 1.5 hours respectively. Then the two solutions were mixed and combined at 4°C for 2 hours to obtain a third mixed solution. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (the amount of the cross-linking agent added was 1.2%) was added to the third mixed solution, and the solution was shaken at 25°C for 10 hours. Then the solution was allowed to stand for 8 hours, and the magnetic Fe3O4 nanoparticles were adsorbed by a magnet. After drying, the immobilized pullulanase was obtained.

[0077] B. Preparation of hydrogel material for embedding α-amylase:

[0078] Add polyethylene glycol diacrylate and polyethylene glycol methacrylate to 75 mL of water, heat to 55° C. and keep stirring, then add α-amylase solution dropwise to obtain a fourth mixed solution; wherein the mass ratio of polyethylene glycol diacrylate to polyethylene glycol methacrylate is 1:0.8; and the mass percentage of the sum of the masses of the two in the fourth mixed solution is 12%.

[0079] The fourth mixed solution was cooled to room temperature, and then 2-hydroxy-2-methylpropiophenone was added as a photoinitiator, and the mixture was shaken for 2.5 hours to obtain a fifth mixed solution, wherein the mass percentage of the photoinitiator was 0.9%;

[0080] The fifth mixed solution was irradiated under a 365 nm ultraviolet lamp for 1.5 h to obtain a hydrogel, and the hydrogel was rinsed with a PBS buffer solution (20 mmol / L PBS, 150 mmol / L NaCl, pH 6.0). Then, glutaraldehyde was added as a cross-linking agent (the mass of glutaraldehyde was 2% of the mass of the hydrogel). After 45 minutes of cross-linking reaction, a crude hydrogel material encapsulating α-amylase was obtained.

[0081] The crude hydrogel material encapsulating α-amylase is crushed and passed through a 100-mesh sieve to obtain the hydrogel material encapsulating α-amylase.

[0082] C. Preparation of gluten short peptides by double enzyme complex enzymolysis:

[0083] The gluten powder is ball-milled to obtain a crude gluten powder; the crude gluten powder is sieved to obtain a gluten powder; the ball milling process includes: adding the gluten powder to anhydrous ethanol for ball milling, the mass ratio of the gluten powder to the anhydrous ethanol is 1:4, 8 10mm agate balls and 30 6mm agate balls are used as grinding balls, the ball mill setting parameters are 250rpm, 45min of ball milling time, and 150 mesh of sieving.

[0084] The gluten powder was mixed with water, and then heated at 80° C. for 2.5 hours to obtain a first mixed slurry; acetic acid-sodium acetate buffer was added to the first mixed slurry, and the pH value was adjusted to 5.5 to obtain a second mixed slurry;

[0085] The temperature of the second mixed slurry was lowered to 55° C., and then the immobilized pullulanase prepared above was added for enzymolysis. The enzymolysis time was 2 h, and the pH value and temperature were kept unchanged to obtain the first enzymolysis solution; wherein the amount of pullulanase used was 1500 U / g raw material.

[0086] Dilute hydrochloric acid and acetylacetone were added to the first enzymatic hydrolysis solution to adjust the pH value to 3.2, and then a hydrogel material encapsulating α-amylase was added, and enzymatic hydrolysis was performed at 45° C. The enzymatic hydrolysis time was 1.5 h, and the pH value and temperature were kept unchanged to obtain a second enzymatic hydrolysis solution; wherein the amount of α-amylase used was 1000 U / g raw material, and the content of acetylacetone was 0.1 mol / L.

[0087] The second enzymatic hydrolysate was subjected to inactivation of enzymes, centrifugation and spray drying to obtain the gluten short peptides of Example 1.

[0088] Embodiment 2

[0089] The steps of Example 2 are basically the same as those of Example 1, except that, in the preparation of the hydrogel material for embedding α-amylase, the mass ratio of polyethylene glycol diacrylate to polyethylene glycol methacrylate used is 1:0.5; and the mass percentage of the sum of the masses of the two in the fourth mixed solution is 12%.

[0090] Embodiment 3

[0091] The steps of Example 3 are basically the same as those of Example 1, except that in the preparation of the hydrogel material for embedding α-amylase, the mass ratio of polyethylene glycol diacrylate to polyethylene glycol methacrylate used is 1:1.5; the mass percentage of the sum of the masses of the two in the fourth mixed solution is 12%.

[0092] Comparative Example 1

[0093] The steps of Comparative Example 1 are substantially the same as those of Example 1, except that acetylacetone is not added in the preparation of gluten short peptides by double enzyme complex enzymolysis.

[0094] Comparative Example 2

[0095] The following steps are involved:

[0096] The gluten powder is ball-milled to obtain a crude gluten powder; the crude gluten powder is sieved to obtain a gluten powder; the ball milling process includes: adding the gluten powder to anhydrous ethanol for ball milling, the mass ratio of the gluten powder to the anhydrous ethanol is 1:4, 8 10mm agate balls and 30 6mm agate balls are used as grinding balls, the ball mill setting parameters are 250rpm, 45min of ball milling time, and 150 mesh of sieving.

[0097] The gluten powder was mixed with water, and then heated at 80° C. for 2.5 hours to obtain a first mixed slurry; acetic acid-sodium acetate buffer was added to the first mixed slurry, and the pH value was adjusted to 5.5 to obtain a second mixed slurry;

[0098] The temperature of the second mixed slurry was lowered to 55° C., and then commercially available pullulanase was added for enzymolysis. The enzymolysis time was 2 h, and the pH value and temperature were kept constant to obtain a first enzymolysis solution. The amount of pullulanase used was 1500 U / g of raw material.

[0099] The pH value of the first enzymatic hydrolyzate was adjusted to 6.0, and then commercially available α-amylase was added, and enzymatic hydrolysis was carried out at 45° C. for 1.5 h, while the pH value and temperature were kept unchanged to obtain a second enzymatic hydrolyzate; wherein the amount of α-amylase used was 1000 U / g raw material.

[0100] The second enzymatic hydrolysate was subjected to inactivation of enzymes, centrifugation and spray drying to obtain gluten short peptides of Comparative Example 2.

[0101] Comparative Example 3

[0102] The following steps are involved:

[0103] The gluten powder is ball-milled to obtain a crude gluten powder; the crude gluten powder is sieved to obtain a gluten powder; the ball milling process includes: adding the gluten powder to anhydrous ethanol for ball milling, the mass ratio of the gluten powder to the anhydrous ethanol is 1:4, 8 10mm agate balls and 30 6mm agate balls are used as grinding balls, the ball mill setting parameters are 250rpm, 45min of ball milling time, and 150 mesh of sieving.

[0104] The gluten powder was mixed with water, and then heated at 80° C. for 2.5 hours to obtain a first mixed slurry; acetic acid-sodium acetate buffer was added to the first mixed slurry, and the pH value was adjusted to 5.5 to obtain a second mixed slurry;

[0105] The temperature of the second mixed slurry was lowered to 55° C., and then commercially available pullulanase was added for enzymolysis. The enzymolysis time was 2 h, and the pH value and temperature were kept constant to obtain a first enzymolysis solution. The amount of pullulanase used was 1500 U / g of raw material.

[0106] The pH value of the first enzymatic hydrolyzate was adjusted to 6.0, and then commercially available α-amylase and cellulase were added, and enzymatic hydrolysis was carried out at 45°C for 1.5 hours, while the pH value and temperature were kept unchanged to obtain a second enzymatic hydrolyzate; wherein the amount of α-amylase was 1000U / g raw material, and the amount of cellulase was 2000U / g raw material.

[0107] The second enzymatic hydrolysate was subjected to inactivation of enzymes, centrifugation and spray drying to obtain gluten short peptides of Comparative Example 3.

[0108] Comparative Example 4

[0109] The following steps are involved:

[0110] A. Preparation of purified pullulanase and immobilized pullulanase:

[0111] The steps of Comparative Example 4 are the same as those of Example 1.

[0112] B. Preparation of gluten short peptides by enzymatic hydrolysis:

[0113] The gluten powder is ball-milled to obtain a crude gluten powder; the crude gluten powder is sieved to obtain a gluten powder; the ball milling process includes: adding the gluten powder to anhydrous ethanol for ball milling, the mass ratio of the gluten powder to the anhydrous ethanol is 1:4, 8 10mm agate balls and 30 6mm agate balls are used as grinding balls, the ball mill setting parameters are 250rpm, 45min of ball milling time, and 150 mesh of sieving.

[0114] The gluten powder was mixed with water, and then heated at 80° C. for 2.5 hours to obtain a first mixed slurry; acetic acid-sodium acetate buffer was added to the first mixed slurry, and the pH value was adjusted to 5.5 to obtain a second mixed slurry;

[0115] The temperature of the second mixed slurry was lowered to 55° C., and then the immobilized pullulanase prepared above was added for enzymolysis. The enzymolysis time was 2 h, and the pH value and temperature were kept unchanged to obtain the first enzymolysis solution; wherein the amount of pullulanase used was 1500 U / g raw material.

[0116] Dilute hydrochloric acid was added to the first enzymolysis solution to adjust the pH value to 3.2, and then commercially available α-amylase and cellulase were added, and enzymolysis was carried out at 45°C for 1.5 hours, while the pH value and temperature were kept unchanged to obtain a second enzymolysis solution; wherein the amount of α-amylase was 1000U / g raw material, and the amount of cellulase was 2000U / g raw material.

[0117] The second enzymatic hydrolysate was subjected to inactivation of enzymes, centrifugation and spray drying to obtain the gluten short peptide of Comparative Example 4.

[0118] Comparative Example 5

[0119] The following steps are involved:

[0120] A. Preparation of purified pullulanase and immobilized pullulanase:

[0121] This step of comparative example 5 is the same as that of embodiment 1.

[0122] B. Preparation of hydrogel material for embedding α-amylase:

[0123] This step of comparative example 5 is the same as that of embodiment 1.

[0124] C. Preparation of gluten short peptides by double enzyme complex enzymolysis:

[0125] The gluten powder is ball-milled to obtain a crude gluten powder; the crude gluten powder is sieved to obtain a gluten powder; the ball milling process includes: adding the gluten powder to anhydrous ethanol for ball milling, the mass ratio of the gluten powder to the anhydrous ethanol is 1:4, 8 10mm agate balls and 30 6mm agate balls are used as grinding balls, the ball mill setting parameters are 250rpm, 45min of ball milling time, and 150 mesh of sieving.

[0126] The gluten powder is mixed with water, and then heated at 80° C. for 2.5 hours to obtain a first mixed slurry; acetic acid-sodium acetate buffer, dilute hydrochloric acid and acetylacetone are added to the first mixed slurry, and the pH value is adjusted to 3.2 to obtain a first mixed slurry;

[0127] The temperature of the first mixed slurry was lowered to 45°C, and then the hydrogel material encapsulating α-amylase prepared above was added for enzymatic hydrolysis. The enzymatic hydrolysis time was 1.5 h, and the pH value and temperature were kept unchanged to obtain the first enzymatic hydrolyzate; wherein the amount of α-amylase used was 1000 U / g raw material.

[0128] The immobilized pullulanase prepared above was added to the first enzymolysis solution, the pH value was adjusted to 5.5, and enzymolysis was carried out at 55° C. for 2 h, while the pH value and temperature were kept constant to obtain a second enzymolysis solution; wherein the amount of pullulanase used was 1500 U / g raw material.

[0129] The second enzymatic hydrolysate was subjected to inactivation of enzymes, centrifugation and spray drying to obtain the gluten short peptides of Comparative Example 5.

[0130] Comparative Example 6

[0131] The steps of Comparative Example 6 are substantially the same as those of Example 4, except that acetylacetone is further added to Comparative Example 6, and the content of acetylacetone is the same as that of Example 1.

[0132] The molecular weight distribution of the peptides of Examples 1 to 3 and Comparative Examples 1 to 6 was measured by GPC. The results are shown in the following table:

[0133]

[0134] Those skilled in the art will readily appreciate other embodiments of the present invention after considering the specification and practicing the disclosure disclosed herein. The present invention is intended to cover any variations, uses or adaptations of the present invention that follow the general principles of the present invention and include common knowledge or customary technical means in the art that are not disclosed by the present invention.

Claims

1. A method for preparing short gluten peptides by double enzyme complex enzymolysis, characterized in that: The following steps are involved: Step 1: ball-milling gluten powder to obtain a crude gluten powder, and sieving the crude gluten powder to obtain gluten powder; Step 2: mixing the gluten powder with water, and then heating at 75° C.-90° C. for 1.5-4 hours to obtain a first mixed slurry; Step 3: adding acetic acid-sodium acetate buffer to the first mixed slurry, and adjusting the pH value to 5.0-6.0 to obtain a second mixed slurry; Step 4: lowering the temperature of the second mixed slurry to 50° C.-65° C., and then adding immobilized pullulanase for enzymolysis to obtain a first enzymolysis solution; during the enzymolysis process of step 4, keeping the pH value and temperature unchanged; Step 5: preparing a hydrogel material for embedding α-amylase; Step 6: adding an acidic solution and acetylacetone to the first enzymatic hydrolyzate, adjusting the pH value to 3.0-3.5, then adding a hydrogel material encapsulating α-amylase, and performing enzymolysis at 40° C.-50° C. to obtain a second enzymatic hydrolyzate; keeping the pH value and temperature unchanged during the enzymatic hydrolysis process in step 6; Step 7: inactivating the enzyme in the second enzymatic hydrolysate, centrifuging and drying to obtain the gluten short peptide; In step 1, the ball milling process includes: adding the gluten powder to anhydrous ethanol for ball milling, wherein the mass ratio of the gluten powder to the anhydrous ethanol is 1:(3.5-5), using 5-10 10 mm agate balls and 25-40 6 mm agate balls as grinding balls, and setting the parameters of the ball mill to a rotation speed of 200-250 rpm and a ball milling time of 30-60 min; In step 2, the mass ratio of the gluten powder to water is 1:(10-15); In step 4, the enzymatic hydrolysis time is 1.5-3h; In step 6, the enzymatic hydrolysis time is 1-2h; The immobilized pullulanase is prepared by the following steps: Step 1-a: adding pullulanase and magnetic Fe3O4 nanoparticles to PBS buffer solution, respectively, shaking for 1-3 hours, and then mixing the two, combining at 4°C for 1.5-3 hours to obtain a third mixed solution; Step 2-a: adding the first cross-linking agent to the third mixed solution, shaking at 20° C.-35° C. for 8-12 hours, and then standing for 8-12 hours, using a magnet to adsorb magnetic Fe3O4 nanoparticles to obtain the immobilized pullulanase; The hydrogel material for embedding α-amylase is prepared by the following steps: Step 1-c: adding polyethylene glycol diacrylate and polyethylene glycol methacrylate to water, heating to 45° C.-60° C. and stirring, and then adding α-amylase solution dropwise to obtain a fourth mixed solution; Step 2-c: lowering the temperature of the fourth mixed solution to room temperature, then adding a photoinitiator, and shaking for 2-4 hours to obtain a fifth mixed solution; Step 3-c: irradiating the fifth mixed solution under a 365 nm ultraviolet lamp for 1-2 hours to obtain a hydrogel; Step 4-c: washing the hydrogel with a PBS buffer solution, then adding a second cross-linking agent, and performing a cross-linking reaction for 30-60 minutes to obtain a crude hydrogel material encapsulating α-amylase; The first cross-linking agent is 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; The second cross-linking agent is glutaraldehyde; Step 7 is as follows: (1) The enzyme inactivation process comprises: heating the second enzymatic hydrolysate to 90°C-95°C and maintaining the temperature for 15-30 minutes; (2) The centrifugation process includes: centrifuging the enzyme-inactivated hydrolyzate at 3500-4500 rpm for 15-30 min; (3) The drying process is spray drying.

2. A gluten short peptide, characterized in that: The gluten short peptide is prepared by the method for preparing gluten short peptide by double enzyme complex enzymolysis as described in claim 1.

3. The gluten short peptide according to claim 2, characterized in that: The content of the gluten short peptides with a molecular weight less than 1000 Da is 89%-98%.

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