Special compound enzyme preparation for barley spent grain small-molecule active peptide

Abstract

The invention relates to the technical field of comprehensive utilization of agricultural by-products, and discloses a special compound enzyme preparation for barley spent grain small-molecule active peptides, which is composed of 30-35% of endo protease, 30-35% of excision peptidase, 20-25% of saccharifying enzyme, 0.4-0.6% of sorbitol, 5-10% of cellulase and the balance of carrier. Through the synergistic effect of specifically compounded endoprotease, exonuclease, saccharifying enzyme, cellulase and stabilizer, efficient hydrolysis and directional cutting of spent grain protein are realized under mild conditions, so that the yield of small-molecule active peptide in a target molecular weight range of 180-5000 Daltons and the proportion of oligopeptide are greatly improved; through coupling of radio frequency pretreatment and a composite enzymatic hydrolysis process, high energy consumption of traditional high-temperature drying is avoided, efficient enzymatic hydrolysis of wet grains without pre-drying is realized, and direct high-valued conversion of the wet grains is realized; the problems that in an existing spent grain treatment technology, the protein extraction rate is low, the yield of small-molecule active peptide is insufficient, the process is complex, energy consumption is high, and the value of by-products is not fully excavated are solved.

Description

Technical Field

[0001] This invention relates to the field of comprehensive utilization technology of agricultural by-products, specifically a compound enzyme preparation for small molecule active peptides in barley and wheat residue. Background Technology

[0002] Beer malt is a major byproduct of the brewing industry, rich in protein, cellulose, hemicellulose, and other components. Currently, wet malt has a high water content and is prone to spoilage, making its disposal a dual pressure on breweries, facing both environmental and cost challenges. In existing technologies, most wet malt is directly used as low-cost animal feed or disposed of in landfills, resulting in extremely low resource utilization value. Although existing studies have attempted to extract proteins or peptides from wheat lees, most processes suffer from the following prominent problems: (1) Low extraction efficiency: Traditional acid-base methods or single-enzyme methods do not completely break down the cell walls of wheat lees, resulting in low protein dissolution rates and easy destruction of peptide activity; (2) Unsatisfactory molecular weight distribution of target products: The proportion of high molecular weight peptides in the hydrolysis products of ordinary proteases is high, while the yield of small molecular weight active peptides, especially oligopeptides, is insufficient, affecting their absorption and functional activity; (3) Complex processes and high energy consumption: If wheat lees are dried before processing, the drying energy consumption is huge; if multi-step separation and enzymatic hydrolysis are used, the process is lengthy and costly; (4) Failure to achieve high-value utilization of all components: Most technologies only focus on protein extraction, ignoring the synergistic processing and utilization of components such as cellulose and hemicellulose in wheat lees. Therefore, developing a special enzyme preparation and supporting process that can efficiently and directionally convert wheat lees protein into high-value-added small molecular weight active peptides, while taking into account low energy consumption and full component utilization, is of great industrial significance. Summary of the Invention

[0003] (a) Technical problems to be solved

[0004] To address the shortcomings of existing technologies, this invention provides a special compound enzyme preparation for small molecule active peptides from barley lees. It has the advantages of efficient synergistic enzymatic hydrolysis, targeted generation of small molecule active peptides, simplified process, low energy consumption, and high raw material utilization. It solves the problems of low protein extraction rate, insufficient yield of small molecule active peptides, complex process and high energy consumption, and insufficient exploitation of by-product value in existing barley lees processing technologies.

[0005] (II) Technical Solution

[0006] To achieve the above objectives, the present invention provides the following technical solution: a special compound enzyme preparation for small molecule active peptides from barley lees, wherein the raw materials and their weight ratios of the compound enzyme preparation are: 30%–35% endopeptidase; 30%–35% exopeptidase; 20%–25% glucoamylase; 0.4%–0.6% sorbitol; 5%–10% cellulase; and the balance being 100% carrier.

[0007] Preferably, the preparation process of the endopeptide is as follows:

[0008] S1.1 Activation and propagation of strains: Select high protease-producing strains of Bacillus subtilis or Aspergillus oryzae, activate them on slant culture medium, transfer them to seed culture medium, and culture them at 35-37℃ and 200-250rpm for 18-24h to the logarithmic growth phase.

[0009] S1.2, Liquid fermentation for enzyme production: Inoculate the seed liquid into the fermenter at an inoculum rate of 7-9%, control the pH at 6.5-7.0, the temperature at 30-32℃, and the dissolved oxygen at >22%, and culture in batches for 36-48 hours until the enzyme activity peak is reached to obtain the fermentation broth;

[0010] S1.3 Enzyme extraction and purification: The fermentation broth was centrifuged, and the supernatant was concentrated by passing it through a 10-30 kDa ultrafiltration membrane. Then, it was further purified by DEAE-Sepharose Fast Flow ion exchange chromatography to obtain a purified enzyme solution. The specific activity of the purified enzyme solution was measured to be ≥390 U / mg.

[0011] S1.4 Freeze-drying and standardization: After the purified enzyme solution is pre-frozen at -40℃ for 4-6 hours, it is vacuum freeze-dried at -20℃ to 0℃ and vacuum degree <10Pa for 24-48 hours. The dried enzyme powder is then pulverized to a particle size ≤150μm to obtain freeze-dried enzyme powder with an enzyme activity ≥20,000U / g.

[0012] Preferably, the preparation process of the exopeptidase is as follows:

[0013] S2.1. Strain screening and culture: Select high-yielding exopeptidase strains of Aspergillus niger or Aspergillus umami, activate them on PDA slant, and inoculate them into solid seed culture medium containing wheat bran and soybean meal, and culture at 28-30℃ for 24-36h.

[0014] S2.2 Solid-state fermentation for enzyme production: Inoculate the seeds with a 5-10% inoculation rate into a sterilized substrate of wheat bran: soybean meal: rice husk = 5:3:2, adjust the moisture content to 60-65%, and incubate at 30-32℃ with ventilation for 48-60 hours, turning the substrate 2-3 times during the period;

[0015] S2.3 Extraction and Concentration: The fermentation material is extracted with 5-10 times the volume of phosphate buffer solution with a pH of 6.0-6.5 at 28-30℃ for 1-2 hours. After plate and frame filtration, the clear liquid is concentrated and desalted by ultrafiltration.

[0016] S2.4 Drying and Standardization: The concentrate is spray-dried for 10-15 seconds under the conditions of inlet air temperature of 160-180℃ and outlet air temperature of 70-80℃ to obtain enzyme powder.

[0017] Preferably, the cellulase is induced to produce enzyme using microcrystalline cellulose or brewer's grains cellulose as a carbon source, and its filter paper enzyme activity is ≥80U / g, and its CMC enzyme activity is ≥500U / g.

[0018] Preferably, the barley residue small molecule active peptide-specific compound enzyme preparation includes the following preparation steps:

[0019] Step 1: Raw material preparation: Prepare the formula proportions of endopeptidase, exopeptidase, glucoamylase, sorbitol, cellulase and carrier;

[0020] Step 2, Raw material pretreatment: Dry heat sterilization of the carrier;

[0021] Step 3: Compound enzyme formulation: The endopeptidase, exopeptidase and glucoamylase components are mixed stepwise according to the formula ratio to obtain premixed enzyme powder;

[0022] Step 4, Final Mixing and Quality Control: The premixed enzyme powder obtained in Step 3 is finally mixed with the remaining components, and the quality control test of the preparation is completed to obtain the compound enzyme preparation.

[0023] Preferably, in step two, the raw material pretreatment involves sterilizing the carrier at 105-120℃ for 1.5-2.5 hours and then naturally cooling it to room temperature before use, with its moisture content controlled at ≤5%.

[0024] Preferably, in step three, the compound enzyme is prepared by mixing endopeptidase, exopeptidase and glycating enzyme in a three-dimensional mixer at 15-20 rpm for 10-15 min to obtain premixed enzyme powder.

[0025] Preferably, in step four, the final mixing involves: putting the premixed enzyme powder obtained in step three into a high-efficiency three-dimensional mixer, first adding cellulase and sorbitol and continuing to mix for 8-10 minutes; finally adding the pretreated carrier and continuing to mix for 15-20 minutes until the uniformity RSD of the sample test is ≤5%, thus obtaining the finished compound enzyme preparation. Samples are then taken for enzyme activity testing and small molecule peptide conversion rate verification.

[0026] Preferably, the enzyme activity of the finished compound enzyme preparation is: total enzyme activity ≥35000U / g.

[0027] Preferably, the compound enzyme preparation is used to verify the conversion rate of small molecule peptides from barley residue:

[0028] S4.1 RF pretreatment of beer lees: Place the wet beer lees with a moisture content of 75-80% in an RF heating device. Under the conditions of a frequency of 27-27.12MHz and an electrode spacing of 8-10cm, control the material temperature to rise to 60-65℃ and maintain it for 8-10min to promote the development of protein secondary structure and at the same time inactivate some miscellaneous bacteria.

[0029] S4.2 Enzymatic hydrolysis: Add compound enzyme preparation at 0.3-0.5% of the weight of wet lees, with a material-to-liquid ratio of 1:3-1:5. Adjust the pH to 6.5-7.0 and enzymatically hydrolyze for 3.5-4.0 hours at 50±2℃. After enzymatic hydrolysis, the content of small molecule active peptides with a molecular weight of 180-5000 Da in the hydrolysate is ≥35%, of which oligopeptides with a molecular weight of <1000 Da account for ≥20%.

[0030] Compared with the prior art, the present invention provides a special compound enzyme preparation of small molecule active peptides from barley lees, which has the following beneficial effects:

[0031] 1. This invention achieves efficient hydrolysis and targeted tailoring of wheat lees protein under mild conditions through the synergistic effect of a specific compound of endopeptidase, exopeptidase, glucoamylase, cellulase, and stabilizer. This significantly increases the yield of small molecule active peptides and the proportion of oligopeptides in the target molecular weight range of 180-5000 Daltons. By coupling radio frequency pretreatment with a compound enzymatic hydrolysis process, the high energy consumption of traditional high-temperature drying is avoided, enabling efficient enzymatic hydrolysis of wet lees without the need for pre-drying, thus facilitating the direct high-value conversion of wet lees. At the same time, this process creates favorable conditions for the subsequent separation and high-value utilization of cellulase components in wheat lees, promoting the resource utilization of all components. Attached Figure Description

[0032] Figure 1 This is a flowchart illustrating the preparation process of the compound enzyme preparation of the present invention. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Please see Figure 1 A special compound enzyme preparation for small molecule active peptides from barley and wheat lees, wherein the raw materials and their weight ratios are as follows: 30%–35% endopeptidase; 30%–35% exopeptidase; 20%–25% saccharifying enzyme; 0.4%–0.6% sorbitol; 5%–10% cellulase (assisting in liquefying the shell); and the balance being 100% carrier (such as maltodextrin).

[0035] Specifically, the raw materials and their functions are shown in Table 1 below:

[0036] Table 1

[0037]

[0038] Specifically, the preparation process of the endopeptide is as follows:

[0039] S1.1 Activation and propagation of strains: Select high protease-producing strains of Bacillus subtilis or Aspergillus oryzae, activate them on slant culture medium, transfer them to seed culture medium, and culture them at 35-37℃ and 200-250rpm for 18-24h to the logarithmic growth phase.

[0040] S1.2, Liquid fermentation for enzyme production: Inoculate the seed liquid into the fermenter at an inoculum rate of 7-9%, control the pH at 6.5-7.0, the temperature at 30-32℃, and the dissolved oxygen at >22%, and culture in batches for 36-48 hours until the enzyme activity peak is reached to obtain the fermentation broth;

[0041] S1.3 Enzyme extraction and purification: The fermentation broth was separated into solid and liquid components by centrifugation or microfiltration. The supernatant was concentrated by ultrafiltration through a 10-30 kDa membrane and then further purified by DEAE-Sepharose Fast Flow ion exchange chromatography to obtain purified enzyme solution. The specific activity of the purified enzyme solution was measured to be ≥390 U / mg.

[0042] S1.4 Freeze-drying and standardization: After pre-freezing the purified enzyme solution at -40℃ for 4-6 hours, it is vacuum freeze-dried at -20℃ to 0℃ and a vacuum degree <10Pa for 24-48 hours. The dried enzyme powder is then pulverized to a particle size ≤150μm, and the enzyme activity is measured. When the enzyme activity of the freeze-dried powder is ≥20,000U / g, it is physically mixed with other raw materials according to the calculated ratio based on the measured enzyme activity, and adjusted to the specified content of endopeptide in the final compound preparation (i.e., 30%-35% by weight).

[0043] The advantages are: by selecting high-yield and specific strains, the high activity and specificity of the enzyme source are ensured; by adopting a controllable fed-batch fermentation and fine purification process, the specific activity (≥390U / mg) and recovery rate of the endopeptide are improved; and by using low-temperature vacuum freeze-drying technology, the enzyme activity can be preserved to the maximum extent, so that the activity and stability of the final enzyme powder meet the industrial application standards (≥20,000U / g), thus providing a core guarantee for the precise formulation and stable efficacy of compound enzyme preparations.

[0044] Specifically, the preparation process of exopeptidase is as follows:

[0045] S2.1. Strain screening and culture: Select high-yielding exopeptidase strains of Aspergillus niger or Aspergillus umami, activate them on PDA slant, and inoculate them into solid seed culture medium containing wheat bran and soybean meal, and culture at 28-30℃ for 24-36h.

[0046] S2.2 Solid-state fermentation for enzyme production: Inoculate the seeds with a 5-10% inoculation rate into a sterilized substrate of wheat bran: soybean meal: rice husk = 5:3:2, adjust the moisture content to 60-65%, and incubate at 30-32℃ with ventilation for 48-60 hours, turning the substrate 2-3 times during the period;

[0047] S2.3 Extraction and Concentration: The fermentation material is extracted with 5-10 times the volume of phosphate buffer (pH 6.0-6.5) at 28-30℃ for 1-2 hours. After plate and frame filtration, the clear liquid is concentrated and desalted by ultrafiltration (5kDa).

[0048] S2.4 Drying and Standardization: The concentrate is spray-dried for 10-15 seconds at an inlet air temperature of 160-180℃ and an outlet air temperature of 70-80℃ to obtain enzyme powder. The enzyme activity is measured. When the enzyme activity of the enzyme powder is ≥15,000U / g, it is physically compounded with other raw materials according to the target enzyme activity to complete the standardization process and ensure that its content in the final compound preparation is stable at 30%-35%.

[0049] The advantages are: by adopting solid-state fermentation technology and using inexpensive agricultural by-products as the main substrate, the cost is low, the energy consumption is small, and it is more suitable for the induction expression of exopeptidase; through optimized extraction and membrane concentration processes, the enzyme is efficiently recovered and rapidly desalted, the process is simple and easy to scale up; by adopting medium-low temperature spray drying, the enzyme activity can be protected to the greatest extent during the instantaneous drying process, resulting in high enzyme powder activity (≥15,000U / g) and good flowability, which facilitates subsequent precise compounding and standardized production.

[0050] Specifically, cellulase is induced to produce enzymes using microcrystalline cellulose or brewer's grains cellulose as a carbon source, with filter paper enzyme activity (FPA) ≥ 80 U / g and CMC enzyme activity ≥ 500 U / g.

[0051] Specifically, the preparation steps for the barley and wheat residue small molecule active peptide compound enzyme preparation are as follows:

[0052] Step 1: Raw material preparation: Prepare the formula proportions of endopeptidase, exopeptidase, saccharifying enzyme (amylase), sorbitol, cellulase (to assist in liquefying the shell) and carrier;

[0053] Step 2, Raw material pretreatment: Dry heat sterilization of the carrier;

[0054] Step 3: Compound enzyme formulation: The endopeptidase, exopeptidase and glucoamylase components are mixed stepwise according to the formula ratio to obtain premixed enzyme powder;

[0055] Step 4, Final Mixing and Quality Control: The premixed enzyme powder obtained in Step 3 is finally mixed with the remaining components, and the quality uniformity and stability of the preparation are confirmed to obtain the compound enzyme preparation.

[0056] Specifically, in step two, the raw material pretreatment involves sterilizing the carrier (maltodextrin) at 105-120℃ for 1.5-2.5 hours with dry heat and allowing it to cool naturally to room temperature before use. The moisture content of the carrier is controlled to be ≤5%. This pretreatment effectively inactivates microorganisms in the carrier and reduces moisture content, preventing the subsequent enzyme preparations from becoming inactive due to moisture absorption or microbial contamination during storage, thus ensuring the biosafety and storage stability of the finished product.

[0057] Specifically, in step three, the compound enzyme is prepared by mixing endopeptidase, exopeptidase, and glucoamylase in a three-dimensional mixer at 15-20 rpm for 10-15 minutes to obtain premixed enzyme powder. This low-speed, short-time preliminary mixing can achieve preliminary homogenization of the three core enzyme components, avoiding the dust and uneven mixing problems that may be caused by direct one-time mixing, and laying a good foundation for subsequent final mixing.

[0058] Specifically, in step four, final mixing involves adding the premixed enzyme powder obtained in step three into a high-efficiency three-dimensional mixer. First, add cellulase and sorbitol and continue mixing for 8-10 minutes. Finally, add the pretreated carrier and continue mixing for 15-20 minutes until the uniformity RSD of the sample is ≤5%, thus obtaining the finished compound enzyme preparation. Samples are then taken for enzyme activity testing and small molecule peptide conversion rate verification (the entire mixing process is strictly controlled with ambient temperature ≤25℃ and relative humidity ≤40%). Through this staged, temperature- and humidity-controlled final mixing process, the uniform dispersion of trace components (such as sorbitol) and the maximum protection of the activity of all enzyme components during the mixing process can be ensured, ultimately resulting in a uniform, stable, and quality-compliant finished compound enzyme preparation.

[0059] Specifically, the enzyme activity test of the finished compound enzyme preparation is as follows: total enzyme activity ≥ 35000 U / g; the enzyme activity test method shall be implemented in accordance with the national standards GB / T23527-2009 and GB 1886.174-2016.

[0060] Specifically, the conversion rate of small molecule peptides from barley grains was verified using compound enzyme preparations:

[0061] S4.1 RF pretreatment of beer lees: Place the wet beer lees with a moisture content of 75-80% in an RF heating device. Under the conditions of a frequency of 27-27.12MHz and an electrode spacing of 8-10cm, control the material temperature to rise to 60-65℃ and maintain it for 8-10min to promote the development of protein secondary structure and at the same time inactivate some miscellaneous bacteria.

[0062] S4.2 Enzymatic hydrolysis: Add compound enzyme preparation at 0.3-0.5% of the weight of wet residue, with a material-to-liquid ratio of 1:3-1:5 (w / v), adjust the pH to 6.5-7.0, and hydrolyze at 50±2℃ for 3.5-4.0 hours. After hydrolysis, the content of small molecule active peptides with a molecular weight of 180-5000 Da in the hydrolysate should be ≥35% (based on total protein), of which oligopeptides with a molecular weight <1000 Da should account for ≥20%. During the hydrolysis process, intermittent stirring should be performed every 15-20 minutes for 3-5 minutes. The total stirring time during the entire hydrolysis cycle is about 40-60 minutes to ensure that the material and enzyme are in full contact and to prevent local overheating or precipitation.

[0063] S4.3 Enzyme inactivation: After enzymatic hydrolysis, raise the temperature to 85-90℃ and maintain it for 10-15 minutes to inactivate the enzyme.

[0064] The advantages are: through the above-mentioned stepwise, gradient, and controlled mixing process, combined with strict raw material pretreatment and environmental control, the uniform dispersion and activity of each enzyme component and trace additive are ensured; finally, through standardized verification, it is proved that the compound enzyme preparation has high enzyme activity and high specificity, and can stably and efficiently convert barley lees into high-value-added small molecule active peptides.

[0065] The compound enzyme preparation of the present invention was applied to the following experiments:

[0066] Example 1 (Standard Process Validation)

[0067] Take 1000g of fresh wet beer lees with a moisture content of 78% (total protein content on a dry basis is 28%) and perform RF pretreatment according to S4.1 (60℃, 10min); after pretreatment, add 3 times the weight of pure water and adjust the pH to 6.8; add 5g (0.5%) of the compound enzyme preparation of this invention, and enzymatically hydrolyze in a 50℃ water bath for 3.5 hours, stirring for 5 minutes every 20 minutes during the process; after the enzymatic hydrolysis is completed, immediately inactivate the enzyme in an 85℃ water bath for 15 minutes; after cooling, centrifuge and collect the supernatant to determine relevant indicators.

[0068] Example 2 (Optimized Enzymatic Hydrolysis Time)

[0069] Except for extending the enzymatic hydrolysis time to 4.0 hours, the other raw materials, dosages, and operating steps are exactly the same as in Example 1.

[0070] Example 3 (Optimization of material-to-liquid ratio and enzyme dosage)

[0071] Take 1000g of wet lees from the same batch as in Example 1, pretreat it with the same RF, add 5 times the weight of pure water (material-to-liquid ratio 1:5), and adjust the pH to 7.0; add 3g (0.3%) of the compound enzyme preparation of the present invention, and enzymatically hydrolyze it in a 50℃ water bath for 4.0 hours, with the stirring method the same as in Example 1; after the enzymatic hydrolysis is completed, inactivate the enzyme, process and measure it in the same way.

[0072] Examples 1-3 were performed in parallel (n=3) and compared with a control group treated with a single commercial protease (with the same amount of enzyme activity). The experimental data are shown in Table 2 below:

[0073] Table 2: Comparison of the effects of compound enzyme preparations on barley lees under different conditions

[0074]

[0075] Note:

[0076] (1) Peptide yield refers to the dry weight of peptides produced per unit weight of wet residue;

[0077] (2) Protein recovery rate = (soluble protein in enzymatic hydrolysate / total protein in raw material) × 100%;

[0078] (3) All data are mean ± standard deviation.

[0079] Table 2 shows that, as verified by Example 1 (standard process), the compound enzyme preparation of the present invention can significantly improve the conversion rate of small molecule peptides (36.8%) and the proportion of oligopeptides (22.4%). All indicators are significantly better than the single enzyme control group, proving the superiority of the synergistic effect of the compound enzyme of the present invention. The experiment of Example 2 (extending the enzymatic hydrolysis time to 4h) verified that the conversion depth of small molecule peptides was further improved under enzymatic hydrolysis, and the content of small molecule peptides and the proportion of oligopeptides were increased. Example 3 (reducing the amount of enzyme but increasing the amount of water and the enzymatic hydrolysis time) achieved the best conversion effect by optimizing the mass transfer conditions at a lower amount of enzyme. The content of small molecule peptides reached 41.2%, the proportion of oligopeptides <1000Da was close to 30%, and the protein recovery rate and product antioxidant activity were the highest, indicating that the process has optimization potential in terms of efficiency and economy.

[0080] In summary, the compound enzyme preparation of the present invention can stably and efficiently catalyze the production of high-value-added small molecule active peptides from barley lees under different process conditions.

[0081] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A special complex enzyme preparation for small molecule active peptides of barley stillage, characterized in that, The raw materials and their weight ratios for the compound enzyme preparation are as follows: 30%–35% endopeptidase; 30%–35% exopeptidase; 20%–25% glucoamylase; 0.4%–0.6% sorbitol; 5%–10% cellulase; and the balance of carrier to 100%.

2. The special complex enzyme preparation for malt small molecule active peptide of barley's stillage according to claim 1, characterized in that: The preparation process of the endopeptidase is as follows: S1.1 Activation and propagation of strains: Select high protease-producing strains of Bacillus subtilis or Aspergillus oryzae, activate them on slant culture medium, transfer them to seed culture medium, and culture them at 35-37℃ and 200-250rpm for 18-24h to the logarithmic growth phase. S1.2, Liquid fermentation for enzyme production: Inoculate the seed liquid into the fermenter at an inoculum rate of 7-9%, control the pH at 6.5-7.0, the temperature at 30-32℃, and the dissolved oxygen at >22%, and culture in batches for 36-48 hours until the enzyme activity peak is reached to obtain the fermentation broth; S1.3 Enzyme extraction and purification: The fermentation broth was centrifuged, and the supernatant was concentrated by passing it through a 10-30 kDa ultrafiltration membrane. Then, it was further purified by DEAE-Sepharose Fast Flow ion exchange chromatography to obtain a purified enzyme solution. The specific activity of the purified enzyme solution was measured to be ≥390 U / mg. S1.4 Freeze-drying and standardization: After the purified enzyme solution is pre-frozen at -40℃ for 4-6 hours, it is vacuum freeze-dried at -20℃ to 0℃ and vacuum degree <10Pa for 24-48 hours. The dried enzyme powder is then pulverized to a particle size ≤150μm to obtain freeze-dried enzyme powder with an enzyme activity ≥20,000U / g.

3. The special complex enzyme preparation for malt small molecule active peptide of barley's stillage according to claim 1, characterized in that: The preparation process of the exopeptidase is as follows: S2.

1. Strain screening and culture: Select high-yielding exopeptidase strains of Aspergillus niger or Aspergillus umami, activate them on PDA slant, and inoculate them into solid seed culture medium containing wheat bran and soybean meal, and culture at 28-30℃ for 24-36h. S2.2 Solid-state fermentation for enzyme production: Inoculate the seeds with a 5-10% inoculation rate into a sterilized substrate of wheat bran: soybean meal: rice husk = 5:3:2, adjust the moisture content to 60-65%, and incubate at 30-32℃ with ventilation for 48-60 hours, turning the substrate 2-3 times during the period; S2.3 Extraction and Concentration: The fermentation material is extracted with 5-10 times the volume of phosphate buffer solution with a pH of 6.0-6.5 at 28-30℃ for 1-2 hours. After plate and frame filtration, the clear liquid is concentrated and desalted by ultrafiltration. S2.4 Drying and Standardization: The concentrate is spray-dried for 10-15 seconds under the conditions of inlet air temperature of 160-180℃ and outlet air temperature of 70-80℃ to obtain enzyme powder.

4. The special complex enzyme preparation for malt small molecule active peptide according to claim 1, characterized in that: The cellulase is induced to produce enzymes using microcrystalline cellulose or the cellulose of brewer's grains as a carbon source, and its filter paper enzyme activity is ≥80U / g, and its CMC enzyme activity is ≥500U / g.

5. The malt spent grain small molecule active peptide specific complex enzyme preparation according to claim 1, characterized in that, The preparation steps include the following: Step 1: Raw material preparation: Prepare the formula proportions of endopeptidase, exopeptidase, glucoamylase, sorbitol, cellulase and carrier; Step 2, Raw material pretreatment: Dry heat sterilization of the carrier; Step 3: Compound enzyme formulation: The endopeptidase, exopeptidase and glucoamylase components are mixed stepwise according to the formula ratio to obtain premixed enzyme powder; Step four, final mixing and quality control: the pre-mixed enzyme powder obtained in step three is mixed with the remaining components, and quality control tests of the preparation are completed to obtain the complex enzyme preparation.

6. The malt spent grain small molecule active peptide specific complex enzyme preparation according to claim 5, characterized in that: In the step two, the raw material pretreatment: the carrier is sterilized by dry heat at 105-120 DEG C for 1.5-2.5 h, and then naturally cooled to room temperature for standby, and the moisture content is controlled to be ≤5%.

7. The special complex enzyme preparation for malt small molecule active peptide according to claim 5, characterized in that: In the step three, the complex enzyme compounding: the endoprotease, exopeptidase and glucoamylase are mixed in a three-dimensional mixer at 15-20 rpm for 10-15 min to obtain a pre-mixed enzyme powder.

8. The special complex enzyme preparation for malt small molecule active peptide according to claim 5, characterized in that: In the step four, the final mixing: the pre-mixed enzyme powder obtained in step three is put into a high-efficiency three-dimensional mixer, and the cellulase and sorbitol are added first and continue to mix for 8-10 min; finally, the pretreated carrier is added and continue to mix for 15-20 min, until the uniformity RSD of the sample detection is ≤5%, and the complex enzyme preparation product is obtained, and the sample is taken for enzyme activity detection and small molecule peptide conversion rate verification.

9. The malt spent grain small molecule active peptide specific complex enzyme preparation according to claim 5, characterized in that: The enzyme activity detection of the complex enzyme preparation product: total enzyme activity ≥35000 U / g.

10. The special complex enzyme preparation for malt small molecule active peptide according to claim 5, characterized in that, The complex enzyme preparation is verified for small molecule peptide conversion rate of barley stillage: S4.1, RF pretreatment of stillage: the beer wet stillage with a water content of 75-80% is placed in a radio frequency heating device, under the conditions of a frequency of 27-27.12 MHz and a plate spacing of 8-10 cm, the material temperature is controlled to rise to 60-65 DEG C and maintain for 8-10 min, to promote the unfolding of protein secondary structure and inactivate part of the bacteria; S4.2, enzymatic reaction: the complex enzyme preparation is added at 0.3-0.5% of the weight of the wet stillage, the solid-liquid ratio is 1:3-1:5, the pH is adjusted to 6.5-7.0, and the enzymatic reaction is carried out at a temperature of 50±2 DEG C for 3.5-4.0 h, and the content of small molecule active peptides with a molecular weight of 180-5000 Da in the enzymatic hydrolysate is ≥35%, and the content of oligopeptides with a molecular weight of <1000 Da is ≥20%.

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