A neuroprotective small-molecule peptide from brewing yellow water and a preparation method and application thereof

Abstract

The application provides a kind of neuroprotective small molecule peptide from brewing yellow water and its preparation method and application, belongs to the technical field of food processing.The preparation method includes yellow water pretreatment, enzymatic hydrolysis-ultrasonic coupling extraction, ultrafiltration fractionation purification, refining and drying and other steps, through the compounding of protease and the cooperation of low-frequency ultrasonic, 1-3kDa small molecule peptide is enriched, the purity of product peptide is greater than or equal to 80%, rich in proline and glycine, containing 12 characteristic active sequences.The small molecule peptide has significant neuroprotective activity, the DPPH clearance rate of 1mg / mL solution is greater than or equal to 75%, can inhibit neuroinflammation, protect nerve cells and improve cognitive impairment.The small molecule peptide can be applied to oral liquid, tablet candy and other neuroprotective functional foods, the preparation process is green and environmentally friendly, and has strong industrialization adaptability, realizes the resource utilization of brewing by-products, provides natural and safe core raw materials for neuroprotective functional foods, and has wide market prospect.

Description

Technical Field

[0001] This invention relates to the field of food processing and functional food technology, specifically to a method for preparing neuroprotective small molecule peptides extracted from yellow water, a byproduct of brewing, and the application of these small molecule peptides in neuroprotective functional foods. Background Technology

[0002] The brewing industry produces tens of millions of tons of "yellow water" as a byproduct every year. It is rich in nutrients such as protein (10%-15%) and polypeptides (5%-8%), but due to its high content of impurities (pigments, polysaccharides, alcohols) and strong odor, it has long been discharged as waste, which not only wastes resources but also causes environmental pollution.

[0003] Meanwhile, neurodegenerative diseases and mental fatigue are becoming increasingly prominent, leading to a strong global market demand for neuroprotective functional foods. However, naturally sourced, highly safe neuroprotective raw materials are scarce. Current methods for extracting and applying yellow water peptides face three major technological bottlenecks: First, low extraction efficiency. Traditional water extraction or single enzymatic hydrolysis processes can only extract 10%-15% of the peptides, and small molecule peptides <3kDa account for less than 30%, resulting in poor intestinal absorption and blood-brain barrier penetration. Second, high purification difficulty. Yellow water contains a large amount of caramel coloring, heteropolysaccharides, and residual alcohol. Traditional purification methods (such as ethanol precipitation) easily lead to loss of peptide activity and are difficult to remove coloring and salts simultaneously. Third, unclear neuroprotective activity. Existing research has not systematically verified the antioxidant and neuroinflammatory inhibition mechanisms of yellow water peptides, which cannot support the development of functional foods.

[0004] While there have been attempts to comprehensively utilize yellow water in existing technologies, most have focused on simply extracting proteins or amino acids. They have not developed an efficient integrated extraction and purification process for neuroprotective small molecule peptides, nor have they clarified their neuroprotective functions and related mechanisms, making it difficult to meet the industrialization needs of functional foods. Summary of the Invention

[0005] The purpose of this invention is to develop a preparation process for neuroprotective small molecule peptides derived from brewing yellow water, so as to achieve comprehensive utilization of brewing yellow water.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention discloses a method for preparing neuroprotective small molecule peptides derived from brewing yellow water, comprising the following steps: (1) Yellow water pretreatment: Fresh yellow water that has been left to stand for 24 hours after brewing strong-aroma baijiu is selected, and after centrifugation and filtration, it is de-alcoholized until the alcohol content is ≤0.5%; finally, adsorption depigmentation is used to obtain pretreated liquid; (2) Enzymatic hydrolysis-ultrasound coupled extraction: Add complex protease to the pretreatment solution, adjust the pH to 8.0, enzymatic hydrolysis at 50℃ for 4h, and simultaneously perform low-frequency ultrasonic treatment; inactivate after enzymatic hydrolysis; (3) Ultrafiltration fractionation purification: A polyether sulfone (PES) ultrafiltration membrane with a molecular weight cutoff of 3 kDa was used. The permeate was collected after filtration. The permeate was desalted to obtain a small molecule peptide purification solution. (4) Refining and drying: The purified solution is concentrated to a solid content of 25%-30%, and then spray-dried to obtain neuroprotective small molecule peptide powder.

[0007] Preferably, the yellow water in step (1) has a pH of 3.5-4.5, an alcohol content of 6%-8%, and a protein content of ≥12%. The treatment process is as follows: centrifugation at 8000 rpm and 4℃ for 15 min, filtration through a 0.22 μm microfiltration membrane, followed by vacuum de-alcoholization at 45℃ and -0.08 MPa until the alcohol content is ≤0.5%. Finally, AB-8 macroporous resin is used for adsorption and depigmentation at 30℃, pH 4.0, and a flow rate of 2 BV / h to obtain a pretreated solution, wherein the pigment removal rate is ≥85% and the peptide loss rate is ≤5%.

[0008] Preferably, the complex protease added in step (2) enzymatic hydrolysis-ultrasound coupling extraction is a mixture of alkaline protease and flavor protease in a 3:1 ratio, with enzyme activities of 20000 U / g and 10000 U / g, respectively. The amount of complex protease added is 2% (w / w). The specific process of enzymatic hydrolysis-ultrasound coupling extraction is as follows: after adding the complex protease, the pH of the treatment solution is adjusted to 8.0, and enzymatic hydrolysis is carried out at 50℃ for 4 hours. During this period, 300W, 20kHz low-frequency ultrasound is used for assistance, with a pulse mode of 5 seconds working and 3 seconds intermittent. After the enzymatic hydrolysis is completed, the enzyme is inactivated by water bath at 95℃ for 10 minutes. The total peptide yield is ≥35%, and the proportion of small molecule peptides <3kDa is ≥60%.

[0009] Preferably, in step (3), the ultrafiltration conditions are filtration at 0.2 MPa, 30 °C, and a flow rate of 1.5 BV / h. The permeate is collected, and the proportion of 1-3 kDa small molecule peptides in the permeate is ≥85%. The permeate is desalted by 732 cation exchange resin at 30 °C and a flow rate of 1 BV / h. After desalting, the salt content is ≤0.1%, and a small molecule peptide purified solution is obtained.

[0010] Preferably, the specific process of refining and drying in step (4) is as follows: the purified liquid is vacuum concentrated to a solid content of 25%-30% at 50°C and -0.09MPa, and then spray-dried at an inlet air temperature of 160°C, an outlet air temperature of 70°C, and a feed rate of 10mL / min to obtain neuroprotective small molecule peptide powder.

[0011] In a second aspect, the present invention provides a neuroprotective small molecule peptide derived from brewing yellow water, prepared by the method described above, wherein the small molecule peptide has a 1-3kDa component accounting for ≥80%, a peptide purity of ≥80%, and a moisture content of ≤5%; and a DPPH free radical scavenging rate of ≥75% and an ABTS free radical scavenging rate of ≥80% at a concentration of 1mg / mL.

[0012] Furthermore, the proportion of proline and glycine in the small molecule peptide is ≥25%.

[0013] In a third aspect, the present invention discloses the application of the neuroprotective small molecule peptides described above in neuroprotective functional foods, the functional foods including oral liquids, compressed candies, or solid beverages.

[0014] Preferably, the oral liquid formulation comprises, by weight percentage: 10% neuroprotective small molecule peptide powder, 0.5% γ-aminobutyric acid, 5% blueberry concentrate, 3% fructooligosaccharides, and 81.5% purified water; the compressed candy formulation comprises, by weight percentage: 20% neuroprotective small molecule peptide powder, 5% phosphatidylcholine, 0.001% vitamin B12, 40% maltitol, 34.999% microcrystalline cellulose, and 0.5% magnesium stearate; the solid beverage formulation comprises, by weight percentage: 15% neuroprotective small molecule peptide powder, 10% whey protein, 8% freeze-dried walnut powder, 5% galactooligosaccharides, 61.5% maltodextrin, and 0.5% steviol glycosides.

[0015] Compared with the prior art, the beneficial effects of the present invention are: Extraction efficiency and purity are significantly improved: Through the integrated pretreatment process and the synergistic effect of enzymatic hydrolysis and ultrasound, the total peptide yield reaches more than 35%, the proportion of 1-3kDa small molecule peptides is ≥80%, and the peptide purity is ≥80%, which is far superior to the traditional process (yield of 10%-15%, proportion of small molecule peptides <30%). The neuroprotective activity is well-defined and potent: the product contains 12 characteristic active sequences (such as Gly-Pro and Pro-His), is rich in proline and glycine (≥25%), and has good blood-brain barrier penetration potential; in vitro experiments show that the DPPH clearance rate of 1 mg / mL peptide solution is ≥75%, the ABTS clearance rate is ≥80%, it can inhibit the secretion of 40%-50% of neuroinflammatory factors, and increase the survival rate of damaged nerve cells to 85%; in vivo experiments have confirmed that it can improve cognitive impairment and shorten the escape latency in mice by 35%; The process is green, environmentally friendly, and industrially adaptable: it uses macroporous resin and ion exchange resin for purification, avoiding organic solvent residue; the core equipment is all conventional equipment for the food industry, and a single production line can achieve a daily output of 1 ton of powder. The raw material is a by-product of brewing, and the comprehensive cost is ≤2000 yuan / ton, which has both resource recycling and environmental benefits. The product exhibits excellent flavor and stability: through the compounding of flavor proteases and subsequent formula optimization, the product is free of yellow water odor and bitterness; with low-temperature processing throughout the process and light-proof and oxygen-barrier packaging, the activity retention rate is ≥80% after 6 months of storage at 25℃. Attached Figure Description

[0016] Figure 1 A bar chart showing the inhibitory effect of the small molecule peptides prepared according to the method of the present invention on neuroinflammatory factors (TNF-α, IL-6). Detailed Implementation

[0017] This invention aims to overcome the technical defects of existing yellow water peptides, such as low extraction efficiency, high purification difficulty, and unclear neuroprotective activity. It provides a method for preparing neuroprotective small molecule peptides derived from brewing yellow water. Through an integrated process of "high-efficiency pretreatment-enzymatic hydrolysis-ultrasonic coupling extraction-precision purification", the method achieves high yield and high purity of small molecule peptides, clarifies their neuroprotective mechanism, and provides natural and safe core raw materials for neuroprotective functional foods.

[0018] The core technical solution of this invention is to directionally extract and enrich small molecule peptides with neuroprotective activity from brewing yellow water. In an exemplary embodiment, the specific steps of the method of this invention are as follows: Yellow water pretreatment (high-efficiency impurity removal and de-alcoholization): Select fresh yellow water that has been left to stand for 24 hours after brewing strong-aroma baijiu. The requirements are pH 3.5-4.5, alcohol content 6%-8%, and protein content ≥12% to avoid spoilage. An integrated process of "centrifugation-microfiltration-macroporous resin adsorption-vacuum de-alcoholization" is adopted: First, centrifugation at 8000 rpm and 4℃ for 15 min removes solids such as lees and yeast cells; then, filtration through a 0.22μm microfiltration membrane retains suspended particles, increasing the transmittance to over 70%; subsequently, vacuum de-alcoholization is performed at 45℃ and -0.08MPa to reduce the alcohol content to below 0.5% (to avoid inhibiting subsequent enzyme activity), while recovering alcohol with a purity ≥90% for reuse in brewing; finally, AB-8 macroporous resin (resin to yellow water volume ratio 1:10) is used to adsorb caramel pigments at 30℃, pH 4.0, and a flow rate of 2 BV / h, achieving a pigment removal rate ≥85% and a peptide loss rate ≤5%, solving the problem of peptide activity loss caused by traditional activated carbon adsorption.

[0019] Enzymatic hydrolysis-ultrasound coupled extraction (improving the yield and activity of small molecule peptides): An alkaline protease (20000 U / g activity) and a flavor protease (10000 U / g activity) were combined in a 3:1 ratio to balance peptide yield and flavor, avoiding the bitterness caused by single-mode enzymatic hydrolysis. 2% (w / w) of the compound protease was added to the pretreatment solution, and the pH was adjusted to 8.0. Enzymatic hydrolysis was carried out in a 50℃ constant temperature water bath for 4 hours. During this process, low-frequency ultrasound was used (300W power, 20kHz frequency, pulse mode: 5s operation / 3s interval) to disrupt the protein's spatial structure through ultrasonic cavitation, accelerating the enzymatic hydrolysis reaction. After enzymatic hydrolysis, the peptides were inactivated by incubating in a 95℃ water bath for 10 minutes to prevent subsequent peptide degradation. At this point, the total peptide yield increased to over 35%, with small molecule peptides (<3kDa) accounting for 60%, far exceeding that of traditional enzymatic hydrolysis processes.

[0020] Ultrafiltration fractional purification (precise enrichment of neuroprotective active peptides): A polyethersulfone (PES) ultrafiltration membrane with a 3kDa molecular weight cutoff is used. Fractional filtration is performed under operating conditions of 0.2 MPa pressure, 30℃ temperature, and 1.5 BV / h. The permeate solution containing small peptides (molecular weight 1-3 kDa ≥ 85%) is collected, while the retained large protein molecules are reused in the enzymatic hydrolysis process, improving raw material utilization. The permeate is then passed through a 732 cation exchange resin (30℃, 1 BV / h) to remove sodium. + K + The presence of salt ions reduces the salt content to below 0.1%, thus avoiding any impact on the taste and stability of subsequent food applications.

[0021] Refining and Drying (Preserving Peptide Activity): Low-temperature vacuum concentration (50℃, -0.09MPa) is used to concentrate the small molecule peptide solution to a solid content of 25%-30%, avoiding peptide activity loss caused by high temperature. A low-temperature spray dryer is used to produce a light yellow, odorless neuroprotective small molecule peptide powder under conditions of inlet air temperature 160℃, outlet air temperature 70℃, and feed rate 10mL / min. The powder has a moisture content ≤5%, peptide purity ≥80%, and the proportion of 1-3kDa small molecule peptides ≥80%.

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments and 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.

[0023] Example 1: Preparation of neuroprotective small molecule peptides Yellow water pretreatment: 100L of fresh yellow water (pH 4.0, alcohol content 7%, protein content 13%) 24h after brewing of strong-aroma baijiu was selected, centrifuged at 8000rpm and 4℃ for 15min, and filtered through a 0.22μm microfiltration membrane; the alcohol content was reduced to 0.3% under vacuum at 45℃ and -0.08MPa; 10L of filtrate was mixed with 1LAB-8 macroporous resin, and adsorbed and depigmented at 30℃, pH 4.0 and flow rate 2BV / h. 9.2L of pretreated solution was collected, with a pigment removal rate of 88% and a peptide loss rate of 4.2%.

[0024] Enzymatic hydrolysis-ultrasound coupled extraction: 1.84 kg of complex protease (1.38 kg of alkaline protease + 0.46 kg of flavor protease) was added to the pretreatment solution, the pH was adjusted to 8.0, and enzymatic hydrolysis was carried out at 50℃ for 4 h; during this period, 300 W, 20 kHz ultrasound was turned on, working for 5 s and then intermittently for 3 s; after inactivation, the enzyme was incubated in a 95℃ water bath for 10 min to obtain 9.0 L of enzymatic hydrolysate.

[0025] Ultrafiltration fractionation purification: A 3kDa PES ultrafiltration membrane was used for filtration at 0.2MPa, 30℃, and a flow rate of 1.5BV / h. 7.5L of permeate was collected (86% of which were 1-3kDa small molecule peptides). The permeate was desalted by 732 cation exchange resin to obtain 7.2L of purified solution with a salt content of 0.08%.

[0026] Refining and drying: Concentrate to 1.8L (solid content 28%) under vacuum at 50℃ and -0.09MPa; spray dry (inlet air 160℃, outlet air 70℃) to obtain 0.5kg of small molecule peptide powder with a moisture content of 4.2%, peptide purity of 82%, and 1-3kDa component accounting for 83%.

[0027] Example 2: Verification of neuroprotective activity I. In vitro validation experiments (a) Determination of antioxidant capacity (DPPH+ABTS free radical scavenging experiment) 1. Experimental Materials Experimental sample: Neuroprotective small molecule peptide powder prepared in Example 1 (sterilely freeze-dried, purity 82%). Control samples: soybean peptides (purity ≥75%, purchased from Sigma-Aldrich), Trolox standard (purity ≥98%, purchased from Solarbio); Experimental reagents: DPPH reagent (Sigma-Aldrich, D9132), ABTS reagent (Solarbio, A8592), anhydrous ethanol (analytical grade), phosphate buffer (PBS, pH 7.4, 0.01 mol / L). Experimental equipment: Microplate reader (Thermo, Multiskan FC), analytical balance, ultrasonic cleaner, centrifuge, pipette, 96-well microplate.

[0028] 2. Experimental Methods (1) Sample solution preparation Small molecule peptide powder and soybean peptide were prepared into sample stock solutions of 1 mg / mL using PBS, and then filtered through a 0.22 μm filter membrane for sterilization. Trolox standards were prepared into a series of standard solutions of 0.1-1.0 mmol / L using anhydrous ethanol.

[0029] (2) DPPH free radical scavenging experiment Take a 96-well plate, add 100 μL of LPPH ethanol solution (0.1 mmol / L) to each well, then add 100 μL of sample solution of different concentrations (diluted to 0.2, 0.4, 0.6, 0.8, 1.0 mg / mL), and add 100 μL of anhydrous ethanol + 100 μL of PBS to the blank control group; Incubate at room temperature in the dark for 30 min, and then measure the absorbance (A value) of each well using a microplate reader at a wavelength of 517 nm. Three parallel wells were set for each concentration, and the clearance rate was calculated according to the formula: DPPH clearance rate (%) = [1 - (A sample - A blank) / A control] × 100%, where A control is the absorbance of DPPH solution + PBS.

[0030] (3) ABTS free radical scavenging experiment First, prepare the ABTS working solution: Mix 7 mmol / L ABTS solution with 2.45 mmol / L potassium persulfate solution at a volume ratio of 1:1, incubate at room temperature in the dark for 12 h, and dilute with anhydrous ethanol to an absorbance (734 nm) of 0.70 ± 0.02. Take a 96-well plate, add 10 μL of sample solution (1 mg / mL) to each well, then add 190 μL of working solution. For the blank control group, add 10 μL of PBS + 190 μL of working solution. Incubate at room temperature in the dark for 10 min, and then measure the absorbance (A value) at a wavelength of 734 nm using an ELISA reader. Each sample has 3 parallel wells. The clearance rate is calculated according to the formula: ABTS clearance rate (%) = [1 - (A sample - A blank) / A control] × 100%, where A control is the absorbance of ABTS working solution + PBS.

[0031] (4) Data processing SPSS 26.0 software was used for statistical analysis to calculate the half-maximum clearance rate (IC). 50 The differences between groups were analyzed using an independent samples t-test, with P < 0.05 considered statistically significant.

[0032] 3. Experimental Results The DPPH radical scavenging rate of 1 mg / mL small molecule peptide solution of this invention is 78%, the ABTS radical scavenging rate is 82%, and the IC50 value is [missing information]. 50 The concentrations were 0.42 mg / mL and 0.38 mg / mL, respectively; while the DPPH scavenging rate of soybean peptides at the same concentration was 58% and the ABTS scavenging rate was 55%. The antioxidant activity of the small molecule peptides of the present invention is significantly better than that of ordinary soybean peptides (P<0.01).

[0033] (II) Experiment on inhibiting neuroinflammation (LPS-induced BV2 microglia model) 1. Experimental Materials Cell line: BV2 microglia (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM high glucose medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin, and passaged in an incubator at 37°C and 5% CO2. Experimental reagents: LPS (lipopolysaccharide, Sigma-Aldrich, L2630, purity ≥99%), small molecule peptide of this invention (10 mg / mL sterile stock solution), TNF-α ELISA kit (Thermo, 88-7340-22), IL-6 ELISA kit (Thermo, 88-7064-22), PBS buffer, trypsin digestion solution (0.25%). Experimental equipment: CO2 incubator (Thermo, 3111), inverted microscope (Olympus, CKX41), microplate reader, centrifuge, and clean bench.

[0034] 2. Experimental Methods (1) Cell grouping and treatment Take BV2 cells in the logarithmic growth phase and administer at a rate of 5 × 10⁻⁶. 4 Cells / well were seeded into 24-well plates and cultured for 24 hours until the cells adhered. The study was divided into three groups: ① blank control group (culture medium only); ② model group (LPS final concentration 1 μg / mL); ③ peptide treatment group (LPS final concentration 1 μg / mL + small molecule peptide final concentration 0.5 mg / mL); each group had 3 parallel wells. After incubating at 37℃ and 5% CO2 for 24 hours, the cell supernatant was collected and frozen at -80℃ for later use.

[0035] (2) Detection of inflammatory factors (ELISA method) Follow the instructions for the TNF-α and IL-6 ELISA kit: serially dilute the standard (0, 15.625, 31.25, 62.5, 125, 250, 500, 1000 pg / mL) and add 100 μL to each well of the ELISA plate; Add 100 μL of sample supernatant, incubate at 37 °C for 2 h, discard the liquid, and wash 5 times with washing buffer; Add 100 μL of biotinylated antibody working solution, incubate at 37°C for 1 h, and wash 5 times; Add 100 μL of enzyme conjugate working solution, incubate at 37°C for 30 min, and wash 5 times; Add 100 μL of substrate solution, incubate in the dark for 15 min, add 50 μL of stop solution, and measure the absorbance at 450 nm using an ELISA reader. The concentrations of TNF-α and IL-6 in the sample were calculated based on the standard curve.

[0036] 3. Experimental Results The concentrations of TNF-α and IL-6 in the supernatant of BV2 cells in the model group were (862.5±45.2) pg / mL and (785.2±38.7) pg / mL, respectively. In the peptide treatment group, the concentration of TNF-α decreased to (474.4±32.1) pg / mL (a decrease of 45%) and the concentration of IL-6 decreased to (408.3±29.5) pg / mL (a decrease of 48%). The differences were statistically significant compared with the model group (P<0.01).

[0037] Example 3: Preparation of Neuroprotective Food (Oral Liquid) Raw materials were weighed according to the following mass ratios: 10 kg of small molecule peptide powder prepared in Example 1, 0.5 kg of γ-aminobutyric acid, 5 kg of blueberry concentrate, 3 kg of fructooligosaccharides, and 81.5 kg of purified water. After dissolving the raw materials, homogenization was performed at 20,000 rpm for 5 min, followed by UHT sterilization at 135℃ for 3 s. The mixture was then aseptically filled (10 mL per vial) and packaged in the dark. Testing showed that each vial contained ≥1000 mg of small molecule peptides, with a DPPH scavenging rate ≥70%, and an activity retention rate of 86% after 6 months of storage at 25℃.

[0038] Any aspects of this invention not described in detail are well-known to those skilled in the art.

[0039] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a neuroprotective small molecule peptide derived from brewing liquid, characterized in that, Includes the following steps: (1) Yellow water pretreatment: Fresh yellow water that has been left to stand for 24 hours after brewing strong-aroma baijiu is selected, and after centrifugation and filtration, it is de-alcoholized until the alcohol content is ≤0.5%; finally, adsorption depigmentation is used to obtain pretreated liquid; (2) Enzymatic hydrolysis-ultrasound coupled extraction: Add complex protease to the pretreatment solution, adjust the pH to 8.0, enzymatic hydrolysis at 50℃ for 4h, and simultaneously perform low-frequency ultrasonic treatment; inactivate after enzymatic hydrolysis. (3) Ultrafiltration fractionation purification: A polyether sulfone (PES) ultrafiltration membrane with a molecular weight cutoff of 3 kDa was used. The permeate was collected after filtration. The permeate was desalted to obtain a small molecule peptide purification solution. (4) Refining and drying: Concentrate the purified solution to a solid content of 25%-30%, and then spray dry to obtain neuroprotective small molecule peptide powder.

2. The preparation method according to claim 1, characterized in that, The yellow water mentioned in step (1) has a pH of 3.5-4.5, an alcohol content of 6%-8%, and a protein content of ≥12%. The treatment process is as follows: centrifugation at 8000 rpm and 4℃ for 15 min, filtration through a 0.22 μm microfiltration membrane, followed by vacuum de-alcoholization at 45℃ and -0.08 MPa until the alcohol content is ≤0.5%. Finally, AB-8 macroporous resin is used for adsorption and depigmentation at 30℃, pH 4.0, and a flow rate of 2 BV / h to obtain a pretreated solution, wherein the pigment removal rate is ≥85% and the peptide loss rate is ≤5%.

3. The preparation method according to claim 1, characterized in that, The complex protease added in step (2) enzymatic hydrolysis-ultrasound coupling extraction is a mixture of alkaline protease and flavor protease in a 3:1 ratio, with enzyme activities of 20000 U / g and 10000 U / g, respectively. The amount of complex protease added is 2% (w / w). The specific process of enzymatic hydrolysis-ultrasound coupling extraction is as follows: after adding the complex protease, the pH of the treatment solution is adjusted to 8.0, and enzymatic hydrolysis is carried out at 50℃ for 4 hours. During this period, 300W, 20kHz low-frequency ultrasound is used for assistance, with a pulse mode of 5 seconds working and 3 seconds intermittent. After the enzymatic hydrolysis is completed, the enzyme is inactivated by water bath at 95℃ for 10 minutes. The total peptide yield is ≥35%, and the proportion of small molecule peptides <3kDa is ≥60%.

4. The preparation method according to claim 1, characterized in that, In step (3), the ultrafiltration conditions are filtration at 0.2 MPa, 30 °C and a flow rate of 1.5 BV / h. The permeate is collected, and the proportion of 1-3 kDa small molecule peptides in the permeate is ≥85%. The permeate is desalted by 732 cation exchange resin at 30 °C and a flow rate of 1 BV / h. After desalting, the salt content is ≤0.1%, and the small molecule peptide purified solution is obtained.

5. The preparation method according to claim 1, characterized in that, The specific process of purification and drying in step (4) is as follows: the purified solution is vacuum concentrated to a solid content of 25%-30% under the conditions of 50℃ and -0.09MPa, and then spray dried under the conditions of 160℃ air inlet, 70℃ air outlet and 10mL / min feed rate to obtain neuroprotective small molecule peptide powder.

6. A neuroprotective small molecule peptide derived from brewing liquid, prepared by the method according to any one of claims 1-5, characterized in that, The small molecule peptides contain ≥80% 1-3kDa components, have a peptide purity ≥80%, and a moisture content ≤5%; at a concentration of 1mg / mL, the DPPH free radical scavenging rate is ≥75%, and the ABTS free radical scavenging rate is ≥80%.

7. The neuroprotective small molecule peptide according to claim 6, characterized in that, The proportion of proline and glycine in the small molecule peptide is ≥25%.

8. The application of the neuroprotective small molecule peptide according to any one of claims 1-5 in neuroprotective functional foods, characterized in that, The functional foods include oral liquids, compressed candies, or solid beverages.

9. The application according to claim 8, characterized in that, The oral liquid formula comprises, by weight percentage: 10% neuroprotective small molecule peptide powder, 0.5% γ-aminobutyric acid, 5% blueberry concentrate, 3% fructooligosaccharides, and 81.5% purified water; the compressed candy formula comprises, by weight percentage: 20% neuroprotective small molecule peptide powder, 5% phosphatidylcholine, 0.001% vitamin B12, 40% maltitol, 34.999% microcrystalline cellulose, and 0.5% magnesium stearate; the solid beverage formula comprises, by weight percentage: 15% neuroprotective small molecule peptide powder, 10% whey protein, 8% freeze-dried walnut powder, 5% galactooligosaccharides, 61.5% maltodextrin, and 0.5% steviol glycosides.

Images