Preparation method of highland barley vinasse polypeptide oral liquid for activating ethanol dehydrogenase

Abstract

The invention relates to the technical field of functional food biology, and discloses a preparation method of highland barley vinasse polypeptide oral liquid for activating ethanol dehydrogenase, highland barley vinasse is used as a raw material, an ultrasonic-assisted alkaline protease enzymolysis technology is adopted, directional enzymolysis is carried out under an optimal process, and active polypeptide components with the molecular weight smaller than 3000Da are cut off through ultrafiltration, so that the highland barley vinasse polypeptide oral liquid is obtained. And then scientifically compounding with a honey extracting solution and a sea-buckthorn diluent. Through identification, the polypeptide component is rich in specific active peptide fragments such as LGAPF and the like. Animal experiments prove that the oral liquid can significantly improve the activity of ethanol dehydrogenase and acetaldehyde dehydrogenase in vivo and effectively reduce serum transaminase and liver lipid peroxidation level; the action mechanism relates to up-regulation of lipid metabolism gene expression and regulation of key intestinal flora abundance such as Muraculum and the like. According to the invention, high-valued utilization of the highland barley vinasse resource is realized, and the prepared product has remarkable comprehensive effects of dispelling the effects of alcohol, protecting liver and improving intestinal microecology.

Description

A method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase Technical Field

[0001] This invention relates to the field of functional food biotechnology, specifically to a method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase. Background Technology

[0002] Bioactive peptides are typically prepared through hydrolysis or microbial fermentation and are considered a valuable resource in the field of nutrition and health care due to their significant health benefits. Barley lees, a major solid-state fermentation byproduct in the production of highland barley-flavored baijiu, contains abundant protein resources and has great potential for development into a low-cost hangover relief and liver protection material. Currently, the utilization of highland barley lees mainly focuses on feed production or simple primary processing, with insufficient high-value utilization.

[0003] Regarding the preparation process of peptides from highland barley lees, conventional extraction methods include the alkali-alcohol method, but different extraction methods have significantly different effects on the yield and bioactivity of peptides. Existing studies have shown that, compared with the single alkali-alcohol method or ordinary enzymatic hydrolysis, ultrasound-assisted extraction often achieves a higher extraction rate. However, determining the optimal enzymatic hydrolysis conditions to obtain specific peptide components with the highest activation rate for alcohol dehydrogenase still requires in-depth process optimization.

[0004] Furthermore, although some peptides are known to have certain antioxidant or hangover-relieving functions, current technologies have not yet provided a complete solution regarding the stability of bioactive peptides of specific molecular weight segments in highland barley lees after in vivo simulated digestion, as well as the mechanism and application of how they comprehensively exert hangover-relieving and liver-protecting effects by regulating liver lipid metabolism-related genes, inhibiting the expression of inflammatory factors, and regulating the structure of intestinal flora. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for preparing an oral liquid of highland barley lees polypeptides that activates alcohol dehydrogenase, thus solving the technical problems of low utilization rate of highland barley lees resources, low protein extraction rate, and unclear or low alcohol dehydrogenase activation activity of the prepared polypeptides in existing technologies.

[0006] The first aspect of this invention provides a method for preparing an oral liquid containing barley lees polypeptides that activate alcohol dehydrogenase, comprising the following steps:

[0007] Step S1, raw material pretreatment: The barley lees raw material is dried and pulverized to obtain barley lees powder with uniform particle size.

[0008] Step S2, enzymatic extraction: Mix the barley lees powder with water, adjust the pH to alkaline, add protease to carry out enzymatic hydrolysis, and use ultrasonic treatment during the reaction. After the reaction, the enzymatic hydrolysate is inactivated to obtain barley lees enzymatic hydrolysate.

[0009] Step S3, ultrafiltration separation: The barley lees enzymatic hydrolysate is centrifuged to remove insoluble residues, and the supernatant is subjected to ultrafiltration separation to retain and collect barley lees polypeptide components with a molecular weight of less than 3000 Da.

[0010] Step S4, compound formulation: prepare excipient base liquid, mix the highland barley wine lees polypeptide component with the excipient base liquid to obtain the highland barley wine lees polypeptide oral liquid.

[0011] The specific conditions for the enzymatic hydrolysis reaction in step S2 are as follows: the pH of the mixture is adjusted to 10.0, and the reaction temperature is controlled at 55℃; the protease is an alkaline protease, and its addition amount is 4.3 KU / g relative to the mass of highland barley distillers' grains. Under these specific pH, temperature, and enzyme addition conditions, the conformation of the alkaline protease is most favorable for binding to the highland barley distillers' grains protein substrate, with sufficient exposure of the enzyme cleavage sites, enabling the targeted hydrolysis of large protein molecules into biologically active short peptide fragments, while avoiding the destruction of the active amino acid sequence due to excessive hydrolysis.

[0012] The enzymatic hydrolysis process described in step S2 employs an ultrasound-assisted alkaline protease hydrolysis method. Utilizing the cavitation, mechanical, and thermal effects generated by ultrasound in the liquid medium, the dense cell wall structure of the barley lees is disrupted, increasing the contact area between the solvent and enzyme with the substrate, reducing mass transfer resistance, thereby improving the protein dissolution rate and the enzymatic hydrolysis rate.

[0013] The enzyme inactivation treatment in step S2 specifically involves boiling the mixture after enzymatic hydrolysis for 10 to 15 minutes; the ultrafiltration separation in step S3 specifically involves pumping the supernatant after centrifugation into a high-pressure flat-panel membrane device and collecting the permeate that has passed through a membrane with a molecular weight cutoff of 3000 Da.

[0014] The enzyme inactivation step denatures and inactivates the protease, terminates the enzymatic reaction, and prevents the polypeptide chain from being continuously cleaved and altering its molecular weight distribution in subsequent processing. Ultrafiltration is performed using a membrane with a molecular weight cutoff of 3000 Da because the polypeptide components of highland barley lees with a molecular weight of less than 3000 Da are more spatially compatible with the binding sites of alcohol dehydrogenase, exhibiting higher ADH activation rate and DPPH free radical scavenging ability compared to larger molecular weight components.

[0015] In a preferred embodiment, in step S2, the ratio of barley lees powder to water is 1:10 to 1:20, and the enzymatic hydrolysis reaction time is 2 to 4 hours.

[0016] In a preferred embodiment, the excipient base liquid in step S4 includes honey extract, which is prepared by mixing honey and water at a volume ratio of 1:5 and extracting at a constant temperature of 80°C for 2 hours. The excipient base liquid also includes sea buckthorn diluent, which is prepared by mixing sea buckthorn pulp and water at a volume ratio of 1:5 and diluting at 40°C. Sea buckthorn pulp is rich in vitamin C and flavonoids, which are heat-sensitive. Low-temperature dilution at 40°C effectively preserves their biological activity and prevents high-temperature oxidation and inactivation, thus enabling a synergistic antioxidant effect with barley lees peptides.

[0017] In a preferred embodiment, the excipient base solution in step S4 is prepared by mixing the honey extract and the sea buckthorn dilution at a volume ratio of 1:1. Specifically, the mixing involves adding the highland barley lees polypeptide component to the excipient base solution and adjusting the volume to a final concentration of 100 mg / mL in the oral solution. This concentration ratio and base solution composition effectively reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in an alcoholic liver injury model and upregulate the expression of the hepatic acetaldehyde dehydrogenase gene.

[0018] A second aspect of the present invention provides a barley lees polypeptide oral liquid prepared by the above method.

[0019] The oral liquid contains a highland barley lees polypeptide component with an alcohol dehydrogenase activation rate of not less than 58.33% and a DPPH free radical scavenging rate of not less than 92.46%.

[0020] The highland barley lees polypeptide component contains polypeptide fragments with the amino acid sequences LGAPF, AGLW, WASL, ALSW, and LGASF.

[0021] The above sequence fragment belongs to the alcohol dehydrogenase activating peptide. Its hydrophobic amino acid residues can bind to the active site or regulatory site of the enzyme through hydrophobic interactions, thereby improving the catalytic efficiency of alcohol dehydrogenase and accelerating ethanol metabolism.

[0022] This invention provides a method for preparing an oral liquid containing barley lees polypeptides that activates alcohol dehydrogenase. It has the following beneficial effects:

[0023] 1. This invention uses ultrafiltration technology to retain and collect barley lees polypeptide components with a molecular weight of less than 3000 Da, removing low-activity large molecular proteins and directionally enriching alcohol dehydrogenase activating peptides containing specific hydrophobic amino acid sequences such as LGAPF and AGLW. Experimental data confirm that this small molecule component has a higher alcohol dehydrogenase activation rate and DPPH free radical scavenging rate than the unfractionated product, and can significantly reduce alcoholic liver damage through a dual mechanism of accelerating alcohol metabolism and inhibiting oxidative stress.

[0024] 2. This invention employs ultrasound-assisted alkaline protease hydrolysis technology. Under specific process conditions of pH 10.0, 55℃, and an enzyme addition of 4.3 KU / g, the cavitation effect and mechanical shear force generated by ultrasound disrupt the dense cell wall structure of highland barley lees, significantly increasing the contact area between the enzyme and the substrate and reducing mass transfer resistance. This process effectively overcomes the low extraction efficiency of traditional enzymatic hydrolysis methods, transforming waste highland barley lees into high-value-added active polypeptide raw materials, thus realizing the high-value utilization of resources.

[0025] 3. This invention scientifically combines highland barley lees polypeptides with honey and low-temperature treated sea buckthorn extract. The sea buckthorn extract is specifically diluted at 40°C, which effectively avoids the destruction of heat-sensitive substances such as vitamin C and flavonoids by high temperatures. This compound system not only improves the sensory flavor of the oral liquid, but also utilizes the antioxidant components in the excipients to produce a synergistic effect with the polypeptides, showing a better effect than the single component in reducing the levels of alanine aminotransferase and aspartate aminotransferase. Attached Figure Description

[0026] Figure 1 is a representative H&E stained section (×200) of liver tissue from mice with chronic alcoholic liver injury in Experiment Example 3 of this invention.

[0027] Figure 2 is a bar chart showing the effect of barley lees polypeptide on serum AST, ALT, and TG levels in mice with chronic alcoholic liver injury in Experiment Example 3 of this invention.

[0028] Figure 3 is a bar chart showing the effect of HBDG-P on the activities of ADH, GPX, MDA, and SOD in the liver of mice with chronic alcoholic liver injury in Experiment Example 3 of this invention.

[0029] Figure 4 is a bar chart showing the effect of HBDG-P on the expression levels of ALDH and IL-6, characteristic liver genes in mice with chronic alcoholic liver injury, in Experiment Example 3 of this invention.

[0030] Figure 5 is a bar chart showing the effect of HBDG-P on the expression levels of lipid metabolism genes AMPK, PPAR-α, and FAS in mice with chronic alcoholic liver injury in Experiment Example 3 of this invention.

[0031] Figure 6 is a heatmap showing the correlation between gut microbiota and biochemical parameters and gene expression at the genus level in Experiment Example 3 of the present invention.

[0032] Figure 7 is a heatmap showing the correlation between gut microbiota, biochemical parameters, and gene expression at the genus level in Experimental Example 3 of the present invention.

[0033] Figure 8 is a schematic diagram of the preparation method steps of the present invention. Detailed Implementation

[0034] The technical solution of the present invention will now be clearly and completely described 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.

[0035] Highland barley lees: a solid-state fermentation byproduct of highland barley light-aroma baijiu production, which is dried at 60℃ before use.

[0036] Alkaline protease: derived from Bacillus subtilis, it is a food-grade proteolytic enzyme preparation with an enzyme activity ≥200,000 U / g and CAS number: 9014-01-1.

[0037] Honey: Commercially available natural multifloral honey.

[0038] Sea buckthorn pulp: 100% pure sea buckthorn pulp, with no additional additives.

[0039] Ethanol: Anhydrous ethanol, analytical grade, CAS No.: 64-17-5.

[0040] Sodium hydroxide: Analytical grade, CAS No.: 1310-73-2, used to adjust pH value.

[0041] Hydrochloric acid: Analytical grade, CAS No.: 7647-01-0, used to adjust pH value.

[0042] β-Nicotinamide adenine dinucleotide: Biochemical reagent grade, CAS No.: 53-84-9, used for the determination of alcohol dehydrogenase activity.

[0043] Ethanol dehydrogenase: Derived from horse liver, lyophilized powder, enzyme activity ≥300 U / mg, CAS No.: 9031-72-5.

[0044] 2,2-Diphenyl-1-picrylhydrazine: Analytical grade, CAS No.: 1898-66-4, used for antioxidant activity determination.

[0045] Example 1: A barley lees polypeptide oral solution that activates alcohol dehydrogenase;

[0046] This embodiment is prepared based on the optimal process parameters optimized through experiments. Referring to Figure 8, the specific steps are as follows:

[0047] Raw material pretreatment: Take highland barley lees, dry them at 60℃, crush and sieve them for later use.

[0048] Enzymatic extraction: Barley lees powder was mixed with water, the pH was adjusted to 10.0, and alkaline protease was added at a rate of 4.3 KU / g. The enzymatic hydrolysis reaction was carried out under ultrasound-assisted conditions at 55℃.

[0049] Separation and purification: After enzymatic hydrolysis, the enzyme was inactivated by boiling, and the supernatant was collected by centrifugation. The supernatant was then subjected to ultrafiltration using a high-pressure flat-plate membrane device to retain and collect the barley lees polypeptide fraction with a molecular weight <3000 Da (3kDa).

[0050] Preparation of auxiliary materials: Honey extract: Weigh an appropriate amount of honey into a beaker, add 5 times the volume of distilled water, and extract in an 80℃ constant temperature water bath for 2 hours to obtain the desired honey extract. Sea buckthorn diluent: Weigh an appropriate amount of sea buckthorn pulp into a beaker, add 5 times the volume of distilled water, and dilute in 40℃ warm water.

[0051] Finished product compounding: Mix honey extract and sea buckthorn dilution in a 1:1 volume ratio to form an excipient base solution, add the above-mentioned highland barley wine lees polypeptide (<3 kDa), make up to a final concentration of 100 mg / mL in the oral solution, and mix well to obtain the final product.

[0052] Comparative Example 1: Preparation method without ultrasound assistance;

[0053] The only difference between this comparative example and Example 1 is that: in step 2, ultrasonic assistance is not used, and enzymatic hydrolysis is carried out by mechanical stirring in a constant temperature water bath at 55°C. The remaining steps are the same as in Example 1.

[0054] Comparative Example 2: Components with different molecular weight cutoffs

[0055] The only difference between this comparative example and Example 1 is that in step 3, components with a molecular weight >10000 Da (10kDa) are collected, while the other steps are the same as in Example 1.

[0056] Experimental Example 1: Optimization of enzymatic hydrolysis process of highland barley lees for polypeptides;

[0057] The effects of extraction pH, extraction temperature, enzyme dosage, extraction time, and material-to-liquid ratio on the activation effect and yield of highland barley lees polypeptide ethanol dehydrogenase were investigated through single-factor experiments. The production process of highland barley lees alcohol-degrading peptides was optimized using response surface methodology.

[0058] Based on screening results, this invention uses ADH activation rate and peptide extraction rate as evaluation indicators to optimize the preparation process of highland barley distiller's grain peptides (HBDG-P) through single-factor experiments and response surface methodology. Ultrafiltration was used to screen for the component with the highest ADH activation rate, and its antioxidant activity was evaluated. Finally, the effects of gastrointestinal digestion on the antioxidant and alcohol-detoxifying activities of HBDG-P were analyzed by measuring ADH activation rate and DPPH free radical scavenging rate.

[0059] The test results showed that, compared with the alkali-alcohol method and the ultrasonic alkaline protease-assisted alkali-alcohol method, the ultrasonic-assisted alkali-alcohol method had the highest extraction rate. Further research indicated that the enzymatic hydrolysis effect was the best after alkaline protease hydrolysis. Based on the results of single-factor tests, with the ADH activation rate as the index, a response surface test was conducted on the extraction process of HBDG-P. Through the test, the optimal parameters for HBDG-P extraction were determined as follows: pH value 10.0, temperature 55 °C, enzyme addition amount 4.3 KU / g. Under these conditions, the ADH activation rate was (39.91 ± 2.63)%.

[0060] After in vitro simulated gastrointestinal digestion, the small molecular polypeptides were superior to the polypeptides without gastrointestinal digestion in terms of both ADH activity and DPPH free radical scavenging activity. Based on the analysis of the above test data, HBDG has the potential to become a low-cost anti-alcoholism material.

[0061] Test Example 2: Isolation and identification of polypeptides from highland barley distiller's grains;

[0062] Ultrafiltration was performed on the highland barley distiller's grains polypeptides obtained under the optimal process conditions using a high-pressure flat membrane device. Qualitative and quantitative detection of the peptide segments in the samples was carried out by LC-MS / MS. At the same time, analysis of the stability, allergenicity and potential toxicity of the peptide segments was conducted, and the identified HBDG-P was used to screen peptide segments with ADH and ALDH activation abilities by means of molecular docking.

[0063] HBDG-P was ultrafiltered to obtain four polypeptide components with different molecular weight ranges: MW > 10KDa, 5KDa < MW < 10KDa, 3KDa < MW < 5KDa, and MW < 3KDa. When the peptide concentrations of each component were the same:

[0064] The ADH activation rates were respectively: 20.33 ± 1.32% (>10KDa), 25.00 ± 1.41% (5 - 10KDa), 42.64 ± 1.45% (3 - 5KDa), and 58.33 ± 2.25% (<3KDa);

[0065] The DPPH free radical scavenging rates were respectively: 51.19 ± 2.05%, 77.4 ± 3.01%, 89.04 ± 2.45%, and 92.46 ± 3.25%. The results showed that the <3KDa component had significantly better activity than other components.

[0066] Peptidomics identification yielded 4263 peptides, ranging in length from 2 to 18 amino acids. Peptides with 2 to 7 amino acid residues accounted for 84.47% of the total peptides, and the molecular weight of HBDG-P was mostly between 400 and 600 Da. Bioactivity assessment of 4236 peptides identified by mass spectrometry using BIOPEP revealed that the HBDG-P peptides contain a large number of antioxidant and anti-inflammatory peptides, demonstrating that barley lees peptides may possess good hepatoprotective activity.

[0067] Based on the characteristics of reported alcohol dehydrogenase activating peptides, HBDG-P was screened and validated through molecular docking, identifying 29 novel peptides with potential ADH and ALDH activation capabilities (including LGAPF, AGLW, WASL, ALSW, and LGASF). Therefore, this demonstrates that highland barley distillers' grains peptides are an excellent source of peptides with high ADH and ALDH activation capabilities, and that highland barley distillers' grains peptides hold promise for intervening in ALI by activating ADH and ALDH.

[0068] Experimental Example 3: Protective effect of oral liquid on alcoholic liver injury in mice

[0069] Experimental grouping and treatment: Mice were divided into Control group (blank control), M group (model group), Y group (positive control group), L group (low-dose group of the polypeptide of the present invention), and H group (high-dose group of the polypeptide of the present invention, using the product of Example 1) to explore the effects of different doses of HBDG polypeptide on the basic indicators of C57BL / 6 and intestinal flora of ALI mice.

[0070] Effects of highland barley lees polypeptides on histopathological changes in liver tissue of mice with chronic alcoholic liver injury:

[0071] To investigate the hepatoprotective effect of HBDG-P, histopathological sections of liver tissue from mice with chronic alcoholic liver injury (ALI) were observed, as shown in Figure 1. In the Control group, the liver plates of the mice were arranged regularly and neatly, with no obvious dilation or compression of the hepatic sinusoids, and no obvious inflammatory cell infiltration; only mild hepatocyte edema was observed (blue arrows). In the M group, the liver tissue showed obvious hepatocyte edema (blue arrows), vascular congestion (red arrows), punctate necrosis of hepatocytes, and focal infiltration of granulocytes (purple arrows), confirming the successful establishment of the ALI model.

[0072] Compared with group M, all HBDG-P dose groups showed varying degrees of repair after administration. The low-dose HBDG-P group showed only minor vascular congestion (red arrow) and a small amount of hepatocyte necrosis, with no other significant abnormalities. HE staining results clearly showed that low molecular weight HBDG-P could effectively reduce hepatocyte edema and fatty degeneration.

[0073] Analysis of serum biochemical indicators of highland barley lees polypeptides in mice with chronic alcoholic liver injury:

[0074] Serum AST, ALT, and TG levels in mice after HBDG-P administration were detected using a kit method, and the results are shown in Figure 2. Compared with the Control group, the levels of AST, ALT, and TG in the M group were significantly increased (P<0.05). Specifically, the serum AST level in the M group was 1.94 times that of the Control group, and the ALT level was 3.56 times that of the Control group, indicating that alcohol exposure leads to hepatocyte damage.

[0075] Compared to group M, the serum AST, ALT, and TG levels in mice in groups Y, L, and H were significantly decreased (P<0.05). Furthermore, the AST and ALT levels in groups L and H were relatively similar, and the inhibitory effects on the increase of AST and ALT levels were stronger than those in the positive control group. This further indicates that HBDG-P can improve liver function damage caused by excessive alcohol consumption.

[0076] Analysis of liver biochemical indicators in mice with chronic alcoholic liver injury by peptides from highland barley lees:

[0077] The effects of different doses of HBDG-P on liver biochemical parameters in mice with chronic alcoholic liver injury were investigated, and the results are shown in Figure 3. In this experiment, compared with group M, the ADH activity in groups L and H was significantly increased, and the differences were statistically significant (P<0.05). This indicates that the protective mechanism of HBDG-P in ALI mice may be related to the regulation of ethanol metabolism enzyme activity.

[0078] To further clarify the effect of HBDG-P in alleviating ALI in mice, the activity levels of oxidative stress markers GPX, MDA, and SOD in liver tissue were measured (see Figure 3). Compared with the Control group, the activities of GPX and SOD in the M group were significantly decreased (P<0.05); conversely, the activities of GPX and SOD in the L and H groups were significantly higher than those in the M group (P<0.05), indicating that HBDG-P can effectively alleviate the decrease in GPX and SOD activities caused by alcohol in a dose-dependent manner. In addition, compared with the Control group, the MDA content in the M group was significantly increased (P<0.05), while the MDA content in each treatment group was significantly lower than that in the M group (P<0.01).

[0079] The above results indicate that HBDG-P can effectively increase GSH concentration and SOD activity, enhance the body's antioxidant capacity, reduce MDA concentration, and inhibit lipid peroxidation, with the H group showing better effects in reducing liver damage.

[0080] Effects of highland barley lees polypeptides on liver gene expression in mice with chronic alcoholic liver injury:

[0081] Based on the relationship between ALDH gene expression and alcoholic liver injury, regulating ALDH gene expression to treat alcoholic liver disease has become a promising research direction. As shown in Figure 4, in the liver, compared with the control group, the mRNA expression level of ALDH gene was significantly decreased in the M group (P<0.05); compared with the M group, different doses of HBDG-P significantly increased the mRNA expression level of ALDH gene (P<0.05).

[0082] Ethanol alters the gut microbiota composition, leading to gut flora imbalance and promoting the production of pro-inflammatory factors. As shown in Figure 4, compared to the Control group, the IL-6 gene expression in the M group was significantly increased (P<0.05), indicating that liver inflammation occurred in mice after gavage administration of alcohol. However, the addition of different doses of HBDG-P resulted in varying degrees of reduction (P<0.05), suggesting that HBDG-P can inhibit the secretion of inflammatory factors in the liver to some extent and has a certain protective effect on the liver.

[0083] Reducing lipid accumulation is a key strategy for alleviating ALI. As shown in Figure 5, compared with group M, both the low-dose HBDG-P and high-dose HBDG-P groups significantly increased the mRNA expression levels of AMPK and PPAR-α genes (P<0.05), with the expression levels in group H being higher than those in group L (P<0.05). FAS is a key enzyme in lipid synthesis. Compared with the control group, the expression level of FAS gene was significantly increased in group M (P<0.05), while after HBDG-P intervention, the expression level of FAS gene decreased in groups L and H (P<0.05). These results indicate that HBDG-P intervention can effectively alleviate alcohol-induced hepatic steatosis in mice with chronic alcoholic liver injury.

[0084] Effects and correlation analysis of highland barley lees polypeptides on gut microbiota in mice with alcoholic liver injury:

[0085] Gut microbiota sequencing analysis revealed that low molecular weight HBDG-P peptides extracted from barley distillers' grains increased the relative abundance of *Muribaculaceae*, *Parabacteroides*, *Lachnospiraceae_NK4A136_group*, and *Muribaculum*, while decreasing the relative abundance of *Prevotellaceae_UCG-001*. This indicates that low molecular weight HBDG-P exerts its effect in alleviating alcoholic liver damage by regulating the microbial community structure.

[0086] Spearman correlation analysis heatmaps elucidated the correlations between key gut microbiota and biochemical parameters and gene expression. As shown in Figure 6, *Muribaculum* was significantly decreased in mice with excessive alcohol consumption and was negatively correlated with serum AST and ALT levels. *Prevotella* was significantly positively correlated with hepatic MDA and significantly negatively correlated with hepatic ADH and GPX activities. Furthermore, *Prevotellaceae_UCG-001* was significantly negatively correlated with SOD activity, and *Lachnospiraceae_NK4A136_group* and *Muribaculum* were significantly positively correlated with ADH activity.

[0087] As shown in Figure 7, Muribaculum showed a significant positive correlation with PPAR-α and AMPK, and a significant negative correlation with IL-1β; Bacteroides showed a significant negative correlation with PPAR-α and AMPK; and Parasutterella showed a significant positive correlation with PPAR-α and AMPK, and a significant negative correlation with IL-1β.

[0088] 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 method for preparing an oral liquid containing barley lees polypeptides that activates alcohol dehydrogenase, characterized in that, Includes the following steps: Step S1, Raw material pretreatment: Dry and pulverize the highland barley lees raw material to obtain highland barley lees powder; Step S2, Enzymatic extraction: Mix the highland barley lees powder with water, adjust the pH to alkaline, add protease to carry out enzymatic hydrolysis, and use ultrasonic treatment during the reaction. After the reaction, inactivate the enzyme in the hydrolysate to obtain highland barley lees enzymatic hydrolysate; Step S3, Ultrafiltration separation: Centrifuge the highland barley lees enzymatic hydrolysate, take the supernatant for ultrafiltration separation, and retain and collect highland barley lees polypeptide components with a molecular weight of less than 3000 Da; Step S4, Compound preparation: Prepare excipient base liquid, mix the highland barley lees polypeptide components with the excipient base liquid to obtain the highland barley lees polypeptide oral liquid.

2. The method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase according to claim 1, characterized in that: The specific conditions for the enzymatic hydrolysis reaction in step S2 are as follows: the pH of the mixture is adjusted to 10.0, and the reaction temperature is controlled at 55℃; the protease is an alkaline protease, and its addition amount is 4.3 KU / g relative to the mass of highland barley lees powder.

3. The method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase according to claim 1, characterized in that: The enzyme inactivation treatment in step S2 specifically involves boiling the mixture after enzymatic hydrolysis for 10 to 15 minutes; and ultrafiltration of the supernatant collected after centrifugation in step S3 using a high-pressure flat-panel membrane device.

4. The method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase according to claim 1, characterized in that: The excipient base liquid mentioned in step S4 includes honey extract. The honey extract is prepared by mixing honey and water at a volume ratio of 1:5 and extracting it in a constant temperature water bath at 80°C for 2 hours.

5. The method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase according to claim 1, characterized in that: The auxiliary base liquid mentioned in step S4 includes sea buckthorn diluent. The sea buckthorn diluent is prepared by mixing sea buckthorn pulp with water at a volume ratio of 1:5 and diluting at 40°C.

6. The method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase according to claim 4, characterized in that: The excipient base liquid mentioned in step S4 is prepared by mixing honey extract and sea buckthorn dilution in a volume ratio of 1:

1.

7. The method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase according to claim 6, characterized in that: The mixing in step S4 specifically involves adding the highland barley lees polypeptide component to the excipient base solution and adjusting the volume to achieve a final concentration of 100 mg / mL in the oral solution.

8. The method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase according to claim 1, characterized in that: In step S2, the ratio of barley lees powder to water is 1:10 to 1:20, and the enzymatic hydrolysis reaction takes 2 to 4 hours.

9. The method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase according to claim 1, characterized in that: The alcohol dehydrogenase activation rate of the highland barley lees polypeptide component in step S3 is not less than 58.33%, and the DPPH free radical scavenging rate is not less than 92.46%.

10. The method for preparing a barley lees polypeptide oral liquid that activates alcohol dehydrogenase according to claim 1, characterized in that: The highland barley lees polypeptide component described in step S3 contains polypeptide fragments with amino acid sequences of LGAPF, AGLW, WASL, ALSW, and LGASF.

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