Activation of ethanol dehydrogenase bird's nest peptide and application thereof

Abstract

The application discloses an activated ethanol dehydrogenase bird's nest peptide and application thereof, and belongs to the technical field of functional polypeptides. The amino acid sequence of the activated ethanol dehydrogenase bird's nest peptide provided by the application is PLLW, can chelate an active center of ethanol dehydrogenase (ADH), helps to prevent oxidation of ADH and further maintain normal protein molecular structure of ADH during alcohol metabolism; the application further provides a bird's nest peptide powder containing the activated ethanol dehydrogenase bird's nest peptide, the bird's nest peptide powder can effectively improve an alcohol catalytic conversion rate of ADH, has a good effect of promoting alcohol metabolism, can be applied to development of various alcoholism-relieving products, and has high market value.

Description

Technical Field

[0001] This invention specifically relates to a bird's nest peptide that activates alcohol dehydrogenase and its application, belonging to the field of functional polypeptide technology. Background Technology

[0002] Alcohol, as a widely popular beverage globally, has gained widespread acceptance among people in various countries. However, excessive drinking is becoming increasingly common, and this problem has gradually evolved into a global challenge posing a serious threat to the health of all humanity. In the normal human metabolic system, approximately 90% of alcohol is converted into acetaldehyde by the liver's alcohol dehydrogenase (ADH), and then rapidly converted into non-toxic acetic acid by aldehyde dehydrogenase (ALDH). Throughout the entire alcohol metabolism process, ADH is considered the most crucial factor limiting the efficiency of alcohol metabolism in the body. However, it is worth noting that excessive alcohol intake, as well as the reactive oxygen species produced during alcohol metabolism, can inhibit the activity of liver ADH. Therefore, enhancing ADH activity is undoubtedly a critical approach to increasing the rate of alcohol metabolism.

[0003] Bird's nest is the nest built by several species of swiftlets in the genus Apodidae using a mixture of saliva and down feathers. In China, bird's nest has a long history of consumption and is considered a precious tonic food, holding an important place in traditional Chinese medicine and high-end catering, with continuously growing market demand. Numerous studies have found that bird's nest has the potential to increase the rate of alcohol metabolism. However, existing bird's nest products are not ideal for relieving hangovers because the proteins in bird's nest have a large molecular structure that is difficult for the intestines to digest and absorb. Therefore, isolating novel alcohol-metabolizing peptides with bioactivity and bioavailability that can enhance ADH activity from bird's nest is of great significance for improving the hangover-relieving effects of bird's nest products and promoting the development of products that promote alcohol metabolism. Summary of the Invention

[0004] This invention provides an activated alcohol dehydrogenase bird's nest peptide and its application. This activated alcohol dehydrogenase bird's nest peptide can chelate the active site of alcohol dehydrogenase (ADH), helping to prevent ADH oxidation and thus maintaining the normal protein molecular structure of ADH during alcohol metabolism. Furthermore, this invention also provides a bird's nest peptide powder containing this activated alcohol dehydrogenase bird's nest peptide, which can effectively improve the alcohol catalytic conversion rate of ADH and has a good effect on promoting alcohol metabolism. It can be applied to the development of hangover relief products and has high market value.

[0005] The technical solution of the present invention is as follows: This invention provides an activated alcohol dehydrogenase bird's nest peptide, wherein the amino acid sequence of the activated alcohol dehydrogenase bird's nest peptide is PLLW (SEQ ID NO:1).

[0006] This invention utilizes enzymatic hydrolysis, ultrafiltration, gel chromatography purification, amino acid sequencing, and computer-aided virtual screening techniques to screen dried bird's nest powder, thereby obtaining a novel activated alcohol dehydrogenase bird's nest peptide (SEQ ID NO:1). This activated alcohol dehydrogenase bird's nest peptide has a molecular weight of 527.3107 Da and exhibits a high affinity for alcohol dehydrogenase (ADH), chelating the active site of ADH and helping to prevent ADH oxidation, thus maintaining the normal protein molecular structure of ADH.

[0007] The activated alcohol dehydrogenase bird's nest peptide provided by this invention can also be applied to the preparation of various products that promote alcohol metabolism.

[0008] The present invention also provides a bird's nest peptide powder that promotes alcohol metabolism, wherein the bird's nest peptide powder contains the above-mentioned bird's nest peptide that activates alcohol dehydrogenase.

[0009] This invention also provides a method for preparing the above-mentioned bird's nest peptide powder that promotes alcohol metabolism, which is obtained by ultrafiltration chromatography from the enzymatic hydrolysate of dried bird's nest powder, including the following specific steps: S1. Weigh out the dried bird's nest powder and place it in water for ultrasonic crushing and even dispersion to obtain bird's nest liquid; S2. Adjust the pH of the bird's nest liquid to 7.5-8.5, add 1-2% of the mass of dried bird's nest powder with trypsin and enzymatically hydrolyze at 40-50℃ for 4 hours, then inactivate the enzyme with boiling water to obtain trypsin hydrolysate. S3. Adjust the pH of the trypsin hydrolysate to 1.5-2.5, add 1-2% of the weight of the dried bird's nest powder with pepsin, and perform enzymatic hydrolysis at 35-40℃ for 4 hours. Then, inactivate the enzyme with boiling water to obtain the pepsin hydrolysate. S4. Centrifuge the enzyme hydrolysate obtained in step S3 after enzyme inactivation, ultrafilter out the components with a molecular weight <3 kDa from its supernatant, and use gel chromatography to separate the polypeptide with the highest ethanol dehydrogenase activation rate. Freeze-dry to obtain the bird's nest peptide powder.

[0010] Furthermore, in step S1, the ratio of dried bird's nest powder to water in the bird's nest liquid is 1:30.

[0011] Furthermore, in step S2, the enzyme activity of trypsin is ≥2500 U / mg.

[0012] Furthermore, in step S3, the pepsin enzyme activity is ≥3000 U / mg.

[0013] Furthermore, the method for separating the polypeptide with the highest alcohol dehydrogenase activation rate described in step S4 includes the following steps: The ultrafiltration fraction with a molecular weight <3 kDa was subjected to column chromatography using an AKTA system equipped with a Superdex™ peptide 10 / 300 GL column, resulting in four elution peaks. The eluents corresponding to the four elution peaks were collected, freeze-dried, and four peptides were obtained. Then, the activation rate of alcohol dehydrogenase was used as the evaluation index, and the peptide with the highest activation rate of alcohol dehydrogenase was screened from the four peptides through in vitro alcohol detoxification experiments. The column chromatography conditions were as follows: sample concentration 20 mg / mL, sample volume 1 mL, flow rate 300 μL / min, one tube collected every 200 μL, and absorbance curves were plotted at 220 nm.

[0014] Furthermore, the in vitro alcohol detoxification experiment described in step S4 includes the following steps: The polypeptide was mixed with water to prepare a polypeptide solution, and then added to a mixture of sodium pyrophosphate buffer solution, oxidized coenzyme I and ethanol. After mixing thoroughly and incubating at 25°C for 5 min, alcohol dehydrogenase was added, and the mixture was shaken to mix. The ultraviolet absorbance at a wavelength of 340 nm was then detected.

[0015] Unlike existing technologies, this invention has the following advantages: 1. This invention provides a bird's nest polypeptide that enhances the activity of alcohol dehydrogenase. The polypeptide has a molecular weight of 527.3107 Da. This polypeptide can chelate the active site of ADH, which helps to prevent the oxidation of ADH and thus maintain the normal protein molecular structure of ADH during alcohol metabolism. It can be applied to the development of hangover relief products.

[0016] 2. The present invention also provides a bird's nest peptide powder containing bird's nest polypeptides that enhance alcohol dehydrogenase activity. This bird's nest peptide powder can effectively activate the alcohol metabolism rate of ADH, promote alcohol metabolism, and is safe and free of toxic side effects, thus having high market economic value.

[0017] 3. The bird's nest polypeptide provided by this invention is extracted from dried bird's nest powder. The raw material is natural and clean. This invention decomposes and separates the polypeptide components with a molecular weight of less than 3 kDa through secondary enzymatic hydrolysis and analyzes and screens out the polypeptide components with the best hangover relief effect. This maximizes the utilization of the nutritional components of bird's nest, broadens the development path of hangover relief products, and improves the economic value and market position of bird's nest. Attached Figure Description

[0018] Figure 1 The absorbance curves are for the gel chromatography column precipitates with molecular weight <3 kDa obtained by ultrafiltration in Examples 1 and 2.

[0019] Figure 2The alcohol dehydrogenase activation rate of the four polypeptides labeled 1-1, 1-2, 1-3, and 1-4 isolated by chromatography in Examples 1 and 2.

[0020] Figure 3 This is a secondary mass spectrum of the amino acid sequence PLLW bird's nest peptide.

[0021] Figure 4 The conformation of PLLW bird's nest peptide with amino acid sequence in ADH pocket, binding mode in ADH active site, and interaction plane diagram are shown.

[0022] Figure 5 The activation rates of alcohol dehydrogenase in EBNP-1 sample of Example 2 and EBNP-2 and EBNP-3 samples of Comparative Examples 1-2 are given. ac indicates significant differences between groups. P <0.05).

[0023] Figure 6 This is the cytotoxicity of EBNP-1 against LO2 cells in Example 2. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and preferred embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Unless otherwise specified, the methods described in the following embodiments are conventional methods.

[0026] This invention provides an activated alcohol dehydrogenase bird's nest peptide, wherein the amino acid sequence of the activated alcohol dehydrogenase bird's nest peptide is PLLW (SEQ ID NO:1).

[0027] This invention utilizes enzymatic hydrolysis, ultrafiltration, gel chromatography purification, amino acid sequencing, and computer-aided virtual screening techniques to screen dried bird's nest powder, thereby obtaining a novel activated alcohol dehydrogenase bird's nest peptide (SEQ ID NO:1). This activated alcohol dehydrogenase bird's nest peptide has a molecular weight of 527.3107 Da and exhibits a high affinity for alcohol dehydrogenase (ADH), chelating the active site of ADH and helping to prevent ADH oxidation, thus maintaining the normal protein molecular structure of ADH.

[0028] The activated alcohol dehydrogenase bird's nest peptide provided by this invention can also be applied to the preparation of various products that promote alcohol metabolism.

[0029] The following specific embodiments further illustrate the activated alcohol dehydrogenase bird's nest peptide of the present invention and its applications: The experimental indicators and methods involved in the following embodiments are as follows: (1) Gel chromatography column chromatography The gel chromatography column used was a Superdex™ peptide 10 / 300 GL gel chromatography column. The chromatography method was as follows: the sample concentration was 20 mg / mL, the sample volume was 1 mL, the flow rate was 300 μL / min, and one tube was collected every 200 μL. The absorbance curves of each chromatographic component were detected under a 220 nm wavelength UV detector and plotted.

[0030] (2) In vitro alcohol metabolism experiment The experimental indicators and methods for the in vitro alcohol detoxification experiment involved in the following examples are as follows: The in vitro ADH activation rate was determined using the modified Waller-Hoch method. The peptide was prepared into a peptide solution with a concentration of 1 mg / mL. In a test tube, 0.1 mL of the peptide solution was taken and mixed with 1.5 mL of sodium pyrophosphate buffer solution (pH 8.8) and 1.0 mL of 27 mmol / L oxidized coenzyme I (NAD). + The polypeptide solution was mixed with 0.5 mL of 11.5% ethanol solution and incubated at 25°C for 5 min. Immediately after mixing, 0.1 mL of 0.25 U / mL alcohol dehydrogenase (ADH) was added, and the mixture was shaken well. The absorbance was measured at 340 nm, with readings taken every 30 s to determine the activation rate of alcohol dehydrogenase. The measurement was repeated for 5 min. The control group used 0.1 mL of ultrapure water instead of the polypeptide solution. The positive group used KD (Kaiwang Jinzun) instead of the polypeptide solution.

[0031] The activation rate of alcohol dehydrogenase is calculated by the following formula: ADH activation rate / % = (E1 - E0) / E0 × 100% In the formula: E1 and E0 represent the enzyme activities of the experimental group and the control group, respectively. The formula for calculating enzyme activity is as follows: E = (E 340 (× 3.2) / (6.2 × Ew) In the formula: E represents the enzyme activity (U), E 340 Ew is the increase in absorbance per minute at 340 nm, Ew is the amount of enzyme in each milliliter of enzyme solution used (mg / mL), 3.2 is the total volume of the reaction solution (mL), and 6.2 is the millimolecular extinction coefficient of NADH at 340 nm.

[0032] Example 1 This embodiment provides a method for obtaining bird's nest peptides activated by alcohol dehydrogenase, comprising the following steps: 1) Preparation of bird's nest peptide powder This embodiment obtains bird's nest peptide powder by enzymatic hydrolysis, ultrafiltration separation, and gel chromatography purification of dried bird's nest powder, including the following steps: S1. Weigh out the dried bird's nest powder and place it in water for ultrasonic crushing and even dispersion. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid. S2. Adjust the pH of the bird's nest liquid to 8.5, add 1% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 50℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate. S3. Adjust the pH of the trypsin hydrolysate to 2.0, add 1% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 37℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate. S4. Centrifuge the enzyme hydrolysate obtained in step S3 after enzyme inactivation, and use an ultrafiltration tube with a molecular weight cutoff of 3 kDa to ultrafilter out the components with a molecular weight <3 kDa from its supernatant. S5. The components with a molecular weight <3 kDa obtained in step S4 were separated by superdex™ peptide 10 / 300 GL gel chromatography column chromatography. The components with the highest alcohol dehydrogenase activation rate were screened by in vitro alcohol detoxification experiment, and then freeze-dried to obtain the bird's nest peptide powder.

[0033] The absorbance curves of the components obtained by gel chromatography in this embodiment under a 220 nm ultraviolet wavelength detector are shown in the figure below. Figure 1 As shown, the fraction with a molecular weight <3 kDa was separated into four polypeptides labeled 1-1, 1-2, 1-3, and 1-4 by gel chromatography; Figure 2 As shown, the four obtained polypeptides were verified by in vitro alcohol dehydrogenation experiments, and polypeptides 1-4 were the components with the highest activation rate of the alcohol dehydrogenase.

[0034] 2) Amino acid sequence identification In this embodiment, liquid chromatography-mass spectrometry (LC-MS / MS) and PEAKS Studio 11 mass spectrometry software were used to identify the amino acid sequence of the bird's nest peptide powder obtained above. The library search conditions for LC-MS / MS and PEAKS Studio 11 mass spectrometry software are as follows: Liquid chromatography (LC) method: The chromatographic column was a NanoViper C18 1.9 μm, 100A, with phase A being 0.1% FA and phase B being 0.1% FA and 80% ACN. The flow rate was 50 nL / min, with a 70-min chromatographic gradient. The specific elution gradient was as follows: 0–1 min, phase B increased uniformly from 8% to 12%; 1–9 min, phase B increased uniformly from 12% to 15%; 9–31 min, phase B increased uniformly from 15% to 25%; 31–38 min, phase B increased uniformly from 25% to 36%; 38–45 min, phase B increased uniformly from 36% to 60%; 45–53 min, phase B increased uniformly from 60% to 95%; 53–60 min, the mobile phase remained unchanged; 60–70 min, phase B decreased uniformly from 95% to 8%.

[0035] Mass spectrometry (MS) method: Primary mass spectrometry parameters: Resolution: 120000; AGC target: Custorm; Maximum IT: Custorm; Scan range: 100 to 1000 m / z. Secondary mass spectrometry parameters: Resolution: 15000; AGC target: Custorm; Maximum IT: Custorm; Cycle time: 3 s; NCE / stepped NCE: 30.

[0036] Search criteria: The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows: Fixed modifications: Carbamidomethyl (C); Variable modifications: Oxidation (M), Acetyl (Peptide N-term); Enzyme: Non-specific; Database: Apodiformes; Peptide Mass Tolerance (PMT): 20 ppm; Fragment Mass Tolerance (FMT): 0.02 Da.

[0037] 3) Molecular docking screening This embodiment uses computer virtual screening technology to screen for potential alcohol dehydrogenase activating peptides from the amino acid sequence obtained in step 2), and includes the following screening conditions and steps: i) For the peptide amino acid sequences obtained by LC-MS / MS and PEAKS Studio 11 mass spectrometry analysis software, firstly, the activity of the peptides was assessed using the PeptideRanker tool, where a score greater than 0.9 was set (indicating that these peptides are likely to have biological activity); the potential toxicity of the peptides was predicted using the ToxinPred tool to exclude peptides with potential application risks; peptides with a score greater than 0.5 were screened using the CPPpred tool (indicating that the peptides have good cell penetration); and novel peptides in the obtained peptide amino acid sequences were identified using the Biopep database. ii) The novel peptides were screened for bioactivity using the molecular docking software Autodock (version 1.5.7) to obtain peptide sequences with strong protein binding ability; the spatial structure of the peptides was drawn using ChemDraw 22 software and energy minimization was performed; the protonation state and hydrogen orientation were optimized in Autodock and saved as a pdbqt file as a ligand file; iii) Obtain the 3D structure of yeast ADH (PDB ID: 5ENV) from the PDB database, remove repetitive sequences and water molecules, and add polar hydrogen atoms. Set the gridbox within the predicted ADH active pocket, with the active centers of ADH at X: -49.821, Y: 43.354, and Z: -19.199; the grid size is 25.5 Å × 17.25 Å × 18.0 Å.

[0038] In this embodiment, 16 new polypeptide sequences were identified from bird's nest peptide powder. Among them, the bird's nest peptide that activates alcohol dehydrogenase and has the strongest interaction with alcohol dehydrogenase and the most potential hangover-relieving effect is the polypeptide with the amino acid sequence PLLW (SEQ ID NO:1). The MS spectrum of this polypeptide is shown below. Figure 3 As shown. The interaction between the activated alcohol dehydrogenase peptide and ADH is as follows. Figure 4 As shown, PLLW can bind to ADH at a rate of -9.5 kcal / mol. This activated alcohol dehydrogenase peptide can enter the active site of ADH, occupy the hydrophobic pocket, and form bonds with the surrounding amino acid residues. The binding is relatively tight, which can effectively prevent its oxidative modification, maintain the normal protein molecular structure of ADH, thereby improving its catalytic efficiency and achieving the effect of sobering up.

[0039] Example 2 This embodiment provides a bird's nest peptide powder that promotes alcohol metabolism. The bird's nest peptide powder contains the bird's nest peptide that activates alcohol dehydrogenase obtained in Example 1.

[0040] The preparation of the bird's nest peptide powder that promotes alcohol metabolism includes the following steps: S1. Weigh out the dried bird's nest powder, place it in water and ultrasonically crush and disperse it evenly. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid. S2. Adjust the pH of the bird's nest liquid to 8.5, add 1% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 50℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate. S3. Adjust the pH of the trypsin hydrolysate to 2.0, add 1% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 37℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate. S4. Centrifuge the enzyme hydrolysate obtained in step S3 after enzyme inactivation, and use an ultrafiltration tube with a molecular weight cutoff of 3 kDa to ultrafilter out the components with a molecular weight <3 kDa from its supernatant. S5. Using Superdex™ peptide 10 / 300 GL gel chromatography column chromatography, the components with a molecular weight <3 kDa obtained in step S4 are screened through in vitro alcohol dehydrogenase experiments to identify the components with the highest alcohol dehydrogenase activation rate, and then freeze-dried to obtain the bird's nest peptide powder.

[0041] The absorbance curves of the components obtained by gel chromatography in this embodiment under a 220 nm ultraviolet wavelength detector are shown in the figure below. Figure 1 As shown, the fraction with a molecular weight <3 kDa was separated into four polypeptides labeled 1-1, 1-2, 1-3, and 1-4 by gel chromatography; Figure 2 As shown, the four obtained polypeptides were verified by in vitro alcohol dehydrogenation experiments, and polypeptides 1-4 were the components with the highest activation rate of the alcohol dehydrogenase.

[0042] Example 3 This embodiment provides a bird's nest peptide powder that promotes alcohol metabolism. The bird's nest peptide powder contains the bird's nest peptide that activates alcohol dehydrogenase obtained in Example 1.

[0043] The preparation of the bird's nest peptide powder that promotes alcohol metabolism includes the following steps: S1. Weigh out the dried bird's nest powder, place it in water and ultrasonically crush and disperse it evenly. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid. S2. Adjust the pH of the bird's nest liquid to 7.5, add 2% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 40℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate. S3. Adjust the pH of the trypsin hydrolysate to 1.5, add 1% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 40℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate. S4. Centrifuge the enzyme hydrolysate obtained in step S3 after enzyme inactivation, and use an ultrafiltration tube with a molecular weight cutoff of 3 kDa to ultrafilter out the components with a molecular weight <3 kDa from its supernatant. S5. The components with a molecular weight <3kDa obtained in step S4 were chromatographically analyzed using a Superdex™ peptide 10 / 300 GL gel chromatography column. The components with the highest alcohol dehydrogenase activation rate were screened through in vitro alcohol metabolism experiments. The components were then freeze-dried to obtain the bird's nest peptide powder that promotes alcohol metabolism.

[0044] The absorbance of the components obtained by gel chromatography under a 220 nm ultraviolet wavelength detector and the results of the in vitro alcohol detoxification experiment in this embodiment are similar to those in Example 2.

[0045] Example 4 This embodiment provides a bird's nest peptide powder that promotes alcohol metabolism. The bird's nest peptide powder contains the bird's nest peptide that activates alcohol dehydrogenase obtained in Example 1.

[0046] The preparation of the bird's nest peptide powder that promotes alcohol metabolism includes the following steps: S1. Weigh out the dried bird's nest powder, place it in water and ultrasonically crush and disperse it evenly. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid. S2. Adjust the pH of the bird's nest liquid to 7.0, add 1.5% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 45℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate. S3. Adjust the pH of the trypsin hydrolysate to 2.5, add 1.5% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 35℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate. S4. Centrifuge the enzyme hydrolysate obtained in step S3 after enzyme inactivation, and use an ultrafiltration tube with a molecular weight cutoff of 3 kDa to ultrafilter out the components with a molecular weight <3 kDa from its supernatant. S5. The components with a molecular weight <3kDa obtained in step S4 were chromatographically analyzed using a Superdex™ peptide 10 / 300 GL gel chromatography column. The components with the highest alcohol dehydrogenase activation rate were screened through in vitro alcohol metabolism experiments. The components were then freeze-dried to obtain the bird's nest peptide powder that promotes alcohol metabolism.

[0047] The absorbance of the components obtained by gel chromatography under a 220 nm ultraviolet wavelength detector and the results of the in vitro alcohol detoxification experiment in this embodiment are similar to those in Example 2.

[0048] Comparative Example 1 This comparative example provides a method for preparing bird's nest peptide powder, including the following steps: S1. Weigh out the dried bird's nest powder, place it in water and ultrasonically crush and disperse it evenly. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid. S2. Adjust the pH of the bird's nest liquid to 8.5, add 1% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 50℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate. S3. Adjust the pH of the trypsin hydrolysate to 2.0, add 1% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 37℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate. S4. Centrifuge the enzyme hydrolysate obtained in step S3 after enzyme inactivation, and use an ultrafiltration tube with a molecular weight cutoff of 3 kD and an ultrafiltration tube with a molecular weight cutoff of 10 kD to ultrafilter out the components with molecular weight between 3 kD and 10 kD from its supernatant. S5. Freeze-dry the components with molecular weights between 3 kD and 10 kD obtained in step S4 to obtain the bird's nest peptide powder.

[0049] Comparative Example 2 This comparative example provides a method for preparing bird's nest peptide powder, including the following steps: S1. Weigh out the dried bird's nest powder, place it in water and ultrasonically crush and disperse it evenly. The ratio of dried bird's nest powder to water is 1:30. The ultrasonic crushing power is 25 kW and the crushing time is 25 seconds to obtain bird's nest liquid. S2. Adjust the pH of the bird's nest liquid to 8.5, add 1% of the mass of dried bird's nest powder with trypsin (enzyme activity ≥2500U / mg) and enzymatically hydrolyze at 50℃ for 4 h, then inactivate the enzyme with boiling water for 10 min to obtain trypsin hydrolysate. S3. Adjust the pH of the trypsin hydrolysate to 2.0, add 1% of the weight of the dried bird's nest powder with pepsin (enzyme activity ≥3000 U / mg) and hydrolyze at 37℃ for 4 h, then boil water to inactivate the enzyme for 10 min to obtain the pepsin hydrolysate. S4. Centrifuge the pepsin hydrolysate obtained in step S3, and use an ultrafiltration tube with a molecular weight cutoff of 10 kD to ultrafilter out the components with a molecular weight >10 kD from its supernatant. S4. Freeze-dry the components with a molecular weight >10 kD obtained in step S3 to obtain the bird's nest peptide powder.

[0050] Performance testing 1) In vitro alcohol dehydrogenase activation experiment The bird's nest peptide powder that promotes alcohol metabolism described in Example 2 was selected for an in vitro alcohol dehydrogenase activation experiment and compared with the bird's nest peptide powder of Comparative Examples 1-2. The three samples were labeled as EBNP-1, EBNP-2, and EBNP-3, respectively.

[0051] The results are as follows Figure 5 As shown, different peptides all have good activation effects on alcohol dehydrogenase. Compared with the bird's nest peptide powder of Comparative Examples 1-2, the bird's nest peptide powder of Example 2 with a molecular weight of less than 3 kDa has the most prominent activation effect on alcohol dehydrogenase. This is due to the full enzymatic hydrolysis of the peptides, which allows the nutrients of bird's nest to be released and utilized to the maximum extent.

[0052] 2) Cytotoxicity test The bird's nest peptide powder that promotes alcohol metabolism, as described in Example 2, was used in a cytotoxicity experiment.

[0053] LO2 cells in logarithmic growth phase were prepared into a homogeneous single-cell suspension, counted, and diluted with culture medium to 10,000 cells / 100 μL. 100 μL of each cell was seeded into 96-well plates and cultured for 12–16 h. The old culture medium was discarded, and different concentrations of peptide-containing culture medium (culture medium:peptide solution = 99:1) were added, and the cells were cultured for another 24 h. Finally, under light-protected conditions, 100 μL of fresh culture medium and 15 μL of 5 mg / mL thiazolyl blue solution were added to each well, and the cells were incubated for 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well, with shaking for 10 min in the dark. The absorbance was measured at 490 nm. Six parallel wells were set up for each sample group, with a PBS control group included. The cell viability was calculated using the following formula: Cell viability (%) = [(A1–A0) / (A2–A0)] × 100% In the formula: A0 is the blank group; A1 is the sample group; A2 is the control group.

[0054] The results are as follows Figure 6 As shown, the peptide promoted the growth of LO2 cells at concentrations of 0.1–10 μg / mL, and was non-toxic to cells at a concentration of 100 μg / mL, with cell viability between 95% and 110%.

[0055] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Application of the activated alcohol dehydrogenase bird's nest peptide with amino acid sequence SEQ ID NO:1 in the preparation of products that promote alcohol metabolism.

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