A method for preparing bone gelatin composite hangover-relieving peptides based on enzymolysis and fermentation technology

Abstract

This invention discloses a method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology, belonging to the field of hangover-relieving peptide preparation technology. The method involves mixing bone gelatin with ultrapure water to swell, followed by enzymatic hydrolysis to obtain the bone gelatin hydrolysate. Food-grade galactooligosaccharide powder is dissolved in chicory root aqueous extract to obtain a complex prebiotic solution. Mixed herbal powders are added to the complex prebiotic solution, extracted, and then food-grade ethanol is added to obtain an ethanol stress-induced pretreated herbal synergistic solution. The bone gelatin hydrolysate and the ethanol stress-induced pretreated herbal synergistic solution are mixed to obtain a mixture. After fermentation, the mixture is centrifuged and freeze-dried to obtain the bone gelatin complex hangover-relieving peptides. This method improves the ADH activation rate, ALDH activation rate, and DPPH scavenging rate of the hangover-relieving peptides.

Description

Technical Field

[0001] This invention relates to the field of hangover-relieving peptide preparation technology, specifically to a method for preparing bone gelatin composite hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology. Background Technology

[0002] Ethanol is the main component of alcoholic beverages. Over 90% of ethanol metabolism occurs in the liver, with the remaining 5-10% excreted directly through respiration, urination, and sweating. Ethanol metabolism in the liver primarily follows three biochemical pathways: the alcohol dehydrogenase pathway, the cytochrome P450 enzyme (CYP2E1) pathway, and the peroxidase pathway. All three pathways convert ethanol into acetic acid. Most of this acetic acid is converted into acetyl-CoA, entering the tricarboxylic acid cycle (TCA cycle), and ultimately breaking down into carbon dioxide and water, releasing energy. In the alcohol dehydrogenase pathway, the rate of ethanol metabolism is limited by the activity of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and the supply of coenzyme NAD+. Therefore, the amount of ethanol metabolized by the liver per hour is relatively fixed (approximately 10-15 grams for adults, equivalent to the alcohol content of a small glass of beer or half a glass of wine). Thus, excessive alcohol consumption in a short period can lead to ethanol accumulation in the body, causing intoxication. The CYP2E1 pathway directly oxidizes ethanol to acetaldehyde, consuming coenzyme II (NADPH) and oxygen in the process. The activity of CYP2E1 can be induced by long-term alcohol consumption (i.e., the enzyme level is elevated in long-term drinkers), leading to a faster rate of ethanol metabolism. However, this also generates more reactive oxygen species (ROS), exacerbating oxidative damage to the liver. The catalase pathway uses hydrogen peroxide (H2O2) as an oxidant to oxidize ethanol to acetaldehyde, but this pathway contributes very little (only about 1% of the metabolic amount) and is usually negligible. Therefore, it can be seen that the key to human ethanol metabolism is the activity of ADH, ALDH, CYP2E1, the supply of coenzymes NAD+ and NADPH, and the supply of oxygen. Since the metabolism of ethanol in the human liver is mainly through the ADH / ALDH metabolic pathway, under the condition of a certain supply of NAD+, enhancing the activity of ADH and ALDH can accelerate the enzymatic reaction rate, thereby accelerating the liver's ethanol metabolism.

[0003] Existing hangover remedies can be categorized into nutritional supplements and herbal remedies and hangover relief ingredients (diuretics, antiemetics, and gastric mucosal protectants). These products alleviate post-drinking discomfort by restoring energy, promoting urination, and relieving vomiting, but they do not affect the body's metabolism of ethanol. Over 90% of alcohol metabolism occurs in the liver, and excessive drinking directly increases the burden on the liver, potentially leading to alcoholic fatty liver, hepatitis, and cirrhosis in the long term. Therefore, existing hangover remedies cannot mitigate this core damage. Besides hangover remedies, there are also some bioactive peptides that alleviate post-drinking discomfort. These bioactive peptides can be categorized into two types: plant-based (corn, peas, grains, nuts, etc.) and animal-based (fish, poultry, eggs, meat, etc.), and are mostly prepared through enzymatic hydrolysis.

[0004] Collagen is a natural structural protein widely found in animal bones and a core component of the bone's organic matrix. It belongs to type I collagen in the collagen family, consistent with the main collagen types found in skin, tendons, and ligaments. Collagen primarily originates from the bones of mammals such as cattle, sheep, and pigs, and is the main organic component of bones besides minerals like calcium hydroxyphosphate. In bones, collagen exists as fibrous bundles, forming a mesh-like framework upon which minerals are deposited, contributing to bone strength and resilience. Its chemical structure is a typical triple helix, formed by three intertwined α-peptide chains. In terms of amino acid composition, it is rich in glycine (approximately one-third), proline, and hydroxyproline, which are crucial for its stable triple helix structure. After extraction and purification, collagen can be made into gelatin. Gelatin has good gelling, emulsifying and stability properties, and is a common additive in the production of jelly, pudding, candy, canned food and meat products (such as sausage and ham). Unlike collagen, in the production process of gelatin, the triple helix structure of collagen is broken and forms a large number of irreversible unwinding irregular coil structures.

[0005] Based on this, the present invention designs a method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology to solve the above problems. Summary of the Invention

[0006] To address the aforementioned shortcomings of existing technologies, this invention provides a method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technologies.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology includes the following steps:

[0009] (1) Enzymatic hydrolysis of bone gelatin

[0010] Bone gelatin was mixed with ultrapure water and swollen, then papain and bromelain were added for enzymatic hydrolysis. After enzymatic hydrolysis, bone gelatin hydrolysate was obtained.

[0011] (2) Ethanol pretreatment of herbal synergistic extract

[0012] Food-grade galactooligosaccharide powder was dissolved in chicory root aqueous extract to obtain a compound prebiotic solution.

[0013] Mix and grind together the herbs, including *Gynostemma pentaphyllum*, ... and *Moringa oleifera* leaves, to obtain a mixed herbal powder.

[0014] Add the mixed herbal powder to the compound prebiotic solution, extract and then add food-grade ethanol. Stir at 45-55℃ for 3-4 hours at a stirring speed of 100-200 rpm. After cooling the system to room temperature, transfer it to a refrigerator and let it stand overnight. The total time for refrigeration and standing is 8-12 hours. After refrigeration, centrifuge and take the supernatant to recover ethanol by vacuum distillation to obtain the herbal synergistic solution for ethanol stress-induced pretreatment.

[0015] (3) Bone gelatin fermentation and drying

[0016] The bone gelatin hydrolysate was mixed with the herbal synergistic solution that had been pretreated with ethanol stress to obtain a mixture. Lactobacillus plantarum and glucose were then added to the mixture for the first step of fermentation.

[0017] After the first fermentation step is completed, activated seed liquid of Kluyveromyces martensii is inoculated at a rate of 3-5%. Food-grade ethanol is added to bring the ethanol concentration in the system to 2-3% for the second fermentation step. After the fermentation step is completed, sterilization and enzyme inactivation are performed. After centrifugation and freeze-drying, bone gelatin complex hangover-relieving peptides are obtained.

[0018] Furthermore, the specific process of swelling in step (1) is as follows:

[0019] Take bone gelatin (with a gel strength >175g), add it to ultrapure water at 20-25℃ to make the material-to-liquid ratio 1:8-1:10, and let it swell for 8-12 minutes.

[0020] Furthermore, the specific process of enzymatic hydrolysis in step (1) is as follows:

[0021] The ratio of papain to bromelain is 1.0:1.2-1.0:2.0. After adding papain and bromelain, maintain the temperature at 53-60℃ for 2-4 hours for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, cool down to 35-39℃ to obtain bone gelatin hydrolysate.

[0022] Furthermore, the preparation method of chicory root aqueous extract in step (2) is as follows:

[0023] Chicory root powder and ultrapure water are mixed at a ratio of 1:10 to 1:12, and extracted at 65-75℃ for 1.5-2.5 hours. After filtration, the chicory root aqueous extract is obtained.

[0024] Furthermore, the preparation method of the compound prebiotic solution in step (2) is as follows:

[0025] Chicory root aqueous extract and food-grade galactooligosaccharide powder are mixed at a dry weight ratio of 2:1-3:1 and completely dissolved to obtain a compound prebiotic solution.

[0026] Furthermore, in step (2), the mixed herbal powder is obtained by mixing and pulverizing the herbs, including *Gynostemma pentaphyllum*, *Gynostemma pentaphyllum* and *Moringa oleifera* leaves in a weight ratio of 5:3:2, and the ratio of the mixed herbal powder to the compound prebiotic solution is 1:6-1:8.

[0027] Furthermore, the specific steps of step (3) are as follows:

[0028] Bone gelatin enzymatic hydrolysate and herbal synergistic solution pretreated with ethanol stress were mixed at a volume ratio of 1:1-2:1. The mixture was then treated at 70-75℃ for 15-20 seconds to obtain the final solution. 0.01g of *Lactobacillus plantarum* powder and 3g-7g of glucose were added directly to the final solution and stirred until a concentration of 10⁻⁶ was achieved. 7 -10 8 CFU / mL, maintain the first fermentation at 36-38℃ for 18-24 hours, during which the pH of the system is 4.5-5.0;

[0029] After the first fermentation is completed, the activated seed liquid of Kluyveromyces martensii is directly inoculated at a rate of 3-5%. At the same time, food-grade ethanol is added to the fermentation system to make the ethanol concentration in the system reach 2-3%. Aerobic culture is carried out using a shaker, and the second fermentation is carried out for 24-36 hours at 28-32℃ and 150-180rpm.

[0030] After the second fermentation is completed, sterilization and enzyme inactivation are performed. After centrifugation and freeze-drying, bone gelatin complex hangover-relieving peptides are obtained.

[0031] Furthermore, the Lactobacillus plantarum powder is selected from Lactobacillus plantarum X3-4BH strain, which is preserved in the Inner Mongolia Engineering Research Center for Quality Identification and Safe Processing of Beef and Mutton at the College of Food Science and Engineering, Inner Mongolia Agricultural University.

[0032] To better achieve the objectives of this invention, this invention also provides a bone gelatin complex hangover-relieving peptide prepared by the above-described preparation method.

[0033] To better achieve the objectives of this invention, the present invention also provides an application of bone gelatin compound hangover-relieving peptides.

[0034] Compared with the prior art, the beneficial effects of this invention are: 1. It effectively improves the ADH activation rate, ALDH activation rate and DPPH clearance rate of bone gelatin compound hangover detoxifying peptide, and the bone gelatin compound hangover detoxifying peptide still maintains its activity after being stored for a period of time.

[0035] 2. A mouse model of alcohol intoxication was established. By observing the time of intoxication and sobering up in mice, serum indicators and liver biochemical indicators, it can be seen that bone gelatin compound alcohol-detoxifying peptides have a good alcohol-detoxifying effect. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a process flow diagram of the preparation of bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology according to the present invention. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0039] Example 1: A method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology, the process flow diagram is as follows. Figure 1 As shown, the specific steps are as follows:

[0040] (1) Enzymatic hydrolysis of bone gelatin

[0041] Take 10g of bone gelatin (the gelatin's freezing power is >175g), add it to 20℃ ultrapure water to make the material-to-liquid ratio 1:10, and let it swell for 8 minutes.

[0042] Then, 11,000 U of protease was added. The protease consisted of papain and bromelain in a ratio of 1.0:1.2. The mixture was then kept at 60°C for 2 hours for enzymatic hydrolysis.

[0043] After the enzymatic hydrolysis is complete, the temperature is lowered to 35°C to obtain bone gelatin enzymatic hydrolysate.

[0044] (2) Ethanol pretreatment of herbal synergistic extract

[0045] Chicory root powder and ultrapure water were mixed at a ratio of 1:10, and the mixture was extracted at 75°C for 1.5 hours. After filtration, the chicory root aqueous extract was obtained.

[0046] Chicory root aqueous extract and food-grade galactooligosaccharide powder were mixed at a dry weight ratio of 2:1 and completely dissolved to obtain a compound prebiotic solution.

[0047] The herbs *Gynostemma pentaphyllum*, *Gynostemma pentaphyllum*, and *Moringa oleifera* leaves were mixed and pulverized in a weight ratio of 5:3:2 to obtain a mixed herbal powder. The mixed herbal powder was added to a compound prebiotic solution at a material-to-liquid ratio of 1:6 and extracted at 65°C for 1 hour. Then, food-grade ethanol was added to adjust the ethanol concentration in the compound prebiotic solution system to 15%. The mixture was stirred at 45°C for 4 hours at a stirring speed of 100 rpm. After cooling the system to room temperature, it was refrigerated at 4°C overnight for a total of 12 hours. After refrigeration, the mixture was centrifuged (5000 rpm, 15 min) and the supernatant was subjected to vacuum distillation to recover ethanol. The treatment temperature was 40°C and the vacuum degree was 0.09 MPa. The ethanol concentration in the supernatant was reduced to ≤2%, and the pH was adjusted to 6.0 to obtain the herbal synergistic solution pretreated with ethanol stress.

[0048] (3) Bone gelatin fermentation and drying

[0049] Bone gelatin enzymatic hydrolysate and herbal synergistic solution pretreated with ethanol stress were mixed at a volume ratio of 1:1, and then treated at 75℃ for 15 seconds to obtain the mixture. 0.01g of *Lactobacillus plantarum* X3-4BH powder and 7g of glucose were then added, resulting in a powder concentration of 10. 12 CFU / g, add the inoculum powder and grape dressing directly to the mixture and stir well to achieve an inoculum concentration of 10. 7 CFU / mL, and the first fermentation was maintained at 38℃ for 18 hours.

[0050] Currently, strain X3-4BH is preserved at the Inner Mongolia Engineering Research Center for Quality Identification and Safe Processing of Beef and Mutton, College of Food Science and Engineering, Inner Mongolia Agricultural University.

[0051] After the first fermentation was completed, activated seed culture of Kluyveromyces martensii was directly inoculated at a rate of 3%. Food-grade ethanol was added to the fermentation system to bring the ethanol concentration to 3%. The system was then aerobically cultured on a shaker at 28°C and 180 rpm for a second fermentation of 24 hours.

[0052] After the second fermentation, the fermentation broth was heated to 90℃ and maintained for 20 minutes to sterilize and inactivate enzymes. Subsequently, it was centrifuged at 5000 rpm for 15 minutes at 4℃, and the supernatant was collected and frozen at -80℃ for 12 hours. After 12 hours, it underwent freeze-drying to obtain bone gelatin complex hangover-relieving peptides.

[0053] Example 2: A method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology, the specific steps of which are as follows:

[0054] (1) Enzymatic hydrolysis of bone gelatin

[0055] Take 10g of bone gelatin (the gelatin's freezing power is >175g), add it to 25℃ ultrapure water to make the material-to-liquid ratio 1:8, and let it swell for 12 minutes.

[0056] Then, 10,000 U of protease was added. The protease consisted of papain and bromelain in a ratio of 1.0:2.0. The mixture was then kept at 53°C for 4 hours for enzymatic hydrolysis.

[0057] After enzymatic hydrolysis is complete, cool the solution to 35-39℃ to obtain bone gelatin hydrolysate.

[0058] (2) Ethanol pretreatment of herbal synergistic extract

[0059] Chicory root powder and ultrapure water were mixed at a ratio of 1:12, and the mixture was extracted at 65°C for 2.5 hours. After filtration, the chicory root aqueous extract was obtained.

[0060] Chicory root aqueous extract and food-grade galactooligosaccharide powder were mixed at a dry weight ratio of 3:1 and completely dissolved to obtain a compound prebiotic solution.

[0061] The herbs *Gynostemma pentaphyllum*, *Gynostemma pentaphyllum*, and *Moringa oleifera* leaves were mixed and pulverized in a weight ratio of 5:3:2 to obtain a mixed herbal powder. The mixed herbal powder was added to a compound prebiotic solution at a material-to-liquid ratio of 1:8 and extracted at 55°C for 2 hours. Then, food-grade ethanol was added to adjust the ethanol concentration in the system to 8%. The mixture was stirred at 55°C for 3 hours at a stirring speed of 200 rpm. After the system was cooled to room temperature, it was refrigerated at 4°C overnight for a total of 8 hours. After refrigeration, the mixture was centrifuged (5000 rpm, 15 min) and the supernatant was collected for vacuum distillation to recover ethanol. The treatment temperature was 50°C and the vacuum degree was 0.08 MPa. The ethanol concentration in the supernatant was reduced to ≤2%, and the pH was adjusted to 6.5 to obtain the herbal synergistic solution pretreated with ethanol stress.

[0062] (3) Bone gelatin fermentation and drying

[0063] Bone gelatin enzymatic hydrolysate and herbal synergistic solution pretreated with ethanol stress were mixed at a volume ratio of 2:1, and then treated at 70℃ for 20 seconds to obtain the mixture. 0.01g of *Lactobacillus plantarum* X3-4BH powder and 3g of glucose were then added to achieve a powder concentration of 10. 12 CFU / g, add the inoculum powder and grape dressing directly to the mixture and stir well to achieve an inoculum concentration of 10. 8CFU / mL, and the first fermentation was maintained at 36℃ for 24 hours.

[0064] After the first fermentation was completed, activated seed culture of Kluyveromyces martensii was directly inoculated at a rate of 5%. Food-grade ethanol was added to the fermentation system to bring the ethanol concentration to 2%. The system was then cultured aerobically using a shaker at 32°C and 150 pm for a second fermentation of 36 hours.

[0065] After the second fermentation, the fermentation broth was heated to 90℃ and maintained for 20 minutes to sterilize and inactivate enzymes. Subsequently, it was centrifuged at 5000 rpm for 15 minutes at 4℃, and the supernatant was collected and frozen at -80℃ for 12 hours. After 12 hours, it underwent freeze-drying to obtain bone gelatin complex hangover-relieving peptides.

[0066] Example 3: A method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology, the specific steps of which are as follows:

[0067] (1) Enzymatic hydrolysis of bone gelatin

[0068] Take 10g of bone gelatin (the gelatin's freezing power is >175g), add it to 22℃ ultrapure water to make the material-to-liquid ratio 1:9, and let it swell for 10 minutes.

[0069] Then, 10050U of protease was added. The protease consisted of papain and bromelain in a ratio of 1.0:1.5. The mixture was then kept at 55°C for 2.5 hours for enzymatic hydrolysis.

[0070] After enzymatic hydrolysis is complete, the temperature is lowered to 37°C to obtain bone gelatin enzymatic hydrolysate.

[0071] (2) Ethanol pretreatment of herbal synergistic extract

[0072] Chicory root powder and ultrapure water were mixed at a ratio of 1:11, and the mixture was extracted at 70°C for 2 hours. After filtration, the chicory root aqueous extract was obtained.

[0073] Chicory root aqueous extract and food-grade galactooligosaccharide powder were mixed at a dry weight ratio of 2.5:1.0 and completely dissolved to obtain a compound prebiotic solution.

[0074] The herbs *Gynostemma pentaphyllum*, *Gynostemma pentaphyllum*, and *Moringa oleifera* leaves were mixed and pulverized in a weight ratio of 5:3:2 to obtain a mixed herbal powder. The mixed herbal powder was added to a compound prebiotic solution at a material-to-liquid ratio of 1:7 and extracted at 60°C for 1.5 hours. Then, food-grade ethanol was added to adjust the ethanol concentration in the system to 10%. The mixture was stirred at 50°C for 3.5 hours at a stirring speed of 150 rpm. After the system was cooled to room temperature, it was refrigerated at 4°C overnight for a total of 10 hours. After refrigeration, the mixture was centrifuged (5000 rpm, 15 min) and the supernatant was collected for vacuum distillation to recover ethanol. The treatment temperature was 45°C and the vacuum degree was 0.085 MPa. The ethanol concentration in the supernatant was reduced to ≤2%, and the pH was adjusted to 6.2 to obtain the herbal synergistic solution pretreated with ethanol stress.

[0075] (3) Bone gelatin fermentation and drying

[0076] Bone gelatin enzymatic hydrolysate and herbal synergistic solution pretreated with ethanol stress were mixed at a volume ratio of 1.5:1.0, and then treated at 72℃ for 18 seconds to obtain the mixture. 0.01g of *Lactobacillus plantarum* X3-4BH powder and 5g of glucose were then added, resulting in a powder concentration of 10. 12 CFU / g, add the inoculum powder and grape dressing directly to the mixture and stir until the inoculum concentration reaches 5.0 × 10⁻⁶. 7 CFU / mL, and the first fermentation was maintained at 37℃ for 20 hours.

[0077] After the first fermentation was completed, activated seed culture of Kluyveromyces martensii was directly inoculated at a rate of 4%. Food-grade ethanol was added to the fermentation system to bring the ethanol concentration to 2.5%. Aerobic culture was carried out using a shaker, and a second fermentation was carried out for 30 hours at 30°C and 160 rpm.

[0078] After the second fermentation, the fermentation broth was heated to 90℃ and maintained for 20 minutes to sterilize and inactivate enzymes. Subsequently, it was centrifuged at 5000 rpm for 15 minutes at 4℃, and the supernatant was collected and frozen at -80℃ for 12 hours. After 12 hours, it underwent freeze-drying to obtain bone gelatin complex hangover-relieving peptides.

[0079] Comparative Example 1: Compared with Example 3, the difference is that no compound prebiotic solution was added during the ethanol pretreatment of the herbal synergistic extract.

[0080] The specific process is as follows:

[0081] The herbs *Gynostemma pentaphyllum*, *Gynostemma pentaphyllum*, and *Moringa oleifera* leaves were mixed and pulverized in a weight ratio of 5:3:2 to obtain a mixed herbal powder. The mixed herbal powder was added to ultrapure water at a material-to-liquid ratio of 1:7 and extracted at 60°C for 1.5 hours. Then, food-grade ethanol was added to adjust the ethanol concentration in the system to 10%. The mixture was stirred at 50°C for 3.5 hours at a stirring speed of 150 rpm. After the system was cooled to room temperature, it was refrigerated at 4°C overnight for a total of 10 hours. After refrigeration, the mixture was centrifuged (5000 rpm, 15 min) and the supernatant was collected for vacuum distillation to recover ethanol. The treatment temperature was 45°C and the vacuum degree was 0.085 MPa. The ethanol concentration in the supernatant was reduced to ≤2%, and the pH was adjusted to 6.2 to obtain the herbal synergistic solution pretreated with ethanol stress.

[0082] The other steps are the same as in Example 3.

[0083] Comparative Example 2: Compared with Example 3, the difference is that food-grade ethanol was not added during the ethanol pretreatment of the herbal synergistic extract.

[0084] The specific process is as follows:

[0085] Mix and pulverize the herbs, including *Gynostemma pentaphyllum*, *Gynostemma pentaphyllum*, and *Moringa oleifera* leaves in a weight ratio of 5:3:2 to obtain a mixed herbal powder. Add the mixed herbal powder to a compound prebiotic solution at a ratio of 1:7. After cooling to room temperature, refrigerate at 4°C overnight for a total of 10 hours to obtain a herbal synergistic liquid.

[0086] The bone gelatin enzymatic hydrolysate and herbal synergist were mixed at a volume ratio of 1.5:1.0, and then treated at 72°C for 18 seconds to obtain the mixture.

[0087] The other steps are the same as in Example 3.

[0088] Comparative Example 3: The difference from Example 3 is that no food-grade ethanol was added during the second fermentation process of bone gelatin fermentation and drying.

[0089] The specific process is as follows:

[0090] After the first fermentation was completed, activated seed culture of Kluyveromyces martensii was directly inoculated at a rate of 4%, and aerobic culture was carried out using a shaker. The second fermentation was carried out for 30 hours at 30°C and 160 rpm.

[0091] The other steps are the same as in Example 3.

[0092] Experimental example:

[0093] (I) Determination of ADH and ALDH activation rates of bone gelatin compound hangover-detoxifying peptides

[0094] The freeze-dried bone gelatin complex alcohol-detoxifying peptides prepared in Examples 1-3 and Comparative Examples 1-3 were used to determine the ADH and ALDH activation rates after storage at -80℃, -20℃, 4℃, and 20℃ for 0d, 7d, 14d, 21d, 28d, 60d, and 90d, respectively, and after being kept at 60℃, 70℃, and 80℃ for 6h.

[0095] (1) Determination of ADH activation rate

[0096] The ADH activation rate assay used a slightly modified method based on the alcohol dehydrogenase test kit (catalog number A083-1-1) from Nanjing Jiancheng Biotechnology Co., Ltd.

[0097] 1. Prepare a 10 mg / mL aqueous solution of freeze-dried bone gelatin compound hangover-relieving peptide powder for later use;

[0098] 2. Prepare a working solution by mixing Reagent 1, Reagent 2, and Reagent 3 working solutions (powder, add 10mL of ultrapure water before use) in the following volume ratios from the alcohol dehydrogenase test kit: Reagent 1: Reagent 2: Reagent 3 working solution = 13:1:15.

[0099] 3. Prepare a 0.2 U / mL solution of powdered ADH (Maclean, catalog number A861433-1.5KU) for later use;

[0100] 4. Add reagents and samples to the 96-well plate according to Table 1, and continuously measure the absorbance at 340 nm using a microplate reader for 30 minutes:

[0101] Table 1. Amounts of reagents and samples used in the experiment for determining ADH activation rate.

[0102]

[0103] 5. Calculate the ADH activation rate using the first derivative at time 0. ADH activation rate = [(first derivative of test well at time 0 - first derivative of blank well at time 0) / first derivative of blank well at time 0] × 100%.

[0104] The ADH activation rate of bone gelatin compound alcohol-detoxifying peptide under different conditions can be obtained. The results are summarized in Table 2.

[0105] Table 2. Summary of ADH activation rate results of bone gelatin compound hangover-detoxifying peptides under different conditions.

[0106]

[0107] As shown in Comparative Example 1 and Example 3, if the herbal synergistic extract is not treated with a compound prebiotic solution, the ADH activation rate of the prepared hangover-relieving peptides decreases significantly, and the decreasing trend with storage time is more pronounced than in the examples. Comparing the data from 90 days of storage at 20°C, Comparative Example 1 showed 36.59%, while Example 3 showed 56.59%, a 20% decrease in activity. This indicates that the compound prebiotic can enable the hangover-relieving peptides to obtain and maintain high ADH activation activity, and its absence leads to insufficient activity generation and poor stability.

[0108] As shown in Comparative Example 2 and Example 3, if ethanol stress-induced pretreatment is not performed during the herbal extraction process, the ADH activation rate of the prepared alcohol-detoxifying peptides is significantly reduced. Comparing data from 90 days of storage at 20°C, Comparative Example 2 showed a rate of 44.22%, a decrease of 12.37% compared to Example 3 (56.59%). This indicates that ethanol stress induction can effectively activate specific active substances related to ADH activation in the herbal raw materials, making a significant contribution to improving this function of the final product.

[0109] As shown in Comparative Example 3 and Example 3, if ethanol is not added as a co-fermentation substrate during the second fermentation process, the ADH activation rate of the prepared alcohol-detoxifying peptides is affected to some extent. Comparing the data from 90 days of storage at 20°C, Comparative Example 3 showed an activity of 43.11%, which is approximately 13.48% lower than that of Example 3. This indicates that the regulation of yeast metabolic pathways by ethanol during the fermentation stage helps to synthesize or convert more components with ADH-activating function.

[0110] (2) Determination of ALDH activation rate

[0111] Similar to the ADH activation rate assay, the test method using the alcohol dehydrogenase test kit from Nanjing Jiancheng Bioengineering was slightly modified by replacing reagent two in the kit with acetaldehyde solution.

[0112] The ALDH activation rate of bone gelatin compound alcohol-detoxifying peptide under different conditions can be obtained. The results are summarized in Table 3.

[0113] Table 3. Summary of ALDH activation rate results of bone gelatin compound hangover-detoxifying peptides

[0114]

[0115] As shown in Comparative Example 1 and Example 3, the ALDH activation rate of the prepared hangover-relieving peptides decreased most significantly without the pretreatment with the compound prebiotic solution. Comparing data from 90 days of storage at 20°C, Comparative Example 1 showed 37.27%, while Example 3 showed 58.80%, representing a 21.53% decrease in activity. This demonstrates that the compound prebiotic has a significant effect on the ALDH activation ability of the hangover-relieving peptides.

[0116] As shown in Comparative Example 2 and Example 3, the ALDH activation rate of the prepared alcohol-detoxifying peptides decreased significantly without ethanol stress-induced pretreatment. Comparing data from 90 days of storage at 20°C, Comparative Example 2 showed an ALDH activation rate of 39.35%, a decrease of 19.45% compared to Example 3 (58.80%). This indicates that ethanol pretreatment plays a crucial role in promoting the production of specific factors from herbal components that effectively activate ALDH.

[0117] As shown in Comparative Example 3 and Example 3, if ethanol is not added during the secondary fermentation, the ALDH activation rate of the prepared alcohol-detoxifying peptide is less affected than in the first two comparative examples. Comparing the data from 90 days of storage at 20°C, Comparative Example 3 showed an activity of 42.23%, which is 16.57% lower than that of Example 3. The ethanol fermentation step clearly enhances ALDH activity, but its impact is slightly less than that of the prebiotic pretreatment and ethanol stress induction steps.

[0118] (II) Determination of DPPH free radical scavenging activity of bone gelatin complex alcohol-detoxifying peptides

[0119] (1) Measurement principle

[0120] DPPH free radicals are deep purple in ethanol, with a maximum absorption wavelength of 517 nm. In the presence of antioxidants, the unpaired electrons of DPPH are neutralized, and the solution color gradually turns pale yellow, with a decrease in absorbance. The scavenging rate is directly proportional to the antioxidant concentration, calculated using the following formula:

[0121] Clearance rate (%) = (1 - (A sample - A control / A blank)) × 100%

[0122] Wherein, A blank is the absorbance of DPPH and solvent, A sample is the absorbance of DPPH and sample, and A control is the absorbance of sample and solvent.

[0123] (2) Reagent preparation

[0124] DPPH powder with a purity greater than 98% was dissolved in anhydrous ethanol to a concentration of 0.2 mM and used immediately after preparation. Freeze-dried bone gelatin compound hangover-relieving peptide powder was prepared into a 10 mg / mL aqueous solution using ultrapure water.

[0125] (3) Measurement method

[0126] Add reagents and samples to a 96-well plate according to Table 4, and measure the absorbance at 517 nm using an ELISA reader.

[0127] Table 4. Experimental Design for Assay of DPPH Free Radical Scavenging Activity of Bone Gelatin Complex Alcohol-Detoxifying Peptides

[0128]

[0129] (4) Measurement results

[0130] The measurement results are shown in Table 5.

[0131] Table 5. Results of DPPH free radical scavenging activity assay of bone gelatin compound hangover-detoxifying peptides

[0132]

[0133] As shown in Comparative Example 1 and Example 3, the DPPH free radical scavenging rate of the prepared hangover-relieving peptides systematically decreased without the addition of the compound prebiotic solution. Comparing data from 90 days of storage at 20°C, the rate was 37.57% in Comparative Example 1 and 50.83% in Example 3, representing a 13.26% decrease in activity. This demonstrates that treatment with the compound prebiotic can enhance the overall antioxidant performance of the hangover-relieving peptides.

[0134] As shown in Comparative Example 2 and Example 3, the DPPH free radical scavenging rate of the prepared alcohol-detoxifying peptides decreased to some extent without ethanol stress-induced pretreatment. Comparing the data from 90 days of storage at 20°C, Comparative Example 2 showed a DPPH free radical scavenging rate of 40.57%, which was 10.26% lower than that of Example 3 (50.83%). This indicates that ethanol pretreatment helps optimize the extraction efficiency of antioxidant components from herbal raw materials.

[0135] As shown in Comparative Example 3 and Example 3, the DPPH free radical scavenging rate of the prepared alcohol-degrading peptides is least affected if ethanol is not added during the secondary fermentation. Comparing the data from 90 days of storage at 20°C, Comparative Example 3 showed a DPPH free radical scavenging rate of 42.57%, which is 8.26% lower than that of Example 3. This indicates that the fermentation process in the presence of ethanol enhances the direct antioxidant capacity of the final product.

[0136] In summary, compared with comparative examples 1-3, the alcohol-detoxifying peptides prepared in Examples 1-3 showed significant improvements in the three core indicators of ADH activation rate, ALDH activation rate, and DPPH scavenging rate. This demonstrates the necessity of the compound prebiotic treatment, ethanol stress induction, and ethanol-assisted fermentation process. Taking the product represented by Example 3 as an example, under 90-day storage conditions ranging from -80℃ to 20℃: the ADH activation rate remained high with a relatively slow decline; the ALDH activation rate showed good maintenance ability; and the DPPH scavenging rate showed extremely high time stability. Overall, the product exhibits good storage stability, and its activity can be effectively maintained during its shelf life.

[0137] (III) Animal experimental verification of bone gelatin compound hangover-detoxifying peptides

[0138] (1) Experimental design

[0139] Thirty-six SPF-grade male C57BL / 6 mice at the age of weeks were purchased from Hohhot Ruisheng Biotechnology Co., Ltd., with the license number SCXK (Yu) 2020-0005 and the certificate number 4100000000000569.

[0140] Feeding conditions: 6 mice per cage, with a 12 / 12h light-dark cycle, a relative humidity of 50%-60%, and a temperature of 20-25°C. The mice were adaptively fed for one week and weighed during the adaptive feeding period. They were randomly assigned into three groups of 12 mice each according to their body weight, including a control group, a model group, and a bone gelatin compound hangover peptide group. The formal experiment started on the 8th day. During the experiment, the mice were allowed to eat and drink freely, the bedding was changed regularly, and the cages were kept clean and dry.

[0141] During the formal experiment, the mice were weighed again. The dose of the bone gelatin compound hangover peptide was 600 mg / kg (BW), and the actual concentration of the bone gelatin compound hangover peptide required for intragastric administration to the mice was calculated according to their body weight. On the 8th day, the mice in the control group were intragastrically administered 0.3 mL of ultrapure water, the mice in the model group were intragastrically administered 0.3 mL of 56-degree Erguotou liquor, and the mice in the bone gelatin compound hangover peptide group were first intragastrically administered the corresponding dose of the bone gelatin compound hangover peptide solution, and then 0.3 mL of 56-degree Erguotou liquor was intragastrically administered 30 minutes later for the righting reflex experiment of the mice. On the 10th day, the mice were fasted but allowed to drink water for 6 hours, and intragastric administration was continued according to the intragastric administration method on the 8th day. After 30 minutes, blood was collected by eye socket puncture and serum was separated (centrifuged at 3500 r / min at 4°C for 15 minutes after standing at room temperature for 30 minutes), the mice were sacrificed by cervical dislocation and dissected, and the livers were taken and stored in liquid nitrogen.

[0142] (2) Observation of general indicators

[0143] (2.1) Experiment on mouse drunkenness and hangover

[0144] After intragastric administration of liquor to the mice, the righting reflex experiment was carried out. Defining the behavior that the mouse can return to the normal position through its own conditioned reflex after being placed on its back with four feet in the air as the righting reflex, and the disappearance of the righting reflex when it cannot right itself three times; defining the process from the start of intragastric administration of liquor to the disappearance of the righting reflex of the mouse as the drunkenness process; and defining the process from the disappearance of the righting reflex of the mouse to the restoration of the righting reflex to normal as the hangover process. Record the drunkenness and hangover times of each mouse.

[0145] (2.2) Organ index

[0146] Dissect the mouse to take the heart, liver, spleen, kidney, and lung for weighing, and calculate the organ index.

[0147] Organ index = (fresh weight of the organ / body weight of the mouse) × 100%

[0148] The observation results of the general indicators are shown in Table 6.

[0149] Table 6 Summary table of the observation results of general indicators

[0150]

[0151] The organ indices of mice in each group are shown in Table 6. Statistical analysis showed that ethanol intake did not affect the organ indices.

[0152] (3) Measurement of serum markers

[0153] According to the instructions of the enzyme-linked immunosorbent assay kit from Shanghai Enzyme-Linked Reagent Company, the ethanol content (mg / dL), AST content (U / L), ALT content (U / g prot), MDA content (nmol / mg prot), SOD content (U / mg prot), GSH content (umol / mg prot), intoxication time (min), and sobering time (min) in mouse serum were measured.

[0154] The results of the serum marker measurements are shown in Table 7.

[0155] Table 7 Summary of Serum Indicator Measurement Results

[0156]

[0157] As shown in Table 7:

[0158] (1) After adaptive feeding, there were no differences in body weight and organ index among the groups of mice (P>0.05). Compared with the model group mice, the intoxication time of mice administered bone gelatin combined with alcohol-detoxifying peptides by gavage was significantly prolonged and the sobering time was significantly shortened (P<0.05), that is, they got drunk slowly and sobered up quickly.

[0159] (2) The results of serum ethanol content, AST and ALT measurements in each group of mice showed that the model group mice failed to metabolize the ingested ethanol in a timely manner and had minor hepatocellular damage. Under the intervention of bone gelatin compound alcohol-detoxifying peptide, the serum ethanol content of mice in the bone gelatin compound alcohol-detoxifying peptide group decreased significantly (P<0.05), AST and ALT were at normal levels, and no hepatocellular damage was found.

[0160] (3) The results of MDA, SOD and GSH measurements in the serum and liver of mice in each group showed that the model group mice had increased peroxides, an imbalance between peroxidation and the oxidation / antioxidant system, and chronic liver damage caused by oxidative stress. Under the intervention of bone gelatin compound alcohol-detoxifying peptides, the peroxidation and oxidation / antioxidant system in the bone gelatin compound alcohol-detoxifying peptide group maintained normal levels and no imbalance was found.

[0161] In summary, the bone gelatin complex hangover-relieving peptides prepared using the methods in Examples 1-3 exhibited the best performance in terms of ADH activation rate, ALDH activation rate, and DPPH free radical scavenging activity, and maintained high activity stability even after storage at different temperatures for a period of time. Animal experiments further confirmed that this product can significantly prolong the intoxication time and shorten the sobering-up time in mice, effectively reduce serum ethanol concentration, and alleviate alcohol-induced oxidative stress and potential liver damage, demonstrating its application potential in hangover relief and liver damage reduction.

[0162] The above 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 foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology, characterized in that, Includes the following steps: (1) Enzymatic hydrolysis of bone gelatin Bone gelatin was mixed with ultrapure water and swollen, then papain and bromelain were added for enzymatic hydrolysis. After enzymatic hydrolysis, bone gelatin hydrolysate was obtained. (2) Ethanol pretreatment of herbal synergistic extract Food-grade galactooligosaccharide powder was dissolved in chicory root aqueous extract to obtain a compound prebiotic solution. Mix and grind together the herbs, including *Gynostemma pentaphyllum*, ... and *Moringa oleifera* leaves, to obtain a mixed herbal powder. Add the mixed herbal powder to the compound prebiotic solution, extract and then add food-grade ethanol. Stir at 45-55℃ for 3-4 hours at a stirring speed of 100-200 rpm. After cooling the system to room temperature, transfer it to a refrigerator and let it stand overnight. The total time for refrigeration and standing is 8-12 hours. After refrigeration, centrifuge and take the supernatant to recover ethanol by vacuum distillation to obtain the herbal synergistic solution for ethanol stress-induced pretreatment. (3) Bone gelatin fermentation and drying The bone gelatin hydrolysate was mixed with the herbal synergistic solution that had been pretreated with ethanol stress to obtain a mixture. Lactobacillus plantarum and glucose were then added to the mixture for the first step of fermentation. After the first fermentation step is completed, activated seed liquid of Kluyveromyces martensii is inoculated at a rate of 3-5%. Food-grade ethanol is added to bring the ethanol concentration in the system to 2-3% for the second fermentation step. After the fermentation step is completed, sterilization and enzyme inactivation are performed. After centrifugation and freeze-drying, bone gelatin complex hangover-relieving peptides are obtained.

2. The method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology according to claim 1, characterized in that, The specific process of swelling in step (1) is as follows: Take bone gelatin (with a gel strength >175g), add it to ultrapure water at 20-25℃ to make the material-to-liquid ratio 1:8-1:10, and let it swell for 8-12 minutes.

3. The method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology according to claim 1, characterized in that, The specific process of enzymatic hydrolysis in step (1) is as follows: The ratio of papain to bromelain is 1.0:1.2-1.0:2.

0. After adding papain and bromelain, maintain the temperature at 53-60℃ for 2-4 hours for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, cool down to 35-39℃ to obtain bone gelatin hydrolysate.

4. The method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology according to claim 1, characterized in that, The preparation method of chicory root aqueous extract in step (2) is as follows: Chicory root powder and ultrapure water are mixed at a ratio of 1:10 to 1:12, and extracted at 65-75℃ for 1.5-2.5 hours. After filtration, the chicory root aqueous extract is obtained.

5. The method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology according to claim 1, characterized in that, The preparation method of the compound prebiotic solution in step (2) is as follows: Chicory root aqueous extract and food-grade galactooligosaccharide powder are mixed at a dry weight ratio of 2:1-3:1 and completely dissolved to obtain a compound prebiotic solution.

6. The method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology according to claim 1, characterized in that, In step (2), the mixed herbal powder is obtained by mixing and pulverizing the herbs, including *Gynostemma pentaphyllum*, *Gynostemma pentaphyllum* and *Moringa oleifera* leaves in a weight ratio of 5:3:

2. The ratio of the mixed herbal powder to the compound prebiotic solution is 1:6-1:

8.

7. The method for preparing bone gelatin complex hangover-relieving peptides based on enzymatic hydrolysis and fermentation technology according to claim 1, characterized in that, The specific steps of step (3) are as follows: Bone gelatin enzymatic hydrolysate and herbal synergistic solution pretreated with ethanol stress were mixed at a volume ratio of 1:1-2:

1. The mixture was then treated at 70-75℃ for 15-20 seconds to obtain the final solution. 0.01g of *Lactobacillus plantarum* powder and 3g-7g of glucose were added directly to the final solution and stirred until a concentration of 10⁻⁶ was achieved. 7 -10 8 CFU / mL, maintain the first fermentation at 36-38℃ for 18-24 hours, during which the pH of the system is 4.5-5.0; After the first fermentation is completed, the activated seed liquid of Kluyveromyces martensii is directly inoculated at a rate of 3-5%. At the same time, food-grade ethanol is added to the fermentation system to make the ethanol concentration in the system reach 2-3%. Aerobic culture is carried out using a shaker, and the second fermentation is carried out for 24-36 hours at 28-32℃ and 150-180rpm. After the second fermentation is completed, sterilization and enzyme inactivation are performed. After centrifugation and freeze-drying, bone gelatin complex alcohol-detoxifying peptides are obtained.

8. A bone gelatin complex hangover-relieving peptide prepared according to any one of claims 1-7.

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