Development and application of a marine protein source product for protecting liver from alcohol

Abstract

The application discloses a kind of development and application of marine protein source alcoholism-preventing liver-protecting product, belong to biotechnology field.Product preparation includes the following steps: (1) to marine organism homogenate liquid first protease is added to carry out enzymolysis, inactivation after enzymolysis is completed, obtain primary enzymolysis liquid;Second protease is added to primary enzymolysis liquid to carry out enzymolysis, inactivation after enzymolysis is completed, obtain secondary enzymolysis liquid.(2) the supernatant of secondary enzymolysis liquid is obtained by centrifugation.(3) the supernatant is concentrated, freeze-drying to obtain target powder.The application uses 8 kinds of marine organisms as raw materials, adopts 7 kinds of protease, and obtains 336 samples by double-enzyme stepwise enzymolysis;Based on the stability of gastrointestinal digestion, amino acid composition and molecular weight distribution, the raw material is determined as sea cucumber, and the alcoholism-preventing liver-protecting product with high stability, high hydrophobic amino acid composition and low molecular weight is obtained by stepwise hydrolysis using alkaline protease and flavour protease.The application is suitable for food health care field.

Description

Technical Field

[0001] This invention relates to the development and application of a marine protein-based product for relieving hangovers and protecting the liver, belonging to the field of biotechnology. Background Technology

[0002] my country is a major producer and consumer of alcoholic beverages globally. According to statistics from the China Alcoholic Drinks Association, in 2021, the total sales revenue of large-scale enterprises in my country's brewing industry reached 868.67 billion yuan. Adding the sales revenue of smaller enterprises, the Chinese alcoholic beverage market is worth at least one trillion yuan. Excessive alcohol consumption is a leading risk factor for liver disease and liver-related deaths. Alcoholic liver disease (ALD) is a range of liver diseases including fatty liver, hepatitis, cirrhosis, and liver cancer. Excessive drinking is not only related to health but also to social harm, particularly traffic accidents. Therefore, developing safe and effective anti-intoxication, hangover relief, and liver-protecting products to address the physical and social problems caused by daily drinking, work-related socializing, and long-term excessive drinking is a national need in the context of the era of health consciousness.

[0003] Alcohol metabolism in the body is divided into oxidative and non-oxidative pathways. The oxidative pathway is the main way of alcohol metabolism, accounting for more than 90%, and depends on the following three enzyme systems: ① alcohol dehydrogenase (ADH) in the cytoplasm; ② the microsomal ethanol oxidation system in the endoplasmic reticulum; ③ catalase in peroxisomes. Approximately 80% of alcohol entering the body is oxidized to acetaldehyde under the catalysis of ADH, and then further oxidized to acetic acid under the action of acetaldehyde dehydrogenase (ALDH), which is activated to acetyl-CoA, and then enters the tricarboxylic acid cycle to be oxidized and decomposed into carbon dioxide and water and excreted from the body. Most products on the market that accelerate alcohol metabolism rely on the addition of traditional Chinese medicines such as turmeric, Japanese raisin tree fruit, and kudzu root, and some even contain ingredients such as naloxone and diuretics. The quality of these products varies greatly, their hangover-relieving effects are unclear, and they may even increase the burden on the liver. Furthermore, the extraction processes for the active plant components of these traditional Chinese medicines are often cumbersome and slow to take effect. Animal-derived small molecule peptides, on the other hand, have the advantages of easy extraction, easy absorption, rapid effect, and high activity, overcoming the shortcomings of existing plant-derived products. However, these animal-derived products are easily hydrolyzed and inactivated in the digestive tract. Therefore, screening for potential functional animal-derived small molecule peptides based on effectively enhancing ADH activity in the liver and accelerating alcohol metabolism, comprehensively considering digestive tract stability, amino acid composition, and molecular weight distribution, and combining evaluations of anti-hangover and hepatoprotective effects, is one of the important approaches to developing truly effective hangover relief and hepatoprotective products.

[0004] The complex marine environment and abundant biodiversity have resulted in a wealth of marine biological resources, characterized by their diversity and sheer numbers. Marine organisms possess a rich array of bioactive substances. Furthermore, due to the extreme conditions of the ocean, such as low temperatures and high salinity, the amino acid composition and sequence of marine proteins differ significantly from those of terrestrial plants and animals. Therefore, marine protein products possess unique biological advantages. However, the development of high-value-added deep processing of marine resources is currently insufficient, and the market lacks hangover relief and liver protection products with high activity and stable digestion in the body, made from animal protein sources. Summary of the Invention

[0005] This invention addresses the shortcomings in diversity, stability, and activity evaluation of marine biological resource-based hangover and liver-protecting products. It provides a method for preparing a marine protein-based hangover and liver-protecting product that exhibits good water solubility, high stability, and significant efficacy. The anti-hangover, hangover-relieving, and liver-protecting activities of the product were comprehensively evaluated. The specific technical solution is as follows:

[0006] This invention uses eight marine organisms with potential hangover-relieving and liver-protecting effects as raw materials and employs seven common proteases. Through stepwise enzymatic hydrolysis combined with high-speed centrifugation and freeze-drying techniques, a highly active and stable small-molecule hangover-relieving and liver-protecting peptide product is prepared.

[0007] This invention provides a hangover relief and liver protection product derived from marine protein sources. The product contains sea cucumber small molecule peptides. In the sea cucumber small molecule peptides, hydrophobic amino acids account for ≥40% of the total amino acid content, and the proportion of those with a molecular weight of less than 1000 Da is ≥45%.

[0008] The present invention also provides a method for preparing the product, comprising the following steps:

[0009] (1) Raw material pretreatment: The marine organisms are cleaned and the meat is extracted. The endogenous enzymes are sterilized by boiling water bath. Distilled water is added in a certain proportion and homogenized using a homogenizer to obtain a homogenate.

[0010] (2) Add the first protease to the homogenate obtained in step (1) for enzymatic hydrolysis. After hydrolysis, inactivate the protease to obtain a primary hydrolysate. Add the second protease to the primary hydrolysate for enzymatic hydrolysis. After hydrolysis, inactivate the protease to obtain a secondary hydrolysate. The ADH activation rate of the secondary hydrolysate is >75%.

[0011] (3) Centrifuge the secondary enzymatic hydrolysate to remove the precipitate and obtain the supernatant; concentrate the supernatant by rotary evaporation and freeze-dry to obtain the target powder for subsequent measurement.

[0012] In one embodiment, in step (1), the marine organism is any one of sea cucumber, oyster, mantis shrimp, squid, scallop, oyster mussel, jellyfish and sardine; the method for preparing the homogenate is as follows: the marine organism is cleaned and the meat is removed, the endogenous enzymes are inactivated by boiling water bath for 20 minutes, distilled water is added at a material-to-liquid ratio of 1:3 (g:mL), and the homogenate is homogenized using a homogenizer.

[0013] In one embodiment, in step (2), the first protease and the second protease are selected from alkaline protease, neutral protease, pepsin, trypsin, papain, flavor protease, and bromelain, respectively, and the first protease and the second protease cannot be the same.

[0014] In one embodiment, in step (2), the amount of protease added in the preparation method is 1000 U / g, which is sufficient to fully enzymatically hydrolyze marine organisms.

[0015] In one embodiment, in step (2), the ADH detection kit used in the preparation method was purchased from Nanjing Jiancheng Biotechnology Research Institute, catalog number A083-2-1.

[0016] In one embodiment, in step (2), the conditions for both the first and second enzymatic hydrolysis in the preparation method are: pH 3.0–10.0, temperature 37–50°C, and time 3 hours. When selecting a protease for enzymatic hydrolysis, the hydrolysis is performed in a water bath with shaking for 3 hours at a suitable pH and temperature. The suitable pH and temperature for alkaline protease are 10.0 and 40°C; for neutral protease, it is 7.0 and 50°C; for pepsin, it is 3.0 and 37°C; for trypsin, it is 8.5 and 37°C; for papain, it is 7.0 and 50°C; for flavor protease, it is 7.5 and 50°C; and for bromelain, it is 7.0 and 50°C.

[0017] In one embodiment, in step (2), the inactivation method in the preparation method is a boiling water bath for 10 to 20 minutes.

[0018] In one embodiment, in step (2), the centrifugation conditions in the preparation method are 4500-5000 rpm for 15-20 min.

[0019] In one embodiment, the obtained product is in powder form, and its color is brown, yellow, or white (depending on the raw materials); its molecular weight is below 3000 Da; its ADH activation rate is >75%, and the ADH activation rate is stable after gastrointestinal digestion; it has certain anti-drunkenness and sobering effects, significantly prolongs the latency period of intoxication in mice and shortens the sleep period; it has a certain effect in alleviating alcoholic liver damage, and can significantly reduce the activity of serum liver function enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST), activate the activity of alcohol metabolism enzymes ADH and ALDH, significantly reduce the accumulation of malondialdehyde (MDA) in the liver, increase the content of superoxide dismutase (SOD) and glutathione (GSH), significantly reduce the content of triglycerides (TG) and total cholesterol (TC), reduce fat accumulation, and this is reflected in the pathological morphology of liver tissue.

[0020] The present invention also provides the application of the method in the preparation of hangover relief and liver protection products.

[0021] Beneficial effects:

[0022] 1. This invention employs a two-enzyme stepwise enzymatic hydrolysis method to fully hydrolyze eight marine organisms. From 336 enzymatically hydrolyzed samples, marine-derived products with hangover-relieving and liver-protecting effects were screened out. The product is a small molecule peptide from sea cucumber, with an ADH activation rate of 118.86%, which is 1.23 times that of the positive control, Haiwang Jinzun.

[0023] 2. The sea cucumber small molecule peptides (SJPs) prepared by this invention are freeze-dried to obtain powder, resulting in a finer final product that is easily soluble in water. In these sea cucumber small molecule peptides, hydrophobic amino acids account for ≥40% of the total amino acid content, and those with a molecular weight below 1000 Da account for ≥45%, exhibiting gastrointestinal digestibility.

[0024] 3. The sea cucumber small molecule peptide hangover relief and liver protection product prepared by this invention has shown effective hangover relief and liver protection effects in animal experiments.

[0025] (1) In an acute mouse alcohol poisoning model, compared with the alcohol model group, high doses of SJPs prolonged the latency period from 9.14 min to 15.11 min, an increase of 1.65 times; and significantly shortened the sleep period from 162.96 min to 65.24 min, an increase of 2.50 times, showing a significant anti-drunkenness and alcohol-relieving effect.

[0026] (2) In an early ALD model in mice, the sea cucumber small molecule peptide product prepared in this invention exhibited significant liver-protective effects, as detailed below:

[0027] The high-dose SJPs group showed reductions of 45.47% and 45.73% in ALT and AST, respectively, compared to the alcohol model group, and reductions of 8.32% and 16.95%, respectively, compared to the positive control group.

[0028] The liver TG and TC contents of mice in the high-dose SJPs group were significantly reduced by 37.74% and 29.41% respectively compared with the alcohol model group (P<0.05), and by 23.26% and 14.29% respectively compared with the positive control group.

[0029] The high-dose SJPs group significantly increased ADH activity in mouse liver by 27.62% and ALDH activity by 80.53% (P<0.05), which were 4.28% and 21.07% higher than those in the positive control group, respectively.

[0030] Compared with the alcohol model group, the high-dose SJPs group showed a significant increase in SOD activity (13.52%), a significant increase in GSH content (60.31%), and a significant decrease in MDA content (36.52%) (P<0.05). Compared with the positive control group, the high-dose SJPs group showed a 4.49% increase in SOD activity, a 7.86% increase in GSH content, and a 4.58% decrease in MDA content.

[0031] 4. The preparation process of the present invention is simple and easy to operate, with mild conditions and high stability. Attached Figure Description

[0032] Figure 1 This study analyzes the stability of ADH activation rate during in vitro simulated gastrointestinal digestion in Examples 2-6 of the present invention.

[0033] Figure 2 This is an in vitro kinetic analysis of ADH in Example 2 of the present invention.

[0034] Figure 3 The modeling process for establishing an early mouse ALD model using the NIAAA method.

[0035] Figure 4 This is the effect of Example 2 of the present invention on the pathological characteristics of mouse liver tissue. Detailed Implementation

[0036] The present invention will be further described below with reference to the embodiments. Unless otherwise specified, all methods are conventional. Unless otherwise specified, the reagents and materials mentioned are commercially available.

[0037] The commercial proteases used in the following examples were purchased from Shanghai Yuanye Biotechnology Co., Ltd., with the following catalog numbers: alkaline protease (S10154), neutral protease (S10013), pepsin (S10028), trypsin (S10032), papain (S10011), flavor protease (S10153), and bromelain (S10009), with an activity range of 1.5 × 10⁻⁶. 4 ~3×10 5 .

[0038] Example 1: Screening of marine biological enzymatic hydrolysis samples with high ADH activation rate

[0039] This invention utilizes diverse marine organisms and a variety of proteases, providing ample sample size. Eight marine organisms (sea cucumber, oyster, mantis shrimp, squid, scallop, razor clam, jellyfish, and sardine) are first hydrolyzed using one of seven enzymes (alkaline protease, neutral protease, pepsin, trypsin, papain, flavor protease, and bromelain), yielding 56 single-enzyme hydrolysates. These 56 samples are then further hydrolyzed using one of the remaining six enzymes, resulting in 336 stepwise double-enzyme hydrolysates. For example, sea cucumber is first hydrolyzed with alkaline protease, then further hydrolyzed with neutral protease, pepsin, trypsin, papain, flavor protease, and bromelain, yielding six samples each. This process can be repeated to obtain 336 samples.

[0040] The above-mentioned enzymatically digested samples were screened based on their ADH activation rate. The ADH activation rate of 336 samples was detected using a publicly reported method. Specifically, the method utilizes the principle of ADH catalyzing the oxidative coenzyme I reaction; ADH catalyzes the dehydrogenation of ethanol to produce acetaldehyde, while NAD... + It is reduced to NADH. NADH has a maximum absorption peak at 340 nm, while other components show no color development. Therefore, the amount of NADH produced can be used to indirectly characterize ADH activity. Following the ADH kit instructions, the protein concentration of the sample and positive control was adjusted to 1 mg / mL using deionized water. 50 μL of sample was mixed with 150 μL of working solution (Reagent 1: Reagent 2: Reagent 3 = 0.65:0.05:0.70), and after equilibration at 37°C for 5 min, 50 μL of ADH (1 U / mL) was added to initiate the reaction. The time was immediately started, and the absorbance was measured at 340 nm using a multi-mode microplate reader. The temperature was set to 37°C, and the absorbance value (OD) at 340 nm was recorded every 10 seconds. 340nm The reaction was carried out for 10 minutes. Distilled water was used as a negative control. The reaction curve was fitted, and the first derivative of the fitted curve at 0 min was calculated, representing the rate of increase of NADH, i.e., the initial reaction rate. All measurements were performed in triplicate. The formula for calculating the ADH activation rate is as follows:

[0041]

[0042] In the formula: V S V0 is the initial reaction rate of the sample; V0 is the initial reaction rate of the negative control.

[0043] Initial screening: The ADH activation rates of 336 samples were arranged from highest to lowest, and the 36 samples with the highest activation rates were selected. Fine screening: The 36 samples were re-prepared using the same method, and their ADH activation rates were tested. The results are shown in Table 1. Among them, 21 samples had higher ADH activation rates than Haiwang Jinzun. The 5 samples with the highest ADH activation rates (preparation methods as shown in Examples 2-6) were selected for further screening. Table 1 shows that samples 1-5 (corresponding to Examples 2-6 of this invention) all had a good effect on improving the in vitro ADH activation rate, significantly greater than the positive control group Haiwang Jinzun (96.42%, P<0.05), at 122.40%, 118.86%, 117.79%, 116.88%, and 117.86%, respectively, which were 26.94%, 23.27%, 22.16%, 21.22%, and 22.24% higher than Haiwang Jinzun, respectively. This suggests that these five examples have the potential to help with sobering up and promoting alcohol metabolism.

[0044] Table 1. ADH activation rates of 136 samples (sample groups sorted from highest to lowest)

[0045]

[0046]

[0047] Note: In the table, "alkali" represents alkaline protease; "wind" represents flavor protease; "stomach" represents pepsin; "pancreas" represents trypsin; "wood" represents papain; "medium" represents neutral protease; and "spinach" represents bromelain. The order of enzymatic digestion is separated by a slash ( / ). Different letters in the same column's superscript indicate significant differences (P<0.05).

[0048] Example 2: Preparation of sea cucumber alkaloid / wind sample

[0049] The preparation method of sea cucumber alkaloid / wind in sample No. 2 in Table 1 is as follows:

[0050] (1) Raw material pretreatment: Remove the internal organs and mouth of the fresh sea cucumber, wash it, cut it into pieces, boil it in water for 20 minutes to inactivate endogenous enzymes, and add 3 times the volume of distilled water to homogenize it.

[0051] (2) Enzymatic hydrolysis: The sea cucumber homogenate was preheated to 40℃, and the pH was adjusted to 10.0 with 1 mol / L sodium hydroxide solution. Alkaline protease was added at a concentration of 1000 U / g sea cucumber (based on the final concentration). The mixture was hydrolyzed in a water bath at 40℃ for 3 hours with shaking. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 minutes. The temperature of the hydrolysate was then adjusted to 50℃, and the pH was adjusted to 7.5 with 1 mol / L hydrochloric acid solution. Flavor protease was added at a concentration of 1000 U / g sea cucumber (based on the final concentration). The mixture was hydrolyzed in a water bath at 50℃ for 3 hours with shaking. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 minutes.

[0052] (3) Centrifuge the enzymatic hydrolysate at 4500 rpm for 15 min, take the supernatant, and concentrate the supernatant by rotary evaporation to about 3 times the volume. Freeze the resulting concentrate at -80℃ for more than 8 h, and then transfer it to a freeze dryer and dry for about 48 h to obtain sea cucumber small molecule peptide powder.

[0053] Example 3: Preparation of shrimp wind / alkali samples

[0054] The preparation method of shrimp venom / alkali for sample No. 1 in Table 1 is as follows:

[0055] (1) Raw material pretreatment: Remove the shells and internal organs of the thawed shrimp, take only the meat, wash it, cut it into pieces, boil it in water for 20 minutes to inactivate endogenous enzymes, and add 3 times the volume of distilled water to homogenize it.

[0056] (2) Enzymatic hydrolysis: The shrimp homogenate was preheated to 50°C, and the pH was adjusted to 7.5 with 1 mol / L sodium hydroxide solution. Flavor protease was added at a concentration of 1000 U / g shrimp (based on the final concentration). The mixture was hydrolyzed in a water bath at 50°C for 3 hours with shaking. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 minutes. The temperature of the hydrolysate was then adjusted to 40°C, and the pH was adjusted to 10.0 with 1 mol / L sodium hydroxide solution. Alkaline protease was added at a concentration of 1000 U / g shrimp (based on the final concentration). The mixture was hydrolyzed in a water bath at 40°C for 3 hours with shaking. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 minutes.

[0057] (3) Centrifuge the enzymatic hydrolysate at 4500 rpm for 15 min, take the supernatant, and concentrate the supernatant by rotary evaporation to about 3 times the volume. Freeze the resulting concentrate at -80℃ for more than 8 h, and then transfer it to a freeze dryer and dry for about 48 h to obtain small molecule peptide powder of shrimp.

[0058] Example 4 Preparation of Squid Alkine / Medium Sample

[0059] The preparation method of squid alkaloid in sample No. 3 in Table 1 is as follows:

[0060] (1) Raw material pretreatment: Remove the skin from the thawed squid, take only the meat, wash it, cut it into pieces, boil it in water for 20 minutes to inactivate endogenous enzymes, and add 3 times the volume of distilled water to homogenize it.

[0061] (2) Enzymatic hydrolysis: The squid homogenate was preheated to 40°C, and the pH was adjusted to 10.0 with 1 mol / L sodium hydroxide solution. Alkaline protease was added at a concentration of 1000 U / g squid (based on the final concentration). The mixture was hydrolyzed in a water bath at 40°C for 3 hours with shaking. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 minutes. The temperature of the hydrolysate was then adjusted to 50°C, and the pH was adjusted to 7.0 with 1 mol / L hydrochloric acid solution. Neutral protease was added at a concentration of 1000 U / g squid (based on the final concentration). The mixture was hydrolyzed in a water bath at 50°C for 3 hours with shaking. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 minutes.

[0062] (3) Centrifuge the enzymatic hydrolysate at 4500 rpm for 15 min, take the supernatant, and concentrate the supernatant by rotary evaporation to about 3 times the volume. Freeze the resulting concentrate at -80℃ for more than 8 h, and then transfer it to a freeze dryer and dry for about 48 h to obtain squid small molecule peptide powder.

[0063] Example 5: Preparation of squid pancreas / skin samples

[0064] The preparation method of pancreas / sugar from sample 4 of the squid in Table 1 is as follows:

[0065] (1) Raw material pretreatment: Remove the skin from the thawed squid, take only the meat, wash it, cut it into pieces, boil it in water for 20 minutes to inactivate endogenous enzymes, and add 3 times the volume of distilled water to homogenize it.

[0066] (2) Enzymatic hydrolysis: The squid homogenate was preheated to 50°C, and the pH was adjusted to 7.0 with 1 mol / L sodium hydroxide solution. Neutral protease was added at a concentration of 1000 U / g squid (based on the final concentration). The mixture was hydrolyzed in a water bath at 50°C for 3 hours with shaking. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 minutes. The temperature of the hydrolysate was then adjusted to 37°C, and the pH was adjusted to 8.5 with 1 mol / L sodium hydroxide solution. Trypsin was added at a concentration of 1000 U / g squid (based on the final concentration). The mixture was hydrolyzed in a water bath at 37°C for 3 hours with shaking. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 minutes.

[0067] (3) Centrifuge the enzymatic hydrolysate at 4500 rpm for 15 min, take the supernatant, and concentrate the supernatant by rotary evaporation to about 3 times the volume. Freeze the resulting concentrate at -80℃ for more than 8 h, and then transfer it to a freeze dryer and dry for about 48 h to obtain squid small molecule peptide powder.

[0068] Example 6: Preparation of sea cucumber pancreas / wind samples

[0069] The preparation method of sea cucumber pancreas / wind in sample No. 5 of Table 1 is as follows:

[0070] (1) Raw material pretreatment: Remove the internal organs and mouth of the fresh sea cucumber, wash it, cut it into pieces, boil it in water for 20 minutes to inactivate endogenous enzymes, and add 3 times the volume of distilled water to homogenize it.

[0071] (2) Enzymatic hydrolysis: The sea cucumber homogenate was preheated to 37°C, and the pH was adjusted to 8.5 with 1 mol / L sodium hydroxide solution. Trypsin was added at a concentration of 1000 U / g sea cucumber (based on the final concentration). The mixture was hydrolyzed in a water bath at 37°C for 3 hours with shaking. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 minutes. The temperature of the hydrolysate was then adjusted to 50°C, and the pH was adjusted to 7.5. Flavor protease was added at a concentration of 1000 U / g sea cucumber (based on the final concentration). The mixture was hydrolyzed in a water bath at 50°C for 3 hours with shaking. After hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 minutes.

[0072] (3) Centrifuge the enzymatic hydrolysate at 4500 rpm for 15 min, take the supernatant, and concentrate the supernatant by rotary evaporation to about 3 times the volume. Freeze the resulting concentrate at -80℃ for more than 8 h, and then transfer it to a freeze dryer and dry for about 48 h to obtain sea cucumber small molecule peptide powder.

[0073] Example 7: Comparative Analysis of Gastrointestinal Digestive Stability, Amino Acid Composition, and Molecular Weight

[0074] The gastrointestinal digestibility, amino acid composition, and molecular weight of the small molecule peptide products prepared in Examples 2-6 were compared and analyzed.

[0075] 1. Gastrointestinal digestive stability

[0076] Simulated digestive solutions (gastric and intestinal juices) were prepared, and their components are shown in Table 2. First, simulated gastric digestion was performed by mixing a 1 mg / mL sample with an equal volume of simulated gastric digestion fluid. Pepsin was added to a final concentration of 2000 U / mL, and the pH was adjusted to 3.0. The mixture was then incubated at 37°C with shaking in a water bath for 2 hours. After digestion, the enzyme was inactivated by boiling in a water bath for 10 minutes. Next, simulated intestinal digestion was performed by mixing the solution with an equal volume of simulated intestinal digestion fluid. Trypsin was added to a final concentration of 100 U / mL, and the pH was adjusted to 7.0. The mixture was then incubated at 37°C with shaking in a water bath for 2 hours. After digestion, the enzyme was inactivated by boiling in a water bath for 10 minutes. After cooling to room temperature, the mixture was centrifuged at 4500 rpm for 15 minutes. The supernatant was collected, concentrated by evaporation, freeze-dried, and stored at -20°C for later use.

[0077] Table 2 Composition of simulated digestive fluid

[0078]

[0079] The ADH activation rate of the samples from Examples 2-6 after gastrointestinal digestion was detected. The experimental results are as follows: Figure 1 As shown. By Figure 1 It can be seen that, after simulated in vitro gastrointestinal digestion, Example 2 has a relatively stable effect overall, with almost no change in ADH activation rate, indicating that it can still maintain a high alcohol-relieving effect after gastrointestinal digestion. However, the ADH activation rate of the samples in Examples 3-6 decreased to varying degrees after gastrointestinal digestion, and they could not maintain a stable alcohol-relieving effect.

[0080] 2. Amino acid composition

[0081] Following the publicly reported detection methods, high performance liquid chromatography was used to analyze the amino acid composition of Examples 2-6, and the results are shown in Table 3.

[0082] Table 3. Amino acid composition (g / 100g) of different embodiments

[0083]

[0084] Note: *Essential amino acids; #Hydrophobic amino acids.

[0085] Some studies have reported that a higher content of hydrophobic amino acids may help produce stable NAD. + This promotes the decomposition of acetaldehyde and plays an important role in promoting the metabolism of ethanol in the liver. The above experimental results show that all five examples after stepwise enzymatic hydrolysis with two enzymes contained a large amount of hydrophobic amino acids, with the hydrophobic amino acid content of Examples 6 and 2 exceeding 40%.

[0086] 3. Molecular weight distribution

[0087] Following the publicly reported detection methods, high performance liquid chromatography was used to analyze the molecular weight distribution of Examples 2-6, and the results are shown in Table 4.

[0088] Table 4 Molecular weight distribution of different embodiments

[0089]

[0090] As shown in Table 4, after stepwise enzymatic hydrolysis using two enzymes, the molecular weight distribution of the hydrolysates was mainly <3000 Da. Among them, in Example 2 (sea cucumber as raw material), the proportion of small molecule active peptides (molecular weight <1000 Da) exceeded 45%, which were more easily absorbed, more stable, and more active.

[0091] Considering the gastrointestinal digestibility, amino acid composition, and molecular weight distribution of Examples 2-6, Example 2 has a higher content of hydrophobic amino acids and a higher proportion of small molecule peptides, and maintains a high ADH activation rate even after gastrointestinal digestion. Therefore, Example 2 is selected as the best alternative for the subsequent development of hangover relief and liver protection products.

[0092] Example 8: In vitro kinetic analysis of ADH

[0093] Referring to the method for detecting ADH activation rate in Example 1, the changes in ADH activation rate under different concentrations as described in Example 2 were analyzed. Following the method of Example 2, a sea cucumber saponin / water product (protein content 16.84%) for alcohol detoxification and liver protection was prepared, and solutions of 0, 0.1, 0.3, 0.5, 0.8, 1, 1.5, 2, and 2.5 mg / mL sea cucumber saponin / water were formulated to intervene in the ADH-catalyzed alcohol oxidation process. The reaction kinetics are as follows: Figure 2 As shown in (A), it can be seen that with the increase of the concentration in Example 2, the OD value at 340 nm increases at a faster rate. Within the monitored 10 min, the reaction curve can be fitted by the following equation:

[0094]

[0095] The parameters of the equation fitting the reaction curve under different concentrations of intervention in Example 2 are as follows: Figure 2 As shown in the table in (A), the first derivative value at 0 min obtained from the fitted equation is the initial reaction rate, which can characterize the relative enzyme activity of ADH. The initial reaction rate without the intervention of Example 2 is recorded as V0, while the initial reaction rate with the intervention of Example 2 is recorded as Vs. The ADH activation rate was calculated according to the method of Example 1. Curve fitting was performed on the corresponding ADH activation rates under different concentrations of the intervention of Example 2, and the results are as follows. Figure 2 As shown in (B), the ADH activation rate increased with increasing concentration in Example 2, exhibiting a good dose-response relationship, indicating that Example 2 effectively intervened in the ADH-catalyzed alcohol oxidation process. The dose-response relationship of Example 2 can be fitted by the following equation:

[0096]

[0097] Based on the above fitting of the ADH-catalyzed alcohol oxidation reaction curve and the establishment of the dose-effect relationship equation, it can be seen that the initial reaction rate without the intervention of Example 2 (aqueous solution of sea cucumber saponin / wind) is 6.16 × 10⁻⁶. -4 The initial response rate reached 13.04 × 10⁻⁵ when treated with 2.5 mg / mL in Example 2. -4 The ADH activation rate increased by 111.67%, which can effectively accelerate the rate of alcohol metabolism.

[0098] Example 9: Verification of the hangover-relieving effect

[0099] Using the sea cucumber small molecule peptides (Stichopus japonicus peptides, SJPs) obtained in Example 2 as experimental material, 60 male C57BL / 6J mice were randomly divided into 6 groups after one week of acclimatization: blank control group, alcohol model group, positive control group, low-dose SJPs group, medium-dose SJPs group, and high-dose SJPs group, with 10 mice in each group. Mice were fasted for 12 hours before the experiment but allowed free access to water. The positive control group was administered 400 mg / kg BW Haiwang Jinzun (converted according to the recommended dosage of Haiwang Jinzun) by gavage. The low, medium, and high-dose SJPs groups were administered 200, 400, and 600 mg / kg BW SJPs by gavage, respectively. The blank control group and alcohol model group were given an equal volume of physiological saline. 30 minutes later, all groups except the blank control group were administered 9.8 mL / kg BW 56° Hongxing Erguotou by gavage, while the blank control group was administered an equal volume of physiological saline by gavage.

[0100] Immediately after administering alcohol to mice, timing was started, and the mice's post-drinking condition was observed using the "righting reflex test (LORR)". When a mouse walked unsteadily or sluggishly, it was placed in a supine position with its back facing down. If the mouse maintained this position for more than 30 seconds, the righting reflex was considered to have disappeared, and the time of disappearance of the righting reflex (latency period of intoxication) was recorded. When a mouse was able to roll over twice consecutively within 60 seconds, the righting reflex was considered to have recovered, and the recovery time of the righting reflex (sobering-up time) and the number of intoxicated mice were recorded. The results are shown in Table 5.

[0101] Table 5. Effects of SJPs intake on LORR in mice

[0102]

[0103] Note: Different letters in the same column's header indicate significant differences (P<0.05).

[0104] Table 5 shows that acute alcohol intake caused LORR in 80% of the model group mice, with an average latency of 9.14 min and a duration of 162.96 min. This indicates that alcohol intake successfully induced acute alcohol poisoning in mice. Ingestion of SJPs and Haiwang Jinzun 30 min before oral administration of baijiu (Chinese liquor) reduced the incidence of LORR, prolonged the latency of LORR, and shortened the duration of LORR to varying degrees. High-dose SJPs prolonged the latency from 9.14 min to 15.11 min and significantly shortened the sleep period from 162.96 min to 65.24 min, reducing the time by more than half (P<0.05). These results indicate that SJPs intake can increase sleep latency, reduce sleep time, and accelerate sobering up.

[0105] Example 10: Verification of Liver Protection Efficacy

[0106] Using the sea cucumber small molecule peptides (Stichopus japonicus peptides, SJPs) obtained in Example 2 as experimental materials, an early ALD model in mice was established using a chronic feeding + acute gavage model (NIAAA). Fifty-four male C57BL / 6J mice were randomly divided into six groups after one week of acclimatization: a blank control group, an alcohol model group, a positive control group, a low-dose SJPs group, a medium-dose SJPs group, and a high-dose SJPs group, with nine mice in each group and three mice per cage. The first five days were the acclimatization period, during which mice were fed ordinary solid feed and water. From days 6 to 12, an alcoholic liquid diet acclimatization period was implemented. Initially, each mouse was given 25 mL of liquid feed (including control feed and alcohol feed) for free access. This was adjusted daily based on the previous day's feed intake to ensure equal caloric intake across groups. The ethanol concentration of the liquid feed was gradually increased from 0% to 5% (v / v). From days 13 to 21, mice were given an alcohol feed with a 5% (v / v) ethanol content (except for the blank group) to establish the early ALD model. Modeling process as follows Figure 3 As shown. The alcohol-containing liquid feed is a commercially available feed purchased from Nantong Trofi Feed Technology Co., Ltd. The blank control feed has the product number TP4030C, and the alcohol-containing feed has the product number TP4030D. TP4030C has the following caloric distribution: fat 35%, protein 18%, carbohydrates 47%. TP4030D has the following caloric distribution: alcohol 28% (alcohol content: 5%, V / V, mL / 100mL), fat 35%, protein 18%, carbohydrates 19%. Alcohol-containing feeds with different alcohol contents were formulated according to the product instructions.

[0107] During this period, food intake was recorded daily. The liquid diet given to each group of mice was adjusted by calculating the minimum calorie intake of the average experimental group mice to ensure that each group of mice consumed the same amount of calories. Each day at a fixed time (4 pm to 5 pm), mice in each group were administered liquid diets by gavage at doses of 400 mg / kg BW Sea King Gold 200, 400, and 600 mg / kg BW SJPs, respectively. The control group and the model group were administered an equal volume of physiological saline by gavage. The liquid diet was changed at a fixed time (5 pm to 6 pm).

[0108] On the morning of day 22 (7:00-9:00 AM), mice in each group were administered BW (Bio-Hyperdimension Neptunia) at doses of 400 mg / kg, 200 mg / kg, 400 mg / kg, and 600 mg / kg, respectively, via gavage. Thirty minutes later, all groups except the control group were administered 12 mL / kg of BW 45% ethanol solution via gavage. The control group was administered an equal-caloric dose of maltodextrin via gavage. After a 9-hour fast, blood was collected from the mice's eyes, followed by isoflurane anesthesia and sacrifice. The livers were harvested, cryopreserved in liquid nitrogen, and used for subsequent biochemical assays.

[0109] The liver index is the ratio of liver weight to body weight in mice, and the results are shown in Table 6.

[0110] Table 6. Effects of SJPs intake on liver weight and liver index in mice.

[0111]

[0112] Note: Different letters in the same column's header indicate significant differences (P<0.05).

[0113] Changes in liver weight and liver index can macroscopically reflect the accumulation of fatty acids in the mouse liver and can infer the degree of liver damage. As shown in Table 6, compared with the control group, the average liver weight and liver index of the model group mice were significantly increased. The intake of SJPs and Haiwang Jinzun could inhibit the increase of liver weight and liver index to varying degrees, and there was no significant difference between Haiwang Jinzun and different doses of SJPs groups.

[0114] Determination of relevant biochemical indicators in mouse serum: Whole blood was incubated at 4°C for 8 hours, then centrifuged at 2000 rpm for 20 minutes at 4°C. The supernatant was collected, and the liver function enzyme-related indicators ALT and AST in mouse serum were determined according to the kit instructions.

[0115] Determination of relevant biochemical indicators in mouse liver: Weigh approximately 100 mg of liver tissue and add 9 volumes of pre-cooled physiological saline at a ratio of 1:9 (mg:μL). Prepare a 10% liver tissue homogenate under ice bath conditions. Centrifuge at 3000 rpm and 4℃ for 10 min, collect the supernatant, and determine the protein concentrations of ADH, ALDH, MDA, SOD, GSH, TG, and TC in mouse liver according to the kit instructions. The results are expressed as protein concentration.

[0116] Histopathological analysis of mouse liver tissue: Liver tissue was fixed in 4% paraformaldehyde solution, then dehydrated, embedded, and sectioned. Sections were stained with hematoxylin and eosin (HE) and mounted. The sections were examined under a microscope at different magnifications for detailed observation.

[0117] The experiment was repeated in triplicate, and the results are expressed as mean ± standard deviation (X±SD). Statistical analysis was performed using SPSS 25.0 software, and graphs were generated using GraphPad Prism 5.0 and Origin 2017 software. One-way ANOVA was used for comparisons of multiple groups, followed by Fisher's least significant difference (LSD) test for multiple comparisons and tests. A p-value < 0.05 was considered statistically significant. Significance was indicated by letter notation, with lowercase letters used to indicate significance from largest to smallest mean. Different letters were used to indicate significant differences (p < 0.05); no notation was used if there were no significant differences between groups.

[0118] Table 7 Effects of SJPs intake on various biochemical indicators in mice.

[0119]

[0120] Note: Different letters in the same column's header indicate significant differences (P<0.05).

[0121] ALT and AST are important indicators for clinically assessing liver injury. As shown in Table 7, compared with the blank control group, the serum ALT and AST activities of mice in the alcohol model group were significantly increased (P<0.05), indicating severe hepatocyte damage and increased hepatocyte membrane permeability. The increased activities of these two enzymes suggest successful modeling. Compared with the alcohol model group, the serum ALT and AST activities of mice in the positive control group and the medium and high dose SJPs groups were significantly decreased (P<0.05). Among them, the ALT and AST activities in the high dose SJPs group were reduced by 45.47% and 45.73% respectively compared with the alcohol model group, and by 8.32% and 16.95% respectively compared with the positive control group, with no significant difference compared with the blank control group (P>0.05). The results indicate that the intervention of medium and high dose SJPs groups has a good repair effect on the permeability and integrity of mouse liver cell membranes, especially the high dose SJPs group has the most significant effect.

[0122] To further understand the accumulation of fatty acids in the liver of mice with early-stage ALD, the TG and TC contents in liver tissue were measured. As shown in Table 7, compared with the control group, the TG content in the liver of mice in the alcohol model group significantly increased from 0.21 mmol / mg prot to 0.53 mmol / mg prot (P<0.05), and the TC content also significantly increased from 0.10 mmol / mg prot to 0.17 mmol / mg prot (P<0.05), indicating that fatty acids accumulated in the liver of mice, and the early-stage ALD model was successfully established. In contrast, the TG and TC contents in the liver of mice in the high-dose SJPs group were significantly reduced by 37.74% and 29.41% respectively compared with the alcohol model group (P<0.05), and by 23.26% and 14.29% respectively compared with the positive control group.

[0123] Alcohol is primarily metabolized in the body via the ADH pathway in the liver. The ADH and ALDH activities required for this pathway are crucial for alcohol metabolism and the rate of blood alcohol clearance. The ADH and ALDH activities in the livers of mice with early-stage ALD were measured, and the results are shown in Table 7. Compared with the blank control group, the ADH and ALDH activities in the livers of mice in the alcohol model group were slightly increased, which is a passive mechanism following alcohol ingestion. Compared with the alcohol model group, both the SJPs dosage group and the positive control group enhanced ADH and ALDH activities to varying degrees. Specifically, the high-dose SJPs group significantly increased ADH activity by 27.62% and ALDH activity by 80.53% (P<0.05), which were 4.28% and 21.07% higher than the positive control group, respectively. These results indicate that high-dose SJPs intervention can increase ADH and ALDH activities in the livers of mice, accelerate ethanol metabolism and decomposition, catalyze the further oxidation of acetaldehyde to acetic acid, and mitigate the direct toxicity of ethanol and its metabolites to the mouse liver, thereby protecting against alcohol-induced liver damage in mice.

[0124] Ethanol-mediated oxidative stress damage depletes antioxidants such as SOD and GSH in the body, leading to impaired fatty acid β-oxidation in hepatocyte mitochondria and the deposition of large amounts of free fatty acids in the liver, resulting in elevated liver TG content. Further lipid peroxidation occurs in the hepatocyte membrane, accompanied by the production of large amounts of MDA. Table 7 shows that compared with the blank control group, the alcohol model group showed a 20.71% decrease in liver GSH activity, a significant 19.69% decrease in SOD activity (P<0.05), and a significant 71.64% increase in MDA content (P<0.05). At this point, the mouse liver was severely damaged, with decreased antioxidant capacity and increased lipid peroxidation products, indicating successful model establishment. Compared with the alcohol model group, the positive control group and each SJPs intervention group showed varying degrees of improvement in liver damage. Among them, the high-dose SJPs group showed a significant increase in SOD activity (13.52%), a significant increase in GSH content (60.31%), and a significant decrease in MDA content (36.52%) compared to the alcohol model group (P<0.05). Compared to the positive control group, it showed an increase in SOD activity (4.49%), an increase in GSH content (7.86%), and a decrease in MDA content (4.58%). The results indicate that SJPs intervention can, to some extent, enhance the antioxidant capacity of mouse liver by increasing hepatic SOD activity, promoting intrahepatic GSH synthesis, regulating hepatic lipid metabolism, alleviating acute alcohol-mediated oxidative stress in the liver, and reducing the production of lipid peroxides (MDA) and the accumulation of large amounts of fat.

[0125] Early signs of alcoholic liver disease include alcoholic fatty liver, which is the accumulation of fat in the liver. Specifically, this manifests as fatty degeneration, where hepatocytes enlarge and contain well-defined, round vacuoles of varying sizes within the cytoplasm. These vacuoles are due to accumulated triglycerides within the hepatocytes, and the large vacuoles push the cell nucleus to one side. Observation of liver sections from mice yielded the following results. Figure 4 As shown in the diagram, the liver capsule of the blank control group was clearly visible, with no obvious hyperplasia. The central vein was located in the center of the liver lobules, surrounded by hepatocytes and sinusoids arranged in a roughly radial pattern. The lobular boundaries were indistinct. Occasionally, fatty degeneration of hepatocytes was observed within the liver lobules (arrows), possibly due to the high lipid content in the liquid diet. In the alcohol model group, numerous fatty degenerations of hepatocytes were visible in the liver parenchyma (arrows), and round vacuoles of varying sizes appeared in the cytoplasm, indicating the appearance of early symptoms of alcoholic liver disease, and the model was successfully established. Simultaneous administration of different doses of SJPs and Haiwang Jinzun (a traditional Chinese medicine) alleviated the symptoms of alcoholic liver disease in mice to varying degrees. The high-dose SJPs group showed a significant reduction in the number of round vacuoles observed by the naked eye, indicating that high-dose SJPs significantly reduced lipid accumulation in the liver.

[0126] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a hangover relief and liver protection product, characterized in that, Includes the following steps: (1) Raw material pretreatment: The marine organisms are cleaned, viscera removed, mouth removed, and meat taken. After enzyme inactivation, distilled water is added and homogenized to obtain a homogenate. (2) Enzymatic hydrolysis: Add protein 1 to the homogenate obtained in step (1) for enzymatic hydrolysis, and inactivate it after enzymatic hydrolysis to obtain a first enzymatic hydrolysate; add protein 2 to the first enzymatic hydrolysate for enzymatic hydrolysis, and inactivate it after enzymatic hydrolysis to obtain a second enzymatic hydrolysate. (3) Centrifuge the secondary enzymatic hydrolysate obtained in step (2) to obtain the supernatant; concentrate and dry the supernatant to obtain the hangover relief and liver protection product; In step (1), the marine organism is a sea cucumber; In step (2), protease 1 is an alkaline protease, and protease 2 is a flavor protease; In step (2), the final concentration of the added protease is 1000 U / g; In step (2), the enzymatic hydrolysis conditions are as follows: after adding alkaline protease, the enzymatic hydrolysis pH is 10.0, the temperature is 40℃, and the time is 3 h; after adding flavor protease, the enzymatic hydrolysis pH is 7.5, the temperature is 50℃, and the time is 3 h.

2. The method according to claim 1, characterized in that, In step (2), the inactivation method is a boiling water bath for 10 to 20 minutes.

3. The method according to claim 2, characterized in that, In step (3), the centrifugation conditions are 4500-5000 rpm for 15-20 min.

4. The hangover relief and liver protection product prepared by the method of claim 1, characterized in that, The product contains sea cucumber small molecule peptides; in the sea cucumber small molecule peptides, hydrophobic amino acids account for ≥40% of the total amino acid content, and the proportion of those with a molecular weight of less than 1000 Da is ≥45%.

5. The application of the method according to any one of claims 1 to 3 in the preparation of hangover relief and liver protection products.

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