Composition for dispelling effects of alcohol and protecting liver and application thereof

Abstract

The invention discloses a composition for dispelling effects of alcohol and protecting liver and application thereof, and belongs to the technical field of biology. The composition comprises the following components in percentage by mass: 0.5-2% of probiotics, 6-20% of beta-glucan, 10-20% of corn peptide and 50-80% of medicinal and edible substances, and the probiotics are mixed bacterial powder prepared by freeze-drying bacillus coagulans BCN019 and lactobacillus salivarius JYLS-372 and then mixing the bacillus coagulans BCN019 and the lactobacillus salivarius JYLS-372 according to a mass ratio of (0.1-1): 1; the medicinal and edible substance is obtained by mixing a hovenia dulcis thunb extract, a kudzuvine root extract and a turmeric extract in a mass ratio of (0.1-1): (0.1-1): (0.1-1). The composition can relieve headache, dizziness and other uncomfortable symptoms caused by drinking; the contents of ethanol and acetaldehyde in the body can be obviously reduced, and the metabolism of the liver is relieved; meanwhile, by improving the intestinal microecology, gastrointestinal discomfort symptoms caused by excessive drinking can be effectively reduced, and hangover reaction is relieved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a hangover-relief and liver-protection composition and application thereof. Background Art

[0002] There are three pathways for alcohol metabolism in the human body: liver, skin and respiratory system; less than 10% of the alcohol will be excreted from the body through urine, breathing, etc., and the rest will gather in the liver and undergo oxidative metabolism through the liver's enzyme system. There are two enzymes in the liver's enzyme system: one is alcohol dehydrogenase, and the other is acetaldehyde dehydrogenase. Alcohol dehydrogenase can remove two hydrogen atoms from alcohol molecules, decomposing ethanol into acetaldehyde, and then remove the two hydrogen atoms from acetaldehyde under the catalysis of acetaldehyde dehydrogenase 2 (ALDH2) in the liver mitochondria, converting acetaldehyde into acetic acid, and then generating carbon dioxide and water through the tricarboxylic acid cycle (TCA cycle).

[0003] Research shows that alcohol dehydrogenase exists in all human bodies, and the amount is basically equal, but many people lack acetaldehyde dehydrogenase. The lack of acetaldehyde dehydrogenase prevents acetaldehyde from being completely decomposed into acetic acid, but acetaldehyde continues to remain in the body, causing people to experience nausea, vomiting, coma and other drunkenness symptoms after drinking. Acetaldehyde can also cause damage to many organs of the human body, especially the liver. Even people who are good at drinking will get drunk if they drink too much and too fast, exceeding the decomposition capacity of acetaldehyde dehydrogenase.

[0004] In order to reduce the physical damage caused by drinking, some people choose to take alcohol-relieving and liver-protecting health products. Currently, the alcohol-relieving beverages on the market are mainly alcohol-relieving tea and alcohol-relieving soup. According to the Chinese medicine alcohol-relieving formula, the Chinese medicine liquid prepared by selecting medicinal materials also has certain effects of sobering up and protecting the liver. For example, patent document CN108126123A discloses a alcohol-relieving and liver-protecting composition, which is composed of Hovenia dulcis, Pueraria lobata, Poria cocos, Atractylodes macrocephala, Phragmites australis, Ligustrum lucidum, Mentha salina, and Dunaliella salina, among which Hovenia dulcis and Pueraria lobata are the main alcohol-relieving and diuretic ingredients. Hovenia dulcis is the seed of Hovenia dulcis, a plant of the Rhamnaceae family, and contains alkaloids, flavonoids, fatty acids, saponins, glycosides, glucose and other ingredients. It has the effects of diuresis, swelling, alcohol-relieving and other effects, and also has a protective effect on liver damage. Pueraria lobata is the flower of wild kudzu or sweet kudzu and powder kudzu of the genus Pueraria of the Leguminosae family, which has the effects of sobering up and invigorating the spleen and stomach. Chinese herbal ingredients have a slow effect in sobering up and take a long time.

[0005] Probiotics refer to a class of living microorganisms that, when ingested in sufficient amounts, can produce clear benefits to human health. In recent years, there have been more and more studies on the potential probiotic effects of probiotics, and studies have shown that probiotics have therapeutic potential for alcohol-induced liver disease. Using probiotics to change the imbalance of the microbiome associated with alcohol use has become a promising direction for the treatment of alcoholic liver disease (ALD). For example, patent document CN1143729A discloses that Lactobacillus plantarum KLDS1.0344 combined with Lactobacillus acidophilus KLDS1.0901 can prevent chronic alcoholic liver damage, positively regulate intestinal flora, and inhibit alcohol-induced liver lipid accumulation and oxidative stress through the gut-liver axis.

[0006] However, the efficacy of hangover products is currently mostly verified in animal models, and no combination has been reported to have a hangover efficacy in a human hangover test, and its efficacy in the human population remains to be verified. Therefore, the development of a safe and healthy hangover and liver protection product that can effectively reduce the incidence of drunkenness and shorten the recovery from drunkenness is still a technical problem that needs to be solved by those skilled in the art. Summary of the invention

[0007] The purpose of the present invention is to provide a hangover and liver protection composition, which can reduce the incidence of drunkenness, shorten the recovery time of drunkenness, and significantly reduce the alcohol content in the blood after drinking, and has significant effects on relieving drunkenness, nourishing the stomach and protecting the liver.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention uses probiotics, medicinal and edible substances, prebiotics and bioactive peptides as main raw materials for compounding, and obtains a safe and reliable alcohol-detoxifying and liver-protecting composition that can reduce alcohol toxicity and has a restorative effect on alcohol-induced liver damage. Specifically, in terms of mass percentage, the composition includes: 0.5-2% probiotics, 6-20% beta-glucan, 10-20% corn peptides, and 50-80% medicinal and edible substances;

[0010] The probiotics are mixed bacteria powder prepared by freeze-drying the expanded cultured Bacillus coagulans BCN019 and Lactobacillus salivarius JYLS-372 at a mass ratio of 0.1 to 1:1;

[0011] The medicine-food homologous substance is a mixture of Hovenia dulcis extract, Pueraria root extract and Curcuma longa extract in a mass ratio of 0.1-1:0.1-1:0.1-1. The Hovenia dulcis extract, Pueraria root extract and Curcuma longa extract are prepared from Hovenia dulcis, Pueraria root and Curcuma longa respectively by water extraction.

[0012] In the above formula, the probiotics are selected from Bacillus coagulans and Lactobacillus salivarius. Bacillus coagulans is a non-intestinal microorganism, and its physiological role in the intestine is the result of secreting a variety of beneficial substances and synergizing with other probiotics in the intestine. The Bacillus coagulans BCN019 used in the present invention has a strong gastrointestinal survival rate and intestinal colonization ability, can achieve anti-fatigue effects by enhancing lactic acid metabolism and reducing blood urea content, and can relieve fatigue after drinking.

[0013] Lactobacillus salivarius belongs to the human protozoan species, is a rare probiotic strain that can improve allergic constitution, and is one of the safe strains listed by the U.S. Food and Drug Administration (FDA). The Lactobacillus salivarius JYLS-372 used in the present invention has a strong gastrointestinal survival rate and intestinal colonization ability, can convert ethanol into ethyl lactate, and has obvious alcohol sobering and liver protection effects.

[0014] The Bacillus coagulans BCN019 and Lactobacillus salivarius JYLS-372 are both biological materials available to the public, see patent documents CN 113789290 A and CN 110791452 A.

[0015] Specifically, the method for expanding the culture of the strain includes: inoculating the activated Bacillus coagulans BCN019 and Lactobacillus salivarius JYLS-372 into a sterile MRS liquid culture medium at a 1% inoculation rate, culturing at 37° C. for 24 hours, and centrifuging at 5000 rpm to obtain the bacterial cells.

[0016] Furthermore, the preparation method of the mixed bacterial powder includes: expanding the strain culture to a concentration of 10 8 ~10 9 CFU / mL, collect the bacteria, resuspend them in lyophilization protectant, freeze-dry and mix them in a mass ratio of 1:1.

[0017] The lyophilization protective agent comprises skim milk powder with a concentration of 5 g / 100 mL and lactose with a concentration of 5 g / 100 mL.

[0018] In the above formula, the bioactive peptide is corn peptide, which is obtained by enzymatic hydrolysis of corn yellow powder with mixed proteases (alkaline protease, neutral protease, papain). Studies have shown that adding corn peptide can better promote the metabolism of alcohol in the body, reduce liver damage, and has a good protective effect on alcoholic liver damage. Mechanism studies have shown that corn peptide can increase the activity of alcohol dehydrogenase, promote ethanol metabolism, inhibit lipid peroxidation, reduce the content of malondialdehyde, and reduce alcoholic liver damage.

[0019] In the above formula, the prebiotic is β-glucan, which can significantly improve intestinal flora disorders, improve acute inflammatory responses caused by lipopolysaccharide, relieve acute inflammation and intestinal homeostasis imbalance caused by alcohol, and thus improve the mental state after drinking. Furthermore, the β-glucan is oat β-glucan.

[0020] In the above formula, the medicinal and edible substances are Hovenia dulcis, Pueraria root and Curcuma longa. The present invention uses water extracts of these three raw materials for compounding. Studies have shown that the mixture has a significant effect on reversing alcoholic liver damage.

[0021] Hovenia dulcis is the mature seed of Hovenia dulcis, Hovenia dulcis and Hovenia dulcis of the Rhamnaceae family. It has the effect of detoxifying alcohol. Studies have shown that its effect of reducing blood ethanol concentration may be related to the following two pathways: (1) inhibiting or reducing the absorption of ethanol by the gastrointestinal tract and enhancing the first-pass effect of ethanol in the gastrointestinal tract; (2) enhancing the activity of alcohol dehydrogenase in the liver and accelerating the decomposition of ethanol in the liver.

[0022] Pueraria root is the dried root of Pueraria lobata or Pueraria lobata and Pueraria lobata of the genus Pueraria of the Leguminosae family. Pueraria root is not only rich in nutrition, but also contains isoflavones such as daidzein and puerarin, and is a natural plant with medicinal and edible properties approved by the Ministry of Health. Puerarin contained in the Pueraria root extract used in the present invention can increase tissue oxygen supply by improving visceral circulation; improve the spasm of renal tubules to increase renal blood flow, increase urine volume, and enhance excretion, thereby reducing the ethanol level in the blood; remove oxygen free radicals and resist lipid peroxidation to restore the abnormal changes in serum viscosity caused by alcohol poisoning to a normal state, improve the patient's systemic symptoms, accelerate the disappearance of poisoning symptoms, and achieve the effect of increasing alcohol tolerance, relieving drunkenness, and alleviating discomfort.

[0023] Turmeric is a plant of the genus Curcuma in the ginger family. It is a food and medicine material. The main active ingredient in turmeric is curcumin. Animal experiments have shown that curcumin has a significant therapeutic and preventive effect on acute alcoholic liver injury in mice. It can significantly prolong the "drunkenness" time of mice caused by alcohol poisoning and significantly shorten the "sobering up" time of mice from "drunkenness" to "awakeness".

[0024] Furthermore, the water extraction method comprises: mixing the raw material and 95-100° C. water at a solid-liquid ratio of 1:10-20, extracting for 2-3 hours, separating the solid and the liquid, collecting the extract, and concentrating and spray-drying to obtain the extract.

[0025] Furthermore, the Hovenia dulcis extract, the Pueraria root extract and the Curcuma longa extract are mixed in a mass ratio of 1-1.2:1-1.5:1 to obtain the medicine-food homologous substance.

[0026] Furthermore, the composition of the alcohol sobering and liver protecting composition includes, by mass percentage: 0.8-1% probiotics, 12-18% β-glucan, 15-18% corn peptide, and 60-70% medicinal and edible substances.

[0027] Furthermore, in terms of mass percentage, the composition of the hangover and liver protection composition includes: 0.8% probiotics, 16-17% β-glucan, 16-17% corn peptides, and 65-66% medicinal and edible substances, wherein Bacillus coagulans BCN019 and Lactobacillus salivarius JYLS-372 are mixed in a mass ratio of 1:1, and Hovenia dulcis extract, Pueraria root extract, and Curcuma longa extract are mixed in a mass ratio of 1:1:1.

[0028] The human hangover tasting test study shows that the hangover and liver protection composition provided by the present invention can relieve headaches, dizziness and other discomfort symptoms caused by drinking, reduce the incidence of drunkenness, shorten the recovery time from drunkenness, significantly reduce the alcohol content in the blood, and has a significant hangover ability. At the same time, it can improve the intestinal microecology, maintain the balance of the intestinal environment, effectively reduce the gastrointestinal discomfort symptoms caused by excessive drinking, alleviate the hangover reaction, and improve liver metabolism.

[0029] Therefore, the present invention provides the use of the alcohol-detoxifying and liver-protecting composition in the preparation of alcohol-detoxifying and liver-protecting medicines or foods.

[0030] The alcohol sobering and liver protecting composition provided by the present invention can be used to prepare alcohol sobering products for preventing and improving excessive drinking, and can also be used to prepare auxiliary treatment products for alcoholic liver and fatty liver caused by alcohol and non-alcohol, and is safe, non-toxic and has reliable efficacy.

[0031] Furthermore, the alcohol sobering up and protecting the liver includes preventing acute alcoholic liver injury. The manifestations of acute alcoholic liver injury include: increase in liver area and accumulation of fat droplets in the liver.

[0032] Furthermore, the alcohol sobering up and protecting the liver includes reducing the alcohol content in breath, the alcohol content in blood, and the acetaldehyde content in blood and saliva after drinking.

[0033] The hangover-relieving and liver-protecting composition provided by the present invention can increase the content of alcohol dehydrogenase and acetaldehyde dehydrogenase in the body, quickly decompose ethanol, promote ethanol metabolism in the liver, significantly reduce the ethanol and acetaldehyde content in the body, increase the body's tolerance to alcohol, increase the amount of alcohol consumed, and enable people to maintain a good drinking experience and feeling, ultimately achieving the purpose of both drinking and health.

[0034] The present invention also provides the use of the alcohol-detoxifying and liver-protecting composition in a drug for preventing or treating flora imbalance or a food for regulating intestinal flora balance. Studies have shown that taking the alcohol-detoxifying and liver-protecting composition provided by the present invention can regulate the intestinal flora of the population and is beneficial to human health, specifically by significantly reducing the abundance of Moryella, an intestinal bacteria related to obesity, and promoting the growth of beneficial bacteria Akkermansia and Ligilactobacillus in the intestinal flora.

[0035] The invention also provides a hangover-relieving and liver-protecting compressed candy, the active ingredient of which is the hangover-relieving and liver-protecting composition.

[0036] Furthermore, the composition is, by mass percentage, 99.6% of the alcohol sobering and liver protecting composition and 0.4% of magnesium stearate.

[0037] Furthermore, the composition of the alcohol-detoxifying and liver-protecting compressed candy is, by mass percentage, 0.4% of Bacillus coagulans BCN019, 0.4% of Lactobacillus salivarius JYLS-372, 21.8% of Hovenia dulcis extract, 21.8% of Pueraria root extract, 21.8% of Curcuma longa extract, 16.7% of corn peptide, 16.7% of β-glucan, and 0.4% of magnesium stearate.

[0038] Furthermore, the preparation method of the alcohol-relieving and liver-protecting compressed candy comprises the following steps:

[0039] Step 1: Bacillus coagulans BCN019, Lactobacillus salivarius JYLS-372, Hovenia dulcis extract, Pueraria root extract, Curcuma longa extract, corn peptide, β-glucan and magnesium stearate are mixed in proportion and passed through an 80-mesh sieve;

[0040] Step 2: The mixture obtained in step 1 is compressed into a tablet candy.

[0041] The present invention has the following beneficial effects:

[0042] The present invention uses probiotics Bacillus coagulans BCN019 and Lactobacillus salivarius JYLS-372, medicinal and edible foods Pueraria extract, Hovenia dulcis extract and Curcuma longa extract, prebiotic β-glucan, and bioactive peptide corn oligopeptide as raw materials to compound a composition with alcohol sobering and liver protecting effects. The composition can relieve headaches, dizziness and other discomfort symptoms caused by drinking; it can significantly reduce the ethanol and acetaldehyde content in the body, reduce liver metabolism, and increase the amount of alcohol consumed; at the same time, it can improve the intestinal microecology, maintain the balance of the intestinal environment, effectively reduce the gastrointestinal discomfort symptoms caused by excessive drinking, reduce hangover reactions, and improve liver metabolism. The medicines and foods made from the composition of the present invention can be used as alcohol sobering products for preventing and improving excessive drinking, and can also be used as auxiliary treatment products for alcoholic liver and fatty liver caused by alcohol and non-alcohol, which are safe, non-toxic, and have reliable efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The effects of each intervention group on the liver area of ​​the zebrafish model of acute alcoholic liver disease, including A, probiotic group; B, bioactive peptide group; C, medicine and food group; D, prebiotic group.

[0044] Figure 2 To investigate the effect of the compound formula on the liver area of ​​the zebrafish model of acute alcoholic liver disease.

[0045] Figure 3 To investigate the effect of the compound prescription on the acetaldehyde dehydrogenase content in the zebrafish model of acute alcoholic liver disease.

[0046] Figure 4 Flowchart for designing a human trial experiment on probiotic hangover-relieving compressed candy tablets.

[0047] Figure 5 Number connection game time 2 h after drinking for the probiotic group and the placebo group.

[0048] Figure 6 The breath alcohol test content at different time points in the population drinking test experiment.

[0049] Figure 7 This is a graph showing changes in serum ethanol content 0.5 hours after drinking in a population experimental drinking test.

[0050] Figure 8 This is a graph showing changes in serum acetaldehyde levels at different time points during a drinking test.

[0051] Fig. 9 The effect of probiotic alcohol detoxification tablets on acetaldehyde content in saliva.

[0052] Fig.10 Effect of probiotic alcohol detoxification tablets on serum liver function.

[0053] Fig.11 To investigate the effects of probiotic alcohol detoxification tablets on differentially characteristic bacteria (genus level).

[0054] Fig.12 The effect of probiotic alcohol detoxification tablets on differential characteristic bacteria (species level). DETAILED DESCRIPTION

[0055] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0056] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0057] Specifically, Bacillus coagulans BCN019 and Lactobacillus salivarius JYLS-372 were purchased from Shandong Zhongke Jiayi Bioengineering Co., Ltd. Preparation of bacterial powder: The activated Bacillus coagulans BCN019 and Lactobacillus salivarius JYLS-372 were inoculated into sterile MRS liquid culture medium at an inoculum volume of 1%, cultured at 37°C for 24 hours, centrifuged at 5000rpm to obtain bacterial cells, and resuspended the bacterial cells with a sterilized mixed solution containing 5% skim milk powder and 5% lactose as a freeze-drying protective agent, and placed in a freeze dryer for freeze drying. Finally, the bacterial powder obtained after freeze drying was counted by the pouring plate method.

[0058] Corn peptide, wheat peptide, and oyster peptide were purchased from Jiangsu Junqi Biotechnology Co., Ltd.

[0059] β-Glucan, inulin and oligofructose were purchased from Xi'an Anao Biotechnology Co., Ltd.

[0060] Hovenia dulcis extract, Pueraria root extract, and Curcuma longa extract were purchased from Zhejiang Tiancao Biotechnology Co., Ltd. Preparation of extracts: The edible and medicinal raw materials were extracted with 95-100°C hot water for 2 h at a solid-liquid ratio of 1:10, and the hot water extraction was repeated 2-3 times after solid-liquid separation. The extracts were combined and concentrated and spray-dried to obtain edible and medicinal extracts.

[0061] Example 1: Screening and verification of active components

[0062] 1. Zebrafish husbandry and model intervention

[0063] The feeding method of zebrafish refers to the Westerfield method. In short, the environmental conditions are 27.5-28.5℃, the light cycle is 14:10 (14h light, 10h dark), the culture water is about 400mg artificial sea salt and 10mg NaHCO3 per liter of reverse osmosis water, the water conductivity is 450-550μS / cm, the pH is 6.8-7.5, and the total hardness (calculated as CaCO3) is 50-100mg / L. Feed once in the morning and evening every day, and feed freshly hatched brine shrimp and solid fish feed alternately. The mating behavior of zebrafish requires the biological clock control of the light cycle, and at the beginning of each light cycle, it enters the state of mating and spawning. The night before mating, the male and female zebrafish are placed in the mating tank in a ratio of 2:1, and the male and female fish are separated by a partition. After the light cycle starts the next day, the partition is removed to allow the male and female fish to mate and spawn naturally. The fish eggs were collected and placed in a culture dish containing embryo culture medium, and developed in a constant temperature biochemical incubator at 28°C. The eggs were observed daily, dead eggs were removed in time, and fresh embryo culture medium was replaced.

[0064] 5dpf zebrafish larvae were randomly selected and divided into a control group, a model group, and an active substance intervention group. All zebrafish in each group were placed in a six-well cell culture plate, 30 per well, and three parallel wells. The control group was cultured with embryo culture medium, the model group was cultured with embryo culture medium containing 350mM ethanol (2% ethanol solution), and the intervention group was cultured with 350mM ethanol and 5μg / mL active substance or 10 4 CFU / mL probiotic embryo culture medium was cultured at 28°C and a photoperiod of 14:10 for 48 h.

[0065] 2. Observation of the histomorphology of zebrafish larvae liver

[0066] Tg zebrafish fry were selected, and the feeding and grouping intervention methods were the same as above. The intervention group was subdivided into 2 probiotic groups: Bacillus coagulans BCN019 and Lactobacillus salivarius JYLS-372; 3 bioactive peptide groups: corn peptides, wheat peptides, and oyster peptides; and 3 medicine-food groups: Hovenia dulcis extract, Pueraria extract, and Curcuma longa extract; 3 prebiotic groups: β-glucan, inulin, and oligofructose.

[0067] After the model intervention, the larvae were anesthetized with 0.02% tricaine for 10-20 seconds, fixed laterally in 4% sodium carboxymethylcellulose, and photographed under bright field and fluorescence conditions of a fluorescence microscope. The relative liver area of ​​the zebrafish larvae in each group was quantified using ImageJ software.

[0068] By directly observing the liver's surface morphology, the protective effects of each component on alcohol-induced liver damage can be compared intuitively and quickly. The liver area calculation results ( Figure 1 ) showed that the liver of the model group was significantly enlarged compared with the control group. It was speculated that the acute alcoholic liver injury model of zebrafish was successfully established after 2% alcohol exposure, and lipid droplets accumulated in the liver. Compared with the control group, the liver area of ​​the model group increased significantly (p < 0.05). Compared with the model group, Bacillus coagulans and Lactobacillus salivarius in probiotics, corn peptides in bioactive peptides, the medicine and food group (Hovenia dulcis, Pueraria and Curcuma longa), and β-glucan in prebiotics significantly improved the enlargement of liver area induced by 2% alcohol exposure (p < 0.05). The remaining components showed a trend of inhibiting the increase of liver area as a whole, but it was not significant, indicating that their protective effect on the enlargement of liver induced by 2% alcohol was general.

[0069] Therefore, in the subsequent formula combination experiments, Bacillus coagulans and Lactobacillus salivarius were selected as probiotics, corn peptides were selected as bioactive peptides, Hovenia dulcis, Pueraria lobata and Curcuma longa were selected as medicine and food, and β-glucan was selected as prebiotics.

[0070] According to the preliminary screening results, prebiotics, bioactive peptides and medicinal and edible substances were selected for compound experiments. 5dpf zebrafish fry were randomly selected and divided into a control group, a model group and an active substance intervention group. The intervention group was further subdivided into a single active substance intervention group: prebiotics, bioactive peptides, medicinal and edible substances (the mass ratio of Hovenia dulcis: Pueraria root: Curcuma longa was 1:1:1) and 7 compound combination intervention groups. The compounding scheme was designed using Minitab Statistical software. Details are shown in Table 1 below.

[0071] Table 1. Compounding scheme design

[0072]

[0073] All zebrafish in all groups were placed in six-well cell culture plates, 30 per well, and three parallel wells. The control group was cultured with embryo culture medium, the model group was cultured with embryo culture medium containing 350mM ethanol (2% ethanol solution), and the intervention group was cultured with embryo culture medium containing 350mM ethanol and 5μg / mL active substances, and cultured for 48h at 28℃ and a light cycle of 14:10.

[0074] The results of direct photographing and observation of Tg zebrafish under a fluorescence microscope and the calculation results of liver area ( Figure 2 ) showed that the prebiotics (β-glucan), bioactive peptides (corn peptides) and food and medicine (Hovenia dulcis: Pueraria root: Curcuma longa = 1:1:1) intervention groups showed significant preventive effects on acute alcoholic liver injury (p < 0.05), and the liver area was close to that of the control group; compared with the model group, schemes 2 and 5 significantly prevented the increase in liver area induced by 2% alcohol exposure (p < 0.05), and the liver area showed a decreasing trend after the other combined schemes, but it was not significant, indicating that they also have a certain effect of reversing alcoholic liver injury.

[0075] 3. Determination of acetaldehyde dehydrogenase (ALDH) in zebrafish larvae

[0076] AB zebrafish fry were selected, and the feeding and grouping intervention methods were the same as above. The relevant alcohol metabolism biochemical indexes, acetaldehyde dehydrogenase (ALDH), were measured according to the steps in the kit instructions.

[0077] Figure 3 The results showed that, except for scheme 1, the other combined schemes showed a trend of increasing ALDH activity after treatment, among which schemes 3 and 5 had significant differences compared with the model group (p < 0.05), and the ALDH activity of scheme 5 was 1.38 times that of the model group.

[0078] In summary, the compound regimen experiment in this study proved that regimen five has a strong ability to sober up and protect the liver.

[0079] 4. Analysis of the synergistic effect of the compound

[0080] The drug interaction coefficient (CDI) was used to analyze the synergistic effect of combined drug use. The calculation formula is as follows:

[0081]

[0082] N AB is the ratio of the parameters of the prebiotics, bioactive peptides and food-drug combination groups to the model group; N A or N B is the ratio of the corresponding parameter between the intervention group and the model group.

[0083] If CDI is less than 1, it indicates that there is a synergistic effect between the drugs; if CDI=1, it indicates that there is an additive effect between the drugs; if CDI>1, it indicates that there is an antagonistic effect between the drugs.

[0084] The CDI values ​​of ALDH indicators of each compounding scheme were calculated according to the formula, and the results are shown in Table 2. The CDI of ALDH of the seven compounding schemes was less than 1, among which the ALDH activity CDI of scheme 5 was 0.41, proving that scheme 5 had a significant synergistic effect. Therefore, the tableting of subsequent population tests was adjusted based on this ratio.

[0085] Table 2. CDI value scoring table for different biochemical indicators in each scheme

[0086]

[0087] Example 2: Preparation of probiotic alcohol-relieving tablets

[0088] The compressed candy provided in this embodiment includes the following raw materials in percentage by mass: 0.4% of Bacillus coagulans BCN019, 0.4% of Lactobacillus salivarius JYLS-372, 21.8% of Hovenia dulcis extract, 21.8% of Pueraria root extract, 21.8% of Curcuma longa extract, 16.7% of corn peptide, 16.7% of β-glucan, and 0.4% of magnesium stearate.

[0089] The method for making alcohol-relieving tablet candy comprises the following steps:

[0090] Step 1: Bacillus coagulans BCN019, Lactobacillus salivarius JYLS-372, Hovenia dulcis extract, Pueraria root extract, Curcuma longa extract, corn peptide, β-glucan and magnesium stearate are mixed in proportion and passed through an 80-mesh sieve;

[0091] Step 2: The mixture obtained in step 1 is pressed into tablet candies, subjected to metal detection, and then boxed and inspected.

[0092] Test Example 1: A human tasting experiment on alcohol-detoxifying tablets

[0093] 1. Experimental plan

[0094] Volunteer inclusion criteria:

[0095] 1) 18-60 years old.

[0096] 2) Healthy adults who fully understand the detailed description of the study and voluntarily agree to participate.

[0097] 3) Ability to tolerate a certain amount of alcohol (calculated based on 60kg, approximately 60mL of 40% alcohol).

[0098] Exclusion criteria:

[0099] 1) People who are allergic or have a history of clinically significant allergy to drugs, alcohol, products or other ingredients.

[0100] 2) People with severe acute or chronic cardiovascular diseases, metabolic diseases, hepatobiliary diseases, pancreatic diseases, muscle diseases, neurological diseases, mental disorders, endocrine diseases, immune diseases, kidney diseases, malignant tumors, lung diseases, and other diseases that require treatment.

[0101] 3) Failure to complete the questionnaire and provide stool on time.

[0102] 4) Failure to take the compressed candy on time.

[0103] 5) Did not participate in the hangover test.

[0104] Finally, 40 healthy subjects who met the above criteria were recruited to participate in a 4-week probiotic compound food tasting experiment. The volunteers were assigned to Group A (probiotic hangover tablet intake → washout → placebo intake) or Group B (placebo intake → washout → probiotic hangover tablet intake). One person dropped out of Group A and six people dropped out of Group B.

[0105] Dosage: Take three times a day (after meals), two capsules each time.

[0106] The duration of taking the probiotic composite compressed candy tablets was one week for each of the probiotic hangover-relieving compressed tablets prepared in Example 2 and the placebo (maltodextrin), followed by a two-week washout period, for a total of four weeks.

[0107] Feces of all volunteers were collected one week before the alcohol experiment (first time), and the second and third feces were collected on the 7th and 28th days, respectively.

[0108] Alcohol tests were conducted after fasting overnight on the 7th and 28th days. The participants were weighed on the day of the test and provided with liquor according to their own weight. The participants ate a meal containing alcohol (standard diet) (alcohol content: 40% v / v, 1mL / kg body weight). Serum and saliva of volunteers were collected at different time points after the meal. Questionnaires (subjective feelings, hangover relief, etc.) were filled out 3h and 24h after the meal. The effects of probiotic tablets on the alcohol sobering ability of healthy volunteers were comprehensively evaluated.

[0109] Specific experimental plan Figure 4 .

[0110] 2. Digital Connection Game

[0111] Drunken game test: After drinking for 2 hours, distribute a pre-printed random table of 1-25 numbers to everyone, and ask the subjects to connect them from small to large in a row with a line, and record the time it takes for each subject to complete the experiment.

[0112] The results are as follows Figure 5 As shown, 2 hours after drinking, the time taken by the probiotic intervention group to play the game (29.49s) was significantly faster than that of the placebo group (35.47s) by nearly 6s (p=0.0034), indicating that taking probiotic candy tablets can restore people's reaction speed after drinking, which proves that the composition has a certain ability to sober up.

[0113] 3. Breath alcohol determination

[0114] During the drinking test experiment, the breath alcohol content (mg / 100mL) of the subjects was measured using a breath alcohol tester at different time points after drinking (15, 30, 60, 120, 180 and 240 minutes).

[0115] like Figure 6 As shown, overall, at 15, 30 and 60 minutes, the breath alcohol content of the probiotic group was significantly lower than that of the placebo group, but there was no significant difference (p>0.05). The breath alcohol of both groups decreased with time and dropped to 0 mg / 100 mL at 240 minutes. Specifically, the breath alcohol content was the highest 15 minutes after drinking. The breath alcohol of the probiotic group was 40.68 mg / 100 mL, which was 16.5% lower than that of the placebo group (48.72 mg / 100 mL). This shows that taking probiotic compressed candy can significantly reduce the breath alcohol content, and this formula has a significant ability to sober up.

[0116] 4. Determination of ethanol content in serum

[0117] A commercial ethanol kit was used to measure the ethanol content in the subjects' serum 0.5 h after drinking.

[0118] The steps for detecting ethanol content in serum are as follows:

[0119] (1) Preparation of the detection working solution (prepared and used immediately): Before use, prepare the detection working solution in a volume ratio of Reagent 1: Reagent 2: Reagent 4 = 10:5:1 according to the usage amount, and mix thoroughly to obtain the detection working solution.

[0120]

[0121] (2) Method: Preheat the microplate reader for more than 30 minutes, adjust the wavelength to 450 nm, and add the following reagents in the 96-well plate in sequence:

[0122]

[0123] Absorbance determination: Mix thoroughly and start timing immediately, measure the absorbance at 450nm at 10s (total time), record it as A1 determination; react accurately at 37℃ for 600s, measure the absorbance at 450nm at 610s (total time), record it as A2 determination; calculate △A=A2-A1.

[0124] formula:

[0125] The ethanol concentration in the subjects' serum 0.5 hours after drinking is Figure 7 As shown. 0.5h after drinking, the ethanol concentration in the serum of the subjects in the probiotic group (4.05μmol / mL) was significantly lower than that in the placebo group (5.40μmol / mL, p=0.0007), a decrease of 27%. This shows that oral administration of probiotic compressed candy can significantly reduce the alcohol content in the blood after drinking, proving that the formula has a significant ability to sober up.

[0126] 5. Effect of probiotic compressed candy on acetaldehyde levels in serum of alcoholic subjects

[0127] The acetaldehyde content in the subjects' serum was determined by liquid chromatography.

[0128] Preparation of acetaldehyde standard solution: Accurately weigh 10 mg of ACH-DNPH standard into a 10 mL brown volumetric flask, dilute to 10 mL with acetonitrile, and shake to obtain a 1000 mg / L ACH-DNPH standard stock solution. Use a pipette to transfer 500, 300, 100, 50, and 20 μL of the standard stock solution into a 10 mL volumetric flask, dilute to the mark with acetonitrile, and shake to obtain a standard solution with a concentration gradient of 50.0, 30.0, 10.0, 5.0, and 2.0 mg / L.

[0129] Preparation of DNPH derivatization reagent stock solution: Accurately weigh 39.63 mg of DNPH high-purity reagent into a 100-mL brown volumetric flask, dilute to 100 mL with acetonitrile, and shake to obtain a DNPH-acetonitrile solution with a concentration of 2 mmol / L.

[0130] Sample preparation: Take 200 μL of serum sample, add 100 mL of 2 mmol / L DNPH-acetonitrile solution, shake and mix, derivatize in a 60°C water bath for 15 min, cool with running water, centrifuge at 8000 r / min, 4°C for 20 min, take the supernatant and filter through a 0.22 μm microporous filter membrane into a brown liquid injection bottle and store in a 4°C refrigerator for later use.

[0131] Preparation of mobile phase: Use a measuring cylinder to measure 180 mL of chromatography grade methanol and 120 mL of pure water, filter through the membrane three times, and then ultrasonicate for 30 minutes.

[0132] Take 20 μL of samples and standards for injection, and use retention time for qualitative analysis and peak area for quantitative analysis.

[0133] Chromatographic conditions: ODS-C18 separation column (150 mm × 4.6 mm, 5 μm); mobile phase:

[0134] Methanol-water (60:40); flow rate: 1.0 mL / min; injection: 20 μL; detection wavelength: 360 nm.

[0135] like Figure 8 As shown, overall, the acetaldehyde content in the blood of the probiotic group was significantly lower than that of the placebo group at 15, 30, 60 and 120 minutes, but there was a significant difference only at 15 and 30 minutes (p<0.05). The acetaldehyde content in the blood of the two groups of subjects decreased with time and dropped to 0μM at 240 minutes. Specifically, the acetaldehyde content in the blood was the highest 30 minutes after drinking. The acetaldehyde content in the blood of the probiotic group was 2.51μM, which was significantly reduced by 45.4% compared with the placebo group (4.60μM) (p<0.0001). This shows that this formula has a significant advantage in promoting the metabolism of acetaldehyde in the body, thereby accelerating alcohol metabolism.

[0136] VI. Effect of Probiotic Compressed Candy on Acetaldehyde Levels in Saliva of Drinking Subjects

[0137] The acetaldehyde content detection method refers to step 5. Sample preparation: Take 200 μL of saliva sample, add 100 μL of 2 mmol / L DNPH-acetonitrile solution, shake and mix, derivatize in a 60°C water bath for 15 minutes, cool with running water, centrifuge at 8000 r / min, 4°C for 20 minutes, take the supernatant and filter through a 0.22 μm microporous filter membrane into a brown liquid injection bottle and store in a 4°C refrigerator for later use.

[0138] The changes of acetaldehyde content in saliva Fig. 9 As shown in the results, the acetaldehyde concentration in the saliva of the group taking probiotic compressed candy was significantly lower than that of the placebo group 1h and 6h after drinking, but there was no statistically significant difference (p>0.1). Among them, 1h after drinking, the acetaldehyde content in the saliva of the probiotic group (2.95μM) was significantly lower than that of the placebo group (3.93μM); 6h after drinking, the acetaldehyde content in the saliva of the probiotic group (1.83μM) was significantly lower than that of the placebo group (3.05μM). This shows that probiotic compressed candy has a certain potential to reduce the acetaldehyde content in saliva after drinking.

[0139] 7. Effects of Probiotic Compressed Candy Tablets on Liver Function

[0140] According to the operating procedures of the commercial kit, the liver function-related enzymes at two test points were detected, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GTT) and alkaline phosphatase (ALP). Serum liver enzymes (ALT, AST, ALP, GGT) are considered to be important markers for the clinical evaluation of liver damage.

[0141] like Fig.10 As shown in the results, compared with the placebo group, taking probiotic compressed candy tablets had no significant effect on serum liver function-related enzymes. This proves that the formula is safe and reliable for population application, does not increase the burden on the liver, and has no liver toxicity or side effects.

[0142] 8. Effects of Probiotic Compressed Candies on Intestinal Flora

[0143] Subordinate level analysis ( Fig.11 ) showed that the intake of probiotic compressed candies significantly reduced the abundance of Moryella genus in human intestinal bacteria (p<0.05) and significantly increased the abundance of Akkermansia.

[0144] A large number of studies have shown that Moryella is positively correlated with high-fat diet and obesity. Therefore, we can infer that this probiotic compressed candy formula has certain potential in regulating lipid metabolism.

[0145] Akkermansia is a recognized new generation of probiotics with significant anti-inflammatory, blood sugar and fat reducing effects. Probiotic compressed candies can significantly enrich Akkermansia in the intestines, indicating that the probiotic compressed candies can promote the growth of beneficial bacteria in the intestines and promote the health of the human body with a balanced intestinal flora.

[0146] In order to further analyze the difference in species of intestinal bacteria between the two groups of mice, we used STAMP analysis to compare the two groups to find the difference in species level. Fig.12As shown. According to the results of differential species analysis, two intestinal microorganisms with significant differences at the species level were screened, namely unclassified_g_Ligilactobacillus and human_gut_metagenome_g_Moryella. The analysis results showed that compared with the placebo group, the probiotic compressed candy significantly increased the abundance of unclassified_g_Ligilactobacillus in the intestinal bacteria, which shows that the probiotic compressed candy rich in Ligilactobacillus has significant survival and colonization ability in the intestine. Consistent with the results found at the genus level, the abundance of human_gut_metagenome_g_Moryella under the Moryella genus in the probiotic group was significantly lower than that in the placebo group.

[0147] The results of the comprehensive study of the effects of probiotic compressed candies on the composition of intestinal bacteria at different levels showed that the intake of probiotic compressed candies can promote the growth of beneficial bacteria Akkermansia and Ligilactobacillus in the intestinal flora and inhibit the enrichment of Moryella, an intestinal bacteria associated with obesity. This proves that the probiotic compressed candies can regulate the intestinal flora of the population and are beneficial to human health.

Claims

1. A composition for sobering up and protecting the liver, characterized in that: The composition includes, by mass percentage, 0.5-2% probiotics, 6-20% beta-glucan, 10-20% corn peptide, and 50-80% edible and medicinal substances, wherein the probiotics are mixed bacterial powder prepared by freeze-drying expanded cultured Bacillus coagulans BCN019 and Lactobacillus salivarius JYLS-372 bacteria in a mass ratio of 0.1-1:1; the edible and medicinal substances are a mixture of a Hovenia dulcis extract, a Pueraria root extract, and a Curcuma longa extract in a mass ratio of 0.1-1:0.1-1:0.1-1, and the Hovenia dulcis extract, the Pueraria root extract, and the Curcuma longa extract are prepared from Hovenia dulcis, Pueraria root, and Curcuma longa, respectively, by water extraction.

2. The alcohol sobering and liver protecting composition according to claim 1, characterized in that: The preparation method of the mixed bacterial powder comprises: expanding the strain culture to a concentration of 10 8 ~10 9 CFU / mL, collect the bacteria, resuspend them in lyophilization protectant, freeze-dry and mix them in a mass ratio of 1:

1.

3. The alcohol sobering and liver protecting composition according to claim 1, characterized in that: The water extraction method comprises: mixing the raw material and 95-100°C water at a solid-liquid ratio of 1:10-20, extracting for 2-3 hours and then separating the solid and the liquid, collecting the extract, and concentrating and spray-drying to obtain the extract; and mixing the Hovenia dulcis extract, the Pueraria root extract, and the Curcuma longa extract at a mass ratio of 1-1.2:1-1.5:1 to obtain the medicinal and edible substance.

4. The alcohol sobering and liver protecting composition according to any one of claims 1 to 3, characterized in that: Calculated by mass percentage, the composition includes: 0.8-1% probiotics, 12-18% beta-glucan, 15-18% corn peptide, and 60-70% medicinal and edible substances.

5. Use of the alcohol sobering and liver protecting composition according to any one of claims 1 to 4 in the preparation of alcohol sobering and liver protecting medicines or foods.

6. The use according to claim 5, characterized in that The alcohol sobering up and liver protecting method includes preventing acute alcoholic liver injury.

7. The use according to claim 5, characterized in that The alcohol sobering up and liver protecting method comprises reducing the alcohol content in breath after drinking, the alcohol content in blood, and the acetaldehyde content in blood and saliva.

8. Use of the alcohol sobering and liver protecting composition according to any one of claims 1 to 4 in a drug for preventing or treating flora imbalance or a food for regulating intestinal flora balance.

9. A candy for sobering up and protecting the liver, characterized in that: The active ingredient is the alcohol sobering and liver protecting composition according to any one of claims 1 to 4.

10. The alcohol-relieving and liver-protecting compressed candy according to claim 9, characterized in that: The composition is as follows, by mass percentage: 99.6% of the hangover-relieving and liver-protecting composition and 0.4% of magnesium stearate. The preparation method of the hangover-relieving and liver-protecting compressed candy comprises the following steps: Step 1: Bacillus coagulans BCN019, Lactobacillus salivarius JYLS-372, Hovenia dulcis extract, Pueraria root extract, Curcuma longa extract, corn peptide, β-glucan and magnesium stearate are mixed in proportion and passed through an 80-mesh sieve; Step 2: The mixture obtained in step 1 is compressed into a tablet candy.

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