WO2020052074A1 - 一种提高肝脏排毒、解酒护肝的提取物及其制备方法 - Google Patents

一种提高肝脏排毒、解酒护肝的提取物及其制备方法 Download PDF

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WO2020052074A1
WO2020052074A1 PCT/CN2018/116635 CN2018116635W WO2020052074A1 WO 2020052074 A1 WO2020052074 A1 WO 2020052074A1 CN 2018116635 W CN2018116635 W CN 2018116635W WO 2020052074 A1 WO2020052074 A1 WO 2020052074A1
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liver
sea cucumber
extract
protease
detoxification
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French (fr)
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徐菀羚
曹凤君
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长春健康未来医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention belongs to the field of health food, and particularly relates to an extract for improving liver detoxification, detoxifying and protecting the liver, and a preparation method thereof.
  • Non-alcoholic fatty liver disease refers to the accumulation of excessive fat in the liver in the form of triglycerides due to multiple causes. It is one of the most important public health problems in the world, and the type of chronic liver disease with the highest incidence in China. It is a common "Fukui disease”. At present, there are more than 150 million people with this disease in China. In 2015, the China Medical Tribune reported that several sample surveys based on the urban population showed that the incidence of fatty liver in adults in China is between 12.5% and 35.4%, and there is a trend of youth. At present, there are at least 200 million people with fatty liver in China. Among them, about 10% of patients will develop cirrhosis, and 12.8% of patients will develop liver cancer after three years.
  • Alcoholic liver disease is a liver disease caused by long-term heavy drinking. It usually appears as fatty liver in the early stage, which can develop into alcoholic hepatitis, liver fibrosis and cirrhosis. Its main clinical features are nausea, vomiting, jaundice, liver enlargement and tenderness. Can be complicated by liver failure and upper gastrointestinal bleeding. Severe alcoholism can induce extensive hepatocyte necrosis and even liver failure. Alcoholic liver disease is one of the common liver diseases in China, which seriously harms the health of people.
  • ethanol After entering the body, ethanol is absorbed by the gastrointestinal tract, and only 2-10% is excreted by the respiratory tract, urine, and sweat glands as prototypes, and the remaining 90-98% is oxidized in the liver; about 80% of ethanol metabolized by the liver is through ethanol dehydrogenase (ADH) is converted to acetaldehyde, and about 20% is converted to acetaldehyde by microsomal ethanol oxidase, and then acetaldehyde is converted to acetic acid by acetaldehyde dehydrogenase (ALDH), enters the tricarboxylic acid cycle, and is oxidized to carbon dioxide and water.
  • ADH ethanol dehydrogenase
  • acetaldehyde can stimulate the secretion of epinephrine, norepinephrine, etc., which can cause symptoms such as red face and red ears, increased heart rate, and increased skin temperature.
  • Human tolerance to alcohol varies by race and individual. 80% of yellow people are alcohol sensitive, and only 5% of white people are alcohol sensitive. This is because most yellow people produce acetaldehyde after drinking alcohol. Fast speed, but slow oxidation to acetic acid, easily cause acetaldehyde accumulation poisoning.
  • Acetaldehyde has a variety of toxicities. First, it is converted to superoxide by xanthine oxidase; second, it causes liver protein secretion and liver enlargement.
  • the current development of hangover products in the market mainly starts from two aspects: one is to inhibit the gastrointestinal absorption of alcohol, strengthen the first-pass effect of ethanol in the gastrointestinal tract, and reduce the concentration of ethanol in blood; Metabolic enzymes or intermediates in the liver, acetaldehyde, accelerate the clearance of ethanol and its metabolites and reduce its cellular damage to tissues.
  • one is to inhibit the gastrointestinal absorption of alcohol, strengthen the first-pass effect of ethanol in the gastrointestinal tract, and reduce the concentration of ethanol in blood
  • Metabolic enzymes or intermediates in the liver, acetaldehyde accelerate the clearance of ethanol and its metabolites and reduce its cellular damage to tissues.
  • many functional products with hangover effects have appeared, but such products have the problem of low absorption and utilization rate, and often do not achieve the real hangover prevention effect.
  • the present invention provides an extract for improving liver detoxification, detoxifying and protecting the liver, which has good absorption, high biological titer, can increase the amount of alcohol consumption and improve the symptoms of alcohol discomfort, and provides the same. Preparation.
  • a method for preparing an extract for improving liver detoxification, hangover and protecting liver includes the following steps:
  • Fresh sea cucumber is softened, cleaned after removing the internal organs, and then protease is added. After heating, the enzyme is hydrolyzed to obtain a sea cucumber active polypeptide solution; 1-3 mass times water is added to the sea cucumber active polypeptide solution for vacuum distillation to obtain sea cucumber. Active polypeptide concentrated solution; spraying and sterilizing the sea cucumber active polypeptide concentrated solution in sequence to obtain the sea cucumber active polypeptide powder;
  • the yeast After the yeast is activated, it is cultured to obtain a seed liquid; the sea cucumber active polypeptide powder of step (1) is added to the seed liquid, and a fermentation liquid is obtained by fermentation and culture; the fermentation liquid is subjected to solid-liquid separation to obtain a supernatant Liquid and yeast; the supernatant is spray-dried and sterilized in order to obtain the extract which can improve liver detoxification and hangover and protect liver.
  • step (1) the time for softening is 1-3h.
  • the protease is a mixture of a microbial-derived protease, an animal protease, and a plant protease; the microbial-derived protease is a complex protease, the animal protease is trypsin, and the plant protease is bromelain.
  • the mass ratio of the complex protease, trypsin, and bromelain is 1-2: 1-2: 1-2.
  • step (1) the heating temperature is 40-60 ° C, and the heating time is 2-3h.
  • step (1) the temperature of the vacuum distillation is 70-80 ° C, and the time of the vacuum distillation is 3-5h.
  • step (1) the inlet temperature of the spray drying is 70-80 ° C
  • the water droplet speed of the spray drying is 20-40 ml / min
  • the outlet temperature of the spray drying is 70-80 ° C.
  • step (2) the ratio of the volume of the seed liquid to the mass of the sea cucumber active polypeptide powder is 0.8L-1.2L: 80g-120g.
  • step (2) the temperature of the fermentation culture is 28-32 ° C, and the time of the fermentation culture is 22-26h.
  • An extract prepared by the method which improves liver detoxification, hangover and protect liver.
  • Sea cucumber has a history of more than 600 million years of survival. It is famous for its “sexual warming and full-featured mountain ginseng”. It ranks first in the “Seafood Eight Treasures", is expensive, and is a well-known nourishing treasure.
  • “Compendium of Compendium of Materia Medica” sea cucumber, sweet and salty, tonifying kidney, replenishing the essence, taking urination, aphrodisiac, and treating dysentery.
  • the medicinal value of sea cucumber It has the effects of calming the liver and burying the sun, calming the nerves, softening and dispersing, and astringent and astringent.
  • Indications include dizziness, tinnitus, tremor of hands and feet, palpitations, insomnia, irritability, seizures, dysentery, breast agglomeration, spontaneous sweating, sweating, nocturnal emission, urination, gastric pain, and eczema sores.
  • the protein content (peptide) in dried sea cucumber body wall is as high as 90%, poly (oligosaccharide) is about 6%, lipid is about 4%, and it contains calcium, magnesium salts, various trace elements and small amounts of nucleic acids. Protein, low fat and low cholesterol, high-quality foods with high vitamins and minerals, sea cucumber body wall also contains a variety of active peptides.
  • sea cucumber According to research by Guangzhou University of Traditional Chinese Medicine, Shenyang Institute of Ecology, Chinese Academy of Social Sciences and other research units, sea cucumber has been proven to be rich in natural collagen, peptides, sphingosine, saponin, mucopolysaccharides, multivitamins, 18 amino acids needed by the human body And iron, phosphorus, manganese, selenium, iodine, calcium, magnesium, zinc and other minerals, a total of more than 50 kinds of nutritional ingredients, is recognized as a good natural nutritional supplement for the body.
  • the mineral content per 100 grams of sea cucumber is: 72mg of calcium, 160mg of magnesium, 0.2mg of zinc, 0.05mg of vitamin B1, 0.4mg of vitamin E, and others: including 18 kinds of amino acids, iron, phosphorus, manganese, selenium, iodine, glutathione More than 50 kinds of nutritional ingredients such as glycine, glycoside and taurine.
  • Active peptides have different functions and biological effects according to their constituent amino acids. Active peptides have a variety of human metabolism and physiological regulation functions, are easy to digest and absorb, and have enhanced immunity, hormone regulation, anti-fatigue, antibacterial, antiviral, and Blood pressure and blood lipid lowering, and high food safety, are currently the most popular research topics and promising functional factors in the international food industry. Extraction of active peptides from sea cucumbers is based on the protein in sea cucumbers, and then specific small peptide separation technology is used to obtain small molecular oligopeptides. At present, the main methods for extracting active peptides from sea cucumber are drying method and enzymatic hydrolysis method.
  • Drying method is to dry or freeze dry fresh sea cucumber or cooked sea cucumber, pulverize, and obtain sea cucumber powder capsules.
  • the disadvantage of this method is fresh
  • the live sea cucumber is not matured, and the prepared sea cucumber powder is difficult to be absorbed and used by humans.
  • the main thing is that this method does not separate the saponin and saponin hydrolase coexisting in the sea cucumber. After the sea cucumber died, the saponin was destroyed by the saponin hydrolase and remained. Body, triterpenoid saponin, glycosyl, or secondary glycoside, its activity is also reduced or disappeared.
  • the enzymatic hydrolysis method was used to prepare sea cucumber mucopolysaccharide in the early stage.
  • the sea cucumber or its fragments can disintegrate the proteoglycan of sea cucumber by enzymolysis.
  • Mucopolysaccharide (HG) which is a sugar chain moiety, is released.
  • HG Mucopolysaccharide
  • using this method to extract active peptides from the sea cucumber destroys the original ecological structure (proteoglycan structure) of the sea cucumber protein.
  • Some people have studied the protein in the sea cucumber. The molecular weight varies, but the whole is most suitable for the needs of the human body.
  • the enzymatic hydrolysis method Can destroy these important natural structures, making its effect on the human body an unknown.
  • the method for preparing an extract for improving liver detoxification, detoxifying and protecting the liver according to the present invention, firstly enzymatically hydrolyze the protein in the sea cucumber to convert it into a smaller molecular weight (less than 1600u, mainly small peptides smaller than 1000u), human body
  • a smaller molecular weight less than 1600u, mainly small peptides smaller than 1000u
  • human body The more easily absorbed active polypeptide form, and then mixed with yeast for fermentation and culture, through yeast metabolism, convert the active polypeptide into an organic active polypeptide form (glucan-peptide chelate) that is closer to its original biological activity, thereby further enhancing
  • the absorption mechanism has been increased, and the biological titer has been increased.
  • the present invention greatly improves the bioavailability of nutrients in sea cucumbers by means of molecular weight interception (taken at 500 Da as the interception unit) and organic conversion, and provides a solution for the most difficult nutrient absorption problem in functional foods at present.
  • the liver detoxification-enhancing, liver-eliminating and liver-protecting extract obtained by the preparation method of the present invention contains a high content of active oligopeptides. After analysis of the amino acid sequence, it is confirmed as a tripeptide (Glu-Cys-Gly, also known as glutathione Peptide, 307Da), two tetrapeptides (Val-Thr-Pro-Tyr, 479Da and Val-Leu-Leu-Tyr, 507Da), and one hexapeptide (Val-Gly-Thr-Val-Glu-Met, 635Da)
  • active oligopeptides can participate in the body's redox reaction and the transport of amino acids into cells, and have the effects of scavenging free radicals, maintaining normal functions of liver cells, and promoting bile acid metabolism; the glutathione can also break down the middle of alcohol metabolism The product acetaldehyde, thereby reducing its damage to the liver, and the synergistic effects of
  • the extract obtained by the method for improving liver detoxification, anti-alcoholic and liver protection is a natural functional food, does not contain any western medicine and hormone components, and has good water rehydration, convenient eating, easy digestion and absorption, and no antigenicity. Safe and stable product quality, suitable for industrial production.
  • This embodiment provides a method for preparing an extract that enhances liver detoxification, hangover and liver protection, and includes the following steps:
  • the sea cucumber active polypeptide concentrated solution is at an inlet temperature of 70 ° C, a water droplet speed of 20ml / min, and effluent. Spray drying at a temperature of 70 ° C, and then sterilizing to obtain the sea cucumber active polypeptide powder;
  • the seed liquid is obtained through expansion culture; 80 g of sea cucumber active polypeptide powder is added to 0.8 L of the seed liquid, and fermented and cultured at 28 ° C for 22 h to obtain a fermentation liquid; the fermentation liquid is subjected to solid-liquid separation, Supernatant and yeast are obtained; the supernatant is spray-dried under the conditions of an inlet temperature of 70 ° C, a water droplet speed of 20ml / min, and an outlet temperature of 70 ° C to obtain a powder, and then adding the powder 5 times
  • the quality cranberry powder is mixed evenly and sterilized to obtain the extract which can improve liver detoxification and hangover and protect liver.
  • This embodiment provides a method for preparing an extract that enhances liver detoxification, hangover and liver protection, and includes the following steps:
  • Fresh sea cucumbers are left for 3 hours, cleaned after removing the internal organs, and then a mixed protease consisting of complex protease, trypsin, and bromelain in a mass ratio of 1: 2: 1 is added, and heated at 60 ° C in a constant temperature water bath for 3 hours to obtain a sea cucumber active peptide solution; 3 times the mass of water was added to the sea cucumber active polypeptide solution, and vacuum distillation was performed at 80 ° C for 5 hours to obtain a sea cucumber active polypeptide concentrated solution; the sea cucumber active polypeptide concentrated solution was at an inlet temperature of 80 ° C, a water droplet speed of 40 ml / min, and effluent Spray drying at a temperature of 80 ° C, and then sterilizing to obtain the sea cucumber active polypeptide powder;
  • the yeast After the yeast is activated, it is expanded to obtain a seed liquid; 120 g of sea cucumber active polypeptide powder is added to 1.2 L of the seed liquid, and fermented and cultured at 32 ° C for 26 h to obtain a fermentation liquid; the fermentation liquid is subjected to solid-liquid separation, The supernatant and the yeast body are obtained; the supernatant is spray-dried under the conditions of an inlet temperature of 80 ° C, a water droplet speed of 40ml / min, and an outlet temperature of 80 ° C, and then sterilization to obtain the improved liver An extract to detoxify and detoxify the liver.
  • This embodiment provides a method for preparing an extract that enhances liver detoxification, hangover and liver protection, and includes the following steps:
  • the yeast After the yeast is activated, it is expanded to obtain a seed liquid; 100 g of sea cucumber active polypeptide powder is added to 1 L of the seed liquid, and fermented and cultured at 30 ° C for 24 hours to obtain a fermentation liquid; the fermentation liquid is subjected to solid-liquid separation to obtain Supernatant and yeast body; spraying and drying the supernatant under the conditions of an inlet temperature of 80 ° C, a water droplet speed of 30ml / min, and an effluent temperature of 80 ° C, followed by sterilization, to obtain the improved liver detoxification Extracts for hangover and liver protection.
  • Acute Alcoholism Experiment The hangover effect of the present invention is analyzed by measuring the blood alcohol concentration, hangover time, and changes in the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) after ethanol is administered to mice.
  • ADH alcohol dehydrogenase
  • ADH acetaldehyde dehydrogenase
  • ICR mice Forty-eight ICR mice were randomly divided into 4 groups, 12 in each group, of which one group was perfused with physiological saline at a dose of 10 mL / kg.bw as a model group, and the other three groups were 0.1 mg / kg.bw and 0.5 mg, respectively. /kg.bw, 1mg / kg.bw gavage the aqueous solution of the sample of Example 3 of the present invention, respectively set as the low-dose group, the middle-dose group, the high-dose group, after 1h, each dose group was 5.75g / kg.bw.
  • mice 50% ethanol was administered by gavage, and the time that the mice remained in a supine posture was set as a drunken time, and the time after which the mice no longer rested and started to move was set as a sobering time.
  • the venous blood of the mice was collected 15 minutes before gavage, 15 minutes after gavage, every 10 minutes of drunkenness, and every 10 minutes after sobering.
  • the serum was collected by centrifugation and stored in a refrigerator at -80 ° C.
  • ELISA was used to detect the activities of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). The experimental data are shown in Table 1.
  • mice Seventy-five ICR mice were randomly divided into five groups of 15 mice, each of which was set as a blank group, a model group, and a low-medium-high-dose group of Example 3 of the present invention (0.1 mg / kg.bw, 0.5 mg / kg.bw, 1mg / kg.bw).
  • the blank group was administrated with distilled water, and the other groups were administrated with 50% ethanol.
  • the blank group and the model group were administrated with 6 mL / kg.bw of distilled water, and the other groups were administrated with a corresponding dose of the aqueous solution of Example 3 of the present invention. Blood samples were taken once a day.
  • ALT serum alanine aminotransferase
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • the maximum drinking amount was 400g / time, and the minimum drinking amount was 20g / time.
  • the total drinking volume of 29 people was 4910g, and the average drinking amount was 169.31g / time / person.
  • the maximum amount of drinking again was 1200g / time, and the minimum amount of drinking was 120g / time.
  • the total drinking amount of 29 people was 16191g, and the average drinking amount was 558.31g / time / person.
  • Example 3 of the present invention at 5 mg / kg for 8 consecutive days, 2 hours after the last intragastric administration, the blank control group was injected intraperitoneally with a blended oil solution, and the remaining groups were injected intraperitoneally with 0.15% CCL 4 blended oil solution. Blood was collected from the eyeballs for 24 hours to determine ALT And AST activity. Take the liver, weigh it to calculate the liver index, prepare liver homogenate, and measure the contents of GSH-Px, SOD, and MDA. The experimental results are shown in Tables 4 and 5.
  • the liver index of the model group was significantly lower than that of the blank group.
  • the liver index of the mice taking Example 3 of the present invention was significantly increased.
  • the levels of ALT and AST in the liver tissue of the model group were significantly increased; compared with the model group, the levels of ALT and AST in the liver tissue of the mice taking Example 3 of the present invention were significantly reduced, and the levels of decline were particularly prominent.
  • Example 3 of the present invention can significantly reduce the inflammatory response caused by experimental liver injury, reduce the levels of ALT, AST activity (P ⁇ 0.01), and MDA in the serum (P ⁇ 0.01); extremely significantly increase GSH in liver cells -Px (reduced glutathione) content (P ⁇ 0.01), which significantly increased the content of SOD in liver cells (P ⁇ 0.01) and decreased the content of MDA (P ⁇ 0.01). It is shown that Example 3 of the present invention has a protective effect on experimental liver injury caused by CCL 4 .
  • the extract of the present invention significantly improves the content and expression of reduced glutathione (GSH-Px) and superoxide dismutase (SOD) in liver cells, and reduces the content of malondialdehyde (MDA) in liver tissues. Therefore, it improves the total antioxidant capacity of the liver, stabilizes the liver cell membrane and maintains its integrity, improves the function of liver cells as a whole, and improves the detoxification of the liver. It can promote the ultrastructure recovery of liver cells, promote the division and proliferation of normal liver cells, and improve the ability of liver cells to synthesize RNA and proteins. Therefore, the extract of the present invention has the effects of protecting normal liver cells, promoting repair of damaged liver cell membranes, and promoting liver metabolism.
  • GSH-Px endogenous reduced glutathione
  • SOD superoxide dismutase
  • Glutathione is a tripeptide composed of glutamic acid, cystine, and glycine. It is the cofactor of glyceraldehyde dehydrogenase and the coenzyme of glyoxalase and triose phosphate dehydrogenase. Participate in the tricarboxylic acid cycle and sugar metabolism in the body, so that the body obtains high energy. Can participate in the body's redox process.
  • glutathione transferase Under the action of glutathione transferase, reduced glutathione can combine with peroxides and free radicals to counteract the destruction of thiol groups by oxidants and protect thiol-containing proteins and thiol-containing enzymes from cell membranes. Damage, but also to fight free radical damage to important organs. Glutathione is abundant in the liver and lens.
  • thiol (-SH) enzymes such as cholinesterase in the body, and protect the body from iodoacetic acid, mustard gas, free radicals (such as superoxide anions), heavy metals (such as mercury, lead),
  • thiol (-SH) enzymes such as cholinesterase
  • iodoacetic acid mustard gas, free radicals (such as superoxide anions), heavy metals (such as mercury, lead)
  • the poisoning of epoxides and other harmful substances which promote the metabolism of sugars, fats and proteins, can also affect the metabolic process of cells. Promote the metabolism of eye tissues, can inhibit the instability of crystal protein thiol groups, prevent the development of cataracts and retinal diseases, and have a good effect of increasing vision.
  • SOD superoxide dismutase

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Abstract

一种提高肝脏排毒、解酒护肝的海参提取物及其制备方法，包括先将海参用蛋白酶酶解，再与酵母菌混合发酵培养。

Description

一种提高肝脏排毒、解酒护肝的提取物及其制备方法 技术领域
本发明属于保健食品领域，具体涉及一种提高肝脏排毒、解酒护肝的提取物及其制备方法。
背景技术
时下的白领人群因工作紧张、压力大，饮酒应酬多，无规律的饮食和休息，加上炒股、供房等精神压力，是脂肪肝的密集区。据法国媒体报道，在50岁以上的“急性肝炎”病人中，43％由药物所致。如阿司匹林、磺胺、红霉素、利福平等都是既常用又有害于肝的药物。即使那些看似安全的药物，也可因误用或滥用而给肝脏埋下隐患。另外，中草药也不安全，如苦杏仁、蟾酥、木薯、广豆根、北豆根、艾叶、毛冬青等中药，剂量大时可引起黄疸、肝区疼痛和肝功能损害。当下，没有肝炎的任何症状，但验血时却表现出单项转氨酶升高的大有人在。不少专家提出食物污染可能难辞其咎，食物污染包括蔬菜、瓜果的农药残留，某些食品添加剂，水源污染，熏烤食物及变质食物等等。食物污染虽然在短时间内不致造成明显危害，但长期慢性毒害作用的积累完全能使肝脏功能受损。中国每12个人里就有一个人肝脏有疾病，肝炎、脂肪肝、酒精肝、肝硬化、肝癌的病人越来越多，保护我们的肝脏，是每一个人迫切的需要。
非酒精性脂肪性肝病(NAFLD)是指多种原因引起的过多脂肪以甘油三酯的形式在肝脏中堆积。是当前全球最重要的公共健康问题之一，也是我国发病率最高的慢性肝病类型，是一种常见的“福贵病”，目前国内该病患者超过1.5亿人。2015年，《中国医学论坛报》的报道显示，几项基于城市人口的抽样调查表明，我国成人脂肪肝发病率在12.5％～35.4％之间，并出现年轻化趋势。目前，我国脂肪肝人群至少有2亿人，其中，约有10％的患者会发展为肝硬化，12.8％的患者在三年以后会发展为肝癌。
酒精性肝病是由于长期大量饮酒导致的肝脏疾病，初期通常表现为脂肪肝，进而可发展成酒精性肝炎、肝纤维化和肝硬化。其主要临床特征是恶心、呕吐、黄疸、可有肝脏肿大和压痛。并可并发肝功能衰竭和上消化道出血等。严重酗酒时可诱发广泛肝细胞坏死，甚至肝功能衰竭。酒精性肝病是我国常见的肝脏疾病之一，严重危害人群健康。近年来酒精性肝病占同期肝病住院患者的比例在不断上升，从1991年的4.2％增至1996年的21.3％；酒精性肝硬化在肝硬化的病因构成比从1999年的10.8％上升到2003年的24.0％。因此，寻求有效防治酒精性肝病的解酒食物或药物是一项关乎民生健康的重要课题。
乙醇进入体内后通过胃肠道吸收，仅有2-10％由呼吸道、尿液和汗腺以原型排出，其余90-98％在肝脏内氧化；经肝脏代谢的乙醇约80％通过乙醇脱氢酶(ADH)转化为乙醛，约20％通过微粒体乙醇氧化酶转化为乙醛，乙醛再经过乙醛脱氢酶(ALDH)转化为乙酸，进入三羧酸循环，氧化成二氧化碳和水。乙醛的生成可刺激肾上腺素、去甲肾上腺素等的分泌，从而引起面红耳赤、心率加快、皮肤温度升高等症状。而人类对酒精的耐受性有种族和个体差异，黄种人中80％为酒精敏感者，白种人中仅有5％为酒精敏感者，这是因为大多数黄种人在饮酒后产生乙醛速度快，而氧化为乙酸的速度慢，容易引起乙醛蓄积中毒。乙醛具有多种毒性，一是通过黄嘌呤氧化酶转变为超氧化物；二是使肝脏分泌性蛋白质潴留造成肝肿大。
根据乙醇的体内代谢特点，目前市面上解酒产品开发主要从两方面着手：一是抑制酒精的胃肠吸收，加强乙醇在胃肠道的首过效应，降低血中乙醇浓度；二是直接作用于肝脏的代谢酶系或代谢中间产物乙醛，加速乙醇及其代谢产物的清除速率，减轻其对组织的细胞损害。由此出现了很多具有解酒作用的功能性产品，但是此类产品均存在吸收利用率低的问题，往往达不到真正的解酒预防效果。
发明内容
为了解决现有技术中存在的上述问题，本发明提供一种吸收好，生物效价高、可以增加饮酒量和改善酒后不适症状的提高肝脏排毒、解酒护肝的提取物， 并提供其制备方法。
本发明的技术方案为：
一种提高肝脏排毒、解酒护肝的提取物的制备方法，包括如下步骤：
(1)制备海参活性多肽粉
鲜海参放至变软，去内脏后清洗干净，然后加入蛋白酶，加热使酶解后，得到海参活性多肽液；向所述海参活性多肽液中加入1-3质量倍水进行真空蒸馏，得到海参活性多肽浓缩液；将所述海参活性多肽浓缩液依次进行喷雾干燥、杀菌，即得所述海参活性多肽粉；
(2)制备提高肝脏排毒、解酒护肝的提取物
酵母菌活化后，经培养，得到种子液；向所述种子液中加入步骤(1)所述海参活性多肽粉，经发酵培养得到发酵液；将所述发酵液进行固液分离，得到上清液和酵母菌体；将所述上清液依次进行喷雾干燥、杀菌，即得所述提高肝脏排毒、解酒护肝的提取物。
进一步地，步骤(1)中，所述放至变软的时间为1-3h。
进一步地，步骤(1)中，所述蛋白酶为微生物来源蛋白酶、动物蛋白酶和植物蛋白酶的混合物；所述微生物来源蛋白酶为复合蛋白酶，所述动物蛋白酶为胰蛋白酶，所述植物蛋白酶为菠萝蛋白酶。
进一步地，所述复合蛋白酶、胰蛋白酶和菠萝蛋白酶的质量比为1-2:1-2:1-2。
进一步地，步骤(1)中，所述加热的温度为40-60℃，所述加热的时间为2-3h。
进一步地，步骤(1)中，所述真空蒸馏的温度为70-80℃，所述真空蒸馏的时间为3-5h。
进一步地，步骤(1)中，所述喷雾干燥的进口温度为70-80℃、所述喷雾干燥的水滴速度为20-40ml/min、所述喷雾干燥的出水温度为70-80℃。
进一步地，步骤(2)中，所述种子液的体积与所述海参活性多肽粉的质量之比为0.8L-1.2L:80g-120g。
进一步地，步骤(2)中，所述发酵培养的温度为28-32℃，所述发酵培养的时间为22-26h。
所述方法制备得到的提高肝脏排毒、解酒护肝的提取物。
海参，有着6亿多年的存活史，因其“性温补、足敌山参”而胜名，在“海味八珍”中居首位，价值昂贵，为久负盛名的滋补珍品。据《本草纲目拾遗》中记载：海参，味甘咸，补肾，益精髓，摄小便，壮阳疗痿，其性温补，足敌人参，故名海参。海参的药用价值：具有平肝潜阳，镇惊安神，软坚散结，收敛固涩的功效。主治眩晕耳鸣，手足振颤，心悸失眠，烦躁不安，惊痫癫狂，瘰疬瘿瘤，乳房结块，自汗盗汗，遗精尿频，崩漏带下，吞酸胃痛，湿疹疮疡。干燥的海参体壁有机成分中蛋白质(多肽)高达90％，多(寡)糖约6％，脂质约4％，且含有钙、镁盐及多种微量元素及少量核酸，是一种高蛋白质、低脂肪和低胆固醇及高维生素和矿物质的优质食品，海参体壁中还含有多种活性多肽。据广州中医药大学、中国社会科学院沈阳生态所等研究单位的研究，海参已经被证明富含天然胶原蛋白、多肽、神经鞘氨醇、皂甙、粘多糖、多种维生素、18种人体需要的氨基酸以及铁、磷、锰、硒、碘、钙、镁、锌等矿物质，共计50多种营养成分，被公认为对身体有益的天然营养滋补品。每100克海参中的矿物质含量为：钙72mg、镁160mg、锌0.2mg、维他命B1 0.05mg、维他命E 0.4mg、其他：包括18种氨基酸、铁、磷、锰、硒、碘、谷胱甘肽、糖苷、牛磺酸等50多种营养成分。
活性多肽根据其组成氨基酸不同，其功能及生物学效应也不同，活性多肽具有多种人体代谢和生理调节功能，易消化吸收，有增强免疫力、激素调节、抗疲劳、抗菌、抗病毒、降血压和降血脂等作用，食用安全性高，是当前国际食品界最热门的研究课题和极具发展前景的功能因子。从海参中提取活性多肽 是以海参中蛋白质为原料，再经过定特定小肽分离技术获得小分子低聚肽。目前从海参中提取活性多肽主要采用的方法有干燥法和酶解法，干燥法是将鲜活海参或熟海参干燥或冷冻干燥、粉碎、制得的海参粉装胶囊，这种方法的弊端在于鲜活海参未经熟化，制得的海参粉难以被人吸收利用，更主要的是该方法未将在海参体内共存的皂甙和皂甙水解酶分离，海参死后皂甙被皂甙水解酶破坏，变为留体、三萜皂甙元、糖基或者次级甙，其活性也随之降低或消失。酶解法早期是用来制取海参粘多糖的，海参或其分割物通过酶解化能使海参的蛋白多糖解体。使作为糖链部分的粘多糖(HG)释放出来。但用这种方法提取海参活性多肽，破坏了海参蛋白的原生态结构(蛋白多糖结构)，有人曾研究过海参体内的蛋白，分子量大小不一，但整体最适于人体的需要，而酶解法能破坏这些重要的天然结构，使其对人体的作用变成了一个未知数。
本发明的有益效果为：
1、本发明所述的提高肝脏排毒、解酒护肝的提取物的制备方法，先将海参中的蛋白质酶解使转化为分子量更小(1600u以下，小于1000u的小肽为主)、人体更易吸收的活性多肽形式，再与酵母混合进行发酵培养，通过酵母的新陈代谢，将活性多肽转化为更接近其原始生物活性的有机活性多肽形式(葡聚糖-多肽螯合物)，从而进一步增强了吸收机制，提高了生物效价。即本发明通过分子量截取(以500Da为截取单位)以及有机转化的方式，大幅提高了海参中营养物质的生物利用度，为目前功能食品中最为棘手的营养成分吸收问题提供了解决思路。
2、本发明制备方法得到的提高肝脏排毒、解酒护肝的提取物含有高含量的活性寡肽，经氨基酸序列分析，确认为一种三肽(Glu-Cys-Gly，又名谷胱甘肽，307Da)、两种四肽(Val-Thr-Pro-Tyr，479Da和Val-Leu-Leu-Tyr，507Da)以及一种六肽(Val-Gly-Thr-Val-Glu-Met，635Da)，这些活性寡肽能参与体内氧化还原反应及氨基酸向细胞内的转运，具有清除自由基、维持肝细胞正常功能以及促进胆酸代谢的作用；所述谷胱甘肽还可以分解酒精代谢的中间产物乙醛，从而减轻其对肝脏的损害，加之其余肽在解毒和抗氧化方面的协同作用，使得本发明具有显著的提高肝脏排毒、解酒护肝的作用，试验证明，本发明疗 效确切，应用于人群可以显著增加饮酒量和改善酒后不适。
3、本发明制备方法得到的提高肝脏排毒、解酒护肝的提取物为天然功能食品，不含任何西药和激素成分，产品复水性好、食用方便、易消化吸收且无抗原性，食用更安全，且产品质量稳定，适合工业化生产。
具体实施方式
下面通过具体实施例，对本发明作进一步说明。
实施例1
本实施例提供一种提高肝脏排毒、解酒护肝的提取物的制备方法，包括如下步骤：
(1)制备海参活性多肽粉
鲜海参放置1h，去内脏后清洗干净，然后加入复合蛋白酶、胰蛋白酶和菠萝蛋白酶按1:1:1质量比组成的混合蛋白酶，40℃恒温水浴加热2h，得到海参活性多肽液；向所述海参活性多肽液中加入1倍质量的水，于70℃进行真空蒸馏3h，得到海参活性多肽浓缩液；将所述海参活性多肽浓缩液在进口温度为70℃、水滴速度为20ml/min、出水温度为70℃的条件下进行喷雾干燥，然后杀菌，即得所述海参活性多肽粉；
(2)制备提高肝脏排毒、解酒护肝的提取物
酵母菌活化后，经扩大培养，得到种子液；向0.8L所述种子液中加入80g海参活性多肽粉，在28℃温度下发酵培养22h得到发酵液；将所述发酵液进行固液分离，得到上清液和酵母菌体；将所述上清液在进口温度为70℃、水滴速度为20ml/min、出水温度为70℃的条件下进行喷雾干燥得到粉末，然后加入所述粉末5倍质量的蔓越莓粉末，混合均匀后杀菌，即得所述提高肝脏排毒、解酒护肝的提取物。
实施例2
本实施例提供一种提高肝脏排毒、解酒护肝的提取物的制备方法，包括如下步骤：
(1)制备海参活性多肽粉
鲜海参放置3h，去内脏后清洗干净，然后加入复合蛋白酶、胰蛋白酶和菠萝蛋白酶按1:2:1质量比组成的混合蛋白酶，60℃恒温水浴加热3h，得到海参活性多肽液；向所述海参活性多肽液中加入3倍质量的水，于80℃进行真空蒸馏5h，得到海参活性多肽浓缩液；将所述海参活性多肽浓缩液在进口温度为80℃、水滴速度为40ml/min、出水温度为80℃的条件下进行喷雾干燥，然后杀菌，即得所述海参活性多肽粉；
(2)制备提高肝脏排毒、解酒护肝的提取物
酵母菌活化后，经扩大培养，得到种子液；向1.2L所述种子液中加入120g海参活性多肽粉，在32℃温度下发酵培养26h得到发酵液；将所述发酵液进行固液分离，得到上清液和酵母菌体；将所述上清液在进口温度为80℃、水滴速度为40ml/min、出水温度为80℃的条件下进行喷雾干燥，然后杀菌，即得所述提高肝脏排毒、解酒护肝的提取物。
实施例3
本实施例提供一种提高肝脏排毒、解酒护肝的提取物的制备方法，包括如下步骤：
(1)制备海参活性多肽粉
鲜海参放置2h，去内脏后清洗干净，然后加入复合蛋白酶、胰蛋白酶和菠萝蛋白酶按2:1:2质量比组成的混合蛋白酶，50℃恒温水浴加热2.5h，得到海参活性多肽液；向所述海参活性多肽液中加入2倍质量的水，于75℃进行真空蒸馏4h，得到海参活性多肽浓缩液；将所述海参活性多肽浓缩液在进口温度为80℃、水滴速度为30ml/min、出水温度为80℃的条件下进行喷雾干燥，然后杀菌，即得所述海参活性多肽粉；
(2)制备提高肝脏排毒、解酒护肝的提取物
酵母菌活化后，经扩大培养，得到种子液；向1L所述种子液中加入100g海参活性多肽粉，在30℃温度下发酵培养24h得到发酵液；将所述发酵液进行固液分离，得到上清液和酵母菌体；将所述上清液在进口温度为80℃、水滴速度为30ml/min、出水温度为80℃的条件下进行喷雾干燥，然后杀菌，即得所述提高肝脏排毒、解酒护肝的提取物。
实验例
1、急性酒精中毒实验通过测定小鼠灌胃乙醇后的血醇浓度、醒酒时间以及乙醇脱氢酶(ADH)、乙醛脱氢酶(ALDH)的活性变化分析本发明的解酒效果。
将48只ICR小鼠随机分为4组，每组12只，其中一组按10mL/kg.bw的剂量灌胃生理盐水作为模型组，另三组分别按0.1mg/kg.bw、0.5mg/kg.bw、1mg/kg.bw的剂量灌胃本发明实施例3样品水溶液，分别设为低剂量组、中剂量组、高剂量组，1h后各剂量组按5.75g/kg.bw的剂量灌胃50％乙醇，小鼠保持横卧不动姿势的时间设定为醉酒时间，之后小鼠不再横卧不动并开始活动的时间设定为醒酒时间。在灌胃前15分钟、灌胃后15分钟、醉酒时间每隔10分钟、醒酒后每隔10分钟各抽取小鼠静脉血，4℃冰箱放置12h后离心留取血清，-80℃冰箱保存，ELISA检测乙醇脱氢酶(ADH)、乙醛脱氢酶(ALDH)的活性，实验数据见表1。
表1 各组小鼠血清乙醇浓度、醒酒时间以及ADH和ALDH活性比较
Figure PCTCN2018116635-appb-000001
注：与模型组相比，*P<0.05，**P<0.01；乙醛脱氨酶的活性以每分钟生成 NADH的nmol表示。
统计学处理统计软件SPSS20.0进行数据分析，定量资料采用均数及标准差表示，组间对比采用单因素方差分析，组间两两比较采用最小差异显著性法(least significance of difference，LSD)，检验水准α＝0.05，当P<0.05时为差异具有统计学意义。
结果表明，本发明各剂量组血清乙醇浓度随剂量增加呈下降趋势，且剂量升高，醒酒时间缩短，各剂量组乙醇脱氢酶(ADH)和乙醛脱氢酶(ALDH)的活性较模型组显著升高，且高剂量组优于低剂量组。综上，说明本发明具有确切的解酒作用。
2、慢性酒精肝损伤实验
将75只ICR小鼠随机分为5组，每组15只，分别设为空白组、模型组、本发明实施例3低中高剂量组(0.1mg/kg.bw、0.5mg/kg.bw、1mg/kg.bw)。空白组灌胃蒸馏水，其它各组灌胃50％乙醇；1h后，空白组和模型组灌胃6mL/kg.bw蒸馏水，其它各组灌胃相应剂量的本发明实施例3样品水溶液，以上操作每天一次，抽取血样，室温静置1-2h后，450r/min离心10min，分离血清，全自动生化分析仪检测血清谷丙转氨酶(ALT)和谷草转氨酶(AST)的活性，11周后处死小鼠，实验数据见表2、表3。
表2 各组小鼠肝脏指数(n＝15，
Figure PCTCN2018116635-appb-000002
)
Figure PCTCN2018116635-appb-000003
表3 各组小鼠肝功指标比较(n＝15，
Figure PCTCN2018116635-appb-000004
)
Figure PCTCN2018116635-appb-000005
Figure PCTCN2018116635-appb-000006
*P<0.05、**P<0.01
统计学处理统计软件SPSS20.0进行数据分析，定量资料采用均数及标准差表示，组间对比采用单因素方差分析，组间两两比较采用最小差异显著性法(least significance of difference，LSD)，检验水准α＝0.05，当P<0.05时为差异具有统计学意义。
结果表明，本发明可有效抑制谷丙转氨酶(ALT)和谷草转氨酶(AST)的活性，减少脂滴，且高剂量组优于低剂量组，说明本发明具有显著的护肝效果。
3、人体解酒实验
1、一般资料
1.1试验对象选择标准
平时饮过酒，近半月内不曾饮酒；年龄18～65岁；身体健康；自愿参加。
1.2试验对象
共29例，全男性，年龄最小20岁，最大61岁，平均年龄29岁。
1.3试验用酒
53°赖茅酒，贵州怀桥酒厂生产。
2、试验方法
2.1首次饮酒
29人全部集中，用同样的菜，一边吃菜一边豪饮53°的赖茅酒，持续时间为1h，尽量让每一个人均达到醉态出现。5h后收集各名受试者饮酒总量和酒后 的感受及项目组织人所观察到的体征。
2.2再次饮酒
29人全部集中，每个受试者按服用本发明实施例3，30min后再次用同样的菜，一边吃菜一边豪饮53°的赖茅酒，持续时间为1h，尽量让每一个人均达到醉态出现。5h后收集各名受试者饮酒总量和酒后的感受及项目组织人所观察到的体征。
3、试验结果
首次最大饮酒量400g/次，最小饮酒量20g/次，29人饮酒总量为4910g，平均饮酒量169.31g/次/人。
再次最大饮酒量1200g/次，最小饮酒量120g/次，29人饮酒总量为16191g，平均饮酒量558.31g/次/人。
4、结果分析
(1)再次饮酒总量较首次增加了11281g，即229％，再次饮酒平均量较首次增加了389g/次/人，即229％。
(2)“增加饮酒量”和“改善酒后不适”的统计结果为：1.8％的人无效，41.03％的人有效，57.17％的人显效，总有效率98.21％。其中，“增加饮酒量”结果：1.48％的人无效，35.52％的人有效，63％的人显效，总有效率98.52％；“改善酒后不适”结果：1.17％的人无效，25.38％的人有效，73.45％的人显效，总有效率98.83％。
4、肝脏排毒实验
ICR小鼠50只随机分为空白对照组、模型组、高、中、低剂量组，空白对照组和模型组每天给予蒸馏水，高、中、低剂量组每天分别给予15mg/kg、10mg/kg、5mg/kg的本发明实施例3，连续8d，末次灌胃2h后，空白对照组腹腔注射调和油溶液，其余各组腹腔注射0.15％CCL 4调和油溶液，24h眼球取血 测定血清中ALT、AST的活性，取肝脏，称重计算肝脏指数，制备肝匀浆，测定GSH-Px、SOD、MDA的含量，实验结果见表4、表5。
统计学处理实验数据以
Figure PCTCN2018116635-appb-000007
表示，结果用单因素方差分析，P<0.05为有显著性差异，P<0.01为有非常显著性差异，P>0.05为无统计学意义。应用SPSS19.0统计软件分析数据。
表4 各组小鼠肝脏指数、血清ALT、AST水平比较
Figure PCTCN2018116635-appb-000008
*P<0.05，**P<0.01
结果表明，与空白组相比，模型组肝脏指数明显降低。与模型组相比，服用了本发明实施例3的小鼠的肝脏指数明显升高。与空白组相比，模型组肝组织ALT、AST水平显著升高；与模型组相比，服用了本发明实施例3的小鼠的肝组织ALT、AST水平显著降低，下降水平尤为突出。
表5 各组小鼠GSH-Px、SOD、MDA水平比较
Figure PCTCN2018116635-appb-000009
*P<0.05，**P<0.01
结果表明，本发明实施例3可显著减轻实验性肝损伤所致炎性反应，降低血清中ALT、AST活性(P﹤0.01)和MDA的含量(P﹤0.01)；极其显著提高肝细胞中GSH-Px(还原型谷胱甘肽)的含量(P﹤0.01)，极其显著提高肝 细胞中SOD的含量(P﹤0.01)及降低MDA的含量(P﹤0.01)。说明本发明实施例3对CCL 4造成的实验性肝损伤有保护作用。
实验结果分析
本发明所述提取物通过显著提高肝细胞中还原型谷胱甘肽(GSH-Px)及超氧化物歧化酶(SOD)的含量及其表达，以及降低肝脏组织丙二醛(MDA)的含量，从而提高肝脏总抗氧化能力，稳定肝细胞膜及保持其完整性，整体上改善肝细胞的功能，提高肝脏排毒作用。并可促进肝细胞超微结构复原，促进正常肝细胞的分裂增殖，提高肝细胞合成RNA及蛋白质的能力。因此，本发明所述提取物具有保护正常肝脏细胞、促进受损害的肝细胞膜修复、促进肝脏代谢的作用。由于本发明显著提高人体组织细胞中内源性还原型谷胱甘肽(GSH-Px)及超氧化物歧化酶(SOD)的含量及其表达，故其部分作用机制与还原型谷胱甘肽(GSH-Px)及超氧化物歧化酶(SOD)相同，即：
(1)谷胱甘肽(GSH)的作用机制。谷胱甘肽是由谷氨酸、胱氨酸及甘氨酸组成的一种三肽，它是甘油醛酸脱氢酶的辅基，又是乙二醛酶及磷酸丙糖脱氢酶的辅酶，参与体内三羧酸循环及糖代谢，使人体获得高能量。能参与体内氧化还原过程。在谷胱甘肽转移酶的作用下，还原型谷胱甘肽能和过氧化物及自由基相结合，以对抗氧化剂对巯基的破坏，保护细胞膜中含巯基的蛋白质和含巯基的酶不被破坏，亦对抗自由基对重要脏器的损害。谷胱甘肽在肝脏和晶状体中含量较多。它能激活和保护各种酶，如体内的胆碱酯酶等巯基(－SH)酶，使机体免受碘乙酸、芥子气、自由基(如超氧阴离子)、重金属(如汞、铅)、环氧化物等有害物质的毒害，从而促进糖类、脂肪及蛋白质代谢，也能影响细胞的代谢过程。促进眼组织的新陈代谢，可抑制晶体蛋白质巯基的不稳定，防止白内障及视网膜疾病的发展，有良好的增加视力的作用。
(2)超氧化物歧化酶(SOD)的作用机制:作为超氧阴离子的整合剂，其在体内进行着以下反应。第一步：作为有害物质的超氧阴离子在SOD的作用下和氢离子反应，生成另一种物质——过氧化氢；第二步：过氧化氢又在过氧化氢酶的作用下和氢离反应，最终生成了一种对人体无害的物质——水。
由于外源性的还原型谷胱甘肽(GSH-Px)及超氧化物歧化酶(SOD)的生物活性及其在人体中的生物利用度均较人体内内源性的相差甚远，故本发明所述提取物内源性还原型谷胱甘肽(GSH-Px)及超氧化物歧化酶(SOD)的作用远强于外源性的实验结果。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (10)

  1. 一种提高肝脏排毒、解酒护肝的提取物的制备方法，其特征在于，包括如下步骤：
    (1)制备海参活性多肽粉
    鲜海参放至变软，去内脏后清洗干净，然后加入蛋白酶，加热使酶解后，得到海参活性多肽液；向所述海参活性多肽液中加入1-3质量倍水进行真空蒸馏，得到海参活性多肽浓缩液；将所述海参活性多肽浓缩液依次进行喷雾干燥、杀菌，即得所述海参活性多肽粉；
    (2)制备提高肝脏排毒、解酒护肝的提取物
    酵母菌活化后，经培养，得到种子液；向所述种子液中加入步骤(1)所述海参活性多肽粉，经发酵培养得到发酵液；将所述发酵液进行固液分离，得到上清液和酵母菌体；将所述上清液依次进行喷雾干燥、杀菌，即得所述提高肝脏排毒、解酒护肝的提取物。
  2. 根据权利要求1所述的提高肝脏排毒、解酒护肝的提取物的制备方法，其特征在于，步骤(1)中，所述放至变软的时间为1-3h。
  3. 根据权利要求1所述的提高肝脏排毒、解酒护肝的提取物的制备方法，其特征在于，步骤(1)中，所述蛋白酶为微生物来源蛋白酶、动物蛋白酶和植物蛋白酶的混合物；所述微生物来源蛋白酶为复合蛋白酶，所述动物蛋白酶为胰蛋白酶，所述植物蛋白酶为菠萝蛋白酶。
  4. 根据权利要求3所述的提高肝脏排毒、解酒护肝的提取物的制备方法，其特征在于，所述复合蛋白酶、胰蛋白酶和菠萝蛋白酶的质量比为1-2:1-2:1-2。
  5. 根据权利要求1所述的提高肝脏排毒、解酒护肝的提取物的制备方法，其特征在于，步骤(1)中，所述加热的温度为40-60℃，所述加热的时间为2-3h。
  6. 根据权利要求1所述的提高肝脏排毒、解酒护肝的提取物的制备方法， 其特征在于，步骤(1)中，所述真空蒸馏的温度为70-80℃，所述真空蒸馏的时间为3-5h。
  7. 根据权利要求1所述的提高肝脏排毒、解酒护肝的提取物的制备方法，其特征在于，步骤(1)中，所述喷雾干燥的进口温度为70-80℃、所述喷雾干燥的水滴速度为20-40ml/min、所述喷雾干燥的出水温度为70-80℃。
  8. 根据权利要求1所述的提高肝脏排毒、解酒护肝的提取物的制备方法，其特征在于，步骤(2)中，所述种子液的体积与所述海参活性多肽粉的质量之比为0.8L-1.2L:80g-120g。
  9. 根据权利要求1所述的提高肝脏排毒、解酒护肝的提取物的制备方法，其特征在于，步骤(2)中，所述发酵培养的温度为28-32℃，所述发酵培养的时间为22-26h。
  10. 权利要求1-9任一项所述方法制备得到的提高肝脏排毒、解酒护肝的提取物。
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