Preparation method of sea cucumber peptide with composite effects of protecting liver and preventing liver cirrhosis

Abstract

The invention belongs to the technical field of preparation of sea cucumber peptides, relates to preparation of sea cucumber small molecule peptides, and relates to a preparation method of sea cucumber peptides with composite effects of protecting liver and preventing liver cirrhosis, and the sea cucumber peptides with various effects are prepared by using fresh and alive sea cucumbers as raw materials through protease hydrolysis, fishy smell removal, deodorization and other processes. The sea cucumber peptide has the advantages of good solubility, good stability, low viscosity and the like. Compared with the transportation function of amino acid, the sea cucumber active oligopeptide has the characteristics of quick absorption, low consumption and difficulty in saturation, can be added into food, health food, medicine and the like, and has the effects of supplementing nutrition, protecting the liver function, preventing liver cirrhosis, supplementing mineral substances, protein and vitamins and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of sea cucumber peptide preparation, relates to the preparation of sea cucumber small molecule peptides, and relates to a method for preparing sea cucumber peptides with the combined effects of protecting liver function and preventing liver cirrhosis. Background Art

[0002] Sea cucumber (Ludwigothurea grisea) belongs to the phylum Echinodermata, class Holothuria, order Scutellaria, and has survived for 50 million years. The organic components of its dried body wall are nearly 90% protein, about 4% lipid, about 6% polysaccharide, and a small amount of nucleic acid. The inorganic components contain calcium, magnesium salts and trace elements such as iron, manganese, zinc, copper, molybdenum, and selenium. It has appeared in people's lives as a health food and nutritional food since ancient times. The Qing Dynasty "Compendium of Materia Medica" records that "sea cucumber is warm and nourishing, and it is as good as ginseng, so it is called sea cucumber; it tastes sweet and salty, nourishes the kidney meridian, benefits the essence, eliminates phlegm and prolongs urination, strengthens yang and cures impotence, and kills sores and insects."

[0003] In recent years, Chinese medicine practitioners have proposed using sea cucumbers alone or in combination to protect liver function, prevent liver cirrhosis, treat tumors, aplastic anemia and diabetes, and have achieved good results. At the same time, sea cucumbers have been widely recognized and used in the treatment of common diseases such as epilepsy, gastric and duodenal ulcers, chronic hepatitis B, chronic nephritis, hypertension, tuberculosis, neurasthenia, venous thrombosis, hemorrhoids, rectal prolapse and constipation, as well as in various medicinal diets or dietary prescriptions formulated for general post-illness or postpartum recovery.

[0004] Compared with drugs, sea cucumber peptides have more advantages in their effects on the human body. Sea cucumber peptides focus on repairing the degenerated cells of the human body, solving a major problem in the world today: the problem of absorption and utilization by the human body. Current studies have shown that some active ingredients in sea cucumber peptides can affect the activity of enzymes related to fat metabolism, thereby playing a role in lowering blood lipid levels. At the same time, the various amino acids and other nutrients contained in sea cucumber peptides are important raw materials for the synthesis of proteins in the human body. Protein is the basic element that constitutes cells. Therefore, the intake of sea cucumber peptides is helpful in promoting growth and development. In addition, certain components in sea cucumber peptides have antioxidant effects, can reduce the damage of oxidative stress to liver cells, and can promote liver cell regeneration, thereby achieving the effect of protecting the liver. Based on the above content, the present invention uses the extracted sea cucumber active peptides to prepare products with the effect of protecting liver function and preventing liver cirrhosis, thereby promoting human health.

[0005] At present, ordinary sea cucumber products on the market are difficult to digest and absorb, have low biological efficacy, and low sea cucumber utilization rate. Therefore, the present invention aims to provide a method for preparing sea cucumber peptides that are rapidly absorbed, low in consumption, not easy to saturate, and have the combined efficacy of protecting liver function and preventing cirrhosis. Summary of the invention

[0006] In view of the above problems, the present invention provides a method for preparing sea cucumber peptides with the combined effects of protecting liver function and preventing liver cirrhosis.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is: A method for preparing sea cucumber peptide with the combined effects of protecting liver function and preventing liver cirrhosis, specifically comprising the following steps: A. Take fresh sea cucumbers, clean them, and freeze them into a hard solid state at a specific temperature; B. Use a meat grinder to mince, grind into a homogenate through a colloidal membrane, and prepare a solution with a substrate concentration of 2%; C. Heat the homogenate and add it to the fermenter after the homogenate is heated. Then, add the enzyme to the hydrolyzate at a rate of 8000 U / g protein. D. After 2 hours of reaction, use the fermentation tank to sterilize the enzyme at high temperature; E. Cooling the sterilized and enzyme-inactivated sea cucumber peptide hydrolyzate to room temperature, centrifuging and collecting the supernatant; F. Filter the supernatant and place it in a rotary evaporator for vacuum concentration until it is 1 / 4 of the original volume; G. Add 95% ethanol solution until the system reaches 60% ethanol content, let stand for 12 hours, and collect the supernatant by centrifugation to obtain sea cucumber protein hydrolyzate; H. Decolorization and deodorization treatment; 1. The sea cucumber protein hydrolyzate through decolorization and deodorization is filtered twice with an ultrafiltration membrane to obtain a sea cucumber small molecule peptide solution; J. Freeze-dry the above solution, detect the sea cucumber small molecule peptide and determine its molecular weight.

[0008] Preferably, the specific temperature in A is -70°C.

[0009] Preferably, the heating condition in C is 80° C., the heating time is 15 min, the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis time is 2 h, and the pH value is not adjusted during the enzymatic hydrolysis process.

[0010] Preferably, the centrifugal speed in E is 6000 r / min, and the centrifugal time is 15 min.

[0011] As a preferred method, H decolorization and deodorization treatment is as follows: a. Add activated carbon with a liquid content of 12% to the filtrate; b. After stirring evenly, let it stand for 4 hours to decolorize and remove the fishy smell; c. Centrifuge to remove activated carbon.

[0012] Preferably, the ultrafiltration membrane is used for filtering: a. Filter for the first time using an ultrafiltration membrane with a molecular weight cutoff of 3000 mesh; b. The filtrate obtained in step a was filtered for a second time using an ultrafiltration membrane with a molecular weight cutoff of 1000 mesh.

[0013] Preferably, freeze-drying in J: a. The freeze-dried powder was dissolved in PBS at pH 7.2.0.1 mol·L-1 and separated using a Sephadex LH 20 gel column; The received eluate was used to detect small molecule peptides and determine their molecular weight using RP-HPLC combined with EMS, and the measured molecular weight was 500-1500.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are: The present invention uses enzymatic hydrolysis technology and fermentation engineering to develop a sea cucumber peptide product that has the functions of protecting liver function and preventing liver cirrhosis, while supplementing nutrition, minerals, proteins, vitamins, etc., to solve the current problem of liver damage in most people, and actively contribute to the development of liver protection and human health. This bioactive peptide product can be added to food, health food, medicine, etc. through different processes; The invention uses fresh sea cucumber as raw material, performs enzymolysis with a specific enzyme amount, kills the enzyme and sterilizes it at high temperature, and extracts sea cucumber active oligopeptides with high biological efficacy through processes such as vacuum concentration by a rotary evaporator, decolorization and deodorization, ultrafiltration membrane filtration, and gel chromatography column separation. The main efficacy of the invention is to protect liver function and prevent liver cirrhosis, and the invention can also assist in the treatment of iron deficiency anemia, atherosclerosis, chronic nephritis, diabetes, hyperlipidemia, and the like. DETAILED DESCRIPTION

[0015] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0016] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments of the following disclosure.

[0017] Example 1, a method for preparing a sea cucumber peptide having a combined effect of protecting liver function and preventing liver cirrhosis, specifically comprising the following steps: A. Take fresh sea cucumbers, clean them, and freeze them into a hard solid state at a specific temperature; B. Use a meat grinder to mince, grind into a homogenate through a colloidal membrane, and prepare a solution with a substrate concentration of 2%; C. Heat the homogenate and add it to the fermenter after the homogenate is heated. Then, add the enzyme to the hydrolyzate at a rate of 8000 U / g protein. D. After 2 hours of reaction, use the fermentation tank to sterilize the enzyme at high temperature; E. Cooling the sterilized and enzyme-inactivated sea cucumber peptide hydrolyzate to room temperature, centrifuging and collecting the supernatant; F. Filter the supernatant and place it in a rotary evaporator for vacuum concentration until it is 1 / 4 of the original volume; G. Add 95% ethanol solution until the system reaches 60% ethanol content, let stand for 12 hours, and collect the supernatant by centrifugation to obtain sea cucumber protein hydrolyzate; H. Decolorization and deodorization treatment; 1. The sea cucumber protein hydrolyzate through decolorization and deodorization is filtered twice with an ultrafiltration membrane to obtain a sea cucumber small molecule peptide solution; J. Freeze-dry the above solution, detect the sea cucumber small molecule peptide and determine its molecular weight.

[0018] Preferably, the specific temperature in A is -70°C.

[0019] Preferably, the heating condition in C is 80° C., the heating time is 15 min, the enzymatic hydrolysis temperature is 50° C., the enzymatic hydrolysis time is 2 h, and the pH value is not adjusted during the enzymatic hydrolysis process.

[0020] Preferably, the centrifugal speed in E is 6000 r / min, and the centrifugal time is 15 min.

[0021] As a preferred method, H decolorization and deodorization treatment is as follows: a. Add activated carbon with a liquid content of 12% to the filtrate; b. After stirring evenly, let it stand for 4 hours to decolorize and remove the fishy smell; c. Centrifuge to remove activated carbon.

[0022] Preferably, the ultrafiltration membrane is used for filtering: a. Filter for the first time using an ultrafiltration membrane with a molecular weight cutoff of 3000 mesh; b. The filtrate obtained in step a was filtered for a second time using an ultrafiltration membrane with a molecular weight cutoff of 1000 mesh.

[0023] Preferably, freeze-drying in J: a. The freeze-dried powder was dissolved in PBS at pH 7.2.0.1 mol·L-1 and separated using a Sephadex LH 20 gel column; The received eluate was used to detect small molecule peptides and determine their molecular weight using RP-HPLC combined with EMS, and the measured molecular weight was 500-1500.

[0024] The above description is only a preferred embodiment of the present invention and does not limit the present invention in other forms. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for preparing a sea cucumber peptide having a combined effect of protecting liver function and preventing liver cirrhosis, characterized in that: The specific steps include: A. Take fresh sea cucumbers, clean them, and freeze them into a hard solid state at a specific temperature; B. Use a meat grinder to mince, grind into a homogenate through a colloidal membrane, and prepare a solution with a substrate concentration of 2%; C. Heat the homogenate and add it to the fermenter after completion, then add the enzyme to the hydrolyzate at a rate of 8000U / g protein; D. After 2 hours of reaction, use the fermentation tank to sterilize the enzyme at high temperature; E. Cooling the sterilized and enzyme-inactivated sea cucumber peptide hydrolyzate to room temperature, centrifuging and collecting the supernatant; F. Filter the supernatant and place it in a rotary evaporator for vacuum concentration until it is 1 / 4 of the original volume; G. Add 95% ethanol solution until the system reaches 60% ethanol content, let stand for 12 hours, and collect the supernatant by centrifugation to obtain sea cucumber protein hydrolyzate; H. Decolorization and deodorization treatment; 1. The sea cucumber protein hydrolyzate through decolorization and deodorization is filtered twice with an ultrafiltration membrane to obtain a sea cucumber small molecule peptide solution; J. Freeze-dry the above solution, detect the sea cucumber small molecule peptide and determine its molecular weight.

2. The method for preparing a sea cucumber peptide having a combined effect of protecting liver function and preventing liver cirrhosis according to claim 1, characterized in that: The specific temperature in A is -70°C.

3. The method for preparing a sea cucumber peptide having a combined effect of protecting liver function and preventing liver cirrhosis according to claim 2, characterized in that: The heating condition in C is 80°C, the heating time is 15 min, the enzymatic hydrolysis temperature is 50°C, the enzymatic hydrolysis time is 2 h, and the pH value is not adjusted during the enzymatic hydrolysis process.

4. The method for preparing a sea cucumber peptide having a combined effect of protecting liver function and preventing liver cirrhosis according to claim 3, characterized in that: The centrifugal speed in E is 6000r / min and the centrifugal time is 15min.

5. The method for preparing a sea cucumber peptide having a combined effect of protecting liver function and preventing liver cirrhosis according to claim 4, characterized in that: H decolorization and deodorization treatment, as follows: a. Add activated carbon with a liquid content of 12% to the filtrate; b. After stirring evenly, let it stand for 4 hours to decolorize and remove the fishy smell; c. Centrifuge to remove activated carbon.

6. The method for preparing a sea cucumber peptide having a combined effect of protecting liver function and preventing liver cirrhosis according to claim 5, characterized in that: I Ultrafiltration Membrane Filtration: a. Filter for the first time using an ultrafiltration membrane with a molecular weight cut-off of 3000 mesh; b. The filtrate obtained in step a was filtered for a second time using an ultrafiltration membrane with a molecular weight cutoff of 1000 mesh.

7. The method for preparing a sea cucumber peptide having a combined effect of protecting liver function and preventing liver cirrhosis according to claim 6, characterized in that: Freeze drying in J: a. The freeze-dried powder was dissolved in PBS (pH 7.2, 0.1 mol·L-1) and separated using a Sephadex LH 20 gel column; b. The received eluate is used to detect small molecule peptides by RP-HPLC combined with EMS and determine their molecular weight, The molecular weight was measured to be 500-1500.