Preparation method of selenium-rich sea cucumber polypeptide

Abstract

The invention discloses a preparation method of selenium-enriched sea cucumber polypeptide, which comprises the following steps of: feeding sea cucumber with selenium-enriched basal feed containing organic selenium, cleaning selenium-enriched sea cucumber intestines, homogenizing, performing enzymolysis treatment, sequentially adding trypsin and papain to obtain enzymatic hydrolysate, centrifuging, taking supernate, and performing freeze drying to obtain the selenium-enriched sea cucumber polypeptide. The selenium-rich sea cucumber polypeptide capable of improving the immunity of the human body is obtained through operation such as membrane separation. The invention has the following beneficial effects: compared with inorganic selenium, organic selenium converted from selenium-enriched yeast can be better absorbed and does not generate toxic and side effects on sea cucumbers and human bodies; the content of selenium and protein in the sea cucumber is increased, and the activity of superoxide dismutase (SOD), catalase (CAT) and acid phosphatase (ACP) is increased; the active polypeptide is extracted from sea intestines of selenium-enriched sea cucumbers, and the prepared active polypeptide has the effect of improving immunity, can be used for preparing health-care products for resisting oxidation and improving immunity, and has excellent social and economic effects.

Description

Technical Field

[0001] The invention belongs to the technical field of marine aquaculture, and in particular relates to a method for preparing a selenium-enriched sea cucumber polypeptide. Background Art

[0002] Selenium is one of the essential trace elements for the human body and plays an important physiological role in many organisms. It is an antioxidant that increases reproduction, immune response and thyroid hormone metabolism. Selenium can improve the antioxidant capacity, immune function and production performance of animals, and also has vision and detoxification functions. Selenium deficiency in humans can lead to a variety of endemic diseases, including Keshan disease, Kashin-Beck disease and goiter. Selenium deficiency in poultry can reduce immunity and egg production, increase early embryonic mortality, exudative diathesis disease and muscular dystrophy. In most parts of the world, selenium intake is lower than the recommended daily intake for humans.

[0003] Sea cucumbers have extremely high nutritional and medicinal value. They are rich in amino acids, fatty acids, vitamins, and a variety of active substances such as polysaccharides, saponins, collagen, and cerebrosides. They have a variety of physiological effects such as anti-oxidation, anti-tumor, anti-coagulation, and regulating the body's immunity. Sea cucumber processed products are very popular among consumers and have broad market prospects. Adding selenium additives to sea cucumber breeding feed can produce selenium-rich sea cucumbers. The results of many studies conducted in the past few years have shown that in countries where selenium intake is lower than the recommended daily intake (for example, the UK's intake is about 50% of the recommended daily intake), supplementing selenium in animal-derived foods (mainly meat, milk and eggs) through animal feed can effectively increase the selenium content of the human body.

[0004] The currently known methods for producing selenium-enriched sea cucumbers include: adding inorganic selenium (sodium selenite), organic selenium (yeast selenium) and nano-selenium to feed, and there are also reports of adding sodium selenite to drinking water. For animals, the best form of selenium to absorb is organic selenium. However, there are also common problems. The different forms of selenium additives and the added dosage have a great impact on the breeding of selenium-enriched sea cucumbers. Different addition dosages have an impact on the growth and immune indicators of sea cucumbers. Excessive additions are toxic to sea cucumbers. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing selenium-enriched sea cucumber polypeptide.

[0006] The technical solution adopted by the present invention is:

[0007] A method for preparing selenium-enriched sea cucumber polypeptide comprises the following steps:

[0008] Step 1: Feeding the sea cucumbers with a basic feed containing organic selenium to obtain selenium-rich sea cucumbers;

[0009] Step 2: Pick out the sea cucumber intestines from the selenium-rich sea cucumber viscera and add water to homogenize;

[0010] Step 3: adding trypsin and papain to the freeze-thaw treatment solution in sequence for enzymatic hydrolysis, and obtaining an enzymatic hydrolyzate after inactivating the enzymes at high temperature;

[0011] Step 4: Centrifuge the enzymatic hydrolyzate, add ethanol to precipitate the polysaccharide, and take the supernatant to obtain selenium-enriched sea cucumber polypeptide.

[0012] Preferably, the selenium concentration in the basal feed is 0.75-1.0 mg / kg.

[0013] Preferably, selenium-enriched yeast whose organic selenium content accounts for more than 75% of the total selenium content is mixed with a gelatin thickener to prepare a granular mixture, wherein the gelatin thickener accounts for 0.2%; and the granular mixture is added to the basic feed.

[0014] Preferably, in step 2, 0.8-1 times the amount of water is added to the sea cucumber intestines, and homogenized at 0-4°C to obtain a homogenate.

[0015] Preferably, in step 3, trypsin is added to the treatment solution for enzymatic treatment at a temperature of 35-55° C. and a pH of 8.0 for 2-10 hours.

[0016] Preferably, in step 3, papain is added to the treatment solution for enzymatic hydrolysis at a temperature of 55-85° C. and a pH of 5.5 for 2-10 hours, and then the enzyme is inactivated at a high temperature of 85-95° C. for 15-30 minutes to obtain an enzymatic hydrolyzate.

[0017] Preferably, in step 4, ethanol is added to centrifuge the enzymatic hydrolyzate to a final ethanol concentration of 60 v / v%, and the supernatant is taken and ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 8000-20000 Da at a temperature of 20-35° C. and a pressure of 0.04-0.007 MPa.

[0018] The advantages and positive effects of the present invention are as follows: organic selenium converted by selenium-enriched yeast can be better absorbed than inorganic selenium, and will not produce toxic side effects on sea cucumbers and human bodies; the selenium element and protein content in sea cucumbers are increased, and the activities of superoxide dismutase (SOD), catalase (CAT) and acid phosphatase (ACP) are increased; active polypeptides are extracted from selenium-enriched sea cucumber intestines as raw materials, and high-value utilization of sea cucumber processing byproducts can be achieved, and the source is abundant and the cost is low;

[0019] Then, the sugar chains in the glycoprotein are removed through enzymatic treatment to release the protein components; the protein components are then hydrolyzed by proteases to form polypeptides, thereby producing selenium-rich sea cucumber polypeptides that improve immunity; active polypeptides are extracted from the sea intestines of selenium-rich sea cucumbers, with abundant raw materials, low cost, simple processing technology, and easy operation, and the resulting active polypeptides have the effect of improving immunity. DETAILED DESCRIPTION

[0020] The invention discloses a method for preparing a selenium-rich sea cucumber polypeptide, and the prepared sea cucumber polypeptide has the effect of improving immunity. The sea cucumber intestine is cleaned and homogenized once, and then trypsin and papain are added for enzymolysis treatment, the obtained enzymolysis liquid is centrifuged, and the supernatant is taken and subjected to membrane separation to obtain the selenium-rich sea cucumber polypeptide for improving human immunity. Active polypeptides are extracted from the sea intestine of the selenium-rich sea cucumber, with abundant raw materials, low cost, simple processing technology, and convenient operation, and the obtained active polypeptide has the effect of improving immunity.

[0021] The preparation process includes the following steps:

[0022] Step 1: Feeding a basic feed supplemented with organic selenium during the sea cucumber breeding process to obtain selenium-enriched sea cucumbers; the selenium concentration in the basic feed for breeding sea cucumbers is 0.75-1.0 mg / kg; selenium-enriched yeast with an organic selenium content of more than 75% of the total selenium content is mixed with a gelatin thickener to prepare a granular mixture, wherein the gelatin thickener accounts for 0.2%; and the granular mixture is added to the basic feed;

[0023] Step 2: Pick out the sea cucumber intestines from the fresh selenium-rich sea cucumber viscera, wash them, add 0.8-1 times the amount of water to the sea cucumber intestines, and homogenize them at 0-4°C to prepare a sea cucumber intestine homogenate;

[0024] Step 3: adding trypsin and papain to the treatment solution in sequence for enzymolysis treatment, and obtaining an enzymolysis solution after high-temperature inactivation of the enzymes; first adding trypsin and then adding papain, first adding trypsin to the treatment solution for enzymolysis treatment at a temperature of 35-55°C and a pH of 8.0 for 2-10 hours, then adding papain for enzymolysis treatment at a temperature of 55-85°C and a pH of 5.5 for 2-10 hours, and then inactivating the enzymes at a high temperature of 85-95°C for 15-30 minutes to obtain a final enzymolysis solution; double enzymolysis is used in the preparation process, the enzymolysis is more sufficient, the sugar chains in the glycoprotein are removed, the protein components are released, and the protein components are fully degraded into polypeptide chains;

[0025] Step 4: Add ethanol, centrifuge the enzymatic hydrolysate to obtain the supernatant, and perform membrane separation and other operations to obtain selenium-rich sea cucumber polypeptides. Use an ultrafiltration membrane with a molecular weight cutoff of 8000-20000Da to perform ultrafiltration treatment at a temperature of 25-30°C and a pressure of 0.05-0.008MPa, and then concentrate and dry to obtain selenium-rich sea cucumber polypeptides.

[0026] The scheme of the present invention is described below in conjunction with embodiments.

[0027] Embodiment 1:

[0028] This embodiment provides a method for producing a selenium-rich sea cucumber polypeptide for improving immunity, which specifically comprises the following steps:

[0029] (1) Feeding a basic feed supplemented with organic selenium during the breeding process of sea cucumbers to obtain selenium-enriched sea cucumbers; the selenium concentration in the basic feed for breeding sea cucumbers is 0.75-1.0 mg / kg; selenium-enriched yeast with an organic selenium content of more than 75% of the total selenium content is mixed with a gelatin thickener to prepare a granular mixture, wherein the gelatin thickener accounts for 0.2%; and the granular mixture is added to the basic feed;

[0030] (2) picking up sea cucumber intestines from fresh selenium-rich sea cucumber viscera, washing them, adding 0.8-1 times the amount of water to the sea cucumber intestines, and homogenizing them at 0-4° C. to prepare a sea cucumber intestine homogenate;

[0031] Add trypsin and papain to the treatment liquid for enzymatic hydrolysis, and obtain an enzymatic hydrolyzate after high-temperature inactivation of the enzymes; add trypsin to the pretreatment liquid for enzymatic hydrolysis at a temperature of 35-55°C and a pH of 8.0 for 2-10 hours. Add papain for enzymatic hydrolysis at a temperature of 55-85°C and a pH of 5.5 for 2-10 hours, and then inactivate the enzyme at a high temperature of 80°C for 12 minutes to obtain an enzymatic hydrolyzate. The amount of papain used is 0.2% of the mass of the pretreatment liquid, and the amount of trypsin used is 0.02% of the mass of the pretreatment liquid;

[0032] (3) Add ethanol, centrifuge the enzymatic hydrolyzate to obtain the supernatant, and perform membrane separation and other operations to obtain selenium-enriched sea cucumber polypeptides. Use an ultrafiltration membrane with a molecular weight cutoff of 8000-20000Da to perform ultrafiltration treatment at a temperature of 25-30°C and a pressure of 0.05-0.008MPa, and then concentrate and dry to obtain selenium-enriched sea cucumber polypeptides.

[0033] Embodiment 2:

[0034] This embodiment provides a method for producing a selenium-rich sea cucumber polypeptide for improving immunity, which specifically comprises the following steps:

[0035] (1) Feeding a basic feed supplemented with organic selenium during the breeding process of sea cucumbers to obtain selenium-enriched sea cucumbers; the selenium concentration in the basic feed for breeding sea cucumbers is 0.25-0.50 mg / kg; mixing selenium-enriched yeast whose organic selenium content accounts for more than 75% of the total selenium content with a gelatin thickener to prepare a granular mixture, wherein the gelatin thickener accounts for 0.2%; and adding the granular mixture to the basic feed;

[0036] (2) picking up sea cucumber intestines from fresh selenium-rich sea cucumber viscera, washing them, adding 0.8-1 times the amount of water to the sea cucumber intestines, and homogenizing them at 0-4° C. to prepare a sea cucumber intestine homogenate;

[0037] (3) Add trypsin and papain to the treatment liquid for enzymatic hydrolysis, and obtain an enzymatic hydrolyzate after high-temperature inactivation of the enzymes; add trypsin to the pretreatment liquid for enzymatic hydrolysis at a temperature of 35-55°C and a pH of 8.0 for 2-10 hours. Add papain for enzymatic hydrolysis at a temperature of 55-85°C and a pH of 5.5 for 2-10 hours, and then inactivate the enzyme at a high temperature of 80°C for 12 minutes to obtain an enzymatic hydrolyzate. The amount of papain used is 0.2% of the mass of the pretreatment liquid, and the amount of trypsin used is 0.02% of the mass of the pretreatment liquid;

[0038] (4) Add ethanol, centrifuge the enzymatic hydrolyzate to obtain the supernatant, and perform membrane separation and other operations to obtain selenium-enriched sea cucumber polypeptides. Use an ultrafiltration membrane with a molecular weight cutoff of 8000-20000Da to perform ultrafiltration treatment at a temperature of 25-30°C and a pressure of 0.05-0.008MPa, and then concentrate and dry to obtain selenium-enriched sea cucumber polypeptides.

[0039] Embodiment 3:

[0040] This embodiment provides a method for producing a selenium-rich sea cucumber polypeptide for improving immunity, which specifically comprises the following steps:

[0041] (1) Feeding a basic feed supplemented with organic selenium during the breeding process of sea cucumbers to obtain selenium-enriched sea cucumbers; the selenium concentration in the basic feed for breeding sea cucumbers is 0-0.20 mg / kg; selenium-enriched yeast whose organic selenium content accounts for more than 75% of the total selenium content is mixed with a gelatin thickener to prepare a granular mixture, wherein the gelatin thickener accounts for 0.2%; and the granular mixture is added to the basic feed;

[0042] (2) picking up sea cucumber intestines from fresh selenium-rich sea cucumber viscera, washing them, adding 0.8-1 times the amount of water to the sea cucumber intestines, and homogenizing them at 0-4° C. to prepare a sea cucumber intestine homogenate;

[0043] (3) Add trypsin and papain to the treatment liquid for enzymatic hydrolysis, and obtain an enzymatic hydrolyzate after high-temperature inactivation of the enzymes; add trypsin to the pretreatment liquid for enzymatic hydrolysis at a temperature of 35-55°C and a pH of 8.0 for 2-10 hours. Add papain for enzymatic hydrolysis at a temperature of 55-85°C and a pH of 5.5 for 2-10 hours, and then inactivate the enzyme at a high temperature of 80°C for 12 minutes to obtain an enzymatic hydrolyzate. The amount of papain used is 0.2% of the mass of the pretreatment liquid, and the amount of trypsin used is 0.02% of the mass of the pretreatment liquid;

[0044] (4) Add ethanol, centrifuge the enzymatic hydrolyzate to obtain the supernatant, and perform membrane separation and other operations to obtain selenium-enriched sea cucumber polypeptides. Use an ultrafiltration membrane with a molecular weight cutoff of 8000-20000Da to perform ultrafiltration treatment at a temperature of 25-30°C and a pressure of 0.05-0.008MPa, and then concentrate and dry to obtain selenium-enriched sea cucumber polypeptides.

[0045] Comparative Example 1:

[0046] The difference between Comparative Example 1 and Example 1 is that selenium is not added during the feeding process, as follows:

[0047] (1) feeding a basic feed during the sea cucumber breeding process; mixing the basic feed for breeding sea cucumbers with a gelatin thickener to form a granular mixture, wherein the gelatin thickener accounts for 0.2%; and adding the granular mixture to the basic feed;

[0048] (2) picking up sea cucumber intestines from fresh selenium-rich sea cucumber viscera, washing them, adding 0.8-1 times the amount of water to the sea cucumber intestines, and homogenizing them at 0-4° C. to prepare a sea cucumber intestine homogenate;

[0049] (3) adding trypsin and papain to the treatment liquid for enzymatic hydrolysis, and then inactivating the enzymes at high temperature to obtain an enzymatic hydrolyzate; adding papain and trypsin to the pretreatment liquid for enzymatic hydrolysis at 38°C and pH 5 for 4 hours, and then inactivating the enzymes at high temperature at 80°C for 12 minutes to obtain an enzymatic hydrolyzate. The amount of papain used is 0.2% of the mass of the pretreatment liquid, and the amount of trypsin used is 0.02% of the mass of the pretreatment liquid;

[0050] (4) Add ethanol, centrifuge the enzymatic hydrolyzate to obtain the supernatant, and perform membrane separation and other operations to obtain selenium-enriched sea cucumber polypeptides. Use an ultrafiltration membrane with a molecular weight cutoff of 8000-20000Da to perform ultrafiltration treatment at a temperature of 25-30°C and a pressure of 0.05-0.008MPa, and then concentrate and dry to obtain selenium-enriched sea cucumber polypeptides.

[0051] Embodiment 4:

[0052] Selenium-enriched sea cucumber peptides increase the number of white blood cells in immunodeficient mice after chemotherapy.

[0053] Fifty C57 mice (weight 20±10g) were randomly divided into five groups, each with 10 mice, half of which were male and half were female, and they were cultured separately. They were blank control group, model control group, high, medium and low dose sea cucumber polypeptide groups.

[0054] Except for the blank control group, the other four groups were all treated with cyclophosphamide, and each mouse was intraperitoneally injected with 25 mg / kg.bw for 4 times.

[0055] Starting from the next day after modeling, each group was gavaged once a day with a gavage volume of 0.25 ml / 10 g.bw for 10 consecutive days. The blank control group and the model control group were given normal saline; the sea cucumber peptide group was given the corresponding sea cucumber peptide component (based on the body surface area conversion relationship between mice and humans, the medium dose group was 200 mg / kg.bw of sea cucumber peptide, the low dose group was 1 / 5 of the medium dose group, and the high dose group was 5 times the medium dose group).

[0056] 24 hours after the last oral administration, the eyeballs of the mice were removed to collect blood, and EDTA-K2 was checked by routine blood test. 500 μl of blood was collected in a blood collection tube, and blood cells were counted using Sysmex XI-1800i blood cell analyzer. The results are shown in Table 1.

[0057] Table 1 Effects of various components of sea cucumber peptide on the number of white blood cells in mice.

[0058]

[0059] Note: Compared with the blank control group *P<0.05, **P<0.01; compared with the model control group ▲P<0.05, ▲▲P<0.01

[0060] As can be seen from Table 1, the selenium-rich sea cucumber polypeptide has a significant effect on increasing the number of white blood cells in immunodeficient mice after chemotherapy, proving that the prepared sea cucumber polypeptide has the effect of assisting in improving immunity.

[0061] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for preparing a selenium-enriched sea cucumber polypeptide, characterized in that: The steps include: Step 1: Feeding the sea cucumbers with a basic feed containing organic selenium to obtain selenium-rich sea cucumbers; Step 2: Pick out the sea cucumber intestines from the selenium-rich sea cucumber viscera and add water to homogenize; Step 3: adding trypsin and papain to the freeze-thaw treatment solution in sequence for enzymatic hydrolysis, and obtaining an enzymatic hydrolyzate after inactivating the enzymes at high temperature; Step 4: Centrifuge the enzymatic hydrolyzate, add ethanol to precipitate the polysaccharide, and take the supernatant to obtain selenium-enriched sea cucumber polypeptide.

2. The method for preparing the selenium-enriched sea cucumber polypeptide according to claim 1, characterized in that: The selenium concentration in the basic feed is 0.75-1.0 mg / kg.

3. The method for preparing the selenium-enriched sea cucumber polypeptide according to claim 2, characterized in that: Selenium-enriched yeast whose organic selenium content accounts for more than 75% of the total selenium content is mixed with a gelatin thickener to prepare a granular mixture, wherein the gelatin thickener accounts for 0.2%; the granular mixture is added to the basic feed.

4. The method for preparing the selenium-enriched sea cucumber polypeptide according to any one of claims 1 to 3, characterized in that: In step 2, 0.8-1 times the amount of water is added to the sea cucumber intestines, and homogenized at 0-4° C. to obtain a homogenate.

5. The method for preparing the selenium-enriched sea cucumber polypeptide according to any one of claims 1 to 3, characterized in that: In step 3, trypsin is added to the treatment solution for enzymatic treatment at a temperature of 35-55° C. and a pH of 8.0 for 2-10 hours.

6. The method for preparing the selenium-enriched sea cucumber polypeptide according to any one of claims 1 to 3, characterized in that: In step 3, papain is added to the treated solution for enzymatic hydrolysis at a temperature of 55-85° C. and a pH of 5.5 for 2-10 hours, and then the enzyme is inactivated at a high temperature of 85-95° C. for 15-30 minutes to obtain an enzymatic hydrolyzate.

7. The method for preparing the selenium-enriched sea cucumber polypeptide according to any one of claims 1 to 3, characterized in that: In step 4, ethanol is added to centrifuge the enzymatic hydrolyzate to a final ethanol concentration of 60 v / v%. The supernatant is taken and ultrafiltration is performed using an ultrafiltration membrane with a molecular weight cutoff of 8000-20000 Da at a temperature of 20-35° C. and a pressure of 0.04-0.007 MPa.

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