A method for preparing aquatic product polysaccharide peptide

Abstract

The present invention relates to the field of aquatic product processing, and specifically to a method for preparing a polysaccharide peptide from aquatic products. The gonads and mucus and body surface mucus of aquatic products are used as raw materials, and the polysaccharide peptides are degraded by using photodynamic technology in combination with low-temperature static irradiation of a cobalt source to obtain small-molecule polysaccharide peptides. A method for preparing polysaccharide peptides from aquatic products, characterized in that the gonads (gonads refer to ovaries or testes), gonadal mucus, and body surface mucus of aquatic products are pretreated and then subjected to enzymatic hydrolysis, enzyme removal and desalting, free protein removal, photodynamic treatment, low-temperature static irradiation, separation and purification, and freeze drying to obtain polysaccharide peptides from aquatic products. The method has simple process, low production cost, and short production cycle. Not only is the polysaccharide peptide extraction rate high, but the polysaccharide peptide also has a small molecular weight, a high DPPH free radical scavenging rate, and good antibacterial properties. Through the preparation of high-value-added products such as polysaccharide peptides, the utilization rate of by-products in the processing of aquatic products is improved.

Description

Technical Field

[0001] The invention relates to the field of aquatic product processing, and in particular to a method for preparing aquatic product polysaccharide peptides. Background Art

[0002] In recent years, the scale of aquatic product breeding in my country has continued to expand, the output has increased year by year, and the aquatic product processing industry has developed rapidly. During the processing process, a large number of low-value by-products are produced, such as fish gonads (fish gonads). Fish testes account for about 5% of the fish body weight, and fish ovaries (i.e., the ovaries of fish after removing eggs) account for 2%-3% of the fish body weight. At present, the processing and utilization rate is extremely low, and most of them are directly discarded, which not only causes a waste of resources, but also brings environmental pollution. In fact, the gonads (ovaries and testes without eggs) of aquatic products are rich in protein and arginine; the gonad mucus and body mucus of fish are rich in polysaccharides (including mucopolysaccharides, hyaluronic acid, glycoproteins, etc.), immunoglobulins and various hydrolytic enzymes. The above substances have medical values ​​such as antibacterial, anti-inflammatory, antioxidant, and skin repair. Extracting antibacterial proteins and polysaccharides from them can not only make full use of resources and increase the added value of fish processing, but also reduce the environmental burden, which is of great significance to promoting the healthy development of the fish processing industry.

[0003] At present, directly extracted polysaccharides have the characteristics of high molecular weight, high viscosity and low solubility, which makes it difficult for them to exert biological effects across the membrane. Therefore, it is particularly important to degrade polysaccharides into small molecular glycopeptides while maintaining their biological activity. Currently, acid degradation, H2O2 degradation, NaNO2 degradation, enzyme degradation and ultrasonic degradation are mostly used, but there are also problems such as difficult to control process conditions, poor product uniformity, loss of certain active groups of polysaccharides, and insufficient reaction. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing aquatic product polysaccharide peptides, which has simple process, low production cost and short production cycle, and not only has a high polysaccharide peptide extraction rate, but also has a small molecular weight of the polysaccharide peptide, a high DPPH free radical scavenging rate and good antibacterial properties.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present invention is: a method for preparing polysaccharide peptides from aquatic products, characterized in that the gonads (gonads refer to ovaries or testes), gonadal mucus, and surface mucus of aquatic products are pretreated and then subjected to enzymatic hydrolysis, enzyme and salt removal, free protein removal, photodynamic treatment, low-temperature static irradiation, separation and purification, and freeze-drying to obtain polysaccharide peptides from aquatic products.

[0006] The above-mentioned method for preparing aquatic product polysaccharide peptide is characterized in that the specific operating steps are as follows:

[0007] (1) Pretreatment: collect one or more of the following (any ratio of two or more) of the ovaries, testes, ovarian mucus, testicular mucus, and body surface mucus of aquatic products, and homogenize for 2 to 8 minutes to obtain a homogenate; stir the homogenate (i.e., material) and acetone (i.e., liquid) at a volume ratio of 1:10 (V / V) at 20 to 30° C. for 2 to 3 hours, and after suction filtration, completely volatilize the acetone (organic reagent) in a fume hood to obtain a defatted solid of the gonad of aquatic products;

[0008] (2) Enzymatic hydrolysis: NaOH solution (i.e., liquid) is added to the defatted solid matter of the gonad of aquatic products (i.e., the material) at a material-liquid ratio of 4.0% to 6.0% (m / V) to adjust the pH value to 10.0 to obtain solution A; alkaline protease is added in an amount of 2.0% to 4.0% (m / V) of solution A to perform a primary enzymatic hydrolysis; after the primary enzymatic hydrolysis is completed, HCl solution is used to adjust the pH value of the solution to 2.0 to obtain solution B; pepsin is added in an amount of 3500 to 4500 U / g of the solid matter in solution B to perform a secondary enzymatic hydrolysis to obtain an enzymatic hydrolyzate;

[0009] (3) Enzyme and salt removal: The enzymatic solution is treated with a polyvinylidene fluoride membrane with a pore size of 0.05 to 0.45 μm, the enzyme and salt are filtered out, and then the crude polysaccharide peptide is obtained by freeze drying;

[0010] (4) Removal of free protein: The crude polysaccharide peptide is added to water to prepare a crude polysaccharide peptide solution of 8% to 15% by mass, and the crude polysaccharide peptide solution is mixed with Sevag reagent in a volume ratio of 1:1, and shaken until there is no free protein between the chloroform layer and the water layer, thereby obtaining the polysaccharide peptide after free protein removal;

[0011] (5) Photodynamic pretreatment of polysaccharide peptides: The polysaccharide peptides obtained by step (4) after free protein removal were photodynamically treated with a visible light source having a wavelength of 405 to 420 nm and an illumination of 50 to 55 mW / cm 2 , irradiation distance 5-6 cm, light intensity 1850 Lux, irradiation 100-150 s, light energy density 5.4-8.1 J / cm 2 , the photosensitivity spectrum function at 405-420nm is 0.030; the polysaccharide peptide after photodynamic pretreatment is obtained;

[0012] (6) Low temperature static irradiation degradation: Use 60 The polysaccharide peptide after photodynamic pretreatment obtained in step (5) of Co-γ ray static irradiation treatment is maintained at an irradiation temperature of 4°C, an irradiation dose of 0.22 to 2.69 kGy, an irradiation time of 2 to 6 min, the polysaccharide peptide (sample) after photodynamic pretreatment is 50 to 100 cm above the ground, and the irradiation non-uniformity is less than 1.5; the polysaccharide peptide after low-temperature static irradiation degradation is obtained;

[0013] (7) Separation and purification: The polysaccharide peptide obtained in step (6) after low-temperature static irradiation degradation was subjected to anion ion exchange column chromatography (DEAE-52, 2.4×30 cm), and gradient eluted with H2O, 0.15 mol / L NaCl solution, and 0.5 mol / L NaCl solution (“gradient elution” means eluting with these three solutions of different concentrations respectively), at a flow rate of 0.8 mL / min; a separated and purified solution was obtained;

[0014] (8) Freeze-drying: The separated and purified solution is desalted and freeze-dried to obtain aquatic product polysaccharide peptides.

[0015] The aquatic product is one of fish (such as sturgeon, etc.) and shellfish (such as abalone, etc.).

[0016] Preferably, the moisture content of the defatted solids in the gonads of the aquatic product in step (1) is 7% to 10%.

[0017] The distance between the polysaccharide peptide (sample) after photodynamic pretreatment and the cobalt source in step (6) is determined by the following formula:

[0018] Calculation of the Γ constant of a radioactive isotope:

[0019]

[0020] In formula 1:

[0021] E represents the absorbed dose of a substance in a certain volume element;

[0022] n represents the mass of the substance in the volume element;

[0023] R represents the radiation area;

[0024] m represents the number of monoenergetic gamma rays of the radioisotope;

[0025] The gamma dose rate at a distance R from a point source is determined by the following formula:

[0026]

[0027] In formula 2:

[0028] P γ : γ dose rate at the distance R from the source, in Gy / h;

[0029] Q: point source activity, unit: Bq;

[0030] Γ: Γ constant of the corresponding radionuclide of the source; unit: Gy.m 2 / (h.Bq);

[0031] d: distance from the point source, in meters;

[0032] 60 The Γ constant of Co is 3.42E-13Gy.m 2 / (h.Bq); the activity of cobalt source (Q) is 1.26×10 16 Bq, the distance (R) between the polysaccharide peptide (sample) after photodynamic pretreatment and the cobalt source is 0.4-1.2 m, and the unit absorbed dose (P γ ) is 2992.50~26932.30Gy / h.

[0033] Preferably, the activity of the cobalt source in the irradiation of step (6) is 1.26×10 16 Bq, the distance between the polysaccharide peptide (sample) after photodynamic pretreatment and the cobalt source is 0.4-1.2 m, and the unit absorbed dose is 2992.50-26932.30 Gy / h.

[0034] Preferably, the extraction rate of the polysaccharide peptides from the aquatic products obtained in step (8) can reach 13.66% to 16.44% (high extraction rate of polysaccharide peptides), the molecular weight of the glycopeptides is reduced to 1.4 to 3.5 kDa (small molecular weight of the polysaccharide peptides), the solubility reaches more than 95%, the essential amino acid content is greater than 15 g / 100 g, the inhibition rate against DPPH is more than 90%, the scavenging rate against hydroxyl radicals is more than 95% (high scavenging rate of DPPH radicals), and the inhibition rate against Escherichia coli at a concentration of 0.1 mg / ml reaches 100% (having good antibacterial properties).

[0035] The beneficial effects of the present invention are as follows: 1. In view of the problems existing in the background technology, the present invention innovatively adopts photodynamic method to pretreat the polysaccharide peptide, and then combines 60 Co-γ rays irradiate and degrade polysaccharide peptides from aquatic products. Photodynamic technology is used in the medical field for photoactivated treatment of tumors, proliferative skin diseases, vascular diseases, etc. In the food field, it is a photosensitizer-mediated non-thermal sterilization technology. So far, its application in the field of macromolecular degradation has not been found. The present invention found that at a specific wavelength of 405-420nm and an illumination of 50-55mW / cm 2 Under the conditions of light, the polysaccharide peptide molecules of aquatic products are highly sensitive to light and can be stimulated and activated. 60 When Co-γ ray static irradiation is used for degradation, the irradiation time can be greatly shortened.

[0036] 2. The present invention utilizes 60 The gamma rays released by Co act on substances, causing degradation of substances by inducing physical and chemical effects such as Compton effect, electron pair effect and free radicals. In the irradiation degradation of aqueous solutions, 60In addition to directly acting on macromolecules to cause degradation, Co-γ rays can also excite water molecules to form highly oxidizing free radicals such as ·OH and ·H, interact with the surrounding organic macromolecules RH, and capture hydrogen on the macromolecules to promote the degradation of macromolecules. Therefore, irradiation technology can effectively degrade natural polysaccharides, improve the biological activity of polysaccharides, and has the advantages of simple operation and high degradation efficiency. However, improper irradiation method, temperature and dose will change the structure of glycopeptides and lose active groups, so it is necessary to select appropriate irradiation methods, temperatures and doses. Compared with dynamic irradiation, the advantages of static irradiation in the present invention are to improve the utilization rate of cobalt sources, greatly shorten the degradation time, reduce irradiation oxidation, and achieve control of the composition and structure of polysaccharide peptides. During static irradiation, the absorbed dose rate remains fixed, which can achieve stable degradation and structural control of polysaccharide peptides. In addition, compared with room temperature 25°C and 18°C, we found that low temperature can effectively inhibit the irradiation oxidation of polysaccharide peptides, so the irradiation temperature of the present invention is selected as 4°C. In terms of dose, through theoretical calculation and experimental verification, we have limited the irradiation distance and dose rate.

[0037] 3. After the gonads and mucus of aquatic products are treated by the method of the present invention, the wet material extraction rate of polysaccharide peptides can reach more than 13.66% (high polysaccharide peptide extraction rate), the molecular weight of polysaccharide peptides is reduced to 1.4-3.5kDa (small molecular weight of polysaccharide peptides), the solubility reaches more than 95%, the essential amino acid content is greater than 15g / 100g, the inhibition rate of DPPH is more than 90%, the scavenging rate of hydroxyl radicals is more than 95% (high DPPH free radical scavenging rate), and the inhibition rate of Escherichia coli at a concentration of 0.1mg / ml reaches 100% (good antibacterial property). The polysaccharide peptides of aquatic products described in the present invention have simple process, low production cost, short production cycle and strong biological activity; not only the polysaccharide peptide extraction rate is high, but also the polysaccharide peptide has a small molecular weight, high scavenging rate of DPPH free radicals and hydroxyl free radicals, and good antibacterial property. DETAILED DESCRIPTION

[0038] The technical content and effects of the present invention are further illustrated by examples below.

[0039] Static irradiation treatment example:

[0040] Preparation of static radiation device: A water well is set under the cobalt source rack, and the cobalt source is placed on the cobalt source rack, which is generally hidden underwater when not in use; the cobalt source rack is connected to the cobalt source lifting system, and the cobalt source rack is located in the irradiation room; the cobalt source is a cobalt 60-γ ray source, and the sample racks are distributed at radioactive intervals around the cobalt source.

[0041] Sample placement: The staff carries a gamma radiator into the irradiation room to place the aquatic product polysaccharide peptide samples to be statically irradiated; the purpose of carrying the gamma radiator is to measure the gamma radiation air absorption dose rate for radiation safety protection (if the dose rate exceeds the alarm value, the gamma radiator will automatically alarm and the operator cannot enter the irradiation room); the dosimeter is placed in the middle of the sample, and the sample is placed in the center of the corresponding sample rack (according to the set irradiation dose, it is placed in the sample rack at the corresponding distance, and the corresponding dose of the corresponding sample rack is the actual measured value of the silver dichromate dosimeter), and the staff leaves the irradiation room;

[0042] Source lifting operation: Start the cobalt source lifting button, and the cobalt source will rise from the water well to the working position;

[0043] Radiation treatment: The sample rack with the sample is placed around the cobalt source (or radioactive source) for static irradiation. The irradiation dose range is 0.22 to 2.69 kGy. The distance between the sample and the cobalt source is 0.4 to 1.2 m. The unit absorbed dose is 2992.50 to 26932.30 Gy / h.

[0044] Source lowering operation: lower the cobalt source into the water well, operate the source lowering button on the console, and the cobalt source (or radioactive source) will automatically lower into the 7-meter-deep water well;

[0045] Sampling: After the irradiation is completed, the source is reduced and the sample is taken out after the irradiation dose rate is within the safe range. The dosimeter is removed for dose detection to obtain the sample after static radiation degradation.

[0046] The distance between the sample and the cobalt source is determined by the following formula:

[0047] The Γ constant of a radioactive isotope represents the dose rate (R / h) caused by an unshielded γ ray emitted from a 1 mCi point source at a distance of 1 cm from the source. The Γ constant is divided into a differential Γ constant and a total Γ constant. The Γ constant calculated for a monoenergetic γ ray of a given radioactive isotope is called a differential Γ constant, and the total Γ constant is called a total Γ constant. i The total Γ constant of radioactive isotopes is referred to as the Γ constant and is equal to Γ i sum. Right now:

[0048]

[0049]

[0050] The above formula can be simplified as:

[0051] In formula 1:

[0052] E represents the absorbed dose of a substance in a certain volume element;

[0053] n represents the mass of the substance in the volume element;

[0054] R represents the radiation area;

[0055] m represents the number of monoenergetic gamma rays of the radioisotope;

[0056] After calculation, 60 The Γ constant of Co is 3.42E-13Gy.m 2 / (h.Bq).

[0057] Calculation of isotropic point source gamma dose rate:

[0058] Assuming that the activity of a point source is Q, the formula for calculating the gamma dose rate at a distance R from the point source is as follows:

[0059]

[0060] In formula 2:

[0061] P γ : γ dose rate at the distance R from the source, in Gy / h;

[0062] Q: point source activity, unit: Bq;

[0063] Γ: Γ constant of the corresponding radionuclide of the source; unit: Gy.m 2 / (h.Bq);

[0064] d: distance from the point source, in meters.

[0065] The activity of the cobalt source in the present invention is 1.26×10 16 Bq, irradiation dose rates at different positions are shown in the following table:

[0066] Table 1 Radiation dose rates at different positions

[0067]

[0068] Table 2 Radiation dose values ​​at different irradiation times (kGy)

[0069]

[0070] Photodynamic Processing Example:

[0071] The polysaccharide peptide liquid after free protein removal was placed under a visible light source with a wavelength of 405-420 nm and a distance of 5-6 cm from the light source, and the polysaccharide peptide liquid was subjected to photodynamic treatment for 100-150 seconds. The light intensity was measured by an illuminometer to be 1850 lux.

[0072] Calculate the illuminance P according to the photometric conversion formula:

[0073]

[0074] In formula 3:

[0075] P: Illuminance, unit: W / m 2 ;

[0076] L: light intensity, unit: lux;

[0077] K m : The maximum value of the spectral luminous efficacy is 683lm / W;

[0078] V(λ): photosensitivity spectrum function at 405-420nm, 0.030.

[0079] The light energy in the polysaccharide peptide solution was calculated using the following formula:

[0080] E=P×t Formula 4

[0081] In formula 4:

[0082] E: Light energy density, unit J / cm 2 ; (Note: E here refers to the "light energy density" in formula 4, not the "absorbed dose of a substance in a certain volume element" in formula 1; because both formulas are usually represented by "E");

[0083] P: Illuminance, unit: W / m 2 ;

[0084] t: Photodynamic treatment time, unit: s.

[0085] Table 3 Irradiation energy at different photodynamic treatment times

[0086]

[0087] Comparative test:

[0088] Taking the same polysaccharide peptide extraction rate and molecular weight as the standard, the degradation effects of dynamic irradiation, static irradiation, and the combination of photodynamic and static irradiation on the polysaccharide peptide liquid were compared and evaluated.

[0089] Table 4 Comparison of degradation effects of different treatment methods

[0090]

[0091] The results in Table 4 show that, compared with dynamic irradiation and static irradiation, the production cycle required for degrading glycopeptide molecules to the same value by combining photodynamic treatment with static irradiation is significantly shortened.

[0092] Embodiment 1:

[0093] A method for preparing aquatic product polysaccharide peptides, the specific operating steps are as follows:

[0094] (1) Pretreatment: collect one or more of the following materials (when there are multiple materials, the raw materials can be in any proportion; that is, there is no special requirement for the amount of each raw material, a certain raw material can be absent, and what can be collected is collected, the same as in the following embodiments), homogenize for 5 minutes to obtain a homogenate; the homogenate (i.e., material) and acetone (i.e., liquid) are stirred at 25° C. for 2 hours at a volume ratio of 1:10 (V / V) at 25° C., and after suction filtration, the acetone (organic reagent) is completely volatilized in a fume hood to obtain a defatted solid of the gonad of aquatic products;

[0095] (2) Enzymolysis: According to the material-liquid ratio of 4.0% (m / V), NaOH solution (i.e., liquid) is added to the defatted solid matter of the gonad of aquatic products (i.e., material) to adjust the pH value to 10.0 to obtain solution A; alkaline protease is added in an amount of 2.0% (m / V) of solution A to perform a primary enzymolysis; after the primary enzymolysis is completed, the temperature is raised to inactivate the enzyme, and the solution is cooled to room temperature; HCl solution is used to adjust the pH value of the solution to 2.0 to obtain solution B; pepsin is added in an amount of 3500 U / g of the solid matter in solution B to perform a secondary enzymolysis, and after the enzymolysis is completed, the temperature is raised to inactivate the enzyme, and the solution is cooled to room temperature to obtain an enzymolysis solution;

[0096] (3) Enzyme and salt removal: The enzymatic hydrolyzate was treated with a polyvinylidene fluoride membrane with a pore size of 0.25 μm, the enzyme and salt were removed by filtration, and then the crude polysaccharide peptide was obtained by freeze drying;

[0097] (4) Removal of free protein: The crude polysaccharide peptide is added to water to prepare an 8% crude polysaccharide peptide solution by mass ratio, and the crude polysaccharide peptide solution is mixed with Sevag reagent by volume ratio of 1:1, and shaken until there is no free protein between the chloroform layer and the water layer, thereby obtaining the polysaccharide peptide after free protein removal;

[0098] (5) Photodynamic pretreatment of polysaccharide peptides: The polysaccharide peptides obtained by step (4) after free protein removal were photodynamically treated with a visible light source having a wavelength of 405 to 420 nm and an illumination of 50 to 55 mW / cm 2 , irradiation distance 5cm, light intensity 1850Lux, irradiation 120s, light energy density (light energy) is 6.5J / cm 2 , the photosensitivity spectrum function at 405-420nm is 0.030; the polysaccharide peptide after photodynamic pretreatment is obtained;

[0099] (6) Low temperature static irradiation degradation: Use 60The polysaccharide peptide after photodynamic pretreatment obtained in the Co-γ ray static irradiation treatment step (5) is 0.8m away from the point cobalt source, the irradiation temperature is maintained at 4°C, the irradiation dose rate is 6733.13Gy / h, the irradiation dose is 0.22kGy, the irradiation time is 2min, the polysaccharide peptide (sample) after photodynamic pretreatment is 50cm above the ground, and the irradiation non-uniformity is less than 1.5; the polysaccharide peptide after low-temperature static irradiation degradation is obtained;

[0100] (7) Separation and purification: The polysaccharide peptide obtained in step (6) after low-temperature static irradiation degradation was subjected to anion ion exchange column chromatography (DEAE-52, 2.4×30 cm), and gradient eluted with H2O, 0.15 mol / L NaCl solution, and 0.5 mol / L NaCl solution (“gradient elution” means eluting with these three solutions of different concentrations respectively), at a flow rate of 0.8 mL / min; a separated and purified solution was obtained;

[0101] (8) Freeze-drying: The separated and purified solution is desalted and freeze-dried to obtain aquatic product polysaccharide peptides.

[0102] The aquatic product glycopeptide extraction rate obtained by the preparation method is 14.22%, the glycopeptide molecular weight is reduced to 3.0kDa, the solubility reaches 97%, the essential amino acid content is 16.01g / 100g, the inhibition rate to DPPH is 94%, the hydroxyl free radical scavenging rate is 96%, and the inhibition rate to Escherichia coli reaches 100% at a concentration of 0.1mg / ml.

[0103] Example 2

[0104] A method for preparing aquatic product polysaccharide peptides, the specific operating steps are as follows:

[0105] (1) Pretreatment: collect one or more of the following (any ratio of two or more) of the ovaries, testes, ovarian mucus, testicular mucus, and body surface mucus of aquatic products (such as sturgeon) except eggs, and homogenize for 3 minutes to obtain a homogenate; the homogenate (i.e., material) and acetone (i.e., liquid) are stirred at 20° C. for 3 hours at a volume ratio of 1:10 (V / V) at a temperature of 20° C., and after filtration, the acetone (organic reagent) is completely volatilized in a fume hood to obtain a defatted solid of the gonad of aquatic products;

[0106] (2) Enzymolysis: According to the material-liquid ratio of 5.0% (m / V), NaOH solution (i.e., liquid) is added to the defatted solid matter of the gonad of aquatic products (i.e., material) to adjust the pH value to 10.0 to obtain solution A; alkaline protease is added in an amount of 3.0% (m / V) of solution A to perform a primary enzymolysis, and the temperature is raised to inactivate the enzyme; after the primary enzymolysis is completed, HCl solution is used to adjust the pH value of the solution to 2.0 to obtain solution B; pepsin is added in an amount of 4000 U / g of the solid matter in solution B to perform a secondary enzymolysis, the temperature is raised to inactivate the enzyme, and the solution is cooled to room temperature for standby use to obtain an enzymolysis solution;

[0107] (3) Enzyme and salt removal: The enzymatic solution was treated with a polyvinylidene fluoride membrane with a pore size of 0.35 μm, the enzyme and salt were filtered out, and then freeze-dried to obtain the crude polysaccharide peptide;

[0108] (4) Removal of free protein: The crude polysaccharide peptide is added to water to prepare a 10% crude polysaccharide peptide solution according to a mass ratio, and the crude polysaccharide peptide solution is mixed with Sevag reagent at a volume ratio of 1:1, and shaken until there is no free protein between the chloroform layer and the water layer, thereby obtaining the polysaccharide peptide after free protein removal;

[0109] (5) Photodynamic pretreatment of polysaccharide peptides: The polysaccharide peptides obtained by step (4) after free protein removal were photodynamically treated with a visible light source having a wavelength of 405 to 420 nm and an illumination of 50 to 55 mW / cm 2 , irradiation distance 5cm, light intensity 1850Lux, irradiation 140s, light energy density 7.6J / cm 2 , the photosensitivity spectrum function at 405-420nm is 0.030; the polysaccharide peptide after photodynamic pretreatment is obtained;

[0110] (6) Low temperature static irradiation degradation: Use 60 The polysaccharide peptide after photodynamic pretreatment obtained in the Co-γ ray static irradiation treatment step (5) is 0.4m away from the point cobalt source, the irradiation dose rate is 26932.30Gy / h, the irradiation temperature is maintained at 4°C, the irradiation dose is 2.69kGy, the irradiation time is 6min, the polysaccharide peptide (sample) after photodynamic pretreatment is 100cm above the ground, and the irradiation non-uniformity is less than 1.5; the polysaccharide peptide after low-temperature static irradiation degradation is obtained;

[0111] (7) Separation and purification: The polysaccharide peptide obtained in step (6) after low-temperature static irradiation degradation was subjected to anion ion exchange column chromatography (DEAE-52, 2.4×30 cm), and gradient eluted with H2O, 0.15 mol / L NaCl solution, and 0.5 mol / L NaCl solution (“gradient elution” means eluting with these three solutions of different concentrations respectively), at a flow rate of 0.8 mL / min; a separated and purified solution was obtained;

[0112] (8) Freeze-drying: The separated and purified solution is desalted and freeze-dried to obtain aquatic product polysaccharide peptides.

[0113] The aquatic product glycopeptide extraction rate obtained by the preparation method can reach 15.98%, the glycopeptide molecular weight is reduced to 1.9kDa, the solubility reaches 97%, the essential amino acid content is 16.85g / 100g, the inhibition rate to DPPH is 94%, the hydroxyl free radical scavenging rate is 96%, and the inhibition rate to Escherichia coli at a concentration of 0.1mg / ml reaches 100%.

[0114] Example 3

[0115] A method for preparing aquatic product polysaccharide peptides, the specific operating steps are as follows:

[0116] (1) Pretreatment: collect one or more of the following (any ratio of two or more) of the ovaries, testes, ovarian mucus, testicular mucus, and body surface mucus of aquatic products (such as sturgeon) except eggs, and homogenize for 8 minutes to obtain a homogenate; the homogenate (i.e., material) and acetone (i.e., liquid) are stirred at 30° C. for 3 hours at a volume ratio of 1:10 (V / V) at room temperature, and after suction filtration, the acetone (organic reagent) is completely volatilized in a fume hood to obtain a defatted solid of the gonad of aquatic products;

[0117] (2) Enzymolysis: According to the material-liquid ratio of 6.0% (m / V), NaOH solution (i.e., liquid) is added to the defatted solid matter of the gonad of aquatic products (i.e., material) to adjust the pH value to 10.0 to obtain solution A; alkaline protease is added in an amount of 4.0% (m / V) of solution A to perform a primary enzymolysis, and the temperature is raised to inactivate the enzyme; after the primary enzymolysis is completed, HCl solution is used to adjust the pH value of the solution to 2.0 to obtain solution B; pepsin is added in an amount of 4500 U / g of the solid matter in solution B to perform a secondary enzymolysis, the temperature is raised to inactivate the enzyme, and the solution is cooled to room temperature for use to obtain an enzymolysis solution;

[0118] (3) Enzyme and salt removal: The enzymatic solution was treated with a polyvinylidene fluoride membrane with a pore size of 0.45 μm, the enzyme and salt were filtered out, and then freeze-dried to obtain the crude polysaccharide peptide;

[0119] (4) Removal of free protein: The crude polysaccharide peptide is added to water to prepare a 15% crude polysaccharide peptide solution by mass ratio, and the crude polysaccharide peptide solution is mixed with Sevag reagent by volume ratio of 1:1, and shaken until there is no free protein between the chloroform layer and the water layer, thereby obtaining the polysaccharide peptide after free protein removal;

[0120] (5) Photodynamic pretreatment of polysaccharide peptides: The polysaccharide peptides obtained by step (4) after free protein removal were photodynamically treated with a visible light source having a wavelength of 405 to 420 nm and an illumination of 50 to 55 mW / cm2 , irradiation distance 6cm, light intensity 1850Lux, irradiation 150s, light energy density 8.1J / cm 2 , the photosensitivity spectrum function at 405-420nm is 0.030; the polysaccharide peptide after photodynamic pretreatment is obtained;

[0121] (6) Low temperature static irradiation degradation: Use 60 The polysaccharide peptide after photodynamic pretreatment obtained in the Co-γ ray static irradiation treatment step (5) is 0.4m away from the point cobalt source, the irradiation dose rate is 26932.30Gy / h, the irradiation temperature is maintained at 4°C, the irradiation dose is 0.99kGy, the irradiation time is 5min, the polysaccharide peptide (sample) after photodynamic pretreatment is 90cm above the ground, and the irradiation non-uniformity is less than 1.5; the polysaccharide peptide after low-temperature static irradiation degradation is obtained;

[0122] (7) Separation and purification: The polysaccharide peptide obtained in step (6) after low-temperature static irradiation degradation was subjected to anion ion exchange column chromatography (DEAE-52, 2.4×30 cm), and gradient eluted with H2O, 0.15 mol / L NaCl solution, and 0.5 mol / L NaCl solution (“gradient elution” means eluting with these three solutions of different concentrations respectively), at a flow rate of 0.8 mL / min; a separated and purified solution was obtained;

[0123] (8) Freeze-drying: The separated and purified solution is desalted and freeze-dried to obtain aquatic product polysaccharide peptides.

[0124] The aquatic product glycopeptide extraction rate obtained by the preparation method can reach 16.44%, the glycopeptide molecular weight is reduced to 1.4kDa, the solubility reaches 98%, the essential amino acid content is 17.68g / 100g, the inhibition rate to DPPH is 95%, the scavenging rate to hydroxyl radicals is 98%, and the inhibition rate to Escherichia coli at a concentration of 0.1mg / ml reaches 100%.

[0125] Example 4

[0126] A method for preparing aquatic product polysaccharide peptides, the specific operating steps are as follows:

[0127] (1) Pretreatment: collect one or more of the following (any ratio of two or more) of the ovaries, testes, ovarian mucus, testicular mucus, and body surface mucus of aquatic products (such as sturgeon) except eggs, and homogenize for 2 minutes to obtain a homogenate; the homogenate (i.e., material) and acetone (i.e., liquid) are stirred at 20° C. for 2 hours at a volume ratio of 1:10 (V / V) at 20° C., and after suction filtration, the acetone (organic reagent) is completely volatilized in a fume hood to obtain a defatted solid of the gonad of aquatic products;

[0128] (2) Enzymolysis: According to the material-liquid ratio of 4.0% (m / V), NaOH solution (i.e., liquid) is added to the defatted solid matter of the gonad of aquatic products (i.e., material) to adjust the pH value to 10.0 to obtain solution A; alkaline protease is added in an amount of 2.0% (m / V) of solution A to perform a primary enzymolysis, and the temperature is raised to inactivate the enzyme; after the primary enzymolysis is completed, HCl solution is used to adjust the pH value of the solution to 2.0 to obtain solution B; pepsin is added in an amount of 3500 U / g of the solid matter in solution B to perform a secondary enzymolysis, after the enzymolysis is completed, the temperature is raised to inactivate the enzyme, and the solution is cooled to room temperature for standby use to obtain an enzymolysis solution;

[0129] (3) Enzyme and salt removal: The enzymatic hydrolyzate was treated with a polyvinylidene fluoride membrane with a pore size of 0.25 μm, the enzyme and salt were removed by filtration, and then the crude polysaccharide peptide was obtained by freeze drying;

[0130] (4) Removal of free protein: The crude polysaccharide peptide is added to water to prepare an 8% crude polysaccharide peptide solution by mass ratio, and the crude polysaccharide peptide solution is mixed with Sevag reagent by volume ratio of 1:1, and shaken until there is no free protein between the chloroform layer and the water layer, thereby obtaining the polysaccharide peptide after free protein removal;

[0131] (5) Photodynamic pretreatment of polysaccharide peptides: The polysaccharide peptides obtained by step (4) after free protein removal were photodynamically treated with a visible light source having a wavelength of 405 to 420 nm and an illumination of 50 to 55 mW / cm 2 , irradiation distance 6cm, light intensity 1850Lux, irradiation 100s, light energy density (light energy) is 5.4J / cm 2 , the photosensitivity spectrum function at 405-420nm is 0.030; the polysaccharide peptide after photodynamic pretreatment is obtained;

[0132] (6) Low temperature static irradiation degradation: Use 60 The polysaccharide peptide after photodynamic pretreatment obtained in the Co-γ ray static irradiation treatment step (5) is 1.2m away from the point cobalt source, the irradiation dose rate is 2992.50Gy / h, the irradiation temperature is maintained at 4°C, the irradiation dose is 1.80kGy, the irradiation time is 4min, the polysaccharide peptide (sample) after photodynamic pretreatment is 75cm above the ground, and the irradiation non-uniformity is less than 1.5; the polysaccharide peptide after low-temperature static irradiation degradation is obtained;

[0133] (7) Separation and purification: The polysaccharide peptide obtained in step (6) after low-temperature static irradiation degradation was subjected to anion ion exchange column chromatography (DEAE-52, 2.4×30 cm), and gradient eluted with H2O, 0.15 mol / L NaCl solution, and 0.5 mol / L NaCl solution (“gradient elution” means eluting with these three solutions of different concentrations respectively), at a flow rate of 0.8 mL / min; a separated and purified solution was obtained;

[0134] (8) Freeze-drying: The separated and purified solution is desalted and freeze-dried to obtain aquatic product polysaccharide peptides.

[0135] The aquatic product glycopeptide extraction rate obtained by the preparation method can reach 13.66%, the glycopeptide molecular weight is reduced to 3.5kDa, the solubility reaches 95%, the essential amino acid content is 15.32g / 100g, the inhibition rate to DPPH is 90%, the hydroxyl free radical scavenging rate is 95%, and the inhibition rate to Escherichia coli at a concentration of 0.1mg / ml reaches 100%.

[0136] Example 5

[0137] A method for preparing aquatic product polysaccharide peptides, the specific operating steps are as follows:

[0138] (1) Pretreatment: collect one or more of the following (any ratio of two or more) of the ovaries, testes, ovarian mucus, testicular mucus, and body surface mucus of aquatic products (such as sturgeon) except eggs, and homogenize for 4 minutes to obtain a homogenate; the homogenate (i.e., material) and acetone (i.e., liquid) are stirred at 25° C. for 3 hours at a volume ratio of 1:10 (V / V) at room temperature, and after suction filtration, the acetone (organic reagent) is completely evaporated in a fume hood to obtain a defatted solid of the gonad of aquatic products;

[0139] (2) Enzymolysis: According to the material-liquid ratio of 6.0% (m / V), NaOH solution (i.e., liquid) is added to the defatted solid matter of the gonad of aquatic products (i.e., material) to adjust the pH value to 10.0 to obtain solution A; alkaline protease is added in an amount of 2.5% (m / V) of solution A to perform a primary enzymolysis, and the temperature is raised to inactivate the enzyme; after the primary enzymolysis is completed, HCl solution is used to adjust the pH value of the solution to 2.0 to obtain solution B; pepsin is added in an amount of 3800 U / g of the solid matter in solution B to perform a secondary enzymolysis, after the enzymolysis is completed, the temperature is raised to inactivate the enzyme, and the solution is cooled to room temperature for standby use to obtain an enzymolysis solution;

[0140] (3) Enzyme and salt removal: The enzymatic solution was treated with a polyvinylidene fluoride membrane with a pore size of 0.05 μm, the enzyme and salt were filtered out, and then freeze-dried to obtain the crude polysaccharide peptide;

[0141] (4) Removal of free protein: The crude polysaccharide peptide is added to water to prepare a 12% crude polysaccharide peptide solution by mass ratio, and the crude polysaccharide peptide solution is mixed with Sevag reagent by volume ratio of 1:1, and shaken until there is no free protein between the chloroform layer and the water layer, thereby obtaining the polysaccharide peptide after free protein removal;

[0142] (5) Photodynamic pretreatment of polysaccharide peptides: The polysaccharide peptides obtained by step (4) after free protein removal were photodynamically treated with a visible light source having a wavelength of 405 to 420 nm and an illumination of 50 to 55 mW / cm 2 , irradiation distance 6cm, light intensity 1850Lux, irradiation 110s, light energy density 6.0J / cm 2 , the photosensitivity spectrum function at 405-420nm is 0.030; the polysaccharide peptide after photodynamic pretreatment is obtained;

[0143] (6) Low temperature static irradiation degradation: Use 60 The polysaccharide peptide after photodynamic pretreatment obtained in the Co-γ ray static irradiation treatment step (5) is 1.0m away from the point cobalt source, the irradiation dose rate is 4309.20Gy / h, the irradiation temperature is maintained at 4°C, the irradiation dose is 0.3kGy, the irradiation time is 6min, the polysaccharide peptide (sample) after photodynamic pretreatment is 60cm above the ground, and the irradiation non-uniformity is less than 1.5; the polysaccharide peptide after low-temperature static irradiation degradation is obtained;

[0144] (7) Separation and purification: The polysaccharide peptide obtained in step (6) after low-temperature static irradiation degradation was subjected to anion ion exchange column chromatography (DEAE-52, 2.4×30 cm), and gradient eluted with H2O, 0.15 mol / L NaCl solution, and 0.5 mol / L NaCl solution (“gradient elution” means eluting with these three solutions of different concentrations respectively), at a flow rate of 0.8 mL / min; a separated and purified solution was obtained;

[0145] (8) Freeze-drying: The separated and purified solution is desalted and freeze-dried to obtain aquatic product polysaccharide peptides.

[0146] The aquatic product glycopeptide extraction rate obtained by the preparation method can reach 13.92%, the glycopeptide molecular weight is reduced to 3.3kDa, the solubility reaches 95%, the essential amino acid content is 15.47g / 100g, the inhibition rate to DPPH is 92%, the scavenging rate to hydroxyl radicals is 95%, and the inhibition rate to Escherichia coli at a concentration of 0.1mg / ml reaches 100%.

[0147] Example 6

[0148] A method for preparing aquatic product polysaccharide peptides, the specific operating steps are as follows:

[0149] (1) Pretreatment: collect one or more of the following (any ratio of two or more) of the ovaries, testes, ovarian mucus, testicular mucus, and body surface mucus of aquatic products (such as sturgeon) except eggs, and homogenize for 6 minutes to obtain a homogenate; the homogenate (i.e., material) and acetone (i.e., liquid) are stirred at 25° C. for 2 hours at a volume ratio of 1:10 (V / V) at room temperature, and after suction filtration, the acetone (organic reagent) is completely evaporated in a fume hood to obtain a defatted solid of the gonad of aquatic products;

[0150] (2) Enzymolysis: According to the material-liquid ratio of 5.0% (m / V), NaOH solution (i.e., liquid) was added to the defatted solid matter of the gonad of aquatic products (i.e., material) to adjust the pH value to 10.0 to obtain solution A; alkaline protease was added in an amount of 3.5% (m / V) of solution A to perform a primary enzymolysis, and after the enzymolysis was completed, the temperature was raised to inactivate the enzyme and the solution was cooled to room temperature; after the primary enzymolysis was completed, HCl solution was used to adjust the pH value of the solution to 2.0 to obtain solution B; pepsin was added in an amount of 4200 U / g of the solid matter in solution B to perform a secondary enzymolysis to obtain an enzymolysis solution;

[0151] (3) Enzyme and salt removal: The enzymatic solution was treated with a polyvinylidene fluoride membrane with a pore size of 0.45 μm, the enzyme and salt were filtered out, and then freeze-dried to obtain the crude polysaccharide peptide;

[0152] (4) Removal of free protein: The crude polysaccharide peptide is added to water to prepare a 14% crude polysaccharide peptide solution by mass ratio, and the crude polysaccharide peptide solution is mixed with Sevag reagent by volume ratio of 1:1, and shaken until there is no free protein between the chloroform layer and the water layer, thereby obtaining the polysaccharide peptide after free protein removal;

[0153] (5) Photodynamic pretreatment of polysaccharide peptides: The polysaccharide peptides obtained by step (4) after free protein removal were photodynamically treated with a visible light source having a wavelength of 405 to 420 nm and an illumination of 50 to 55 mW / cm 2 , irradiation distance 6cm, light intensity 1850Lux, irradiation 130s, light energy density 7.0J / cm 2 , the photosensitivity spectrum function at 405-420nm is 0.030; the polysaccharide peptide after photodynamic pretreatment is obtained;

[0154] (6) Low temperature static irradiation degradation: Use 60 The polysaccharide peptide after photodynamic pretreatment obtained in the Co-γ ray static irradiation treatment step (5) is 0.6m away from the point cobalt source, the irradiation dose rate is 11970.00Gy / h, the irradiation temperature is maintained at 4°C, the irradiation dose is 1.35kGy, the irradiation time is 3min, the polysaccharide peptide (sample) after photodynamic pretreatment is 75cm above the ground, and the irradiation non-uniformity is less than 1.5; the polysaccharide peptide after low-temperature static irradiation degradation is obtained;

[0155] (7) Separation and purification: The polysaccharide peptide obtained in step (6) after low-temperature static irradiation degradation was subjected to anion ion exchange column chromatography (DEAE-52, 2.4×30 cm), and gradient eluted with H2O, 0.15 mol / L NaCl solution, and 0.5 mol / L NaCl solution (“gradient elution” means eluting with these three solutions of different concentrations respectively), at a flow rate of 0.8 mL / min; a separated and purified solution was obtained;

[0156] (8) Freeze-drying: The separated and purified solution is desalted and freeze-dried to obtain aquatic product polysaccharide peptides.

[0157] The aquatic product glycopeptide extraction rate obtained by the preparation method can reach 14.36%, the glycopeptide molecular weight is reduced to 2.5kDa, the solubility reaches 97%, the essential amino acid content is 16.38g / 100g, the inhibition rate to DPPH is 94%, the hydroxyl free radical scavenging rate is 96%, and the inhibition rate to Escherichia coli at a concentration of 0.1mg / ml reaches 100%.

[0158] The upper and lower limits and interval values ​​of the process parameters (such as wavelength, illumination, time, dosage, etc.) listed in the present invention can all realize the present invention, and the embodiments are not listed one by one here.

Claims

1. A method for preparing aquatic product polysaccharide peptides, characterized in that The gonads, gonad mucus and body surface mucus of aquatic products are pretreated and then subjected to enzymatic hydrolysis, enzyme and salt removal, free protein removal, photodynamic treatment, low-temperature static irradiation, separation and purification, and freeze-drying to obtain aquatic product polysaccharide peptides; The specific steps are as follows: (1) Pretreatment: collecting one or more of the following: ovaries, testes, ovarian mucus, testicular mucus, and body mucus of aquatic products, and homogenizing for 2 to 8 minutes to obtain a homogenate; the homogenate and acetone are stirred at 20 to 30° C. for 2 to 3 hours at a volume ratio of 1:10 (V / V) between the feed and the liquid; and after filtration, the acetone is completely volatilized to obtain a defatted solid of the gonad of aquatic products; (2) Enzymatic hydrolysis: according to the solid-liquid ratio of 4.0% to 6.0% (m / V), NaOH solution is added to the defatted solids of the gonads of aquatic products to adjust the pH value to 10.0 to obtain solution A; alkaline protease is added in an amount of 2.0% to 4.0% (m / V) of solution A to perform a primary enzymatic hydrolysis; after the primary enzymatic hydrolysis, HCl solution is used to adjust the pH value of the solution to 2.0 to obtain solution B; pepsin is added in an amount of 3500 to 4500 U / g of the solids in solution B to perform a secondary enzymatic hydrolysis to obtain an enzymatic solution; (3) Enzyme and salt removal: The enzymatic solution is treated with a polyvinylidene fluoride membrane with a pore size of 0.05 to 0.45 μm, the enzyme and salt are filtered out, and then the crude polysaccharide peptide is obtained by freeze drying; (4) Removal of free protein: The crude polysaccharide peptide is added to water to prepare a crude polysaccharide peptide solution of 8% to 15% by mass, and the crude polysaccharide peptide solution is mixed with Sevag reagent in a volume ratio of 1:1, and shaken until there is no free protein between the chloroform layer and the water layer, thereby obtaining the polysaccharide peptide after free protein removal; (5) Photodynamic pretreatment of polysaccharide peptides: The polysaccharide peptides obtained in step (4) after free protein removal were photodynamically treated with a visible light source having a wavelength of 405 to 420 nm and an illumination of 50 to 55 mW / cm 2 , irradiation distance 5-6 cm, light intensity 1850 Lux, irradiation 100-150 s, light energy density 5.4-8.1 J / cm 2 , the photosensitivity spectrum function at 405-420nm is 0.030; the polysaccharide peptide after photodynamic pretreatment is obtained; (6) Low temperature static irradiation degradation: Use 60 The polysaccharide peptide after photodynamic pretreatment obtained in step (5) of Co-γ ray static irradiation treatment is maintained at 4°C, the irradiation dose is 0.22-2.69 kGy, the irradiation time is 2-6 min, the polysaccharide peptide after photodynamic pretreatment is 50-100 cm above the ground, and the irradiation non-uniformity is less than 1.5; the polysaccharide peptide after low-temperature static irradiation degradation is obtained; (7) Isolation and purification: The polysaccharide peptide obtained in step (6) after low-temperature static irradiation degradation was subjected to anion exchange column chromatography, and gradient elution was performed with H2O, 0.15 mol / L NaCl solution, and 0.5 mol / L NaCl solution at a flow rate of 0.8 mL / min; Obtaining a separated and purified solution; (8) Freeze-drying: The separated and purified solution is desalted and freeze-dried to obtain aquatic product polysaccharide peptides.

2. The method for preparing a polysaccharide peptide from aquatic products according to claim 1, characterized in that The aquatic product is one of fish and shellfish.

3. The method for preparing a polysaccharide peptide from aquatic products according to claim 1, characterized in that The water content of the defatted solid matter of the gonads of aquatic products in step (1) is 7% to 10%.

4. The method for preparing a polysaccharide peptide from aquatic products according to claim 1, characterized in that The distance between the polysaccharide peptide and the cobalt source after photodynamic pretreatment in step (6) is determined by the following formula: Calculation of the Γ constant of a radioactive isotope: In formula 1: E represents the absorbed dose of a substance in a certain volume element; n represents the mass of the substance in the volume element; R represents the radiation area; m represents the number of monoenergetic gamma rays of the radioisotope; The gamma dose rate at a distance R from a point source is determined by the following formula: In formula 2: P γ : γ dose rate at the distance R from the source, in Gy / h; Q: point source activity, unit: Bq; Γ: Γ constant of the corresponding radionuclide of the source; unit: Gy.m 2 / (h.Bq); d: distance from the point source, in meters.

5. The method for preparing aquatic product polysaccharide peptide according to claim 1, characterized in that The activity of the cobalt source in the irradiation step (6) is 1.26×10 16 Bq, the distance between the polysaccharide peptide and the cobalt source after photodynamic pretreatment was 0.4-1.2 m, and the unit absorbed dose was 2992.50-26932.30 Gy / h.

6. The method for preparing aquatic product polysaccharide peptide according to claim 1, characterized in that The extraction rate of the aquatic product polysaccharide peptide obtained in step (8) can reach 13.66% to 16.44%, the molecular weight of the polysaccharide peptide is reduced to 1.4 to 3.5 kDa, the solubility reaches more than 95%, the essential amino acid content is greater than 15 g / 100 g, the inhibition rate of DPPH is more than 90%, the scavenging rate of hydroxyl radicals is more than 95%, and the inhibition rate of Escherichia coli at a concentration of 0.1 mg / ml reaches 100%.

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