CN113151387B - Cod skin collagen peptide with oxidation resistance and immunity enhancement functions and preparation method thereof - Google Patents
Cod skin collagen peptide with oxidation resistance and immunity enhancement functions and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P37/02—Immunomodulators
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P39/06—Free radical scavengers or antioxidants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention relates to the technical field of animal food processing, in particular to cod skin collagen peptide with antioxidant and immunity enhancing functions and a preparation method thereof. The preparation method of the cod skin collagen peptide provided by the invention comprises the following steps: taking cod skin as a raw material, and performing two-step enzymolysis on the pretreated raw material to prepare an zymolyte; in the two-step enzymolysis, the enzyme used in the first step of enzymolysis is composite protease, the composite protease is trypsin and neutral protease, and the enzyme used in the second step of enzymolysis is flavourzyme. The collagen peptide prepared by the method has high content of small molecular peptide, and has excellent functions of resisting oxidation and enhancing immunity.
Description
Technical Field
The invention relates to the technical field of animal food processing, in particular to cod skin collagen peptide with antioxidant and immunity enhancing functions and a preparation method thereof
Background
The cod skin contains a large amount of collagen and has good nutritional value, but the cod skin is used as leftovers of aquatic enterprises in cod processing, is often discarded or used for making low-value products such as fish meal and the like, has low comprehensive utilization level, and causes waste of a large amount of protein resources and environmental pressure. At present, the cod skin collagen peptide has better processing characteristics such as water absorption, oil absorption, water retention, foaming and the like, and meanwhile, the cod skin collagen peptide has researches on beautifying, skin care, calcium absorption promotion and the like, but the researches on oxidation resistance and immunity improvement of the cod skin collagen peptide are less, particularly a protein peptide segment with a specific effect is not reported at present.
Disclosure of Invention
The invention aims to provide cod skin collagen peptide which has good functions of resisting oxidation and enhancing immunity. The invention also aims to provide a preparation method and application of the cod skin collagen peptide.
The invention aims to develop a cod skin collagen peptide with oxidation resistance and immunity enhancement functions, and develops a process for producing the cod skin collagen peptide with oxidation resistance and immunity enhancement functions by taking cod skin as a raw material according to characteristics of the raw material and composition characteristics of the cod skin. Polypeptide products of proteins in cod skin after enzymolysis are quite complex in composition, and comprise a large number of polypeptides with unknown sequences and unknown functions, and active peptides with specific functions may comprise a plurality of peptides with different amino acid compositions and different molecular weights, so that the peptides are difficult to distinguish through certain common characteristics. These all pose great difficulties in the development of cod skin collagen peptides with specific functions. Many fish skin collagen peptides in the prior art do not have specific functions, and the fish skin collagen peptides with better functions of resisting oxidation and improving immunity are fewer. In addition, in the prior art, acid and alkali are needed to be added to adjust the pH value in the enzymolysis process, which not only increases the complexity and cost of the process, but also may affect the functional characteristics of the product and increase the ash content of the product. In the process of research and development, multiple single enzymolysis and compound enzymolysis are tried, although peptides with small molecular weight can be obtained by a plurality of methods, the obtained peptides have no functions of resisting oxidation and improving immunity, or have poor functions, and some methods need to rely on acid and alkali to repeatedly adjust the pH. Among the methods, the invention determines a process for developing collagen peptide which is suitable for cod skin, does not depend on acid or alkali for pH adjustment, and has the advantages of clear peptide segment characteristics, good flavor, processing characteristics, obvious oxidation resistance and immunity improvement through a large number of experiments. The protein peptide can be widely applied to foods with special diet, nutrition, function and the like.
Specifically, the invention provides the following technical scheme:
the invention provides a preparation method of cod skin collagen peptide, which comprises the following steps: taking cod skin as a raw material, and performing two-step enzymolysis on the pretreated raw material to prepare an zymolyte; in the two-step enzymolysis, the enzyme used in the first step of enzymolysis is composite protease, the composite protease is trypsin and neutral protease, and the enzyme used in the second step of enzymolysis is flavourzyme.
The invention discovers that the content of the peptide with the functions of resisting oxidation and enhancing immunity in the enzymolysis product can be obviously improved by adopting the two-step enzymolysis method, and the functions of resisting oxidation and enhancing immunity of the prepared cod skin collagen peptide are improved.
Preferably, in the compound protease, the mass ratio of trypsin to neutral protease is (3-1): 1, the enzyme activity of the trypsin is 100,000-1,000,000U/g, and the enzyme activity of the neutral protease is 100,000-650,000U/g. Within the range of the mixture ratio, trypsin and neutral protease can better cooperate, and meanwhile, the first step of enzymolysis can be better connected and matched with the second step of enzymolysis, so that the oxidation resistance and the immunity enhancing function of the cod skin collagen peptide are improved.
Specifically, in the first step of enzymolysis, the dosage of the compound protease is 0.2-2g/100g of raw material protein, and the enzymolysis condition is enzymolysis for 2-5h at 45-60 ℃;
in the second step of enzymolysis, the dosage of the flavourzyme is 0.1-1g/100g of raw material protein, the enzyme activity of the flavourzyme is 80,000-250,000U/g, and the enzymolysis condition is 50-60 ℃ for enzymolysis for 1-3 h.
By adopting the two-step enzymolysis method, the cod skin protein can be fully degraded into the small molecular peptide, and meanwhile, the high content of the peptide with the functions of resisting oxidation and enhancing immunity in the small molecular peptide is ensured. Through identification, high-content polypeptides with sequences of DINFDL (Asp-Ile-Asn-Phe-Asp-Leu), GYDAPPN (Gly-Tyr-Asp-Ala-Pro-Pro-Asn) and EKANGAT (Glu-Lys-Ala-Asn-Gly-Ala-Thr) can be obtained through the enzymolysis, and the polypeptides have excellent functions of resisting oxidation and enhancing immunity.
The preparation method of the invention also comprises the step of pretreating the cod skin before enzymolysis, and specifically, the pretreatment comprises the following steps: and (3) steaming the minced cod skin at the temperature of 100-121 ℃ for 1-3h, and performing ultrasonic treatment at the temperature of 70-75 ℃ and 60-90kHz for 20-30min to obtain the raw material protein liquid.
Preferably, the protein content of the raw material protein solution (raw material protein) is adjusted to 8-15% by mass, and then enzymolysis is performed.
The high-temperature treatment is combined with the ultrasonic treatment, so that the contact between the protein raw material and the enzyme is improved, the enzymolysis efficiency can be better improved, and the use amount of the enzyme is reduced.
The preparation method also comprises the steps of carrying out enzyme deactivation treatment on the zymolyte after enzymolysis, and then carrying out filtration and separation of target peptide.
The enzyme deactivation is preferably carried out at 95-100 deg.C.
Preferably, the filtering comprises: firstly, filtering by using a ceramic membrane with the pore diameter of nano grade, filtering the filtrate by using a nanofiltration membrane, and collecting trapped fluid; the filtration can remove macromolecular impurities and remove micromolecular salts;
and sequentially carrying out ultrafiltration on the trapped fluid by ultrafiltration membranes with the aperture of 5000 daltons and 2000 daltons to obtain a protein peptide solution with the molecular weight of less than 2000 daltons.
The protein peptide product subjected to the ceramic membrane nano-scale filtration has better solubility, the nanofiltration membrane removes inorganic salt contained in the raw material, the content of sodium ions in the product is reduced, the obtained product has better taste, the activity of the protein peptide product can be further improved by the ultrafiltration membrane separation and purification, and the functionality is stronger.
Preferably, the isolation of the target peptide comprises:
separating the protein peptide liquid with the molecular weight less than 2000 by SephadexG-15 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 3 rd elution peak is collected; then RP-HPLC reversed-phase high performance liquid chromatography is used for separation, and the separation conditions are as follows: using a C18 chromatographic column, taking an aqueous solution containing 0.1% TFA as a mobile phase A and an acetonitrile solution containing 0.1% TFA as a mobile phase B, and performing separation by gradient elution: 0-5min, 5% mobile phase B; 5-45min, 5-45% of mobile phase B; 45-55min, 45-5% of mobile phase B, the flow rate is 1mL/min, and the separated protein peptide solution is collected for 6-11 min.
As a preferred embodiment of the present invention, the preparation method of the cod skin collagen peptide comprises the following steps:
(1) pretreatment of raw materials: mincing cod skin into fish skin fragments, cooking at 100-121 deg.C for 1-3h, adjusting temperature to 70-75 deg.C, and performing ultrasonic treatment with ultrasonic generator at 60-90kHz for 20-30min to change tissue structure of cod skin protein to obtain cod skin protein solution;
(2) enzymolysis: regulating the protein content in the cod skin protein solution to 8-15%, adding protease according to the weight percentage of 0.3-3.0% of the protein weight in the protein solution for step-by-step enzymolysis, firstly, performing enzymolysis reaction for 2-5h at 45-60 ℃ by using 0.2-2.0% of composite protease (composed of trypsin and neutral protease with the enzyme activity unit ratio of (1-30): 1-6.5); then 0.1-1.0% flavourzyme (the enzyme activity is 80,000-; after the enzymolysis is finished, preserving the heat for 10 to 20 minutes at the temperature of between 95 and 100 ℃, and cooling to room temperature;
(3) and (3) filtering: carrying out graded filtration treatment on the separation liquid obtained in the step (2), firstly filtering by using a ceramic membrane with a nano-grade pore diameter to remove macromolecular impurities, then carrying out filtration treatment on the filtrate by using a nanofiltration membrane to remove micromolecular inorganic salts in the separation liquid, and collecting trapped liquid;
treating the trapped fluid collected in the above step by two-step ultrafiltration method, performing ultrafiltration with ultrafiltration membrane with aperture of 5000 Dalton, separating protein and polypeptide with molecular weight less than 5000 Dalton, and separating protein peptide with molecular weight less than 2000 Dalton with ultrafiltration membrane with aperture of 2000 Dalton;
(4) separation of target peptide: taking protein peptide liquid with molecular weight less than 2000, firstly carrying out SephadexG-15 gel separation, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 3 rd elution peak is collected; concentrating, and freeze-drying to obtain cod skin protein peptide; then RP-HPLC reversed phase high performance liquid chromatography is used for 1 separation, and the separation conditions are as follows: using a C18 chromatographic column, taking an aqueous solution containing 0.1% TFA as a mobile phase A and an acetonitrile solution containing 0.1% TFA as a mobile phase B, and performing separation by gradient elution: 0-5min, 5% mobile phase B; 5-45min, 5-45% of mobile phase B; 45-55min, 45-5% of mobile phase B, the flow rate is 1mL/min, and the separated protein peptide solution is collected for 6-11 min;
(5) concentrating and freeze-drying the peptide solution obtained in the step (4) to obtain codfish skin protein peptide powder with a specific function; the main components of the protein peptide are determined by LC-MS/MS, and the content of the peptide with the amino acid sequence of DINFDL, GYDAPPN and EKANGAT is more than or equal to 60 percent.
In the preparation method, high-frequency ultrasonic pretreatment is combined, multiple compound proteases are used for carrying out stepwise enzymolysis under the condition of not adding any acid and alkali for assisting hydrolysis, and three-membrane separation, gel separation and reversed-phase HPLC separation technologies are combined, so that the preparation method is a set of efficient and simple functional protein peptide preparation method taking the cod skin as a raw material. The protein peptide prepared by the method has high content of polypeptide (DINFDL, GYDAPPN and EKANGAT) with specific amino acid sequence, and has excellent oxidation resistance and immunity enhancing function.
Further, the invention provides a cod skin collagen peptide prepared by the preparation method of the cod skin collagen peptide.
Preferably, the cod skin collagen peptide contains functional peptides with the mass percentage content of more than or equal to 60%, and the sequence of the functional peptides is shown in any one of SEQ ID NO. 1-3.
The invention also provides a functional polypeptide which has an amino acid sequence shown in any one of SEQ ID NO. 1-3.
The invention also provides application of the cod skin collagen peptide or the functional polypeptide in preparation of medicines, foods or food additives.
Preferably, the pharmaceutical product or the food product has an antioxidant or immunity-enhancing function.
The present invention also provides a pharmaceutical product, food or food additive comprising said cod skin collagen peptide or said functional polypeptide.
The medicine, food or food additive can contain auxiliary materials allowed in the field of medicine or food besides the cod skin collagen peptide or the functional polypeptide.
The invention has the beneficial effects that: the cod skin collagen peptide is prepared by taking cod skin as a raw material, the collagen peptide prepared by the method has high content of small molecular peptides, the content proportion of peptides with molecular weights less than 2000 is more than 90%, the content of peptides with sequences of DINFDL, GYDAPPN and EKANGAT is more than 60%, and the collagen peptide has high purity and safety and has excellent functions of resisting oxidation and enhancing immunity.
The preparation method of the cod protein peptide, which is developed by the invention, does not add acid or alkali to adjust the pH value in the whole processing process, so that the product keeps better functional characteristics and has low ash content. The preparation method provided by the invention also has the advantages of high efficiency and simplicity.
Drawings
FIG. 1 is a diagram showing the detection of cod skin protein peptide in example 1 of the present invention.
FIG. 2 is a graph showing the detection of cod skin protein peptide in example 2 of the present invention.
FIG. 3 is a graph showing the detection of cod skin protein peptide in example 3 of the present invention.
FIG. 4 is a LC-MS graph of the cod skin protein peptide of example 1 of the present invention.
FIG. 5 is a LC-MS graph of the cod skin protein peptide of example 2 of the present invention.
FIG. 6 is a LC-MS graph of the cod skin protein peptide of example 3 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The activity unit contents of trypsin, neutral protease and flavourzyme used in the following examples were 150,000U/g, 350,000U/g and 180,000U/g, respectively.
In the following examples, the separation conditions of reverse phase HPLC are specifically as follows:
liquid phase system: shimadzu LC-16;
a detector: an ultraviolet detector is 220 nm;
mobile phase: aqueous solution containing 0.1% TFA, B-acetonitrile solution containing 0.1% TFA;
column: cosmosil 5C18-PAQ column (4.6X 250mm, Nacalai Tesque, Kyoto, Japan);
flow rate: 1 mL/min;
elution procedure: 0-5min, 5% mobile phase B; 5-45min, 5-45% of mobile phase B; 45-55min, 45-5% of mobile phase B.
Example 1 preparation of cod skin collagen peptide having antioxidant and immunity enhancing effects (1)
The embodiment provides a preparation method of cod skin collagen peptide with oxidation resistance and immunity enhancement, which comprises the following steps:
(1) selecting cod skin, placing cod skin in 1.0 times of water meeting drinking water sanitary standard, steaming at 121 deg.C for 60 min, cooling to 70 deg.C, and treating with ultrasonic generator (60KHz) for 30min to obtain cod skin protein solution;
(2) regulating the protein content in the cod skin protein solution to be 8%, adding protease according to the weight percentage of 1.2% of the weight of the protein in the cod skin protein solution for step-by-step enzymolysis, adding compound protease (trypsin and neutral protease in a mass ratio of 1: 1) in the weight of the protein in the first step of enzymolysis, and performing enzymolysis reaction for 2 hours at the temperature of 50 ℃; secondly, adding flavourzyme with the weight of 0.2 percent of the weight of the protein for enzymolysis, and carrying out enzymolysis reaction for 1h at the temperature of 55 ℃; preserving the heat for 10 minutes at 100 ℃, and cooling to room temperature;
(3) carrying out graded filtration treatment on the separation liquid obtained in the step (2), firstly filtering by using a ceramic membrane with a nano-grade aperture to remove macromolecular impurities and proteins, then separating again by using a nanofiltration membrane, and collecting trapped liquid;
(4) treating the retentate obtained in the step (3) by a two-step ultrafiltration method, performing ultrafiltration by using an ultrafiltration membrane with the aperture of 5000 daltons, separating proteins and polypeptides with the molecular weight of less than 5000 daltons, and separating protein peptides with the molecular weight of less than 2000 daltons by using an ultrafiltration membrane with the aperture of 2000 daltons;
(5) taking protein peptide liquid with molecular weight less than 2000, firstly carrying out Sephadex G-15 gel separation, wherein an eluent is deionized water, an elution peak is detected at 280nm, and a 3 rd elution peak is collected; concentrating, and freeze drying to obtain cod protein peptide powder; then RP-HPLC reversed phase high performance liquid chromatography is used for 1 time of separation, and the separated protein peptide solution collected for 6.8-8.4 minutes is taken.
(6) Concentrating and freeze-drying the peptide solution obtained in the step (5) to obtain cod protein peptide powder with a specific function; the measurement results of the cod protein peptide powder are shown in fig. 1; the main component of the protein peptide was determined by LC-MS/MS (fig. 4), and the content of the peptide whose amino acid sequence was gydapn was 60.1%.
The present example also provides the cod skin collagen peptide prepared by the above method.
Example 2 preparation of cod skin collagen peptide having antioxidant and immunity enhancing effects (2)
The embodiment provides a preparation method of cod skin collagen peptide with oxidation resistance and immunity enhancement, which comprises the following steps:
(1) selecting cod skin, placing cod skin in 3.0 times of water meeting drinking water sanitary standard, steaming at 115 deg.C for 120 min, cooling to 75 deg.C, and treating with ultrasonic generator (75KHz) for 25min to obtain cod skin protein solution;
(2) regulating the protein content in the cod skin protein solution to 10%, adding protease according to the weight percentage of 1.3% of the protein weight in the cod skin protein solution for step-by-step enzymolysis, adding composite protease (trypsin and neutral protease with the mass ratio of 2: 1) with the protein weight of 0.8% in the first step of enzymolysis, and performing enzymolysis reaction for 3 hours at 55 ℃; secondly, adding flavourzyme with the weight of 0.5 percent of the weight of the protein for enzymolysis, and carrying out enzymolysis reaction for 2 hours at the temperature of 60 ℃; preserving the heat for 20 minutes at 95 ℃, and cooling to room temperature;
(3) carrying out graded filtration treatment on the separation liquid obtained in the step (2), firstly filtering by using a ceramic membrane with a nano-grade aperture to remove macromolecular impurities and proteins, treating the separation liquid by using a nanofiltration membrane, and collecting trapped liquid;
(4) treating the trapped fluid obtained in the step (3) by a two-step ultrafiltration method, performing ultrafiltration by using an ultrafiltration membrane with the aperture of 5000 daltons, separating proteins and polypeptides with the molecular weight of less than 5000 daltons, and separating protein peptides with the molecular weight of less than 2000 daltons by using the ultrafiltration membrane with the aperture of 2000 daltons;
(5) taking protein peptide liquid with molecular weight less than 2000, firstly carrying out Sephadex G-15 gel separation, wherein an eluent is deionized water, an elution peak is detected at 280nm, and a 3 rd elution peak is collected; concentrating, and freeze drying to obtain cod protein peptide powder; then RP-HPLC reversed-phase high performance liquid chromatography is used for 1 time of separation, and the separated protein peptide solution collected for 9.5-10.5 minutes is taken out.
(6) Concentrating and freeze-drying the peptide solution obtained in the step (5) to obtain cod protein peptide powder with a specific function; the measurement results of the cod protein peptide powder are shown in fig. 2; the main component of the protein peptide was determined by LC-MS/MS (FIG. 5), and the content of the peptide whose amino acid sequence was EKANGAT was 62.8%.
The present example also provides the cod skin collagen peptide prepared by the above method.
Example 3 preparation of cod skin collagen peptide having antioxidant and immunity enhancing effects (3)
The embodiment provides a preparation method of cod skin collagen peptide with oxidation resistance and immunity enhancement, which comprises the following steps:
(1) selecting cod skin, placing cod skin in 5 times of water meeting drinking water sanitary standard, treating at 105 deg.C for 180 min, cooling to 70 deg.C, and treating with ultrasonic generator (90KHz) for 30min to obtain cod skin protein solution;
(2) regulating the protein content in the cod skin protein solution to 13%, adding protease according to the weight percentage of 1.5% of the protein weight in the cod skin protein solution for stepwise enzymolysis, adding composite protease (trypsin and neutral protease in a mass ratio of 3: 1) in an amount of 0.9% of the protein weight in the first step of enzymolysis, and performing enzymolysis reaction for 4 hours at 45 ℃; secondly, adding flavourzyme with the weight of 0.6 percent of the weight of the protein for enzymolysis, and carrying out enzymolysis reaction for 3.0 hours at the temperature of 50 ℃; preserving the heat for 10 minutes at 100 ℃, and cooling to room temperature;
(3) carrying out graded filtration treatment on the separation liquid obtained in the step (2), firstly filtering by using a ceramic membrane with a nano-grade aperture to remove macromolecular impurities and proteins, treating the separation liquid by using a nanofiltration membrane, and collecting trapped liquid;
(4) treating the trapped fluid obtained in the step (3) by a two-step ultrafiltration method, carrying out ultrafiltration by using an ultrafiltration membrane with the aperture of 5000 daltons, firstly separating proteins and polypeptides with the molecular weight of less than 5000 daltons, and then separating protein peptides with the molecular weight of less than 2000 daltons by using an ultrafiltration membrane with the aperture of 2000 daltons;
(5) taking protein peptide liquid with molecular weight less than 2000, firstly carrying out Sephadex G-15 gel separation, wherein an eluent is deionized water, an elution peak is detected at 280nm, and a 3 rd elution peak is collected; concentrating, and freeze drying to obtain cod protein peptide powder; separating by RP-HPLC reversed-phase high performance liquid chromatography for 1 time, and collecting separated protein peptide solution after 6.2-7.3 min;
(6) concentrating and freeze-drying the peptide solution obtained in the step (5) to obtain cod protein peptide powder with a specific function; the measurement results of the cod protein peptide powder are shown in fig. 3; the main component of the protein peptide was determined by LC-MS/MS (fig. 6), and the content of the peptide whose amino acid sequence was DINFDL was 67.1%.
The present example also provides the cod skin collagen peptide prepared by the above method.
Experimental example 1 measurement of antioxidant Activity of cod skin collagen peptide
Test samples: samples 1 to 3 are cod skin protein peptide powder having a specific function prepared in step (6) of examples 1 to 3, respectively, sample 4 is cod skin protein powder obtained by freeze-drying the cod skin protein solution obtained in step (1) of example 1, sample 5 is cod skin protein peptide powder obtained by freeze-drying in step (3) of example 1, sample 6 is cod skin protein peptide powder obtained by freeze-drying in step (4) of example 1, sample 7(DINFDL), sample 8 (gydapn), sample 9(EKANGAT), and polypeptides artificially synthesized according to amino acid sequences DINFDL, gydapn, EKANGAT, respectively.
The antioxidant activity was measured as follows:
(1) ability to scavenge DPPH free radicals: taking 1.5mL of 20 mu g/mL sample, adding 1.5mL of 99.5% ethanol and 0.675mL of 0.02% DPPH ethanol solution, mixing, oscillating, mixing uniformly, carrying out water bath in a dark place at room temperature for 30min, and detecting the light absorption value of the system at 517 nm. The lower the light absorption value, the stronger the DPPH free radical scavenging ability of the system. The blank control is to change 1.5mL of sample solution to 1.5mL of deionized water.
DPPH radical scavenging capacity { (blank absorbance-sample absorbance)/blank absorbance } × 100 { (blank absorbance-sample absorbance)/blank absorbance }
(2) Determination of superoxide anion radical scavenging capacity: taking 4.5mL of 50mmol/L (pH8.2) Tris-HCl buffer solution, adding 2mL distilled water into a test tube, adding 1mL of cod skin protein peptide solution with different concentrations, fully mixing, adding 1mL distilled water instead of a sample solution into a blank group, reacting at a constant temperature of 25 ℃ for 20min, adding 0.5mL of a pyrogallol solution with a concentration of 3mmol/L, quickly shaking up, measuring absorbance at a wavelength of 325nm (taking a buffer solution as a reference solution), reading once every 30s within 4min, and calculating the average oxidation rate within 4 min. Each concentration group was measured 3 times in parallel, and the average value was taken, and the measurement results were expressed in terms of clearance, and the calculation formula was:
clearance (%) - (k) 0 -k 1 )/k0×100％
In the formula: k is a radical of 0 -reaction rate at the time of auto-oxidation of pyrogallol; k is a radical of 1 Reaction rate on the autoxidation of pyrogallol after addition of the sample solution.
As shown in Table 1, the results show that the cod skin protein peptides of examples 1-3 have a good antioxidant activity, and the DPPH radical scavenging activity is more than 90% and O content is more than 20 μ g/mL 2 - The free radical scavenging capacity reaches more than 70 percent, and the peptide is a better antioxidant peptide.
TABLE 1 measurement results of antioxidant Activity of cod protein peptide
Experimental example 2 functional assay of cod skin protein peptide for enhancing immunity
Test samples: samples 1 to 3 are cod protein peptide powders having specific functions prepared in step (6) of examples 1 to 3, respectively.
1. Design of model of immune hypofunction
96 BAC mice with weight of 18-22g are purchased from the department of medicine of Beijing university, Beijing, and are randomly divided into 8 groups after being adapted to be raised for 7 days, and each group is divided into 12 BAC mice, and the BAC mice are fed for 60 days according to the following steps: (1) sample 1 low dose group: performing intragastric administration according to the cod skin protein peptide of 20mg/kg of the body weight of a mouse every day; (2) sample 1 high dose group: performing intragastric administration according to 100mg/kg of cod skin protein peptide every day; (3) sample 2 low dose group: performing intragastric administration according to the cod skin protein peptide of 20mg/kg of the body weight of a mouse every day; (4) sample 2 high dose group: performing intragastric administration according to 100mg/kg of cod skin protein peptide every day; (5) sample 3 low dose group: performing intragastric administration of the cod skin protein peptide according to the weight of 20mg/kg of the mouse every day; (6) sample 3 high dose group: performing intragastric administration according to 100mg/kg of cod skin protein peptide every day; (7) model blank set: physiological saline; (8) blank control group: physiological saline. 1-6 groups of samples with different doses, 1-3 cod skin protein peptide solution dissolved by normal saline, are perfused for 1-59 days, and (7) and (8) groups of samples are perfused with equal volume of normal saline, 1-7 groups of mice are injected with 200mg/Kg of cyclophosphamide in the abdominal cavity on the 60 th day, 8 groups of mice are injected with equal volume of normal saline in the abdominal cavity, after eating and water cut off for 12 hours, the mice are killed by blood sampling of eyeballs, and the blood biochemical and blood routine results are detected.
2. Results of the experiment
As shown in Table 2, the white blood cell count in the blood of the mice can evaluate the inflammatory state in vivo, reflecting the intensity of the immune response. Low dose cod skin collagen peptide group mouse leukocyte concentration (2.476 + -0.16X 10) 9 /L，P<0.01) is significantly higher than the immunocompromised group. The main factor in which significant changes occur is the concentration of lymphocytes (2.086. + -. 0.25X 10) 9 /L，P<0.01). Mice gavaged with low doses of cod skin collagen peptide showed a significant increase in the number of leukocytes. Meanwhile, the ingestion of the cod skin collagen peptide has no influence on the concentration of red blood cells, hemoglobin and platelets in the blood of the mice, which indicates that the cod skin collagen peptide has no side effect on other indexes in the conventional blood detection. The above results indicate that the cod skin collagen peptide can regulate the immunity of mice by improving the level of lymphocytes in blood leukocytes.
TABLE 2 measurement results of immunity enhancing activity of cod protein peptide
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (5)
1. The cod skin collagen peptide is characterized by comprising functional peptides with the mass percentage content of more than or equal to 60%, and the sequence of the functional peptides is shown in any one of SEQ ID NO. 1-3.
2. The method for preparing cod skin collagen peptide according to claim 1, comprising the steps of:
(1) pretreatment of raw materials: cooking the minced cod skin at the temperature of 100-121 ℃ for 1-3h, and performing ultrasonic treatment at the temperature of 70-75 ℃ and the temperature of 60-90kHz for 20-30min to obtain a raw material protein solution;
(2) enzymolysis: performing two-step enzymolysis on the raw material protein solution obtained in the step (1), wherein in the first step of enzymolysis, composite protease is used for enzymolysis for 2-5 hours at the temperature of 45-60 ℃, and in the composite protease, the mass ratio of trypsin to neutral protease is (3-1): 1, the enzyme activity of the trypsin is 100,000-1,000,000U/g, the enzyme activity of the neutral protease is 100,000-650,000U/g, and the dosage of the composite protease is 0.2-2g/100g of raw material protein; then, carrying out second-step enzymolysis by using flavourzyme at the temperature of 50-60 ℃, wherein the time of the second-step enzymolysis is 1-3h, the using amount of the flavourzyme is 0.1-1g/100g of the raw material protein, and the enzyme activity of the flavourzyme is 80,000-250,000U/g; after the enzymolysis is finished, preserving the heat for 10-20 minutes at the temperature of 95-100 ℃, and cooling to room temperature;
(3) and (3) filtering: carrying out graded filtration treatment on the zymolyte obtained in the step (2), firstly filtering by using a ceramic membrane with a nano-grade aperture, filtering the filtrate by using a nanofiltration membrane, and collecting trapped fluid; sequentially ultrafiltering the trapped fluid by ultrafiltration membranes with aperture of 5000 Dalton and 2000 Dalton to obtain protein peptide solution with molecular weight less than 2000;
(4) separation of target peptide: separating the protein peptide liquid with the molecular weight of less than 2000 obtained in the step (3) by SephadexG-15 gel, wherein the eluent is deionized water, the elution peak is detected at 280nm, and the 3 rd elution peak is collected; then RP-HPLC reversed-phase high performance liquid chromatography is used for separation, and the separation conditions are as follows: using a C18 chromatographic column, taking an aqueous solution containing 0.1% TFA as a mobile phase A and an acetonitrile solution containing 0.1% TFA as a mobile phase B, and performing separation by gradient elution: 0-5min, 5% mobile phase B; 5-45min, 5-45% of mobile phase B; 45-55min, 45-5% of mobile phase B, the flow rate is 1mL/min, and the separated protein peptide solution is collected for 6-11 min;
(5) concentrating and freeze-drying the peptide solution obtained in the step (4) to obtain the codfish skin protein peptide powder with specific functions, wherein the content of the peptide with the amino acid sequence of DINFDL, GYDAPPN or EKANGAT is more than or equal to 60%.
3. The functional polypeptide with the functions of resisting oxidation and enhancing immunity is characterized in that the amino acid sequence of the functional polypeptide is shown in any one of SEQ ID NO. 1-3.
4. Use of the cod skin collagen peptide of claim 1 or the functional polypeptide of claim 3 for the preparation of a medicament, food or food additive;
the medicine or the food has the functions of resisting oxidation or enhancing immunity.
5. A pharmaceutical, food or food additive comprising the cod skin collagen peptide of claim 1 or the functional polypeptide of claim 3.
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| CN114796089B (en) * | 2022-05-26 | 2024-05-14 | 格莱康美生物医学技术(北京)有限公司 | Application of umbilical cord mesenchymal stem cell exosome combined rhodiola rosea polypeptide in maintaining beauty and keeping young |
| CN114990095B (en) * | 2022-06-28 | 2024-05-24 | 天津天隆江大生物科技有限公司 | Complex enzyme preparation for producing antifreeze protein polypeptide and application thereof |
| CN116284341B (en) * | 2023-02-20 | 2024-05-03 | 中国石油大学(华东) | Preparation and application of deep sea fish skin collagen peptide with low immunogenicity, blood pressure reduction and oxidation resistance |
| CN117653562A (en) * | 2023-11-27 | 2024-03-08 | 广州亚丽化妆品有限公司 | Collagen peptide composition with anti-aging and immunity improving effects and preparation method thereof |
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