CN112195214A - Fish skin collagen polypeptide and preparation method and application thereof - Google Patents
Fish skin collagen polypeptide and preparation method and application thereof Download PDFInfo
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- CN112195214A CN112195214A CN202010903691.1A CN202010903691A CN112195214A CN 112195214 A CN112195214 A CN 112195214A CN 202010903691 A CN202010903691 A CN 202010903691A CN 112195214 A CN112195214 A CN 112195214A
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- 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
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- 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
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
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Abstract
The invention belongs to the technical field of food biology, and discloses a fish skin collagen polypeptide and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) immobilizing Bacillus subtilis alkaline protease (BAP) with Egg Shell Membrane (ESM) as carrier to obtain ESM-BAP; (2) carrying out enzymolysis on fish skin collagen by using ESM-BAP, separating by using a 1-5 kDa ultrafiltration membrane, and screening and verifying by using a human bone marrow mesenchymal stem cell experiment and a zebra fish animal experiment to obtain the fish skin collagen polypeptide with high anti-osteoporosis activity. The fish skin collagen polypeptide raw material obtained by the invention is derived from fish processing waste, has high safety and no side effect, and reduces the pollution to the environment to a certain extent while greatly improving the value of fish by-products. The obtained collagen polypeptide is safe and reliable, has high anti-osteoporosis activity, and can be used in various fields such as food and medical treatment.
Description
Technical Field
The invention relates to the technical field of food biology, in particular to a preparation method of fish skin collagen polypeptide with anti-osteoporosis activity.
Background
Statistically, at least 2 million people worldwide are afflicted with osteoporosis and related diseases caused thereby, including mainly the elderly and postmenopausal women. The fundamental reason of osteoporosis is abnormal bone metabolism in the body caused by aging, diseases, drugs and other factors, increased bone absorption, decreased bone mass and abnormal change of bone microstructure, thereby causing increased bone fragility and easy fracture. Therefore, the key to treating osteoporosis is to inhibit bone resorption and promote bone formation. In addition to drug therapy, a reasonable diet is also a key to the treatment of osteoporosis. Research shows that the collagen polypeptide can be used as a food supplement to stimulate joints to promote cartilage tissues to produce substances such as type II collagen, hyaluronic acid, proteoglycan and the like, so that the low bone density disease is improved. Compared with the traditional medicine treatment, the method is safer and more reliable, and has no side effect influence.
The collagen polypeptide is prepared by hydrolyzing collagen with amino acid sequences with different biological activities under the action of acid, alkali, enzyme and the like, and the hydrolysis action promotes the exposure of the active center of the collagen peptide so as to ensure that the collagen peptide has the function of resisting osteoporosis. Generally, collagen polypeptides are mostly prepared from mammals or aquatic organisms, the preparation of collagen polypeptides from mammals is limited by policy and religious belief, and the preparation of collagen polypeptides from aquatic organisms is not limited by the above factors, and has abundant resources, low finished products and high bioactivity.
Tilapia is one of important freshwater aquaculture species in China, the total amount of the Tilapia is over 162 ten thousand tons, but in the processing process of Tilapia, 60-70% of leftovers such as fish scales, fish skins and fish bones are wasted, the content of collagen in the fish skins can reach more than 80% of the total amount of protein at most, and the fat content is extremely low. Therefore, the collagen polypeptide prepared from the fish skin collagen is accepted by people in the fields of food and medical treatment, and the influence on the environment is reduced to a certain extent while the value of fish by-products is greatly improved.
At present, the methods for preparing fish skin collagen polypeptide mainly comprise a chemical method, a recombinant DNA method, a direct extraction method and a protein hydrolysis method. But the chemical method can generate byproducts such as waste, waste material and the like in the preparation and synthesis process, and causes certain pollution to the environment; the direct extraction method has higher cost and low extraction rate; the polypeptide peptide chain prepared by the recombinant DNA method is longer, and the bioactivity is lower; the protein hydrolysis method has short time consumption, less loss of nutrient substances and no pollution to the environment, so the optimal method for preparing the fish skin collagen polypeptide is the protein hydrolysis method. Patent CN103805665 discloses a preparation method of deep sea fish skin collagen polypeptide, which comprises the steps of carrying out enzymolysis on a crude collagen liquid product at 55 ℃ by using compound protease, using an ultrafiltration membrane for multiple times for filtration, adsorbing, decoloring, removing fishy smell, carrying out nanofiltration on the obtained enzymolysis liquid, drying and finally preparing powdery collagen polypeptide. The preparation method combines enzymolysis and ultrafiltration separation processes, can manually control reaction conditions, and obtain a product with better physicochemical properties, but the adsorption effect of the composite activated carbon is not ideal, and the product has a certain bitter taste. Patent 201810408287.X is improved on the basis, collagenase and flavourzyme are selected for proteolysis, and modified activated carbon is used for adsorption, so that the method improves the adsorption efficiency and reduces the bitter taste of the product, but the enzymolysis time is longer, and the method is not suitable for large-scale production. In addition, most studies on the activity of collagen polypeptides are directed to antioxidant activity, and methods for preparing collagen polypeptides with anti-osteoporosis activity are few at present. Therefore, the determination of a preparation method of collagen polypeptide with anti-osteoporosis activity is the direction of the development of the current collagen polypeptide industry.
Disclosure of Invention
Aiming at the defects and shortcomings of the prior art, the invention aims at providing a preparation method of fish skin collagen polypeptide with anti-osteoporosis activity.
The purpose of the invention is realized by the following technical scheme:
a preparation method of fish skin collagen polypeptide comprises the following preparation steps:
(1) immobilizing alkaline protease (BAP) of Bacillus subtilis with eggshell membrane (ESM) as carrier to obtain ESM-BAP;
(2) adding fish skin collagen into an alkaline buffer solution, adding ESM-BAP for enzymolysis, inactivating enzyme after the enzymolysis is finished, centrifuging to obtain a supernatant, separating the supernatant through a 1-5 kDa ultrafiltration membrane to obtain fish skin collagen polypeptide, and freeze-drying to obtain collagen polypeptide powder.
Preferably, the preparation method of the ESM-BAP comprises the following steps: and (3) taking the eggshell membrane to be oscillated and soaked in the genipin solution, then washing off redundant genipin on the eggshell membrane, adding the bacillus subtilis alkaline protease solution under the oscillating condition of constant-temperature water bath, and carrying out crosslinking reaction to obtain the ESM-BAP.
Preferably, the temperature of the crosslinking reaction is 25-50 ℃ and the time is 1-4 h.
Preferably, the concentration of the genipin is 0.5% -1.0%, and the concentration of BAP is 1-8 mg/mL.
Preferably, the concentration of the genipin is 0.75 +/-0.15%, and the concentration of the BAP is 5 +/-1 mg/mL.
Preferably, the pH value of the enzymolysis is 7.5-10.5, and the enzymolysis temperature is 25-75 ℃.
Preferably, the pH value of the enzymolysis is 9.5 +/-1.0, and the enzymolysis temperature is 55 +/-10 ℃.
Preferably, the fish skin is tilapia fish skin, and the molecular weight cut-off of the ultrafiltration membrane is 1-3 kDa.
The fish skin collagen polypeptide prepared by the method is applied to preparing anti-osteoporosis medicines or health care products.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) all raw materials used by the collagen polypeptide prepared by the invention are wastes in the fish processing industry, are safe and reliable, and are beneficial to resource utilization of the wastes.
(2) The invention uses ESM as a carrier to immobilize BAP enzyme, and ESM has rich source, easy acquisition and high safety.
(3) The invention uses ESM to fix BAP to prepare ESM-BAP, and greatly improves the enzyme activity of BAP while maintaining the affinity and catalytic efficiency of BAP to a substrate.
(4) The anti-osteoporosis activity of the fish skin collagen polypeptide prepared by the invention is higher than that of a disodium etidronate medicament.
(5) The collagen polypeptide prepared by the invention can be applied to various fields such as food, medicine and the like, has good application prospect, simple process technology and easy mass production.
Drawings
FIG. 1 is a photograph of samples of ESM and ESM-BAP obtained in example 1.
FIG. 2 shows the results of the experiment for inducing osteogenic differentiation using the collagen polypeptide hBMSC obtained in example 1.
FIG. 3 is a staining pattern of alizarin red S on the skull of zebra fish in the animal experiment of the collagen polypeptide zebra fish obtained in example 1 (the concentrations of the collagen polypeptide are 1mg/mL, 0.5mg/mL and 0.2mg/mL, respectively).
FIG. 4 is a graph showing the effect of the collagen polypeptide obtained in example 1 and etidronate Disodium (DI) drug on Dexamethasone (DE) -induced zebrafish osteoporosis young fish (P1(1-3:50ug/mL, 100ug/mL, 150ug/mL) and P3 (4-6: 50ug/mL, 100ug/mL, 150ug/mL)) (a: improvement rate of bone mineralization in skull bone, b: calculation of cumulative optical density of skull bone).
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto. The subtilisin alkaline protease (BAP) used in the present invention was purchased from Novoxin (Japan) Co., Ltd.
Example 1
(1) Soaking egg shell in 20% acetic acid solution for 24 hr, removing egg shell, rinsing with ultrapure water to neutral to obtain Egg Shell Membrane (ESM), and preserving in pH 7.4 phosphoric acid buffer solution at 4 deg.C;
(2) weighing a certain amount of ESM prepared in the step (1), shaking and soaking in 0.75% genipin for 3h, washing off redundant genipin on the ESM by using ultrapure water, adding a BAP solution of 5mg/mL under the condition of shaking in a constant-temperature water bath at 45 ℃, crosslinking for 2h, and washing by using PBS to obtain ESM-BAP;
(3) placing tilapia fishskin collagen in 20mmol/L Na2CO3-NaHCO3Adding the ESM-BAP prepared in the step (2) into a buffer system, adjusting the pH value to 9.5, and adjusting the enzymolysis temperature to 55 ℃. After the enzymolysis is finished, inactivating the enzyme for 10min at 100 ℃, and centrifuging to take the supernatant;
(4) and (4) carrying out ultrafiltration separation on the supernatant obtained in the step (3) by using 5kDa, 3kDa and 1kDa ultrafiltration membranes to obtain 3 tilapia skin collagen polypeptide components P5(3 kDa-5 kDa), P3(1 kDa-3 kDa) and P1(1 kDa) with different molecular weights. Freeze-drying to obtain collagen polypeptide powder;
(5) performing an osteogenic differentiation experiment induced by human mesenchymal stem cells (hBMSC) on the tilapia skin collagen polypeptides with 3 different molecular weights prepared in the step (4): the hbmscs cells were harvested by trypsinization (sixth generation), suspended in 0.4mL growth medium and incubated in incubator for 24h (degree of fusion 80%). Respectively preparing three concentrations of 1mg/mL, 0.5mg/mL and 0.2mg/mL for P1, P3 and P5 samples, adopting a special osteogenic differentiation induction culture solution (with or without adding a test sample with a certain concentration) to induce osteogenic differentiation, adopting an alizarin red S staining kit to stain cells after continuously inducing for 15 days, and observing calcium nodules (red). And then carrying out zebra fish animal experiments: setting zebra fish as a normal control group (treated by fish culture water containing 0.5% DMSO), a model group (treated by dexamethasone water solution administration at 10 mu mol/mL), and a positive control group (treated by etidronate disodium at 25 mu g/mL and dexamethasone water solution administration at 10 mu mol/mL); experimental group (collagen polypeptide samples + 10. mu. mol/mL dexamethasone in water). The zebra fish is cultured at 28.5 +/-0.5 ℃ to 10dpf, the medicine is changed every other day before 6dpf, and the zebra fish starts to feed at 7 dpf. And (4) screening the fish skin collagen polypeptide with the highest anti-osteoporosis activity according to the test result.
As can be seen from fig. 2, the deeper red calcium nodules indicate the better differentiation of osteoblasts. Compared with the Control group, the three concentrations of P1 and P3 can promote the formation of osteoblast calcium nodules and are dose-dependent, and P5 only has the effect of promoting osteogenesis at the concentration of 1 mg/mL. Therefore, P1 and P3 were selected for subsequent experiments to further screen for anti-osteoporotic fish skin collagen active peptides of optimal molecular weight.
As can be seen from FIG. 3, alizarin red S can be combined with calcium salt to form red color, and bone mineralization area can be observed and detected according to the result of alizarin red S staining solution.
As can be seen from FIG. 4, the DE group was able to significantly reduce the bone mineralization area and the cumulative optical density of the skull of the zebra fish juvenile fish, indicating that DE has an inhibitory effect on bone formation. The DI drug group, P1, P3 component of ESM-BAP all had an improvement effect on bone formation inhibition of zebra fish juvenile fish, and the P1 component results were similar to those of the DI drug group.
Example 2
0.1, 0.25, 0.5, and 1% genipin were used in step (2), respectively, and the remaining conditions were the same as in example 1.
Example 3
The temperature of the oscillating conditions of the constant temperature water bath in the step (2) was 25 ℃, 35 ℃, 45 ℃, 55 ℃ and 65 ℃ respectively, and the rest conditions were the same as those of the example 1.
Example 4
The BAP concentrations added in step (2) were 1, 2, 5, 8, 10mg/mL, respectively, and the rest of the conditions were the same as in example 1.
Example 5
The cross-linking time in step (2) was 1, 2, 4, 6, 8h, respectively, and the rest of the conditions were the same as in example 1.
Example 6
The enzymolysis temperature in the step (3) is respectively 25, 35, 45, 55, 65, 75 and 85 ℃, and the rest conditions are the same as those in the embodiment 1.
Example 7
The pH values of the enzymolysis in the step (3) are 5.5, 6.5, 7.5, 8.5, 9.5, 10.5 and 11 respectively, and the other conditions are the same as those in the example 1.
Tables 1 to 4 show the recovery rate of the enzyme activity, the enzyme load of ESM and the enzyme activity load of ESM-BAP prepared in examples 1 to 5, respectively. As can be seen from tables 1-4, the optimum preparation condition of ESM-BAP is 0.75% genipin concentration, the crosslinking temperature is 45 ℃, the enzyme concentration is 5mg/mL, the crosslinking time is 2h, the recovery rate of enzyme activity is up to 63.36%, the enzyme loading amount is 319.69mg/g, and the enzyme activity loading amount is 30039.94U/g.
TABLE 1
TABLE 2
TABLE 3
TABLE 4
Tables 5-6 are the ESM-BAP catalytic performances prepared in examples 6-7, respectively. As can be seen from tables 5-6, the optimum catalytic pH of ESM-BAP was 9.5 and the optimum catalytic temperature was 55 ℃.
TABLE 5
TABLE 6
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A preparation method of fish skin collagen polypeptide is characterized by comprising the following preparation steps:
(1) immobilizing alkaline protease (BAP) of Bacillus subtilis with eggshell membrane (ESM) as carrier to obtain ESM-BAP;
(2) adding the fish skin collagen into an alkaline buffer solution, adding ESM-BAP for enzymolysis, inactivating enzyme after the enzymolysis is finished, centrifuging to obtain a supernatant, and separating the supernatant through a 1-5 kDa ultrafiltration membrane to obtain the fish skin collagen polypeptide.
2. The method of claim 1, wherein the ESM-BAP is prepared by:
and (3) taking the eggshell membrane to be oscillated and soaked in the genipin solution, then washing off redundant genipin on the eggshell membrane, adding the bacillus subtilis alkaline protease solution under the oscillating condition of constant-temperature water bath, and carrying out crosslinking reaction to obtain the ESM-BAP.
3. The method according to claim 2, wherein the crosslinking reaction is carried out at a temperature of 25 to 50 ℃ for 1 to 4 hours.
4. The preparation method according to claim 2, wherein the genipin concentration is 0.5% -1.0% and the BAP concentration is 1-8 mg/mL.
5. The preparation method according to claim 2, wherein the genipin concentration is 0.75 ± 0.15%, and the BAP concentration is 5 ± 1 mg/mL.
6. The preparation method according to any one of claims 1 to 5, wherein the pH value of the enzymolysis is 7.5 to 10.5, and the enzymolysis temperature is 25 to 75 ℃.
7. The preparation method of claim 6, wherein the pH value of the enzymolysis is 9.5 +/-1.0, and the enzymolysis temperature is 55 +/-10 ℃.
8. The preparation method of claim 6, wherein the skin of tilapia mossambica is the skin of tilapia mossambica, and the molecular weight cut-off of the ultrafiltration membrane is 1-3 kDa.
9. A fish skin collagen polypeptide prepared by the method of any one of claims 1 to 8.
10. The use of the fish skin collagen polypeptide of claim 9 in the preparation of an anti-osteoporosis medicament or health product.
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