CN116284341A - Preparation and application of deep sea fish skin collagen peptide with low immunogenicity, blood pressure reduction and oxidation resistance - Google Patents
Preparation and application of deep sea fish skin collagen peptide with low immunogenicity, blood pressure reduction and oxidation resistance Download PDFInfo
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- CN116284341A CN116284341A CN202310139849.6A CN202310139849A CN116284341A CN 116284341 A CN116284341 A CN 116284341A CN 202310139849 A CN202310139849 A CN 202310139849A CN 116284341 A CN116284341 A CN 116284341A
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Abstract
The invention provides a preparation method and application of a low-immunogenicity deep-sea fish skin micromolecular collagen peptide with blood pressure reduction and oxidation resistance. The preparation method of the collagen peptide comprises the following steps: physical pretreatment of fish skin, degreasing and impurity protein removal; removing terminal peptide; obtaining micromolecular collagen peptide by a composite proteolysis-ultrafiltration membrane coupling process; purifying by chromatography to obtain antihypertensive and antioxidant small molecule collagen peptide; the bacteria and endotoxin are inactivated by radiation, and finally the low-immunogenicity micromolecular collagen peptide for reducing blood pressure and resisting oxidation is obtained. According to the invention, substances such as terminal peptide, hetero protein, endotoxin and the like are removed by various means, so that the immunogenicity of collagen peptide is greatly reduced, and the stimulation and side reaction to organisms are reduced. The collagen peptide with low immunogenicity has the characteristics of small molecular weight, high purity, high antihypertensive activity, strong antioxidant activity, low immunogenicity and good safety, and can be used in the fields of food, health care products and medicine.
Description
Technical Field
The invention relates to the field of marine biotechnology product development and application, in particular to preparation and application of a deep-sea fish skin small-molecule collagen peptide with low immunogenicity, blood pressure lowering and antioxidant activities.
Background
Collagen peptide is a hydrolysis product of collagen, has a plurality of biological activities which are not possessed by collagen, has small molecular weight and is easier to be absorbed by human bodies than collagen, and reported effects include antioxidation, anti-tumor, blood pressure reduction, liver protection, calcium absorption promotion, hormone level regulation, beauty skin care and the like, and is rapidly developed at home and abroad as an emerging biological active substance, and is widely applied to the fields of functional foods, health care products, medical treatment, cosmetics and the like.
Hypertension is a global public health problem and is the cause of many diseases such as stroke, myocardial infarction, heart failure, aneurysms and the like, and greatly influences the health of people. Angiotensin converting enzyme (angiotensin converting enzyme, ACE) is a dipeptidyl peptidase that regulates blood pressure via the renin-angiotensin system and kallikrein-kinin system (KKS). ACE is capable of catalyzing the production of angiotensin I to produce angiotensin II which leads to vasoconstriction, while inactivating bradykinin having vasodilatory action, leading to an increase in blood pressure. Therefore, inhibiting ACE activity can effectively reduce blood pressure, prevent and treat hypertension and related diseases. Common ACE inhibitor drugs include captopril, benazepril, enalapril, lisinopril and the like, and the drugs have side effects such as dysgeusia, cough, rash, headache and the like after being taken for a long time. Compared with chemical synthetic medicines, the ACE inhibitory peptide obtained by the food-borne protein has the characteristics of safer, milder, easy absorption and no side effect.
The metabolism of human body can generate a lot of active oxygen and free radicals, and oxidative stress injury and free radical metabolic disturbance can cause cell injury, and cause diseases such as arteriosclerosis, diabetes, asthma, skin aging, organism aging and the like. Some synthetic antioxidants are used for food preservation and the like, and are more active than natural antioxidants, but also have certain side effects such as damage to DNA and toxicity. The bioactive peptide has proved to have good antioxidant activity, and is safe and nontoxic, and has great significance for maintaining human health and preventing diseases.
Most of the collagen peptide products used at the present stage are derived from terrestrial animals, such as pigs, cows and the like. Compared with collagen peptide derived from terrestrial animals, collagen peptide derived from deep sea fish skin has advantages such as easy extraction, low heavy metal and toxin content, low immunogenicity, no risk of zoonosis, no religious customs constraint, etc. The offal fish skin produced in the deep sea fish processing process has collagen content accounting for 70-80% of the total protein, is rich in source and low in cost, and provides a new idea for developing novel collagen peptide products. In addition, the collagen peptide with high added value is extracted from the marine processing leftovers, so that the resources can be reasonably utilized, the problem of environmental pollution is avoided, and the blue marine economy and the green sustainable development of China are facilitated.
At present, various bioactive collagen peptide products exist in China, but the problems of larger molecular weight and wide molecular weight distribution index generally exist, so that the content of effective active ingredients is low, and the purity of the product is not high. In addition, collagen itself has a certain immunogenicity, and can stimulate the body, which is mainly derived from terminal peptide sequences with non-helical structures, including N-terminal and C-terminal amino acid sequences. In addition, residual foreign proteins, bacteria, endotoxins, etc. in the production process may also elicit immune responses in the organism, which all limit the application of collagen peptide products in the organism. At present, antioxidant and antihypertensive micromolecular collagen peptide products derived from deep sea fishes with low immunogenicity are not reported yet.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a low-immunogenicity deep-sea fish skin small-molecule collagen peptide with antihypertensive and antioxidant activities and a preparation method thereof, which can be applied to food, health care products and medical related applications.
In order to achieve the above object, the technical scheme of the present invention includes the following steps:
(1) Pretreatment of fish skin: the deep sea fish skin is cleaned by a wire brush, soaked in softened water for 3 hours, repeatedly washed by softened water under high pressure for a plurality of times, and drained. Then shearing the clean fish skin to obtain the fish skin with proper size. Adding sodium hydroxide alkali solution into the fish skin to treat for 12-24 hours, removing fat and foreign protein in the fish skin, and washing the fish skin to be neutral by using softened water;
(2) Terminal peptide removal: the treated fish skin is processed according to the feed liquid ratio of 1: 15-1: 20 is added into 0.5M acetic acid, pepsin with the concentration of 60-100U/mL is added, and the magnetic stirring treatment is carried out for 12-72 h. Adjusting the pH value to be neutral, and dialyzing the reaction solution by using a 100kDa dialysis bag;
(3) The collagen peptide is obtained by a composite enzymolysis-ultrafiltration coupling process: the pH value of the liquid is adjusted to be neutral, and the composite protease with the mass of 1-3% of the fish skin is added for enzymolysis for 2-24 hours at the temperature of 40-60 ℃ to obtain the crude extract of the collagen peptide. Preserving the temperature of the crude extract at 95-100 ℃ for 10-20 min, and inactivating enzyme. And passing through ultrafiltration membrane with molecular weight cutoff of 1kDa, retaining the filtered component, and freeze drying to powder;
(4) Purification of bioactive small molecule collagen peptide: the collagen peptide powder obtained in the last step is dissolved by ultrapure water to prepare a solution of 10-20 mg/mL, and sephadex G25 molecular sieve chromatography is used for separating and purifying the collagen peptide powder, and the peak components are collected and combined. Selecting the component with highest activity, further refining and purifying by using a reversed phase chromatography C18 column, and collecting each peak component. Finally, the collagen peptide component with ACE inhibition activity and antioxidation activity is obtained, and freeze-dried.
(5) Bacteria and endotoxin removal: and performing freeze drying to obtain active micromolecular collagen peptide powder, performing irradiation treatment on the active micromolecular collagen peptide powder by using 60Co, inactivating endotoxin, and sterilizing and packaging to obtain the low-immunogenicity deep sea fish skin micromolecular collagen peptide finished product with blood pressure reducing and antioxidant activities.
Preferably, the alkali solution in the step (1) uses sodium hydroxide solution with the concentration of 0.01-0.02M, and the ratio of fish skin to alkali solution is 1:10 to 1:20, the treatment time is 12-24 hours.
Preferably, the acetic acid solution in step (2) is used at a concentration of 0.5M, feed to feed ratio 1: 15-1: the pepsin concentration is 60-100 IU/mL.
Preferably, the compound protease in the step (3) uses neutral protease and bromelain, and the mass ratio of the neutral protease to bromelain is 1: 2-1: 4, the enzymolysis temperature is 40-60 ℃ and the enzymolysis time is 2-24 h. Ultrafiltration membranes use a membrane with a molecular weight cut-off of 1kDa, retaining the filtered components.
Preferably, the molecular sieve chromatography in step (4) uses a Sephadex G25 packing material, ultrapure water as the mobile phase, and the linear flow rate is 0.8ml/min, and the detection is performed at a wavelength of 220 nm. Reverse phase chromatography using XTerra Prep MS C18 prepared column, mobile phase a as 5% acetonitrile in water with 0.1% TFA, B as 90% acetonitrile in water with 0.1% TFA, equilibrate column with 100% solution a for 0-5min, eluting with 5-20min, rising linearly to 100% solution B, and detecting elution peak at 220nm wavelength.
Preferably, in step (6) is taken 60 Co irradiation treatment deactivates endotoxin.
The invention also provides the low-immunogenicity deep-sea fish skin small molecule collagen peptide prepared by the preparation method.
The low-immunogenicity collagen peptide provided by the invention has small molecular weight and narrow distribution range, is concentrated between 300 Da and 600Da, and has an average molecular weight of 500Da.
The invention also provides application of the low-immunogenicity collagen peptide in reducing blood pressure and resisting oxidization.
Compared with the prior art, the invention has the following beneficial effects:
in the preparation of the micromolecular deep sea fish skin collagen peptide, various methods for reducing immunogenicity are adopted, such as pretreatment of sodium hydroxide, and impurity particles, lipids and non-collagen proteins in the fish skin are removed; the terminal peptide sequence is the factor which is most easy to trigger the immune response of the organism, and pepsin treatment is used for removing collagen terminal peptide, so that the immune response possibly caused by enzymolysis products is reduced; sterilization and radiation treatment inactivate bacteria and endotoxins, further reducing the immunogenicity of collagen peptides.
The small molecular collagen peptide obtained by the invention has smaller molecular weight and more concentrated distribution range. The composite enzymolysis-ultrafiltration coupling process ensures that the molecular weight range of the collagen peptide obtained by the invention is concentrated at 300-600Da, which is far lower than that of products on the market, has higher biological activity and more complete organism absorption, and also avoids immune reaction possibly caused by biological macromolecules entering the body.
And (3) refining and purifying by molecular sieve chromatography and reverse chromatography to obtain the antihypertensive and antioxidant small molecular collagen peptide with the highest activity. Most of collagen peptide products in the market are products obtained by direct enzymolysis, and the collagen peptide products are not further purified, have wide molecular weight distribution, are complex in components, contain more impurities and have low activity, and can possibly cause side effects. Some pure peptide products synthesized according to known sequences have high purity and strong activity, but can only be obtained by chemical synthesis, and have high cost, large pollution and impossible mass production. The collagen peptide component obtained by purifying the product contains the component with the highest biological activity, and has the advantages of convenient preparation, low price, simple purification steps, easy amplification, full cost reduction and ocean waste resource utilization.
The deep sea fish skin micromolecular collagen peptide with low immunogenicity is prepared by the method, has high-efficiency antihypertensive and antioxidant activities, has the advantages of simple process flow, low cost, mild reaction, small pollution and environmental friendliness, and can be applied to the fields of special medical raw materials and medical assurance.
Drawings
FIG. 1 antioxidant capacity of collagen peptides of different molecular weights;
in the figure: dpph radical; abts radical; c. superoxide anion radical; d. lipid peroxides
FIG. 2 inhibitory effect of collagen peptides of different molecular weights on ACE;
FIG. 3 purification of collagen peptide elution peaks by Sephadex G-25 molecular sieve;
FIG. 4 purification of elution peaks of collagen peptide by reverse HPLC on an XTerra Prep MS C18 column;
FIG. 5 effect of small molecule collagen peptides on ACE inhibition and antioxidant effect after gastrointestinal digestion;
figure 6 cytotoxicity test of small molecule collagen peptides.
In the figure: a. physiological saline group 72h cell growth case b. collagen peptide group 24h cell growth case c. collagen peptide group 72h cell growth case
Detailed Description
The following embodiments better illustrate the present invention. The present invention is not limited to the following examples.
Example 1: preparation of deep sea fish skin collagen peptide
(1) Pretreatment of fish skin: the deep sea fish skin is brushed with steel wires to clean the back fish tissue, soaked in softened water for 3 hours, repeatedly washed with softened water under high pressure for many times, and drained. Then shearing the clean fish skin to obtain the fish skin with proper size. According to the feed liquid ratio of the fish skin to the alkali solution of 1:10 to 1:20 adding 0.01-0.02M sodium hydroxide solution, treating for 12-24 hours, removing fat and foreign protein in the fish skin, and washing the fish skin to be neutral by using softened water;
(2) Terminal peptide removal: the treated fish skin is processed according to the feed liquid ratio of 1: 15-1: 20 is added into 0.5M acetic acid, pepsin with the concentration of 60-100U/mL is added, the magnetic stirring treatment is carried out for 12-72 h, terminal peptide is removed, the reaction solution is dialyzed by a 100KD dialysis bag, and trapped liquid is reserved;
(3) The composite enzymolysis-ultrafiltration membrane coupling process comprises the following steps: and regulating the pH value of the liquid to be neutral, continuously adding compound protease accounting for 1-3% of the weight of the fish skin, and carrying out enzymolysis for 2-24 hours at 40-60 ℃ to obtain the collagen peptide crude extract. Preserving the temperature of the crude extract at 95-100 ℃ for 10-20 min, and inactivating enzyme. And (3) passing the crude extract through an ultrafiltration membrane with the molecular weight cut-off of 1K, centrifugally collecting filtrate, and freeze-drying. The hydrolysis degree is controlled by regulating and controlling the enzymolysis time, and collagen peptide products with average molecular weights ranging from 500D to 15000D can be obtained by ultrafiltration membranes with different molecular weights.
(4) Purification of bioactive small molecule collagen peptide: the collagen peptide powder obtained in the last step is dissolved by ultrapure water to prepare a solution of 10-20 mg/mL, and sephadex Sephadex xG25 molecular sieve chromatography is used for separating and purifying the collagen peptide powder, and the peak components are collected and combined. After measuring the biological activity, selecting the component with the highest activity for freeze drying. And further refined and purified using reverse phase chromatography XTerra Prep MS C18 column, and the peak fractions were collected. Finally, the collagen peptide component with ACE inhibition and antioxidation activities is obtained.
(5) Bacteria and endotoxin removal: freeze drying to obtain collagen peptide powder 60 Co irradiation treatment, endotoxin inactivation, sterilization and packaging to obtain the low-immunogenicity micromolecular collagen polypeptide finished product with blood pressure lowering and antioxidant activities.
Example 2: comparison of antioxidant Activity of cod skin collagen peptides of different average molecular weights
According to the report of the literature, the small molecular weight collagen peptide has better functions of resisting organism and cell peroxidation and the like, and has better development prospect in the fields of food health care and medicine development. The molecular weight of the collagen peptide obtained by the invention is 300-600Da, the average molecular weight is 500Da, and compared with other collagen peptides with larger average molecular weight, the collagen peptide has better antioxidant activity. The antioxidant activity of the collagen peptide products having average molecular weights of 500Da, 1000Da, 3000Da, 7000Da and 15000Da, respectively, obtained by controlling the degree of hydrolysis, was measured.
(1) DPPH radical scavenging Activity
Collagen peptides of the above different molecular weights were dissolved in 2mL deionized water, respectively. To each sample, 2mL of DPPH solution prepared with methanol was added, and after the temperature was kept at 37℃for 30 minutes in an oven, the absorbance was measured. The same volume of sample solution was replaced with 2mL of ethanol solution as a negative control. The characteristic absorbance peak of DPPH radical is 517nm, the change of absorbance at 517nm is detected, and DPPH clearance is calculated according to the following formula.
A 0 The absorbance when no sample was added, and a was the absorbance after the addition of the sample.
DPPH radical scavenging ability of collagen peptide samples of different molecular weights at 1mg/ml concentration is shown in FIG. 1 a. It can be seen that collagen peptides with different molecular weights have better free radical scavenging ability, and the small molecular collagen peptide with the average molecular weight of 500Da is optimal.
(2) ABTS radical scavenging activity
Collagen peptides of the above different average molecular weights were dissolved in 2ml deionized water, respectively. To each sample tube was added 2mL of ABTS solution formulated with potassium disulfate, while a blank set was set. The sample was replaced with 2ml deionized water as a negative control. The characteristic absorption peak of the ABTS radical was 728nm, and the absorbance at 728nm was selected in this experiment and ABTS clearance was calculated according to the following formula.
A 0 The absorbance when no sample was added, and a was the absorbance after the addition of the sample.
ABTS radical scavenging ability of collagen peptide samples of different molecular weights at 1mg/ml concentration is shown in fig. 1 b. It can be seen that all collagen peptides possess a better ABTS radical scavenging ability, with small molecular collagen peptides with an average molecular weight of 500Da being the best.
(3) Superoxide anion radical scavenging activity
The collagen peptides with different molecular weights are respectively dissolved in 2mL of deionized water, and the scavenging capacity of superoxide anion free radicals is measured by adopting an NADH method. 1mL of pre-prepared nitrotetrazolium chloride (NBT) and 1mL of NADH were mixed well, 1mL of collagen peptide solutions of different average molecular weight sizes were added, and then 1mL of Phenazine Methosulfate (PMS) solution was added to the reaction mixture to initiate the reaction. After incubation for 5 minutes at 25 ℃, the absorbance was measured at 560nm for the corresponding blank.
The antioxidant capacity of the collagen peptide samples with different molecular weights at 1mg/ml is shown in FIG. 1c, and it can be seen that the collagen peptides with different molecular weights have better scavenging capacity of superoxide anions than the collagen peptide samples without the collagen peptide, wherein the small molecular peptides with the average molecular weight of 500Da have the best effect on scavenging free radicals.
(4) Lipid peroxidation radical scavenging Activity
Collagen peptides of the above different molecular weights were dissolved in 2mL of deionized water, respectively, and 2mL of collagen peptide solution, 30 μl of linoleic acid, 2mL of absolute ethanol, and 100 μl of tween 20 were added to 870 μl of PBS solution. After preparation, a test tube is wrapped by tinfoil, a light-shielding environment is provided, and the test tube is placed in a constant-temperature water bath kettle at 37 ℃ and incubated for 12 hours. mu.L of the reaction mixture was removed, and 4.75mL of 75% ethanol, 100. Mu.L of 30% ammonium thiocyanate solution, and 100. Mu.L of 20mmol/L ferrous chloride solution (prepared using 3.5% HCl) were sequentially added. And (3) uniformly mixing the added reagent by using a vortex oscillator, and measuring data at 500nm wavelength every 24 hours. Ascorbic acid was used as positive control and deionized water was used as negative control.
The antioxidant capacity of the collagen peptide samples with different molecular weights at 1mg/ml is shown in figure 1d, and compared with the collagen peptide samples without the collagen peptide, the collagen peptide samples with different molecular weights have better lipid peroxide free radical scavenging capacity, and the effect of the collagen peptide samples is equivalent to that of ascorbic acid.
Combining the above various types of radical scavenging experiments, a small molecule peptide with an average molecular weight of 500Da is advantageous over other sizes of collagen peptides.
Example 2: comparison of the ACE enzyme inhibition Effect of cod skin collagen peptides of different average molecular weights
In order to verify the inhibition effect of the collagen peptide on the ACE enzyme activity, an in vitro FAPGG (N- [3- (2-furyl) acryloyl ] -L-phenylalanyl-glycyl-glycine) substrate method is adopted to verify the inhibition effect of the collagen peptide on the ACE enzyme activity. To 100. Mu.L of 0.1U ACE solution, 90. Mu.L of FAPGG solution and 40. Mu.L of collagen peptide solution were added, and absorbance at 340nm was measured immediately with a microplate reader. After incubation at 37℃for 30min, absorbance at 340nm was again measured and ACE inhibition was assessed. As shown in fig. 2, it can be seen that the small molecular collagen peptide having a molecular weight of 500Da has an optimal ACE enzyme inhibitory effect.
Example 3: biological Activity of collagen peptide before and after chromatography purification
(1) And (3) separating the micromolecular collagen peptide obtained by the composite enzymolysis-ultrafiltration coupling process by using a molecular sieve Sephadex G-25 gel. The sample was prepared at a concentration of 20mg/mL, with ultrapure water as elution buffer, at a linear flow rate of 0.8mL/min, and detected at 220nm (FIG. 3). The elution peaks were collected and tested for ACE inhibition and ABTS radical scavenging ability, which showed that the last elution peak, G, was the most active and significantly improved over the sample before purification (Table 1).
TABLE 1 ACE inhibition and ABTS free radical scavenging Capacity of collagen peptide component after molecular sieve purification
(2) The collagen peptide was further purified using reverse phase high performance liquid chromatography. The lyophilized collagen peptide molecular sieve fractions were redissolved in ultrapure water and separated using XTerra Prep MS C18 column from Waters company. At a flow rate of 2mL/min, mobile phase A was 5% acetonitrile in water containing 0.1% TFA, mobile phase B was 90% acetonitrile in water containing 0.1% TFA. The column was equilibrated with 100% solution A for 0-5min, linearly raised to 100% solution B for 5-20min, and the elution peak was detected at 220nm (FIG. 4). The components were collected and tested by ACE inhibition and ABTS radical scavenging experiments, component 3 showed the highest biological activity (table 2).
TABLE 2 ACE inhibition and ABTS free radical scavenging Capacity of collagen peptide component after purification by C18 reverse phase chromatography
Example 4: effect of small molecule collagen peptide gastrointestinal digestion on biological Activity
Dissolving the purified small molecular collagen peptide in 10mL of deionized water, adding 1M HCl prepared in advance to adjust pH=2, adding pepsin (5% w/w), incubating in a shaking water bath kettle at 37 ℃ for 1h, heating the reaction solution to 100 ℃, keeping for 30 minutes, and inactivating enzyme. After cooling to room temperature, 1M NaHCO was added 3 Adjusting pH to 5.3, adding trypsin (5% w/w), adding 2M NaOH to adjust pH=7.5, incubating in a shaking water bath kettle at 37 ℃ for 2 hours, heating the reaction liquid to 100 ℃ for inactivating for 20 minutes, cooling to room temperature, centrifuging at 5000rpm/min, taking supernatant, and detecting ACE inhibition and ABTS free radical scavenging capacity change before and after gastrointestinal digestion of collagen peptide.
As can be seen from fig. 5, the ACE inhibition and free radical scavenging capacity did not change much or increased after simulated gastrointestinal digestion of the collagen peptide before and after purification. It is presumed that the collagen peptide active ingredient is not degraded after being treated with the gastrointestinal digestive juice, or that the degraded small molecular weight oligopeptide is still bioactive.
Example 4: small molecule collagen peptide immunogenicity test
(1) Test of immunogenicity of Small molecule collagen peptides
Referring to the medical industry standard of the people's republic of China, the immunogenicity evaluation method YY/T1465.1-2016 of medical instruments uses an in vitro T lymphocyte transformation experiment to carry out immunogenicity analysis on the collagen peptide after purification and treatment. Fresh sterile spleen cell suspension is taken, and the cell concentration is adjusted to 2X 10 6 cell/mL. The effect of collagen peptide on lymphocyte proliferation was detected using a microplate reader. 200. Mu.L of cell suspensionAdding collagen peptide with final concentration of 5 μg/mL, culturing in a carbon dioxide incubator at 37deg.C for 3 days, centrifuging, removing supernatant, adding thiazole blue (MTT) 50 μl with concentration of 1mg/mL, continuously culturing for 6 hr, adding 150 μL of LDMSO, shaking the plate, detecting absorbance at 570nm wavelength, and referencing wavelength of 630nm. The positive control group used 5. Mu.g/mL Canavalia gladiata.
Experiments prove that the small molecular collagen peptide does not generate specific or non-specific immune response to lymphocytes, and the cell proliferation rate is not higher than that of a negative control group, so that the prepared small molecular collagen peptide does not cause specific or non-specific immune response to lymphocytes of an organism.
(2) Test of cytotoxicity of Small molecule collagen peptide
Small molecule collagen peptide cytotoxicity assays were performed using mouse fibroblast NIH 3T 3. The results show that the small molecular collagen peptide is nontoxic to cells, can significantly promote fibroblast proliferation, and the cell proliferation rate can reach 20% at 72h (figure 6).
(3) Small molecule collagen peptide intradermal stimulation test
Taking micromolecular collagen peptide freeze-dried powder, dissolving the micromolecular collagen peptide freeze-dried powder in 0.9% sodium chloride injection, and oscillating for 5 minutes. After the healthy rabbits are fixed, the fur on the two sides of the spine of the back is cut off, and the fur is carefully removed by using a clipper. The positive control solutions prepared with different concentrations of small molecule collagen peptide solution were injected intradermally with a syringe at the left side of each rabbit spinal column by 0.2mL, and with 0.9% saline and 0.2mL vegetable oil at the right side. The condition of each injection site was recorded after 24 hours, 48 hours and 72 hours after injection. The results show that no erythema and edema appear at the collagen peptide injection site, which indicates that the stimulation of the low-immunogenicity small molecule collagen peptide to the skin meets the test requirements.
(4) Acute systemic toxicity test of small molecular collagen peptide
Healthy mice, a standard diet, were prepared and randomly divided into test and control groups of at least 5 mice each. Small molecule collagen peptide solutions were prepared using 0.9% sodium chloride injection and sterilized by filtration through a 0.22 μm sterile filter. The small molecule collagen peptide solution with different concentration is injected into the rat tail vein at the injection speed of not more than 0.1mL/s. Sterile saline served as control, vegetable oil served as positive control, and was compared to the commercially available collagen peptide product. The status, toxic performance and mortality of all animals were observed and recorded 4, 24, 48 and 72 hours after injection. As a result, it was found that in the 72h observation period, the positive control group showed an obvious toxic reaction, and the collagen peptide injection group showed no more mice reaction than the negative control group, which is superior to the commercial collagen peptide product group, and it was considered that the atelopeptide small molecule collagen peptide did not produce acute systemic toxicity.
Example 5: animal experiment of antihypertensive by small molecular collagen peptide
And constructing a renal artery stenosis type hypertension model mouse, and taking the mice with successful modeling after 28-30 days for testing. Small molecule collagen peptide solutions were prepared using 0.9% sodium chloride injection and sterilized by filtration through a 0.22 μm sterile filter. Mice were randomly grouped and injected with small molecule collagen peptide solution via tail vein, 0.9% physiological saline as negative control, captopril as positive control. And dynamically observing and recording blood pressure waveforms, systolic pressure, diastolic pressure, average pressure and heart rate in real time. The results are shown in Table 3, and the small molecular collagen peptide group and the captopril group have obvious blood pressure reducing effect on mice, and the blood pressure reducing effect of the small molecular collagen peptide is higher than that of the captopril group.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (2)
1. A preparation method of a low-immunogenicity, antihypertensive and antioxidant deep-sea fish skin small-molecule collagen peptide comprises the following steps:
(1) Pretreatment of fish skin: the deep sea fish skin is cleaned by a wire brush, soaked in softened water for 3 hours, repeatedly washed by softened water under high pressure for a plurality of times, and drained. Then shearing the clean fish skin to obtain the fish skin with proper size. Adding sodium hydroxide alkali solution into the fish skin to treat for 12-24 hours, removing fat and foreign protein in the fish skin, and washing the fish skin to be neutral by using softened water;
(2) Terminal peptide removal: the treated fish skin is processed according to the feed liquid ratio of 1: 15-1: 20 is added into 0.5M acetic acid, pepsin with the concentration of 60-100U/mL is added, and the magnetic stirring treatment is carried out for 12-72 h. Adjusting the pH value to be neutral, and dialyzing the reaction solution by using a 100kDa dialysis bag;
(3) The collagen peptide is obtained by a composite enzymolysis-ultrafiltration coupling process: the pH value of the liquid is adjusted to be neutral, and the composite protease with the mass of 1-3% of the fish skin is added for enzymolysis for 2-24 hours at the temperature of 40-60 ℃ to obtain the crude extract of the collagen peptide. Preserving the temperature of the crude extract at 95-100 ℃ for 10-20 min, and inactivating enzyme. And passing through ultrafiltration membrane with molecular weight cutoff of 1kDa, retaining the filtered component, and freeze drying to powder;
(4) Purification of bioactive small molecule collagen peptide: the collagen peptide powder obtained in the last step is dissolved by ultrapure water to prepare a solution of 10-20 mg/mL, and sephadex G25 molecular sieve chromatography is used for separating and purifying the collagen peptide powder, and the peak components are collected and combined. Selecting the component with highest activity, further refining and purifying by using a reversed phase chromatography C18 column, and collecting each peak component. Finally, the collagen peptide component with ACE inhibition activity and antioxidation activity is obtained, and freeze-dried.
(5) Bacteria and endotoxin removal: and performing freeze drying to obtain active micromolecular collagen peptide powder, performing irradiation treatment on the active micromolecular collagen peptide powder by using 60Co, inactivating endotoxin, and sterilizing and packaging to obtain the low-immunogenicity deep sea fish skin micromolecular collagen peptide finished product with blood pressure reducing and antioxidant activities.
Wherein the alkali solution in the step (1) uses sodium hydroxide solution with the concentration of 0.01-0.02M, and the ratio of the fish skin to the alkali solution is 1:10 to 1:20, the treatment time is 12-24 hours.
The acetic acid solution in the step (2) is used with the concentration of 0.5M, and the feed liquid ratio is 1: 15-1: the pepsin concentration is 60-100 IU/mL.
The compound protease in the step (3) uses neutral protease and bromelain, and the mass ratio of the neutral protease to the bromelain is 1: 2-1: 4, the enzymolysis temperature is 40-60 ℃ and the enzymolysis time is 2-24 h. Ultrafiltration membranes use a membrane with a molecular weight cut-off of 1kDa, retaining the filtered components.
In the step (4), molecular sieve chromatography was carried out using a Sephadex G25 packing material and ultrapure water as a mobile phase at a linear flow rate of 0.8ml/min at a wavelength of 220 nm. Reverse phase chromatography using XTerra Prep MS C18 prepared column, mobile phase a as 5% acetonitrile in water with 0.1% tfa, B as 90% acetonitrile in water with 0.1% tfa, equilibrate column with 100% a for 0-5min, eluting with 5-20min, rising linearly to 100% B, and detecting elution peak at 220nm wavelength.
In the step (6), take 60 Co irradiation treatment deactivates endotoxin.
2. The low-immunogenicity small molecular collagen peptide prepared by the preparation method according to claim 1 and application thereof in the fields of food, health care products and medical use.
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