CN111635920A - Method for preparing donkey bone collagen peptide powder by two-stage bionic enzymatic hydrolysis technology - Google Patents
Method for preparing donkey bone collagen peptide powder by two-stage bionic enzymatic hydrolysis technology Download PDFInfo
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- CN111635920A CN111635920A CN202010434230.4A CN202010434230A CN111635920A CN 111635920 A CN111635920 A CN 111635920A CN 202010434230 A CN202010434230 A CN 202010434230A CN 111635920 A CN111635920 A CN 111635920A
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- bone
- enzymolysis
- collagen peptide
- donkey
- peptide powder
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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
-
- 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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- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method for preparing donkey bone collagen peptide powder by a secondary bionic enzymatic hydrolysis technology. The method not only obtains the collagen peptide with bioactivity, but also improves the collagen yield, and the donkey bone collagen peptide obtained by simulating gastrointestinal tract digestion better conforms to the physiological environment of organisms, is easy to absorb and has good biological effect.
Description
Technical Field
The invention relates to the field of extraction of bone collagen peptide, in particular to a method for preparing donkey bone collagen peptide powder by a two-stage bionic enzymatic hydrolysis technology.
Background
China is the country which most nourishes donkeys in the world, the annual stock number is about 1000 thousands, the donkeys have mild temperament, strong tolerance, long service life and small food intake, and the donkeys integrate the three functions of medicine, tonifying and eating. The donkey meat has high protein content, lower fat content than other livestock meat, about 1.1 percent of mineral content and lower cholesterol content, has the characteristics of light and delicious taste, unique flavor and the like, and is used as a delicious dish on dining tables and leisure; donkey hide has high medicinal value since ancient times and is the main raw material for preparing donkey-hide gelatin of "national medicine treasure"; the donkey bone and donkey skin contain the same ossein peptide, so the donkey bone and donkey skin have the same effects of enriching blood, beautifying, regulating intestines and stomach and enhancing immunity as donkey hide, and also have the effects of strengthening tendons and bones, preventing and treating osteoporosis and the like because the donkey bone and donkey skin are bone substances. The donkey bone has high density and excellent bone quality, the content of collagen in the bone is high, and the donkey bone collagen peptide is a very suitable small molecular bone collagen peptide extraction raw material, so that a natural energy treasure house is provided for the extraction of active small molecular peptide, and the development of donkey bone collagen peptide also becomes a direction for improving the added value of the donkey industry.
The collagen peptide has high health care effect because the collagen peptide is a micromolecular protein, compared with common collagen, the collagen peptide has higher absorption rate, the absorption utilization rate of edible collagen peptide can reach 100 percent, and the collagen peptide can be actively absorbed by human body without barriers and can be fully utilized by body tissues. After being fully absorbed and utilized by a human body, the collagen can be synthesized into skin collagen to play a certain beautifying role, and the collagen has very good hydrophilicity and can provide a plurality of whitening, moisturizing and nourishing effects for the skin, so that the aims of improving the skin quality and beautifying are fulfilled; the collagen can promote the formation of bones and strengthen the collagen structure under the low calcium level, thereby improving the bone strength and achieving the effect of preventing osteoporosis; the collagen peptide keeps the muscles and bones soft and elastic, strengthens the lubrication degree of the muscles and bones during movement and friction, reduces the incidence rate of arthritis and can avoid joint degeneration; the collagen can improve anxiety and insomnia states and treat neurasthenia; the collagen is helpful for strengthening the functions of various tissues and organs and improving the immunity of the human body.
With the development of the donkey industry, people pay more and more attention to the donkey industry, the bone collagen peptide which simulates the artificial gastrointestinal environment and is prepared by adopting the bionic enzymolysis technology and suitable for being absorbed by human bodies is provided, and the problems of dyspepsia and abdominal distension, poor digestion and absorption and the like caused by poor spleen and stomach, weakness and poor digestion function of the people who are not good in spleen and stomach due to large molecular weight and high viscosity of the collagen are reduced.
Disclosure of Invention
In order to solve the problems, the invention provides a method for preparing donkey bone collagen peptide powder by a two-stage bionic enzymatic hydrolysis technology, which comprises the steps of crushing donkey bones, degreasing, simulating the physiological environment conditions of human stomach and intestines, and producing donkey bone collagen peptide which is more in line with the absorption and biological effects of a human body by adopting a two-stage biological enzymatic hydrolysis production process. The method not only obtains the collagen peptide with bioactivity, but also improves the collagen yield, and the donkey bone collagen peptide obtained by simulating gastrointestinal tract digestion better conforms to the physiological environment of organisms, is easy to absorb and has good biological effect.
In order to achieve the purpose of the present invention, the present invention provides a preparation method of donkey bone collagen peptide powder, comprising the following steps: ultrasonic extraction, high-pressure homogenization, primary biological enzymolysis and secondary biological enzymolysis.
The invention has the beneficial effects that:
firstly, the method simulates the physiological environment of the stomach and the intestine of a human, and the donkey bone collagen peptide is prepared by adopting a two-stage bionic enzymolysis technology, so that the bone collagen peptide which is more in line with the absorption and biological effects of the human body can be produced, and the absorption utilization rate of the collagen peptide is improved.
Secondly, the donkey bone collagen peptide prepared by the method not only ensures the bioactivity of the effective components of the product, enlarges the application range and the use effect of the product, but also avoids worrying about toxic and side effects.
Thirdly, the method puts forward the weak links of the animal husbandry and the breeding industry in China at present, and has the important effect of solving the practical production difficulty.
Fourthly, the application range of the donkey bone is widened, the utilization rate and the added value of the donkey bone are improved, the application direction and the value of the product are increased, and the donkey bone has good market prospect.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
Fig. 1 shows a flow chart of the method for preparing donkey bone collagen peptide powder by the two-stage bionic enzymatic hydrolysis technology.
FIGS. 2-1, 2-2 and 2-3 show the results of measuring the content of collagen peptide and amino acid in the donkey bone collagen peptide powder according to the present invention. Wherein, figure 2-1 shows the donkey bone collagen peptide amino acid content determination map; fig. 2-2 shows the measurement result of proline (Pro) content in donkey bone collagen peptide (λ ═ 440 nm); fig. 2 to 3 show the results of measuring the content of 16 amino acids such as aspartic acid (Asp) in the donkey bone collagen peptide (λ 570 nm).
FIG. 3 shows the result of SDS-PAGE electrophoresis of donkey bone collagen peptide, wherein, lane 1 is protein MARKER; lane 2 is the donkey bone collagen peptide prepared in example 1; lane 3 is yak bone peptide; lane 4 is the donkey bone collagen peptide prepared in example 1. Lanes 2 and 4 are both donkey bone collagen peptide prepared in example 1, and 2 samples are spotted for parallel experiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more clearly shown, embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.
As shown in fig. 1, the preparation method of the donkey bone collagen peptide powder provided by the invention comprises the following steps: bone paste preparation (bone breaking, crushing and degreasing), ultrasonic extraction, high-pressure homogenization, filtration, primary biological enzymolysis, secondary biological enzymolysis, centrifugation, enzymatic hydrolysate collection, molecular interception, filtration, concentration, drying and packaging to obtain a finished product.
The method for preparing the donkey bone collagen peptide simulates the physiological environment of the stomach and the intestine of a human body, and the donkey bone collagen peptide is prepared by adopting a two-stage bionic enzymolysis technology, so that the donkey bone collagen peptide which is more in line with the absorption and biological effects of the human body can be produced, and the absorption utilization rate of the collagen peptide is improved; not only ensures the biological activity of the effective components of the product, enlarges the application range and the use effect of the product, but also avoids worrying about the generation of toxic and side effects
The invention is realized by the following technical scheme:
the preparation method of the donkey bone collagen peptide powder is characterized by comprising the following steps: ultrasonic extraction, high-pressure homogenization, primary biological enzymolysis and secondary biological enzymolysis.
In a preferred embodiment of the invention, the extraction process further comprises a step selected from one or more of: breaking bone, crushing, degreasing, filtering, collecting enzymolysis liquid, intercepting molecules, filtering, concentrating, drying and packaging to obtain a finished product.
In a preferred embodiment of the present invention, the preparation method comprises the steps of:
(1) osteoclasts;
(2) crushing;
(3) degreasing;
(4) ultrasonic extraction;
(5) homogenizing under high pressure;
(6) filtering;
(7) first-order biological enzymolysis (simulating artificial gastric juice);
(8) secondary biological enzymolysis (simulating human factory intestinal juice);
(9) and collecting the enzymolysis liquid.
In a preferred embodiment of the present invention, in the step (1) osteoclasts, the donkey bone is broken to obtain a broken donkey bone; preferably, the donkey bone is firstly crushed and then cleaned, and the crushed and cleaned donkey bone is obtained; preferably, the donkey bones are crushed into blocks of 2-6 cm; preferably, the washing is performed several times, preferably 2 times, with purified water.
In a specific embodiment of the present invention, in the crushing in the step (2), the crushed and washed donkey bone obtained in the step (1) is ground into bone cement by using bone cement to obtain processed bone cement; preferably, the bone paste contains 60-75% of water, and the fineness of the bone paste is 60-80 meshes; more preferably, the bone paste contains 70% of water, and the fineness of the bone paste is 80 meshes.
In a specific embodiment of the present invention, in the step (3), in the degreasing, multiple volumes of water are added to the treated bone paste obtained in the step (2), and the mixture is cooked, cooled and degreased to obtain degreased bone paste and cooking liquor; preferably, 2.0-5.0 times of water is added, and more preferably, purified water is added; preferably, the mixture is cooked for 2 to 4 hours; preferably, the cooking is carried out in a high-pressure cooking kettle at the temperature of 121 ℃ and under the pressure of 0.2 MPa; preferably, the temperature is cooled at-10 ℃ to 0 ℃; more preferably, 2.5 times of purified water is added into the treated bone cement, and the bone cement is cooked for 2 hours and cooled to remove fat at the temperature of minus 5 ℃.
In a preferred embodiment of the present invention, in the step (4) of ultrasonic extraction, the degreased bone paste obtained in the step (3) and the cooking liquor are extracted in an ultrasonic extractor to obtain an ultrasonic extract.
In a specific embodiment of the present invention, the ultrasonic power is 500-.
In one embodiment of the invention, the extraction is carried out under stirring, preferably at a stirring speed of 1200 to 2000r/min, more preferably at a stirring speed of 1500 r/min.
In a specific embodiment of the invention, the ultrasonic extraction time is 20-50min, and more preferably, the ultrasonic extraction time is 30 min.
In a specific embodiment of the invention, the ultrasonic power is 650w/L, the stirring speed is 1500r/min, and the ultrasonic time is 30 min.
In a preferred embodiment of the present invention, in the step (5) of high-pressure homogenization, the ultrasonic extract obtained in the step (4) is centrifuged to obtain a supernatant; taking the supernatant fluid to carry out homogenizing and shearing in a high-pressure homogenizer to obtain homogenized fluid after high-pressure homogenization.
In a specific embodiment of the present invention, the homogenization is repeated 3 to 5 times, and more preferably, the homogenization is repeated 3 times.
In one embodiment of the invention, centrifugation is carried out at a temperature of 2 to 4 ℃ for 10 to 30 min.
In a specific embodiment of the present invention, the centrifugation rotation speed is 2000-.
In a particular embodiment of the invention, the homogeneous shearing is carried out at a shearing pressure of 5000-7000Pa, preferably 6000 Pa;
in one embodiment of the invention, the homogenizing shear is carried out at a homogenizing rate of 300-700ml/min, preferably 500 ml/min.
In a specific embodiment of the invention, the ultrasonic extract is centrifuged for 20min at 4000r/min at 2-4 ℃ in a centrifuge to obtain a supernatant; taking the supernatant, homogenizing and shearing in a high-pressure homogenizer at shearing pressure of 5000-7000Pa and homogenizing speed of 500ml/min, and repeating for 3 times.
In a preferred embodiment of the present invention, in the step (6) filtration, the homogenized liquid obtained in the step (5) after the high-pressure homogenization is filtered.
In one embodiment of the invention, the filtration is carried out in 5 μm, 3 μm, 1 μm stages and the filtrate is collected.
In a preferred embodiment of the invention, in the step (7), in the first-stage biological enzymolysis (simulated artificial gastric juice), the filtrate obtained in the step (6) is adjusted to a pH value of between 1.5 and 2.5 by acid or alkali, preferably, the pH value is 2.0; adding pepsin (10 ten thousand U/g) according to the mass percent of 1.6-2.5% of the bone mud feeding amount, carrying out enzymolysis for 2.0-2.5 h at 37 +/-0.5 ℃; obtaining enzymolysis liquid after the first-stage biological enzymolysis.
In a particular embodiment of the invention, the acid is selected from one or more of hydrochloric acid, phosphoric acid, sulfuric acid, oxalic acid, acetic acid and citric acid, preferably HCl.
In a particular embodiment of the invention, the base is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate and sodium bicarbonate and triethanolamine, preferably NaOH.
In a particular embodiment of the invention, the pH is adjusted with 2.0mol/L HCl and/or 2mol/L NaOH.
In a particular embodiment of the invention, 2.0% pepsin (10 ten thousand U/g) is added; preferably, enzymolysis is carried out for 2.0 h-2.5 h at 37 ℃ +/-0.5 ℃.
In a specific embodiment of the invention, heating to 90-100 ℃ to inactivate enzyme to obtain enzymolysis liquid after primary biological enzymolysis; preferably, the enzyme is inactivated for 15-20 min, more preferably, the enzyme is inactivated for 15 min.
In a preferred embodiment of the invention, in the secondary biological enzymolysis (simulating human factory intestinal juice) in step (8), the pH value of the enzymolysis solution obtained after the primary biological enzymolysis in step (7) is adjusted to 7.5-8.5 by using a buffer solution, preferably, the pH value is adjusted to 8.0; adding trypsin (4000U/g) according to the amount which is 1.6-2.5 percent of the mass percentage of the bone mud; obtaining enzymolysis liquid after the second-stage biological enzymolysis.
In a particular embodiment of the invention, the buffer is a phosphate buffer.
In a particular embodiment of the invention, 2.0% pancreatin (4000U/g) is added; more preferably, enzymolysis is carried out for 2.0 h-2.5 h at 50 ℃ +/-0.5 ℃.
In a specific embodiment of the invention, heating to 90-100 ℃ to inactivate enzyme to obtain enzymolysis liquid after secondary biological enzymolysis; preferably, the enzyme is inactivated for 15-20 min, more preferably, the enzyme is inactivated for 15 min.
In a preferred embodiment of the present invention, in the step (9), collecting the enzymolysis liquid, centrifuging the enzymolysis liquid obtained in the step (8) after the secondary biological enzymolysis in a centrifuge, and collecting the enzymolysis supernatant; preferably, the centrifugation is carried out for 20-50min at 2-4 ℃ and 6000-8000 r/min, more preferably, the centrifugation is carried out for 30min at 7000 r/min.
In one embodiment of the present invention, the preparation method further comprises the step (10) of molecular entrapment: classifying and intercepting the polypeptides with different molecular weights in a hollow fiber ultrafilter from the collected enzymolysis supernatant obtained in the step (9) to obtain intercepted polypeptide liquids with different molecular weight ranges; preferably, the molecular cut-off ranges are 8000Da, 5000Da, 3000Da, 1000 Da.
In a specific embodiment of the present invention, the preparation method further comprises the step (11) of filtering: filtering the polypeptide liquid with different molecular weight ranges obtained in the step (10) through a 0.1 mu m filter to obtain filtrate.
In a specific embodiment of the present invention, the preparation method further comprises the step (12) of concentrating and drying: adjusting the pH value of the filtrate obtained in the step (11) to be neutral, then concentrating, and sending the filtrate to a concentrated solution storage tank when the Baume degree is 15-20 ℃; conveying the protein peptide stock solution in the concentrated solution storage tank to a centrifugal sprayer, atomizing the liquid, and drying the liquid into powder by using high temperature in a drying tower; preferably, the pH is adjusted by 2.0mol/L HCl and/or 2mol/L NaOH.
In a specific embodiment of the present invention, the preparation method further comprises the step (13) of packaging: drying to obtain peptide powder, and packaging to obtain the final product.
Example 1 preparation of donkey bone collagen peptide
TABLE 1 reagents and instruments for preparation of donkey bone collagen peptide
| Reagent/instrument | Model/specification | Manufacturer of the product |
| Bone breaking machine | PG-300 | Mechanical spring city food factory |
| Bone mud mill | GN-80 | Mechanical spring city food factory |
| Ultrasonic extractor | Scientz-50T | NINGBO SCIENTZ BIOTECHNOLOGY Co.,Ltd. |
| High-speed refrigerated centrifuge | TGC-16M | HUNAN MICHAEL EXPERIMENTAL APPARATUS Co.,Ltd. |
| High-pressure homogenizer | ATS-AH08-100 | ANTOS NANO TECHNOLOGY (SUZHOU) Co.,Ltd. |
| Pepsin | 9001-75-6 | solarbio |
| Trypsin | T8151 | solarbio |
The preparation method of the donkey bone collagen peptide comprises the following steps:
1. osteoclast: taking 10kg of fresh donkey bone, crushing the donkey bone into small bone residues of about 2cm in an osteoclast machine, then sending the donkey bone into a cleaning machine, and cleaning for 2 times by using purified water to obtain the crushed and cleaned donkey bone.
2. Crushing: grinding the crushed and cleaned donkey bone with bone cement to form bone cement, and obtaining treated bone cement; wherein the bone paste contains 70% of water, and the fineness of the bone paste is 80 meshes.
3. Degreasing: adding 2.5 times of purified water into the treated bone mud, and stewing in a high-pressure stewing kettle at 121 ℃ and 0.2Mpa for 2h at-5 ℃ for low-temperature cooling and degreasing to obtain degreased bone mud and a stewing liquid.
4. Ultrasonic extraction: ultrasonically extracting the degreased bone mud and the cooking liquor in an ultrasonic extractor for 30min to obtain an ultrasonic extract, wherein the ultrasonic power is 650w/L, and the stirring speed is 1500 r/min.
5. High-pressure homogenization: centrifuging the ultrasonic extract in a refrigerated centrifuge at 4000r/min at 2-4 ℃ for 20 min; taking the supernatant, homogenizing and shearing in a high-pressure homogenizer at shearing pressure of 5000-7000Pa and homogenizing speed of 500ml/min for 3 times to obtain homogenized liquid after high-pressure homogenization.
6. And (3) filtering: the homogenized solution after high pressure homogenization is filtered in a grading way at 5 mu m, 3 mu m and 1 mu m, and the filtrate is collected.
7. First-order biological enzymolysis (simulated artificial gastric juice): and (3) adjusting the pH value of the obtained filtrate to 2.0 by using 2.0mol/L HCl and 2mol/L NaOH, adding 200g of pepsin (10 ten thousand U/g) according to the amount of 2.0 percent of the material percentage, namely the mass percentage of the bone paste, and carrying out enzymolysis for 2.0-2.5 h at the temperature of 37 +/-0.5 ℃. Heating to 100 deg.C, inactivating enzyme for 15min to obtain enzymatic hydrolysate after first-stage biological enzymolysis.
8. Secondary biological enzymolysis (simulating human factory intestinal juice): and (2) adjusting the pH value of the enzymatic hydrolysate subjected to the primary biological enzymolysis to 7.5-8.0 by using a phosphate buffer solution, adding 200g of trypsin (4000U/g) according to the material percentage which is 2.0% of the mass percentage of the bone mud obtained in the step (2), and carrying out enzymolysis for 2.0-2.5 h at 50 +/-0.5 ℃. Heating to 100 deg.C, inactivating enzyme for 15min to obtain enzymatic hydrolysate after secondary biological enzymolysis.
9. Centrifuging and collecting enzymolysis liquid: and (3) centrifuging the enzymolysis liquid after the secondary biological enzymolysis in a high-capacity low-temperature centrifuge at the temperature of 2-4 ℃ at 7000r/min for 30min, collecting enzymolysis supernatant, and storing in a liquid storage tank.
10. Molecular interception: and (3) putting the collected enzymolysis supernate into a hollow fiber ultrafilter, and intercepting polypeptide liquid with the molecular weight of below 5000 Da.
11. And (3) filtering: the trapped polypeptide solution was filtered through a 0.1 μm filter to obtain a filtrate.
12. Concentrating and drying: and (3) adjusting the pH value of the filtrate to be neutral through 2.0mol/L HCl and 2mol/L NaOH, then conveying the filtrate to a concentrator for concentration, and conveying the filtrate to a concentrated solution storage tank when the Baume degree is 15-20 degrees. And (3) conveying the protein peptide stock solution in the concentrated solution storage tank to a centrifugal sprayer through a pipeline, atomizing the liquid through the centrifugal sprayer rotating at a high speed, and instantly drying the liquid into powder by utilizing high temperature in a drying tower.
13. Packaging: drying into powder peptide powder, quantitatively packaging with a specific packaging bag at a ratio of 250 g/bag, and packaging to obtain the final product.
Example 2 donkey bone collagen peptide and amino acid content determination thereof
The donkey bone collagen peptide content (Folin phenol method) and the amino acid content (amino acid analyzer) obtained by the method of example 1 were measured.
TABLE 2 detection reagent and instrument for small molecule peptide content and amino acid content
1. The specific method for measuring the content of the donkey bone collagen peptide comprises the following steps:
I. preparation of control solutions
Bovine serum albumin control (batch No. PB10056)0.30g was weighed precisely, and water was added to 100ml to prepare a solution containing bovine serum albumin 0.03mg per 1.0 ml.
Preparation of sample to be examined
0.5g of donkey bone collagen peptide powder prepared in example 1 was taken, and water was added to 100 ml. Precisely taking 1ml of protein peptide solution into a 25-volume flask, adding water to a constant volume of 25ml, and taking 3 samples to be detected in parallel.
Preparation of the Standard Curve
Precisely measuring 0.0 ml, 0.1 ml, 0.3 ml, 0.5 ml, 0.7 ml and 0.9ml of the reference substance solution prepared in the step I, respectively placing the reference substance solution in test tubes with scales, adding water to 1ml, respectively adding 1ml of alkaline copper solution (mixed solution of copper sulfate pentahydrate, potassium tartrate and sodium hydroxide), shaking uniformly, adding 4ml of the test solution of Folin phenol, immediately mixing uniformly, placing in a water bath at 55 ℃ for 10 minutes, and measuring the absorbance at the wavelength of 650 nm; meanwhile, a No. 0 tube is used as a blank control, the absorbance is used as a vertical coordinate, and the concentration of the control solution is used as a horizontal coordinate to draw a standard curve.
And IV, precisely measuring 1.0ml of the collagen peptide sample to be detected prepared in the step II, measuring the concentration of the sample solution to be detected from the standard curve according to a method under the preparation item of the standard curve in the step III from the addition of the alkaline copper test solution, and multiplying the concentration by the dilution times to obtain the polypeptide content of the collagen peptide solution prepared in the example 1.
V. the results are shown in Table 3. As can be seen from Table 3, the content of the marker polypeptide in the donkey bone collagen peptide powder obtained by the invention is more than or equal to 550 mg/g.
TABLE 3 detection results of donkey bone collagen peptide content and amino acid content
2. The specific method for measuring the amino acid content of the donkey bone collagen peptide comprises the following steps:
I. solution preparation:
a.6mol/L hydrochloric acid, concentrated hydrochloric acid and water 1+ 1.
B. Buffer solution preparing table
TABLE 4 buffer solution preparation table
Wherein pH indicates that the corresponding reagent solution is also used for adjusting the pH value of the buffer solution, and the pH value of the buffer solution a is 3.45; the pH value of the buffer solution B is 10.85; the pH of the sample dilution was 2.20.
C. Preparation of ninhydrin solution
1 liter potassium sodium buffer formulation (pH 5.51): 272.0g of sodium acetate trihydrate; 196.0g of potassium acetate; 200ml of acetic acid; deionized water was made to a volume of 1 liter.
Formulation of 1 l ninhydrin solution: 20g of ninhydrin; 2g of phenol; 600ml of methanol; 400ml of potassium-sodium buffer solution; 0.2g ascorbic acid was added per 1000ml ninhydrin solution. The addition method of the reducing agent comprises the following steps: pouring the prepared ninhydrin into a ninhydrin reagent bottle, blowing nitrogen from the bottom of the bottle for 3 minutes, adding a reducing agent, shaking up, blowing nitrogen from the bottom of the bottle for 3 minutes, covering a ninhydrin reagent bottle cover, closing a bottom blowing valve and an air outlet valve, and opening an air inlet valve and an air outlet valve of a protective gas.
Instruments and equipment: a vacuum pump, a constant temperature drying box, a hydrolysis tube (a pressure-resistant screw cap glass tube or a hard glass tube, the volume is 20-30 mL, the hydrolysis tube is washed clean by deionized water and dried), a vacuum drier (the temperature can be adjusted), and an amino acid automatic analyzer
Sample treatment
A. Accurately weighing 0.0200g of donkey bone collagen peptide powder obtained in example 1, and placing the donkey bone collagen peptide powder in a hydrolysis tube; adding 20mL of 6mol/L hydrochloric acid into a hydrolysis tube, adding 3-4 drops of newly distilled phenol, putting the hydrolysis tube into a refrigerant, freezing for 3-5 min, connecting to an exhaust tube of a vacuum pump, vacuumizing (nearly 0Pa), and then filling high-purity nitrogen; vacuumizing and filling nitrogen again, repeating the steps for three times, and sealing or screwing down the screw cap in the nitrogen filling state. And (3) placing the sealed hydrolysis tube in a constant-temperature drying box at 110 +/-1 ℃, hydrolyzing for 22h, taking out and cooling.
B. And opening the hydrolysis tube, filtering the hydrolysate, washing the hydrolysis tube with deionized water for multiple times, and transferring all the hydrolysate to a 50mL volumetric flask for constant volume with the deionized water. Sucking 1mL of filtrate into a 5mL volumetric flask, drying at 40-50 ℃ by using a vacuum dryer, dissolving the residue with 1-2 mL of water, drying again, repeating the steps twice, evaporating to dryness, dissolving with 1mL of sample diluent, and filtering by using a 0.45-micron filter membrane for measurement by an instrument.
Measurement of
0.200mL of the mixed amino acid standard was accurately aspirated, and the sample was diluted to 5mL with a sample diluent of 5.00 nmol/50. mu.L, and the amino acid content of the sample measurement solution was measured by a external standard method using an automatic amino acid analyzer as an amino acid standard for on-machine measurement.
V. the detection results are shown in figure 2-1, figure 2-2 and figure 2-3. The result shows that the donkey bone collagen peptide powder obtained by the invention contains 17 amino acids: pro, Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Phe, His, Lys and Arg, wherein the amino acid comprises 7 essential amino acids, and the total amount of the amino acid is more than or equal to 800 mg/g.
Example 3 measurement of molecular weight of donkey bone collagen peptide
The molecular weight range of the donkey bone collagen peptide obtained in example 1 was determined by SDS-PAGE and compared with that of a commercially available yak bone peptide powder (batch No. 20171003, manufacturer: national peptide Biotech (Beijing) Co., Ltd.).
TABLE 4 collagen peptide molecular weight detection reagent and instrument
The specific experimental steps are as follows:
1. glue making
TABLE 5
The glue volume ratio of 3 glues is about: 4:1.5:1, preparing the separation glue and the interlayer glue, carefully injecting purified water to seal the glue, pouring out the covering water layer after the glue is completely polymerized, and then sucking residual water by using filter paper.
2. Sample treatment: after the sample was concentrated, 1ml of the loading buffer was added and boiled in hot water for 5 minutes.
3. Sample application: the mark (marker) and the sample are added into each spot by 15 mu l, which contains about 15 mu g of small molecule peptide.
4. Glue running: the first three hours voltage 50(20-30mA), the second two hours 90(30 mA).
5. Fixing: adding the glue into the fixing solution, fixing for 20 minutes, and repeatedly cleaning with purified water.
6. Dyeing: staining solution was added for overnight staining.
7. And (3) decoloring: discarding the staining solution, adding a decolorizing solution, decolorizing for 40 min on a shaking table (60 revolutions), and repeatedly cleaning with purified water.
8. Protein bands were visualized.
9. Preparing a reagent:
9.1 acrylamide: acrylamide 48g, N, N-methylene-bisacrylamide 1.5g, and water was added to make the volume to 100 ml.
9.2 gel buffer: 0.3g of SDS and 36.4g of Tris alkali (Tris-base), adding water to a constant volume of 100ml, and adjusting the pH value to 8.45 by hydrochloric acid to obtain the finished product.
9.3 Anode buffer: 12.11g of Tris alkali (Tris-base), adding water to a constant volume of 500ml, and adjusting the pH value to 8.9 by hydrochloric acid to obtain the finished product.
9.4 cathode buffer: 6.06g of Tris alkali (Tris-base), 8.96g of Tricine and 0.5g of SDS, and adding water to a constant volume of 500ml to obtain the finished product.
9.5 Loading buffer: SDS 0.8g, glycerin 4g, beta-mercaptoethanol 0.2g, bromophenol blue 0.01g, add 0.5mol/LTris-HClpH6.8 to dissolve to 10ml, get final product.
9.6 stationary liquid: 50ml of ethanol, 10ml of glacial acetic acid and 40ml of water are mixed evenly to obtain the product.
9.7 staining solution: 4ml of phosphoric acid, 20g of ammonium sulfate, 2500.05g of Coomassie brilliant blue and 50ml of ethanol, and adding water to a constant volume of 250ml to obtain the compound.
9.8 decolorizing solution: 45ml of ethanol, 10ml of glacial acetic acid and 45ml of water are mixed evenly to obtain the product.
9.910% APS: 1g of ammonium persulfate is dissolved in 10ml of water.
10. As a result:
as shown in the SDS-PAGE electrophoresis result of the donkey bone collagen peptide shown in figure 3, the molecular weight of the donkey bone collagen peptide obtained by the invention is about 5000D after molecular interception, and a band is obvious, and the molecular weight of the yak bone collagen peptide is between 5000 and 15000 Da. Therefore, after the molecular interception, the molecular weight of the donkey bone collagen peptide is more concentrated than that of the yak bone peptide and is lower than that of the yak bone peptide, and the biological activity of the donkey bone collagen peptide is equivalent to or higher than that of the yak bone peptide.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. The preparation method of the donkey bone collagen peptide powder is characterized by comprising the following steps: ultrasonic extraction, high-pressure homogenization, primary biological enzymolysis and secondary biological enzymolysis.
2. The method for preparing donkey bone collagen peptide powder according to claim 1, characterized in that the extraction process further comprises one or more steps selected from the following: breaking bone, crushing, degreasing, filtering, collecting enzymolysis liquid, intercepting molecules, filtering, concentrating, drying and packaging to obtain a finished product.
3. The preparation method of donkey bone collagen peptide powder according to claim 1 or 2, characterized in that it comprises the following steps:
(1) osteoclasts;
(2) crushing;
(3) degreasing;
(4) ultrasonic extraction;
(5) homogenizing under high pressure;
(6) filtering;
(7) first-order biological enzymolysis (simulating artificial gastric juice);
(8) secondary biological enzymolysis (simulating human factory intestinal juice);
(9) and collecting the enzymolysis liquid.
4. The method for preparing donkey bone collagen peptide powder according to any one of claims 1 to 3, wherein in the step (1) osteoclasts, donkey bones are broken to obtain broken donkey bones; preferably, the donkey bone is firstly crushed and then cleaned, and the crushed and cleaned donkey bone is obtained; preferably, the donkey bones are crushed into blocks of 2-6 cm; preferably, washing with purified water is performed a plurality of times, preferably 2 times;
preferably, in the crushing in the step (2), the crushed and cleaned donkey bone obtained in the step (1) is ground into bone cement by using bone cement mill, so as to obtain processed bone cement; preferably, the bone paste contains 60-75% of water, and the fineness of the bone paste is 60-80 meshes; more preferably, the bone paste contains 70% of water, and the fineness of the bone paste is 80 meshes;
preferably, in the step (3), in the degreasing, multiple volumes of water are added into the treated bone paste obtained in the step (2), and the mixture is cooked, cooled and degreased to obtain degreased bone paste and cooking liquor; preferably, 2.0-5.0 times of water is added, and more preferably, purified water is added; preferably, the mixture is cooked for 2 to 4 hours; preferably, the cooking is carried out in a high-pressure cooking kettle at the temperature of 121 ℃ and under the pressure of 0.2 MPa; preferably, the temperature is cooled at-10 ℃ to 0 ℃; more preferably, 2.5 times of purified water is added into the treated bone cement, and the bone cement is cooked for 2 hours and cooled to remove fat at the temperature of minus 5 ℃.
5. The method for preparing donkey bone collagen peptide powder according to any one of claims 1 to 4, characterized in that in the step (4) of ultrasonic extraction, the degreased bone paste obtained in the step (3) and the cooking liquor are extracted in an ultrasonic extractor to obtain an ultrasonic extract;
preferably, the ultrasonic power is 500-;
preferably, the extraction is carried out under stirring, preferably, the stirring speed is 1200-2000 r/min, and more preferably, the stirring speed is 1500 r/min;
preferably, the ultrasonic extraction time is 20-50min, and more preferably, the ultrasonic extraction time is 30 min;
more preferably, the ultrasonic power is 650w/L, the stirring speed is 1500r/min, and the ultrasonic time is 30 min.
6. The method for preparing donkey bone collagen peptide powder according to any one of claims 1 to 5, characterized in that, in the step (5) of high pressure homogenization, the ultrasonic extract obtained in the step (4) is centrifuged to obtain supernatant; taking the supernatant fluid to carry out homogenizing and shearing in a high-pressure homogenizer to obtain homogenized fluid after high-pressure homogenization;
preferably, repeatedly homogenizing for 3-5 times, more preferably, repeatedly homogenizing for 3 times;
preferably, centrifuging for 10-30 min at the temperature of 2-4 ℃;
preferably, the centrifugal rotation speed is 2000-6000r/min, more preferably, the centrifugal rotation speed is 4000 r/min;
preferably, the homogeneous shearing is carried out at a shearing pressure of 5000-7000Pa, preferably 6000 Pa;
preferably, the homogenizing shear is carried out at a homogenizing speed of 300-700ml/min, preferably 500 ml/min;
more preferably, the ultrasonic extract is placed in a centrifuge, and centrifuged for 20min at the temperature of 2-4 ℃ and 4000r/min to obtain a supernatant; taking the supernatant, homogenizing and shearing in a high-pressure homogenizer at shearing pressure of 5000-7000Pa and homogenizing speed of 500ml/min, and repeating for 3 times.
7. The method for preparing donkey bone collagen peptide powder according to any one of claims 1 to 6, characterized in that in the step (6) filtration, the homogenized liquid obtained in the step (5) after high pressure homogenization is filtered;
preferably, the filtration is carried out in 5 μm, 3 μm, 1 μm stages and the filtrate is collected.
8. The preparation method of donkey bone collagen peptide powder according to any one of claims 1 to 7, characterized in that, in the step (7) of first-stage biological enzymolysis (simulated artificial gastric juice), the pH value of the filtrate obtained in the step (6) is adjusted to 1.5-2.5 by acid or alkali, preferably the pH value is 2.0; adding pepsin (10 ten thousand U/g) according to the mass percent of 1.6-2.5% of the bone mud feeding amount, carrying out enzymolysis for 2.0-2.5 h at 37 +/-0.5 ℃; obtaining enzymolysis liquid after primary biological enzymolysis;
preferably, the acid is selected from one or more of hydrochloric acid, phosphoric acid, sulphuric acid, oxalic acid, acetic acid and citric acid, preferably HCl;
preferably, the base is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate and sodium bicarbonate and triethanolamine, preferably NaOH;
more preferably, the pH is adjusted with 2.0mol/L HCl and/or 2mol/L NaOH;
preferably, 2.0% pepsin (10 ten thousand U/g) is added; preferably, enzymolysis is carried out for 2.0 h-2.5 h at 37 +/-0.5 ℃;
preferably, heating to 90-100 ℃ to inactivate the enzyme to obtain enzymatic hydrolysate after primary biological enzymolysis; preferably, the enzyme is inactivated for 15-20 min, more preferably, the enzyme is inactivated for 15 min.
9. The method for preparing donkey bone collagen peptide powder according to any one of claims 1 to 8, characterized in that in the secondary biological enzymolysis (simulating human factory intestinal juice) in the step (8), the pH value of the enzymolysis liquid obtained in the step (7) after the primary biological enzymolysis is adjusted to 7.5-8.5 by using a buffer solution, preferably, the pH value is adjusted to 8.0; adding trypsin (4000U/g) according to the amount which is 1.6-2.5 percent of the mass percentage of the bone mud; obtaining enzymolysis liquid after secondary biological enzymolysis;
preferably, the buffer is a phosphate buffer;
preferably, 2.0% pancreatin (4000U/g) is added; more preferably, enzymolysis is carried out for 2.0 h-2.5 h at 50 ℃ +/-0.5 ℃;
preferably, heating to 90-100 ℃ to inactivate the enzyme to obtain enzymatic hydrolysate after secondary biological enzymolysis; preferably, the enzyme is inactivated for 15-20 min, more preferably, the enzyme is inactivated for 15 min.
10. The method for preparing donkey bone collagen peptide powder according to any one of claims 1 to 9, characterized in that, in the step (9), the enzymolysis liquid obtained in the step (8) after the secondary biological enzymolysis is centrifuged in a centrifuge, and the enzymolysis supernatant is collected; preferably, centrifuging at 2-4 ℃ and 6000-8000 r/min for 20-50min, more preferably at 7000r/min for 30 min;
preferably, the preparation method further comprises the step (10) of molecular entrapment: classifying and intercepting the polypeptides with different molecular weights in a hollow fiber ultrafilter from the collected enzymolysis supernatant obtained in the step (9) to obtain intercepted polypeptide liquids with different molecular weight ranges; preferably, the molecular cut-off range is 8000Da, 5000Da, 3000Da, 1000 Da;
preferably, the preparation method further comprises the step (11) of filtering: filtering the polypeptide liquid with different molecular weight ranges intercepted in the step (10) by a 0.1 mu m filter to obtain filtrate;
preferably, the preparation method further comprises the step (12) of concentrating and drying: adjusting the pH value of the filtrate obtained in the step (11) to be neutral, then concentrating, and sending the filtrate to a concentrated solution storage tank when the Baume degree is 15-20 ℃; conveying the protein peptide stock solution in the concentrated solution storage tank to a centrifugal sprayer, atomizing the liquid, and drying the liquid into powder by using high temperature in a drying tower; preferably, the pH value is adjusted by 2.0mol/L HCl and/or 2mol/L NaOH;
preferably, the preparation method further comprises the step (13) of packaging: drying to obtain peptide powder, and packaging to obtain the final product.
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