CN108913749A - Active peptide nanometre collagen and preparation method thereof - Google Patents
Active peptide nanometre collagen and preparation method thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
-
- 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]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of active peptide nanometre collagens and preparation method thereof.The preparation method of the active peptide nanometre collagen is included the following steps using live stock and fowl bone as primary raw material：S1 cleaning；S2 is arranged；S3 prepares livestock and poultry bone meal or livestock and poultry ossein；S4 livestock and poultry bone meal or the fermentation of livestock and poultry ossein；S5 sterilizing；S6 micro-filtration, ultrafiltration；S7 vacuum freeze drying.The active peptide nanometre collagen obtained with this method can diffuse to deep skin, make collagen increased activity in skin, promote the metabolism of skin histology, with the beauty treatments effect such as whitening, moisturizing, wrinkle resistant, reparation, nutrition, weight-reducing, it can also promote the maturation and mineralising of osteocyte, improve bone strength, promote mineral absorption, play the role of effectively preventing to osteoporosis, it can be used as medicine, health care product, cosmetic material to use, promotional value with higher and good application prospect.
Description
Technical field
The present invention relates to biological products processing technique fields, more particularly to a kind of active peptide nanometre collagen and its preparation side
Method.
Background technique
China is Production of Livestock and Poultry and sale big country, nearly 60,000,000 tons of meat total output, accounts for about whole world meat total output
A quarter ranks first in the world.In meat production, nearly 20,000,000 tons of all kinds of live stock and fowl bones are generated every year.As people consume
Horizontal raising, the demand to meat products constantly increase, and the yield of live stock and fowl bone also continues to increase, but most of live stock and fowl bone does not have
It is fully used, not only causes huge waste, also result in environmental pollution.
It is divided into four kinds according to the production method of the general nanometre collagen of technology type：Acid decomposition, caustic leaching process, high temperature pyrolysis
Method and biologic enzymolysis method.The simple process and low cost of acid system and alkaline process processing, but salt content is higher in product, products obtained therefrom
Relative molecular mass distribution is wider, is not suitable for the food-grade collagen polypeptide of production high-quality.And both methods is in production
The immersion for needing the long period in the process requires the material of equipment relatively high.High temperature pyrolytic cracking (HTP) product salt content is lower, but
Hydrolysis time is long, needs operation with high pressure, and the relative molecular mass distribution of products obtained therefrom is wider, is not suitable for the food of production high-quality
Grade collagen polypeptide, and since both methods needs to impregnate for a long time in process of production, the material of equipment is required all
It is relatively high.High temperature pyrolytic cracking (HTP) product salt content is low, but hydrolysis time is long, needs operation with high pressure, and the average molecular of products obtained therefrom
Mass Distribution is uneven and not easy to control, is also not suitable for the food-grade collagen polypeptide of production high-quality.
Summary of the invention
The first object of the present invention is to provide a kind of preparation method of nano peptide active collagen.
The invention discloses a kind of preparation methods of active peptide nanometre collagen, using live stock and fowl bone as primary raw material, including
Following steps：
S1 cleaning；
S2 is arranged；
S3 prepares livestock and poultry bone meal or livestock and poultry ossein；
S4 livestock and poultry bone meal or the fermentation of livestock and poultry ossein；
S5 sterilizing；
S6 micro-filtration, ultrafiltration；
S7 vacuum freeze drying.
As one of technical solution specifically preferred according to the invention, the preparation method of active peptide nanometre collagen, using live stock and fowl bone
As primary raw material, include the following steps：
S1 cleaning：Live stock and fowl bone is rinsed with flowing water, removes surface contaminants and watery blood；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of live stock and fowl bone after cleaning；
S3 prepares livestock and poultry bone meal：Live stock and fowl bone after arrangement is broken into livestock and poultry osseous granules, is rinsed with flowing water, is drained standby
With；Livestock and poultry osseous granules after the draining normal pressure in 90~100 DEG C of water boils 4~12 hours, then pressure 0.1~
0.3MPa, it pressure cook 5~8 hours under conditions of 110~125 DEG C, pulls out, the livestock and poultry osseous granules after obtaining degreasing；After degreasing
Livestock and poultry osseous granules be dried to moisture content and reach 2~6wt%；Then it crushes, is sieved, obtain livestock and poultry bone meal；
The fermentation of S4 livestock and poultry bone meal：Livestock and poultry bone meal is sterilized；Into the livestock and poultry bone meal after sterilizing plus water, preparation become live stock and fowl bone
The livestock and poultry bone meal solution of 20~40g/L of powder concentration；Zymophyte is made an addition into livestock and poultry bone meal according to the inoculum concentration of 2~4% (mL/mL)
In solution, after 35~37 DEG C ferment 24~48 hours, fermentation liquid is collected；
S5 sterilizing：By fermentation liquid after 110~120 DEG C sterilize 10~30 minutes, 40~50 DEG C are naturally cooled to, centrifugation 20
~40 minutes, take supernatant；
S6 micro-filtration, ultrafiltration：By supernatant micro-filtration, the little particle of suspension is removed, collects micro-filtrate；Then molecular cut off is used
Micro-filtrate is separated for the ultrafiltration membrane of 2~10kDa, collects ultrafiltration permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometer
Collagen.
As the two of technical solution specifically preferred according to the invention, the preparation method of active peptide nanometre collagen, using live stock and fowl bone
As primary raw material, include the following steps：
S1 cleaning：Live stock and fowl bone is rinsed with flowing water, removes surface contaminants and watery blood；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of live stock and fowl bone after cleaning；
S3 prepares livestock and poultry ossein：Live stock and fowl bone after arrangement is broken into livestock and poultry osseous granules, is rinsed with flowing water, is drained standby
With；Livestock and poultry osseous granules after the draining normal pressure in 90~100 DEG C of water boils 4~12 hours, then pressure 0.1~
0.3MPa, it pressure cook 5~8 hours under conditions of 110~125 DEG C, pulls out, the livestock and poultry osseous granules after obtaining degreasing；By degreasing
Livestock and poultry osseous granules afterwards are immersed in the hydrochloric acid of 0.25~0.65mol/L of molar concentration, after impregnating 1~6 hour, pull live stock and fowl bone out
Particle simultaneously removes surface hydrochloric acid with flowing water；Continue to be placed in the sodium hydrate aqueous solution of mass fraction 0.01~0.1%, soak
Bubble pulls livestock and poultry osseous granules out and removes surface sodium hydrate aqueous solution with flowing water after 1~2 hour, the poultry after obtaining desalination
Fowl osseous granules；Livestock and poultry osseous granules after desalination are dried to moisture content and reach 2~6wt%；Then it crushes, is sieved, obtain livestock and poultry
Ossein；
The fermentation of S4 livestock and poultry ossein：Livestock and poultry ossein is sterilized；Into the livestock and poultry ossein after sterilizing plus water, preparation become live stock and fowl bone
The livestock and poultry ossein solution of plain 20~40g/L of concentration；Zymophyte is made an addition into livestock and poultry ossein according to the inoculum concentration of 2~4% (mL/mL)
In solution, after 35~37 DEG C ferment 24~48 hours, fermentation liquid is collected；
S5 sterilizing：By fermentation liquid after 110~120 DEG C sterilize 10~30 minutes, 40~50 DEG C are naturally cooled to, centrifugation 20
~40 minutes, take supernatant；
S6 micro-filtration, ultrafiltration：By supernatant micro-filtration, the little particle of suspension is removed, collects micro-filtrate；Then molecular cut off is used
Micro-filtrate is separated for the ultrafiltration membrane of 2~10kDa, collects ultrafiltration permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometer
Collagen.
As the three of technical solution specifically preferred according to the invention, the preparation method of active peptide nanometre collagen, using live stock and fowl bone
As primary raw material, include the following steps：
S1 cleaning：Live stock and fowl bone is rinsed with flowing water, removes surface contaminants and watery blood；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of live stock and fowl bone after cleaning；
S3 prepares livestock and poultry ossein：Live stock and fowl bone after arrangement is broken into livestock and poultry osseous granules, is rinsed with flowing water, is drained standby
With；Livestock and poultry osseous granules after the draining normal pressure in 90~100 DEG C of water boils 4~12 hours, then pressure 0.1~
0.3MPa, it pressure cook 5~8 hours under conditions of 110~125 DEG C, pulls out, the livestock and poultry osseous granules after obtaining degreasing；By degreasing
Livestock and poultry osseous granules afterwards are immersed in the hydrochloric acid of 0.25~0.65mol/L of molar concentration, after impregnating 1~6 hour, pull live stock and fowl bone out
Particle simultaneously removes surface hydrochloric acid with flowing water；Continue to be placed in the sodium hydrate aqueous solution of mass fraction 0.01~0.1%, soak
Bubble pulls livestock and poultry osseous granules out and removes surface sodium hydrate aqueous solution with flowing water after 1~2 hour, the poultry after obtaining desalination
Fowl osseous granules；Livestock and poultry osseous granules after desalination are dried to moisture content and reach 2~6wt%；Then it crushes, is sieved, obtain livestock and poultry
Ossein；
The fermentation of S4 livestock and poultry ossein：The Chinese herbal medicine of livestock and poultry ossein weight 1~4% is added into livestock and poultry ossein, uniform mixing obtains
Expect to mixing and stirring；Material sterilizing will be mixed and stirred；To mixing and stirring in material after sterilizing plus water, prepares to become and mix and stir mixing for material 20~40g/L of concentration
With material solution；Zymophyte is made an addition to according to the inoculum concentration of 2~4% (mL/mL) and is mixed and stirred in material solution, in 35~37 DEG C of fermentations 24
After~48 hours, fermentation liquid is collected；The Chinese herbal medicine is made of the raw material of following proportion：30~50wt% of the root of Dahurain angelica, cordate houttuynia 10
~30wt%, surplus are fructus amomi；
S5 sterilizing：By fermentation liquid after 110~120 DEG C sterilize 10~30 minutes, 40~50 DEG C are naturally cooled to, centrifugation 20
~40 minutes, take supernatant；
S6 micro-filtration, ultrafiltration：By supernatant micro-filtration, the little particle of suspension is removed, collects micro-filtrate；Then molecular cut off is used
Micro-filtrate is separated for the ultrafiltration membrane of 2~10kDa, collects ultrafiltration permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometer
Collagen.
The live stock and fowl bone is one of frontal bone, ox bone, pig bone, fish-bone, camel bone, dog bone, deer bone.
Preferably, it in step S4 fermentation process, to livestock and poultry ossein solution or mixes and stirs in material solution according to 10~15g/L's
Sucrose is added in additive amount, then adds zymophyte and ferments.
Preferably, while adding sucrose, inorganic salts are added according to the additive amount of 0.1~1g/L, the inorganic salts are
One of calcium chloride, zinc chloride, magnesium chloride, stannous chloride, frerrous chloride or a variety of mixtures.
It is highly preferred that the inorganic salts are calcium chloride.
Preferably, the zymophyte is bacillus cereus and/or lactobacillus bulgaricus.As specifically preferred according to the invention
Embodiment, the zymophyte be by bacillus cereus and lactobacillus bulgaricus with volume ratio 1:The Mixed Microbes of 1 composition.
The second object of the present invention is to provide a kind of active peptide nanometre collagen, using any of the above-described kind of active peptide nanometre glue
Former preparation method is process.
The beneficial effect of the present invention compared with the existing technology is：
1, the method that the present invention uses high-temperature heat treatment and high voltage driving IC in skimming processes, it is easy to operate, to equipment
Requirement it is also relatively low, without using organic solvent, be suitble to industrial application；
2, the present invention is by way of acidleach and alkali leaching, by the inorganic mineral salt around collagen it is free go out material bone,
The ossein using collagenous fiber bundle as principal mode is obtained, while being adjusted to suitable yeasting, so that zymophyte metabolite
Collagenous fiber bundle is degraded, significant hydrolysis effect is generated；
3, the present invention adds the Chinese herbal medicine of the root of Dahurain angelica, cordate houttuynia and fructus amomi composition during fermentation, is reducing active peptide
While nanometre collagen bitterness mouthfeel and fishy smell, while Chinese herbal medicine fermentating metabolism product promotes ossein, active peptide is improved
The curative effect of nanometre collagen；In some embodiments, sucrose and inorganic salts calcium chloride, sucrose be also added during fermentation
Carbon source abundant is provided for the fermentation of thallus, calcium chloride may promote the generation of fermentating metabolism product Collagenase and mention
Its high activity；
4, it is worth noting that, in particularly preferred embodiment of the present invention, bacillus cereus and Bao Jiali are used
The Mixed Microbes of sub- lactobacillus composition are as zymophyte, but there are certain antagonisms for both bacterium.Inventor is by waxy bud
Spore bacillus and lactobacillus bulgaricus composition Mixed Microbes as zymophyte, have been surprisingly found that, obtained active peptide nanometre collagen
Obvious effect enhancing, thus it is speculated that may be that lactic acid is generated using the sugar metabolism in fermentation liquid due to lactobacillus bulgaricus, create
Acidic environment, is conducive to hydrolysis of the mixed fermentation bacterium metabolite to collagen；Judge lactobacillus bulgaricus bacterium solution and
The mode of antagonism between bacillus cereus；In MRS plating medium, 1mL lactobacillus bulgaricus bacterium solution is added and applies
Cloth is uniform, 37 DEG C constant temperature incubation 48 hours, after bacterium colony to be formed, then with oese picking bacillus cereus, on bacterium colony
Face rows dry S-shaped, then after cultivating 48 hours under the conditions of 37 DEG C.Observation has bacterium colony to generate in S-shaped, and the two has slight antagonism to make
With.
The active peptide nanometre collagen obtained with this method can diffuse to deep skin, increase collagen activity in skin
By force, promote the metabolism of skin histology, there is the beauty treatments effect such as whitening, moisturizing, wrinkle resistant, reparation, nutrition, weight-reducing, also
It can promote the maturation and mineralising of osteocyte, improve bone strength, promote mineral absorption, osteoporosis is played and is effectively prevented
Effect can be used as medicine, health care product, cosmetic material and use, promotional value with higher and good application prospect.
Specific embodiment
Raw material is described below in embodiment：
Frontal bone is purchased from Shanghai Tao Pu animal products market.
Bacillus cereus is purchased from the first biological medicine company Co., Ltd in Anyang source, strain concentration 108cfu/mL。
Lactobacillus bulgaricus is purchased from Shaanxi Teng Mai biotechnology Co., Ltd, strain concentration 108cfu/mL。
Sucrose, No. CAS：57-50-1.
Calcium chloride, No. CAS：10043-52-4.
The root of Dahurain angelica, place of production Anhui are purchased from Bozhou Bai Chuan medicine company sale Co., Ltd.
Cordate houttuynia, place of production Sichuan are purchased from Bozhou City Yuan Shengtang pharmaceutcal corporation, Ltd.
Fructus amomi is purchased from Bozhou City Lang Yuan pharmaceutcal corporation, Ltd.
In the case where the present invention does not illustrate, the concrete operations of vacuum freeze drying are in embodiment：Pre-freeze temperature
- 80 DEG C, the pre-freeze time 1 hour, -70 DEG C of cryogenic temperature, absolute pressure 100Pa, sublimation drying 48 hours of degree.
Embodiment 1
The preparation method of active peptide nanometre collagen is included the following steps using frontal bone as primary raw material：
S1 cleaning：Frontal bone flowing water is rinsed 20 minutes, surface contaminants and watery blood are removed；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of frontal bone after cleaning；
S3 prepares frontal bone powder：It is 5mm × 10mm × 3mm frontal bone particle that frontal bone after arrangement, which is broken into size, with stream
Dynamic water rinses 10 minutes, drained and standby；Frontal bone particle after the draining normal pressure in 100 DEG C of water boils 8 hours, is then pressing
Power 0.1MPa, it pressure cook 6 hours under conditions of 121 DEG C, pulls out, the frontal bone particle after obtaining degreasing；By the frontal bone after degreasing
Grain is dried to moisture content in 60 DEG C and reaches 3wt%；Then it crushes, sieves with 100 mesh sieve, obtain frontal bone powder；
The fermentation of S4 frontal bone powder：Frontal bone powder is sterilized 5 minutes under conditions of microwave power 20kW, microwave frequency 2450MHz；
Into the frontal bone powder after sterilizing plus water, preparation become the frontal bone powder solution of frontal bone powder concentration 40g/L；By bacillus cereus according to
The inoculum concentration of 4% (mL/mL) makes an addition in frontal bone powder solution, after 37 DEG C ferment 36 hours, collects fermentation liquid；
S5 sterilizing：After ten minutes in 121 DEG C of sterilizings by fermentation liquid, 40 DEG C are naturally cooled to, with 6000 revs/min of centrifugations 20
Minute, take supernatant；
S6 micro-filtration, ultrafiltration：The microfiltration membranes that supernatant is passed through to 0.15 μm of aperture at operating pressure 0.2MPa, remove and suspend
Little particle, collect micro-filtrate；Then micro-filtrate is separated with the ultrafiltration membrane that molecular cut off is 10kDa, collects ultrafiltration
Permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometer
Collagen.
Embodiment 2
The preparation method of active peptide nanometre collagen is included the following steps using frontal bone as primary raw material：
S1 cleaning：Frontal bone flowing water is rinsed 20 minutes, surface contaminants and watery blood are removed；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of frontal bone after cleaning；
S3 prepares frontal bone element：It is 5mm × 10mm × 3mm frontal bone particle that frontal bone after arrangement, which is broken into size, with stream
Dynamic water rinses 10 minutes, drained and standby；Frontal bone particle after the draining normal pressure in 100 DEG C of water boils 8 hours, is then pressing
Power 0.1MPa, it pressure cook 6 hours under conditions of 121 DEG C, pulls out, the frontal bone particle after obtaining degreasing；By the frontal bone after degreasing
Grain is immersed in the hydrochloric acid of molar concentration 0.49mol/L, and the solid-to-liquid ratio of frontal bone particle and hydrochloric acid is 1：5 (g/mL) impregnate 6 hours
Afterwards, it pulls frontal bone particle out and removes surface hydrochloric acid with flowing water；The sodium hydroxide for continuing to be placed in mass fraction 0.05% is water-soluble
In liquid, the solid-to-liquid ratio of frontal bone particle and sodium hydrate aqueous solution is 1：3 (g/mL) pull frontal bone particle out and are used in combination after impregnating 2 hours
Flowing water removes surface sodium hydrate aqueous solution, the frontal bone particle after obtaining desalination；By the frontal bone particle after desalination in 60 DEG C
It is dried to moisture content and reaches 3wt%；Then it crushes, sieves with 100 mesh sieve, obtain frontal bone element；
The fermentation of S4 frontal bone element：Frontal bone element is sterilized 5 minutes under conditions of microwave power 20kW, microwave frequency 2450MHz；
Into the frontal bone element after sterilizing plus water, preparation become the frontal bone element solution of frontal bone element concentration 40g/L；By bacillus cereus according to
The inoculum concentration of 4% (mL/mL) makes an addition in frontal bone element solution, after 37 DEG C ferment 36 hours, collects fermentation liquid；
S5 sterilizing：After ten minutes in 121 DEG C of sterilizings by fermentation liquid, 40 DEG C are naturally cooled to, with 6000 revs/min of centrifugations 20
Minute, take supernatant；
S6 micro-filtration, ultrafiltration：The microfiltration membranes that supernatant is passed through to 0.15 μm of aperture at operating pressure 0.2MPa, remove and suspend
Little particle, collect micro-filtrate；Then micro-filtrate is separated with the ultrafiltration membrane that molecular cut off is 10kDa, collects ultrafiltration
Permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometer
Collagen.
Embodiment 3
The preparation method of active peptide nanometre collagen is included the following steps using frontal bone as primary raw material：
S1 cleaning：Frontal bone flowing water is rinsed 20 minutes, surface contaminants and watery blood are removed；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of frontal bone after cleaning；
S3 prepares frontal bone element：It is 5mm × 10mm × 3mm frontal bone particle that frontal bone after arrangement, which is broken into size, with stream
Dynamic water rinses 10 minutes, drained and standby；Frontal bone particle after the draining normal pressure in 100 DEG C of water boils 8 hours, is then pressing
Power 0.1MPa, it pressure cook 6 hours under conditions of 121 DEG C, pulls out, the frontal bone particle after obtaining degreasing；By the frontal bone after degreasing
Grain is immersed in the hydrochloric acid of molar concentration 0.49mol/L, and the solid-to-liquid ratio of frontal bone particle and hydrochloric acid is 1：5 (g/mL) impregnate 6 hours
Afterwards, it pulls frontal bone particle out and removes surface hydrochloric acid with flowing water；The sodium hydroxide for continuing to be placed in mass fraction 0.05% is water-soluble
In liquid, the solid-to-liquid ratio of frontal bone particle and sodium hydrate aqueous solution is 1：3 (g/mL) pull frontal bone particle out and are used in combination after impregnating 2 hours
Flowing water removes surface sodium hydrate aqueous solution, the frontal bone particle after obtaining desalination；By the frontal bone particle after desalination in 60 DEG C
It is dried to moisture content and reaches 3wt%；Then it crushes, sieves with 100 mesh sieve, obtain frontal bone element；
The fermentation of S4 frontal bone element：The Chinese herbal medicine of frontal bone element weight 2% is added into frontal bone element, uniform mixing obtains mixing and stirring material；
Material will be mixed and stirred to sterilize 5 minutes under conditions of microwave power 20kW, microwave frequency 2450MHz；Add to mixing and stirring in material after sterilizing
Water is prepared to become to mix and stir and expects that concentration 40g/L's mixes and stirs material solution；Bacillus cereus is added according to the inoculum concentration of 4% (mL/mL)
It is added on and mixes and stirs in material solution, after 37 DEG C ferment 36 hours, collect fermentation liquid；The Chinese herbal medicine by following proportion raw material group
At：Root of Dahurain angelica 50wt%, cordate houttuynia 30wt%, surplus are fructus amomi；The root of Dahurain angelica, cordate houttuynia, fructus amomi are mixed according to the ratio up to Chinese herbal medicine；
S5 sterilizing：After ten minutes in 121 DEG C of sterilizings by fermentation liquid, 40 DEG C are naturally cooled to, with 6000 revs/min of centrifugations 20
Minute, take supernatant；
S6 micro-filtration, ultrafiltration：The microfiltration membranes that supernatant is passed through to 0.15 μm of aperture at operating pressure 0.2MPa, remove and suspend
Little particle, collect micro-filtrate；Then micro-filtrate is separated with the ultrafiltration membrane that molecular cut off is 10kDa, collects ultrafiltration
Permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometer
Collagen.
Embodiment 4
The preparation method of active peptide nanometre collagen is included the following steps using frontal bone as primary raw material：
S1 cleaning：Frontal bone flowing water is rinsed 20 minutes, surface contaminants and watery blood are removed；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of frontal bone after cleaning；
S3 prepares frontal bone element：It is 5mm × 10mm × 3mm frontal bone particle that frontal bone after arrangement, which is broken into size, with stream
Dynamic water rinses 10 minutes, drained and standby；Frontal bone particle after the draining normal pressure in 100 DEG C of water boils 8 hours, is then pressing
Power 0.1MPa, it pressure cook 6 hours under conditions of 121 DEG C, pulls out, the frontal bone particle after obtaining degreasing；By the frontal bone after degreasing
Grain is immersed in the hydrochloric acid of molar concentration 0.49mol/L, and the solid-to-liquid ratio of frontal bone particle and hydrochloric acid is 1：5 (g/mL) impregnate 6 hours
Afterwards, it pulls frontal bone particle out and removes surface hydrochloric acid with flowing water；The sodium hydroxide for continuing to be placed in mass fraction 0.05% is water-soluble
In liquid, the solid-to-liquid ratio of frontal bone particle and sodium hydrate aqueous solution is 1：3 (g/mL) pull frontal bone particle out and are used in combination after impregnating 2 hours
Flowing water removes surface sodium hydrate aqueous solution, the frontal bone particle after obtaining desalination；By the frontal bone particle after desalination in 60 DEG C
It is dried to moisture content and reaches 3wt%；Then it crushes, sieves with 100 mesh sieve, obtain frontal bone element；
The fermentation of S4 frontal bone element：The Chinese herbal medicine of frontal bone element weight 2% is added into frontal bone element, uniform mixing obtains mixing and stirring material；
Material will be mixed and stirred to sterilize 5 minutes under conditions of microwave power 20kW, microwave frequency 2450MHz；Add to mixing and stirring in material after sterilizing
Water is prepared to become to mix and stir and expects that concentration 40g/L's mixes and stirs material solution；Sugarcane is added according to the additive amount of 10g/L into mix solution
Then bacillus cereus will be made an addition to according to the inoculum concentration of 4% (mL/mL) and be mixed and stirred in material solution by sugar, in 37 DEG C of fermentations 36
After hour, fermentation liquid is collected；The Chinese herbal medicine is made of the raw material of following proportion：Root of Dahurain angelica 50wt%, cordate houttuynia 30wt%, surplus
For fructus amomi；The root of Dahurain angelica, cordate houttuynia, fructus amomi are mixed according to the ratio up to Chinese herbal medicine；
S5 sterilizing：After ten minutes in 121 DEG C of sterilizings by fermentation liquid, 40 DEG C are naturally cooled to, with 6000 revs/min of centrifugations 20
Minute, take supernatant；
S6 micro-filtration, ultrafiltration：The microfiltration membranes that supernatant is passed through to 0.15 μm of aperture at operating pressure 0.2MPa, remove and suspend
Little particle, collect micro-filtrate；Then micro-filtrate is separated with the ultrafiltration membrane that molecular cut off is 10kDa, collects ultrafiltration
Permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometer
Collagen.
Embodiment 5
The preparation method of active peptide nanometre collagen is included the following steps using frontal bone as primary raw material：
S1 cleaning：Frontal bone flowing water is rinsed 20 minutes, surface contaminants and watery blood are removed；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of frontal bone after cleaning；
S3 prepares frontal bone element：It is 5mm × 10mm × 3mm frontal bone particle that frontal bone after arrangement, which is broken into size, with stream
Dynamic water rinses 10 minutes, drained and standby；Frontal bone particle after the draining normal pressure in 100 DEG C of water boils 8 hours, is then pressing
Power 0.1MPa, it pressure cook 6 hours under conditions of 121 DEG C, pulls out, the frontal bone particle after obtaining degreasing；By the frontal bone after degreasing
Grain is immersed in the hydrochloric acid of molar concentration 0.49mol/L, and the solid-to-liquid ratio of frontal bone particle and hydrochloric acid is 1：5 (g/mL) impregnate 6 hours
Afterwards, it pulls frontal bone particle out and removes surface hydrochloric acid with flowing water；The sodium hydroxide for continuing to be placed in mass fraction 0.05% is water-soluble
In liquid, the solid-to-liquid ratio of frontal bone particle and sodium hydrate aqueous solution is 1：3 (g/mL) pull frontal bone particle out and are used in combination after impregnating 2 hours
Flowing water removes surface sodium hydrate aqueous solution, the frontal bone particle after obtaining desalination；By the frontal bone particle after desalination in 60 DEG C
It is dried to moisture content and reaches 3wt%；Then it crushes, sieves with 100 mesh sieve, obtain frontal bone element；
The fermentation of S4 frontal bone element：The Chinese herbal medicine of frontal bone element weight 2% is added into frontal bone element, uniform mixing obtains mixing and stirring material；
Material will be mixed and stirred to sterilize 5 minutes under conditions of microwave power 20kW, microwave frequency 2450MHz；Add to mixing and stirring in material after sterilizing
Water is prepared to become to mix and stir and expects that concentration 40g/L's mixes and stirs material solution；Sugarcane is added according to the additive amount of 10g/L into mix solution
Calcium chloride is added according to the additive amount of 0.5g/L in sugar, then will add bacillus cereus according to the inoculum concentration of 4% (mL/mL)
It is added on and mixes and stirs in material solution, after 37 DEG C ferment 36 hours, collect fermentation liquid；The Chinese herbal medicine by following proportion raw material group
At：Root of Dahurain angelica 50wt%, cordate houttuynia 30wt%, surplus are fructus amomi；The root of Dahurain angelica, cordate houttuynia, fructus amomi are mixed according to the ratio up to Chinese herbal medicine；
S5 sterilizing：After ten minutes in 121 DEG C of sterilizings by fermentation liquid, 40 DEG C are naturally cooled to, with 6000 revs/min of centrifugations 20
Minute, take supernatant；
S6 micro-filtration, ultrafiltration：The microfiltration membranes that supernatant is passed through to 0.15 μm of aperture at operating pressure 0.2MPa, remove and suspend
Little particle, collect micro-filtrate；Then micro-filtrate is separated with the ultrafiltration membrane that molecular cut off is 10kDa, collects ultrafiltration
Permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometer
Collagen.
Embodiment 6
The preparation method of active peptide nanometre collagen is included the following steps using frontal bone as primary raw material：
S1 cleaning：Frontal bone flowing water is rinsed 20 minutes, surface contaminants and watery blood are removed；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of frontal bone after cleaning；
S3 prepares frontal bone element：It is 5mm × 10mm × 3mm frontal bone particle that frontal bone after arrangement, which is broken into size, with stream
Dynamic water rinses 10 minutes, drained and standby；Frontal bone particle after the draining normal pressure in 100 DEG C of water boils 8 hours, is then pressing
Power 0.1MPa, it pressure cook 6 hours under conditions of 121 DEG C, pulls out, the frontal bone particle after obtaining degreasing；By the frontal bone after degreasing
Grain is immersed in the hydrochloric acid of molar concentration 0.49mol/L, and the solid-to-liquid ratio of frontal bone particle and hydrochloric acid is 1：5 (g/mL) impregnate 6 hours
Afterwards, it pulls frontal bone particle out and removes surface hydrochloric acid with flowing water；The sodium hydroxide for continuing to be placed in mass fraction 0.05% is water-soluble
In liquid, the solid-to-liquid ratio of frontal bone particle and sodium hydrate aqueous solution is 1：3 (g/mL) pull frontal bone particle out and are used in combination after impregnating 2 hours
Flowing water removes surface sodium hydrate aqueous solution, the frontal bone particle after obtaining desalination；By the frontal bone particle after desalination in 60 DEG C
It is dried to moisture content and reaches 3wt%；Then it crushes, sieves with 100 mesh sieve, obtain frontal bone element；
The fermentation of S4 frontal bone element：The Chinese herbal medicine of frontal bone element weight 2% is added into frontal bone element, uniform mixing obtains mixing and stirring material；
Material will be mixed and stirred to sterilize 5 minutes under conditions of microwave power 20kW, microwave frequency 2450MHz；Add to mixing and stirring in material after sterilizing
Water is prepared to become to mix and stir and expects that concentration 40g/L's mixes and stirs material solution；Sugarcane is added according to the additive amount of 10g/L into mix solution
Calcium chloride is added according to the additive amount of 0.5g/L in sugar, then adds lactobacillus bulgaricus according to the inoculum concentration of 4% (mL/mL)
It is added on and mixes and stirs in material solution, after 37 DEG C ferment 36 hours, collect fermentation liquid；The Chinese herbal medicine by following proportion raw material group
At：Root of Dahurain angelica 50wt%, cordate houttuynia 30wt%, surplus are fructus amomi；The root of Dahurain angelica, cordate houttuynia, fructus amomi are mixed according to the ratio up to Chinese herbal medicine；
S5 sterilizing：After ten minutes in 121 DEG C of sterilizings by fermentation liquid, 40 DEG C are naturally cooled to, with 6000 revs/min of centrifugations 20
Minute, take supernatant；
S6 micro-filtration, ultrafiltration：The microfiltration membranes that supernatant is passed through to 0.15 μm of aperture at operating pressure 0.2MPa, remove and suspend
Little particle, collect micro-filtrate；Then micro-filtrate is separated with the ultrafiltration membrane that molecular cut off is 10kDa, collects ultrafiltration
Permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometer
Collagen.
Embodiment 7
The preparation method of active peptide nanometre collagen is included the following steps using frontal bone as primary raw material：
S1 cleaning：Frontal bone flowing water is rinsed 20 minutes, surface contaminants and watery blood are removed；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of frontal bone after cleaning；
S3 prepares frontal bone element：It is 5mm × 10mm × 3mm frontal bone particle that frontal bone after arrangement, which is broken into size, with stream
Dynamic water rinses 10 minutes, drained and standby；Frontal bone particle after the draining normal pressure in 100 DEG C of water boils 8 hours, is then pressing
Power 0.1MPa, it pressure cook 6 hours under conditions of 121 DEG C, pulls out, the frontal bone particle after obtaining degreasing；By the frontal bone after degreasing
Grain is immersed in the hydrochloric acid of molar concentration 0.49mol/L, and the solid-to-liquid ratio of frontal bone particle and hydrochloric acid is 1：5 (g/mL) impregnate 6 hours
Afterwards, it pulls frontal bone particle out and removes surface hydrochloric acid with flowing water；The sodium hydroxide for continuing to be placed in mass fraction 0.05% is water-soluble
In liquid, the solid-to-liquid ratio of frontal bone particle and sodium hydrate aqueous solution is 1：3 (g/mL) pull frontal bone particle out and are used in combination after impregnating 2 hours
Flowing water removes surface sodium hydrate aqueous solution, the frontal bone particle after obtaining desalination；By the frontal bone particle after desalination in 60 DEG C
It is dried to moisture content and reaches 3wt%；Then it crushes, sieves with 100 mesh sieve, obtain frontal bone element；
The fermentation of S4 frontal bone element：The Chinese herbal medicine of frontal bone element weight 2% is added into frontal bone element, uniform mixing obtains mixing and stirring material；
Material will be mixed and stirred to sterilize 5 minutes under conditions of microwave power 20kW, microwave frequency 2450MHz；Add to mixing and stirring in material after sterilizing
Water is prepared to become to mix and stir and expects that concentration 40g/L's mixes and stirs material solution；Sugarcane is added according to the additive amount of 10g/L into mix solution
Calcium chloride is added according to the additive amount of 0.5g/L in sugar, then makes an addition to zymophyte according to the inoculum concentration of 4% (mL/mL) and mixes and stirs
Expect in solution, the zymophyte is by bacillus cereus and lactobacillus bulgaricus with volume ratio 1:The Mixed Microbes of 1 composition, in
After 37 DEG C ferment 36 hours, fermentation liquid is collected；The Chinese herbal medicine is made of the raw material of following proportion：Root of Dahurain angelica 50wt%, cordate houttuynia
30wt%, surplus are fructus amomi；The root of Dahurain angelica, cordate houttuynia, fructus amomi are mixed according to the ratio up to Chinese herbal medicine；
S5 sterilizing：After ten minutes in 121 DEG C of sterilizings by fermentation liquid, 40 DEG C are naturally cooled to, with 6000 revs/min of centrifugations 20
Minute, take supernatant；
S6 micro-filtration, ultrafiltration：The microfiltration membranes that supernatant is passed through to 0.15 μm of aperture at operating pressure 0.2MPa, remove and suspend
Little particle, collect micro-filtrate；Then micro-filtrate is separated with the ultrafiltration membrane that molecular cut off is 10kDa, collects ultrafiltration
Permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometer
Collagen.
Test case 1
The white-skinned face function of Examples 1 to 7 active peptide nanometre collagen is evaluated.Made with Murine B 16 Melanoma Cells
Increasing of the active peptide nanometre collagen to melanoma cells is judged using melanin content and tyrosinase activity as index for model
Grow inhibiting effect.
Melanin content detection
By murine melanoma cells B16 strain (being purchased from Wuhan Pu Nuosai Life Science Co., Ltd, article No. CL-0029) in
RPMI-1640 culture medium (be purchased from Gibco company, contain 10% calf serum, 105U/L penicillin, 105μ g/L streptomysin), 37
DEG C, 5%CO2Incubator in cultivate, passage in every 3 days is primary.
The Murine B 16 Melanoma Cells of logarithmic growth phase, it is complete with RPMI-1640 after the digestion of 0.25% pancreatin
It is 1 × 10 that culture medium, which is made into density,5The cell suspension of a/mL, is inoculated in 6 orifice plates, and every hole 2mL is placed in 37 DEG C, 5%CO2Training
Support culture in case.After adhere-wall culture 24 hours, culture solution is abandoned, is received to every mLRPMI-1640 culture medium addition 0.025mg active peptide
Rice glue original work are test group, and control group is isometric RPMI-1640 culture medium.After culture 48 hours, harvest cell carries out black
Pigment content measurement.With the sodium hydrate aqueous solution lytic cell of the 1mol/L containing 10%DMSO, ultrasonic disruption 30 minutes, in
90 DEG C after water bath processing 2 hours, are centrifuged 15 minutes with 3000 revs/min, and absorbance value is surveyed in microplate reader 450nm at, is calculated carefully
Born of the same parents' melanin relative amount.
Melanin genesis relative amount (%)=A1/A0× 100%.In formula, A1It is the absorbance of test group, A0It is control group
Light absorption value.
Each processing group experiment is repeated 3 times.Take its average value as final testing result.
Tyrosinase activity detection
The Murine B 16 Melanoma Cells of logarithmic growth phase are cultivated after the digestion of 0.25% pancreatin with RPMI-1640
Basigamy is 1 × 10 at density5The cell suspension of a/mL, is inoculated in 6 orifice plates, and every hole 2mL is placed in 37 DEG C, 5%CO2Incubator
Interior culture.After adhere-wall culture 24 hours, culture solution is abandoned, adds 0.025mg active peptide nanometre glue to every mLRPMI-1640 culture medium
Original work are test group, and control group is isometric RPMI-1640 culture medium.After culture 48 hours, harvest cell carries out tyrosine
Enzymatic activity measurement.The phosphate buffer of cell pH 6.8, molar concentration 0.1mol/L are rinsed twice, every hole adds 300 μ L
Buffer containing 5% volume fraction Triton X-100, multigelation, ultrasonication in ice bath obtain clasmatosis liquid；It takes
2mL clasmatosis liquid preheats 10 minutes in 37 DEG C, 500 μ L of 0.1%L-DOPA solution is rapidly added, in spectrophotometer after shaking
At wavelength 475nm, absorbance value is read when 0 minute and 30 minutes, calculates inhibitory activity against tyrosinase.
Inhibitory activity against tyrosinase (%)=(A '30-A’0)/(A30-A0In the formula of) × 100%, A '0And A '30It is test group
Absorbance at 0 minute and 30 minutes；A0And A30It is absorbance of the control group at 0 minute and 30 minutes.
Each processing group experiment is repeated 3 times.Take its average value as final testing result.Specific test result is shown in Table 1.
The white-skinned face function test result table of 1 active peptide nanometre collagen of table
Test case2
Osteoporosis is that bone amount is reduced in unit volume, and the microstructure of bone is destroyed, so as to cause the increasing of bone brittleness
Add a kind of bone metabolic disease of even fracture.The treatment osteoporosis function of Examples 1 to 7 active peptide nanometre collagen is commented
Valence.
The secondary culture of rat bone marrow mesenchymal stem cells and induction：Rat bone marrow mesenchymal stem cells BMSCs (is purchased from
Shanghai Bang Jing Industrial Co., Ltd., article No. CS-0001-0067) with 1 × 105The density of a/mL is inoculated in culture plate, in
DMEM-F12 culture medium (it is purchased from Shanghai Bo Sheng Biotechnology Co., Ltd, containing 10% fetal calf serum, 105U/L penicillin, 105μ
G/L streptomysin), in 37 DEG C, 5%CO2Incubator in cultivated, change the liquid once within three days, grow into 80% to cell~
When 90% fusion, is rinsed twice with the phosphate buffer of pH 6.8, molar concentration 0.1mol/L, disappeared with 0.25% pancreas enzyme -EDTA
Change 1~2 minute, complete medium be added and terminates digestion, with suction pipe piping and druming at single cell suspension, move into centrifuge tube, 20 DEG C with
250 revs/min are centrifuged 5 minutes, supernatant are abandoned, according to 1：2 ratio carries out secondary culture.
The detection of alkali formula phosphatase activity
It takes well-grown to reach the rat bone marrow mesenchymal stem cells BMSCs of the third generation, single cell suspension is made after digestion
And concentration is adjusted to 1 × 105A/mL is inoculated in 24 orifice plates, every hole l mL；Culture added control group culture after 24 hours respectively
Base and test group culture medium, to every mLRPMI-1640 culture medium addition 0.1mg active peptide nanometre collagen as test group, control
Group is isometric RPMI-1640 culture medium.It is placed in 37 DEG C, 5%CO2Incubator in, in the 14th day measurement alkali formula phosphatase
Activity.Culture solution in hole is sucked, is rinsed twice with the phosphate buffer of pH 6.8, molar concentration 0.1mol/L, l mL is added and contains
The buffer of 2% volume fraction Triton X-100 is cracked in 4 DEG C of refrigerator overnights, is centrifuged 5 points in 4 DEG C with 12000 revs/min
Clock takes supernatant；It takes 30 μ L supernatants in 96 orifice plates, is measured according to alkali formula Phosphatase Kit specification, microplate reader surveys each pipe
Absorbance calculates alkali formula phosphatase according to alkali formula Phosphatase Kit (being purchased from Beijing Zhong Sheng bioengineering Hitek Ltd)
Activity.
The detection of calcium element
By mesenchymal stem cell BMSCs with 1 × 105The density of a/mL is inoculated in 6 orifice plates, respectively with control group and
Test group culture solution culture changes the liquid once for every 3 days, in the 14th day collection cell culture supernatant, with 250 revs/min of centrifugations 15
Minute, supernatant is taken, is contained with enzyme-linked immunization (ELISA) (offer of Shanghai Ren Jie Biotechnology Co., Ltd) detection osteocalcin
Amount.
Each processing group experiment is repeated 3 times, and takes its average value as final testing result.Specific test result is shown in Table 2.
The treatment osteoporosis function test result table of 2 active peptide nanometre collagen of table
From above-mentioned test case it is found that no matter 2 active peptide nanometre collagen of embodiment whitening effect or treats osteoporosis
Effect is better than embodiment 1, and reason may be to affect zymophyte generation containing high-content inorganic mineral salt in live stock and fowl bone
The contact of Collagenase and collagen in bone.In the present invention by way of acidleach and alkali leaching, by the nothing around collagen
The free material bone out of machine mineral salt, obtains the ossein using collagenous fiber bundle as principal mode, while being adjusted to suitable fermentation ring
Border generates significant hydrolysis effect so that zymophyte metabolite degrades collagenous fiber bundle.It is worth noting that, implementing
In example 7, the Mixed Microbes formed using bacillus cereus and lactobacillus bulgaricus are as zymophyte, but both bacterium are deposited
In certain antagonism.The Mixed Microbes that inventor forms bacillus cereus and lactobacillus bulgaricus as zymophyte,
It has been surprisingly found that, the effect of obtained active peptide nanometre collagen significantly increases, thus it is speculated that may be since lactobacillus bulgaricus utilizes
Sugar metabolism in fermentation liquid generates lactic acid, creates acidic environment, is conducive to mixed fermentation bacterium metabolite to collagen
Hydrolysis.
It should be appreciated that although this specification is described in terms of embodiments, but not each embodiment only includes one
A independent technical solution, for the sake of this narrating mode of specification is used for the purpose of clearly, those skilled in the art should be incited somebody to action
As a whole, the technical solutions in the various embodiments may also be suitably combined for specification, and forming those skilled in the art can
With the other embodiments of understanding.
Claims (10)
1. the preparation method of active peptide nanometre collagen, which is characterized in that using live stock and fowl bone as primary raw material, including following step
Suddenly：
S1 cleaning；
S2 is arranged；
S3 prepares livestock and poultry bone meal or livestock and poultry ossein；
S4 livestock and poultry bone meal or the fermentation of livestock and poultry ossein；
S5 sterilizing；
S6 micro-filtration, ultrafiltration；
S7 vacuum freeze drying.
2. the preparation method of active peptide nanometre collagen according to claim 1, which is characterized in that using live stock and fowl bone as master
Raw material is wanted, is included the following steps：
S1 cleaning：Live stock and fowl bone is rinsed with flowing water, removes surface contaminants and watery blood；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of live stock and fowl bone after cleaning；
S3 prepares livestock and poultry bone meal：Live stock and fowl bone after arrangement is broken into livestock and poultry osseous granules, is rinsed with flowing water, it is drained and standby；
Livestock and poultry osseous granules after the draining normal pressure in 90~100 DEG C of water boils 4~12 hours, then 0.1~0.3MPa of pressure,
Pressure cook 5~8 hours, are pulled out under conditions of 110~125 DEG C, the livestock and poultry osseous granules after obtaining degreasing；By the live stock and fowl bone after degreasing
Particle is dried to moisture content and reaches 2~6wt%；Then it crushes, is sieved, obtain livestock and poultry bone meal；
The fermentation of S4 livestock and poultry bone meal：Livestock and poultry bone meal is sterilized；Into the livestock and poultry bone meal after sterilizing plus water, preparation are dense as live stock and fowl bone powder
Spend the livestock and poultry bone meal solution of 20~40g/L；Zymophyte is made an addition into livestock and poultry bone meal solution according to the inoculum concentration of 2~4% (mL/mL)
In, after 35~37 DEG C ferment 24~48 hours, collect fermentation liquid；
S5 sterilizing：By fermentation liquid after 110~120 DEG C sterilize 10~30 minutes, 40~50 DEG C are naturally cooled to, centrifugation 20~40
Minute, take supernatant；
S6 micro-filtration, ultrafiltration：By supernatant micro-filtration, the little particle of suspension is removed, collects micro-filtrate；It then is 2 with molecular cut off
The ultrafiltration membrane of~10kDa separates micro-filtrate, collects ultrafiltration permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometre collagen.
3. the preparation method of active peptide nanometre collagen according to claim 1, which is characterized in that using live stock and fowl bone as master
Raw material is wanted, is included the following steps：
S1 cleaning：Live stock and fowl bone is rinsed with flowing water, removes surface contaminants and watery blood；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of live stock and fowl bone after cleaning；
S3 prepares livestock and poultry ossein：Live stock and fowl bone after arrangement is broken into livestock and poultry osseous granules, is rinsed with flowing water, it is drained and standby；
Livestock and poultry osseous granules after the draining normal pressure in 90~100 DEG C of water boils 4~12 hours, then 0.1~0.3MPa of pressure,
Pressure cook 5~8 hours, are pulled out under conditions of 110~125 DEG C, the livestock and poultry osseous granules after obtaining degreasing；By the livestock and poultry after degreasing
Osseous granules are immersed in the hydrochloric acid of 0.25~0.65mol/L of molar concentration, after impregnating 1~6 hour, are pulled livestock and poultry osseous granules out and are used in combination
Flowing water removes surface hydrochloric acid；Continue to be placed in the sodium hydrate aqueous solution of mass fraction 0.01~0.1%, it is small to impregnate 1~2
Shi Hou pulls livestock and poultry osseous granules out and removes surface sodium hydrate aqueous solution with flowing water, the livestock and poultry osseous granules after obtaining desalination；
Livestock and poultry osseous granules after desalination are dried to moisture content and reach 2~6wt%；Then it crushes, is sieved, obtain livestock and poultry ossein；
The fermentation of S4 livestock and poultry ossein：Livestock and poultry ossein is sterilized；Into the livestock and poultry ossein after sterilizing plus water, preparation are dense as live stock and fowl bone element
Spend the livestock and poultry ossein solution of 20~40g/L；Zymophyte is made an addition into livestock and poultry ossein solution according to the inoculum concentration of 2~4% (mL/mL)
In, after 35~37 DEG C ferment 24~48 hours, collect fermentation liquid；
S5 sterilizing：By fermentation liquid after 110~120 DEG C sterilize 10~30 minutes, 40~50 DEG C are naturally cooled to, centrifugation 20~40
Minute, take supernatant；
S6 micro-filtration, ultrafiltration：By supernatant micro-filtration, the little particle of suspension is removed, collects micro-filtrate；It then is 2 with molecular cut off
The ultrafiltration membrane of~10kDa separates micro-filtrate, collects ultrafiltration permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometre collagen.
4. the preparation method of active peptide nanometre collagen according to claim 1, which is characterized in that using live stock and fowl bone as master
Raw material is wanted, is included the following steps：
S1 cleaning：Live stock and fowl bone is rinsed with flowing water, removes surface contaminants and watery blood；
S2 is arranged：Reject muscle, tendon forceps, cartilage, spongy bone and the periosteum of live stock and fowl bone after cleaning；
S3 prepares livestock and poultry ossein：Live stock and fowl bone after arrangement is broken into livestock and poultry osseous granules, is rinsed with flowing water, it is drained and standby；
Livestock and poultry osseous granules after the draining normal pressure in 90~100 DEG C of water boils 4~12 hours, then 0.1~0.3MPa of pressure,
Pressure cook 5~8 hours, are pulled out under conditions of 110~125 DEG C, the livestock and poultry osseous granules after obtaining degreasing；By the livestock and poultry after degreasing
Osseous granules are immersed in the hydrochloric acid of 0.25~0.65mol/L of molar concentration, after impregnating 1~6 hour, are pulled livestock and poultry osseous granules out and are used in combination
Flowing water removes surface hydrochloric acid；Continue to be placed in the sodium hydrate aqueous solution of mass fraction 0.01~0.1%, it is small to impregnate 1~2
Shi Hou pulls livestock and poultry osseous granules out and removes surface sodium hydrate aqueous solution with flowing water, the livestock and poultry osseous granules after obtaining desalination；
Livestock and poultry osseous granules after desalination are dried to moisture content and reach 2~6wt%；Then it crushes, is sieved, obtain livestock and poultry ossein；
The fermentation of S4 livestock and poultry ossein：The Chinese herbal medicine of livestock and poultry ossein weight 1~4% is added into livestock and poultry ossein, uniform mixing is mixed
And material；Material sterilizing will be mixed and stirred；To mixing and stirring in material after sterilizing plus water, prepares to become to mix and stir and expect that 20~40g/L's of concentration mixes and stirs material
Solution；Zymophyte is made an addition to according to the inoculum concentration of 2~4% (mL/mL) and is mixed and stirred in material solution, in 35~37 DEG C of fermentations 24~48
After hour, fermentation liquid is collected；The Chinese herbal medicine is made of the raw material of following proportion：30~50wt% of the root of Dahurain angelica, cordate houttuynia 10~
30wt%, surplus are fructus amomi；
S5 sterilizing：By fermentation liquid after 110~120 DEG C sterilize 10~30 minutes, 40~50 DEG C are naturally cooled to, centrifugation 20~40
Minute, take supernatant；
S6 micro-filtration, ultrafiltration：By supernatant micro-filtration, the little particle of suspension is removed, collects micro-filtrate；It then is 2 with molecular cut off
The ultrafiltration membrane of~10kDa separates micro-filtrate, collects ultrafiltration permeate；
S7 vacuum freeze drying：The ultrafiltration permeate vacuum freeze drying that collection is obtained, obtains the active peptide nanometre collagen.
5. the preparation method of active peptide nanometre collagen according to claim 1, which is characterized in that the live stock and fowl bone is goose
One of bone, ox bone, pig bone, fish-bone, camel bone, dog bone, deer bone.
6. the preparation method of active peptide nanometre collagen according to claim 3, which is characterized in that in step S4 fermentation process
In, sucrose is added according to the additive amount of 10~15g/L into livestock and poultry ossein solution, then adds zymophyte and ferments.
7. the preparation method of active peptide nanometre collagen according to claim 4, which is characterized in that in step S4 fermentation process
In, sucrose is added according to the additive amount of 10~15g/L to mixing and stirring in material solution, then adds zymophyte and ferments.
8. the preparation method of active peptide nanometre collagen according to claim 6 or 7, which is characterized in that in addition sucrose
Meanwhile inorganic salts are added according to the additive amount of 0.1~1g/L, the inorganic salts are calcium chloride, zinc chloride, magnesium chloride, protochloride
One of copper, frerrous chloride or a variety of mixtures.
9. the preparation method of active peptide nanometre collagen according to any one of claim 2~4, which is characterized in that described
Inorganic salts are calcium chloride；The zymophyte is bacillus cereus and/or lactobacillus bulgaricus.
10. active peptide nanometre collagen, which is characterized in that use active peptide nanometre collagen according to any one of claims 1 to 9
Preparation method be process.
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