CN101787078A - Collagen polypeptide, preparation method and application thereof - Google Patents
Collagen polypeptide, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a collagen polypeptide, a preparation method and application thereof. The collagen polypeptide comprises the following components in percentage by weight: 45-55 w/w% of components with molecular weight of 6500+/-100 Da, 20-25 w/w% of components with molecular weight of 1300+/-100 Da, and 25-35 w/w% of components with molecular weight of below 1000 Da. The nitrogen solubility index of the collagen polypeptide is 98.5-99.9%. The preparation method disclosed by the invention comprises the following steps: mixing the raw materials with complex enzyme A, and hydrolyzing.
Description
Technical field
The invention belongs to the enzyme technology field, relate in particular to a kind of collagen polypeptide and its production and use.
Background technology
In numerous animal proteinums, collagen protein has very high using value.According to there being the people to analyze, to high Mammals, the nearly altogether collagen protein more than 50,000,000,000 tons of nature is a kind of main renewable resources from the low invertebrates of waiting.
Collagen protein mainly is present in the animal body, is the main protein that constitutes animal connective tissue, and collagen protein still is the main component of extracellular matrix (ECM), accounts for 85% of collegen filament solids.In vertebrates, account for the 25%-30% of body internal protein total amount, be equivalent to 6% of body weight.Mainly be contained in bone, skin and tendon etc.Contain a large amount of glycine in the collagen protein, account for 27% of total amino acid, the content of proline(Pro) and oxyproline is also high especially, account for 14%, these two kinds is the peculiar amino acid of collagenic protein, and essential amino acids content such as tryptophane, tyrosine and methionine(Met) are low, and therefore, collagen protein belongs to incomplete protein.Collagen protein is white in color, and is a kind of polysaccharide protein, contains a spot of semi-lactosi and glucose.Collagen protein has very strong stretching force, and is water insoluble, can be dissolved in the diluted acid of tepor.
Up to the present, it is found that 27 kinds of dissimilar collagen proteins, wherein separate easily is come out and output is higher has only 5 kinds of collagen proteins.The I collagen type extensively is present in the reticular tissue such as skin, bone and blood vessel of animal, accounts for more than 90% of whole collagen contents, and it is lower to be easy to extraction and cost, application that can be a large amount of; The collagen of other type such as III, IV, V etc. are preparation under study for action only, all can't mass production owing to cost an arm and a leg.The I collagen type claims tropocollagen again, and its molecule is clavate, and molecular weight is about 300,000.Its basic structure is three strands of chains, is made up of two α 1 (I) and a α 2 (I) chain, and each gang α chain contains 1056 amino-acid residues, and three strands of α chains are in the dextrorotation mode, mutually three bursts of superhelixs of coiled.Further find that all dissimilar collagen all forms in the mode of triple helix, difference only is polypeptide fragment that various stage casing triple helix forms difference to some extent, thereby is folded into different three-D space structures.
Collagen protein is a kind of very important protein of human body, mainly is present in the reticular tissue, and be the main component of human body skin, account for skin dry weight 70%-80%.The growth of skin, reparation and nutrition all be unable to do without collagen protein.Collagen protein makes cell become plentiful, thereby makes skin full, keeps skin elasticity with moist, and it is moist to keep skin, skin health two big crucial---crease-resistant with preserve moisture all relevant with collagen protein.The medical research prompting: the oxyproline in the collagen protein is the crucial thing that human body skin forms.Organic 70%-80% is a collagen protein in the bone, when bone generates, at first must synthesize the framework that competent collagen fabric is formed bone, and therefore, the someone claims that collagen protein is the bone in the bone.Collagen protein also is the main component of eyes cornea, but forms with crystallized form.But, after about 25 years old, the speed that the intravital collagen protein of people runs off just begins to accelerate, supply with not as good as consume, add uviolizing and intravital oxygenizement, all may destroy the structure of collagen protein, allow it lose original elastic force, the lax reason that occurs ahead of time of wrinkle and facial skin that Here it is.Therefore, in time replenish healthy most important to human body of the collagen protein that runs off.
Collagen protein has superior biocompatibility and biodegradability and uses very extensive as natural Biological resources.And collagen protein source is very extensive, can be by animal byproduct, make as the scales of the dead meal of skin, bone, leather processing factory, fish, fish-skin etc.Fully utilize these byproducts and can reduce environmental pollution, its preparation is also relatively easy, thereby great application prospect is arranged.
Collagen protein in Application in Food relatively early can make the tender degree increase of product, elasticity improvement etc. as early stage adding pigskin in meat product as the quality improver of meat product.As functional food and protective foods in recent years collagen protein and degraded product thereof be used at large in functional food and the protein drinks, particularly its degradation product does not almost have allergy protein, body can absorb fully.Japanese health ministry is introduced it to the market as " heath food "; Collagen protein can also be as a kind of protective foods of replenishing the calcium, because is the launch vehicle that the calcium in the blood plasma is transported to osteocyte from collagen protein in the blood plasma through proline(Pro), therefore take the photograph the enough collagen proteins of people, just can guarantee normal calcareous requirement to a certain extent, the wangkai professor of preventing disease institute of Hong Kong University points out, if lack collagen protein, prevent and treat osteoporosis with replenishing the calcium, Duo calcium also can't improve again.The collagen protein powder that is processed into can directly add in coffee, tea drink, beer, fruit juice, the grape wine, and is edible very convenient.There is not the possibility that infects mad cow disease and other relative disease in EFSA (EFSA) even confirmed that the collagen protein in animal skeleton source also is safe yet.
What collagen protein can keep vessel wall elasticity, prevents embolism, improve cartilage and ligament lubricates, alleviates ankylosis etc., its effect in treating aspects such as corium damage or defective, beauty and shaping, improvement sleep, enhancing body immunizing power has obtained common recognition, science and technology development is in recent years quickened and has been expanded collagen protein in medically new application.As: in order to increase the application of collagen protein in pharmaceutical industries, traditional technology is with high temperature, chemical reaction etc., but these technology can be destroyed protein usually, also can increase the insecurity that it is used in human body.Now the scientist of John Hopkins university mixes collagen protein and the small-molecule substance that the is called collagen protein imitation polypeptide mode by physical mixed in research and changes the various character of collagen protein, thereby brings in the new Application Areas of medical field.
The collagen molecules outside exists a large amount of carboxyls and hydroxyl, has a large amount of natural moisturizing factors such as glycine simultaneously, can improve histiocytic water storage capacity, makes skin moisture-keeping; Collagen solution also has very strong radiation resistance, thereby is widely used in cosmetic industry, nowadays much all contains collagen protein in the makeup such as the facial mask of selling in the market, eye cream, protective skin cream.Collagen protein is used in the makeup can prevent skin aging, anti-aging in advance, repairs or alleviates symptoms such as wrinkle, pouch, black eye, color spot.Zhengwei Mao etc. studies show that glutaraldehyde is used for the crosslinked of collagen protein, can improve the biologically stable of collagen protein face greatly, thereby enlarge the application of collagen protein in makeup.But along with the increase of degree of crosslinking, water-retaining capacity and turgidity but can reduce.In addition, collagen protein promotes effects such as growth hormone secretion and muscle growth, the U.S. breast of chest enlarge, weight-reducing in addition.
At present collagen protein is the health ﹠ beauty products that receives an acclaim very much, and use range is three big main flows with shaping medical science, nutritional supplement, skin care products, just with inject, oral, wiping is application.And wherein with the injection effect preferably but cost an arm and a leg; Wiping then can't absorb too greatly because of molecular weight; The oral collagen of hydrolysis, though moderate cost and effect are better, in fact oral collagen protein can be subjected to stomach acids destroy, can't absorb, so oral collagen protein is also little for the help of skin in fact.And different purposes, the relative molecular mass of collagen protein there is different requirements: require relative molecular mass usually below 2000Da merely in makeup, to allow in the hydrolytic collagen porous human skin as trophicity skin type raw material; And the hair care category makeup also should have certain film-forming properties usually except that requiring hydrolytic collagen to have the moisture retention, so the relative molecular mass general requirement of hydrolytic collagen is higher.Therefore, inquiring into the collagen hydrolysate method is to widen the main channel that it is used in makeup.
China has generally adopted Production by Enzymes animal Collagen Hydrolysate at present, though tentatively solved above problem, also improve and promoted collagen protein application and development to a certain extent, but still there are a lot of problems, as: 1. the collagen molecules amount is big, poorly soluble, can't directly be absorbed by cell; 2. existing collagen peptide complex manufacturing, cost height, and the quality instability of product; 3. the enzymatic production process of existing collagen peptide, hydrolysis is not thorough, and raw material availability is low; Though can making, the technology of chemistry, physics method collagen protein hydrolysis comparatively fully can destroy amino acid such as tryptophane, Serine, and resulting hydrolysate biologically active not substantially; 4. existing collagen peptide still has bitter taste, fishy smell, and is difficult to remove; 5. existing collagen peptide can be by stomach en-and trypsin hydrolyzing after entering human body, most of loss of activity.
Therefore, this area presses for provides a kind of free from extraneous odour, bitter taste, and stay-in-grade collagen polypeptide also needs to provide the preparation method of a kind of degree of hydrolysis height, technology described collagen polypeptide simple, with low cost.
Summary of the invention
The present invention aims to provide a kind of collagen polypeptide.
Another object of the present invention provides the preparation method of described collagen polypeptide.
A further object of the present invention provides the purposes of described collagen polypeptide.
In a first aspect of the present invention, a kind of collagen polypeptide is provided, in the collagen polypeptide gross weight, molecular weight is at the ingredients constitute 45-55w/w% of 6500Da ± 100Da, molecular weight is at the ingredients constitute 20-25w/w% of 1300 ± 100Da, the ingredients constitute 25-35w/w% of molecular weight below 1000Da; Nitrogen solubility index NSI is 98.5-99.9%.
In another preference, in the collagen polypeptide gross weight, crude protein content 90-95w/w%, ash content 3-5w/w%, sugar, moisture and lipid content 2-4w/w%, acid-soluble protein content 99.5-99.9w/w%.
In another preference, described collagen polypeptide can both solution water under all pH, the solution clarification, be faint yellow-khaki, show slightly acidic; Described collagen polypeptide free from extraneous odour, no bitter taste; Very stable to heat, 100 ℃ are boiled the nothing precipitation and separate out; Preserve 6 months content under the room temperature and do not have considerable change.
In another preference, described collagen polypeptide prepares by following step:
(I) at pH9-10,50-60 ℃ was mixed 6-10 hour with starting material and prozyme A; In the gross weight of solid substance, the amount of prozyme A is 2-5w/w%; Described starting material are selected from following one or more: animal skin, animal hoof tendon, animal bone, animal scale and shell; Described prozyme A is made of following composition: in the gross weight of the dry of prozyme A, M-Zyme 55-65w/w%, papoid 5-10w/w%, trypsinase 10-15w/w%, Quimotrase 2-5w/w%, Sumizyme MP 10-15w/w%, kethepsin 0.02-0.05w/w% and bromeline 2-5w/w%; With
(II) removes by filter insolubles and obtains collagen polypeptide solution.
In a second aspect of the present invention, a kind of preparation method of aforesaid collagen polypeptide provided by the invention is provided, described method comprises step:
(1) with starting material and prozyme A at pH9-10,50-60 ℃ was mixed 6-10 hour; In the gross weight of solid substance, the amount of prozyme A is 2-5w/w%; Described starting material are selected from following one or more: animal skin, animal hoof tendon, animal bone, animal scale and shell; Described prozyme A is made of following composition: in the gross weight of the dry of prozyme A, M-Zyme 55-65w/w%, papoid 5-10w/w%, trypsinase 10-15w/w%, Quimotrase 2-5w/w%, Sumizyme MP 10-15w/w%, kethepsin 0.02-0.05w/w% and bromeline 2-5w/w%; With
(2) remove by filter insolubles and obtain collagen polypeptide solution.
In another preference, described starting material are selected from following one or more: fish-skin, pigskin, pig's feet, beef tendon, fish scale, chicken bone, fish-bone.
In another preference, described step (1) is: at pH9-10,50-60 ℃ was mixed after 6-10 hour, 75-85 ℃ of insulation 30 minutes with starting material and prozyme A.
In another preference, the starting material in the step (1) are Powdered or pasty state.
In another preference, comprise step between step (1) and (2): make 15-20 ℃ of enzyme reaction solution pH5-6, leave standstill.
In a third aspect of the present invention, a kind of purposes of aforesaid collagen polypeptide provided by the invention is provided, described collagen polypeptide is used for preparation and removes free radical, oxidation resistant composition; Or be used to prepare the composition that promotes the cell growth; Or be used to prepare the composition of anticancer growth; Or be used to prepare and absorb and/or the composition of water conservation; Or be used to prepare the composition of oil suction; Or be used for preparation and be used for anticoagulant composition.
In view of the above, the invention provides a kind of free from extraneous odour, bitter taste, stay-in-grade collagen polypeptide also provides the preparation method of a kind of degree of hydrolysis height, technology described collagen polypeptide simple, with low cost.
Description of drawings
The M-Zyme of different concns is to the hydrolysis ability of collagen protein among Fig. 1 prozyme A.
The prozyme A of Fig. 2 different amounts is to the hydrolysis ability of collagen protein.
The process flow diagram of Fig. 3 collagen polypeptide preparation.
The gel chromatography figure of Fig. 4 collagen polypeptide.
Fig. 5 collagen polypeptide is to normal cell (dog renal epithelial cell MDCK) proliferation function.
Fig. 6 collagen polypeptide is to the effect of human embryonic lung's fibroblasts proliferation.
Fig. 7 collagen polypeptide is to the inhibition effect of liver cancer cell; Wherein
A is human liver cancer cell HepG2 (growing state when not adding collagen polypeptide), and B is human liver cancer cell HepG2 (growing state when adding collagen polypeptide).
Fig. 8 collagen polypeptide is to the restraining effect of liver cancer cell (HepG2) growth.
The comparison of the retentiveness of Fig. 9 collagen polypeptide and G ﹠ W.
The gel chromatography figure of Figure 10 collagen polypeptide before and after stomach, tryptic digestion; Wherein
A is the gel chromatography figure before the digestion, and B is the gel chromatography figure behind gastric pepsin digestion, and C is the gel chromatography figure behind stomach en-, tryptic digestion.
Embodiment
The contriver is through extensive and deep research, be surprised to find that hydrolysis of animal skin, animal hoof tendon, animal bone, animal scale and shell obtain collagen polypeptide to use prozyme A effectively, resulting collagen polypeptide does not have bitter taste, peculiar smell, the hydrolysis degree height, the molecular weight of its nearly all component does not have total free aminoacids below 6500Da.The collagen polypeptide that the present invention obtains can be removed free radical, anti-oxidant; Can promote the cell growth; Can grow by anticancer; Can absorb moisture and/or keep moisture; Can oil suction; Also has anticoagulant effect.
Definition
As used herein, " prozyme A " is the composition that is made of following composition, gross weight in the dry of prozyme A, M-Zyme 55-65w/w%, papoid 5-10w/w%, trypsinase 10-15w/w%, Quimotrase 2-5w/w%, Sumizyme MP 10-15w/w%, kethepsin 0.02-0.05w/w% and bromeline 2-5w/w%;
Used M-Zyme is the DNA of Bacillus licheniformis L-25 M-Zyme and this M-Zyme of encoding among the prozyme A, multiple animal proteinum and vegetable-protein all there are very strong hydrolysis ability and very high hydrolysis efficiency, in publication number is the Chinese patent application of CN1361279A detailed description is arranged, the related content in this patent is incorporated among the application.All the other proteolytic enzyme can obtain from commercially available channel.
As used herein, " collagen protein " is meant the main protein that is contained in skin, hoof tendon, bone and the scale and shell of animal, and mainly refers to wherein contained I collagen type, or by the prepared powder that is rich in collagen protein of these materials.
As used herein, " starting material " are meant the skin that comes from the non-living body animal, hoof tendon, bone and the scale and shell that contains collagen protein, and described animal is selected from birds, fish or Mammals, preferably, is selected from chicken, duck, dove, pig, ox, horse or sheep.
As used herein, the composition of term " pharmaceutically acceptable " or " acceptable on the bromatology " is applicable to people and/or animal and does not have excessive bad side reaction (as toxicity, stimulation and transformation reactions), the material of rational benefit/risk ratio is promptly arranged.
Collagen polypeptide
The invention provides a kind of collagen polypeptide, in the collagen polypeptide gross weight, molecular weight is at the ingredients constitute 45-55w/w% of 6500Da ± 100Da, and molecular weight is at the ingredients constitute 20-25w/w% of 1300 ± 100Da, the ingredients constitute 25-35w/w% of molecular weight below 1000Da; Nitrogen solubility index NSI is 98.5-99.9%.
Collagen polypeptide provided by the invention also has following feature:
1) be a kind of oyster white-white powder, crude protein content 90%-95%, ash content 3%-5%, sugar and moisture content 2%-4%, its nitrogen solubility index NSI is 98.5%-99.9%, acid-soluble protein content 99.5%-99.9%;
2) be a kind of peptide composition, the polypeptide molecular weight of each component is all below 6.5kDa in the composition, and wherein molecular weight accounts for 48.5% about 6500Da, and molecular weight accounts for 22.5% between 1300Da, molecular weight accounts for 29.0% below 1000Da, do not have total free aminoacids substantially and produce;
3) free from extraneous odour, no bitter taste;
4) can both solution water under all pH, the solution clarification, be faint yellow-khaki, show slightly acidic;
5) very stable to heat, 100 ℃ are boiled the nothing precipitation and separate out;
6) at room temperature preserve 6 months content and do not have considerable change.
Collagen polypeptide provided by the invention also has following function:
1) has strong anti-oxidant activity, the removing ability of hydroxy radical qiao and DPPH free radical all is significantly higher than vitamins C, can reach 94.8%, 59.2% and 86.5% respectively the clearance rate of hydroxy radical qiao, ultra-oxygen anion free radical and DPPH free radical;
2) normal somatic cell has good increment effect, and cancer cells is also had very strong inhibition effect, can reach 26.5% and 238.4% respectively to the proliferation rate of normal cell MDCK and HLF, can reach 32.4% to the inhibiting rate of cancer cells Hep-G2;
3) good water-absorbent, oil absorbency are arranged, and retentiveness, emulsifying property and emulsifying stability are preferably arranged;
4) effect of stronger anticoagulant is arranged;
5) strong stomach en-and trypsin-resistant are arranged, still have very strong activity after digesting and assimilating in vivo.
Collagen polypeptide provided by the invention can be liquid or liquid concentrate.
The preparation method
The invention provides a kind of preparation method of collagen polypeptide, it comprises step: through adding prozyme A hydrolysis after the pre-treatment, hydrolysate is crossed ion exchange resin desalination, decolouring through behind the micro-filtration, finally makes collagen polypeptide with starting material.
In a preferred embodiment of the invention, in the gross weight of solid substance, the dosage of prozyme A adds by the 2-5w/w% of solid content.
In a preferred embodiment of the invention, the hydrolysis of being adopted is at 50 ℃-60 ℃, under the pH9-10 condition hydrolysis 6-10 hour.
In another preferred embodiment of the present invention, the described starting material that prepare the method for collagen polypeptide are the skin of animal, are example with fish-skin or pigskin, may further comprise the steps:
1) pre-treatment of raw material I: fish-skin or unhairing pigskin are cleaned and the degrease processing, to remove other impurity on fat and surface; More preferably, behind the rejecting subcutaneous lipids, soak with sodium chloride solution;
2) pre-treatment of raw material II: fish-skin or the unhairing pigskin handled through step 1) are cut into fritter, soak with sodium hydroxide solution again, further remove the impurity on fat and surface;
3) pre-treatment of raw material III: process step 2) fish-skin or the unhairing pigskin piece of handling is milled to uniform soup compound or mashed prod;
4) enzymic hydrolysis: at pH9-10, under 50 ℃-60 ℃ and prozyme A mixing 6-10 hour, according to the total solid meter, prozyme A amount is 2%-5% (m/m) with the soup compound of step 3) gained or mashed prod; More preferably, after hydrolysis 6-10 hour, be warming up to 75 ℃-85 ℃ rapidly, and be incubated 30 minutes;
5) separate: reduce the temperature to 15 ℃-20 ℃ of reaction solution, regulate pH5.0-6.0, carry out membrane filtration, remove insoluble solid substance residue, collect collagen polypeptide solution.
In another preferred embodiment of the present invention, employed starting material are hoof tendons of animal, are example with pig's feet or beef tendon, may further comprise the steps:
1) pre-treatment of raw material I: with pig's feet or beef tendon with stripping and slicing after, carry out alkali and clean, to remove the impurity on fat and surface; More preferably, be with sodium bicarbonate (sodium-chlor that contains 1%-3%, m/v) solution soaking;
2) pre-treatment of raw material II: pig's feet or the beef tendon fritter handled through step 1) are milled to uniform soup compound or mashed prod;
3) enzymic hydrolysis: with step 2) soup compound of gained or mashed prod be at pH9-10, and under 50 ℃-60 ℃ and prozyme A mixing 6-10 hour, according to the total solid meter, prozyme A amount is 2%-5% (m/m); More preferably, after hydrolysis 6-10 hour, be warming up to 75 ℃-85 ℃ rapidly, and be incubated 30 minutes;
4) separate: reduce the temperature to 15 ℃-20 ℃ of reaction solution, regulate pH5.0-6.0, carry out membrane filtration, remove insoluble solid substance residue, collect collagen polypeptide solution.
In another preferred embodiment of the present invention, employed starting material are bone or scale and shells of animal, are example with fish scale, chicken bone or fish-bone, may further comprise the steps:
1) pre-treatment of raw material I: fish scale, chicken bone or fish-bone are carried out alkali clean, with dope and other impurity of removing the surface; More preferably, soak with sodium hydrogen carbonate solution;
2) pre-treatment of raw material II: fish scale, chicken bone or the fish-bone handled through step 1) with after being slit into fritter, are used sour decalcification; More preferably, be with 30 ℃ of-35 ℃ of stirrings of hydrochloric acid;
3) pre-treatment of raw material III: process step 2) fish scale, chicken bone or the fish-bone piece of handling is milled to uniform mud shape thing or mashed prod;
4) enzymic hydrolysis: at pH9-10, under 50 ℃-60 ℃ and prozyme A mixing 6-10 hour, according to the total solid meter, prozyme A amount is 2%-5% (m/m) with the soup compound of step 3) gained or mashed prod; More preferably, after hydrolysis 6-10 hour, be warming up to 75 ℃-85 ℃ rapidly, and be incubated 30 minutes;
5) separate: reduce the temperature to 15 ℃-20 ℃ of reaction solution, regulate pH5.0-6.0, carry out membrane filtration, remove insoluble solid substance residue, collect collagen polypeptide solution.
More preferably, after the step 5) of above-mentioned each preference, can also comprise following one or more steps:
6) purifying: desalination and decolouring;
7) concentrate: with thin film evaporation or vacuum concentration collagen polypeptide solution is concentrated, make solid content reach 15%-20%;
8) drying: concentrated solution is made the collagen polypeptide powder through lyophilize, spraying drying or vacuum drying.
Purposes
Provided by the invention have good characteristic and multiple bioactive collagen polypeptide can be used for various technical fields, comprises food, beverage, makeup, makeup, medicines and health protection and industrial application.
Collagen polypeptide provided by the invention can be used to remove free radical, anti-oxidant aspect, can make protective foods, beverage, makeup or pharmaceuticals with anti-oxidant activity; Or be used for food fresh keeping; Or make to have and take care of hair and the hair-cream agent of moisture-keeping function or face cream with antioxygenation; Or make prevention and treatment because the medicine of the caused disease of radical damage.
Collagen polypeptide provided by the invention can be used for platelet aggregation-against and prevention and treatment thrombosis.
Collagen polypeptide provided by the invention can be used for the anticancer growth, can make to have medicine or the healthcare products that the prevention cancer cells formed or be used for the anticancer growth.
Collagen polypeptide provided by the invention can be used to promote normal somatic cell growth, can make the cosmetics such as face cream, emulsifiable paste, emulsion of Pear Power, beauty treatment effect; Or make the cosmetic health product or the medicine of Pear Power, beauty treatment effect; Or make cosmetic health product or medicine with the U.S. newborn effect of chest enlarge.
It is above-mentioned when every when collagen polypeptide provided by the invention is applied to, collagen polypeptide and the mixing of " pharmaceutically acceptable " or " acceptable on the bromatology " carrier can be obtained composition, described composition is selected from pharmaceutical composition, food compositions or makeup.
Formulation for composition of the present invention has no particular limits, and can be any formulation that is applicable to that Mammals is taken; Preferably, described formulation can be selected from: granule, capsule, tablet, powder agent, oral liquid, suspension or emulsion.
When making pharmaceutical composition, be that collagen polypeptide with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about the 0.4g/ kg body weight, and in most of the cases be no more than about 5g/ kg body weight, preferably this dosage is about 0.6g/ kg body weight-Yue 3.0g/ kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
The above-mentioned feature that the present invention mentions, or the feature that embodiment mentions can arbitrary combination.All features that this case specification sheets is disclosed can with any composition forms and usefulness, each feature that is disclosed in the specification sheets can anyly provide the alternative characteristics of identical, impartial or similar purpose to replace.Therefore removing has special instruction, and the feature that is disclosed only is the general example of equalization or similar features.
Major advantage of the present invention is:
1, the present invention starts with from the raw material composition, has opened up the novel process of collagen polypeptide preparation, has realized the inward nature and the technological innovation of product concept.
2, Du Te compound protease has special restriction enzyme site, the hydrolysate homogeneous, stable, do not have a hydrophobicity ends exposed substantially, thereby at the bitter taste of fundamentally having eliminated collagen polypeptide and stink, debitterize, the deodorization process of very complicated have been avoided, improve the yield of product greatly, reduced production cost.
3, its solvability of prepared collagen polypeptide and viscosity are not subjected to pH and Temperature Influence, good water-absorbent is arranged, oil absorbency, and retentiveness is preferably arranged, emulsifying property and emulsifying stability, molecular weight is all below 6500Da, molecular weight is accounting for more than 50% between the 300-1300Da, no total free aminoacids produces, can be well directly by skin absorption and utilization, therefore, the prepared collagen polypeptide of the present invention is expected to solve and improves present stage collagen protein and a series of problems of using of collagen hydrolysate peptide, can be widely used in beauty treatment, health care, food, a plurality of fields such as medicine.
4, the prepared collagen polypeptide of the present invention has good increment effect to normal somatic cell, and cancer cells also there is very strong inhibition effect, can also effectively suppress hematoblastic gathering, strong oxidation-resistance is arranged, after digesting in vivo, absorbing, its biological activity does not have tangible minimizing, so the prepared collagen polypeptide of the present invention is a kind of very fine multifunctional bio-active peptide, and very high using value is arranged.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise all percentage ratio, ratio, ratio or umber by weight.
Unit in the percent weight in volume among the present invention is well-known to those skilled in the art, for example is meant the weight of solute in 100 milliliters solution.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Experiment condition:
1. material
The skin of animal such as pigskin, fish-skin, or the hoof tendon of animals such as pig's feet, beef tendon, or bone or the scale and shell of animal such as fish scale, chicken bone, fish-bone are collected by our company oneself; Stomach en-(1: 3000), trypsin 1: 250), give birth to the worker available from Shanghai; Experimental cell strain MDCK and HLF are available from Invitrogen company, and HepG2 is available from ATCC company; The DMEM substratum, the RPM1640 substratum, Glutamine, cell cultures with non-essential amino acid all available from Invitrogen company; Cell cultures uses foetal calf serum available from HyClone company; MTT is available from Ameresco company.
2. reagent
The experiment agents useful for same is commercially available analytical reagent.
3. used Measurement for Biochemistry is routine techniques in this area among the present invention.In following examples, unless specified otherwise, all experimental implementation are all carried out according to related Sections in following laboratory manual or the document or part, comprising: Cheng Jinrui etc., Chinese food industrial standards compilation (second edition); Zhao Yongfang etc., Measurement for Biochemistry principle and application thereof (second edition); Zhu Jian etc., Biochemistry Experiment [M]; D.L Spector etc., cell experiment guide (upper and lower).
Prozyme A is to the hydrolysis of collagen protein
In the 40-80w/w% scope, adjust the percentage concentration of M-Zyme among the prozyme A gradually successively, then according to the step 1) described in the embodiment 2 (1) to step 4), or the step 1) described in the embodiment 2 (2) is to step 3), or the step 1) described in the embodiment 2 (3) to step 4) is implemented the hydrolysis (amount of prozyme A according to the 3w/w% of material quantity add) of prozyme A to collagen protein, after hydrolysis finishes, got enzymolysis solution 5ml12000rpm centrifugal 5 minutes, get supernatant liquor and measure soluble protein content, and calculate degree of hydrolysis DH according to following formula with Folin-phenol method:
In the formula: N
2---the soluble protein content of enzymolysis solution behind the enzyme digestion reaction, mg/mL;
N
1---the soluble protein content of solution before the enzyme digestion reaction, mg/mL;
N
0---participate in the total protein concentration (add-on of the crude protein content * raw material of raw material, the crude protein content of raw material is measured according to Kjeldahl determination) of the collagen protein of enzyme digestion reaction, mg;
V---enzyme digestion reaction liquid is long-pending, mL.
Get the centrifugal back of the above gained enzymolysis solution of 2ml supernatant again, mix with 2ml 20% trichoroacetic acid(TCA) (TCA), vibration evenly, after leaving standstill 5 minutes, the centrifugal 10min of 10000rpm gets supernatant liquor, measures soluble protein content with Folin-phenol method, be acid-soluble protein content in the enzymolysis solution, the degree of acid-soluble protein calculates according to following formula:
Acid-soluble protein degree %=Ns/N * 100
In the formula: acid-soluble protein content in the Ns-enzymolysis solution, mg/ml;
Soluble protein content in the N-enzymolysis solution, mg/ml.
In the scope of 2-5w/w% (amount of prozyme A is with respect to the per-cent number of material quantity), change the addition of prozyme A gradually successively, then according to the step 1) described in the embodiment 2 (1) to step 4), or the step 1) described in the embodiment 2 (2) is to step 3), or the step 1) described in the embodiment 2 (3) to step 4) is implemented the hydrolysis (in prozyme A the content of M-Zyme be 60wt%) of prozyme A to collagen protein, hydrolysis is measured the degree of hydrolysis of enzymolysis solution and the degree of acid-soluble protein respectively according to above same method after finishing.
Above gained result as depicted in figs. 1 and 2.As can be seen from Figure 1, M-Zyme has very strong hydrolysis ability to collagen protein, and when M-Zyme content was in the 55-65% scope among the prozyme A, the degree of hydrolysis of enzymolysis solution and the degree of acid-soluble protein all can reach 100%.After the content of M-Zyme among the prozyme A is lower than 55%, the content of acid-soluble protein reduces rapidly in the enzymolysis solution, M-Zyme content is 40% o'clock, and acid-soluble protein content is reduced to about 60%, and this explanation M-Zyme has crucial effects to the generation of collagen polypeptide.As can be seen from Figure 2, in the scope of 2-5% prozyme A addition, all can be fast, effectively, Collagen Hydrolysate produces collagen polypeptide completely.
The preparation of collagen polypeptide
(1) skin with animals such as pigskin, fish-skins is raw material
Skin with animals such as pigskin, fish-skins is raw material, and the preparation collagen polypeptide carries out according to following steps, and technical process as shown in Figure 3.
1) pre-treatment of raw material I: with fish-skin or unhairing pigskin, clean up with tap water, reject subcutaneous incidental fat, then according to solid-to-liquid ratio 1: 1-1: 3 (m/v) add the sodium-chlor (m/v) of 1%-3%, be heated to 40 ℃-50 ℃, and place under this condition and to soak 30-45 minute, the liquid that inclines, and once more it is cleaned up with tap water;
2) pre-treatment of raw material II: the fish-skin of handling through step 1) or unhairing pigskin cut with cutting machine be slit into fritter, then according to solid-to-liquid ratio 1: 1-1: 3 (m/v) add 1% sodium hydroxide (m/v), be heated to 70 ℃-80 ℃, and place under this condition and to soak 20-30 minute, the liquid that inclines, and once more it is cleaned up with tap water;
3) pre-treatment of raw material III: through step 2) fish-skin of handling or unhairing pigskin piece and water are according to solid-to-liquid ratio 3: 1-4: 1 mixes (m/v), with mincer or colloidal mill it is milled to uniform soup compound or mashed prod;
4) enzymic hydrolysis: the soup compound or the mashed prod of step 3) gained are joined in the retort, and then the tap water of 3-5 times of volume of adding, after fully mixing, regulate pH to 9-10, be warming up to 50 ℃-60 ℃, add prozyme A (m/m), mix the back under this condition hydrolysis 6-10 hour according to the 2%-5% of solid content, be warming up to 75 ℃-85 ℃ then rapidly, and be incubated 30 minutes;
5) separate: reduce the temperature to 15 ℃-20 ℃ of reaction solution, regulate pH to 5.0-6.0, and left standstill 30 minutes, carry out membrane filtration then, remove insoluble solid substance residue, collect collagen polypeptide solution;
6) purifying: spent ion exchange resin HZ016, D345 and macroporous adsorbent resin HD-8 carry out purifying to collagen polypeptide, desalination and decolouring;
7) concentrate: with thin film evaporation or vacuum concentration collagen polypeptide solution is concentrated, make solid content reach 15%-20%;
8) drying: concentrated solution is made the collagen polypeptide powder through lyophilize, spraying drying or vacuum drying.
(2) the hoof tendon with animals such as pig's feet, beef tendons is a raw material
Hoof tendon with animals such as pig's feet, beef tendons is a raw material, and the preparation collagen polypeptide carries out according to following steps, and technical process as shown in Figure 3.
1) pre-treatment of raw material I: pig's feet or beef tendon are cut with cutting machine be slit into fritter earlier, clean up with tap water, again according to solid-to-liquid ratio 1: 1-1: 3 (m/v) add the sodium bicarbonate (sodium-chlor that contains 1%-3% of 3%-5%, m/v), be heated to 75 ℃-85 ℃, and place under this condition and to soak 45-60 minute, the liquid that inclines, and once more it is cleaned up with tap water;
2) pre-treatment of raw material II: pig's feet or the beef tendon fritter handled through step 1), with water according to solid-to-liquid ratio 3: 1-4: 1 mixes (m/v), with mincer or colloidal mill it is milled to uniform soup compound or mashed prod;
3) enzymic hydrolysis: with step 2) soup compound of gained or mashed prod join in the retort, and then the tap water of 3-5 times of volume of adding, after fully mixing, regulate pH to 9-10, be warming up to 50 ℃-60 ℃, add prozyme A (m/m), mix the back under this condition hydrolysis 6-10 hour according to the 2-5% of solid content, be warming up to 75 ℃-85 ℃ then rapidly, and be incubated 30 minutes;
4) separate: reduce the temperature to 15 ℃-20 ℃ of reaction solution, regulate pH to 5.0-6.0, and left standstill 30 minutes, carry out membrane filtration then, remove insoluble solid substance residue, collect collagen polypeptide solution;
5) purifying: spent ion exchange resin HZ016, D345 and macroporous adsorbent resin HD-8 carry out purifying to collagen polypeptide, desalination and decolouring;
6) concentrate: with thin film evaporation or vacuum concentration collagen polypeptide solution is concentrated, make solid content reach 15%-20%;
7) drying: concentrated solution is made the collagen polypeptide powder through lyophilize, spraying drying or vacuum drying.
(3) bone or the scale and shell with animals such as fish scale, chicken bone, fish-bones is raw material
Bone or scale and shell with animals such as fish scale, chicken bone, fish-bones are raw material, and the preparation collagen polypeptide carries out according to following steps, and technical process as shown in Figure 3.
1) pre-treatment of raw material I: with fish scale, chicken bone or fish-bone, clean up with tap water, then according to solid-to-liquid ratio 1: 1-1: 3 (m/v) add the sodium bicarbonate (m/v) of 1%-3%, be heated to 40 ℃-50 ℃, and place under this condition and to soak 30-45 minute, the liquid that inclines, and once more it is cleaned up with tap water;
2) pre-treatment of raw material II: fish scale, chicken bone or the fish-bone handled through step 1) cut with cutting machine be slit into fritter, then according to solid-to-liquid ratio 1: 3-1: 5 (m/v) add the hydrochloric acid of 0.5-2N, be heated to 30 ℃-35 ℃, and place under this condition and to stir 45-50 minute, the liquid that inclines, and once more it is cleaned up with tap water;
3) pre-treatment of raw material III: through step 2) fish scale, chicken bone or the fish-bone piece of handling and water are according to solid-to-liquid ratio 3: 1-4: 1 mixes (m/v), with bone cutter or bone mud machine it is milled to uniform mud shape thing or mashed prod;
4) enzymic hydrolysis: the soup compound or the mashed prod of step 3) gained are joined in the retort, and then the tap water of 3-5 times of volume of adding, after fully mixing, regulate pH to 9-10, be warming up to 50 ℃-60 ℃, add prozyme A (m/m), mix the back under this condition hydrolysis 6-8 hour according to the 2%-5% of solid content, be warming up to 75 ℃-85 ℃ then rapidly, and be incubated 30 minutes;
5) separate: reduce the temperature to 15 ℃-20 ℃ of reaction solution, regulate pH to 5.0-6.0, and left standstill 30 minutes, carry out membrane filtration then, remove insoluble solid substance residue, collect collagen polypeptide solution;
6) purifying: spent ion exchange resin HZ016, D345 and macroporous adsorbent resin HD-8 carry out purifying to collagen polypeptide, desalination and decolouring;
7) concentrate: with thin film evaporation or vacuum concentration collagen polypeptide solution is concentrated, make solid content reach 15%-20%;
8) drying: concentrated solution is made the collagen polypeptide powder through lyophilize, spraying drying or vacuum drying.
Prepared thus collagen polypeptide solution be clarifying faint yellow-khaki liquid, free from extraneous odour, no bitter taste are slightly acidic (pH5-6).
Prepared thus collagen polypeptide white powder-off-white powder, free from extraneous odour, crude protein content 90%-95% (Kjeldahl determination), ash content 3%-5%, sugar and moisture content 2%-4%, its nitrogen solubility index NSI is 98.5%-99.9%, acid-soluble protein content 99.5%-99.9%, can be dissolved in fully in the water of any pH, solution be clarifying faint yellow-khaki liquid, also be slightly acidic.
The mensuration of collagen polypeptide molecular weight distribution
Adopt gel filtration chromatography to analyze the molecular weight distribution of collagen polypeptide.Gel filtration chromatography is separated component of mixture according to the molecular weight size, and according to the retention volume of each component, the reference standard spectrogram can calculate the molecular weight size of each component in the sample.
Retention volume with each standard protein in the standard spectrogram is an X-coordinate, logarithm with each standard protein molecular weight is an ordinate zou, and the drawing standard curve is tried to achieve analysis formula lgY=-0.1775X+6.4976, Y is the molecular weight (Da) of component in the formula, and X is the retention volume of component.
Get 25 ℃ of 2ml collagen polypeptide solution (embodiment 2 is prepared), the centrifugal 10min of 10000rpm, supernatant liquor get 100ul and carry out the HPLC analysis with behind the micro-filtrate membrane filtration of 0.2um.Chromatographic condition is: 0.05M phosphate buffered saline buffer, 0.15M NaCl, pH7.0; 0.5ml/min, 25 ℃; UV-detector, 220nm; Last sample 100ul; Superdex
TMThe 7510/300GL chromatographic column.
The gained color atlas calculates the molecular weight of each component according to the retention volume of each component in the spectrogram as shown in Figure 4, goes out each percentages of ingredients content according to the calculated by peak area of each component in the spectrogram, and the result is as shown in table 1.
The molecular weight of each component and degree in table 1 collagen polypeptide
Molecular weight (Da) | Degree (%) |
??6500Da | ??48.5 |
??1300Da | ??22.5 |
??≤1000Da | ??29.0 |
From Fig. 4 and table 1 as can be seen collagen hydrolysate polypeptide and amino acid whose ratio account for the overwhelming majority, polypeptide molecular weight is all below 6.5kDa, wherein molecular weight accounts for 48.5% about 6500Da, molecular weight accounts for 22.5% between 1300Da, molecular weight accounts for 29.0% below 1000Da, do not have total free aminoacids substantially and produce.
The anti-oxidant activity of collagen polypeptide
1) collagen polypeptide is to the scavenging(action) of hydroxyl radical free radical (OH)
With reference to Fenton reaction system model, utilize H
2O
2With Fe
2+Mix producing OH, but because OH has very high reactive behavior, the survival time is short, if add Whitfield's ointment in system, just can catch OH effectively, and produce coloured product.This product has strong absorption at wavelength 510nm place, has the analyte of removing the OH function if add in reaction system, just can compete OH with Whitfield's ointment, and coloured product growing amount is reduced.Adopt the fixation response time method, therefore, can be used for removing effect (the Sabu MC of assess sample to OH, Smitha K, et al.Anti-diabetic activity of green tea polyphenols and their role in reduceingoxidative stress in experimental diabetes[J] .J.Ethnopharmacol, 2002,83:109-116.).
In test tube, add 0.5ml 9mM Whitfield's ointment-ethanolic soln successively, the collagen polypeptide sample solution of 0.5ml 10mg/ml (used collagen polypeptide is that embodiment 2 is prepared), 0.5ml 9mM Fe
2+Solution (now joining), 3.5ml distilled water adds 5ml 8.8mM H at last
2O
2Start the Fenton reaction, shake up the back and measure absorbance A in the 510nm place
1The distilled water of getting 0.5ml replaces 9mM Fe
2+The measured absorbancy of solution is A
2Getting the measured absorbancy of 0.5ml distilled water replacement sample is A
3The clearance rate P of hydroxyl radical free radical (%) can calculate according to formula (1).
In the formula: P-clearance rate, %;
A
1The reacted light absorption value of-sample solution;
A
2-the distilled water of getting 0.5ml replaces 9mM Fe
2+Light absorption value behind the solution;
A
3-get the light absorption value that 0.5ml distilled water replaces sample.
Measured thus collagen polypeptide is 86.5% to the clearance rate of hydroxy radical qiao.
2) collagen polypeptide is to ultra-oxygen anion free radical (O
2 -) scavenging(action)
The rapid autoxidation of pyrogallol energy under alkaline condition discharges ultra-oxygen anion free radical, and ultra-oxygen anion free radical quickens autoxidation, generates coloured intermediate product simultaneously.Intermediate product be accumulated in hysteresis 40s to 45s the time become the good linear relation with the time, coloured product has strong photoabsorption at the 420nm place, thereby can be used for estimating tried thing remove the ability of ultra-oxygen anion free radical (Zou Guolin etc. the improvement [J] of the measuring method for activity of a kind of SOD---pyrogallol autoxidation method. biological chemistry is made progress with biophysics, 1986,4:71-73).
Getting the 10mM PBS damping fluid of 4.5ml pH8.3 mixes with the collagen polypeptide sample solution of 100 μ l10mg/ml (used collagen polypeptide is prepared for embodiment 2), in 25 ℃ of constant temperature 15min, get pyrogallol (with the preparation of 10mM HCl solution) the 100 μ l that mixed solution 3ml adds 45mM, shake up, reaction 3min measures light absorption value A at wavelength 420nm
1The clearance rate P of ultra-oxygen anion free radical (%) can calculate according to formula (2).
In the formula: P-clearance rate, %;
A
1The reacted light absorption value of-sample solution;
A
2The light absorption value of-4.5ml PBS solution+100 μ l HCl (10mM)+100 μ l distilled water;
A
3The light absorption value of-4.5ml PBS solution+100 μ l pyrogallol solution+100 μ l distilled water.
Measured thus collagen polypeptide is 59.2% to the clearance rate of ultra-oxygen anion free radical.
3) collagen polypeptide is to the DPPH measured by esr technique
DPPH (1, the bitter diazanyl of 1-hexichol-2-) is a kind of stable free radical.Its ethanolic soln is purple, is 517nm at the visible region maximum absorption band.When adding free-radical scavengers in the DPPH solution, solution colour shoals, and the absorbancy at 517nm place diminishes, and the degree that degree that absorbancy diminishes and free radical are eliminated is linear.Therefore, can be used to detect the removing situation of free radical, thereby estimate the resistance of oxidation of certain material.Its ability represents that with inhibiting rate inhibiting rate is big more, resistance of oxidation strong more (Lu Donglian etc. the 23 kinds of Chinese medicine Study on Antioxidant Activities in Mount Taibai district [J]. research and development of natural products, 2001,13:20-22.).
Compound concentration is 1 * 10
-4The DPPH ethanolic soln of mol/L keeps in Dark Place.Take by weighing an amount of collagen polypeptide sample (used collagen polypeptide is prepared for embodiment 2), and be configured to the solution of 10mg/ml with 95% ethanol.At the 517nm place, measure according to the method application of sample in the following calculation formula (3), react 30min under the room temperature, every sample repeats 3 times, averages, and calculates free radical scavenging activity P.
In the formula: P-clearance rate, %;
A
1The light absorption value of-2ml DPPH solution+2ml sample solution;
A
2-2ml solution to be measured+2ml alcoholic acid light absorption value;
A
3-2ml DPPH solution+2ml alcoholic acid light absorption value.
Measured thus collagen polypeptide is 94.8% to the clearance rate of DPPH free radical.
Under identical concentration and condition, relatively prepared collagen peptide and the Vc of this patent removes DPPH, OH and O respectively
2 -The ability of three kinds of free radicals the results are shown in Table shown in 2.
The comparison of table 2 collagen polypeptide and Vc anti-oxidant activity
Annotate: collagen polypeptide concentration and Vc concentration are 10mg/mL, and Vc all carries out according to the described method of present embodiment the mensuration of three kinds of radical scavenging activities.
As can be seen from Table 2, collagen polypeptide all is significantly higher than Vc to the removing ability of three kinds of free radicals under identical condition, and the collagen polypeptide of this explanation this patent has good anti-oxidant activity.
The effect of collagen polypeptide cell growth
This experimental selection mtt assay comes the short proliferation function of study sample pair cell and the restraining effect of pair cell, and MTT[3-(4,5-dimethylthiazol-2-yi)-2,5-dipenyl terazolium bromide], be called for short tetrazolium salts, have another name called tetrazolium bromide; Be pale yellow powder, can be reduced to blue Formazam by Hu desaturase in the viable cell plastosome, after the appropriate solvent dissolving, its absorbancy is relevant with viable count, thus the index of conduct detection viable cell.(Huang Quanyong, Huang Juen, Huang Peichun.The activity of renal cells under the hydrocortisone of MTT detection different concns.The medical science selected works, 2005,10 (1): 8-10; Russell C A, Vindel ov L L.Optimization andcomparison of the MTT assay and the 3H-TdR assay for the detection of IL-2in helper T cell precursor assay.J Immunol Method, 1998,217 (1-2): 165-175.).
1) collagen polypeptide is to the proliferation function of dog renal epithelial cell (mdck cell)
The cultivation of cell: dog renal epithelial cell (mdck cell) is incubated at the MEM nutrient solution that contains 10% new-born calf serum (FCS), 37 ℃, 5%CO
2Cultivate in the incubator, change nutrient solution 2-3 time weekly,, go down to posterity or inoculation culture with 0.25% trypsinase-0.2%EDTA working fluid digestion cell dispersion.
The active detection: the mdck cell of vitro culture through digestion, piping and druming, centrifugal after, with the MEM nutrient solution that contains 10% new-born calf serum cell concn is transferred to 3 * 10
4Individual/ml, be inoculated in the 96 porocyte culture plates, the 100ul/ hole, every group establish 6 parallel.Cultivate after 24 hours, (concentration is 30 * 2 to renew the collagen polypeptide sample that adds the 100ul different concns behind the bright MEM substratum 100ul
-7-30 * 2
-1, used collagen polypeptide is prepared by embodiment 2), with the PBS damping fluid in contrast, continue to cultivate 48 hours.After cultivate finishing, remove substratum, add the MTT solution 50ul of 1mg/ml, 37 ℃ hatch 3 hours after, remove supernatant liquor, add the 150ul dimethyl sulfoxide (DMSO), micro oscillator vibration 10 minutes treats that precipitation fully after the dissolving, surveys A with enzyme-linked immunosorbent assay instrument
570Value.The cell proliferation rate calculation formula is as follows:
Cell proliferation rate=[(A
570Test group-A
570Control group)/A
570Control group] * 100%
The gained result as shown in Figure 5, from Fig. 5 more as can be seen, collagen polypeptide has significant proliferation function to normal cell MDCK.Increase along with collagen polypeptide concentration, the proliferation rate of pair cell also increases thereupon, when concentration is 1.88mg/ml, appreciation rate reaches maximum 26.5%, afterwards along with the increase of collagen polypeptide concentration, proliferation rate reduces gradually, and this explanation collagen polypeptide does not present the dependence effect of medicine to the increment effect of normal cell MDCK.
2) collagen peptide is to the research of human embryonic fibroblast's (HLF) proliferation function
The cultivation of cell: the human embryonic lung becomes fiber epithelial cell (HLF) to be incubated at the MEM nutrient solution that contains 10% new-born calf serum (FCS), 37 ℃, 5%CO
2Cultivate in the incubator, change nutrient solution 2-3 time weekly,, go down to posterity or inoculation culture with 0.25% trypsinase-0.2%EDTA working fluid digestion cell dispersion.
The active detection: the HLF cell of vitro culture through digestion, piping and druming, centrifugal after, with the MEM nutrient solution that contains 10% new-born calf serum cell concn is transferred to 3 * 10
4Individual/ml, be inoculated in the 96 porocyte culture plates, the 100ul/ hole, every group establish 4 parallel.Cultivate after 24 hours, (concentration is 30 * 2 to renew the collagen polypeptide sample that adds the 100ul different concns behind the bright MEM substratum 100ul
-7-30 * 2
-1, used collagen polypeptide is prepared by embodiment 2), with the PBS damping fluid in contrast, continue to cultivate 48 hours.After cultivate finishing, remove substratum, add the MTT solution 50ul of 1mg/ml, 37 ℃ hatch 3 hours after, remove supernatant liquor, add the 150ul dimethyl sulfoxide (DMSO), micro oscillator vibration 10 minutes treats that precipitation fully after the dissolving, surveys A with enzyme-linked immunosorbent assay instrument
570Value.The cell proliferation rate calculation formula is as follows:
Cell proliferation rate=[(A
570Test group-A
570Control group)/A
570Control group] * 100%
The gained result as shown in Figure 6, from Fig. 6 more as can be seen, collagen polypeptide becomes fiber epithelial cell (HLF) that very significant proliferation function is arranged to the human embryonic lung.Increase along with collagen polypeptide concentration, the proliferation rate of pair cell also increases thereupon, when concentration is 7.5mg/ml, appreciation rate reaches maximum 238.4%, afterwards along with the increase of collagen polypeptide concentration, proliferation rate reduces gradually, and this explanation collagen polypeptide becomes the increment effect of fiber epithelial cell (HLF) also not present the dependence effect of medicine to the human embryonic lung.
3) collagen polypeptide is to the cancer cells restraining effect
The cultivation of cell: liver cancer cell (Hep-G2 cell) is incubated at the RPM I RPMI-1640 that contains 10% new-born calf serum (FCS), 37 ℃, 5%CO
2Cultivate in the incubator, change nutrient solution 2-3 time weekly,, go down to posterity or inoculation culture with 0.25% trypsinase-0.2%EDTA working fluid digestion cell dispersion.
The active detection: the liver cancer cell of vitro culture through digestion, piping and druming, centrifugal after, with the RPM I RPMI-1640 that contains 10% new-born calf serum cell concn is transferred to 4 * 10
5Individual/ml, be inoculated in the 96 porocyte culture plates, the 100ul/ hole, every group establish 6 parallel.Cultivate after 24 hours, (concentration is 30 * 2 for the collagen polypeptide sample of adding 100ul different concns
-7-30 * 2
-1, used collagen polypeptide is prepared by embodiment 2), with the PBS damping fluid in contrast, continue to cultivate 24 hours.After cultivate finishing, remove substratum, add the MTT solution 50ul of 1mg/ml, 37 ℃ hatch 3 hours after, remove supernatant liquor, add the 150ul dimethyl sulfoxide (DMSO), micro oscillator vibration 10 minutes treats that precipitation fully after the dissolving, surveys A with enzyme-linked immunosorbent assay instrument
570Value.The cell proliferation rate calculation formula is as follows:
The gained result as shown in Figure 7 and Figure 8.As can be seen from Figure 7, collagen polypeptide has the obvious suppression effect to liver cancer cell growth, and after adding collagen polypeptide sample cell was cultivated, not only cell count reduced in a large number, and bigger variation also takes place cellular form.The cancer cells form is the shuttle shape before the application of sample, and cellular form is not obvious behind the application of sample, presents the decline sign, and collagen polypeptide has demonstrated the inhibition activity of good liver cancer cell.
As shown in Figure 8, increase along with collagen polypeptide concentration, inhibiting rate to liver cancer cell also increases thereupon, when collagen polypeptide concentration is 1.45mg/ml, its inhibiting rate to liver cancer cell is 6.3%, when concentration reached 11.6mg/mL, its inhibiting rate to liver cancer cell can be up to 32.4%.
Fiber adhesion albumen plays important effect in the cancer metastasis process.It is generally acknowledged that the restraining effect of cancer metastasis is adhered to its anticancer and to soak into the fiber adhesion albumen and the ln of basilar membrane relevant, just relevant with the formation of anticancer new vessel.Collagen polypeptide and fiber adhesion albumen have very high affinity, so collagen polypeptide has very big influence to the cancer metastasis process.
The hydratability of collagen polypeptide
(1) water-absorbent
Water-absorbent is meant the ability of proteinaceous product absorption or intake water, represents with the gram number or the milliliter number of every gram product adsorption moisture usually.
Accurately take by weighing 1g collagen polypeptide (used collagen polypeptide is prepared by embodiment 2), be tiled in the culture dish that diameter is 10cm, weigh, the quality of record culture dish this moment is m
1Place T=32 ℃ then, the constant temperature of RH=90%, constant humidity (adopt oversaturated potassium nitrate solution controlling moisture, its relative humidity in the time of 30 ℃ is about 89%) incubator in, when 6h, measure the quality of a culture dish, stop to measure when the quality of culture dish no longer increases, the quality that write down culture dish this moment is m
2, then the water-absorbent of sample is calculated as follows:
Water-absorbent (g/g)=(m
2-m
1)/1.0
Collagen protein or casein with same quality replace collagen polypeptide, and in contrast, the gained result is as shown in table 4, as can be seen from the table, collagen polypeptide has very strong water-absorbent, and its water-absorbent has improved 1.7 times than collagen protein, is caseic 1.8 times.
Table 4 collagen polypeptide and collagen protein and caseic absorptive comparison
The sample title | Water-absorbent (g/g) |
Collagen protein | ??0.06 |
Collagen polypeptide | ??0.16 |
Casein | ??0.09 |
(2) water-retentivity
Water-retentivity is meant that proteinaceous product keeps the ability of moisture, represents water-retentivity with the moisture survival rate usually.
Accurately take by weighing 1.0g collagen polypeptide (used collagen polypeptide is prepared by embodiment 2), after fully dissolving with a spot of distilled water, be settled to 100ml, get 1ml sample liquid then, it is tiled in the culture dish that diameter is 10cm, weighs, the quality of record culture dish this moment is m
1, placing then in the incubator of constant temperature, constant humidity (adopt oversaturated sodium bromide solution controlling moisture, its relative humidity at 40 ℃ is 53.2+/-0.5%) of T=40 ℃ of RH=54%, the quality every 10min measures a culture dish is recorded as m
2, and calculate the moisture survival rate of sample liquid successively according to following formula.
Moisture survival rate=(m
2/ m
1) * 100%
Glycerine or water with same quality replace collagen polypeptide, in contrast, the gained result as shown in Figure 9, as can be seen from the figure, collagen polypeptide can keep a certain amount of moisture at certain condition under still, retentiveness is preferably arranged, and its retentiveness is a little less than glycerine, and this ability is subjected to the influence of environmental change very little.
The oil absorbency of collagen polypeptide
Oil absorbency is meant the ability of proteinaceous product adsorbed oil, represents by the milliliter number with every gram proteinaceous product adsorbed oil, generally uses centrifugal determination.
Accurately measure the 2.0ml refining oil, put into the graduated centrifuge tube of 5ml, take by weighing 0.3g collagen polypeptide sample again and join (used collagen polypeptide is prepared by embodiment 2) in the centrifuge tube, the even 2min of vortex oscillation, behind the static 30min, with the centrifugal 25min of the speed of 100rpm/min, write down the volume of free oil, then oil absorbency is calculated as follows:
Oil absorbency (ml/g)=(volume of 2.0-free oil)/0.3
Collagen protein or soybean protein isolate with same quality replace collagen polypeptide, in contrast, the gained result is as shown in table 5, as can be seen from the table, collagen polypeptide has very high oil absorbency, improved 20% than collagen protein, exceeded 38% than soybean protein isolate, the prepared collagen polypeptide of this explanation the present invention has distinctive molecular structure and size.The collagen polypeptide oil absorbency of people's enzymolysis gained such as Jia Dongying is 1.37ml/g.The polypeptide oil absorbency of this experiment gained is than the raising that has 33.6%.(Yao opens for Jia Dongying, Wang Wenxian, Zhang Mingrang. many skins of collagen protein Study on Functional Properties. and Food science .2001,22 (6): 21-24.)
The comparison of table 5 collagen polypeptide and collagen protein and soybean protein isolate oil absorbency
Kind | Oil absorbency (ml/g) |
Collagen protein | ??1.52 |
Kind | Oil absorbency (ml/g) |
Collagen polypeptide | ??1.83 |
Soybean protein isolate | ??1.33 |
The emulsifying property of collagen polypeptide and emulsifying stability
1) emulsifying property
Emulsifying property is meant the area (cm that the protein (or polypeptide) of unit mass can the stabilize oil water termination
2/ g), represent that the opacity method is adopted in this experiment with EAI.
Take by weighing a certain amount of collagen polypeptide (used collagen polypeptide is prepared by embodiment 2), use 0.2mol/L, the dissolving of pH7.0 sodium phosphate buffer also is configured to 1% solution (w/v), ratio according to 0.025L/L adds soybean salad oil again, use clarifixator, refiner or electronic stirring make it form the emulsion of homogeneous, respectively get 100 times of the 99ml distilled water dilutings of emulsion of the new configuration of 1ml then respectively at 0min and 30min, getting the 0.1%SDS that the diluted emulsion of 1ml joins 39ml again dilutes, 4000 times of final dilutions, last solution is carried out light absorption value measure (measuring 9 times averages) under 500nm, calculate the EAI value according to following formula at last.
EAI=2 * T * (A * extension rate)/(C * Φ * 100)
In the formula:
EAI---emulsifying activity;
T——2.303;
Protein concentration in the protein water soln before C---emulsion forms, g/ml;
Φ---the volume fraction of oil in the emulsion, 0.025;
A---solution is at the light absorption value at 500nm place.
2) emulsifying stability
Emulsifying stability is meant that protein keeps water-oil interface and mix the unseparated emulsification property anti-adaptability to changes of condition to external world, represents with ESI.
Get 100 times of the 99ml distilled water dilutings of emulsion of the new configuration of 1ml, getting the 0.1%SDS that the diluted emulsion of 1ml joins 39ml again dilutes, after 4000 times of the final dilutions, measure the light absorption value of dilution back emulsion every 30min, and calculate the ESI value according to following formula at the 500nm place.
ESI=A
0×t/(A
0-A
30)
In the formula:
ESI---emulsifying stability, min;
A
0---the light absorption value of rapidly diluted emulsion behind the homogeneous;
A
30---the light absorption value of emulsion behind static 30min;
T---the time, 30min;
Collagen protein or casein with same quality replace collagen polypeptide, and in contrast, the gained result is as shown in table 6, and as can be seen from the table, collagen polypeptide still has emulsifying property and emulsifying stability preferably at certain condition.
The comparison of table 6 collagen polypeptide and collagen protein and caseic emulsifying property, emulsifying stability
Sample | Emulsifying property (cm 2/g) | Emulsifying stability (min) |
Collagen polypeptide | ??10.87 | ??160.9 |
Collagen protein | ??14.09 | ??325.4 |
Casein | ??13.23 | ??304.1 |
Comprehensive above embodiment 2-embodiment 7 as can be known, the collagen polypeptide that the present invention does preparation has good anti-oxidant activity, can reach 94.8%, 59.2% and 86.5% respectively to the clearance rate of hydroxy radical qiao, ultra-oxygen anion free radical and DPPH free radical; Normal somatic cell there is good increment effect, and cancer cells is also had very strong inhibition effect, can reach 26.5% and 238.4% respectively, can reach 32.4% the inhibiting rate of cancer cells Hep-G2 to the proliferation rate of normal cell MDCK and HLF; Good water-absorbent, oil absorbency are arranged, and retentiveness, emulsifying property and emulsifying stability are preferably arranged; Also has molecular weight all below 6500Da, molecular weight is accounting for more than 50% between the 300-1300Da, can be well by skin absorption and utilization, therefore, the prepared collagen polypeptide of the present invention is expected to solve and improves present stage collagen protein and a series of problems of using of collagen hydrolysate peptide, can be widely used in a plurality of fields such as beauty treatment, health care, food, medicine.
Embodiment 9
The slow blood aggregation activity of collagen polypeptide
Glycoprotein on the platelet membrane (GP II b/ III a), Fibrinogen, calcium ion, GP II b and GP III a are separated from each other and are distributed in the platelet membrane surface, two kinds of glycoprotein interactions form mixture becomes fibrinogenic acceptor, and Fibrinogen is combined on the activated blood platelet, at Ca
2+Participate in realizing down aggreation.Therefore, can compare ADP inductive platelet aggregation result who reacts and the blank that does not add sample by detecting the adding sample, working sample is to the inhibition activity of platelet aggregation.
Get fresh rabbit blood 0.45ml, with collagen polypeptide solution by mixing (v/v), timing blood aggegation time at 9: 1.3.8% Trisodium Citrate (m/v) or collagen solution with equal volume replace collagen polypeptide, write down the complete agglutinative time of blood respectively, the results are shown in Table shown in 7, as can be seen from the table, collagen polypeptide has stronger anti-blood agglutination activity, and its specific activity collagen protein improves 25%.
The comparison of the anti-blood agglutination of table 7 collagen polypeptide and collagen protein and Trisodium Citrate
Antithrombotics | The slow setting collection time (min) |
Collagen protein | ??20 |
Collagen polypeptide | ??25 |
Trisodium Citrate | ??60 |
Collagen polypeptide is to stomach en-and tryptic resistance
This experiment adopts in-vitro simulated digestion to investigate collagen polypeptide to stomach en-and tryptic resistance.
(1) stomach en-external digestion
In 25ml tool plug test tube, add 50mg collagen polypeptide (used collagen polypeptide is that embodiment 2 is prepared), 5ml distilled water, pH to 2.5 is transferred with hydrochloric acid in the dissolving back, and the ratio according to the 2000U/g collagen polypeptide adds stomach en-then, places 37 ℃ of water-baths 3 hours, reaction finishes the back and adds 2ml 20%TCA termination reaction, in 10000rpm, 25 ℃ of following centrifugal 5min get supernatant 100ul and carry out HPLC analysis (being undertaken by the method for being chatted among the embodiment 3) with reaction solution.
(2) trypsinase external digestion
In 25ml tool plug test tube, add 50mg collagen polypeptide (used collagen polypeptide is that embodiment 2 is prepared), 5ml distilled water, pH to 8.0 is transferred with hydrochloric acid in the dissolving back, and the ratio according to the 2000U/g collagen polypeptide adds trypsinase then, places 37 ℃ of water-baths 3 hours, reaction finishes the back and adds 2ml 20%TCA termination reaction, in 10000rpm, 25 ℃ of following centrifugal 5min get supernatant 100ul and carry out HPLC analysis (being undertaken by the method for being chatted among the embodiment 3) with reaction solution.
With the collagen polypeptide without stomach en-, tryptic digestion is contrast, relatively digests the variation of forward and backward collagen polypeptide molecular weight distribution, and the result is shown in Figure 10 and table 8.From figure, the table as can be seen, collagen polypeptide external digestion forward and backward peak type and each components contents all do not have obvious variation, after this says that collagen polypeptide of the present invention digests in vivo, absorbs, its biological activity does not have tangible minimizing, this proves that further the prepared collagen polypeptide of the present invention is a kind of very fine multifunctional bio-active peptide, has very high using value.
The degree of table 8 collagen polypeptide each molecular weight component before and after stomach en-and tryptic digestion
The above only is preferred embodiment of the present invention, be not in order to limit essence technology contents scope of the present invention, essence technology contents of the present invention is broadly to be defined in the claim scope of application, any technology entity or method that other people finish, if it is defined identical with the claim scope of application, also or a kind of change of equivalence, all will be regarded as being covered by among this claim scope.
Claims (10)
1. collagen polypeptide, it is characterized in that in the collagen polypeptide gross weight, molecular weight is at the ingredients constitute 45-55w/w% of 6500Da ± 100Da, molecular weight is at the ingredients constitute 20-25w/w% of 1300 ± 100Da, the ingredients constitute 25-35w/w% of molecular weight below 1000Da; Nitrogen solubility index NSI is 98.5-99.9%.
2. as claimed in claim 1, it is characterized in that, in the collagen polypeptide gross weight, crude protein content 90-95w/w%, ash content 3-5w/w%, sugar, moisture and lipid content 2-4w/w%, acid-soluble protein content 99.5-99.9w/w%.
3. collagen polypeptide as claimed in claim 1 is characterized in that, described collagen polypeptide can both solution water under all pH, the solution clarification, be faint yellow-khaki, show slightly acidic; Described collagen polypeptide free from extraneous odour, no bitter taste; Very stable to heat, 100 ℃ are boiled the nothing precipitation and separate out; Preserve 6 months content under the room temperature and do not have considerable change.
4. collagen polypeptide as claimed in claim 1 is characterized in that, described collagen polypeptide prepares by following step:
(I) with starting material and prozyme A at pH9-10,50-60 ℃ was mixed 6-10 hour; In the gross weight of solid substance, the amount of prozyme A is 2-5w/w%; Described starting material are selected from following one or more: animal skin, animal hoof tendon, animal bone, animal scale and shell; Described prozyme A is made of following composition: in the gross weight of the dry of prozyme A, M-Zyme 55-65w/w%, papoid 5-10w/w%, trypsinase 10-15w/w%, Quimotrase 2-5w/w%, Sumizyme MP 10-15w/w%, kethepsin 0.02-0.05w/w% and bromeline 2-5w/w%; With
(II) remove by filter insolubles and obtain collagen polypeptide solution.
5. the preparation method as the arbitrary described collagen polypeptide of claim 1-4 is characterized in that, described method comprises step:
(1) with starting material and prozyme A at pH9-10,50-60 ℃ was mixed 6-10 hour; In the gross weight of solid substance, the amount of prozyme A is 2-5w/w%; Described starting material are selected from following one or more: animal skin, animal hoof tendon, animal bone, animal scale and shell; Described prozyme A is made of following composition: in the gross weight of the dry of prozyme A, M-Zyme 55-65w/w%, papoid 5-10w/w%, trypsinase 10-15w/w%, Quimotrase 2-5w/w%, Sumizyme MP 10-15w/w%, kethepsin 0.02-0.05w/w% and bromeline 2-5w/w%; With
(2) remove by filter insolubles and obtain collagen polypeptide solution.
6. preparation method as claimed in claim 5 is characterized in that, described starting material are selected from following one or more: fish-skin, pigskin, pig's feet, beef tendon, fish scale, chicken bone, fish-bone.
7. preparation method as claimed in claim 5 is characterized in that, described step (1) is: at pH9-10,50-60 ℃ was mixed after 6-10 hour, 75-85 ℃ of insulation 30 minutes with starting material and prozyme A.
8. preparation method as claimed in claim 5 is characterized in that, the starting material in the step (1) are Powdered or pasty state.
9. preparation method as claimed in claim 5 is characterized in that, comprises step between step (1) and (2): make 15-20 ℃ of enzyme reaction solution pH5-6, leave standstill.
10. the purposes as the arbitrary described collagen polypeptide of claim 1-4 is characterized in that, described collagen polypeptide is used for preparation and removes free radical, oxidation resistant composition; Or be used to prepare the composition that promotes the cell growth; Or be used to prepare the composition of anticancer growth; Or be used to prepare and absorb and/or the composition of water conservation; Or be used to prepare the composition of oil suction; Or be used for preparation and be used for anticoagulant composition.
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