CN101445821A - Method for preparing deer byproduct collagen and functional collagen protein taken as medical and function foods - Google Patents
Method for preparing deer byproduct collagen and functional collagen protein taken as medical and function foods Download PDFInfo
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- CN101445821A CN101445821A CNA2008102097627A CN200810209762A CN101445821A CN 101445821 A CN101445821 A CN 101445821A CN A2008102097627 A CNA2008102097627 A CN A2008102097627A CN 200810209762 A CN200810209762 A CN 200810209762A CN 101445821 A CN101445821 A CN 101445821A
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Abstract
The invention relates to a method for preparing active collagen and functional collagen protein by taking deer byproduct (deer bone and deerskin) as raw material. The method adopts micro-grinding and high pressure homogenate to treat the raw material, and an acid-enzyme process to extract deer collagen; purified collagen can be obtained by washing and drying. Locating zymohydrolysis is adopted to prepare the active collagen protein, and the functional collagen protein is obtained by utilizing the membrane separation technique, desalinization, concentration and drying technology. The molecular weight of the collagen prepared by the invention is more than 100KDa, and the collagen biological activity; the molecular weight of the prepared collagen protein is less than 5KDa, and the collagen protein has the functions of antifatigue and immunity regulation. The adopted method has the characteristics of being low in cost, suitable for commercial process, high in production efficiency and controllable in production. The invention can be applied to the fields such as biomaterial, medicine, health-care food, cosmetics, etc.
Description
Technical field: the present invention relates to medical active deer horn glue former and functional foodstuff and the former proteic preparation method of the active deer horn glue of used for cosmetic.
Background technology: acid system or enzyme process are adopted in the extraction of collagen usually.The preparation of collagen protein can be used heating, alkaline process and enzyme process.The present invention handles deer byproduct earlier through the broken and high-pressure homogeneous assisted extraction method of micro mist, the composition that will extract is discharged as far as possible, by the method for acetic acid and biological enzyme formulation combination treatment, extract collagen wherein again, after washing and drying, can be used as biomaterial and medical raw material.Raw material after the preparation employing of collagen protein is high-pressure homogeneous makes through biological enzyme directional enzymatic technology, utilizes membrane separation technique, and desalination or warp concentrate and drying can prepare the collagen protein with antifatigue and immunoloregulation function.Adopt method preparing product of the present invention to have the industrialization degree height, mild condition, safe characteristics.To improve present deer product especially the deer byproduct added value and utilize effect to have a good application prospect.
Summary of the invention: the object of the present invention is to provide a kind of method that separation and Extraction collagen and preparation have antifatigue and regulate the bioactivity collagen of immunologic function from deer product by product, it is characterized in that its preparation process is:
A. the raw materials pretreatment raw material is deer byproducts such as the deer bone of degreasing and deerskin.Pretreated gordian technique is that raw material is broken and high-pressure homogeneously become small particle through micro mist, adopts high pressure homogenizer to handle 1min at 50MPa, sieves, and separates the raw material that obtains handling well.
B. the extraction of collagen: all operations all carries out below the condition at 40 ℃.Raw material adds in the 0.5M acetum of 10-15 times of volume, regulates pH value to 2.0～3.0, adds 0.5%-1% gastric pepsin digestion 48h, regulates pH value to 7, removes stomach en-, and Digestive system is centrifugal, and (35,000g 45min), collects supernatant liquor.Slowly adding solid NaCl in solution, to make ultimate density be 4.1%, slowly stirs 12hr continuously.Precipitate centrifugal (7000g, 45min) collect the back, is dissolved in the 0.5M acetum again, it is centrifugal that (35,000g 45min) removes precipitation, at same solution dialysis 2d, changes dialyzate every day 2 times.Until in dialyzate, dripping AgNO
3Solution does not have precipitation and produces, and the back solution lyophilize of will dialysing so far can obtain highly purified collagen.
C. the preparation of collagen protein: bio-enzyme degradation: the temperature of equal raw material is adjusted to 50 ℃～65 ℃, adding acid or alkali makes its pH value 8.0～8.5, the protease A that adding accounts for raw materials quality 3%～6% is hydrolyzed and constantly stirring, adding alkali makes the pH value of reaction solution maintain between 8.0～8.5 hydrolysis 1～5h.Hydrolysis finishes the back and adds acid for adjusting pH value to 4.2～4.6, and the enzyme that goes out of heating up is lived, and is cooled to centrifugal 20～40min after the room temperature, gets supernatant liquor.
D. membrane sepn: adopt the ultra-filtration membrane of 30KDa molecular weight, enzymolysis product is carried out membrane sepn, obtain the collagen protein of molecular weight＜5KDa, the collection membrane filtrate.
E. desalination is adorned post with anion-cation exchange resin, get membrane filtration fluid enzymolysis product with the flow velocity of 8～12 times of column volume/h by cationic, anionic exchange resin, the collagen protein of desalting refinement, i.e. finished product.Also can concentrate or dry it.
The collagen product has the essential characteristic of collagen, purity〉90%, molecular weight〉100KDa.The collagen that uses method of the present invention to obtain is tasteless, and dry back is a white, and no supersensitivity has cavernous structure.Its preparation method and condition have advantages such as technology is simple, easy handling, cycle weak point.The product of the present invention's preparation is made up of special amino acid, and wherein glycine is the former main amino acid of deer horn glue, accounts for about 30%, and other content are proline(Pro), oxyproline, L-glutamic acid, L-Ala than higher amino acid successively.Its biological property mainly shows with function: (1) low antigenicity: have immunogenic protein with other and compare, the immunogenicity of collagen is very low.(2) biodegradable: under the effect of collagenase, the peptide bond of collagen will be hydrolyzed and interrupt gradually, and the collagen peptide chain break causes the destruction of spirane structure immediately, causes collagen by the thorough hydrolysis of proteolytic enzyme, the biodegradable of Here it is collagen.Why collagen can be utilized as the organ transplantation material, just is being based on this characteristic.(3) biocompatibility: i.e. good interaction between collagen and host cell and the tissue.(4) cell adapted property and cel l proliferation: collagen helps the survival and the growth of cell, can not only promote the proliferation and differentiation of cell, and the division function of improving cell is also produced effect.(5) good mechanical performance: natural collagen spirane structure closely plays an important role to high-intensity mechanical property, in organism, collagen is the major protein component that intensity is provided for reticular tissue, thereby can in very large range satisfy the requirement of human body to physical strength.
Collagen to the present invention's preparation carries out SDS-PAGE vertical electrophoresis molecular weight determination, and 3 bands are arranged behind the electrophoresis, and molecular weight is 116,000Da, 140, and 000Da and 200 is more than the 000Da.Collagen to the present invention's preparation is analyzed with automatic analyzer for amino acids, proof has collagen characteristic amino acid, and wherein glycine accounts for 1/3 of amino acid sum, oxyproline and proline content height, possess (Gly-Pro-Hyp) n collagen pattern, illustrate that the collagen triple-helix structure is complete.
The former protein product purity of deer horn glue〉95%, molecular weight＜5KDa.The collagen protein that uses method of the present invention to obtain is tasteless.Its preparation method and condition have advantages such as technology is simple, easy handling, cycle weak point.Its functional performance mainly shows: anti-ageing, antitumor, antifatigue, hypotensive, anti-oxidant, hair care skin care, beauty treatment, reparation stomach etc.
Collagen protein to the present invention's preparation carries out molecular weight determination, and method and result are as follows: molecular weight determination adopts gel chromatography.Gel is selected Sephdex-G25 for use, and product application of sample amount 0.1ml, elutriant are the phosphate buffered saline buffer of concentration 0.1mol/L, pH value 7.0, flow velocity 1ml/min.The molecular weight standard product are bovine serum albumin (68000), N,O-Diacetylmuramidase (14400), VB
12(1355), tryptophane (204), the molecular weight of hydrolysate is below 5000Da.Immunoregulation effect to collagen protein carries out proving test, experimental result shows that each dosage group all can significantly increase the weight of mouse immune organ, cellular immune function, humoral immune function and monokaryon-macrophage phagocytic function to mouse also all have promoter action, show to have immunoregulation effect.Has the obvious anti-fatigue function by mouse anti-reflecting fatigue evidence collagen proteins such as blood lactic acid, blood urea nitrogen, swimming with a load attached to the body times.
Claims (6)
1, the deer horn glue of biologically active is former and have antifatigue and regulate the former proteic preparation method of deer horn glue of immunity function, it is characterized in that its preparation process is:
A. the raw materials pretreatment raw material is deer (spotted deer, red deer, the hybridization of plum horse deer, wapiti and reinder an etc.) by product, comprises deer bone, deerskin etc.Pretreated gordian technique is that raw material is broken through micro mist, makes raw meal particle size＜0.2mm.
B. the extraction all operations of collagen all carries out below the condition at 40 ℃.Raw material is broken through micro mist, immerses in the 0.5M acetum of 10-15 times of volume, and is high-pressure homogeneous through 30-100Mpa, regulates pH value to 2.0～3.0, add 0.5%-1% gastric pepsin digestion 48h, regulate pH value to 7, remove stomach en-, Digestive system centrifugal (35,000g 45min), collects supernatant liquor.Slowly adding solid NaCl in solution, to make ultimate density be 4.1%, slowly stirs 12hr continuously.Precipitate centrifugal (7000g, 45min) collect the back, is dissolved in the 0.5M acetum again, it is centrifugal that (35,000g 45min) removes precipitation, at same solution dialysis 2d, changes dialyzate every day 2 times.Until in dialyzate, dripping AgNO
3Solution does not have precipitation and produces, and the back solution lyophilize of will dialysing so far can obtain highly purified collagen.
C. the preparation of collagen protein is adjusted to 50 ℃～65 ℃ with the temperature of equal raw material, adding acid or alkali makes its pH value 8.0～8.5, the protease A that adding accounts for raw materials quality 3%～6% is hydrolyzed and constantly stirring, adds alkali and makes the pH value of reaction solution maintain between 8.0～8.5 hydrolysis 1～5h.Hydrolysis finishes the back and adds acid for adjusting pH value to 4.2～4.6, and the enzyme that goes out of heating up is lived, and is cooled to centrifugal 20～40min after the room temperature, gets supernatant liquor.
D. membrane sepn adopts the ultra-filtration membrane of 1-3 ten thousand molecular weight, and enzymolysis product is carried out membrane sepn, obtains the collagen protein of molecular weight＜5KDa, the collection membrane filtrate.
E. desalination is adorned post with anion-cation exchange resin, get membrane filtration fluid enzymolysis product with 8～12 times of column volumes/hour flow velocity by cationic, anionic exchange resin, the collagen protein of desalting refinement, i.e. finished product.Also can concentrate or dry it.
2, the preparation method of deer product collagen according to claim 1, it is broken to it is characterized in that the raw materials pretreatment stage need be carried out micro mist, pickling is after high-pressure homogeneous (50Mpa), make raw material particle size as far as possible little (＜0.2mm), so that better enzymolysis therefrom extracts more collagen and collagen protein.
3, the preparation method of deer product collagen according to claim 1 is characterized in that all operations all carries out 40 ℃ of following conditions, prevents the sex change of collagen.
4, deer product collagen is characterized in that it is to isolate collagen by claim 1,2,3 described preparation methods, and product has the essential characteristic of collagen, the purity of collagen〉90%, molecular weight〉100KDa.Product has good biocompatibility, degradability, reduced immunogenicity function, and be absorbed easily in vivo, wetting ability is strong, nontoxic, security good.
5, collagen protein preparation method according to claim 1, the enzyme that hydrolysis is used can be compound in the following enzyme one or both: Neutrase, Alcalase, Potamex, neutral protease 1398 and trypsinase, wherein, use the effect of Alcalase better separately.
6, according to claim 1,5 described collagen protein preparation methods, separate the collagen protein that obtains and have purity 95%, molecular weight＜5KDa.Have immunoregulation effect and anti-fatigue effect.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101803738A (en) * | 2010-03-24 | 2010-08-18 | 郑彬 | Method for preparing deer bone frozen powder by ultrasonic degradation |
| CN101628937B (en) * | 2009-08-12 | 2012-02-29 | 江苏省海洋资源开发研究院 | Purification technology of macromolecular collagen protein |
| CN103173510A (en) * | 2011-12-21 | 2013-06-26 | 中国科学院大连化学物理研究所 | Preparation method of high-hydroxyproline-content deer collagen peptide |
| CN103416736A (en) * | 2012-05-14 | 2013-12-04 | 宋甲祥 | Nutritious food with functions of activating and regenerating brain cells and production technology |
| CN104371910A (en) * | 2014-10-31 | 2015-02-25 | 王选明 | Method for extracting collagen from deer bones |
| CN111235202A (en) * | 2018-11-29 | 2020-06-05 | 中国中医科学院医学实验中心 | Deer bone protein extract and preparation method and application thereof |
| CN111358707A (en) * | 2019-04-18 | 2020-07-03 | 吉林省东鳌鹿业科技开发有限公司 | Deer collagen peptide toning lotion and preparation method thereof |
| CN111548409A (en) * | 2020-05-21 | 2020-08-18 | 内蒙古元本生物医药科技有限公司 | Extraction process of animal fresh skin collagen polypeptide |
| CN111812333A (en) * | 2020-06-29 | 2020-10-23 | 吉林省东北亚生物科技有限公司 | Method for comparing activity of ligamentum cervi natural peptide and enzymatic hydrolysis peptide on resisting rheumatoid arthritis |
-
2008
- 2008-12-24 CN CNA2008102097627A patent/CN101445821A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101628937B (en) * | 2009-08-12 | 2012-02-29 | 江苏省海洋资源开发研究院 | Purification technology of macromolecular collagen protein |
| CN101803738A (en) * | 2010-03-24 | 2010-08-18 | 郑彬 | Method for preparing deer bone frozen powder by ultrasonic degradation |
| CN101803738B (en) * | 2010-03-24 | 2012-04-18 | 郑彬 | Method for preparing deer bone frozen powder by ultrasonic degradation |
| CN103173510A (en) * | 2011-12-21 | 2013-06-26 | 中国科学院大连化学物理研究所 | Preparation method of high-hydroxyproline-content deer collagen peptide |
| CN103416736A (en) * | 2012-05-14 | 2013-12-04 | 宋甲祥 | Nutritious food with functions of activating and regenerating brain cells and production technology |
| CN105505769A (en) * | 2014-10-31 | 2016-04-20 | 王选明 | Technology for extracting collagen protein from deer bones |
| CN104371910A (en) * | 2014-10-31 | 2015-02-25 | 王选明 | Method for extracting collagen from deer bones |
| CN104371910B (en) * | 2014-10-31 | 2016-08-24 | 世茂（苏州）生物科技有限公司 | A kind of method extracting collagen from deer bone |
| CN105505769B (en) * | 2014-10-31 | 2018-12-07 | 内蒙古神元康肽生物工程有限公司 | A kind of technique for extracting collagen from deer bone |
| CN111235202A (en) * | 2018-11-29 | 2020-06-05 | 中国中医科学院医学实验中心 | Deer bone protein extract and preparation method and application thereof |
| CN111235202B (en) * | 2018-11-29 | 2022-03-08 | 中国中医科学院医学实验中心 | Deer bone protein extract and preparation method and application thereof |
| CN111358707A (en) * | 2019-04-18 | 2020-07-03 | 吉林省东鳌鹿业科技开发有限公司 | Deer collagen peptide toning lotion and preparation method thereof |
| CN111548409A (en) * | 2020-05-21 | 2020-08-18 | 内蒙古元本生物医药科技有限公司 | Extraction process of animal fresh skin collagen polypeptide |
| CN111812333A (en) * | 2020-06-29 | 2020-10-23 | 吉林省东北亚生物科技有限公司 | Method for comparing activity of ligamentum cervi natural peptide and enzymatic hydrolysis peptide on resisting rheumatoid arthritis |
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Application publication date: 20090603 |