CN1663961A - Technology for separating and purifying lactoferritin from cow colostrum - Google Patents
Technology for separating and purifying lactoferritin from cow colostrum Download PDFInfo
- Publication number
- CN1663961A CN1663961A CN 200410080264 CN200410080264A CN1663961A CN 1663961 A CN1663961 A CN 1663961A CN 200410080264 CN200410080264 CN 200410080264 CN 200410080264 A CN200410080264 A CN 200410080264A CN 1663961 A CN1663961 A CN 1663961A
- Authority
- CN
- China
- Prior art keywords
- lactoferrin
- resin
- whey
- separation
- exchange
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides a method for preparing lactoferrin from cow's colostrums. The lactoferrin has great potential application in food and medicine industry for its multiple biological function. The method is characterized by the low production cost, simple process, low investment for device, non pollution, high purity and maintaining active and so on. The method comprises the following steps: (1) activating the weakly-acidic cationic ion-exchange resin to make it to be Na or K type; (2) defatting the colostrums and setting casei; (3) mixing the resin and whey at proper temperature and pH value, stirring lightly for 2-20 hours; (4) separating the resin and whey with filter cloth, washing the resin with deionized water to remove the whey and impurity protein; (5) eluting the lactoferrin with proper salt solution; (6)desalting the eluent with ultrafiltration; (7)drying the ultrafiltrate and concentrate with freeze drying and getting the lactoferrin; (8) analyzing the product purity with SDS-PAGE electrophoresis.
Description
The present invention relates to a kind of from bovine coloctrum the method for separation and purification lactoferrin, particularly set up the new technology that a cover is fit to the suitability for industrialized production lactoferrin.It is low, simple to operate, pollution-free that this technology has a production cost, do not change characteristics such as lactoferrin activity, and the lactoferrin purity of production can reach more than 85%, and the yield of lactoferrin is more than 70%.
Lactoferrin (Lactoferrin, be called for short Lf) be a kind of functional factor that is rich in first Ruzhong, it is found in animal milk by Sorensen etc. in nineteen thirty-nine at first, nineteen fifty-three Polis etc. have obtained its raw product when the preparation lactoperoxidase, nineteen sixty Johansson and Montreuil etc. isolate Lf first from human milk.Lf gains the name because of being found in milk at first, studies show that afterwards, it also extensively is present in the inside and outside juice such as tear, saliva, sweat, pancreatic juice, bile, seminal fluid of animal body, just content is higher in milk, as the Lf mass concentration in the bovine coloctrum is 1g/L, and content is very little in other secretory product.Recent studies proves that Lf has many unique biological functions: strengthen the transmission and the absorption of iron; The germ resistance of wide spectrum; Immunization; Antioxygenation; Promote the balance of intestinal microflora; As somatomedin; Anti-inflammatory; Antiviral; Anticancer disease effect etc.Research to the separation of Lf, processing, comprehensive utilization has become food circle, particularly the focus be concerned about of dairy products circle.
Lf is a kind of iron associativity glycoprotein, belongs to Transferrins,iron complexes family.The main body of molecule is that nearly 700 amino-acid residues constitute, relative molecular mass is about 80, the single chain polypeptide of 000Da, and connect 1～2 glycoside, its composition comprises seminose, semi-lactosi, N-acetylgalactosamine, sialic acid, rock alginic acid etc.Cow's milk Lf is made of 703 amino acid, and iso-electric point is 8.0.
Because Lf has some outstanding physiological functions, people just try to adopt the whole bag of tricks to prepare Lf since nineteen sixty, though Lf content is abundant in the human milk, relies on and extract Lf and unrealistic from human milk.Commercial Lf is separation and Extraction from cow's milk, particularly bovine coloctrum and whey usually, and aboundresources can be realized mass-producing, commercialization production.The earlier stage processing method of material is basic identical, all be adopt the fresh bovine colostrum by centrifugal slough fat (4 ℃, 3000rpm 30min) obtains skimming milk, after the skimming milk dilution, adjust the pH value to 4.6 (caseic iso-electric point) of skimming milk to wherein adding the 1M hydrochloric acid soln while stirring, room temperature is placed 30min, centrifugal (4 ℃ of the caseins of removing, 3000rpm, 30min), the supernatant that obtains is exactly the whey (or directly caseic by product whey is produced in utilization) that we need, and separates Lf with it.
The Isolation and Purification of Lactoferrin method is more, and common have adsorption chromatography, affinity chromatography, immobilization monoclonal antibody method, ultrafiltration process, salting-out process, an ion exchange method etc.Adsorption chromatography is to utilize solid-phase adsorbent by combining with protein molecule, utilizes the otherness of different substances and sorbent material bonding force and they are separated, and its shortcoming is that loading capacity is little, efficient is low; Affinity chromatography: affinity chromatography is a kind of emerging isolation technique, and it is used for producing highly purified Lf, this method good separating effect purity height, and there is not the biological activity loss, be to produce one of effective means of pharmaceutical high purity Lf, its shortcoming is the cost costliness, is difficult to suitability for industrialized production; The immobilization monoclonal antibody method: this method is actually the high specific association reaction that utilizes between the Ag-Ab and realizes the separation of target compound.The immune chromatograph that is loaded with monoclonal antibody is a kind of avidity chromatography, the realization that it is successful the step separation of Lf, the rate of recovery reaches 97%, the advantage of this method is a good separating effect purity height, antibody is fixed reusable, its shortcoming is the complicated process of preparation of post, and antibody cost costliness (1000 dollars/gram) is difficult to suitability for industrialized production; Ultrafiltration process: ultrafiltration is a kind of basic membrane separation technique, its principle is the separating of macromolecular substance that can realize differing molecular quality and shape of molecule according to the membrane pore size difference, because providing not heat or do not undergo phase transition, ultrafiltration process carries out the concentrating of macromole mass component, isolating chance, be fit to very much the separation of the functional component of thermo-sensitivity, but the low requirement that is difficult to satisfy production of the pollution of film and product purity.Salting-out process: salting-out process normally utilizes the saturated ammonium sulphate lactoferrin, although this method production cost is simple, has brought pollution owing to add ammonium sulfate in the whey, and the purity of product is also lower, has restricted its application.Ion exchange method: this method principle is according to the bonding force different separation that realize target substance of ion-exchanger to different ions or ionic compound.Grove uses DEAE Mierocrystalline cellulose anionite-exchange resin to isolate purer Lf composition the earliest, and after this, he develops the phosphorylated cotton exchange chromatography again, and by Foley etc. this method is improved, and the purity of the Lf that obtains is about 81%; The carboxymethyl cation-exchange chromatography is applied to Lf and newborn catalatic the separation.Resin can be regenerated with 0.2MNaOH and 0.2MHCl, and this method is more effective than the DEAE fibre method.To the greatest extent the possessor produces lactoferrin attempting the utilization ion exchange method, but because kind, performance difference, the give-and-take conditions difference of selected resin, fails to realize fully suitability for industrialized production.
The objective of the invention is to: set up the method that a cover is fit to industrialization separation and purification lactoferrin from bovine coloctrum, the lactoferrin purity of purifying can reach more than 85%.
Comprehensive documents and materials both domestic and external as can be known, the Isolation and Purification of Lactoferrin complex process, the purity of product is lower, the price height of product is difficult to commercialization.The present invention selects the carboxymethyl Zeo-karb for use, adopts the method for exchange in batches, has successfully set up the method for suitable suitability for industrialized production lactoferrin in conjunction with ultrafiltration desalination and freeze drying process.The principal feature of this technology is:
1. selected resin meets the food sanitation production requirement, and behind the separation and purification lactoferrin, raw dairy can continue to utilize, and does not have any pollution.
2. the technology of this technology is simple, and facility investment is low, is fit to suitability for industrialized production.
3. the lactoferrin produced of this technology has kept the activity of lactoferrin preferably, and the lactoferrin purity of producing is higher, can satisfy the needs of food and medical industry.
Concrete implementation condition and invention production program
1, the bovine coloctrum centrifugal degreasing (3000rpm 30min), transfers pH to 4.6 with the HCl of 1M, the precipitation casein, centrifugal (3000rpm, 30min), whey is standby;
2, resin activation takes by weighing a certain amount of resin as required, soaks (under the normal temperature 1-2 days, or boiling water 1-2 hour) with deionized water, is soaked in the 10%NaCl solution again, is mixed with Na type resin;
3, with 1MNaOH whey pH is transferred to 5-8, add Na type Zeo-karb,,,, use 0.1-15% salts solution (NaCl, KCl, CaCl again with deionized water eccysis foreign protein with filter cloth suction filtration separation resin in 0-50 ℃ of stirring 2-20h
2, MgCl
2, NaCO
3Deng) the wash-out resin, elutriant ultrafiltration desalination.The concentrated solution lyophilize gets lactoferrin.
4, available ELISA method detects the exchange situation of lactoferrin in the whey, detects the wash-out situation of resin.Product carries out electrophoresis through SDS-PAGE, analyzes its purity with gel imaging system.
Be elaborated the present invention in order more clearly to illustrate below in conjunction with embodiment:
Embodiment 1: take by weighing 2g CM-SEPHAROSE FF (PHARMACIA) resin, add the 200mL deionized water and soak 24h, collect resin and be soaked in 30min in 10% the NaCl solution, be prepared into Na type resin.Resin is added (the pH value is 6.9) in the 5L skimming milk clear liquid, stir exchange 8 hours gently in 4 ℃.With filter cloth separated and collected resin, pH, color, smell, the sense organ of the skimmed whey after the process exchange all do not change.
With the resin pH that collects is 6.9 washed with de-ionized water, removing foreign protein, and then with 5% salts solution wash-out resin, mode with suction filtration is separated resin with elutriant, collect elutriant and carry out the ultrafiltration desalination, the molecular weight cut-off of ultra-filtration membrane is 3-5 ten thousand, and ultrafiltration pressure is 0.15-0.2MPa.Through ultrafiltration
Lactoferrin concentrated solution after the desalination carries out lyophilize, collects product 4.01g.Product is 86% through electrophoretic analysis purity, and yield is 69%.
Embodiment 2: take by weighing 30g SEPABEADS FP-CM13 (MITSUBISHI KASEI) Zeo-karb and activate, after balance, adorn pillar (diameter 10cm), with peristaltic pump 2L is contained 10%KCl solution and cross post, cross post with deionized water then, be prepared into K type Zeo-karb.Then the normal whey of 80L degreasing (pH6.6,4 ℃) is exchanged by pillar with the 6L/h flow velocity, the whey colour through exchanging, taste does not all change.
The pillar that exchanged is crossed post with deionized water earlier, remove whey and foreign protein, then containing 12%KCl solution with 15L crosses post wash-out lactoferrin, collects elutriant 15L.Elutriant gets lactoferrin 7.2g after ultrafiltration desalination and lyophilize, lactoferrin purity is 81% after testing, and yield is 73%.
Embodiment 3: take by weighing 5g CM-SEPHADEX C-50 (PHARMACIA) resin, add the 400mL deionized water and soak 24h, collect resin and be soaked in 30min in 10% the NaCl solution, be prepared into Na type resin 230mL.Resin is added (the pH value is 6.8) in the 20L skimmed colostrum whey liquid, stir exchange 12 hours gently in 4 ℃.With filter cloth separated and collected resin, pH, color, smell, the sense organ of the skimmed whey after the process exchange all do not change.
With the resin pH that collects is 6.8 washed with de-ionized water, to remove foreign protein, then earlier with 1% salts solution wash-out resin, to remove in conjunction with not firm foreign protein, use 5% salts solution wash-out resin again, resin is separated with elutriant, collect elutriant and carry out the ultrafiltration desalination with the mode of suction filtration, the molecular weight cut-off of ultra-filtration membrane is 3-5 ten thousand, and ultrafiltration pressure is 0.15-0.2MPa.Concentrated solution after the ultrafiltration desalination carries out lyophilize, collects lactoferrin product 14.5g.Product is 92% through electrophoretic analysis purity, and yield is 65%.Present embodiment adopts two-step approach wash-out resin, and the purity of product is greatly improved, but the yield of product descends to some extent (yield of product is that 1g/L calculates with first Ruzhong lactoferrin content).
Claims (6)
1, Isolation and Purification of Lactoferrin technology in the bovine coloctrum, this invention are that the utilization ion-exchange techniques prepares a kind of purity and can reach lactoferrin finished product more than 85%, and the present invention has its distinctive technical superiority.It is characterized in that the inventor has set up the method for the suitable suitability for industrialized production lactoferrin of a cover, the activatory weakly acidic cation-exchange resin is mixed with skimmed colostrum (or colostrum whey), in 0-50 ℃ of scope, stir 2-20h gently, with resin isolation, remove foreigh protein removing with filter cloth, use the salts solution (NaCl of 0.1%-15% then with deionized water wash-out resin, KCl, CaCl
2, MgCl
2, NaCO
3Deng) the wash-out resin, the elutriant molecular weight cut-off is the membrane ultrafiltration desalination of 30000-50000, gets the lactoferrin product after the concentrated solution lyophilize.It is easy that this method has production technique, and equipment is simple, pollution-free, and by product is convenient to be utilized, and keeps the lactoferrin activity, to characteristics such as HUMAN HEALTH are harmless.
2, according to claim 1, feature of the present invention is a utilization ion-exchange techniques separation and purification lactoferrin, and utilization ultrafiltration desalting technology and freeze-drying method prepare lactoferrin.
3, according to claim 1, the selected resin of the present invention is a weakly acidic cation-exchange resin, and cation exchange groups is a carboxymethyl, and selected resin meets the food sanitation production requirement, and give-and-take conditions are 0-50 ℃, pH5-8, time 2-20h, stirring velocity 200r/min.
3, according to claim 1, select filter cloth (aperture 100-600 order) separation resin for use, adopt ultrafiltration desalination (molecular weight cut-off is 3-5 ten thousand) method, have characteristics such as quick and convenient, meet the suitability for industrialized production requirement.Adopt Freeze Drying Technique, can keep the activity of lactoferrin preferably.
4, according to claim 1, the technology of the present invention is produced lactoferrin, and it is characterized in that: production technique is easy, and equipment is simple, cost of investment is low, and energy consumption is low, environmentally safe, first whey behind the separation lactoferrin, the change of no color, taste can continue development and use.
5, according to claim 1,4 described, the present invention has set up the method for the perfect separation and purification lactoferrin of a cover, and it is low to have a cost, simple to operate, pollution-free, do not change characteristics such as lactoferrin activity, the lactoferrin purity of producing can reach more than 85%, and the yield of lactoferrin is more than 70%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410080264 CN1663961A (en) | 2004-09-29 | 2004-09-29 | Technology for separating and purifying lactoferritin from cow colostrum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410080264 CN1663961A (en) | 2004-09-29 | 2004-09-29 | Technology for separating and purifying lactoferritin from cow colostrum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1663961A true CN1663961A (en) | 2005-09-07 |
Family
ID=35035295
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410080264 Pending CN1663961A (en) | 2004-09-29 | 2004-09-29 | Technology for separating and purifying lactoferritin from cow colostrum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1663961A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101477079A (en) * | 2009-01-06 | 2009-07-08 | 中国农业大学 | Active gel electrophoresis method for lactalbumin |
| CN101362792B (en) * | 2008-09-25 | 2011-07-20 | 上海交通大学 | Affinity separation polymer of lactoferrin and affinity purification method of lactoferrin |
| CN101294931B (en) * | 2008-06-24 | 2012-01-25 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting content of beta-lactoglobulin in cow's milk |
| CN101256168B (en) * | 2008-04-22 | 2012-01-25 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for testing peroxidase content in cow's milk |
| CN102898516A (en) * | 2012-10-26 | 2013-01-30 | 浙江大学 | Method for purifying lactoferrin from milk serum by using an expanded bed adsorption technology |
| CN104109204A (en) * | 2013-04-16 | 2014-10-22 | 武汉禾元生物科技有限公司 | Method for separating and purifying recombinant human lactoferrins from paddy rice seeds |
| CN105566489A (en) * | 2015-12-10 | 2016-05-11 | 无锡科捷诺生物科技有限责任公司 | Method for preparing lactoferrin with different iron saturation degrees |
| CN110973345A (en) * | 2019-12-26 | 2020-04-10 | 吉林大学 | Method for continuously separating and preparing functional lactoprotein in colostrum |
| CN113105542A (en) * | 2021-04-15 | 2021-07-13 | 黑龙江飞鹤乳业有限公司 | Preparation method of lactoferrin |
| CN113387413A (en) * | 2021-05-19 | 2021-09-14 | 中核四0四有限公司 | Ion exchange method for uranium-containing wastewater treatment in nitric acid and carbonic acid mixed system |
-
2004
- 2004-09-29 CN CN 200410080264 patent/CN1663961A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101256168B (en) * | 2008-04-22 | 2012-01-25 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for testing peroxidase content in cow's milk |
| CN101294931B (en) * | 2008-06-24 | 2012-01-25 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting content of beta-lactoglobulin in cow's milk |
| CN101362792B (en) * | 2008-09-25 | 2011-07-20 | 上海交通大学 | Affinity separation polymer of lactoferrin and affinity purification method of lactoferrin |
| CN101477079A (en) * | 2009-01-06 | 2009-07-08 | 中国农业大学 | Active gel electrophoresis method for lactalbumin |
| CN102898516A (en) * | 2012-10-26 | 2013-01-30 | 浙江大学 | Method for purifying lactoferrin from milk serum by using an expanded bed adsorption technology |
| WO2014169760A1 (en) * | 2013-04-16 | 2014-10-23 | 武汉禾元生物科技有限公司 | Method for separating and purifying recombined human lactoferrin from rice seeds |
| CN104109204A (en) * | 2013-04-16 | 2014-10-22 | 武汉禾元生物科技有限公司 | Method for separating and purifying recombinant human lactoferrins from paddy rice seeds |
| CN105566489A (en) * | 2015-12-10 | 2016-05-11 | 无锡科捷诺生物科技有限责任公司 | Method for preparing lactoferrin with different iron saturation degrees |
| CN105566489B (en) * | 2015-12-10 | 2021-07-30 | 无锡科捷诺生物科技有限责任公司 | Method for preparing lactoferrin with different iron saturation degrees |
| CN110973345A (en) * | 2019-12-26 | 2020-04-10 | 吉林大学 | Method for continuously separating and preparing functional lactoprotein in colostrum |
| CN110973345B (en) * | 2019-12-26 | 2022-02-25 | 吉林大学 | Method for continuously separating and preparing functional lactoprotein in colostrum |
| CN113105542A (en) * | 2021-04-15 | 2021-07-13 | 黑龙江飞鹤乳业有限公司 | Preparation method of lactoferrin |
| CN113105542B (en) * | 2021-04-15 | 2023-06-30 | 黑龙江飞鹤乳业有限公司 | Lactoferrin preparation method |
| CN113387413A (en) * | 2021-05-19 | 2021-09-14 | 中核四0四有限公司 | Ion exchange method for uranium-containing wastewater treatment in nitric acid and carbonic acid mixed system |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0253395B2 (en) | A process for producing bovine lactoferrin in high purity | |
| US5516675A (en) | Separation of lactoperoxidase, secretory component and lactoferrin from milk or whey with a cation exchange resin | |
| CA2288184C (en) | Method for treating a lactic raw material containing gmp | |
| CN100417329C (en) | Complete separating process for fresh liquid milk | |
| EP0348508B1 (en) | Process for separating and purifying lactoferrin from milk using sulfate compound | |
| JPH03502921A (en) | Method for extracting pure fractions of lactoperoxidase and lactoferrin from whey | |
| CA2188654C (en) | Modified milk growth factor | |
| CN106699869A (en) | Angiogenin-enriched milk fractions | |
| CN1663961A (en) | Technology for separating and purifying lactoferritin from cow colostrum | |
| EP0418704B1 (en) | Process of separating, purifying and recovering milk proteins capable of binding iron | |
| CHEN et al. | Microfiltration affinity purification of lactoferrin and immunoglobulin G from cheese whey | |
| JP5709353B2 (en) | Novel milk protein fraction and its use for the prevention or treatment of chronic inflammatory diseases | |
| CN100558242C (en) | A kind of from colostrum the method for inductrialized separation of purified lactoferrins | |
| CN101948896B (en) | Integrated preparation method of multi-function milk casein active peptides | |
| EP0443763B1 (en) | Formulated milk for infants analogous to human milk | |
| CN109705208A (en) | A kind of technique of single step chromatography preparation high-purity vWF ELISA | |
| CN106793793A (en) | The separation of the soluble protein of the mixture from the casein containing aggregation | |
| WO2002028194A1 (en) | Process for recovering proteins from whey protein containing feedstocks | |
| JP2985158B2 (en) | Recovery method of highly active lactenin fraction | |
| CN104513305B (en) | A kind of purification process of lactalbumin | |
| CN1274716C (en) | Method for extracting albumin transferrin with iron supplemented and antibacterial function | |
| CN1166688C (en) | Method of separating lactotransferrin from whey liquid | |
| CN116836257A (en) | Osteopontin purification method | |
| JPH02117366A (en) | Treatment of whey for production of low phosphorus whey protein and low salt deproteinized whey | |
| JP2963081B2 (en) | Method for separating and purifying lactoferrin from milk and its by-products |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Ma Chen Document name: Notice of first review |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |