CN1663961A - Separation and Purification Technology of Lactoferrin in Bovine Colostrum - Google Patents

Abstract

Lactoferrin has a variety of biological functions and has great application potential in the food and medical industries. The present invention establishes a method suitable for industrialization to separate and purify lactoferrin from bovine colostrum. The produced lactoferrin has the characteristics of low cost, simple process, low equipment investment, no pollution, high product purity, and good activity retention. . The specific method is: ① activate the weakly acidic cation exchange resin, and prepare it into Na or K type; ② skim the colostrum, and precipitate the casein for later use; ③ mix the resin and whey at a suitable pH and temperature, and stir gently for 2-20 hours ; ④ Use a filter cloth to separate the resin from the whey, and wash the resin with deionized water to remove whey and impurities; ⑤ Elute the lactoferrin with a suitable salt solution; ⑥ The eluent is desalted by ultrafiltration; ⑦ Ultrafiltration The concentrated solution is dried by freeze-drying method to obtain lactoferrin product; ⑧Using SDS-PAGE electrophoresis to analyze the product purity.

Description

Separation and Purification Technology of Lactoferrin in Bovine Colostrum

The invention relates to a method for separating and purifying lactoferrin from bovine colostrum, in particular establishing a set of new technologies suitable for industrial production of lactoferrin. The technology has the characteristics of low production cost, simple operation, no pollution, and does not change the activity of lactoferrin. The purity of the produced lactoferrin can reach more than 85%, and the yield of lactoferrin is more than 70%.

Lactoferrin (Lactoferrin, referred to as Lf) is a functional factor rich in colostrum. It was first discovered in animal milk by Sorensen et al. in 1939, and was obtained by Polis et al. in the preparation of lactoperoxidase in 1953. Its crude product, Johansson and Montreuil first isolated Lf from human milk in 1960. Lf got its name because it was first discovered in milk. Later studies showed that it also widely exists in the internal and external secretions of animals, such as tears, saliva, sweat, pancreatic juice, bile, semen, etc., but its content in milk is relatively low. High, such as the mass concentration of Lf in bovine colostrum is 1g/L, but the content in other secretions is very small. Modern studies have proved that Lf has many unique biological functions: enhancing iron delivery and absorption; broad-spectrum antibacterial properties; immune effects; antioxidant effects; promoting the balance of intestinal flora; as a growth factor; anti-inflammatory; antiviral ; Anti-cancer effect, etc. The research on the separation, processing and comprehensive utilization of Lf has become a hot spot in the food industry, especially in the dairy industry.

Lf is an iron-binding glycoprotein that belongs to the transferrin family. The main body of the molecule is a single peptide chain consisting of about 700 amino acid residues and a relative molecular mass of about 80,000 Da, which is connected with 1 or 2 glycosides, and its composition includes mannose, galactose, N-acetyl half Lactosamine, sialic acid, fucoic acid, etc. Milk Lf is composed of 703 amino acids with an isoelectric point of 8.0.

Since Lf has some extraordinary physiological functions, people have tried various methods to prepare Lf since 1960. Although human milk is rich in Lf, it is not realistic to rely on extracting Lf from human milk. Commercial Lf is usually isolated and extracted from bovine milk, especially bovine colostrum and whey, which are rich in resources and can achieve large-scale and commercial production. The pre-processing method of the material is basically the same. Fresh bovine colostrum is used to remove fat by centrifugation (4°C, 3000rpm, 30min) to obtain skim milk. After diluting the skim milk, add 1M hydrochloric acid solution to it while stirring to adjust the skim milk. pH value to 4.6 (the isoelectric point of casein), placed at room temperature for 30min, centrifuged to remove casein (4°C, 3000rpm, 30min), the obtained supernatant is the whey we need (or directly use the by-product of cheese production whey), which was used to isolate Lf.

There are many separation and purification methods for lactoferrin, the common ones are adsorption chromatography, affinity chromatography, immobilized monoclonal antibody method, ultrafiltration method, salting out method, ion exchange method and so on. Adsorption chromatography is to use solid-phase adsorbents to combine with protein molecules, and to separate them by using the differences in the binding forces of different substances and adsorbents. The disadvantages are small adsorption capacity and low efficiency; affinity chromatography: affinity chromatography It is an emerging separation technology, which is used to produce high-purity Lf. This method has good separation effect and high purity without loss of biological activity. It is one of the most effective methods for producing high-purity Lf for medicine. Its disadvantage is that it is expensive , Difficult to industrial production; Immobilized monoclonal antibody method: This method actually uses the highly specific binding reaction between antigen-antibody to achieve the separation of the target. Immunochromatography loaded with monoclonal antibody is a kind of affinity chromatography, which successfully realizes the one-step separation of Lf, and the recovery rate reaches 97%. The disadvantage is that the preparation process of the column is complicated, the antibody cost is expensive (1000 US dollars/gram), and it is difficult to industrialize production; ultrafiltration method: ultrafiltration is a basic membrane separation technology, and its principle is that different molecular weights can be achieved according to different membrane pore sizes. Separation of macromolecular substances with molecular shapes, because ultrafiltration provides the opportunity to concentrate and separate macromolecular mass components without heating or phase change, it is very suitable for the separation of heat-sensitive functional components, but the membrane Pollution and low product purity are difficult to meet the requirements of production. Salting-out method: The salting-out method usually uses saturated ammonium sulfate to precipitate lactoferrin. Although the production cost of this method is simple, the addition of ammonium sulfate in whey brings pollution and the product has low purity, which restricts its application. Ion exchange method: The principle of this method is to achieve the separation of target substances according to the different binding forces of ion exchangers to different ions or ionic compounds. Grove first used DEAE cellulose anion exchange resin to separate relatively pure Lf components. After that, he developed phosphocellulose exchange chromatography and improved this method by Foley et al. The purity of Lf obtained was about 81%. Carboxymethyl cation exchange chromatography was applied to the separation of Lf and lactate catalase. The resin can be regenerated with 0.2M NaOH and 0.2M HCl, which is more effective than the DEAE fiber method. Although someone is trying to use the ion exchange method to produce lactoferrin, due to the different types, properties and exchange conditions of the selected resins, industrial production has not been fully realized.

The purpose of the present invention is to establish a method suitable for industrial separation and purification of lactoferrin from bovine colostrum, and the purity of the purified lactoferrin can reach more than 85%.

Based on domestic and foreign literature, it can be known that the separation and purification process of lactoferrin is complex, the product has low purity, high product price, and is difficult to commercialize. The present invention selects carboxymethyl cation exchange resin, adopts batch exchange method, and combines ultrafiltration desalination and freeze-drying techniques to successfully establish a method suitable for industrial production of lactoferrin. The main features of this technology are:

①The selected resin meets the food hygiene production requirements. After the separation and purification of lactoferrin, the raw milk can be continuously used without any pollution.

②The technique of this technology is simple, the equipment investment is low, and it is suitable for industrialized production.

③The lactoferrin produced by this technology can better maintain the activity of lactoferrin, and the produced lactoferrin has higher purity, which can meet the needs of food and medical industries.

Specific Implementation Conditions and Invention Production Scheme

1. Bovine colostrum was centrifuged and defatted (3000rpm, 30min), adjusted to pH 4.6 with 1M HCl, precipitated casein, centrifuged (3000rpm, 30min) to obtain whey for later use;

2. Resin activation, weigh a certain amount of resin as needed, soak in deionized water (1-2 days at room temperature, or 1-2 hours in boiling water), and then soak in 10% NaCl solution to prepare Na-type resin;

3. Adjust the whey pH to 5-8 with 1M NaOH, add Na-type cation exchange resin, stir at 0-50°C for 2-20 hours, filter the resin with filter cloth, wash with deionized water to remove impurities, and then use 0.1 -15% salt solution (NaCl, KCl, CaCl ₂ , MgCl ₂ , NaCO _{3 ,} etc.) to elute the resin, and the eluate is desalted by ultrafiltration. The concentrate is freeze-dried to obtain lactoferrin.

4. The exchange of lactoferrin in whey can be detected by ELISA method, and the elution of resin can be detected. The product was electrophoresed by SDS-PAGE, and its purity was analyzed by gel imaging system.

In order to illustrate the present invention more clearly, describe in detail below in conjunction with embodiment:

Embodiment 1: Weigh 2g of CM-SEPHAROSE FF (PHARMACIA) resin, add 200mL of deionized water to soak for 24h, collect the resin and soak in 10% NaCl solution for 30min, and prepare Na-type resin. The resin was added to 5L of skimmed whey (pH 6.9), and exchanged with gentle stirring at 4°C for 8 hours. The resin was separated and collected with a filter cloth, and the pH, color, smell and sensory of the exchanged skim whey did not change.

The collected resin is washed with deionized water with a pH of 6.9 to remove foreign proteins, and then the resin is eluted with 5% salt solution, and the resin and the eluent are separated by suction filtration, and the eluent is collected for ultra- Filtration and desalination, the molecular weight cut-off of the ultrafiltration membrane is 30,000-50,000, and the ultrafiltration pressure is 0.15-0.2MPa. Ultrafiltered

The desalted lactoferrin concentrate was freeze-dried, and 4.01 g of the product was collected. The purity of the product analyzed by electrophoresis was 86%, and the yield was 69%.

Embodiment 2: Take by weighing 30g SEPABEADS FP-CM13 (MITSUBISHI KASEI) cation exchange resin and carry out activation, install column (diameter 10cm) after balancing, with peristaltic pump, 2L containing 10% KCl solution is passed through the column, and then passed through with deionized water Column, prepared as K-type cation exchange resin. Then 80L of non-fat normal milk whey (pH6.6, 4°C) was exchanged through the column at a flow rate of 6L/h, and the color and taste of the exchanged whey remained unchanged.

The exchanged column was first passed through the column with deionized water to remove whey and impurity proteins, and then 15 L of 12% KCl solution was used to elute the lactoferrin through the column, and 15 L of the eluent was collected. After the eluate was desalted by ultrafiltration and freeze-dried, 7.2 g of lactoferrin was obtained. The purity of lactoferrin was detected to be 81%, and the yield was 73%.

Embodiment 3: Weigh 5g of CM-SEPHADEX C-50 (PHARMACIA) resin, add 400mL of deionized water and soak for 24h, collect the resin and soak in 10% NaCl solution for 30min, and prepare 230mL of Na-type resin. The resin was added to 20L of defatted colostrum whey (pH value was 6.8), and exchanged with gentle stirring at 4°C for 12 hours. The resin was separated and collected with a filter cloth, and the pH, color, smell and sensory of the exchanged skim whey did not change.

Wash the collected resin with deionized water with a pH of 6.8 to remove impurity proteins, then first elute the resin with 1% salt solution to remove unbound impurity proteins, and then elute the resin with 5% salt solution , the resin and eluent are separated by suction filtration, and the eluent is collected for ultrafiltration and desalination. The molecular weight cut-off of the ultrafiltration membrane is 30,000-50,000, and the ultrafiltration pressure is 0.15-0.2MPa. The concentrated solution after ultrafiltration and desalination was freeze-dried to collect 14.5 g of lactoferrin products. The purity of the product analyzed by electrophoresis was 92%, and the yield was 65%. The present embodiment adopts two-step method to elute the resin, and the purity of the product has been greatly improved, but the yield of the product has decreased (the yield of the product is calculated as 1 g/L of lactoferrin content in colostrum).

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%.