Method for separating and purifying immunoglobulin A, immunoglobulin G and lactoferrin from bovine colostrum in industrializing way

A technology for separation and purification of immunoglobulins, which is applied in the field of industrial separation and purification of immunoglobulin A, immunoglobulin G and lactoferrin, can solve the problems of inability to carry out industrial amplification, purification of immunoglobulins, and restrictions on industrial production, and achieve High production efficiency, pollution reduction, high biological activity and high purity effects

Active Publication Date: 2010-06-09
HEILONGJIANG KANPURE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] With the increasing demand for high-tech immune products in the market, the current development trend of bovine colostrum industry is to separate and purify the rich immune factors and growth factors in it, but the separation technology and equipment restrict the progress of industrial production. Many reports on the separation of active components of bovine colostrum are mostly limited to some classic methods commonly used in the laboratory, such as salting out and gel chromatography. Although these methods are feasible in the laboratory, they cannot be scaled up industrially. practical application value
With the continuous development of science and technology, the emergence of industrial membrane technology and industrial chromatography technology has made it possible to separate proteins industrially. However, in what order the components are separated, what membrane technology and chromatography technology are used, etc. There are mutual constraints and influences between the links. At present, there is no efficient and continuous industrial separation and purification of immunoglobulin A, immunoglobulin G and lactoferrin from bovine colostrum.

Method used

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  • Method for separating and purifying immunoglobulin A, immunoglobulin G and lactoferrin from bovine colostrum in industrializing way
  • Method for separating and purifying immunoglobulin A, immunoglobulin G and lactoferrin from bovine colostrum in industrializing way
  • Method for separating and purifying immunoglobulin A, immunoglobulin G and lactoferrin from bovine colostrum in industrializing way

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Industrial separation and purification of immunoglobulin A, immunoglobulin G and lactoferrin from bovine colostrum

[0025] (1) Preparation of whey

[0026] Selection of bovine colostrum raw materials: select the milk produced within 48 hours after giving birth;

[0027] Degreasing: continuous disc centrifuge degreasing, temperature 45°C, speed 5500rpm;

[0028] Whey preparation: Use acetic acid to adjust the pH of skim milk to 4.6, keep it warm at 40°C for 20 minutes, use a horizontal centrifuge to separate casein, and then go through a continuous disc clarifier centrifuge to remove casein particles. The whey pH was adjusted to 6.0.

[0029] (2) Prepare lactoferrin

[0030] Microfiltration sterilization: use French TAMI ceramic membrane with a pore size of 0.45um for microfiltration sterilization, operating pressure 0.1-0.2Mpa, operating temperature 20-35°C, tangential flow rate 4-6m / s, sterilization rate 100%;

[0031] Cation exchange chromatography: sel...

Embodiment 2

[0037] The identification of the lactoferrin product that embodiment 2 method of the present invention obtains

[0038] Determination of Sodium Dodecyl Sulfate Polyacrylamide Electrophoresis (SDS-PAGE)

[0039]The lactoferrin product that embodiment 1 is obtained is measured through sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE), and the lactoferrin of gained is a single band, as figure 2 As shown, the single strip on column 1 is the lactoferrin band with a relative molecular weight of 80000Da.

[0040] HPLC (High Performance Liquid Chromatography) Determination

[0041] HPLC conditions of use: TSK-G3000PWxl gel chromatography column is selected, mobile phase: 0.02mol / L, pH 6.8 phosphate buffer, the prepared mobile phase needs to use pure water, and the mobile phase is filtered through a 0.22μm microporous filter before use. Membrane suction filtration to prevent insoluble particles in the salt from damaging the equipment, and ultrasonic degassing for 20 m...

Embodiment 3

[0046] The identification of the immunoglobulin A product that embodiment 3 method of the present invention obtains

[0047] Determination of Sodium Dodecyl Sulfate Polyacrylamide Electrophoresis (SDS-PAGE)

[0048] The immunoglobulin A (SigA) product obtained in Example 1 was determined by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE), and the obtained SigA band was clear, and the relative molecular weight was 390000Da.

[0049] Western blot detection

[0050] Western blot detection method: cut 2 pieces of 3MM ultra-thick filter paper and a piece of nitrocellulose membrane (NC membrane), the size of which is about gel > filter paper ≥ NC membrane. The NC membrane was equilibrated with deionized water for 30 minutes, and the cut 3MM ultra-thick filter paper and NC membrane were soaked in the transfer buffer for 5 minutes. After SDS-PAGE is over, remove the gel and pre-equilibrate in deionized water for 5 minutes. On the semi-dry electroporation instrument,...

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Abstract

The invention provides a method for separating and purifying immunoglobulin A, immunoglobulin G and lactoferrin from bovine colostrum in an industrializing way, which comprises the following steps of: (1) degreasing the bovine colostrum and separating casein to prepare whey; (2) after micro-filtering the whey for degerming, carrying out cation exchange chromatography to obtain a first fast flow liquid, eluting a chromatographic column to obtain eluent, ultra-filtering, concentrating and desalting the eluent by using a first ceramic membrane, and freezing and drying to obtain the lactoferrin; (3) carrying out anion exchange chromatography on the first fast flow liquid to obtain a second fast flow liquid, eluting the chromatographic column to obtain eluent, ultra-filtering, concentrating and desalting the eluent by using a second ceramic membrane, and freezing and drying to obtain the immunoglobulin A; and (4) ultra-filtering, concentrating and desalting the second fast flow liquid by using a third ceramic membrane, and obtaining the immunoglobulin G by using low-temperature spray drying. By utilizing the method, the immunoglobulin A, the immunoglobulin G and the lactoferrin can be separated and purified from the bovine colostrumin efficiently and continuously in the industrializing way.

Description

technical field [0001] The invention relates to a method for industrially separating and purifying immunoglobulin A, immunoglobulin G and lactoferrin from bovine colostrum. Background technique [0002] Bovine colostrum naturally contains a large number of functional components with different physiological activities, and is one of the food resources with the most abundant immune factors in nature. Among them, immunoglobulin has the highest content in bovine colostrum, is the most similar to human immunoglobulin, has homology, and has the greatest effect on human health. Immunoglobulin is mainly divided into the following categories: immunoglobulin G ( IgG), immunoglobulin A (SigA), immunoglobulin M (IgM), lactoferrin (LF), etc. The main functions of immunoglobulin include regulating the body's gastrointestinal function, improving the body's immunity and promoting the body's recovery. The role of SigA in several immunoglobulins is particularly important, because the main i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/34C07K1/18C07K16/04C07K14/79
Inventor 段旭煌赵红宇
Owner HEILONGJIANG KANPURE BIOTECH
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