CN111116706A

CN111116706A - Purification preparation method for extracting whey protein in breast milk

Info

Publication number: CN111116706A
Application number: CN201811286097.1A
Authority: CN
Inventors: 梁鑫淼; 叶贤龙; 肖思佳; 闫竟宇; 郭志谋; 张华林
Original assignee: Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
Current assignee: Taizhou Medical City Guoke Huawu Biomedical Technology Co ltd
Priority date: 2018-10-31
Filing date: 2018-10-31
Publication date: 2020-05-08

Abstract

The invention relates to a process in the technical field of biology, in particular to a method for extracting and purifying total whey protein from natural breast milk, and specifically relates to a method for preparing breast milk total whey protein by applying an ion exchange chromatography technology. In particular to the processes of membrane separation, low-temperature high-speed centrifugation, casein removal by isoelectric point precipitation, ion exchange chromatography refining and concentration drying. The invention takes breast milk as a raw material, prepares a whey solution by adopting membrane separation, low-temperature high-speed centrifugation and isoelectric point precipitation, and refines and prepares components such as high-abundance casein by utilizing a middle-low pressure purification system through ion exchange chromatography. The whey protein dry powder prepared by the invention has good solubility and higher purity; the production process has the advantages of simple operation of the preparation process, high efficiency and the like.

Description

Purification preparation method for extracting whey protein in breast milk

Technical Field

The invention relates to a process in the technical field of biology, in particular to a method for extracting and purifying total whey protein from natural breast milk, and specifically relates to a method for preparing breast milk total whey protein by applying an ion exchange chromatography technology.

Background

The breast milk contains all nutrients necessary for the growth and development of the newborn and plays an important role in the growth and healthy development of the newborn. The protein in the breast milk is classified into casein, whey protein and the like, the ratio of the whey protein to the casein is about 80: 20-70: 30 in 40 days before the lactation period, and about 60: 40-50: 50 in 40 days after the lactation period.

Whey protein is a protein contained in whey separated after removal of casein. Whey protein contains more immunoglobulins which have the functions of regulating and immunizing the body. Whey protein, one of the most important components in breast milk, plays a very important role in the healthy growth of infants and young children. Therefore, the deep research on the whey protein by using a proteomics method can comprehensively and deeply understand the whey protein, the research on the whey protein by using a proteomics technology is beneficial to the prevention and treatment of diseases, and the method has important significance on the healthy development of infants.

However, because of the existence of various high-abundance proteins (such as casein, etc.) as well as lipids, lactose, etc. in the breast milk, the expression and identification of surrounding low-abundance proteins are interfered, and the repeatability and resolution of the electrophoresis result are reduced. How to minimize or avoid the influence of high-abundance proteins and other components on the identification of whey proteins becomes one of the key steps in the study of whey proteomics.

Disclosure of Invention

The invention aims to provide a method for extracting and purifying total whey protein from natural breast milk, wherein the purified total whey protein contains no casein component and can be used for analyzing low-abundance proteins in the breast milk. In particular to a method for preparing breast milk total whey protein by applying ion exchange chromatography technology. Is used for reducing or avoiding the interference of various high-abundance proteins (such as casein) in breast milk, lipids, lactose and the like on the identification of surrounding low-abundance proteins. Is a key step in the subsequent research of whey proteomics.

In order to achieve the above purpose, the purification preparation method for extracting whey protein from breast milk of the invention comprises the following steps:

(1) pretreatment: removing casein from skim milk by membrane separation and low temperature high speed centrifugation, adjusting pH to 4.6-4.8 isoelectric point with 0.5-1M acetic acid, and precipitating to obtain clear whey solution.

(2) Loading the whey solution onto an ion exchange chromatography column equilibrated with a buffer solution of 0-10mS/cm in conductivity and pH4.0-5.0 at a flow rate of 150-; after the sample loading is finished, washing the chromatographic column by using a buffer solution with the conductivity of 0-10mS/cm and the pH value of 4.0-5.0 until the absorption value of 280nm reaches a baseline, performing linear gradient elution by using the buffer solution with the conductivity of 10-90mS/cm and the pH value of 4.0-5.0, wherein the elution volume is 10-50 times of the column volume, collecting fractions at the peak, merging the fractions qualified through SDS-PAGE purity detection, concentrating and drying into powder, and obtaining the refined whey protein.

Furthermore, the membrane separation system used in the step (1) is a membrane separation membrane with molecular weight cut-off of 0.22-0.65 μm or 500-1000kD, and after filtration, the membrane separation system is washed and filtered by purified water, and the trapped fluid and the permeate are collected. The trapped fluid is ultracentrifuged at 8000-.

Further, the membrane type used in the membrane separation in step (1) can be hollow fiber type, roll type, tube type or plate and frame type; the washing and filtering times in the membrane separation are 3-6 times;

further, the matrix of the separation material used in the ion exchange chromatography technology used in the step (2) is agarose, silica gel or high molecular polymer; the grain diameter is 40-100 μm; the ligand may have the formula

、

Or

；

The sample loading volume is 10-50 times of the column volume; the flow rate of the loading and elution of the chromatographic operation can be 150-500 cm/h; the mode of chromatographic purification of the target component is a binding mode, and the target component is eluted by using a linear gradient of a buffer solution with the salt concentration of 0-1M, wherein the elution volume is 10-50 times of the column volume.

Further, the concentration and drying mode used in the purification process of step (2) may be freeze drying, vacuum drying or spray drying.

Based on the technical scheme, the invention has the beneficial effects that:

(1) by adopting a method combining membrane separation, isoelectric precipitation and ultracentrifugation, various high-abundance proteins (such as casein), lipids, lactose and other components can be effectively removed, and interference on subsequent research on whey proteomics is avoided. The tangential flow membrane separation technology adopted by the invention is not the traditional dead-end filtration technology, so that the influence of larger shearing force on the stability of the protein is avoided, and the influence on the result of proteomic identification is avoided. The membrane separation mode can enrich the whey protein and improve the treatment efficiency.

(2) The invention adopts the isoelectric precipitation method to extract the whey protein, uses acetic acid to adjust the pH value instead of the traditional hydrochloric acid, and ensures the stability of the extracted whey protein.

(3) The ion exchange chromatography technology is adopted to prepare the total whey protein of the breast milk, so that the purity of a target object and the content of effective components can be obviously improved, and a high-purity sample for subsequent research on whey proteomics can be provided.

Drawings

FIG. 1 is a flow chart of a method of purifying human milk whey protein according to the present invention;

FIG. 2 is a graph comparing the distribution of proteins in whey of example 2 with the proteins in isoelectric precipitation;

FIG. 3 is a comparison of the pure whey protein of example 3 with the crude whey protein;

figure 4 is a graph comparing the pure whey protein of example 4 with the crude whey protein.

Detailed Description

The present invention will be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.

Example 1

The breast milk sample 1L was weighed and filtered. Installing the 750kD ultrafiltration membrane on a membrane treatment system, setting the flow rate to be 400 rpm, setting the flow rate at a circulating end to be 1.5L/min, setting the average flow rate at a permeation end to be 15 ml/min, collecting a permeate, and washing and filtering by using purified water for 5 times when the volume of a liquid at the circulating end is reduced to 80-100 ml each time, wherein 50 ml is used each time; collecting trapped fluid to obtain the breast milk total protein extract.

Example 2

Weighing 16g of the breast milk total protein extract in example 1, adding 100ml of water, shaking fully, mixing uniformly, and centrifuging at 10000 r/min and 4 ℃ for 10 min for degreasing. Adjusting pH of the water solution of the middle layer to 4.6 with 1 mol/L acetic acid, mixing, centrifuging at 10000 r/min 4 deg.C for 10 min, collecting supernatant and precipitate, performing SDS-PAGE analysis (see figure 2), filtering with 0.45 μm filter membrane, and purifying the supernatant with ion exchange chromatography column.

Example 3

Connecting a cation exchange chromatography S column (5 mL of column volume) to a chromatographic system, setting the flow rate to be 4 mL/min (240 cm/h), pumping the filtered supernatant in the example 2 through the balanced S column by a system pump, eluting a target component by adopting a linear gradient of 0-1M sodium chloride concentration, eluting by 16 times of column volume, collecting flow-through (LC) and elution fractions (F1, F2, F3 and F4) for SDS-PAGE analysis (see figure 3), and merging the qualified fractions after detection to obtain the refined whey protein. Placing the refined whey protein in a centrifugal concentrator, vacuumizing and drying until the water is completely removed, and collecting dry powder. The purity of the prepared whey protein is about 90%, and the yield of the whey protein is about 85%.

Example 4

Connecting an anion exchange chromatography Q column (column volume 5 mL) to a chromatographic system, setting the flow rate to be 4 mL/min (240 cm/h), pumping the filtered supernatant in the example 2 through the balanced Q column by a system pump, eluting a target component by using 0-1M sodium chloride concentration, eluting by 16 times of the column volume, collecting flow through (LC) and elution fractions (F1, F2, F3 and F4) for SDS-PAGE analysis (see figure 4), and merging the qualified fractions to obtain the refined whey protein. Placing the refined solution in an ion exchange chromatography centrifugal concentrator, vacuumizing and drying until all water is removed, and collecting dry powder. The purity of the prepared whey protein is about 84%, and the yield of the whey protein is about 89%.

The above results (as shown in fig. 2, 3 and 4) indicate that most of various kinds of whey proteins are present in the supernatant after isoelectric precipitation and centrifugation, and casein is mainly present in the precipitate; the whey protein can be effectively purified and separated by two ion exchange chromatography techniques. The results fully show that the whey protein prepared by the method has the advantages of high purity and high yield. As shown in table 1.

Table 1 comparison of whey protein purity and yield in the inventive examples and prior art preparation methods

Method of producing a composite material	Whey protein purity (%)	Whey protein yield (%)
			Example 3	90	85
Example 4	84	89
			Prior Art	65-75	40-60

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A purification preparation method for extracting whey protein in breast milk is characterized by comprising the following steps:

(1) pretreatment: removing casein from skim milk by membrane separation and low temperature high speed centrifugation degreasing, adjusting pH to 4.6-4.8 isoelectric point with 0.5-1M acetic acid, precipitating to obtain clear whey solution;

2. The method as claimed in claim 1, wherein the membrane separation system used in step (1) is a membrane separation membrane with a molecular weight cut-off of 0.22-0.65 μm or 500-1000kD, and after filtration, the membrane separation membrane is washed with purified water to collect the retentate and the permeate; the trapped fluid is ultracentrifuged at 8000-.

3. The method for purifying and preparing whey protein in breast milk according to claim 1, wherein the membrane type used in the membrane separation in step (1) can be hollow fiber type, roll type, tube type or plate and frame type.

4. The method for purifying and preparing whey protein in breast milk according to claim 1, wherein the number of washing and filtering times in the membrane separation in step (1) is 3-6.

5. The method for purifying and preparing whey protein in breast milk according to claim 1, wherein the matrix of the separation material used in the ion exchange chromatography technique used in step (2) is agarose, silica gel or high molecular polymer; the grain diameter is 40-100 μm; the ligand may have the formula

、

Or

；

6. The method for purifying and preparing whey protein in breast milk according to claim 1, wherein the concentration and drying in step (2) can be freeze drying, vacuum drying or spray drying.