Unlock instant, AI-driven research and patent intelligence for your innovation.

A kind of purification method of lactoperoxidase in milk

A technology of lactoperoxidase and purification method, applied in the directions of oxidoreductase, biochemical equipment and methods, enzymes, etc., can solve the problems of cumbersome operation method, complicated operation and high cost

Active Publication Date: 2021-04-27
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the purification methods of these studies generally use at least two different exchange columns, which are complicated, time-consuming, and costly.
At present, there are still some methods to separate LPO, but the operation method is more cumbersome. For example, to separate and purify LPO, 1.9-2.5mol L can be added to the crude rennet whey -1 (NH 4 ) 2 SO 4 Salt out, followed by calcium phosphate gel chromatography, can be further purified in silica calcite gel, and finally from 4mol L -1 K 2 HPO 4 crystallization

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of purification method of lactoperoxidase in milk
  • A kind of purification method of lactoperoxidase in milk
  • A kind of purification method of lactoperoxidase in milk

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0057] The preparation method of the present invention will be further described in detail in conjunction with specific examples below. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies realized based on the above contents of the present invention are covered within the scope of protection intended by the present invention.

[0058] The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents and materials used in the following examples can be obtained from commercial sources unless otherwise specified.

[0059] Instrument and reagent used in the embodiment:

[0060] Instruments: AKTA Pure Protein Purifier (GE Healthcare), Magnetic Stirrer (SCILOGEX), Peristaltic Pump (Langer), Discover12m-PES 0.2μm Hollow Fiber Column (WaterSep), Discover12m-PES 30...

Embodiment 1

[0065] Example 1: Selection of hydrophobic chromatography prepacked column conditions

[0066] (1) Sample pretreatment

[0067] Prepare skim milk: centrifuge 1 L of whole fresh milk at 2500 rpm and 25° C. for 15 min in a 500 mL centrifuge bottle. The upper separation layer containing the cream was discarded, and the skim milk was poured into a 1 L beaker.

[0068] Preparation of whey: Dissolve 1 g of rennet in 5 mL of ultrapure water to prepare a rennet solution with a concentration of 0.2 g / mL. Add 400 μL of rennet solution to 1 L of skim milk and mix well. Heat at 30°C for 60min to coagulate and precipitate the casein, and centrifuge at 4000rpm and 25°C for 15min in a 500mL centrifuge bottle. Finally, pour the whey into a clean 2L beaker for later use, and discard the precipitated coagulated casein.

[0069] (2) Ammonium sulfate precipitates miscellaneous proteins

[0070] Take 1L of deionized water and dissolve 750g of ammonium sulfate to prepare a saturated ammonium s...

Embodiment 2

[0086] Embodiment 2: the purification method of lactoperoxidase in cow's milk

[0087] According to the same conditions of (1) sample pretreatment and (2) ammonium sulfate precipitation of miscellaneous proteins in Example 1, sample pretreatment and ammonium sulfate precipitation of miscellaneous proteins were carried out.

[0088] (3) Preparation of starting buffer and elution buffer

[0089] Starting buffer: 50mM NaH 2 PO 4 ,50% (NH 4 ) 2 SO 4 ,2mM CaCl 2 ,pH 7.0;

[0090] Elution buffer: 50mM NaH 2 PO 4 ,2mM CaCl 2 ,pH 7.0;

[0091] (4) Purified lactoperoxidase

[0092] A hydrophobic chromatography prepacked column Octyl Sepharose 4FF (purchased from GE, codenamed GE HiTrapOctyl FF) was used as the purification filler, and the purification process was as follows:

[0093] A.5 column volume ultrapure water washes the chromatography column;

[0094] B. 5 column volumes of 100% starting buffer equilibrate the chromatographic column;

[0095] C. the clarified ammo...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
particle diameteraaaaaaaaaa
pore sizeaaaaaaaaaa
molecular weightaaaaaaaaaa
Login to View More

Abstract

A method for purifying lactoperoxidase in milk. The present invention adopts an agarose gel hydrophobic chromatography column to purify lactoperoxidase in liquid milk by one-step hydrophobic chromatography, which omits multi-step and complicated extraction and purification steps compared with the prior art, and is fast, efficient and low-cost advantages, and the purity of the purified lactoperoxidase is greater than or equal to 90%, the protein is dissolved in the elution buffer of pH = 7.0, and can be used for subsequent experiments or operations without removing ammonium sulfate.

Description

technical field [0001] The present invention relates to a fast purification method of lactoperoxidase in liquid milk (such as cow's milk), more specifically adopts fast protein purification chromatography technology, purifies lactoperoxidase in liquid milk through one-step hydrophobic chromatography, It belongs to the technical field of protein purification. Background technique [0002] Lactoperoxidase (lactoperoxidase, LPO, EC 1.11.1.7), as one of the endogenous enzymes in emulsion, widely exists in mammalian mammary glands, salivary glands, lacrimal glands and their respective secretions (milk, saliva, tears). LPO has high thermal stability and can be used as one of the evaluation indicators for ultra-pasteurization. LPO is an effective antibacterial agent, which can not only reduce the bacterial flora in milk and milk products, but also prolong the life of frozen raw milk by adding hydrogen peroxide and thiocyanate to activate the lactoperoxidase system (LPOS). It also...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/08
CPCC12N9/0065C12Y111/01007
Inventor 王加启张养东郑楠李松励赵圣国
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI