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A method for isolating lactoperoxidase from bovine whey

A technology of lactoperoxidase and milk, which is applied in the direction of oxidoreductase, biochemical equipment and methods, enzymes, etc., can solve the problems such as difficult to achieve selective separation, and achieve large-scale separation, which is easy to realize, good in safety, and easy to operate simple effect

Active Publication Date: 2017-12-08
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Literature (Billakanti J.M. and Fee C.J., Biotechnol. Bioeng. 2009, 103: 1155–1163) used polyacrylamide-based cation-exchange crystal gel media with carboxyl functional groups to separate lactoferrin from milk and whey by chromatography. The purity of the obtained lactoferrin was 90%. However, because the isoelectric point and molecular weight of lactoferrin and lactoperoxidase are very close, it is difficult to achieve selective separation with the existing carboxyl-bearing cation exchange gel
So far, there is no research report on the separation of lactoperoxidase from whey, skimmed milk, homogenized milk, etc. by crystal gel chromatography

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Take polymethacrylic acid with a diameter of 10 mm, a height of 40 mm, embedded cellulose crystal gel microspheres (average particle size is about 900 μm, accounting for 25% of the crystal gel matrix skeleton), and a sulfonic acid functional group. Hydroxyethyl ester cation exchange chimeric crystal gel media bed column (effective porosity 84%, maximum porosity 95%, pore diameter about 0.1-300 μm, adsorption capacity for lysozyme model protein 1.1 mg / mL), at 20 mM pH 5.8 Phosphate (Na 2 HPO 4 / NaH 2 PO 4 ) solution is a buffer solution, use 30 mL buffer solution to equilibrate the cation exchange chimeric gel bed column; take 8 mL whey, dilute 2 times with buffer solution, load the sample at a flow rate of 10 cm / min, and then wash with buffer solution Unadsorbed impurities were removed, and 0.05, 0.5, and 2 M NaCl eluents (buffer solution configuration) were used for step-by-step elution, and the target elution peaks were collected, desalted and freeze-dried to obtai...

Embodiment 2

[0029] Take polymethacrylic acid with a diameter of 10 mm, a height of 38 mm, embedded cellulose crystal gel microspheres (average particle size is about 900 μm, accounting for 25% of the crystal gel matrix skeleton), and a sulfonic acid functional group. Hydroxyethyl ester cation exchange chimeric crystal gel medium bed column (effective porosity 83%, maximum porosity 94%, pore diameter about 0.1-300 μm, saturated adsorption capacity for lysozyme model protein 5 mg / mL), at 10 mM pH 6 Phosphate (Na 2 HPO 4 / NaH 2 PO 4 ) solution is a buffer solution, equilibrate the cation exchange chimeric gel bed column with 45 mL buffer solution; take 4 mL whey, dilute 4 times with buffer solution, load the sample at a flow rate of 0.5cm / min, and then use buffer solution Rinse to remove unadsorbed impurities, use 0.05, 0.5, and 2 M NaCl eluents (buffer solution configuration) for step-by-step elution, collect target elution peaks, desalt and freeze-dry to obtain lactoperoxidase. By SDS ...

Embodiment 3

[0031] Take polymethacrylic acid with a diameter of 10 mm, a height of 52 mm, embedded cellulose crystal gel microspheres (average particle size is about 900 μm, accounting for 30% of the crystal gel matrix skeleton), and a sulfonic acid functional group. Hydroxyethyl ester cation exchange chimeric crystal gel media bed column (effective porosity 80%, maximum porosity 94%, pore diameter about 0.1-300 μm, adsorption capacity for lysozyme model protein 3.4 mg / mL), at 10 mM pH 9 Glycine-sodium hydroxide solution was used as the buffer solution, and the cation exchange chimeric gel bed column was equilibrated with 20 mL of buffer solution; 5.1 mL of whey was taken, diluted 3 times with buffer solution, and loaded at a flow rate of 1 cm / min. Then rinse with a buffer solution to remove unadsorbed impurities, use 0.075, 0.15, 1, and 2 M NaCl eluents (buffer solution configuration) for step-by-step elution, collect the target elution peaks, desalt and freeze-dry to obtain Lactoperoxid...

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Abstract

The invention relates to a method for separating lactoperoxidase from bovine whey and belongs to the technical field of biochemical engineering. According to the method, the bovine whey serves as a raw material liquid, cation exchange chimeric composite crystal gel with multistage pores serves as a separating medium, and conventional treatment, such as crystal gel chromatography, bed column balancing, sampling and adsorbing, buffer solution flushing, saliferous eluent stepwise elution, subsequent desalting and freeze drying, is carried out, so that high-purity lactoperoxidase with the purity of 98% is obtained through separating, and the yield can reach 92%. The cation exchange chimeric composite crystal gel medium has very good selectivity to lactoferrin and lactoperoxidase, which are difficultly separated through conventional chromatography, in the whey. The method has the advantages that steps are few, the operation is simple, the separation is rapid, the cost is low, and the obtained lactoperoxidase is high in purity and yield and has broad application prospects in the respects of high-value utilization of whey and separation of active protein.

Description

technical field [0001] The invention belongs to the technical field of biochemical industry, and in particular relates to a method for isolating lactoperoxidase from bovine whey. Background technique [0002] Lactoperoxidase is an oxidoreductase secreted by mammalian mammary glands, salivary glands, lacrimal glands, etc. It has many functions such as participating in the body's antibacterial, degrading harmful substances such as amines and phenols, and protecting cells from peroxidation. It has important applications in the fields of fresh-keeping and cold storage of foods such as dairy products and meat, and daily necessities such as toothpaste. Whey is a by-product of the dairy industry, and its high-value utilization has been a common concern around the world for many years. The content of lactoperoxidase in bovine whey is high, and it is an important source of lactoperoxidase in industry. [0003] However, there are many kinds of proteins in whey. The existing separati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/08
CPCC12N9/0065C12Y111/01007
Inventor 贠军贤潘毛毛姚善泾林东强张颂红陈良代斌
Owner ZHEJIANG UNIV OF TECH
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