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Scale-scale purification of whey protein

A purification method and technology of whey protein, applied in the direction of albumin peptide, animal/human protein, animal/human peptide, etc., can solve the problems of low selectivity, low efficiency, many operation steps, etc., to reduce production cost, low cost, etc. cost, the effect of improving production efficiency

Inactive Publication Date: 2011-12-21
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] After searching the patent literature, it was found that Chinese Patent Application No. 200810118961.7 "Method for Separation and Purification of Heterogeneous and Homologous Whey Proteins" uses two-step methods of hydrophobic chromatography and gel filtration chromatography to purify α-lactalbumin, first using a hydrophobic layer The crude product of the target protein is obtained by analysis, and then purified by gel filtration chromatography; this purification method has many steps, complicated operation and low efficiency
Chinese Patent Application No. 200910161153.3 "Method for Separation and Determination of Whey Protein" adopts the method of ultra-high performance liquid chromatography column with ethylene bridged hybrid (BEH) particles as filler to separate α-lactalbumin and β-lactoglobulin Separation; U.S. Patent No. 7524932 is pretreated under acidic conditions, and anion exchange method and gradient elution are used to purify α-lactalbumin; U.S. Patent No. 5503864 is a method that combines isoelectric point precipitation and membrane filtration. Low sex, low product purity
These methods either have many steps, or are difficult to scale up to achieve large-scale production

Method used

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  • Scale-scale purification of whey protein
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  • Scale-scale purification of whey protein

Examples

Experimental program
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Effect test

Embodiment 1

[0013] Take the reconstituted milk powder, add deionized water at a ratio of 1:8 (w / v), centrifuge and degrease, take the whey and divide it into multiple parts, and adjust the CaCl 2 The final concentration is 0.0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4M, and then heated to the temperature: 70°C, 75°C, 80°C, 85°C, 90°C, 95°C for 10min, 20min, 30min, 40min, 50min, 60min; after the reaction is completed, centrifuge each tube at 4°C, 9800rpm for 10min, transfer the supernatant to a new tube and add NaCO 3 , to remove CaCl by centrifugation 2 , with 200mL pH 7.0, 10mM sodium phosphate buffer solution to wash the precipitate three times and dissolve it. 13.5% SDS-PAGE was used to analyze the composition of supernatant and precipitated protein, and the supernatant was determined for protein quantification. The total content of total α-lactalbumin and β-lactoglobulin in the concentrated sample can reach more than 99%. Table 1 shows whey samples at different concentrations of CaCl ...

Embodiment 2

[0024] The whey pretreatment sample prepared in Example 1 was adjusted to pH 7.4 with 1M HCl or NaOH solution, and loaded onto a Q Sepharose Fast Flow anion-exchange chromatography column (11x22cm) pre-equilibrated with 50mM, pH 7.4 Tris-HCl buffer , flow rate 200ml / ml; after sample loading, wash the column with 50mM, pH 7.4 Tris-HCl buffer until the absorption signal at 280nm reaches the baseline, and elute with NaCl-50mM Tris-HCl (pH 7.4) buffer with a conductivity of 10.2ms / cm . 1.5L of recombinant human α-lactalbumin fraction was collected with a recovery rate of 76%; the purity of recombinant human α-lactalbumin detected by high performance liquid chromatography was 94.1%; 1.2L of bovine α-lactalbumin peak was collected and recovered by the process The yield is 79.2%, and the purity of bovine α-lactalbumin detected by high performance liquid chromatography is 90.1%; the bovine β-lactoglobulin peak 2L is collected, and the process recovery rate is 85.5%, and the purity of ...

Embodiment 3

[0026] The whey pretreatment sample prepared in Example 1 was adjusted to pH 6.4 with 1M HCl or NaOH solution, and loaded onto a Q Sepharose Fast Flow anion-exchange chromatography column (11x22cm) pre-equilibrated with 50mM, pH6.4 Tris-HCl buffer , the flow rate is 200ml / ml; after loading the sample, wash the column with 50mM, pH6.4 Tris-HCl buffer until the absorption signal at 280nm reaches the baseline, and then wash with NaCl-50mM Tris-HCl (pH6.4) buffer with a conductance of 10.2ms / cm take off. 1.6L of recombinant human α-lactalbumin fraction was collected with a recovery rate of 72%; the purity of recombinant human α-lactalbumin detected by HPLC was 91.2%; bovine α-lactalbumin peak 1.3L was collected with a recovery rate of 75.3%, the purity of bovine α-lactalbumin detected by HPLC is 87.3%; the bovine β-lactoglobulin peak 2.1L is collected, the recovery rate is 90.7%, the purity of bovine β-lactoglobulin detected by HPLC is 75.8% %.

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Abstract

A large-scale purification method of whey protein in the field of bioengineering technology. Human α-lactalbumin recombinant milk is used as raw material, and after pretreatment and purification of the protein, recombinant human α-lactalbumin, recombinant human α-lactalbumin, and Bovine alpha-lactalbumin and bovine beta-lactoglobulin. The invention can purify recombinant human α-lactalbumin rapidly, conveniently, at low cost and in large quantities, reduces production cost and improves production efficiency.

Description

technical field [0001] The invention relates to a method in the technical field of bioengineering, in particular to a large-scale purification method of whey protein. Background technique [0002] α-lactalbumin widely exists in mammalian milk and is the regulatory subunit of β-1,4-galactosidyl transferase, which catalyzes the synthesis of lactose. The amino acid composition of α-lactalbumin contains a large amount of amino acids necessary for human nutrition, and has important nutritional value. Human α-lactalbumin multimers or folding variants can induce tumor cell apoptosis and are expected to be developed into clinical drugs for the treatment of cancer. Alpha-lactalbumin is a type of whey protein. Chinese patent 200610076242 establishes a production method for recombinantly cloning large-scale livestock with transhuman α-lactalbumin gene, laying the foundation for large-scale production of recombinant human α-lactalbumin for use as clinical medicine and functional food....

Claims

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Application Information

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IPC IPC(8): C07K14/76C07K14/47C07K1/18
Inventor 李荣秀王一翟东改李雪霍晨曦颜远伟
Owner SHANGHAI JIAO TONG UNIV
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