# US 7,465,467 B2 · Process for the extraction, purification and enzymatic modification of soy 7S globulin α' subunit for use as hypocholesterolemizing agent

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- Number: US 7,465,467 B2
- Document ID: US7465467B2
- Status: Granted
- Kind: B2
- Application number: 10/502,582
- Filed: 2003-01-27
- Published: 2005-07-28
- Granted: 2008-12-16
- Earliest claimed: 2002-01-29
- Assignees: Indena S.p.A.
- Inventors: Marcello Duranti; Paolo Morazzoni
- Web page: https://patentgrep.com/patent/US7465467B2

## CPC

- A61K 38/168: {from plants}
- A23J 1/14: from leguminous or other vegetable seeds; from press-cake or oil-bearing seeds
- A23J 3/16: from soybean
- A23L 33/185: Vegetable proteins
- A61P 3/02: Nutrients, e.g. vitamins, minerals
- A61P 3/06: Antihyperlipidemics
- Y02E 50/10: Biofuels, e.g. bio-diesel
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A23J: PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

A process for the extraction, purification and enzymatic modification of β-conglycinin α′ subunit, wherein β-conglycinin is selectively extracted from ground, defatted soy, then precipitated by treatment with aqueous ethanol; the enriched fraction is then subjected to Metal Affinity Chromatography (MAC) in denaturant conditions to obtain the α′ subunit, which is treated with chymotrypsin, then subjected to a further MAC to recover the amino-terminal region of this polypeptide (MW 28,000 Da).

## Claims

### Claim 1

A process for the selective extraction and purification of soy β-conglycinin α′ subunit, which process comprises the following steps:
a) extraction of defatted ground soy with a sodium bisulfite aqueous solution at slightly acidic pH to obtain a β-conglycinin-enriched soluble protein fraction;
b) precipitation of the β-conglycinin fraction from step a) by treatment with ethanol;
c) purification of the precipitated fraction from step b) by Metal Affinity Chromatography (MAC) under denaturant conditions, to isolate the α′ subunit; and
d) precipitation of the α′ subunit with an organic solvent.

### Claim 2 (depends on 1)

The process as claimed in claim 1, comprising enzymatic treatment of the α′ subunit from step c) with a proteolytic enzyme and further purification by MAC chromatography.

### Claim 3 (depends on 1)

The process as claimed in claim 1 wherein the extraction is carried out with 15 volumes of a 0.98 g/L sodium bisulfite solution at pH 6.4.

### Claim 4 (depends on 1)

The process as claimed in claim 1 wherein the precipitation of step b) is carried out with 40% ethanol.

### Claim 5 (depends on 1)

The process as claimed in claim 1 wherein MAC in step c) is carried out on a matrix conjugated with zinc or nickel.

### Claim 6 (depends on 5)

The process as claimed in claim 5 wherein the matrix is conjugated with zinc.

### Claim 7 (depends on 5)

The process as claimed in claim 5 wherein the matrix consists of agarose-iminodiacetic acid.

### Claim 8 (depends on 1)

The process as claimed in claim 1 wherein the denaturing agent used in MAC of step c) is urea.

### Claim 9 (depends on 2)

The process as claimed in claim 2 wherein the proteolytic enzyme for the enzymatic treatment is chymotrypsin.

### Claim 10 (depends on 1)

The process as claimed in claim 1 wherein the precipitating solvent for the α′ subunit in step d) is acetone.

### Claim 11 (depends on 1)

The process as claimed in claim 1 wherein the β-conglycinin-enriched fraction and the α′ subunit are stabilized by freeze-drying.