# US 7,608,697 B2 · Protein hydrolysates enriched in peptides having a carboxy terminal proline residue

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- Number: US 7,608,697 B2
- Document ID: US7608697B2
- Status: Granted
- Kind: B2
- Application number: 10/433,747
- Filed: 2001-12-06
- Published: 2004-12-02
- Granted: 2009-10-27
- Earliest claimed: 2000-12-07
- Assignees: Dsm IP Assets B.V.
- Inventors: Luppo Edens; Robertus Antonius Mijndert Van Der Hoeven; Veronique Delest
- Web page: https://patentgrep.com/patent/US7608697B2

## CPC

- G01N 33/573: for enzymes or isoenzymes
- A23J 3/16: from soybean
- A23J 3/343: {of dairy proteins}
- A23J 3/344: {of casein}
- A23J 3/346: {of vegetable proteins}
- A23L 2/02: containing fruit or vegetable juices
- A23L 2/84: using microorganisms or biological material, e.g. enzymes
- A23L 33/18: Peptides; Protein hydrolysates
- A23L 33/40: {Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula}
- C12H 1/003: {by a biochemical process}
- C12Q 1/37: involving peptidase or proteinase
- G01N 33/56961: {Plant cells or fungi}
- G01N 2333/38: from Aspergillus
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)
- C12H: PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES (simulation ageing by flavouring C12G3/06)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method of enzymatically producing a protein hydrolysate from a protein substrate is described, wherein a proline-specific endoprotease or a composition containing a proline-specific endoprotease and optionally a subtilisin or a metallo endoprotease, and other enzymes such as carboxypeptidases, is used to produce a protein hydrolysate enriched in peptide fragments having a carboxy terminal proline residue. Such protein hydrolysates may be used as such or to reduce bitterness in foods nutritionally supplemented by protein hydrolysates, as well as to produce hydrolysate containing foodstuffs having low antigenicity.

## Claims

### Claim 1

A protein hydrolysate which comprises peptides, wherein the molar fraction of peptides carrying a carboxy terminal proline is more than two times the molar fraction of proline in a protein substrate used to generate the protein hydrolysate, and wherein the protein substrate is selected from the group consisting of whey, casein and soy.

### Claim 2 (depends on 1)

A protein hydrolysate according to claim 1, wherein the molar fraction of peptides carrying a carboxy terminal proline is at least three times the molar fraction of proline in the protein substrate.

### Claim 3 (depends on 1)

A protein hydrolysate according to claim 1, wherein the average peptide length is from 3 to 9 amino acids.

### Claim 4 (depends on 1)

A protein hydrolysate according to claim 1, wherein at least 10% of the protein substrate is hydrolysed.

### Claim 5

A protein hydrolysate comprising peptides selected from the group consisting of
a whey hydrolysate wherein the molar fraction of peptides carrying a carboxy terminal proline is at least 8%;
a casein hydrolysate wherein the molar fraction of peptide carrying a carboxy terminal proline is at least 25%; and
a soy hydrolysate wherein the molar fraction of peptide carrying a carboxy terminal proline is at least 20%.

### Claim 6 (depends on 1)

A food or drink which comprises a protein hydrolysate according to claim 1.

### Claim 7 (depends on 6)

The food or drink of claim 6 which comprises from 5% to 10% (w/v) of the protein hydrolysate.

### Claim 8 (depends on 6)

The food or drink of claim 6 that has reduced bitterness and/or low antigenicity.

### Claim 9 (depends on 8)

The food or drink of claim 8 which comprises an infant formula.

### Claim 10

A method of enzymatically producing a protein hydrolysate from a protein substrate, which method comprises incubating the protein substrate with sufficient proline-specific endoprotease to produce a protein hydrolysate which comprises peptides, wherein the molar fraction of peptides carrying a carboxy terminal proline is more than two times the molar fraction of proline in the protein substrate used to generate the protein hydrolysate, and wherein the protein substrate is selected from the group consisting of whey, casein and soy.

### Claim 11 (depends on 10)

A method according to claim 10, wherein said incubating is performed either sequentially or concomitantly with the proline-specific endoprotease and one or more other endoprotease(s).

### Claim 12 (depends on 11)

A method according to claim 11, wherein the other endoprotease(s) is a serine or a metallo-endoprotease or a combination of a serine and a metallo-endoprotease.

### Claim 13 (depends on 10)

A method according to claim 10, wherein the method further comprises recovering the protein hydrolysate without ultrafiltration or a microfiltration.

### Claim 14 (depends on 12)

A method according to claim 12, wherein the other endoprotease(s) is a combination of subtilisin and carboxypeptidase.

### Claim 15 (depends on 7)

A method according to claim 7, wherein the molar fraction of peptides carrying a carboxy terminal proline in the protein hydrolysate is more than three times the molar fraction of proline in the protein substrate.

### Claim 16 (depends on 10)

A method according to claim 10, wherein the method employs at least 150 milli-units of proline-specific endoprotease per gram substrate.

### Claim 17 (depends on 10)

A method according to claim 10, wherein the proline-specific endoprotease has a pH optimum below 7.

### Claim 18 (depends on 10)

A method according to claim 10, wherein the proline-specific endoprotease is from A. niger.

### Claim 19 (depends on 5)

A food or drink which comprises a protein hydrolysate according to claim 5.

### Claim 20 (depends on 19)

A food or drink of claim 19 which comprises from 5% to 10% (w/v) of the protein hydrolysate.

### Claim 21 (depends on 19)

A food or drink of claim 19 which has reduced bitterness and/or low antigenicity.

### Claim 22 (depends on 10)

A method according to claim 10, wherein the average peptide length is from 3 to 9 amino acids.

### Claim 23 (depends on 10)

A method according to claim 10, wherein at least 10% of the protein substrate is hydrolysed.