# US 6,991,824 B2 · Expression of human milk proteins in transgenic plants

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- Number: US 6,991,824 B2
- Document ID: US6991824B2
- Status: Granted
- Kind: B2
- Application number: 10/077,381
- Filed: 2002-02-14
- Published: 2003-04-17
- Granted: 2006-01-31
- Earliest claimed: 2001-02-06
- Assignees: Ventria Bioscience
- Inventors: Ning Huang; Raymond L. Rodriguez; Frank E. Hagie
- Web page: https://patentgrep.com/patent/US6991824B2

## CPC

- G01N 33/5759: {involving compounds localised on the membrane of tumour or cancer cells}
- A23K 10/30: from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms (obtained by microbiological or biochemical processes, e.g. using yeasts or enzymes, A23K10/10)
- A23K 20/147: Polymeric derivatives, e.g. peptides or proteins
- A23L 7/198: {Dry unshaped finely divided cereal products, not provided for in groups A23L7/117 - A23L7/196 and A23L29/00, e.g. meal, flour, powder, dried cereal creams or extracts}
- A23L 7/20: Malt products (preparation of malt for brewing C12C)
- A23L 33/105: Plant extracts, their artificial duplicates or their derivatives
- A23L 33/19: Dairy proteins
- B82Y 30/00: Nanotechnology for materials or surface science, e.g. nanocomposites
- C07K 14/415: from plants
- C07K 14/485: Epidermal growth factor [EGF], i.e. urogastrone
- C07K 14/65: Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
- C07K 14/79: Transferrins, e.g. lactoferrins, ovotransferrins
- C07K 14/8125: {Alpha-1-antitrypsin}
- C12N 9/0065: {acting on hydrogen peroxide as acceptor (1.11)}
- C12N 9/2462: {Lysozyme (3.2.1.17)}
- C12N 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N 15/8222: {Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation}
- C12N 15/8234: {Seed-specific, e.g. embryo, endosperm}
- C12N 15/8257: {for the production of primary gene products, e.g. pharmaceutical products, interferon}
- G01N 33/5758: {involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites}
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- G01N 2333/4725: Mucins, e.g. human intestinal mucin
- G01N 2500/02: Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
- 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)
- A23K: FODDER
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION

## Abstract

The invention is directed to food and food additive compositions comprising one or more human milk proteins produced in the seeds of a transgenic plant and methods of making the same. The invention is further directed to improved infant formula comprising such food supplement composition.

## Claims

### Claim 1

A nutritionally enhanced food comprising
one or more plant-derived food ingredients, and
as an additive, a seed composition containing a flour, extract, or malt obtained from mature monocot seeds, and wherein said flour, extract, or malt contains seed-produced human milk proteins in substantially unpurified form selected from
(i) lysozyme or lactoferrin, and one or more seed-produced human milk proteins selected from the group consisting of lactoferricin, epidermal growth factor, insulin-like growth factor-1, lactadherin, kappa-casein, haptocorrin, lactoperoxidase, and alpha-1-antitrypsin, -or
(ii) lysozyme and lactoferrin, and optionally one or more seed-produced human milk proteins selected from the group consisting of lactoferricin, epidermal growth factor, insulin-like growth factor-1, lactadherin, kappa-casein, haptocorrin, lactoperoxidase, and alpha-1-antitrypsin.

### Claim 2 (depends on 1)

The food of claim 1, wherein said the seed-produced protein(s) are lysozyme and lactoferrin.

### Claim 3 (depends on 2)

The food of claim 2, which is an infant formula, wherein said the seed-produced proteins are lysozyme, in an amount between 0.03 to 0.3 g protein/liter formula, and lactoferrin, in an amount between 0.3 to 3 g/liter formula.

### Claim 4 (depends on 1)

The food of claim 1, wherein the seed composition comprises between 0.1 to 20% of the total solid weight of the food.

### Claim 5 (depends on 4)

The food of claim 4, which is an infant milk formula, where the seed composition contains a seed extract or malt obtained from mature seeds of rice or barley.

### Claim 6

An ingestible monocot-seed composition containing a flour, extract, or malt obtained from mature monocot seeds, and wherein said flour, extract, or malt contains one or more seed-produced human milk proteins in substantially unpurified form, selected from
(i) lysozyme or lactoferrin, and one or more seed-produced human milk proteins selected from the group consisting of lactoferricin, epidermal growth factor, insulin-like growth factor-1, lactadherin, kappa-casein, haptocorrin, lactoperoxidase, and alphf-1-antitrypsin, or
(ii) lysozyme and lactoferrin, and optionally one or more seed-produced human milk proteins selected from the group consisting of lactoferricin, epidermal growth factor, insulin-like growth factor-1, lactadherin, kappa-casein, haptocorrin, lactoperoxidase, and alpha-1-antitrypsin.

### Claim 7 (depends on 6)

The composition of claim 6, said the seed-produced proteins are lysozyme and lactoferrin.

### Claim 8 (depends on 6)

The composition of claim 6, wherein when said composition contains an extract, at least one of the human proteins is present in the extract in an amount greater than 1 mg/gram dry weight of extract.

### Claim 9 (depends on 6)

The composition of claim 6, wherein
(a) the flour is prepared by milling mature monocot seeds;
(b) the extract is prepared by suspending milled flour in a buffered aqueous medium; and
(c) the malt is prepared by (i) steeping barley seeds to a desired water content, (ii) germinating the steeped barley, (iii) drying the germinated seeds, under conditions effective to stop germination, (iv) crushing the dried seeds, (v) optionally, adding crushed seeds from a non-barley monocot plant, (vi) forming a mixture of crushed seeds in water, and (vii) malting the crushed seed mixture until a desired malt is achieved, where at least one of the barley or non-barley monocot seeds contain such milk protein(s).

### Claim 10 (depends on 9)

The composition of claim 9, wherein step (v) includes adding to the crushed dried barley seeds, mature rice transgenic seeds that produce a milk protein.