# US 11,439,166 B2 · Ground meat replicas

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- Number: US 11,439,166 B2
- Document ID: US11439166B2
- Status: Granted
- Kind: B2
- Application number: 17/531,460
- Filed: 2021-11-19
- Published: 2022-03-31
- Granted: 2022-09-13
- Earliest claimed: 2014-03-31
- Assignees: Impossible Foods Inc.
- Applicants: Impossible Foods Inc.
- Inventors: Ranjani Varadan; Sergey Solomatin; Celeste Holz-Schietinger; Elysia Cohn; Ariel Klapholz-Brown; Jennifer Woan-Yi Shiu; Aniket Kale; Jessica Karr; Rachel Fraser
- Web page: https://patentgrep.com/patent/US11439166B2

## CPC

- A23L 13/43: {Addition of vegetable fats or oils; Addition of non-meat animal fats or oils; Addition of fatty acids}
- A23L 13/422: {Addition of natural plant hydrocolloids, e.g. gums of cellulose derivatives or of microbial fermentation gums}
- A23L 13/426: {Addition of proteins, carbohydrates or fibrous material from vegetable origin other than sugars or sugar alcohols (A23L13/422 takes precedence)}
- C12N 15/81: for yeasts
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- C07K 14/805: Haemoglobins; Myoglobins
- C12N 1/165: Yeast isolates
- C12N 9/1029: {transferring groups other than amino-acyl groups (2.3.1)}
- C12N 9/88: Lyases (4.)
- C12N 15/815: {for yeasts other than Saccharomyces}
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- 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
- 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)

## Abstract

This document relates to ground meat replicas, and more particularly to plant-based products that mimic ground meat, including the fibrousness, heterogeneity in texture, beefy flavor, and red-to-brown color transition during cooking of ground meat.

## Claims

### Claim 1

A recombinant Pichia pastoris organism comprising:
eight stably integrated genes encoding recombinant heme biosynthesis enzymes; and
a stably integrated nucleic acid encoding a recombinant heme-containing protein selected from leghemoglobin and myoglobin,
wherein the heme biosynthesis enzymes are Pichia pastoris ALA synthase, Pichia pastoris ALA dehydratase, Pichia pastoris porphobilinogen deaminase, Pichia pastoris uroporphyrinogen III synthase, Pichia pastoris uroporphyrinogen III decarboxylase, Pichia pastoris coproporphyrinogen oxidase, Pichia pastoris protoporphyrinogen III oxidase, and Pichia pastoris ferrochelatase.

### Claim 2 (depends on 1)

The recombinant Pichia pastoris organism of claim 1, wherein at least one of the eight genes and/or the recombinant heme-containing protein is expressed by a methanol inducible promoter.

### Claim 3 (depends on 2)

The recombinant Pichia pastoris organism of claim 2, wherein the methanol inducible promoter is pAOXI.

### Claim 4 (depends on 1)

The recombinant Pichia pastoris organism of claim 1, wherein the heme-containing protein is a mammalian myoglobin.

### Claim 5 (depends on 1)

The recombinant Pichia pastoris organism of claim 1, wherein the heme-containing protein is a Glycine max leghemoglobin.

### Claim 6 (depends on 2)

A method of producing a consumable heme-containing protein, the method comprising:
fermenting the recombinant Pichia pastoris organism of claim 2 in a fermentation broth, and inducing expression of the heme-containing protein with methanol.

### Claim 7 (depends on 6)

The method of claim 6, comprising centrifuging cells of the Pichia pastoris organism expressing the heme-containing protein.

### Claim 8 (depends on 6)

The method of claim 6, comprising lysing cells of the Pichia pastoris organism expressing the heme-containing protein to produce a cell lysate.

### Claim 9 (depends on 8)

The method of claim 8, comprising filtering the cell lysate to produce a soluble lysate.

### Claim 10 (depends on 9)

The method of claim 9, comprising concentrating the heme-containing protein in the soluble lysate.

### Claim 11 (depends on 6)

The method of claim 6, comprising centrifuging cells of the Pichia pastoris organism expressing the heme-containing protein, lysing cells of the Pichia pastoris organism expressing the heme-containing protein to produce a cell lysate, filtering the cell lysate to produce a soluble lysate, and concentrating the heme-containing protein in the soluble lysate.

### Claim 12 (depends on 6)

The method of claim 6, wherein the heme-containing protein is a mammalian myoglobin.

### Claim 13 (depends on 6)

The method of claim 6, wherein the heme-containing protein is a Glycine max leghemoglobin.

### Claim 14

A method for preparing a food additive comprising recombinant heme-containing protein, the method comprising:
providing a recombinant Pichia pastoris organism comprising: eight stably integrated genes encoding recombinant heme biosynthesis enzymes; and a stably integrated gene encoding a recombinant heme-containing protein selected from leghemoglobin and myoglobin under the control of a methanol inducible promoter, wherein the recombinant heme biosynthesis enzymes comprise Pichia pastoris ALA synthase, Pichia pastoris ALA dehydratase, Pichia pastoris porphobilinogen deaminase, Pichia pastoris uroporphyrinogen III synthase, Pichia pastoris uroporphyrinogen III decarboxylase, Pichia pastoris coproporphyrinogen oxidase, Pichia pastoris protoporphyrinogen III oxidase, and Pichia pastoris ferrochelatase;
fermenting the recombinant Pichia pastoris organism in a fermentation broth;
inducing expression of the heme-containing protein with methanol;
purifying the recombinant heme-containing protein; and
combining the purified recombinant heme-containing protein with a buffered solution.