Compositions comprising digestive enzymes

• US 11,142,754 B2
• Filed: 03/02/2021
• Issued: 10/12/2021
• Est. Priority Date: 08/07/2019
• Status: Active Grant

• Current Assignee: Clara Foods, Co.
• Original Assignee: Clara Foods, Co.
• Inventors: Redfearn, Halle, Kshirsagar, Harshal, Mahadevan, Kritika, Chapeaux, Alexandre, Rutherford-Jenkins, Wesley, Kreps, Joel Andrew, Joshi, Isha
• Primary Examiner(s): Desai, Anand U
• Application Number:
  US17/190,173
• Publication Number:
  US 20210189369A1
• Time in Patent Office: 224 Days
• Field of Search: None
• CPC Class Codes: C12N 9/50 Proteinases , e.g. Endopeptidases (3.4.21-3.4.25)
• CPC Class Codes: C12N 9/6481 Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12Y 304/23001 Pepsin A (3.4.23.1)

Abstract

Provided herein are compositions with enhanced protein specific activity, protein combinations and methods for the preparation thereof.

Claims

1. A composition comprising a recombinant pepsin polypeptide, wherein (a) the composition is free from animal-derived proteins, (b) the pepsin polypeptide is in a proteolytic ally inactive form which does not self-digest, (c) the composition has a pH of 5.4 or higher, and (d) the composition has a specific activity of from 500 FCC units/mg total protein to less than 30,000 FCC units/mg total protein;

wherein the composition is in powdered form.

2. The composition of claim 1, wherein the composition comprising the proteolytically inactive pepsin polypeptide form which does not self-digest is stable for at least 6 months at room temperature or for at least 6 months at 4°

C.

3. The composition of claim 2, wherein the composition has a moisture content of less than about 10%.

4. The composition of claim 1, wherein the recombinant pepsin polypeptide has a specific activity of from 500 FCC units/mg total protein to 20,000 FCC units/mg total protein at pH 2.

5. The composition of claim 1, wherein the composition has a pH of 6.0 or higher.

6. The composition of claim 1, wherein the powdered form can be solubilized in an aqueous medium to create a liquid composition having a concentration of the recombinant pepsin polypeptide that is at least 20 g per liter.

7. The composition of claim 6, wherein the proteolytically inactive pepsin polypeptide form which does not self-digest is stable in the liquid composition for at least 30 days at a temperature of 4°

C.

8. The composition of claim 1, wherein the recombinant pepsin polypeptide comprises an amino acid sequence of a sheep, pig, cow, human, zebu, yak, Central European red deer, or goat pepsin.

9. The composition of claim 1, wherein the recombinant pepsin polypeptide comprises SEQ ID NO:

10, or an amino acid sequence with at least 90% identity thereto.

10. The composition of claim 1, wherein the recombinant pepsin polypeptide is produced in a Pichia species, a yeast, a filamentous fungi, a Saccharomyces species, a bacterium, a Trichoderma species, or an Aspergillus species.

11. The composition of claim 1, wherein the composition comprises less than 5% of contaminating proteins.

12. The composition of claim 11, wherein the contaminating protein is a non-pepsin protein.

13. The powdered composition of claim 1, produced by a method comprising steps of:

a. providing a microorganism expressing a recombinant pepsinogen, wherein the expressed pepsinogen is secreted by the microorganism into a growth media;

b. harvesting the growth media and removing the microorganismal cells therefrom to obtain a liquid starting material;

c. lowering the pH of the liquid starting material to less than pH 4.0 to obtain an activated pepsin composition;

d. raising the activated pepsin composition to a pH of 5.4 or higher to obtain the composition comprising the recombinant pepsin polypeptide; and

e. isolating the recombinant pepsin polypeptide from protein and small molecules in the liquid starting material to obtain the powdered composition.

14. The powdered composition of claim 13, wherein the recombinant pepsinogen comprises any one of SEQ ID NO:

1 to SEQ ID NO;

9, or an amino acid sequence with at least 90% identity thereto.

22. A method for producing a consumable composition, the method comprising:

providing the high-activity stable pepsin powdered composition of claim 1; and

combining the high-activity stable pepsin powdered composition with a food or beverage ingredient,

thereby producing a consumable composition.

23. A food or beverage product comprising the high-activity stable pepsin powdered composition of claim 1 and a food or beverage ingredient.

15. A method for producing a high-activity stable pepsin powdered composition comprising steps of:

a. providing a microorganism expressing a recombinant pepsinogen, wherein the expressed pepsinogen is secreted by the microorganism into its growth media;

b. harvesting the growth media and removing the microorganismal cells therefrom to obtain a liquid starting material;

c. lowering the pH of the liquid starting material to less than pH 4.0 to obtain an activated pepsin composition;

d. raising the activated pepsin composition to a pH of 5.4 or higher to obtain a high-activity stable pepsin composition; and

e. isolating the stable pepsin polypeptide from protein and small molecules in the liquid starting material to obtain the high-activity stable pepsin powdered composition having a specific activity of less than 10,000 FCC units/mg total protein;

wherein step (e) is performed after steps (c) and (d).

16. The method of claim 15, wherein the high-activity stable pepsin composition comprises a pepsin polypeptide having an amino acid sequence of a sheep, pig, cow, human, zebu, yak, Central European red deer, or goat pepsin.

17. The method of claim 15, wherein the microorganism is a Pichia species, a yeast, a filamentous fungus, a Saccharomyces species, a bacterium, a Trichoderma species, or an Aspergillus species.

18. The method of claim 15, wherein the high-activity stable pepsin powdered composition comprises less than 5% of contaminating proteins.

19. The method of claim 18, wherein the contaminating protein is a non-pepsin protein.

20. The method of claim 15, wherein the recombinant pepsinogen comprises any one of SEQ ID NO:

1 to SEQ ID NO;

9, or an amino acid sequence with at least 90% identity thereto.

21. The method of claim 15, wherein the high-activity stable pepsin powdered composition comprises at least 500 FCC units/mg total protein.