COMPOSITIONS COMPRISING DIGESTIVE ENZYMES
Provided herein are compositions with enhanced protein content, compositions with functional proteins, protein combinations and methods for the preparation thereof.
1 . A method for producing a consumable composition comprising combining a high-activity stable pepsin powdered composition with one or more consumable ingredients 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 from 500 FCC units/mg total protein to 50,000 FCC units/mg total protein;
(f) combining the high-activity stable pepsin powdered composition with the one or more consumable ingredients to produce a consumable composition; and
wherein step (e) is performed after steps (c) and (d).
2 . The method of claim 1 , 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.
3 . The method of claim 2 , 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.
4 . The method of claim 2 , wherein the high-activity stable pepsin powdered composition comprises at least 5000 FCC units/mg total protein.
5 . The method of claim 1 , wherein the microorganism is a Pichia species, a yeast, a filamentous fungus, a Saccharomyces species, a bacterium, a Trichoderma species, or an Aspergillus species.
6 . The method of claim 1 , wherein the high-activity stable pepsin powdered composition comprises less than 5% of contaminating proteins.
7 . A recombinant ovalbumin (rOVA) composition comprising:
an rOVA comprising an N-linked glycan, wherein the N-linked glycan comprises 11 or fewer mannose units, wherein the rOVA composition is produced by:
(i) expressing a recombinant ovalbumin (rOVA) in a host cell, wherein the rOVA is secreted or expressed by the host cell into a liquid media;
(ii) harvesting the liquid media containing secreted or expressed rOVA;
(iii) purifying the secreted or expressed rOVA by separating the host cell from the liquid media;
(iv) adjusting the final pH of the rOVA to between about 3.5 and about 7.0 to generate the rOVA composition.
8 . The rOVA composition of claim 7 , wherein the N-linked glycan comprises 5-11 mannose units or 9-11 mannose units.
9 . The rOVA composition of claim 7 , wherein the mannose units comprise:
about 40% of Mannose 9, about 47% of Mannose 10, or about 13% of Mannose 11; or
about 40% of Mannose 9, about 47% of Mannose 10, and about 13% of Mannose 11.
10 . The rOVA composition of claim 7 , wherein the mannose units are linked to an N-acetyl glucosamine.
11 . The rOVA composition of claim 7 , wherein the N-linked glycan does not comprise a galactose unit or wherein a glycosylation pattern of the rOVA is devoid of N-linked galactose units.
12 . The rOVA composition of claim 7 , wherein the rOVA comprises:
a glycosylation, an acetylation, or a phosphorylation pattern different from native ovalbumin (nOVA); or
a glycosylation and a phosphorylation pattern different from nOVA.
13 . The rOVA composition of claim 7 , wherein the rOVA is: mono- or di-glycosylated; or is not phosphorylated.
14 . The rOVA composition of claim 7 , wherein the amino acid sequence of rOVA lacks an N-terminal methionine.
15 . The rOVA composition of claim 7 , wherein the rOVA composition provides an improved characteristic compared to a native ovalbumin (nOVA) composition comprising nOVA, wherein the improved characteristic is selected from: a foaming, a gelling, and a binding functional characteristic.
16 . The rOVA composition of claim 15 , wherein the improved characteristic is foam stability or foam capacity, wherein foam stability of the rOVA composition is:
greater than a foam stability of the nOVA composition;
greater than 100% of the foam stability of the nOVA composition;
greater than about 150% of the foam stability of the nOVA composition;
greater than about 200% of the foam stability of the nOVA composition;
greater than a foam capacity of the nOVA composition; or
greater than about 100% of the foam capacity of the nOVA composition.
17 . The rOVA composition of claim 7 , wherein the rOVA comprises an amino acid sequence having at least 70% sequence identity to a sequence selected from: SEQ ID NOs: 1-74.
18 . The rOVA composition of claim 7 , wherein the rOVA composition is dried or powdered.
19 . The rOVA composition of claim 7 , wherein the rOVA composition is soluble in water.
20 . A food product comprising the rOVA composition of claim 7 .
21 . A recombinant ovalbumin (rOVA) composition comprising:
an rOVA comprising an N-linked glycan, wherein the N-linked glycan comprises 11 or fewer mannose units.