MILK CLOTTING ASPARTIC PROTEASE ENZYME COMPOSITION

Abstract

A liquid or dried granulated milk clotting aspartic protease enzyme composition comprising added polypeptides/proteins. The polypeptides/proteins may be animal-derived (e.g. whey, lactalbumin, transferrin, casein, ovalbumin, gelatin, blood), vegetable-derived (soy, pea, corn, potato, hemp, rice, wheat, peanut, sun flower, rape seed) or algae proteins (e.g. spirulina). Addition of protein in several instances increases activity of the enzyme and simultaneously improves physical stability.

Claims

1-16. (canceled)

17. A method for making a liquid milk clotting aspartic protease enzyme composition, comprising: obtaining a liquid milk clotting aspartic protease enzyme sample having a milk clotting aspartic protease enzyme strength of from 25 IMCU/g to 30,000 IMCU/g, wherein milk clotting aspartic protease enzyme constitutes at least 70% by weight of the total amount of proteins present in the sample having a size bigger than 10 kDa as determined by SDS-PAGE; and adding to the sample an amount of a polypeptide formulation comprising polypeptides longer than 10 amino acid residues, wherein the polypeptides are not milk clotting aspartic protease enzymes and are not enzymes that degrade the milk clotting aspartic protease enzymes, wherein the amount of added polypeptide formulation results in a concentration of said polypeptides in the composition of from 0.01% to 10% (w/w) and is effective to obtain a liquid milk clotting aspartic protease enzyme composition having a milk clotting aspartic protease enzyme strength of from 26 IMCU/g to 30,100 IMCU/g of the composition, wherein the milk clotting aspartic protease enzyme strength of the composition is at least 1% higher than the milk clotting aspartic protease enzyme strength of the sample when measured after storage for one week at 5 C.

18. The method of claim 17, wherein the milk clotting aspartic protease enzyme comprises chymosin (EC 3.4.23.4).

19. The method of claim 17, wherein the milk clotting aspartic protease enzyme comprises Camelius dromedarius chymosin comprising the Camel_chymosin amino acid sequence shown in FIG. 5 or a variant thereof comprising an amino acid sequence having at least 90% sequence identity thereto.

20. The method of claim 17, wherein the milk clotting aspartic protease enzyme comprises bovine pepsin comprising the Cow_pepsin amino acid sequence shown in FIG. 5 or a variant thereof comprising an amino acid sequence having at least 90% sequence identity thereto.

21. The method of claim 17, wherein said polypeptides are proteins longer than 75 amino acid residues.

22. The method of claim 17, wherein said polypeptides comprise one or more selected from whey proteins, alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, bovine serum albumin, soy proteins, pea proteins, corn proteins, potato proteins, hemp proteins, rice proteins, spirulina proteins, wheat proteins, peanut proteins, sun flower proteins, rape seed proteins, blood proteins, and algae proteins.

23. The method of claim 22, wherein said polypeptides comprise one or more selected from whey proteins, alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, and bovine serum albumin.

24. The method of claim 17, wherein the milk clotting aspartic protease enzyme strength of the composition is at least 10% higher than the milk clotting aspartic protease enzyme strength of the sample when measured after storage for one week at 5 C.

25. The method of claim 17, wherein the amount of added polypeptide formulation results in a concentration of said polypeptides in the composition of from 0.5% to 4% (w/w) of the composition.

26. A liquid milk clotting aspartic protease enzyme composition obtained by a method according to claim 17.

27. The method of claim 17, further comprising drying and granulating the composition to obtain a dried granulated milk clotting aspartic protease enzyme composition.

28. A dried granulated milk clotting aspartic protease enzyme composition obtained by a method according to claim 27.

29. A liquid milk clotting aspartic protease enzyme composition, comprising: (a) milk clotting aspartic protease enzyme at a strength of from 25 IMCU/g to 30,000 IMCU/g of the composition; (b) polypeptides longer than 10 amino acid residues at a concentration from 0.01% to 10% (w/w) of the composition, wherein the polypeptides are not milk clotting aspartic protease enzymes and comprise one or more selected from whey proteins, alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, bovine serum albumin, soy proteins, pea proteins, corn proteins, potato proteins, hemp proteins, rice proteins, spirulina proteins, wheat proteins, peanut proteins, sun flower proteins, rape seed proteins, blood proteins, and algae proteins; and (c) a salt in a concentration of from 1 to 350 g/kg, wherein the pH of the composition is from 2 to 8.

30. A liquid milk clotting aspartic protease enzyme composition of claim 29, wherein the milk clotting aspartic protease enzyme comprises chymosin (EC 3.4.23.4).

31. A liquid milk clotting aspartic protease enzyme composition of claim 29, wherein: (i) said polypeptides comprise one or more selected from whey proteins, alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin and bovine serum albumin, (ii) the concentration of said polypeptides is from 0.5% to 4% (w/w) of the composition, (iii) the sum of the amounts of milk clotting aspartic protease enzyme and said polypeptides, determined by SDS-PAGE, is greater than 50% (w/w) of the total amount of proteins and/or polypeptides longer than 10 amino acid residues present in the composition; and (iv) the total weight of the composition is from 100 g to 3000 kg.

32. A method for making a food or feed product comprising adding an effective amount of a milk clotting aspartic protease enzyme composition of claim 29 to the food or feed ingredient(s).

33. A dried granulated milk clotting aspartic protease enzyme composition comprising: (a) milk clotting aspartic protease enzyme at a strength of from 25 IMCU/g to 30,000 IMCU/g of the composition; (b) polypeptides longer than 10 amino acid residues at a concentration from 0.01% to 10% (w/w) of the composition, wherein the polypeptides are not milk clotting aspartic protease enzymes and comprise one or more selected from whey proteins, alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, bovine serum albumin, soy proteins, pea proteins, corn proteins, potato proteins, hemp proteins, rice proteins, spirulina proteins, wheat proteins, peanut proteins, sun flower proteins, rape seed proteins, blood proteins, and algae proteins; and (c) a salt, wherein the pH of the composition when suspended in water is from 2 to 8.

34. A dried milk clotting aspartic protease enzyme composition of claim 33, wherein the milk clotting aspartic protease enzyme comprises chymosin (EC 3.4.23.4).

35. A dried milk clotting aspartic protease enzyme composition of claim 33, wherein: (i) said polypeptides comprise one or more selected from whey proteins, alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin and bovine serum albumin, (ii) the concentration of said polypeptides is from 0.5% to 4% (w/w) of the composition, (iii) the sum of the amounts of milk clotting aspartic protease enzyme and said polypeptides, determined by SDS-PAGE, is greater than 50% (w/w) of the total amount of proteins and/or polypeptides longer than 10 amino acid residues present in the composition; and (iv) the total weight of the composition is from 0.5 to 50 kg.

36. A method for making a food or feed product comprising adding an effective amount of a milk clotting aspartic protease enzyme composition of claim 34 to the food or feed ingredient(s).

Description

DRAWINGS

[0095] FIG. 1: In FIG. 1 herein is shown SDS-PAGE data for different samples of bovine chymosin (CHY-MAX, Chr. Hansen A/S) and mucorpepsin (Hannilase, Chr. Hansen A/S). For both CHY-MAX and Hannilase are Lane 2 (named Feed) unpurified samples essentially taken from the fermentation media and Lane 3 are purified samples.

[0096] FIG. 2: Shows some of the results of inversion experiments as discussed herein to determine herein relevant physical stability. See working Example herein for further details.

[0097] FIG. 3: Conformational stability of an enzyme is illustrated.

[0098] FIG. 4: An alignment of herein relevant different milk clotting chymosin sequences from different mammalian species (cow, buffalo, goat, sheep, camel and pig). All the sequences of FIG. 4 are public available.

[0099] FIG. 5: An alignment of herein relevant commercially available different milk clotting aspartic protease enzymes sequences from different mammalian or fungal species (camel chymosin, cow chymosin, cow pepsin, fungal mucor pepsin and fungal Endothia pepsin). All the sequences of FIG. 5 are public available.

DETAILED DESCRIPTION OF THE INVENTION

Milk Clotting Aspartic Protease Enzyme

[0100] The discussion of specific embodiments/examples of herein relevant milk clotting aspartic protease enzymes below is relevant for all the aspects of the invention as discussed herein.

[0101] In a preferred embodiment, the milk clotting aspartic protease enzyme is a milk-clotting enzyme selected from the group consisting of chymosin (EC 3.4.23.4), pepsin (EC 3.4.23.1) and mucorpepsin (EC 3.4.23.23).

[0102] As discussed in working Examples hereinthe herein relevant increase in the specific activity and strength were most significant for the bovine and camel chymosin compositions.

[0103] Accordingly, in a preferred embodiment the milk clotting aspartic protease enzyme is chymosin (EC 3.4.23.4).

[0104] A preferred milk clotting aspartic protease enzyme is Camelius dromedarius chymosin as described in e.g. WO02/36752A2 (Chr. Hansen). It may herein alternatively be termed camel chymosin and the publically known mature polypeptide amino acid sequence is shown in FIG. 5 herein.

[0105] As known in the artit is routine work for the skilled person to make variants (i.e. amino acid modifications) of an enzyme of interest without significantly changing the characteristics of the enzyme.

[0106] Accordingly, in a preferred embodiment the milk clotting aspartic protease enzyme is Camelius dromedarius chymosin comprising the polypeptide amino acid sequence shown in FIG. 5 herein (termed Camel_chymosin) or a variant of Camelius dromedarius chymosin, wherein the variant comprises a polypeptide sequence which has at least 90% (preferably at least 95%, more preferably at least 99%) sequence identity with the camel chymosin polypeptide amino acid sequence shown in FIG. 5 herein.

[0107] A preferred milk clotting aspartic protease enzyme is bovine chymosin. It may herein alternatively be termed cow chymosin and the publically known mature polypeptide amino acid sequence is shown in FIG. 5 herein.

[0108] Accordingly, in a preferred embodiment the milk clotting aspartic protease enzyme is bovine chymosin comprising the polypeptide amino acid sequence shown in FIG. 5 herein (termed Cow_chymosin) or a variant of bovine chymosin, wherein the variant comprises a polypeptide sequence which has at least 90% (preferably at least 95%, more preferably at least 99%) sequence identity with the bovine chymosin polypeptide amino acid sequence shown in FIG. 5 herein.

[0109] A preferred milk clotting aspartic protease enzyme is bovine pepsin. It may herein alternatively be termed cow pepsin and the publically known mature polypeptide amino acid sequence is shown in FIG. 5 herein.

[0110] Accordingly, in a preferred embodiment the milk clotting aspartic protease enzyme is bovine pepsin comprising the polypeptide amino acid sequence shown in FIG. 5 herein (termed Cow_pepsin) or a variant of bovine pepsin, wherein the variant comprises a polypeptide sequence which has at least 90% (preferably at least 95%, more preferably at least 99%) sequence identity with the bovine pepsin polypeptide amino acid sequence shown in FIG. 5 herein.

[0111] A preferred milk clotting aspartic protease enzyme is Mucor pepsin (see e.g. EP0805866B1 (Harboe et al, Chr. Hansen A/S, Denmark)). The publically known mature polypeptide amino acid sequence is shown in FIG. 5 herein.

[0112] Accordingly, in a preferred embodiment the milk clotting aspartic protease enzyme is Mucor pepsin comprising the polypeptide amino acid sequence shown in FIG. 5 herein (termed Mucor) or a variant of Mucor pepsin, wherein the variant comprises a polypeptide sequence which has at least 90% (preferably at least 95%, more preferably at least 99%) sequence identity with the Mucor pepsin polypeptide amino acid sequence shown in FIG. 5 herein.

[0113] A preferred milk clotting aspartic protease enzyme is Endothia pepsin. The publically known mature polypeptide amino acid sequence is shown in FIG. 5 herein.

[0114] Accordingly, in a preferred embodiment the milk clotting aspartic protease enzyme is Endothia pepsin comprising the polypeptide amino acid sequence shown in FIG. 5 herein (termed Endothia) or a variant of Endothia pepsin, wherein the variant comprises a polypeptide sequence which has at least 90% (preferably at least 95%, more preferably at least 99%) sequence identity with the Endothia pepsin polypeptide amino acid sequence shown in FIG. 5 herein.

Added Polypeptide/Proteins

[0115] As discussed abovestep (b) of the method of the first aspect reads: [0116] step (b): adding a suitable amount of a polypeptide formulation comprising polypeptides longer than 10 amino acids to the sample of step (a), wherein the polypeptides are not milk clotting aspartic protease enzymes and not enzymes that degrade the aspartic protease enzymes, to get a liquid milk clotting aspartic protease enzyme composition,

[0117] As discussed aboveitem (II) of the liquid milk clotting aspartic protease enzyme composition of the fifth aspect and item (II) of the dried granulated milk clotting aspartic protease enzyme composition of the sixth aspect reads: [0118] (II): not milk clotting aspartic protease enzyme polypeptides longer than 10 amino acids in a concentration from 0.01% to 10% (w/w) of the composition;

[0119] The discussion of specific embodiments/examples of herein relevant polypeptides longer than 10 amino acids below is relevant for all the aspects of the invention as discussed herein.

[0120] In the present context, it is evident that in step (b) of the method of the first aspect it is not preferred to add enzymes that degrade the aspartic protease enzymes and the term not enzymes that degrade the aspartic protease enzymes of step (b) should be understood in relation to this.

[0121] Preferably, the polypeptides longer than 10 amino acids are at least one polypeptide selected from the group of polypeptides consisting of: whey proteins, alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, bovine serum albumin, soy proteins, pea proteins, corn proteins, potato proteins, hemp proteins, rice proteins, spirulina proteins, wheat proteins, peanut proteins, sun flower proteins, rape seed proteins, blood proteins and algae proteins.

[0122] More preferably, the polypeptides longer than 10 amino acids are at least one polypeptide selected from the group of polypeptides consisting of: whey proteins, alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin and bovine serum albumin.

[0123] Within the group immediately above it is preferred that the polypeptides longer than 10 amino acids are at least one polypeptide selected from the group of polypeptides consisting of: whey proteins, alpha lactalbumin, beta-lactoglobulin, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin and bovine serum albumin.

[0124] In a preferred embodimentpolypeptides longer than 10 amino acids are polypeptides longer than 25 amino acids, more preferably polypeptides longer than 40 amino acids.

[0125] As discussed in working Examples hereinherein relevant positive results were obtained by addition of e.g. whey protein, ovalbumin and BSA, which herein all may be characterized as relatively large proteins.

[0126] As known in the artthe term peptide may be distinguished from the term protein on the basis of size, which as and as an arbitrary benchmark may be understood to be approximately 50 or fewer amino acids.

[0127] Said in other words, a polypeptide longer than 50 amino acids may normally in the art be understood to be a protein.

[0128] Accordingly, a polypeptide longer than 50 amino acids may herein alternatively be termed a protein.

[0129] In a preferred embodimentpolypeptides longer than 10 amino acids are proteins longer than 50 amino acids, more preferably proteins longer than 75 amino acids, even more preferably proteins longer than 150 amino acids.

[0130] It may even be preferred that polypeptides longer than 10 amino acids are proteins longer than 300 amino acids.

First AspectA Method for Making a Liquid Milk Clotting Aspartic Protease Enzyme Composition

[0131] As discussed abovethe first aspect of the invention relates to a method for making a liquid milk clotting aspartic protease enzyme composition, wherein the method comprises the steps of: [0132] (a): obtaining a purified liquid milk clotting aspartic protease enzyme sample comprising: [0133] (i): a strength of from 25 IMCU/g to 30,000 IMCU/g of the sample; and [0134] (ii): wherein at least 70% of the total amounts of proteins with a size bigger than 10 kDa, determined by SDS-PAGE, in the purified sample are milk clotting aspartic protease enzyme; and [0135] (b): adding a suitable amount of a polypeptide formulation comprising polypeptides longer than 10 amino acids to the sample of step (a), wherein the polypeptides are not milk clotting aspartic protease enzymes and not enzymes that degrade the aspartic protease enzymes, to get a liquid milk clotting aspartic protease enzyme composition, wherein the composition comprises: [0136] (I): milk clotting aspartic protease enzyme at a strength of from 26 IMCU/g to 30,100 IMCU/g of the composition; and [0137] (II): the in step (b) added not milk clotting aspartic protease enzyme polypeptides longer than 10 amino acids in a concentration from 0.01% to 10% (w/w) of the composition; and
wherein the strength (IMCU/g of the composition) in (I) is at least 1% higher than the strength in (i) (IMCU/g of the sample), measured after one week of storage at 5 C.

[0138] In a preferred embodiment, the strength (IMCU/g of the composition) in (I) is at least 3% higher than the strength in (i) (IMCU/g of the composition), measured after one week of storage at 5 C.; more preferably the strength (IMCU/g of the composition) in (I) is at least 7% higher than the strength in (i) (IMCU/g of the composition), measured after one week of storage at 5 C.; even more preferably the strength (IMCU/g of the composition) in (I) is at least 10% higher than the strength in (i) (IMCU/g of the composition), measured after one week of storage at 5 C.; and most preferably the strength (IMCU/g of the composition) in (I) is at least 15% higher than the strength in (i) (IMCU/g of the composition), measured after one week of storage at 5 C.

[0139] Preferred examples/embodiments of milk clotting aspartic protease enzymes are described above.

[0140] Preferred examples/embodiments of polypeptides longer than 10 amino acids are described above.

[0141] It is preferred that the enzyme strength in item (i) is a strength of from 100 IMCU/g of the sample to 10,000 IMCU/g of the sample, more preferably a strength of from 500 IMCU/g of the sample to 6000 IMCU/g of the sample.

[0142] It is preferred that the enzyme strength in item (I) is a strength of from 100 IMCU/g of the composition to 10,000 IMCU/g of the composition, more preferably a strength of from 500 IMCU/g of the composition to 6000 IMCU/g of the composition.

[0143] Preferably, the purified sample of step (a)(ii) is a sample, wherein at least 80% of the total amounts of proteins with a size bigger than 10 kDa, determined by SDS-PAGE, in the purified sample are milk clotting aspartic protease enzyme;

[0144] more preferably, wherein at least 90% of the total amounts of proteins with a size bigger than 10 kDa, determined by SDS-PAGE, in the purified sample are milk clotting aspartic protease enzyme;

[0145] even more preferably wherein at least 95% of the total amounts of proteins with a size bigger than 10 kDa, determined by SDS-PAGE, in the purified sample are milk clotting aspartic protease enzyme.

[0146] It may be preferred that at least 99% of the total amounts of proteins with a size bigger than 10 kDa, determined by SDS-PAGE, in the purified sample are milk clotting aspartic protease enzyme.

[0147] As known to the skilled personin the present context it is routine work for the skilled person to obtain purified liquid milk clotting aspartic protease enzyme sample as discussed hereinfor instance by use of suitable chromatography (e.g. column chromatography) isolation procedures. As such chromatography is well known to the skilled person are it is therefore not necessary to describe chromatography procedures as such in details herein.

[0148] For instance, WO02/36752A2 (Chr. Hansen) describes a recombinant method to produce Camelius dromedarius chymosin (Camel chymosin) using Aspergillus cells (preferably Aspergillus niger) as production host cells.

[0149] It is also known to use other cells as production host cellssuch as e.g. yeast cell, where an example is e.g. Kluyveromyces cells (for instance Kluyveromyces lactis).

[0150] Accordingly, it may be preferred that the purified sample of step (a)(ii) is a sample obtained from recombinant production of the milk clotting aspartic protease enzyme in fungal or yeast production host cells, such as e.g. Aspergillus cells or Kluyveromyces cells.

[0151] As known in the artMucorpepsin derived from Rhizomucor miehei may preferably be produced by use of Rhizomucor miehei as production host cell.

[0152] In relation to item (II)it is preferred that the in step (b) added not milk clotting aspartic protease enzyme polypeptides longer than 10 amino acids is in a concentration from 0.05% to 8% (w/w) of the composition; more preferably in a concentration from 0.1% to 7% (w/w) of the composition; even more preferably in a concentration from 0.25% to 5% (w/w) of the composition and most preferably in a concentration from 0.5% to 4% (w/w) of the composition (such as e.g. in a concentration from 1% to 3% (w/w) of the composition).

Fifth and/or Sixth AspectA Liquid and/or Dried Milk Clotting Aspartic Protease Enzyme Composition

[0153] As discussed abovethe fifth aspect of the invention relates to a liquid milk clotting aspartic protease enzyme composition comprising: [0154] (I): milk clotting aspartic protease enzyme at a strength of from 25 IMCU/g to 30,000 IMCU/g of the composition; [0155] (II): not milk clotting aspartic protease enzyme polypeptides longer than 10 amino acids in a concentration from 0.01% to 10% (w/w) of the composition; and [0156] (III): a salt in a concentration from 1 to 350 g/kg and wherein the pH of the composition is from 2 to 8; and [0157] (x): wherein the polypeptides longer than 10 amino acids of item (II) are at least one polypeptide selected from the group of polypeptides consisting of: whey proteins, alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, bovine serum albumin, soy proteins, pea proteins, corn proteins, potato proteins, hemp proteins, rice proteins, spirulina proteins, wheat proteins, peanut proteins, sun flower proteins, rape seed proteins, blood proteins and algae proteins.

[0158] As discussed abovethe sixth aspect of the invention relates to a dried granulated milk clotting aspartic protease enzyme composition comprising: [0159] (I): milk clotting aspartic protease enzyme at a strength of from 25 IMCU/g to 30,000 IMCU/g of the composition; [0160] (II): not milk clotting aspartic protease enzyme polypeptides longer than 10 amino acids in a concentration from 0.01% to 10% (w/w) of the composition; and [0161] (III): a salt and wherein the pH of the composition suspended in water is from 2 to 8; and [0162] (x): wherein the polypeptides longer than 10 amino acids of item (II) are at least one polypeptide selected from the group of polypeptides consisting of: whey proteins, alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, bovine serum albumin, soy proteins, pea proteins, corn proteins, potato proteins, hemp proteins, rice proteins, spirulina proteins, wheat proteins, peanut proteins, sun flower proteins, rape seed proteins, blood proteins and algae proteins.

[0163] For both the liquid and the dried compositionpreferred examples/embodiments of milk clotting aspartic protease enzymes are described above.

[0164] For both the liquid and the dried compositionpreferred examples/embodiments of polypeptides longer than 10 amino acids are described above.

[0165] For both the liquid and the dried compositionit is preferred that the enzyme strength in item (I) is a strength of from 100 IMCU/g of the composition to 10,000 IMCU/g of the composition, more preferably a strength of from 500 IMCU/g of the composition to 6000 IMCU/g of the composition.

[0166] For both the liquid and the dried compositionin relation to item (II), it is preferred the not milk clotting aspartic protease enzyme polypeptides longer than 10 amino acids is in a concentration from 0.05% to 8% (w/w) of the composition; more preferably in a concentration from 0.1% to 7% (w/w) of the composition; even more preferably in a concentration from 0.25% to 5% (w/w) of the composition and most preferably in a concentration from 0.5% to 4% (w/w) of the composition (such as e.g. in a concentration from 1% to 3% (w/w) of the composition).

[0167] For the liquid compositionthe salt in item (iii) is preferably in a concentration from 10 to 300 g/kg, more preferably is in a concentration from 25 to 250 g/kg.

[0168] As known to the skilled personfor the dried composition the salt concentration in item (iii) may be relatively highsuch as e.g. from 50% (w/w) to 99.9% (w/w) or such as e.g. from 80% (w/w) to 99% (w/w).

[0169] For both the liquid and the dried compositionit is preferred that the salt is an inorganic saltpreferably wherein the inorganic salt is selected from the group of NaCl, KCl, Na.sub.2SO.sub.4, (NH.sub.4).sub.2SO.sub.4, K.sub.2HPO.sub.4, KH.sub.2PO.sub.4, Na.sub.2HPO.sub.4 or NaH.sub.2PO.sub.4 or a combination thereof. Most preferably, the salt is NaCl.

[0170] Both the liquid and the dried composition may comprise further additives/compounds such as e.g. a preservative.

[0171] As known to the skilled personpreservative may generally be added in a concentration sufficient to prevent microbial growth during shelf life of the product.

[0172] Examples of preservatives may be e.g. weak organic acids such as formate, acetate, lactate, propionate, malate, benzoate, sorbate or fumarate. Parabens (alkyl esters of para-hydroxybenzoate) may also be used as preservative. Glycerol or propanediol has also been described as preservatives.

[0173] Both the liquid and the dried compositionit is preferred that the pH is from 3 to 7, more preferably that the pH is from 4 to 6.5 and even more preferably that the pH is from 5 to 6.

[0174] Preferably, the liquid composition is an aqueous composition, for instance an aqueous solution. As used herein an aqueous composition or aqueous solution encompasses any composition or solution comprising water, for instance at least 20 wt % of water, for instance at least 40 wt % of water.

[0175] It may be preferred that the liquid composition as described herein has a total weight of from 10 g to 10,000 kg, such as e.g. from 100 g to 3000 kg.

[0176] It may be preferred that the dried granulated composition as described herein has a total weight of from 0.25 g to 200 kg, such as e.g. from 0.5 g to 50 kg.

[0177] It is preferred that the composition is a liquid milk clotting aspartic protease enzyme composition as described herein.

[0178] For both the liquid and the dried compositiona preferred embodiment is: [0179] (y): wherein the sum of the amounts of aspartic protease enzyme of item (I) and polypeptides longer than 10 amino acids of item (II), determined by SDS-PAGE, are higher than 50% (w/w) of the total amount of proteins and/or polypeptides longer than 10 amino acids in the composition; more preferably [0180] (y): wherein the sum of the amounts of aspartic protease enzyme of item (I) and polypeptides longer than 10 amino acids of item (II), determined by SDS-PAGE, are higher than 70% (w/w) of the total amount of proteins and/or polypeptides longer than 10 amino acids in the composition; even more preferably [0181] (y): wherein the sum of the amounts of aspartic protease enzyme of item (I) and polypeptides longer than 10 amino acids of item (II), determined by SDS-PAGE, are higher than 80% (w/w) of the total amount of proteins and/or polypeptides longer than 10 amino acids in the composition; and most preferably [0182] (y): wherein the sum of the amounts of aspartic protease enzyme of item (I) and polypeptides longer than 10 amino acids of item (II), determined by SDS-PAGE, are higher than 90% (w/w) of the total amount of proteins and/or polypeptides longer than 10 amino acids in the composition.

[0183] In the present contextthe skilled person will know or may routinely determine (e.g. based on specific relevant amino acid sequences) the origin of the polypeptides longer than 10 amino acids of item (x).

[0184] Accordingly, the skilled person may therefore also routine determine (e.g. via SDS-PAGE) if item (y) is fulfilled in a herein relevant composition of interest.

Seventh Aspect and Eight Aspect

[0185] As discussed abovethe seventh aspect of the invention relates to a liquid milk clotting aspartic protease enzyme composition comprising milk clotting aspartic protease enzyme at a strength of from 1600 IMCU/g to 30,000 IMCU/g of the composition and a salt in a concentration from 1 to 350 g/kg and wherein the pH of the composition is from 2 to 8.

[0186] An eight aspect of the invention relates to a dried granulated milk clotting aspartic protease enzyme composition comprising milk clotting aspartic protease enzyme at a strength of from 1600 IMCU/g to 30,000 IMCU/g of the composition and a salt and wherein the pH of the composition suspended in water is from 2 to 8.

[0187] For both the liquid and the dried compositionpreferably, the strength is a strength of from 2000 IMCU/g to 15,000 IMCU/g of the composition, such as from 3000 IMCU/g to 10,000 IMCU/g of the composition or such as from 4000 IMCU/g to 8000 IMCU/g of the composition.

Ninth AspectA Method for a Method for Making a Food or Feed Product

[0188] As discussed abovea milk clotting aspartic protease enzyme composition as described herein may be used according to the arte.g. to make a milk based product of interest (such as e.g. a cheese product).

[0189] As discussed abovethe ninth aspect of the invention relates to a method for making a food or feed product comprising adding an effective amount of a milk clotting aspartic protease enzyme composition of any of fifth to eight aspect or any herein relevant embodiments thereof to the food or feed ingredient(s) and carrying out further manufacturing steps to obtain the food or feed product.

[0190] Preferably, the food or feed product is a milk based product and wherein the method comprises adding an effective amount of the isolated chymosin polypeptide variant as described herein to milk and carrying our further manufacturing steps to obtain the milk based product.

[0191] The milk may e.g. be sheep milk, goat milk, buffalo milk, yak milk, lama milk, camel milk or cow milk.

[0192] The milk based product may e.g. be a fermented milk product, a quark or a cheese.

[0193] It may be preferred that the method for making a food or feed product of the fourth aspect or herein relevant embodiments thereof is a method, wherein a milk clotting aspartic protease enzyme composition first have been stored according to the method for storage of a milk clotting aspartic protease enzyme of the third aspect and thereafter added to the food or feed ingredient(s) according to the method for making a food or feed product of the fourth aspect.

EXAMPLES

Example 1

Determination of Specific Milk-Clotting Activity

4.1 Determination of Clotting Activity

[0194] Milk clotting activity was determined using the REMCAT method, which is the standard method developed by the International Dairy Federation (IDF method).

[0195] Milk clotting activity is determined from the time needed for a visible flocculation of a standard milk substrate prepared from a low-heat, low fat milk powder with a calcium chloride solution of 0.5 g per litre (pH6.5). The clotting time of a milk-clotting enzyme sample is compared to that of a reference standard having known milk-clotting activity and having the same enzyme composition by IDF Standard 110B as the sample. Samples and reference standards were measured under identical chemical and physical conditions. Variant samples were adjusted to approximately 3 IMCU/ml using an 84 mM acetic acid pH 5.5 buffer. Hereafter, 200 l enzyme was added to 10 ml preheated milk (32 C.) in a glass test tube placed in a water bath, capable of maintaining a constant temperature of 32 C.1 C. under constant stirring.

[0196] The total milk-clotting activity (strength) of a milk-clotting enzyme is calculated in International Milk-Clotting Units (IMCU) per ml relative to a standard having the same enzyme composition as the sample according to the formula:

[00001]$\mathrm{Strength}.Math..Math.\mathrm{in}.Math..Math.\mathrm{IMCU}.Math.\text{/}.Math.\mathrm{ml}=\frac{\mathrm{Sstandard}\mathrm{Tstandard}\mathrm{Dsample}}{\mathrm{Dstandard}\mathrm{Tsample}}$ [0197] Sstandard: The milk-clotting activity of the international reference standard for rennet. [0198] Tstandard: Clotting time in seconds obtained for the standard dilution. [0199] Dsample: Dilution factor for the sample [0200] Dstandard: Dilution factor for the standard [0201] Tsample: Clotting time in seconds obtained for the diluted rennet sample from addition of enzyme to time of flocculation

4.2 Determination of Total Protein Content

[0202] Total protein content was determined using the Pierce BCA Protein Assay Kit from Thermo Scientific following the instructions of the providers.

4.3 Calculation of Specific Clotting Activity

[0203] Specific clotting activity (IMCU/mg total protein) was determined by dividing the clotting activity (IMCU/ml) by the total protein content (mg total protein per ml).

Example 2

Enzyme Preparations

[0204] Bovine chymosin (CHY-MAX, Chr. Hansen A/S) or camel chymosin (CHY-MAX M, Chr. Hansen A/S) were recombinantly expressed in Aspergillus niger (roughly as described in WO02/36752A2). The enzymes were purified by chromatography technology. Mucorpepsin (Hannilase, Chr. Hansen A/S) derived from Rhizomucor miehei was produced by use of Rhizomucor miehei as production host cell and purified by chromatography technology. For all the purified enzyme samplesat least 90% of the total amounts of proteins with a size bigger than 10 kDa, determined by SDS-PAGE, in the purified sample were the relevant milk clotting aspartic protease enzymes.

[0205] All enzyme samples were prepared by mixing an exact volume for a stock solution of the enzyme with a solution of the additive and adding buffer to a final volume. In this manner the concentration of enzyme protein is kept constant for all prepared samples. Buffer composition was: 0.25 M sodium acetate, 20 mM sodium phosphate, 2.0 M sodium chloride, pH 5.7, 5 mM methionine, and 35 mM sodium benzoate. The strength was from 200 to 1200 IMCU/ml. The composition was added different polypeptides such as Hammersten casein, WPC80, Lacprodan Alpha 10, Lacprodan Alpha 20, etc.

[0206] WPC80 is a commercial preparation of dried whey protein concentrate with 80% protein. Lacprodan Alpha 10 and Lacprodan Alpha 20 are commercial preparations of whey protein isolate containing 43% and 60% alpha lactalbumin of total protein content, respectively.

Example 3

Physical Stability of Camel Chymosin

[0207] Liquid formulations of industrial enzymes are subjected to physical forces from unit operations such as pumping, stirring and filtration over membranes. During transportation of partly filled containers sloshing around of liquid formulation may also contribute to this. Shear stress and increased exposure of enzyme to the water-air interface may induce denaturation and concomitant loss of enzyme activity.

[0208] Physical stability of an enzyme or protein sample can be tested by repeatable shaking a sample of the enzyme in a test tube having high head space to sample volume ratio. The stability of different aspartic proteases towards shaking was investigated by inverting a 2 ml sample filled in a 10 ml tube in a rotary device for 1 hour (see FIG. 2 herein). For each solution, relative milk clotting activity was measured after 1 hour of vertical inversion (at room temperature) and compared to a non-inverted control having the exact same composition. Results were expressed as retained activity which is obtained by diving activity of the inverted sample with the activity of the non-inverted control sample.

Results:

[0209] Vertical inversion for 1 hr of a sample of camel chymosin results in an activity loss of more than 30% cf. Table 1 (No addition). Addition of PEG8000 to a concentration of 0.015% was found to protect camel chymosin and resulting in no loss of activity upon vertical inversion. When the sample of camel chymosin contained Hammersten casein, Alpha 10, Alpha 20 or WPC 80 at a concentration of either 0.5% w/v or 1.0% w/v practically no loss upon vertical inversion was seen.

[0210] The protecting effect of Alpha 20 and WPC 80 was found to decrease gradually when their concentration was decreased below 0.1%. A formulation of camel chymosin with either acid casein hydrolysate or whey permeate did not increase stability of the enzyme as the loss in activity upon vertical inversion was the same as for the untreated control sample (Table 3).

[0211] Conclusion: The results show that certain proteins added to a preparation of camel chymosin can increase the physical stability of the enzyme.

TABLE-US-00001 TABLE 1 Retained activity of camel chymosin samples subjected to vertical inversion for 1 hr (Internal ref number: EXP-13-AD2681). Compound Retained activity No addition 67% (3%) PEG (0.015%) 99% (0%) Glycerol (6%) 67% (4%) 0.5% Hammersten casein 97% (0%) 1% Hammersten casein 97% (1%) 0.5% Casein hydrolysate (Merck) 67% (4%) 1% Casein hydrolysate (Merck) 74% (5%) 0.5% Alpha 10 (Prve 1) 98% (0%) 1% Alpha 10 (Prve 1) 98% (0%) 0.5% Alpha 20 (Prve 2) 98% (0%) 1% Alpha 20 98% (0%) 0.5% Permeat 64% (17%) 1% Permeat 69% (1%) 0.1% WPC 80 100% (0%) 1% WPC 80 99% (0%)

Example 4

Specific Activity of Camel Chymosin

Results:

[0212] The inversion experiments were designed so the exact same amount of enzyme protein was added in each experiment and all formulations were made up to the same volume. If composition of the formulation did not influence enzymatic activity, one would expect to find the same enzymatic activity of all control samples, i.e. samples not inverted. However, this was not the case. Samples containing PEG8000 were 4-5% higher in activity in good accordance with recently submitted patent application IN5103DK00. Samples containing Hammerstein casein, Alpha 20 or WPC 80 had 13-18% higher activity compared to the sample without additives (no addition) even though the same amount of enzyme protein was applied (Table 2). This shows that the presence milk proteins in the formulation of camel chymosin increase the specific activity of the enzyme. The same conclusion is made from Table 3 which shows the activity of compositions containing Alpha 20 and WPC80 at five different concentrations. Table 5 show activity of the composition, activity index with no addition=100%, and the retained activity of a sample subjected to vertical inversion for 1 hr.

[0213] Addition of polypeptides to a composition of camel chymosin has two effects on the enzyme: Increase in specific activity and an increased physical stability of the enzyme. Both properties correlate well with the dosage of polypeptide as seen from Table 3. In Table 3 it is found that when the concentration of Alpha 20 is decreased from 0.5 to 0.01%, the enzyme activity drops from index 115 to index 102 which is almost the same level as the untreated control. At a concentration of 0.01% Alpha 20 the physical stability (retained activity) of the enzyme is the same as in the control experiment (no addition).

TABLE-US-00002 TABLE 2 Activity of CHY-MAX M added different polypeptides. Activity index relative to untreated control (no addition) is shown in percentage (Internal ref number: EXP-13-AD2681). Compound Activity (IMCU/ml) No addition 1124 (13.3) 100% PEG (0.015%) 1163 (6.8) 104% Glycerol (6%) 1069 (12.0) 95% 0.5% Hammersten casein 1251 (12.3) 111% 1% Hammersten casein 1275 (25.0) 113% 0.5% Casein hydrolysate (Merck) 1112 (12.3) 99% 1% Casein hydrolysate (Merck) 1102 (19.8) 98% 0.5% Alpha 10 (Prve 1) 1282 (8.6) 114% 1% Alpha 10 (Prve 1) 1305 (9.9) 116% 0.5% Alpha 20 (Prve 2) 1287 (5.0) 115% 1% Alpha 20 (Prve 2) 1323 (1.2) 118% 0.5% Permeat (Prve 3) 1147 (15.6) 102% 1% Permeat (Prve 3) 1127 (32.2) 100% 0.1% WPC 80 (Prve 4) 1248 (15.3) 111% 1% WPC 80 (Prve 4) 1287 (2.8) 115%

TABLE-US-00003 TABLE 3 Activity of CHY-MAX M added different polypeptides. Activity index relative to untreated control (no addition) is shown in percentage. Column to the right show retained activity of camel chymosin samples subjected to vertical inversion for 1 hr (Internal ref number: EXP-13-AD2681). Compound Activity (IMCU/ml) Retained activity No addition 1102 (19.6) 100% 76% (4%) PEG, 0.015% 1153 (12.4) 105% 101% (2%) Alpha 20, 0.5% 1269 (13.0) 115% 100% (1%) Alpha 20, 0.25% 1224 (8.5) 111% 100% (1%) Alpha 20, 0.1 1162 (1.2) 105% 100% (0%) Alpha 20, 0.05% 1134 (7.2) 103% 97% (0%) Alpha 20, 0.01% 1129 (1.0) 102% 79% (0%) WPC 80, 0.5% 1235 (13.9) 112% 101% (1%) WPC 80, 0.25% 1227 (10.2) 111% 98% (0%) WPC 80, 0.1% 1155 (14.3) 105% 100% (1%) WPC 80, 0.05% 1141 (7.3) 104% 95% (2%) WPC 80, 0.01% 1112 (5.4) 101% 80% (1%)

[0214] Conclusion: Addition of polypeptides to a composition of camel chymosin has two effects on the enzyme: Increase in specific activity and an increased physical stability of the enzyme. Both properties correlate well with the dosage of polypeptide.

Example 5

Specific Activity of Camel Chymosin, Bovine Chymosin, Mucor Pepsin L and Mucor Pepsin XL

[0215]

TABLE-US-00004 TABLE 4 Activity of milk clotting enzymes added different polypeptides (Internal ref number: EXP-13-AD2690). Standard deviation in absolute numbers is in parenthesis. Compound CHY-MAX M CHY-MAX Hannilase L Hannilase XP Control 1066 (18.7) 100% 1117 (11.7) 100% 985 (4.0) 100% 1041 (2.3) 100% Alpha 20, 1108 (6.2) 104% 1143 (2.7) 102% 983 (9.4) 99% 1035 (7.1) 100% 0.1% Alpha 20, 1192 (20.1) 111% 1173 (3.3) 105% 994 (7.6) 100% 1044 (2.3) 100% 0.5% Alpha 20, 1225 (6.8) 116% 1191 (3.0) 107% 1,007 (11.0) 101% 1057 (5.1) 102% 1.0% WPC80 0.1% 1086 (11.4) 103% 1131 (9.1) 101% 980 (10.2) 101% 1030 (13.5) 100% WPC80 0.5% 1156 (11.0) 110% 1151 (7.2) 102% 995 (9.8) 101% 1034 (2.5) 99% WPC80 1.0% 1196 (20.5) 114% 1170 (15.6) 104% 997 (3.7) 102% 1047 (9.7) 101%

[0216] Conclusion: The results in table 2-4 show that addition of polypeptides to a formulation of aspartic proteases increase the specific activity.

Example 6

[0217] The table below show results from tests performed similar to Example 3 abovebut using different protein formulations. All proteins shown in the table were added to a final content of 1% w/w and with gliadin as only exception gave clear solutions. Physical stability was tested according to example 3. Samples were stored for 1 year at 5 C. and 37 C., respectively, and stability was tested during storage period. The column End of storage shows remaining activity after 1 yearthe number was calculated by fitting a single exponential function to all data points.

[0218] As known in the artthe term peptone refers to proteins digested by proteolysis.

[0219] As known in the artthe term tryptone refers to proteins digested by the protease trypsin.

TABLE-US-00005 TABLE 5 Activity of CHY-MAX M added different polypeptides at 1% w/w. Activity index relative to untreated control (no addition) is shown in percentage. Column Retained Activity show retained activity of camel chymosin samples subjected to vertical inversion for 1 hr. Column End of Storage show activity after storage at 5 C. and 37 C. Standard deviation is shown in parenthesis. (Internal ref number: EXP-14-AE8307) Activity Retained End of storage Compound IMCU/ml/Index activity (5 C./37 C.) 1 Control (no addition) 389 (3.8) 100% 36% (2.4) 101%/14% 2 18332 Peptone (vegetable) 400 (11.1) 106% 95% (3.8) 91%/13% 3 51841 Peptone (vegetable) acid 418 (1.5) 109% 99% (4.6) 94%/13% hydrolysate 4 19942 Peptone (vegetable), no 1 422 (5.5) 108% 97% (0.6) 93%/15% 5 61854 Peptone (vegetable), no 2 423 (3.7) 109% 99% (1.4) 93%/13% 6 92976 Peptone special (vegetable) 423 (1.8) 110% 93% (7.1) 93%/13% 7 29185 Proteose Peptone (vegetable) 428 (3.0) 110% 97% (0.6) 94%/12% 8 16922 Tryptone (vegetable) 431 (2.9) 111% 96% (0.6) 93%/13% 9 12331 Tryptose (vegetable) 428 (2.0) 111% 94% (5).sup. 96%/15% 10 05138 Vegetable Extract 412 (9.8) 109% 98% (2.3) 100%/14% 11 04316 Vegetable Extract no 1 414 (3.1) 108% 97% (5.1) 97%/15% 12 49869 Vegetable Extract no 2 409 (2.3) 106% 99% (3.5) 97%/15% 13 07436 Vegetable hydrolysate no 2 411 (5.8) 107% 101% (2.5) 98%/14% 14 67381 Vegetable Infusion powder 403 (5.9) 103% 99% (2.9) 102%/13% 15 95757 Vegetable Special Infusion 411 (1.6) 106% 98% (6.4) 99%/18% powder 16 83059 Peptone from potatoes 403 (1.9) 104% 99% (1.5) 100%/13% 17 93491 Peptone from broad bean 425 (10.2) 108% 99% (1.4) 96%/12% 18 93492 Peptone from wheat 427 (1.2) 110% 100% (2.2) 100%/13% 19 90765 Peptone from soybean, 408 (11.2) 108% 100% (2.3) 102%/18% enzymatic digest 20 70178 Peptone from soybean, 401 (0.3) 104% 92% (2.3) 98%/14% enzymatic digest 21 S1674 Soy protein acid hydrolysate 432 (13.7) 109% 88% (1.3) 106%/18% 22 87972 Peptone from soybean, 400 (7.0) 102% 91% (0.6) 106%/21% enzymatic digest 23 96174 Peptone from pea 404 (2.9) 104% 98% (1.3) 98%/10% 25 49760 GLUTEN HYDROLYSATE F. 432 (5.1) 111% 95% (2).sup. 107%/16% MAIZE 26 Ovalbumin 464 (4.9) 121% 99% (1.1) 97%/14% 27 Gelatin 475 (6.0) 124% 99% (1.7) 101%/16% 28 Bovine serum albumin 487 (8.1) 128% 100% (2.6) 104%/15% 29 PEG8000 (0.015%) 443 (1.8) 115% 98% (0.1) 104%/15%

Example 7

[0220] Extracts of plant proteins were prepared by suspending 2 g sample in 40 ml brine consisting of: 12% NaCl, 20 g/L NaAc anhydrous, 2.5 g/L NaH2PO4 anhydrous, and 10 g/L Na-benzoate in water, pH 5.4-5.8. After mixing for 2 hours on rotating device the suspension was centrifuged and the supernatant pH adjusted to 5.4-5.8 and filtered through a 0.45 m syringe filter. The extracts were used for preparing formulations of CHY-MAX M by mixing with an exact measured and equal volume of a stock solution the enzyme to give samples having same concentration of enzyme proteins. In this example, extracts of 27 different plant proteins were tested in three groups with each group prepared on different days.

[0221] The activity was measured one day after sample preparation; the column titled activity shown activity in IMCU/ml and relative to untreated control (no addition). Physical stability was tested according to example 3 with the only difference that in present example a different rotary device was used for vertical inversion of the samples (Multi RS-60 from Biosan at 32 rpm for 1 hour). The change in rotary device may have resulted in increased physical stress of the samples since retained activity of untreated sample was only 10% compared to ca. 70% in preceding examples. Samples were stored for 1 year at 5 C. and 37 C., respectively, and stability was tested during storage period. The column End of storage shows remaining activity after 1 yearthe number was calculated by fitting a single exponential function to all data points.

TABLE-US-00006 TABLE 6 Activity of CHY-MAX M added different polypeptides. Activity index relative to untreated control (no addition) is shown in percentage. Column Retained Activity show retained activity of camel chymosin samples subjected to vertical inversion for 1 hr. Column End of Storage show activity after storage at 5 C. and 37 C. Standard deviation is shown in parenthesis. Internal ref number: Group 1/EXP-14-AF3604. Activity Retained End of storage Compound IMCU/ml/Index activity (5 C./37 C.) No addition (only Brine) .sup.405 (6.8) 100% 10% (1.4) 96%/22% 0.015% PEG 8000 481.9 (6.5) 119% 99% (3.1) 95%/21% 1 Nutralys F 85 F 450.4 (6).sup. 111% 96% (1) 87%/17% (Roquette) 2 Nutralys F85G 421.6 (4.6) 104% 102% (0.5) 91%/20% (Roquette) 3 Nutralys F 85 M 419.9 (13.8) 104% 102% (0.9) 89%/10% (Roquette) 4 Nutralys S 85 F 415.2 (6.2) 103% 102% (0.2) 87%/19% (Roquette) 5 Nutralys W 461.3 (11.3) 114% 100% (0.4) 87%/14% (Roquette) 6 Solulys 048E 399.5 (12.4) 99% 103% (1).sup. 94%/8% (Roquette) 7 Solulys 095E 412.3 (2.6) 102% 100% (1.8) 91%/9% (Roquette) 8 Tubermine Fv Proteine 403.4 (5.7) 100% 96% (0) 94%/15% De Pomme Des Terre (Roquette) 9 Tubermine GP Proteine .sup.411 (3.3) 101% 90% (0.7) 95%/17% De Pomme Des Terre (Roquette)

TABLE-US-00007 TABLE 7 Activity of CHY-MAXM added different polypeptides. Activity index relative to untreated control (no addition) is shown in percentage. Column Retained Activity show retained activity of camel chymosin samples subjected to vertical inversion for 1 hr. Column End of Storage show activity after storage at 5 C. and 37 C. Standard deviation is shown in parenthesis. Internal ref number: Group 2/EXP-14-AF3604. Activity Retained End of storage Compound IMCU/ml/Index activity (5 C./37 C.) No addition (only Brine) 395.9 (10.8) 100% 7% (1.25) 93%/24% No addition (only Brine) .sup.385 (3.7) 97% 7.5% (0.49) No addition (only Brine) 396.3 (4.9) 100% 6.3% (0.54) 0.015% PEG 8000 457.8 (5.6) 116% 98.9% (1.13) 99%/24% 10 Alburex E 361 G 511.1 (1).sup. 129% 92.7% (0.84) 95%/21% (Roquette) 11 Gluten De Ble Supra 423.2 (2.2) 107% .sup.90% (2.83) 92%/17% Vital (Roquette) 12 Lysamine GP Proteine 391.4 (13.6) 99% 77.3% (0.88) 95%/14% De Pois (Roquette) 13 Corn Gluten 359.1 (18.7) 91% 97.3% (0.28) 103%/22% (Roquette) 14 rteprotein 440.4 (3.8) 111% 98.3% (1.36) 86%/19% (Naturdrogeriet A/S) 15 Sojaprotein .sup.427 (1.1) 108% 106.8% (6.59) 88%/17% (Naturdrogeriet A/S) 16 Vegan Ris protein 352.4 (4.3) 89% 96.5% (2.22) 114%/28% (Naturdrogeriet A/S) 17 Sojaprotein 437 (6) 110% 94.2% (2.41) 89%/18% Ketolyse A/S 18 Hamp protein complex 422.7 (3.2) 107% 98.2% (2.66) 100%/20% kologisk (Nybogaard A/S)

TABLE-US-00008 TABLE 8 Activity of CHY-MAX M added different polypeptides. Activity index relative to untreated control (no addition) is shown in percentage. Column Retained Activity show retained activity of camel chymosin samples subjected to vertical inversion for 1 hr. Column End of Storage show activity after storage at 5 C. and 37 C. Standard deviation is shown in parenthesis. Internal ref number: Group 3/EXP-14-AF3604. Activity Retained End of storage Compound IMCU/ml/Index activity (5 C./37 C.) No addition (only Brine) 419.5 (1.1) 100% 8.8% (2.38) 97%/28% 0.015% PEG 8000 478.1 (2.9) 114% 102.8% (1.12) 100%/29% 19 Spirulina pulver kologisk 484.8 (9.4) 116% .sup.97% (0.17) 57%/1% (Dinsundhed.Net Aps) 20 Plant force rice protein 413.4 (5).sup. 99% 87.5% (1.45) 96%/17% (Third Wave Nutrition) 21 Superfruict Hemp protein 447.8 (4.1) 107% 97.7% (1.81) 97%/20% (Superfruit Scandinavia AB) 22 Hampe protein kologisk 421.2 (1.3) 100% 101.2% (2.12) 98%/23% (Nybogaard A/S) 23 Plant force Rice protein 412.7 (9.8) 98% 84.1% (5.77) 99%/18% (Third Wave Nutrition) 24 Nutralys T 65 M 454.5 (8.5) 108% 100.1% (0.34) 88%/14% (Roquette) 25 LAB 4460 Nutralys PEA 451.9 (13.1) 108% 97.2% (0.1) 90%/19% XF EXP (Roquette) 26 LAB 4462 Nutralys PEA 436.8 (10.5) 104% 99.9% (0.45) 93%/16% BF EXP (Roquette) 27 Gluten De MAIS 58E 402.9 (0.5) 96% 65.5% (1.33) 97%/20% (Roquette)

REFERENCES

[0222] 1: EP2333056A1 (DSM, date of filing Dec. 4, 2007) [0223] 2: WO2012/127005A1 (DSM) [0224] 3: DE1492060A1 (Nordmark-Werke GmbH, published in 1969)