Patent Description:
Enzymatic coagulation of milk by milk-clotting enzymes, such as chymosin and pepsin, is one of the most important processes in the manufacture of cheeses. Enzymatic milk coagulation is a two-phase process: a first phase where a proteolytic enzyme, chymosin or pepsin, attacks κ-casein, resulting in a metastable state of the casein micelle structure and a second phase, where the milk subsequently coagulates and forms a coagulum.

Chymosin (EC <NUM>. <NUM>) and pepsin (EC <NUM>. <NUM>), the milk clotting enzymes of the mammalian stomach, are aspartic proteases belonging to a broad class of peptidases.

Mucorpepsin (EC <NUM>. <NUM>) is a milk clotting enzyme derived from the fungus Rhizomucor miehei.

Commercial relevant milk-clotting enzyme products are often liquid compositions and in the art is described numerous different ways to try to stabilize the milk-clotting enzyme in the product - e.g. to improve storage stability or specific activity of the enzyme.

For instance, <CIT>describes that formate, acetate, lactate, propionate, malate, fumarate or propanediol may increase stability of aspartic protease enzyme in a liquid composition/product.

<CIT>) describes a stable liquid chymosin composition comprising inorganic salt in a concentration of <NUM>-<NUM>/kg and a preservative such as formate, acetate, lactate, propionate, malate, benzoate, sorbate or fumarate, glycol (ethanediol), propylene glycol (propanediol), glycerol, erythritol, xylitol, mannitol, sorbitol, inositol or galactitol. The highest strength of the described chymosin compositions is <NUM> IMCU/ml (see e.g. page <NUM>, lines <NUM>-<NUM>).

Polyethylene glycol (PEG) is a polymer of ethylene oxide - it may alternatively be termed polyoxyethylene (POE). PEG is commercially available over a wide range of molecular weights such as from <NUM>/mol to <NUM>,<NUM>,<NUM>/mol.

<CIT>) discloses a method for making a pepsin composition by adding Polyethylene glycol (PEG) with a molecular weight of <NUM>-<NUM> at a concentration of <NUM>-<NUM> wt% (corresponds to <NUM> to <NUM> ppm).

A problem to be solved by the present invention is to provide a novel milk clotting aspartic protease enzyme (e.g. chymosin) composition, wherein the aspartic protease has increased physical stability and possibly specific activity.

The solution is based on that the present inventors have identified that by adding suitable polypeptide/protein formulations as set forth in claim <NUM> to different aspartic protease enzymes, namely chymosin from Camelius dromedarius, one significantly improves the physical stability and possibly the specific activity of the enzyme compositions.

Liquid formulations of industrial enzymes are subjected to physical forces (such as shaking) during e.g. transportation and physical stability of an enzyme composition can be tested by repeatable shaking (e.g. via inversion) a sample in a test tube having high head space to sample volume ratio.

As discussed in working Examples herein - to camel chymosin (CHY-MAX® M, Chr. Hansen A/S) were added numerous different polypeptide/protein formulations and a number of these polypeptide/protein formulations significantly increased the physical stability of camel chymosin.

As discussed in working Examples herein - to bovine chymosin (CHY-MAX®, Chr. Hansen A/S), camel chymosin (CHY-MAX® M, Chr. Hansen A/S) and mucorpepsin (Hannilase®, Chr. Hansen A/S) were added numerous different polypeptide/protein formulations and a number of these polypeptide/protein formulations significantly increased the specific activity of the enzyme compositions.

The increase in the specific activity was most significant for the bovine and camel chymosins.

As understood by the skilled person in the present context - specific activity of a milk clotting aspartic protease enzyme composition relates to activity/IMCU per total amount of milk clotting aspartic protease enzyme protein in the composition.

As described in working Examples herein - addition of e.g. whey protein formulations to a purified chymosin sample gave an enzyme composition with significant higher IMCU/ml strength - i.e. a composition with higher specific activity.

For instance, addition of <NUM>,<NUM>% (w/w) of whey protein concentrate formulation WPC <NUM> gave around <NUM>% increase of the strength in the camel chymosin composition as such. Without being limited to theory - it was a surprise to the present inventors that it was possible to increase the strength of e.g. a chymosin composition/product by simply adding a suitable amount of e.g. whey proteins.

It is here relevant to note that casein hydrolysate did not significantly increase the physical stability and/or the specific activity.

As known to the skilled person - in a casein hydrolysate has been performed a hydrolysis of casein.

Without being limited to theory - it is believed that in such a casein hydrolysate formulation a significant amount of the polypeptides are of less than <NUM> amino acids.

Said in other words and without being limited to theory - it is believed that the herein relevant increased physical stability and specific activity effects are obtained when there are used polypeptides longer than <NUM> amino acids.

As known in the art - the term peptide may be distinguished from the term protein on the basis of size, which as an arbitrary benchmark may be understood to be approximately <NUM> or fewer amino acids.

Said in other words, a polypeptide longer than <NUM> amino acids may normally in the art be understood to be a protein.

Accordingly, a polypeptide longer than <NUM> amino acids may herein alternatively be termed a protein.

As discussed in working Examples herein - the herein relevant increased/improved physical stability and possibly specific activity effects were related to the amount of polypeptide/protein composition added to chymosin - where no significant positive effect was obtained if less than <NUM>% (w/w) was added.

Without being limited to theory - it is believed that addition of polypeptide/protein as set forth in claim <NUM> provide increased conformational stability to the chymosins and this could explain the observed increased physical stability and possibly increased specific activity observed in working Examples herein.

Without being limited to theory - it is believed that in the prior art it has not been described or suggested that addition of polypeptide/protein may increase the stability of aspartic protease milk-clotting enzymes such as e.g. chymosins - in particular it has not been described that conformational stability may be increased.

Conformational stability of an enzyme is illustrated in <FIG> herein.

As known in the art - loss of conformation equals loss of activity of the enzyme - i.e. less specific activity of the enzyme.

Without being limited to theory - loss of conformation may increase denaturation/precipitation of the enzyme and thereby give less physical stability as discussed herein.

As known in the art - milk clotting aspartic protease enzymes may be seen as structurally relatively similar.

As known in the art - different natural wildtype milk clotting aspartic protease polypeptide sequences obtained from different mammalian or fungal species (such as e.g. bovines, camels, sheep, pigs, or mucor) are having a relatively high tertiary structural similarity.

In <FIG> herein this is provided an alignment of herein relevant different milk clotting chymosin sequences from different mammalian species (cow, buffalo, goat, sheep, camel and pig) - as can be seen in <FIG> they have a close sequence relationship and are known to have a very high tertiary structural similarity.

In <FIG> herein there is provided 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).

It may be said that the <NUM> different sequences of <FIG> are not highly identical - but as known to the skilled person all these <NUM> different milk clotting aspartic protease enzymes are known to have a high tertiary structural similarity.

As discussed above and shown in working Examples herein - the herein relevant improved/increased stability/activity effects have been demonstrated for bovine chymosin, camel chymosin and mucorpepsin. Only enzyme compositions comprising camel chymosin as set forth in the claims are according to the present invention.

Without being limited to theory - it is believed that there is no significant technical reason to believe that the herein relevant improved/increased stability effect should not be relevant for milk clotting aspartic protease enzymes in general - as discussed above, they are known to have a high tertiary structural similarity and as understood by the skilled person in the present context this tertiary structural similarity makes it plausible that the herein described polymer-enzyme interaction to get improved stability would be a general class effect of the structural similar herein relevant milk clotting aspartic protease enzymes.

As discussed herein, numerous of polypeptide/protein formulations were tested by the present inventors (see working Examples herein) and a number of these significantly increased the specific activity of the enzyme compositions.

In <FIG> 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 <NUM> (named "Feed") unpurified samples essentially taken from the fermentation media and Lane <NUM> are purified samples.

In <FIG> herein can be seen that there are two bands in the migration range <NUM>-<NUM> kDa for e.g. CHY-MAX® in purified sample of lane <NUM>.

Without being limited to theory - it is believed that these two bands represent two different glycosylated forms of CHY-MAX®.

As understood by the skilled person in the present context - both of these different glycosylated forms are milk clotting aspartic protease enzymes, since they both have milk-clotting enzymatic activity.

The term "IMCU/g of the composition" in item I relates to IMCU enzyme activity per gram of the composition as such.

A first aspect of the invention relates to a liquid milk clotting aspartic protease enzyme composition comprising according to claim <NUM>.

In the present context - the skilled person will know or may routinely determine (e.g. based on specific relevant amino acid sequences) the origin of the polypeptides longer than <NUM> amino acids wherein the polypeptides longer than <NUM> amino acids are at least one polypeptide selected from the group consisting of: alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, 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.

As understood by the skilled person in the present context - the term "g/kg" in relation to item III relates to gram salt per kg of the composition as such.

A second aspect of the invention relates to a liquid milk clotting aspartic protease enzyme composition which must fall within the ambit of claim <NUM> comprising milk clotting aspartic protease enzyme at a strength of from <NUM> IMCU/g to <NUM> IMCU/g of the composition and a salt in a concentration from <NUM> to <NUM>/kg and wherein the pH of the composition is from <NUM> to <NUM>.

A milk clotting aspartic protease enzyme composition as described herein may be used according to the art - e.g. to make a food or feed product of interest (such as e.g. a milk based product of interest that e.g. could be a cheese product).

Accordingly, a third aspect of the invention relates to a method for making a food or feed product according to claim <NUM> comprising adding an effective amount of a milk clotting aspartic protease enzyme composition of any of first to second 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.

All definitions of herein relevant terms are in accordance of what would be understood by the skilled person in relation to the herein relevant technical context.

The term "milk-clotting enzyme" refers to an enzyme with milk-clotting enzymatic activity - i.e. an active milk-clotting enzyme. The milk-clotting activity may be expressed in International Milk-Clotting Units (IMCU) per ml or IMCU per g. The skilled person knows how to determine herein relevant milk-clotting enzymatic activity. In working Example <NUM> herein is provided an example of a standard method to determine milk-clotting enzymatic activity and specific milk-clotting enzymatic activity. As known in the art - specific clotting activity (IMCU/mg total protein) is determined by dividing the clotting activity (IMCU/ml) by the total protein content (mg total protein per ml).

The term "Sequence Identity" relates to the relatedness between two amino acid sequences.

For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined according to the art and preferably determined using the Needleman-Wunsch algorithm (<NPL>) as implemented in the Needle program of the EMBOSS package (<NPL>), preferably version <NUM>. <NUM> or later. The optional parameters used are gap open penalty of <NUM>, gap extension penalty of <NUM>, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: <MAT>.

The term "variant" means a peptide having milk-clotting enzymatic activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding <NUM>-<NUM> amino acids adjacent to an amino acid occupying a position.

The amino acid may be natural or unnatural amino acids - for instance, substitution with e.g. a particularly D-isomers (or D-forms) of e.g. D-alanine could theoretically be possible.

Embodiment of the present invention is described below, by way of examples only.

As discussed in working Examples herein - the herein relevant increase in the specific activity and strength were most significant for the camel chymosin compositions.

The milk clotting aspartic protease enzyme is a Camelius dromedarius chymosin (EC <NUM>.

A preferred milk clotting aspartic protease enzyme is Camelius dromedarius chymosin as described in e.g. <CIT>). It may herein alternatively be termed camel chymosin and the publicly known mature polypeptide amino acid sequence is shown in <FIG> herein.

As known in the art - it 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.

Accordingly, the milk clotting aspartic protease enzyme is Camelius dromedarius chymosin comprising the polypeptide amino acid sequence shown in <FIG> herein (termed "Camel_chymosin") or a variant of Camelius dromedarius chymosin, wherein the variant comprises a polypeptide sequence which has at least <NUM>% (preferably at least <NUM>%, more preferably at least <NUM>%) sequence identity with the camel chymosin polypeptide amino acid sequence shown in <FIG> herein.

The polypeptides longer than <NUM> amino acids are at least one polypeptide selected from the group of polypeptides consisting of: alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, 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.

More preferably, the polypeptides longer than <NUM> amino acids are at least one polypeptide selected from the group of polypeptides consisting of: alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alphas2-casein, beta-casein, kappa-casein, ovalbumin and gelatin.

Within the group immediately above it is preferred that the polypeptides longer than <NUM> amino acids are at least one polypeptide selected from the group of polypeptides consisting of: alpha lactalbumin, beta-lactoglobulin, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin and gelatin.

In a preferred embodiment - polypeptides longer than <NUM> amino acids are polypeptides longer than <NUM> amino acids, more preferably polypeptides longer than <NUM> amino acids.

As discussed in working Examples herein - herein 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.

As known in the art - the 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 <NUM> or fewer amino acids.

In a preferred embodiment - polypeptides longer than <NUM> amino acids are proteins longer than <NUM> amino acids, more preferably proteins longer than <NUM> amino acids, even more preferably proteins longer than <NUM> amino acids.

It may even be preferred that polypeptides longer than <NUM> amino acids are proteins longer than <NUM> amino acids.

As discussed above - the first aspect of the invention relates to a liquid milk clotting aspartic protease enzyme composition according to claim <NUM>. For the liquid composition - the milk clotting aspartic protease is a camel chymosin.

For the liquid composition - "polypeptides longer than <NUM> amino acids" are indicated in claim <NUM>.

For the liquid composition - it is preferred that the enzyme strength in item I is a strength of from <NUM> IMCU/g of the composition to <NUM> IMCU/g of the composition, more preferably a strength of from <NUM> IMCU/g of the composition to <NUM> IMCU/g of the composition.

For the liquid composition - in relation to item II, it is preferred the polypeptides longer than <NUM> amino acids is in a concentration from <NUM>% to <NUM>% (w/w) of the composition; more preferably in a concentration from <NUM>% to <NUM>% (w/w) of the composition; even more preferably in a concentration from <NUM>% to <NUM>% (w/w) of the composition and most preferably in a concentration from <NUM>% to <NUM>% (w/w) of the composition (such as e.g. in a concentration from <NUM>% to <NUM>% (w/w) of the composition).

For the liquid composition - the salt in item III is preferably in a concentration from <NUM> to <NUM>/kg, more preferably is in a concentration from <NUM> to <NUM>/kg.

For the liquid composition - it is preferred that the salt is an inorganic salt - preferably wherein the inorganic salt is selected from the group of NaCl, KCI, Na<NUM>SO<NUM>, (NH<NUM>)<NUM>SO<NUM>. K<NUM>HPO<NUM>, KH<NUM>PO<NUM>, Na<NUM>HPO<NUM> or NaH<NUM>PO<NUM> or a combination thereof.

The liquid composition may comprise further additives/compounds such as e.g. a preservative.

As known to the skilled person - preservative may generally be added in a concentration sufficient to prevent microbial growth during shelf life of the product.

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.

The liquid composition - it is preferred that the pH is from <NUM> to <NUM>, more preferably that the pH is from <NUM> to <NUM> and even more preferably that the pH is from <NUM> to <NUM>.

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 <NUM> wt % of water, for instance at least <NUM> wt % of water.

It may be preferred that the liquid composition as described herein has a total weight of from <NUM> to <NUM>, such as e.g. from <NUM> to <NUM>.

For the liquid composition - a preferred embodiment is:.

In the present context - the skilled person will know or may routinely determine (e.g. based on specific relevant amino acid sequences) the origin of the polypeptides longer than <NUM> amino acids wherein the polypeptides longer than <NUM> amino acids of item II are at least one polypeptide selected from the group of polypeptides consisting of: alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, 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.

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.

As discussed above - the second aspect of the invention relates to a liquid milk clotting aspartic protease enzyme composition which must fall within the ambit of claim <NUM> comprising milk clotting aspartic protease enzyme at a strength of from <NUM> IMCU/g to <NUM> IMCU/g of the composition and a salt in a concentration from <NUM> to <NUM>/kg and wherein the pH of the composition is from <NUM> to <NUM>.

For the liquid composition - preferably, the strength is a strength of from <NUM> IMCU/g to <NUM> IMCU/g of the composition, such as from <NUM> IMCU/g to <NUM> IMCU/g of the composition or such as from <NUM> IMCU/g to <NUM> IMCU/g of the composition.

As discussed above - a milk clotting aspartic protease enzyme composition as described herein may be used according to the art - e.g. to make a milk based product of interest (such as e.g. a cheese product).

As discussed above - the third aspect of the invention relates to a method for making a food or feed product according to claim <NUM> comprising adding an effective amount of a milk clotting aspartic protease enzyme composition of the first or second aspect to the food or feed ingredient(s) and carrying out further manufacturing steps to obtain the food or feed product.

Preferably, the food or feed product is a milk based product and wherein the method comprises adding an effective amount of the composition according to claim <NUM> to milk and carrying our further manufacturing steps to obtain the milk based product.

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

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

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

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 <NUM>,<NUM> per litre (pH ≈ <NUM>,<NUM>). 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 <NUM> IMCU/ml using an <NUM> acetic acid pH <NUM> buffer. Hereafter, <NUM>µl enzyme was added to <NUM> preheated milk (<NUM>) in a glass test tube placed in a water bath, capable of maintaining a constant temperature of <NUM> ± <NUM> under constant stirring.

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: <MAT>.

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

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).

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 <CIT>). 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 samples - at least <NUM>% of the total amounts of proteins with a size bigger than <NUM> kDa, determined by SDS-PAGE, in the purified sample were the relevant milk clotting aspartic protease enzymes.

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: <NUM> sodium acetate, <NUM> sodium phosphate, <NUM> sodium chloride, pH <NUM>, <NUM> methionine, and <NUM> sodium benzoate. The strength was from <NUM> to <NUM> IMCU/ml. The composition was added different polypeptides such as Hammersten casein, WPC80, Lacprodan Alpha <NUM>, Lacprodan Alpha <NUM>, etc..

WPC80 is a commercial preparation of dried whey protein concentrate with <NUM>% protein. Lacprodan Alpha <NUM> and Lacprodan Alpha <NUM> are commercial preparations of whey protein isolate containing <NUM>% and <NUM>% alpha lactalbumin of total protein content, respectively.

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.

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 <NUM> sample filled in a <NUM> tube in a rotary device for <NUM> hour (see <FIG> herein). For each solution, relative milk clotting activity was measured after <NUM> 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.

Vertical inversion for <NUM> hr of a sample of camel chymosin results in an activity loss of more than <NUM>% cf. Table <NUM> (No addition). Addition of PEG8000 to a concentration of <NUM>% 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 <NUM>, Alpha <NUM> or WPC <NUM> at a concentration of either <NUM>% w/v or <NUM>% w/v practically no loss upon vertical inversion was seen.

The protecting effect of Alpha <NUM> and WPC <NUM> was found to decrease gradually when their concentration was decreased below <NUM>%. 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 <NUM>).

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

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 <NUM>-<NUM>% higher in activity in good accordance with recently submitted patent application IN5103DK00. Samples containing Hammerstein casein, Alpha <NUM> or WPC <NUM> had <NUM> - <NUM>% higher activity compared to the sample without additives (no addition) even though the same amount of enzyme protein was applied (Table <NUM>). 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 <NUM> which shows the activity of compostions containing Alpha <NUM> and WPC80 at five different concentrations. Table <NUM> show activity of the composition, activity index with 'no addition' = <NUM>%, and the retained activity of a sample subjected to vertical inversion for <NUM> hr.

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 <NUM>. In Table <NUM> it is found that when the concentration of Alpha <NUM> is decreased from <NUM> to <NUM>%, the enzyme activity drops from index <NUM> to index <NUM> which is almost the same level as the untreated control. At a concentration of <NUM>% Alpha <NUM> the physical stability (retained activity) of the enzyme is the same as in the control experiment (no addition).

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

As known in the art - the term "peptone" refers to proteins digested by proteolysis. As known in the art - the term "tryptone" refers to proteins digested by the protease trypsin.

Extracts of plant proteins were prepared by suspending <NUM> sample in <NUM> brine consisting of: <NUM> % NaCl, <NUM>/L NaAc anhydrous, <NUM>/L NaH2PO4 anhydrous, and <NUM>/L Na-benzoate in water, pH <NUM> - <NUM>. After mixing for <NUM> hours on rotating device the suspension was centrifuged and the supernatant pH adjusted to <NUM> - <NUM> and filtered through a <NUM> 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 <NUM> different plant proteins were tested in three groups with each group prepared on different days.

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 <NUM> with the only difference that in present example a different rotary device was used for vertical inversion of the samples (Multi RS-<NUM> from Biosan at <NUM> rpm for <NUM> hour). The change in rotary device may have resulted in increased physical stress of the samples since retained activity of untreated sample was only <NUM>% compared to ca. <NUM>% in preceding examples. Samples were stored for <NUM> year at <NUM> and <NUM>, respectively, and stability was tested during storage period. The column 'End of storage' shows remaining activity after <NUM> year - the number was calculated by fitting a single exponential function to all data points.

Claim 1:
A liquid milk clotting aspartic protease enzyme composition comprising:
I: milk clotting aspartic protease enzyme at a strength of from <NUM> IMCU/g to <NUM> IMCU/g of the composition;
II: polypeptides longer than <NUM> amino acids in a concentration from <NUM>% to <NUM>% (w/w) of the composition, wherein the polypeptides longer than <NUM> amino acids are at least one polypeptide selected from the group consisting of: alpha lactalbumin, beta-lactoglobulin, transferrin, lactoperoxidase, casein, alpha-s1-casein, alpha-s2-casein, beta-casein, kappa-casein, ovalbumin, gelatin, 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
III: a salt in a concentration from <NUM> to <NUM>/kg and wherein the pH of the composition is from <NUM> to <NUM>; and
wherein the milk clotting aspartic protease enzyme is chymosin (EC <NUM>.<NUM>),
wherein the milk clotting aspartic protease enzyme is Camelius dromedarius chymosin comprising the polypeptide amino acid sequence shown in Figure <NUM> herein (termed "Camel_chymosin") or a variant of Camelius dromedarius chymosin, wherein the variant comprises a polypeptide sequence which has at least <NUM>%, preferably at least <NUM>%, more preferably at least <NUM>%, sequence identity with the camel chymosin polypeptide amino acid sequence shown in Figure <NUM> herein, wherein the International Milk Clotting Units (IMCU) are determined as set forth in the description.