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Title:
CHYMOSIN FORMULATION
Document Type and Number:
WIPO Patent Application WO/2012/127005
Kind Code:
A1
Abstract:
The present invention relates to a liquid formulation comprising chymosin at a high strength.

Inventors:
DEKKER PETRUS JACOBUS THEODORUS (NL)
HOLLANDER DEN JEROEN (NL)
Application Number:
EP2012/055131
Publication Date:
September 27, 2012
Filing Date:
March 22, 2012
Export Citation:
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Assignee:
DSM IP ASSETS BV (NL)
DEKKER PETRUS JACOBUS THEODORUS (NL)
HOLLANDER DEN JEROEN (NL)
International Classes:
A23C19/032; A23L29/00
Domestic Patent References:
WO1990015866A11990-12-27
WO1990015865A11990-12-27
WO1996019582A11996-06-27
WO1995029999A21995-11-09
WO2007118838A12007-10-25
WO2007118838A12007-10-25
Foreign References:
US20100009033A12010-01-14
FR2140642A11973-01-19
Attorney, Agent or Firm:
GRIEKEN PLOOSTER, VAN, Izabella, Johanna et al. (DSM Intellectual Property, P.O. Box 130, AC Echt, NL-6100, NL)
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Claims:
CLAIMS

1. A liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative.

2. A composition according to claim 1 , wherein said chymosin has a strength of at least 1 100 IMCU/ml.

3. A composition according to claim 1 or 2, wherein said chymosin is bovine or cameloid chymosin.

4. A composition according to any one of claims 1 to 3, wherein said chymosin is recombinant chymosin.

5. A composition according to any one of claims 1 to 4, wherein said chymosin is not chromatographically purified.

6. A composition according to any one of claims 1 to 5, wherein said salt has a concentration of 2-75, 2-70, 2-65 or 2-60 g/kg.

7. A composition according to any one of claims 1 to 6, wherein said salt is an inorganic salt.

8. A composition according to any one of claims 1 to 7, wherein said salt is selected from the group of NaCI, KCI, Na2S04, (NH4)2S04, K2HP04, KH2P04, Na2HP04 or NaH2P04 or a combination thereof.

9. A composition according to any one of claims 1 to 8, wherein said preservative is (i) a polyalcohol or (ii) an organic acid or any a combination thereof.

10. A composition according to claim 9, wherein said polyalcohol is glycerol or propanediol or a combination thereof.

11. A composition according to claim 8, wherein said polyol is present in a concentration of 100-500 g/kg.

12. A composition according to claim 9, wherein said organic acid is benzoic acid, sorbic acid, acetic acid or lactic acid or an alkyl esters of para-hydroxybenzoate.

13. A composition according to claim 10, wherein said organic acid is present in a concentration of 3-5 g/kg.

14. A closed container comprising at least 1 liter of a composition according to any one of claims 1 to 13.

15. A method for preparing a composition according to any one of claims 1 to 13 comprising the steps of mixing chymosin at a strength of at least 1000 IMCU/ml with salt in a concentration of 2-100 g/kg and a preservative and optionally storing said composition at or higher than 4 °C.

16. A method for preparing cheese, comprising adding a composition according to any one of claims 1 to 13 to milk, allowing the milk to coagulate, obtaining a curd and processing the curd into cheese.

Description:
CHYMOSIN FORMULATION

Field of the invention

The present invention relates to a liquid formulation comprising chymosin at a high strength.

Background of the invention

It is well known that the preparation of cheese involves the use of an aspartic protease. The aspartic protease causes the milk to coagulate, resulting in a solid curd which is further processed into cheese. The most frequently used aspartic protease for cheese production is chymosin (group 3.4.23.4 according to the Enzyme Nomenclature, 1992 of the International Union of Biochemistry and Molecular Biology, IUBMB).

Chymosin can be recovered from animals, e.g. from the stomach of calf, camel, buffalo, lamb and seal. Alternatively, the gene or cDNA coding for chymosin from one of these animals can be cloned and overexpressed in a host organism. Well known host organisms that have been used for chymosin over-expression in the past are from Aspergillus, Kluyveromyces, Trichoderma, Escherichia coli, Pichia, Saccharomyces, Yarrowia, Neurospora or Bacillus.

In the cheese making industry, liquid compositions comprising chymosin are often used. Such liquid compositions typically contain certain additives to obtain a desired stability. One can distinguish between physical, enzymatic, and microbial stability. The physical stability is a measure for the rate at which aggregates or turbidity is formed in the composition. If this rate is high, the physical stability is low. The enzymatic stability is a measure for the rate at which the activity of the enzyme decreases. If activity decrease is high, the enzymatic stability is low. The microbial stability is a measure for the rate at which microorganisms can proliferate and grow in the composition. If the proliferation rate is high, the microbial stability is low. For a commercial enzyme product it is essential that enzymatic, physical and microbial stability are all high, to withstand the storage of the enzyme preparation at moderate temperatures (4-20 degrees Celsius) for a prolonged period of time (1-2 years). An enzyme preparation that does not comply to this is regarded as inferior.

In the art a number of liquid chymosin formulations have been described that result in a relatively stable chymosin, both physical, enzymatic and microbial. These liquid formulations always comprise of a relatively low strength (<1000 IMCU/ml) and a relatively high (inorganic) salt concentration (>100 g/kg) combined with a preservative (commonly benzoate). All commercially available liquid chymosin preparations have these properties.

Traditionally, extraction of chymosin and production of rennet from calf stomachs begins by extracting, for several days, chopped or macerated stomachs with a 10% sodium chloride solution. Additional salt up to a total of 16-18% is introduced followed by filtration and clarification. The suspension is adjusted to pH5.0 to activate prochymosin to chymosin and the enzyme strength is standardized. Consequently, all commercial calf rennet preparations will contain a high inorganic salt content, and a low strength.

When chymosin was developed using recombinant DNA technology, formulation of the product had to mimic as closely as possible the traditional calf rennet. Therefore, extra inorganic salt was added to the formulation, generally to 130-150 g/kg and the same low strength was supplied as with the animal rennets. Salt may also be added to increase the microbial stability of the final product. Consequently, all commercial fermentation- produced chymosin (FPC) is currently formulated with a high salt content, and at a low strength.

WO9015865 describes the development of a purification protocol for chymosin using a phenyl sepharose column chromatography step. Chymosin is eluted from the column with water or with dilute salt such as 50 mM phosphate (approximately 6 g/kg). The eluted chymosin is made 17% NaCI for commercial food grade use. Apparently it is thought in the art that this extra inorganic salt is required to obtain a commercial food grade chymosin composition. From example II it is clear that the highest amount of chymosin activity is 198.4 CHU/I. CHU/I is an internal unit of the Christian Hansen company. According to WO 96/19582 (page 15, lines 5-7) 67 CHU/ml corresponds to 195 IMCU/ml. An amount of 198.4 CHU/I corresponds to 0.1984 CHU/ml and according to the WO 96/19582 information, to 0.6 IMCU/ml. The WO 90/15865 subject matter does not relate to high strength chymosin formulations. W09529999 describes the development of a purification protocol for chymosin from crude calf rennet extract using an anion exchange column chromatography. The application is especially directed to the separation of chymosin and bovine pepsin. Again, it is explicitly mentioned that salt has to be added: "The combined chymosin fractions were salted gently until 19 °Be" (example 4, page 44, lines 19-21), and "To the resulting combined eluted chymosin and bovine pepsin solutions NaCI was added to 18 °Be for commercial food grade liquid rennets or for further downstream processing use" (example 6; page 48, lines 33-35). The unit "°Be" (Baume degrees) originally represented the percent by mass of sodium chloride in water at 60 degrees Fahrenheit (15.5 °C); 19 °Be corresponds to approximately 19% (w/w) NaCI in water. Liquid formulations having high strength chymosin and relative low salt content are not described in this prior art.

WO07118838 describes a microbial stable liquid composition comprising an aspartic protease and a low concentration of organic acid. It is described that different combinations of salt, organic acid and poly-alcohol will result in a microbial stable formulation. Stability of the formulation is tested with the so called "challenge test", where microbial growth is followed in time. No test and mentioning of the enzymatic and physical stability of the enzyme preparation were mentioned in WO2007/1 18838. Also, no high strength formulations (>1000 IMCU/ml) were prepared and tested in this patent application.

At present a stable liquid chymosin preparation of >1000 IMCU/ml is not available commercially. An enzymatic, physical and microbial stable high strength chymosin would, however, have several advantages. Dosage of the enzyme could be reduced in the cheese making process, and therefore stock volume of the enzyme can be reduced, both at the enzyme supplier and the cheese maker. Also transport costs per activity unit will be reduced since a smaller volume has to be shipped. Furthermore, formulation costs can be reduced since a smaller volume of preservatives and other additives are required. Consequently, since the volume of chymosin added to the cheese milk will be lower, also the amount of preservatives and other additives that end up in the cheese matrix, or the whey, will be reduced. Because of the reduced shipping volume, reduced use of chemicals for formulation and reduced impact of preservatives and other additives in the final product and in waste streams, a higher strength liquid chymosin preparation will have lower environmental impact than current liquid chymosin preparations. From this it will be clear that a liquid chymosin preparation with a higher strength compared to current commercial chymosin preparations will have an advantage for both the enzyme manufacturer and the cheese producer. Preferably such a liquid formulation will have a strength higher than 1000 IMCU/ml and is enzymatically, physically and microbially stable. The absence of a commercial liquid chymosin formulation with a high strength although regular strength chymosin is commercially available for many years, indicates that it difficult to obtain a high strength chymosin formulation that is sufficiently stable.

Description of the Figures

Figure 1 : Enzymatic stability versus salt concentration. Squares represent a chymosin preparation with an initial strength of 1230 IMCU/ml: triangles represent a chymosin preparation with an initial strength of 1070 IMCU/ml; diamonds represent a chymosin preparation with an initial strength of 670 IMCU/ml.

Summary of the invention

The purpose of this invention is to provide a liquid chymosin preparation comprising a strength of at least 1000 IMCU/ml, a salt concentration between 2 and 100 g/kg and a preservative in a concentration sufficient to prevent microbial growth during shelf life of the product. Liquid chymosin formulations with such high strength and having more than 100 g/kg salt will have decreased physical stability. Liquid chymosin formulations with such high strength and having less than 2 g/kg salt will have decreased enzymatic stability. The preservative is required to prevent microbial spoilage during shelf life of the enzyme.

Detailed description of the invention

The present invention provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative.

Commercial chymosin is available as liquid and as a granulate. The invention provides a liquid composition. 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. Preferably, a composition according to the invention comprises at least 50, 60, 70 or 80 wt % of water. More preferably, the composition of the invention comprises at least 85, 90 or 95 wt % of water. In a most preferred embodiment, the invention provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said composition comprises at least 96, 97, 98 or 99 wt % of water.

With "chymosin" is typically meant an aspartic protease, group 3.4.23.4 according to the Enzyme Nomenclature, 1992 of the International Union of Biochemistry and Molecular Biology, IUBMB. Chymosin is naturally produced by gastric chief cells in juvenile mammals. Chymosin is the main enzymatic component in rennet. Calf rennet is obtained of the lining of the abomasum (the fourth and final, chamber of the stomach) of young, unweaned calves.

By the term "IMCU" is understood International Milk Clotting Units. One IMCU equals about 0.126 nmol of bovine chymosin B (e.g. Maxiren or CHY-MAX). The strength of a milk clotting enzyme (such as chymosin enzyme present in a composition of the present invention) is determined as the milk clotting activity (IMCU per ml or per gram). Following the addition of diluted coagulant to a standard milk substrate, the milk will flocculate. The milk clotting time is the time period from addition of the coagulant until formation of visible flocks or flakes in the milk substrate. The strength of a coagulant sample is found by comparing the milk clotting time for the sample to that of a reference standard, a normal. This is expressed in IDF standard 157A: 1997 which gives the IMCU definition: The total milk-clotting activity of the first batch of calf chymosin reference standard powder has once and for all been set at 1000 International Milk-Clotting Units per gram (IMCU/g). Further preparations of reference standards will be set relative to the previous reference. IMCU principle: Determination of the time needed for visible flocculation of renneted standard milk substrate with 0.05% calcium chloride, pH6.5. IMCU/ml of a sample is determined by comparison of the clotting time to that of a standard having known milk clotting activity and having the same enzyme composition of the sample. A liquid composition according to the invention comprises chymosin at a strength of at least 1000 IMCU/ml, preferably at least 1 100, more preferably at least 1200 or 1250 and most preferably at least 1300, 1400 or 1500 IMCU/ml.

In one of its embodiments, the invention therefore provides a liquid composition comprising:

chymosin at a strength of at least 1 100, more preferably 1200 or 1250 and most preferably at least 1300 IMCU/ml,

salt in a concentration of 2-100 g/kg, and

a preservative.

In an even more preferred embodiment, the chymosin has a strength of at least 1400 or 1500 IMCU/ml.

The examples described herein provide a method for obtaining chymosin with an activity in the claimed amounts. One can for example obtain chymosin in the claimed strength by concentrating -for example by ultrafiltration- a chymosin preparation having an activity below 1000 IMCU/ml.

Chymosin or rennet can be obtained from different species comprising calf, camel, buffalo, sheep, etc. Bovine chymosin can be produced recombinantly in E. coli, Aspergillus, Kluveromyces, Trichoderma, Escherichia coli, Pichia, Saccharomyces, Yarrowia, Neurospora or Bacillus as alternative resource. Also chymosin from other species may be produced recombinantly. Recombinant camel chymosin is also available commercially and is included in this invention.

In a preferred embodiment, the invention provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said chymosin is bovine or cameloid chymosin.

Preferably, the chymosin is produced by a genetically modified micro-organism, wherein said micro-organism is provided with a nucleic acid sequence encoding for example bovine or cameloid chymosin. The nucleic acid sequence is operably linked to regulatory sequences such as to obtain expression of the for example bovine or cameloid chymosin in a micro organism. During fermentation the micro organism will produce the chymosin. The chymosin in a composition according to the invention is thus preferably a fermentation produced chymosin. In a more preferred embodiment, the invention thus provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said chymosin is recombinant chymosin. Or alternatively, the invention thus provides a liquid composition comprising: chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said chymosin is fermentation produced chymosin. In a most preferred embodiment, the invention provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said chymosin is recombinant bovine chymosin or recombinant cameloid chymosin.

Chymosin and more specific bovine chymosin, extracted from calves stomachs is a combination of chymosin A, B and C. The ratio between chymosin A, B and C depends on the genotype of the used calves. However, it is clear that stomach extracts from many calves are combined and that there is always a combination of chymosin A, B and C.

As described above, the chymosin present in a composition according to the invention is preferably recombinant or fermentation produced chymosin. In such a case preferably only one type of chymosin is cloned and overexpressed. In a further preferred embodiment, a composition according to the invention comprises chymosin B.

In one of its embodiment, the invention thus provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said chymosin is recombinant chymosin B. Or alternatively, the invention thus provides a liquid composition comprising: chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said chymosin is fermentation produced chymosin B. In a most preferred embodiment, the invention provides a liquid composition comprising: chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said chymosin is recombinant bovine chymosin B or recombinant cameloid chymosin B.

Chymosin and rennet are generally used for coagulation of milk protein in the manufacturing of cheese.

Also genetically engineered chymosin is included in this invention. Typically one or more amino acids within the protein sequence of chymosin may be replaced in an engineered chymosin. Overall up to 10% of amino acids in the wild type chymosin protein sequence may be replaced in an engineered chymosin. Also chymosin that is modified by other means is regarded as part of this invention. For example, chymosin may be chemically modified by alkylation, acylation, peroxidation or other means. Also glycosylated, prenylated or addition of other side-groups to chymosin is regarded as modified chymosin and is included in this invention.

The chymosin may be of animal origin. Preferably, the chymosin is produced by a micro-organism (a microbially or fermentation produced chymosin).

The microorganism may for instance be Aspergillus, Kluyveromyces, Trichoderma, Escherichia coli, Pichia, Saccharomyces, Yarrowia, Neurospora or Bacillus In a preferred embodiment, the micro organism is Aspergillus or Kluyveromyces. Even more preferably, the micro organism is Kluyveromyces.

In a preferred embodiment, the chymosin in a liquid composition according to the invention is produced by a micro organism and the produced chymosin is present in the supernatant. The supernatant is separated from the fermentation broth and the chymosin present in the supernatant is used as such i.e. without subjecting the chymosin to chromatography. The invention thus provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said chymosin is not chromatographically purified.

The composition may have any suitable pH. In a preferred embodiment, the composition has a pH of less than 7, preferably less than 6. Preferably, the pH is at least at least 3, preferably at least 4, preferably at least 5. The pH may for instance be between 5.0 and 6.0. The invention therefore provides a liquid composition comprising: chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein the pH of the composition is less than 7, preferably less than 6 but at least 3, preferably at least 4, preferably at least 5 and most preferred the pH is between 5.0 and 6.0.

In yet another aspect of the invention, a composition according to the invention is a sterile composition, e.g. before packaging the composition is sterile filtered. The invention therefore also provides a sterile liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

- a preservative.

Preferably, a composition according to the invention has acceptable enzymatic stability, physical stability and microbial stability.

The physical stability is a measure for the rate at which aggregates or turbidity is formed in the composition. If this rate is high, the physical stability is low.

The enzymatic stability is a measure for the rate at which the activity of the enzyme decreases. If activity decrease is high, the enzymatic stability is low.

The microbial stability is a measure for the rate at which microorganisms can proliferate and grow in the composition. If the proliferation rate is high, the microbial stability is low.

The microbial properties of a composition can be expressed by the standard plate count, number of yeasts and number of moulds using well-defined standard procedures. For instance, the standard plate count can be≤ 100 in 1 ml, the yeast count can be≤ 10 in 1 ml and the mould count can be≤ 10 in 1 ml. As compositions are often stored prior to use, it is desirable that the plate count, number of yeasts and number of moulds remain below certain boundary values, for instance the values mentioned above, for a prolonged period, for instance for a period of at least 3 months.

Preferably, the standard plate count is≤ 100 in 1 ml; the yeast count is≤ 10 in 1 ml; and the mould count is≤ 10 in 1 ml.

As used herein, the standard plate count is determined according to ISO 4833: 1991 (E) (Microbiology - General guidance for the enumeration of micro-organisms - Colony count technique at 30°C). The yeast count is determined according to ISO 7954: 1987 (E) (Microbiology - General guidance for enumeration of yeasts and moulds - Colony count technique at 25°C).

The moulds count is determined according to ISO 7954: 1987 (E) (Microbiology - General guidance for enumeration of yeasts and moulds - Colony count technique at 25°C).

Preferably, the standard plate count remains ≤ 100 in 1 ml, the yeast count remains≤ 10 in 1 ml and the mould count remains≤ 10 in 1 ml during a period of at least 4 months, preferably at least 6 months, preferably at least 9 months, preferably at least 12 months, preferably at least 18 months, preferably at least 24 months, when the composition is stored in a closed container at a temperature of 4 °C in the dark.

In an embodiment of the invention, the standard plate count remains≤ 100 in 1 ml, the yeast count remains≤ 10 in 1 ml and the mould count remains≤ 10 in 1 ml during a period of at least 4 months, preferably at least 6 months, preferably at least 9 months, preferably at least 12 months, preferably at least 18 months, preferably at least 24 months, when the composition is stored in a closed container at a temperature of 30 °C in the dark.

In an embodiment of the invention, the enzymatic activity decreases at most 5% during a period of at least 4 months, preferably at least 6 months, preferably at least 9 months, preferably at least 12 months, preferably at least 18 months, preferably at least 24 months, when the composition is stored in a closed container at a temperature of 4 °C in the dark.

Physical stability of an enzyme preparation can be measured using the measurement of the turbidity of the solution. Turbidity can be measured used a turbidimeter, like the one that is described in Example 2 herein, after homogenization of the sample and can be expressed in NTU.

In an embodiment of the invention, the turbidity increases at most with 20 NTU during a period of 4 months, preferably at least 6 months, preferably at least 9 months, preferably at least 12 months, preferably at least 18 months, preferably at least 24 months, when the composition is stored in a closed container at a temperature of 4 °C in the dark.

For a commercial enzyme product it is essential that enzymatic, physical and microbial stability are all high, to withstand the storage of the enzyme preparation at moderate temperatures (4-20 degrees Celsius) for a prolonged period of time (1-2 years). With "shelf life" is meant the time that enzymes are normally stored before expiration. A normal shelf life for commercial liquid preparations of chymosin is one or two years under typical storage temperature of 4-8 degrees Celsius, and is mainly limited by a decrease in enzymatic activity due to enzymatic instability. Obviously, it is essential that microbial stability is preserved during storage of the enzyme, and that physical and enzymatic stability is as high as possible. A formulation that will increase the physical and enzymatic stability of an enzyme, as described herein, will increase the shelf life of a product. Such formulation is commercially interesting since less enzyme reaches expiration date and has to be discarded.

As described above, the invention provides a liquid composition comprising: chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative.

The term "salt" as used herein in refers to an inorganic salt which decomposes in ions upon solution in water.

The invention therefore provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said salt is an inorganic salt.

The salt ions are divided in cations and anions. For cations any metal ion may be suitable as long as it does not interfere with the application of the chymosin preparation or the food-grade status of the final dairy product. Suitable cations comprise sodium, potassium, magnesium, ammonium or others. Preferably the cation is a sodium ion. The anion may be any negatively charged ion comprising chloride, sulfate, phosphate, carbonate or others. Preferred inorganic salts are NaCI, KCI, Na 2 S0 4 , (NH 4 ) 2 S0 4 , K 2 HP0 4 , KH 2 P0 4 , Na 2 HP0 4 or NaH 2 P0 4 . The composition may contain one or more inorganic salts, i.e. in one of its embodiments, the invention provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said salt is selected from the group of NaCI, KCI, Na 2 S0 4 , (NH 4 ) 2 S0 4 , K 2 HP0 4 , KH 2 P0 4 , Na 2 HP0 4 or NaH 2 P0 4 or a combination thereof. Preferably the anion is a chloride ion. Most preferably the salt is sodium chloride.

In its main embodiment, the used amounts of (inorganic) salt are in the range of 2-100 g/kg. However, it is further desired to reduce especially the upper limit of the 2-100 g/kg range. More preferred embodiments are (inorganic) salt in a concentration of 2-80 g/kg, 2-75 g/kg, 2-70 g/kg, 2-65 g/kg or even 2-60 g/kg. In one of its aspects the invention provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-75, more preferably 2-70, 2-65 or 2-60 g/kg, and a preservative.

The salt ranges mentioned herein refer to the sum (i.e. total) concentrations of inorganic salt in the composition.

Preferred embodiments are:

a liquid composition comprising:

chymosin at a strength between 1000 and 1250 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative,

a liquid composition comprising:

chymosin at a strength of at least 1250 IMCU/ml

salt in a concentration of 2-90 g/kg, and

a preservative,

a liquid composition comprising:

chymosin at a strength between 1250 and 1500 IMCU/ml

salt in a concentration of 2-90 g/kg, and

a preservative,

a liquid composition comprising:

chymosin at a strength of at least 1500 IMCU/ml

salt in a concentration of 2-75 g/kg, and

a preservative.

With "preservative" is meant an addition to the formulation that preserves the microbial stability during shelf life of the product. A solution is microbial stable when no growth of micro-organisms occurs during the shelf-life of the product. Traditionally preservatives can be weak organic acids such as formate, acetate, lactate, propionate, malate, benzoate, sorbate or fumarate. It will be understood that these compounds are the anions of the corresponding organic acids (formic acid, acetic acid, lactic acid, propionic acid malic acid and fumaric acid), and that these compounds may be supplemented to the composition as the organic acid or the salt thereof. The salt may for instance be a potassium salt, a sodium salt or a calcium salt. Parabens (alkyl esters of para-hydroxybenzoate) may also be used as preservative. The concentration of the organic acid in the enzyme formulation depends on the efficacy of the prevention of microbial growth, and can be adjusted dependent on the pH, salt concentration, storage temperature and presence of other preservatives in the formulation.

The presence of an organic acid is however not essential to preserve the microbial stability of an enzyme preparation. WO2007/1 18838 describes a microbial stable liquid composition comprising an aspartic protease and a low concentration of organic acid. It is described that different combinations of salt, organic acid and poly- alcohol will result in a microbial stable formulation. A preservative may also be a poly- alcohol in a sufficiently high concentration to prevent microbial growth during shelf life of the enzyme.

In one of its embodiments, the invention therefore provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said preservative is (i) a polyalcohol or (ii) an organic acid or any a combination thereof.

In a preferred embodiment, the composition comprises a polyalcohol as a preservative. The polyalcohol can function to decrease the water activity of the composition. Decreasing the water activity can assist in achieving a desired microbial stability. Any suitable polyalcohol may be used. The polyalcohol may for instance be ethylene glycol (ethanediol), propylene glycol (propanediol), glycerol, erythritol, xylitol, mannitol, sorbitol, inositol, galactitol. Preferably, the polyalcohol is glycerol, sorbitol or propanediol, more preferably glycerol or propanediol. The composition may comprise one or more polyalcohols. In preferred embodiment, the polyalcohol is glycerol or propanediol or a combination thereof and most preferred the polyalcohol is glycerol. The concentration of the polyalcohol can easily be selected by the skilled person to be high enough to prevent microbial growth without the addition of other preservatives. In a preferred embodiment, the preservative is a polyalcohol which is present in an amount of 100-500 g/kg. The invention thus provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said preservative is a polyalcohol in a concentration of 100-500 g/kg.

Further preferred embodiments are:

a liquid composition comprising:

chymosin at a strength between 1000 and 1250 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said preservative is a polyalcohol in a concentration of 100-500 g/kg.

a liquid composition comprising:

chymosin at a strength of at least 1250 IMCU/ml

salt in a concentration of 2-90 g/kg, and

a preservative, wherein said preservative is a polyalcohol in a concentration of 100-500 g/kg.

a liquid composition comprising:

chymosin at a strength between 1250 and 1500 IMCU/ml

salt in a concentration of 2-90 g/kg, and

a preservative, wherein said preservative is a polyalcohol in a concentration of 100-500 g/kg.

a liquid composition comprising:

chymosin at a strength of at least 1500 IMCU/ml

salt in a concentration of 2-75 g/kg, and

a preservative, wherein said preservative is a polyalcohol in a concentration of 100-500 g/kg.

In yet another preferred embodiment, a composition according to the invention comprises an organic acid as a preservative. As mentioned above, traditionally preservatives can be weak organic acids such as formate, acetate, lactate, propionate, malate, benzoate, sorbate or fumarate. It will be understood that these compounds are the anions of the corresponding organic acids (formic acid, acetic acid, lactic acid, propionic acid malic acid and fumaric acid), and that these compounds may be supplemented to the composition as the organic acid or the salt thereof. The salt may for instance be a potassium salt, a sodium salt or a calcium salt. Parabens (alkyl esters of para-hydroxybenzoate) may also be used as preservative. The concentration of the organic acid in the enzyme formulation depends on the efficacy of the prevention of microbial growth, and can be adjusted dependent on the pH, salt concentration, storage temperature and presence of other preservatives in the formulation.

The invention therefore also provides a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said preservative is an organic acid and wherein said organic acid is benzoic acid, sorbic acid, acetic acid or lactic acid or an alkyl esters of para-hydroxybenzoate. Preferably, said organic acid is present in a concentration of 3 to 5 g/kg.

Further preferred embodiments are:

a liquid composition comprising:

chymosin at a strength between 1000 and 1250 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative, wherein said preservative is an organic acid and wherein said organic acid is benzoic acid, sorbic acid, acetic acid or lactic acid or an alkyl esters of para-hydroxybenzoate. Preferably, said organic acid is present in a concentration of 3 to 5 g/kg.

a liquid composition comprising:

chymosin at a strength of at least 1250 IMCU/ml

salt in a concentration of 2-90 g/kg, and

a preservative, wherein said preservative is an organic acid and wherein said organic acid is benzoic acid, sorbic acid, acetic acid or lactic acid or an alkyl esters of para-hydroxybenzoate. Preferably, said organic acid is present in a concentration of 3 to 5 g/kg.

a liquid composition comprising:

chymosin at a strength between 1250 and 1500 IMCU/ml

salt in a concentration of 2-90 g/kg, and

a preservative, wherein said preservative is an organic acid and wherein said organic acid is benzoic acid, sorbic acid, acetic acid or lactic acid or an alkyl esters of para-hydroxybenzoate. Preferably, said organic acid is present in a concentration of 3 to 5 g/kg.

a liquid composition comprising:

chymosin at a strength of at least 1500 IMCU/ml

salt in a concentration of 2-75 g/kg, and

a preservative, wherein said preservative is an organic acid and wherein said organic acid is benzoic acid, sorbic acid, acetic acid or lactic acid or an alkyl esters of para-hydroxybenzoate. Preferably, said organic acid is present in a concentration of 3 to 5 g/kg.

A composition according to the invention can comprise additional components such as an antioxidant or a colorant e.g. caramel.

In a preferred embodiment, the composition comprises an antioxidant, preferably methionine, cysteine, glutathione, yeast extract, ascorbate or sulphite. Other useful antioxidants for chymosin are casamino acids, whey protein, casein peptone and cysteic acid. Preferably, the composition comprises at least approximately 0.1 g/l to approximately 1 g/l of methionine. A suitable concentration of methionine is 0.75-1.1 g/l.

Most preferred compositions are:

- a liquid composition comprising:

o chymosin at a strength of at least 1000 IMCU/ml

o salt in a concentration of 2-100 g/kg, and

o a preservative, wherein said chymosin is fermentation produced chymosin, preferably chymosin B

-a liquid composition comprising:

o chymosin at a strength between 1000 and 1250 IMCU/ml o salt in a concentration of 2-100 g/kg, and

o a preservative, wherein said chymosin is fermentation produced chymosin, preferably chymosin B

-a liquid composition comprising:

o chymosin at a strength between 1000 and 1250 IMCU/ml o salt in a concentration of 2-100 g/kg, and

o a preservative, wherein said chymosin is fermentation produced bovine or cameloid chymosin, preferably chymosin B

- a liquid composition comprising: o chymosin at a strength of at least 1250 IMCU/ml

o salt, preferably NaCI, in a concentration of 2-90 g/kg, and o a preservative, wherein said chymosin is fermentation produced chymosin, preferably chymosin B

- a liquid composition comprising:

o chymosin at a strength of at least 1250 IMCU/ml

o salt, preferably NaCI, in a concentration of 2-90 g/kg, and o a preservative, wherein said chymosin is fermentation produced bovine or cameloid chymosin, preferably chymosin B a liquid composition comprising:

o chymosin at a strength between 1250 and 1500 IMCU/ml o salt, preferably NaCI, in a concentration of 2-90 g/kg, and o a preservative, wherein said chymosin is fermentation produced chymosin, preferably chymosin B

a liquid composition comprising:

o chymosin at a strength between 1250 and 1500 IMCU/ml o salt, preferably NaCI, in a concentration of 2-90 g/kg, and o a preservative, wherein said chymosin is fermentation produced bovine or cameloid chymosin, preferably chymosin B a liquid composition comprising:

o chymosin at a strength of at least 1500 IMCU/ml

o salt, preferably NaCI, in a concentration of 2-75 g/kg, and o a preservative, wherein said chymosin is fermentation produced chymosin, preferably chymosin B

a liquid composition comprising:

o chymosin at a strength of at least 1500 IMCU/ml

o salt, preferably NaCI, in a concentration of 2-75 g/kg, and o a preservative, wherein said chymosin is fermentation produced bovine or cameloid chymosin, preferably chymosin B

Any of the above described liquid compositions is preferably a final chymosin product ready for sale and commercial use in which composition the standard plate count is < 100 in 1 ml, yeast count is < 10 in 1 ml and mould count is < 10 in 1 ml. The composition according to the invention can be packaged in any suitable closed container. Accordingly, the invention further provides a closed and/or sealed container containing any of the above described compositions, i.e. a liquid composition comprising:

chymosin at a strength of at least 1000 IMCU/ml

salt in a concentration of 2-100 g/kg, and

a preservative or any of the above described specific embodiments. Preferably the closed and/or sealed container comprises at least 1 , 2 or 5 liter of a composition according to the invention. In a more preferred embodiment, the closed and/or sealed container comprises at least 10 or 15 liter of a composition according to the invention.

The invention further comprises a method for preparing a composition as described herein comprising the steps of

- optionally concentrating chymosin to a strength of at least 1000 IMCU/ml

- mixing chymosin at a strength of at least 1000 IMCU/ml with salt in a concentration of 2-100 g/kg and a preservative.

The invention further provides the use of the composition according to the invention as a coagulant in the production of cheese.

The invention further provides a process for preparing cheese, comprising, (i) supplementing milk with a composition according to the invention, to effect coagulation of the milk, wherein a curd is obtained; and (ii) processing the curd into cheese.

The invention will now be elucidated with reference to the following examples, without, however, being limited thereto.

Example 1

Testing stability of chymosin at high salt concentration

All experiments were performed starting from a Maxiren 600 preparation, which is a commercial enzyme preparation of a recombinant produced bovine chymosin that can be obtained from DSM Food-Specialties (Delft, the Netherlands). In order to develop a chymosin formulation with a higher strength than usual, Maxiren 600 (-600 IMCU/ml) was concentrated until >1000 IMCU/ml. The Maxiren 600 preparation was concentrated by means of ultrafiltration on a 10 kD polyethersulfone membrane (Prep/Scale TM TFF, Millipore Corporation)

Maxiren 600 contains 130-150 g NaCI/kg product. In order to test the effect of salt on the physical and enzymatic stability of chymosin in a high-strength formulation we varied the NaCI concentration in the ultrafiltrated Maxiren by the addition of extra NaCI, or removal of salt using continuous diafiltration with demineralised water. Using this method several chymosin formulations having different strengths and different NaCI concentrations were made. All formulations were stored overnight at 4 degrees Celcius, before filtration over a 0.45 urn filter (Pall Acrodisc GHP/Glass Fiber Membrane Filter). Any aggregate that is formed in the formulation will be removed in this step. The remaining chymosin activity in the filtrate was measured using the IMCU test.

From the results it becomes clear that the stability of chymosin formulations with higher strength (>1000 IMCU/ml) is very much dependent on the salt concentration in the formulation. Chymosin becomes less stable at higher strength and higher salt concentrations. As can be seen in Figure 1 , a formulation of 670 IMCU/ml is still stable up to 170 g NaCI/kg (diamonds). A formulation of 1070 IMCU/ml however already becomes instable at a NaCI concentration higher than 120 g/kg (triangles), while a formulation of 1230 IMCU/ml is unstable at salt concentrations exceeding 100 g/kg (squares). At high strength and high salt concentration, chymosin partly precipitates and is removed from solution by the filtration step. This is an unexpected result since until now all commercial liquid chymosin and calf rennet preparations are formulated at a high salt concentration (typically 130-150 g/kg). From our result it becomes clear that it is essential to lower the salt concentration if one wants to prevent precipitation of chymosin from high-strength (>1000 IMCU/ml) formulations.

Example 2

Testing physical stability of chymosin at different salt concentrations

An Accelerated Physical Stability Test (APST) was performed on the high strength Maxiren samples. In this test the enzyme preparation is stressed, and will form aggregates when the physical stability is poor. To test the effect of a low salt concentration on the stability of chymosin, we purified Maxiren 600 by hydrophobic interaction chromatography. Specifically Maxiren 600 was applied at lowered pH (4) on a chromatography column (1.6 cm I.D, 18 cm bed height butyl-Sepharose gel (GE Healthcare)) at 100 cm/hr. Subsequently the column was washed with an equilibration buffer (phosphate buffer, pH 4 including 0.2 M Na2S04). Chymosin was eluted using a 50 mM phosphate buffer containing no additional salt and elevated pH (pH =6.5). The eluate fraction was concentrated by means of ultrafiltration (as in example 1) to obtain a high chymosin strength. To preserve the microbial stability of the sample, glycerol at 50% (w/w) was added to the sample. Final strength of the chymosin preparation was 1520 IMCU/g. For the stability experiment NaCI was added to this chymosin preparation at 1 , 2, 5, 10, 20, 50, 100, and 150 g/kg. The pH of the formulation was set at pH5.4 after salt dissolution. For this test the turbidity was measured using a "Hach Turbidimeter - 2100N IS" using cuvettes with a volume of 30 ml. The liquid sample was measured undiluted. The cuvette was filled up with the sample (volume is about 30 ml) and placed in the measuring compartment. The Hach meter automatically measured the turbidity of the sample using a wavelength of 860 nm. The units of turbidity are expressed in NTU.

A 30 ml_ sample was put into a vial with a magnetic stirring bar and the vial was closed. In a Binder cooled incubator KB1 15 a multipoint magnetic stirrer was placed and the vials were placed in the incubator on a magnetic stirrer plate. Stir-setting was set at 150 rpm. The incubator was programmed for a temperature cycle of 6 hours at -4°C and 6 hours at +35°C. This cycle was repeated 14 times, meaning that the samples were incubated for 7 days. Turbidity was measured again after incubation. The difference between the turbidity before and after the APST test is a measure for the physical stability of the chymosin preparation.

The results of this experiment are shown in table 1. From these results it becomes clear that physical instability occurs in both the samples that have no or little salt addition, and the samples that have salt added at >100 g/kg. High strength chymosin preparations were stabile when salt was added to the formulation from 2 until 100 g/kg.

Table 1 NTU

NaCI cone (g/kg) Before APST After APST

0 7.61 24.0

1 7.54 22.8

2 7.76 10.7

5 7.70 10.1

10 7.24 13.5

20 6.73 9.95

50 5.85 13.3

100 5.96 8.57

150 5.40 49.1

The results from Example 1 and 2 show that a specific window of salt concentrations is required for the stabilization of chymosin. At a strength of >1000 IMCU/ml, a liquid formulation of chymosin should contain 2-100 g/kg salt to prevent precipitation of the enzyme. This is in contrast to chymosin formulations at lower strength (e.g. 600 IMCU/ml) that are stable also at higher salt concentrations

Example 3

Long term enzymatic stability of high-strength chymosin formulations

Maxiren 600 was purified by hydrophobic interaction chromatography as described in Example 2. Glycerol (50%(w/w)) was added as preservative, pH was adjusted to pH 5.4 or pH 5.8 and final strength of the formulation was set at 1200 IMCU/ml. Sodium chloride was added to the formulation at 0, 50 and 100 and g/kg. Formulated chymosin samples were stored at 4, 20 and 30 degrees Celsius for 1 , 2, 3 and 4 weeks. After this storage period the IMCU activity of each sample was determined in duplicate and compared to the activity of a sample in the same formulation that was stored at -20 °C.

Under the conditions in this procedure, the enzyme activity degradation is assumed to follow a first order model:

In (ACT time) = In (ACT 0) + K x time (K<0)

Where ACT time is the activity after storage, ACT 0 is the activity in the control sample, K is the inactivation constant in week-1 and time is measured in weeks. Using linear least squares regression, the inactivation constants for each condition are calculated from the regression line with an appropriate statistical program (e.g. SAS). Inactivation constants calculated from the fitted regression for the different samples are shown in Table 2

Table 2: Inactivation constants of different high strength chymosin formulations

Inactivation constant (weeks- 1)

From the results in these examples it becomes clear that the high strength formulations with highest storage stability do contain NaCI but not more than 100 g/kg. Apparently salt is required to obtain high enzymatic stability of chymosin, and the absence of salt dramatically decreases enzymatic stability (Example 3).

Examples 1 and 2 furthermore show that at high salt concentrations a high strength formulation becomes physically unstable. Consequently, the preferred formulation for chymosin at high strength (>1000 IMCU/ml) will have 2-100 g/kg salt.