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Title:
LIQUID COMPOSITION COMPRISING AN ASPARTIC PROTEASE HAVING IMPROVED ENZYME STABILITY
Document Type and Number:
WIPO Patent Application WO/2017/089613
Kind Code:
A1
Abstract:
The present invention relates to liquid Rhizomucor miehei compositions having improved microbiological stability and enzymatic stability.

Inventors:
STAPERSMA, Pietsje (Herenwaltje 10D, 8911 HN Leeuwarden, 8911 HN, NL)
KEVELAM, Jan (Laan van Kernhem 151, 6718 HR Ede, 6718 HR, NL)
Application Number:
EP2016/079022
Publication Date:
June 01, 2017
Filing Date:
November 28, 2016
Export Citation:
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Assignee:
CSK FOOD ENRICHMENT B.V. (PO Box 225, 8901 BA Leeuwarden, 8901 BA, NL)
International Classes:
C12N9/96; A23C19/032
Domestic Patent References:
WO2012127005A12012-09-27
WO1994024880A11994-11-10
WO2007118838A12007-10-25
WO2003100048A12003-12-04
Foreign References:
EP2333056A12011-06-15
EP2333056B12014-05-07
EP1365019A12003-11-26
Other References:
SIRMA YEGIN ET AL: "Aspartic proteinases fromspp. in cheese manufacturing", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 89, no. 4, 3 December 2010 (2010-12-03), pages 949 - 960, XP019880543, ISSN: 1432-0614, DOI: 10.1007/S00253-010-3020-6
ISO 7954, 1987
Attorney, Agent or Firm:
HGF LIMITED (GLASGOW) (1 City Walk, Leeds Yorkshire LS11 9DX, LS11 9DX, GB)
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Claims:
Claims

I. A liquid composition comprising (i) a Rhizomucor miehei aspartic protease; (ii) an inorganic salt; and (iii) propionic acid and a propionate salt in a total concentration of at least 0.1 mol/l;

wherein the liquid composition has a pH within the range of 4.00 - 4.75.

2. The liquid composition according to claim 1 having a pH of 4.3-4.7.

3. The liquid composition according to any one of the preceding claims, wherein the liquid

composition comprises sorbate, benzoate or alkyl esters of parahydroxybenzoate provided that the sum concentration of sorbate, benzoate and alkyl esters of parahydroxybenzoate is less than 0.010 mol/l and wherein preferably the liquid composition comprises no added sorbate, benzoate or alkyl esters of parahydroxybenzoate.

4. The liquid composition according to any one of the preceding claims having the following

microbiological properties: standard plate count < 100 in 1 ml; yeast count < 10 in 1 ml; mould count < 10 in 1 ml.

5. The liquid composition according to any one of the preceding claims comprising propionic acid and a propionate salt in a total concentration of at least 0.2 mol/l.

6. The liquid composition according to any one of the preceding claims wherein the inorganic salt comprises sodium chloride, and preferably wherein the liquid composition comprises sodium chloride in a concentration of 100-200 g/L.

7. The liquid composition according to any one of the preceding claims, having a pH within the range of 4.5 - 4.7.

8. A method of storing a liquid composition as defined in any one of the preceding claims, the method comprising storing the composition at a temperature of 25 °C or less for a storage period of at least one month

9. The method of claim 8 wherein the liquid composition retains at least 90% of its initial aspartic protease activity throughout the storage period.

10. The method of claim 8 or 9 wherein the liquid composition is stored in a container that is non- sterile or which has not been sterilized prior to filling.

II. The method of any one of claims 8 to 10 wherein the liquid composition is stored in a container that is in connection with piping which has previously carried animal rennet, and which may therefore comprise traces of animal rennet.

12. The method according to any one of claims 8 to 11, wherein the liquid composition is stored at a temperature of 14 °C or less, more preferably at a temperature of 10 °C or less.

13. Use of the liquid composition as defined in any one of claims 1-7 for producing cheese.

14. Use of the liquid composition according to claim 12 in a factory wherein on the same production lines, in addition or alternatively to the liquid composition, animal rennet compositions are used in producing cheese.

15. Use of the liquid composition according to any one of claims 12 or 14 wherein prior to cheese production the liquid composition is stored at a temperature of 14 °C or less, more preferably at a temperature of 10 °C or less, most preferably at a temperature of 7 °C or less.

16. Use of propionic acid or of a propionate in the manufacture of a liquid composition further comprising (i) a Rhizomucor miehei aspartic protease and (ii) an inorganic salt, for improving the enzymatic stability of said liquid composition.

Description:
LIQU ID COMPOSITION COM PRISING AN ASPARTIC PROTEASE HAVING IM PROVED E NZYME STABILITY Field of the invention

The present invention relates to a liquid composition comprising an aspartic protease and an organic acid, and associated methods and uses.

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. Aspartic proteases can be recovered from animals, e.g. from the stomach of calf, camel, and seal. Liquid aspartic protease compositions recovered from animals are herein referred to as animal rennet. Aspartic proteases can also be produced by micro-organisms, especially by Rhizomucor, or by host strains such as Aspergillus or Kluyveromyces (these will be referred to as microbial rennet). A well-known and commonly used microbial rennet is obtained from Rhizomucor miehei.

In the cheese making industry, liquid compositions comprising the aspartic protease are often used. Such liqu id compositions typically contain certain additives to obtain a desired stability. One can distinguish between enzymatic stability and microbial stability. The enzymatic stability is a measure for the rate at which the activity of the enzyme decreases. The microbial stability is a measure for the rate at which microorganisms can proliferate and grow in the composition.

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. It is well known to use sorbate or benzoate as a preservative to obtain a desired microbial stability. Parabens (alkyl esters of parahydroxybenzoate) can also be used.

However, there is a desire for products containing no or smaller amounts of sorbates, benzoates and pa rabens. WO2007118838 aims to provide a composition which has a good microbial stability in the absence of sorbates, benzoates and parabens. In one embodiment, WO2007118838 discloses a liquid composition comprising (i) an aspartic protease; and (ii) an inorganic salt and/or a polyalcohol, in which composition the sum concentration of sorbate, benzoate and alkyl esters of parahydroxybenzoate is less than 0.010 mol/l; the standard plate count < 100 in 1 ml; yeast count < 10 in 1 ml; and mould count < 10 in 1 ml. It should be noted, however, that the use of polyalcohols is not preferred because of their legislative status.

In another embodiment, WO2007118838 discloses a liquid composition comprising an aspartic protease and a compound selected from formate, acetate, lactate, propionate, malate, or fumarate. The use of acetate is disclosed as preferred. Claim 1 of EP2333056B1, a European family member of

WO2007118838, claims a liquid composition comprising (i) a Rhizomucor miehei aspartic protease; and (ii) an inorganic salt; and (iii) a compound selected from formate, acetate, lactate, propionate, malate or fumarate in which composition the sum concentration of sorbate, benzoate and alkyl esters of parahydroxybenzoate is less than 0.010 mol/l; the standard plate count < 100 in 1 ml; yeast count < 10 in 1 ml; mould count < 10 in 1 ml, and wherein the pH is between 4.8 and 5.5, and in which the concentration of formate, acetate, lactate, propionate, malate or fumarate or combination thereof is at least 0.1 mol/l. WO2007118838 teaches that liquid composition comprising an aspartic protease, acetate and an inorganic salt having pH levels below 5.0 display poor enzymatic stability (see, for example, page 13).

Summary of the invention

The present inventors found that the microbial stability of a liquid composition comprising a Rhizomucor miehei aspartic protease, an inorganic salt and an organic acid was significantly improved if the pH of the composition was set to below the pKa of the organic acid. For example, if acetic acid was employed as organic acid, the microbial stability of the composition was found to be improved at pH = 4.6 as compared with the same composition having a pH of 5.0.

Surprisingly, it was found that if acetate was at least partially replaced by propionate, microbiological stability of the composition would further improve.

Further surprisingly, the present inventors found that if acetate was at least partially replaced with propionate in a liquid Rhizomucor miehei composition, the enzyme stability could be significantly improved during storage over a wide temperature and pH range. Indeed, within a preferred pH range of 4.00 - 4.75, more preferably within a pH range of 4.3 - 4.7, most preferably within a pH range of 4.5 - 4.7, an optimal balance has been achieved between microbial stability and enzymatic stability.

Favourably, within these pH ranges and especially at 4.5 < pH < 4.7 the composition can be robustly buffered using approximately equimolar concentrations of propionic acid and a propionate salt (preferably sodium propionate or potassium propionate or a mixture thereof.

In a first aspect, therefore, the present invention relates to a liquid composition comprising (i) a Rhizomucor miehei aspartic protease; and (ii) an inorganic salt; and (iii) propionic acid and a propionate salt in a total concentration of at least 0.1 mol/l, wherein the pH is within the range of 4.00 - 4.75.

The liquid composition may further contain formate, acetate, lactate, malate or fumarate provided that the total concentration of formate, acetate, lactate, propionate, malate or fumarate or combination thereof preferably does not exceed the total concentration of propionic acid and the propionate salt by more than 50%. Thus, by way of example, if the total concentration of propionic acid and the propionate salt in the liquid composition will be 0.2 mol/l, the total concentration of formate, acetate, lactate, propionate, malate or fumarate or combination thereof preferably may not exceed 0.3 mol/l.

Preferably the sum concentration of sorbate, benzoate and alkyl esters of parahydroxybenzoate in the combination is less than 0.010 mol/l.

In another aspect, the invention provides a use of propionate in the manufacture of a liquid composition further comprising (i) a Rhizomucor miehei aspartic protease and (ii) an inorganic salt for improving the enzymatic stability thereof.

The liquid composition preferably has a pH within the range of 4.0 - 4.75, even more preferably 4.3 - 4.7 and most preferably 4.5 - 4.7. These pH ranges represent preferred features of all aspects and embodiments of the invention. In a further aspect, the present invention relates to a liquid composition comprising (i) a Rhizomucor miehei aspartic protease; (ii) an inorganic salt; and (iii) propionic acid and a propionate salt in a total concentration of at least 0.1 mol/l; wherein the liquid composition has a temperature of 25 °C or less and a pH within the range of 4.00 - 4.75.

Preferably the composition has a temperature of 14 °C or lower, more preferably 10 °C or lower, most preferably 7 °C or lower. It should be noted that these temperatures represent preferred features of all aspects and embodiments of the invention.

Preferably, in said liquid composition the sum concentration of sorbate, benzoate and alkyl esters of parahydroxybenzoate is less than 0.010 mol/l. This 0.010 mol/l upper limit to the sum concentration of these preservatives represents preferred features of all aspects and embodiments of the invention.

Preferably, said liquid composition has the following microbiological properties: standard plate count < 100 in 1 ml; yeast count < 10 in 1 ml; mould count < 10 in 1 ml. Preferably the composition has a pH within the range of 4.3 - 4.75, more preferably 4.5 - 4.7, and suitably a pH of 4.6, for example. These pH ranges represent preferred features of all aspects and embodiments of the invention.

In yet another aspect, the present invention provides a method of storing a liquid composition comprising:

(i) a Rhizomucor miehei aspartic protease;

(ii) an inorganic salt; and

(iii) propionic acid and a propionate salt in a total concentration of at least 0.1 mol/l, at a temperature of 25 °C or less and at a pH within the range of 4.00 - 4.75, for a storage period of at least one month, wherein the liquid composition retains at least 90% of its initial aspartic protease activity throughout the storage period.

Preferably, in said liquid composition the sum concentration of sorbate, benzoate and alkyl esters of parahydroxybenzoate is less than 0.010 mol/l.

Preferably, said liquid composition retains the following microbiological properties throughout the storage period : standard plate count < 100 in 1 ml; yeast count < 10 in 1 ml; mould count < 10 in 1 ml. In a further aspect, the invention provides a use of propionic acid and a propionate salt in a total concentration of at least 0.1 mol/l in a liquid composition further comprising a Rhizomucor miehei aspartic protease and an inorganic salt for preserving the microbiological stability for a storage period of at least one month and, in addition, keeping the aspartic protease activity of the liquid composition to at least 90% of its initial value throughout the storage period, wherein the liquid composition is stored at a temperature of 25 °C or less and has a pH within the range of 4.00 - 4.75.

As used herein, the expression "preserving the microbiological stability", and linguistic variations thereof, preferably means "retaining the following microbiological properties of the liquid composition throughout the relevant storage period: standard plate count < 100 in 1 ml; yeast count < 10 in 1 ml; mould count < 10 in 1 ml."

Most preferably, in said liquid composition the sum concentration of sorbate, benzoate and alkyl esters of parahydroxybenzoate is less than 0.010 mol/l.

It is noted that in one embodiment of WO2007118838, cf. page 8, storage of a liquid composition comprising an aspartic protease is disclosed, the composition having less than 5% decrease of enzymatic activity during a period of at least 4 months when stored in a closed container at 4 or 30 °C in the dark. Furthermore, in another embodiment of WO2007118838, storage of a liquid composition comprising an aspartic protease is disclosed, wherein standard plate count remains <= 100 in 1 ml, yeast count remains <= 10 in 1 ml and mould count remains < 10 in 1 ml during a period of at least 4 months when stored in a closed container at 4 or 30 °C in the dark. However, firstly these embodiments are very broad and do not disclose relevant details such as which type of aspartic protease is provided, whether the composition comprises an in organic salt or a polyalcohol, which organic acid is comprised at which concentration, what is the pH of the composition etc. Secondly these embodiments all relate to storage conditions comprising a closed container which is sterilized prior to filling. By contrast, the liquid compositions according to the present invention can be filled into containers which have not been previously sterilized and are still well-protected against loss of microbial stability, thus avoiding microbiological spoilage even when non-sterile conditions are used. In comparison with the preferred compositions of EP23333056B1 (cf. claim 3), the liquid compositions according to the present invention are especially more robust in challenge tests (see below for explanation) involving microorganisms selected from the group consisting of Staphylococcus subsp., Chromohalobacter subsp. and Debaryomyces hansenii subsp, in particular at storage temperatures of 25 °C or lower, more preferably at 14 °C or lower, yet more preferably at 10 °C or lower, most preferably at 7 °C or lower. Interestingly, it has been found that it is generally easier to reduce the viable count of these spoilage micro-organisms in liquid Rhizomucor miehei compositions by propionate (or acetate) at storage temperatures which in the absence of an added organic acid would be beneficial for their growth (e.g. 25 °C) than at lower storage temperatures at which the microorganisms can survive but not grow rapidly (e.g. 5°C). However it was found that within the claimed pH range (involving pH < pKa of propionic acid) in the liquid composition comprising propionate the above-indicated microorganisms, especially Staphylococcus subsp. and/or Debaryomyces hansenii, are much better inhibited or killed at 5 °C than in corresponding liquid compositions having pH > pKa. Significant differences were observed in microbiological stability at a storage temperature of 5 °C between liquid Rhizomucor miehei compositions having pH 4.6 (according to present invention) or having a higher pH (e.g. according to claim 3 of EP23333056B1).

Given that Staphylococcus subsp. or Chromohalobacter subsp. may be present in animal rennet compositions, use of the liquid composition according to the present invention in producing cheese provides great advantages in those cheese production facilities where cross contamination risks occur involved with use of liquid animal rennet and liquid Rhizomucor miehei compositions in the same production line, especially if the same piping is used for transporting both types of liquid coagulant compositions into a cheese vat. Thus, in an especially preferred embodiment there is provided a method of storing a liquid composition comprising

(i) a Rhizomucor miehei aspartic protease;

(ii) an inorganic salt; and

(iii) propionic acid and a propionate salt in a total concentration of at least 0.1 mol/l and having a pH within the range of 4.00 - 4.75

at a temperature of 25 °C or lower (preferably 14 °C or lower, more preferably 10 °C or lower or most preferably 7 °C or lower) during a storage period of at least 1 month (suitably at least 3 months, and optionally at least 6 months), wherein the liquid composition retains at least 90% of its initial aspartic protease activity throughout the storage period; and

wherein the liquid composition is stored in a container that has not been sterilized prior to filling and/or wherein the liquid composition is stored in a container that is in connection with piping comprising traces of animal rennet.

Preferably, in said liquid composition the sum concentration of sorbate, benzoate and alkyl esters of parahydroxybenzoate is less than 0.010 mol/l.

Preferably, said liquid composition retains the following microbiological properties throughout the storage period: standard plate count < 100 in 1 ml; yeast count < 10 in 1 ml; mould count < 10 in 1 ml. Most preferably, in said liquid composition the sum concentration of sorbate, benzoate and alkyl esters of parahydroxybenzoate is less than 0.010 mol/l.

In a further aspect, the invention provides use of a liquid composition comprising

(i) a Rhizomucor miehei aspartic protease;

(ii) an inorganic salt; and

(iii) propionic acid and a propionate salt in a total concentration of at least 0.1 mol/l and having a pH within the range of 4.00 - 4.75 (preferably within the range of 4.3 - 4.75, more preferably within the range of 4.5 - 4.7) for producing cheese in a factory wherein on the same production lines, in addition or alternatively to the liquid composition, animal rennet compositions are used in producing cheese and wherein the liquid composition is preferably stored at a temperature of 25 °C or lower (preferably of 10 °C or lower, most preferably 7 °C or lower).

Suitably the use is on a production line in which both the composition and animal rennet compositions are used in producing cheese.

As discussed above, the microbiological properties throughout the storage period can suitably be characterized by the feature: "standard plate count < 100 in 1 ml; yeast count < 10 in 1 ml; mould count < 10 in 1 ml". However, in preferred embodiments of the present invention the microbiological properties of the liquid composition can be assessed by a so-called 'challenge test', whereby the liquid composition is inoculated with one or more known micro-organisms at predetermined inoculation levels, wherein the colony count density (in cfu/mL of the liquid composition) for the specific micro-organism(s) that has been purposefully added to the liquid composition is determined by plating on a suitable growth medium and under suitable growth conditions (e.g. as discussed above and in the examples below): immediately after inoculation (at t= tO) and

after storing the liquid composition for a certain storage period (at t = t2, wherein t2 - tO equals the storage period) which is preferably 4 weeks (but alternative times can be used), at a certain storage temperature which is preferably set at 25 °C or even more preferably at 5 °C.

The known micro-organisms used in the challenge test preferably comprise Staphylococcus pasteuri and/or Debaryomyces hansenii. A challenge test assesses the ability of a composition to prevent growth of (and in some cases actively kill or render incapable of replication) the introduced microorganisms.

In preferred embodiments of the present invention the microbiological properties of the liquid composition is such that in a challenge test as described above (e.g. a challenge test using

Staphylococcus pasteuri), the colony count density at t = t2 is equal to or lower than the colony count density at t = tO. More preferably, the colony count density at t=t0 is preferably about 1000 cfu/ml and the colony count density at t=t2 is preferably < 10 cfu/ml.

The expression "propionic acid and a propionate salt in a total concentration of at least 0.1 mol/l" means that the composition comprises propionic acid in both its undissociated and its dissociated forms and that the total concentration of propionic acid in all of said forms is at least 0.1 mol/l. In preferred embodiments of the present invention both propionic acid and propionate salt have been provided to the composition in order to obtain the desired concentration of propionic acid and a propionate salt. For ease of demonstration, the following experiment can be taken in mind: 7.4 g (0.1 mol) of pure propionic acid and 9.6 g (0.1 mol) of pure anhydrous sodium propionate are dissolved in water to obtain a total volume of 1 litre. In the language of this patent application, the resulting aqueous solution comprises propionic acid and a propionate salt in a total concentration of 0.2 mol/l. In some embodiments the propionic acid and propionate salt can be, for example, in a molar ratio of between 1:3 and 3:1 of the acid form to the salt form. In some preferred embodiments approximately equimolar amounts of propionic acid and propionate salt are provided. In the expression "propionic acid and a propionate salt", the term "propionate salt" may relate to any monovalent, divalent or trivalent metal salts of acetate, or any combination of a plurality of such salts. Preferred monovalent metal salts of propionate comprise potassium propionate and sodium propionate. Preferred divalent metal salts of propionate comprise iron (II) propionate, magnesium propionate and calcium propionate. Preferred trivalent metal salts of acetate comprise iron (iii) propionate. In preferred embodiments the "propionic acid and a propionate salt" is preferably propionic acid and a propionate salt selected from the group consisting of sodium propionate and potassium propionate, more preferably propionic acid and sodium propionate.

Regarding the expression "the sum concentration of sorbate, benzoate and alkyl esters of para- hydroxybenzoate", it will be understood that sorbic acid as well as salts of sorbic acid contribute to the concentration of sorbate; benzoic acid as well as salts of benzoic acid contribute to the concentration of benzoate; alkyl esters of para-hydroxybenzoate as well as salts of alkyl esters of para-hydroxybenzoate contribute to the concentration of alkyl esters of parahydroxybenzoate. Accordingly, as used herein, the sum concentration of sorbate, benzoate and alkyl esters of para-hydroxybenzoate refers to the sum concentration of sorbic acid, salts of sorbic acid, benzoic acid, salts of benzoic acid, alkyl esters of parahydroxybenzoate and salts of alkyl esters of para-hydroxybenzoate in the composition. Examples of salts of sorbic acid are sodium sorbate, potassium sorbate, and calcium sorbate. Examples of salts of benzoic acid are sodium benzoate, potassium benzoate and calcium benzoate. Examples of alkyl esters of para-hydroxybenzoates are methyl-p- hydroxybenzoate, ethyl-p-hydroxybenzoate and propyl-p- hydroxybenzoate. Examples of salts of alkyl esters of para-hydroxybenzoates are sodium salt of methyl- p- hydroxybenzoate, the sodium salt of ethyl-p-hydroxybenzoate, and the sodium salt of propyl-p- hydroxybenzoate.

The expression "enzyme activity" and "aspartic protease activity" can be used interchangeably herein and preferably are expressed as IMCU per ml of the liquid composition. In a preferred embodiment, the enzyme activity is at least 100 IMCU per ml of the liquid composition, preferably at least 200 IMCU per ml of composition, preferably at least 500 IMCU per ml of composition. There is no specific upper limit for the enzyme activity. The enzyme activity may be below 5000 IMCU per ml of composition, for instance less than 2000 IMCU per ml, for instance less than 1000 IMCU per ml of composition. IMCU refers to International Milk Clotting Unit, defined by the International Dairy Federation (IDF), protocol 176: 1996.

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).

Optionally the composition comprises a polyalcohol. 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, where a polyalcohol is present, the polyalcohol is glycerol, sorbitol or propanediol, more preferably glycerol or propanediol. The composition may comprise one or more polyalcohols. In a preferred embodiment, the composition comprises less than 40 g/l of a polyalcohol or of a combination of polyalcohols. It will be understood that it is possible that at least one polyalcohol is present in a preferred concentration as defined herein. It is also possible that a combination of polyalcohols is present in a concentration as defined herein. If a combination is of polyalcohols is employed, the concentration refers to the sum concentration of inorganic salts. In some embodiments of the present invention the polyalcohol comprises glycerol and/or propanediol. However, it is generally preferred that the liquid composition is free of an added polyalcohol.

According to the invention the use of benzoate, sorbate or para-hydroxybenzoate is not necessary or benzoate, sorbate or para-hydroxybenzoate can be used in smaller quantities. In a preferred embodiment, the composition according to the invention comprises less than 0.010 mol/l of benzoate, preferably less than 0.005 mol/l, preferably less than 0.002 mol/l, preferably less than 0.001 mol/l, preferably less than 0.0005 mol/l, preferably less than 0.0001 mol/l, preferably less than 0.00005 mol/l, preferably less than 0.00001 mol/l, preferably no detectable amount. In a further preferred embodiment, the sum concentration of sorbate, benzoate and alkyl esters of para-hydroxybenzoates in the composition according to the invention is less than 0.010 mol/l, preferably less than 0.005 mol/l, preferably less than 0.002 mol/l, preferably less than 0.001 mol/l, preferably less than 0.0005 mol/l, preferably less than 0.0001 mol/l, preferably less than 0.00005 mol/l, preferably less than 0.00001 mol/l, preferably no detectable amount. It is known that benzoate, sorbate and parahydroxybenzoate may be used to kill the microorganisms after a fermentation. Accordingly, small amounts of these compounds may be present in the composition resulting from such killing step.

Any suitable inorganic salt may be used. The inorganic salt may for instance be a salt which comprises a cation selected from the group consisting of (CH 3 ) N + , NH 4 + , K + , Na + , Ca 2+ and Mg 2+ and an anion selected from the group consisting of SO4 2" , CI " , Br " , N03 " , CIO4 " and SCN " . Preferred inorganic salts are NaCI, KCI, Na 2 S0 4 or CaCI 2 . The composition may contain one or more inorganic salts. In a preferred embodiment, the composition comprises at least 80 g/l of an inorganic salt or of a combination of inorganic salts, preferably at least 100 g/l, more preferably at least 120 g/l, more preferably at least 140 g/l. An increased salt concentration has the effect that the water activity of the composition is increased, which can assist to achieve a desired microbial stability. It will be understood that it is possible that at least one inorganic salt is present in a preferred concentration as defined herein. It is also possible that a combination of inorganic salts is present in a concentration as defined herein. If a combination is of inorganic salts is employed, the concentration refers to the sum concentration of inorganic salts. In a preferred embodiment, the composition comprises at least 80 g/l of NaCI, preferably at least 100 g/l, more preferably at least 120 g/l, more preferably at least 140 g/l. The upper limit to the salt concentration is determined by the solubility limit of the salt in water at a certain desired storage temperature of the liquid composition. It is especially preferred that the liquid composition comprises NaCI in a concentration of 130-180 g/l, most preferably 140-160 g/l.

In an embodiment, the liquid composition further comprises a reducing agent, preferably methionine or ascorbic acid or a mixture thereof. The liquid composition may comprise at least 1 g/l of methionine, preferably at least 2 g/l, more preferably at least 5 g/l, for instance less than 100 g/l, for instance less than 30 g/l. However, the presence of a reducing agent is not a prerequisite for keeping sufficient enzymatic stability, especially if the liquid composition is kept at a temperature of 10 °C or below, or preferably at 7 °C or below. Thus in a preferred embodiment, the liquid composition comprises less than 1 g/l of methionine and/or less than 50 g/l of ascorbic acid. In every embodiment of the invention and most preferably in the absence of methionine concentrations of 1 g/l or higher, the liquid composition is preferably not kept at a temperature of 15 °C or higher for a period of 1 week or longer, most preferably for a period of 12 hours or longer.

The liquid composition can be prepared according to any known method. Known methods are presented in EP1365019, W003/100048 and WO2007/118838. A culture of Rhizomucor miehei can be cultured as described in EP1365019. At the end of fermentation the broth can be cooled, the fungus killed off and separated from the liquid using a membrane filter press and polish filtration. The milk clotting protease was subsequently purified using chromatography as described in W003/100048. WO2007/118838, for example, describes a suitable basic method of making a microbial rennet composition comprising acetate, and the method comprises providing a fermentation broth containing microorganisms for prod ucing the protease and supernatant containing the protease; separating the supernatant from the fermentation broth; purifying the separated supernatant, to obtain a purified solution containing the microbial rennet; add ing one or more additives to the purified solution. After the additives have been added, WO2007/118838 expla ins that the composition must be filtered. In the context of the present invention the filtration step can optionally be omitted because of the intrinsic microbiological stability of the composition. Examples

Challenge tests involving liquid Rhizomucor miehei compositions

Materials:

Ref 1: a commercially available Rhizomucor miehei composition (Fromase ® 220 TL BF, ex DSM

Food Specialties, The Netherlands), having a pH of 5.4-6.1, and comprising sodium chloride in a concentration of 140-180 g/l. In such compositions the sum concentration of acetate and acetic acid has been determined to be 1.0 - 1.3 % (wt/wt) as determined using H PLC.

Ref 2: a commercially available Rhizomucor miehei composition (Milase ® 750, ex CSK Food

En richment BV), 18 wt % sodium chloride, having a pH of 5.0 and comprising no added organic acids. Ref 3: a commercially available Rhizomucor miehei composition (Milase ® 750, ex CSK Food Enrichment BV) , 18 wt % sodium chloride, having a pH of 5.0 and comprising 0.5 wt.% sodium benzoate.

Ex 1: a liquid composition obtainable by mixing a Milase ® concentrate (1400 +/- 100 IMCU/ml), 15 wt % sodium chloride, composition with sodium acetate and setting the pH to 4.6. Sodium acetate was added in a concentration of 2.0% (wt/wt). The sum concentration of acetate and acetic acid was 1.2 % (wt/wt), as determined using HPLC.

Ex 2: a liquid composition obtainable by mixing a Milase ® concentrate (1400 +/- 100 IMCU/ml), 15 wt % sodium chloride, composition with sodium propionate and setting the pH to 4.6 using HCI. Sodium propionate was added in an amount of 2.0% (wt/wt). The sum concentration of propionate and propionic acid was 1.2 % (wt/wt), as determined using HPLC.

The enzyme activity of the Fromase ® composition, Ref 1, is about 220 IMCU/ml and the enzyme activity of the Milase ® compositions, Refs 2 and 3, is about 750 IMCU/ml, as indicated in the product names. The enzyme activity of Milase ® compositions Ex 1 and 2, is 1400 +/- 100 IMCU/ml.

It should be noted that various Rhizomucor miehei aspartic protease formulations are available commercially (e.g. in concentrated and dilute forms) and can readily be formulated as required. PCMA Agar (non-specific growth medium used herein for determining viable cell count of

Staphylococcus pasteuri and Chromohalobacter):

Mix 42g Plate Count Agar (Oxoid CM325) and 2.4g skim milk powder (Oxoid L31) into 2400 g of demineralized water, leave to soak for 15 mins and sterilize for 15 min at 121 °C. The resulting PCMA agar can be kept for 3 weeks at 4-7 °C.

OGY Agar (specific growth medium for yeasts and moulds used herein for determining viable cell count of Debaryomyces hansenii)

Mix 88.8g Oxytetracycline-Glucose-Yeast Extract Agar (Oxoid CM 545) into 2400 mL, leave to soak for 15 mins and sterilize for 10 min at 121 °C. The resulting OGY agar can be kept for 3 weeks at 4-7 °C.

Dilution medium for preparing lOx, lOOx, lOOOx etc. dilutions: Ringer Oxoid BR52 (4 tablets in 2L water; sterilize for 15 min at 121 °C).

The following strains were used for inoculating these compositions in the examples given below:

Staphylococcus pasteuri isolated from a liquid animal rennet

Chromohalobacter subsp. isolated from a liquid animal rennet

Debaryomyces hansenii isolated from a liquid animal rennet and being a common spoilage organism that can be found in industrial brining solutions for Gouda cheese

Viable cell counts for these micro-organisms were determined as follows:

for Staphylococcus pasteuri and Chromohalobacter : add 1 mL of sample (appropriately diluted into Ringer) onto a petri dish with 90 mm diameter and add 15 mL molten PCMA agar having a temperature of 45 °C. Mix well and leave for 3 days at 30 °C. Count the number of colonies and multiply by the dilution factor to obtain the colony count density (also referred to as "viable cell count") in cfu/mL. The number of colonies that can be counted should be less than 300 per plate.

for Debaryomyces hansenii: add 1 mL of sample (appropriately diluted into Ringer) onto a petri dish with 90 mm diameter and add 15 mL molten OGY agar having a temperature of 45 °C. Mix well and leave for 5 days at 25 °C. Count the number of colonies and multiply by the dilution factor to obtain the colony count density (also referred to as "viable cell count") in cfu/mL. The number of colonies that can be counted should be less than 200 per plate.

Viable cell counts of these micro-organisms were determined in the liquid compositions before and after incubation at 5 or 25 °C for 4 weeks in closed plastic bottles which were used as received (i.e. not sterilized before use). The results are depicted in Tables 1 and 2. In Table 1, "before" = before storage of the compositions and just after inoculation of the liquid compositions; "after" = after storing the liquid composition for 4 weeks at the indicated temperatures; 0E1 means no colony forming units counted on a 10-fold dilution plate. Table 1. Viable cell counts of spoilage microorganisms relevant for cheese industry, in liquid

Rhizomucor miehei compositions both comprising acetate and having different pH values, before and after incubation during 4 weeks at different temperatures.

Effectiveness of four different liquid Rhizomucor miehei compositions in preventing outgrowth or killing of three different spoilage microorganisms relevant for the cheese industry.

Herein, ++ indicates complete killing after 4 weeks; +/- indicates > 1 log reduction of viable cell count after 4 weeks but no complete killing achieved( cfu/ml > 1E1 after 4 weeks' incubation); -- indicates < 1 log reduction of viable cell count after 4 weeks. It can be concluded that Exl, the liquid Rhizomucor miehei composition according to the invention and comprising acetate at pH 4.6, has better microbiological properties than Refl, a Rhizomucor miehei composition according to the invention and comprising acetate at 5.4 < pH < 6.1. Furthermore, Ex2, comprising propionate instead of acetate, has further improved microbiological properties in that complete killing of all tested microorganisms was achieved after 4 weeks.

In addition, enzyme stability tests were performed wherein milk clotting activity (IMCU/ml) was performed before and after storing liquid Rhizomucor miehei compositions at 6 and 25 °C. Enzymatic stability decreased more strongly in compositions having pH 4.6 and comprising acetate as compared with other compositions comprising no additives or comprising acetate and having pH 5.0. However, interestingly, when stored for 6 months at a temperature of 6 °C, decrease of enzymatic stability was 5% or less at each pH value (4.6 or 5.0) for each variant tested. Furthermore it appeared that propionate appeared to have a stabilizing effect on the enzyme, as compared with acetate at the same pH.

Table 3. Enzyme stability after storage

Sample Storage temperature Residual enzyme activity

after 6 months' storage (%)

pH 5.0, no additives 5 °C > 95

25 °C 65

pH 5.0, 0.4% sodium acetate 5 °C > 95

25 °C 65

pH 4.6, 2% sodium acetate 5 °C > 95

25 °C 58

pH 4.6, 2% sodium 5 °C > 95

propionate

25 °C 78