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WO2012127005A1 - Formulation de chymosine - Google Patents

Formulation de chymosine Download PDF

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Publication number
WO2012127005A1
WO2012127005A1 PCT/EP2012/055131 EP2012055131W WO2012127005A1 WO 2012127005 A1 WO2012127005 A1 WO 2012127005A1 EP 2012055131 W EP2012055131 W EP 2012055131W WO 2012127005 A1 WO2012127005 A1 WO 2012127005A1
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WO
WIPO (PCT)
Prior art keywords
chymosin
salt
concentration
imcu
strength
Prior art date
Application number
PCT/EP2012/055131
Other languages
English (en)
Inventor
Petrus Jacobus Theodorus Dekker
DEN Jeroen HOLLANDER
Original Assignee
Dsm Ip Assets B.V.
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Filing date
Publication date
Application filed by Dsm Ip Assets B.V. filed Critical Dsm Ip Assets B.V.
Publication of WO2012127005A1 publication Critical patent/WO2012127005A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)
    • C12Y304/23004—Chymosin (3.4.23.4), i.e. rennin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0326—Rennet produced by fermentation, e.g. microbial rennet; Rennet produced by genetic engineering

Definitions

  • the present invention relates to a liquid formulation comprising chymosin at a high strength.
  • 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.
  • liquid compositions comprising chymosin are often used. Such liquid compositions typically contain certain additives to obtain a desired 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.
  • liquid chymosin formulations 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides a liquid composition comprising:
  • the invention provides a liquid composition.
  • the liquid composition is an aqueous composition, for instance an aqueous solution.
  • 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.
  • 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.
  • the invention provides a liquid composition comprising:
  • composition comprises at least 96, 97, 98 or 99 wt % of water.
  • 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.
  • IMCU 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).
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • chymosin from other species may be produced recombinantly.
  • Recombinant camel chymosin is also available commercially and is included in this invention.
  • the invention provides a liquid composition comprising:
  • chymosin is bovine or cameloid chymosin.
  • 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.
  • the chymosin in a composition according to the invention is thus preferably a fermentation produced chymosin.
  • the invention thus provides a liquid composition comprising:
  • the invention thus provides a liquid composition comprising: chymosin at a strength of at least 1000 IMCU/ml
  • the invention provides a liquid composition comprising:
  • 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.
  • 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.
  • a composition according to the invention comprises chymosin B.
  • the invention thus provides a liquid composition comprising:
  • the invention thus provides a liquid composition comprising: chymosin at a strength of at least 1000 IMCU/ml
  • the invention provides a liquid composition comprising: chymosin at a strength of at least 1000 IMCU/ml
  • 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.
  • chymosin 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.
  • 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.
  • 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
  • the micro organism is Aspergillus or Kluyveromyces. Even more preferably, the micro organism is Kluyveromyces.
  • 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:
  • the composition may have any suitable pH.
  • the composition has a pH of less than 7, preferably less than 6.
  • 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
  • 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.
  • 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:
  • 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.
  • the standard plate count can be ⁇ 100 in 1 ml
  • the yeast count can be ⁇ 10 in 1 ml
  • the mould count can be ⁇ 10 in 1 ml.
  • 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.
  • 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).
  • the standard plate count remains ⁇ 100 in 1 ml
  • the yeast count remains ⁇ 10 in 1 ml
  • 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.
  • the standard plate count remains ⁇ 100 in 1 ml
  • the yeast count remains ⁇ 10 in 1 ml
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention provides a liquid composition comprising: chymosin at a strength of at least 1000 IMCU/ml
  • salt refers to an inorganic salt which decomposes in ions upon solution in water.
  • the invention therefore provides a liquid composition comprising:
  • the salt ions are divided in cations and anions.
  • 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.
  • 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:
  • 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.
  • the anion is a chloride ion.
  • the salt is sodium chloride.
  • 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.
  • the invention provides a liquid composition comprising:
  • the salt ranges mentioned herein refer to the sum (i.e. total) concentrations of inorganic salt in the composition.
  • a liquid composition comprising:
  • a liquid composition comprising:
  • a liquid composition comprising:
  • a liquid composition comprising:
  • 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.
  • 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
  • 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.
  • 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.
  • the invention therefore provides a liquid composition comprising:
  • preservative is (i) a polyalcohol or (ii) an organic acid or any a combination thereof.
  • 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.
  • the polyalcohol is glycerol, sorbitol or propanediol, more preferably glycerol or propanediol.
  • the composition may comprise one or more polyalcohols.
  • 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.
  • the preservative is a polyalcohol which is present in an amount of 100-500 g/kg.
  • the invention thus provides a liquid composition comprising:
  • preservative is a polyalcohol in a concentration of 100-500 g/kg.
  • a liquid composition comprising:
  • preservative is a polyalcohol in a concentration of 100-500 g/kg.
  • a liquid composition comprising:
  • preservative is a polyalcohol in a concentration of 100-500 g/kg.
  • a liquid composition comprising:
  • preservative is a polyalcohol in a concentration of 100-500 g/kg.
  • a liquid composition comprising:
  • preservative is a polyalcohol in a concentration of 100-500 g/kg.
  • a composition according to the invention comprises an organic acid as a preservative.
  • 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.
  • 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:
  • 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.
  • organic acid is present in a concentration of 3 to 5 g/kg.
  • a liquid composition comprising:
  • 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.
  • organic acid is present in a concentration of 3 to 5 g/kg.
  • a liquid composition comprising:
  • 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.
  • organic acid is present in a concentration of 3 to 5 g/kg.
  • a liquid composition comprising:
  • 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.
  • organic acid is present in a concentration of 3 to 5 g/kg.
  • a liquid composition comprising:
  • 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.
  • organic acid is present in a concentration of 3 to 5 g/kg.
  • composition according to the invention can comprise additional components such as an antioxidant or a colorant e.g. caramel.
  • the composition comprises an antioxidant, preferably methionine, cysteine, glutathione, yeast extract, ascorbate or sulphite.
  • 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.
  • 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.
  • compositions are:
  • chymosin is fermentation produced chymosin, preferably chymosin B
  • o chymosin at a strength between 1000 and 1250 IMCU/ml o salt in a concentration of 2-100 g/kg
  • chymosin is fermentation produced chymosin, preferably chymosin B
  • o chymosin at a strength between 1000 and 1250 IMCU/ml o salt in a concentration of 2-100 g/kg
  • chymosin is fermentation produced bovine or cameloid chymosin, preferably chymosin B
  • liquid composition comprising: o chymosin at a strength of at least 1250 IMCU/ml
  • chymosin preferably NaCI, in a concentration of 2-90 g/kg, and o a preservative, wherein said chymosin is fermentation produced chymosin, preferably chymosin B
  • chymosin 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:
  • 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:
  • 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:
  • chymosin 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:
  • 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:
  • 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
  • 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.
  • Maxiren 600 is a commercial enzyme preparation of a recombinant produced bovine chymosin that can be obtained from DSM Food-Specialties (Delft, the Netherlands).
  • 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.
  • the NaCI concentration in the ultrafiltrated Maxiren was varied 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.
  • 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.
  • 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.
  • Example 1 and 2 show that a specific window of salt concentrations is required for the stabilization of chymosin.
  • 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
  • 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.
  • 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.
  • 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
  • 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.

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Abstract

La présente invention concerne une formulation liquide comprenant de la chymosine à une concentration élevée.
PCT/EP2012/055131 2011-03-22 2012-03-22 Formulation de chymosine WO2012127005A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015007638A1 (fr) * 2013-07-18 2015-01-22 Chr. Hansen A/S Composition d'enzyme protéase aspartique pour le caillage du lait
WO2016005587A1 (fr) * 2014-07-11 2016-01-14 Chr. Hansen A/S Composition d'enzyme protéase à acide aspartique pour le caillage du lait
WO2017089613A1 (fr) * 2015-11-26 2017-06-01 Csk Food Enrichment B.V. Composition liquide comprenant une protéase aspartique ayant une stabilité enzymatique améliorée
WO2018228966A1 (fr) 2017-06-15 2018-12-20 Dsm Ip Assets B.V. Granulés d'enzyme congelés

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FR2140642A1 (fr) * 1971-06-10 1973-01-19 Baxter Laboratories Inc
WO1990015866A1 (fr) * 1989-06-13 1990-12-27 Genencor International, Inc. Procedes pour la recuperation de chymosine produite naturellement
WO1990015865A1 (fr) 1989-06-13 1990-12-27 Genencor International, Inc. Recuperation et purification de chymosine
WO1995029999A2 (fr) 1994-05-03 1995-11-09 Chr. Hansen A/S Procede de separation d'enzymes de caillage du lait, et compositions stables de presure
WO1996019582A1 (fr) 1994-12-21 1996-06-27 Chr. Hansen A/S Rennine derivee d'un microbe presentant une activite de coagulation du lait accrue et son procede de production
WO2007118838A1 (fr) 2006-04-13 2007-10-25 Dsm Ip Assets B.V. Composition liquide comprenant une protease aspartique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2140642A1 (fr) * 1971-06-10 1973-01-19 Baxter Laboratories Inc
WO1990015866A1 (fr) * 1989-06-13 1990-12-27 Genencor International, Inc. Procedes pour la recuperation de chymosine produite naturellement
WO1990015865A1 (fr) 1989-06-13 1990-12-27 Genencor International, Inc. Recuperation et purification de chymosine
WO1995029999A2 (fr) 1994-05-03 1995-11-09 Chr. Hansen A/S Procede de separation d'enzymes de caillage du lait, et compositions stables de presure
WO1996019582A1 (fr) 1994-12-21 1996-06-27 Chr. Hansen A/S Rennine derivee d'un microbe presentant une activite de coagulation du lait accrue et son procede de production
WO2007118838A1 (fr) 2006-04-13 2007-10-25 Dsm Ip Assets B.V. Composition liquide comprenant une protease aspartique
US20100009033A1 (en) * 2006-04-13 2010-01-14 Hann Andre De Liquid composition comprising an aspartic protease

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015007638A1 (fr) * 2013-07-18 2015-01-22 Chr. Hansen A/S Composition d'enzyme protéase aspartique pour le caillage du lait
RU2684457C2 (ru) * 2013-07-18 2019-04-09 Кхр. Хансен А/С Композиция молокосвертывающего фермента аспарагиновой протеазы
US10779552B2 (en) 2013-07-18 2020-09-22 Chr. Hansen A/S Stabilization of chymosin by polymers
US11234446B2 (en) 2013-07-18 2022-02-01 Chr. Hansen A/S Stabilization of chymosin by polymers
WO2016005587A1 (fr) * 2014-07-11 2016-01-14 Chr. Hansen A/S Composition d'enzyme protéase à acide aspartique pour le caillage du lait
CN106661567A (zh) * 2014-07-11 2017-05-10 科.汉森有限公司 凝乳天冬氨酸蛋白酶组合物
CN106661567B (zh) * 2014-07-11 2021-08-13 科.汉森有限公司 凝乳天冬氨酸蛋白酶组合物
WO2017089613A1 (fr) * 2015-11-26 2017-06-01 Csk Food Enrichment B.V. Composition liquide comprenant une protéase aspartique ayant une stabilité enzymatique améliorée
WO2018228966A1 (fr) 2017-06-15 2018-12-20 Dsm Ip Assets B.V. Granulés d'enzyme congelés

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