WO2002074098A1

WO2002074098A1 - Cheese-making process

Info

Publication number: WO2002074098A1
Application number: PCT/EP2002/003345
Authority: WO
Inventors: Claus Dambmann; Peter Budtz
Original assignee: Dsm Ip Assets B.V.
Priority date: 2001-03-21
Filing date: 2002-03-21
Publication date: 2002-09-26
Also published as: EP1370147A1

Abstract

The present invention describes a process for making cheese, comprising: a) adding a endoprotease having a specificity for peptide bonds on the amino or carboxyl side of one or two amino acids and the ability to hydrolyze one or more bonds in the partial amino acid sequence between positions 106 and 131 in K-casein-casein to milk, b) incubating so as to partially hydrolyze K-casein-casein in the milk, and c) during or after step b) conditions causing clotting of the milk.

Description

Cheese-making process

Technical field

This invention relates to a cheese-making process.

Background Art

In the production of cheese it is necessary to coagulate the cheese-milk to be able to separate the cheese matters e.g. casein from the whey. Products containing chymosin, which is a milk-coagulating enzyme isolated from the fourth stomach of calf, have for many years been used for this purpose. Shortage of calf stomachs has in the last decades resulted in intense research for other milk coagulating enzymes. Today also bovine pepsin, porcine pepsin as well as microbial enzymes are used commercially. All these known milk-clotting enzymes are characterized by having specificity for the peptide bond between residues 105 (phenylalanine) and 106 (methionine) or a bond adjacent to that in κ-casein. This means that by employing these enzymes in cheese-making, the κ-casein is split at the junction between para-κ-casein and the macro-peptide moiety called glyco-macro-peptide (GMP) carrying the negative charges. When this occurs the macro-peptide diffuses into the whey, its stabilizing effect on the casein micelle is lost, and the casein micelles can start to aggregate once sufficient of their κ-casein has been hydrolysed. For further elaboration on the enzymatic coagulation of milk see e.g. D.G. Dalgleish in Advanced Dairy Chemistry vol 1 ed by P.F. Fox Elsevier. London, 1992. It is an object of this invention to provide a novel cheese-making process using a different proteolytic enzyme from known methods. It is also an object of the invention to provide a method having a higher cheese yield.

Summary of the invention In this invention it is surprisingly found that it is not necessary to hydrolyse the peptide bond between residues 105 (phenylalanine) and 106 (methionine) or a bond adjacent to that in κ-casein to effect clotting of cheesemilk. It is found that other specific proteases hydrolysing bonds between bond 105 and 106 and the glycosylated part of the κ-casein-casein will lead to clotting, especially at high calcium concentration. By clotting the milk in this way, a larger part of theκ-casein is retained in the cheese and a higher yield can be obtained.

Accordingly, the invention provides a process for making cheese, comprising: a) adding a endo-protease having a specificity for peptide bonds on the amino or carboxyl side of one or two amino acids and the ability to hydrolyze one or more bonds in the partial amino acid sequence between positions 106 and 131 in κ-casein-casein to milk, b) incubating so as to partially hydrolyze κ-casein-casein in the milk, and c) during or after step b) conditions causing clotting of the milk. The endo-protease of the present invention was able to cause clotting of the milk. No additional protease was to be added for that purpose.

WO 91 /13553 (Novo Nordisk A/S) discloses that a certain specific protease derived from Bacillus licheniformis does not cause any clotting of milk, thus teaching away from the invention. As will be described below, we have now surprisingly found that the same specific protease can be used for milk clotting by addition of a calcium salt.

Detailed disclosure of the invention Cheesemaking

Any type of milk, in particular milk from ruminants such as cows, sheep, goats, buffalos or camels, may be used as the starting material in the process of the invention, e.g. as reconstituted milk, whole milk, concentrated whole milk or low fat milk.

The milk may be concentrated in various ways such as by evaporation or spray- drying, but is preferably concentrated by membrane-filtration, i.e. ultra-filtration in which molecules with a molecular weight of up to 20,000 Dalton are allowed to pass the membrane, optionally with dia-filtration before or after ultra-filtration in which molecules of a molecular weight of up to 500 Dalton are allowed to pass the membrane. For a more detailed description of the ultra-filtration process, see for instance Quist et al., Beretning fra Statens Mejeriforsog, 1986.

A starter culture may be added to the milk before or simultaneously with the addition of the coagulation inducing enzyme described in the present lo invention. The starter culture is a culture of lactic acid bacteria used, in conventional cheese making, to ferment the lactose present in the milk and to cause further decomposition of the clotted casein into smaller peptides and free amino acids as a result of their production of proteases and peptidases. The starter culture may be added in amounts which are conventional for the present purpose, i.e. typically amounts of about 1 *E4 to 1 *E5 bacteria/g of cheese milk, and may be added in the form of freeze-dried, frozen or liquid cultures. When the milk employed in the process of the invention is concentrated milk, it is preferred to add the starter culture after concentrating the milk, although this is not an absolute requirement, as the starter culture bacteria will be retained during filtration.

After adding the enzyme giving cause to clotting as described in the present invention the subsequent steps in the cheese-making process, i.e. further salting, pressing, and ripening of the curd, may be conducted in the traditional way of producing cheese, e.g. as described by R.Scott, Cheesemaking in Practice, 2^nd Ed., Elsevier,

London, 1986.

It is at present contemplated that most types of cheese may advantageously be prepared by the process of the invention.

Proteases

A suitable protease for cheese-making at the same time should be able to split off the glycosylated part of κ-casein, causing clotting, and not hydrolyse the precipitated casein to a substantial degree. The inventors have found that this requirement is met by an endo-protease having a specificity for peptide bonds on the amino or carboxyl side of one or two amino acids and the ability to hydrolyze one or more bonds in the partial amino acid sequence between positions 106 and 131 in κ-casein, which is known to have the following sequence:

Met(106)-Ala-lle-Pro-Pro-Lys-Lys-Asn-Gln-Asp-Lys-Thr-Glu-lle-Pro-Thr- lle-Asn-Thr-lle-Ala-Ser-Gly-Glu-Pro-Thr(131 ) Any specific endo-protease with specificity for one of the amino acids occurring in the sequence between methionine (106) and threonine (131) in κ-casein will meet the requirement. Similar to chymosin, such proteases would induce coagulation of the casein micelles during the cheese making process. The size of theκ-casein retained in the cheese would be larger than the para-κ-casein obtained with chymosin. This directly means that the total amount of protein retained in the cheese will be larger and thus that cheese yield will be increased. The exact increase % will depend on the cleavage site of the endo-protease in κ-casein and experimental parameters like processing conditions and cheese-milk composition.

A preferred type of protease specifically cleaves at the carboxyl side of glutamic acid (with slow hydrolysis at the carboxyl side of aspartic acid) and thus hydrolyzes the glutamic acid (118) - isoleucine (119) bond in κ-casein. One such protease derived from B.licheniformis is described in Svendsen, I. and Breddam, K., Eur.J.Biochem., 204, 165-171 (1992), and in WO 91/13553 (Novo Nordisk A/S). Another protease with this specificity derived from Streptomyces griseus is described in Yoshida, N. et al., J. 5 Biochem. (Tokyo) 104, 451-456 (1988).

Another preferred type of protease cleaves at the carboxyl side of both Glu and Asp. Examples of such proteases are those derived from Staphylococcus aureus, Actinomyces sp., and Streptomyces thermovulgaris.

A third preferred group of proteases cleaves specifically at the amino side of o Lys, derived e.g. from Achromobacter or Myxobacter protease II.

A fourth preferred group of proteases have trypsin-like specificity, i.e. they cleave specifically at the carboxyl side of Lys and Arg. Examples are trypsin (e.g. bovine and porcine trypsin) and microbial trypsin-like proteases derived from

Streptomyces griseus, Streptomyces fradiae, Streptomyces erythreus and Fusarium 5 oxysporum (US 5288627, to Novo Nordisk A/S).

Yet another group of preferred proteases are specific for the carboxyl side of glycine and thus hydrolyze the glycine (128) - glutamic acid (129) bond in κ-casein. One example is protease IV from papaya latex, see Buttle.D.J. et al.. FEBS Letters. 260 no. 2, 195-197 (1990). Typical reaction conditions are 5-30 minutes at 20-40°C. 0 It is important for the stability of the curd that a further hydrolysis of the precipitated casein does not take place. Therefore, no other protease should be added in the process.

Milk clotting. 5 Milk clotting can be achieved, e.g. by addition of a soluble calcium salt such as calcium chloride, e.g. adding 2-3 mM to a total calcium concentration of 4-6 mm.

Legend to the Figure:

Figure 1 : overlaid and background corrected chromatograms of the clear 0 supernatants of the κ-casein solutions after incubaton with Maxiren, Protease XIV, protease XXI or without any additions. Experimental details are given in example 5. Examples

Example 1

Miniature cheeses were produced as described by Shakeel-Ur-Rehman et al. (Protocol for the manufacture of miniature cheeses in Lait, 78 (1998), 607-620). Raw cows milk was pasteurised by heating for 30 minutes at 63°C. The pasteurised milk was transferred to wide mouth plastic centrifuge bottles (200ml per bottle) and cooled to 31 °C. Subsequently, 0J2 ml of starter culture DS 5LT1 (DSM Gist B.V., Delft, The Netherlands) was added to each of the 200 ml of pasteurised milk in the centrifuge bottles and the milk was ripened for 20 minutes. Than, CaCI₂ (132 μl of a 1 mol.l^"1 solution per 200ml ripened milk) was added, followed by addition of the coagulant (0.04 IMCU per ml). The milk solutions were held for 40-50 minutes at 31 °C until a coagulum was formed. The coagulum was cut manually by cutters of stretched wire, spaced 1 cm apart on a frame. Healing was allowed for 2 minutes followed by gently stirring for 10 minutes. After that, the temperature was increased gradually to 39°C over 30 minutes under continuous stirring of the curd / whey mixture. Upon reaching a pH of 6.2 the curd / whey mixtures were centrifuged at room temperature for 60 minutes at 1 JOOg. The whey was drained and the curds were held in a water bath at 36°C. The cheeses were inverted every 15 minutes until the pH had decreased to 5.2-5.3 and were then centrifuged at room temperature at 1 JOOg for 20 minutes. After further whey drainage the cheeses were weighed . The cheese yield was calculated as follows: Cheese yield (%) = {(cheese weight) / (total milk weight)} * 100%.

Example 2.

The milk clotting activity of several endo-proteases was determined in IMCU

(International Milk Clotting Units) according to the international IDF (International Dairy Federation) standard 157A:1997. The activity of the commercial is given in the table below in International milk clotting units (IMCU).

The observation of milk clotting for these endo-proteases demonstrates that endo- proteases with a specificity for proteolysis of κ-casein in the region between residues 105 (phenylalanine) and 131 (threonine) other than that of chymosin can be used for milk clotting, opening a novel route for cheese making.

Example 3 Mini cheeses were produced using the proteases XIV and XXI that were tested positively in example 2 described before. The commercial rennet Maxiren (DSM Gist BV, Delft, The Netherlands) was used as the control.

Experimental results are shown in the table below:

Table 1 : Experimental data and cheese yields for the preparation of mini cheeses using three different proteases. Experimental details are given in the text of example 3.

The numbers in the table 1 show that cheese can be obtained in excellent yields using endo-proteases XIV and XXI instead of chymosin (Maxiren). The experimental error is too large to allow an unambiguous conclusion about relative cheese yields. A yield increase for the endo-proteases XIV and XXI would, however, be in line with theoretical calculations.

Example 4

Purified κ-casein (obtained from Sigma) was dissolved to an end concentration of 3.3 mg/ml in 20 mM Tris.HAc, pH 6.5. Separate solutions were prepared to which either protease XIV or protease XXI (both obtained from Sigma) or chymosin (Maxiren) were added, all to an end concentration of 0.04 IMCU / ml. In a control experiment no enzyme was added. The samples were incubated at 32 °C for 30 minutes. Precipitate was formed between 1-3 minutes after enzyme addition in all solutions except for the control experiment. Samples of 10 μl of the clear supernatants were subjected to HPLC size exclusion chromatography, using a TSK3000 column (obtained from TosoHaas) using a buffer containing 0.1M NaPj and 0.2M NaCI (pH7.0) as the eluent at a flow rate of 1.0 ml/minute and using UV detection (280 nm). Chromatograms were recorded for all four solutions. Liquid Chromatography - Mass spectra (LC/MS) analyses were recorded for the supernatants as follows. LC/MS was performed using an ion trap mass spectrometer (LCQ classic, Thermoquest, Breda, The Netherlands) coupled to a P4000 pump (Thermoquest, Breda, the Netherlands) in characterising the κ-casein supernatant solutions. The peptides formed were separated using a PEPMAP C₁₈ 300A (MIC-15-03-C18-PM, LC Packings, Amsterdam, The Netherlands) column in combination with a gradient of 0.1% formic acid in Milli Q water (Millipore, Bedford, MA, USA; solution A) and 0.1% formic acid in acetonitrile (solution B) for elution. The gradient started at 90% of solution A and increased to 40% of solution B in 45 minutes and was kept at the latter ratio for another 5 minutes. The injection volume used was 50 μl, the flow rate was 50 μl/min and the column temperature was maintained ambient. The protein concentration of the injected sample was approx. 50 μg/ml.

Detailed information on the individual peptides was obtained by using the "scan dependent" MS/MS algorithm, which is a characteristic algorithm for an ion trap mass spectrometer.

Full scan analysis was followed by zoom scan analysis for the determination of the charge state of the most intense ion in the full scan mass range. Subsequent MS/MS analysis of the latter ion resulted in partial peptide sequence information, which could be used for database searching using the SEQUEST application from Xcalibur Bioworks (Thermoquest, Breda, The Netherlands). Databanks used were extracted from the OWL.fasta databank, available at the NCBI (National Centre for Biotechnology informatics), containing bovine caseines only for this particular application.

The chromatograms, recorded for the clear κ-casein solutions and corrected for background, are shown in figure 1. The blanc experiment shows a strong peak in the chromatogram for the intact κ-casein. No significant other protein peaks are present. After incubation with protease XIV, protease XXI or chymosin (Maxiren), the κ-casein peak has completely disappeared, indicating its quantitative precipitation. Mass spectrometrix analysis of the clear supernatant solution obtained after incubation with Maxiren showed a double peak with a mass of approximately 6700 Dalton, most probably being the κ-casein derived GMP (residues nr. 106-169), but to large to identify by MS/MS fragmentation and database searching. After digestion of this peptide using the endo-protease endo Asp-N, only three peptides were generated, which could be identified to cover the κ-casein amino acid sequence 106-169, by using databank searching. This unambiguously identified the GMP in the supernatant solution. LC-MS analysis of the supernatant of the protease XIV and protease XXI incubations, a paek at MW of approximately 5700 dalton was found; in addition, several lower MW weights peptides were observed. The 5700-dalton peak corresponds to the κ-casein derived peptide obtained after cleavage at lysinel 16 of κ-casein; most of the smaller peptides could be assigned to specific sequences on this cleaved peptide, indicating that the peptide is further degraded. The presence of this peptide indicates that both proteases (XIV and XXI) cleave the κ-casein bond at a specific peptide bond different from the one cleaved by chymosin but still leading to K- casein insolubilization. This example therefore shows that such alternative proteases can be used to strongly reduce the solubility of κ-casein in aqueous solutions offering a route for enzyme mediated coagulation of κ-casein containing casein micelles resulting in cheese making (as described in example 3). The alternative cleavage of κ-casein will obviously result in a higher amount of casein incorporation in the cheese compared to the chymosin mediated proteolytic process. Therefore, the cheese making process with these alternative proteases will give increased cheese yield, the actual cheese yield depending on process parameters during the cheese making process.

Claims

1. A process for making cheese, comprising: a) adding a endo-protease having a specificity for peptide bonds on the amino or carboxyl side of one or two amino acids and the ability to hydrolyze one or more bonds in the partial amino acid sequence between positions 106 and 131 in κ-casein in milk, b) incubating so as to partially hydrolyze κ-casein in the milk, and c) during or after step b) conditions causing clotting of the milk.

2. The process of Claim 1 , wherein the endo-protease is specific for peptide bonds on the carboxyl side of glutamic acid.

3. The process of Claim 2, wherein the endo-protease is derived from a strain of Bacillus licheniformis.

4. The process of Claim 1 , wherein the endo-protease is specific for peptide bonds at the carboxyl side of glycine.

5. The process of Claim 4, wherein the endo-protease is papaya proteinase IV.

6. The process of any preceding claim wherein the clotting is done at a total calcium concentration above 4 mM.

7. The process of any preceding claim, comprising adding lactic acid bacteria as a starter culture before or simultaneously with the addition of the endo-protease.

8. The process of any preceding claim wherein the incubation is continued to 60-80%) hydrolysis of κ-casein.

9. The process of any preceding claim with an increased cheese yield compared to a process in which chymosin is used a the coagulant.

0. Use of endo-protease having a specificity for peptide bonds on the amino or carboxyl side of one or two amino acids and the ability to hydrolyze one or more bonds in the partial amino acid sequence between positions 106 and 131 in κ-casein in milk, in cheesemaking.