EP0027813A1 - A method for destabilizing milk-clotting enzymes - Google Patents

Abstract

One method of destabilizing the proteolytic activity of microbial milk curdling enzymes used in cheese production is to subject the enzyme to an oxidation treatment to a degree that does not significantly reduce the curdling activity. milk in comparison with the untreated enzyme and which makes it possible to substantially preserve the storage stability of the enzyme.

Description

A Method for Destabilizing Milk-Clotting Enzymes

The present invention relates to a method for destabilizing proteolytic activity of microbial milk-clotting enzymes for use in cheese production .

Microbial milk-clotting enzymes (microbial coagulants) are known, for example enzymes produced by Mucor miehei (vide for example, U.S. Patent No. 3,988, 207 and Danish published Patent Specification No . 138 , 999) , Mucor pusillus (vide, for example, U. S . Patents Nos . 3, 151 ,039 and 3, 212,905) , and Endothia parasitica (vide, for example, U . S . Patent No. 3,275,453) . In the production of cheese, using microbial coagulant, it is desirable to have an optimum effect with respect to the clotting of the milk to obtain a curd formation which is as close as possible to the curd formation obtained using calf rennet. The milk-clotting effect is a specific aspect of the proteolytic activity of a coagulant, and a general requirement for a coagulant to be useful in cheese production is that it has a high milk-clotting proteolytic activity as compared to its general proteolytic activity. In this regard, calf rennet shows a unique combination of properties .

In addition to showing a high milk-clotting activity as compared to its general proteolytic activity, calf rennet has the advantage that, its activity is easily destroyed by heating to temperatures above 50°C . This feature is important in the production of cooked cheese (for example Emmenthaler) and in whey processing, as both the preparation of cooked cheese and the whey processing comprise stages of heating to above 50°C, so that the resulting product is substantially free from any residual proteolytic enzymatic activity derived from the calf rennet. This , again, means that in the further utilization of the whey, e.g. for baby food, as a substrate for fermentation purposes, as a diet for calves, etc. , there is no deterioration of the desired functional properties of the whey proteins due to any residual proteolytic. activity derived from the calf rennet, and in the cooked cheeses , there is no deterioration of taste and aroma due to degradation of the cheese constituents . In non-cooked cheeses , calf rennet shows a limited proteolytic activity which is favourable during the maturation of the cheese . In contrast to calf rennet, microbial coagulants show a higher degree of stability and, hence, tend to show undesired residual proteolytic activity in both cheeses made using such coagulants and in whey resulting from such cheese production. It would therefore be desirable to be able to modify the microbial coagulants so as to obtain a reduction of their residual proteolytic activity in whey and cheese, but at the same time retaining a satisfactory milk-clotting activity and storage stability of the microbial coagulants permitting normal storage and manipulation thereof.

According to the invention, it has now been found that such a destabilization of microbial coagulants so as to conform their characteristics more closely with the desired characteristics of calf rennet may be obtained by subjecting the microbial coagulants to an oxidation treatment to an extent which does not substantially reduce the milk-clotting activity as compared with the untreated enzyme and which does not substantially reduce the storage stability of the enzyme at normal storage conditions . For example, the oxidation treatment of the invention may be carried out to such an extent that the enzyme retains at the most about 30% of its original milkclotting activity upon heat treatment for 1 hour at 60°C and pH 6.0, with satisfactory retention of the storage stability of the enzyme. (The statement that the enzyme retains at the most about 30% of its original milk-clorring activity upon heat treatment for 1 hour at 60°C and pH 6.0 does not necessarily imply that in the particular use of the enzyme, a heat treatment for 1 hour at 60°C and pH 6.0 will be involved. The statement is only to be taken as a practical quantitative test for the determination of a desired measure of destabilization obtained according to the invention) .

As appears from the below experimental results, a destabilization of the proteolytic activity has been obtained concomitantly with a substantial retainment of the storage stability of the enzyme, as measured by its milk-clotting capability, at reasonable storage conditions (e. g. refrigerator, about 4°C) . It is believed that the effect of the treatment according to the invention is a general destabilization of the enzyme, but that the effect of this destabiliza tion becomes increasingly pronounced at increasing temperatures , which means that the effect of the destabilization is not noted to any substantial effect during reasonable storage and in the use of the enzyme, but becomes very pronounced during heat treatments subsequent to the use of the enzyme for curd formation.

According to the invention, the oxidation treatment is carried out to such an extent that the enzyme retains at the most about 30% of its original milk-clotting activity upon heat treatment for 1 hour at 60°C and pH 6.0. The extent of the oxidation treatment is determined by various parameters , such as treatment pH, temperature, duration of the treatment, etc. and is adjusted in such a way that the enzyme resulting from the treatment is capable of losing at least about 70% of its original milk-clotting activity upon the above-mentioned heat treatment for 1 hour at 60°C and pH 6.0.

The oxidation treatment is suitably performed in an aqueous solution of the enzyme. This solution may suitably be a fermentation broth from the production of the enzyme, or a concentrate therof. Evidently, the destabilization treatment should be performed at such a stage that it is not followed by any production stage involving conditions which might prematurely elicit the destabilization. It is preferred that the aqueous solution in which the treatment is performed contains sodium chloride in a concentration of between 0.1 and 20% w/v. It is believed that a sodium chloride concentration of

10 - 20% w/v contributes to the protection of the enzyme during the oxidation treatment, thereby resulting in an improved storage stability of the product.

The pH of the enzyme solution during the oxidation treatment is suitably in the range of 3 - 7, preferably in the range of 3 - 5. The pH adjustment is normally performed by addition of a suitable organic or inorganic acid.

in practice, the oxidation is usually performed by adding a chemical compound of high oxidation potential to the enzyme-containing solution . While, in principle, any chemical compound of high oxi dation potential would be contemplated as useful for the method, especially suitable compounds of high oxidation potentials which are known to be useful for modification of proteins (vide, e. g. Methods in Enzymology 25, (1972) , 393 - 400) are perchlorates , perborates, perbromates, and peroxides .

These compounds are suitably added to the reaction mixture in a concentration of 0.1 - 3% w/v.

A preferred oxidizing agent for use in the method is hydrogen peroxide which is suitably added to the enzyme solution to a concentration between 0.1 and 3%. In the most preferred embodiments discussed below, the concentration to which H₂O is added is about 1% w/v. It is believed that this low concentration contributes to the improved storage stability of the product.

The oxidation treatment may suitably be performed at any temperature between 0 and 40°C , but is, in most cases, preferably performed without any heating of the enzyme solution, in other words , at ambient temperature of about 20°C.

The period of time with which the enzyme is incubated with the oxidation agent is preferably at least 1 hour and may be up to several hours or days . At otherwise identical conditions to obtain the destabilization, the storage stability of the product is better, the shorter the treatment time is .

While the oxidation treatment can be relatively easily adjusted to result in the desired destabilization of the proteolytic activity of the enzyme, it should be ensured that the enzyme, in spite of its capability of destabilization induced by the oxidation treatment, will retain sufficient storage stability to permit storage for reasonable periods and manipulation in the manner conventional to milk- clotting enzyme preparations . It has been found that the storage stability of the enzyme treated with oxidation agent is to a very high degree dependent upon the effective removal of any surplus of the oxidizing agent. If surplus of the oxidizing agent is not effectively removed after the desired degree of oxidation treatment, the resulting enzyme preparation will tend to have a poor storage stability.

According to the invention , the removal of the oxidizing agent used is preferably performed by adding a reducing agent or a reduction-catalysing enzyme in suffient amount to bring any residual amount of oxidizing agent, for example H₂O₂, down to below 1 ppm in the reaction mixture. When the oxidizing agent is H₂O₂, the residual amount thereof can be assessed by means of, e. g. ,

"Peroxide Test Strips" from Merck, Darmstadt. It is believed that the restriction that any residual content of oxidizing agent should be brought down to below 1 ppm in the reaction mixture can be considered a quite general condition for obtainment of a desired storage stability of the modified enzyme, irrespective of the identity of the oxidizing agent used.

When a reducing agent is used for the removal of the oxidizing agent, this may be any conventional reducing agent capable of reacting with the oxidizing agent used, for example sodium bisulphite , ascorbic acid, hypochlorites, or sodium nitrite. While, due to the intended end use for milk-clotting to prepare cheese for human consumption, all reagents used should be so chosen that in the concentrations in which they may reappear in the final product for consumption, they are physiologically acceptable, it can be generally stated that due to the relatively small amounts which will be involved, the selection of both oxidizing agent and reducing agent is relatively uncritical.

According to the invention it has been found especially advantageous to use, as the agent for removing surplus of oxidizing agent, a reduction-catalyzing enzyme, in particular catalase. Catalases are well-known enzymes which may be isolated from various sources , such as beef liver and bacterial and fungal sources , vide, for example, "The Enzymes" , editor P. Boyer, Academic Press, New

York, 1975. Another reduction-catalysing enzyme useful for the removal of surplus of oxidizing agent is peroxidase, vide The Enzymes , above . When using a reduction-catalysing enzyme, in particular catalase, for removing a surplus of hydrogen peroxide, a practical mode of operation is to add small portions of catalase (for example 0.1 ml o f the catalase solution used in Example 1) at 1 hour intervals until the residual hydrogen peroxide level, as assessed, for example, by means of the above-metioned test strips, has been decreased to 1 ppm or less .

Prior to the removal of the surplus of oxidizing agent, the pH of the reaction mixture is suitably adjusted to favour the removal by the particular agent used therefor. When the removal is performed by means of catalase, the pH of the reaction mixture is suitably adjusted to about 4.5 - 7 prior to the addition of catalase. In general, it is preferred to adjust the pH to the upper end of this interval, for example to a value in the range of 6 - 7.

The modification treatment of the present invention comprises several parameters, and the particular value of each parameter in the set of parameters to obtain optimum results will depend on various factors, including the origin of the enzyme to be modified, the identity of the oxidizing agent, and the identity of the agent used for removal of surplus of oxidizing agent.

It has been found that when the enzyme is Mucor miehei proteinase , the oxidizing agent is H₂O₂, and the agent used for removal of surplus of H₂O₂ is catalase, the sodium chloride concentration of the reaction mixture is preferably of the order of 10% w/v or lower, the H₂O₂ concentration in preferably of the order of 1% w/v or lower, the temperature of the incubation with H₂O₂ is preferably as high as possible while still permitting obtainment of a reasonable treatment yield of milk-clotting activity, typically in the range of 20 - 40°C, and the incubation time is preferably several days (but may, at higher temperatures , have to be shortened somewhat in order to obtain a reasonable treatment yield) . The pH at the treatment with catalase is, as mentioned above, preferably in the higher end of the range stated, for example about 6.6, and the catalase addition is preferably adjusted for efficient removal of H₂O₂ down to a residual concentration of 1 ppm or less . Particular treatments which have resulted in very attractive modified enzymes appear from the examples .

The modified enzyme prepared by the method of the present invention can suitably be characterized by the features stated in claims 17 through 21. The enzymes are suitably packed and shipped in the same way as conventional microbial coagulant compositions , but it will be preferred to avoid too high temperatures during the storage, in order to avoid unintended eliciting of their destabilization. Thus , storage under reasonably cool conditions , preferably refrigeration conditions , will be optimum. When using the enzymes of the present invention, the normal concentrations thereof as conventionally used in the particular application are employed, and also in all other regards, the enzymes are used in the normal way known for microbial coagulants .

The destabilization induced by the method of the present invention can, such as mentioned above, be ascertained by the 1 hour heating at 60°C, pH 6.0 test. However, in accordance with what has been stated above, this does not necessarily mean that increased temperature is the only extreme condition which will elicit the destabilization of the enzymes . On the contrary, it must be presumed that an induced destabilization is a general destabilization which will express itself in any extreme condition, including, as the most typical example apart from the high temperature, extreme pH conditions , in particular very low pH values and very high pH values such as pH values below 2 or above 9. Of course, the influence of a particular parameter on the stability condition of the enzyme will depend upon the duration of the said influence, and in this regard, for example, a short period at a very extreme pH may correspond to a longer period at a less extreme pH .

It is also contemplated that the destabilization will occur to a sufficient extent even under the not very extreme conditions prevailing in cheese under long term (e . g. 2 months and above) ripening, which means that the destabilized enzyme will show a generally increased usefulness for almost any cheese production .

The Anson units referred to above are determined in the wellknown manner as described in J. Gen. Physiol. 22, 79 (1938) .

The CHL method for determining milk-clotting activity (expresse in CHU units) is performed as described in J. Dairy Res . , 43, 85, (1976) , G.A.L. Rothe, N. H. Axelsen, P. Jøhnk, P. Foltmann: "Immunochemical, Chromatographic and Milk-Clotting Activity for Quantification of Milk-Clotting Enzymes in Bovine Rennets" .

Example 1.

An enzyme solution of Mucor miehei produced milk-clotting enzyme (Rennilase 14 from Novo Industri A/S, Copenhagen : commercial product containing enzyme in a concentration corresponding to about 64 CHU/ml as measured by the CHL-method described above, proteins, carbohydrates , and sodium chloride) was admixed with solid sodium chloride and hydrochloric acid solution and thereafter made up to 97 ml with water, the final sodium chloride concentration being 10%, the final pH being 4.4, and the final concentration of the enzyme being 10 CHU/ml. At a temperature of 20°C, 3.3 ml of 30% H₂O₂ solution was added, and the reaction solution was allowed to stand at 20°C for 41 hours . Thereafter, the pH was adjusted to 6.6 by addition of 5% sodium hydroxide solution, and

0.1 ml of beef liver catalase (obtainable from Worthington Biochemical Corp . , catalase concentration 30,000 IU/ml) was added. The resulting mixture was incubated for 1 hour at room temperature (with cooling to keep the temperature at room temperature) . As assessed by means of "Peroxide Test Strips" , the H₂O₂ concentration was still above 1 ppm, and an additional 0.1 ml of the catalase solution was added, with subsequent incubation for 1 hour at room temperature. Thereafter, the H₂O₂ concentration was found to be below 1 ppm. The milk-clotting activity of the thus treated Mucor meihei enzyme was measured according to the CHL-method described above . The milk-clotting activity was found to be 104% of the initial enzyme activity . Thereafter, the pH of a portion of the mixture was adjusted to 6.0 with hydrochloric acid, and the mixture was heated at 60°C for 1 hour. After this heating, the milkclotting activity was found to be 27% of the activity prior to heating. Another portion of the mixture was stored for 14 days at 4°C . It was found that the milk-clotting activity was thereafter 103% of the activity prior to the storage period.

Example 2.

The same procedure as in Example 1 was followed with the exception that the temperature at the H₂O₂ incubation was 40°C, and the adjustment of pH, prior to the catalase treatment, was to pH 4.6. The yield of milk-clotting activity was 92%, compared to the initial activity. The milk-clotting activity retained upon heat treatment for 1 hour at 60°C and pH 6.0 was 24%. The storage stability was he same as in Example 1.

Example 3.

The same procedure as described in Example 1 was followed with the following exceptions : The pH at the H₂O₂ incubation was 3.6, the incubation temperature was 40°C, the incubation time was 1 hour, and the amount of the catalase solution added was 0.1 ml.

The yield of milk-clotting treatment was 106%, as compared to the initial activity, and the milk- clotting activity retained upon heat treatment for 1 hour at 60°C, pH 6.0, was 33%. The milk-clotting activity after 14 days of storage at 4°C was 105% of the activity prior to the storage period.

The portion-wise addition of catalase performed in the examples is the preferred manner of addition of peroxide-neutralizing enzyme as peroxides in themselves have a tendency to destroy such enzymes . Also the addition of the catalase under mild conditions (such as slowly, portion-wise, and with wooling) is believed to contribute to the storage stability of the product.

Claims

Claims .

1. A method for destabilizing proteolytic activity of microbial milkclotting enzymes for use in cheese production, comprising subjecting the enzyme to an oxidation treatment to an extent which does not substantially reduce the milk clotting activity as compared with the untreated enzyme and which permits substantial retention of the storage stability of the enzyme.

2. A method as claimed in claim 1 in which the oxidation treatment is carried out to such an extent that the enzyme retains at the most about 30% of its original milk-clotting activity upon heat treatment for 1 hour at 60°C and pH 6.0.

3. A method as claimed in claim 1 and 2 in which the enzyme preparation subjected to the oxidation treatment is an aqueous solution of the enzyme.

4. A metod as claimed in claim 3 in which the aqueous solution contain sodium chloride in a concentration of between 0.1 and 20% w/v .

5. A method as claimed in any of claims 3 and 4 in which the pH of the enzyme solution is 3 - 7, preferably 3 - 5.

6. A method as claimed in any of the preceding claims in which the oxidation is performed by adding a chemical compound of high oxidation potential.

7. A method as claimed in claim 6 in which the chemical compound is selected from the group consisting of perchlorates, perborates , perbromates, and peroxides.

8. A method as claimed in claim 7 in which the compound is added to the reaction mixture in a concentration of 0.1 - 3 w/v.

9. A method as claimed in claim 8 in which the oxidation is performed by adding H₂O₂ to a concentration of 0.1 - 3%, preferably about 1%

10. A method as claimed in any of the preceding claims in which the temperature at the oxidation treatment is between 0 and 40°C, preferably about 20°C .

11. A method as claimed in any of the preceding claims in which the enzyme is treated with the oxidizing agent for a period of at least 1 hour.

12. A method as claimed in any of the preceding claims in which the reaction mixture, subsequent to the oxidation treatment, is subjected to removal of any surplus of the oxidizing agent used.

13. A method as claimed in claim 12 in which the removal of the surplus of the oxidizing agent used is performed by adding a reducing agent or a reduction-catalysing enzyme.

14. A method as claimed in claim 13 in which the oxidizing agent is a peroxide, and the reduction of the peroxide is performed by adding, as the reduction-catalysing enzyme, catalase.

15. A method as claimed in any of claims 12 - 14 in which, prior to the removal of the surplus of oxidizing agent, the pH of the reaction mixture is adjusted to favour the removal of the oxidizing agent.

16. A method as claimed in claim 15 in which the oxidizing agent is a peroxide, and the removal thereof is performed by means of catalase, comprising adjusting the pH of the reaction mixture, prior to the addition of catalase, to about 4.5 - 6.5.

17. A method as claimed in claim 13 in which the removal of the surplus of the oxidizing agent is performed by adding a reducting catalyzing enzyme, comprising performing the removal by adding the enzyme in portions , with incubation following the addition of each portion .

18. A method as claimed in claim 17 in which the oxidizing agent is H₂O₂ and the reduction catalyzing enzyme is catalase.

19. A method as claimed in any of claims 12 - 18 in which the oxidizing agent is removed to such an extent that any residual amount thereof does not exceed 1 ppm, calculated on the reaction mixture .

20. A microbial milk-clotting enzyme for use in cheese production and showing a milk-clotting activity of at least 20 CHU units/mg enzyme protein and a proteolytic activity, as measured in milli¬

Anson units , which is at the most 1/10 of the milk- clotting activity expressed in CHU units, the enzyme having been made capable of losing at least about 70% of its original milk-clotting activity upon heat treatment for 1 hour at 60°C and pH 6.0 by an oxidation treatment.

21. An enzyme as claimed in claim 20 which shows a milk-clotting activity of about 26 CHU units/mg of enzyme protein and a proteolytic activity of about 1,6 milliAnson units .

22. An enzyme as claimed in claim 20 or 21 which is a Mucorderived enzyme.

23. An enzyme as claimed in claim 22 which is a Mucor mieheiderived enzyme .

24. A Mucor-derived milk-clotting enzyme for use in cheese production, the enzyme having been capable of losing at least 70% of its original milk- clotting activity upon heat treatment for 1 hour at 60°C and pH 6.0 by an oxidation treatment.

25. An enzyme as claimed in any of claims 20 - 24 in an aqueous solution containing sodium chloride, proteins , and carbohydrates .

26. An enzyme composition for use in cheese production and comprising, an aqueous solution of a milk-clotting enzyme, sodium chloride, proteins , and carbohydrates , the enzyme being an enzyme as claimed in any of claims 20 - 25.