Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
• • 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 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/99—Enzyme inactivation by chemical treatment

Definitions

• 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 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) .
• 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
• Endothia parasitica vide, for example, U . S . Patent No. 3,275,453 .
• 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.
• calf rennet shows a unique combination of properties .
• calf rennet 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.
• 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 .
• 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 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.
• 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.
• 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
• 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.
• the oxidation is usually performed by adding a chemical compound of high oxidation potential to the enzyme-containing solution .
• a 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 .
• 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%.
• the concentration to which H 2 O 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 .
• 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.
• 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 H 2 O 2 , down to below 1 ppm in the reaction mixture.
• a reducing agent or a reduction-catalysing enzyme in suffient amount to bring any residual amount of oxidizing agent, for example H 2 O 2 , down to below 1 ppm in the reaction mixture.
• the oxidizing agent is H 2 O 2
• the residual amount thereof can be assessed by means of, e. g. ,
• a reducing agent 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.
• sodium bisulphite sodium bisulphite
• ascorbic acid sodium nitrite
• sodium nitrite sodium nitrite
• 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.
• 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
• Another reduction-catalysing enzyme useful for the removal of surplus of oxidizing agent is peroxidase, vide The Enzymes , above .
• 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 .
• the pH of the reaction mixture 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.
• 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.
• the sodium chloride concentration of the reaction mixture is preferably of the order of 10% w/v or lower, the H 2 O 2 concentration in preferably of the order of 1% w/v or lower, the temperature of the incubation with H 2 O 2 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 H 2 O 2 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.
• 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.
• this does not necessarily mean that increased temperature is the only extreme condition which will elicit the destabilization of the enzymes .
• 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.
• 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 .
• 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 CHL method for determining milk-clotting activity is performed as described in J. Dairy Res . , 43, 85, (1976) , G.A.L. Rothe, N. H. Axelsen, P. J ⁇ hnk, P. Foltmann: "Immunochemical, Chromatographic and Milk-Clotting Activity for Quantification of Milk-Clotting Enzymes in Bovine Rennets" .
• 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.
• a temperature of 20°C 3.3 ml of 30% H 2 O 2 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
• 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 H 2 O 2 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 2 The same procedure as described in Example 1 was followed with the following exceptions : The pH at the H 2 O 2 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.
• 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.

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• 1980
  • 1980-04-25 JP JP50099780A patent/JPS56500520A/ja active Pending
  • 1980-04-25 WO PCT/DK1980/000025 patent/WO1980002225A1/en not_active Application Discontinuation
  • 1980-11-04 EP EP19800900832 patent/EP0027813A1/en not_active Withdrawn

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