• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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  • 引用文献
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  • 优先权
    • 专利摘要:
    1. A METHOD OF DESTABILIZATION OF A MICROBIAL ENLASTIC ENZYME by chemical modification carried out by treating a rennet enzyme with a modifying agent at an optimum temperature in an aqueous medium, characterized in that, in order to reduce thermal stability, an oxidizing agent from the group of free halogen groups is used to reduce thermal stability. - chlorine, bromine or iodine or their derivatives, in which the halogen has an oxidation number of 1-3, and the mass ratio of oxidizer to the total protein content in the reaction mixture is em 0.01-1.0. 2. Method pop. 1, distinguished by the fact that the microbial rennet enzyme is treated with an oxidizing agent at pH 5–9 to the level of destabilization with a loss of CO activity up to 50%, preferably up to 30%. 3. A method according to claims 1.2, characterized in that the enzyme produced by Mucor miehei is used as a microbial rennet enzyme.
    公开号:SU1109054A3
    申请号:SU802908403
    申请日:1980-04-08
    公开日:1984-08-15
    发明作者:Браннер-Йоргенсен Свен；Шнайдер Палле；Эйгтвед Петер
    申请人:Ново Индустри А/С (Фирма)；
    IPC主号:
    专利说明:

    with
    about
    SP 4
    This invention relates to a method for destabilizing a microbial rennet enzyme.
    In the production of cheese, milk is rolled to separate the curd from the whey. For these purposes, products containing rennin, which is a milk-coagulating enzyme of the calf's stomach, are used.
    Known Several substitutes for calf rennet, including known microbial rennet from Mucor miehei and Mucor pusillus iH.
    Rennet produced by Mucor miehei is preferred in cheese making because of its low cost, low nonspecific proteolytic activity and great similarity to rennin in relation to sensitivity to calcium ion. In addition, Mucor miehei is distinguished by its storage stability of rennet, which is explained by its high thermal stability.
    Some pasteurized whey is used as an additive to whole milk, for example, in the form of a powder to obtain enriched milk, for example det (; someone nutrition. Pasteurized whey obtained and cheese made with Mucor miehei rennet has a low level of activity of the rennet due to high thermal stability of the latter, produced by Mucor miehei.
    There is a method of destabilization of microbial raw enzyme by chemical modification, carried out by treating rennet with a modifying agent at the optimum temperature in an aqueous medium, where potassium C23 cyanate is used as a modifying agent.
    The resulting carbamylated product is thermally less stable. However, destabilization of the carbamylated enzyme is too small ().
    I
    The object of the invention is to reduce the thermal stability of the microbial rennet.
    The goal is achieved by the fact that according to the method of destabilization of the Microbial rennet enzyme by chemical modification carried out by treating the enzyme with a modifying agent at the optimum temperature in an aqueous medium, an oxidizing agent from the group of free halogens — chlorine, bromine or iodine — or their derivatives, is used as a modifying agent in which halogen has an oxidation number of 1-3, and the mass ratio of oxidant to the total protein content in the reaction mixture is 0.01-1.0.
    The microbial rennet enzyme is treated with an oxidizing agent at a pH of 5–9 to a destabilization level of 5–11 ° C with a loss of activity of up to 50%, preferably up to 30%, and the enzyme produced by Mucor miehei is used as a microbial rennet.
    The degree of destabilization of rennet modified according to the invention is sufficient to satisfy the requirements for the use of whey and does not adversely affect the storage stability of rennet preparations.
    Under ideal conditions, the enzyme can be inactivated at the appropriate (high) temperature so that the residual enzyme activity decreases as a function of time along the exponential decay curve, i.e. characterized by an easily identifiable durability period and this half period is a function of temperature). The half life of TII-J can be calculated by the formula
    (t, -t) 1.n2
    T,
    / t In Hell-ln Aj
    where a. - enzyme activity, measured after heating to a certain temperature during time t ;,;
    A2 - enzyme activity, measured after heating to the same temperature for a time t.
    All other things being equal, the half-life will be shorter, the higher the temperature. For many enzymes, a change in the pH of the enzyme solution and the strength, as well as the presence of some salts, can significantly affect the half life. Chemical modification of a specific enzyme causes thermal destabilization of the enzyme, therefore the degree of destabilization is n ° C, if the initial (unmodified) and modified enzymes have the same half-period at. N and (Nn) C, respectively. However, the destabilization values are to some extent approximated by the approximate nature of the half-period value. All destabilization values given in this description. It has been measured at pH 6.0, because the measurement results depend on the pH value. Usually, the treatment of the enzyme according to the invention is accompanied by a loss of activity. It has been established that, for economic reasons, destabilization should not be carried out at a stage corresponding to a loss of activity on average more than 50%, preferably at a stage with a loss of 30% and more preferably with a loss of activity of 10% change, Usually the destabilization is carried out to the level of tOC and with loss of activity less than 10%. Hypochlorite, for example sodium hypochlorite, hypobromide such as sodium hypobromide, free chlorine, N-chlorosuccinimide, chloramine T and trichloroisocyanuric acid are used as oxylating agents that can be used in the method according to the invention. The oxidizing agent should be applied in such a concentration that the desired degree of destabilization is achieved in a reasonable time, which can be calculated from several minutes to 48 hours or more in those cases when the reaction proceeds without interruption by cooling. If the concentration of the oxidant is too low, then destabilization is too weak, if the concentration of the oxidant is very high, then the loss of activity is too great. The optimum concentrations of the oxidizing agent usually correspond to the weight ratio between the oxidizing agent and the total amount of protein contained in the enzyme preparation, and is 3-30 hours of oxide per 100 parts of total protein. If the microbial rennet farm preparation is purified to a high degree of single activity, the amount of oxidant can be reduced to 1 g per 100 g of total protein. The pH values at which the reaction proceeds can vary widely, i.e. from about 3 to 10, which mainly depends on the oxidizer. For example, if the oxidizing agent is hypochlorite, then the preferred limit of pH values is 5-9 and more preferably 6-8. The reaction temperature is not critical if it is maintained at a certain level, i.e. below 30 ° C, when the stability of the enzyme is satisfactory. However, the stability of the enzyme can be enhanced by the addition of known protein stabilizing agents, for example, sodium chloride in a coli. 5-20% of the preparation of the enzyme, or sorbitol in amounts customary for the stabilization of enzymes. Rennet modified according to the invention is capable of producing traditional children's cheese, similar in its qualities to Sams and Danbo cheese and at least with the same properties as the corresponding unmodified rennet. The destabilization operation can and preferably should be carried out as a final operation for the production of a microbial rennet enzyme. The pH of the microbial enzyme solution prepared for normal end operations prior to administration is set at a given level for treatment (for example, 7), then the rennet is mixed, stirring at room temperature, with an oxidizing solution (3-30 hours, for example 20 hours hypochlorite per 100 hours total protein. The mixture is then left for an hour to achieve the desired level of destabilization. The reaction can be interrupted by the addition of activated carbon or a reducing agent, such as sodium sulfite, after the completion of the appropriate reaction period. The destabilized enzyme product is then subjected to conventional end operations, i.e. filtering the establishment of pH and single enzymatic activity to standard levels, etc. Example 1. The starting material is rennet enzyme concentrate (the culture fluid is concentrated to an activity approximately corresponding to a 1% solution of pure enzyme, an 18% solution of sodium chloride is added to the crude concentrate). This end of HTpiar is called Rennilas-46. In two series of samples, including three portions (25 MP), prepared by mixing 12.5 MP of rennilase-46 and 12.5 MP of water, the pH is adjusted to 7.0, 8.0 and 9.0, respectively, by adding 1 n, NaOH and to 0.4 mp of 2.25 M sodium hypochlorite (NaGlO) solution is added to each of these mixtures, while the pH values are kept constant at the indicated level. The first series, including three portions, is placed in a refrigerator overnight (approximately 18 hours). The second series, which includes three portions, is left at room temperature for 2 hours. The decrease in pH is measured after the indicated periods of time. The portions are then diluted with a mixture of 0.2 ml of the mixture with 50 ml of water and at the same time the pH is adjusted to 6.0 with a mixture of sodium acetate and acetic acid. The effect of sodium hypochlorite on the residual enzyme activity of the pH is given in Table. 1. The diluted samples are then heat treated at 55 ° C for 30 minutes at pH 6.0, after which the residual activity is measured. The effect of heat treatment on the residual activity of hypochlorite-modified rennet is given in Table. 2. The effect of heat treatment on the level of destabilization of the enzyme modified by hypochlorite is given in Table. 3. PRI mme R 2. The pH value of three portions of rennilase-Ab, 150 ml each, is adjusted to 5.0, 6.0, and 7.0, respectively. Each of the three portions is divided into three parts, to each of which 0.8, 1.2, and 1.6 mp of a commercial 2.25 M solution of NaOCl are added, respectively, while the pH is kept constant at the above values. These nine samples are then stored at room temperature () for 20 hours, after which a sample (10 ml) is taken from each sample. To each such sample, 0.4, 0.6 or 0.8 ml of 1 M Na, SOj solution are added, respectively, to remove excess hypochlorite, 0.2 ml of the samples thus treated are diluted with 50 ml of water and adjusted to pH 6.0 a mixture of acetic acid and sodium acetate. Diluted samples with a set pH value are subjected to heat treatment at 55 ° C for 30 minutes. The results of destabilization of rennet enzyme hypochlorite under conditions of pH 5.0-7.0 and t 18-20 ° C are given in Table. 4. Example 3. At 600 ml of renylase-46 at a temperature of about establish a pH value of 6.0 to 4 n. NaOH. This batch is divided into 6 hours with 100 MP each. O, 0.8, 1.2, 1.6, 2.0, and 2.4 ml of commercial 2.25 M NaOCl solution were added to each of these 6 parts, respectively, and the pH was adjusted to 7.0. After extraction, about 15 hours, about 2 ml of 1 M are added to eliminate excessive hypochlorite. The addition of sulfite does not change the stability of the rennet. The change in activity is measured. Samples of 5 ml are diluted 10-fold with 0.1 N acetate buffer pH 6.0, after which the diluted samples are subjected to heat treatment at 60 ° C for 15 minutes. The residual activity after heat treatment is determined and a half-life and destabilization are calculated. The results are presented in table.5. Example 4. The starting material for this example is rennelase-46 without 18% NaCl. Based on the indicated starting material, five samples of 200 g each with an addition of 0.5; ten; 15 and 20% NaCl, respectively. The pH is adjusted to 6.0 with 4 n. NaOH. Samples of 100 ml each are taken from each of the five samples. To each such sample, add 1 MP commercial 2.25 n. NaOCl at. Then for all five samples set pH 7.0 with 4 n. NaOH and stored at about. After a storage time (about 20 hours), the change in activity is determined. Samples of 5 wi are diluted 10-fold with O, 1 M acetate buffer with a pH of 6.0, after which the diluted samples with a steady-state pH value are subjected to heat treatment at 60 ° C for 15 minutes. Residual activity is determined (the sample not subjected to heat treatment is analyzed in parallel as a control). The test results are shown in Table. 6. Example 5. Prepare two portions of a mixture of 25 ml of rennilase-46 and 20 g of ice water, adjust to pH 7.0 with 4 n. NaOH, 33 and 66 mg of N-chlorosuccinimide, respectively, are added to these two cyMs, dissolved in 5 mc ice water, while the pH is constantly maintained at 7.0. After 2 hours, 0.2 ml of these portions were diluted with 50 ml of water, while the pH was adjusted to 6.0 using a mixture of sodium acetate and acetic acid. The residual activity after the reaction of rennet enzyme with N-chlorosuccinimide is as follows: when using 33 mg of N-chlorosuccinimide 99%, and when using 66 mg - 96.5%. It is heat-treated at 55 ° C for 30 minutes at pH 6.0. After that, the residual activity is determined and the half life and destabilization are calculated. The results are shown in Table. 7. „, g Example 6. Prepare a solution of NaOBr (about 2 M), adding dropwise bromine to 10 ml of stirred 4N. NaOH when cooled in an ice bath. 25 ml of Renilase-46 is mixed with 25 ml of water and the pH is adjusted to 7.5 with one-normal NaOH solution. Within 25 hours, 0.5 ml of sodium hypobromide solution is added to the 25 m of the obtained solution, while maintaining a pH of 7.3-7.8 with the addition of 1N. HC1. After about 30 minutes, the loss of activity and thermal stability are determined. The activity loss is about 20%, the residual activity after heat treatment for 20 minutes at and pH 6.0 is 14%, which corresponds to a half-life of about 7 minutes or destabilization at about 8 ° C. Example 7. 50 ml of water is diluted with 50 ml . The mixture was cooled in an ice bath and adjusted to pH 7.0 with 1N. NaOH. About 0 is then introduced, 1 g of free chlorine drops to a pH of 3.8, the liquid becomes turbid, and then clear. The reaction mixture was left for 48 min for 10 minutes and then 4 MP of 1 M Naj SO solution was added to remove excess chlorine. 0.2 ml of the sample before and after the introduction of chlorine is diluted with 50 ml of water and adjusted to pH 6.0 with a mixture of sodium acetate and acetic acid. The loss of activity associated with the reaction between the rennet and chlorine is 21 After heating the rennet treated with chlorine for 30 minutes, the residual activity is. This is 63%, which corresponds to a half-life of about 45 minutes or destabilization of about. Example 8. Prepare two portions of 25 ml of rennilase-46 and 25 g of ice water. Set pH 7.0 4 N. NaOH. To these mixtures, 60 and 120 mg of trichloroisocyanuric acid are added, respectively, and the pH is simultaneously adjusted to 7.0. After 4 hours, 0.2 ml of the mixtures were diluted with 50 MP of water and the pH was adjusted to 6.0 with a mixture of sodium acetate and acetic acid. Diluted samples are heat treated at 55 ° C for 30 minutes. The results of destabilization of the rennet enzyme trichloroisocyanuric acid are given in table. 8, Example 9. Two 25 ml portions of renilase 25 ml are mixed with 20 g of ice water for each batch and adjusted to pH 7.0 with 4 n. NaOH. Then, 70 and 140 mg of chloramine T dissolved in 5 ml of ice water are added to each of these portions, respectively, and the pH is simultaneously adjusted to 7.0. After about 2 hours, 0.2 ml of each sample is diluted with 50 ml of water while adjusting the pH to 6.0 with a mixture of sodium acetate and acetic acid. Diluted samples with the established pH are subjected to heat treatment at 55 ° C for 30 minutes. The results of destabilization of the enzyme chloramine-T are given in table. 9. Example 10. Rennilase-46 is diluted 3-fold without addition of NaCl. 20 ml of diluted rennilase-46 is adjusted to pH 8.5 and cooled to 0 ° C, after which 0.7 ml of a 0.05 M solution of iodine in 0.25 M potassium iodide is added. After about 15 minutes, the pH is adjusted to 6.0 with the addition of 0.1 M NaHSO. For analysis, this sample is diluted and at the same time the pH is adjusted to 6.0 with a mixture of acetic acid and sodium acetate. The sample is then heated for 30 minutes. Obsa activity was 42.6%; the residual activity after heat treatment is 35%, the half-life at and pH 6.0 is 20 minutes, which corresponds to a destabilization of approximately 5 ° C (a control sample gives the same half-period at 65 ° C). Example 11. The 3g of protease produced by the strain of microorganisms Mucor pusillus (nutrient medium, 1: 2,20000) is dissolved in 30 ml of 10% NaCl and adjusted to pH 6.5 at. 15% NaOCl was added. 50 µl three times with an interval of 30 min. After stirring for 3 h, the mixture was kept at 16 h and then the pH was adjusted to 5.0. Samples are taken to determine activity and thermal stability. In tab. 10 shows the effect of 20-hour storage on the level of destabilization. Example 12. Renilase-46 is the starting material, in which instead of 18% NaCl 6% is added. 1 l of the starting material is divided into 4 hours by 250. ml. The temperature is maintained and the pH is adjusted to 6.5 in three parts with 4 n. NaOH. To these three parts, 1, 1.2 and 1.4% v / v 10.5% (by weight) NaOCl were added, respectively, then the pH was adjusted to 7.5 with 4 n. NaOH and samples are incubated in a thermostat at 5 ° C. . After the reaction time (3.27 and 53 hours, respectively), 50 ml samples are taken from each of the parts treated with hypochlorite and 250 mg (0.5% by weight by volume) of activated carbon are added to each sample. One day after the introduction of activated carbon, the samples are filtered and tested for stability and activity of the rennet enzyme (1st definition). Samples after the addition of hypochlorite are held for 9 days after which they are again tested for stability and activity of the rennet enzyme (2nd definition ). 1 4Ш After a 4-day hold at 5 ° C and 2 days at 25 ° C, the rennet enzyme activity is again determined and the total activity is calculated (3rd determination). The results are summarized in table. 11 and 12. From table. Figures 11 and 12 show that there is no significant change in destabilization or total activity with an increase in reaction time from 3 hours to 53 hours. Example 13. Five samples of rennilase-46, 25 ml each are prepared at pH 2.0, 2.5, 3 , 0, 3.5 and 6, O, respectively. To each of these samples, 0.04 g of sodium hypochlorite is added, while the pH is maintained at the indicated level. Samples are stored at 5 ° C for 6 days, after which the residual, precise activity and thermostability are analyzed at and pH 6.0. The results are presented in Table 143. Example 14. The starting material is rennilase-46, modified in two respects: the culture nutrient medium is concentrated to an activity corresponding to a 1.6% solution of pure enzyme, 9% NaCl is added to the crude concentrate . Thus, 5952 kg of renylase-46 are modified on the pilot plant. The pH is adjusted to 6.4 with 34% NaOH and 10 kg of a 12.5% (by volume) solution of NaOCl is added to the enzyme. During this treatment, the pH is increased to 8.0. The pH value of 7.4 is adjusted by means of 37% g-HCl. After 18 h when the pH value is reduced to 6.9. Then 1% activated carbon is added at 4-5 ° C. After 26 hours, filtration was carried out through a filter press and destabilization was again measured, after which the surface layer was concentrated under reduced pressure at pH 6.7 and NaCl was added to a total content of 18%. The half-life of the final product is 11 minutes, and destabilization is 1bC. Thus, the implementation of the invention reduces the thermal stability of the rennet, keeping it within the required interval.
    1109054-12
    ----. ". ,R
    similar pH through Residual active-. occupation, Z
    pH .-.
    2 h t8 h 2 h 18 h
    7.0 6.9 6.9 86.3 83.1 8.0 7.6 7.6 86.1 81.7 9.0 8.2 8.2 78.4
    Baseline Residual activity of race, Z, series, pH
    . J
    716,34,2
    816,34,3
    932,314,6 Etalon 98,7
    Original. Half period T |. , Destabilization, €, the value of minutes in
    pH through
    2 h tS h 2 h 18 h
     Table 1
    . Table2
    Table 3
    II.5 11.5
    18.4
    12 12 ft
    eleven
    11 10
    10.8
    Table 5
    T a b l and c a 7
    PolupertyDestipod T 1/2 for acclimation, C tivnost. durability, min
    41.5
    23.6
    10 10 15.6 26.4
    70
    96.5 19.2 140 77.9 8.5
    T a b c a 8
    93.0
    35 16
    9 10 81 5
    Table 9
    10 12
    Table 10
    权利要求:
    Claims (3)
    [1]
    1. A process DESTABILIZATION microbial rennet by chemical modification carried out by treating sychuzh- (Nogo enzyme modifying agent at an optimal temperature in an aqueous medium, characterized in that, to reduce the thermal stability, as a modifying agent is an oxidant from the group of free halogens - chlorine, bromine or iodine or their derivatives, in which the halogen has an oxidation number of 1-3, and the mass ratio of the oxidizing agent to the total protein content in the reaction mixture is 0.01-1, 0.
    [2]
    2. The method of pop. 1, characterized in that the treatment of the microbial rennet with an oxidizing agent is carried out at a pH of 5-9 to a level of destabilization of 5-11 ° C with a loss of activity of up to 50Z, preferably up to 30Z.
    [3]
    3. The method according to claims 1, 2, characterized in that the enzyme produced by Mucor miehei is used as a microbial rennet enzyme.
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    同族专利:
    公开号 | 公开日
    IT1188922B|1988-01-28|
    SE8002640L|1980-10-10|
    IT8048334D0|1980-04-03|
    US4357357A|1982-11-02|
    FR2453897B1|1984-02-24|
    AR222534A1|1981-05-29|
    NZ193349A|1982-02-23|
    DE3012924C2|1983-04-21|
    GB2045772B|1983-02-23|
    BE882689A|1980-10-08|
    NL191064C|1995-01-02|
    AU5711880A|1980-10-16|
    NL8002050A|1980-10-13|
    CA1160970A|1984-01-24|
    SE446539B|1986-09-22|
    NL191064B|1994-08-01|
    AU525451B2|1982-11-04|
    DE3012924A1|1980-10-30|
    CH650022A5|1985-06-28|
    DK145679A|1980-10-10|
    FI66728C|1984-12-10|
    FI801072A|1980-10-10|
    DD149999A5|1981-08-12|
    FI66728B|1984-08-31|
    GB2045772A|1980-11-05|
    ES8103931A1|1981-04-01|
    ES490363A0|1981-04-01|
    FR2453897A1|1980-11-07|
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    US3275453A|1964-04-21|1966-09-27|Pfizer & Co C|Milk-curdling enzyme elaborated by endothia parasitica|
    US3886288A|1970-06-15|1975-05-27|Swift & Co|Cheese manufacture using highly active proteolytic enzymes|
    US4086139A|1976-04-09|1978-04-25|Gb Fermentation Industries Inc.|Differential inactivation of amylase in amylase-protease mixtures|
    JPS56500520A|1979-04-25|1981-04-23|DK145779A|1979-04-09|1980-10-10|Novo Industri As|PROCEDURE FOR THERMAL DESTABILIZATION OF MICROBIAL OSTELOEBE|
    EP0027834A1|1979-10-24|1981-05-06|Rapidase B.V.|Thermally sensitive microbial rennet, a process for its preparation and its application for cheese preparation|
    DK267480A|1980-06-23|1981-12-24|Novo Industri As|PROTEOLYTIC AGENT FOR PROTEIN HYDROOLYSIS AND PROTEIN HYDROOLYSIS PROCESS|
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    JPH0218834B2|1982-04-08|1990-04-26|Meito Sangyo Kk|
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    DK145679A|DK145679A|1979-04-09|1979-04-09|PROCEDURE FOR THERMAL DESTABILIZATION OF MICROBIAL OSTELOEBE|LV930263A| LV5284A3|1979-04-09|1993-04-23|Method of destabilizing enzyme of microbial origin in abomasum|
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