Method for enhancing stability of neutral proteases in preservation

Abstract

The invention belongs to the technical field of enzyme treatment, and particularly relates to a method for enhancing the stability of neutral protease in preservation. The method disclosed by the invention comprises the following steps: adding stabilizers, namely anhydrous calcium chloride, sorbitol and potassium sorbate into neutral protease, and preserving at a certain pH value and temperature. By adopting the method disclosed by the invention, the stability of the neutral protease can be remarkably enhanced, the stability of the neutral protease is enhanced by adding stabilizers including anhydrous calcium chloride, potassium sorbate and sorbitol into the neutral protease, and further, the pH value of the neutral protease added with the stabilizers is adjusted, so that the stability of the neutral protease is improved. And storing the neutral protease mixed material in an environment at the temperature of 25 DEG C or below, wherein the enzyme activity residual rate of the neutral protease is 90% or above.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme treatment, and particularly relates to a method for enhancing the stability of neutral protease. Background Art

[0002] Neutral protease is one of the earliest protease preparations discovered by humans and applied to industrial production. Neutral protease is widely used in the baking industry, soy protein isolate, yeast extract, beer, textile industry, leather industry, feed industry, medicine, etc. The existing neutral protease in China is mainly powder preparation, but the colony of powder preparation is difficult to control, and it needs to be dissolved when used, and there will be residues. For some high-level hygiene, it is difficult to meet the hygiene standards. Since neutral protease is an active substance and a protease, it can decompose itself, and the enzyme activity decreases quickly. The enzyme activity will lose 50% in one week under room temperature conditions.

[0003] Therefore, it is necessary to design a method for enhancing the stability of neutral proteases during storage in view of the above-mentioned instability of neutral proteases. Summary of the invention

[0004] In order to solve the above-mentioned technical problems, the present invention provides a method for enhancing the stability of neutral protease. The present invention provides a stabilizer for preserving liquid neutral protease, which can preserve the enzymatic activity of neutral protease for a long time under liquid conditions. The technical scheme of this experiment is: the stabilizer for preserving liquid neutral protease in this experiment is prepared by mixing and stirring calcium chloride, potassium sorbate, and sorbitol. The use of sorbitol can make the neutral protease molecules maintain their stability in aqueous solution and not easily denatured and lose activity; the use of potassium sorbate controls the number of colonies to facilitate meeting the requirements of food conditions; the use of calcium chloride can activate the activity of neutral protease and improve enzyme activity. This stabilizer can keep the activity of neutral protease at 90% for 4 months when the liquid neutral protease is stored at ≤25°C, forming a stable product process formula, which is convenient for application in food, leather, textiles and other aspects.

[0005] A method for enhancing the stability of neutral protease during storage, the method comprising adding a stabilizer to the neutral protease.

[0006] The stabilizer mentioned above includes at least one of a preservative, a water absorbent and a sugar.

[0007] The above-mentioned neutral protease is a liquid neutral protease.

[0008] The preservative is any one of potassium sorbate, sorbic acid, benzoic acid, sodium benzoate, calcium propionate, and ethyl p-hydroxybenzoate;

[0009] The above-mentioned water absorbing agent is anhydrous calcium chloride;

[0010] The sugar mentioned above is any one of glucose, maltose, fructose, dextran, xylitol, erythritol, oligofructose, isomerized lactose, sucrose, trehalose and sorbitol.

[0011] Preferably, the stabilizer comprises a water absorbent and sugars, the water absorbent is anhydrous calcium chloride, and the amount of anhydrous calcium chloride added is 0.07-0.13% of the mass of the neutral protease;

[0012] The sugar is sorbitol, and the added amount of the sorbitol is 40-80% of the mass of the liquid neutral protease.

[0013] More preferably, the stabilizer comprises anhydrous calcium chloride, sorbitol, potassium sorbate and glucose, and the amount of anhydrous calcium chloride added is 0.07-0.13% of the mass of the neutral protease;

[0014] The amount of sorbitol added is 40-80% of the mass of the liquid neutral protease;

[0015] The weight of potassium sorbate is 0.12-0.3% of the weight of the neutral protease, and the weight of the glucose is 0.05-0.2% of the weight of the neutral protease.

[0016] More preferably, the above-mentioned stabilizer includes a water absorbent and sugars;

[0017] The water absorbent is anhydrous calcium chloride, and the amount of anhydrous calcium chloride added is 0.07-0.13% of the mass of the neutral protease;

[0018] The sugars are sorbitol, sucrose and trehalose, and the added amount of sorbitol is 40-80% of the mass of the liquid neutral protease;

[0019] The added amount of sucrose is 10%-25% of the mass of the liquid neutral protease, and the added amount of trehalose is 2%-5% of the mass of the liquid neutral protease.

[0020] The stabilizer includes anhydrous calcium chloride, sorbitol, potassium sorbate, glucose, sucrose and trehalose;

[0021] The amount of anhydrous calcium chloride added is 0.07-0.13% of the mass of neutral protease;

[0022] The amount of sorbitol added is 40-80% of the mass of the liquid neutral protease;

[0023] The weight of potassium sorbate is 0.12-0.3% of the weight of neutral protease;

[0024] The weight of glucose is 0.05-0.2% of the weight of neutral protease;

[0025] The amount of sucrose added is 10%-25% of the mass of liquid neutral protease;

[0026] The amount of trehalose added is 2%-5% of the mass of the liquid neutral protease. 8. The method for enhancing the stability of the neutral protease during storage according to claim 1, comprising the following steps:

[0027] Take the neutral protease concentrate, add anhydrous calcium chloride and potassium sorbate while stirring, the amount of anhydrous calcium chloride added is 0.07-0.13% of the mass of the neutral protease, the weight of potassium sorbate is 0.12-0.3% of the mass of the neutral protease, stir until dissolved, add sorbitol, the amount of sorbitol added is 40-80% of the mass of the liquid neutral protease, and continue stirring until the materials are fully mixed.

[0028] A pH regulator is added to the fully mixed neutral protease mixture to adjust the pH to 5.00-6.00; the pH regulator is any one of glacial acetic acid and citric acid; and the pH-adjusted neutral protease mixture is stored at 0-25°C.

[0029] The beneficial effect of the present invention is that the method of the present invention can significantly enhance the stability of the neutral protease. The method of the present invention is to add stabilizers such as anhydrous calcium chloride, potassium sorbate and sorbitol to the neutral protease to enhance its stability. Further, the pH of the neutral protease to which the stabilizer is added is adjusted, and then the neutral protease mixture is stored in an environment below 25°C, and the residual enzyme activity rate of the neutral protease is above 90%. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention, but the present invention is not limited thereby.

[0031] Example 1

[0032] Take 2000g of neutral protease concentrate (pH is 6.45), add 2g of anhydrous calcium chloride and 4g of potassium sorbate while stirring, add 1000g of sorbitol after stirring until dissolved, and continue stirring for 10min. Use glacial acetic acid to adjust the pH according to the table below and detect the enzyme activity. Then divide each group of samples into three groups, one group is placed at room temperature (temperature 15-30℃, the room temperature is the same below), one group is placed at 25℃, and the other group is oscillated in a water bath at 37℃. The enzyme activity detection method refers to the Folin method for the determination of protease activity in GB / T23527-2009. If there is no special explanation, this method is used for the following enzyme activity tests.

[0033] pH and enzyme activity of each group of liquid neutral protease (16.2.27)

[0034] name Acetic acid pH adjustment Enzyme activity 0 6.45 40221 1 6.44 27196 2 6.01 26853 3 5.51 27196 4 5.06 26902

[0035] Results of 4 enzyme activities and pH tests under three different conditions

[0036]

[0037] The room temperature was about 15-20℃ from February to April 2016; and about 20-30℃ from May to June 2016. During the whole experiment, the appearance of each experimental group was clear and transparent, without any odor. From the data in Table 2, it can be seen that the storage temperature has a great influence on the liquid neutral protease. The higher the storage temperature, the faster the enzyme activity decreases. After being placed at 25℃ for 4 months, the enzyme activity loss was about 10%. And the enzyme activity residue was relatively high under various storage conditions when the pH was 5.00-5.50. Combined with the particularity of the liquid neutral protease itself, the storage temperature should be less than 25℃.

[0038] The liquid neutral protease of the invention comprises the following raw materials: 50% sorbitol, 0.2% potassium sorbate, 0.1% anhydrous calcium chloride, the pH value of the enzyme solution is adjusted to be controlled at 5.00-6.00 by using glacial acetic acid-sodium acetate, the storage temperature is ≤25°C, and the enzyme activity residual rate is above 90%.

[0039] Comparative Example 1

[0040] Take 2000g of neutral protease concentrate (pH 6.45), add 2g of anhydrous calcium chloride and 4g of potassium sorbate while stirring, and continue stirring for 10 minutes. Use glacial acetic acid to adjust the pH according to the table below, let it stand at 25°C, and test the enzyme activity.

[0041] name Acetic acid pH adjustment Enzyme activity 1 6.45 40221 2 6.01 40363 3 5.51 40555 4 5.02 40679

[0042] After standing for a period of time at 25°C, the enzyme activity results were as follows:

[0043]

[0044] It can be seen from the above table that the enzyme activities of all groups without adding sorbitol decreased rapidly, and the enzyme activity decreased by about 40% after being placed at 25°C for one month.

[0045] Comparative Example 2

[0046] Take 2000 g of neutral protease concentrate (detected pH is 6.45), add 4 g of potassium sorbate while stirring, stir until dissolved, add 1000 g of sorbitol, and continue stirring for 10 min.

[0047] name Acetic acid pH adjustment Enzyme activity 1 6.44 25996 2 6.01 25553 3 5.51 25496 4 5.06 25902

[0048] Stored at 25°C, the test results are as follows:

[0049]

[0050]

[0051] Comparative Example 3

[0052] Take 2000 g of neutral protease concentrate (detected pH is 6.45), add 2 g of anhydrous calcium chloride while stirring, stir until dissolved, add 1000 g of sorbitol, and continue stirring for 10 minutes.

[0053] The test results are shown in the following table:

[0054]

[0055] After being placed at 25°C for one month, the appearance of each group became turbid and had an odor, and microscopic examination revealed bacteria.

[0056] Example 2

[0057] Verification experiment

[0058] Effect of sorbitol addition on storage stability of liquid neutral protease

[0059] Add 0.2% potassium sorbate to liquid neutral protease, adjust the pH to 5.00 with glacial acetic acid-sodium acetate, add sorbitol to the enzyme solution according to the table below, stir evenly, place at room temperature, and track the changes in enzyme activity. The unit of enzyme activity is u / g.

[0060] Trial Groups

[0061] serial number Liquid neutral protease, g Sorbitol, g 1 100 0 2 100 40 3 100 60 4 100 80

[0062] After two weeks, the enzyme activity and pH of each group were tested. The test results are as follows:

[0063] Changes of neutral protease activity and pH at different sorbitol additions

[0064]

[0065] From the above table, we can see that the amount of sorbitol added has a great influence on the activity of liquid neutral protease. After being placed at room temperature for 1 month, the activity of 1# enzyme lost about 50%, and the activity of other groups with sorbitol added did not change significantly; after being placed at room temperature for another three months, the residual activity of 1# enzyme was only 17%, and the higher the amount of sorbitol added in other groups, the higher the residual activity. When the amount of sorbitol added was higher than 40%, the residual activity rate was higher than 90%, and when the amount of sorbitol added was 80%, the residual activity rate reached 100%.

[0066] Example 3

[0067] Take 2000g of neutral protease concentrate (pH is 6.45), add 1.4g of anhydrous calcium chloride and 2.4g of potassium sorbate while stirring, stir until dissolved, add 800g of sorbitol, and continue stirring for 10 minutes. Adjust the pH to 5.12 with glacial acetic acid, and test the enzyme activity. Then divide each group of samples into three groups, one group is placed at room temperature (15℃-30℃), one group is kept at 25℃, and the other group is refrigerated at 15℃. The enzyme activity detection method refers to the Folin method for the determination of protease activity in GB / T23527-2009. If there is no special explanation, this method is used for the following enzyme activity tests.

[0068] Enzyme activity and pH test results under three different conditions

[0069]

[0070] As can be seen from the table, according to the mass ratio of liquid neutral protease, 0.07% anhydrous calcium chloride and 0.2% sorbic acid are added.

[0071] Potassium and 50% sorbitol were placed at 25°C for 120 days, and the residual enzyme activity was about 94%.

[0072] Example 4

[0073] Take 2000g of neutral protease concentrate (pH is 6.45), add 2.6g of anhydrous calcium chloride and 6g of potassium sorbate while stirring, stir until dissolved, add 1600g of sorbitol, and continue stirring for 10min. Adjust the pH to 5.15 with glacial acetic acid, and test the enzyme activity. Then divide each group of samples into three groups, one group is placed at room temperature (15℃-30℃), one group is kept at 25℃, and the other group is refrigerated at a constant temperature of 15℃. The enzyme activity detection method refers to the Folin method for the determination of protease activity in "GB / T23527-2009". If there is no special explanation, this method is used for the following enzyme activity tests.

[0074] The results of enzyme activity and pH test of each group of liquid neutral protease under three different conditions

[0075]

[0076] Example 5

[0077] Take 2000g of neutral protease concentrate (pH is 6.45), add 2g of anhydrous calcium chloride and 4g of potassium sorbate while stirring, add 300g of sucrose and 60g of trehalose after stirring until dissolved, and continue stirring for 10min. Use glacial acetic acid to adjust the pH to 5.11 and detect the enzyme activity. Then divide each group of samples into three groups, one group is placed at room temperature (15℃-30℃), one group is kept at 25℃, and the other group is refrigerated at a constant temperature of 15℃. The enzyme activity detection method refers to the Folin method for the determination of protease activity in "GB / T 23527-2009". Unless otherwise specified, this method is used for the following enzyme activity tests.

[0078] Enzyme activity and pH test results under three different conditions

[0079]

[0080]

[0081] Example 6

[0082] Take 2000g of neutral protease concentrate (pH 6.45), add 2g of anhydrous calcium chloride and 4g of potassium sorbate while stirring, stir until dissolved, add 300g of sucrose, 60g of trehalose, 900g of sorbitol, and 100g of glucose, and continue stirring for 10 minutes. Use glacial acetic acid to adjust the pH to 5.11 and detect the enzyme activity. Then divide each group of samples into three groups, one group is placed at room temperature (15℃-30℃), one group is kept at 25℃, and the other group is refrigerated at a constant temperature of 15℃. The enzyme activity detection method refers to the Folin method for the determination of protease activity in "GB / T 23527-2009". Unless otherwise specified, this method is used for the following enzyme activity tests.

[0083] Enzyme activity and pH test results under three different conditions

[0084]

Claims

1. A method for enhancing the stability of a neutral protease during storage, characterized in that: A stabilizer is added to the neutral protease.

2. The method for enhancing the stability of neutral protease during storage according to claim 1, characterized in that: The stabilizer includes at least one of a preservative, a water absorbent and a sugar.

3. The method for enhancing the stability of neutral protease during storage according to claim 1, characterized in that: The neutral protease is a liquid neutral protease.

4. The method for enhancing the stability of neutral protease during storage according to claim 2, characterized in that: The preservative is any one of potassium sorbate, sorbic acid, benzoic acid, sodium benzoate, calcium propionate, and ethyl p-hydroxybenzoate; The water absorbing agent is anhydrous calcium chloride; The sugar is any one of glucose, maltose, fructose, dextran, xylitol, erythritol, oligofructose, isomerized lactose, sucrose, trehalose and sorbitol.

5. The method for enhancing the stability of neutral protease during storage according to claim 2, characterized in that: The stabilizer comprises a water absorbent and sugars, wherein the water absorbent is anhydrous calcium chloride, and the amount of anhydrous calcium chloride added is 0.07-0.13% of the mass of the neutral protease; The sugar is sorbitol, and the added amount of the sorbitol is 40-80% of the mass of the liquid neutral protease.

6. The method for enhancing the stability of neutral protease during storage according to claim 5, characterized in that: The stabilizer comprises anhydrous calcium chloride, sorbitol, potassium sorbate and glucose, and the amount of anhydrous calcium chloride added is 0.07-0.13% of the mass of the neutral protease; The amount of sorbitol added is 40-80% of the mass of the liquid neutral protease; The weight of the potassium sorbate is 0.12-0.3% of the weight of the neutral protease, and the weight of the glucose is 0.05-0.2% of the weight of the neutral protease.

7. The method for enhancing the stability of neutral protease during storage according to claim 2, characterized in that: The stabilizer includes a water absorbent and sugars; The water absorbing agent is anhydrous calcium chloride, and the amount of anhydrous calcium chloride added is 0.07-0.13% of the mass of the neutral protease; The sugars are sorbitol, sucrose and trehalose, and the added amount of sorbitol is 40-80% of the mass of the liquid neutral protease; The added amount of sucrose is 10%-25% of the mass of the liquid neutral protease, and the added amount of trehalose is 2%-5% of the mass of the liquid neutral protease.

8. The method for enhancing the stability of neutral protease during storage according to claim 2, characterized in that: The stabilizer includes anhydrous calcium chloride, sorbitol, potassium sorbate, glucose, sucrose and trehalose; The amount of anhydrous calcium chloride added is 0.07-0.13% of the mass of neutral protease; The amount of sorbitol added is 40-80% of the mass of the liquid neutral protease; The weight of potassium sorbate is 0.12-0.3% of the weight of neutral protease; The weight of glucose is 0.05-0.2% of the weight of neutral protease; The amount of sucrose added is 10%-25% of the mass of liquid neutral protease; The amount of trehalose added is 2%-5% of the mass of the liquid neutral protease.

9. The method for enhancing the stability of neutral protease during storage as claimed in claim 1, comprising the following steps: Take the neutral protease concentrate, add anhydrous calcium chloride and potassium sorbate while stirring, the amount of anhydrous calcium chloride added is 0.07-0.13% of the mass of the neutral protease, the weight of potassium sorbate is 0.12-0.3% of the mass of the neutral protease, stir until dissolved, add sorbitol, the amount of sorbitol added is 40-80% of the mass of the liquid neutral protease, and continue stirring until the materials are fully mixed.

10. The method for enhancing the stability of neutral protease during storage according to claim 7, characterized in that: A pH regulator is added to the fully mixed neutral protease mixture to adjust the pH to 5.00-6.00; the pH regulator is any one of glacial acetic acid and citric acid; and the pH-adjusted neutral protease mixture is stored at 0-25°C.

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