Preparation method and application of high-temperature-resistant amylase

Abstract

The invention provides a preparation method and application of high-temperature-resistant amylase, and relates to the technical field of amylase production. The preparation method of the high-temperature-resistant amylase mainly comprises the following steps: selecting heat-resistant bacillus licheniformis, inoculating a seed solution of the heat-resistant bacillus licheniformis to a liquid fermentation culture medium consisting of okra freeze-dried powder, bran, yeast extract, sodium nitrate, peptone, monopotassium phosphate and sodium chloride, and culturing; subsequently, mixing nickel nitrate modified okra freeze-dried powder, sodium borohydride, calcium chloride and disodium hydrogen phosphate. According to the method, the defects in the prior art are overcome, the enzymatic activity of the prepared amylase is effectively improved, meanwhile, culture medium raw materials and subsequent raw materials are adopted for treatment, the high-temperature-resistant effect and activity of the amylase produced by the heat-resistant bacillus licheniformis are effectively improved, and the high-temperature steam desizing effect of the amylase is comprehensively improved.

Description

Technical Field

[0001] The invention relates to the technical field of amylase production, and in particular to a preparation method of a high-temperature resistant amylase and application thereof. Background Art

[0002] Bioenzyme is a new type of biocatalyst developed with the advancement of biotechnology. Compared with traditional catalysts, it has the advantages of high efficiency, low energy consumption, and environmental friendliness.

[0003] Desizing refers to the process of removing the size on the fabric. Most of the existing desizing processes use the following methods: hot water desizing, enzyme desizing, alkali desizing, and oxidant desizing. Among them, hot water desizing takes a long time and consumes a lot of energy overall. Alkali desizing and oxidant desizing use certain chemical raw materials, which will cause damage to the fabric fibers to a certain extent and affect the quality of the final product. On this basis, enzyme desizing has become a popular desizing method.

[0004] Most of the existing enzyme desizing methods use amylase for desizing. Generally, the fabric is immersed in amylase solution and then piled at 40-50°C for 1-2 hours to fully hydrolyze the starch. Although the desizing effect is good and the damage to the fabric is small, it is time-consuming overall.

[0005] In order to improve the desizing efficiency, the prior art often uses a hot steam process to accelerate desizing during enzyme desizing. However, high temperature treatment can easily reduce or inactivate the activity of amylase, so continuous or semi-continuous amylase steam desizing is often used, that is, multiple additions of amylase and multiple steam desizing treatments are performed, which can reduce the overall desizing time to a certain extent, but also increases the complexity of the operation and is also prone to incomplete desizing. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a method for preparing a high-temperature resistant amylase and its application. The existing heat-resistant Bacillus licheniformis is cultured in liquid and subsequently modified to effectively improve the high-temperature resistance and activity of the amylase produced by the heat-resistant Bacillus licheniformis, and comprehensively improve the effect of high-temperature steam desizing of the amylase.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented through the following technical solutions:

[0008] A method for preparing a thermostable amylase, comprising the following steps:

[0009] S1. Seed solution preparation: selecting thermotolerant Bacillus licheniformis to prepare seed solution;

[0010] S2. Prepare liquid fermentation medium according to the following mass percentages: 5.0%-7.0% of okra freeze-dried powder, 3.0%-6.0% of bran, 0.1%-0.2% of yeast extract, 0.05%-0.1% of sodium nitrate, 0.1%-0.15% of peptone, 0.05%-0.15% of potassium dihydrogen phosphate, 0.1%-0.2% of sodium chloride, and the rest is distilled water;

[0011] S3, liquid culture: inoculate the seed liquid into the liquid fermentation medium and ferment for 60-72 hours to obtain a fermentation material for standby use;

[0012] S4, amylase separation: filter the fermented material, and the filtrate is a crude amylase solution for standby use;

[0013] S5. Pretreatment of crude amylase solution: Take okra freeze-dried powder and add it to nickel nitrate solution, stir and mix to obtain a mixed solution, add the mixed solution to the crude amylase solution and continue stirring for 20-40 minutes, then add sodium borohydride, calcium chloride and disodium hydrogen phosphate, stir evenly, let stand for 4-6 hours and then centrifuge, and take the centrifuged supernatant as the thermostable amylase solution.

[0014] Preferably, the number of viable bacteria in the seed solution in step S1 is 10 6 -10 10 Pieces / mL.

[0015] Preferably, the method for preparing freeze-dried okra powder in step S2 comprises the following steps:

[0016] S2-1, washing the fresh okra to remove surface impurities, then adding water to grind into pulp, and obtaining okra pulp for later use;

[0017] S2-2, freeze the okra slurry at -2°C - -8°C for 10-20h, take it out and thaw it naturally, freeze-dry it, grind it through a 60-mesh sieve, and obtain okra freeze-dried powder.

[0018] Preferably, in step S3, the amount of seed liquid inoculated into the liquid fermentation medium is 3%-5% of the mass of the liquid fermentation medium, the fermentation temperature is 35±2°C, and the ventilation rate in the first 10-12 hours is 0.07m / (m·min), the ventilation rate in the middle period is 0.1-0.12m / (m·min), and the ventilation rate in the last 10-20 hours of fermentation, i.e., the late period, is 0.08m / (m·min).

[0019] Preferably, the mass concentration of the nickel nitrate solution in step S5 is 2%-4%, and the solid-liquid ratio of the freeze-dried okra powder to the nickel nitrate solution is 1:1.2-1.5.

[0020] Preferably, in step S5, the amounts of the mixed solution, sodium borohydride, calcium chloride and disodium hydrogen phosphate added to the crude amylase solution are 5%-10%, 2%-4%, 1% and 1% of the crude amylase solution respectively, and the standing temperature is 2-4°C.

[0021] Preferably, the centrifugation condition in step S5 is at 4° C. and at a speed of 6000-8000 r / min for 20-25 min.

[0022] Preferably, the thermostable amylase is applied to the fabric desizing process, and the application method is to dilute the thermostable amylase with water at 10-15 mL / L, then impregnate the printed and dyed textiles alone or mixed with other auxiliaries, and pass hot steam for desizing treatment.

[0023] The present invention provides a preparation method of a thermostable amylase and application thereof, which has the advantages of:

[0024] The invention adds okra freeze-dried powder to the culture medium of heat-resistant bacillus licheniformis, can effectively promote the proliferation of bacillus licheniformis and the production of amylase, and further plays a role of high temperature protection for amylase. When the crude amylase solution is subsequently treated, the okra freeze-dried powder oxidized by nickel nitrate is added, and sodium borohydride, calcium chloride and disodium hydrogen phosphate are jointly added to further improve the activity of amylase, comprehensively improve the high temperature resistance of amylase, make the prepared amylase still maintain a certain activity when the temperature is higher than 95 DEG C, can effectively realize fabric desizing, and improve the overall desizing efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a line graph showing the enzyme activity detection of amylases prepared in Examples 1-2 of the present invention and Comparative Examples 1-4 at different temperatures;

[0026] Figure 2 It is a line graph of enzyme activity detection of amylase prepared in Examples 1-2 of the present invention and Comparative Examples 1-4 at different pH values. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The thermotolerant Bacillus licheniformis used below is Bacillus licheniformis F1, with a deposit number of CCTCC No. M20211387, purchased from Wuhan University of Technology.

[0029] Embodiment 1:

[0030] Preparation of thermostable amylase:

[0031] (1) The thermotolerant Bacillus licheniformis was inoculated into LB liquid medium and cultured at 35±2℃ in a constant temperature shaker (160r / min) for 4h. The number of viable bacteria in the seed liquid was 10 8 Pieces / mL.

[0032] (2) Wash the fresh okra to remove surface impurities, add water and grind into slurry, freeze at -6°C for 14 h, take out and thaw naturally, freeze-dry, grind through a 60-mesh sieve, and obtain okra freeze-dried powder for later use;

[0033] (3) preparing a liquid fermentation medium according to the following mass percentages: 5.0% of okra freeze-dried powder, 3.0% of bran, 0.1% of yeast extract, 0.05% of sodium nitrate, 0.1% of peptone, 0.05% of potassium dihydrogen phosphate, 0.1% of sodium chloride, and the remainder being distilled water; and sterilizing at 121° C. for 15 min after mixing to obtain a liquid fermentation medium;

[0034] (4) inoculating 4% of the mass of the seed liquid into the liquid fermentation medium, fermenting at 35±2° C. for 70 h, and controlling the ventilation rate during the fermentation from 0 to 10 h to 0.07 m / (m·min), the ventilation rate during the fermentation from 11 to 55 h to 0.1 m / (m·min), and the ventilation rate during the fermentation from 56 to 70 h to 0.08 m / (m·min), and obtaining the fermentation material for use;

[0035] (5) adding freeze-dried okra powder to a 3% nickel nitrate solution at a material-liquid ratio of 1:1.3, stirring and mixing to obtain a mixed material;

[0036] (6) Filter the fermented material, and the filtrate is a crude amylase solution. Add a mixed solution of 8% of the total mass of the crude amylase solution, 3% of sodium borohydride, 1% of calcium chloride and 1% of hydrogen phosphate to the crude amylase solution, let it stand at 4°C for 5 hours, and then centrifuge it at a speed of 8000 r / min for 20 minutes, and take the supernatant as a clarified amylase solution for later use;

[0037] (7) Add 50% ammonium sulfate to the clarified amylase solution, let stand at 4°C overnight for 8 h, then centrifuge at 5000 rpm for 5 min, and collect the precipitate as amylase.

[0038] Embodiment 2:

[0039] Preparation of thermostable amylase:

[0040] The specific steps are the same as those of Example 1, except that the liquid fermentation medium is configured as follows: 7.0% okra freeze-dried powder, 6.0% bran, 0.2% yeast extract, 0.1% sodium nitrate, 0.15% peptone, 0.15% potassium dihydrogen phosphate, 0.2% sodium chloride, and the rest is distilled water.

[0041] Comparative Example 1:

[0042] Preparation of amylase:

[0043] The specific steps are the same as those of Example 1, except that the liquid fermentation medium is configured as follows: 3.0% bran, 0.1% yeast extract, 0.05% sodium nitrate, 0.1% peptone, 0.05% potassium dihydrogen phosphate, 0.1% sodium chloride, and the rest is distilled water.

[0044] Comparative Example 2:

[0045] Preparation of amylase:

[0046] The steps (1) to (4) are the same as those in Example 1;

[0047] (5) Filter the fermented material, and the filtrate is a crude amylase solution. Add 3% sodium borohydride, 1% calcium chloride and 1% hydrogen phosphate to the crude amylase solution, let it stand at 4°C for 5 hours, and then centrifuge it at 8000 r / min for 20 minutes. Take the supernatant as the clarified amylase solution for later use;

[0048] (6) Add 50% ammonium sulfate to the clarified amylase solution, let stand at 4°C overnight for 8 h, then centrifuge at 5000 rpm for 5 min, and collect the precipitate as amylase.

[0049] Comparative Example 3:

[0050] Preparation of thermostable amylase:

[0051] (1) The thermotolerant Bacillus licheniformis was inoculated into LB liquid culture medium and cultured at 35±2°C in a constant temperature shaker (160 r / min) for 4 h. The viable bacterial count of the seed liquid was 108 cells / mL.

[0052] (2) preparing a liquid fermentation medium according to the following mass percentages: 3.0% bran, 0.1% yeast extract, 0.05% sodium nitrate, 0.1% peptone, 0.05% potassium dihydrogen phosphate, 0.1% sodium chloride, and the remainder being distilled water, and sterilizing at 121° C. for 15 min after mixing to obtain a liquid fermentation medium;

[0053] (3) inoculating 4% of the mass of the seed liquid into the liquid fermentation medium, fermenting at 35±2° C. for 70 h, and controlling the ventilation rate during the fermentation from 0 to 10 h to be 0.07 m / (m·min), the ventilation rate during the fermentation from 11 to 55 h to be 0.1 m / (m·min), and the ventilation rate during the fermentation from 56 to 70 h to be 0.08 m / (m·min), and obtaining the fermentation material for use;

[0054] (4) adding chitosan to a 3% nickel nitrate solution at a material-liquid ratio of 1:1.3, stirring and mixing to obtain a mixture;

[0055] (5) Filter the fermented material, and the filtrate is a crude amylase solution. A mixed solution of 8% of the total mass of the crude amylase solution, 3% of sodium borohydride, 1% of calcium chloride and 1% of hydrogen phosphate are added to the crude amylase solution, and the mixture is allowed to stand at 4° C. for 5 h, and then centrifuged at 8000 r / min for 20 min, and the supernatant is taken as a clarified amylase solution for later use;

[0056] (6) Add 50% ammonium sulfate to the clarified amylase solution, let stand at 4°C overnight for 8 h, then centrifuge at 5000 rpm for 5 min, and collect the precipitate as amylase.

[0057] Comparative Example 4:

[0058] Preparation of amylase:

[0059] (1) The thermotolerant Bacillus licheniformis was inoculated into LB liquid culture medium and cultured at 35±2°C in a constant temperature shaker (160 r / min) for 4 h. The viable bacterial count of the seed liquid was 108 cells / mL.

[0060] (2) preparing a liquid fermentation medium according to the following mass percentages: 3.0% bran, 0.1% yeast extract, 0.05% sodium nitrate, 0.1% peptone, 0.05% potassium dihydrogen phosphate, 0.1% sodium chloride, and the remainder being distilled water, and sterilizing at 121° C. for 15 min after mixing to obtain a liquid fermentation medium;

[0061] (3) inoculating 4% of the mass of the seed liquid into the liquid fermentation medium, fermenting at 35±2° C. for 70 h, and controlling the ventilation rate during the fermentation from 0 to 10 h to be 0.07 m / (m·min), the ventilation rate during the fermentation from 11 to 55 h to be 0.1 m / (m·min), and the ventilation rate during the fermentation from 56 to 70 h to be 0.08 m / (m·min), and obtaining the fermentation material for use;

[0062] (4) Filter the fermented material, and the filtrate is a crude amylase solution. Add 3% sodium borohydride, 1% calcium chloride and 1% hydrogen phosphate to the crude amylase solution, let it stand at 4°C for 5 hours, and then centrifuge it at 8000 r / min for 20 minutes. Take the supernatant as the clarified amylase solution for later use;

[0063] Detection:

[0064] 1. Refer to GB1886.174-2024 "National Food Safety Standard Food Additives Enzyme Preparations for Food Industry" and use ultraviolet spectrophotometry to determine the amylase activity.

[0065] 1.1. Detection of the enzymatic activity of each group of amylases at different temperatures: 1 mL of clear amylase solution at pH 6.0 and temperatures of 20°C, 30°C, 50°C, 80°C, 90°C, and 100°C, the amount of enzyme required to liquefy 1 g of soluble starch in 1 h is one enzyme activity unit, U / mL.

[0066] The specific results are shown in Table 1 below:

[0067] Table 1

[0068]

[0069] 1.2. Detection of amylase activity in each group under different pH conditions: 1 mL of clear amylase solution is used to liquefy 1 g of soluble starch in 1 h at pH 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 and temperature 60°C. The amount of enzyme required is one enzyme activity unit, U / mL.

[0070] The specific results are shown in Table 2 below:

[0071] Table 2

[0072]

[0073]

[0074] From the above test, it can be seen that the clarified amylase solutions prepared in Example 1 and Example 2 have high enzyme activity at 20-80°C, and also have certain enzyme activity at 100°C. At the same time, the enzyme activity is relatively stable in the pH range of 5.0-10.0.

[0075] 2. Desizing effect of clarified amylase solutions in each group:

[0076] The same batch of sized textiles (cotton and linen fibers) were selected for desizing by immersion method. The above-mentioned clarified amylase solutions were selected respectively, and the soaking solution was prepared according to the dosage of 12 mL / L clarified amylase solution. The immersion solution was steamed for 30 minutes at a pH of 6.0 and a temperature of 80°C. The desizing rate of each group of textiles was then checked. The specific results are shown in Table 3 below:

[0077] Table 3

[0078]

[0079] It can be seen from Table 3 above that the clarified amylase solutions prepared in Example 1 and Example 2 can achieve a higher desizing efficiency in a short time.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a thermostable amylase, characterized in that: The preparation method of the thermostable amylase comprises the following steps: S1. Seed solution preparation: selecting thermotolerant Bacillus licheniformis to prepare seed solution; S2. Prepare liquid fermentation medium according to the following mass percentages: 5.0%-7.0% of okra freeze-dried powder, 3.0%-6.0% of bran, 0.1%-0.2% of yeast extract, 0.05%-0.1% of sodium nitrate, 0.1%-0.15% of peptone, 0.05%-0.15% of potassium dihydrogen phosphate, 0.1%-0.2% of sodium chloride, and the rest is distilled water; S3, liquid culture: inoculate the seed liquid into the liquid fermentation medium and ferment for 60-72 hours to obtain a fermentation material for standby use; S4, amylase separation: filter the fermented material, and the filtrate is a crude amylase solution for standby use; S5. Pretreatment of crude amylase solution: Take okra freeze-dried powder and add it to nickel nitrate solution, stir and mix to obtain a mixed solution, add the mixed solution to the crude amylase solution and continue stirring for 20-40 minutes, then add sodium borohydride, calcium chloride and disodium hydrogen phosphate, stir evenly, let stand for 4-6 hours and then centrifuge, and take the centrifuged supernatant as the thermostable amylase solution.

2. The method for preparing a thermostable amylase according to claim 1, characterized in that: The number of viable bacteria in the seed solution in step S1 is 10 6 -10 10 Pieces / mL.

3. The method for preparing a thermostable amylase according to claim 1, characterized in that: The preparation method of okra freeze-dried powder in step S2 comprises the following steps: S2-1, washing the fresh okra to remove surface impurities, then adding water to grind into pulp, and obtaining okra pulp for later use; S2-2, freeze the okra slurry at -2°C - -8°C for 10-20h, take it out and thaw it naturally, freeze-dry it, grind it through a 60-mesh sieve, and obtain okra freeze-dried powder.

4. The method for preparing a thermostable amylase according to claim 1, characterized in that: In step S3, the amount of seed liquid inoculated into the liquid fermentation medium is 3%-5% of the mass of the liquid fermentation medium, the fermentation temperature is 35±2°C, the ventilation rate in the first 10-12 hours is 0.07m / (m·min), the ventilation rate in the middle period is 0.1-0.12m / (m·min), and the ventilation rate in the last 10-20 hours of fermentation, i.e., the late period, is 0.08m / (m·min).

5. The method for preparing a thermostable amylase according to claim 1, characterized in that: The mass concentration of the nickel nitrate solution in step S5 is 2%-4%, and the solid-liquid ratio of the okra freeze-dried powder to the nickel nitrate solution is 1:1.2-1.

5.

6. The method for preparing a thermostable amylase according to claim 1, characterized in that: In step S5, the mixed solution, sodium borohydride, calcium chloride and disodium hydrogen phosphate are added to the crude amylase solution in amounts of 5%-10%, 2%-4%, 1% and 1% of the crude amylase solution respectively, and the standing temperature is 2-4°C.

7. The method for preparing a thermostable amylase according to claim 1, characterized in that: The centrifugal conditions in step S5 are 4° C. and 6000-8000 r / min for 20-25 min.

8. Use of the high temperature resistant amylase prepared by the preparation method according to any one of claims 1 to 7 in a fabric desizing process.

9. The use according to claim 8, characterized in that: The application method comprises the following steps: diluting the high temperature resistant amylase with water at a rate of 10-15 mL / L, and then impregnating the printed and dyed textiles alone or in a mixture with other auxiliaries, and then introducing hot steam for desizing treatment.

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