Method for preparing pullulanase by fermenting bacillus subtilis

Abstract

The application provides a method for preparing pullulanase by fermentation of bacillus subtilis, and the preparation method ends in a specific pH environment through a strain activation process, a primary fermentation process and a fed-batch fermentation process, so that a pullulanase-containing fermentation liquor with an enzyme activity of 550 U / mL or more can be obtained. In the fermentation process, the pH change range is strictly controlled to determine the end point of the fermentation, so that the high enzyme activity of the pullulanase is ensured, and a more time-effective end point determination method is provided for production.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for preparing pullulanase using Bacillus subtilis fermentation. Background Technology

[0002] Pullulanase is an important starch debranching enzyme that specifically hydrolyzes the glycosidic bonds of amylopectin and pullulan polysaccharides to form amylose and maltotriose. It can synergistically increase the glucose or maltose content of starch with saccharifying enzymes or amylases, making it crucial in the starch processing industry. Furthermore, pullulanase also has a significant market and application potential in the feed and textile industries.

[0003] However, obtaining highly active pullulanase remains a challenge for the development of this application field. Summary of the Invention

[0004] Therefore, it is necessary to provide a method for preparing pullulanase by fermentation using Bacillus subtilis, which can ensure the high activity of pullulanase in the fermentation broth.

[0005] The present invention adopts the following technical solution:

[0006] This invention provides a method for preparing pullulanase using Bacillus subtilis fermentation, comprising the following steps: inoculating Bacillus subtilis into an activation medium for activation to obtain an activated bacterial culture broth; transferring the activated bacterial culture broth into a fermentation medium, controlling the culture conditions: temperature 36-38℃, pH 5.6-5.8, to carry out primary fermentation and obtain a primary fermentation product broth; continuously adding feed medium to the primary fermentation product broth for fed-batch fermentation until the pH of the fermentation broth rises to 5.9-6.1, stopping the feeding and ending the fermentation, and transferring the broth to a tank to obtain the pullulanase product broth.

[0007] Preferably, both the fermentation medium and the fed medium contain glucose, molasses, starch, corn steep liquor, urea, and silicone oil, and the concentrations of glucose and molasses in the fermentation medium are lower than those in the fed medium, while the concentrations of the other components in the fermentation medium are higher than those in the corresponding components in the fed medium.

[0008] Preferably, the fermentation medium comprises: 5 wt% glucose, 5 wt% molasses, 5 wt% starch, 3 wt% corn steep liquor, 2 wt% urea, 0.6 wt% ammonium dihydrogen phosphate, 0.3 wt% dipotassium hydrogen phosphate, 0.02 wt% silicone oil, and the balance being water.

[0009] Preferably, the composition of the supplemental culture medium includes: 10 wt% glucose, 10 wt% molasses, 2 wt% corn steep liquor, 1 wt% urea, 0.3 wt% ammonium dihydrogen phosphate, 0.1 wt% dipotassium hydrogen phosphate, 0.01 wt% silicone oil, and the balance being water.

[0010] Preferably, the primary fermentation controlled culture conditions are: temperature 36-38℃, pH 5.6-5.8, rotation speed 400-800 r / min, and air volume 1-1.5 vvm.

[0011] Preferably, during the primary fermentation process, ammonia water is used to adjust the pH to 5.6–5.8.

[0012] Preferably, the activation medium is LB medium, which comprises: 1 wt% tryptone, 0.5 wt% yeast extract, 1 wt% sodium chloride, and the balance being water, with a pH of 7.0.

[0013] Preferably, the primary fermentation time is 6 hours, the fed-batch fermentation time is 18-20 hours, and the residual sugar is maintained at ≤3.5% during the fed-batch process.

[0014] The present invention also provides a pullulanase fermentation product prepared using the above method. The enzyme activity of the pullulanase fermentation product, as tested in a vat, reaches 550 U / mL.

[0015] Compared with the prior art, the core advantage of this invention is:

[0016] The preparation method of this invention relies on specific process conditions, including activation of Bacillus subtilis strains capable of producing pullulanase, primary fermentation, fed-batch fermentation to a specific pH environment, to obtain a pullulanase-containing fermentation broth with an enzyme activity of over 550 U / mL. This method strictly controls the pH range during fermentation to determine the endpoint, ensuring high pullulanase activity and providing a more timely endpoint determination method for production. This effectively controls the fermentation cycle and helps reduce production costs. Detailed Implementation

[0017] The technical concept of this invention is to provide a method for preparing pullulanase by fermentation using Bacillus subtilis. The pH value is used to determine the end point of fermentation, which effectively avoids the decrease in pullulanase activity in the fermentation product liquid, ensures that pullulanase with high activity is obtained from the fermentation liquid, and effectively controls the fermentation cycle, thereby reducing production costs.

[0018] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. The following embodiments are only used to illustrate the present invention, and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0019] The detection method for pullulanase in fermentation broth is as follows: refer to GB 1886.174-2016 National Food Safety Standard - Determination of pullulanase activity in food additives and enzyme preparations for food industry.

[0020] Example 1

[0021] This embodiment provides a method for preparing pullulanase using Bacillus subtilis fermentation, comprising the following steps:

[0022] (1) Seed activation:

[0023] Commercially available Bacillus subtilis, which can produce pullulanase, was inoculated into an activation medium for activation culture to obtain a bacterial culture solution.

[0024] In this step, the activation medium is LB medium, which includes the following components by weight percentage: 1% tryptone, 0.5% yeast extract, 1% sodium chloride, and the balance water, pH 7.0, sterilized at 121°C for 30 min.

[0025] (2) Primary fermentation:

[0026] The bacterial culture broth from step (1) is inoculated into the fermentation medium for liquid deep fermentation to obtain a fermentation broth containing pullulanase.

[0027] In this step, the culture conditions for submerged fermentation are: temperature 37℃, pH adjusted to 5.75 with ammonia, rotation speed 600 r / min, and air volume 1-1.5 vvm.

[0028] In this step, the fermentation medium comprises the following components by weight percentage: 5% glucose, 5% molasses, 5% starch, 3% corn steep liquor, 2% urea, 0.6% ammonium dihydrogen phosphate, 0.3% dipotassium hydrogen phosphate, 0.02% silicone oil, and the balance being water.

[0029] (3) Fed-in-place fermentation:

[0030] After 6 hours of fermentation, fed-batch culture medium was started, and the residual sugar was maintained at ≤3.5% during the feeding process for 18 hours.

[0031] In this step, the feed medium comprises the following components by weight percentage: 10% glucose, 10% molasses, 2% corn steep liquor, 1% urea, 0.3% ammonium dihydrogen phosphate, 0.1% dipotassium hydrogen phosphate, 0.01% silicone oil, and the balance being water.

[0032] (4) Determine the fermentation endpoint:

[0033] Fermentation lasts approximately 24 hours. Once the pH rises to 5.90, feeding is stopped and fermentation is terminated.

[0034] Under this process, pullulanase activity continuously increases during fermentation, and the pullulanase activity in the fermentation broth tested in the tank was 586 U / mL.

[0035] Example 2

[0036] This embodiment provides a method for preparing pullulanase using Bacillus subtilis fermentation. The method steps are basically the same as those in Example 1, with the only difference being:

[0037] (3) Fed fermentation: After 6 hours of fermentation, fed culture medium was started. The residual sugar was maintained at ≤3.5% during the feeding process, and the feeding time was 19 hours.

[0038] (4) Determine the fermentation endpoint:

[0039] Fermentation lasts approximately 25 hours. Once the pH rises to 6.0, feeding is stopped and fermentation is terminated.

[0040] Under this process, pullulanase activity continuously increases during fermentation, and the pullulanase activity in the fermentation broth tested in the tank was 620 U / mL.

[0041] Example 3

[0042] This embodiment provides a method for preparing pullulanase using Bacillus subtilis fermentation. The method steps are basically the same as those in Example 1, with the only difference being:

[0043] (3) Fed fermentation: After 6 hours of fermentation, fed culture medium was started. The residual sugar was maintained at ≤3.5% during the feeding process, and the feeding time was 20 hours.

[0044] (4) Determine the fermentation endpoint:

[0045] Fermentation lasts approximately 26 hours. Once the pH rises to 6.1, feeding is stopped and fermentation is terminated.

[0046] Under this process, pullulanase activity continuously increases during fermentation, and the pullulanase activity in the fermentation broth tested in the tank was 660 U / mL.

[0047] Comparative Example 1

[0048] This comparative example provides a method for preparing pullulanase using Bacillus subtilis fermentation. The method steps are basically the same as those in Example 1, with the only difference being:

[0049] (4) Determine the fermentation endpoint:

[0050] When the pH rises to 5.8, stop feeding and end the fermentation process.

[0051] Under this process, the enzyme activity of pullulanase in the fermentation broth tested in the tank was 424 U / mL.

[0052] Comparative Example 2

[0053] This comparative example provides a method for preparing pullulanase using Bacillus subtilis fermentation. The method steps are basically the same as those in Example 1, with the only difference being:

[0054] (3) Fed fermentation: After 6 hours of fermentation, fed culture medium was started. The residual sugar was maintained at ≤3.5% during the feeding process, and the feeding time was 20 hours.

[0055] (4) Determine the fermentation endpoint:

[0056] When the pH rises to 6.2, stop feeding and end the fermentation process.

[0057] Under this process, the enzyme activity of pullulanase in the fermentation broth tested in the tank was 458 U / mL.

[0058] Comparative Example 3

[0059] This comparative example provides a method for preparing pullulanase using Bacillus subtilis fermentation. The method steps are basically the same as those in Example 1, with the only difference being:

[0060] (3) Fed fermentation: After 6 hours of fermentation, fed culture medium was started. The residual sugar was maintained at ≤3.5% during the feeding process, and the feeding time was 20 hours.

[0061] (4) Determine the fermentation endpoint:

[0062] When the pH rises to 6.3, stop feeding and end the fermentation process.

[0063] Under this process, the enzyme activity of pullulanase in the fermentation broth tested in the tank was 237 U / mL.

[0064] The results of the above experimental examples regarding fed-batch fermentation and pH control at the fermentation endpoint show that: in a fermentation system containing glucose, molasses, corn steep liquor, urea, and silicone oil, the pH range at the primary fermentation stage, the fed-batch fermentation stage, and the fermentation endpoint of Bacillus subtilis, which can produce pullulanase, has a significant impact on the enzyme activity of pullulanase in the obtained fermentation broth. The results show that only within the pH range of 5.9 to 6.1 can the enzyme activity of pullulanase fermentation products tested in the tank be guaranteed to reach above 550 U / mL.

[0065] It should be noted that the above embodiments are only for further elaboration and explanation of the technical solution of the present invention, and are not intended to further limit the technical solution of the present invention. The method of the present invention is only a preferred embodiment and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing pullulanase using Bacillus subtilis fermentation, characterized in that, The steps include the following: Bacillus subtilis was inoculated into an activation medium for activation to obtain an activated bacterial culture solution. The activated bacterial culture broth was transferred into a fermentation medium, and the culture conditions were controlled as follows: temperature 36~38℃, pH 5.6~5.8, to carry out primary fermentation and obtain primary fermentation product broth. Feed culture medium was continuously added to the primary fermentation product liquid for fed fermentation until the pH of the fermentation liquid rose to 5.9-6.

1. Feeding was then stopped and fermentation was terminated. The liquid was then discharged from the tank to obtain pullulanase product liquid. The fermentation medium consists of: 5 wt% glucose, 5 wt% molasses, 5 wt% starch, 3 wt% corn steep liquor, 2 wt% urea, 0.6 wt% ammonium dihydrogen phosphate, 0.3 wt% dipotassium hydrogen phosphate, 0.02 wt% silicone oil, with the remainder being water. The composition of the supplemental culture medium is as follows: 10 wt% glucose, 10 wt% molasses, 2 wt% corn steep liquor, 1 wt% urea, 0.3 wt% ammonium dihydrogen phosphate, 0.1 wt% dipotassium hydrogen phosphate, 0.01 wt% silicone oil, with the remainder being water; The primary fermentation lasts for 6 hours, and the fed-batch fermentation lasts for 18-20 hours, with the residual sugar maintained at ≤3.5% during the fed-batch process.

2. The method for preparing pullulanase by fermentation of Bacillus subtilis according to claim 1, characterized in that, The controlled culture conditions are: temperature 36~38℃, pH 5.6~5.8, rotation speed 400~800 r / min, and air volume 1~1.5vvm.

3. The method for preparing pullulanase by fermentation of Bacillus subtilis according to claim 2, characterized in that, During the primary fermentation process, ammonia water is used to adjust the pH to 5.6-5.

8.

4. The method for preparing pullulanase by fermentation using Bacillus subtilis according to claim 1, characterized in that, The activation medium is LB medium, with the following composition: 1 wt% tryptone, 0.5 wt% yeast extract, 1 wt% sodium chloride, and the remainder is water, with a pH of 7.0.