Method for improving high-temperature survival rate of bacillus cereus

Abstract

The invention discloses a method for improving the high-temperature survival rate of bacillus cereus. The method comprises the following steps: adding a stabilizer into a bacterial liquid obtained by fermenting the bacillus cereus; the stabilizer is a sterile aqueous solution prepared from one or more of trehalose, 2-nitrogen hexanone, gallic acid, glycerol, 3-phenylpropionic acid, cane sugar and L-alanine. By adding the stabilizer, the high-temperature thallus survival rate of the bacillus cereus is improved, the application field of the bacillus cereus is widened, and the limitation of temperature on the application of the bacillus cereus is effectively solved.

Description

Technical Field

[0001] The invention relates to a method for improving the high-temperature survival rate of Bacillus cereus, and belongs to the field of biotechnology. Background Art

[0002] Bacillus cereus can produce antibacterial substances, inhibit the reproduction of harmful microorganisms, and has the characteristics of decomposing organic matter and inhibiting the growth of harmful microorganisms, thus having a certain ability to prevent infection. Therefore, Bacillus cereus can be added to agricultural fertilizers to inhibit the growth of harmful microorganisms. However, the survival rate of Bacillus cereus is significantly reduced under high temperature conditions, and its metabolic activity is weakened, which will affect its fermentation ability and probiotic effect during the composting process. Therefore, finding a method for improving the survival rate of Bacillus cereus at high temperatures can improve its application in agriculture. Summary of the invention

[0003] In view of the problems existing in the prior art, the present invention provides a method for improving the survival rate of Bacillus cereus at high temperature. The method of the present invention can reduce the activity loss of Bacillus cereus at high temperature.

[0004] The method for improving the high temperature survival rate of Bacillus cereus of the present invention comprises adding a stabilizer into the bacterial liquid after the fermentation of Bacillus cereus.

[0005] The stabilizer is a sterile aqueous solution prepared from one or more of trehalose, 2-azolopyrrolidone, gallic acid, glycerol, 3-phenylpropionic acid, sucrose, and L-alanine, and the mass concentration of the stabilizer added is 0.1-8%.

[0006] Furthermore, the stabilizer is trehalose, added at a concentration of 0.1-0.5%; or the stabilizer is 2-azolopyrrolidone, added at a concentration of 0.1-0.5%; or the stabilizer is glycerol, added at a concentration of 3-8%; or the stabilizer is 3-phenylpropionic acid, added at a concentration of 0.1-0.5%; or the stabilizer is sucrose, added at a concentration of 3-8%; or the stabilizer is L-alanine, added at a concentration of 0.1-0.3%.

[0007] Furthermore, the stabilizer is composed of L-alanine and 2-azocetone.

[0008] The concentration of L-alanine in the system is 0.1-0.3%, and the concentration of 2-azolopyridine is 0.1-0.5%.

[0009] Most preferably, the concentration of L-alanine in the system is 0.2%, and the concentration of 2-azolopyridine is 0.1%.

[0010] The method for improving the high temperature survival rate of Bacillus cereus of the present invention specifically comprises the following steps: Step 1: Streak the Bacillus cereus glycerol stock on an LB agar plate and culture it in a 37°C incubator for 24 h. Select a single colony and transfer it to a test tube containing 5 mL of LB liquid culture medium. Culture it in a 37°C incubator for 24 h to obtain the fermentation broth.

[0011] Step 2: Inoculate the Bacillus cereus test tube fermentation broth into 200 mL of LB liquid culture medium at a volume ratio of 5%, and culture in a 37° C. incubator for 24-48 hours to obtain the Bacillus cereus fermentation broth.

[0012] Step 3: The Bacillus cereus fermentation liquid obtained in step 2 is centrifuged at 4° C. and 10,000 rpm for 20 min to obtain Bacillus cereus fermentation cells; the obtained cells are resuspended in sterile water and a stabilizer is added.

[0013] The Bacillus cereus can be purchased conventionally.

[0014] The LB medium contains: 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L sodium chloride. When preparing the agar medium, add 2 g / L agar.

[0015] The beneficial effects of the present invention are embodied in: The invention provides a method for improving the high-temperature survival rate of Bacillus cereus, and optimizes the type and amount of stabilizer added. The optimized composite stabilizer formula comprises 0.1% of 2-azocetone by mass concentration and 0.2% of L-alanine by mass concentration, and the survival rate of Bacillus cereus cells can be improved by 1.67 times at a high temperature of 50°C.

[0016] The prior art has not paid attention to the survival rate of Bacillus cereus at high temperatures. The present invention improves the high-temperature stability of Bacillus cereus by adding a stabilizer, broadens the application field of Bacillus cereus, and effectively solves the limitation of temperature on its application. DETAILED DESCRIPTION

[0017] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. In the following examples, various processes and methods not described in detail are conventional methods known in the art. The specific embodiments of the present invention described below are to supplement the purpose, technical solutions and effects of the present invention, but they are not intended to limit the scope of protection of the present invention. Within the scope of the present invention, simple changes, modifications or replacements can be made, which are all considered to be within the scope of protection of the present invention. Example 1: Fermentation preparation of Bacillus cereus

[0018] Streak the strain of Bacillus cereus glycerol preservation tube on LB agar medium plate, culture in 37℃ incubator for 24h, select single colony into test tube containing 5mL LB medium, culture in 37℃ incubator for 24h to obtain Bacillus cereus test tube fermentation liquid. Take 1mL of the above test tube fermentation liquid, inoculate it into 200mL liquid LB medium, culture in 37℃ incubator for 48h to obtain Bacillus cereus fermentation liquid. Take the above fermentation liquid and centrifuge it at 4℃ and 10000rpm for 20min to obtain bacteria. Example 2: Selection of heat stabilizer

[0019] Single factor optimization of stabilizers: trehalose, 2-azolophenone, glycerol, gallic acid, 3-phenylpropionic acid, sucrose, L-alanine, and the protective effect of the added types and amounts on the activity of Bacillus cereus under high temperature conditions. For the convenience of experimental operation, 10 mL of Bacillus cereus fermentation broth was taken, and the cells were obtained by centrifugation at 4 ° C and 10000 rpm for 20 min. The cells were resuspended in 5 mL of sterile water. The types of stabilizers added and the final mass concentration of the system are shown in Table 1. Sterile water was added to make up to 10 mL to prepare a mixed solution. The control group was without stabilizer.

[0020] The mixed solutions of each group were placed in incubators at 30°C, 45°C and 50°C respectively, incubated for 12-24 hours, and then taken out and the OD was measured using an enzyme-labeled instrument. 600 The value was used to calculate the survival rate of Bacillus cereus under high temperature conditions. The formula is as follows: A% = (A 1 -A 0 ) / A 0 ×100% Among them, A% is the survival rate of Bacillus cereus at high temperature, A 0 A is the number of viable bacteria in the control group after the culture is completed. 1 It is the number of viable bacteria in the sample after cultivation at different high temperatures, that is, comparing the increase rate of the number of viable bacteria in each experimental group compared with the control group.

[0021]

[0022] Under different stabilizers and different addition amounts, the survival improvement rates of Bacillus cereus at different temperatures are shown in Table 2. The addition of stabilizers can significantly improve the survival rate of Bacillus cereus in a high temperature environment. The survival rates of trehalose at 0.3%, 2-azolopyridine at 0.1%, L-alanine at 0.2%, 3-phenylpropionic acid at 0.5% and sucrose at 0.1% are all improved. Compared with the control group without protective agent, the high temperature survival rate of the bacteria is increased by up to 1.49 times.

[0023] Example 3: Combination of Stabilizers

[0024] The stabilizer compound addition was optimized, and trehalose (system mass concentration 0.3%), 2-azolopialdehyde (system mass concentration 0.1%), and L-alanine (system mass concentration 0.2%) with the highest bacterial survival rate were selected for combination and cultured at high temperatures of 30°C, 45°C, and 50°C, respectively.

[0025] Take 10 mL of Bacillus cereus fermentation broth, centrifuge at 4°C and 10,000 rpm for 20 min to obtain the bacterial cells, resuspend the bacterial cells in 5 mL of sterile water, add the stabilizer compound as shown in Table 3, and make up to 10 mL with sterile water to prepare a mixed solution. The control group was a control group without stabilizer.

[0026] The mixed solution was placed in an incubator at 30°C, 45°C and 50°C, incubated for 12-24 hours, and then the OD was measured using an enzyme-labeled instrument. 600 The numerical value was obtained and the survival rate of Bacillus cereus under high temperature conditions was calculated.

[0027] The results are shown in Table 3. At 30°C, 45°C and 50°C, in the compound combinations of different protective agents, when 0.2% L-alanine and 0.1% 2-azocetone were added, the high-temperature survival rate of the bacteria was increased by up to 1.67 times compared with the group without protective agent, which significantly improved the survival rate of Bacillus cereus at high temperature.

[0028]

Claims

1. A method for improving the high temperature survival rate of Bacillus cereus, characterized in that: A stabilizer is added to the bacterial liquid after fermentation of Bacillus cereus; the stabilizer is a sterile aqueous solution prepared from one or more of trehalose, 2-azolopyrrolidone, gallic acid, glycerol, 3-phenylpropionic acid, sucrose and L-alanine.

2. The method according to claim 1, characterized in that: The mass concentration of the stabilizer in the system is 0.1-8%.

3. The method according to claim 1, characterized in that: The stabilizer is trehalose, and the added concentration is 0.1-0.5%.

4. The method according to claim 1, characterized in that: The stabilizer is 2-azocetone, and the added concentration is 0.1-0.5%.

5. The method according to claim 1, characterized in that: The stabilizer is glycerol, and the added concentration is 3-8%.

6. The method according to claim 1, characterized in that: The stabilizer is 3-phenylpropionic acid, and the added concentration is 0.1-0.5%.

7. The method according to claim 1, characterized in that: The stabilizer is sucrose, and the added concentration is 3-8%.

8. The method according to claim 1, characterized in that: The stabilizer is L-alanine, and the added concentration is 0.1-0.3%.

9. The method according to claim 1, characterized in that: The stabilizer is composed of L-alanine and 2-azocetone.

10. The method according to claim 9, characterized in that: The concentration of L-alanine in the system is 0.2%, and the concentration of 2-azolopyridine is 0.1%.

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