A culture medium for culturing Brevibacillus laterosporus and its application

Abstract

The present invention discloses a culture medium for culturing Brevibacillus laterosporus and its application, which relates to the field of bioengineering. The fermentation medium comprises the following components: molasses 12 - 16 g / L, beef powder 6 - 10 g / L, magnesium sulfate 0.6 - 1 g / L, KH2PO4 0.3 - 0.7 g / L, and K2HPO4 1 - 3 g / L. By using the culture medium provided by the present invention for fermentative culturing of Brevibacillus laterosporus, the viable bacteria content and spore rate of the fermentation broth of Brevibacillus laterosporus can be effectively improved. Therefore, this culture medium can be applied to improve the fermentation level of Brevibacillus laterosporus, laying a foundation for the large-scale preparation and application of Brevibacillus laterosporus microcapsule preparations. The bacterial liquid cultured by using this culture medium can be used for preventing and controlling various plant diseases such as rice bacterial blight and tomato bacterial wilt, and can alleviate the problem of poor biological control effect of microbial agents.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and particularly to a culture medium for culturing Brevibacillus laterosporus and its application. Background Art

[0002] Brevibacillus laterosporus, belonging to the phylum Firmicutes and the genus Brevibacillus, is a rod-shaped spore-forming bacterium that can produce its unique canoe-shaped parasporal body. All along, the special morphological characteristics of Brevibacillus laterosporus have attracted the attention of many scientific researchers. In the mid-20th century, Hannay observed fixed, spore-embedded cells and spores using optical and electron microscopes and reached the following conclusion: The parasporal body forms before sporulation and remains attached to one side of the spore after sporulation. It is widely distributed in nature and has been isolated from materials such as soil, volcanic mudflows, fresh water, sea water, insects, leaf surfaces, carob beans, compost, milk, cheese, honey, starchy foods, wastewater from rubber product factories, animal furs, and quails. Brevibacillus laterosporus is a microbial resource with both antibacterial and insecticidal properties and medical value. Brevibacillus laterosporus has a variety of biological activities and exhibits broad-spectrum antibacterial activity, especially against bacteria and fungi. Recent whole-genome sequencing results show that Brevibacillus laterosporus also has the potential to produce substances such as polyketides, non-ribosomal peptides, and toxins. It can also produce active substances such as aminopeptidase inhibitors, glutaric acid 7-ACA acyltransferase, thrombin inhibitors, and lysine, showing good application prospects. The antibacterial property of Brevibacillus laterosporus is related to the antibacterial peptides it produces. Regarding the insecticidal activity of Brevibacillus laterosporus, there is more research abroad, and commercial biological insecticides have been launched. A variety of Brevibacillus laterosporus are pathogenic to different invertebrates such as insects, nematodes, and mollusks.

[0003] The insecticidal activity of Brevibacillus laterosporus is mainly due to the toxicity of the proteins contained in the parasporal bodies to a variety of insects. After contacting or invading the target, different substances are produced at different stages of the cell growth cycle, thus playing a toxic role. The spores of Brevibacillus laterosporus strains contain nematicidal compounds that can inhibit the egg hatching and larval development of nematodes. Therefore, Brevibacillus laterosporus has become a biological control agent for parasitic nematodes. Brevibacillus laterosporus also has functions such as phosphorus and potassium solubilization. Research shows that it is suitable for the development and application of bacterial fertilizers for crops and can increase the content of available phosphorus in the soil and the yield of crops to a certain extent. It has been found that Brevibacillus laterosporus can degrade organophosphorus pesticides such as isocarbophos and omethoate. More and more substances have also been found to be degraded by Brevibacillus laterosporus. The most typical example is the degradation of polyvinyl alcohol into acetate, and it can also produce various enzymes such as lignin peroxidase, laccase, aminopyrine-N-demethylase, NADH-DCIP reductase, and malachite green reductase. It can be seen that Brevibacillus laterosporus has the characteristics of being developed into a biological control agent for preventing and controlling insects, nematodes, mollusks, and plant pathogens. At the same time, its characteristics of phosphorus and potassium solubilization and biodegradation of substances such as chemical pesticides endow it with the ability of soil bioremediation.

[0004] Develop a culture medium to improve the fermentation level of Brevibacillus laterosporus, and provide a new fermentation process suitable for large-scale fermentation, which can lay a foundation for the large-scale preparation and application of Brevibacillus laterosporus microcapsule preparations. Summary of the Invention

[0005] The purpose of the present invention is to provide a culture medium for culturing Brevibacillus laterosporus and its application to solve the problems existing in the above-mentioned prior art. The culture medium provided by the present invention can effectively improve the fermentation level of Brevibacillus laterosporus and lay a foundation for the large-scale preparation and application of Brevibacillus laterosporus microcapsule preparations.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides a culture medium for culturing Brevibacillus laterosporu, and the culture medium comprises the following components: molasses 12 - 16 g / L, beef powder 6 - 10 g / L, magnesium sulfate 0.6 - 1 g / L, KH 2 PO 4 0.3 - 0.7 g / L, and K 2 HPO 4 1 - 3 g / L.

[0008] Preferably, the culture medium specifically comprises the following components: molasses 14.2 g / L, beef powder 8.9 g / L, magnesium sulfate 0.8 g / L, KH 2PO 4 0.5 g / L and K 2 HPO 4 2 g / L.

[0009] The present invention also provides the application of the above-mentioned culture medium in the fermentation culture of Brevibacillus brevis.

[0010] The present invention also provides a method for improving the fermentation level of Brevibacillus brevis, which uses the above-mentioned culture medium to carry out fermentation culture on the Brevibacillus brevis.

[0011] Furthermore, the conditions for the fermentation culture are: pH is 6 - 7, temperature is 31 - 35 °C, rotation speed is 180 - 220 r / min, inoculum amount is 3 - 5%, and culture time is 45 - 50 h.

[0012] The present invention also provides a fermentation broth of Brevibacillus brevis prepared according to the above-mentioned method.

[0013] The present invention also provides a Brevibacillus brevis preparation, which is prepared by mixing the above-mentioned fermentation broth, compound starch and silica white.

[0014] Furthermore, the mass ratio of the fermentation broth, the compound starch and the silica white is 5:9:1.

[0015] The present invention also provides the application of the above-mentioned fermentation broth or Brevibacillus brevis preparation in preventing and controlling plant bacterial diseases:

[0016] Furthermore, the plant bacterial diseases include rice bacterial blight and solanaceous bacterial wilt.

[0017] The present invention discloses the following technical effects:

[0018] 1. The components of the fermentation culture medium of the present invention are selected based on the characteristics of Brevibacillus brevis, mainly beef powder, molasses, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate. The raw materials are easily available, the cost is low, and the preparation is simple.

[0019] 2. The fermentation process control of the present invention is simple, easy to operate, and the effect is obvious. Under this fermentation process, the bacterial cell yield and spore rate can be greatly improved.

[0020] 3. The fermentation culture broth of the present invention has the characteristics of high activity, high viable bacteria content and high spore rate, which can increase the preservation time of the subsequent preparation and enhance the efficacy stability of the preparation. Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is the staining results of the experimental group (B) with the addition amount of magnesium sulfate being 0.8 g / L and the blank control group (A) in Experimental Example 1;

[0023] Figure 2 It is the physical diagram of the plate count of the experimental groups at 29 °C (A) and 35 °C (B) in Experimental Example 2;

[0024] Figure 3 It is the prevention and control effect diagram of tomato bacterial wilt in Example 6. Among them, the four plants on the left are the treatment group with the 1300-fold dilution of the fermentation broth in Example 1, the four plants in the middle are the water treatment group, and the four plants on the right are the streptomycin treatment group. Detailed implementation manners

[0025] Now, the various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0027] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes the preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0028] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0029] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0030] The Bacillus brevis used in the following examples was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms under the deposit number CGMCC No. 17435.

[0031] LB medium: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L.

[0032] Example 1

[0033] (1) Activation of the target strain: Inoculate Bacillus brevis on LB solid medium and culture it in an incubator at 30 °C for 24 h.

[0034] (2) Seed solution: Pick a single colony and inoculate it into LB liquid medium, and culture it at 30 °C and 200 r / min for 12 h to obtain the seed solution.

[0035] (3) Shake flask fermentation: Inoculate the seed solution into the fermentation medium and carry out fermentation culture. The culture conditions are: pH 6.5, temperature 35 °C, rotation speed 200 r / min, liquid loading 50 mL / 250 mL, inoculation amount 4%, and culture time 48 h.

[0036] Fermentation medium: molasses 14.2 g / L, beef powder 8.9 g / L, magnesium sulfate 0.8 g / L, KH 2 PO 4 0.5 g / L, K 2 HPO 4 2.0 g / L.

[0037] Example 2

[0038] (1) Activation of the target strain: Inoculate Bacillus brevis on LB solid medium and culture it in an incubator at 30 °C for 24 h.

[0039] (2) Seed solution: Pick a single colony and inoculate it into LB liquid medium, and culture it at 30 °C and 200 r / min for 12 h to obtain the seed solution.

[0040] (3) Shake flask fermentation: Inoculate the seed liquid into the fermentation medium and carry out fermentation culture. Culture conditions: pH is 6, temperature is 31 °C, rotation speed is 180 r / min, liquid loading is 100 mL / 250 mL, inoculation amount is 3%, and culture time is 45 h.

[0041] Fermentation medium: Molasses 12.0 g / L, beef powder 10.0 g / L, magnesium sulfate 0.6 g / L, KH 2 PO 4 0.7 g / L, K 2 HPO 4 1.0 g / L.

[0042] Example 3

[0043] (1) Activation of the target strain: Inoculate Brevibacillus laterosporus on the LB solid medium and culture it in an incubator at 30 °C for 24 h.

[0044] (2) Seed liquid: Pick a single colony and inoculate it into the LB liquid medium, and culture it at 30 °C and 200 r / min for 12 h to obtain the seed liquid.

[0045] (3) Shake flask fermentation: Inoculate the seed liquid into the fermentation medium and carry out fermentation culture. Culture conditions: pH is 7, temperature is 35 °C, rotation speed is 220 r / min, liquid loading is 50 mL / 250 mL, inoculation amount is 5%, and culture time is 50 h.

[0046] Fermentation medium: Molasses 16.0 g / L, beef powder 6.0 g / L, magnesium sulfate 1.0 g / L, KH 2 PO 4 0.3 g / L, K 2 HPO 4 3.0 g / L.

[0047] Comparative Example 1

[0048] Same as Example 1, the only difference is that the fermentation medium in Example 1 is replaced with LB medium.

[0049] Comparative Example 2

[0050] Same as Example 1, the only difference is that the fermentation medium in Example 1 is replaced with Medium A.

[0051] Medium A: Corn starch 20 g / L, silkworm chrysalis powder 25 g / L, yeast extract powder 10 g / L, corn steep liquor 10 g / L.

[0052] Comparative Example 3

[0053] Same as Example 1, the only difference is that the fermentation medium in Example 1 is replaced with Medium B.

[0054] Medium B: 28.65 g / L of sucrose, 17.04 g / L of beef extract, 13.01 g / L of magnesium ions, 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride.

[0055] Comparative Example 4

[0056] Same as Example 1, except that the fermentation medium in Example 1 was replaced with Medium C.

[0057] Medium C: 30 g / L of sucrose, 8 g / L of yeast extract paste, 12 g / L of peptone, MgSO 4 0.7 g / L, KH 2 PO 4 2.5 g / L, MnSO 4 0.1 g / L, CaCO 3 8 g / L.

[0058] The viable cell content and spore rate after fermentation culture in Example 1 and Comparative Examples 1-4 were counted, and the results are shown in Table 1. It can be seen from Table 1 that when culturing Brevibacillus brevis with the medium of this example, the viable cell content and spore rate have been greatly improved compared with LB medium, Medium A-C.

[0059] Table 1 Detection results of viable cell content and spore rate after fermentation culture in Example 1 and Comparative Examples 1-3

[0060]

[0061]

[0062] Experimental Example 1

[0063] The addition amounts of magnesium sulfate in the fermentation medium of Example 1 were adjusted to 0 g / L, 0.4 g / L, 0.8 g / L, 1.2 g / L and 1.6 g / L respectively, and other steps were the same as those in Example 1. Brevibacillus brevis was fermented and cultured respectively. After fermentation culture, the viable cell content was counted, and the results are shown in Table 2. The results show that when the addition amount of magnesium sulfate is 0.8 g / L, the viable cell content is the highest, and the spore rate is significantly increased compared with the blank control ( Figure 1 ).

[0064] Table 2 Effects of different addition amounts of magnesium sulfate on viable cell content

[0065] Magnesium sulfate addition amount (g / L) Bacterial content (cfu / mL) 0.4 <![CDATA[9.78×10 11 > 0.8 <![CDATA[2.19×10 12 > 1.2 <![CDATA[1.05×10 12 > 1.6 <![CDATA[7.89×10 11 > 0 (blank) <![CDATA[1.17×10 11 >

[0066] Experimental Example 2

[0067] The shaking flask fermentation culture temperature in step (3) of Example 1 was adjusted to 29°C, 31°C, 33°C, 35°C, 37°C, and 39°C respectively. Other steps were the same as in Example 1, and Bacillus brevis fermentation culture was carried out separately. After fermentation culture, the viable cell content was counted. The results are shown in Table 3. The physical pictures of the plate counts for the experimental groups at 29°C and 35°C are shown in Figure 2 . The results showed that the viable cell content reached the highest at 35°C, and the spore formation time was advanced. The cell content of Bacillus brevis decreased significantly at temperatures below 29°C.

[0068] Table 3 Effect of temperature on viable cell content

[0069] Temperature Bacterial content (cfu / mL) 29℃ <![CDATA[3.19×10 3 > 31℃ <![CDATA[1.05×10 12 > 33℃ <![CDATA[2.10×10 12 > 35℃ <![CDATA[2.14×10 12 > 37℃ <![CDATA[7.79×10 11 > 39℃ <![CDATA[4.15×10 11 >

[0070] Experimental Example 3

[0071] The carbon source molasses in the fermentation medium of Example 1 was replaced with glycerol, lactose, mannose, sucrose, maltose, and glucose respectively. Other steps were the same as in Example 1, and Bacillus brevis fermentation culture was carried out separately. After fermentation culture, the viable cell content was counted. The results are shown in Table 4. The results showed that when molasses was added as the carbon source, the viable cell content in the fermentation broth was significantly higher than that of other monosaccharides and polysaccharides.

[0072] Table 4 Effect of different carbon sources on viable cell content

[0073] Carbon source Bacterial content (cfu / mL) Blank <![CDATA[9.26×10 10 > Molasses <![CDATA[2.16×10 12 > Glycerol <![CDATA[2.21×10 11 > Lactose <![CDATA[3.17×10 11 > Mannose <![CDATA[8.56×10 10 > Sucrose <![CDATA[2.26×10 10 > Maltose <![CDATA[9.76×10 10 > Glucose <![CDATA[9.55×10 10 >

[0074] Example 4

[0075] The fermentation broth prepared in step (3) of Example 1 was processed by the microcapsule process together with composite starch (the mass ratio of starch to dextrin was 1:5) and silica white (where the mass ratio of fermentation broth, composite starch, and silica white was 5:9:1), and the preparation product was obtained after drying at 50°C.

[0076] Comparative Example 5

[0077] Same as Example 2, the difference was only that the fermentation broth was replaced with the fermentation broth prepared in step (3) of Comparative Example 4.

[0078] The viable cell content of the preparations prepared in Example 4 and Comparative Example 5 was counted, and the cell survival rate was calculated according to formula (1). The results are shown in Table 5. The results showed that after the fermentation broth prepared in Example 1 was processed into a preparation, the viable cell content remained at 10 11 cfu / mL level, meeting the registration requirements for microbial pesticides.

[0079]

[0080] Table 5 Viable Bacterial Content and Survival Rate of the Formulation Products in Example 4 and Comparative Example 5

[0081] Preparation Viable bacterial content (cfu / mL) Survival rate (%) Comparative example 5 <![CDATA[3.21×10 10 > 88.97 Example 4 <![CDATA[4.30×10 11 > 97.72

[0082] Control Effect of Example 5 Against Bacterial Blight of Rice

[0083] 1. Pathogenic indicator bacterium: Xanthomomas oryzae pv. oryzae, donated by Shanghai Jiao Tong University.

[0084] Pathogenic bacterium culture medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl.

[0085] 2. Experimental methods and results

[0086] Six disinfected rice seeds were sown in each PVC flower pot. When the rice grew to the three-leaf and one-heart stage, the pathogenic bacterium was sprayed and inoculated on the leaves and roots. After 24 hours, the fermentation broth of Brevibacillus brevis prepared in Example 1 and Comparative Examples 1-4 (diluted 1300 times with water, 100 mL / pot) was added respectively. The control groups were added with equal amounts of clear water and carbendazim respectively. When obvious disease symptoms appeared, the length of the rice disease spots was counted and the control effect was calculated to obtain Table 6. From the results in Table 6, it can be seen that the disease in the clear water group was severe, while the control effect of the carbendazim treatment reached 40.29%, and the control effect of the Brevibacillus brevis fermentation broth treatment group reached 45.56%, which was better than that of the chemical agent carbendazim.

[0087] Table 6 Control Effects of Different Treatment Groups Against Bacterial Blight of Rice

[0088]

[0089] Note: Different letters represent significant differences.

[0090] Control Effect of Example 6 Against Bacterial Wilt of Tomato

[0091] 1. Pathogenic indicator bacterium: Ralstonia solanacearum, donated by Shanghai Jiao Tong University.

[0092] Pathogenic indicator bacterium culture medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl.

[0093] 2. Experimental methods and results

[0094] Sow 6 disinfected tomato seeds in each PVC flower pot. After the tomato seedlings emerge, transplant them, with 1 plant in each PVC flower pot. When the tomato plants grow four cotyledons, use a syringe to inoculate the tomato roots with the Ralstonia solanacearum pathogen, and then add the dilution of the Brevibacillus laterosporus fermentation broth prepared in Example 1 and Comparative Examples 1-4 (as shown in Treatment Groups 3-8 in Table 1). The control groups were respectively added with an equal amount of clear water and streptomycin sulfate. After 100 days, investigate the disease index, and the disease index was calculated according to formula (2). For the treatment groups of the 1300-fold dilution of the fermentation broth in Example 1, clear water, and streptomycin sulfate, the control effect is shown in Figure 3 .

[0095]

[0096] Through data statistics, Table 7 was obtained. It can be seen from the results in Table 7 that the disease occurred severely in the clear water control group, while the control effect of the streptomycin treatment reached 46.34%, and the control effect of the treatment group with the 1300-fold dilution of the fermentation broth in Example 1 reached 51.93%, and the effect was better than that of the chemical agent streptomycin in terms of control.

[0097] Table 7 Control Effects of Different Treatment Groups on Tomato Bacterial Wilt

[0098]

[0099] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a culture medium in increasing the spore formation rate of Brevibacillus laterosporu during fermentation culture, characterized in that, The culture medium consists of the following components: molasses 14.2 g / L, beef powder 8.9 g / L, magnesium sulfate 0.8 g / L, KH 2 PO 4 0.5 g / L, and K 2 HPO 4 2 g / L.

2. A method for increasing the spore formation rate of Brevibacillus laterosporu during fermentation culture, characterized in that, using the culture medium to perform fermentation culture on the Brevibacillus laterosporu; The culture medium consists of the following components: molasses 14.2 g / L, beef powder 8.9 g / L, magnesium sulfate 0.8 g / L, KH 2 PO 4 0.5 g / L, and K 2 HPO 4 2 g / L.

3. The method according to claim 2, characterized in that, the conditions for the fermentation culture are: pH is 6 - 7, temperature is 31 - 35 °C, rotation speed is 180 - 220 r / min, inoculum size is 3 - 5%, and culture time is 45 - 50 h.

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