Bacillus inhibitor as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of microbial preparations, and discloses a bacillus inhibitor and a preparation method thereof, and the bacillus inhibitor comprises bacillus or fermentation liquor thereof and inhibitor chemical components. The preparation method comprises the following steps: carrying out first, second and third step-by-step fermentation on the bacillus strains to obtain high-concentration fermentation liquor, adding the chemical components of the inhibitor, and uniformly mixing to obtain the product. The bacillus inhibitor disclosed by the invention can be used for remarkably reducing the conversion of spores into vegetative bodies, improving the spore rate and prolonging the shelf life of microbial inoculum products.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial preparations, and in particular relates to a Bacillus inhibitor and a preparation method and application thereof. Background Art

[0002] Bacillus products, with their superior growth-promoting and disease-resistant properties, dominate the market of microbial fertilizers and microbial pesticides in my country, accounting for more than 80%. Bacillus products mainly include Bacillus subtilis, Bacillus velez, Bacillus licheniformis, Bacillus megaterium, Bacillus siamese, etc. These strains are widely used in microbial fertilizers and microbial pesticides, solving problems such as soil compaction and resistance to continuous cropping, and there is no substitute.

[0003] There are many types of Bacillus products currently circulating on the market. Some products are mainly composed of nutritious living microorganisms, while others are mainly composed of dormant spores. There are problems such as low concentration of live spores or nutritious living organisms, short shelf life, and multiple fermentation and proliferation leading to functional attenuation of strains, which seriously affect the functional microorganisms in the soil and directly reduce the economic benefits of agriculture. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a Bacillus inhibitor and a preparation method and application thereof.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0006] A Bacillus inhibitor comprises Bacillus or its fermentation liquid and inhibitor chemical components.

[0007] The above-mentioned Bacillus inhibitor, preferably, the Bacillus genus includes Bacillus velezensis XY40-1 or Bacillus siamensis KD50, the Bacillus velezensis XY40-1 was deposited in the China Center for Type Culture Collection on March 29, 2022, with a deposit number of CCTCC M 2022342; the Bacillus siamensis KD50 was deposited in the China Center for Type Culture Collection on March 10, 2023, with a deposit number of CCTCC NO: M 2023289.

[0008] Preferably, among the above-mentioned Bacillus inhibitors, the spore concentration of Bacillus velez XY40-1 is 15-20 billion CFU / mL, and the spore concentration of Bacillus siamensis KD50 is 10-15 billion CFU / mL.

[0009] The above-mentioned Bacillus inhibitor, preferably, the chemical components of the Bacillus inhibitor include 0.005-0.01 g / L copper sulfate, 0.05%-0.1% magnesium sulfate, 4-7 g / L calcium phosphate, and 600-1000 μmol / L pyridoxal phosphate.

[0010] As a general inventive concept, the present invention also provides a method for preparing the above-mentioned Bacillus inhibitor, comprising the following steps:

[0011] (1) inoculating a Bacillus strain on LB solid medium for activation culture to obtain an activated strain;

[0012] (2) inoculating the activated strain into a seed culture medium for primary fermentation to obtain a primary seed solution;

[0013] (3) subjecting the primary seed liquid to secondary fermentation to obtain a secondary seed liquid;

[0014] (4) subjecting the secondary seed liquid to tertiary fermentation;

[0015] (5) After the third-stage fermentation is completed, the culture liquid is added with the inhibitor chemical components, mixed evenly, and then packaged into a liquid dosage form to obtain the Bacillus inhibitor.

[0016] In the above preparation method, preferably, in step (1), the formula of the LB solid culture medium is: 8-10 g / L peptone, 5-6 g / L yeast extract, 8-10 g / L sodium chloride, 15-18 g / L agar, and pH is 7.2-7.3;

[0017] In step (2), the formula of the seed culture medium is: 18-20 g / L peptone, 8-10 g / L sodium chloride, 8-10 g / L yeast extract powder, and the pH is 7.2-7.3;

[0018] In step (3) and step (4), the formula of the culture medium used in the secondary fermentation and the tertiary fermentation is: 0.8-1% soybean meal powder, 0.3-0.5% ammonium sulfate, 0.4-0.5% yeast extract powder, 0.8-1% glucose, 0.5-0.6% starch, 0.1-0.2% sodium chloride, 0.3-0.5% potassium dihydrogen phosphate, and pH 7.2-7.3.

[0019] In the above preparation method, preferably, in step (1), the activation culture temperature is 28 to 30° C. and the time is 24 to 48 hours.

[0020] In the above preparation method, preferably, in step (2), the process conditions of the primary fermentation include: liquid volume 60-70%, inoculation amount 2%-5%, culture temperature 28-30°C, and shaking culture at 180-200 rpm for 18-20 hours.

[0021] In the above preparation method, preferably, in step (3), the process conditions of the secondary fermentation include a liquid volume of 60-70%, an inoculation amount of 8%-10%, a culture temperature of 28-30°C, and a shaking culture at 180-200 rpm for 18-20 hours.

[0022] In the above preparation method, preferably, in step (4), the process conditions of the tertiary fermentation include: liquid volume 60-70%, inoculation amount 8%-10%, culture temperature 28-30°C, and shaking culture at 150-180rpm for 22-24h.

[0023] As a general inventive concept, the present invention also provides a use of the above-mentioned Bacillus inhibitor or the Bacillus inhibitor prepared by the above-mentioned preparation method in extending the shelf life of a bacterial agent.

[0024] Furthermore, the Bacillus inhibitor is added to the microbial agent and sealed for use under anaerobic conditions or an air content of less than 5%.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention studies a microbial inhibitor containing Bacillus velezensis XY40-1 or Bacillus siamensis KD50, and finds that the Bacillus inhibitor can significantly reduce the conversion of spores into vegetative bodies, thereby obtaining a higher spore rate. At the same time, the survival period of dormant spores can be stabilized without causing spore death, thereby extending the shelf life of microbial products, reducing the need for frequent production and storage, ensuring a more stable supply of products in the market, directly reducing production costs, and improving economic benefits. The present invention can be used in the field of microbial preparation technology.

[0027] (2) The Bacillus inhibitors of the present invention do not need to be refrigerated or stored at low temperatures, making logistics more economical and convenient, and significantly reducing transportation and storage costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the colony morphology of Bacillus velezensis on the front of the XY40-1 plate;

[0029] Figure 2 This is the colony morphology of Bacillus siamensis on the front of the KD50 plate;

[0030] Figure 3 Phylogenetic tree analysis of 16S rDNA sequence of Bacillus velez XY40-1 constructed by the neighbor-joining method;

[0031] Figure 4 Phylogenetic tree analysis of 16S rDNA sequence of Bacillus siamensis KD50 constructed by neighbor-joining method;

[0032] Figure 5 It is a statistical line chart of the shelf life of the inhibitor containing Bacillus Velezii XY40-1;

[0033] Figure 6 This is a statistical line chart of the shelf life of products containing siam Bacillus KD50 inhibitors. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0035] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0036] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0037] Example 1: Isolation of bacteria

[0038] The isolation method of Bacillus velezensis XY40-1 involved in the present invention is: taking pepper plants from the Hunan Research Base in Changsha, disinfecting the surfaces of the leaves of the pepper plants, and then using tissue culture method and dilution coating plate method to isolate the endophytic bacteria in the leaves.

[0039] The isolation method of Bacillus siamensis KD50 involved in the present invention is: collecting rapeseed soil from the demonstration garden of Hunan Academy of Agricultural Sciences, and separating and purifying bacteria in the soil by using a dilution coating plate method.

[0040] Figure 1 and Figure 2These are the colony morphologies of Bacillus velezensis XY40-1 and Bacillus siamensis KD50 on the front of the plate.

[0041] Example 2: Identification of bacteria

[0042] The strains XY40-1 and KD50 were identified by 16S rRNA identification technology. The 16S rRNA gene of strain XY40-1 was amplified using bacterial universal primers (sequences shown in SEQ ID: 1 and SEQ ID: 2). After PCR amplification, the PCR product was detected by electrophoresis using 1% agarose gel; the gene product obtained by PCR was sequenced, and the resulting sequence was entered into the NCBI database and compared and analyzed using BLAST software. The 16SrRNA gene fragment of strain XY40-1 obtained by PCR amplification is shown in SEQ ID: 3. The sequence analysis and comparison showed that the similarity between strain XY40-1 and Bacillus velezensis reached 99.79%. Strains with higher homology to the strains were selected, and multiple sequence alignment was performed using MEGA 7.0 software to establish a phylogenetic tree, see Figure 3 As shown, combined with the morphological characteristics and 16S rRNA gene sequence determination results, it was found that strain XY40-1 was closest to Bacillus velezensis. Therefore, strain XY40-1 was identified as Bacillus velezensis and named Bacillus velezensis XY40-1. It was deposited in the China Type Culture Collection, Wuhan (Wuhan University), with the strain collection number CCTCC M 2022342 and the preservation time of March 29, 2022.

[0043] Similarly, bacterial universal primers (sequences shown in SEQ ID: 1 and SEQ ID: 2) were used to amplify the 16S rRNA gene of strain KD50. After PCR amplification, the PCR product was detected by electrophoresis using 1% agarose gel; the gene product obtained by PCR was sequenced, and the resulting sequence was entered into the NCBI database and compared and analyzed using BLAST software. The 16S rRNA gene fragment of strain KD50 obtained by PCR amplification is shown in SEQ ID: 4. The sequence analysis and comparison showed that strain KD50 had the highest similarity with Bacillus siamensis. Strains with higher homology to the strains were selected, and multiple sequence alignment was performed using MEGA 7.0 software to establish a phylogenetic tree, see Figure 4As shown, combined with the morphological characteristics and 16S rRNA gene sequence determination results, it was found that strain KD50 was closest to Bacillus siamensis. Therefore, strain KD50 was identified as Bacillus siamensis and named Bacillus siamensis KD50. It was deposited in the China Type Culture Collection, Wuhan (Wuhan University), with the strain collection number CCTCC NO:M 2023289 and the preservation time of March 10, 2023.

[0044] SEQ ID: 1: 27F 5'-AGAGTTTGATCCTGGCTCAG-3';

[0045] SEQ ID: 2: 1492R 5'-TACGGCTACCTTGTTACGACTT-3';

[0046]

[0047]

[0048] Example 3: Preparation method of Bacillus inhibitor

[0049] The Bacillus inhibitor involved in this embodiment includes Bacillus velezensis XY40-1 fermentation broth and inhibitor chemical components, wherein the spore concentration of Bacillus velezensis XY40-1 is 18 billion CFU / mL, and the chemical components include 0.008 g / L copper sulfate, 0.06% magnesium sulfate, 5 g / L calcium phosphate, and 900 μmol / L pyridoxal phosphate.

[0050] The preparation method of the Bacillus inhibitor in this embodiment comprises the following steps:

[0051] (1) Strain activation: Bacillus Velez XY40-1 was inoculated on LB solid medium and cultured at 28° C. for 24 h to obtain activated Bacillus Velez; wherein the formula of LB solid medium is: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, pH 7.2;

[0052] (2) Primary fermentation (50 L): The activated Bacillus Velezii XY40-1 was inoculated into a seed culture medium for primary fermentation, with a liquid volume of 70%, an inoculum size of 5%, a culture temperature of 28° C., and a shaking culture at 190 rpm for 20 h to obtain a primary seed solution of Bacillus Velezii; wherein the formula of the seed culture medium is: 18 g / L peptone, 10 g / L sodium chloride, 10 g / L yeast extract powder, and a pH of 7.2;

[0053] (3) Secondary fermentation (500 L): The primary seed liquid was subjected to secondary fermentation, with a liquid volume of 70%, an inoculation amount of 10%, and a culture temperature of 29° C. The culture was shaken at 190 rpm for 20 h to obtain a secondary seed liquid of Bacillus Velez. The formula of the culture medium used in the secondary fermentation was: 0.9% soybean meal, 0.4% ammonium sulfate, 0.4% yeast extract powder, 0.8% glucose, 0.5% starch, 0.1% sodium chloride, and 0.3% potassium dihydrogen phosphate, with a pH of 7.2;

[0054] (4) Tertiary fermentation (50t): The secondary seed liquid was subjected to tertiary fermentation, with a liquid volume of 65%, an inoculation amount of 10%, a culture temperature of 30°C, and a shaking culture at 180rpm for 24h. The formula of the culture medium used in the tertiary fermentation was: 0.9% soybean meal, 0.4% ammonium sulfate, 0.4% yeast extract powder, 0.8% glucose, 0.5% starch, 0.1% sodium chloride, 0.3% potassium dihydrogen phosphate, pH of 7.2, and the concentration of Bacillus Velezii obtained by fermentation was 18 billion CFU / mL.

[0055] (5) The high-concentration bacterial solution after fermentation was added with inhibitor component A (0.008 g / L copper sulfate, 0.06% magnesium sulfate, 5 g / L calcium phosphate, 900 μmol / L pyridoxal phosphate), mixed and packaged into liquid dosage forms to obtain Bacillus inhibitor A containing Bacillus Velezii XY40-1.

[0056] Example 4: Preparation method of Bacillus inhibitor

[0057] The Bacillus inhibitor involved in this embodiment is a Bacillus inhibitor A containing Siamese Bacillus KD50, including Siamese Bacillus KD50 fermentation broth and inhibitor chemical components, wherein the spore concentration of Siamese Bacillus KD50 is 12 billion CFU / mL, and the chemical components include 0.008 g / L copper sulfate, 0.06% magnesium sulfate, 5 g / L calcium phosphate, and 900 μmol / L pyridoxal phosphate. The preparation method is the same as that in Example 3.

[0058] Comparative Example 1:

[0059] In this comparative example, the same fermentation method as in Example 3 was used to prepare Bacillus Velezii. The high-concentration bacterial solution after fermentation was added with inhibitor B component (calcium chloride 0.008 g / L, magnesium sulfate 0.06%, calcium phosphate 5 g / L, pyridoxal phosphate 900 μmol / L), mixed and packaged into a liquid dosage form, namely, Bacillus inhibitor B containing Bacillus Velezii XY40-1.

[0060] Comparative Example 2:

[0061] In this comparative example, the same fermentation method as in Example 4 was used to prepare Siamese Bacillus bacterial liquid. The high-concentration bacterial liquid after fermentation was added with inhibitor B component (calcium chloride 0.008 g / L, magnesium sulfate 0.06%, calcium phosphate 5 g / L, pyridoxal phosphate 900 μmol / L), mixed and packaged into a liquid dosage form, i.e., a Bacillus inhibitor B containing Siamese Bacillus bacterial liquid.

[0062] Comparative Example 3:

[0063] In this comparative example, the same fermentation method as in Example 3 was used to prepare Bacillus Velez subtilis. The high-concentration bacterial solution after fermentation was added with inhibitor C component (0.008 g / L copper sulfate, 0.06% manganese sulfate, 5 g / L calcium phosphate, 900 μmol / L pyridoxal phosphate), mixed and packaged into a liquid dosage form, namely, Bacillus inhibitor C containing Bacillus Velez subtilis XY40-1.

[0064] Comparative Example 4:

[0065] In this comparative example, the same fermentation method as in Example 4 was used to prepare a Siamese Bacillus bacterial solution. The high-concentration bacterial solution after fermentation was added with an inhibitor C component (0.008 g / L copper sulfate, 0.06% manganese sulfate, 5 g / L calcium phosphate, 900 μmol / L pyridoxal phosphate), mixed and packaged into a liquid dosage form, i.e., a Bacillus inhibitor C containing a Siamese Bacillus bacterial solution.

[0066] Comparative Example 5:

[0067] In this comparative example, the same fermentation method as in Example 3 was used to prepare Bacillus Velezii. The high-concentration bacterial solution after fermentation was added with inhibitor D component (0.008 g / L copper sulfate, 0.06% magnesium sulfate, 5 g / L calcium nitrate, 900 μmol / L pyridoxal phosphate), mixed and packaged into a liquid dosage form, namely, Bacillus inhibitor D containing Bacillus Velezii XY40-1.

[0068] Comparative Example 6:

[0069] In this comparative example, the same fermentation method as in Example 4 was used to prepare a Siamese Bacillus bacterial solution. The high-concentration bacterial solution after fermentation was added with the inhibitor D component (0.008 g / L copper sulfate, 0.06% magnesium sulfate, 5 g / L calcium nitrate, 900 μmol / L pyridoxal phosphate), mixed and packaged into a liquid dosage form, namely, the Bacillus inhibitor D containing Siamese Bacillus bacterial solution.

[0070] Comparative Example 7:

[0071] In this comparative example, the same fermentation method as in Example 3 was used to prepare Bacillus Velez subtilis. The inhibitor E component (0.008 g / L copper sulfate, 0.06% magnesium sulfate, 5 g / L calcium phosphate, 900 μmol / L ascorbic acid) was added to the high-concentration bacterial solution after the fermentation was completed, mixed and packaged into a liquid dosage form, namely, the Bacillus inhibitor E containing Bacillus Velez subtilis XY40-1.

[0072] Comparative Example 8:

[0073] In this comparative example, the same fermentation method as in Example 4 was used to prepare a Siamese Bacillus bacterial solution. The high-concentration bacterial solution after fermentation was added with the inhibitor E component (0.008 g / L copper sulfate, 0.06% magnesium sulfate, 5 g / L calcium phosphate, 900 μmol / L ascorbic acid), mixed and packaged into a liquid dosage form, namely, the Bacillus inhibitor E containing Siamese Bacillus bacterial solution.

[0074] Analysis of the effect of Bacillus inhibitors

[0075] The spore quality of the Bacillus inhibitors with different formulations in Examples 3-4 and Comparative Examples 1 to 8 was dynamically monitored under anaerobic conditions for multiple storage periods. Figure 5and Figure 6 These are the statistical line graphs of the shelf life of Bacillus velez XY40-1 and Bacillus siamese KD50 inhibitors. Figure 5 and Figure 6 The analysis shows that for Bacillus Velez and Bacillus siamense, the effects of inhibiting the conversion of spores into vegetative bodies, increasing the spore rate and extending the shelf life of the bacterial solution are A>E>C>D>B from best to worst, that is, the inhibitor composition ratio of Bacillus Velez and Bacillus siamense is: copper sulfate 0.008g / L, magnesium sulfate 0.06%, calcium phosphate 5g / L, pyridoxal phosphate 900μmol / L.

[0076] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. A Bacillus inhibitor, characterized in that: The inhibitor includes Bacillus or its fermentation broth, and inhibitor chemical components.

2. The Bacillus inhibitor according to claim 1, characterized in that The Bacillus genus includes Bacillus velezensis XY40-1 or Bacillus siamensis KD50, the Bacillus velezensis XY40-1 was deposited in the China Center for Type Culture Collection on March 29, 2022, with a deposit number of CCTCC M 2022342; the Bacillus siamensis KD50 was deposited in the China Center for Type Culture Collection on March 10, 2023, with a deposit number of CCTCC NO: M 2023289.

3. The Bacillus inhibitor according to claim 2, characterized in that Among the Bacillus inhibitors, the spore concentration of Bacillus velez XY40-1 is 15-20 billion CFU / mL, and the spore concentration of Bacillus siamensis KD50 is 10-15 billion CFU / mL.

4. The Bacillus inhibitor according to claim 1, characterized in that The chemical components in the bacillus inhibitor include 0.005-0.01 g / L of copper sulfate, 0.05%-0.1% of magnesium sulfate, 4-7 g / L of calcium phosphate, and 600-1000 μmol / L of pyridoxal phosphate.

5. A method for preparing a Bacillus inhibitor according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) inoculating a Bacillus strain on LB solid medium for activation culture to obtain an activated strain; (2) inoculating the activated strain into a seed culture medium for primary fermentation to obtain a primary seed solution; (3) subjecting the primary seed liquid to secondary fermentation to obtain a secondary seed liquid; (4) subjecting the secondary seed liquid to tertiary fermentation; (5) After the third-stage fermentation is completed, the culture liquid is added with the inhibitor chemical components, mixed evenly, and then packaged into a liquid dosage form to obtain the Bacillus inhibitor.

6. The preparation method according to claim 5, characterized in that: In step (1), the formula of LB solid medium is: peptone 8-10 g / L, yeast extract 5-6 g / L, sodium chloride 8-10 g / L, agar 15-18 g / L, pH 7.2-7.3; In step (2), the formula of the seed culture medium is: 18-20 g / L peptone, 8-10 g / L sodium chloride, 8-10 g / L yeast extract powder, and the pH is 7.2-7.3; In step (3) and step (4), the formula of the culture medium used in the secondary fermentation and the tertiary fermentation is: 0.8-1% soybean meal powder, 0.3-0.5% ammonium sulfate, 0.4-0.5% yeast extract powder, 0.8-1% glucose, 0.5-0.6% starch, 0.1-0.2% sodium chloride, 0.3-0.5% potassium dihydrogen phosphate, and pH 7.2-7.

3.

7. The preparation method according to claim 5, characterized in that: In step (1), the activation culture is carried out at a temperature of 28 to 30° C. and for a time of 24 to 48 hours.

8. The preparation method according to claim 5, characterized in that: In step (2), the process conditions of the primary fermentation include: liquid volume 60-70%, inoculation amount 2%-5%, culture temperature 28-30°C, shaking culture at 180-200 rpm for 18-20 hours; In step (3), the process conditions of the secondary fermentation include a liquid volume of 60-70%, an inoculation amount of 8%-10%, a culture temperature of 28-30° C., and a shaking culture at 180-200 rpm for 18-20 hours; The process conditions of the tertiary fermentation include: liquid volume 60-70%, inoculation amount 8%-10%, culture temperature 28-30°C, and shaking culture at 150-180 rpm for 22-24 hours.

9. Use of the Bacillus inhibitor according to any one of claims 1 to 4 or the Bacillus inhibitor prepared by the preparation method according to any one of claims 5 to 8 in extending the shelf life of a bacterial agent.

10. The use according to claim 9, characterized in that Add the Bacillus inhibitor to the microbial agent and seal it for use under anaerobic conditions or under air content below 5%.

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