A Bacillus-like Inhibitor, Its Preparation Method and Application

Abstract

This invention belongs to the field of microbial preparation technology. It discloses a Bacillus inhibitor and its preparation method. The Bacillus inhibitor comprises Bacillus spp. or its fermentation broth, and inhibitor chemical components. The preparation method involves performing primary, secondary, and tertiary fermentation of a Bacillus spp. strain to obtain a high-concentration fermentation broth, followed by mixing with the added inhibitor chemical components. The Bacillus inhibitor of this invention can significantly reduce the conversion of spores to vegetative cells, increase the spore rate, and extend the shelf life of the microbial preparation product.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparation technology, and particularly relates to a Bacillus inhibitor, its preparation method, and its application. Background Technology

[0002] Bacillus products, with their superior growth-promoting and disease-resistant properties, dominate the Chinese microbial fertilizer and pesticide market, accounting for over 80% of the market. Major Bacillus products include Bacillus subtilis, Bacillus belye, Bacillus licheniformis, Bacillus megaterium, and Bacillus sianniae. These strains are widely used in microbial fertilizers and pesticides, solving problems such as soil compaction and continuous cropping resistance, and have no substitutes.

[0003] Currently, there are many types of Bacillus products on the market. Some products are mainly composed of live microorganisms, while others are mainly composed of dormant spores. These products have problems such as low concentration of live spores or live microorganisms, short shelf life, and functional decay of strains due to repeated fermentation and proliferation. These problems seriously affect the effectiveness of functional microorganisms in the soil and directly reduce agricultural economic benefits. 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 background art above, and to provide a Bacillus inhibitor, its preparation method and application.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0006] A Bacillus-based inhibitor, the inhibitor comprising Bacillus spp. or its fermentation broth, and an inhibitor chemical component.

[0007] Preferably, the Bacillus genus mentioned above includes Bacillus velezensis XY40-1 or Bacillus siamensis KD50. Bacillus velezensis XY40-1 was deposited at the China Center for Type Culture Collection (CCTCC) on March 29, 2022, with accession number CCTCC M 2022342; Bacillus siamensis KD50 was deposited at the CCTC on March 10, 2023, with accession number CCTCC NO:M 2023289.

[0008] Preferably, among the above-mentioned Bacillus inhibitors, the spore concentration of Bacillus belyss XY40-1 is 150-200 billion CFU / mL, and the spore concentration of Bacillus sicca KD50 is 100-150 billion CFU / mL.

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

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

[0011] (1) Bacillus strains were inoculated onto LB solid medium for activation culture to obtain activated strains;

[0012] (2) The activated strain was inoculated into a seed culture medium for primary fermentation to obtain a primary seed liquid;

[0013] (3) The primary seed liquid is subjected to secondary fermentation to obtain secondary seed liquid;

[0014] (4) The secondary seed liquid is subjected to tertiary fermentation;

[0015] (5) After the tertiary fermentation is completed, the culture medium is added to the inhibitor chemical components, mixed well, and then packaged into a liquid dosage form to obtain the Bacillus inhibitor.

[0016] In the above preparation method, preferably, in step (1), the LB solid culture medium formula 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 7.2-7.3.

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

[0018] In steps (3) and (4), the culture medium used for secondary and tertiary fermentation has the following formula: 0.8-1% soybean meal powder, 0.3-0.5% ammonium sulfate, 0.4-0.5% yeast extract, 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 28-30℃ and the time is 24-48h.

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

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

[0022] In the above preparation method, preferably, the process conditions for the three-stage fermentation in step (4) include: liquid volume of 60-70%, inoculum volume of 8%-10%, culture temperature of 28-30℃, and shaking culture at 150-180 rpm for 22-24 hours.

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

[0024] Furthermore, Bacillus inhibitors can be added to microbial agents and sealed for use under anaerobic conditions or with an air content of less than 5%.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) This invention studies microbial inhibitors containing Bacillus velezensis XY40-1 or Bacillus siamensis KD50. It was found that the Bacillus inhibitor can significantly reduce the conversion of spores into vegetative cells, obtain a higher spore rate, stabilize the survival period of dormant spores without causing spore death, extend the shelf life of microbial products, reduce the need for frequent production and storage, ensure a more stable supply of products in the market, directly reduce production costs, improve economic benefits, and can be used in the field of microbial preparation technology.

[0027] (2) The Bacillus inhibitors of the present invention do not require refrigeration or low-temperature storage, making logistics more economical and convenient, and significantly reducing transportation and storage costs. Attached Figure Description

[0028] Figure 1 The colony morphology of Bacillus velezensis on the front side of XY40-1 agar plates;

[0029] Figure 2 The colony morphology of Bacillus siamensis on the front side of KD50 plates;

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

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

[0032] Figure 5 Line graph showing the shelf life statistics of Bacillus bereaves XY40-1 inhibitors;

[0033] Figure 6 Line graph showing the shelf life of products containing Bacillus sicca KD50 inhibitors. Detailed Implementation

[0034] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0035] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

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

[0037] Example 1: Isolation of bacteria

[0038] The method for isolating Bacillus velezensis XY40-1 involved in this invention is as follows: take pepper plants from the Xiangyan Base in Changsha City, disinfect the leaves of the pepper plants, and then isolate the endophytic bacteria from the leaves using tissue culture and dilution plating methods.

[0039] The method for isolating Bacillus siamensis KD50 involved in this invention is as follows: rapeseed soil was collected from the demonstration garden of Hunan Academy of Agricultural Sciences, and the bacteria in the soil were isolated and purified using the dilution plating method.

[0040] Figure 1 and Figure 2The images show the colony morphology on the front of Bacillus velezensis XY40-1 and Bacillus siamensis KD50 plates, respectively.

[0041] Example 2: Identification of bacteria

[0042] Species identification of strains XY40-1 and KD50 was performed using 16S rRNA identification technology. The 16S rRNA gene of strain XY40-1 was amplified using universal bacterial primers (sequences shown in SEQ ID:1 and SEQ ID:2). After PCR amplification, the PCR products were detected by electrophoresis on a 1% agarose gel. The PCR-obtained gene product was sequenced, and the resulting sequence was entered into the NCBI database and analyzed using BLAST software. The 16S rRNA gene fragment of strain XY40-1 obtained by PCR amplification is shown in SEQ ID:3. Sequence analysis showed that strain XY40-1 had 99.79% similarity to Bacillus velezensis. Strains with high homology were selected, and multiple sequence alignment was performed using MEGA 7.0 software to construct a phylogenetic tree, as shown in [see figure]. Figure 3 As shown, based on morphological characteristics and 16S rRNA gene sequence determination, strain XY40-1 was found to be most closely related to Bacillus velezensis. Therefore, strain XY40-1 was identified as Bacillus velezensis and named Bacillus velezensis XY40-1. It is deposited at the China Center for Type Culture Collection, Wuhan (Wuhan University), with accession number CCTCC M 2022342 and deposit date of March 29, 2022.

[0043] Similarly, the 16S rRNA gene of strain KD50 was amplified using universal bacterial primers (sequences shown in SEQ ID:1 and SEQ ID:2). After PCR amplification, the PCR products were detected by electrophoresis on a 1% agarose gel. The gene products obtained by PCR were sequenced, and the resulting sequences were entered into the NCBI database and analyzed using BLAST software. The 16S rRNA gene fragment of strain KD50 obtained by PCR amplification is shown in SEQ ID:4. Sequence analysis showed that strain KD50 had the highest similarity to Bacillus siamensis. Strains with high homology were selected, and multiple sequence alignment was performed using MEGA 7.0 software to construct a phylogenetic tree, as shown in [see figure]. Figure 4As shown, based on morphological characteristics and 16S rRNA gene sequence determination, strain KD50 was found to be most similar to Bacillus siamensis. Therefore, strain KD50 was identified as Bacillus siamensis and named Bacillus siamensis KD50. It is deposited at the China Center for Type Culture Collection, Wuhan (Wuhan University), with accession number CCTCC NO: M 2023289, and the deposit date is 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 inhibitors

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

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

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

[0052] (2) Primary fermentation (50L): The activated Bacillus berleis XY40-1 was inoculated into the seed culture medium for primary fermentation. The liquid volume was 70%, the inoculum volume was 5%, the culture temperature was 28℃, and the culture was shaken at 190rpm for 20h to obtain the primary seed culture of Bacillus berleis. The seed culture medium formula was: 18g / L peptone, 10g / L sodium chloride, 10g / L yeast extract powder, and pH 7.2.

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

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

[0055] (5) After the high-concentration bacterial solution is fermented, add inhibitor component A (copper sulfate 0.008 g / L, magnesium sulfate 0.06%, calcium phosphate 5 g / L, pyridoxal phosphate 900 μmol / L), mix well, and then dispense into a liquid dosage form to obtain Bacillus inhibitor A containing Bacillus belyss XY40-1.

[0056] Example 4: Preparation method of Bacillus inhibitors

[0057] The Bacillus inhibitor involved in this embodiment is Bacillus inhibitor A containing Bacillus sicca KD50, which includes Bacillus sicca KD50 fermentation broth and inhibitor chemical components. The spore concentration of Bacillus sicca 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. Its preparation method is the same as in Example 3.

[0058] Comparative Example 1:

[0059] This comparative example uses the same fermentation method as Example 3 to prepare Bacillus belyss. After the high concentration of bacterial solution after fermentation is completed, inhibitor component B (0.008 g / L calcium chloride, 0.06% magnesium sulfate, 5 g / L calcium phosphate, and 900 μmol / L pyridoxal phosphate) is added, mixed well, and then packaged into a liquid dosage form, which is Bacillus belyss XY40-1 Bacillus inhibitor B.

[0060] Comparative Example 2:

[0061] This comparative example uses the same fermentation method as Example 4 to prepare Bacillus sicca bacterial culture. After the high concentration of bacterial culture is fermented, inhibitor component B (0.008 g / L calcium chloride, 0.06% magnesium sulfate, 5 g / L calcium phosphate, and 900 μmol / L pyridoxal phosphate) is added, mixed well, and then packaged into a liquid dosage form, which is Bacillus inhibitor B containing Bacillus sicca bacterial culture.

[0062] Comparative Example 3:

[0063] This comparative example uses the same fermentation method as Example 3 to prepare Bacillus belyss. After the high concentration of bacterial solution after fermentation is completed, inhibitor component C (copper sulfate 0.008 g / L, manganese sulfate 0.06%, calcium phosphate 5 g / L, pyridoxal phosphate 900 μmol / L) is added, mixed well, and then packaged into a liquid dosage form, which is Bacillus belyss XY40-1 Bacillus inhibitor C.

[0064] Comparative Example 4:

[0065] This comparative example uses the same fermentation method as Example 4 to prepare Bacillus sicca bacterial culture. After the high concentration of bacterial culture is fermented, inhibitor component C (copper sulfate 0.008 g / L, manganese sulfate 0.06%, calcium phosphate 5 g / L, pyridoxal phosphate 900 μmol / L) is added, mixed well, and then packaged into a liquid dosage form, which is Bacillus inhibitor C containing Bacillus sicca bacterial culture.

[0066] Comparative Example 5:

[0067] This comparative example uses the same fermentation method as Example 3 to prepare Bacillus belyss. After the high concentration of bacterial solution is fermented, inhibitor component D (copper sulfate 0.008 g / L, magnesium sulfate 0.06%, calcium nitrate 5 g / L, pyridoxal phosphate 900 μmol / L) is added, mixed well, and then packaged into a liquid dosage form, which is Bacillus belyss XY40-1 Bacillus inhibitor D.

[0068] Comparative Example 6:

[0069] This comparative example uses the same fermentation method as Example 4 to prepare Bacillus sicca bacterial culture. After the high concentration of bacterial culture is fermented, inhibitor component D (copper sulfate 0.008 g / L, magnesium sulfate 0.06%, calcium nitrate 5 g / L, pyridoxal phosphate 900 μmol / L) is added, mixed well, and then packaged into a liquid dosage form, which is Bacillus inhibitor D containing Bacillus sicca bacterial culture.

[0070] Comparative Example 7:

[0071] This comparative example uses the same fermentation method as Example 3 to prepare Bacillus belyss. After the high concentration of bacterial solution is fermented, inhibitor component E (copper sulfate 0.008 g / L, magnesium sulfate 0.06%, calcium phosphate 5 g / L, ascorbic acid 900 μmol / L) is added, mixed well, and then packaged into a liquid dosage form, which is Bacillus belyss XY40-1 Bacillus inhibitor E.

[0072] Comparative Example 8:

[0073] This comparative example uses the same fermentation method as Example 4 to prepare Bacillus sicca bacterial culture. After the high concentration of bacterial culture is fermented, inhibitor component E (copper sulfate 0.008 g / L, magnesium sulfate 0.06%, calcium phosphate 5 g / L, ascorbic acid 900 μmol / L) is added, mixed well, and then packaged into a liquid dosage form, which is Bacillus inhibitor E containing Bacillus sicca bacterial culture.

[0074] Efficacy analysis of Bacillus inhibitors

[0075] Dynamic monitoring of spore quality was conducted at multiple storage periods under anaerobic conditions for different formulations of Bacillus inhibitors in Examples 3-4 and Comparative Examples 1-8. Figure 5and Figure 6 Line graphs showing the shelf life statistics of Bacillus vesiculosus XY40-1 and Bacillus sicca KD50 inhibitors, respectively. Figure 5 and Figure 6 Analysis shows that for Bacillus belyss and Bacillus sicca, the effects of inhibiting spore conversion to vegetative cells, increasing spore rate, and extending shelf life of the bacterial culture, from best to worst, are A > E > C > D > B. That is, the ratio of the inhibitor group for combining Bacillus belyss and Bacillus sicca is: copper sulfate 0.008 g / L, magnesium sulfate 0.06%, calcium phosphate 5 g / L, and pyridoxal phosphate 900 μmol / L.

[0076] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. However, it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A Bacillus inhibitor, characterized in that, The inhibitors include those containing Bacillus belye ( Bacillus velezensis Fermentation broth of XY40-1 or containing Bacillus sicca ( Bacillus siamensis The fermentation broth of KD50 and the inhibitor chemical components; the Bacillus belesiensis ( Bacillus velezensis XY40-1 was deposited at the China Center for Type Culture Collection on March 29, 2022, with accession number CCTCC NO: M 2022342; the described Bacillus sicca ( Bacillus siamensis KD50 was deposited at the China Center for Type Culture Collection on March 10, 2023, with accession number CCTCC NO: M 2023289; the inhibitor's chemical composition includes copper sulfate 0.005~0.01 g / L, magnesium sulfate 0.05%~0.1%, calcium phosphate 4~7 g / L, and pyridoxal phosphate 600~1000 μmol / L; among the Bacillus inhibitors, the spore concentration of Bacillus belyss XY40-1 is 150-200 billion CFU / mL, and the spore concentration of Bacillus sicca KD50 is 100-150 billion CFU / mL.

2. A method for preparing a Bacillus inhibitor as described in claim 1, characterized in that, Includes the following steps: (1) Bacillus strains were inoculated onto LB solid medium for activation culture to obtain activated strains; (2) The activated strain was inoculated into a seed culture medium for primary fermentation to obtain a primary seed liquid; (3) The primary seed liquid is subjected to secondary fermentation to obtain secondary seed liquid; (4) The secondary seed liquid is subjected to tertiary fermentation; (5) After the three-stage fermentation is completed, the culture medium is added to the inhibitor chemical components, mixed well, and then packaged into a liquid dosage form to obtain the Bacillus inhibitor.

3. The preparation method according to claim 2, characterized in that, In step (1), the LB solid medium formula 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 seed culture medium formula is: 18~20 g / L peptone, 8~10 g / L sodium chloride, 8~10 g / L yeast extract, pH 7.2~7.3; In steps (3) and (4), the culture medium used for secondary and tertiary fermentation has the following formula: 0.8-1% soybean meal powder, 0.3-0.5% ammonium sulfate, 0.4-0.5% yeast extract, 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.

4. The preparation method according to claim 2, characterized in that, In step (1), the activation culture temperature is 28~30 ℃ and the time is 24~48 h.

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

6. The use of a Bacillus inhibitor as described in claim 1, or a Bacillus inhibitor prepared by the method described in any one of claims 2 to 5, in extending the shelf life of the bacterial agent, wherein, The bacterial strain in the inoculum is Bacillus belysinus (B. belysinus). Bacillus velezensis XY40-1 or Bacillus sicca ( Bacillus siamensis KD50.

7. The application as described in claim 6, characterized in that, Bacillus inhibitors are added to a microbial inoculant and sealed for use under anaerobic conditions or with an air content below 5%. The strain in the microbial inoculant is *Bacillus belesiensis*. Bacillus velezensis XY40-1 or Bacillus sicca ( Bacillus siamensis KD50.

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