Culture media for inhibiting the production of secondary metabolites of Bacillus and their applications

Abstract

This invention relates to the field of microbial technology, and particularly to a culture medium for inhibiting the formation of secondary metabolites in Bacillus and its application. This invention provides a culture medium for inhibiting the formation of secondary metabolites in Bacillus and a method for identifying Bacillus. Experiments have shown that the culture medium provided by this invention can significantly reduce the production of secondary metabolites in Bacillus. The cultured colonies can be accurately identified directly using a formic acid treatment method, which is simple and rapid. It significantly improves the quality of the identification mass spectrum, lowers the baseline, increases peak intensity, and improves the accuracy of identification.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a culture medium for inhibiting the generation of secondary metabolites of Bacillus and its application. Background Technology

[0002] Bacillus sp. is a group of bacteria widely distributed in nature, with nearly a hundred species discovered. It has important applications in agriculture, food, environmental protection, medicine, and industry. Currently, the most widely used species include Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus velezensis. In agricultural production, Bacillus can fix atmospheric nitrogen, improve soil fertility, and promote crop growth. Some Bacillus species can also produce antibiotics and toxins for the prevention and control of plant diseases. In the food industry, Bacillus is used as a leavening agent, such as in the production of fermented foods like soy sauce, miso, and natto. The antibacterial substances produced by Bacillus can also be used for food preservation, extending shelf life. In environmental protection, Bacillus can degrade organic pollutants and is used for the bioremediation of soil and water bodies. In the pharmaceutical field, some Bacillus species can produce antibiotics. Bacillus preparations can be used to treat certain intestinal diseases, such as diarrhea. In industrial production, Bacillus can produce a variety of enzymes and is widely used in enzyme preparation production.

[0003] Isolating and identifying Bacillus species with potential applications from nature is one of the most crucial steps in developing their value. Currently, identification remains a challenge. Bacillus identification typically employs biochemical methods, including 16S rRNA sequencing. Bacterial biochemical identification is based on the biochemical reactions that occur during bacterial metabolism. Different bacterial species possess different enzyme systems, resulting in varying abilities to break down substrates and different metabolic products. Detecting these metabolic products allows for the identification of bacterial species. While bacterial biochemical identification is a classic and widely used method, it has some drawbacks:

[0004] 1. Time-consuming: Traditional biochemical identification processes usually take several days to several weeks to complete because bacteria need to grow under specific culture conditions, and some biochemical reactions also require a long time to show results.

[0005] 2. Cumbersome operation: It requires the preparation of various different culture media and reagents, and multiple inoculation, culture and testing. The whole process requires a high level of skill and experience and is easily affected by the differences in the operator's skills.

[0006] 3. High degree of subjectivity in result interpretation: The results of some biochemical reactions may not be entirely accurate and may depend on the operator's experience and judgment, which may lead to subjectivity in the results.

[0007] 4. Low identification accuracy: For Bacillus, the biochemical characteristics of different species of Bacillus are very similar or completely consistent. That is, different bacteria may produce the same biochemical reaction results, which greatly reduces the accuracy of identification (that is, traditional biochemical identification methods can only distinguish Bacillus with obvious biochemical characteristics, forming groups of Bacillus, and it is difficult to accurately identify them to the species).

[0008] 16S rRNA sequencing is a classic method for microbial identification. However, the 16S rRNA sequences of Bacillus are very similar, so this method can only distinguish Bacillus by complex groups, not by species. Currently, the main method for accurate identification of Bacillus is whole-genome sequencing. This method has high accuracy, but the detection cost is high, making it difficult to popularize. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapidly developing mass spectrometry analysis technique that has been widely used in microbial identification. It has advantages such as speed, accuracy, simple operation, and high throughput. Its basic principle is to lyse the bacterial cells with formic acid to extract the standard protein of the test strain. The protein and the matrix crystals are then irradiated with a laser to ionize and form a specific mass spectrum. This spectrum is then compared with fingerprints in a database to identify the species of microorganism. Extraction of standard proteins from bacterial strains is a crucial step in MALDI-TOF MS pretreatment and a significant factor affecting MALDI-TOF MS identification results. Bacillus bacteria almost always produce a large number of secondary metabolites during growth, typically manifesting as colonies containing abundant mucus after culture. Since the standard proteins collected by MALDI-TOF are primarily ribosomal proteins, these extracellular secreted secondary metabolites are not required for MALDI-TOF MS identification and will severely impact the quality of the mass spectrometry peaks, resulting in low peak intensity, high baseline, and a small number of peaks. This manifests as low identification scores and inaccurate identification. Summary of the Invention

[0009] In view of this, the present invention provides a culture medium for inhibiting the generation of secondary metabolites of Bacillus and its application. The present invention provides a culture medium for inhibiting the generation of secondary metabolites of Bacillus and a method for identifying Bacillus. Experiments have shown that the culture medium provided by the present invention can significantly reduce the production of secondary metabolites of Bacillus. The cultured colonies can be accurately identified directly using a formic acid treatment method, which is simple and rapid. It significantly improves the quality of the identification mass spectrum, lowers the baseline, increases peak intensity, and improves the identification accuracy.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] The present invention provides a culture medium for inhibiting the generation of secondary metabolites of Bacillus. The culture medium, in a 1L system, comprises the following components: 5-10g potato extract powder, 15-20g agar powder, 20-40g anhydrous glucose, 0.05-0.5g metal cation compound, and water.

[0012] The metal cation compounds include MnSO4·H2O and / or FeSO4·7H2O.

[0013] In some specific embodiments of the present invention, the pH value of the culture medium includes 5.0 to 6.0.

[0014] The present invention also provides the application of the culture medium in inhibiting the generation of secondary metabolites of Bacillus.

[0015] In some specific embodiments of the present invention, the Bacillus includes one or more of Bacillus velezensis, Bacillus amyloliquefaciens, Bacillus subtilis, or Bacillus licheniformis.

[0016] The present invention also provides the application of the culture medium in the preparation of a kit for identifying Bacillus.

[0017] The present invention also provides a method for inhibiting the generation of secondary metabolites of Bacillus, including culturing Bacillus using the culture medium.

[0018] In some specific embodiments of the present invention, the culture temperature includes 28°C.

[0019] In some specific embodiments of the present invention, the culture time includes 24 to 48 hours.

[0020] This invention also provides a method for identifying Bacillus, comprising the following steps:

[0021] Step 1: Culture the test strain in the culture medium described above;

[0022] Step 2: Extract standard proteins from the test strain, identify the Bacillus using the MALDI-TOF MS mass spectrometry platform, obtain identification scores, and determine the biological classification of the Bacillus.

[0023] In some specific embodiments of the present invention, the method for extracting the standard protein includes: taking a single colony of the test strain and spreading it on a sample spot, covering it with 1-2 μL of a 50%-70% formic acid solution, air-drying it, adding a matrix solution to cover the sample spot, and obtaining the standard protein after the sample spot is completely dried. Preferably, the matrix solution includes a supersaturated solution of α-cyano-4-hydroxycinnamic acid, and its solvent includes an aqueous solution containing 2.5% trifluoroacetic acid and 50% acetonitrile.

[0024] In some specific embodiments of the present invention, the method of judgment includes: an identification score ≥ 9.5 indicates a subspecies level of certain probability at the species level; an identification score ≥ 9.0 but < 9.5 indicates certain probability at the species level; an identification score ≥ 6.0 but < 9.0 indicates a species level of certain probability at the genus level; and a score < 6.0 indicates an unreliable result.

[0025] The present invention also provides a product comprising the culture medium.

[0026] This invention includes, but is not limited to, the following beneficial effects:

[0027] This invention utilizes a culture medium containing potato extract powder, agar powder, and anhydrous glucose, with the addition of a 0.05-0.5 g / L metal cation compound. This significantly inhibits the mucus production of Bacillus subtilis, resulting in a significantly higher mass spectrometry identification score compared to media without the metal cation compound or media containing NA, BA, or LB, thus achieving species-level identification. The Bacillus subtilis identification method provided by this invention is simple, rapid, produces high-quality mass spectra with low baseline, high peak intensity, and high accuracy. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0029] Figure 1 The colony morphology of Bacillus belysin was cultured using the method described in Example 1.

[0030] Figure 2 The colony morphology of Bacillus belye cultured using the method in Example 2 is shown.

[0031] Figure 3 The colony morphology of Bacillus belye cultured using the method in Example 3 is shown.

[0032] Figure 4 The colony morphology of Bacillus belye cultured using the method in Example 4 is shown.

[0033] Figure 5 The colony morphology of Bacillus belye cultured using the method in Example 5 is shown.

[0034] Figure 6 The colony morphology of Bacillus belye cultured using the method of Example 6 is shown.

[0035] Figure 7 The colony morphology of Bacillus belye cultured using the method of Example 7 is shown.

[0036] Figure 8 The colony morphology of Bacillus belye cultured using the method of Example 8 is shown.

[0037] Figure 9 The colony morphology of Bacillus belyssus cultured using the method of Example 9 is shown.

[0038] Figure 10 The colony morphology of Bacillus belye cultured using the method of Example 10 is shown.

[0039] Figure 11 The colony morphology of Bacillus belye cultured using the method of Comparative Example 1 is shown.

[0040] Figure 12 The colony morphology of Bacillus belye cultured using the method in Comparative Example 2 is shown.

[0041] Figure 13 The colony morphology of Bacillus belyssus cultured using the method in Comparative Example 3 is shown.

[0042] Figure 14 The colony morphology of Bacillus belyssus cultured using the method in Comparative Example 4 is shown.

[0043] Figure 15 The colony morphology of Bacillus belyssus cultured using the method in Comparative Example 5 is shown.

[0044] Figure 16 The colony morphology of Bacillus belye cultured using the method of Comparative Example 6 is shown.

[0045] Figure 17 The colony morphology of Bacillus belyssus cultured using the method in Comparative Example 7 is shown.

[0046] Figure 18 The colony morphology of Bacillus belyssus cultured using the method of Comparative Example 8 is shown.

[0047] Figure 19 The mass spectrometry results of Bacillus belyssus identified using the methods in Examples 1-8 are shown.

[0048] Figure 20 The mass spectrometry results of Bacillus belyssus identified using comparative examples 1, 2, 4, 6-8 are shown.

[0049] Figure 21The colony morphology of Bacillus amyloliquefaciens cultured using the method of Example 11 is shown.

[0050] Figure 22 The colony morphology of Bacillus subtilis cultured using the method of Example 11 is shown.

[0051] Figure 23 The colony morphology of Bacillus licheniformis cultured using the method of Example 11 is shown.

[0052] Figure 24 The mass spectrometry results of different species of myxobacterial bacteria identified using the method in Example 9 are shown. Detailed Implementation

[0053] This invention discloses a culture medium for inhibiting the generation of secondary metabolites from Bacillus and its applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0054] Terminology Explanation:

[0055] Mass spectrum: A mass spectrum is obtained using a mass spectrometer and is a graphical representation of mass spectrometry analysis. It shows the relationship between the mass / charge ratio (m / z) of different molecules or molecular fragments in a sample and their relative abundance (signal intensity). In a mass spectrum, the horizontal axis (x-axis) usually represents the m / z value, while the vertical axis (y-axis) represents the relative abundance or signal intensity.

[0056] Baseline: This refers to the signal level when no molecular signal is detected. The baseline represents the instrument's background noise level and is typically displayed as a relatively flat line at the bottom of the mass spectrum. Baseline stability is crucial for the accuracy and reliability of mass spectrometry analysis.

[0057] Microbial secondary metabolites are compounds with specific biological activities produced by microorganisms during their growth and reproduction, in addition to the primary metabolites used for growth and maintaining basic life activities. These secondary metabolites do not usually participate directly in the growth and reproduction of microorganisms, but they play important roles in microbial competition for survival, environmental adaptation, defense mechanisms, and interactions with the host.

[0058] The method for culturing and identifying mucus-producing Bacillus provided by this invention includes the following steps:

[0059] 1. Prepare the culture medium:

[0060] Weigh out 5-10g of potato starch, 15-20g of agar powder, and 20-40g of anhydrous glucose using a balance. Add 0.05-0.5g of a metal cation compound additive, such as MnSO4·H2O or FeSO4·7H2O. Add 1L of purified water. Adjust the pH of the culture medium to 5.0-6.0 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution. Sterilize at 115℃ for 20-30 minutes or at 121℃ for 15-20 minutes. Cool the culture medium to about 50℃, pour it into sterile culture dishes, and allow it to cool and solidify before use.

[0061] 2. Inoculation: For plate inoculation, streak a single colony of the bacteria to be identified onto an agar plate. Alternatively, spread 0.05-0.1 mL of bacterial suspension or the sample to be tested onto an agar plate.

[0062] 3. Incubation: Incubate at 28℃ for 24-48 hours.

[0063] 4. Observe the morphology of the colonies on the plate and visually inspect the morphology of the colonies and the production of mucus after incubation.

[0064] 5. Mass spectrometry pretreatment: Formic acid treatment method is adopted. The operation steps are as follows: (1) Pick a single colony after agar plate culture and spread it on the sample spot; (2) Cover it with 1~2μL of 50%~70% formic acid solution; (3) After air drying, add an appropriate amount of matrix solution to cover the sample spot (the matrix solution is a supersaturated solution of α-cyano-4-hydroxycinnamic acid, and the solvent is an aqueous solution containing 2.5% trifluoroacetic acid and 50% acetonitrile). After the sample spot is completely dry, it is ready for use.

[0065] 6. Mass spectrometry identification:

[0066] Identification was performed using the Autof ms1000 mass spectrometry platform. Specific parameter settings and experimental methods are as follows:

[0067] Nitrogen ultraviolet excitation source with a wavelength of 337 nm;

[0068] Mass acquisition range 2000~20000 Da, linear positive ion operation mode;

[0069] Ion source voltage 1: 20 kV, ion source voltage 2: 17~20 kV, electron lens voltage: 5~10 kV;

[0070] The delay acquisition time is between 30 and 400 ns; the laser frequency is between 40 and 60 Hz; and the detector voltage is below -5 kV.

[0071] Detector voltage setting: The lowest voltage value at which the detector responds to the matrix HCCA as the background peak;

[0072] The number of laser bombardments for acquiring the spectrum was set between 40 and 140.

[0073] Instrument calibration: Instrument calibration was performed using a mixture of standard strain Escherichia coli DH5α, myoglobin, and ribonuclease.

[0074] Mass spectrometry identification uses automated acquisition software for mass spectrometry to automatically acquire and compare spectra.

[0075] 7. Analysis of mass spectrometry identification results

[0076] Taking the standard strain *Bacillus belyssus* (CICC 24093) as an example, the differences in identification results and mass spectra between the cultured strain of this invention and those obtained using NA, BA, and LB agar plates were compared. Identification results were determined according to the Autof MS1000 scoring criteria: a score ≥9.5 indicated a subspecies level of certain probability at the species level; a score ≥9.0 but <9.5 indicated a certain probability at the species level; a score ≥6.0 but <9.0 indicated a certain probability at the genus level; and a score <6.0 indicated an unreliable result.

[0077] The method for culturing mucinous Bacillus provided by this invention can significantly reduce the production of secondary metabolites of Bacillus. The cultured colonies can be accurately identified directly using formic acid treatment, which is simple and rapid. It significantly improves the quality of the identification mass spectrum, lowers the baseline, increases peak intensity, and improves identification accuracy.

[0078] Unless otherwise specified, the culture medium for inhibiting the generation of secondary metabolites of Bacillus provided in this invention, as well as the raw materials and reagents used in its application, are all commercially available.

[0079] The present invention will be further illustrated below with reference to the embodiments:

[0080] Example 1

[0081] 1. Culture medium preparation: Weigh 5g of potato extract powder (purchased from OXOID, 500g / bottle), 15g of agar powder (purchased from OXOID, 500g / bottle), and 20g of anhydrous glucose. Add 0.05g of MnSO4·H2O and 1L of purified water. Adjust the pH of the culture medium to 5.5 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution. Sterilize at 121℃ for 15 minutes. Cool the culture medium to approximately 50℃, pour it into sterile culture dishes, and allow it to cool and solidify before use.

[0082] 2. Inoculation: Plate inoculation was performed using the culture medium containing the bacteria to be identified. The strain selected for this experiment was Bacillus belyssus (CICC 24093), purchased from the China Industrial Microbial Culture Collection Center.

[0083] 3. Incubation: Incubate at 28℃ for 48 hours.

[0084] 4. Observation of colony morphology on plate: Visually inspect the size and shape of colonies and the amount of mucus produced after incubation.

[0085] 5. Mass spectrometry pretreatment: Formic acid treatment method is used.

[0086] 6. Mass spectrometry identification: Identification was performed using the MALDI-TOF MS mass spectrometry platform.

[0087] Example 2

[0088] The amount of MnSO4·H2O added was changed to 0.1 g / L, and the remaining steps were performed as in Example 1.

[0089] Example 3

[0090] The amount of MnSO4·H2O added was changed to 0.3 g / L, and the remaining steps were performed as in Example 1.

[0091] Example 4

[0092] Replace the amount of MnSO4·H2O added with 0.5 g / L, and perform the remaining steps as in Example 1.

[0093] Example 5

[0094] Replace MnSO4·H2O with FeSO4·7H2O at a rate of 0.05 g / L, and perform the remaining steps as in Example 1.

[0095] Example 6

[0096] Replace MnSO4·H2O with FeSO4·7H2O at a rate of 0.1 g / L, and follow the same steps as in Example 1.

[0097] Example 7

[0098] Replace MnSO4·H2O with FeSO4·7H2O at a rate of 0.3 g / L, and perform the remaining steps as in Example 1.

[0099] Example 8

[0100] Replace MnSO4·H2O with FeSO4·7H2O at a rate of 0.5 g / L, and follow the same steps as in Example 1.

[0101] Example 9

[0102] Adjust the pH of the culture medium to 5.0, and perform the remaining steps as in Example 1.

[0103] Example 10

[0104] Adjust the pH of the culture medium to 6.0, and perform the remaining steps as in Example 1.

[0105] Example 11

[0106] The Bacillus belysin in Example 1 was replaced with other mucinous Bacillus species: Bacillus amyloliquefaciens (CICC 20229), Bacillus subtilis (CICC 10721), and Bacillus licheniformis (ATCC 14580), and the rest of the operation was the same as in Example 1.

[0107] Comparative Example 1

[0108] Culture medium preparation: Weigh 5g potato extract powder, 15g agar powder, and 20g anhydrous glucose. Add 1L purified water and adjust the pH of the culture medium to 5.5 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution. Sterilize at 121℃ for 15 minutes. Cool the culture medium to about 50℃, pour it into sterile culture dishes, and allow it to cool and solidify before use. The remaining operations are the same as in Example 1.

[0109] For example 2

[0110] The amount of MnSO4·H2O added in Example 1 was changed to 0.005 g / L, and the rest of the operation was the same as in Example 1.

[0111] Comparative Example 3

[0112] The amount of MnSO4·H2O added in Example 1 was changed to 1g / L, and the rest of the operation was the same as in Example 1.

[0113] Comparative Example 4

[0114] The MnSO4·H2O in Example 1 was replaced with FeSO4·7H2O, and the amount added was changed to 0.005 g / L. The rest of the operation was the same as in Example 1.

[0115] Comparative Example 5

[0116] The MnSO4·H2O in Example 1 was replaced with FeSO4·7H2O, the amount added was changed to 1g / L, and the rest of the operation was the same as in Example 1.

[0117] Comparative Example 6

[0118] Testing the role of LB agar plates, a standard bacterial culture medium, in the identification of Bacillus cultures.

[0119] LB agar plate preparation: Weigh 10g peptone, 15g agar powder, 5g yeast extract, and 10g sodium chloride. Add 1L purified water and adjust the pH of the medium to 7.0 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution. Sterilize at 121℃ for 15 minutes. Cool the medium to approximately 50℃ and pour it into sterile culture dishes. Allow the medium to cool and solidify before use. The remaining procedures are the same as in Example 1.

[0120] Comparative Example 7

[0121] Testing the role of standard bacterial culture medium NA agar plates in the identification of Bacillus cultures.

[0122] Preparation of NA agar plates: Weigh 5g peptone, 15g agar powder, 3g beef extract powder, and 5g sodium chloride. Add 1L of purified water. Adjust the pH of the culture medium to 7.0 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution. Sterilize at 121℃ for 15 minutes. The remaining procedures are the same as in Example 1.

[0123] Comparative Example 8

[0124] Testing the role of BA agar plates, a standard bacterial culture medium, in the identification of Bacillus cultures.

[0125] Preparation of BA agar plates: Weigh 15g peptone, 15g agar powder, 4g beef extract powder, 4g yeast extract powder, and 5g sodium chloride. Add 1L of purified water. Adjust the pH of the culture medium to 7.2 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution. Sterilize at 121℃ for 15 minutes. Cool to approximately 55℃, add 5%~10% sterile defibrinated sheep blood, mix well, and pour into sterile culture dishes. Allow the culture medium to cool and solidify before use. The remaining procedures are the same as in Example 1.

[0126] Comparative Example 9

[0127] The pH of the culture medium in Example 1 was adjusted to 4.5, and the remaining operations were the same as in Example 1. The results were then observed.

[0128] Comparative Example 10

[0129] The pH of the culture medium in Example 1 was adjusted to 6.5, and the remaining operations were the same as in Example 1. The results were then observed.

[0130] Example of effect

[0131] Bacillus belyssus (CICC 24093) was cultured and identified using the experimental methods described in Examples 1-10 and Comparative Examples 1-10. The experimental results are shown in Table 1. Figures 1-18 As shown.

[0132] Table 1. Effects of different culture media on the identification results of Bacillus belyssus.

[0133]

[0134]

[0135] The results showed that in Comparative Example 9, the culture medium did not solidify after sterilization and could not be used for subsequent experiments. In Comparative Example 10, visible precipitate was found after the culture medium was prepared, making it unsuitable for subsequent experiments. Therefore, the pH of the culture medium was set to 5-6. In Comparative Examples 3 and 5, Bacillus bacteria did not grow after culture, making subsequent experiments impossible.

[0136] From the perspective of colony morphology ( Figures 1-18 In Examples 1-10, no obvious mucus was observed in the colonies. Comparative Example 1 showed that culturing Bacillus using potato extract powder, agar powder, and anhydrous glucose could inhibit mucus production, but not significantly. The addition of 0.05-0.5 g / L FeSO4·7H2O or MnSO4·H2O significantly inhibited Bacillus mucus production.

[0137] Based on the identification results (Table 1), the culture medium containing potato extract powder, agar powder, and anhydrous glucose, and the mass spectrometry identification score of the medium containing 0.05~0.5 g / L of metal cation compounds, was significantly higher than that of the culture medium without metal cation compounds and the NA, BA, and LB media. All of these results achieved species-level identification, and the identification results were correct.

[0138] From the mass spectrum ( Figures 19-20 The peak abundance of the spectrum collected in the example was significantly higher than that in the comparative example, the obtained spectrum baseline was lower, and the spectrum peaks were more stable, with abundant characteristic peaks appearing in the range of 2000~14000Da. In contrast, the spectrum baseline obtained using the comparative example was unstable and showed a bulging phenomenon.

[0139] In Example 11, the *Bacillus belyssus* inoculated in Example 1 was replaced with other myxotrophic bacilli, namely *Bacillus amyloliquefaciens* (CICC 20229), *Bacillus subtilis* (CICC 10721), and *Bacillus licheniformis* (ATCC14580), while the other steps remained unchanged. The experimental results are shown in Table 2. Figures 21-24 .

[0140] Table 2. Identification of different species of myxobolus bacilli

[0141]

[0142] Based on the identification results (Table 2), the culture medium containing potato extract powder, agar powder, and anhydrous glucose, along with the addition of 0.05~0.5 g / L of metal cation compounds, showed high mass spectrometry identification scores. All three types of myxobacteria were identified at the species level, and the identification results were correct.

[0143] From the perspective of colony morphology ( Figures 21-23The culture medium consisted of potato extract powder, agar powder, and anhydrous glucose. The addition of 0.05~0.5 g / L of a metal cation compound significantly inhibited the mucus production of Bacillus.

[0144] From the mass spectrum ( Figure 24 The three myxobacterial bacilli obtained had low baselines, relatively stable peaks, and high peak abundance, with abundant characteristic peaks appearing in the range of 2000~14000 Da.

[0145] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of culture medium in inhibiting the generation of secondary metabolites in Bacillus; The culture medium consists, for 1 L of system, of the following components: 5-10g potato starch, 15-20g agar powder, 20-40g anhydrous glucose, 0.05-0.5g metal cation compound and water; The metal cation compound is MnSO4·H2O and / or FeSO4·7H2O; The pH value of the culture medium is 5.0~6.0; The Bacillus is selected from Bacillus velezensis ( Bacillus velezensis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus subtilis ( Bacillus subtilis ), or Bacillus licheniformis ( Bacillus licheniformis ). The secondary metabolite is mucus.

2. A method for inhibiting the generation of secondary metabolites of Bacillus, characterized in that, This includes culturing Bacillus using culture media; Based on a 1L system, the culture medium consists of the following components: 5-10g potato extract powder, 15-20g agar powder, 20-40g anhydrous glucose, 0.05-0.5g metal cation compound, and water; The metal cation compound is MnSO4·H2O and / or FeSO4·7H2O; The pH value of the culture medium is 5.0~6.0; The Bacillus species is selected from Bacillus belysinus (B. belysinus). Bacillus velezensis ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Bacillus subtilis ( Bacillus subtilis ) or Bacillus licheniformis ( Bacillus licheniformis ); The culture temperature was 28°C; The secondary metabolite is mucus.

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