Culture medium for inhibiting generation of secondary metabolite of bacillus and application of culture medium

Abstract

The invention relates to the technical field of microorganisms, in particular to a culture medium for inhibiting generation of bacillus secondary metabolites and application of the culture medium. The invention provides a culture medium for inhibiting generation of secondary metabolites of bacillus and an identification method of bacillus. Experiments show that the culture medium provided by the invention can significantly reduce the generation of secondary metabolites of bacillus, and the cultured bacterial colony can be accurately identified by directly adopting a formic acid treatment method, so that the method is simple and rapid. The quality of the identified mass spectrum is obviously improved, the baseline is reduced, the peak intensity is improved, and the identification accuracy is improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a culture medium for inhibiting the generation of secondary metabolites of bacillus and an application thereof. Background Art

[0002] Bacillus sp. is a type of bacteria widely found in nature, with nearly 100 species discovered. It has important application value in agriculture, food, environmental protection, medicine and industry. At present, the most widely used ones are Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus velezensis, etc. In agricultural production, Bacillus can fix nitrogen in the atmosphere, improve soil fertility and promote crop growth. Some Bacillus can also produce antibiotics and toxins for the prevention and treatment of plant diseases. In the food industry, Bacillus is used as a fermentation agent, such as in the production of fermented foods such as soy sauce, miso and natto. The antibacterial substances produced by Bacillus can also be used to preserve food and extend the shelf life of food. In terms of environmental protection, Bacillus can degrade organic pollutants and be used for bioremediation of soil and water bodies. In the field of medicine, some Bacillus can produce antibiotics. Bacillus preparations can be used to treat certain intestinal diseases, such as diarrhea, etc. In the field of industrial production, Bacillus can produce a variety of enzymes and are widely used in the production of enzyme preparations.

[0003] Isolating and identifying Bacillus with potential application value from nature is one of the most critical steps to develop its value. At present, there is a problem of difficult identification. Bacillus is usually identified by biochemical methods and 16S rRNA sequencing. Bacterial biochemical identification, the principle of bacterial biochemical identification is based on the biochemical reactions carried out by bacteria during metabolism. Different types of bacteria have different enzyme systems, so their ability to decompose substrates and metabolites are also different. By detecting these metabolites, the type of bacteria can be identified. Although bacterial biochemical identification is a classic and widely used identification method, it has some disadvantages:

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

[0005] 2. Complicated operation: It is necessary to prepare a variety of different culture media and reagents, and perform multiple inoculations, cultures, and tests. The entire process requires a high degree of skill and experience and is easily affected by differences in operator technique.

[0006] 3. The interpretation of results is highly subjective: The results of certain biochemical reactions may not be completely correct and need to rely on the operator's experience and judgment, which may lead to subjective results.

[0007] 4. Low identification accuracy: For Bacillus, the biochemical characteristics of different species 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 obviously different biochemical characteristics, forming groups of Bacillus, which are difficult to accurately identify to species).

[0008] 16S rRNA sequencing is a classic method for microbial identification, but the 16S rRNA sequences of Bacillus are very similar. This method can only distinguish Bacillus according to complex groups, and it is difficult to distinguish species. At present, the main method for accurate identification of Bacillus is whole genome sequencing. This method has high accuracy, but the detection cost is high and it is difficult to popularize. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology is a mass spectrometry analysis technology that has developed rapidly in recent years. It has been widely used in the identification of microorganisms. It has the advantages of being fast, accurate, simple, fast, and high-throughput. Its basic principle is to lyse the bacteria with formic acid, extract the standard protein of the strain to be tested, and use laser irradiation to form crystals formed by protein and matrix to ionize them to form a specific mass spectrum, which is then compared with the fingerprint spectrum in the database to identify the species of the microorganism. The extraction of strain standard protein is a key operation in MALDI-TOF MS pre-treatment and an important factor affecting the MALDI-TOF MS identification results. A large number of secondary metabolites are almost always produced during the growth of Bacillus, which is usually manifested as a large amount of mucus in the cultured colonies. Since the standard proteins collected by MALDI TOF are mainly intracellular ribosomal proteins, and these extracellular secondary metabolites are not required for MALDI-TOF MS identification, they will seriously affect the quality of the mass spectrum peak graph, resulting in low peak intensity, high baseline, and small number of peaks. This is manifested as a low identification score and inability to accurately identify. Summary of the invention

[0009] In view of this, the present invention provides a culture medium for inhibiting the production of secondary metabolites of Bacillus and its application. The present invention provides a culture medium for inhibiting the production of secondary metabolites of Bacillus and a method for identifying Bacillus. The present invention has found through experiments that the culture medium provided by the present invention can significantly reduce the production of secondary metabolites of Bacillus, and the colonies after culture can be directly treated with formic acid to achieve accurate identification, which is simple and fast. The quality of the identification mass spectrum is significantly improved, the baseline is reduced, the peak intensity is increased, and the identification accuracy is improved.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0012] The metal cation compound includes MnSO4.H2O and / or FeSO4.7H2O.

[0013] In some specific embodiments of the present invention, the pH value of the culture medium is comprised between 5.0 and 6.0.

[0014] The invention also provides 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 invention also provides application of the culture medium in preparing a kit for identifying bacillus.

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

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

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

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

[0021] Step 1, culturing the strain to be tested using the culture medium;

[0022] Step 2: extracting the standard protein of the strain to be tested, identifying the Bacillus using a MALDI-TOF MS mass spectrometry platform, obtaining an identification score, and determining the biological classification of the Bacillus.

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

[0024] In some specific embodiments of the present invention, the judgment method includes: the identification score ≥9.5 is considered to be a subspecies level with certainty at the species level; the identification score ≥9.0 and <9.5 is considered to be a species level with certainty at the species level; the identification score ≥6.0 and <9.0 is considered to be a species level with certainty at the genus level; and <6.0 is an unreliable result.

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

[0026] The present invention includes but is not limited to providing the following beneficial effects:

[0027] The invention adopts a culture medium of potato powder, agar powder, and anhydrous glucose, and adds 0.05-0.5 g / L of metal cation compounds to significantly inhibit the mucus of bacillus, and the mass spectrometry identification score is significantly higher than that of a culture medium without adding metal cation compounds and NA, BA, and LB culture medium, thereby achieving species level identification. The bacillus identification method provided by the invention is simple and rapid, has high quality of identification mass spectrogram, low baseline, high peak intensity, and high identification accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0029] Figure 1 The colony morphology of Bacillus Velezii cultured by the method of Example 1 is shown;

[0030] Figure 2 The colony morphology of Bacillus Velezii cultured by the method of Example 2 is shown;

[0031] Figure 3 The colony morphology of Bacillus Velezii cultured by the method of Example 3 is shown;

[0032] Figure 4 The colony morphology of Bacillus Velezii cultured by the method of Example 4 is shown;

[0033] Figure 5 The colony morphology of Bacillus Velezii cultured by the method of Example 5 is shown;

[0034] Figure 6 The colony morphology of Bacillus Velezii cultured by the method of Example 6 is shown;

[0035] Figure 7 The colony morphology of Bacillus Velezii cultured by the method of Example 7 is shown;

[0036] Figure 8 The colony morphology of Bacillus Velezii cultured by the method of Example 8 is shown;

[0037] Fig. 9 The colony morphology of Bacillus Velezii cultured by the method of Example 9 is shown;

[0038] Fig.10 The colony morphology of Bacillus Velezii cultured by the method of Example 10 is shown;

[0039] Fig.11 The colony morphology of Bacillus Velezii cultured by the method of Comparative Example 1 is shown;

[0040] Fig.12 The colony morphology of Bacillus Velezii cultured by the method of Comparative Example 2 is shown;

[0041] Fig.13 The colony morphology of Bacillus Velezii cultured by the method of Comparative Example 3 is shown;

[0042] Fig.14 The colony morphology of Bacillus Velezii cultured by the method of Comparative Example 4 is shown;

[0043] Fig.15 The colony morphology of Bacillus Velezii cultured by the method of Comparative Example 5 is shown;

[0044] Fig.16 The colony morphology of Bacillus Velezii cultured by the method of Comparative Example 6 is shown;

[0045] Fig.17 The colony morphology of Bacillus Velezii cultured by the method of Comparative Example 7 is shown;

[0046] Fig.18 The colony morphology of Bacillus Velezii cultured by the method of Comparative Example 8 is shown;

[0047] Fig.19 The mass spectrometry results of identifying Bacillus Velezii using the methods of Examples 1 to 8 are shown;

[0048] Fig. 20 The mass spectrometry results of identifying Bacillus Velezii using the methods of Comparative Examples 1, 2, 4, 6-8 are shown;

[0049] Fig.21The colony morphology of Bacillus amyloliquefaciens cultured by the method of Example 11 is shown;

[0050] Fig. 22 The colony morphology of Bacillus subtilis cultured by the method of Example 11 is shown;

[0051] Fig.23 The colony morphology of Bacillus licheniformis cultured by the method of Example 11 is shown;

[0052] Fig.24 1 and 2 show the mass spectrometry results of identifying different species of mucoid Bacillus using the method of Example 9. DETAILED DESCRIPTION

[0053] The present invention discloses a culture medium for inhibiting the production of secondary metabolites of Bacillus and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0054] Terminology explanation:

[0055] Mass Spectrum: A mass spectrum is obtained by a mass spectrometer and is a graphical representation of mass spectrometry. 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: refers to the signal level when no molecular signal is detected. The baseline represents the background noise level of the instrument, which is usually displayed as a relatively flat line at the bottom of the mass spectrometer. The stability of the baseline is crucial to the accuracy and reliability of mass spectrometry analysis.

[0057] Microbial secondary metabolites: refers to a class of compounds with specific biological activity produced by microorganisms during their growth and reproduction, in addition to primary metabolites used for growth and maintenance of basic life activities. These secondary metabolites usually do not directly participate in the growth and reproduction of microorganisms, but they play an important role in the survival competition, environmental adaptation, defense mechanism and interaction with the host of microorganisms.

[0058] The method for culturing and identifying mucoid Bacillus provided by the present invention comprises the following steps:

[0059] 1. Prepare culture medium:

[0060] Use a balance to weigh 5-10g potato powder, 15-20g agar powder, 20-40g anhydrous glucose, add metal cation compound additives, such as 0.05-0.5g MnSO4·H2O, FeSO4·7H2O, etc., add 1L purified water, adjust the pH of the 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 121℃ for 15-20 minutes. Cool the medium to about 50℃, pour it into a sterile culture dish, and set it aside after it cools and solidifies.

[0061] 2. Inoculation: Take a single colony of the bacteria to be identified and streak it on an agar plate. Or take 0.05~0.1mL of bacterial suspension or the sample to be tested and spread it on an agar plate.

[0062] 3. Cultivation: Cultivate in a constant temperature incubator at 28°C for 24 to 48 hours.

[0063] 4. Observe the colony morphology on the plate and visually inspect the colony morphology and mucus production after culture.

[0064] 5. Mass spectrometry pretreatment: The formic acid treatment method is used. The operation steps are as follows: (1) Pick a single colony cultured on an agar plate and apply it to the sample point; (2) Cover it with 1-2 μL of 50%-70% formic acid solution; (3) After air drying, add an appropriate amount of matrix solution to cover the sample point (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). Wait until the sample point is completely dry and set aside.

[0065] 6. Mass spectrometry identification:

[0066] The Autof ms1000 mass spectrometry platform was used for identification. The specific parameter settings and experimental methods are as follows:

[0067] Nitrogen ultraviolet excitation light source, wavelength is 337nm;

[0068] The mass collection range is between 2000 and 20000Da, and the linear positive ion operation mode;

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

[0070] The delayed 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: Taking matrix HCCA as the background peak, the lowest voltage value at which the detector responds to it is obtained;

[0072] The number of laser shots for collecting spectra was set between 40 and 140.

[0073] Instrument calibration: A mixture of standard strain Escherichia coli DH5a and myoglobin and ribonuclease RNase was used for instrument calibration.

[0074] Mass spectrometry identification uses mass spectrometer automatic acquisition software to automatically collect and compare spectra.

[0075] 7. Analysis of mass spectrometry results

[0076] Taking the standard strain Bacillus Velezii (CICC 24093) as an example, the mass spectrometry identification results and mass spectra of the cultured NA, BA, and LB agar plates were compared. The identification results were determined according to the scoring criteria of Autof ms1000, with scores ≥9.5 being classified as the subspecies level with a species level of confidence; scores ≥9.0 and <9.5 being classified as the species level of confidence; scores ≥6.0 and <9.0 being classified as the species level with a genus level of confidence; and <6.0 being an unreliable result.

[0077] The method for cultivating mucus-producing Bacillus provided by the present invention can significantly reduce the production of secondary metabolites of Bacillus, and the cultured colonies can be directly treated with formic acid to achieve accurate identification, which is simple and fast. The quality of the identification mass spectrum is significantly improved, the baseline is reduced, the peak intensity is increased, and the identification accuracy is improved.

[0078] Unless otherwise specified, the culture medium for inhibiting the production of secondary metabolites of Bacillus provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0079] The present invention will be further described below in conjunction with embodiments:

[0080] Example 1

[0081] 1. Preparation of culture medium: Weigh 5g potato powder (purchased from OXOID, specification 500g / bottle), 15g agar powder (purchased from OXOID, specification 500g / bottle), 20g anhydrous glucose, add 0.05g MnSO4·H2O, add 1L 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 about 50℃, pour it into a sterile culture dish, and set aside after the culture medium cools and solidifies.

[0082] 2. Inoculation: Plate inoculation, take the bacteria to be identified and inoculate the culture medium. The strain selected for this experiment was Bacillus Velezii (CICC 24093) purchased from the China Industrial Microbiological Culture Collection Administration Center.

[0083] 3. Cultivation: Cultivate in a constant temperature incubator at 28°C for 48 hours.

[0084] 4. Observation of colony morphology on plates: Visually inspect the size and shape of colonies and the production of mucus after culture.

[0085] 5. Mass spectrometry pretreatment: formic acid treatment method was used.

[0086] 6. Mass spectrometry identification: Use MALDI-TOF MS mass spectrometry platform for identification.

[0087] Example 2

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

[0089] Example 3

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

[0091] Example 4

[0092] The amount of MnSO4·H2O added was changed to 0.5 g / L, and the remaining steps were carried out according to Example 1.

[0093] Example 5

[0094] MnSO4·H2O was replaced with FeSO4·7H2O in an amount of 0.05 g / L, and the remaining steps were carried out as in Example 1.

[0095] Example 6

[0096] MnSO4·H2O was replaced with FeSO4·7H2O in an amount of 0.1 g / L, and the remaining steps were carried out as in Example 1.

[0097] Example 7

[0098] MnSO4·H2O was replaced with FeSO4·7H2O in an amount of 0.3 g / L, and the remaining steps were carried out according to Example 1.

[0099] Example 8

[0100] MnSO4·H2O was replaced with FeSO4·7H2O in an amount of 0.5 g / L, and the remaining steps were carried out as in Example 1.

[0101] Example 9

[0102] The pH of the culture medium was adjusted to 5.0, and the remaining steps were carried out according to Example 1.

[0103] Example 10

[0104] The pH of the culture medium was adjusted to 6.0, and the remaining steps were carried out according to Example 1.

[0105] Embodiment 11

[0106] The Bacillus Velez inoculated in Example 1 was replaced by other mucoid Bacillus: Bacillus amyloliquefaciens (CICC 20229), Bacillus subtilis (CICC 10721) and Bacillus licheniformis (ATCC 14580), and the remaining operations were the same as in Example 1.

[0107] Comparative Example 1

[0108] Preparation of culture medium: Weigh 5g potato powder, 15g agar powder, 20g anhydrous glucose, add 1L purified water, adjust the pH of the culture medium to 5.5 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution, and sterilize at 121°C for 15 minutes. Cool the culture medium to about 50°C, pour it into a sterile culture dish, and set it aside after cooling and solidification. The rest of the operation is the same as in Example 1.

[0109] For Example 2

[0110] The addition amount of MnSO4·H2O in Example 1 was changed to 0.005 g / L, and the remaining operations were the same as in Example 1.

[0111] Comparative Example 3

[0112] The addition amount of MnSO4·H2O in Example 1 was changed to 1 g / L, and the remaining operations were the same as in Example 1.

[0113] Comparative Example 4

[0114] The MnSO4·H2O in Example 1 was replaced with FeSO4·7H2O, and the addition amount was changed to 0.005 g / L. The remaining operations were the same as in Example 1.

[0115] Comparative Example 5

[0116] The MnSO4·H2O in Example 1 was replaced with FeSO4·7H2O, and the addition amount was changed to 1 g / L. The remaining operations were the same as in Example 1.

[0117] Comparative Example 6

[0118] Testing the effect of conventional bacterial culture medium LB agar plate on Bacillus culture and identification

[0119] Preparation of LB agar plate: weigh 10g of peptone, 15g of agar powder, 5g of yeast extract powder, and 10g of sodium chloride, add 1L of purified water, adjust the pH of the medium to 7.0 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution, and sterilize at 121°C for 15 minutes. Cool the medium to about 50°C and pour it into a sterile culture dish. After the medium is cooled and solidified, it is ready for use. The rest of the operation is the same as in Example 1.

[0120] Comparative Example 7

[0121] Testing the effect of conventional bacterial culture medium NA agar plate on the culture and identification of Bacillus

[0122] Preparation of NA agar plate: weigh 5 g of peptone, 15 g of agar powder, 3 g of beef extract powder, 5 g of sodium chloride, add 1 L of purified water, adjust the medium pH to 7.0 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution, and sterilize at 121° C. for 15 minutes. The rest of the operation is the same as in Example 1.

[0123] Comparative Example 8

[0124] Testing the effect of conventional bacterial culture medium BA agar plate on the culture and identification of Bacillus

[0125] Preparation of BA agar plate: weigh 15g of peptone, 15g of agar powder, 4g of beef extract powder, 4g of yeast extract powder, 5g of sodium chloride, add 1L of purified water, adjust the pH of the medium to 7.2 with 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution, and sterilize at 121°C for 15 minutes. Cool to about 55°C, add 5% to 10% sterile defibrinated sheep blood, mix well, pour into a sterile culture dish, and cool and solidify the culture medium for use. The rest of the operation is 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, and the results were 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, and the results were observed.

[0130] Effect example

[0131] The experimental methods in Examples 1 to 10 and Comparative Examples 1 to 10 were used to culture and identify Bacillus velez (CICC 24093). The experimental results are shown in Table 1. Figures 1 to 18 shown.

[0132] Table 1 Effects of different culture media on identification results of Bacillus velez

[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, the culture medium had visible precipitates after preparation and could not be used for subsequent experiments. Therefore, the pH of the culture medium was set to 5-6. In Comparative Examples 3 and 5, the Bacillus did not grow after culture and could not be used for subsequent experiments.

[0136] From the colony morphology ( Figures 1 to 18 ), the colonies of Examples 1 to 10 had no obvious mucus; the results of Comparative Example 1 showed that the culture medium using potato extract powder, agar powder, and anhydrous glucose to culture Bacillus could inhibit the production of mucus, but not significantly; adding 0.05 to 0.5 g / L of FeSO4·7H2O or MnSO4·H2O had a significant inhibitory effect on Bacillus mucus.

[0137] Judging from the identification results (Table 1), the culture medium using potato extract powder, agar powder, anhydrous glucose and adding 0.05-0.5 g / L metal cation compounds had a significantly higher mass spectrometry identification score than the culture medium without adding metal cation compounds and NA, BA, and LB culture media, all of which achieved species level identification and the identification results were correct.

[0138] From the mass spectrum ( Figures 19 and 20 ), the peak abundance of the spectrum collected by the embodiment is significantly higher than that of the comparative example, the obtained spectrum baseline is low, the spectrum peak is relatively stable, and there are abundant characteristic peaks in the range of 2000~14000Da, while the spectrum baseline obtained by using the comparative example is unstable and has a bulging phenomenon.

[0139] Example 11 The Velez subtilis inoculated in the above Example 1 was replaced by other mucoid Bacillus, Bacillus amyloliquefaciens (CICC 20229), Bacillus subtilis (CICC 10721) and Bacillus licheniformis (ATCC14580), respectively, and the other steps remained unchanged. The analysis experimental results are shown in Table 2. Figures 21 to 24 .

[0140] Table 2 Identification of different species of mucoid Bacillus

[0141]

[0142] From the identification results (Table 2), the culture medium using potato extract powder, agar powder, anhydrous glucose and adding 0.05~0.5g / L metal cation compounds gave a higher mass spectrometry identification score. The three types of mucoid Bacillus were all identified at the species level, and the identification results were correct.

[0143] From the perspective of colony morphology ( Figures 21 to 23), the culture medium is made of potato extract powder, agar powder, and anhydrous glucose, and adding 0.05~0.5g / L metal cation compounds has a significant inhibitory effect on Bacillus mucus.

[0144] From the mass spectrum ( Fig.24 ), the chromatograms obtained by the three mucus-producing Bacillus had a low baseline, relatively stable spectral peaks, and high spectral peak abundance, with abundant characteristic peaks appearing in the range of 2000~14000Da.

[0145] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A culture medium for inhibiting the production of secondary metabolites of Bacillus, characterized in that: Based on a 1L system, the culture medium includes the following components: 5-10g potato powder, 15-20g agar powder, 20-40g anhydrous glucose, 0.05-0.5g metal cation compound, and water; The metal cation compound includes MnSO4.H2O and / or FeSO4.7H2O.

2. The culture medium according to claim 1, characterized in that The pH value of the culture medium is between 5.0 and 6.

0.

3. Use of the culture medium as claimed in claim 1 or 2 in inhibiting the production of secondary metabolites of Bacillus.

4. The use according to claim 3, characterized in that The Bacillus includes one or more of Bacillus velezensis, Bacillus amyloliquefaciens, Bacillus subtilis or Bacillus licheniformis.

5. Use of the culture medium as claimed in claim 1 or 2 in preparing a kit for identifying Bacillus.

6. A method for inhibiting the production of secondary metabolites of Bacillus, characterized in that: The method comprises culturing Bacillus using the culture medium as claimed in claim 1 or 2.

7. The method according to claim 6, characterized in that The culture temperature includes 28°C.

8. A method for identifying Bacillus, characterized in that: The steps include: Step 1, culturing the strain to be tested using the culture medium as claimed in claim 1 or 2; Step 2: extracting the standard protein of the strain to be tested, identifying the Bacillus using a MALDI-TOF MS mass spectrometry platform, obtaining an identification score, and determining the biological classification of the Bacillus.

9. The identification method according to claim 8, characterized in that: The judgment method includes: the identification score ≥9.5 is the subspecies level that is certain at the species level; the identification score ≥9.0 and <9.5 is the species level that is certain at the species level; the identification score ≥6.0 and <9.0 is the species level that is certain at the genus level; <6.0 is an unreliable result.

10. A product, characterized in that Comprising the culture medium as described in claim 1 or 2.

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