A culture medium for parabacillus breeae and application thereof

Abstract

This invention relates to microbial culture. It provides a *Bacillus parabregas* culture medium and its applications, wherein the medium comprises at least one of glycerol, yeast extract, soybean peptone, corn steep liquor powder, ammonium sulfate, magnesium sulfate, and calcium carbonate. The *Bacillus parabregas* culture medium of this invention utilizes glycerol as a carbon source and yeast extract, soybean peptone, etc., as nitrogen sources for large-scale liquid fermentation of *Bacillus parabregas*, achieving a total bacitracin content of over 10.5 g / L. The proportions of each component can be controlled by feeding. Using this medium for *Bacillus parabregas* fermentation results in high bacitracin yield and controllable composition. The medium formulation is simple, inexpensive, readily available, safe, and environmentally friendly. The fermentation process is simple and controllable, with a short fermentation cycle, facilitating industrial-scale production and possessing high application value.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, specifically to a Bacillus parabrittlesis culture medium and its application. Background Technology

[0002] Tyrothricin is a mixture of polypeptides produced by the bacterium Bacillus brevis. It mainly consists of two types of components: cyclic polypeptides tyrocidines (Trc) and their analogues tryptocidine (Tpc) and phenycidine (Phc), and neutral linear polypeptides gramicidins. Tyrothricin is composed of 50%–70% tyrocidines and 25%–50% gramicidins.

[0003] The first polypeptide antibiotic isolated by Hotohkiss and Dubos in 1941 was tyrothricin, produced by a soil-dwelling Bacillus strain. It was initially named *Bacillus brevis*, with the preservation code ATCC 8185. Based on its solubility in acetone ether, tyrothricin was divided into soluble gramicidin and insoluble tyrocidine. Gramicidin is a neutral compound without free basic or acidic groups and has antibacterial activity against many Gram-positive bacteria. Tyrocidine has free basic groups and can cause lysis of Gram-negative bacteria. Both tyrocidine and gramicidin have been shown to be less effective against susceptible pathogens in the host, and their use as antibiotics is limited because they also cause hemolysis of red blood cells.

[0004] In 1949, Craig et al. analyzed recrystallized tyramine using countercurrent distribution technology and separated it into three closely related peptide components, which they named tyrocidines A, B, and C. Their amino acid sequences were subsequently determined. Since the first reports of brevicin abroad in the 1940s, scholars and companies both domestically and internationally have conducted extensive research on brevicin, mainly focusing on preparation methods and optimization of fermentation conditions, with fewer reports on strain screening. Current research reports that the fermentation levels of brevicin are generally 0.3–2 g / L.

[0005] Therefore, optimizing the fermentation process of the strain can significantly improve the bacitracin production capacity and the proportion of each component of Bacillus brevis, thereby increasing production efficiency and reducing production costs, and has important application value. Summary of the Invention

[0006] The present invention aims to at least partially solve one of the technical problems in the related art.

[0007] Therefore, a first aspect of the present invention provides a culture medium for Bacillus parabriquettes. According to an embodiment of the present invention, the culture medium comprises at least one of glycerol, yeast extract, soybean peptone, corn steep liquor powder, ammonium sulfate, magnesium sulfate, and calcium carbonate.

[0008] The *Bacillus parabrittlesis* culture medium of the present invention utilizes glycerol as a carbon source, yeast extract, soybean peptone, and corn steep liquor powder as nitrogen sources, ammonium sulfate and magnesium sulfate as growth factors, and calcium carbonate as an environmental stabilizer. Under these conditions, the strains grow normally. Large-scale liquid fermentation of *Bacillus parabrittlesis* can be carried out, with the total amount of the metabolite bacitracin reaching over 10.5 g / L. The proportions of each component can be controlled by feeding. Using this culture medium for *Bacillus parabrittlesis* fermentation results in high bacitracin yield and controllable composition. The culture medium formula is simple, inexpensive, readily available, safe, and environmentally friendly. The fermentation process is simple and controllable, with a short fermentation cycle, which is conducive to industrial-scale production and has high application value.

[0009] According to an embodiment of the present invention, the concentration of glycerol is 5-50 g / L, preferably 30 g / L.

[0010] According to an embodiment of the present invention, the concentration of the yeast extract is 1-30 g / L, preferably 15 g / L.

[0011] According to an embodiment of the present invention, the concentration of soybean peptone is 2-60 g / L, preferably 38 g / L.

[0012] According to an embodiment of the present invention, the concentration of the corn steep liquor powder is 2-40 g / L, preferably 23 g / L.

[0013] According to an embodiment of the present invention, the concentration of ammonium sulfate is 0.1 to 5 g / L, preferably 1.1 g / L.

[0014] According to an embodiment of the present invention, the concentration of magnesium sulfate is 0-6 g / L, preferably 0.2 g / L.

[0015] According to an embodiment of the present invention, the concentration of calcium carbonate is 0-6 g / L, preferably 2 g / L.

[0016] According to an embodiment of the present invention, the pH of the culture medium is 6.5 to 8.5, preferably 7.5.

[0017] In a second aspect of the invention, the invention provides the use of the culture medium described in the first aspect in the culture of Bacillus parabrittleis.

[0018] In a third aspect, the present invention provides a method for culturing Bacillus parabrachium. According to an embodiment of the present invention, the method includes: inoculating the Bacillus parabrachium seed culture into the culture medium described in the first aspect for fermentation culture.

[0019] According to an embodiment of the present invention, during the fermentation process, a nitrogen source is added to control the total nitrogen concentration of the fermentation broth to be no less than 500 ppm.

[0020] According to some preferred embodiments of the present invention, during the fermentation process, a nitrogen source is added to control the total nitrogen concentration of the fermentation broth to be 500-1000 ppm.

[0021] According to an embodiment of the present invention, the dissolved oxygen content is above 20% during the fermentation process.

[0022] According to an embodiment of the present invention, the temperature during the fermentation process is 35℃~37℃.

[0023] According to an embodiment of the present invention, the fermentation process takes 2 to 5 days, preferably 3 days.

[0024] According to an embodiment of the present invention, the Bacillus parabrachii seed culture is obtained by inoculating the Bacillus parabrachii into a liquid seed culture medium and culturing it at 37°C to obtain the seed culture.

[0025] In some embodiments of the present invention, amino acids are added during the fermentation process; preferably, the amino acid is at least one of tyrosine and tryptophan. Adding amino acids can increase the yield of breviculin.

[0026] In a third aspect, the present invention provides a method for preparing breviculin. According to an embodiment of the present invention, the method comprises: obtaining it by aerobic fermentation using *Bacillus parabrevicus* in the culture medium described in the first aspect.

[0027] In a fourth aspect, the present invention provides a method for regulating the production of breviculin. According to an embodiment of the present invention, the method includes: culturing *Bacillus parabrevicus* using the culture medium described in the first aspect, and using a biological buffer to control the pH value during the culturing process.

[0028] According to an embodiment of the present invention, the brevicin includes at least one of the following: brevicin A1, brevicin B, and brevicin C.

[0029] According to an embodiment of the present invention, the biological buffer includes at least one of the following: NaOH, MES, MOPS, TES, and Tris.

[0030] According to an embodiment of the present invention, the adjustment includes adjusting the ratio of bacitracin A1, bacitracin B, and bacitracin C in the total bacitracin.

[0031] According to an embodiment of the present invention, when the pH is adjusted to 5.5 to 6.5, the proportion of bacitracin A1 in the culture product is 75% to 100%.

[0032] According to an embodiment of the present invention, when the pH is adjusted to 6.8 to 7.4, the proportion of short bacillus casein B in the cultured product is 65% to 100%.

[0033] According to an embodiment of the present invention, when the pH is adjusted to 7.8 to 8.4, the proportion of short bacillus casein C in the culture product is 65% to 100%.

[0034] According to some specific embodiments of the present invention, when the pH is adjusted to 5.8 to 6.0, the proportion of bacitracin A1 in the culture product is 75% to 82%.

[0035] According to some specific embodiments of the present invention, when the pH is adjusted to 6.8 to 7.0, the proportion of short bacillus casein B in the culture product is 65% to 80%.

[0036] According to some specific embodiments of the present invention, when the pH is adjusted to 7.8 to 8.0, the proportion of short bacillus casein C in the culture product is 65% to 85%.

[0037] According to an embodiment of the present invention, when the pH is adjusted to 5.5 to 6.5 by MES, the proportion of short bacitracin A1 in the culture product is 75% to 100%.

[0038] According to an embodiment of the present invention, when the pH is adjusted to 6.9–7.4 by MOPS, the proportion of short bacillus casein B in the culture product is 65%–100%.

[0039] According to an embodiment of the present invention, when the pH is adjusted to 7.8–8.4 by Tris, the proportion of short bacillus casein C in the culture product is 65%–100%.

[0040] According to some specific embodiments of the present invention, when the pH is adjusted to 5.8-6.0 by MES, the proportion of short bacitracin A1 in the culture product is 75%-82%.

[0041] According to some specific embodiments of the present invention, when the pH is adjusted to 6.8 to 7.0 by MOPS, the proportion of short bacillus casein B in the culture product is 65% to 80%.

[0042] According to some specific embodiments of the present invention, when the pH is adjusted to 7.8-8.0 by Tris, the proportion of short bacillus casein C in the culture product is 65%-85%.

[0043] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0044] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0045] Figure 1 This refers to the changes in the proportions of the fermentation components in this embodiment of the invention. Detailed Implementation

[0046] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0047] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0048] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0049] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0050] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0051] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0052] In this article, all reagent concentrations refer to the final concentration of the reagent in the culture medium. For example, in the epidermal organoid culture medium, the concentration of glycerol is 5-50 g / L, which means that the final concentration of glycerol in the Bacillus parabriquets culture medium is 5-50 g / L.

[0053] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0054] Reagent source:

[0055] Table 1

[0056] Reagent Name Reagent source glycerin Malaysian Oils and Fats Company yeast extract Angel Yeast Co., Ltd. Soy peptone Angel Yeast Co., Ltd. Corn liquor dry powder Shanghai Yuantai Biotechnology Co., Ltd. ammonium sulfate Zhejiang Juhua Group Co., Ltd. Magnesium sulfate Zhejiang Juhua Group Co., Ltd. Calcium carbonate Changzhou Calcium Carbonate Co., Ltd.

[0057] Example 1

[0058] 1.1 Activation of microbial strains

[0059] Glycerol tubes containing the working strain *Brevibacillus parabrevis* HDCC00278 (deposited on July 4, 2024, at the China General Microbiological Culture Collection Center, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, accession number CGMCC NO.31168) were thawed at room temperature, inoculated onto nutrient agar plates using the streak method, and incubated at 37°C for 24 hours to obtain activated and revived bacterial colonies.

[0060] 1.2 Primary Seed Preparation

[0061] Prepare the primary seed culture medium according to the following formula: 10 g / L yeast extract, 8 g / L peptone, 4 g / L corn steep liquor, and 1.5 g / L potassium dihydrogen phosphate. Adjust the pH of the primary seed culture medium to 7.0 and then sterilize it at 121℃~123℃ for 30 min.

[0062] Take a portion of the activated and revived bacterial culture from 1.1, scrape off a loopful of colony with an inoculation loop, and then inoculate it into a 500ml Erlenmeyer flask containing 100ml of the primary seed liquid culture medium. After sealing, place the flask on a shaker at 37℃ and 220rpm for 16–30 hours of liquid culture.

[0063] 1.3 Secondary Seed Preparation

[0064] Prepare the secondary seed culture medium according to the following formula: 20 g / L yeast extract, 17 g / L peptone, 8 g / L corn steep liquor, and 1.5 g / L potassium dihydrogen phosphate. Adjust the pH of the secondary seed culture medium to 7.0 and then sterilize it at 121℃~123℃ for 30 min.

[0065] Take the qualified primary seeds from part 1.2 and inoculate them at a ratio of 3.0% (V / W) into a seed tank containing 10L of the secondary seed liquid culture medium. Set the culture temperature to 37℃, dissolved oxygen to be greater than 20%, air flow rate to 1vvm, initial stirring speed to 200rpm, and culture for 8-15h.

[0066] 1.4 Fermentation Culture

[0067] Prepare the fermentation medium according to the following formula: 30 g / L glycerol, 38 g / L soybean peptone, 15 g / L yeast extract, 23 g / L corn steep liquor powder, 1.1 g / L ammonium sulfate, 0.2 g / L magnesium sulfate, and 2 g / L CaCO3. Adjust the pH of the fermentation medium to 7.0 and then sterilize it at 121℃~123℃ for 30 min.

[0068] Take the qualified secondary seed culture from part 1.3 and inoculate it at a ratio of 10% (v / w) into a 50L fermenter containing 27L of liquid fermentation medium. Incubate at 37℃, dissolved oxygen above 30%, air flow rate 1.0 vvm, initial stirring speed 200 rpm, for 3 days. During incubation, if the dissolved ammonium concentration drops below 500 ppm, begin adding 20% ​​yeast extract solution, controlling the dissolved ammonium concentration to 500–1000 ppm.

[0069] 1.5 HPLC determination of breviculin yield and component ratio

[0070] After fermentation, 1 ml of the fermentation broth was taken and diluted to a 25 ml volumetric flask with anhydrous ethanol. The flask was then soaked and sonicated for 60 min. After centrifugation, the supernatant was filtered through a 0.45 μm microporous membrane and analyzed by HPLC. The HPLC detection method was a standard procedure in the field. The chromatographic column was a C18 analytical column (4.6 mm × 250 mm, 5 μm); the mobile phase was acetonitrile-0.1% formic acid; the elution conditions were shown in Table 1; the flow rate was 1.0 mL / min; the column temperature was 25 ℃; the detection wavelength was 190 nm; and the injection volume was 10 μL.

[0071] Table 1

[0072] Time (min) 0.1% formic acid Acetonitrile 0 60％ 40％ 10 34％ 66％ 15 10％ 90％ 15.01 60％ 40％ 20 60％ 40％

[0073] Based on the USP standard for breviculin, the potency of breviculin was calculated to be 8.5 g / L. The HPLC peak area ratios of each component in breviculin were as follows: brevicin A1 23.1%, brevicin A2 3.3%, brevicin C1 6.2%, brevicin C2 6.1%, brevicin tyrosin A 5.4%, brevicin tyrosin B 23.6%, brevicin tyrosin C 22.2%, brevicin tyrosin D 6.5%, and brevicin tyrosin E 2.9%.

[0074] Example 2

[0075] 2.1 Preparation of primary and secondary seeds

[0076] The preparation methods for primary and secondary seeds in this embodiment are the same as in Example 1.

[0077] 2.2 Fermentation Culture

[0078] The fermentation medium formula was the same as in Example 1. The pH of the medium was adjusted to 7.0 and then sterilized. The sterilization conditions were 121℃~123℃ for 30 min.

[0079] Fermentation culture: Take the qualified secondary seed culture from section 2.1 and inoculate it at a ratio of 10% (V / W) into a 50L fermenter containing 27L of the described fermentation medium. The culture temperature is 37℃, dissolved oxygen is controlled at 5%–10%, air flow rate is 1.0 vvm, initial stirring speed is 200 rpm, and the culture is carried out for 3 days. It should be noted that no feed is added during this fermentation culture process.

[0080] 2.3 HPLC determination of breviculin yield and the proportion of its components

[0081] In this embodiment, the HPLC detection method and experimental conditions for the yield and proportion of breviculin were the same as in Example 1. The detection results showed that under these conditions, the yield of breviculin in the fermentation broth was 0 g / L.

[0082] Example 3

[0083] 3.1 Preparation of primary and secondary seeds

[0084] The preparation methods for primary and secondary seeds in this embodiment are the same as in Example 1.

[0085] 3.2 Fermentation Culture

[0086] The fermentation medium formula was the same as in Example 1. The pH of the medium was adjusted to 7.0 and then sterilized. The sterilization conditions were 121℃~123℃ for 30 min.

[0087] Fermentation culture: Take the qualified secondary seed from part 3.1 and inoculate it into a 50L fermenter containing 29.5L of fermentation medium at a ratio of 2% (V / W). The culture temperature is 37℃, dissolved oxygen is controlled to be greater than 20%, air flow rate is 1.0 vvm, initial stirring speed is 200 rpm, and culture for 4 days.

[0088] 3.3 HPLC determination of breviculin yield and the proportion of its components

[0089] In this embodiment, the HPLC detection method and experimental conditions for the yield and proportion of breviculin were the same as in Example 1. The results showed that under these conditions, the yield of breviculin in the fermentation broth was 6.6 g / L, and the proportions of the components in the breviculin were as follows: brevicin A1 24.5%, brevicin A2 4.1%, brevicin C1 3.6%, brevicin C2 5.8%, brevicin tyrosin A 5.7%, brevicin tyrosin B 23.9%, brevicin tyrosin C 23.1%, brevicin tyrosin D 6.0%, and brevicin tyrosin E 3.0%.

[0090] Example 4

[0091] 4.1 Preparation of primary and secondary seeds

[0092] The preparation methods for primary and secondary seeds in this embodiment are the same as in Example 1.

[0093] 4.2 Fermentation Culture

[0094] The fermentation medium formula was the same as in Example 1. The pH of the medium was adjusted to 7.0 and then sterilized. The sterilization conditions were 121℃~123℃ for 30 min.

[0095] Fermentation culture: Secondary seed culture from section 4.1 that has passed the initial culture was inoculated at a ratio of 10% (v / w) into a 50L fermenter containing 27L of fermentation medium. The culture temperature was 37℃, dissolved oxygen was controlled to be greater than 20%, air flow rate was 1.0 vvm, and the initial stirring speed was 200 rpm. The culture was carried out for 3 days. During the culture process, a nitrogen source (20% yeast extract) was added, and a strong alkali (NaOH aqueous solution) was used to control the pH at 5.8–6.0.

[0096] 4.3 HPLC determination of breviculin yield and the proportion of its components

[0097] In this embodiment, the HPLC detection method and experimental conditions for the yield and proportion of breviculin were the same as in Example 1. The results showed that under these conditions, the yield of breviculin in the fermentation broth was 6.4 g / L, and the proportions of the components in the breviculin were as follows: brevicin A1 79.5%, brevicin A2 0.8%, brevicin C1 0.7%, brevicin C2 0.6%, brevicin tyrosin A 0.3%, brevicin tyrosin B 8.7%, brevicin tyrosin C 8.3%, brevicin tyrosin D 0.6%, and brevicin tyrosin E 0.6%.

[0098] Example 5

[0099] 5.1 Preparation of primary and secondary seeds

[0100] The preparation methods for primary and secondary seeds in this embodiment are the same as in Example 1.

[0101] 5.2 Fermentation Culture

[0102] The fermentation medium formula was the same as in Example 1. The pH of the medium was adjusted to 7.0 and then sterilized. The sterilization conditions were 121℃~123℃ for 30 min.

[0103] Fermentation culture: Secondary seed cultured from section 5.1 that passed the initial culture was inoculated at a ratio of 10% (v / w) into a 50L fermenter containing 27L of fermentation medium. The culture temperature was 37℃, dissolved oxygen was controlled to be greater than 20%, air flow rate was 1.0 vvm, and the initial stirring speed was 200 rpm. The culture was carried out for 3 days. During the culture process, a nitrogen source (20% yeast extract) was added, and a strong alkali (NaOH aqueous solution) was used to maintain the pH at 6.8–7.0.

[0104] 5.3 HPLC determination of breviculin yield and the proportion of its components

[0105] In this embodiment, the HPLC detection method and experimental conditions for the yield and proportion of breviculin were the same as in Example 1. The results showed that under these conditions, the yield of breviculin in the fermentation broth was 7.4 g / L, and the proportions of the components in the breviculin were as follows: brevicin A1 6.9%, brevicin A2 2.0%, brevicin C1 1.1%, brevicin C2 0.8%, brevicin tyrosin A 0.5%, brevicin tyrosin B 68.2%, brevicin tyrosin C 15.3%, brevicin tyrosin D 0.6%, and brevicin tyrosin E 0.1%.

[0106] Example 6

[0107] 6.1 Preparation of primary and secondary seeds

[0108] The preparation methods for primary and secondary seeds in this embodiment are the same as in Example 1.

[0109] 6.2 Fermentation Culture

[0110] The fermentation medium formula was the same as in Example 1. The pH of the medium was adjusted to 7.0 and then sterilized. The sterilization conditions were 121℃~123℃ for 30 min.

[0111] Fermentation culture: Secondary seed cultured from section 6.1 that passed the initial culture was inoculated at a ratio of 10% (v / w) into a 50L fermenter containing 27L of optimized liquid fermentation medium. The culture temperature was 37℃, dissolved oxygen was controlled to be greater than 20%, air flow rate was 1.0 vvm, and the initial stirring speed was 200 rpm. The culture was carried out for 3 days. During the culture process, a nitrogen source (20% yeast extract) was added, and a strong alkali (NaOH aqueous solution) was used to maintain the pH at 7.8–8.0.

[0112] 6.3 HPLC determination of breviculin yield and the proportion of its components

[0113] In this embodiment, the HPLC detection method and experimental conditions for the yield and proportion of breviculin were the same as in Example 1. The results showed that under these conditions, the yield of breviculin in the fermentation broth was 6.8 g / L. The proportions of the components in the breviculin were as follows: brevicin A1 10.5%, brevicin A2 0.5%, brevicin C1 1.2%, brevicin C2 1.1%, brevicin tyrosin A 0.2%, brevicin tyrosin B 18.2%, brevicin tyrosin C 67.3%, brevicin tyrosin D 0.5%, and brevicin tyrosin E 0.3%.

[0114] Example 7

[0115] 7.1 Preparation of primary and secondary seeds

[0116] The preparation methods for primary and secondary seeds in this embodiment are the same as in Example 1.

[0117] 7.2 Fermentation Culture

[0118] The fermentation medium formula was the same as in Example 1. The pH of the medium was adjusted to 7.0 and then sterilized. The sterilization conditions were 121℃~123℃ for 30 min.

[0119] Fermentation culture: Secondary seed culture from section 7.1, having passed the initial culture, was inoculated at a ratio of 10% (V / W) into a 50L fermenter containing 27L of fermentation medium. The culture temperature was 37℃, dissolved oxygen was controlled to be greater than 20%, air flow rate was 1.0 vvm, and the initial stirring speed was 200 rpm. The culture was carried out for 3 days. During the fermentation process, tryptophan was added in a pulsed manner. Pulsed addition refers to adding 0.1% of the total volume of tryptophan every 12 hours after the logarithmic growth phase, until the end of the fermentation. Other examples involving pulsed addition follow the same principle.

[0120] 7.3 HPLC determination of breviculin yield and the proportion of its components

[0121] In this embodiment, the HPLC detection method and experimental conditions for the yield and proportion of breviculin were the same as in Example 1. The results showed that under these conditions, the yield of breviculin in the fermentation broth was 6.5 g / L. The proportions of the components in breviculin were as follows: brevicin A1 32.6%, brevicin A2 30.6%, brevicin C1 7.2%, brevicin C2 0.6%, brevicin tyrosin A 0.3%, brevicin tyrosin B 15.2%, brevicin tyrosin C 11.6%, brevicin tyrosin D 0.7%, and brevicin tyrosin E 0.8%. Breviculin contains tryptophan and other structures, and feeding promotes its production.

[0122] Example 8

[0123] 8.1 Preparation of primary and secondary seeds

[0124] The preparation methods for primary and secondary seeds in this embodiment are the same as in Example 1.

[0125] 8.2 Fermentation Culture

[0126] The fermentation medium formula was the same as in Example 1. The pH of the medium was adjusted to 7.0 and then sterilized. The sterilization conditions were 121℃~123℃ for 30 min.

[0127] Fermentation culture: Secondary seed cultured from section 8.1 that passed the initial culture was inoculated at a ratio of 10% (V / W) into a 50L fermenter containing 27L of optimized liquid fermentation medium. The culture temperature was 37℃, dissolved oxygen was controlled to be greater than 20%, air flow rate was 1.0 vvm, and the initial stirring speed was 200 rpm. The culture was carried out for 3 days. Tyrosine was added in a pulsed manner during the fermentation process.

[0128] 8.3 HPLC determination of breviculin yield and the proportion of its components

[0129] In this embodiment, the HPLC detection method and experimental conditions for the yield and proportion of brevicin were the same as in Example 1. The results showed that under these conditions, the yield of brevicin in the fermentation broth was 6.2 g / L. The proportions of the components in brevicin were as follows: brevicin A1 6.7%, brevicin A2 1.2%, brevicin C1 21.4%, brevicin C2 28.1%, brevicin tyrosine A 1.2%, brevicin tyrosine B 16.7%, brevicin tyrosine C 13.8%, brevicin tyrosine D 3.4%, and brevicin tyrosine E 4.8%. Tyrosine, as a precursor, promoted the production of brevicin C2.

[0130] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0131] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A culture medium for Bacillus parabrittlesis, characterized in that, It includes at least one of glycerol, yeast extract, soybean peptone, corn steep liquor powder, ammonium sulfate, magnesium sulfate, and calcium carbonate.

2. The culture medium according to claim 1, characterized in that, The concentration of the glycerol is 5-50 g / L, preferably 30 g / L; Optionally, the concentration of the yeast extract is 1-30 g / L, preferably 15 g / L; Optionally, the concentration of soybean peptone is 2-60 g / L, preferably 38 g / L; Optionally, the concentration of the corn steep liquor powder is 2-40 g / L, preferably 23 g / L; Optionally, the concentration of ammonium sulfate is 0.1–5 g / L, preferably 1.1 g / L; Optionally, the concentration of the magnesium sulfate is 0-6 g / L, preferably 0.2 g / L; Optionally, the concentration of calcium carbonate is 0-6 g / L, preferably 2 g / L; Optionally, the pH of the culture medium is 6.5 to 8.5, preferably 7.

5.

3. The use of the culture medium according to any one of claims 1 to 2 in the culture of Bacillus parabrittlesis.

4. A method for culturing Bacillus parabrittlesis, characterized in that, include: The seed culture of Bacillus parashortiae is inoculated into the culture medium according to any one of claims 1 to 2 for fermentation culture.

5. The cultivation method according to claim 7, characterized in that, During the fermentation process, a nitrogen source is added to control the total nitrogen concentration of the fermentation broth to be no less than 500 ppm; Preferably, during the fermentation process, a nitrogen source is added to control the total nitrogen concentration of the fermentation broth to be 500–1000 ppm.

6. The cultivation method according to claim 4, characterized in that, During the fermentation process, the dissolved oxygen level is above 20%. Optionally, the temperature during the fermentation process is 35℃~37℃; Optionally, the fermentation process takes 2 to 5 days, preferably 3 days.

7. The cultivation method according to claim 6, characterized in that, The Bacillus parabrachii seed culture was obtained by inoculating the Bacillus parabrachii into a liquid seed culture medium and culturing it at 37°C.

8. A method for preparing breviculin, characterized in that, include: Obtained by aerobic fermentation of Bacillus parashortia in the culture medium described in any one of claims 1 to 2.

9. A method for regulating the production of breviculin, characterized in that, include: Bacillus parabrittleis was cultured using the culture medium according to any one of claims 1 to 2, and the pH value during the culture process was controlled using a biological buffer.

10. The method according to claim 9, characterized in that, The brevicin includes at least one of the following: brevicin A1, brevicin B, and brevicin C; Optionally, the biological buffer includes at least one of the following: MES, MOPS, TES, and Tris; Optionally, the adjustment includes changing the ratio of bacitracin A1, bacitracin B, and bacitracin C in the total bacitracin; Optionally, when the pH is adjusted to 5.5–6.5, the proportion of bacitracin A1 in the culture product is 75%–100%. Optionally, when the pH is adjusted to 6.9–7.4, the proportion of short bacillus casein B in the culture product is 65%–100%. Optionally, when the pH is adjusted to 7.8–8.4, the proportion of short bacillus casein C in the culture product is 65%–100%.

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