Brevibacillus brevis as well as fermentation medium and fermentation method thereof

Abstract

The invention provides bacillus brevis HDCC00067, the preservation number of the bacillus brevis HDCC00067 is CGMCC (China General Microbiological Culture Collection Center) No.35553, and the capability of producing brevibacterium peptide A is obviously higher than that of the prior art. The applicant also finds that the yield of the brevibacitracin A can be obviously improved when amino acid precursor substances are added into the fermentation culture medium, especially when the concentrations of L-tryptophan, D-leucine and D-valine are controlled to be within a certain range. By controlling pH and dissolved oxygen in a tank fermentation culture medium and optimizing a feeding control strategy of a mixed solution of a carbon source and a nitrogen source, the content of the brevibacitracin A in fermentation liquor is further greatly increased and reaches 7.5 g / L or above, and the brevibacitracin A can be used for large-scale industrial production.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a short-bracted Bacillus, its fermentation culture medium, and its fermentation method. Background Technology

[0002] Gramicidin A is a linear antimicrobial peptide synthesized by Bacillus brevis. It consists of 15 alternating L- and D-amino acids. The antimicrobial activity of gramicidin A stems from its ability to increase bacterial cell wall permeability, thereby inhibiting or even killing cellular metabolic activities; hence, it is known as an ionic antibiotic. The antifungal, antiviral, and antitumor effects of gramicidin A have gradually gained recognition and acceptance from industry experts and scholars, and its development and utilization are underway. While extracting large quantities of antimicrobial peptides, it is also being used in the health and pharmaceutical industries. Therefore, gramicidin A has broad application prospects in the pharmaceutical industry.

[0003] Short-chain bacitracin A is produced via a non-ribosomal pathway through non-ribosomal peptide synthase. Japanese researcher Tomonori NAKAI explored the optimal culture medium for the synthesis of linear short-chain bacitracin by Bacillus brevis, achieving a maximum yield of 20.3 μg / mL. However, this yield is difficult to achieve on a large scale, so further methods to increase the yield are needed to realize industrialization. Summary of the Invention

[0004] To improve the ability of Bacillus brevis to produce bacitracin A, this invention provides a strain of Bacillus brevis, HDCC00067, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 8, 2025, with accession number CGMCC No. 35553. Its 16S rRNA is shown in SEQ ID No. 1.

[0005] The present invention also provides a use of the aforementioned Bacillus brevis for preparing bacitracin A; or for preparing products containing bacitracin A.

[0006] Products containing bacitracin A include, but are not limited to, antibacterial agents, animal feed, and raw materials.

[0007] The present invention also provides a fermentation product containing the aforementioned Bacillus pseudobrevibacterium.

[0008] Preferably, the fermentation product is a fermentation broth, seed broth, culture broth, or bacterial suspension.

[0009] The present invention also provides a use of the fermentation broth for preparing gramicidin A; or for preparing a product containing gramicidin A.

[0010] The present invention also provides a fermentation method for producing bacitracin A using the aforementioned Bacillus brevis.

[0011] Preferably, fermentation is carried out in a fermentation medium containing an assimilable carbon source and / or nitrogen source.

[0012] Preferably, the fermentation medium also includes inorganic salts.

[0013] Preferably, the fermentation medium also includes an antifoaming agent.

[0014] Preferably, the fermentation medium includes: soluble starch, yeast extract, methyl oleate, soybean meal, sodium chloride, calcium chloride, ammonium sulfate, and potassium dihydrogen phosphate.

[0015] Preferably, the fermentation medium also includes amino acid precursors. Preferably, the amino acid precursors include L-tryptophan, D-leucine, and D-valine.

[0016] Preferably, the seed culture solution is inoculated into a fermentation medium for fermentation culture, wherein the fermentation culture includes dissolved oxygen control and feeding control.

[0017] Preferably, the dissolved oxygen level is always maintained above 35%.

[0018] Preferably, dissolved oxygen is maintained at 45±5%.

[0019] Preferably, the fermentation culture is carried out at 37°C.

[0020] Preferably, during the fermentation process, a mixture of carbon and nitrogen sources, as well as a foaming agent, is fed in.

[0021] Preferably, the process begins 36 hours after seed culture inoculation, with the addition of a mixture of carbon and nitrogen sources, as well as a foaming agent.

[0022] Preferably, during the fermentation process, the pH of the fermentation broth is maintained at 7.0 ± 0.3.

[0023] Preferably, the fermentation culture includes dissolved oxygen control and / or feeding control and / or pH control; Among them, dissolved oxygen can be selectively maintained at 35% or above, or at 45±5%; Among them, the feeding can be selectively a mixture of carbon source and nitrogen source, as well as a foaming agent; The pH can be selectively maintained at 7.0 ± 0.3. "Selectively" means either selected or not selected.

[0024] Preferably, during the fermentation process, the stirring speed is 150-400 rpm.

[0025] Preferably, during the fermentation process, the aeration rate is 0.8~1.1 vvm.

[0026] The present invention also provides a fermentation culture medium for Bacillus brevis, comprising L-tryptophan, D-leucine, and D-valine.

[0027] Preferably, the culture medium contains 1-3 g / L of L-tryptophan, 1-3 g / L of D-leucine, and 0.5-2 g / L of D-valine, based on mass concentration.

[0028] Preferably, the concentrations of L-tryptophan are 1~2.5 g / L, D-leucine are 1~2.5 g / L, and D-valine are 0.5~1.5 g / L.

[0029] Preferably, the concentrations of L-tryptophan are 2-2.5 g / L, D-leucine are 2-2.5 g / L, and D-valine are 1-1.5 g / L. The beneficial effects of this invention are as follows: The Bacillus brevis HDCC00067 provided by this invention has a higher ability to produce bacitracin A than the prior art.

[0030] Adding amino acid precursors to the fermentation medium, especially controlling the concentrations of L-tryptophan, D-leucine, and D-valine within a certain range, can significantly increase the yield of bacitracin A.

[0031] By controlling the pH and dissolved oxygen in the fermentation medium and optimizing the feeding control strategy of the carbon and nitrogen source mixture, the content of bacitracin A in the fermentation broth was further significantly increased to over 7.5 g / L, making it suitable for large-scale industrial production. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples are all commercially available products that can be purchased on the market.

[0034] The present invention will be further described below through embodiments, but these descriptions are not intended to further limit the scope of the invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the present invention still fall within the protection scope of the present invention.

[0035] Soil samples were obtained from Moganshan Scenic Area in Hangzhou City. After isolation, screening, purification, and selection, the strain of this invention was obtained. The 16S rRNA of this strain is shown in SEQ ID No. 1. It was identified as *Bacillus brevis* and named *Bacillus brevis* HDCC00067. As an example, this strain was used in the following embodiments. SEQ ID No.1 Unless otherwise specified, the culture medium used in the following examples consists of the following components: The composition of LB solid plate medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 2% agar, and the balance is water. The pH of the medium before sterilization is 7.2-7.3.

[0036] The composition of LB solid slant culture medium is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar, and the balance is water. The pH value of the culture medium before sterilization is 7.2-7.3.

[0037] The seed culture medium consists of 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the remainder is water. The pH of the culture medium before sterilization is 7.2–7.3.

[0038] Example 1 Preparation of effective inoculum source: Take the working strain cryopreservation tube and inoculate it onto LB solid slant medium. Incubate it at 37℃ and 50% relative humidity for 1 day. Immediately collect fresh and mature mycelial growths to use as effective inoculum source. Shake flask seed culture: Place approximately 1 cm of solid slant mycelial growth into the flask. 2 Inoculate into 50 ml of seed culture medium and incubate at 37 ℃ and 250 rpm for 24 h to obtain shake flask seed culture solution; Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 5% and cultured at 37℃ and 250rpm for 5 days to obtain a fermentation broth containing bacitracin A. The fermentation medium was formulated as follows: soluble starch 30 g / L, yeast extract 10 g / L, methyl oleate 10 g / L, soybean meal 15 g / L, sodium chloride 3 g / L, calcium chloride 4 g / L, ammonium sulfate 1.5 g / L, potassium dihydrogen phosphate 0.5 g / L, with the remainder being water. The pH of the medium before sterilization was 7.2.

[0039] Sample processing and detection: Take 1 ml of fermentation broth, add 5 ml of anhydrous methanol, mix well, sonicate for 1 h, mix again, centrifuge, filter, and take the supernatant for HPLC analysis. The content of bacitracin A in the fermentation broth was 4.82 g / L.

[0040] Example 2 Seed culture medium was obtained according to Example 1.

[0041] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 5% and cultured at 37℃ and 250rpm for 5 days to obtain a fermentation broth containing bacitracin A. The fermentation medium was formulated as follows: soluble starch 30 g / L, yeast extract 10 g / L, methyl oleate 10 g / L, soybean meal 15 g / L, sodium chloride 3 g / L, calcium chloride 4 g / L, ammonium sulfate 1.5 g / L, L-tryptophan 1.9 g / L, potassium dihydrogen phosphate 0.5 g / L, with the remainder being water. The pH of the medium before sterilization was 7.2.

[0042] Sample processing and detection: Take 1 ml of fermentation broth, add 5 ml of anhydrous methanol, mix well, sonicate for 1 h, mix again, centrifuge, filter, and take the supernatant for HPLC analysis. The content of bacitracin A in the fermentation broth was 4.93 g / L.

[0043] Example 3 Seed culture medium was obtained according to Example 1.

[0044] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 5% and cultured at 37℃ and 250rpm for 5 days to obtain a fermentation broth containing bacitracin A. The fermentation medium was formulated as follows: soluble starch 30 g / L, yeast extract 10 g / L, methyl oleate 10 g / L, soybean meal 15 g / L, sodium chloride 3 g / L, calcium chloride 4 g / L, ammonium sulfate 1.5 g / L, L-tryptophan 2.1 g / L, D-valine 1 g / L, potassium dihydrogen phosphate 0.5 g / L, with the remainder being water. The pH of the medium before sterilization was 7.2.

[0045] Sample processing and detection: Take 1 ml of fermentation broth, add 5 ml of anhydrous methanol, mix well, sonicate for 1 h, mix again, centrifuge, filter, and take the supernatant for HPLC analysis. The content of bacitracin A in the fermentation broth was 5.23 g / L.

[0046] Example 4 Seed culture medium was obtained according to Example 1.

[0047] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 5% and cultured at 37℃ and 250rpm for 5 days to obtain a fermentation broth containing bacitracin A. The fermentation medium was formulated as follows: soluble starch 30 g / L, yeast extract 10 g / L, methyl oleate 10 g / L, soybean meal 15 g / L, sodium chloride 3 g / L, calcium chloride 4 g / L, ammonium sulfate 1.5 g / L, L-tryptophan 2 g / L, D-leucine 2 g / L, D-valine 1 g / L, potassium dihydrogen phosphate 0.5 g / L, with the remainder being water. The pH of the medium before sterilization was 7.2.

[0048] Sample processing and detection: Take 1 ml of fermentation broth, add 5 ml of anhydrous methanol, mix well, sonicate for 1 h, mix again, centrifuge, filter, and take the supernatant for HPLC analysis. The content of bacitracin A in the fermentation broth was 6.17 g / L.

[0049] As shown in Examples 1-3 and Example 4, adding amino acid precursors to the culture medium can affect the potency. The degree of influence varies depending on the type of amino acid added. Adding L-tryptophan, D-leucine, and D-valine promotes potency improvement. Furthermore, Example 4 was repeated multiple times, but the concentrations of L-tryptophan (1-2.5 g / L), D-leucine (1-2.5 g / L), and D-valine (0.5-1.5 g / L) were adjusted. The content of bacitracin A in the fermentation broth was measured to be 5.09-6.21 g / L.

[0050] Example 5 Preparation of effective inoculum source: Take the working strain cryopreservation tube and inoculate it onto LB solid slant medium. Incubate it at 37℃ and 50% relative humidity for 1 day. Immediately collect fresh and mature mycelial growths to use as effective inoculum source. Seed culture in jars: Place approximately 1 cm of solid slant culture medium... 2 Inoculate into 50 ml of seed culture medium and culture at 37 ℃ and 250 rpm for 24 h to obtain shake flask seed culture solution. Then, inoculate the shake flask seed culture solution into a 10 L seed tank at a volume ratio of 0.2% and culture at 37 ℃, 300 rpm and 0.6 vvm for 14 h to obtain tank seed culture solution. Tank fermentation culture: The tank seed culture was inoculated into the above fermentation medium at a ratio of 3% of the fermentation medium volume. The culture was incubated at 37℃ with stirring at 150-400 rpm and aeration at 0.8-1.1 vvm. Stirring and dissolved oxygen were controlled in tandem to maintain dissolved oxygen above 35%. Starting 36 hours after inoculation, a mixture of carbon and nitrogen sources, along with a foaming agent, was fed in, maintaining the pH within the range of 7.0±0.3 until 1-2 days before the end of the culture period. The culture cycle was 120 hours, yielding a fermentation broth containing bacitracin A. The fermentation medium formula (containing amino acid precursors and defoamer) consists of: 30 g / L soluble starch, 10 g / L yeast extract, 10 g / L methyl oleate, 15 g / L soybean meal, 3 g / L sodium chloride, 4 g / L calcium chloride, 1.5 g / L ammonium sulfate, 2 g / L L-tryptophan, 2 g / L D-leucine, 1 g / L D-valine, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L THIX-298 defoamer, and the balance being water. The pH of the medium before sterilization is 7.2.

[0051] Sample processing and detection: Take 1 ml of fermentation broth, add 5 ml of anhydrous methanol, mix well, sonicate for 1 h, mix again, centrifuge, filter, and take the supernatant for HPLC analysis. The content of bacitracin A in the fermentation broth was 7.57 g / L.

[0052] Comparative Example 1 Seed culture medium was obtained according to Example 1.

[0053] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 5% and cultured at 37℃ and 250rpm for 5 days to obtain a fermentation broth containing bacitracin A. The fermentation medium was formulated as follows: soluble starch 30 g / L, yeast extract 10 g / L, methyl oleate 10 g / L, soybean meal 15 g / L, sodium chloride 3 g / L, calcium chloride 4 g / L, ammonium sulfate 1.5 g / L, L-tryptophan 3 g / L, D-leucine 2 g / L, D-valine 6 g / L, potassium dihydrogen phosphate 0.5 g / L, with the remainder being water. The pH of the medium before sterilization was 7.2.

[0054] Sample processing and detection: Take 1 ml of fermentation broth, add 5 ml of anhydrous methanol, mix well, sonicate for 1 h, mix again, centrifuge, filter, and take the supernatant for HPLC analysis. The content of bacitracin A in the fermentation broth was 4.34 g / L.

[0055] Comparative Example 2 Seed culture medium was obtained according to Example 1.

[0056] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 5% and cultured at 37℃ and 250rpm for 5 days to obtain a fermentation broth containing bacitracin A. The fermentation medium was formulated as follows: soluble starch 30 g / L, yeast extract 10 g / L, methyl oleate 10 g / L, soybean meal 15 g / L, sodium chloride 3 g / L, calcium chloride 4 g / L, ammonium sulfate 1.5 g / L, L-tryptophan 4 g / L, D-leucine 2 g / L, D-valine 1 g / L, potassium dihydrogen phosphate 0.5 g / L, with the remainder being water. The pH of the medium before sterilization was 7.2.

[0057] Sample processing and detection: Take 1 ml of fermentation broth, add 5 ml of anhydrous methanol, mix well, sonicate for 1 h, mix again, centrifuge, filter, and take the supernatant for HPLC analysis. The content of bacitracin A in the fermentation broth was 4.03 g / L.

[0058] Comparative Example 3 Seed culture medium was obtained according to Example 1.

[0059] Shake-flask fermentation: The seed culture was inoculated into the fermentation medium at a volume ratio of 5% and cultured at 37℃ and 250rpm for 5 days to obtain a fermentation broth containing bacitracin A. The fermentation medium was formulated as follows: soluble starch 30 g / L, yeast extract 10 g / L, methyl oleate 10 g / L, soybean meal 15 g / L, sodium chloride 3 g / L, calcium chloride 4 g / L, ammonium sulfate 1.5 g / L, L-tryptophan 4 g / L, D-leucine 4 g / L, D-valine 2 g / L, potassium dihydrogen phosphate 0.5 g / L, with the remainder being water. The pH of the medium before sterilization was 7.2.

[0060] Sample processing and detection: Take 1 ml of fermentation broth, add 5 ml of anhydrous methanol, mix well, sonicate for 1 h, mix again, centrifuge, filter, and take the supernatant for HPLC analysis. The content of bacitracin A in the fermentation broth was 3.23 g / L.

[0061] Comparative Example 4 Preparation of effective inoculum source: Take the working strain cryopreservation tube and inoculate it onto LB solid slant medium. Incubate it at 37℃ and 50% relative humidity for 1 day. Immediately collect fresh and mature mycelial growths to use as effective inoculum source. Seed culture in jars: Place approximately 1 cm of solid slant culture medium... 2 Inoculate into 50 ml of seed culture medium and culture at 37 ℃ and 250 rpm for 24 h to obtain shake flask seed culture solution. Then, inoculate the shake flask seed culture solution into a 10 L seed tank at a volume ratio of 0.2% and culture at 37 ℃, 300 rpm and 0.6 vvm for 14 h to obtain tank seed culture solution. Tank fermentation culture: The tank seed culture broth was inoculated into the above fermentation medium at a ratio of 3% of the fermentation medium volume and cultured at 37℃, stirring at 250 rpm and aeration at 1.0 vvm. No pH or dissolved oxygen control was performed and no feed was added. After 120 h, a fermentation broth containing bacitracin A was obtained. The fermentation medium formula (containing amino acid precursors and defoamer) consists of: 30 g / L soluble starch, 10 g / L yeast extract, 10 g / L methyl oleate, 15 g / L soybean meal, 3 g / L sodium chloride, 4 g / L calcium chloride, 1.5 g / L ammonium sulfate, 2 g / L L-tryptophan, 2 g / L D-leucine, 1 g / L D-valine, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L THIX-298 defoamer, and the balance being water. The pH of the medium before sterilization is 7.2.

[0062] Sample processing and detection: Take 1 ml of fermentation broth, add 5 ml of anhydrous methanol, mix well, sonicate for 1 h, mix again, centrifuge, filter, and take the supernatant for HPLC analysis. The content of bacitracin A in the fermentation broth was 5.07 g / L.

[0063] As shown in Example 4 and Comparative Examples 1-3, the content of L-tryptophan, D-leucine, and D-valine in the culture medium affects the potency. Appropriate concentrations, such as 1-2.5 g / L for L-tryptophan, 1-2.5 g / L for D-leucine, and 0.5-1.5 g / L for D-valine, can significantly improve the potency. Inappropriate concentrations have a weak effect on potency improvement and may even decrease it. Furthermore, as shown in Example 5 and Comparative Example 4, adjusting fermentation parameters during fermentation can improve the potency. By controlling pH and dissolved oxygen and supplementing the feed, the potency can be increased from 5.07 g / L to 7.57 g / L.

Claims

1. A strain of Bacillus brevis HDCC00067, characterized in that, It is deposited at the China General Microbiological Culture Collection Center on August 8, 2025, with accession number CGMCC No. 35553.

2. The use of the *Bacillus brevis* according to claim 1, characterized in that: Used to prepare short bacillus peptide A; Alternatively, it can be used to prepare products containing gramicidin A.

3. A fermentation product containing the Bacillus brevis of claim 1.

4. The fermentation product according to claim 3, characterized in that: The fermentation product is a fermentation broth, seed broth, culture broth, or bacterial suspension.

5. A use of the fermentation broth as described in claim 4, characterized in that: Used to prepare short bacillus peptide A; Alternatively, it can be used to prepare products containing gramicidin A.

6. A fermentation method for producing bacitracin A using Bacillus brevis as described in claim 1.

7. The method according to claim 6, characterized in that: The seed culture is inoculated into a fermentation medium for fermentation culture, the fermentation medium including an assimilable carbon source and / or nitrogen source.

8. The method according to claim 7, characterized in that: The fermentation medium also includes L-tryptophan, D-leucine, and D-valine.

9. The method according to claim 7 or 8, characterized in that: The fermentation culture includes dissolved oxygen control and / or feeding control and / or pH control; Among them, dissolved oxygen can be selectively maintained at 35% or above, or at 45±5%; Among them, the feeding can be selectively a mixture of carbon source and nitrogen source, as well as a foaming agent; The pH can be selectively maintained at 7.0 ± 0.

3.

10. A fermentation medium for Bacillus brevis, characterized in that: It includes amino acid precursors, or L-tryptophan, D-leucine, and D-valine.

11. The fermentation medium according to claim 10, characterized in that: According to mass concentration, the culture medium contains 1-3 g / L of L-tryptophan, 1-3 g / L of D-leucine, and 0.5-2 g / L of D-valine. Alternatively, L-tryptophan is 1~2.5 g / L, D-leucine is 1~2.5 g / L, and D-valine is 0.5~1.5 g / L; Alternatively, L-tryptophan should be 2-2.5 g / L, D-leucine 2-2.5 g / L, and D-valine 1-1.5 g / L.