Bacillus velezensis and application thereof in preparation of surfactin

Abstract

The invention relates to the field of microorganisms, in particular to bacillus velezensis and application of the bacillus velezensis in preparation of surfactin, the preservation number is CCTCC NO: M 20251689, the bacillus velezensis is a wild type bacillus velezensis strain, and the surfactin yield of the bacillus velezensis is 2-3 times higher than that of a conventional wild strain. At the shake flask level, the yield of the surfactin reaches 10.4 g / L, when fed-batch fermentation is carried out in a 5L fermentation tank, the yield reaches 22.3 g / L, the yield and production intensity are higher than the currently known wild fungus fermentation level, and the method has huge industrial application potential.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a Bacillus belye and its use in the preparation of surfactants. Background Technology

[0002] Surfactin is a novel biosurfactant with excellent surface activity, biodegradability, antibacterial activity, and biocompatibility, showing broad application prospects in petroleum extraction, pharmaceuticals, agriculture, and daily chemical industries. Surfactin is a lipopeptide biosurfactant whose cyclic structure consists of seven amino acid residues (Leu, Glu, Asp, and Val) linked to a long β-hydroxy fatty acid chain (usually C12–C17) via lactone bonds, making it an amphoteric molecule.

[0003] Surfactants are typically synthesized by Bacillus bacteria, with Bacillus subtilis being the most commonly used chassis strain. Other Bacillus species genetically closely related to Bacillus subtilis, such as Bacillus belyceae and Bacillus amyloliquefaciens, have also been developed for production. Reported high-yielding strains are usually genetically modified and belong to the category of genetically engineered bacteria. On the one hand, due to policy and safety constraints, surfactants produced by recombinant engineered bacteria may not be applicable to pharmaceutical, food, and other applications; on the other hand, mutagenesis or genetic modification also requires a naturally high-yielding surfactant strain as a chassis cell. Therefore, obtaining a naturally high-yielding surfactant strain is essential. However, most reported wild-type Bacillus surfactant yields are between 0.2-1 g / L during shake-flask fermentation; during scale-up production, yields are typically below 10 g / L, limiting industrial application. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide Bacillus belyssus and its use in the preparation of surfactants, in order to solve the problems in the prior art.

[0005] To achieve the above and other related objectives, this invention provides a strain of Bacillus belyssus, with accession number CCTCC NO: M 20251689.

[0006] The present invention also provides a microbial agent containing Bacillus velezensis and / or its metabolites.

[0007] The present invention also provides a method for preparing surfactants, comprising the following steps: inoculating the seed culture of Bacillus velezensis or the bacterial agent into a fermentation medium for culture, and obtaining a fermentation broth containing surfactants after fermentation is completed.

[0008] As described above, the *Bacillus belyceae* strain of the present invention and its use in the preparation of surfactants have the following beneficial effects: It is a wild-type *Bacillus belyceae* strain, and its surfactant yield is 2-3 times higher than that of conventional wild-type strains. At the shake-flask level, the surfactant yield reaches 10.4 g / L; in a 5L fermenter with fed-batch fermentation, the yield reaches 22.3 g / L. The yield and production intensity are higher than the currently known fermentation levels of wild-type strains, demonstrating significant potential for industrial application. Attached Figure Description

[0009] Figure 1 The colony morphology is shown as Bacillus belyssus Bv251.

[0010] Figure 2 The image shows the HPLC analysis results of the surfactant standard.

[0011] Figure 3 The image shows the LC-MS analysis results of the surfactant standard. Figure 4 , Figure 6 , Figure 8 , Figure 10 The following are HPLC analysis results of Bacillus belyss Bv251 fermentation broth from Examples 3 to 6, displayed sequentially.

[0012] Figure 5 , Figure 7 , Figure 9 , Figure 11 The following figures sequentially show the LC-MS analysis results of Bacillus belyssus Bv251 fermentation broth from Examples 3 to 6. Detailed Implementation

[0013] The first aspect of this invention provides a strain of Bacillus velezensis, deposited at the China Center for Type Culture Collection (CCTCC), with the strain name: Bacillus velezensis Bv251, accession number: CCTCC NO: M20251689, deposit date: July 24, 2025, and deposit address: Wuhan University, Wuhan, China.

[0014] The Bacillus velezensis described is a Gram-positive bacterium with a colony morphology typical of Bacillus velezensis, characterized by being round, smooth, opaque, milky white, viscous, and moist in texture; after culturing for more than 36 hours, obvious wrinkles and protrusions appear in the center of the colony.

[0015] The *Bacillus velezensis* species described herein is capable of producing surfactants. The surfactant production yield of *Bacillus velezensis* can reach up to 22 g / L or higher, for example, 22.3 g / L.

[0016] The Bacillus velezensis strain was obtained by screening high-surfactant-producing strains of Bacillus isolated from soil samples, cultured in test tubes and shake flasks, and analyzed by HPLC of the fermentation broth.

[0017] The present invention also provides a microbial agent containing Bacillus velezensis and / or its metabolites.

[0018] In one embodiment, the bacterial agent is a liquid bacterial agent.

[0019] The present invention also provides the use of the Bacillus velezensis in the preparation of surfactants.

[0020] Surfactants are not single compounds, but a family of homologues sharing a common core structure. Their core structure consists of a cyclic structure composed of seven amino acid residues (Leu, Glu, Asp, and Val) linked by a lactone bond to a long β-hydroxy fatty acid chain (usually C12–C17). Their classification is primarily based on two key structural differences: the length of the fatty acid chain and the type of the seventh amino acid residue (X). Based on these two variables, surfactants are mainly divided into the following major categories:

[0021] Surfactin A (major type): The fatty acid chain is usually a straight-chain β-hydroxy fatty acid with C12 to C17 (such as β-OH-C14, β-OH-C15). The seventh amino acid is leucine.

[0022] Surfactin B: The fatty acid chain is usually a C14 or C15 straight-chain β-hydroxy fatty acid (similar to A). The seventh amino acid is valine.

[0023] Surfactin C: The fatty acid chain is usually a C15 or C16 straight-chain β-hydroxy fatty acid (slightly longer than A / B). The seventh amino acid is isoleucine (Ile).

[0024] Other variations:

[0025] Fatty acid chain length variants: Within each major class (A, B, C), there are also homologues with different fatty acid chain lengths (such as C12, C13, C14, C15, C16, C17). For example: [Ile7]Surfactin C15 (the seventh position is Ile, and the fatty acid chain is C15), [Leu7]Surfactin C13 (the seventh position is Leu, and the fatty acid chain is C13).

[0026] Branched-chain fatty acid variants: In rare cases, fatty acid chains may have methyl branches (such as iso-C15, anteiso-C15), which can also produce variants with slightly different properties.

[0027] Amino acid substitution variants: Except for position 7, amino acids at other positions may also be substituted in very rare ways (such as Leu2 being replaced by Ile), but this is not usually the main classification method.

[0028] In some embodiments of the present invention, the Bacillus velezensis is used to prepare a surfactant of type Surfactin A.

[0029] The present invention also provides a method for preparing surfactants, comprising the following steps: inoculating the seed culture of Bacillus velezensis or the bacterial agent into a fermentation medium for culture, and obtaining a fermentation broth containing surfactants after fermentation is completed.

[0030] The inoculation amount of the *Bacillus velezensis* seed culture is 0.5–15% of the fermentation medium volume. For example, the inoculation amount of the *Bacillus velezensis* seed culture is 0.5–1%, 1–1.5%, 1.5–2%, 2–5%, 5–10%, or 10–15% of the fermentation medium volume. Preferably, the inoculation amount of the *Bacillus velezensis* seed culture is 0.5–7% of the fermentation medium volume.

[0031] In some embodiments of the present invention, the fermentation medium comprises a carbon source, a nitrogen source, and inorganic salts.

[0032] The carbon source is selected from sucrose, glucose, fructose, brown sugar, xylose, maltose, starch, dextrin, glycerol, cellulose, cellulose hydrolysate, molasses, or any combination of one or more of the foregoing. Based on the total volume of the fermentation medium, the concentration of the carbon source is 10–80 g / L, for example, 10–20 g / L, 20–30 g / L, 30–40 g / L, 40–50 g / L, 50–60 g / L, 60–70 g / L, or 70–80 g / L.

[0033] The nitrogen source is selected from yeast extract, peptone (e.g., soybean peptone or tryptone), corn steep liquor, urea, ammonium sulfate, ammonium chloride, sodium nitrate, potassium nitrate, monosodium glutamate, leucine, soybean flour, soybean meal, peanut flour, cottonseed flour, or any combination of one or more of the foregoing substances, such as a combination of tryptone and corn steep liquor. Based on the total volume of the fermentation medium, the concentration of the nitrogen source is 10–50 g / L, for example, 10–20 g / L, 20–30 g / L, 30–40 g / L, or 40–50 g / L.

[0034] The inorganic salt is selected from Ca. 2+ Fe 2+ Mn 2+ Mg 2+ The solution is a hydrochloride or sulfate buffer, or a phosphate (disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, or potassium dihydrogen phosphate) buffer system. The concentration of inorganic salts is 0.01–20 g / L based on the total volume of the fermentation medium, or the pH is 7.0–7.5.

[0035] In some embodiments of the present invention, the fermentation medium further includes amino acids at a concentration of 8–15 g / L, for example, 8–10 g / L, 10–11 g / L, 11–13 g / L, or 13–15 g / L. The amino acids are selected from leucine, isoleucine, glutamic acid, aspartic acid, and valine; preferably, the amino acid is leucine.

[0036] In some embodiments of the present invention, the fermentation medium further includes an antifoaming agent. The antifoaming agent is selected from polyether-based antifoaming agents, vegetable oil-based antifoaming agents, organosilicon-based antifoaming agents, or a combination of the above three components. Based on the total volume of the fermentation medium, the volume fraction of the antifoaming agent is 1–3‰, for example, 1–1.5‰, 1.5–2‰, 2–2.5‰, or 2.5–3‰.

[0037] In some embodiments of the present invention, the polyether-based defoamer is, for example, a defoamer comprising a polyoxypropylene-polyoxyethylene block copolymer.

[0038] The vegetable oil defoamer is a defoamer containing one or more of the following substances: soybean oil, rapeseed oil, corn oil, rice bran oil, sesame oil, walnut oil, and castor oil.

[0039] The organosilicon defoamer is a defoamer containing silicone oil or modified silicone oil.

[0040] In some embodiments of the present invention, the fermentation medium comprises 40-80 g / L glucose, 10-30 g / L tryptone, 5-15 g / L corn steep liquor, 0.6-1.0 g / L potassium dihydrogen phosphate, 2-2.6 g / L disodium hydrogen phosphate, 0.05-0.15 g / L manganese chloride, 0.05-0.15 g / L calcium chloride, 0.1-0.3 g / L magnesium sulfate, 0.1-0.3 g / L ferrous sulfate, and 10-14 g / L leucine.

[0041] In some embodiments of the present invention, the fermentation medium comprises 40-80 g / L glucose, 10-30 g / L tryptone, 5-15 g / L corn steep liquor, 3.5-4.5 g / L potassium dihydrogen phosphate, 11-13 g / L disodium hydrogen phosphate, 0.8-1.2 g / L magnesium sulfate, 0.2-0.6 g / L ferrous sulfate, and a polyether defoamer with a volume fraction of 1-3‰. The culture medium is sterilized by filtration or autoclaving after preparation.

[0042] In some embodiments of the present invention, the culture temperature is 20–45°C. Specifically, the culture temperature is, for example, 20–25°C, 25–30°C, 30–35°C, 35–38°C, or 38–45°C.

[0043] In some embodiments of the present invention, the culture method is oscillation culture. The oscillation speed is 100-600 rpm. For example, the oscillation speed is 100-150 rpm, 150-200 rpm, 200-250 rpm, 250-300 rpm, 300-350 rpm, 350-400 rpm, 400-500 rpm, or 500-600 rpm.

[0044] In some embodiments of the present invention, the addition of feed culture medium to the culture system is also included.

[0045] In some embodiments of the present invention, the fed-batch culture medium includes a carbon source and a nitrogen source, based on the total volume of the feeding medium. The concentration of the carbon source is 500–1000 g / L, and the concentration of the nitrogen source is 100–200 g / L. The types of carbon and nitrogen sources in the fed-batch culture medium can be the same as those in the fermentation culture medium.

[0046] In some embodiments of the present invention, the supplemental culture medium further includes amino acids at a concentration of 8–15 g / L, for example, 8–10 g / L, 10–11 g / L, 11–13 g / L, or 13–15 g / L. The amino acids are selected from leucine, isoleucine, glutamic acid, aspartic acid, and valine; preferably, the amino acid is leucine.

[0047] In one embodiment, the supplemental culture medium comprises 700-900 g / L glucose, 100-200 g / L tryptone, and 8-12 g / L leucine.

[0048] In some embodiments of the present invention, air is introduced into the culture system at a rate of 3 to 7 L / min.

[0049] In some embodiments of the present invention, the pH value of the system during fermentation is 6 to 8, preferably 6.5 to 7.5.

[0050] During fermentation, the system's dissolved oxygen (DO) value is above 20%, for example, 20-40%.

[0051] Fermentation time is 10–96 hours. The specific processing time can be adjusted as needed, for example, 10–24 hours, 24–36 hours, 36–48 hours, 48–60 hours, 60–72 hours, or 72–96 hours.

[0052] In some embodiments of the present invention, the method for preparing surfactants includes the following steps:

[0053] 1) Inoculate the seed culture of Bacillus velezensis into the fermentation medium and culture for 3-5 hours at a speed of 100-300 rpm and an aeration rate of 4-5 L / min;

[0054] 2) After the culture is completed, increase the rotation speed to 400-600 rpm, the aeration rate to 5.5-7 L / min, and add feed medium until the culture is completed. During the fermentation process, maintain dissolved oxygen above 30% and pH at 6.8-7.2.

[0055] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0056] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0057] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0058] Example 1: Isolation, screening and identification of Bacillus

[0059] 1. Isolation of Bacillus

[0060] (1) On February 5, 2025, at a depth of 5-10 cm near the roots of the lush vegetation in the green belt of Zhengbo Road, Fengxian District, Shanghai, a soil sample of about 2-3 g was dug out with a sterile spatula and sealed in a sterile 5 mL centrifuge tube.

[0061] (2) Take 1g of soil sample, add 10mL of sterile water, mix thoroughly, and allow to stand for separation. Collect the supernatant and dilute serially to a concentration of 10. -1 ~10 -5 Soil leachate was heated in an 80°C metal bath for 20 minutes to kill vegetative cells and enrich dormant spores. After static cooling for 30 minutes, 10 samples were sequentially aspirated. -3 ~10 -5 100 μL of soil leachate was spread onto LB agar plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar powder, pH 7.0, sterilized at 121℃ for 20 min), and incubated at 37℃ for 24 h. Single colonies of Bacillus were picked up with a sterile pipette tip and streaked onto LB agar plates. After incubation at 37℃ for 24 h, the plates were stored at 4℃.

[0062] Screening of Bacillus

[0063] (1) Initial screening in test tubes

[0064] On LB solid plates, pick single colonies with typical Bacillus colony morphology characteristics (nearly round, opaque, milky white or grayish white, with biofilm) and inoculate them into test tubes containing 3 mL of LB liquid medium. Incubate at 37°C and 200 rpm for 36 h.

[0065] After cultivation, 1 mL of fermentation broth was centrifuged at 12000 rpm for 3 min, filtered through a 0.22 μm filter, and then analyzed by high-performance liquid chromatography (HPLC) to determine the surfactant content in the fermentation broth. The HPLC conditions for surfactant determination were as follows: Agilent ZORBAX Eclipse XDB-C18 column, column temperature 35℃, mobile phase of trifluoroacetic acid, methanol, and water in a ratio of 1:900:100, flow rate 1 mL / min, injection volume 10 μL, and detection wavelength 214 nm. The HPLC analysis results of the surfactant standard (brand: Adamas, original code: 112801A, purchased from Shanghai Titan Technology Co., Ltd., exploration code: 015138967) are as follows. Figure 2 As shown, the LC-MS analysis results are as follows: Figure 3 As shown. Figure 2 In the study, the retention time of surfactants with different configurations ranged from 7 to 35 minutes. Figure 3 In the formula, m / z values ​​of 994, 1008, 1022, 1036, and 1050 correspond to C12-surfactin, C13-surfactin, C14-surfactin, C15-surfactin, and C16-surfactin, respectively; and 1016, 1030, 1044, 1058, and 1072 correspond to the monosodium salt forms of C12-surfactin, C13-surfactin, C14-surfactin, C15-surfactin, and C16-surfactin ([M+Na)). + ).

[0066] Strains with surfactant content above 300 mg / L were subjected to shake-flask fermentation for secondary screening. In the initial screening using test tubes, *Bacillus belyssiensis* Bv251 showed a surfactant yield of 0.6 g / L.

[0067] (2) Shaking flask and sieving

[0068] The shake-flask rescreening medium consisted of: 50 g / L glucose, 20 g / L ammonium sulfate, 10 g / L corn steep liquor, 0.82 g / L potassium dihydrogen phosphate, and 2.49 g / L disodium hydrogen phosphate. The medium was streaked onto LB agar plates using an inoculation loop and incubated at 37°C for 16 h. A single colony of *Bacillus belye* Bv251 from an LB agar plate was picked and inoculated into a test tube containing 3 mL of LB medium. The culture was incubated overnight at 37°C and 200 rpm for 16 h to obtain the activated seed culture. 500 μL of the seed culture was inoculated into a 500 mL Erlenmeyer flask containing 50 mL of the shake-flask rescreening medium and incubated at 37°C and 200 rpm for 72 h. After fermentation, 1 mL of the fermentation broth was centrifuged at 12000 rpm for 3 min, filtered through a 0.22 μm filter, diluted 20-fold with methanol, and analyzed by high-performance liquid chromatography (HPLC) to determine the surfactant content in the fermentation broth. Strains with a surfactant content of 1.5 g / L or higher were preserved. During shake-flask re-screening, *Bacillus belye* Bv251 showed a surfactant yield of 2.6 g / L.

[0069] 2. Identification of Bacillus belyssus Bv251

[0070] (1) Colony morphology observation: After inoculating the isolated strains into LB liquid medium and activating for 24 h, the bacterial suspension was serially diluted to a suitable concentration and spread onto LB solid plates. The plates were then incubated at 37°C for 24 h, and the colony morphology was observed. The colony morphology of *Bacillus belyssiensis* Bv251 was typical of *Bacillus belyssiensis*, characterized by round, smooth, opaque, milky-white, viscous colonies with obvious wrinkles and protrusions in the center, and a moist, viscous texture. The colony morphology is as follows: Figure 1 As shown, the bacteria, after Gram staining, appear blue-purple under an optical microscope, exhibiting the staining color of Gram-positive bacteria, and their morphology is rod-shaped.

[0071] (2) Molecular biological identification: The obtained Bacillus bereaves Bv251 was subjected to 16S rDNA sequence analysis. Using Bv251 colonies as DNA templates, PCR amplification was performed using universal primers 27F and 1492R, the primer sequences of which are shown in SEQ ID NO:1-2. The DNA products obtained by PCR amplification were sequenced by Shanghai Sangon Biotech, and the sequencing results are shown in SEQ ID NO:3. After NCBI BLAST alignment, the 16S rDNA sequence showed 99.93% homology with Bacillus bereaves.

[0072] Because Bacillus species are closely related, bacterial classification using 16S rDNA typically yields low resolution. Therefore, sequencing of the gyrB gene is necessary for accurate classification and identification. PCR amplification was performed using gyrBF and gyrBR primers, the primer sequences of which are shown in SEQ ID NO:4-5. The DNA products obtained from PCR amplification were sequenced by Shanghai Sangon Biotech, and the sequencing results are shown in SEQ ID NO:6. NCBI BLAST alignment showed that the gyrB gene sequence had 99.96% homology with Bacillus belesii.

[0073] Based on the morphological characteristics of the strain and the sequencing comparison results, the selected strain can be identified as Bacillus velezensis.

[0074] The above-mentioned Bacillus velezensis Bv251 was deposited at the China Center for Type Culture Collection, and the proposed classification name is Bacillus velezensis.

[0075] Example 2: Surfactant shake-flask fermentation

[0076] The fermentation medium consisted of: 60 g / L glucose, 20 g / L ammonium sulfate, 10 g / L corn steep liquor, 0.82 g / L potassium dihydrogen phosphate, 2.49 g / L disodium hydrogen phosphate, 0.1 g / L manganese chloride, 0.1 g / L calcium chloride, 0.2 g / L magnesium sulfate, and 0.2 g / L ferrous sulfate.

[0077] Bacillus belye Bv251, stored at -80℃, was streaked onto LB agar plates and incubated at 37℃ for 16 h. A single colony of Bacillus belye Bv251 from an LB agar plate was picked and inoculated into a test tube containing 3 mL of LB medium. The culture was incubated overnight at 37℃ and 200 rpm for 16 h to obtain activated seed culture. 500 μL of the seed culture was inoculated into a 500 mL Erlenmeyer flask containing 50 mL of the above fermentation medium and incubated at 37℃ and 200 rpm for 72 h. After fermentation, 1 mL of fermentation broth was centrifuged at 12000 rpm for 3 min, filtered through a 0.22 μm filter, diluted 20-fold with methanol, and analyzed by high-performance liquid chromatography (HPLC) to determine the surfactant content in the fermentation broth. Three replicates were performed, and the average yield was calculated.

[0078] The surfactant yield was measured to be 3.2 g / L under the conditions described in Example 2.

[0079] Example 3: Surfactant shake-flask fermentation

[0080] The fermentation medium consisted of: 60 g / L glucose, 20 g / L tryptone, 10 g / L corn steep liquor, 0.82 g / L potassium dihydrogen phosphate, 2.49 g / L disodium hydrogen phosphate, 0.1 g / L manganese chloride, 0.1 g / L calcium chloride, 0.2 g / L magnesium sulfate, and 0.2 g / L ferrous sulfate. The remaining fermentation conditions were the same as in Example 2.

[0081] The surfactant yield was determined to be 4.8 g / L under the conditions described in Example 3. The HPLC analysis results of the product are as follows: Figure 4 As shown, the LC-MS analysis results are as follows: Figure 5 As shown. Figure 4 In this study, the retention time of surfactants was 7-35 minutes, compared to... Figure 2 Consistent, Figure 5 In the middle, m / z and Figure 3 The consistency with the molecular weights of surfactants with different carbon chain lengths indicates that the product is a surfactant. Figure 4 Central main peak and Figure 2 The inconsistency is related to the isomerism of side chains and amino acids.

[0082] Example 4: Surfactant shake-flask fermentation

[0083] The fermentation medium consisted of: 60 g / L glucose, 20 g / L tryptone, 10 g / L corn steep liquor, 0.82 g / L potassium dihydrogen phosphate, 2.49 g / L disodium hydrogen phosphate, 0.1 g / L manganese chloride, 0.1 g / L calcium chloride, 0.2 g / L magnesium sulfate, 0.2 g / L ferrous sulfate, and 12 g / L leucine. The remaining fermentation conditions were the same as in Example 2.

[0084] The surfactant yield was determined to be 10.4 g / L under the conditions described in Example 4. The HPLC analysis of the product is as follows: Figure 6 As shown, the LC-MS analysis results are as follows: Figure 7 As shown. Figure 6 In this study, the retention time of surfactants was 7-35 minutes, compared to... Figure 2 Consistent, Figure 7 In the middle, m / z and Figure 3 The consistency with the molecular weights of surfactants with different carbon chain lengths indicates that the product is a surfactant. Figure 6 Central main peak and Figure 2 The inconsistency is related to the isomerism of side chains and amino acids. (Comparison) Figure 6 and Figure 4 This indicates that the addition of leucine to the fermentation medium not only led to increased yield but also reduced the proportion of C14-surfactin and increased the proportion of C15-surfactin.

[0085] Example 5: Surfactant-fed fermentation

[0086] The initial culture medium consisted of: 20 g / L glucose, 20 g / L tryptone, 20 g / L corn steep liquor, 4.1 g / L potassium dihydrogen phosphate, 12.5 g / L disodium hydrogen phosphate, 1 g / L magnesium sulfate, 0.4 g / L ferrous sulfate, and 2‰ (v / v) polyether defoamer (Dow DOWFAX DF-103 defoamer).

[0087] The feeding medium consisted of 800 g / L glucose and 150 g / L tryptone.

[0088] Take Bacillus belye Bv251 stored at -80℃, streak it on an LB agar plate, and incubate it at 37℃ for 16 hours. Pick a loopful of Bacillus belye Bv251 colony from the LB agar plate and inoculate it into a test tube containing 3 mL of LB medium. Incubate at 37℃ and 200 rpm for 9 hours to obtain the activated primary seed culture.

[0089] Take 500 μL of primary seed culture and inoculate it into a 500 mL Erlenmeyer flask containing 100 mL of the above fermentation medium. Incubate at 37 °C and 200 rpm for 9 h to obtain secondary seed culture.

[0090] 100 mL of the secondary seed culture was inoculated into a 5 L bioreactor containing 2 L of initial culture medium. The initial fermentation speed was 200 rpm and the aeration rate was 4 L / min. After 4 hours of incubation, the speed was increased to 500 rpm and the aeration rate was 6 L / min, while simultaneously feeding culture medium was started for 36 hours. Dissolved oxygen was maintained above 30% during fermentation, and the pH was maintained at 7.0 using sodium hydroxide.

[0091] The surfactant yield was determined to be 17.3 g / L under the conditions described in Example 4. The HPLC analysis of the product is as follows: Figure 8 As shown, the LC-MS analysis results are as follows: Figure 9 As shown. Figure 8 In this study, the retention time of surfactants was 7-35 minutes, compared to... Figure 2 Consistent, Figure 9 In the middle, m / z and Figure 3 The consistency with the molecular weights of surfactants with different carbon chain lengths indicates that the product is a surfactant. Figure 8 Central main peak and Figure 2 The inconsistency is related to the isomerism of side chains and amino acids.

[0092] Example 6: Surfactant-fed fermentation

[0093] The initial culture medium consisted of: 30 g / L glucose, 20 g / L tryptone, 20 g / L corn steep liquor, 4.1 g / L potassium dihydrogen phosphate, 12.5 g / L disodium hydrogen phosphate, 1 g / L magnesium sulfate, 0.4 g / L ferrous sulfate, and 2‰ defoamer.

[0094] The supplemental culture medium consisted of 800 g / L glucose, 150 g / L tryptone, and 10 g / L leucine.

[0095] Other fermentation conditions are the same as in Example 4.

[0096] The surfactant yield was determined to be 22.3 g / L under the conditions described in Example 6. The HPLC analysis of the product is as follows: Figure 10 As shown, the LC-MS analysis results are as follows: Figure 11 As shown. Figure 10 In this study, the retention time of surfactants was 7-35 minutes, compared to... Figure 2 Consistent, Figure 11 In the middle, m / z and Figure 3 The consistency with the molecular weights of surfactants with different carbon chain lengths indicates that the product is a surfactant. Figure 10 Central main peak and Figure 2 The inconsistency is related to the isomerism of side chains and amino acids. (Comparison) Figure 10 and Figure 8 This indicates that the addition of leucine to the fermentation medium not only led to increased yield but also reduced the proportion of C14-surfactin and increased the proportion of C15-surfactin.

[0097] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

[0098] The sequences used in this invention are as follows:

[0099] Seq ID No: 1

[0100] AGAGTTTGATCCTGGCTCAG

[0101] Seq ID No: 2

[0102] GGTTACCTTGTTACGACTT

[0103] Seq ID No:3

[0104] TCGCGGACTGCCTATACTGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGAT

[0105] GTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACT

[0106] CCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAA

[0107] GGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGT

[0108] AACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTG

[0109] GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATG

[0110] GACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGC

[0111] TCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTA

[0112] ACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAG

[0113] CGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGA

[0114] AAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAG

[0115] AGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCA

[0116] GTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGC

[0117] GAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGG

[0118] GGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACG

[0119] GTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATG

[0120] TGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCCTGACAATC

[0121] TAGGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAG

[0122] CTCGTGTCGTGAGATGTTGGGTTAAGTCCCCGCAACGAGCGCAACCCTTGATCTTAGTTG

[0123] CCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGG

[0124] GATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACCGTGCTACAATGGAC

[0125] AGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTT

[0126] CGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCA

[0127] GCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGA

[0128] GTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGAC

[0129] CGGA

[0130] Seq ID No:4

[0131] TGCTTCATACGATCCAAGGC

[0132] Seq ID No:5

[0133] CTTGTGGTGTGTTTTGTTCACTCAT

[0134] Seq ID No:6

[0135] GTGGAGGCATTGACCACGATACCTTACATCAAGCAGGAATGTTCCGTGTGGAAAA

[0136] CGGTACGTTAGTGAAGTGAAGAAATGAGGTGAGCGATTGTATATTCATTTAGGTGACG

[0137] ACTTTGTCGTTTCAACACGCGATATCGTCGGCATTTTTGACTGCAAGGTCAATGTATCG

[0138] CCGATCGTTGAAGAATTTCTGGACAGACAGAAAGAAAAAGTAGTGCCGTCCGTAAACG

[0139] GCACGCCAAAGTCTATCGTAGTCACGACTGAGAATATATATTACTCTCCCTTATCTTCC

[0140] GGCACACTGAAGAAACGTGCGCAATTTATGTTAGAAATAGATTCTTAGCGGATCTTTA

[0141] CAATGAATATATCGTTTAAGAAAAGTGTAGGTGAATGTGCATGGCTATGGAACAGCAG

[0142] CAAAATAGTTATGATGAGAATCAGATACAGGTATTAGAAGGTTTGGAAGCTGTTCGAA

[0143] AGAGACCGGGGATGTACATCGGATCAACTAACAGCAAAGGCCTTCACCACTTGGTGTG

[0144] GGAAATCGTCGACAACAGTATTGACGAAGCCCTGGCCGGTTATTGTACAGATATTAAC

[0145] ATCGAGATTGAAAAAGATAACAGCATTACCGTTAAGGACAACGGGCGCGGAATTCCG

[0146] GTCGGTATCCAGGAGAAGATGGGCCGCCCTGCGGTTGAAGTCATCATGACCGTTCTCC

[0147] ACGCCGGCGGTAAATTTGACGGAAGCGGATATAAAGTATCCGGCGGTCTTCACGGTGT

[0148] AGGGGCGTCTGTCGTAAACGCCTTGTCGACCACTCTTGACGTTACGGTTCATCGTGACG

[0149] GAAAAATCCACTATCAGGCGTACGAGCGCGGTGTACCTGTGGCCGATCTTGAAGTGAT

[0150] CGGTGATACTGATAAGACCGGAACGATTACGCACTTCGTTCCGGATCCGGAAATTTTC

[0151] AAAGAAACAACCGAATACGACTATGACCTGCTTTCAAACCGTGTCCGGGAATTGGCCT

[0152] TCCTGACAAAAGGTGTAAACATCACGATTGAAGACAAACGTGAAGGACAAGAACGGA

[0153] AAAACGAGTACCACTACGAAGGCGGAATCAAAAGCTATGTTGAGTACTTAAACCGTTC

[0154] CAAAGAAGTCGTTCATGAAGAGCCGATTTATATCGAAGGCGAGAAAGACGGCATAAC

[0155] GGTTGAAGTTGCATTGCAATACAACGACAGCTATACAAGCAATATTTATTCTTTCACAA

[0156] ATAATATCAACACATACGAAGGCGGCACGCACGAAGCCGGATTTAAAACCGGTCTGAC

[0157] CCGTGTTATAAACGACTATGCAAGAAGAAAAGGGATTTTCAAAGAAAATGATCCGAAT

[0158] TTAAGCGGGGATGATGTGAGGGAAGGGCTGACTGCCATTATTTCAATTAAGCACCCTG

[0159] ATCCGCAATTCGAAGGGCAGACGAAAACGAAGCTCGGCAACTCCGAAGCGAGAACGA

[0160] TCACTGATACGCTGTTTTCTTCTGCGCTGGAAACATTCCTTCTTGAAAATCCGGACTCA

[0161] GCCCGCAAAATCGTTGAAAAAGGTTTAATGGCCGCAAGAGCGCGGATGGCAGCGAAA

[0162] AAAGCGCGGGAATTGACCCGCCGCAAAAGTGCGCTTGAGATTTCCAATCTGCCGGGCA

[0163] AACTGGCGGACTGTTCTTCTAAAGATCCGAGCATTTCCGAGCTGTATATCGTAGAGGGT

[0164] GACTCTGCGGGCGGATCAGCGAAACAGGGACGGGACCGTCATTTCCAAGCCATTCTGC

[0165] CGCTGCGCGGTAAGATTCTGAACGTTGAGAAAGCCAGACTTGATAAGATTCTCTCAAA

[0166] CAATGAGGTCAGATCAATGATCACGGCCCTCGGAACAGGAATCGGAGAAGATTTTAAT

[0167] CTTGAAAAAGCGCGTTATCATAAAGTGGTCATCATGACGGATGCCGATGTTGACGGCG

[0168] CCCACATCAGAACGCTTTTATTAACGTTCTTCTACAGATACATGCGGGAAATCATCGAA

[0169] AACGGCTATGTCTACATTGCCCAGCCGCCGCTTTATAAAGTGCAGCAGGGAAAACGGG

[0170] TGGAATACGCTTATAATGATAAGCAGCTTGATGAGCTGTTAAAAGAACTTCCGCAATC

[0171] ACCTAAGCCCGGCCTCCAGCGTTATAAAGGTCTTGGAGAAATGAACGCGACTCAGCTT

[0172] TGGGAAACGACAATGGACCCTGCGACCAGAACGCTTCTGCAAGTCAATCTTGAAGATG

[0173] CAATGGACGCTGACGAGACTTTTGAAATGCTGATGGGTGACAAAGTAGAACCGCGGAG

[0174] AAACTTCATAGAAGCAAACGCCAGATACGTGAAAAATCTTGATATTTAACCAAGAAAG

[0175] CCTTATTTTCGTTAAGAAATAAGGCTTTTTTCTCAAAGCGGCTTCTAAAGCCTCTGAAA

[0176] ACCCGCAAAACCCGTATATTCTACTAAATTAATGTGTATAAACACAAGTTTATTGATAT

[0177] AATGGAGAATAGTGAATCGTATTGAAGATCATAATGGACAATCTACTCCCACATTAT

[0178] TTCA

Claims

1. A strain of Bacillus velezensis, characterized in that, The accession number is: CCTCC NO: M20251689.

2. The Bacillus velezensis according to claim 1, wherein the Bacillus velezensis is used to produce surfactants, and the Bacillus velezensis produces surfactants at a yield of 22 g / L or more.

3. A microbial agent, characterized in that, The bacterial agent contains Bacillus velezensis as described in claim 1 or 2 and / or its metabolites.

4. Use of the Bacillus velezensis as described in claim 1 or 2 in the preparation of surfactants; preferably, the surfactant is a Surfactin A type surfactant.

5. A method for preparing a surfactant, characterized in that, The method includes the following steps: inoculating the seed culture of Bacillus velezensis as described in any one of claims 1 to 2 or the inoculum as described in claim 3 into a fermentation medium for culture, and obtaining a fermentation broth containing surfactant after fermentation is completed.

6. The method according to claim 5, characterized in that, The inoculation amount of the Bacillus velezensis seed culture is 0.5-15% of the fermentation medium volume.

7. The method according to claim 5, characterized in that, The fermentation medium includes a carbon source, a nitrogen source, and inorganic salts. The carbon source is selected from sucrose, glucose, fructose, brown sugar, xylose, maltose, starch, dextrin, glycerol, cellulose, cellulose hydrolysate, molasses, or any combination of one or more of the aforementioned substances. Based on the total volume of the fermentation medium, the concentration of the carbon source is 10–80 g / L. Alternatively, the nitrogen source is selected from yeast extract, peptone, corn steep liquor, urea, ammonium sulfate, ammonium chloride, sodium nitrate, potassium nitrate, monosodium glutamate, leucine, soybean flour, soybean meal, peanut flour, cottonseed flour, or any combination of one or more of the foregoing substances, such as a combination of tryptone and corn steep liquor; the concentration of the nitrogen source is 10 to 50 g / L based on the total volume of the fermentation medium. Alternatively, the inorganic salt is selected from Ca. 2+ Fe 2+ Mn 2+ Mg 2+ The hydrochloride or sulfate, or a phosphate buffer system; Based on the total volume of the fermentation medium, the concentration of inorganic salts is 0.01–20 g / L, or the pH is 7.0–7.

5.

8. The method according to claim 7, characterized in that, The fermentation medium also includes an antifoaming agent; preferably, the antifoaming agent is selected from any one or more of polyether antifoaming agents, vegetable oil antifoaming agents, and organosilicon antifoaming agents; preferably, the volume fraction of the antifoaming agent is 1 to 3‰ based on the total volume of the fermentation medium. Alternatively, the fermentation medium may also include amino acids, with a concentration of 8–15 g / L; preferably, the amino acids are one or more of leucine, isoleucine, glutamic acid, aspartic acid, or valine.

9. The method according to claim 5, characterized in that, The preparation method further includes any one or more of the following features: 1) The incubation temperature is 20–45℃; 2) The culture method is shaking culture; preferably, the shaking speed is 100-600 rpm; 3) It also includes adding feed medium to the culture system; preferably, based on the total volume of the feed medium, the feed medium includes a carbon source and a nitrogen source, the concentration of the carbon source is 500-1000 g / L, and the concentration of the nitrogen source is 100-200 g / L; more preferably, the feed medium also includes amino acids, the concentration of which is 8-15 g / L; even more preferably, the amino acids are selected from any one or more of leucine, isoleucine, glutamic acid, aspartic acid, or valine; 4) It also includes introducing air into the culture system at a rate of 3–7 L / min; 5) The pH value of the system during fermentation is 6-8; 6) During the fermentation process, the dissolved oxygen value of the system is above 20%.

10. The method according to claim 5, characterized in that, The method for preparing the surfactant includes the following steps: 1) Inoculate the seed culture of Bacillus velezensis as described in claim 1 or 2 into the fermentation medium and culture for 3 to 5 hours at a rotation speed of 100 to 300 rpm and an aeration rate of 4 to 5 L / min; 2) After the culture is completed, increase the rotation speed to 400-600 rpm, the aeration rate to 5.5-7 L / min, and add feed medium until the culture is completed. During the fermentation process, maintain dissolved oxygen above 20% and pH at 6.8-7.2.

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