Intestine source bacillus licheniformis and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGDONG RONGDA BIOENG CO LTD
- Filing Date
- 2025-12-19
- Publication Date
- 2026-04-28
Smart Images
Figure CN121930987A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology; specifically, it relates to an enterogenic Bacillus licheniformis and its applications. Background Technology
[0002] Microecological preparations, as a type of biological additive, have attracted much attention due to their unique ecological regulatory functions and their role in promoting animal intestinal health. Bacillus licheniformis, as a common feed microecological preparation, has been widely studied and applied in feed additives in the "post-antibiotic" era. As one of the microbial feed additives permitted for use in my country's feed additive catalog, Bacillus licheniformis's function in animal husbandry often extends beyond simply inhibiting pathogenic microorganisms in the intestines through competitive site occupation and oxygen depletion. More importantly, it exerts its effects in the animal gut through its related functional metabolites or byproducts, thereby promoting animal growth and improving production performance. Lichenin, as a member of the surfactant family produced by Bacillus licheniformis metabolism, is a class of small molecule polypeptides with special biological activities, exhibiting significant effects in antibacterial, antiviral, and immunomodulatory aspects. It can disrupt bacterial cell walls, inhibit the growth of harmful bacteria, and activate the host's immune system, enhancing the body's resistance. Due to its unique physiological activity, it has important application value in livestock and poultry health breeding and can serve as a highly efficient, safe, and environmentally friendly biological additive to promote the sustainable development of animal husbandry.
[0003] However, significant differences exist in the metabolic characteristics and bioactivity of Bacillus licheniformis from different sources, which greatly limits their practical application in animal husbandry. Therefore, screening for Bacillus licheniformis strains with efficient lichenin-like lipopeptide production capabilities and optimizing fermentation conditions to improve the yield and activity of these peptides has become a key research focus. In the gut microbiota, there is a close symbiotic relationship between enteric probiotics and the host. Enteric Bacillus licheniformis, as a member of the gut microbiota, possesses unique ecological functions and metabolic characteristics that give it broad application prospects in animal husbandry. By isolating, selecting, and fermenting enteric Bacillus licheniformis, its potential for producing lichenin-like cyclic lipopeptides can be further explored, providing new ideas and methods for the development of animal husbandry and agriculture. Summary of the Invention
[0004] To overcome the shortcomings of existing technologies, this invention aims to select enterogenic Bacillus licheniformis strains with the ability to efficiently produce lichenin-like cyclic lipopeptides from the intestinal environment through innovative isolation, selection, and fermentation technologies, and to provide a scientific basis for their practical application in animal husbandry through comprehensive evaluation of biological characteristics and analysis of metabolites.
[0005] One of the objectives of this invention is achieved through the following technical solution:
[0006] A strain of enteric Bacillus licheniformis, taxonomically named Bacillus licheniformis RD508, with accession number GDMCC No: 64606, accession date May 11, 2024, and depositary classification as Bacillus licheniformis, is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.
[0007] Preferably, the sequence of the housekeeping gene gyrB of Bacillus licheniformis RD508 is shown in SEQ ID No. 8.
[0008] This invention also provides the application of the described enterogenic Bacillus licheniformis in the preparation of lipopeptides. Specifically, the lipopeptide is lichenin.
[0009] The present invention has, but is not limited to, the following beneficial effects:
[0010] This invention provides an enteric Bacillus licheniformis strain that exhibits heat and bile salt tolerance, and significantly increases the production of lipopeptide active substances, particularly lichenin, through metabolism. A second objective of this invention is to provide an application for this enteric Bacillus licheniformis strain, which, along with its metabolites, can be used to prepare microecological and post-biotic preparations, serving as a substitute for feed antibiotics. A third objective of this invention is to provide a method for selecting high-yielding lichenin-producing enteric Bacillus licheniformis strains, involving the targeted isolation and screening of heat- and bile salt-tolerant strains from healthy piglet feces, combined with protoplast ARTP mutagenesis, to select functional Bacillus licheniformis strains with high product tolerance and lichenin-producing metabolic activity, as well as a chromatographic method for preparing lichenin-like lipopeptide compounds. Attached Figure Description
[0011] To clearly illustrate the specific embodiments of the present invention and certain detection techniques used in the experiments, the implementation schemes and the techniques used will be described below, mainly through the accompanying drawings.
[0012] Figure 1 Phylogenetic tree diagram of strain BL02.
[0013] Figure 2 Inhibition zones (partial) of supernatant culture broth from 96 microplates of different bacterial strains.
[0014] Figure 3 The RD508 strain is based on a phylogenetic tree of the housekeeping gene gyrB.
[0015] Specific implementation methods
[0016] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are not intended to limit the invention, but only to illustrate it. Unless otherwise specified, the experimental methods used in the following embodiments are performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. All quantitative experiments in the following embodiments are performed in triplicate, and the results are averaged.
[0017] Example 1:
[0018] Isolation, screening and identification of starting strain BL02
[0019] 1. Pretreatment and bacterial isolation of fecal samples from healthy piglets
[0020] Sample pretreatment: In a sterile operating table, take 5-10g of feces from healthy piglets at different time points and add it to an Erlenmeyer flask containing 50 mL of sterile PBS (0.1mol / L, pH 7.4, purchased from Sangon Biotech (Shanghai) Co., Ltd.). Add 5 sterile glass beads, shake at 150 rpm for 30 min, filter with gauze, and transfer the filtrate to a new sterile Erlenmeyer flask. To specifically screen for strains that can adapt to high temperatures, continue shaking the Erlenmeyer flask containing the contents at 150 rpm for 60 min in a 40-45℃ water bath.
[0021] Isolation of strains: After shaking incubation, the water bath solution was serially diluted 10-fold with sterile PBS buffer to 10⁻⁶. -3 Different dilutions of the original treatment solution were spread on bile salt tolerance medium containing 0.2% and incubated at 40-50℃ for 1-3 days until obvious single colonies grew. Single strains with obvious Bacillus colony characteristics were selected and re-streaked on isolation medium for purification.
[0022] The bile salt tolerance medium described above is an isolated medium with the addition of 0.25% porcine bile salt, namely 12.0g tryptone, 8.0g yeast extract, 2.0g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 2.5g porcine bile salt, 20.0g agar powder, 1000 mL deionized water, pH 7.0±0.2, sterilized at 121℃ for 20min.
[0023] Initial screening using antibacterial confrontation: Since *Bacillus licheniformis* is a facultative anaerobic bacterium, its metabolic mechanism allows it to utilize oxygen for aerobic respiration and also obtain energy and metabolize antibacterial substances (such as lichenin) under anaerobic conditions through fermentation or nitrate respiration. However, some *Bacillus* genera are strictly aerobic, such as *Bacillus subtilis*, whose growth and spore germination are highly dependent on the presence of free oxygen. In an anaerobic environment, its metabolic activity is inhibited, and only a portion of its vegetative cells can survive, unable to reproduce or perform metabolic activities. Therefore, *Clostridium perfringens* ATCC13124 was used as an indicator bacterium. An indicator bacterium suspension was prepared through anaerobic confrontation culture, and confrontation plates containing the indicator bacterium suspension were prepared. Single colonies purified by streaking were then agaricated onto these confrontation plates and cultured in an anaerobic chamber at 42°C. Based on the differences in the ability of different strains to reproduce and metabolize under anaerobic conditions, strains that can grow well under anaerobic conditions at 40℃ and form a confrontation inhibition zone were selected. The confrontation inhibition ability was preliminarily ranked according to the ratio of the diameter of the inhibition zone to the colony diameter (C). The larger the ratio, the stronger the confrontation inhibition activity.
[0024] The aforementioned indicator bacterial suspension refers to inoculating indicator bacteria ATCC13124 into a broth containing 150 mL of brain and heart infusion, and then incubating it statically in an anaerobic chamber at 30°C for 24 hours.
[0025] The aforementioned confrontation plate refers to a modified Clostridium perfringens medium (CW medium), containing 10.0 g / L peptone, 52.0 g / L brain heart infusion broth (purchased from Guangdong Huankai Microbial Technology Co., Ltd.), 5.0 g / L glucose, 20.0 g / L agar powder, 1000 mL deionized water, and pH 7.2. 100 μL of the bacterial culture is evenly spread onto the surface of the CW medium in an anaerobic operating room. After full absorption, it is placed in a 4°C refrigerator for 30 minutes; this is the confrontation plate.
[0026] The aforementioned confrontation culture refers to the process of planting the isolated and purified strain on the surface of a confrontation plate, and then placing the plate containing the indicator bacteria and the isolated strain in an anaerobic culture box and incubating it overnight at 42°C, observing and measuring the inhibition ratio.
[0027] Table 1. Oppositional antibacterial activity of different strains – inhibition ratio (partial)
[0028] strain number Antibacterial ratio B / C 01 1.2 02 2.0 09 1.5 15 1.3 26 1.5 28 1.6 40 1.6 54 1.3 95 1.6 102 1.4
[0029] Note: If there is no antagonistic antibacterial activity, the antibacterial ratio H / C is 1.0.
[0030] The results showed that among the 120 selected strains subjected to confrontation culture, most strains had an inhibition ratio of 1.0, and 10 strains had obvious inhibition zones, with strain 02 showing the best inhibition ratio.
[0031] Shake-flask fermentation and secondary screening: The 10 strains with the highest inhibition ratio (H / C) were transferred to test tube slant (containing LB medium, 18×180 mm test tubes), incubated at 42℃ for 24 h, and then eluted with 5 mL of sterile physiological saline to prepare a bacterial suspension (colony count of 1×10⁻⁶). 9 (CFU / mL), and finally the bacterial suspension was transferred to a shake flask for fermentation culture. The shake flask fermentation conditions were as follows: 50 mL of shake flask fermentation medium was added to a 250 mL Erlenmeyer flask, and the culture was carried out at 42℃ and 220 rpm for 48 h with shaking.
[0032] The above fermentation medium contains (g / L): 50.0g sugarcane molasses, 3.0g sucrose, 60.0g corn steep liquor, 2.0g ammonium sulfate, 2.5g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 1.0g sodium chloride, pH 7.0±0.5, and is sterilized at 121℃ for 20min.
[0033] After fermentation, the sample was centrifuged at 12,000 rpm for 5 min, the biomass (wet weight of the strain) was calculated, and the supernatant was collected. The antibacterial properties of the fermentation supernatant against the indicator bacterium ATCC13124 and the content of lichenin-like cyclic lipopeptides were detected by agar diffusion method and HPLC method, respectively.
[0034] The agar diffusion method described above refers to taking 200 μL of indicator bacteria culture in an anaerobic operating room and spreading it evenly on the surface of a medium containing CW, punching holes (6.0 mm), adding 50 μL of supernatant, quickly placing it in an anaerobic culture box, and placing it in a 4℃ refrigerator for 2 hours to allow the sample to be fully absorbed into the culture medium. Then, the anaerobic culture box containing glass plates is placed in a 42℃ culture box and incubated overnight. The inhibition zone is then observed and measured.
[0035] The steps for determining the content of lichenin lipopeptides using the HPLC method described above are briefly described below:
[0036] ① Sample pretreatment: Take 8 mL of fermentation broth, centrifuge at 12,000 rpm for 8 min to remove bacterial cells; adjust the pH of the supernatant to 2.0 with 6 mol / L hydrochloric acid, and let it stand overnight at 4ºC; then centrifuge at 12,000 rpm for 8 min to remove the supernatant, and vacuum dry the precipitate to obtain crude lipopeptide.
[0037] The crude product was resuspended in 1.6 mL of methanol, extracted by standing for 1–2 h, and finally centrifuged at 12,000 rpm for 2 min. The supernatant was filtered through a 0.45 μm organic filter membrane and used for HPLC detection.
[0038] ② HPLC detection method: Lichenin lipopeptides were detected using a Shimadzu LC2030C column. The column was C18 (4.6 μm × 250 mm, 5 μm).
[0039] Mobile phase A: An aqueous solution containing 0.1% trifluoroacetic acid (TFA). Take 1 ml of TFA, dilute to 1000 ml with ultrapure water, and filter through a 0.45 μm filter membrane.
[0040] Mobile phase B: An acetonitrile solution containing 0.1% trifluoroacetic acid (TFA).
[0041] The injection volume was 20 μL, the column temperature was 40ºC, the flow rate was 1.0 mL / min, and the UV detection wavelength was 215 nm.
[0042] The mobile phase gradient is set as follows:
[0043] Time / min Mobile phase A Mobile phase B 0~5 90% 10% 5~35 90%→50% 10%→50% 35~45 50% 50% 45~50 50%→90% 50%→10%
[0044] Note: Lichenin is a multi-component homologue, and a self-prepared lichenin sample was used as a control. Its structure is similar to that of surfactant, which is also a cyclic lipopeptide, and surfactant (CAS: 24730-31-2) can also be used as a basic control.
[0045] Table 2. Diameter of inhibition zones and lichenin content in supernatants of different bacterial strains
[0046] strain number Biomass (wet weight of cells / 100mL) Diameter of the inhibition zone (mm) Crude lipopeptides (mg / 100mL) Lichenin derivatives (mg / 100mL) 01 10.5 6.3 105.2 12.3 02 13.6 13.5 212.2 33.5 09 10.3 8.3 115.6 10.5 15 10.6 7.5 108.4 12.6 26 11.5 8.2 121.1 15.3 28 10.5 9.8 133.3 13.1 40 10.1 8.6 123.0 12.3 54 10.8 11.6 155.5 14.1 95 11.4 10.2 138.6 11.1 102 11.1 11.0 142.1 12.8
[0047] Note: Inhibition zone diameter (mm) = Inhibition zone outer diameter (mm) - Ore diameter (6.0mm)
[0048] Strains with good biomass, large agar inhibition zone diameter, and the highest content of lichenin-like cyclic lipopeptides were selected for preservation.
[0049] 2. Molecular identification of the strain
[0050] Using the colonies of strain 02 isolated and screened above as templates, the 16S rDNA partial sequence was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), sequence shown in SEQ ID No. 1, and 1492R (5'-TACGGCTACCTTGTTACGACTT-3'), sequence shown in SEQ ID No. 2. The reaction system was as follows: 12.5 μL Pre-mix Ex Taq (Takara), 1 μL 27F primer (10 μmol / L), 1 μL 1492R primer (10 μmol / L), 0.5 μL LA18 genomic DNA template, and 10 μL ultrapure water. PCR conditions: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 57℃ annealing for 40 s, 72℃ extension for 1 min 30 s, for 30 cycles; final extension at 72℃ for 10 min, and storage at 4℃. After obtaining the specific amplification product, the target fragment was recovered using a PCR recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing results were analyzed and aligned using BLAST in the NCBI nucleic acid library (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and a phylogenetic tree was constructed.
[0051] >BL02_16S rDNA, sequence shown in SEQ ID No. 3
[0052]
[0053] The results are as follows Figure 1 The data shows that strain 02 is most closely related to the genus *Bacillaceae*. Phylogenetic analysis was performed using 16S rDNA sequences of homologous strains with high similarity. A phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 4.0. Strain 02 is most closely related to *Bacillus licheniformis*, but evolutionarily distant from the genus *Heindrickella* (formerly *Weizmannella*). Furthermore, it clusters with *Bacillus subtilis* and *Bacillus sp.*, indicating that strain 02 is evolutionarily closely related to *Bacillus licheniformis*. Based on its colony and cell morphology, strain 02 can be definitively identified as *Bacillus licheniformis*, and is renumbered as BL02, or simply *Bacillus licheniformis* BL02.
[0054] This is the starting strain obtained from the isolation—Bacillus licheniformis BL02.
[0055] Example 2:
[0056] Selection and preservation of superior mutant strain Bacillus licheniformis RD508
[0057] 1. Preparation of protoplasts
[0058] 1. Protoplast preparation
[0059] (1) Activation of the starting strain
[0060] The strain BL02 obtained above was used as the starting strain and was activated by two subcultures on LB slant medium.
[0061] (2) Preparation of the starting strain suspension
[0062] The bacterial strain activated on the slant was washed with sterile physiological saline to prepare a bacterial suspension, which was then thoroughly vortexed to mix. The bacterial cells were collected by centrifugation at 12000 r / min, 4℃ for 5 min, and washed twice with sterile physiological saline, followed by two washes with hypertonic solution SMM. The supernatant was discarded by centrifugation, and the final bacterial suspension was resuspended in SMM solution to obtain a bacterial suspension. The colony count was adjusted to approximately 1.0 × 10⁻⁶. 8 CFU / mL (SMM hypertonic solution composition includes: 0.5 mol / L sucrose, 0.02 mol / L MgCl2·6H2O, 0.02 mol / L maleic acid, prepared with distilled water, pH 7.0, sterilized at 121℃ for 22 min, and stored at 4℃ for later use).
[0063] (3) Preparation of protoplasts
[0064] Cell digestion was performed using a combination of enzymes. Specifically, lysozyme and snailase were used for combined digestion. A certain amount of filtered and sterilized enzyme stock solution (prepared with SMM solution) was added to the above-mentioned bacterial suspension to prepare a bacterial suspension containing enzymes. The suspension was placed on a shaker and reacted at 50-100 r / min. The cells were examined under a microscope every 5 min to observe the formation of protoplasts. When about 90% of the cells were transformed into protoplasts, the cells were collected by centrifugation at 2000-3000 rpm, washed twice with SMM, and resuspended in an appropriate amount of SMM. The concentration of lysozyme (purchased from Sangon Biotech (Shanghai) Co., Ltd.) used was 0.05%-0.5%, with an optimal concentration of 0.05%-0.15%; the concentration of snailase (purchased from Sangon Biotech (Shanghai) Co., Ltd.) used was 0.05%-0.5%, with an optimal concentration of 0.05%-0.15%; the enzymatic hydrolysis temperature range was 24-35℃, with an optimal temperature of 26-30℃; the enzymatic hydrolysis time was 10-90 min, with an optimal time of 10-30 min. Under optimized conditions, the final protoplast concentration reached greater than 0.9 × 10⁻⁶. 8 The number of cells / mL was 95.0% or higher, and the protoplast formation rate was also the best, reaching 24.3%.
[0065] 2. ARTP mutagenesis in protoplasts
[0066] (1) Determination of ARTP mutagenesis parameters
[0067] Take 10-20 μL of protoplast suspension and spread it evenly on the upper surface of a metal slide. After drying, transfer the protoplast suspension slide to the stage using tweezers. Use high-purity helium as the working gas for the plasma. Set the power supply to 80W, irradiation distance to 4 mm, plasma temperature to 26-30℃, and gas flow rate to 10 L / min. Treat the bacterial slides with different treatment groups. The treatment times for each group are 0 (control), 10, 20, 30, 40, 50, and 60 s, with three replicates for each group. After treatment, transfer the slides to EP tubes containing SMM solution, shake to elute and form a new protoplast suspension, spread it on regeneration plates, and incubate at 42℃ until single colonies grow. Count the colonies. Calculate the lethality rate using the following method:
[0068] Lethality % = (Number of unmutated colonies - Number of mutagenized colonies) / Number of unmutated colonies × 100%
[0069] The results showed that, compared with the untreated strain (treatment time 0 s), the lethality rates of BL02 protoplast suspension after ARTP mutagenesis were 60.6% (treatment time 10 s), 80.5% (treatment time 20 s), and 90.9% (treatment time 30 s), respectively. Furthermore, the lethality rate almost reached 100% after treatment of 40 s or more. Therefore, to ensure sufficient cell survival while preventing mutagenesis damage, a lethality rate of approximately 90% was selected for ARTP treatment. Thus, the optimal treatment time for ARTP on the BL02 strain was determined to be 30 s.
[0070] (3) Screening of mutagenic strains
[0071] Primary screening using 96-well plates: From the regenerated ARTP mutagenesis plates, select 280 colonies with larger, lighter blue to lighter red circles (approximately 640 colonies) from the larger colonies on the plates. These colonies are then transferred to 96-well microplates containing microplate fermentation medium and incubated at 37-42℃ with shaking at 250 rpm for 72 h. After incubation, centrifuge the 96-well microplates at 6000 rpm for 15 min, and use the supernatant to detect their antibacterial activity using the agar diffusion method.
[0072] The microplate culture medium composition includes: glucose 10 g / L, corn steep liquor 10 g / L, KH2PO4 5 g / L, Na2HPO4 5.0 g / L, (NH4)2HPO4 8.0 g / L, FeSO4·4H2O 0.01 g / L, MgSO4·7H2O 0.21 g / L, pH 7.0±0.5, sterilized at 121℃ for 20 min, ready for use.
[0073] like Figure 2 As shown, using the starting strain BL02 as a control, 12 strains with an inhibition zone increase of more than 10% and good clarity of the transparent zone were selected for further shake-flask fermentation and screening. The fermentation medium and culture conditions were the same as described above.
[0074] Table 3. Shake-flask fermentation secondary screening of different mutant strains
[0075] strain number Crude lipopeptides (mg / 100mL) Lichenin derivatives (mg / 100mL) BL02 215.3 32.5 02 315.6 30.5 06 515.5 90.6 21 501.0 45.6 36 369.0 32.0 49 355.6 31.5 65 358.4 36.6 76 321.1 35.1 101 333.3 41.1 124 323.3 33.2 144 345.5 34.1 215 338.6 40.1 242 342.1 40.8
[0076] The results, as shown in Table 3, indicate that the crude lipopeptide yield of strain 5-06 was 139.4% higher than that of the original strain, and significantly higher than that of other strains. Simultaneously, its total lichenin content and its proportion of total crude lipopeptides were also higher than those of the original strain. Strain 06 was preserved by slant culture and glycerol culture, and designated RD508.
[0077] 3. Identification of RD508 strain based on the housekeeping gene gyrB
[0078] First, the genome of RD508 was extracted using a bacterial genomic DNA extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and a partial sequence of its gyrB gene was amplified using gyrB housekeeping gene primers. The PCR amplification primer sequences are as follows:
[0079] gyrB-F: The sequence is shown in SEQ ID No. 4.
[0080] 5-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA
[0081] gyrB-R: The sequence is shown in SEQ ID No. 5.
[0082] 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3'
[0083] The reaction system is as follows:
[0084] 12.5 μL of Pre-mix Ex Taq (Takara), 1 μL each of gyrB-F / R primers, 0.5 μL of genomic template, and 10 μL of ultrapure water.
[0085] PCR conditions: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 57℃ annealing for 40 s, 72℃ extension for 90 s, for 30 cycles, with a final extension at 72℃ for 10 min, and storage at 4℃. After obtaining the specific amplification product, the target fragment was recovered using a PCR recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.
[0086] The sequencing primers are as follows:
[0087] S-gyrB-F: 5-GAAGTCATCATGACCGTTCTGCA-3, sequence as shown in SEQ ID No. 6.
[0088] S-gyrB-R: 5-AGCAGGGTACGGATGTGCGAGCC-3, sequence as shown in SEQ ID No. 7.
[0089] The sequencing results of strain RD508 are shown below:
[0090] >RD508_gyrB, sequence as shown in SEQ ID No. 8
[0091]
[0092] Sequencing results were analyzed and aligned using BLAST in the NCBI nucleic acid library (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that the closest phylogenetic relationship to strain RD508 is with the genus *Bacillus* (family *Bacillusaceae*). Phylogenetic analysis was performed on sequences from homologous strains with high similarity, and a phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 4.0 (e.g.,...). Figure 3 (As shown).
[0093] Strain RD508 clusters with strains of the genus *Bacillus*, and is most closely related to some branches of the genus *Bacillus licheniformis*, with a similarity of up to 99.91%, indicating that RD508 and *Bacillus licheniformis* are evolutionarily closely related. Based on its colony and cell morphology, RD508 can be definitively identified as *Bacillus licheniformis*, designated *Bacillus licheniformis* RD508, or simply *Bacillus licheniformis* RD508.
[0094] 4. Verification of genetic stability
[0095] The superior mutant strain obtained above, *Bacillus licheniformis* RD508, was passaged every 3 days for 10 generations. Shake-flask fermentation was performed every other generation, and the contents of crude lipopeptides and lichenin in the fermentation broth were measured to assess the stability of the strain during passage. The results showed that the contents of crude lipopeptides and lichenin in the fermentation broth of *Bacillus licheniformis* RD508 did not change significantly during passage, indicating good genetic stability.
[0096] The genetically stable superior mutant strain Bacillus licheniformis RD508 was deposited on May 11, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC; address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, China, postcode: 510075). The accession number of strain RD508 is GDMCC No: 64606.
[0097] Example 3:
[0098] Fermentation application of Bacillus licheniformis RD508
[0099] 1. Seed culture
[0100] (1) Activation of strain
[0101] First, RD508 glycerol bacteria were inoculated onto LB agar slants (18×180 mm) and incubated at 42°C for 24 h; this is the F1 generation. Then, the bacterial strain was washed off the slants with 5 mL of sterile physiological saline to prepare a bacterial suspension, which was then transferred to an LB flask containing LB medium and incubated at 42°C for 12 h; this is the F2 generation activated strain.
[0102] (2) Shake flask seed culture
[0103] The activated culture of the eggplant flask strain was washed with 20 mL of sterile water to prepare a bacterial suspension, which was then transferred to a 5 L shake flask containing 2000 mL of nutrient broth. The suspension was then incubated in a constant temperature shaking incubator at 220 r / min and 42℃ for 16 h to further increase the total number of colonies and enhance the viability of the strain.
[0104] (3) Seed culture expansion
[0105] Seed culture was expanded using seed tanks in shake flasks. Seed flasks were transferred at an inoculum of 5% to seed tanks containing seed culture medium. The control procedures were as follows: tank pressure: 0.06 MPa; tank temperature: 37-42℃; initial stirring: 250 r / min; dissolved oxygen (DO) was adjusted to 20-50% by controlling stirring; aeration was maintained for 12 h; and the transfer criterion was a colony count > 2 × 10⁻⁶. 9 CFU / mL.
[0106] The seed culture medium described above comprises the following components (g / L): glucose 25, yeast extract 20, ammonium sulfate 5.0, ammonium dihydrogen phosphate 2.0, ferrous sulfate 0.05, magnesium sulfate 0.02, and zinc sulfate 0.01, with the pH adjusted to 7.0 ± 0.5. The medium is then autoclaved at 121°C for 20 min.
[0107] 2. Fermentation production of lichenin-like cyclic lipopeptides
[0108] The seed culture was transferred to a fermenter containing fermentation medium, with a transfer volume of 20%. During the initial fermentation stage, the aeration rate in the fermenter was controlled at 150-200 mL / min, initial stirring was maintained at 250 r / min, the temperature was controlled at 37-42℃, and the initial total sugar concentration was controlled at 100-160 g / L. Dissolved oxygen (DO) was controlled at 20-50% through stirring. Throughout the process, the pH of the fermentation broth was controlled to 6.0-6.5 by adding 20% ammonia water, and the total sugar content in the fermenter was maintained at 10-20 g / L by adding molasses. Fermentation was stopped after 48 h.
[0109] The fermentation medium consisted of the following components (g / L): molasses 100, sucrose 20, yeast extract 10, corn steep liquor 50, ammonium sulfate 20, potassium dihydrogen phosphate 25, disodium hydrogen phosphate 15, ferrous sulfate 0.05, magnesium sulfate 0.02, zinc sulfate 0.01, folic acid 10 mg / L, biotin 2 mg / L, and deionized water to bring the final volume to 1000 L. The medium was autoclaved at 121°C for 20 min.
[0110] 3. Fermentation of control strain
[0111] The starting strain BL02 was used instead of RD508 for fermentation under the same conditions, and the yield of lichenin was detected using the same method.
[0112] 4. Comparison of the content of lichenin-type cyclic lipopeptides
[0113] The results showed that the lichenin content in the fermentation broth of strain RD508 reached 1235.3 mg / L, while the lichenin yield of the starting strain BL02 was only 355.3 mg / L, demonstrating a very good ability to synthesize and accumulate lichenin.
[0114] 5. Preparation of postbiotic formulations of cyclic lipopeptide active products of lichenin
[0115] After fermentation, the pH of the fermentation broth was adjusted to 2.0-2.5 with 6 mol / L hydrochloric acid and acidified for 3-6 hours. Then, the pH was adjusted back to 5.5-6.5 with sodium hydroxide, and the temperature was raised to 80-100℃ and heated for 30 minutes to completely inactivate the bacterial cells. The mixture was then evaporated and concentrated to a dry matter content of 35-45%, and spray-dried to obtain a post-biotic preparation of high lichenin-type cyclic lipopeptide active products.
[0116] Example 4:
[0117] Chromatographic preparation of lichenin-type cyclic lipopeptides
[0118] Lichenin was purified and prepared by high-performance liquid chromatography.
[0119] First, the fermentation broth was pretreated: the supernatant was collected by centrifugation, then the pH was adjusted to 2.0 with 6 mol / L hydrochloric acid and allowed to stand overnight at 4ºC. Following this, the broth was centrifuged again for 8 min, the supernatant was removed, and the precipitate was dried under vacuum to obtain the crude lipopeptide. Subsequent purification was performed using a two-step hydrophobic process, the specific steps of which are as follows:
[0120] 1. Preparation of lichenin by primary purification (crude purity)
[0121] Chromatographic column: 50*250mm DAC50 column (purchased from Jiangsu Hanbang Technology Co., Ltd.)
[0122] Packing material: Kromasil Phenyl 10μm (purchased from Guangzhou Ruibai Instrument Technology Co., Ltd.)
[0123] Detection wavelength: 215nm
[0124] Flow rate: 60 mL / min
[0125] Take 0.4 g of crude product, dissolve it in 18 mL of DMSO, filter it through a 0.45 μm filter membrane, and inject the sample.
[0126] Mobile phase A: Acetonitrile
[0127] Mobile phase B: 30 mM sodium dihydrogen phosphate aqueous solution (adjusted to pH 3.0 with phosphoric acid)
[0128] Washing method:
[0129] Time (min) A% B% 0 20 80 15 20 80 75 50 50 80 60 40 80 60 40
[0130] HPLC analysis was performed on the collected solution, and fractions containing the target stream were combined.
[0131] This process is repeated 5 times.
[0132] 2. Secondary purification (refining) of lichenin.
[0133] Column: 50*250mm DAC50 column; Packing material: Kromasil C8 10μm
[0134] Detection wavelength: 215nm
[0135] Flow rate: 60 mL / min
[0136] Take 1 / 3 of the target stream obtained from the first purification, dilute it with pure water by half, and then load the sample using a pump.
[0137] Mobile phase A: Acetonitrile
[0138] Mobile phase B: 0.1% aqueous acetic acid solution
[0139] Washing method:
[0140] Time (min) A% B% 0 27 73 10 27 73 80 47 53 85 60 40 100 60 40
[0141] HPLC analysis was performed on the collected solution, and the fractions with a purity greater than 95% were combined. For fractions with a purity less than 95%, this method was used to purify them again until the purity was greater than 95%.
[0142] 3. Concentration and drying:
[0143] After removing acetonitrile by rotary evaporation under reduced pressure from the collected liquid with a purity greater than 95%, the lichenin was freeze-dried to obtain pure lichenin.
[0144] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. An enteric Bacillus licheniformis, characterized in that... The enteric Bacillus licheniformis is Bacillus licheniformis RD508, with accession number GDMCC No: 64606. The depository is Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province.
2. The enteric Bacillus licheniformis as described in claim 1, characterized in that, The sequence of the housekeeping gene gyrB of Bacillus licheniformis RD508 is shown in SEQ ID No.
8.
3. The application of the enterogenic Bacillus licheniformis strain as described in claim 1 in the preparation of lipopeptide substances.
4. The application as described in claim 3, characterized in that, The lipopeptide substance mentioned is lichenin.