Bacillus licheniformis and screening method and application thereof

Abstract

A strain of Bacillus licheniformis, its screening method and application, in the field of microbial fermentation technology, wherein the Bacillus licheniformis is classified as Bacillus licheniformis ( Bacillus licheniformis BL-2501, with accession number CCTCC NO: M20251206, accession date May 28, 2025. In this invention, BL-2501, using corn stalks as a carbon source for fermentation, achieved degradation rates of 38.52% for NDF and 31.61% for ADF. During the fermentation of corn stalks to prepare feed, it effectively increased the crude protein content and reducing sugar content of the feed, and significantly improved the sulfur-containing amino acid content, with methionine and cysteine ​​reaching 5.88 mg / g and 0.73 mg / g, respectively, thus significantly improving the nutritional value of the feed.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a strain of Bacillus licheniformis, its screening method, and its application. Background Technology

[0002] Cellulose is a primary product of photosynthesis in terrestrial ecosystems and one of the most abundant renewable biological resources in the biosphere. Its structure consists of linear homopolymers of glucose units linked by β-1,4 glycosidic bonds. It is the most important component of lignocellulose and, together with hemicellulose and lignin, forms the basic skeletal structure of plant cell walls. Due to its wide availability, low cost, and environmental friendliness, lignocellulose has become an important raw material for biomass energy and bio-based materials. Enzymatic hydrolysis to convert it into usable small-molecule sugars is one of the key technologies for achieving efficient utilization of biomass resources.

[0003] Cellulose degradation relies on the synergistic action of a cellulase system, primarily including endoglucanase, exoglucanase, and β-glucosidase. Plants, animals, and microorganisms in nature can all produce these degrading enzymes. Among them, microbial cellulases have attracted widespread attention due to their ease of acquisition, high yield, and suitability for industrial production. Compared to fungi, bacteria, especially Bacillus, have advantages such as short growth cycles, simple culture conditions, short fermentation cycles, and low industrial energy consumption. Furthermore, they can secrete various lignocellulose-degrading enzymes, including cellulase and xylanase, thus showing promising applications in feed, energy, and bioconversion.

[0004] However, natural strains typically have low cellulose degradation capabilities, necessitating the use of mutagenesis breeding to obtain high-yield enzyme mutants. In recent years, ARTP (atmospheric pressure room temperature plasma) mutagenesis technology has been widely applied in microbial breeding due to its high mutation efficiency and ease of operation, enabling the rapid construction of large-scale mutant libraries. However, ARTP mutagenesis usually produces a large number of mutants. If traditional plate screening and individual strain verification methods are still used, not only is the screening throughput low and the cycle long, but it is also difficult to achieve rapid enrichment of superior mutants, thus limiting the full realization of the advantages of highly efficient ARTP mutagenesis.

[0005] Currently, commonly used microbial screening methods mainly rely on the CMC plate clear zone method and shake-flask fermentation to verify enzyme activity. While these methods are simple to operate, they suffer from low throughput, low efficiency, and long cycles, making them unsuitable for high-throughput rapid screening of superior mutant strains. Furthermore, traditional methods often require the individual cultivation and testing of large numbers of strains, resulting in high labor intensity and susceptibility to human factors during the screening process. Droplet microfluidics technology enables independent culture, detection, and sorting at the single-cell level, offering advantages such as high screening throughput, low reagent consumption, and high automation. When combined with ARTP mutagenesis, it holds promise for rapid screening of large-scale mutant libraries and enrichment of superior mutant strains. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Bacillus licheniformis. This strain can efficiently synthesize cellulase and xylanase, and when fermenting corn stalks, it can effectively increase the content of sulfur-containing amino acids in the fermentation products.

[0007] Another object of the present invention is to provide applications of the above-mentioned strains.

[0008] Another objective of this invention is to provide a rapid microbial screening method. By integrating efficient mutagenesis with high-throughput screening for strain selection, the screening efficiency of cellulose-degrading strains can be significantly improved, the breeding cycle shortened, and their industrial applications promoted.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A strain of Bacillus licheniformis, characterized in that: the taxonomic name of the Bacillus licheniformis is Bacillus licheniformis (… Bacillus licheniformis BL-2501, deposited at the China Center for Type Culture Collection, accession number CCTCCNO: M20251206, deposited on May 28, 2025, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0011] The above-mentioned Bacillus licheniformis mutant strain BL-2501 is used in the preparation of biomass degradation agents or feed fermentation agents containing lignocellulose.

[0012] This strain can be used to efficiently degrade cellulose and hemicellulose components in lignocellulosic raw materials.

[0013] In application, when this strain ferments straw to prepare feed, it can increase the content of crude protein, true protein, total amino acids, reducing sugars, and sulfur-containing amino acids in the feed. The mutant strain obtained through CMC-Na single carbon source screening has stronger cellulose degradation and substrate utilization capabilities, which can increase the release level of available carbon sources in straw, promote the accumulation of cell biomass and microbial protein synthesis, thereby increasing the content of true protein, total amino acids, and sulfur-containing amino acids in the fermentation products.

[0014] Therefore, when Bacillus licheniformis BL-2501 is used as a feed fermentation agent, it can be used to increase the content of crude protein, true protein, total amino acids, methionine, cysteine ​​and / or reducing sugar in the fermentation products.

[0015] A method for screening Bacillus licheniformis mutant strains, characterized by comprising the following steps: (1) Mutagenesis of strains: The starting strain of Bacillus licheniformis was mutagenized by ambient pressure room temperature plasma (ARTP) to construct a mutant library; (2) Droplet preparation: The mutant library is processed into a single-cell suspension, a culture medium containing cellulose or its derivatives as the sole carbon source is used as the aqueous phase, and the fluorinated oil is used as the oil phase. Single-cell droplets are generated through a microfluidic system. (3) Indirect signal detection and screening: The generated droplets are cultured in situ. The growth signal of single cells in the droplets is detected to indirectly reflect their ability to degrade and utilize cellulose substrates. The target droplets are collected by high-throughput sorting based on the signal intensity. (4) Strain recovery and verification: The collected target droplets were demulsified and recovered to obtain candidate strains with high cellulose degradation capabilities.

[0016] Preferably, the conditions for ARTP mutagenesis in step (1) are: radio frequency power 100 W, helium flow rate 10.0 SLM, irradiation distance 2 mm, and mutagenesis time based on the treatment time corresponding to a lethality of about 90%.

[0017] Furthermore, the sole carbon source in step (2) is sodium carboxymethyl cellulose (CMC-Na), which has a mass fraction of 0.4-0.6% in the aqueous phase.

[0018] Furthermore, the growth signal in step (3) includes at least one of OD signal, bright-field turbidity signal and fluorescence signal.

[0019] Preferably, when the detection signal includes a fluorescence signal, a metabolic indicator, preferably 5 μg / mL resazurin, is added to the aqueous phase in step (2).

[0020] Preferably, the threshold for high-throughput sorting in step (3) is set to filter droplets with signal intensity in the top 1‰ to 1%, preferably the top 1‰.

[0021] Furthermore, the verification in step (4) also includes: determining the degradation ability of the candidate strain on neutral detergent fiber (NDF) and acid detergent fiber (ADF) in corn stalks.

[0022] The mutant library bacterial suspension, after being treated with optimal ARTP conditions and restored to culture, was ultrasonically dispersed (950 W rated power, 2% amplitude, 5 s sonication, 5 s interval, total time 1-2 min). The treatment time was optimized by microscopic observation to obtain a single-cell suspension suitable for droplet embedding. If necessary, incompletely dispersed cell clusters were removed by filtration through a 5 μm filter membrane. The bacterial suspension was washed three times with minimal culture medium (KH2PO4: 3.0 g; K2HPO4: 3.0 g; (NH4)2SO4: 2.0 g; MgSO4·7H2O: 0.2 g; NaCl: 0.5 g), and the OD was adjusted accordingly. 600 Adjust to 0.05-0.1.

[0023] Using the minimum culture medium as the basal medium, 0.5% CMC-Na is added as a carbon source to construct the droplet culture aqueous phase; in a preferred embodiment, 5 μg / mL can also be added as a metabolic indicator.

[0024] The oil phase is a fluorinated oil, preferably Novec 7500 or HFE-7500; the oil phase contains a surfactant, which is a fluorinated oil surfactant suitable for oil-in-water droplet systems, preferably added at 1.0% to 3.0% (w / w), more preferably at 1.5% to 2.5% (w / w).

[0025] Furthermore, the oil phase and the aqueous phase were added separately to a microsyringe, and droplets were prepared using a microfluidic droplet generation chip.

[0026] Furthermore, the injection flow rate ratio of the oil phase to the water phase is 2:1 to 5:1, preferably 2.5:1 to 4:1.

[0027] In a preferred embodiment, the aqueous phase flow rate is 150–250 μL / h, and the oil phase flow rate is 400–900 μL / h; more preferably, the aqueous phase flow rate is 180–220 μL / h, and the oil phase flow rate is 500–800 μL / h. Under these conditions, a homogeneous droplet system with a diameter of approximately 15–30 μm is formed for single-cell embedding and subsequent culture and screening.

[0028] A droplet culture aqueous phase containing mutant bacterial cells and 0.5% CMC-Na (with an optional 5 μg / mL resazurin as a metabolic indicator) was added to the aqueous phase injection bottle of the MISS cell droplet microfluidic system. An oil phase containing 2.0% (w / w) Novec 7500 fluorinated surfactant was added to the oil phase injection bottle. The stirring speed of the aqueous phase injection bottle was set to 35 r / min to ensure uniform bacterial dispersion. The droplet preparation program was started to automatically generate single-cell droplets. The droplets were placed in the system's in-situ culture module and incubated at 37 ℃ for 12–16 h.

[0029] Furthermore, after the culture is completed, the growth of bacteria in the droplets is detected. The detection methods include OD detection mode, bright field turbidity detection mode and / or fluorescence detection mode. When using fluorescence detection mode, resazurin is preferably used as a metabolic indicator, and the excitation wavelength is set to 560 nm and the emission wavelength to 590 nm for detection.

[0030] Droplets were sorted according to the detection signal intensity, and those with signal intensities in the top 1‰ to 1% were selected, preferably in the top 1‰. The target droplets were collected into 96-well plates using the system's automatic sorting function. A demulsifier (perfluorooctanol, added by volume to the oil phase system containing the droplets, at a concentration of 5% to 30% of the total droplet volume, preferably 10% to 20%) was then added. After adding the demulsifier, the plates were gently shaken or mixed for 30 seconds to 2 minutes to break the droplets and release the embedded microbial cells. The plates were then allowed to stand and separate into layers, or the aqueous and oil phases were separated by centrifugation. The aqueous phase was collected, spread onto LB plates, and incubated at 37 °C for 12–16 h to obtain single colonies.

[0031] The present invention has the following technical effects: In this invention, Bacillus licheniformis BL-2501 exhibits excellent ability to degrade cellulose and hemicellulose. In fermentation using corn stalks as a carbon source, the degradation rates of neutral detergent fiber (NDF) and acid detergent fiber (ADF) reached 38.52% and 31.61%, respectively. During the single-strain fermentation of corn stalks to prepare feed, it effectively increased the crude protein content and the proportion of true protein in the feed, while also increasing the reducing sugar content. Most importantly, the content of sulfur-containing amino acids in the feed was significantly improved, with methionine and cysteine ​​reaching 5.88 mg / g and 0.73 mg / g, respectively, significantly enhancing the nutritional value of the feed. Attached Figure Description

[0032] Figure 1 : Lethality curve of the original strain BL of this invention over mutagenesis time.

[0033] Figure 2 : Colony morphology diagram of strain bl-101 ​​in this invention.

[0034] Figure 3 Gram staining image of strain bl-101 ​​in this invention. Detailed Implementation

[0035] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0036] Example 1 Screening, mutagenesis and identification of strains The starting strain BL used in this invention was isolated from decaying straw samples collected near Shuanghe Street, Rongchang District, Chongqing. The specific isolation steps are as follows: The decaying straw sample was placed in a sterile sampling bag. After being brought back to the laboratory, obvious impurities were removed. 10g of the sample was weighed and added to 90 mL of sterile physiological saline. The mixture was shaken at 37 ℃ and 180 r / min for 30 min to fully release microorganisms attached to the sample surface and interior, obtaining a sample suspension. The sample suspension was treated in an 80 ℃ water bath for 10 min to enrich thermostable spore-forming bacteria. After 10-fold serial dilution, 100 µL of the treated suspension was spread onto LB agar plates and incubated at 37 ℃ for 24 h. Single colonies with different morphologies and good growth were picked and repeatedly streaked for purification to obtain the purified strain.

[0037] The purified strains were inoculated onto Congo red selection medium containing CMC-Na and cultured at 37 °C for 24–48 h. After staining with 0.1% Congo red solution for 15–30 min, the colonies were destained with 1 mol / L NaCl solution for 15–30 min, and the formation of a clear zone around the colony was observed. The strain with the largest clear zone and stable colony growth was selected as a candidate strain for cellulose degradation, named BL, and used as the original strain for subsequent ARTP mutagenesis breeding.

[0038] Single colonies of Bacillus licheniformis were inoculated into LB medium and cultured at 37 ℃ and 250 r / min for 10–12 h until the logarithmic growth phase (OD50). 600 (≈0.8–1.0). Collect the bacterial culture by centrifugation, wash 2–3 times with sterile physiological saline, resuspend, and adjust the bacterial concentration to OD0.8. 600 ≈0.6–0.8, to ensure the uniformity of the mutagenesis treatment.

[0039] Take 20 μL of bacterial suspension and spread it evenly on a sterile sample plate. Place it in an ARTP mutagenesis instrument for processing. The mutagenesis conditions are as follows: radio frequency (RF) power 100 W, helium flow rate 10.0 SLM, irradiation distance 2 mm, and mutagenesis time set to 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, and 300 s, respectively. Each group is set up with 3 replicates. Immediately after mutagenesis, the bacterial cells were resuspended in LB medium and transferred to LB liquid medium, where they were incubated at 37 °C with shaking for 2–4 h. The recovered bacterial suspension was then serially diluted and plated on LB plates. After incubation at 37 °C for 12–16 h, colony counts were performed, survival rates were calculated, and lethality curves were plotted. The lethality calculation formula is as follows: Fatal rate = (A - B) / A × 100% Where A represents the colony count (CFU / mL) of the untreated control group, and B represents the colony count (CFU / mL) of the ARTP-treated group.

[0040] The mortality rate curve is shown below. Figure 1 As shown, the survival rate decreased with prolonged treatment time, reaching approximately 90% at 240 s. This was considered the optimal mutagenesis condition, and Bacillus licheniformis was subjected to ARTP mutagenesis under these conditions to construct a mutant library for subsequent droplet microfluidic screening.

[0041] The mutant library culture, after being treated with optimal ARTP conditions and restored to culture, was ultrasonically dispersed (950 W rated power, 2% amplitude, 5 s sonication, 5 s intervals, total time 2 min). The treatment time was optimized using microscopy to obtain a single-cell suspension suitable for droplet embedding. If necessary, incompletely dispersed cell clusters were removed by filtration through a 5 μm filter. The culture was washed three times with minimal culture medium (KH₂PO₄: 3.0 g; K₂HPO₄: 3.0 g; (NH₄)₂SO₄: 2.0 g; MgSO₄·7H₂O: 0.2 g; NaCl: 0.5 g), and the OD₂O₃ content was adjusted. 600 Adjust to 0.05-0.1.

[0042] Using a minimal culture medium as the basal medium, 0.5% CMC-Na was added as a carbon source to construct the droplet culture aqueous phase; 5 μg / mL resazurin was added as a metabolic indicator. Fluorinated oil, preferably Novec 7500, was used as the oil phase, containing a fluorinated oil surfactant suitable for oil-in-water droplet systems at a concentration of 2.0% (w / w). The droplet culture aqueous phase containing mutant bacterial cells and 0.5% CMC-Na was added to the aqueous phase injection bottle of the MISS cell droplet microfluidic system, and the Novec 7500 oil phase containing 2.0% (w / w) fluorinated oil surfactant was added to the oil phase injection bottle. The stirring speed of the aqueous phase injection bottle was set to 35 r / min to ensure uniform dispersion of the bacterial cells in the aqueous phase. The droplet preparation program was started, allowing the aqueous and oil phases to enter the droplet generation chip through their respective channels, forming oil-in-water single-cell droplets under the shearing action of the oil phase. The oil-to-water phase injection flow rate ratio was 3:1, with an aqueous phase flow rate of 200 μL / h and an oil phase flow rate of 600 μL / h. Under these conditions, a homogeneous droplet system with a diameter of approximately 15–30 μm was formed. The generated droplets were collected and placed in the system's in-situ culture module, where they were cultured at 37 ℃ for 14 h. After culture, the bacterial growth within the droplets was detected using OD detection mode, bright-field turbidity detection mode, and / or fluorescence detection mode. When using fluorescence detection mode, the excitation wavelength was set to 560 nm and the emission wavelength to 590 nm.

[0043] Droplets were sorted based on their signal intensity, and those with the highest signal intensities (top 1‰) were selected. The target droplets were automatically collected into 96-well plates using the system's sorting function. A demulsifier (perfluorooctanol, added by volume to the oil phase containing the droplets, at 15% of the total droplet volume) was then added. After adding the demulsifier, the plates were gently shaken or mixed for 1 min to break the droplets and release the embedded microbial cells. The plates were then allowed to separate into layers or centrifuged to separate the aqueous and oil phases. The aqueous phase was collected, spread onto LB plates, and incubated at 37 °C for 14 h to obtain single colonies. The droplet selection results are shown in Table 1.

[0044] Table 1. Number of positive droplets and recovered strains obtained from microfluidic screening

[0045] The total number of droplets generated was approximately 6.3 × 10⁻⁶. 5Approximately 612 positive droplets were obtained through screening, and 184 bacterial strains were recovered after demulsification. The number of recovered strains was lower than the number of positive droplets, mainly due to the failure of some cells to recover successfully during demulsification and resuscitation. Since the droplet culture system uses CMC-Na as the sole carbon source, the enhanced cell growth and metabolic signals within the droplets indicate that the cells can more effectively utilize CMC-Na degradation products, thus serving as an indirect screening indicator of cellulose degradation capacity. Subsequent screening and verification were conducted using Congo red transparent zone assays, enzyme activity assays, and straw degradation experiments.

[0046] Example 2 Screening of mutant strains and verification of their cellulose degradation ability: Step 1, strain activation: The candidate strain recovered in Example 1 was streaked onto LB solid medium and cultured at 37 °C for 12–16 h to obtain well-grown single colonies.

[0047] Step 2, Inoculation: Pick a single colony and inoculate it onto the surface of a solid culture medium containing CMC-Na. Inoculate multiple strains evenly on each plate, and set up the original strain BL as a control.

[0048] Step 3, Preparation of CMC solid culture medium: Prepare the culture medium according to the following formula: KH2PO4 3.0 g, K2HPO4 3.0 g, (NH4)2SO4 2.0 g, MgSO4·7H2O 0.2 g, NaCl 0.5 g, CMC-Na 10.0 g, agar 15.0 g, distilled water 1000 mL, adjust the pH to 7.0, sterilize at 121 ℃ for 20 min, cool and pour into plates for later use.

[0049] Step 4, Cultivation: Place the inoculated plates in a 37 ℃ constant temperature incubator for 24 h to allow the colonies to grow fully and produce degradation.

[0050] Step 5, staining treatment: After the culture is completed, stain the plate with 0.1% Congo red solution for 15–30 min to stain the undegraded CMC areas.

[0051] Step 6, Decolorization treatment: After discarding the dye solution, add 1 mol / L NaCl solution for decolorization for 15–30 min to form a transparent zone in the degradation area.

[0052] Step 7, Data Measurement: Use a fully automated colony counter (icount22) to photograph and measure the colony diameter and the diameter of the clear zone, and calculate the ratio of clear zone to colony diameter to evaluate the cellulose degradation ability of the strain.

[0053] Step 8, strain screening: Strains with good growth status, large transparent zone diameter, and high transparent zone / colon diameter ratio were selected as candidate dominant strains for secondary screening and subsequent fermentation verification. Specific transparent zone statistics are shown in Table 2.

[0054] Table 2: Results of the transparent zone (10 best plants)

[0055] Compared with the original strain BL, the diameter of the transparent zone and the ratio of transparent zone to colony diameter of the selected mutant strains were significantly improved. Among them, the transparent zone to colony diameter ratio of mutant strain bl-101 ​​reached 4.86, which was 129.25% higher than that of the original strain, and it performed the best.

[0056] Step 9: Inoculate the 10 dominant bacterial strains obtained from the above clear zone screening into LB liquid medium and culture at 37 ℃ and 250 r / min for 10-12 h to prepare seed culture; Step 10, Fermentation medium preparation: Prepare the fermentation medium according to the following formula: KH2PO4: 3.0 g; K2HPO4: 3.0 g; (NH4)2SO4: 2.0 g; MgSO4·7H2O: 0.2 g; NaCl: 0.5 g; CMC-Na: 10.0 g; distilled water: 1000 mL; adjust the pH to 7.0, sterilize at 121 ℃ for 20 min and set aside.

[0057] Step 11, Fermentation culture: Inoculate the seed liquid into the above fermentation culture medium at an inoculation rate of 1%, and culture with shaking at 37 ℃ and 250 r / min for 24 h.

[0058] Step 12, preparation of crude enzyme solution: After fermentation, centrifuge the fermentation broth at 8000 r / min for 15 min and take the supernatant as crude enzyme solution for later use.

[0059] The steps for cellulase assay are as follows: Step 1, reagent preparation: Prepare 0.1 mol / L acetic acid solution (take 0.6 mL of glacial acetic acid and add ultrapure water to make up to 100 mL), 0.1 mol / L sodium acetate solution (weigh 0.82 g of sodium acetate, dissolve in water and make up to 100 mL), and acetate-sodium acetate buffer (pH 5.5) (weigh 13.95 g of anhydrous sodium acetate, dissolve in water, add 1.70 mL of glacial acetic acid, make up to 2000 mL, and adjust the pH to 5.5); weigh 1.5 g of CMC-Na and add it to 80 mL of the above buffer solution, heat and stir until completely dissolved, and make up to 100 mL after 30 min to obtain 15 g / L CMC-Na substrate solution, which is stored at 0–4 ℃ protected from light for later use; DNS reagent is commercially available.

[0060] Step 2, prepare enzyme solution: Centrifuge the fermentation broth at 8000 r / min and 4 ℃ for 10 min, take the supernatant as crude enzyme solution, and dilute it 5 times with distilled water for later use.

[0061] Step 3, substrate preheating: Add 100 μL of CMC-Na substrate solution to a 1.5 mL centrifuge tube and preheat in a 37 ℃ water bath for 5 min.

[0062] Step 4, Enzymatic reaction: Add 100 μL of diluted enzyme solution to the experimental group and 100 μL of ultrapure water to the blank control group. React in a water bath at 37 ℃ for 30 min. Set up 3 replicates for each sample.

[0063] Step 5, terminate the reaction: After the reaction is complete, quickly add 600 μL of DNS reagent to terminate the reaction.

[0064] Step 6, color development: Place the reaction tube in a 100 ℃ metal bath or boiling water bath for 5 min.

[0065] Step 7, Cooling and Volume Adjustment: After removal, immediately cool to room temperature, add 1200 μL of distilled water to each tube to bring the total volume to 2.0 mL and mix thoroughly.

[0066] Step 8, Measure absorbance: Measure the absorbance of each sample at 540 nm.

[0067] Step 9, Enzyme activity calculation: Establish a standard curve using glucose as the standard, and calculate the amount of reducing sugar generated based on the absorbance value. Cellulase activity is defined as the amount of enzyme required to catalyze the generation of 1 μmol of glucose equivalent reducing sugar per minute as 1 U. The result is expressed as U / mL.

[0068] Method for determining xylanase activity: Step 1, reagent preparation: Prepare 0.1 mol / L acetic acid solution (take 0.6 mL of glacial acetic acid and add ultrapure water to make up to 100 mL), 0.1 mol / L sodium acetate solution (weigh 0.82 g of sodium acetate, dissolve in water and make up to 100 mL), and acetate-sodium acetate buffer (pH 5.5) (weigh 13.95 g of anhydrous sodium acetate, dissolve in water, add 1.70 mL of glacial acetic acid, make up to 2000 mL, and adjust the pH to 5.5); weigh 1.0 g of xylan and add it to 80 mL of the above buffer solution, heat and stir until completely dissolved, and make up to 100 mL after 30 min to obtain 10 g / L xylan substrate solution, which is stored at 0–4 ℃ protected from light for later use; DNS reagent is commercially available.

[0069] Step 2, prepare enzyme solution: Centrifuge the fermentation broth at 8000 r / min and 4 ℃ for 10 min, take the supernatant as crude enzyme solution, and dilute it 5 times with distilled water for later use.

[0070] Step 3, substrate preheating: Add 100 μL of xylan substrate solution to a 1.5 mL centrifuge tube and preheat in a 37 ℃ water bath for 5 min.

[0071] Step 4, Enzymatic reaction: Add 100 μL of diluted enzyme solution to the experimental group and 100 μL of ultrapure water to the blank control group. React in a water bath at 37 ℃ for 30 min. Set up 3 replicates for each sample.

[0072] Step 5, terminate the reaction: After the reaction is complete, quickly add 600 μL of DNS reagent to terminate the reaction.

[0073] Step 6, color development: Place the reaction tube in a 100 ℃ metal bath or boiling water bath for 5 min.

[0074] Step 7, Cooling and Volume Adjustment: After removal, immediately cool to room temperature, add 1200 μL of distilled water to each tube to bring the total volume to 2.0 mL and mix thoroughly.

[0075] Step 8, Measure absorbance: Measure the absorbance of each sample at 540 nm.

[0076] Step 9, Enzyme Activity Calculation: A standard curve was established using xylose as the standard. The amount of reducing sugar generated was calculated based on the absorbance value. Xylanase activity was defined as the amount of enzyme required to catalyze the generation of 1 μmol of xylose equivalent reducing sugar per minute, which is 1 U. The results are expressed as U / mL. The test results are shown in Table 3.

[0077] Table 3: Enzyme activities of candidate mutant strains against cellulose and xylan

[0078] Shake-flask fermentation results showed that the cellulase and xylanase activities of all candidate mutant strains were higher than those of the original strain. Specifically, the cellulase activity of the original strain BL was 5.37 U / mL, while the highest cellulase activity was observed in the mutant strain bl-7 (34.43 U / mL), and the highest xylanase activity was observed in bl-45 (33.96 U / mL). The cellulase activity of the mutant strain bl-101 ​​reached 34.33 U / mL, a 5.39-fold increase compared to the original strain. Simultaneously, the xylanase activity of bl-101 ​​reached 31.87 U / mL, a 4.14-fold increase compared to the original strain, both at relatively high levels.

[0079] Overall, the clear zone screening results can reflect the cellulose degradation potential of candidate mutant strains. However, the size of the clear zone is affected by factors such as cell growth, enzyme diffusion, and staining conditions, and its relationship with liquid fermentation enzyme activity is not entirely linear. Therefore, this invention further combines liquid fermentation enzyme activity and corn straw degradation rate to comprehensively evaluate candidate strains.

[0080] Example 3: Verification method for the ability of mutant strains to degrade NDF and ADF in corn stalks To further verify the application effect of the Bacillus licheniformis mutant strain obtained by screening in this invention on actual lignocellulose substrates, corn stalks were used as substrates to evaluate their degradation ability on neutral detergent fiber (NDF) and acid detergent fiber (ADF).

[0081] Step 1: Pretreatment of straw raw materials Corn stalks were air-dried, pulverized, and passed through a 40-mesh sieve. They were then dried at 60°C to constant weight and set aside for later use. Since corn stalks are a natural lignocellulose raw material, even after pulverization, mixing, and packaging by weight, there may still be slight differences in the initial NDF and ADF contents among different treatment samples. Therefore, the NDF and ADF contents of the samples before fermentation for each treatment were measured, and the corresponding NDF degradation rates and ADF degradation rates were calculated accordingly.

[0082] Step 2: Preparation of fermentation medium Prepare an inorganic salt culture medium according to the following formula: 3.0 g KH₂PO₄, 3.0 g K₂HPO₄, 2.0 g (NH₄)₂SO₄, 0.2 g MgSO₄·7H₂O, 0.5 g NaCl, and 1000 mL distilled water were added to each bottle, using pretreated corn stalks as the sole carbon source. The mixture was sterilized at 121 °C for 20 min and then cooled for later use.

[0083] Step 3: Seed liquid preparation The original strain BL and 10 dominant mutant strains were inoculated into LB liquid medium and cultured with shaking at 37 ℃ and 250 r / min for 10–12 h to prepare seed culture.

[0084] Step 4: Inoculation and Fermentation Culture Seed culture was inoculated into a culture system containing corn straw at a 3% inoculation rate and cultured with shaking at 37 ℃ and 180 r / min for 48–72 h, preferably 48 h. A blank control group, the original strain BL group, and treatment groups of each dominant mutant strain were set up, with 3 replicates for each treatment.

[0085] Step 5: Collection and treatment of fermentation residue After fermentation, the culture system was filtered through filter paper or filter bags, and the residue was collected. The residue was washed three times with distilled water to remove soluble substances and residual cells, and then dried at 60 °C to constant weight. The dry weight of the residue was recorded.

[0086] Step 6: Calculation of NDF and ADF degradation rates The Van Soest fiber analysis method was used to determine the content of neutral detergent fiber (NDF) and acid detergent fiber (ADF) in straw samples before fermentation and residues after fermentation. The degradation rate was calculated based on the absolute mass change of fiber components in the samples before and after fermentation. NDF degradation rate (%) = [Mass of NDF in the sample before fermentation - Mass of NDF in the residue after fermentation] / Mass of NDF in the sample before fermentation × 100% ADF degradation rate (%) = [Mass of ADF in the sample before fermentation - Mass of ADF in the residue after fermentation] / Mass of ADF in the sample before fermentation × 100% The mass of NDF or ADF in the sample before fermentation = initial dry weight of straw × corresponding content (%). The mass of NDF or ADF in the fermentation residue = dry weight of residue × corresponding content (%).

[0087] The calculation results are shown in Table 4.

[0088] Table 4: Ability of candidate strains to degrade corn straw cellulose

[0089] Based on the combined results of clear zone screening, liquid fermentation enzyme activity, and degradation using corn straw as the actual substrate, both bl-61 and bl-101 ​​exhibited strong cellulose and xylan degradation capabilities. Specifically, bl-61 showed a clear zone / colony diameter ratio, cellulase activity of 4.33 U / mL, and xylanase activity of 30.14 U / mL, while bl-101 ​​showed ratios of 4.86 U / mL, 34.33 U / mL, and 31.87 U / mL, respectively. Both were significantly superior to the original strain BL. Further degradation verification using corn straw as the actual substrate showed that bl-101 ​​achieved NDF and ADF degradation rates of 38.52% and 31.61%, respectively, while bl-61 achieved NDF and ADF degradation rates of 37.81% and 31.86%, respectively, both at relatively high levels. Therefore, bl-61 and bl-101 ​​were selected as key candidate mutants for subsequent verification of the nutritional properties of corn straw fermentation.

[0090] Example 4 Effects of the dominant mutant strain bl-101 ​​on protein levels, methionine and cysteine ​​content, and fiber composition of corn straw fermentation products: To further verify the application effect of the Bacillus licheniformis mutant strain bl-101 ​​obtained in this invention on the nutritional improvement of straw fermentation, corn straw was used as the substrate to evaluate its effects on dry matter, crude protein, true protein, total amino acids, methionine, cysteine, NDF, ADF, hemicellulose, lignin, and reducing sugars in the fermentation products: Step 1: Pretreatment of straw raw materials The method for pretreatment of corn stalk raw materials is described in step 1 of Example 3.

[0091] Step 2: Preparation of fermentation medium Prepare a low-nitrogen starter-type inorganic salt medium according to the following formula: KH₂PO₄ 3.0 g, K₂HPO₄ 3.0 g, (NH₄)₂SO₄ 1.0 g, MgSO₄·7H₂O 0.2 g, NaCl 0.5 g, and distilled water 1000 mL. Use pretreated corn straw as the sole primary carbon source. Add 5.000 g of straw and 100 mL of the above medium to each bottle, sterilize at 121 °C for 20 min, and cool for later use. To ensure the start-up growth and early enzyme production capacity of the strain in the straw fermentation system, and to minimize the interference of exogenous nitrogen sources on the determination results of crude protein, true protein, and amino acids, 1.0 g / L ammonium sulfate is used as the starter nitrogen source in the fermentation system.

[0092] Step 3: Seed liquid preparation The seed culture preparation methods for the original strain BL and the screened strains bl-101 ​​and bl-61 are described in step 3 of Example 3.

[0093] Step 4: Inoculation and Fermentation Culture The following groups were set up: a straw blank group, an original strain BL+ straw group, and a screened strain bl-101+ straw group. bl-61, whose cellulase and xylanase activities were comparable to bl-101 ​​during the screening process, was selected as the bl-61+ straw group as another control. Each treatment was performed in triplicate. The original strain BL+ straw group, bl-61+ straw group, and screened strain bl-101+ straw group were cultured according to the inoculation method and culture conditions described in step 4 of Example 3. The straw blank group was added with an equal volume of sterile physiological saline, without inoculation, and placed under the same conditions until the sampling time, serving as the uninoculated control group.

[0094] Step 5: Collection and processing of fermentation samples After cultivation, each treatment system was thoroughly mixed without liquid-solid separation or washing. A portion of the sample was used for reducing sugar determination, while the remaining sample was dried at 60 °C to constant weight. The dry matter content was recorded, and the dried sample was then used for the determination of crude protein, true protein, total amino acids, methionine, cysteine, NDF, ADF, and lignin.

[0095] Step 6: Determination of dry matter, crude protein, and true protein. Each treated sample was dried at 60 ℃ to constant weight, and the dry matter content was determined. Crude protein content was determined using the Kjeldahl method to determine total nitrogen content, and the crude protein content was calculated using N×6.25. True protein content was determined using the trichloroacetic acid precipitation method: the sample was weighed, added to a 12% trichloroacetic acid solution, allowed to stand at 4 ℃ for 30 min, centrifuged, the precipitate was collected, and its nitrogen content was determined; the true protein content was then calculated using N×6.25.

[0096] Step 7: Determination of total amino acids, methionine, and cysteine. Total amino acids were determined by acid hydrolysis-amino acid analysis: approximately 100 mg of dried and pulverized sample was weighed, 6 mol / L hydrochloric acid was added, the mixture was sealed with nitrogen, and hydrolyzed at 110 °C for 24 h. After deacidification under reduced pressure, the hydrolysate was diluted to a final volume and filtered. The total amino acid content was then determined by HPLC.

[0097] Methionine and cysteine ​​were determined using the performic acid oxidation-acid hydrolysis method: approximately 100 mg of sample was weighed, freshly prepared performic acid was added, and the mixture was oxidized at 0–4 °C in the dark for 16 h to oxidize methionine to methionine sulfone and cysteine ​​to sulfoalanine; then hydrobromic acid was added to terminate the oxidation, followed by the addition of 6 mol / L hydrochloric acid, and the mixture was hydrolyzed at 110 °C for 24 h. After deacidification under reduced pressure, volume adjustment, and filtration, the mixture was determined by HPLC.

[0098] Step 8: NDF, ADF, and ADL determination The contents of neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were determined using the filter bag method, referring to the Van Soest method. Hemicellulose content was calculated as the difference between NDF and ADF, and lignin content was expressed as ADL.

[0099] Hemicellulose content = NDF - ADF Lignin content = ADL.

[0100] Step 9: Determination of reducing sugars Referring to Miller's method with modifications, 5.0 g of the treated sample after thorough mixing was weighed, added to 50 mL of purified water, soaked for 12 h, allowed to stand for 10 min, and the supernatant was collected. The reducing sugar content was determined using the 3,5-dinitrosalicylic acid method, and the results are expressed in mg / g. The test results are shown in Table 5 (all indicators are based on dry matter).

[0101] Table 5: Effects of different treatment groups on the nutrient composition of corn straw fermentation products

[0102] Table 5 shows that compared with the control group, the crude protein, true protein, total amino acid, methionine, and cysteine ​​contents of the original strain BL+ straw group, bl-61+ straw group, and bl-101+ straw group were all increased. Among them, the crude protein, true protein, and total amino acid contents of the bl-101+ straw group reached 8.84%, 7.91%, and 73.62 mg / g, respectively, all higher than the other groups; its methionine and cysteine ​​contents reached 5.88 mg / g and 0.73 mg / g, respectively, significantly higher than the 4.06 mg / g and 0.37 mg / g of the bl-61+ straw group. Meanwhile, the reducing sugar content of the bl-101+ straw group reached 75.67 mg / g, significantly higher than the 44.62 mg / g of the bl-61+ straw group and the 38.91 mg / g of the BL+ straw group, the highest among all treatment groups. Regarding fiber composition, the NDF, ADF, hemicellulose, and lignin contents of the bl-101+ straw group decreased to 54.13%, 39.19%, 14.94%, and 3.48%, respectively, all lower than those of the straw blank group and the BL+ straw group. These results indicate that both bl-61 and bl-101 ​​can improve the nutritional composition of corn straw fermentation products, but bl-101 ​​shows greater performance in increasing the contents of reducing sugars, total amino acids, methionine, and cysteine. This may be because bl-101 ​​has strong cellulose degradation and substrate utilization capabilities, which can increase the release level of available carbon sources in straw, promote the accumulation of microbial biomass and microbial protein synthesis, thereby increasing the contents of true protein, total amino acids, and sulfur-containing amino acids in the fermentation products. Considering both fiber degradation ability and the nutritional improvement effect of fermentation products, bl-101 ​​was ultimately selected as the target dominant mutant strain for subsequent identification, preservation, and application verification.

[0103] Example 5 Genetic stability analysis of mutant strains Step 1, strain activation: The dominant mutant strain bl-101 ​​was streaked from the glycerol storage tube onto LB solid medium and incubated at 37 ℃ for 12–16 h to obtain single colonies.

[0104] Step 2, Seed culture preparation: Pick a single colony and inoculate it into LB liquid medium, and culture it at 37 ℃ and 250 r / min for 10-12 h to prepare the seed culture.

[0105] Step 3, continuous subculture: Inoculate the seed culture into fresh LB liquid medium at an inoculum of 1%, and culture with shaking at 37 ℃ and 250 r / min for 10–12 h, which is recorded as F1 generation; then continuously subculture with the same inoculum to F5 generation (F2–F5), and each generation is cultured under the same conditions.

[0106] Step 4, Fermentation culture: Inoculate each generation (F1–F5) culture medium with CMC-Na as carbon source at an inoculation rate of 1% and culture with shaking at 37 ℃ and 250 r / min for 24–72 h.

[0107] Step 5, preparation of crude enzyme solution: After fermentation, centrifuge the culture medium at 8000 r / min and 4 ℃ for 10 min, and take the supernatant as crude enzyme solution for later use.

[0108] Step 6, Enzyme activity assay: The cellulase activity of each generation of strains was determined using the DNS method. The specific assay steps are the same as those in Example 2.

[0109] Step 7, Result Calculation: Using the enzyme activity of the F1 generation strain as a control, calculate the enzyme activity retention rate of each generation strain. The calculation formula is as follows: Enzyme activity retention rate (%) = (enzyme activity of each generation / enzyme activity of F1 generation) × 100%.

[0110] Step 8, Result Determination: If the enzyme activity retention rate of each generation of the strain does not decrease significantly and the fluctuation range is small during continuous subculturing, then the mutant strain is determined to have good genetic stability. The results are shown in Table 6.

[0111] Table 6: Genetic stability analysis of mutant strain bl-101

[0112] The results are shown in Table 6. The enzyme activity retention rate of strain bl-101 ​​in each generation ranged from 90.63% to 101.08%, without significant fluctuations or a downward trend, indicating that this mutant strain has good genetic stability.

[0113] The strain bl-101 ​​was numbered BL-2501 and then identified: (1) Identification of colony morphology: BL-2501 was streaked onto LB agar and incubated at 37 °C for 24 h. Colony morphology was observed: colonies were milky white, rough and opaque, with irregular edges and a slightly raised center, approximately 5–6 mm in diameter, consistent with typical Bacillus colony characteristics (see...). Figure 2Under a microscope, the bacteria appear as rods, arranged singly or in short chains, with a cell size of approximately 0.8–1.0 μm × 2–3 μm. No obvious gelatinous substance was observed between the cells, indicating good growth and no contamination by other bacteria.

[0114] (2) Gram staining: Freshly cultured BL-2501 cells were used to prepare Gram stain slides, which were then stained according to standard procedures: crystal violet staining for 1 min, iodine fixation for 1 min, alcohol destaining for 10–15 s, safranin counterstaining for 30 s, rinsing with water, and air-drying. Microscopic observation showed that BL-2501 cells were purple rod-shaped, arranged singly or in short chains, consistent with the characteristics of Gram-positive bacilli (see...). Figure 3 ).

[0115] (3) Molecular identification Genomic DNA was extracted from BL-2501, and the 16S rRNA gene sequence was amplified and sequenced. Sequencing results were compared with the NCBI database BLAST, showing that this strain is related to... Bacillus licheniformis The reference strain showed high homology. Based on colony morphology and Gram staining results, this strain was identified as *Bacillus licheniformis* (…). Bacillus licheniformis BL-2501.

[0116] The strain BL-2501 was biopreserved and its taxonomic name is Bacillus licheniformis (Bacillus licheniformis). Bacillus licheniformis BL-2501, deposited at the China Center for Type Culture Collection, accession number CCTCC NO: M20251206, deposited on May 28, 2025, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

Claims

1. A strain of Bacillus licheniformis, characterized by: The classification name of the Bacillus licheniformis is Bacillus licheniformis ( Bacillus licheniformis BL-2501, deposited at the China Center for Type Culture Collection, accession number CCTCCNO: M20251206, deposited on May 28, 2025, at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. The application of the Bacillus licheniformis strain BL-2501 as described in claim 1 in the preparation of biomass degradation agents or feed fermentation agents containing lignocellulose.

3. The application as described in claim 2, characterized in that: When Bacillus licheniformis BL-2501 is used as a feed fermentation agent, it is used to increase the content of crude protein, true protein, total amino acids, methionine, cysteine ​​and / or reducing sugar in the fermentation products.

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