Bacillus pumilus and application thereof

Abstract

The invention relates to bacillus pumilus and application thereof. The bacillus pumilus DX-fK is obtained through a chemical mutagenesis method, the bacillus pumilus DX-fK can efficiently degrade straw, the bacillus pumilus DX-fK is used as a substrate to produce feed alternative protein, and therefore the bacillus pumilus DX-fK can be applied industrially and has good industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain of Bacillus pumilus and its applications. Background Technology

[0002] As a major livestock country, China's annual demand for feed protein has exceeded 130 million tons, with soybean meal accounting for more than 60%. However, the harsh reality is that my country's soybean self-sufficiency rate is less than 20%, and imports reached 91.08 million tons in 2022, costing US$61.2 billion. This structural contradiction is becoming increasingly prominent under the dual constraints of scarce arable land resources (only 1.36 mu per capita) and low planting efficiency (soybean yield of only 130 kg per mu).

[0003] my country is also a major agricultural country, producing approximately 800-1 billion tons of straw annually, equivalent to more than 20% of the global total. However, improper handling of straw can lead to serious environmental problems. Open burning of straw releases large amounts of PM2.5 and greenhouse gases, exacerbating smog and climate change; direct burial and returning straw to the field damages soil structure, causes outbreaks of crop diseases and pests, leading to reduced crop yields and increased pesticide use. Therefore, the scientific development and utilization of straw resources can not only protect the rural ecological environment and promote sustainable and coordinated agricultural development, but also alleviate the shortage of rural feed supplies and help farmers reduce costs and increase efficiency.

[0004] Crop straw is rich in lignin, cellulose, and hemicellulose, which intertwine to form a dense structure that hinders enzyme and microbial degradation, resulting in low digestibility for livestock. Furthermore, straw is low in protein and has poor palatability, making it difficult to provide sufficient nutrition for livestock growth. Bacillus pumilus, as a probiotic, has demonstrated numerous benefits in livestock farming. It can secrete various digestive enzymes, improve feed utilization efficiency, promote nutrient absorption, directly improve livestock growth efficiency, enhance immune regulation and disease resistance, optimize intestinal microecological balance, and maintain a healthy gut microbiota. Bacillus pumilus can also provide livestock with high-quality bacterial protein, rich in essential amino acids, and can be used to produce feed-grade protein substitutes.

[0005] Currently, the mainstream process for producing feed alternative proteins from crop straw is based on the technical route of "extreme pretreatment + enzymatic hydrolysis and fermentation". Its core process includes the following key steps: First, the straw is pretreated with a 2-4% sulfuric acid or sodium hydroxide solution at a high temperature of 180-220℃ and a high pressure of 0.8-1.5MPa for 2-4 hours to destroy the lignin-cellulose complex structure; then, cellulase (approximately 20-40 FPU / g substrate) is used for enzymatic hydrolysis at 50℃ and pH 4.8 for 12-24 hours to obtain a degradation solution containing reducing sugars such as glucose and xylose; finally, bacterial strains are inoculated for fermentation, and the microbial protein product is harvested after a culture period of 3-5 days.

[0006] This process system faces significant technical and economic bottlenecks: 1) The pretreatment stage requires special pressure-resistant reactors and consumes a large amount of steam, with equipment depreciation and energy costs accounting for over 45% of the total cost; 2) Wastewater generated from acid and alkali pretreatment requires the construction of a wastewater treatment system, increasing the water treatment cost by approximately 300-500 yuan per ton of product; 3) The entire process takes 5-7 days, with pretreatment and enzymatic hydrolysis accounting for over 60% of the time window. More seriously, the use of strong acids and alkalis leads to severe equipment corrosion (annual maintenance costs reach 15-20% of the equipment value), and the phenolic compounds (such as vanillin and p-hydroxybenzoic acid) produced by lignin degradation have a significant inhibitory effect on subsequent fermentation microorganisms, resulting in a 30-40% decrease in fermentation efficiency.

[0007] Therefore, it is of great significance to select and breed short-lived Bacillus strains that can effectively degrade straw and have high protein content, as well as to develop a simple and low-cost method for producing feed alternative proteins. Summary of the Invention

[0008] To address the shortcomings of the prior art mentioned above, this invention provides a strain of Bacillus pumilus DX-fK1 that efficiently degrades straw and produces high levels of protein. Another object of this invention is to provide the application of the aforementioned Bacillus pumilus fK1 in the production of feed alternative proteins using straw.

[0009] Solution for solving the problem

[0010] On the one hand, the present invention provides a mutant Bacillus pumilus strain, classified as Bacillus pumilus, strain number DX-fK1; it was deposited on April 25, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.34350, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

[0011] Preferably, the 16S rRNA gene sequence of the Bacillus pumilus is shown in SEQ ID NO.1.

[0012] On one hand, the present invention provides a microbial agent comprising the above-mentioned Bacillus pumilus and / or the fermentation broth of the Bacillus pumilus.

[0013] On the one hand, the present invention provides the application of the above-mentioned Bacillus pumilus in the degradation of straw from lignin raw materials.

[0014] Preferably, the lignin raw material is a straw-based raw material.

[0015] Preferably, the straw-based raw materials include one or more of wheat straw, corn straw, rice straw, soybean straw, and cotton straw.

[0016] On the one hand, the present invention provides the application of the above-described Bacillus pumilus in the preparation of feed protein.

[0017] On one hand, the present invention provides a method for preparing feed protein, which involves fermenting lignin raw materials with the aforementioned Bacillus pumilus to obtain feed protein, and optionally, further includes a step of separating the feed protein.

[0018] Preferably, the lignin raw material is a straw-based raw material.

[0019] Preferably, the straw includes one or more of wheat straw, corn straw, rice straw, soybean straw, and cotton straw.

[0020] The effects of the invention

[0021] This invention utilizes the existing Bacillus pumilus strain (strain number: ACCC02145) and employs MNNG and EMS mutagenesis methods to obtain a mutagenic Bacillus pumilus DX-fK1 capable of efficiently degrading straw. Its straw degradation rate can reach over 45%, and it also possesses advantages such as high protein content and a short growth cycle. The Bacillus pumilus DX-fK1 of this application can use crop straw as a substrate, achieving a crude protein content between 14% and 16% after fermentation. This realizes the transformation of agricultural waste into valuable resources, revolutionizing the traditional agricultural protein production model, promoting self-sufficiency in protein raw materials in my country, and serving as an important pathway to achieving my country's circular economy and sustainable agricultural development. Simultaneously, it precisely addresses the two major challenges of low resource utilization rates of agricultural waste and the shortage of traditional agricultural protein in my country, enhancing the overall productivity and competitiveness of Chinese agriculture, and possessing significant strategic importance. Attached Figure Description

[0022] Figure 1 This is a colony morphology diagram of Bacillus pumilus fK1 of the present invention on LB agar plates;

[0023] Figure 2 This is a growth curve diagram of Bacillus subtilis fK1 of the present invention;

[0024] Figure 3 This is a graph showing the protein concentration curve of Bacillus pumilus fK1 in this invention;

[0025] Figure 4 This is a schematic diagram showing the straw degradation efficiency results of Bacillus subtilis fK1 in this invention;

[0026] Figure 5 This is a schematic diagram showing the crude protein content of straw fermented by Bacillus pumilus fK1 according to the present invention. Detailed Implementation

[0027] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0028] Microbial protein offers significant advantages as a feed protein. From a production perspective, its production cycle is short, completed in just a few days; it boasts high space utilization and is unaffected by environmental or climatic constraints, allowing for flexible adjustment of production scale based on demand and achieving rapid and stable supply. Raw material selection is extremely flexible, utilizing various organic wastes; production does not rely on arable land; and with the aid of a circulating water system, it effectively reduces pressure on natural resources. Furthermore, compared to traditional treatment methods, fermenting straw consumes less energy, emits less carbon, and does not pollute soil or water bodies. Nutritionally, microbial protein is rich in protein, ranging from 20% to 40%, with a complete amino acid profile. Its essential amino acid ratio (EAA / TAA) is approximately 47%, and the essential amino acid to non-essential amino acid ratio (EAA / NEAA) is approximately 0.91, resulting in high digestibility and absorption. It is also rich in various vitamins and minerals. In contrast, soybean meal contains methionine as a limiting amino acid and anti-nutritional factors such as trypsin inhibitors, which affect animal digestibility and absorption. Bacillus pumilus in the natural environment can secrete enzymes that degrade cellulose and hemicellulose, but its ability to degrade straw is weak and takes a long time. Based on this, the inventors, through extensive experimental research, discovered a new mutagenic Bacillus pumilus strain. Through various mutagenesis methods, gene mutations in Bacillus pumilus can be effectively induced. Combined with specific screening plates, mutant strains that efficiently degrade straw can be bred. These strains can efficiently degrade straw, and the fermentation produces a high protein content, providing a new and effective technical means for straw degradation with promising industrial application prospects.

[0029] As used herein, the terms “Bacillus pumilus DX-fK1”, “Bacillus pumilus fK1”, and “fK1” are used interchangeably and all refer to the Bacillus pumilus DX-fK1 of this invention, which has the accession number CGMCC NO.34350.

[0030] As used in this article, the terms “wild-type Bacillus pumilus”, “original strain WT that has not undergone mutagenesis screening”, and “WT” are used interchangeably and all refer to Bacillus pumilus with the number ACCC02145.

[0031] On the one hand, the present invention provides a mutant Bacillus pumilus strain, classified as Bacillus pumilus, strain number DX-fK1, which is a Gram-positive spore-forming bacillus, an aerobic bacterium, and is cultured at a temperature of 15-45℃, and grows rapidly on cellulose / lignin selection medium.

[0032] Preservation instructions:

[0033] Collection Number: CGMCC NO.34350;

[0034] Preservation institution code: CGMCC - China General Microbiological Culture Collection Center;

[0035] Deposit date: April 25, 2025;

[0036] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China;

[0037] Classification and naming: Bacillus pumilus;

[0038] Preservation status: Alive.

[0039] In this invention, the Bacillus DX-fK1 is obtained by chemical mutagenesis of the existing Bacillus pumilus (strain number: ACCC02145).

[0040] In one embodiment, the 16S rRNA gene sequence of the Bacillus pumilus is shown in SEQ ID NO.1.

[0041] SEQ ID NO.1:

[0042]

[0043] The *Bacillus pumilus* fK1 provided by this invention can grow rapidly and stably using inexpensive culture media, with a short fermentation cycle and high cellulose degradation efficiency. This bacterium can be used for the biological degradation of straw, improving straw utilization and possessing high economic and ecological value.

[0044] On one hand, the present invention provides a microbial agent comprising the above-mentioned Bacillus pumilus and / or the fermentation broth of the Bacillus pumilus.

[0045] On the one hand, the present invention provides the application of the above-mentioned Bacillus pumilus in the degradation of straw from lignin raw materials.

[0046] In one embodiment, the lignin raw material is a straw-based raw material.

[0047] In one embodiment, the straw-based raw materials include one or more of wheat straw, corn straw, rice straw, soybean straw, and cotton straw.

[0048] On the one hand, the present invention provides the application of the above-described Bacillus pumilus in the preparation of feed protein.

[0049] On one hand, the present invention provides a method for preparing feed protein, which involves fermenting lignin raw materials with the aforementioned Bacillus pumilus to obtain feed protein, and optionally, further includes a step of separating the feed protein.

[0050] In one embodiment, the lignin raw material is a straw-based raw material.

[0051] In one embodiment, the straw includes one or more of wheat straw, corn straw, rice straw, soybean straw, and cotton straw.

[0052] The present invention will be further described below through specific embodiments. Unless otherwise specified, "%" represents a mass percentage. The materials and reagents used in the following embodiments are all commonly used materials or reagents in the art, and can be obtained commercially or synthesized by known methods. Experimental methods in the following embodiments without specified conditions are generally performed according to conventional experimental conditions or the conditions recommended by the manufacturer of the relevant reagent (kit).

[0053] The formulations of the various culture media used in this invention are as follows:

[0054] The LB medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and deionized water to a final volume of 1 L. Autoclave at 121°C for 20 min. For solid medium, an additional 18 g / L of agar needs to be added.

[0055] Straw screening plate culture medium: straw powder 3g / L, potassium dihydrogen phosphate 3g / L, sodium chloride 0.5g / L, dipotassium hydrogen sulfate 7.3g / L, magnesium sulfate heptahydrate 0.5g / L, ammonium sulfate 5g / L, copper chloride dihydrate 0.68g / L, trace element stock solution 5ml / L, agar 15g / L. Sterilize at 121℃ for 20min.

[0056] Straw liquid culture medium: straw powder 3g / L, potassium dihydrogen phosphate 3g / L, sodium chloride 0.5g / L, dipotassium hydrogen sulfate 7.3g / L, magnesium sulfate heptahydrate 0.5g / L, ammonium sulfate 5g / L, copper chloride dihydrate 0.68g / L, trace element stock solution 5ml / L. Sterilize at 121℃ for 20min.

[0057] Preparation method of cellulose screening medium: 3 g / L sodium carboxymethyl cellulose, 2 mL / L sodium nitrate solution (concentration 1 mol / L), 0.5 g / L urea, 0.5 g / L ammonium chloride, 1 g / L disodium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 1 mL / L each of trace elements, and 18 g / L agar powder. In this invention, the cellulose screening medium provided by this invention needs to be autoclaved at 115°C for 30 min. After sterilization, when the medium is cooled to 60°C, 1 / 100 volume of Congo red solution is added, mixed well, and then poured into plates.

[0058] The cellulose fermentation medium is prepared as follows: sodium carboxymethyl cellulose 3 g / L, sodium nitrate solution 2 mL / L (concentration 1 mol / L), urea 0.5 g / L, ammonium chloride 0.5 g / L, disodium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1.2 g / L, magnesium sulfate heptahydrate 0.5 g / L, and trace elements 1 mL / L each. The cellulose fermentation medium provided by this invention requires autoclaving at 115°C for 30 minutes.

[0059] The fermentation medium formula is as follows: each liter of medium contains 50g straw powder, 2g potassium dihydrogen phosphate, 2g dipotassium hydrogen phosphate, 5g sodium chloride, 1g magnesium chloride hexahydrate, 2g ammonium chloride, 0.5g calcium chloride, and 1mL trace elements.

[0060] The trace element formula is as follows: boric acid 210 mg / L, cobalt chloride hexahydrate 100 mg / L, manganese chloride tetrahydrate 350 mg / L, zinc chloride 280 mg / L, cobalt chloride hexahydrate 75 mg / L, sodium molybdate dihydrate 100 mg / L, copper chloride dihydrate 10 mg / L, and ferrous sulfate heptahydrate 15 mg / L. Each of the above trace elements should be prepared separately and stored individually.

[0061] The formula for the straw fermentation broth culture medium is as follows: 3g ammonified straw, 2mL sodium nitrate solution (concentration 1mol / L), 0.5g / L urea, 0.5g / L ammonium chloride, 1g / L disodium hydrogen phosphate, 1.2g / L dipotassium hydrogen phosphate, 0.5g / L magnesium sulfate heptahydrate, and 1mL of each trace element. The volume of liquid culture medium in each shake flask is 60mL.

[0062] The method for producing ammonified straw is as follows: dilute 25% ammonia water to 2-4%, add 1-3 mL of diluted ammonia water to 1 g of crushed straw, mix thoroughly, place in a sealed container to prevent ammonia leakage, and heat in an oven at 60-100℃ for 1-3 days.

[0063] The process for producing shredded straw is as follows: long sections of straw (5-20cm) are shredded using a shredder, producing shredded straw with a length of 3-5mm. The shredded straw can also be puffed using an extruder. It is then dried at 50-85℃ before use.

[0064] The preparation method of the reagent of the present invention is as follows:

[0065] Preparation of phosphate buffer: First, prepare 0.2 mol / L potassium dihydrogen phosphate and 0.2 mol / L disodium hydrogen phosphate, then mix them in a ratio of 39:61. Sterilize at 121℃ for 20 min.

[0066] Preparation of 0.85% physiological saline: Add 0.85g of sodium chloride to a final volume of 100mL with deionized water. Sterilize at 121℃ for 20min.

[0067] Preparation of MNNG stock solution: Accurately weigh 5 mg MNNG into a sterile test tube, add 500 μL DMSO to dissolve, and filter the solution using a 0.22 μm sterile filter to obtain an MNNG stock solution with a concentration of 10 mg / ml.

[0068] Preparation of 5% Na2S2O3 solution: Weigh 1g Na2S2O3·5H2O and dissolve it in 19mL of distilled water, then filter to remove bacteria; or obtain it by diluting 1M Na2S2O3·5H2O 5 times.

[0069] Preparation of Congo Red solution: Weigh 2.53g of Congo Red powder and dissolve it in 100mL of distilled water. After complete dissolution, filter the solution through a 0.22μm filter membrane for sterilization and store at room temperature away from light.

[0070] Preparation of sodium hydroxide solution: Weigh 400g of sodium hydroxide solid, dissolve it in water, and after cooling to room temperature, dilute it with water to 1L.

[0071] Preparation of boric acid absorption solution: Weigh 20g of boric acid, dissolve in water and dilute to 1L. Weigh 0.1g of methyl red, dissolve in ethanol and dilute to 100mL. Weigh 0.5g of bromocresol green, dissolve in ethanol and dilute to 100mL. Add 1mL of methyl red ethanol solution and 1mL of bromocresol green ethanol solution to the boric acid aqueous solution, mix well and store protected from light.

[0072] Example 1: Obtaining Bacillus pumilus DX-fK1 through mutagenesis

[0073] (1) Obtaining the original strain

[0074] Bacillus pumilus, strain number ACCC02145, was purchased from the China Agricultural Microbial Culture Collection Center. The purchased Bacillus pumilus was inoculated into LB liquid medium for resuscitation and cultured, and then streaked onto LB solid medium. Single clones were selected for PCR and sequencing identification. PCR primer information is shown in Table 1, PCR reaction system in Table 2, and PCR procedure in Table 3.

[0075] Table 1

[0076]

[0077] Table 2

[0078] name Volume (μL) PCR enzyme Mix 25 Forward primer 27F (10 μM) 1.5 Reverse primer 1492R (10 μM) 1.5 Template monoclonal colonies 2 Sterilized water 20

[0079] Table 3

[0080]

[0081] PCR products were subjected to agarose gel electrophoresis. Samples showing a single band of the correct size (approximately 1500 bp for 27F / 1492R) were selected for sequencing. Sequence alignment was performed on samples with normal sequencing data, and the sequencing results are shown in SEQ ID NO. 2.

[0082] SEQ ID NO.2:

[0083]

[0084] (2) Obtaining the mutant strain DX-fK1

[0085] Glyceryl bacteria, identified as straw-degrading bacteria through strain identification, were streaked onto straw screening plates and incubated at 37℃ for 24–48 h. The activated strain was then inoculated at 100 μL into straw liquid culture medium and incubated at 37℃ with shaking at 150 rpm.

[0086] After culturing in straw liquid medium for a period of time, the culture can be transferred to LB medium for further cultivation. The bacterial density is estimated by measuring the OD value of the bacterial solution. (1OD≈1×10⁻⁶) x One bacterium, recommended OD 600 =0.2~0.8, X value needs to be tested. The testing method is as follows: measure the optical density of a series of bacterial suspensions with known bacterial counts, and construct an optical density-bacterial count standard curve. Then, use the optical density measured for the sample solution to find the corresponding bacterial count from the standard curve. When constructing the standard curve, the bacterial count can be performed using the plate colony counting method, and blank culture medium can be used uniformly for dilution and zeroing. MNNG mutagenesis is then performed.

[0087] Concentration gradient experiment: Take 500 μL of bacterial suspension and add 20 / 40 / 60 / 80 / 100 / 120 / 140 / 160 / 180 μL of MNNG stock solution respectively. Make up to 1 mL with phosphate buffer, mix thoroughly by pipetting, and treat at 30℃ and 150 rpm for 20 min in the dark. After the reaction, centrifuge at 8000g for 5 min, discard the supernatant, collect the bacterial cells, wash twice with physiological saline, and resuspend in an equal volume (500 μL). Add the treated sample to straw screening plate medium.

[0088] Glyceryl bacteria, identified as straw-degrading bacteria through strain identification, were streaked onto straw screening plates and incubated at 37℃ for 24–48 h. The activated strain was then inoculated at 100 μL into straw liquid culture medium and incubated at 37℃ with shaking at 150 rpm.

[0089] After culturing in straw liquid medium for a period of time, the culture can be transferred to LB medium for further cultivation. The bacterial density is estimated by measuring the OD value of the bacterial solution (1OD≈1×10⁻⁶). x For each bacterium, an OD600 of 0.2–0.8 is preferred (X value needs to be tested). The testing method is as follows: Measure the optical density of a series of bacterial suspensions with known bacterial counts to create an optical density-bacterial count standard curve. Then, use the optical density measured for the sample solution to find the corresponding bacterial count from the standard curve. When creating the standard curve, the bacterial count can be performed using the plate count method, and a blank culture medium can be used uniformly for dilution and zeroing.

[0090] The mutagenized bacterial culture was diluted by a dilution factor of 1×10⁻⁶. -2 1×10 -4 1×10 -6 1×10 -8 The strains were spread onto cellulose selection medium and incubated at 37°C for 24–48 hours. The clear zones on the cellulose selection medium were observed, and strains with larger clear zones were selected and cultured together in LB medium, followed by EMS mutagenesis.

[0091] Concentration gradient experiment: 500 μL of bacterial suspension was placed in a 1.5 mL EP tube, and then resuspended in EMS at concentrations of 0, 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 35 μL, 40 μL, 45 μL, and 50 μL, respectively. The mixture was then incubated at 30 °C with shaking at 150 rpm for 30 min. After the reaction, the mixture was centrifuged at 8000 g for 5 min, the supernatant was discarded, and the bacterial cells were collected. The cells were washed twice with 1 mL of 5% Na₂S₂O₃, and then once with phosphate buffer. After centrifugation at 8000 g for 5 min, the supernatant was discarded, and the treated samples were serially diluted to 10⁻⁶ with phosphate buffer. 2 cuf / mL, 10 1 The bacterial suspension was diluted to cuf / mL (ignoring cell loss during treatment). 100 μL of the diluted bacterial suspension was spread onto straw plates and incubated at 37°C for 48 h before colony counting. The lethality rate was calculated using the sample without EMS as a control.

[0092] The mutagenized bacterial culture was diluted by a dilution factor of 1×10⁻⁶. -2 1×10 -4 1×10 -6 1×10 -8 The bacteria were spread onto cellulose selection medium and incubated at 37°C for 24–48 hours. The clear zones on the cellulose selection medium were observed, and strains with larger clear zones (DX-fK1) were selected and cultured in LB medium, then stored at -80°C. The colony morphology of *Bacillus pumilus* DX-fK1 from this invention on LB plates is shown. Figure 1 As shown.

[0093] The 16S rRNA sequence of Bacillus pumilus DX-fK1 was amplified using the above-mentioned PCR primers, system and program. After sequencing identification, the sequence is shown in SEQ ID NO.1. The DX-fK1 obtained by mutagenesis in this invention is Bacillus pumilus.

[0094] Example 2: Determination of growth curve and cell protein of Bacillus pumilus DX-fK1

[0095] (1) Determining the bacterial growth curve

[0096] The DX-fK1 strain stored at -80℃ was inoculated into LB liquid medium for resuscitation and cultured at 37℃ with shaking for 24 hours at a speed of 100-200 rpm.

[0097] The obtained Bacillus pumilus culture was centrifuged at 5000 rpm for 5 minutes at room temperature. The supernatant was discarded, and the culture was resuspended in sterile water. The culture was then centrifuged again, the supernatant was discarded, and the culture was resuspended in sterile water.

[0098] The OD value of the bacterial solution was measured using a spectrophotometer at a wavelength of 600 nm. The OD value of the bacterial solution was adjusted to 0.8–1.2 using sterile water.

[0099] The obtained *Bacillus pumilus* bacterial suspension was inoculated into cellulose fermentation medium at a volume of 3%–6% of the fermentation medium. Fermentation was carried out at 35–40℃ with a shaking speed of 100–200 rpm for 5 days. Samples were taken every 24 hours to detect bacterial OD values ​​and protein content. Bacterial OD values ​​were measured at 600 nm using a visible light spectrophotometer; the bacterial OD values ​​are shown in Table 4. The growth curve of *Bacillus pumilus* DX-fK1 in cellulose fermentation medium is shown below. Figure 2 As shown.

[0100] Table 4

[0101]

[0102] (2) The Bradford method was used to determine bacterial protein.

[0103] Remove the Bradford staining solution and equilibrate to room temperature. Remove the BSA sample and thaw at room temperature. Prepare BSA protein standards at different concentration gradients: 0 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, and 400 μg / mL.

[0104] Take 250 μL of sample and add an equal volume of 1M NaOH solution, then heat in a metal bath at 100℃ for 15 min. Take 5 μL of sample and add it to 250 μL of G250 solution, mix quickly, transfer the entire solution to a cuvette, add 2 mL of ddH2O, and mix well.

[0105] Take 255 μL of G250 without the sample into a cuvette, add 2 mL of ddH2O, and use it to zero the spectrophotometer. Set the spectrophotometer to 595 nm and measure the absorbance of the protein standard after zeroing. Construct a standard curve equation based on the absorbance and protein concentration. Measure the absorbance of the cellulose fermentation medium sample using the same method, and calculate the protein concentration based on the standard curve equation. The protein concentration results are shown in Table 5. The protein concentration curve of Bacillus pumilus DX-fK1 in the cellulose fermentation medium is shown in Table 5. Figure 3 As shown.

[0106] Table 5

[0107]

[0108]

[0109] Example 3: Fermentation of straw using Bacillus pumilus DX-fK1 to produce feed

[0110] The preserved Bacillus pumilus strain DX-fK1 and the original strain WT, which had not undergone mutagenesis screening, were inoculated into LB broth medium and cultured overnight in a shaker at 37°C and 100–200 rpm.

[0111] The cultures of two strains of *Bacillus pumilus* were centrifuged at 5000 rpm for 5 minutes. The supernatant was discarded, sterile water was added, and the mixture was vortexed. The mixture was then centrifuged again at 5000 rpm for 5 minutes. The supernatant was discarded, and sterile water was added to adjust the bacterial OD value to 0.9–1.1 at 600 nm. The *Bacillus pumilus* suspension was inoculated into the straw fermentation broth at a volume ratio of 3–6%. Fermentation conditions were 37°C, centrifugation speed of 160–200 rpm, and fermentation time of 5 days.

[0112] After fermentation, collect the solid product by centrifugation at 9000 rpm for 5–10 minutes, and discard the supernatant. After drying in an oven at 85℃, it can be used as feed for livestock such as cattle and sheep.

[0113] The results of this fermentation were tested:

[0114] After fermentation, collect the solid product by centrifugation at 9000 rpm for 5-10 minutes. Discard the supernatant, add sterile water, shake to mix, and centrifuge again. Repeat this centrifugation process 5 times to remove salt ions from the culture medium.

[0115] The obtained solid product was placed in an 85°C oven and dried for 1 hour. After drying, it was removed and cooled to room temperature. The mass of the weighing instrument was measured (accurate to 0.1 g). This operation was repeated until the mass difference of consecutive weighings did not exceed 1% of the sample mass.

[0116] After the sample is completely dried, it is weighed using an electronic balance, and the straw degradation rate is calculated. The formula is:

[0117]

[0118] Table 6 shows the straw degradation rates of the unmutated original strain WT (control group) and Bacillus pumilus DX-fK1 (sample) (results from three independent fermentation experiments). The straw degradation efficiency of Bacillus pumilus DX-fK1 is shown in the table below. Figure 4 As shown, using the unmutated original strain WT as a control, the Bacillus pumilus DX-fK1 strain of this invention exhibited a straw degradation rate of over 45% after fermentation, while the unmutated original strain WT had a straw degradation rate of approximately 20%. Compared to the control, the straw degradation rate of strain fK1 was significantly increased.

[0119] Table 6

[0120]

[0121] The crude protein content of the dried samples was determined using the Kjeldahl method.

[0122] Weigh 0.5–1 g of sample into a digestion tube, add 2 tablets of Kjeldahl nitrogen analyzer, and add 12–20 mL of sulfuric acid. Place the Kjeldahl flask on an electric furnace and begin heating at 200°C. After the sample has charred and the foam has disappeared, increase the temperature to 420°C and continue heating for approximately 2 hours. Remove the flask and allow it to cool to room temperature. Once the sample digest has cooled, use an automatic Kjeldahl nitrogen analyzer according to the operating instructions. The procedure is as follows: Add 20 mL of water and 80 mL of sodium hydroxide solution, and automatically distill for 5 minutes. Absorb the distillate condensate with 20–200 mL of boric acid absorbent. Prepare a 40 mM sulfuric acid titration solution and titrate the boric acid absorbent solution until the solution changes from blue-green to gray-red, indicating the endpoint.

[0123] Blank determination: Accurately weigh 0.5g of sucrose and perform a blank determination according to the above experimental method. The volume of sulfuric acid titration solution consumed should not exceed 0.1mL.

[0124] Calculate the crude protein content, expressed as a mass fraction w, and present the value as a percentage (%), using the following formula:

[0125]

[0126] In the formula:

[0127] V2—The volume of sulfuric acid titrant consumed in titrating the sample, in milliliters (mL);

[0128] V1—The volume of sulfuric acid titrant consumed in the titration of the blank, in milliliters (mL);

[0129] c — Concentration of the sulfuric acid titration solution, in moles per liter (mol / L);

[0130] m — the mass of the sample, in grams (g);

[0131] V—Total volume of the sample digest solution, in milliliters (mL);

[0132] V' — The volume of digestion solution used during distillation, in milliliters (mL);

[0133] 14 — Molar mass of nitrogen, in grams per mole (g / mol);

[0134] 6.25 — Average coefficient for converting nitrogen to crude protein.

[0135] Each experimental group requires at least two parallel samples for measurement, and the arithmetic mean of the results is taken as the measurement result, expressed to two decimal places. The crude protein content results of the fermented straw by *Bacillus pumilus* and the unmutated original strain of this invention are shown in Table 7 (results from three independent fermentation experiments) and as shown in... Figure 5 .

[0136] Table 7

[0137]

[0138] The *Bacillus pumilus* fK1 strain of this invention, after fermentation, has a crude protein content between 14% and 16%, while the original strain without mutagenesis has a crude protein content of 6% to 8%. Compared with the control group, the crude protein content of fermented straw by strain fK1 is significantly increased.

[0139] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. A mutagenic Bacillus pumilus strain, characterized in that: It is classified as Bacillus pumilus and the strain number is DX-fK1; it was deposited in the General Microbiology Center of China Culture Collection Administration on April 25, 2025, with the deposit number CGMCC NO.34350, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

2. The Bacillus pumilus according to claim 1, characterized in that The Bacillus pumilus 16S rRNA gene sequence is shown in SEQ ID NO.

1.

3. A bacterial agent, characterized in that Comprising the Bacillus pumilus according to claim 1 or 2 and / or the fermentation broth of the Bacillus pumilus.

4. Use of the Bacillus pumilus according to claim 1 or 2 in degrading straw as a lignin raw material.

5. The application according to claim 4, characterized in that: The lignin raw material is a straw raw material.

6. The application according to claim 5, characterized in that The straw raw materials include one or more of wheat straw, corn straw, rice straw, soybean straw, and cotton straw.

7. Use of the Bacillus pumilus according to claim 1 or 2 in the preparation of feed protein.

8. A method for preparing feed protein, characterized in that: The method of obtaining feed protein by fermenting lignin raw material with the Bacillus pumilus according to claim 1 or 2, optionally further comprises the step of separating the feed protein.

9. The method according to claim 8, characterized in that The lignin raw material is a straw raw material.

10. The method according to claim 9, characterized in that The straw includes one or more of wheat straw, corn straw, rice straw, soybean straw, and cotton straw.

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