Bacillus licheniformis strain KIBZ5, bacterial agent and application thereof

Abstract

The present application belongs to the field of microbial technology, and particularly relates to a kind of bacillus licheniformis KIBZ5 strain, bacterial agent and application thereof.The bacillus licheniformis (Bacillus licheniformis) Bacillus paralicheniformis ) KIBZ5 of the present application is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.38695.The bacillus licheniformis KIBZ5 strain of the present application has the characteristics of non-toxicity and non-hemolyticity, and has biological safety;It also has the functions of improving the blood glucose regulation of type 2 diabetes mice, reducing insulin resistance, reducing the level of inflammation in vivo, improving the composition of intestinal flora and enhancing the function of intestinal barrier.The present application provides a new idea for treating type 2 diabetes.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Bacillus licheniformis strain KIBZ5, its inoculum, and its application. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by insulin resistance or progressive decline in pancreatic β-cell function. Its global prevalence continues to rise, making it a serious public health problem. Currently, commonly used oral hypoglycemic agents for the clinical treatment of type 2 diabetes include metformin, sulfonylureas, and alpha-glucosidase inhibitors (such as acarbose). While these oral hypoglycemic agents have some efficacy, long-term use can lead to a series of risks, including gastrointestinal adverse reactions, liver and kidney toxicity, and hypoglycemia.

[0003] In recent years, the link between gut microbiota and metabolic health has received widespread attention. Existing research indicates that gut microbiota imbalance is closely related to the occurrence and development of type 2 diabetes mellitus (T2DM), and probiotic intervention, as a means of regulating gut microbiota, provides a new approach for the adjunctive treatment of T2DM. Currently, some Lactobacillus species (…) Lactobacillus Strains of *Lactobacillus reuteri*, *Lactobacillus plantarum*, and *Bifidobacterium* have been reported to have hypoglycemic potential. However, other microbial genera, such as *Bacillus* (…), have been reported to have hypoglycemic potential. Bacillus Research on T2DM regulation is relatively limited, and the hypoglycemic effect and gastrointestinal tolerance of existing strains still need to be improved. Summary of the Invention

[0004] To address the lack of existing microbial agents capable of improving type 2 diabetes mellitus (T2DM) and the intestinal environment, this invention provides a Bacillus licheniformis KIBZ5 strain, a microbial agent, and their applications, specifically including the following technical solutions: This invention provides a type of Bacillus licheniformis ( Bacillus paralicheniformis KIBZ5, the Bacillus licheniformis KIBZ5, was deposited on May 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC), at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 38695.

[0005] The present invention also provides a microbial agent comprising Bacillus licheniformis KIBZ5 as described above.

[0006] Preferably, the microbial agent comprises a solid microbial agent or a liquid microbial agent; the effective viable concentration of Bacillus licheniformis KIBZ5 in the solid microbial agent is 1×10⁻⁶. 8 ~1×1011 CFU / g; The effective viable bacterial concentration of Bacillus licheniformis KIBZ5 in the liquid bacterial agent is 1×10⁻⁶. 8 ~1×10 11 CFU / mL.

[0007] Preferably, the bacterial agent comprises Bacillus licheniformis KIBZ5 lyophilized powder.

[0008] This invention also provides a method for preparing a microbial agent, comprising: Fermentation was carried out on Bacillus licheniformis KIBZ5 as described above to obtain fermentation samples; Centrifuge the fermentation sample, collect the precipitate, and obtain wet bacterial cells; The wet mycelium was pre-frozen to obtain pre-frozen mycelium blocks; The pre-frozen bacterial blocks were dried and pulverized to obtain Bacillus licheniformis KIBZ5 freeze-dried powder.

[0009] The present invention also provides a probiotic preparation, the preparation comprising Bacillus licheniformis KIBZ5 as described above, a bacterial agent, or a bacterial agent prepared by a preparation method.

[0010] Preferably, the formulation includes oral lyophilized powder formulation, enteric-coated capsules, or microcapsules.

[0011] This invention also provides the application of Bacillus licheniformis KIBZ5, bacterial agents, bacterial agents prepared by the above-described method, or probiotic preparations in the preparation of any one or more of the following products: 1) To prepare products for the treatment and / or improvement of type 2 diabetes; 2) Prepare products that improve intestinal barrier function; 3) Prepare products that regulate intestinal flora; 4) Prepare products that reduce inflammation in the body.

[0012] Preferably, the preparation of products for treating and / or improving type 2 diabetes includes the preparation of products for improving insulin resistance.

[0013] Preferably, the preparation of products that improve intestinal barrier function includes the preparation of products that upregulate the expression of ZO-1, Claudin-1, and Occludin tight junction proteins; the preparation of products that improve intestinal flora includes the preparation of products enriched with Lactobacillus spp. in the gut ( Lactobacillus ) and / or inhibit desulfovibrioceae in the gut ( Desulfovibrionaceae ) products.

[0014] The beneficial effects of this invention are as follows: This invention discloses a Bacillus licheniformis (Bacillus licheniformis) Bacillus paralicheniformisThe *Bacillus licheniformis* strain KIBZ5 was deposited on May 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 38695. This invention obtained a *Bacillus licheniformis* strain KIBZ5 through screening. This strain exhibits both therapeutic effects on type 2 diabetes and effects on regulating intestinal function. It is also non-toxic, non-hemolytic, and biosafety in nature. Experiments demonstrated that oral administration of the *Bacillus licheniformis* strain KIBZ5 to type 2 diabetic mice significantly improved their blood glucose regulation, reduced insulin resistance, decreased inflammation levels, enhanced intestinal barrier function, improved gut microbiota composition, and increased the proportion of beneficial bacteria. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 The colony morphology of strain KIBZ5 is shown from left to right on NA medium, the back of a blood agar plate, and the front of a blood agar plate. Figure 2 SEM images of KIBZ5 and the survival rate of KIBZ5 in simulated gastric juice at pH 2 or 1% bile salt solution for 2 hours. Figure 3 The results show the biosafety of KIBZ5; where A represents the elements of the biosafety experimental design; B-F represent the effects of KIBZ5 on mouse body weight, TBIL content, ALT content, AST content, and GGT content, respectively; and G represents schematic diagrams of tissue sections from the heart, liver, lungs, spleen, and kidneys of normal control mice and KIBZ5-treated mice. Figure 4 The results show the effects of heat-inactivated KIBZ5 (KBP) and non-inactivated KIBZ5 (LBP) on type 2 diabetic mice; where A represents the experimental design elements; B~E represent the results of measuring body weight, body weight change, blood glucose level, and area under the curve for each group of mice. Figure 5 The results of blood glucose-related indicators in mice of each group are shown below; from left to right, they are fasting blood glucose level, fasting insulin level, insulin resistance (HOMA-IR) level, glycated hemoglobin (HbA1c) level, and glucagon-like peptide-1 (GLP-1) level. Figure 6The results of inflammation and endotoxin index detection in mice of each group are shown; from left to right, the levels of serum tumor necrosis factor (TNF-α), serum endotoxin (LPS), and interleukin-1β (IL-1β) are shown. Figure 7 The results of intestinal barrier function related indicators in mice of each group are shown. Among them, A is the result of the expression level of ZO-1, Claudin-1 and Occludin proteins in mice of each group, and B~D are the results of the difference analysis of the expression levels of ZO-1, Claudin-1 and Occludin proteins in mice of each group, respectively. Figure 8 A 16S rRNA circular dendrogram was used to analyze the differences in gut microbiota between the KBP and LBP groups of mice. Figure 9 LDA plot for species differences in gut microbiota between KBP and LBP mice; Figure 10 The results show the alpha diversity in the gut microbiota of mice in the HFD and LBP groups; from left to right, the results show the ACE index, Chao1 index, and Shannon index. Figure 11 The staining results of liver tissues from mice in the HFD and LBP groups. Biological Preservation Instructions This invention provides a type of Bacillus licheniformis ( Bacillus paralicheniformis KIBZ5 was deposited on May 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCCNo. 38695. Detailed Implementation

[0017] This invention provides a type of Bacillus licheniformis ( Bacillus paralicheniformis KIBZ5, the Bacillus licheniformis KIBZ5, was deposited on May 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC), at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 38695.

[0018] The *Bacillus licheniformis* KIBZ5 strain described in this invention appears grayish-white or off-white on NA medium, with a dry, rough, and dull surface and obvious wrinkles. On blood agar plates, the *Bacillus licheniformis* KIBZ5 strain appears as a circular colony with a darker, opaque central area, and exhibits no hemolysis.

[0019] The Bacillus licheniformis KIBZ5 described in this invention exhibits a typical rod shape under electron microscopy, with blunt or slightly straight ends. The bacterial cell surface is full and intact, without obvious collapse or damage, and is arranged in single, paired, or short chain patterns.

[0020] The Bacillus licheniformis KIBZ5 strain described in this invention exhibits a survival rate of over 75% after treatment in simulated gastric juice (pH=2.0) and high concentration of bile salts (1%) for 2 h, indicating that the Bacillus licheniformis KIBZ5 strain has excellent tolerance to the gastrointestinal environment and meets the conditions for use as a probiotic.

[0021] The present invention also provides a microbial agent comprising Bacillus licheniformis KIBZ5 as described above.

[0022] In one embodiment, the microbial agent includes a solid microbial agent or a liquid microbial agent. In one embodiment, the effective viable concentration of *Bacillus licheniformis* KIBZ5 in the solid microbial agent is 1 × 10⁻⁶. 8 ~1×10 11 CFU / g. As one embodiment, the effective viable concentration of Bacillus licheniformis KIBZ5 in the solid bacterial agent can be 1×10⁻⁶. 8 CFU / g, 1×10 9 CFU / g, 1×10 10 CFU / g and 1×10 11 The CFU / g value is any one or the median of any two values. As one embodiment, the effective viable concentration of Bacillus licheniformis KIBZ5 in the liquid bacterial agent is 1 × 10⁻⁶. 8 ~1×10 11 CFU / mL. As one embodiment, the effective viable concentration of Bacillus licheniformis KIBZ5 in the liquid bacterial agent can be 1×10⁻⁶ CFU / mL. 8 CFU / mL, 1×10 9 CFU / mL, 1×10 10 CFU / mL and 1×10 11The CFU / mL value is any one or the median of any two values. As one embodiment, the solid bacterial agent includes *Bacillus licheniformis* KIBZ5 lyophilized powder. This invention experimentally verified that lyophilized powder containing live cells can positively affect indicators such as blood glucose, insulin resistance, and intestinal environment in type 2 diabetic mice, while inactivated lyophilized powder no longer possesses these functions. Therefore, the bacterial agent of this invention exerts its effect through live *Bacillus licheniformis* KIBZ5. Thus, the dosage form of the bacterial agent of this invention is not limited to lyophilized powder; other preparations containing live *Bacillus licheniformis* KIBZ5 can produce similar effects to lyophilized powder. These other preparations containing live *Bacillus licheniformis* KIBZ5 include, but are not limited to, any one or more of fermentation broth, bacterial suspension, and mycelium.

[0023] This invention also provides a method for preparing a microbial agent, comprising: Fermentation of Bacillus licheniformis KIBZ5 was carried out to obtain fermentation samples; Centrifuge the fermentation sample, collect the precipitate, and obtain wet bacterial cells; The wet mycelium was pre-frozen to obtain pre-frozen mycelium blocks; The pre-frozen bacterial blocks were dried and pulverized to obtain Bacillus licheniformis KIBZ5 freeze-dried powder.

[0024] This invention involves fermenting *Bacillus licheniformis* KIBZ5 to obtain a fermented sample. In one embodiment, the fermentation temperature is 28-42℃. In another embodiment, the fermentation temperature can be any one or the midpoint of any two values ​​from 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, and 42℃. In one embodiment, the fermentation endpoint is defined as a viable bacterial count of 1×10⁻⁶ in the fermentation broth. 10 ~2×10 10 The concentration of CFU / mL and the spore formation rate under microscopic examination are greater than 90%. As one embodiment, the viable cell count in the fermentation broth can be 1×10⁻⁶. 10 CFU / mL and 2×10 10 CFU / mL can be any one or the midpoint of any two point values. As one embodiment, the spore formation rate of the fermentation broth can be any one or the midpoint of any two point values ​​from 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0025] After obtaining the fermentation broth sample, the sample is centrifuged, and the precipitate is collected to obtain wet bacterial cells. As one embodiment, the centrifugation includes refrigerated centrifugation. As one embodiment, the centrifugation speed is 4000-5000 r / min. As one embodiment, the centrifugation speed can be any one or an intermediate value of any two of 4000 r / min, 4500 r / min, and 5000 r / min. As one embodiment, the centrifugation time is 10-15 min. As one embodiment, the centrifugation time can be any one or an intermediate value of any two of 10 min, 11 min, 12 min, 13 min, 14 min, and 15 min.

[0026] After obtaining the wet bacterial cells, the wet bacterial cells are pre-frozen to obtain pre-frozen bacterial blocks. As one embodiment, the pre-freezing treatment includes: adding a freeze-drying protectant to the wet bacterial cells, mixing, and preparing a bacterial suspension; adding the bacterial suspension to a freeze-drying pan, immersing it in liquid nitrogen, and waiting for the bacterial suspension to completely freeze to obtain pre-frozen bacterial blocks. As one embodiment, the mass ratio of the freeze-drying protectant to the wet bacterial cells is 1~2:1. As another embodiment, the mass ratio of the freeze-drying protectant to the wet bacterial cells can be any one of 1:1 and 2:1, or any intermediate value between two points. The liquid nitrogen pre-cooling operation of this invention allows the bacterial suspension to form tiny glassy ice crystals, preventing large ice crystals from piercing the cell wall.

[0027] After obtaining the pre-frozen mycelial blocks, the pre-frozen mycelial blocks are dried and pulverized to obtain lyophilized Bacillus licheniformis KIBZ5 powder. As one embodiment, the drying includes sublimation drying. As one embodiment, the sublimation drying includes sublimation drying using a lyophilizer. As one embodiment, the cold trap temperature for sublimation drying is -50℃ to -58℃. As one embodiment, the cold trap temperature for sublimation drying can be any one or any two values ​​between -50℃, -51℃, -52℃, -53℃, -54℃, -55℃, -56℃, -57℃, and -58℃. As one embodiment, the vacuum degree for sublimation drying is 10~100 mT. In one embodiment, the vacuum degree of the sublimation drying can be any one or an intermediate value of any two of the following: 10 mT, 20 mT, 30 mT, 40 mT, 50 mT, 60 mT, 70 mT, 80 mT, 90 mT, and 100 mT. In one embodiment, the sublimation drying time is 36–40 h. In another embodiment, the sublimation drying time can be any one or an intermediate value of any two of the following: 36 h, 37 h, 38 h, 39 h, and 40 h. In one embodiment, the particle size after pulverization is 50–70 mesh. In another embodiment, the particle size after pulverization can be any one or an intermediate value of any two of the following: 50 mesh, 60 mesh, and 70 mesh.

[0028] The present invention also provides a probiotic preparation, the preparation comprising Bacillus licheniformis KIBZ5 as described above, a bacterial agent, or a bacterial agent prepared by a preparation method.

[0029] As one implementation method, the probiotic preparation includes, but is not limited to, oral lyophilized powder preparations, enteric-coated capsules, or microcapsules. The dosage form of the probiotic preparation of the present invention can protect the probiotic components, achieve targeted intestinal delivery, and improve the stability and bioavailability of the probiotics.

[0030] The present invention also provides the application of Bacillus licheniformis KIBZ5, bacterial agents, bacterial agents prepared by the above-described preparation method, or probiotic preparations in the preparation of any one or more of the following products: 1) preparing products for treating and / or improving type 2 diabetes; 2) preparing products for improving intestinal barrier function; 3) preparing products for regulating intestinal flora; 4) preparing products for reducing inflammation in the body.

[0031] As one embodiment, the preparation of products for treating and / or improving type 2 diabetes includes the preparation of products that improve insulin resistance. As one embodiment, the preparation of products that improve intestinal barrier function includes the preparation of products that upregulate the expression of ZO-1, Claudin-1, and Occludin tight junction proteins. As one embodiment, the preparation of products that improve gut microbiota includes the preparation of products enriched with Lactobacillus spp. in the gut (…). Lactobacillus ) and / or inhibit desulfovibrioceae in the gut ( Desulfovibrionaceae ) products.

[0032] As one embodiment, the preparation of products that improve insulin resistance includes products that improve HOMA-IR levels, fasting insulin levels, blood glucose levels, glycated hemoglobin levels, and glucagon-like peptide levels. As another embodiment, the preparation of products that reduce systemic inflammation includes products that reduce the expression levels of one or more of the proteins TNF-α, LPS, and IL-1β.

[0033] In one embodiment, the product includes health supplements and / or pharmaceuticals. In another embodiment, the health supplement includes health supplements that help regulate gut microbiota and / or health supplements that help maintain healthy blood sugar levels.

[0034] In one embodiment, the product includes live Bacillus licheniformis KIBZ5. In another embodiment, the effective concentration of the live bacteria is as described above and will not be repeated here. Through examples, this invention has found that the lyophilized form of Bacillus licheniformis KIBZ5 has a significant therapeutic effect on type 2 diabetic mouse models, while the heat-inactivated lyophilized form of KIBZ5 has no significant effect, indicating that its hypoglycemic effect depends on the metabolic activity of the live bacteria.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a Bacillus licheniformis KIBZ5 strain, its inoculum, and its applications, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0036] In this invention, the NA solid culture medium is prepared as follows: 10.0 g of peptone, 3.0 g of beef extract powder, 5.0 g of NaCl and 15.0 g of agar are added to each liter of distilled water.

[0037] The NB medium is prepared as follows: add 10 g of peptone, 3 g of beef extract powder and 5 g of NaCl to each liter of distilled water.

[0038] The freeze-drying protectant, by mass percentage, consists of: 10% skim milk powder, 10% glucomannan, 5.1% monosodium glutamate, and the remainder being sterile water.

[0039] All experimental data obtained in this invention were statistically analyzed using GraphPad Prism 8.0 software. One-way ANOVA was used for comparisons among multiple groups, and Tukey's test was used for post-hoc multiple comparisons. Independent samples t-tests were used for comparisons between two groups. Data are presented as mean ± standard deviation (...). )express. The difference is considered statistically significant.

[0040] Unless otherwise specified, the methods used in this invention are all conventional operations in the field, and the products used in this invention are all conventional commercially available products.

[0041] Example 1: Isolation and Screening of Strain KIBZ5 1. Isolation and purification of strain KIBZ5: Fresh Dendrobium officinale stems were obtained from the Dendrobium Industry Development Center (Dendrobium Research Institute) in Longling County, Baoshan City. The fresh Dendrobium officinale stems were soaked in 75% ethanol for 2 minutes, and then disinfected by soaking in 3% sodium hypochlorite for 10 minutes. The disinfected Dendrobium officinale stems were then aseptically washed three times to obtain Dendrobium officinale stem samples.

[0042] The Dendrobium officinale stem sample was placed in a mortar, and sterile water was added to the mortar for grinding to obtain Dendrobium officinale ground material. The Dendrobium officinale ground material was transferred to a beaker, and sterile water was added to make up to 20 mL. The mixture was stirred with a magnetic stirrer for 1 h to obtain Dendrobium officinale grinding liquid. The mixture was allowed to stand until it separated into layers, and the supernatant was collected for later use.

[0043] Weigh 100 µL of supernatant and mix according to 10 -1 10 -2 10 -3 10 -4 10 -5 The strain was serially diluted to different concentrations, with 100 µL of each diluted sample spread onto the surface of NA solid medium. The medium containing different concentrations of supernatant was then incubated at 29°C for 7 days. After 7 days, based on the morphological characteristics of the strain in NA medium, the strain was purified using the streak plate method. After three generations of purification, a purified strain was obtained and named KIBZ5.

[0044] 2. Appearance of the strain Single colonies of KIBZ5 were selected and streaked onto NA medium and blood agar plates, respectively, and incubated at 37°C for 24 hours. A schematic diagram of the strain's appearance on NA solid medium is shown below. Figure 1 As shown on the left, the colony morphology on the back and front of the blood agar plate is as follows. Figure 1 As shown in the middle and right figures.

[0045] Depend on Figure 1 As can be seen, KIBZ5 strain colonies on NA medium are 1-5 mm in diameter, grayish-white or off-white in color, with a dry, rough, dull surface and obvious wrinkles. Observing the reverse side of KIBZ5 strain on blood agar plates (through the bottom of the petri dish) is mainly to eliminate interference from light reflection from the front side and to more clearly observe hemolysis. From the reverse side, the colonies are usually round, with a darker and opaque central area, indicating that the bacterium does not produce hemolysis.

[0046] Depend on Figure 2 As seen in the scanning electron microscope image on the left, the KIBZ5 strain exhibits a typical rod-like shape with blunt or slightly straight ends. The cell surface is plump and intact, without obvious collapse or damage, indicating that the cell structure remained intact during treatment. In the field of view shown in the image, the KIBZ5 strain is arranged singly, in pairs, or in short chains.

[0047] 3. Bile salt tolerance test and gastric juice tolerance test Preparation of test bacterial suspension: After activating and culturing the test strain KIBZ5, take an appropriate amount of culture medium, centrifuge at 3000×g for 10 min, collect the bacterial cells, wash the bacterial cells twice with sterile phosphate buffer (PBS, pH 7.2), resuspend in PBS, and adjust the bacterial suspension concentration to approximately 10. 8 ~10 9 CFU / mL, for later use.

[0048] Artificial gastric fluid: Based on sterile PBS, the pH was adjusted to 2.0 with hydrochloric acid, and pepsin was added until the final concentration of pepsin was 3.0 g / L. The mixture was then filtered through a 0.22 μm filter membrane for sterilization to obtain artificial gastric fluid.

[0049] Tolerance test: 1.0 mL of the test bacterial culture was added to 9.0 mL of artificial gastric fluid and incubated at 37°C with shaking (e.g., 140 r / min). Samples were taken at different time points (e.g., 0, 2, 4 h), and the viable bacterial count was determined using the plate count method. The survival rate was calculated, and the results are as follows: Figure 2 As shown on the right.

[0050] Bile salt tolerance test: The bacterial culture was inoculated at a volume percentage of 1% into a liquid culture medium containing 1% by mass of bile salts, and incubated at 37°C. Viable bacterial counts were measured periodically, and the survival rate of the strain was calculated. Results are shown below. Figure 2 As shown on the right.

[0051] Results evaluation: The tolerance of the strain was evaluated by the degree of decrease in survival rate. Generally, a high survival rate (e.g., >50% or a decrease in viable bacteria count of no more than one order of magnitude) after 3 hours of treatment with artificial gastric fluid was considered a standard of good tolerance.

[0052] Depend on Figure 2 As shown in the results on the right, strain KIBZ5 maintained extremely high survival rates after 2 h of treatment in simulated gastric juice (pH=2.0) and high-concentration bile salts (1%). Its survival rate in gastric juice was 77.2%, and in bile salts it was 81.3%, both exceeding 75%. This indicates that this strain possesses excellent tolerance to the gastrointestinal environment and is suitable as a potential probiotic candidate strain.

[0053] Example 2: Classification of strain KIBZ5 The purified strain KIBZ5 obtained in Example 1 was activated on NA plate medium and sent to Pasenno for 16S rDNA gene sequencing and identification.

[0054] After extracting the genomic DNA of strain KIBZ5 using a bacterial genomic DNA extraction kit (Solarbio D1600), the 16S rRNA of strain KIBZ5 was amplified using the PCR amplification system shown below.

[0055] The PCR amplification primers are as follows: Upstream primer 27F (SEQ ID NO:1): 5'-AGAGTTTGATCCTGGCTCAG-3'; Downstream primer 1492R (SEQ ID NO:2): 5'-CTACGGCTACCTTGTTACGA-3'; The PCR amplification system is as follows: 1 μL genomic DNA, 5 μL 10×Buffer, 1 μL Taq polymerase, 1 μL dNTP, 1.5 μL each of upstream and downstream primers, and 39 μL ddH2O.

[0056] The PCR reaction parameters are as follows: Pre-denaturation at 95℃ for 5 min, denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 1 min 30 s, and final extension at 72℃ for 7 min, for a total of 35 cycles.

[0057] The reaction products obtained by PCR amplification were detected by 1% agarose gel electrophoresis, and the expected 16S rRNA band was successfully amplified.

[0058] The 16S rRNA of the endophytic bacterium KIBZ5 was sequenced using an ABI3730-XL sequencer. The assembled sequence file was compared with data in the 16S database of the NCBI website using the NCBI Blast program to obtain the species information with the highest sequence similarity to the target species. The result is the preliminary identification of the 16S rRNA sequencing assembly result of the strain KIBZ5, as shown in SEQ ID NO:3 below.

[0059] SEQ ID NO:3:

[0060] 16S rRNA sequencing results showed that strain KIBZ5 was similar to Bacillus licheniformis. Bacillus paralicheniformis With a similarity of 99.55%, strain KIBZ5 was identified as a Bacillus licheniformis. (Source: NCBI database) Bacillus paralicheniformis The accession number for the 16S rRNA is CP133705.1.

[0061] Example 3: Preparation of freeze-dried powder of Bacillus licheniformis strain KIBZ5 Take Bacillus licheniformis ( Bacillus paralicheniformis KIBZ5 single colonies were amplified and cultured in NB liquid medium to OD. 600 A seed culture was obtained when the concentration of viable cells reached 0.6 or higher. The seed culture was then inoculated into a fermenter at an inoculum volume of 3%–10% for liquid fermentation. The fermentation was continued until the viable cell count in the fermentation broth reached 1 × 10⁶. 10 ~2×10 10 Fermentation was stopped when the concentration of CFU / mL was reached and the spore formation rate under microscopic examination was greater than 90%, and the fermentation broth sample was obtained.

[0062] The fermentation broth sample was centrifuged in a refrigerated centrifuge. The centrifugation parameters were: rotation speed 4500 r / min, centrifugation time 15 min. After centrifugation, the precipitate was collected to obtain wet bacterial cells.

[0063] A freeze-drying protectant was added to the obtained wet bacterial cells at a mass ratio of 1:1. After adding the freeze-drying protectant, the mixture was stirred thoroughly to obtain a bacterial suspension. The freeze-drying tray was immersed in liquid nitrogen until the bacterial suspension was completely frozen and vitrified, resulting in pre-frozen bacterial blocks. The freeze-drying tray was then removed and quickly transferred to a freeze dryer. The liquid nitrogen pre-cooling process allows the bacterial suspension to form tiny glassy ice crystals, preventing large ice crystals from piercing the cell walls.

[0064] Turn on the freeze dryer to sublimate the pre-frozen mycelium blocks, obtaining dried mycelium cakes. Control the shelf temperature and vacuum level of the freeze dryer to sublimate the moisture in the material. Specific parameters are set as follows: cold trap temperature -50℃ to -58℃, vacuum level 10 to 100 mT, and drying time 36 to 40 hours.

[0065] After drying, the dried mycelium cake is pulverized under low temperature and passed through a 60-mesh sieve to obtain Bacillus subtilis. Bacillus paralicheniformis KIBZ5 freeze-dried powder finished product.

[0066] Example 4: Intervention experiment of Bacillus licheniformis KIBZ5 lyophilized powder on type 2 diabetic mice. 1. Laboratory animals and their housing conditions Animal strain: SPF-grade male C57BL / 6J mice, 6 weeks old, weighing 18~22 g.

[0067] Purchase source: Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0068] Rearing environment: Temperature 22±2℃, humidity 50%±10%, 12h light / 12h dark cycle.

[0069] Feed and water: Normal maintenance feed or high-fat feed, free access to water.

[0070] 2. Establishment of a type 2 diabetic mouse model Adaptive feeding: After one week of adaptive feeding, SPF-grade male C57BL / 6J mice were randomly divided into a normal treatment group and a model group. There were 16 mice in the normal treatment group and 24 mice in the model group.

[0071] Model group: Mice were induced to follow a high-fat diet by being fed a high-fat diet (60 kcal% fat, product number: D12492, purchased from Research Diets Inc.) for 6 consecutive weeks. After 6 weeks of high-fat feeding, mice were fasted but allowed free water for 12 hours, and were intraperitoneally injected with streptozotocin for 3 consecutive days. Blood samples were collected from the tail vein of the mice 72 hours after the last injection and on day 7. Fasting blood glucose levels were measured using a blood glucose meter. A fasting blood glucose level ≥11.1 mmol / L on both occasions was considered the standard for successful establishment of the type 2 diabetes model. The type 2 diabetes model was successfully established in this example.

[0072] The streptozotocin was dissolved in 0.1 mol / L citrate buffer to prepare a pH 4.2 STZ solution, which was then injected. Each mouse was injected with streptozotocin at a dose of 35 mg / kg / day.

[0073] Mice in the normal treatment group were fed a standard maintenance diet.

[0074] 3. Experimental grouping and intervention plan Grouping: Mice in the model group were randomly divided into 3 groups (n=8 per group) based on blood glucose and body weight. The normal treatment group was divided into 2 groups (n=8 per group). The treatment groups are shown below: 1. KIBZ5 Live Bacteria Group (LBP): Mice in the model group were fed a high-fat diet daily, and were also treated by gavage with streptozotocin solution and KIBZ5 lyophilized powder solution. Streptozotocin (STZ) solution: The dosage of STZ solution is 35 mg / kg / day, based on the mass of streptozotocin. KIBZ5 lyophilized powder solution: The dosage of Bacillus licheniformis KIBZ5 solution is 2 × 10⁻⁶, based on the effective viable count of Bacillus licheniformis KIBZ5. 8CFU / unit / day. The KIBZ5 lyophilized powder solution is prepared by dissolving KIBZ5 lyophilized powder in sterile physiological saline.

[0075] 2. KIBZ5 Heat-Inactivated Group (KBP): Mice in the model group were fed a high-fat diet daily and were also administered streptozotocin solution and heat-inactivated KIBZ5 lyophilized powder solution by gavage. Streptozotocin solution: The amount of STZ solution was 35 mg / kg / day, based on the mass of streptozotocin. Heat-inactivated KIBZ5 lyophilized powder solution: The bacterial count was equal to that in the KIBZ5 lyophilized powder solution of the LBP group. The preparation method of the heat-inactivated KIBZ5 lyophilized powder solution was as follows: the KIBZ5 live bacterial suspension was heated in an 80℃ water bath for 30 min, and used after verifying the absence of live bacterial growth on NA plates.

[0076] The preparation method of the KIBZ5 live bacterial suspension is as follows: KIBZ bacteria are activated and expanded under aseptic conditions to obtain fresh culture. The bacterial growth on the culture medium is then washed off with sterile physiological saline, and the cells are collected by centrifugation. The collected cells are then fully resuspended in phosphate buffer, and the suspension is ensured to be homogeneous through vortexing. The suspension is then heat-treated at approximately 80°C to kill any residual vegetative cells. Finally, the concentration is roughly adjusted using turbidimetry as needed, and viable cell counts are performed using the pour plate method to determine the accurate viable cell concentration (CFU / mL), thus obtaining the KIBZ5 live bacterial suspension. Strict aseptic operation must be maintained throughout the entire preparation process to ensure the purity and accuracy of the suspension.

[0077] 3. Model Group (HFD): Mice in the model group were fed a high-fat diet daily and were treated with streptozotocin solution and distilled water by gavage. Streptozotocin solution: The amount of STZ solution was 35 mg / kg / day based on the mass of streptozotocin. Distilled water: The volume was the same as the lyophilized powder solution in the LBP group.

[0078] Mice in the LBP, KBP, and HFD groups were treated using the above method, and were administered the drug by gavage for 4 consecutive weeks, once daily.

[0079] 4. Normal control group (NC): Mice that had been treated normally were fed a normal diet and received a gavage treatment: distilled water: the same volume as the LBP group, gavage for 14 consecutive days.

[0080] 5. KIBZ5 lyophilized powder group (KIBZ5): Mice that had undergone normal treatment were fed a regular diet and then received a gavage treatment: a KIBZ5 lyophilized powder solution, administered at a dose of 1×10⁻⁶ based on the effective viable count of KIBZ5 bacteria. 10 The dose was CFU / kg / day, approximately 100 times the human dose, administered via gavage for 14 consecutive days.

[0081] 4. Biosafety testing experiments This experiment used mice from the NC group and the KIBZ5 group to conduct a biosafety test. The experimental procedure is as follows: During the gavage period, the daily body weight, behavioral status, and mortality of mice in the NC and KIBZ5 groups were observed. Fourteen days after gavage, blood samples were collected from mice to detect liver function indicators; the detection methods are shown in Table 1. After euthanasia, the mice were dissected to observe the gross morphology of the heart, liver, spleen, lungs, and kidneys. H&E-stained sections of each tissue were subjected to pathological evaluation. The results are summarized in [Table 1]. Figure 3 middle.

[0082] Depend on Figure 3 As shown in Figure B, there was no significant difference in weight gain curves between the KIBZ5 group and the NC group (i.e., the Control group shown in the figure). Figure 3 As observed in C-F, the activities of key enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum were not significantly increased compared with the control group. Figure 3 As observed in the G-test, the size, color, and texture of all organs in both the KIBZ5 and NC groups were normal, with no congestion, necrosis, or tumors. Cells were arranged in an orderly manner, without pathological changes such as inflammatory cell infiltration, cell edema, vacuolar degeneration, or fibrosis. In summary, within 14 days, 10 10 At a gavage dose of CFU / kg / d, the KIBZ5 strain can be considered practically non-toxic.

[0083] 5. KIBZ5 intervention effectiveness testing experiment This experiment used mice from the LBP, KBP, and HFD groups as samples for subsequent testing.

[0084] Sample collection: Three days before the end of the intervention, mice in each group were tested for oral glucose tolerance.

[0085] After the intervention in each group of mice, they were fasted for 12 h, and blood was collected from the orbital venous plexus. Glycated hemoglobin was measured using a portion of whole blood. Another portion of the blood was allowed to stand at room temperature for 30 min, then centrifuged at 4℃ and 3000 rpm for 15 min to separate the serum. The serum was aliquoted and stored at -80℃ for the detection of fasting insulin levels, glucagon-like peptide-1 levels, lipopolysaccharide content, interleukin-1β content, and tumor necrosis factor-α levels in mice. The detection methods are shown in Table 1. The effects of each treatment on blood glucose levels in type 2 diabetic mice are shown in Table 2. Figure 4 The results of each treatment on the detection of blood glucose-related indicators in mice are shown in the figure. Figure 5 The results of each treatment on the detection of inflammation and endotoxin markers in mice are shown in the figure. Figure 6 .

[0086] Depend on Figure 4As shown in Figures B and C, the HFD group mice experienced a continuous decrease in body weight due to metabolic disturbances and energy expenditure caused by hyperglycemia. In contrast, the LBP treatment group showed a slower rate of body weight loss compared to the HFD group, suggesting that LBP improved the metabolic state. Figure 4 As shown in Figures D and E, the peak blood glucose level in the oral glucose tolerance test (OGTT) was significantly lower than that in the model group, and the rate of decline was significantly faster. The AUC value was significantly reduced. This is direct evidence that LBP improves diabetes. This example... Figure 4 In terms of D and E, "blood glucose level" refers to a series of blood glucose values ​​directly measured at various time points during the oral glucose tolerance test (OGTT) on an empty stomach and after glucose administration. "Area under the blood glucose curve" is a derived comprehensive indicator calculated by integrating these OGTT time point blood glucose values ​​using the trapezoidal method. It reflects the overall blood glucose exposure level and fluctuation trend throughout the entire test period. The two are not additional tests independent of OGTT. The former is the basic data of OGTT, and the latter is its deeply integrated result. Using them together can utilize the gold standard of OGTT 2-hour blood glucose for diagnosis, and can also quantify the degree of glucose intolerance more precisely through the area under the curve, thereby comprehensively assessing the overall impact of intervention measures on the body's glucose homeostasis.

[0087] Depend on Figure 5 It is evident that, compared to the HFD group, LBP significantly reduced fasting blood glucose, glycated hemoglobin (HbA1c), and insulin resistance index (HOMA-IR) in T2DM mice, while increasing GLP-1 and fasting insulin secretion. Furthermore, the results from the LBP group showed significant differences compared to the HFD group. P <0.05). This indicates that gavage treatment with the KIBZ5 strain can improve glycemic homeostasis and reduce insulin resistance in mice.

[0088] Depend on Figure 6 As can be seen, compared with the HFD group, the LBP group was able to reduce serum LPS, IL-1β, and TNF-α levels. This indicates that gavage treatment with the KIBZ5 strain can alleviate inflammation and endotoxin levels in mice.

[0089] After euthanizing the mice, they were dissected, and the pancreas, colon, liver, and other tissues were separated. Pancreatic and colonic tissues (each 0.5 cm × 0.5 cm) were fixed in 4% paraformaldehyde for pathological sections and immunofluorescence staining. Paraffin sections of colonic tissue were used to detect the expression levels of ZP-1, Claudin-1, and Occludin proteins. Cecal contents (0.5 cm × 0.5 cm) were flash-frozen in liquid nitrogen and stored at -80°C for intestinal flora analysis. The detection methods are shown in Table 1. The results of intestinal barrier function testing in each group of mice are as follows: Figure 7 As shown.

[0090] Depend on Figure 7As can be seen, compared with the HFD group, the LBP group increased the expression levels of tight junction proteins such as ZO-1, Claudin-1, and Occludin, with significant differences in expression levels. The KBP group, however, did not produce the above effects. Therefore, KIBZ5 gavage treatment significantly restored the damaged intestinal barrier function in type 2 diabetic mice.

[0091] Table 1. Detection Indicators and Experimental Methods

[0092] Gut microbiota analysis: DNA extraction: Total DNA was extracted from the cecal contents of mice in the KBP, LBP and HFD groups using a fecal genomic DNA extraction kit (purchased from QIAGEN QIAampFast DNA Stool Mini Kit).

[0093] PCR amplification: Using extracted DNA as a template, the V3-V4 hypervariable region of the 16S rRNA gene was amplified. The primers used for this amplification were: Forward primer 338F (SEQ ID NO:4): 5'-ACTCCTACGGGAGGCAGCA-3'; Reverse primer 806R (SEQ ID NO:5): 5'-GGACTACHVGGGTWTCTAAT-3'.

[0094] Sequencing: The paired-end 250 bp sequences of the V3-V4 hypervariable regions of the amplified KBP and LBP 16S rRNA genes were sequenced using the Illumina MiSeq / NovaSeq high-throughput sequencing platform. Intergroup comparisons were performed using one-way ANOVA combined with Tukey's multiple test. P <0.05 was considered significant. LDA effect analysis (LEfSe) was performed on the gut microbiota of mice, with an LDA score threshold set at >2.0. The results of the gut microbiota difference analysis are as follows: Figure 8 As shown, the LDA score results of the differential analysis of gut microbiota are as follows: Figure 9 As shown.

[0095] Depend on Figure 7 It is evident that, compared with the KBP group, the LBP group significantly altered the gut microbiota structure of type 2 diabetic mice, with a decrease in the relative abundance of harmful bacteria and an increase in the relative abundance of probiotics / beneficial bacteria; the KBP group had a weak effect on the gut microbiota structure, with no significant changes.

[0096] Depend on Figure 8 It is evident that, compared to the KBP group, the LBP group was able to more effectively regulate the gut microbiota and significantly enrich Lactobacillus spp. ( LactobacillusBeneficial bacteria such as ) and inhibit desulfovibrio ( Desulfovibrionaceae The abundance of harmful bacteria such as ).

[0097] The V3-V4 hypervariable regions of the 16S rRNA gene amplified into the HFD and LBP groups were sequenced at both ends (250 bp) using the Illumina MiSeq / NovaSeq high-throughput sequencing platform. Sequence quality control, noise reduction, and OTU clustering based on 97% similarity were performed using QIIME2 software. The ACE index, Chao1 index, and Shannon index were then calculated to assess alpha diversity. The alpha diversity results are shown below. Figure 10 As shown.

[0098] Depend on Figure 10 It is evident that, compared to the HFD group, the LBP group treated with KIBZ5 lyophilized bacterial solution via gavage significantly improved the ACE index, Shannon index, and Chao1 index, and increased the alpha diversity of the gut microbiota in type 2 diabetic mice, with statistically significant differences. P <0.0001), which reshaped the gut microbiota.

[0099] Left lateral lobe liver tissue was harvested from mice in the HFD and LBP groups. A portion was embedded in OCT embedding medium, flash-frozen in liquid nitrogen, and stored at -80℃ for Oil Red O staining. Another portion was fixed in 4% paraformaldehyde, embedded in paraffin, and used for PAS staining. The remaining liver tissue was homogenized (100 mg tissue + 1 mL RIPA lysis buffer), centrifuged at 12000 rpm for 15 min at 4℃, and the supernatant was used for MDA detection. Protein concentration was determined using the BCA method for correction. Staining results are shown below. Figure 11 As shown in Table 2, the test results are as follows.

[0100] Table 2 Results of liver marker detection in mice of the HFD and LBP groups

[0101] Note: In Table 2, express P <0.05, LPB is statistically different from HFD.

[0102] Depend on Figure 11 As can be seen, LBP improved liver lipid metabolism in type 2 diabetic mice, reducing the number of positive areas stained with Oil Red chromatogram. Table 2 shows that, compared with the HFD group, the LBP group mice had an increased liver glycogen ratio and reduced levels of the lipid peroxide malondialdehyde (MDA).

[0103] In summary, this invention has obtained a Bacillus licheniformis strain, KIBZ5, through screening. This strain is non-toxic, non-hemolytic, and biosafety in nature. Experiments have demonstrated that oral administration of the KIBZ5 strain to type 2 diabetic mice significantly improves their blood glucose regulation, reduces insulin resistance, alleviates inflammation, improves gut microbiota composition, and increases the proportion of beneficial bacteria. This invention provides a new approach for the treatment of type 2 diabetes.

[0104] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of Bacillus licheniformis ( Bacillus paralicheniformis KIBZ5, characterized in that, The Bacillus licheniformis KIBZ5 was deposited on May 15, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 38695.

2. A microbial agent, characterized in that, The bacterial agent includes Bacillus licheniformis KIBZ5 as described in claim 1.

3. The microbial agent as described in claim 2, characterized in that, The microbial agent includes solid or liquid microbial agents; The effective viable bacterial concentration of Bacillus licheniformis KIBZ5 in the solid bacterial agent is 1×10⁻⁶. 8 ~1×10 11 CFU / g; The effective viable bacterial concentration of Bacillus licheniformis KIBZ5 in the liquid bacterial agent is 1×10⁻⁶. 8 ~1×10 11 CFU / mL.

4. The microbial agent as described in claim 2, characterized in that, The microbial agent includes Bacillus licheniformis KIBZ5 lyophilized powder.

5. A method for preparing a microbial agent, characterized in that, include: Fermentation of the Bacillus licheniformis KIBZ5 according to claim 1 was performed to obtain a fermentation sample; Centrifuge the fermentation sample, collect the precipitate, and obtain wet bacterial cells; The wet mycelium was pre-frozen to obtain pre-frozen mycelium blocks; The pre-frozen bacterial blocks were dried and pulverized to obtain Bacillus licheniformis KIBZ5 freeze-dried powder.

6. A probiotic preparation, characterized in that, The formulation includes Bacillus licheniformis KIBZ5 as described in claim 1, the bacterial agent as described in any one of claims 2 to 4, or the bacterial agent prepared by the preparation method described in claim 5.

7. The probiotic preparation according to claim 6, characterized in that, The formulations include oral lyophilized powder formulations, enteric-coated capsules, or microcapsules.

8. The use of Bacillus licheniformis KIBZ5 according to claim 1, the bacterial agent according to any one of claims 2 to 4, the bacterial agent prepared by the preparation method according to claim 5, or the probiotic preparation according to claim 6 or 7 in the preparation of any one or more of the following products: 1) To prepare products for the treatment and / or improvement of type 2 diabetes; 2) Prepare products that improve intestinal barrier function; 3) Prepare products that regulate intestinal flora; 4) Prepare products that reduce inflammation in the body.

9. The application as described in claim 8, characterized in that, The preparation of products for treating and / or improving type 2 diabetes includes the preparation of products for improving insulin resistance.

10. The application as described in claim 8, characterized in that, The preparation of products that improve intestinal barrier function includes the preparation of products that upregulate the expression of ZO-1, Claudin-1, and Occludin tight junction proteins; the preparation of products that improve intestinal flora includes the preparation of products enriched with Lactobacillus spp. in the gut ( Lactobacillus ) and / or inhibit desulfovibrioceae in the gut ( Desulfovibrionaceae ) products.

Images