A polysaccharide-producing irpex lacteus strain and application thereof

Abstract

The application discloses a polysaccharide-producing white-cystogloea strain and application thereof. The fungal strain is preserved in the China General Microbiological Culture Collection Center, and is named as white-cystogloea (Gloeophyllum sepiarum) TL-8 strain with a preservation number of CGMCC No. 40595. Irpex lacteus .

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a polysaccharide-producing strain of *Alternaria alternata* and its applications. Background Technology

[0002] Fungal polysaccharides can be obtained from wild fruiting bodies, through artificial cultivation, or through mechanically aerated and stirred fermentation. Wild-harvested fruiting bodies are of high quality and rich in secondary metabolites. However, the yield of wild fruiting bodies is limited by geographical location, climate, and environmental resources, making it difficult to meet market demand. Artificial cultivation of large fresh fungi, such as various mushrooms, wood ear fungus, silver ear fungus, and Ganoderma lucidum, greatly satisfies market demand, and fungal polysaccharides are further obtained through extraction. However, the cultivation of some fungi requires large amounts of timber; for example, Poria cocos requires pine wood for mass production. Mechanically aerated and stirred fermentation of fungal polysaccharides significantly shortens the fermentation cycle and allows for unrestricted raw material preparation, making it an effective method for rapidly obtaining functional polysaccharides.

[0003] The biological functions of polysaccharides from medicinal and edible fungi have attracted considerable attention from researchers, such as Ganoderma lucidum polysaccharides, Phellinus linteus polysaccharides, Tremella fuciformis polysaccharides, Poria cocos polysaccharides, and Hericium erinaceus polysaccharides. Extensive research has been conducted on their anti-inflammatory, antioxidant, antitumor, hypoglycemic, hypotensive, hepatoprotective, and immune-enhancing effects. However, research on *Bletilla striata* polysaccharides is relatively scarce. The main findings include: the intracellular polysaccharide component ILN3A of *Bletilla striata* may serve as a candidate anti-inflammatory drug for the clinical treatment of induced membranous glomerulonephritis; and the presence of an active polysaccharide with anti-glomerular mesangial cell (GMC) proliferation activity. *Bletilla striata* polysaccharides have been shown to regulate immune activity by preventing or improving certain characteristic immune responses in mercuric chloride-induced polyclonal activation in female CBA mice. However, other biological functions of *Bletilla striata* polysaccharides, such as antitumor activity, require further exploration and application. Furthermore, there are no reports on the repair of colonic inflammation by *Bletilla striata* extracellular polysaccharides. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide a polysaccharide-producing strain of *Alternaria alternata* and its applications.

[0005] This invention involves collecting dead branches and decaying wood with small fruiting bodies or white mycelia from the wild. The surface sample of the dead branches and decaying wood is then crushed using a sterile knife and cultured and screened on potato juice (PDA) medium, PDA slant medium, and fermentation basic medium to obtain fungal strains. After further screening, a fungal strain with an extracellular polysaccharide yield of 5.94 g / L was obtained. This fungal strain was deposited on May 24, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 40595, and named *Irpex lacteus* TL-8 strain.

[0006] This invention successfully constructed a mouse colitis model through DSS induction and then explored the repairing effect of high and low doses of *Bacillus thuringiensis* extracellular polysaccharide (ILP) on mouse colitis using 5-aminosalicylic acid as a positive control. The initial adaptive feeding, the mid-term colitis model construction and inflammatory symptom observation, the later-term mouse repair, gavage, weight measurement, behavioral observation, and the final dissection experiment were all completed in the laboratory. Other samples were sent to a third-party biotechnology company for testing. The results showed that *Bacillus thuringiensis* extracellular polysaccharide (ILP) played a consistent role in repairing weight loss due to colitis in mice, promoting tissue healing, maintaining intestinal microbiota balance, and adjusting the expression of serum inflammatory factors and active enzymes, demonstrating excellent biological activity. The low-dose polysaccharide L-ILP (100 mg / kg BW) showed superior anti-inflammatory activity compared to the positive control drug 5-aminosalicylic acid (100 mg / kg BW) and also superior biological activity compared to the high-dose polysaccharide H-ILP (300 mg / kg BW), indicating its potential for further exploration. The experimental results of this invention show that the extracellular polysaccharide ILP of *Bacillus thuringiensis* can regulate the colonic flora of inflamed mice, reducing the abundance of *Akkermansia* and *Bacteroides* species induced by inflammation, while increasing the abundance of *Lactobacillus* species. This may be one of the mechanisms by which colonic inflammation is repaired. *Bacillus thuringiensis* polysaccharide ILP can also increase SOD enzyme activity and IL-10 content, reduce myeloperoxidase (MPO) activity and the expression levels of IL-1β, TNF-α, and MDA, thereby curing colonic inflammation in mice.

[0007] That is, the polysaccharide-producing *Alternaria alternata* strain of the present invention can be used to prepare a colitis repair agent. Specifically, the colitis repair agent is prepared by fermenting the extracellular polysaccharide produced by the fungal strain. Preferably, the fermentation conditions are as follows: the polysaccharide-producing *Alternaria alternata* strain is inoculated into the fermentation medium at an inoculum of 3%, and cultured at 30°C and 160 r / min for 8 days; the fermentation medium is: lactose 16.0 g / L, glucose 24.0 g / L, soybean residue powder 7.5 g / L, yeast extract 5.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 1.0 g / L, pH 5.0. Preferably, the extracellular polysaccharide is a mixture of polysaccharide ILP1 and polysaccharide ILP2. More preferably, the extracellular polysaccharide ILP1 contains 20% by weight, and the extracellular polysaccharide ILP2 contains 80% by weight. More preferably, the polysaccharide ILP1 contains mannose, glucose, galactose, glucosamine, xylose, and arabinose, with the weight percentage contents of mannose, glucose, galactose, glucosamine, xylose, and arabinose in polysaccharide ILP1 being 93.25%, 4.85%, 0.74%, 0.58%, 0.47%, and 0.11%, respectively; and the polysaccharide ILP2 contains mannose, glucose, galactose, glucosamine, xylose, and galacturonic acid, with the weight percentage contents of mannose, glucose, galactose, glucosamine, xylose, and galacturonic acid in polysaccharide ILP2 being 96.11%, 2.74%, 0.44%, 0.45%, 0.10%, and 0.15%, respectively.

[0008] The aforementioned extracellular polysaccharides can also be used to prepare colitis repair agents. The polysaccharide-producing *Alternaria alternata* strain was inoculated into a fermentation medium at a 3% inoculum and cultured at 30°C and 160 rpm for 8 days to obtain the colitis repair agent. The fermentation medium consisted of: lactose 16.0 g / L, glucose 24.0 g / L, soybean residue powder 7.5 g / L, yeast extract 5.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 1.0 g / L, and pH 5.0. Attached Figure Description

[0009] Figure 1 Infrared dilution spectrum of extracellular polysaccharide ILP1;

[0010] Figure 2 Infrared dilution spectrum of extracellular polysaccharide ILP1;

[0011] Figure 3This study demonstrates the restorative effect of extracellular polysaccharide ILP (Illuminati leucocytogenes) on weight loss in inflammatory mice. In the figure: CTRL, control group; DSS, inflammatory model group; 5-ASA, 5-aminosalicylic acid treatment group; H-ILP, high-dose ILP treatment group; L-ILP, low-dose ILP treatment group.

[0012] Figure 4 Hematoxylin-eosin staining was used to visualize colonic tissue damage and repair in inflammatory mice.

[0013] Figure 5 The table shows the expression of serum inflammatory factors and active enzymes in different groups of a mouse colon model. In the figure: A, IL-1β; B, TNF-α; C, MPO; D, IL-10; E, MDA; F, SOD and IL-4. * indicates p<0.05.

[0014] Figure 6 To investigate the protective effect of ILP (extracellular polysaccharide) administered by gavage on the intestinal microbial diversity of mice; In the figure: A, ACE index; B, Chao index; C, Simpson index; D, Shannon index. Data are mean ± standard deviation (n=6), and one-way ANOVA was used.

[0015] Figure 7 The effect of extracellular polysaccharide ILP of *Alternaria alternata* on the phylum-level microbial community of mouse colon;

[0016] Figure 8 The effect of extracellular polysaccharide ILP of *Bacillus thuringiensis* on the colonic microbial community in mice.

[0017] Figure 9 The effect of ILP (extracellular polysaccharide of *Bacillus thuringiensis*) on the genus-level community of colonic microbes in mice. Detailed Implementation

[0018] 1. Strains screening

[0019] Culture media: Strain screening medium: Potato juice (PDA) medium, sterilized at 121℃ for 25 min, cooled to approximately 60℃, then 1.0 g / L streptomycin sulfate was added. Slant medium: PDA slant, sterilized at 121℃ for 25 min. Basic fermentation medium: 1.0 g yeast extract, 2.0 g glucose, 0.2 g potassium dihydrogen phosphate, 0.1 g magnesium sulfate, 0.02 g ferrous chloride, 100 mL tap water, poured into 250 mL Erlenmeyer flasks, natural pH, sealed with 8 layers of gauze and cotton filling, sterilized at 121℃ for 25 min. All culture media materials were commercially available.

[0020] (1) Sample collection

[0021] In the forest at an altitude of 600-800m in Zhupo Village, Yingzuijie Nature Reserve, Huitong County, Hunan Province, dead branches and decaying wood with small fruiting bodies or white mycelium were collected, sealed in aseptic edible mushroom bags, and brought back to the laboratory for storage at 4℃ for later use.

[0022] (2) Preliminary screening of bacterial strains

[0023] Shake the screening medium while hot, pour into plates, and allow to cool and solidify. Take 20-30 glass beads and place them in a 250mL Erlenmeyer flask, along with 90mL of sterile physiological saline. Using a sterile knife, crush 10-20g of the surface sample of dead branches and decayed wood, add it to the prepared sterile physiological saline, and shake in a shaker at 200 rpm for 30 minutes. Then dilute to 10⁻¹⁰ using the tenfold dilution method. -6 At a dilution of 10... -3 -10 -6 Take 0.1 mL of each sample and spread it onto a plate. Spread 3-6 plates for each gradient. Incubate the plates upside down in a 28℃ incubator for 3-6 days, observing at least once a day. When the colony diameter is greater than 1.0 cm, observe the changes in colony morphology and discard colored colonies (black, green, red, brown, yellow, etc.). Select white filamentous colonies and inoculate them onto test tube slant. Incubate the slant at 28℃ until bacterial growth appears. Store the slant in a 4℃ refrigerator for later use.

[0024] (3) Shake-flask fermentation and double screening

[0025] The initial screening slant was inoculated into the fermentation medium and cultured at 28℃ with shaking at 160 rpm. Mycelial growth and the color of the fermentation broth were observed daily. After 7 days of fermentation, the fermentation broth was filtered with qualitative filter paper. The filtrate was used for extracellular polysaccharide extraction and determination, and the filter residue was used for intracellular polysaccharide extraction and determination. Strains with high extracellular polysaccharide production were selected for further fermentation, and the process was repeated three times to screen for the optimal polysaccharide-producing strain.

[0026] (4) Extraction of crude extracellular polysaccharides from strains

[0027] The fermentation broth filtrate was centrifuged at 10,000g for 8 minutes. 2 mL of the supernatant was collected, and 6 mL of industrial alcohol was added. The mixture was precipitated overnight at 4°C. After centrifugation at 10,000g for another 8 minutes, the supernatant was discarded. The alcohol on the surface of the precipitate was removed with filter paper. The precipitate was reconstituted with 2 mL of distilled water, and the polysaccharide content was determined. Strains with excellent polysaccharide fermentation performance were screened.

[0028] (5) Determination of polysaccharides

[0029] The crude polysaccharide content in edible fungi was determined according to the method in NY / T 1676—2008. Glucose was used as a standard, and the extracellular polysaccharide content in the fermentation broth was determined using the phenol-sulfuric acid method. The glucose standard curve was obtained as follows:

[0030] Y = 0.1182·OD 490nm -0.003(R) 2 =0.9984)

[0031] The curve has good fit and can be used for polysaccharide determination.

[0032] (6) Identification of superior strains

[0033] Strains with excellent performance in fermenting extracellular polysaccharides were sent to Shanghai Bioengineering Co., Ltd. for molecular identification. A phylogenetic tree was constructed by comparing the strains with the gene bank to determine the species.

[0034] (7) Optimization of culture medium formulation and culture conditions for the fermentation of extracellular polysaccharides by superior strains

[0035] Under the initial conditions of the basal fermentation medium, single-factor optimization was performed on the medium components (such as nitrogen source, carbon source, inorganic salts, etc.) and culture conditions (such as temperature, shaking speed, pH value, fermentation time, etc.). Based on the results of the single-factor optimization, factors with significant influence were screened and optimized through orthogonal experiments to determine the optimal fermentation conditions for extracellular polysaccharides. The experimental data were repeated three times, and significance analysis was performed using SPSS20 software.

[0036] (8) Optimization of fermentation conditions by orthogonal experiment

[0037] Based on the results of single-factor experiments, L9(3) was designed. 3 An orthogonal experiment was conducted, selecting three factors (complex carbon source concentration, complex nitrogen source concentration, and fermentation time) that significantly affected polysaccharides in single-factor experiments. The data were analyzed using SPSS 20.0 software.

[0038] Based on the above steps, the optimal strain TL-8 was determined. TL-8 was identified as belonging to the genus *Irpex*, and presumably *Irpex lacteus*. The optimized fermentation conditions were determined as follows: inoculum size 3%, cultured at 30℃ and 160 rpm for 8 days; fermentation medium: lactose 16.0 g / L, glucose 24.0 g / L, soybean residue powder 7.5 g / L, yeast extract 5.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 1.0 g / L, pH 5.0. After fermentation under the optimized conditions, the optimal strain TL-8 produced 5.94 g / L of *Irpex lacteus* extracellular polysaccharides. This optimal strain TL-8 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40595 and named *Irpex lacteus* strain TL-8.

[0039] 2. Analysis of extracellular polysaccharides of *Alternaria alternata*

[0040] 2.1 Isolation and purification of extracellular polysaccharides from *Alternaria alternata*

[0041] (1) DEAE C-52 ion exchange column chromatography:

[0042] Column packing and equilibration: Soak 100g of DEAE C-52 ion-exchange cellulose in 70% ethanol-water for 24-48 hours, then slowly pack it into a 2.6cm·40cm chromatography column, ensuring the exchanger liquid level is within a certain range. The plastic float was used to drop the buffer solution, and then equilibrated with more than 20 times the volume of distilled water. The flow rate of the chromatography pump was controlled at 1 mL / min.

[0043] Sample loading, elution, and collection: Dissolve the freeze-dried polysaccharide powder (after removing protein) in distilled water to a polysaccharide concentration of 20 mg / mL; carefully load 1 mL (approximately 20 mg of polysaccharide) of the polysaccharide solution using a pipette; after the polysaccharide solution has largely penetrated the exchange resin layer, add 8-10 mL of distilled water using a dropper; elute with 0.2 mol / L sodium chloride solution, controlling the elution flow rate of the chromatography pump at 1 mL / min; collect one tube of eluent for every 10 mL of eluent.

[0044] Detection and recovery of polysaccharides: Take 0.5 mL of each collected solution and add 3 times the volume of 95% ethanol. Observe the precipitate and turbidity. Visually track the polysaccharide activity peak. At the same time, determine the polysaccharide content of each eluent using the sulfuric acid-phenol method. Summarize the polysaccharide activity peaks and precipitate them with 3 times the volume of 95% ethanol. After centrifugation, collect the precipitate or dialyze and freeze-dry it into powder. Weigh the powder and calculate the polysaccharide purity. Store in a sealed container at room temperature for later use.

[0045] (2) Dextran G-100 column chromatography:

[0046] Column packing and equilibration: Soak 50g of G-100 dextran gel in 70% ethanol-water for 24-48 hours, then slowly pack it into a 2.6cm·50cm chromatography column, ensuring the gel surface is level with water. The plastic float was used to drop the buffer solution, and then equilibrated with more than 20 times the volume of distilled water. The flow rate of the chromatography pump was controlled at 0.5 mL / min.

[0047] Sample loading, elution, and collection: Dissolve the freeze-dried polysaccharide powder obtained after DEAE C-52 ion exchange chromatography in distilled water to a polysaccharide concentration of 20 mg / mL; load 1 mL (approximately 20 mg of polysaccharide) of the polysaccharide solution onto the gel layer using a pipette; after the polysaccharide solution has basically penetrated into the gel layer, add 8-10 mL of distilled water using a dropper; continue eluting with distilled water, controlling the elution flow rate of the chromatography pump at 0.3-0.5 mL / min; collect one tube of eluent every 10 mL.

[0048] Detection and recovery of polysaccharides: The polysaccharide content of each tube of eluent was determined by the sulfuric acid-phenol method. The main active peaks of polysaccharides were summarized, freeze-dried for 3-5 days to obtain powder, accurately weighed and the polysaccharide purity was calculated. The powder was sealed and stored at room temperature for later use.

[0049] In DEAE-52 ion-exchange column chromatography, the extracellular polysaccharide of *Alternaria alternata* exhibits an absorption peak at a volume of 90-110 mL when eluted with pure water. This polysaccharide is defined as ILP1. Since ILP1 can be eluted with pure water, it exhibits characteristics of a neutral polysaccharide. With increasing sodium chloride concentration in the eluent, at a sodium chloride concentration of 0.2 mol / L, another polysaccharide absorption peak is observed at a volume of 410-490 mL. This polysaccharide is defined as ILP2. Since ILP2 cannot be eluted with pure water, a higher concentration of sodium chloride is required, indicating that ILP2 exhibits acidic characteristics. Subsequently, when the sodium chloride concentration is continuously increased to 1 mol / L, no polysaccharide elution occurs. Therefore, it is inferred that the extracellular polysaccharide of *Alternaria alternata* has two components, with ILP1 accounting for approximately 20% and ILP2 accounting for approximately 80%. This suggests that the extracellular polysaccharide of *Alternaria alternata* is primarily an acidic polysaccharide.

[0050] The elution curves of ILP1 separated and purified using dextran gel G-100 show that ILP1 elutes in 40-50 mL of ultrapure water, indicating that ILP1 has a relatively large molecular weight, and small molecule impurities and other low molecular weight substances are separated. The elution curves of ILP2 separated and purified using dextran gel G-100 show that ILP2 elutes in 50-70 mL of ultrapure water, indicating that ILP2 also has a relatively large molecular weight. From the purification steps, it can be seen that trichloroacetic acid has the highest impurity removal rate, increasing the purity of the crude polysaccharide from 31.46% to 68.13%. This indicates that ethanol not only precipitates polysaccharides, but also causes pure insoluble substances such as proteins to settle. The final polysaccharide recovery rate after purification is only 10.29%. The final polysaccharide purity reaches 96.74%, with a white color. The purified polysaccharide can be used for molecular weight determination, infrared spectroscopy group analysis, and polysaccharide monosaccharide component analysis.

[0051] 2.2 Determination of the molecular weight of extracellular polysaccharides ILP1 and ILP2 of *Alternaria alternata*

[0052] The relative molecular weights of ILP1 and ILP2 are shown in Table 1. The relative molecular weights of ILP1 and ILP2 are 41753 Da and 61470 Da, respectively, after fitting with the logarithmic curve of the standard molecular weight.

[0053] Table 1. Relative molecular weights of ILP1 and ILP2

[0054]

[0055] 2.3 Monosaccharide composition of the extracellular polysaccharides ILP1 and ILP2 of *Alternaria alternata*

[0056] The monosaccharide composition of ILP1 and ILP2 is shown in Table 2. As can be seen from Table 2, the main monosaccharides of ILP1 and ILP2 are mannose and glucose, with mannose accounting for over 90%. The next main components are galactose, glucosamine, and xylose. The difference is that ILP1 also contains a small amount of arabinose (0.11%), while ILP2 also contains a small amount of galacturonic acid (0.15%).

[0057] Table 2 Monosaccharide composition of extracellular polysaccharides ILP1 and ILP2

[0058]

[0059] 2.4 Infrared spectroscopy and functional group analysis of extracellular polysaccharides ILP1 and ILP2 from *Alternaria alternata*.

[0060] The red spectra of the extracellular polysaccharides ILP1 and ILP2 of *Bacillus thuringiensis* are shown below. Figure 1 and Figure 2 As can be seen from the figure, both ILP1 and ILP2 are between 3200 and 3500 cm. -1 A strong absorption peak is observed at this location, with a broad and robust peak shape, which is characteristic of the -OH absorption peak of polysaccharides. Both ILP1 and ILP2 show absorption peaks between 1640 and 1870 cm⁻¹. -1 It exhibits a relatively strong C=O characteristic absorption peak; in the range of 1000–1300 cm⁻¹ -1 The presence of an absorption peak indicates the presence of a CO stretching vibration glycosidic bond (COC) and a hydroxyl group (C-OH) in the compound.

[40] ILP1 and ILP2 are at 500-600cm -1 The absorption within the range indicates that the compound has a pyran ring skeleton structure.

[41] Since both ILP1 and ILP2 are within the range of 650–1000 cm... -1 The presence of multiple absorption peaks indicates the presence of =CH groups in the compound. However, ILP1 has more complex functional groups than ILP2. ILP1 peaks at 2935.82 cm⁻¹. -1 The absorption peak indicates that the compound has CH stretching vibrations of saturated carbon atoms; ILP1 is at 1413.32 cm⁻¹. -1 The absorption peaks indicate that the compound has saturated carbon atoms with -CH3 or -CH2 atoms; ILP1 is at 2142.82 cm⁻¹. -1 The absorption peak indicates that the compound has accumulated unsaturated double bonds C=C=C or O=C=NH; ILP1 is at 910.80 cm⁻¹. -1 974.42cm -1 The presence of an absorption peak indicates that the compound has an R-CH=CH2 terminal group. The unsaturated double bond group characteristic of *Bacillus thuringiensis* suggests that ILPs have good antioxidant activity, with ILP1 being superior to ILP2.

[0061] 3. Treatment of DSS-induced colitis in mice by Irpex lacteus extracellular polysaccharide

[0062] 3.1 Experimental materials

[0063] (1) Experimental materials: Irpex lacteus extracellular polysaccharide ILP, the purity of the polysaccharides (ILP1, ILP2) is greater than 95%; prepared and purified by laboratory fermentation

[0064] (2) 50 10-week-old female ICR mice, Hunan Silaike Jingda Experimental Animal Co., Ltd., Experimental Animal License No.: SCXK (Xiang) 2019-0004.

[0065] (3) Dextran sulfate sodium (DSS), Dalian Meilun Biotechnology Co., Ltd.

[0066] (4) 5-Aminosalicylic acid (5-ASA), Dalian Meilun Biotechnology Co., Ltd.

[0067] (5) Malondialdehyde (MDA), Item No. YX-W-A401, Specification: 100 tubes / 96 samples.

[0068] (6) Experimental kits: The information of the kits is shown in Table 3.

[0069] Table 3 Experimental kits

[0070]

[0071] 3.2 Experimental methods (the experimental design is shown in Table 4)

[0072] (1) Construction of experimental animals and DSS inflammation model

[0073] The mouse experiment is divided into three periods: adaptation period, inflammation model period, and inflammation treatment period, each period lasts for 7 days, for a total of 21 days. To ensure that each group has at least 6 replicated mice and prevent abnormal changes in the number of mice during feeding, 50 10-week-old female ICR mice were purchased and numbered with ear tags. The mice were kept in a stable environment (room temperature of about 23-25°C, day-night alternation cycle of 12h). During the experiment, all mice were given sufficient feed and clean drinking water. Subsequently, the mice were randomly divided into 5 groups (Table 4, n=6).

[0074] Adaptation period: The mice were fed ad libitum for 7 days to adapt to the environment.

[0075] Inflammation model period: The control group drank distilled water ad libitum, and the other groups drank 3% dextran sulfate sodium (DSS) ad libitum for 7 days.

[0076] During the inflammation treatment period: the control group had free access to distilled water and were given saline by gavage, the DSS group was given distilled water by gavage, and the other groups were given the corresponding treatment reagents by gavage, for 7 days.

[0077] Table 4 Experimental Design

[0078]

[0079] (2) Detection of inflammatory factors in serum

[0080] On day 21 of the experiment, after the experiment, the mice were fasted for 12 hours, then anesthetized with ether and blood was collected from the eyeballs. The blood was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected and stored at -80°C. The expression levels of inflammatory factors and antioxidant enzymes in mouse serum were measured according to the ELISA kit instructions. The inflammatory factor assay was performed by sending samples to Beijing Biomarker Technologies Co., Ltd.

[0081] (3) Observation of colon tissue: Colon tissue specimens were obtained from dissected mice, fixed with 4% formaldehyde, sectioned, stained with hematoxylin and eosin (H&E), dehydrated with ethanol, and sealed with neutral glue. The samples were sent to Wuhan Saiweier Biotechnology Co., Ltd. for HE staining and post-processing. The histological morphology and tissue inflammation of the colon were observed and analyzed under a microscope, and photographs were taken and recorded.

[0082] (4) Colonic contents microbial community analysis

[0083] The colon of mice anesthetized with ether was removed, its length measured, and its contents extracted, labeled, and placed in liquid nitrogen. Sample DNA was extracted and amplified using the MN NucleoSpin 96 DNA kit, following the kit's instructions, targeting the highly variable V3-V4 region of the bacterial 16S rRNA gene. Solexa PCR, Nanodrop quantification, and sample mixing were then performed. Finally, the column was purified, gel recovered, and subjected to MiSeq high-throughput sequencing (Illumina, NoveSeq 6000, USA). Microbial community analysis of the colon samples was conducted at Biomarker Technologies (Wuhan).

[0084] (5) Data Processing and Analysis

[0085] Data variance was analyzed using SPSS 21 with Levene's and Student's tests, and homogeneity of variance was confirmed. Origin 2021 Pro was used to plot mouse body weight changes, colon length, serum levels of various immune factors, MPO and SOD enzyme levels, and colonic microbial abundance at different levels. *p<0.05 indicates statistical significance.

[0086] 3.3 Results

[0087] 3.3.1 Effects of ILP (intracellular polysaccharide) on mouse growth

[0088] After a 7-day acclimatization period, the overall growth of the mice in all five groups was normal. There was no significant difference in body weight among the randomly assigned mice, which was approximately 25.15 ± 1.09 g. Figure 3 Four days after modeling, except for the control group which showed normal weight gain, the other experimental groups experienced weight loss. By day 7, the average weight of the control group (CTRL) was 26.99 ± 1.69 g, while the average weight of the mice in the four modeling groups was 24.89 ± 1.48 g. The difference in weight between the modeling groups and the control group was highly significant, and some mice in each modeling group experienced hematochezia. This indicates that the inflammation induced by sodium dextran sulfate (DSS) for 7 days was successful. During the inflammation treatment period, the weight of all modeling groups continued to decrease within 3 days of treatment. Starting from day 3 of treatment, the weight of mice in the 5-aminosalicylic acid positive treatment group (ASA), the low-dose L-ILP treatment group, and the high-dose L-ILP treatment group stopped decreasing, while the weight of mice in the DSS group continued to decrease to 20.49 ± 0.67 g. At this point, the body weight of the mice was 28.84±1.13g in the control group, 23.56±0.75g in the 5-ASA group, 23.56±1.15g in the H-ILP group, and 23.99±1.45g in the L-ILP group. The body weight of the control group mice was significantly different from that of the DSS, 5-ASA, L-ILP, and H-ILP groups (p<0.05). The body weight of the mice in the 5-ASA, L-ILP, and H-ILP treatment groups was significantly different from that of the DSS inflammation model group. This indicates that 5-ASA, L-ILP, and H-ILP have a highly significant restorative effect on body weight loss in inflamed mice. Among them, the restorative effect of L-ILP was superior to that of H-ILP and 5-ASA.

[0089] 3.3.2 Effects of *Bacillus thuringiensis* polysaccharide ILP on the repair of inflamed mouse colon tissue

[0090] Colitis primarily manifests as symptoms such as rectal bleeding, constipation, diarrhea, and abdominal pain. Its essence lies in physiological pathological changes within the colonic tissue. HE staining results of mouse colonic tissue are shown below. Figure 4 .from Figure 4 It can be seen that histological examination showed DSS ( Figure 4 b) In treated mice, some goblet cells disappeared from the colon tissue, U-shaped crypts were severely deformed, inflammatory lymphocyte infiltration was severe, and physiological pathology was severe. Inflammatory mice showed good repair after 7 days of treatment with 5-ASA, L-ILP, and a low-dose H-ILP group. Figure 4(c, d, e), among which L-ILP has the best repair effect.

[0091] 3.3.3 Effects of polysaccharide ILP on inflammatory factors and the activities of two enzymes in mouse serum

[0092] In addition to symptoms such as bloody stools, diarrhea, and colonic tissue necrosis, colitis in mice also profoundly affects the expression and synthesis of inflammatory factors and related enzymes. The effects of the five experimental groups (n=6) on inflammatory factors and active enzymes are shown in the table below. Figure 5 .from Figure 5 The results showed that after 7 days of natural repair, the DSS-induced inflammation model group exhibited significant differences in all indicators compared to the normally growing CTRL group (p<0.05). Serum SOD enzyme activity and IL-10 expression levels were significantly decreased, while myeloperoxidase (MPO) activity, IL-1β, TNF-α, and MDA expression levels were significantly increased, indicating that natural repair was unlikely to reach the expected values. Compared with the 5-ASA, H-ILP, and L-ILP treatment groups, the DSS group showed significant improvements in SOD, MPO, IL-1β, IL-10, TNF-α, and MDA indicators, indicating that 5-ASA and L-ILP have significant repair effects on DSS-induced colonic inflammation, with L-ILP showing better repair effects than H-ILP, and H-ILP better than 5-ASA. In particular, the inflammatory repair effect of *Bacillus subtilis* extracellular polysaccharide (ILP) was superior to that of the positive control drug (5-ASA), showing potential development value.

[0093] 3.3.4 Protective effect of polysaccharide ILP on mouse gut microbiota

[0094] (1) Effects of polysaccharide ILP on α-diversity of colonic microbiota in mice

[0095] Polysaccharide ILPs not only repaired inflammatory markers in mouse serum but also affected the colonic microbiota. The α-diversity of the V3-V4 region of 16S rRNA sequenced from colonic contents samples is shown in [reference needed]. Figure 6Compared with the CTRL group, the abundance and diversity of colonic microbiota (Simpson, Shannon, Ace, Chao1) in the DSS inflammation model group were significantly reduced (p<0.05). After treatment with 5-ASA, H-ILP, and L-ILP, the Simpson, Shannon, Ace, and Chao1 indices in the treatment group were significantly increased compared with the DSS group (p<0.05), with L-ILP showing the best repair effect, followed by 5-ASA, and then H-ILP. In particular, the Simpson and Shannon indices of 5-ASA, H-ILP, and L-ILP in the treatment group were close to those in the normal CTRL group. It can be seen that the extracellular polysaccharide of *Bacillus albus* plays a significant repairing role in the restoration of intestinal microbiota diversity in inflammatory mice at low doses (100 mg / kg BW).

[0096] (2) Effects of polysaccharide ILP on community composition at the phylum level of colonic microbiota in mice

[0097] The composition of the colonic microbiota of mice in each experimental group is shown in the figure. Figure 7 . Figure 7 A shows the top ten microbial phyla. In normal mice, the colonic microbiota is mainly composed of Firmicutes, Bacteroides, and Proteobacteria, which are the three major phyla. These three phyla account for 86.66% of all microorganisms. After DSS-induced inflammation, the abundance of Firmicutes, Bacteroides, and Proteobacteria decreased to 74.78%, while the abundance of Verrucomicrobiota significantly increased from 0.004% to 17.13% (p<0.05). After treatment with 5-ASA, H-ILP, and L-ILP, the abundance of Firmicutes, Bacteroides, and Proteobacteria increased again to 83.19%, 81.88%, and 86.14%, respectively. Meanwhile, the abundance of Verrucomicrobiota decreased to 8.89%, 9.05%, and 3.42%. The abundance of 5-ASA, H-ILP, and L-ILP in the treatment group was significantly different from that in the DSS inflammation group in terms of Firmicutes and Verrucous microbes (p<0.05). Low-dose L-ILP showed the best repair effect.

[0098] (3) Effects of polysaccharide ILP on the composition of colonic microbial community in mice

[0099] Further refine the analysis of microbial community classification levels, Figure 8The study revealed that at the class level, the colon of normal mice is mainly distributed with Clostridia, Bacteroidia, Bacilli, Gammaproteobacteria, and Desulfovibrionia, among others. Figure 8 A) The proportions of the total microorganisms were 38.95%, 34.32%, 6.19%, 6.40%, and 4.57%, respectively. After DSS-induced inflammation in mice, the proportions of Clostridium, Bacteroides, Bacillus, Bacillus, Bacillus, and Desulfovibrio in the colon were 21.92%, 36.32%, 9.36%, 6.40%, and 7.11%, respectively. The abundance of Clostridium decreased most significantly (p<0.05), with a loss of 43.72%. The abundance of Verrucomicrobiota increased significantly from 0.004% to 17.16%. After treatment with 5-ASA, H-ILP, and L-ILP, the proportions of Clostridium, Bacteroides, Bacillus, Bacillus, Bacillus, and Desulfovibrio in the colon of inflamed mice were significantly increased, basically approaching the proportions of the colonic microbiota in normal mice. In particular, low doses of L-ILP showed an abundance of 49.15% in Clostridia. Figure 8 B) The percentage of the normal group was 38.95% higher than that of the normal group; the percentage of Verrucomicrobiota decreased to 3.42%, significantly lower than that of the DSS group (17.16%). This indicates that polysaccharide ILP has a significant effect on the repair of the colonic microbiota in mice.

[0100] (4) Effects of polysaccharide ILP on the composition of colonic microbial community in mice

[0101] The colonic microbiota and its proportions at the genus level in normal mice were as follows: Unclassified *Muribaculaceae* (15.27±0.26%), Unclassified *Lachnospiraceae* (11.66±0.39%), *Lachnospiraceae* NK4A136 group (8.94±0.37%), *Lactobacillus* (3.61±0.08%), *Oscillospiraceae* (3.53±0.08%), and other genera (…). Figure 9In mice, DSS-induced inflammation resulted in a complete imbalance in their gut microbiota, notably an increase in Akkermansia from 0.02% to 17.17%, Bacteroides from 1.45% to 15.24%, and a decrease in Lactobacillus from 3.618% to 1.54%. Treatment with 5-ASA, H-ILP, and L-ILP in the inflamed mice showed significant restoration of Akkermansia, Bacteroides, and Lactobacillus flora. Specifically, low-dose L-ILP reduced Akkermansia abundance to 3.43%, Bacteroides abundance to 6.62%, and Lactobacillus abundance to 2.44%. The inflammation symptoms in the mouse colon were improved.

[0102] The extracellular polysaccharide ILP of *Bacillus thuringiensis* can regulate the colonic flora of inflamed mice, reducing the abundance of *Akkermansia* and *Bacteroides* species induced by inflammation, while increasing the abundance of *Lactobacillus* species. This may be one of the mechanisms by which colonic inflammation is repaired. *Bacillus thuringiensis* polysaccharide ILP can also increase SOD enzyme activity and IL-10 levels, and decrease myeloperoxidase (MPO) activity and the expression levels of IL-1β, TNF-α, and MDA, thus curing colonic inflammation in mice.

Claims

1. A type of white-sac-shaped tooth fungus ( Irpex lacteus The application of TL-8 in the preparation of colon inflammation repair agents is characterized by, The *Bacillus subtilis* TL-8 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 40595. The application involves preparing a colon inflammation repair agent using the extracellular polysaccharide produced from the fermentation culture of *Bacillus subtilis* TL-8. The extracellular polysaccharide is a mixture of polysaccharides ILP1 and ILP2. The extracellular polysaccharide ILP1 contains 20% by weight, and the extracellular polysaccharide ILP2 contains 80% by weight. The ILP1 polysaccharide contains mannose, glucose, galactose, glucosamine, xylose, and arabinose. The weight percentage contents of galactose, glucosamine, xylose, and arabinose in polysaccharide ILP1 are 93.25%, 4.85%, 0.74%, 0.58%, 0.47%, and 0.11%, respectively; the weight percentage contents of mannose, glucose, galactose, glucosamine, xylose, and galacturonic acid in polysaccharide ILP2 are 96.11%, 2.74%, 0.44%, 0.45%, 0.10%, and 0.15%, respectively.

2. The application as described in claim 1, characterized in that, The fermentation culture conditions were as follows: *Bacillus thuringiensis* TL-8 was inoculated into the fermentation medium at an inoculum rate of 3% and cultured at 30℃ and 160 r / min for 8 days; the fermentation medium was: lactose 16.0 g / L, glucose 24.0 g / L, soybean residue powder 7.5 g / L, yeast extract 5.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 1.0 g / L, pH 5.

0.

3. A method for preparing a colon inflammation repair agent, characterized in that, The method involves inoculating the *Bacillus thuringiensis* TL-8 as described in claim 1 into a fermentation medium at an inoculation rate of 3%, and culturing it at 30°C and 160 r / min for 8 days to obtain a colon inflammation repair agent. The fermentation medium is composed of: lactose 16.0 g / L, glucose 24.0 g / L, soybean residue powder 7.5 g / L, yeast extract 5.0 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 1.0 g / L, and a pH of 5.0.

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