The invention relates to a strain capable of efficiently utilizing 3apos; bacteroides vulgaris of sialyllactose and application of bacteroides vulgaris for improving intestinal health

Abstract

The invention discloses a common bacteroides efficiently utilizing 3 '-sialic acid lactose and application of the common bacteroides to improvement of intestinal health, the taxonomic name of the common bacteroides N059 is Phocaecola vulgaris, the common bacteroides N059 is preserved in the general microbiological center of the China Committee for Culture Collection of Microorganisms, the preservation number is CGMCC No.46910, and the preservation date is December 11, 2025. The bacteroides vulgaris N059 disclosed by the invention can efficiently and specifically metabolize and utilize the 3 '-sialic acid lactose, has good probiotic characteristics, is suitable for being used as probiotics and prebiotics (such as the 3'-sialic acid lactose) for combined application, has better technical effects of improving intestinal health, relieving colitis and reducing enteritis-related inflammatory factors, and achieves the effects of regulating intestinal florae, improving intestinal health and improving intestinal health. The traditional Chinese medicine composition can be widely applied to animal breeding industry and related fields of food, medicines and the like.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically, to a strain of Bacteroides that efficiently utilizes 3'-sialic acid lactose and its application in improving gut health. Background Technology

[0002] As the importance of gut microbiota in human health grows, interest in interventions that can regulate gut microbiota and their interactions with the host is also increasing. Besides diet and fecal microbiota transplantation, probiotics are the most well-known and widely recognized substances capable of regulating the human gut microbiota. *Bacteroides commonis* is one of the most common *Bacteroides* species in the gut microbiota of healthy adults, possessing significant potential for oligosaccharide utilization and immunomodulation, but it has not been systematically developed as a gut microbiota regulator. Furthermore, existing probiotics (such as *Lactobacillus* and *Bacillus*) have relatively limited functions, only utilizing simple sugars, and currently lack *Bacteroides* strains with clear safety assessments and functional validation. Therefore, *Bacteroides commonis* holds promise as a next-generation probiotic candidate for improving gut health and reducing the incidence of diarrhea. Based on this background, screening for safe *Bacteroides commonis* and combining it with 3'-sialyl lactose (3'-SL) could represent a novel gut microbiota intervention strategy under green antibiotic alternatives. Summary of the Invention

[0003] The purpose of this invention is to provide a strain of common Bacteroides that efficiently utilizes 3'-sialic acid lactose and its application in improving intestinal health.

[0004] According to a first aspect of the invention, a strain of *Bacteroides commonis* N059 is provided, the taxonomic name of which is *Bacteroides commonis*. Phocaeicola vulgatus It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46910 and deposit date of December 11, 2025.

[0005] In some embodiments, Bacteroides commonis N059 is a Gram-negative bacillus without spores. On KVLB agar supplemented with 5% sterile defibrinated sheep blood, the colony morphology is milky white, round and raised, and has a smooth and moist surface.

[0006] In some implementations, Bacteroides commonis N059 can metabolize 3'-sialyl lactose into sialic acid and lactose within 24 hours.

[0007] According to a second aspect of the present invention, a microbial agent is provided, which is a liquid agent containing Bacteroides N059 or a solid agent containing Bacteroides N059.

[0008] In some embodiments, the viable count of Bacteroides commonis N059 in the liquid bacterial agent is 10. 8 ~10 9 CFU / mL, the viable count of Bacteroides N059 in the solid bacterial agent is 10. 7 -10 8 CFU / mL.

[0009] According to a third aspect of the present invention, a method for preparing a microbial inoculant is provided, wherein the preparation of a liquid inoculant containing Bacteroides commonis N059 comprises the following steps: The stock solution of *Bacteroides vulgaris* N059 was streaked onto KVLB solid medium for activation and cultured anaerobically at 37°C for 48 h. Single colonies of *Bacteroides vulgaris* N059 were picked and inoculated into KVLB liquid medium and incubated overnight. The resulting bacterial suspension was centrifuged at 5000 rpm for 10 min, washed twice with sterile KVLB liquid medium, and finally the bacterial precipitate was dispersed in physiological saline to obtain a live bacterial suspension of *Bacteroides vulgaris* N059 with a concentration of 10%. 8 -10 9 CFU / mL, to obtain a liquid bacterial agent containing Bacteroides N059; A method for preparing a solid bacterial agent containing Bacteroides commonis N059 includes the following steps: The stock solution of *Bacteroides vulgaris* N059 was streaked onto KVLB solid medium for activation and cultured anaerobicly at 37°C for 48 h. Single colonies of *Bacteroides vulgaris* N059 were picked and inoculated into KVLB liquid medium and cultured anaerobicly at 37°C for 24 h. The resulting bacterial suspension was centrifuged at 12000 rpm for 2 min, and the cells were collected, resuspended in sterile PBS, and the cell concentration was adjusted to 10-1. 8 ~10 9 CFU / mL, with added protectant, and subjected to low-temperature spray drying. The parameters for low-temperature spray drying are: inlet air temperature of 85-95℃, outlet air temperature of 42-48℃, and the protectant ratio is: 10% skim milk powder, 8% maltodextrin, 2% glycerol, 0.1% ascorbic acid, and the remainder is deionized water.

[0010] According to a fourth aspect of the present invention, a microecological preparation is provided, which contains Bacteroides commonis N059 or the above-mentioned microbial agents. The microecological preparation is prepared by microencapsulation using a polymeric substance as a base material and a live bacterial solution of Bacteroides commonis N059 as a core material.

[0011] According to a fifth aspect of the present invention, a synbiotic preparation is provided, which is prepared by microencapsulation using a polymeric substance as a base material and a live bacterial solution of prebiotics and Bacteroides var. mongolica N059 as a core material. The prebiotic is 3'-sialyl lactose, and the polymeric substance is gelatin or sodium alginate.

[0012] According to a sixth aspect of the present invention, the use of the above-mentioned common Bacteroides N059, or the above-mentioned microbial agent, or the above-mentioned probiotic preparation, or the above-mentioned synbiotic preparation in the preparation of a drug for reducing the level of intestinal inflammatory factors is provided.

[0013] According to a seventh aspect of the present invention, the application of the above-mentioned common Bacteroides N059, or the above-mentioned microbial agent, or the above-mentioned microecological preparation, or the above-mentioned synbiotic preparation in the preparation of a product for regulating intestinal flora, wherein the product is food or feed.

[0014] The beneficial effects of this invention are as follows: The common Bacteroides N059 of this invention can efficiently and specifically metabolize and utilize 3'-sialic acid lactose, and has good probiotic properties. It is suitable for use as a probiotic in combination with prebiotics (such as 3'-sialic acid lactose). It has good technical effects in improving intestinal health, relieving colitis, and reducing enteritis-related inflammatory factors. It achieves the purpose of regulating intestinal flora, enhancing intestinal barrier function, and improving diarrhea. It can be widely used in animal husbandry and related fields such as food and medicine. Attached Figure Description

[0015] Figure 1 This is a growth curve diagram of the common Bacteroides N059 of the present invention.

[0016] Figure 2 This is a TLC image of the supernatant of Bacteroides N059 fermentation at different sampling times according to the present invention.

[0017] Figure 3 This is a colony morphology diagram of the common Bacteroides N059 of the present invention.

[0018] Figure 4 This is a Gram staining image of the common Bacteroides N059 of the present invention.

[0019] Figure 5 This is a phylogenetic tree diagram of Bacteroides N059 based on the 16S rRNA gene, which is the present invention.

[0020] Figure 6 This is a comparison graph of the rate of change in mouse body weight under different treatments in Example 9 of the present invention.

[0021] Figure 7Figure A shows a comparison of the effects of different treatments on the length of mouse colons in Example 9 of the present invention. Figure B shows a comparison of the lengths of mouse colons under different treatments.

[0022] Figure 8 This is a comparison chart of the Disease Activity Index (DAI) of mice under different treatments in Example 9 of the present invention.

[0023] Figure 9 This is a comparison chart of serum inflammatory factor levels (TNF-α, IL-1β and IL-6) in mice treated differently in Example 9 of the present invention.

[0024] Figure 10 HE staining images of colon tissue from mice subjected to different treatments in Example 9 of the present invention.

[0025] Figure 11 This is a comparison diagram of the expression levels of tight junction proteins (Claudin-1, Occludin, ZO-1) in mice under different treatments in Example 9 of the present invention. Detailed Implementation

[0026] The present invention is further described in detail through specific implementation examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are commercially available.

[0027] Example 1: Screening and preservation of Bacteroides vulgaris strain N059

[0028] 1.1 Screening of Bacteroides vulgaris strain N059

[0029] Sample collection: Select healthy calves aged 1-2 months, collect calf fecal samples using sterile swabs, and place them in sterile phosphate buffer (PBS, pH=7.2) supplemented with 0.1% L-cysteine. The samples should be quickly sent to the laboratory for experiments.

[0030] Dilution and Coating: Take the well-mixed stock solution and perform serial dilutions of 10-fold with PBS containing 0.1% L-cysteine ​​(10... -1 -10 -8 After that, 100µL of the diluted fecal sample solution was taken from each sample and spread onto KVLB agar plates, and anaerobically incubated at 37℃ for 48 h.

[0031] Single colony isolation and purification: Observe and record colony characteristics, pick suspected Bacteroides colonies with an inoculation loop, purify for two generations until single colonies are obtained.

[0032] Assessment of Bacteroides' ability to metabolize 3'-sialic acid lactose: a. Carbon-free medium (DC) formulation: per 1L of medium, add 20.0g peptone, 5.0g yeast extract, 5.0g sodium chloride, 0.05g dipotassium hydrogen phosphate, 0.05g potassium dihydrogen phosphate, 1.0g L-cysteine, 10mg heme chloride, and 12mg vitamin K1. Add water to a final volume of 1L, adjust the pH to 7.0, and sterilize at 121℃ for 15 min.

[0033] b. The *Bacteroides* strains isolated and purified above were enriched overnight in KVLB liquid medium. The bacterial suspensions were then inoculated at 1% (v / v) into DC + sterile water, DC + glucose (10% w / v), and DC + 3'-SL (10% w / v), respectively. After anaerobic incubation at 37°C for 48 h, their OD values ​​were detected using a microplate reader. 600nm The absorbance values ​​were measured. DC+H2O was used as a negative control, and DC+glucose as a positive control. The metabolic utilization capacity of the above-mentioned Bacteroides for 3'-SL was determined based on the changes in absorbance values.

[0034] 1.2 Results

[0035] A total of 18 Bacteroides strains were isolated from the feces of healthy calves. Among them, 7 strains were capable of metabolizing 3'-SL (+++ or ++++). Bacteroides vulgatus N059 showed the best growth in a medium with 3'-SL as the sole carbon source (sole ++++). In conclusion, Bacteroides vulgatus N059 is a Bacteroides strain capable of efficiently and specifically metabolizing and utilizing 3'-SL.

[0036] Table 1. Evaluation results of the 3'-SL metabolic capacity of Bacteroides

[0037] Note: The growth status of Bacteroides is indicated by - and +: - (OD600nm < 0.2), + (0.2 < OD600nm < 0.4), ++ (0.4 < OD600nm < 0.6), +++ (0.6 < OD600nm < 0.8), ++++ (OD600nm > 0.8).

[0038] 1.3 Preservation

[0039] The above-mentioned common Bacteroides Phocaeicola vulgatus N059 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46910 and deposit date of December 11, 2025.

[0040] Example 2: 3'-SL metabolic utilization characteristics of Bacteroides commonis N059

[0041] 2.1 Growth curve determination of Bacteroides commonis N059: One loopful of *Bacteroides var. sarcodactylis* N059 culture was picked from a glycerol tube stored at -80°C and streaked onto a KVLB agar plate for activation. The culture was incubated at 37°C for 48 h. Single colonies were picked and incubated overnight on KVLB liquid medium. The culture was then inoculated at 1% (v / v) into 3 mL centrifuge tubes containing DC + sterile water, DC + glucose (10% w / v), and DC + 3'-SL (10% w / v). The cultures were anaerobic at 37°C. Fermentation broth was collected at 12 different time points (0, 2, 4, 6, 8, 10, 12, 24, 30, 36, 48, and 72 h), and its OD value was measured using a microplate reader. 600nm The absorbance value is used to determine the growth status of Bacteroides N059.

[0042] 2.2 Thin Layer Chromatography (TLC) Analysis of Metabolites

[0043] The dynamic changes in the 3'-SL composition of *Bacteroides vulgaris* N059 during 3'-SL fermentation were analyzed using TLC. Fermentation broths collected at 0, 6, 12, 24, 36, and 48 h were centrifuged at 12000 rpm for 2 min, and the supernatant was collected. 4 μL of the supernatant was applied to a thin-layer chromatography plate using a capillary tube, with 4 μL of 1% 3'-SL, 1% sialic acid, 1% β-lactose, 1% D-galactose, and 1% glucose standards as controls. A developing solvent was prepared at a ratio of n-butanol:acetic acid:water = 2:1:1 (v / v / v / v). 0.6 g of N-diamine dihydrochloric acid was added to 190 mL of methanol and 10 mL of sulfuric acid, and the mixture was stirred thoroughly to prepare the colorimetric reagent. Finally, the developed thin-layer chromatography plate was sprayed with this colorimetric reagent and heated at 110 °C for 10 min for color development.

[0044] 2.3 Results:

[0045] The growth curve of Bacteroides vulgaris N059 is shown in the figure. Figure 1 .Depend on Figure 1 It can be seen that Bacteroides var. mongolica N059 can rapidly increase its bacterial count in 6-12 h in a medium with 1% 3'-SL as the sole carbon source, enter the logarithmic phase, and enter the stationary phase after 36 h, with the OD value reaching its maximum value, which is similar to the growth in a medium with 1% glucose as the sole carbon source.

[0046] The metabolic kinetics of 3'-SL in Bacteroides vulgaris N059 were analyzed by TLC. The results are shown in [Figure number missing]. Figure 2.Depend on Figure 2 It was observed that at 0 h, the 3'-SL component of the fermentation supernatant was almost identically distributed on the plate to the 3'-SL standard after separation by thin-layer chromatography. After 24 h, 3'-SL could be utilized by Bacteroides vulgaris N059, with only a small amount of glucose and galactose remaining in the supernatant. In summary, Bacteroides vulgaris N059 possesses a highly efficient ability to metabolize 3'-SL.

[0047] Example 3 Identification of Bacteroides commonis N059

[0048] 3.1 Morphological identification: like Figure 3 As shown, Bacteroides var. ...

[0049] 3.2 Gram staining: like Figure 4 As shown, using Gram staining, Bacteroides var. mongolica N059 was determined to be a Gram-negative bacterium with rod-shaped cells and no spores.

[0050] 3.3 Identification of 16S rRNA

[0051] a. Using 1 mL of purified Bacteroides N059 culture medium as a template, amplification was performed using universal primers for the bacterial 16S rRNA gene: 27F (as shown in SEQ ID NO:1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (as shown in SEQ ID NO:2: 5'-GGTTACCTTGTTACGACTT-3') (Shanghai Sangon Biotech Co., Ltd., China).

[0052] b. The total volume of the PCR reaction was 25 μL, including 12.5 µL of 2×Taq PCR Master Mix (Nanjing Novizan Biotechnology Co., Ltd., China), 2 µL of DNA, 1 µL of 27F, 1 µL of 1492R, and 8.5 µL of ddH2O. Amplification was performed using a T100 thermal cycler (Bio-Rad, China). The PCR reaction program was: 95℃ for 3 min; 95℃ for 30 s, 60℃ for 20 s, 72℃ for 1 min, for a total of 34 cycles.

[0053] c. The amplified PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing to obtain the valid sequence of Bacteroides vulgaris N059 (SEQ ID NO:3). A phylogenetic tree was constructed by comparing the sequence with sequences in the GenBank database using BLAST. Figure 5 As shown.

[0054] Depend on Figure 5 Based on the NCBI accession numbers and publicly available sequence data of the listed strains, N059 was compared with these strains using 16S rRNA gene sequence alignment to clarify its phylogenetic position. The alignment results showed that N059 formed a tight cluster with the aforementioned *Phocaeicoia vulgatus* ATCC 8482 (accession number NR 074515.1), with a similarity of up to 100%. Therefore, N059 was identified as *Phocaeicoia vulgatus*.

[0055] Example 4 Safety assessment of Bacteroides commonis N059

[0056] 4.1 Evaluation Methods

[0057] DNA was extracted from *Bacteroides commonis* N059 using a bacterial DNA purification kit, and the extracted and purified DNA was then quantified using a TBS-380 fluorescence spectrometer. Genomic DNA was fragmented using a Covaris M220 focused acoustic shearing device, yielding DNA fragments of approximately 400 bp. At least 1 μg of high-quality genomic DNA was sent to Shanghai Lingen Biotechnology Co., Ltd. for library preparation and sequencing. The library was constructed using the NEXTflex® Rapid DNA-Seq kit, and the prepared library was subjected to paired-end sequencing (2 × 150 bp) on an Illumina Hiseq X Ten system. Bioinformatics analysis of the obtained data was performed using an Illumina next-generation sequencing platform.

[0058] (1) Detection of virulence factors:

[0059] After quality control of the whole genome data, clean reads were obtained and then de novo assembled to obtain the genome sequence. ORF prediction was performed based on the assembly results to obtain protein sequence files. Subsequently, using the virulence factor protein sequences included in VFDB (Virulence Factor Database) as a reference database, homology alignment analysis was performed on the above protein sequences. The alignment parameters were set as follows: sequence similarity not less than 80%, alignment coverage not less than 80%, E-value not higher than 1e-10, and hit results meeting the thresholds were retained. Redundancy removal and functional classification were performed on the hit results to obtain the virulence factor prediction profile of Bacteroides commonis N059. Furthermore, the distribution of candidate virulence factor genes on the chromosome can be statistically analyzed and visualized using genomic location information for subsequent safety feature description.

[0060] (2) Screening for resistance genes:

[0061] Based on the completion of genome assembly and gene prediction, and the acquisition of protein sequence files, the CARD (Comprehensive Antibiotic Resistance Database) was used as a reference database for antibiotic resistance genes. Homology comparisons of antibiotic resistance genes were performed on the protein sequences of *Bacteroides vulgaris* N059. The CRAD database was compared using the rgi tool, with results marked as "strict" selected as the entries for alignment. The candidate antibiotic resistance genes obtained from the screening were categorized and summarized, and their corresponding resistance mechanism types and potentially involved antibiotic classes were output. A predicted spectrum of antibiotic resistance genes for N059 was also compiled.

[0062] (3) MIC detection

[0063] The minimum inhibitory concentration (MIC) of *Bacteroides vulgaris* N059 was determined using the anaerobic broth microdilution method. Frozen *Bacteroides vulgaris* N059 strain was streaked onto KVLB solid medium and anaerobically cultured at 37°C for 48 h to obtain fresh colonies. Single colonies were picked and cultured overnight anaerobically at 37°C on KVLB liquid medium. The bacterial culture was then diluted in KVLB liquid medium to an OD600nm of 0.1 (approximately 10⁻⁶). 7 (CFU / mL), then diluted 100 times for later use. Different antibiotics were added to a culture medium suitable for anaerobic bacteria growth in a serial dilution of two-fold. After incubation at 37°C under anaerobic conditions for 48 h, the results were interpreted, and the lowest drug concentration with no visible growth was taken as the MIC value.

[0064] 4.2 Results:

[0065] (1) The virulence factor annotation results showed that no key virulence factors commonly found in enteropathogenic bacteria were detected in Bacteroides vulgaris N059. Its predicted virulence-related genes were mainly structurally conserved proteins or basic functional proteins related to immune barriers. No virulence genes related to specific pathogenicity or tissue invasion were found, indicating that the strain has high biosafety at the genomic level.

[0066] (2) The results of antibiotic resistance gene screening showed that the resistance-related genes carried by this bacterium mainly belong to the common endogenous or adaptive resistance elements in intestinal anaerobic bacteria, including antibiotic resistance genes related to β-lactams, macrolides, lincosamides and tetracyclines, and the overall resistance risk is low.

[0067] (3) The MIC test results are shown in Table 2. The bacteria showed low MIC values ​​for metronidazole, clindamycin and fluoroquinolone antibiotics. No abnormally high MIC phenotypes were observed for the other tested antibiotics, indicating that the strain has controllable drug resistance characteristics under multiple antibiotic conditions.

[0068] Table 2. MIC Results of Bacteroides vulgaris N059

[0069]

[0070] The above results indicate that Bacteroides commonis N059 exhibits good biosafety characteristics in in vitro safety evaluation, providing a technical basis for its application in improving gut health.

[0071] Example 5: Method for preparing a liquid bacterial agent containing Bacteroides commonis N059

[0072] The stock solution of *Bacteroides vulgaris* N059, preserved in glycerol supplemented with 0.1% L-cysteine, was streaked onto KVLB solid medium for activation. After anaerobic incubation at 37°C for 48 h, single colonies were picked and inoculated into KVLB liquid medium for overnight incubation. The resulting *Bacteroides vulgaris* N059 bacterial suspension was centrifuged at 5000 rpm for 10 min, washed twice with sterile KVLB liquid medium, and finally the bacterial pellet was dispersed in physiological saline to obtain a viable bacterial suspension with a concentration of 10%. 8 -10 9 CFU / mL means a liquid bacterial agent containing Bacteroides N059.

[0073] Example 6: Method for preparing a solid bacterial agent containing Bacteroides commonis N059

[0074] 6.1 Preparation of live bacterial suspension:

[0075] The activated Bacteroides var. ... 8 ~10 9 CFU / mL was used to obtain a concentrated bacterial suspension.

[0076] 6.2 Add protective agent:

[0077] The above-mentioned concentrated bacterial suspension was mixed with a protectant at a volume ratio of 1:3 and used as feed for low-temperature spray drying. The total solids content of the feed was controlled at 15%-25% (w / w). The mixture was stirred evenly and allowed to stand at 4°C for 0.5-2 hours to allow for full penetration, thereby improving the survival rate of the bacteria during the spray drying process. The protectant formulation is as follows: 10% skim milk powder, 8% maltodextrin, 2% glycerol, 0.1% ascorbic acid, and the remainder being deionized water.

[0078] 6.3 Low-temperature spray drying:

[0079] The premixed feed was subjected to low-temperature spray drying under fully enclosed anaerobic conditions or under nitrogen protection. Specific parameters were as follows: inlet air temperature controlled at 85-95℃, outlet air temperature controlled at 42-48℃. After drying, the resulting powder was immediately cooled to room temperature under nitrogen protection and then packaged in light-proof aluminum foil bags or other oxygen-barrier packaging materials under low humidity conditions to obtain a solid bacterial agent containing Bacteroides commonis N059, which was stored at 4℃.

[0080] 6.4 Viable Bacterial Count Detection:

[0081] Viability testing of the solid bacterial agent containing Bacteroides commonis N059 revealed that, after low-temperature spray drying, the viable count of Bacteroides commonis N059 in the solid bacterial agent could reach 10. 7 -10 8 CFU / mL, and the viable count remained at 10 after 6 months of storage at 4°C. 6 CFU / mL or higher, meeting the requirements for the number of live probiotics in microecological preparations or feed additives.

[0082] Example 7: Preparation of Probiotic Preparations

[0083] 7.1 Preparation of live bacterial suspension of Bacteroides N059:

[0084] Activated Bacteroides vulgaris N059 was added to KVLB liquid medium and anaerobically cultured at 37°C for 24 h. The resulting bacterial suspension was centrifuged at 5000 rpm for 10 min, washed twice with sterile KVLB liquid medium, and finally the bacterial pellet was dispersed in physiological saline to obtain a live bacterial suspension with a concentration of 10%. 8 -10 9 CFU / mL.

[0085] 7.4 Cryo-microcapsule encapsulation:

[0086] To improve bacterial stability, the above-mentioned live Bacteroides N059 bacterial suspension was microencapsulated. The specific procedure is as follows: (1) Mix the live bacterial solution of Bacteroides N059 with 2% sodium alginate solution at a volume ratio of 1:3 and stir evenly to form a mixture of Bacteroides N059 and sodium alginate. (2) The solution was dripped into a 1.5% calcium chloride solution at a constant rate using a syringe to form gel microspheres, which were then solidified for 15 min. (3) Collect the gel microspheres and place them in 0.5% chitosan solution (pH=6.0) for 10 min to encapsulate them and form microcapsules; (4) After pre-freezing the encapsulated microcapsules at -40℃ for 4 h, they were freeze-dried at low temperature. The procedure was: -50℃, 10Pa, 24-36 h. (5) The obtained powder is collected under sterile conditions and sealed with nitrogen to obtain a microecological preparation, which is stored at -20℃ away from light.

[0087] The microecological preparation prepared by this invention is a microcapsule with high mechanical strength and biodegradability, which can enhance the resistance of Bacteroides N059 cells to the acidic environment of the gastrointestinal tract, and at the same time significantly improve its colonization rate and biological activity in the intestine.

[0088] Example 8 Preparation of Synbiotic Formulation

[0089] 8.1 Preparation of live bacterial suspension of Bacteroides N059: Activated Bacteroides vulgaris N059 was added to KVLB liquid medium and anaerobically cultured at 37°C for 24 h. The resulting bacterial suspension was centrifuged at 5000 rpm for 10 min, washed twice with sterile KVLB liquid medium, and finally the bacterial pellet was dispersed in physiological saline to obtain a live bacterial suspension with a concentration of 10%. 8 -10 9 CFU / mL.

[0090] 8.2 Preparation of 3'-SL solution: Weigh 5 g of purified 3'-SL and dissolve it in 100 mL of water to ensure a final concentration of 5%. Filter the solution through a 0.22 μm filter membrane for sterilization.

[0091] 8.3 Preparation of Synbiotic Premix: Common Bacteroides N059 was mixed with 5% 3'-SL solution at a volume ratio of 1:1 to obtain a synbiotic premix.

[0092] 8.4 Cryo-microcapsule encapsulation: To improve bacterial cell stability, the above-mentioned synbiotic premix was microencapsulated. The specific process is as follows: (1) Mix the synbiotic premixed solution and 2% sodium alginate solution at a volume ratio of 1:3 and stir evenly to form a synbiotic-sodium alginate mixture; (2) The solution was dripped into a 1.5% calcium chloride solution at a constant rate using a syringe to form gel microspheres, which were then solidified for 15 min. (3) Collect the gel microspheres and place them in 0.5% chitosan solution (pH=6.0) for 10 min to encapsulate them and form microcapsules; (4) After pre-freezing the encapsulated microcapsules at -40℃ for 4 h, they were freeze-dried at low temperature. The procedure was: -50℃, 10Pa, 24-36 h. (5) The obtained powder was collected under sterile conditions and sealed with nitrogen to obtain the synbiotic preparation, which was stored at -20℃ away from light.

[0093] The prebiotic 3'-SL in the synbiotic preparation prepared in this invention can directionally promote the colonization of Bacteroides commonis N059 in the intestine, thereby optimizing the intestinal microbiota, inhibiting the proliferation of pathogens, and effectively reducing intestinal infection and inflammatory response.

[0094] 8.5 Viable Bacteria Detection: The prepared synbiotic formulation was tested for viability, and the results showed that the viable count of Bacteroides commonis N059 in the synbiotic formulation could reach 10. 7 -10 8 With a CFU / mL concentration, the viability rate reaches over 85%, and the stability of 3'-SL is guaranteed, achieving the goal of targeted release in the intestine.

[0095] Example 9: Synbiotic formulation alleviates DSS-induced colitis in mice

[0096] 9.1 Experimental Procedure for Synbiotic Preparations to Alleviate Colitis in Mice

[0097] Thirty male C57BL / 6 mice aged 6-8 weeks, weighing 18-20g, were selected. The mice were randomly divided into 5 groups of 6 mice each. Except for the control group, all other groups received 2.5% DSS solution on day 7, while the control group received normal water intake, continuing this treatment for 7 days.

[0098] a. Experimental Groups: Control group: Normal water intake.

[0099] DSS group: Drink 2.5% DSS solution on days 7-14, and drink normal water at other times.

[0100] DSS+N059 group: 200 μL of fresh N059 live bacteria solution was administered by gavage daily from day 0 to 7, with a concentration of 10. 8 -10 9 CFU / mL, drink normal water during the period, and drink 2.5% DSS solution from day 7 to day 14.

[0101] DSS+3'-SL group: 200 μL of fresh 3'-SL (5% concentration) was administered by gavage daily from day 0 to day 14, with normal water intake during this period. From day 7 to day 14, 2.5% DSS solution was administered orally.

[0102] DSS+ N059+3'-SL group (i.e., DSS+synbiotic group): 200 μL of fresh N059 live bacteria solution was administered by gavage daily from day 0 to 7, with a concentration of 10. 8 -10 9 CFU / mL, and administered 200 μL of fresh 3'-SL at a concentration of 5% by gavage daily from day 0 to 14, with normal drinking water during this period, and 2.5% DSS solution ingested from day 7 to 14.

[0103] b. Experimental effect evaluation items:

[0104] (1) Assessment of mouse body weight change rate During the experiment, the mice's weight, food intake, and water intake were recorded daily to assess their growth.

[0105] (2) Length and pathological assessment of mouse colon After the experiment, the mice were euthanized, their colons were removed, and their length was measured.

[0106] HE staining was used to observe the infiltration of inflammatory cells in the colonic epithelium and the integrity of the mucosa.

[0107] (3) Detection of inflammatory factors in mouse colon The effects of synbiotics on immune regulation were assessed by detecting the levels of inflammatory factors (TNF-α, IL-6, IL-1β) in mouse serum and the expression levels of tight junction proteins (Claudin-1, Occludin, ZO-1) in colon tissue using quantitative real-time qPCR.

[0108] 7.2 Experimental Results

[0109] Supplementation with Bacteroides var. coli N059 and synbiotic (N059+3'-SL) both alleviated DSS-induced colitis in mice. The synbiotic group showed better results than the Bacteroides var. coli N059 supplementation alone, but supplementation with 3'-sialic acid lactose alone had no significant effect. Specific results are as follows: Figures 6-11 As shown.

[0110] Depend on Figures 6-11 It can be seen that supplementing with synbiotics, rather than 3'-SL or common Bacteroides N059 alone, significantly reversed weight loss. Figure 6 ), shortened colon ( Figure 7 ), lower DAI score ( Figure 8 ), while maintaining the integrity of the colonic mucosa ( Figure 10 Furthermore, the synbiotic supplementation group significantly reversed the DSS-induced inhibition of tight junction proteins Claudin-1, Occludin, and ZO-1. Figure 11 ), and alleviates intestinal inflammation by reducing pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. Figure 9 The above demonstrates that the combined use of Bacteroides N059 and 3'-sialic acid lactose can alleviate DSS-induced colitis in mice, reduce inflammatory factor levels, specifically by restoring weight loss and colonic shortening symptoms in mice, regulating intestinal microecology, and enhancing intestinal barrier function.

[0111] The above descriptions are merely some embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A strain of Bacteroides commonis N059, characterized in that, The taxonomic name of the Bacteroides commonis N059 is Bacteroides commonis. Phocaeicola vulgatus It is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46910 and deposit date of December 11, 2025.

2. The *Bacteroides commonis* strain N059 according to claim 1, characterized in that, The Bacteroides N059 is a Gram-negative bacillus without spores. On KVLB agar supplemented with 5% sterile defibrinated sheep blood, its colonies are milky white, round, raised, and have a smooth, moist surface.

3. The *Bacteroides commonis* strain N059 according to claim 1, characterized in that, The *Bacteroides commonis* N059 can metabolize 3'-sialyl lactose into sialic acid and lactose within 24 hours.

4. A microbial inoculant, characterized in that, The microbial agent is a liquid agent containing Bacteroides N059 as described in claim 1 or a solid agent containing Bacteroides N059 as described in claim 1.

5. The microbial agent according to claim 4, characterized in that, The viable count of Bacteroides commonis N059 in the liquid bacterial agent is 10. 8 ~10 9 CFU / mL, the viable count of Bacteroides N059 in the solid bacterial agent is 10. 7 -10 8 CFU / mL.

6. The method for preparing the microbial inoculant according to claim 4, characterized in that, The preparation of a liquid bacterial agent containing *Bacteroides commonis* N059 as described in claim 1 comprises the following steps: The stock solution of *Bacteroides vulgaris* N059 was streaked onto KVLB solid medium for activation and cultured anaerobically at 37°C for 48 h. Single colonies of *Bacteroides vulgaris* N059 were picked and inoculated into KVLB liquid medium, incubated overnight, and then centrifuged at 5000 rpm for 10 min. The bacterial suspension was washed twice with sterile KVLB liquid medium. Finally, the bacterial pellet was dispersed in physiological saline to obtain a live bacterial suspension of *Bacteroides vulgaris* N059 with a concentration of 10%. 8 -10 9 CFU / mL, to obtain a liquid bacterial agent containing Bacteroides N059; A method for preparing a solid bacterial agent containing *Bacteroides commonis* N059 as described in claim 1, characterized by comprising the following steps: The stock solution of *Bacteroides vulgaris* N059 was streaked onto KVLB solid medium for activation and cultured anaerobicly at 37°C for 48 h. Single colonies of *Bacteroides vulgaris* N059 were picked and inoculated into KVLB liquid medium and cultured anaerobicly at 37°C for 24 h. The resulting bacterial suspension was centrifuged at 12000 rpm for 2 min, and the cells were collected, resuspended in sterile PBS, and the cell concentration was adjusted to 10-1. 8 ~10 9 The concentration of CFU / mL was increased, and a protective agent was added. The mixture was then subjected to low-temperature spray drying. The parameters for the low-temperature spray drying were: inlet air temperature of 85-95℃ and outlet air temperature of 42-48℃. The protective agent consisted of: 10% skim milk powder, 8% maltodextrin, 2% glycerol, 0.1% ascorbic acid, and the remainder being deionized water.

7. A microecological preparation, characterized in that, The microecological preparation contains Bacteroides var. mongolica N059 as described in any one of claims 1-3 or the microbial agent as described in claim 4. The microecological preparation is prepared by microencapsulation using a high molecular weight substance as the base material and a live bacterial solution of Bacteroides var. mongolica N059 as described in any one of claims 1-3 as the core material. The high molecular weight substance is gelatin or sodium alginate.

8. A synbiotic preparation, characterized in that, The synbiotic preparation uses a high molecular weight substance as the base material and a live bacterial liquid of Bacteroides commonis N059 as described in any one of claims 1-3 as the core material, and is prepared by microencapsulation. The prebiotic is 3'-sialyl lactose, and the high molecular weight substance is gelatin or sodium alginate.

9. The use of the common Bacteroides N059 according to any one of claims 1 to 3, or the microbial agent according to any one of claims 4 to 5, or the probiotic preparation according to claim 7, or the synbiotic preparation according to claim 8, in the preparation of a drug for reducing the level of intestinal inflammatory factors.

10. The application of the common Bacteroides N059 according to any one of claims 1 to 3, or the microbial agent according to any one of claims 4 to 5, or the microecological preparation according to claim 7, or the synbiotic preparation according to claim 8, in the preparation of a product for regulating intestinal flora, wherein the product is food or feed.

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