Use of a probiotic complex preparation containing long bifidobacterium exopolysaccharide in the prevention and treatment of colitis

Abstract

The application discloses application of a probiotic compound preparation containing Bifidobacterium longum exopolysaccharide in prevention and treatment of colitis. In the application, the potential of the Bifidobacterium longum exopolysaccharide in preparation of prevention and / or treatment of ulcerative colitis is first proposed, and it is found that the Bifidobacterium longum exopolysaccharide can produce a strong synergistic effect in combination with Lactobacillus acidophilus, and can produce an effect of inhibiting intestinal inflammation of a DSS-induced colitis model mouse.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a probiotic compound preparation containing Bifidobacterium longum extracellular polysaccharide in the prevention and treatment of colitis. Background Technology

[0002] Ulcerative colitis (UC) is a chronic inflammatory disease affecting the colon and rectum, primarily manifesting as abdominal pain, diarrhea, and rectal bleeding. There is currently no highly effective cure for ulcerative colitis. Statistics show that the incidence of ulcerative colitis is increasing year by year, and its course is lengthy, with a risk of developing colon cancer, thus attracting widespread attention. Currently, the etiology of ulcerative colitis is not fully understood, but researchers generally believe that factors such as immunity, genetics, gut microbiota, and environment contribute to the progression of UC. Gut microbiota dysbiosis is a typical clinical manifestation of UC patients, and regulating the gut microbiota through probiotic therapy can alleviate clinical symptoms such as abdominal pain and diarrhea in UC patients. However, the current limitations of probiotic therapies in clinical practice lie in the viability of live bacteria strains and the potential risk of bacteremia.

[0003] Prebiotics refer to organic substances that are not digested and absorbed by the host but can selectively promote the metabolism and proliferation of beneficial bacteria in the body, thereby improving the host's health. The combination of prebiotics and probiotics is a common form of commercially available compound probiotic formulations. Postbiotics are a collective term for the metabolites of probiotics after processing, including the bacterial cells and metabolites. Recent studies have found that the bioactivity of postbiotics on host health may be comparable to that of probiotics, especially in the treatment of gastrointestinal diseases. However, there are currently no products developed or applied using this technology. Summary of the Invention

[0004] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. To this end, this invention provides the application of a probiotic compound preparation containing *Bifidobacterium longum* extracellular polysaccharide in the prevention and treatment of colitis. This invention is the first to propose the potential of *Bifidobacterium longum* extracellular polysaccharide in the preparation of products for the prevention and / or treatment of ulcerative colitis, and discovers that it can produce a strong synergistic effect with *Lactobacillus acidophilus*, resulting in the inhibition of intestinal inflammation in a DSS-induced colitis model mouse.

[0005] In a first aspect, the present invention provides a composition comprising Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus.

[0006] Concerns about the viability of live bacterial strains are a major problem in the application of probiotics in existing technologies. However, this is no longer an issue with the combination of *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* in this invention. First, the combination in this invention does not carry the risk of causing life-threatening diseases such as bacteremia, as with single probiotic therapies. Second, *Bifidobacterium longum* extracellular polysaccharide, as a metabiotic, also has the same efficacy as prebiotics and can selectively promote the metabolism and proliferation of beneficial bacteria in the body, thereby improving host health. Therefore, the use of this combination greatly promotes the development of novel metabiotic-probiotic combined biological agents, and has significant clinical significance and application prospects for the treatment of gastrointestinal diseases and other diseases.

[0007] In some embodiments of the present invention, the Bifidobacterium longum is Bifidobacterium longum XZ01.

[0008] In some embodiments of the present invention, the Lactobacillus acidophilus is Lactobacillus acidophilus CIP 76.13.

[0009] In some embodiments of the present invention, the extracellular polysaccharide of Bifidobacterium longum is composed of mannose, glucose, rhamnose and galactose, wherein the molar ratio of mannose, glucose, rhamnose and galactose is 11.85:0.46:5.60:0.68.

[0010] In some embodiments of the present invention, the extracellular polysaccharide of Bifidobacterium longum has an α-(1→6) glycosidic bond and a pyranose ring structure.

[0011] In some embodiments of the present invention, the method for extracting the extracellular polysaccharide of Bifidobacterium longum is as follows:

[0012] After centrifuging the culture of Bifidobacterium longum XZ01, the supernatant was collected, the enzyme was inactivated by heating in a water bath, the concentration was reduced under reduced pressure, ice-cold ethanol was added, the mixture was allowed to stand overnight, and the precipitate was collected by centrifugation to obtain the extracellular polysaccharide of Bifidobacterium longum XZ01.

[0013] In some embodiments of the present invention, the method for extracting the extracellular polysaccharide of Bifidobacterium longum further includes deproteinization, decolorization and purification processes.

[0014] In some embodiments of the present invention, the deproteinization operation uses Sevag reagent, and the operation method is described in the instruction manual.

[0015] In some embodiments of the present invention, the decolorization is performed using macroporous adsorption resin.

[0016] In some embodiments of the present invention, the macroporous adsorption resin is AB-8 macroporous adsorption resin.

[0017] In some embodiments of the present invention, the purification process is carried out sequentially using a DEAE cellulose-52 ion-exchange cellulose chromatography column and a dextran gel chromatography column.

[0018] In some embodiments of the present invention, the eluent used in the purification process for DEAE cellulose-52 ion exchange cellulose chromatography is a NaCl solution.

[0019] In some embodiments of the present invention, the elution concentrations of the NaCl solution are 0, 0.05, 0.1, 0.3, and 0.5 mol / L, respectively.

[0020] In some embodiments of the present invention, after NaCl elution, dialysis is required, with a molecular weight cutoff of 3500 Da.

[0021] In some embodiments of the present invention, the extracellular polysaccharide component of Bifidobacterium longum is the component obtained after elution with 0 mol / L NaCl.

[0022] In some embodiments of the present invention, the extracellular polysaccharide fraction of Bifidobacterium longum is subjected to dextran gel chromatography.

[0023] In some embodiments of the present invention, the eluent for the dextran gel chromatography is a 0.1 mol / L NaCl solution.

[0024] In some embodiments of the present invention, the ratio of Bifidobacterium longum extracellular polysaccharide to Lactobacillus acidophilus in the composition is 180–220 μg dry weight: 0.5 × 10⁻⁶ μg. 9 ~1.5×10 9 CFU.

[0025] In some embodiments of the present invention, the ratio of Bifidobacterium longum extracellular polysaccharide to Lactobacillus acidophilus in the composition is 200 μg dry weight: 1 × 10⁻⁶. 9 CFU.

[0026] A second aspect of the invention provides the use of the composition described in the first aspect of the invention in the preparation of a medicament for the prevention and / or treatment of colitis.

[0027] In some embodiments of the present invention, the effective content of the composition in the drug is 1 to 100 wt%.

[0028] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0029] In some embodiments of the present invention, the pharmaceutically acceptable excipients include, but are not limited to: solvents, emulsifiers, softeners, antioxidants, preservatives, chelating agents, pH adjusters, thickeners, penetration enhancers, and light-blocking agents.

[0030] In some embodiments of the present invention, the drug is applicable to animals, and the animals are selected from humans, cats, cattle, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice; preferably, the animals are humans.

[0031] A third aspect of the invention provides the use of the composition described in the first aspect of the invention in the preparation of food.

[0032] In some embodiments of the present invention, the food has the function of regulating immunity.

[0033] In some embodiments of the present invention, the regulation of immunity includes:

[0034] (1) Inducing the production of Treg immune cells;

[0035] (2) Inhibit the formation of macrophages.

[0036] In some embodiments of the present invention, the effective content of the composition is 1 to 100 wt%.

[0037] A fourth aspect of the invention provides the use of the composition described in the first aspect of the invention in the preparation of intestinal flora regulators.

[0038] In some embodiments of the present invention, the effective content of the composition is 1 to 100 wt%.

[0039] The beneficial effects of this invention are:

[0040] This invention is the first to propose the application of a probiotic compound preparation containing Bifidobacterium longum extracellular polysaccharide in the prevention and treatment of colitis. In vivo experiments have verified that Bifidobacterium longum extracellular polysaccharide can produce a strong synergistic effect with Lactobacillus acidophilus, thereby inhibiting intestinal inflammation in DSS-induced colitis model mice. Attached Figure Description

[0041] Figure 1 This is a permeation gel chromatogram of the S-PES-1 component of Bifidobacterium longum extracellular polysaccharide.

[0042] Figure 2Analysis of the monosaccharide composition of S-EPS-1, an extracellular polysaccharide of Bifidobacterium longum. A is the liquid chromatogram of the mixed monosaccharide standard PMP derivatization; B is the liquid chromatogram of the monosaccharide composition of S-EPS-1. The peaks in the figure correspond to: 1: mannose; 2: rhamnose; 3: glucuronic acid; 4: galacturonic acid; 5: glucose; 6: galactose; 7: xylose; 8: arabinose; 9: fucose.

[0043] Figure 3 The UV absorption spectra of the total extracellular polysaccharide C-EPS and the extracellular polysaccharide S-EPS-1 component of Bifidobacterium longum are shown.

[0044] Figure 4 The infrared absorption spectrum shows the S-EPS-1 component of Bifidobacterium longum extracellular polysaccharide.

[0045] Figure 5 The extracellular polysaccharide S-EPS-1 component of Bifidobacterium longum 1 H NMR spectrum.

[0046] Figure 6 The extracellular polysaccharide S-EPS-1 component of Bifidobacterium longum 13 C NMR spectrum.

[0047] Figure 7 The HSQC spectrum of the S-EPS-1 component of Bifidobacterium longum extracellular polysaccharide.

[0048] Figure 8 This is the COSY map of the S-EPS-1 component of Bifidobacterium longum extracellular polysaccharide.

[0049] Figure 9 The experimental procedure for the use of Bifidobacterium longum extracellular polysaccharide and its combination with Lactobacillus acidophilus in animal experiments.

[0050] Figure 10 The effect of Bifidobacterium longum extracellular polysaccharide on body weight and DAI score in mice with enteritis (N=7-8).

[0051] Figure 11 The effect of Bifidobacterium longum extracellular polysaccharide on colon length in mice with enteritis (N=7-8, * compared with NC group, * indicates P<0.05, ** indicates P<0.01; # compared with BLZPS+LA group, # indicates P<0.05, ## indicates P<0.01).

[0052] Figure 12 The effect of Bifidobacterium longum extracellular polysaccharide on spleen weight in mice with enteritis (N=7-8, * compared with NC group, * indicates P<0.05, ** indicates P<0.01; # compared with BLZPS+LA group, # indicates P<0.05, ## indicates P<0.01).

[0053] Figure 13 The effect of Bifidobacterium longum extracellular polysaccharide on colonic pathology in mice with enteritis.

[0054] Figure 14 The effect of Bifidobacterium longum extracellular polysaccharide on the proportion of Treg cells in the lamina propria of the colon in mice with enteritis.

[0055] Figure 15 The effect of Bifidobacterium longum extracellular polysaccharide on the proportion of macrophages in the lamina propria of the colon in mice with enteritis.

[0056] Figure 16 To investigate the effect of Bifidobacterium longum extracellular polysaccharide on the diversity of intestinal flora in mice with enteritis. Detailed Implementation

[0057] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0058] Experimental materials

[0059] In the following examples, the Bifidobacterium longum used was Bifidobacterium longum XZ01 (GDMCC NO:61618), and the Lactobacillus acidophilus used was Lactobacillus acidophilus CIP 76.13.

[0060] Resuscitation culture and identification of Bifidobacterium longum and Lactobacillus acidophilus

[0061] (1) Recovery and culture of the strain:

[0062] In a clean bench, wipe the cryovials of the strain with absorbent cotton soaked in 75% alcohol. After the alcohol has dried, unwrap the sealing film, heat the opening of the tube with an alcohol lamp, open the cryovial, and add 500 μL of sterile water to the lyophilized strain powder. After dissolving and mixing, use a sterile inoculation loop to pick up the bacterial solution and streak it onto a Columbia blood agar plate. Incubate at 37°C under anaerobic conditions for 48 hours.

[0063] After 48 hours of incubation, a single colony was picked from the streak plate and transferred to a new blood agar plate for further incubation at 37°C under anaerobic conditions for 48 hours.

[0064] (2) Strain identification:

[0065] Extracting bacterial genomic DNA from the cultured strain involves using a bacterial genomic DNA extraction kit and following the instructions to extract the genomic DNA from the strain. After obtaining the strain's DNA, its concentration and purity are tested; an OD260 / OD280 ratio within the range of 1.7-1.9 is considered acceptable.

[0066] PCR amplification: PCR amplification is performed using qualified DNA as a template.

[0067] The PCR amplification system is shown in Table 1.

[0068] Table 1 PCR amplification system

[0069] Components content Taq PCR Master Mix 12.5μL DNA template 0.5μL Upstream primer (F) 1μL Downstream primer (R) 1μL sterile water 10μL Total volume 25μL

[0070] The upstream primer was 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1);

[0071] The downstream primer was 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2).

[0072] The PCR amplification program was as follows: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 1 min, 55℃ annealing for 1 min, 72℃ extension for 1.5 min, for 30 cycles; 72℃ final extension for 10 min. The amplified products were sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rRNA sequencing to confirm the bacterial species.

[0073] Extraction and purification of extracellular polysaccharides from Bifidobacterium longum

[0074] The confirmed Bifidobacterium longum XZ01 was cultured in MRS liquid medium at 37°C for 24 h. After two generations of activation, it was inoculated into MRS liquid medium at an inoculum of 2% (v / v) for expansion culture at 37°C for 48 h.

[0075] After culturing for 48 hours, the fermentation broth was centrifuged at 8000 rpm for 30 minutes at 4°C to remove the bacterial cells. The supernatant was collected and incubated in a water bath at 100°C for 15 minutes to inactivate the enzymes. The supernatant was then concentrated under reduced pressure to 1 / 5 of its original volume, and three volumes of ice-cold ethanol were added to the concentrate. The mixture was then incubated overnight at 4°C. The next day, the precipitate was centrifuged at 8000 rpm for 30 minutes at 4°C, collected, and reconstituted with an appropriate amount of ultrapure water to obtain a crude extract of extracellular polysaccharides from *Bifidobacterium longum* XZ01.

[0076] The crude extracellular polysaccharide extract of *Bifidobacterium longum* XZ01 was deproteinized using Sevag reagent. Specifically, 1 / 5 volume of Sevag reagent (chloroform: n-butanol = 4:1) was added to the extract, followed by vigorous shaking for 15 min, centrifugation at 4500 rpm for 30 min at 4°C, and collection of the supernatant (polysaccharide solution). This process was repeated until the lower protein layer completely disappeared. The deproteinized solution was transferred to a dialysis bag (molecular weight cutoff: 8000 Da) and dialyzed with purified water for 48 h to remove small molecule impurities, changing the water every 4 h. After dialysis, the extract was freeze-dried to obtain the crude extracellular polysaccharide extract of *Bifidobacterium longum* XZ01, sealed, and stored in a desiccator.

[0077] The crude extract of extracellular polysaccharides from *Bifidobacterium longum* XZ01 was decolorized using AB-8 macroporous adsorption resin. Before decolorization, the AB-8 macroporous adsorption resin was activated. An appropriate amount of resin was weighed into a 2L glass beaker, and sufficient 95% ethanol was added for 12 hours to allow it to fully swell. The resin was then repeatedly rinsed with distilled water until the effluent was clear and free of alcohol odor. The washed resin was dried in a 50℃ oven and then set aside for use. The amount of macroporous adsorption resin used for decolorization was 0.3 g / mL, the concentration of the crude extract of extracellular polysaccharides from *Bifidobacterium longum* XZ01 was 7 mg / mL, and the adsorption time was 3 hours.

[0078] Purification was performed using a DEAE cellulose-52 ion-exchange cellulose chromatography column. The specific steps were as follows: An appropriate amount of DEAE Cellulose 52 ion-exchange cellulose was weighed into a glass beaker, and sufficient ultrapure water was added to swell it. After removing suspended particles, the beaker was treated with 0.5 mol / L NaOH solution for 1 hour, followed by repeated washing with ultrapure water until neutral. Then, it was treated with 0.5 mol / L HCl solution for 1 hour, and washed with ultrapure water until neutral. The chromatography column was 2.6 × 50 cm in size. After cleaning, it was vertically fixed on an iron stand, and 1 / 3 column volume of ultrapure water was added. The outlet was opened. The packing material was then slowly poured into the column along a glass rod, allowing it to settle naturally. The column was gently tapped with a soft rod to remove air bubbles. Packing material (DEAE cellulose-52 ion-exchange cellulose) was repeatedly added until it reached 5 cm from the top of the column. The column was then packed, and a constant flow pump was connected. The column was equilibrated with ultrapure water at a flow rate of 1.0 mL / min. 400 mg of the decolorized crude extracellular polysaccharide extract of *Bifidobacterium longum* XZ01 was weighed and dissolved thoroughly in 10 mL of ultrapure water. After centrifugation at 4500 rpm for 15 min, the supernatant was collected and filtered through a 0.45 μm filter membrane. The sample was loaded, and elution was initiated using a constant flow pump. The eluents were successively 0, 0.05, 0.1, 0.3, and 0.5 mol / L NaCl solutions at a flow rate of 1.0 mL / min, with 10 mL collected per tube. The collected samples were analyzed using the sulfuric acid-phenol method, and elution curves were plotted. Based on the elution curves, the 0 mol / L peak fraction was collected. The collected eluents were concentrated under reduced pressure and transferred to dialysis bags (3500 Da). Dialysis was performed with distilled water for 24 h, with the distilled water replaced every 2 h. After dialysis, the dialysate was freeze-dried to obtain a 0 mol / L polysaccharide gradient (yield 36%), which was then stored in a desiccator for later use.

[0079] Further purification was performed using Sephacryl S-300HR dextran gel chromatography. Sephacryl S-300HR was used as pretreated packing material, stored in 20% ethanol, and washed with ultrapure water before use. The chromatography column was 1.6 × 90 cm, cleaned, and vertically fixed on an iron stand. One-third column volume of ultrapure water was added, and the outlet was opened. The packing material was then slowly poured into the column along a glass rod, allowing it to settle naturally. The column was gently tapped with a soft rod to remove air bubbles. Packing material was repeatedly added until the appropriate height was reached. Once the packing surface was calm, a constant flow pump was connected, and the dextran gel column was rinsed with 5 column volumes of 0.1 mol / L NaCl solution to remove residual ethanol and further compact the gel packing. 50 mg of the polysaccharide fraction obtained from the 0 mol / L gradient purified by DEAE 52 was weighed and dissolved thoroughly in 10 mL of ultrapure water. After centrifugation at 4500 rpm for 15 min, the supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was loaded as the sample, and a constant flow pump was started for elution. The eluent was 0.1 mol / L NaCl solution, the flow rate was 0.5 mL / min, and 4 mL was collected from each tube. The collected sample solution was analyzed using the sulfuric acid-phenol method in separate tubes, and an elution curve was plotted. Based on the elution curve, the eluent below the elution peak was collected. The eluent was concentrated, dialyzed, and lyophilized to obtain purified Bifidobacterium longum XZ01 extracellular polysaccharide.

[0080] The relative molecular weight of S-EPS-1 polysaccharides was determined using gel permeation chromatography (GPC), and their purity was also verified. Specifically, different molecular weight dextran standards were injected sequentially, and their retention times (TR) were recorded. A standard curve was plotted with the TR of each dextran standard on the x-axis and the logarithm of the corresponding molecular weight (Mw) on the y-axis. The regression equation between Lg(Mw) and TR was obtained as follows: lg(Mw) = -0.7765TR + 10.371, with a correlation coefficient R0. 2=0.9942. The chromatographic conditions were as follows: The chromatographic system used was a gel chromatography-differential chromatography-multi-angle laser light scattering system. The liquid chromatography system was a U3000 (Thermo, USA), the differential detector was an Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was a DAWN HELEOSⅡ (Wyatt technology, CA, USA). Size exclusion columns (Ohpak SB-805HQ (300×8 mm), Ohpak SB-804HQ (300×8 mm), and Ohpak SB-803HQ (300×8 mm) in series were used. The column temperature was 45℃, the injection volume was 100 μL, the mobile phase A (0.02% NaN3, 0.1M NaNO3) flow rate was 0.4 mL / min, and the elution gradient was 1 mL / min isocratic for 100 min.

[0081] The results are as follows Figure 1 As shown in the figure, the HPGPC chromatogram of S-EPS-1 shows a single and symmetrical peak, indicating that the relative molecular mass distribution of this component is relatively uniform, and it is a homogeneous polysaccharide with high purity. Based on the linear regression equation, the retention time TR = 5.921 min was used to calculate the relative molecular mass of S-EPS-1 to be 6.38 × 10⁻⁶. 5 Da.

[0082] The monosaccharide composition of S-EPS-1 was identified. The monosaccharide composition of S-EPS-1 was determined using PMP pre-column derivatization-high performance liquid chromatography. The specific steps were as follows: 5.0 mg of S-EPS sample was weighed into a stoppered reaction tube, 2 mL of trifluoroacetic acid (TFA, 3M) was added, and the tube was sealed. The mixture was then hydrolyzed in an oil bath at 120 °C for 6 h. After complete hydrolysis, the mixture was cooled for 5 min, concentrated under reduced pressure with methanol until dry (repeated 3 times to remove residual TFA), and then dissolved in 800 μL of ultrapure water. The completely hydrolyzed polysaccharide solution was transferred to a 1.5 mL centrifuge tube. 100 μL of the completely hydrolyzed polysaccharide solution was taken, and 100 μL each of 0.5 M PMP methanol solution and 0.3 M NaOH solution were added. After mixing, the mixture was reacted in a 70 °C water bath for 30 min. After cooling, 105 μL of 0.3 M HCl solution was added for neutralization, and then 200 μL of ultrapure water was added for dilution. Subsequently, 600 μL of chloroform solution was added, vortexed, and centrifuged (10000 rpm, 15 min). The lower chloroform layer was discarded, and the process was repeated three times to remove excess PMP. The upper aqueous layer was collected, filtered through a 0.45 μm filter membrane, and analyzed by HPLC. The chromatographic conditions were as follows: Shimadzu LC-20AT high-performance liquid chromatograph, Symmetry C18 column (Waters, 4.6 × 250 mm), UV detector, 0.05 M phosphate buffer (pH 6.7)-acetonitrile (v / v 83:17), flow rate 1.0 mL / min, and injection volume 20 μL. By comparing the peak time and peak area of ​​each standard monosaccharide (after derivatization), the S-EPS-1 components were qualitatively and quantitatively analyzed to determine the monosaccharide composition and molar ratio of S-EPS-1.

[0083] The results are as follows Figure 2 As shown.

[0084] The liquid chromatogram of the S-EPS-1 component ( Figure 2 A) Peak diagram of derivatized products from mixed monosaccharide standards ( Figure 2 B) By comparison, based on retention time, S-EPS-1 is mainly composed of mannose, glucose, and small amounts of rhamnose and galactose. Based on peak area, the molar ratio of each monosaccharide is mannose:rhamnose:glucose:galactose = 11.85:0.46:5.60:0.68. Furthermore, database comparison reveals that the specific proportions of monosaccharides in S-EPS-1 of this invention differ somewhat from those of polysaccharides in existing technologies, suggesting that it may be a novel bacterial extracellular polysaccharide structure.

[0085] Ultraviolet (UV) spectroscopy was used to analyze whether the purified polysaccharide sample contained nucleic acids and proteins. The specific procedure was as follows: total extracellular polysaccharide C-EPS (i.e., crude polysaccharide before DEAE column chromatography) and polysaccharide fraction S-EPS-1 were each prepared into 1 mg / mL solutions using ultrapure water. A full-wavelength UV scan was performed in the 190-400 nm wavelength region, and absorption peaks at 260 nm and 280 nm were observed to determine whether the purified polysaccharide sample contained nucleic acids and proteins.

[0086] The results are as follows Figure 3 As shown.

[0087] According to the ultraviolet scanning spectrum of S-EPS-1 in the wavelength range of 190-800nm, it can be seen that S-EPS-1 has no ultraviolet absorption at 260nm and 280nm, which indicates that S-EPS-1 does not contain protein and nucleic acid, and it has been purified relatively thoroughly.

[0088] Simultaneously, the total sugar content in the S-EPS fraction was determined using glucose as a standard via the phenol-sulfuric acid method. The protein content of S-EPS-1 was determined using bovine serum albumin as a standard, following the instructions in the BCA kit.

[0089] The results showed that S-EPS-1 had a high sugar content of 99.20±1.21%, and no nucleic acids or proteins were detected, consistent with the results of ultraviolet scanning.

[0090] Take an appropriate amount of dried Bifidobacterium extracellular polysaccharide S-EPS and compress it into tablets. Perform infrared spectroscopy using ATR-FTIR at wavenumbers of 4000-4000 cm⁻¹. -1 Infrared scanning is performed within the range.

[0091] The infrared absorption spectrum of Bifidobacterium longum extracellular polysaccharide S-EPS-1 is as follows: Figure 4 As shown.

[0092] As can be seen from the image, at 3296cm -1 A strong, broad stretching absorption peak is observed at 2932 cm⁻¹, which is caused by the abundant -OH stretching vibrations present in the sample and is a characteristic absorption peak of polysaccharides. Similarly, at 2932 cm⁻¹... -1 The CH stretching vibration absorption peak, often caused by the CH in hexoses, is another characteristic absorption peak of polysaccharides. (1645 cm⁻¹) -1 The strong absorption peak at 1375 cm⁻¹ corresponds to the absorption peak of water in the sugar molecule, indicating that S-EPS-1 contains a certain amount of bound water. -1 The absorption peak is generated by the CO stretching vibration and CH bending vibration. Additionally, at 1023 cm⁻¹... -1The strong absorption peak at 518 cm⁻¹ indicates the presence of α-(1→6) glycosidic bonds and pyranose in S-EPS-1. -1 The absorption peak at 904 cm⁻¹ further confirms the presence of glycosidic bonds. Meanwhile, the absorption peak at 904 cm⁻¹... -1 The absorption peak observed is due to the asymmetric vibration of the pyranose ring, suggesting the presence of α-D-glucose in the structure; while the 809 cm⁻¹ peak... -1 The absorption peak at that point is very likely a characteristic absorption of the mannose ring or galactopyranose, consistent with the above analysis of the monosaccharide composition of S-EPS-1.

[0093] Typically, amide bonds in proteins are located at 1541 cm⁻¹. -1 A characteristic absorption peak will appear, but S-EPS-1 shows no absorption in this band, indicating that the purified polysaccharide does not contain protein, consistent with the above results. In addition, S-EPS-1 shows absorption at 890 cm⁻¹. -1 There was no absorption at any point, indicating that the monosaccharide it contains does not have a β configuration.

[0094] 30 mg of lyophilized Bifidobacterium longum extracellular polysaccharide S-EPS-1 was taken, dissolved in 0.55 mL of D2O by vortexing, and then lyophilized. This process was repeated three times to remove protons from the sample. Finally, the sample was completely dissolved in 0.55 mL of D2O, centrifuged at 10000 rpm for 5 min, and the supernatant was filtered through a 0.45 μm filter membrane and transferred to a 5 mm NMR tube. Detection was performed using 600 MHz superconducting NMR. 1 D NMR signal ( 1 H NMR and 13 C NMR and two-dimensional NMR (HSQC, HMBC and COSY) spectra.

[0095] The results are as follows Figures 5-8 As shown in Table 2.

[0096] according to 1 H NMR spectrum ( Figure 5 It can be observed that there are a total of 7 proton signals in the anomeric proton region of S-EPS-1, indicating that S-EPS-1 contains 7 types of sugar residues, which are named A, B, C, D, E, F, and G. Further combining... 13 C NMR spectrum ( Figure 6 According to the HSQC spectrum ( Figure 7 ) and COSY spectrum ( Figure 8 ) for each sugar residue in S-EPS-1 1 H and 13 The other signals of C were assigned (as shown in Table 2).

[0097] Table 2S-EPS-1 1 H and13 Chemical shift of C (ppm)

[0098]

[0099] Extracellular polysaccharides of Bifidobacterium longum and their effects in combination with Lactobacillus acidophilus in animal experiments

[0100] (1) Preparation of Bifidobacterium longum extracellular polysaccharide and Bifidobacterium longum extracellular polysaccharide-Lactobacillus acidophilus mixed solution:

[0101] Bifidobacterium longum extracellular polysaccharide solution: Take 100 mg of S-EPS-1 purified in the above example and add it to PBS solution to prepare a 1 mg / mL Bifidobacterium longum extracellular polysaccharide solution. Filter it through a 0.22 μm microporous membrane for later use.

[0102] Bifidobacterium longum extracellular polysaccharide-Lactobacillus acidophilus mixed solution: After rapidly thawing frozen Lactobacillus acidophilus CIP 76.13 at 37℃, centrifuge at 4500rpm for 15min, wash twice with PBS, centrifuge again, and resuspend the cells in 2mL of the above 1mg / mL Bifidobacterium longum extracellular polysaccharide S-EPS-1 solution for later use.

[0103] (2) Establishment of a mouse model of colitis:

[0104] Forty-five six-week-old female C57BL / 6 mice were randomly divided into six groups (n=7-8 per group) according to body weight after one week of acclimatization: control group (Control), model group (NC), 5-aminosalicylic acid group (5-ASA), Bifidobacterium longum XZ01 extracellular polysaccharide group (BL-ZPS), Lactobacillus acidophilus group (LA), and Bifidobacterium longum XZ01 extracellular polysaccharide combined with Lactobacillus acidophilus group (BLZPS+LA). The control and model groups were administered 200 μL of PBS by gavage for nine days prior to the experiment. Mice in the 5-ASA, BL-ZPS, LA, and BLZPS+LA groups were administered 6 mg / mL of 5-aminosalicylic acid solution, Bifidobacterium longum extracellular polysaccharide solution, Lactobacillus acidophilus solution, and a mixture of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus solution by gavage daily. The gavage dose of 5-aminosalicylic acid solution was 50 mg of 5-aminosalicylic acid dry weight / kg (i.e., 8.33 mL of 5-aminosalicylic acid solution). The gavage dose for the BL-ZPS group was 200 μg / animal / day (200 μL, concentration 1 mg / mL), and for the LA group it was 1 × 10⁻⁶ mg / mL. 9 CFU / animal / day. The BLZPS+LA group was administered a mixed solution of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus via gavage, with the gavage dose meeting the requirements of 200 μg / animal / day for Bifidobacterium longum extracellular polysaccharide S-EPS-1 and 10 μg / animal / day for Lactobacillus acidophilus. 9The dosage was based on CFU / mouse / day. After 9 days, the drinking water of the three groups other than the normal control group was replaced with drinking water containing 3% DSS to induce colitis modeling. Two days after the modeling was completed, the mice were sacrificed and their tissues (colon and spleen) were collected.

[0105] The experimental procedure is as follows Figure 9 As shown.

[0106] During the experiment, changes in the mice's body weight were monitored and recorded.

[0107] The DAI value is obtained by comprehensively scoring the percentage of weight loss and disease activity index of diseased animals. The breakdown is as follows: 0 points for unchanged weight, 1-5 points for 1 point, 5-10 points for 2 points, 10-15 points for 3 points, and greater than 15 points for 4 points; 0 points for normal stool consistency, 2 points for loose stool, and 4 points for diarrhea; and 0 points for normal stool, 2 points for occult blood, and 4 points for overt bleeding. The DAI value is obtained by dividing the total score of these three results by 3.

[0108] HE staining was performed on the obtained mouse samples.

[0109] The proportions of Treg cells and macrophages were detected using cell loss assay. The specific steps were as follows: After dissecting the mice, the mesentery and Peyre's nodes were removed with ophthalmic scissors. The colon was opened, and the contents were rinsed with pre-cooled PBS. The colon was cut into 0.5 cm pieces and placed in 8 mL of PBS, then vigorously shaken manually. Centrifugation was performed, and the supernatant was discarded. The colon was further minced to facilitate enzyme digestion. The cells were placed in 1640 medium containing 4 mL of enzymes (2 mg / mL collagenase II, 2 mg / mL collagenase IV, and 1 mg / mL DNase I), and digested at 37°C and 100 rpm for 30 min (using a constant temperature shaking water bath). Centrifugation was then performed at 650 g for 5 min, the supernatant was discarded, and the cells were resuspended in PBS and passed through a 70 μm mesh sieve. The resuspended cell suspension was separated using Percoll, centrifuged at 800 g for 20 min, and the intermediate white turbid layer was aspirated. The cells were resuspended in 1640 medium containing 10% FBS and 1% antibiotics. After counting, the cells were seeded into 96-well plates, approximately 1 × 10⁶ cells per well. 6 Each cell.

[0110] To detect the proportion of Treg cells by flow cytometry, the cells were first resuspended in 100 μL of PBS, and 50 μL of prepared CD4+ and CD25+ solutions were added. The cells were incubated at room temperature in the dark for 25 min. After extracellular staining incubation, the cells were washed with 2 mL of PBS, centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in 500 μL of permeabilization and fixation buffer. The mixture was then incubated at 4°C in the dark for 50 min. The cells were washed with 1 mL of fixation and permeabilization buffer, centrifuged, and the supernatant was discarded. This process was repeated once. The cells were resuspended in 100 μL of PBS, and 50 μL of Anti-Mouse / RatFoxp3PE (FOXP3 diluted 1:20 with PBS) was added. The cells were incubated at 4°C in the dark for 30 min. The cells were washed with 1 mL of fixation and permeabilization buffer, centrifuged, and the supernatant was discarded. The cells were then resuspended and loaded onto the flow cytometry.

[0111] For flow cytometry analysis of macrophage proportions, cells were first incubated with FCR blocking reagent (FcRblock) at 4°C for 5-10 min, followed by surface staining with F4 / 80, CD11b, and CD86 at 4°C for 30 min. Cells were washed 2-3 times with PBS, and then fixed with cell fixative at room temperature in the dark for 30 min. Cells were centrifuged at 150g for 5 min, the fixative was discarded, and the cells were resuspended in 2 mL of 10×Intracellular Staining Permeabilization Wash Buffer diluted 10-fold with ddH2O. The cells were centrifuged at 150g for 5 min, the supernatant was discarded, and this step was repeated 2-3 times. Cells were then resuspended in 100 μL of 1×Intracellular Staining Permeabilization Wash Buffer, CD206 antibody was added, and the cells were incubated at room temperature in the dark for 30 min. After incubation, cells were washed 2-3 times with 2 mL of 1×Intracellular Staining Permeabilization Wash Buffer, and then resuspended in 500 μL of cell staining buffer before loading the flow cytometer.

[0112] Simultaneously, intestinal contents were collected from mice in each group for 16S rRNA gene sequencing of the gut microbiota to analyze the effects of a mixed solution of *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* on the gut microbiota. The Stool DNA Kit was used to extract intestinal microbiota DNA; please refer to the instruction manual for specific procedures. The purity of the extracted DNA was determined by 1% agarose gel electrophoresis. The DNA concentration of the extracted sample was measured using a Nanodrop 2000 nucleic acid analyzer. Based on the concentration, the DNA was diluted with DEPC water to 1 ng / μL.

[0113] The PCR amplification of 16S rRNA was performed in the same manner as in the above examples, and the PCR amplification products were sequenced by Beijing Novogene Technology Co., Ltd.

[0114] Based on the above results, the King's formula was further used to determine whether the combined effect of Bifidobacterium longum 1 extracellular polysaccharide and Lactobacillus acidophilus has a synergistic effect in improving intestinal inflammation and regulating immune cells.

[0115] The formula for calculating synergistic effect is:

[0116] Among them, E A Indicates the effect of drug A, E B Indicates the effect of drug B, E A+B This indicates the combined effect of medication.

[0117] Q<1 indicates that the two drugs have an antagonistic effect when used together, while Q>=1 indicates that the two drugs have a synergistic effect when used together.

[0118] The results are as follows Figures 10-16 As shown.

[0119] Using a classic DSS-induced enteritis mouse model, this study employed a prophylactic pre-administration regimen of 9 days. The in vivo anti-inflammatory activity of *Bifidobacterium longum* extracellular polysaccharide (S-EPS-1) and its combination with *Lactobacillus acidophilus* was analyzed by observing changes in body weight, inflammation scores, and pathological morphology during the administration period. The results showed that pre-gavage administration of 200 μg / mouse / day of S-EPS-1 and a mixture of S-EPS-1 and *Lactobacillus acidophilus* alleviated weight loss and DAI intestinal inflammation scores in enteritis mice, while also mitigating DSS-induced colonic shortening and spleen enlargement. Specifically, the combination of S-EPS-1 and *Lactobacillus acidophilus* showed a synergistic effect of 1.14 on colonic length and 1.68 on spleen weight. The spleen is an important immune organ. DSS induces spleen enlargement, stimulates the immune response, and exacerbates intestinal inflammation. However, administration of Bifidobacterium longum extracellular polysaccharide and its combination with Lactobacillus acidophilus resulted in a decrease in spleen weight, suggesting that Bifidobacterium longum extracellular polysaccharide has a certain immunosuppressive function. Simultaneously, HE staining results showed that, compared to the model group, administration of Bifidobacterium longum extracellular polysaccharide and its combination with Lactobacillus acidophilus reduced local colonic mucosal epithelial loss, decreased lymphocyte infiltration in the lamina propria, increased goblet cell number, and restored the intestinal barrier in mice. This also demonstrates the direct regulatory effect of Bifidobacterium longum extracellular polysaccharide combined with Lactobacillus acidophilus on intestinal pathological morphology.

[0120] Analysis of the composition of Treg immune cells in the lamina propria of mice with DSS-induced enteritis using flow cytometry revealed that *Bifidobacterium longum* extracellular polysaccharide and its combination with *Lactobacillus acidophilus* can regulate the Treg immune cells in the mouse gut, exhibiting in vivo immunomodulatory functions. Furthermore, synergistic analysis of the combined effects of *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* showed that the combination of *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* stimulated the proportion of anti-inflammatory Treg cells, with a synergistic index of 2.78 (strong synergy). This suggests that *Bifidobacterium longum* extracellular polysaccharide and its combination with *Lactobacillus acidophilus* have a good anti-inflammatory function under inflammatory conditions and influence the differentiation of the important Treg immune cell subset.

[0121] Analysis of macrophage composition in the lamina propria of DSS-induced enteritis mice using flow cytometry revealed that *Bifidobacterium longum* extracellular polysaccharide and its combination with *Lactobacillus acidophilus* could inhibit the proportion of macrophages. Synergy analysis showed that the synergy index of the combination of *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* was 1.07 (synergistic effect). This suggests that *Bifidobacterium longum* extracellular polysaccharide and its combination with *Lactobacillus acidophilus* can synergistically inhibit the formation of inflammation-induced innate immune macrophages, thereby suppressing inflammation.

[0122] Decreased gut microbiota diversity is a typical feature of intestinal inflammation. Based on 16S rRNA gene sequencing, the gut microbiota diversity of the intestinal contents of mice with DSS-induced enteritis was analyzed. The results showed that Bifidobacterium longum extracellular polysaccharide and its combination with Lactobacillus acidophilus could alleviate the decrease in microbiota diversity caused by DSS-induced enteritis, which suggests that it may act as a gut microbiota regulator to alleviate intestinal inflammation by improving microbiota diversity.

[0123] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of the composition in the preparation of a medicament for the prevention and / or treatment of colitis; The composition is a combination of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus; The extracellular polysaccharide of Bifidobacterium longum is composed of mannose, glucose, rhamnose and galactose, and the molar ratio of mannose, glucose, rhamnose and galactose is 11.85:0.46:5.60:0.

68. The Lactobacillus acidophilus is Lactobacillus acidophilus CIP 76.13; The Bifidobacterium longum is Bifidobacterium longum XZ01, accession number: GDMCC NO:61618; In the composition, the ratio of Bifidobacterium longum extracellular polysaccharide to Lactobacillus acidophilus is 180~220 μg dry weight: 0.5 × 10⁻⁶. 9 ~1.5×10 9 CFU.

2. The use according to claim 1, characterized in that, The extracellular polysaccharide of Bifidobacterium longum contains α-(1→6) glycosidic bonds and a pyranose ring structure.

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