Use of b. longum exopolysaccharide or co-incubation extract with probiotic for improving inflammation

Abstract

The application discloses application of Bifidobacterium longum exopolysaccharide or a co-incubation extractive solution of the Bifidobacterium longum exopolysaccharide and probiotics in improving inflammation. The application finds that the Bifidobacterium longum exopolysaccharide and the co-incubation extractive solution of the Bifidobacterium longum exopolysaccharide and Lactobacillus acidophilus can inhibit LPS-induced TLR4 / NfkB (P65) signal path activation, inhibit the polarization of macrophages from M0 to M1, thereby inhibiting inflammatory reactions, and the inhibiting effect of the co-incubation extractive solution of the Bifidobacterium longum exopolysaccharide and Lactobacillus acidophilus is more obvious. Therefore, the Bifidobacterium longum exopolysaccharide and the co-incubation extractive solution of the Bifidobacterium longum exopolysaccharide and Lactobacillus acidophilus can be used for preparing drugs for inhibiting inflammatory reactions, and are helpful to the treatment of immune and chronic inflammatory diseases and the development of related drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of Bifidobacterium longum extracellular polysaccharide or its co-incubation extract with probiotics in improving inflammation. Background Technology

[0002] The innate immune response, also known as the nonspecific or acquired immune response, is the host's first line of defense against invading pathogens. Pathogen-associated pattern molecules (PAMPs) are a class of molecules expressed by pathogens during infection. The innate immune system's recognition of PAMPs largely depends on innate immune cells, such as Toll-like receptors (TLRs) on macrophages. Except for TLR3, the recognition of PAMPs by all TLRs activates signaling through the MyD88-dependent pathway, leading to the activation and nuclear translocation of nuclear factor-κB (NfκB). NfκB signaling results in AP-1 activation, which in turn leads to the transcription of pro-inflammatory genes, including pro-inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and IL-12, as well as the transcription of many chemokines. Therefore, targeting and inhibiting the TLR4 / NfκB inflammatory pathway, thereby suppressing the production of pro-inflammatory M1 macrophages, holds promise for improving the severity of the inflammatory response.

[0003] Bifidobacterium longum is an important beneficial gut microbiome. Studies have found that the extracellular polysaccharides of the Bifidobacterium longum XZ01 strain can enhance immune activity and improve insulin resistance, but whether it can inhibit inflammatory responses has not been reported. Furthermore, although some studies have found that dietary polysaccharides can be metabolized by the strain to produce small molecule acids, thereby regulating the TLR4 / NfkB inflammatory pathway, affecting macrophage polarization, and ultimately regulating inflammation, there is currently limited information on polysaccharides that can be metabolized by the strain and regulate inflammation. Exploring this information would contribute to the development of anti-inflammatory drugs and has significant clinical implications for the treatment of immune and chronic inflammatory diseases. Summary of the Invention

[0004] The purpose of this invention is to provide the application of Bifidobacterium longum extracellular polysaccharide or its co-incubation extract with probiotics in improving inflammation.

[0005] The first objective of this invention is to provide the use of Bifidobacterium longum extracellular polysaccharide in the preparation of medicaments for inhibiting inflammatory responses.

[0006] A second objective of this invention is to provide the use of the co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus in the preparation of a drug for inhibiting inflammatory responses.

[0007] A third objective of this invention is to provide a drug for suppressing inflammatory responses.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention discloses a co-incubation extract of *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* by co-incubating, centrifuging, filtering, and extracting with ethyl acetate and water-saturated n-butanol. Experiments on the *Bifidobacterium longum* extracellular polysaccharide, the *Lactobacillus acidophilus* extract, and the co-incubation extract of *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* showed that the *Bifidobacterium longum* extracellular polysaccharide and the co-incubation extract of *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* have an inhibitory effect on inflammatory responses, and the effect of the co-incubation extract of *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* is significantly better than that of *Bifidobacterium longum* extracellular polysaccharide. Therefore, this invention applies for protection of the following applications of the *Bifidobacterium longum* extracellular polysaccharide and the co-incubation extract of *Bifidobacterium longum* extracellular polysaccharide.

[0010] This invention seeks protection for the use of Bifidobacterium longum extracellular polysaccharide in the preparation of drugs that inhibit inflammatory responses.

[0011] Specifically, the inflammatory response is an inflammatory response caused by macrophage polarization towards M1 and / or activation of the TLR4 / NfkB (P65) signaling pathway. The extracellular polysaccharide of *Bifidobacterium longum* described in this invention can inhibit macrophage polarization from M0 to M1 and inhibit the activation of the TLR4 / NfkB (P65) signaling pathway, thereby inhibiting the inflammatory response.

[0012] In this invention, the polarization of macrophages from M0 to M1 and the activation of the TLR4 / NfkB(P65) signaling pathway are induced by lipopolysaccharide (LPS).

[0013] Specifically, the extracellular polysaccharide of the *Bifidobacterium longum* has a sugar content of 99.20 ± 1.21% and a relative molecular mass of 6.38 × 10⁻⁶. 5 Da.

[0014] Specifically, 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.

[0015] Specifically, the preparation method of the extracellular polysaccharide of Bifidobacterium longum is as follows: collect the fermentation culture broth of Bifidobacterium longum, centrifuge to remove the bacterial cells in the culture broth, inactivate the enzymes in the culture broth, precipitate the polysaccharide and remove the protein, and then purify to obtain the extracellular polysaccharide of Bifidobacterium longum.

[0016] More specifically, the method for preparing the extracellular polysaccharide of Bifidobacterium longum includes the following steps:

[0017] S1. Culture Bifidobacterium longum in a culture medium and collect the fermentation broth;

[0018] S2. Centrifuge to remove the bacterial cells from the fermentation broth, collect the broth supernatant, inactivate the enzymes in the broth supernatant, precipitate the polysaccharide and remove the protein, and purify to obtain Bifidobacterium longum extracellular polysaccharide.

[0019] Specifically, the Bifidobacterium longum strain is Bifidobacterium longum strain XZ01, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 61618.

[0020] Specifically, the culture medium is MRS liquid medium, and after inoculation, it is placed at 37°C and cultured in an anaerobic environment for 48 hours.

[0021] Specifically, the centrifugation conditions were 8000 rpm, 30 min, and 4℃.

[0022] Specifically, the enzyme inactivation method is as follows: place the supernatant in a water bath at 100°C for 15 minutes to inactivate the enzyme.

[0023] Specifically, the polysaccharide precipitation process involves: concentrating the supernatant after enzyme inactivation under reduced pressure to 1 / 10 of its original volume, adding 3 times the volume of ice-cold ethanol to the concentrate, and allowing it to stand overnight at 4°C; the next day, centrifuging the ethanol-precipitated portion and collecting the precipitate, then reconstituted it with an appropriate amount of ultrapure water to obtain the crude extract of Bifidobacterium longum extracellular polysaccharides; the centrifugation conditions are 8000 rpm, 30 min, and 4°C.

[0024] Specifically, protein removal includes: using Sevage reagent to deproteinize the crude extract of Bifidobacterium longum extracellular polysaccharides; the specific procedure is as follows: add 1 / 5 volume of Sevage reagent (chloroform: n-butanol = 4:1, v / v) to the crude extract, shake vigorously for 15 min, centrifuge at 4500 rpm and 4℃ for 30 min, and collect the supernatant polysaccharide solution; repeat the above steps until the protein layer completely disappears; transfer the protein-free solution to a dialysis bag (molecular weight cutoff: 8000 Da) and dialyze with purified water for 48 h to remove small molecule impurities, changing the water every 4 h during the process; after dialysis, freeze-dry to obtain the crude extract of extracellular polysaccharides, seal and store in a desiccator.

[0025] Specifically, the purification includes DEAE cellulose-52 ion exchange column purification and Sephacryl S-300HR dextran gel purification.

[0026] The present invention also claims protection for the use of the co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus in the preparation of a drug for inhibiting inflammatory responses.

[0027] Specifically, the inflammatory response is an inflammatory response caused by macrophage polarization to M1 and / or activation of the TLR4 / NfkB (P65) signaling pathway.

[0028] Specifically, the Lactobacillus acidophilus strain is Lactobacillus acidophilus CIP 76.13.

[0029] Specifically, the preparation method of the co-incubation extract is as follows: centrifuge the culture medium after co-culturing Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus, collect the supernatant and filter it with a filter membrane, extract the filtrate twice with ethyl acetate and water-saturated n-butanol respectively, evaporate to dryness, rinse with water to form a paste and freeze dry to obtain the freeze-dried product of co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus.

[0030] Specifically, Bifidobacterium longum extracellular polysaccharide was dissolved in liquid culture medium at a mass-to-volume ratio of 1%. At the same time, Lactobacillus acidophilus was inoculated into the liquid culture medium containing Bifidobacterium longum extracellular polysaccharide at an inoculation rate of 2% (v / v). The mixture was placed in an anaerobic incubator and cultured with shaking at 100 rpm for 48 h. After centrifugation at 4500 rpm for 40 min, the supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was extracted twice with equal volumes of ethyl acetate and water-saturated n-butanol. After rotary evaporation, the extract was washed off with water and lyophilized for later use to obtain the lyophilized extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus co-incubation.

[0031] Specifically, the liquid culture medium is MRS culture medium.

[0032] The present invention also provides a drug for inhibiting inflammatory responses, the drug containing a co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus.

[0033] The present invention has the following beneficial effects:

[0034] This invention, through experiments using Bifidobacterium longum extracellular polysaccharide, Lactobacillus acidophilus extract, and a co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus, found that the Bifidobacterium longum extracellular polysaccharide and the co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus described in this invention can inhibit LPS-induced activation of the TLR4 / NfkB(P65) signaling pathway and inhibit macrophage polarization from M0 to M1, thereby suppressing the inflammatory response. Furthermore, the inhibitory effect of the Bifidobacterium longum extracellular polysaccharide + Lactobacillus acidophilus co-incubation extract is more significant. Therefore, the Bifidobacterium longum extracellular polysaccharide and the co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus described in this invention can be used to prepare drugs that inhibit inflammatory responses, contributing to the treatment of immune and chronic inflammatory diseases and the development of related drugs. Attached Figure Description

[0035] Figure 1 This is a high-performance permeation gel chromatogram of the extracellular polysaccharide S-PES-1 from Bifidobacterium longum.

[0036] Figure 2 The results show the monosaccharide composition analysis of Bifidobacterium longum extracellular polysaccharide S-EPS-1; Figure A is the liquid chromatogram of the mixed monosaccharide standard PMP derivatization; Figure 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.

[0037] Figure 3 The UV absorption spectra of total extracellular polysaccharide C-EPS and extracellular polysaccharide S-EPS-1 of Bifidobacterium longum are shown.

[0038] Figure 4 The infrared absorption spectrum of Bifidobacterium longum extracellular polysaccharide S-EPS-1.

[0039] Figure 5 The extracellular polysaccharide S-EPS-1 of Bifidobacterium longum 1 H NMR spectrum.

[0040] Figure 6 The extracellular polysaccharide S-EPS-1 of Bifidobacterium longum 13 C NMR spectrum.

[0041] Figure 7 The HSQC map of Bifidobacterium longum extracellular polysaccharide S-EPS-1.

[0042] Figure 8 This is the COSY map of the extracellular polysaccharide S-EPS-1 of Bifidobacterium longum.

[0043] Figure 9 The results of flow cytometry analysis of LPS-induced macrophage M0 to M1 polarization.

[0044] Figure 10 The results of Western blot analysis on the effects of Bifidobacterium longum extracellular polysaccharide S-EPS-1 (BLEPS), Lactobacillus acidophilus extract (L.acidophilusIE), and Bifidobacterium longum extracellular polysaccharide S-EPS-1 + Lactobacillus acidophilus co-incubation extract (L.acidophilus+BLEPSIE) on protein expression in the TLR4 / NfkB (P65) signaling pathway are presented.

[0045] Figure 11This figure presents the quantitative results of the effects of Bifidobacterium longum extracellular polysaccharide S-EPS-1 (BLEPS), Lactobacillus acidophilus extract (L. acidophilus IE), and the co-incubation extract of Bifidobacterium longum extracellular polysaccharide S-EPS-1 and Lactobacillus acidophilus (L. acidophilus+BLEPSIE) on protein expression in the TLR4 / NfkB (P65) signaling pathway. Figure A shows the effect of BLEPS on TLR4 expression; Figure B shows the effect of BLEPS on P65 expression; Figure C shows the effect of L. acidophilus IE on TLR4 expression; Figure D shows the effect of L. acidophilus IE on P65 expression; Figure E shows the effect of L. acidophilus+BLEPS IE on TLR4 expression; Figure F shows the effect of L. acidophilus+BLEPS IE on P65 expression. *p < 0.05, **p < 0.01, ***p < 0.001. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0047] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0048] The Bifidobacterium longum used in this invention is Bifidobacterium longum strain XZ01, which is a subsp. longum of Bifidobacterium longum, deposited by the Pharmaceutical Analysis Laboratory of the School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University; this strain is also deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 61618.

[0049] The Lactobacillus acidophilus strain used in this invention is CIP 76.13, which was deposited by the Pharmaceutical Analysis Laboratory of the School of Pharmaceutical Sciences, Sun Yat-sen University (Shenzhen).

[0050] The MRS culture medium used in this invention was purchased from Qingdao Haibo Biotechnology Co., Ltd., catalog number HB0384-5; the bacterial genomic DNA extraction kit was purchased from Tiangen Biotech Co., Ltd., catalog number DP302-02; the Taq PCR Mix premix was purchased from Sangon Biotech Co., Ltd., catalog number B639295-0001; and PBS was purchased from Guangzhou Yizibang Bio-instrument Co., Ltd., brand name GIBCO, catalog number C10010500BT.

[0051] The following products were purchased from Dako Biotech Co., Ltd.: 10× Intracellular Staining Permeabilization Wash Buffer (BioLegend, catalog number 421002); Fixation Buffer (BioLegend, catalog number 420801); FcRblock (BioLegend, catalog number 101319); APC anti-mouse F4 / 80 (BioLegend, catalog number 123116); PE anti-mouse CD206 (MMR) (BioLegend, catalog number 141706); and PerCP / Cyanine 5.5 Anti-Mouse CD86 (BioLegend, catalog number 105028).

[0052] Example 1: Resuscitation culture and identification of Bifidobacterium longum and Lactobacillus acidophilus

[0053] This invention involves the resuscitation culture and identification of Bifidobacterium longum strain XZ01 and Lactobacillus acidophilus strain CIP 76.13, respectively. The specific process is as follows:

[0054] 1. Resuscitation and Cultivation

[0055] In a clean bench, wipe the cryovials of strain XZ01 / CIP 76.13 with cotton wool 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, add 500 μL of sterile water to the lyophilized strain powder, and 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 h.

[0056] After 48 hours, a single colony was picked from the streak plate and cultured on a new blood agar plate. The culture was carried out at 37°C under anaerobic conditions for 48 hours to complete the resuscitation culture.

[0057] 2. Strain identification

[0058] Extraction of bacterial genomic DNA: Genomic DNA of the bacterial strain was extracted using a bacterial genomic DNA extraction kit, following the instructions. After obtaining the strain's DNA, its concentration and purity were tested; an OD260 / OD280 ratio within the range of 1.7–1.9 was considered acceptable.

[0059] PCR amplification system and conditions: The upstream and downstream primers, Taq PCR Master Mix and sterile water were thawed on ice. Before use, each reagent was mixed by pipetting. The PCR amplification system was prepared in a sterile enzyme-inactivated PCR tube according to Table 1. After preparation, the PCR tube was briefly centrifuged to remove the reaction liquid adhering to the wall. Then, PCR amplification was performed. The primer sequences and amplification conditions used for amplification are shown in Tables 2 and 3, respectively.

[0060] Table 1 PCR amplification system

[0061]

[0062] Table 2 16S rRNA amplification primer sequences

[0063]

[0064] Table 3 PCR amplification conditions

[0065]

[0066] The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rRNA sequencing. Sequencing comparison revealed that the XZ01 strain revived in this invention is *Bifidobacterium longum* subsp. *longum*, and the CIP 76.13 strain revived in this invention is *Lactobacillus acidophilus*.

[0067] Example 2: Extraction, purification and analysis of extracellular polysaccharides from Bifidobacterium longum

[0068] 1. Obtaining crude extract of extracellular polysaccharides from Bifidobacterium longum XZ01

[0069] The identified XZ01 strain, preserved at -80℃, was taken out and inoculated into MRS liquid medium. After culturing at 37℃ for 24 hours and activating for two generations, it was inoculated into MRS liquid medium at a 2% (v / v) inoculum for expansion culture to obtain fermentation broth. The culture temperature was 37℃ and the culture time was 48 hours.

[0070] After culturing for 48 hours, the fermentation broth was centrifuged (8000 rpm, 30 min, 4℃) to remove the cells. The sterile supernatant was collected and placed in a water bath at 100℃ for 15 min to inactivate the enzymes in the fermentation broth. Subsequently, the supernatant was concentrated under reduced pressure to 1 / 5 of its volume. Three times the volume of ice-cold ethanol was added to the concentrate, and the mixture was allowed to stand overnight at 4℃. The next day, the precipitate was collected by centrifugation (8000 rpm, 30 min, 4℃) and reconstituted with an appropriate amount of ultrapure water to obtain the crude extract of XZ01 extracellular polysaccharides.

[0071] The polysaccharide crude extract was deproteinized using Sevag reagent. The specific procedure was as follows: 1 / 5 volume of Sevag reagent (chloroform: n-butanol = 4:1, v / v) was added to the crude extract, and the mixture was shaken vigorously for 15 min. After centrifugation at 4500 rpm and 4℃ for 30 min, the supernatant polysaccharide solution was collected. The above steps were repeated until the protein layer completely disappeared. The protein-free 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, with the water changed every 4 h. After dialysis, the sample was freeze-dried to obtain the crude extracellular polysaccharide extract, sealed, and stored in a desiccator.

[0072] 2. Decolorization treatment of extracellular polysaccharides from Bifidobacterium longum subsp. XZ01

[0073] The crude extract of extracellular polysaccharides from Bifidobacterium longum 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.

[0074] 3. DEAE cellulose-52 purification

[0075] Purification was performed using a DEAE cellulose-52 ion-exchange cellulose chromatography column. 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, it was first treated with 0.5 mol / L NaOH solution for 1 h, then repeatedly washed with ultrapure water until neutral, and then treated with 0.5 mol / L HCl solution for 1 h, and washed with ultrapure water until neutral.

[0076] Column packing: The chromatography column is 2.6×50cm. After cleaning, fix it vertically on the iron stand. Add 1 / 3 column volume of ultrapure water and open the outlet. Then, slowly pour the packing material into the chromatography column along the glass rod and let it settle naturally. Gently tap the column with a soft rod to remove air bubbles. Then, repeatedly add packing material until it is 5cm away from the top of the chromatography column. Stop packing the column and connect the constant flow pump. Equilibrate with ultrapure water at a flow rate of 1.0mL / min.

[0077] Sample loading: Weigh 400 mg of decolorized crude polysaccharide and dissolve it completely in 10 mL of ultrapure water. After centrifugation at 4500 rpm for 15 min, take the supernatant and filter it through a 0.45 μm filter membrane; load the sample and turn on the constant flow pump to start elution;

[0078] Elution: The eluent was successively 0, 0.05, 0.1, 0.3, and 0.5 mol / L NaCl solution, at a flow rate of 1.0 mL / min, and 10 mL was collected from each tube;

[0079] Detection: The collected sample solution was detected using the sulfuric acid-phenol method in a diaphragm tube, and elution curves were plotted.

[0080] Collection: Based on the elution curve, fractions from different peak segments were combined. The collected eluents were concentrated under reduced pressure and transferred to dialysis bags (3500 Da). Dialysis was performed with distilled water for 24 hours, with the distilled water being changed every 2 hours. After dialysis, the dialysate was freeze-dried to obtain polysaccharides of different components, which were then stored in a desiccator for later use. Further purification was performed using Sephacryl S-300HR dextran gel.

[0081] After decolorization, the polysaccharides were eluted with NaCl at different concentration gradients, resulting in three distinct elution peaks, which were named EPS-1, EPS-2, and EPS-3 in order of elution. EPS-1 is an uncharged neutral polysaccharide, while EPS-2 and EPS-3 are acidic polysaccharides with a certain amount of negative charge. The polysaccharides were then further purified using gel chromatography.

[0082] 4. Sephacryl S-300HR dextran gel purification

[0083] Sephacryl S-300HR is a pretreated packing material, stored in 20% ethanol, and should be washed with ultrapure water before use;

[0084] Column packing: The chromatography column is 1.6×90cm. After cleaning, it is vertically fixed on the iron stand. Add 1 / 3 column volume of ultrapure water and open the outlet. Then, slowly pour the packing material into the chromatography column along the glass rod and let it settle naturally. Gently tap the column with a soft rod to remove air bubbles. Then, repeatedly add packing material to the appropriate height. After the packing liquid surface is calm, connect the constant flow pump and rinse the dextran gel column with 5 column volumes of 0.1mol / L NaCl solution to remove residual ethanol in the packing material and further compact the gel column packing material.

[0085] Sample loading: Weigh 50 mg of each component obtained by DEAE 52 purification, dissolve them completely in 10 mL of ultrapure water, centrifuge at 4500 rpm for 15 min, collect the supernatant and filter it through a 0.45 μm filter membrane; load the sample and turn on the constant flow pump to start elution;

[0086] 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;

[0087] Detection: The collected sample solution was detected using the sulfuric acid-phenol method in a diaphragm tube, and elution curves were plotted.

[0088] Collection: Based on the elution curve, collect the eluent under the elution peak; concentrate, dialyze, and freeze-dry the eluent to obtain purified extracellular polysaccharides.

[0089] Four components were obtained from the Sephacryl S-300HR dextran gel purification, namely S-EPS-1 to S-EPS-4. Among them, the S-EPS-1 component had a higher yield and sugar content. It was used as the research object to determine its chemical composition, molecular weight and monosaccharide composition.

[0090] 5. Analysis

[0091] The relative molecular weight of S-EPS-1 polysaccharides was determined using high-performance gel permeation chromatography (HPGPC), 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; Chromatographic conditions: The chromatographic system used was a gel chromatography-differential-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 DAWNHELEOSⅡ (Wyatt technology, CA, USA); the gel size exclusion columns were Ohpak SB-805HQ (300×8mm), Ohpak SB-804HQ (300×8mm), and Ohpak SB-803HQ (300×8mm) in series; the column temperature was 45℃, the injection volume was 100μL, the mobile phase A (0.02% NaN3, 0.1M NaNO3) flow rate was 0.4mL / min, and the elution gradient was 1mL / min isocratic for 100min.

[0092] High-performance permeation gel chromatogram of the S-PES-1 component of Bifidobacterium longum extracellular polysaccharide is shown below. Figure 1 As shown. By Figure 1 The permeate gel chromatogram of S-EPS-1 shows a single and symmetrical peak, indicating a relatively uniform molecular weight distribution and high purity homogeneous polysaccharide. Based on the linear regression equation, the retention time TR = 5.921 min was used to calculate the relative molecular weight of S-EPS-1 to be 6.38 × 10⁻⁶. 5 Da.

[0093] The monosaccharide composition of S-EPS-1 was determined using a PMP pre-column derivatization-high performance liquid chromatography (HPLC) method. The specific steps were as follows: 5.0 mg of S-EPS-1 sample was weighed into a stoppered reaction tube, 2 mL of trifluoroacetic acid (TFA, 3M) was added, and the tube was sealed. Hydrolysis was carried out in an oil bath at 120 °C for 6 h. After complete hydrolysis, the solution was cooled for 5 min, and methanol was added to concentrate the solution under reduced pressure to dryness (repeated three times to remove residual TFA). Then, 800 μL of ultrapure water was added to dissolve the solution, and 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 solution was reacted in a 70 °C water bath for 30 min. After cooling, 105 μL of 0.3 M NaOH solution was added. Neutralize with HCl solution, then dilute with 200 μL of ultrapure water; subsequently add 600 μL of chloroform solution, vortex to mix, and centrifuge (10000 rpm, 15 min). Discard the lower chloroform layer, repeat three times to remove excess PMP; collect the upper aqueous layer, filter through a 0.45 μm filter membrane, and analyze using HPLC. Chromatographic conditions: Shimadzu LC-20AT HPLC system, Symmetry C18 (Waters, 4.6 × 250 mm) column, UV detector, 0.05 M phosphate buffer (pH 6.7)-acetonitrile (83:17 v / v), flow rate 1.0 mL / min, injection volume 20 μL. Qualitative and quantitative analysis of S-EPS-1 components was performed by comparing the peak time and peak area of ​​each standard monosaccharide (after derivatization), yielding the monosaccharide composition and molar ratio of S-EPS-1.

[0094] The analysis results of the monosaccharide composition of Bifidobacterium longum extracellular polysaccharide S-EPS-1 are as follows: Figure 2 As shown, Figure 2 In the figure, A represents the liquid chromatogram of the PMP-derived mixed monosaccharide standard. Figure 2 B in the figure represents the liquid chromatogram of the S-EPS-1 monosaccharide composition. The liquid chromatogram of the S-EPS-1 component ( Figure 2 Peak diagram of A) and derivatized products of mixed monosaccharide standards (in the figure) Figure 2 Comparing with B) in the previous section, 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 revealed that the specific proportions of monosaccharides in S-EPS-1 differ somewhat from those in existing polysaccharides, suggesting it may be a novel bacterial extracellular polysaccharide structure.

[0095] 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 range, and absorption peaks at 260 nm and 280 nm were observed to determine whether the purified polysaccharide sample contained nucleic acids and proteins.

[0096] The UV absorption spectra of the total extracellular polysaccharide C-EPS and the extracellular polysaccharide S-EPS-1 fraction of Bifidobacterium longum are shown in the figure below. Figure 3 As shown in the figure, based on the ultraviolet scanning spectrum of S-EPS-1 in the wavelength range of 190–800 nm, it can be seen that S-EPS-1 has no ultraviolet absorption at 260 nm and 280 nm, indicating that S-EPS-1 does not contain protein and nucleic acid, and that it has been purified relatively thoroughly.

[0097] Meanwhile, 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. The results showed that the sugar content of S-EPS-1 was high, at 99.20 ± 1.21%, and no nucleic acids or proteins were detected, consistent with the UV scanning results.

[0098] Take an appropriate amount of dried Bifidobacterium extracellular polysaccharide S-EPS-1 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.

[0099] The infrared absorption spectrum of Bifidobacterium longum extracellular polysaccharide S-EPS-1 is shown below. Figure 4 As shown in the figure, 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, a peak is observed 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, the 1023 cm⁻¹ peak... -1 The strong absorption peak at 518 cm⁻¹ indicates the presence of α-(1→6) glycosidic bonds and pyranose in S-EPS-1. -1The 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.

[0100] 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. Furthermore, 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.

[0101] 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 and COSY) spectra.

[0102] Bifidobacterium longum extracellular polysaccharide S-EPS-1 component 1 H NMR spectrum, 13 The results of the C NMR spectrum, HSQC spectrum, and COSY spectrum are as follows: Figures 5-8 As shown, S-EPS-1's 1 H and 13 The chemical shift (ppm) assignments for C are shown in Table 4. 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 4).

[0103] Table 4S-EPS-1 1 H and 13 Chemical shift of C (ppm)

[0104]

[0105] Example 3: Preparation of co-incubated extract and anti-inflammatory experiment

[0106] 1. Preparation of Lactobacillus acidophilus extract and co-incubation extract

[0107] Bifidobacterium longum extracellular polysaccharide (S-EPS-1) was dissolved in liquid culture medium at a mass-to-volume ratio of 1%. At the same time, Lactobacillus acidophilus was inoculated into the liquid culture medium containing Bifidobacterium longum extracellular polysaccharide at an inoculation rate of 2% (v / v). The medium was placed in an anaerobic incubator and cultured with shaking at 100 rpm for 48 h. After centrifugation at 4500 rpm for 40 min, the supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was extracted twice with equal volumes of ethyl acetate and water-saturated n-butanol. After rotary evaporation, the extract was washed off with water and lyophilized for later use to obtain the lyophilized extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus co-incubation.

[0108] The preparation of Lactobacillus acidophilus extract is the same as above, except that Bifidobacterium longum extracellular polysaccharide (S-EPS-1) is not added.

[0109] 2. Construction of the RAW264.7 macrophage inflammation model

[0110] Resuscitation and Culture of RAW264.7 Macrophages: RAW264.7 cell lines were removed from a vertical cryogenic freezer and heated in a 37°C water bath with rapid shaking to thaw the cells. The thawed cell solution was transferred to a 15mL sterile centrifuge tube containing enzymes, and 2mL of DMEM medium containing 10% FBS and 1% penicillin-streptomycin solution was added. The cells were centrifuged at 1000rpm for 3min. After centrifugation, the supernatant was aspirated, and 1mL of medium was added and mixed thoroughly. The mixed cell solution was transferred to a culture dish containing 5mL of medium, and the dish was gently shaken horizontally to mix the cell solution. The cells were observed under a microscope to ensure they were well mixed. The cells were incubated at 37°C, 5% CO2, and saturated humidity. Cells were passaged when the cell density reached 80%–90%.

[0111] M0 to M1 intervention experiment: RAW264.7 macrophages in logarithmic growth phase were divided into groups of 4*102 5The cells were seeded at a density of 2 mL of culture medium per well in 6-well plates and allowed to adhere for 24 h. The old culture medium was discarded, and culture medium containing 10 and 20 μg / mL of S-EPS-1, Lactobacillus acidophilus extract, and S-EPS-1 + Lactobacillus acidophilus co-incubation extract were added respectively. After half an hour, 100 ng / mL of LPS was added. The control without LPS and the control with LPS but without drugs (S-EPS-1, Lactobacillus acidophilus extract or S-EPS-1 + Lactobacillus acidophilus co-incubation extract) were retained and cultured for another 24 h. After incubation, the cells were washed three times with pre-cooled PBS, and then 200 μL / well of RIPA reagent containing phosphatase and protease inhibitors was added for protein extraction. The experiment was repeated to collect cells for flow cytometry analysis.

[0112] 3. Flow cytometry analysis

[0113] Macrophage proportions were detected using flow cytometry: First, FcRblock incubation was performed at 4°C for 5–10 min; then, F4 / 80 and CD86 surface staining was performed at 4°C for 30 min; cells were washed 2–3 times with PBS, and 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 cells were resuspended in 2 mL of 10×Intracellular Staining Permeabilization Wash Buffer diluted 10-fold with ddH2O, centrifuged at 150g for 5 min, and the supernatant was discarded. This step was repeated 2–3 times; cells were resuspended in 100 μL of 1×Intracellular Staining Permeabilization Wash Buffer, CD206 antibody was added, and 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 cells were resuspended in 500 μL of cell staining buffer before loading onto the flow cytometer.

[0114] 4. Western blot

[0115] (1) Extraction, determination and denaturation of total protein:

[0116] Remove the collected cells from the incubator and place them on ice. Use a pipette to aspirate the culture medium and wash the cells three times with pre-cooled PBS solution. Then, add 200 μL of cell lysis buffer (RIPA:PMSF = 50:1, v / v, with one tablet of phosphatase inhibitor per 10 mL) to lyse the cells. After standing for 5 min, scrape the cells off the well wall with a cell scraper and transfer them to a sterile 1.5 mL EP tube to remove enzymes. Mix the lysis buffer and place the tube on ice for 30 min. Then, centrifuge at 4 °C and 14,000 rpm for 30 min. After centrifugation, transfer the supernatant to a sterile 1.5 mL EP tube to remove enzymes.

[0117] Protein assays were performed according to the BCA kit instructions. A standard curve was plotted with standard concentration on the x-axis and absorbance on the y-axis. The protein concentration of the test solution was calculated based on the regression equation of the standard curve. Finally, the protein sample and loading buffer were mixed thoroughly at a volume ratio of 4:1, boiled at 100°C for 10 min to denature the protein, and then aliquoted and stored at -80°C.

[0118] (2) SDS-PAGE electrophoresis:

[0119] After preparing 10% separating gel and 5% stacking gel, vertically insert the comb into the stacking gel and wait for it to solidify. Then, transfer the glass plate to the electrophoresis tank, pour the electrophoresis solution into the tank, remove the comb, and load the sample. Add 5 μL of marker to both wells. First, use electrophoresis at 90V for 15 min to allow the sample to reach the top of the separating gel, then use electrophoresis at 120V for 1 h to allow the sample to reach the bottom of the separating gel. Finally, end the electrophoresis.

[0120] (3) Transfer:

[0121] After electrophoresis, remove the glass plate, take out the gel block, cut off the stacking gel, and soak the separating gel in the transfer buffer. Cut a PVDF membrane to the appropriate size, activate it with methanol solution for 3-5 minutes, and then wash it before use. Soak the sponge and filter paper thoroughly in the transfer buffer, and then lay the sponge, three layers of filter paper, separating gel, PVDF membrane, three layers of filter paper, and sponge in the following order from negative to positive. Clamp the transfer clamp and place it in the electrophoresis tank. Pour the transfer buffer into the tank and surround the electrophoresis tank with ice packs. Perform the transfer at 100V for 55 minutes.

[0122] (4) Blocking and antibody incubation:

[0123] After the transfer was complete, the electroporation clamp was removed, the PVDF membrane was taken out, and washed several times with TBST buffer. The membrane was then placed in BSA blocking buffer and blocked at room temperature for 2 hours. After blocking, the PVDF membrane was cut according to the marker markings, and the cut bands were placed in the corresponding antibodies (TLR4, p65, β-actin) and incubated overnight at 4°C. After incubation, the bands were washed three times with TBST buffer for 10 minutes each time. The washed bands were then incubated with the corresponding secondary antibody for 2 hours, and finally washed several times with TBST buffer.

[0124] (5) Development:

[0125] The ECL chemiluminescence solution was prepared according to the instruction manual, using a volume ratio of solution A to solution B of 1:1. The developing solution was then placed in a multi-functional imager, an appropriate amount of developing solution was added, and photographs were taken and recorded. Using β-actin as an internal reference, the bands were analyzed using ImageJ for quantitative analysis to obtain quantitative results.

[0126] 5. Experimental Results

[0127] Flow cytometry analysis results of LPS-induced macrophage M0 to M1 polarization are as follows: Figure 9 As shown, by Figure 9 It is known that the extracellular polysaccharide S-EPS-1 (BLEPS) of Bifidobacterium longum and the co-incubation extract of S-EPS-1 and Lactobacillus acidophilus (BLEPS IE) can inhibit the polarization from M0 to M1. Moreover, the co-incubation extract has a more significant effect than the extracellular polysaccharide of Bifidobacterium longum. The Lactobacillus acidophilus extract has no effect on the LPS-induced polarization from M0 to M1.

[0128] The Western blot results and quantification of the effects of Bifidobacterium longum extracellular polysaccharide S-EPS-1 (BLEPS), Lactobacillus acidophilus extract (L. acidophilus IE), and Bifidobacterium longum extracellular polysaccharide S-EPS-1 + Lactobacillus acidophilus co-incubation extract (L. acidophilus + BLEPS IE) on protein expression in the TLR4 / NfkB (P65) signaling pathway are as follows: Figure 10 and 11 As shown. Figure 11 In the figure, A represents the effect of BLEPS on TLR4 expression; Figure 11 In the figure, B represents the effect of BLEPS on P65 expression; Figure 11 In the figure, C represents the effect of L. acidophilus IE on TLR4 expression; Figure 11 D in the figure represents the effect of L. acidophilus IE on P65 expression; Figure 11E in the figure represents the effect of L. acidophilus+BLEPS IE on TLR4 expression; Figure 11 F in the figure represents the effect of L. acidophilus+BLEPS IE on p65 expression. Figure 10 and Figure 11 It was found that the extracellular polysaccharide of Bifidobacterium longum, the extract of Lactobacillus acidophilus, and the extract of Bifidobacterium longum extracellular polysaccharide + Lactobacillus acidophilus co-incubation can significantly inhibit the activation of the LPS-induced TLR4 / NfkB(P65) signaling pathway, thereby inhibiting the inflammatory response. Among them, the inhibitory effect of the extract of Bifidobacterium longum extracellular polysaccharide + Lactobacillus acidophilus co-incubation was more obvious.

[0129] Based on the above results, the present invention further used the King's formula to determine whether the combined effect of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus has a synergistic effect in improving inflammation.

[0130] The formula for calculating synergistic effect is:

[0131] Among them, E A Indicates the effect of drug A, E B Indicates the effect of drug B, E A+B Indicates the effect of combined drug use;

[0132] 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.

[0133] Calculations showed that for TLR4, the Q value of the extract co-incubated with *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* in the high-dose (40 μg / mL) group was 1.384, which is greater than 1, indicating a synergistic effect between the two. Similarly, for NfkB(P65), the Q value of the extract co-incubated with *Bifidobacterium longum* extracellular polysaccharide and *Lactobacillus acidophilus* in the medium-dose (20 μg / mL) group was 1.07, also greater than 1, indicating a synergistic effect between the two.

[0134] 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. The application of the co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus in the preparation of anti-inflammatory drugs, characterized in that, The Bifidobacterium longum strain is Bifidobacterium longum XZ01, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 61618; the Lactobacillus acidophilus strain is Lactobacillus acidophilus CIP 76.

13.

2. The application according to claim 1, characterized in that, The preparation method of the co-incubation extract is as follows: the culture medium after co-culturing Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus is centrifuged, the supernatant is collected and filtered with a filter membrane, the filtrate is extracted twice with ethyl acetate and water-saturated n-butanol respectively, evaporated to dryness, and then the paste is washed out with water and freeze-dried to obtain the freeze-dried product of co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus.

3. A drug for anti-inflammatory purposes, characterized in that, The drug contains a co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus; the Bifidobacterium longum is Bifidobacterium longum strain XZ01, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 61618; the Lactobacillus acidophilus is Lactobacillus acidophilus strain CIP 76.

13.

4. The drug according to claim 3, characterized in that, The preparation method of the co-incubation extract is as follows: the culture medium after co-culturing Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus is centrifuged, the supernatant is collected and filtered with a filter membrane, the filtrate is extracted twice with ethyl acetate and water-saturated n-butanol respectively, evaporated to dryness, and then the paste is washed out with water and freeze-dried to obtain the freeze-dried product of co-incubation extract of Bifidobacterium longum extracellular polysaccharide and Lactobacillus acidophilus.

Images