Preparation method and application of ginseng-sourced probiotic fermentation polysaccharide

Abstract

The invention relates to the technical field of biology, in particular to probiotic fermented polysaccharide GPS-F sourced from ginseng, the probiotic fermented polysaccharide GPS-F is prepared from ginseng polysaccharide extract through fermentation of a strain of L.B, and the invention discloses a preparation method and application of the probiotic fermented polysaccharide GPS-F. The probiotic fermented polysaccharide GPS-F is prepared from the ginseng polysaccharide extract through fermentation of the strain of L.B. The invention also discloses a GPS-F scanning electron microscope structure of the probiotic fermented polysaccharide derived from ginseng, and methylation and nuclear magnetism results. The obtained probiotic fermented polysaccharide GPS-F derived from ginseng has good Shigella flexneri resistance activity, and can be further used for development of related treatment products.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of ginseng-derived probiotic fermentation polysaccharide GPS-F. Background Art

[0002] In my country, ginseng has a long history of use in traditional Chinese medicine decoctions and diets. It is a precious Chinese medicine with both medicinal and edible properties, and is known as the "King of Herbs." The 2020 edition of the Chinese Pharmacopoeia stipulates that ginseng is the dried root and rhizome of Panax ginseng C.A.Mey., a plant of the Araliaceae family. It has the effects of replenishing vital energy, restoring the meridians, and regulating immunity. Traditional Chinese Medicine believes that deficiency of vital energy is a major cause of tumors. Traditional Chinese Medicine often treats clinical tumors through the important principle of strengthening the body's vital energy, and ginseng is the most commonly used anti-tumor Chinese medicine for strengthening the body's vital energy in clinical practice. Ginseng polysaccharides are the most abundant active ingredient in ginseng, and have significant immunomodulatory effects and the ability to promote probiotics.

[0003] Previous research has focused on the intrinsic structure and functional characteristics of plant polysaccharides. The use of plant polysaccharides is often accompanied by changes in microbial composition. Probiotic fermentation of the original polysaccharides is often accompanied by the production of new bioactive polysaccharides. The impact of probiotic fermentation on the structure and function of polysaccharides is also gaining increasing attention. During the fermentation process, enzymes, organic acids, free radicals, and other substances in the microbial environment can alter the sugar chain structure of polysaccharides, reducing their molecular weight and modifying their monosaccharide composition, thereby affecting their bioactivity and enriching their efficacy. Furthermore, because ginseng is a valuable medicinal herb, its larger molecular weight precludes biosynthesis. The more clearly defined sugar chain structure and smaller molecular weight facilitate subsequent artificial synthesis, alleviating ginseng shortages and improving economic efficiency. Therefore, studying post-fermentation polysaccharide fragments not only provides a deeper understanding of new material bases but also facilitates the subsequent biosynthesis of smaller molecular weight active polysaccharides.

[0004] In the human intestine, Lachnospiraceae bacteria are the main users of polysaccharides, which play a vital role in host health. A large number of literature reports on the application of Lachnospiraceae bacteria in anti-colon tumor immunity and anti-colon inflammatory response, and they are also considered to be the next generation of probiotics for development. The short-chain fatty acids produced by the metabolism of polysaccharides by Lachnospiraceae bacteria have a significant effect on intestinal health and immune regulation. In addition, the low-molecular-weight polysaccharides produced in this process also have biological activities related to cancer development, immunotherapy and pathogen resistance. However, there is currently no method for the preparation, structural analysis and application of subsequent small-molecule polysaccharide fragments after fermentation of ginseng polysaccharides by Lachnospiraceae bacteria.

[0005] In view of this, it is necessary to provide a preparation method and application of ginseng-derived probiotic fermentation polysaccharide GPS-F to meet the above needs. Summary of the Invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a preparation method and application of ginseng-derived probiotic fermentation polysaccharide GPS-F.

[0007] The invention discloses a ginseng-derived probiotic fermented polysaccharide GPS-F. The ginseng-derived probiotic fermented polysaccharide GPS-F is prepared by fermenting a ginseng polysaccharide extract GPS with Lachnospiraceae bacterium. The Lachnospiraceae bacterium is from the German National Culture Collection DSMZ and has a preservation number of DMS24404.

[0008] The preparation steps of the ginseng-derived probiotic fermented polysaccharide GPS-F are as follows:

[0009] (1) Preparation of ginseng polysaccharide extract GPS;

[0010] (2) Activation and cultivation of Lachnospiraceae bacteria;

[0011] (3) Ginseng polysaccharide extract GPS was added to the bacterial liquid of Lachnospiraceae bacteria for fermentation, centrifugation was performed, the supernatant was collected, water extraction and alcohol precipitation were performed, and the supernatant was freeze-dried under reduced pressure to obtain crude polysaccharide after fermentation.

[0012] (4) The crude polysaccharide obtained in step (3) is eluted by ion exchange column chromatography, and the eluate is collected; and then purified by gel chromatography column to obtain ginseng-derived probiotic fermentation polysaccharide GPS-F.

[0013] The ginseng-derived probiotic fermented polysaccharide GPS-F, the ginseng polysaccharide extract in the preparation step (1) is prepared by the following steps:

[0014] (1) Ginseng slices were ground into powder, and then refluxed with water for extraction, and filtered; the residue was further refluxed with water for extraction, filtered, and the filtrates were combined, concentrated, and ethanol was added, and the mixture was allowed to settle overnight;

[0015] (2) Separate the precipitate and dissolve it in water;

[0016] (3) Chloroform and n-butanol were mixed in a volume ratio of 5:1 to form Sevage reagent, and Sevage reagent was added to the precipitate. The mixture was shaken thoroughly, vortexed, and centrifuged. The mixture was allowed to stand and separated into three layers. The top layer was taken and the above process was repeated 5-10 times until no protein layer was precipitated. The mixture was then freeze-dried under reduced pressure to obtain the ginseng polysaccharide extract GPS.

[0017] The ginseng-derived probiotic fermented polysaccharide GPS-F is prepared by activating and culturing the Lachnospiraceae bacterium in step (2) by: dipping the first-generation bacterial solution of the Lachnospiraceae bacterium in the storage tube, streaking a plate, and then culturing it overnight at 37°C in an anaerobic workstation; picking a single colony on the plate, inoculating it, and activating it in a liquid culture medium for 24 hours to obtain a second-generation bacterial solution, taking the second-generation bacterial solution and adding it to the liquid culture medium for passage, aspirating the bacterial solution every 2 hours, detecting its absorbance value at OD600, drawing a 24-hour bacterial growth curve, and determining the bacterial logarithmic growth phase and plateau phase. Finally, the concentration of the Lachnospiraceae bacterium in the liquid culture medium is 1.5x108CFU / ml.

[0018] The ginseng-derived probiotic fermented polysaccharide GPS-F is prepared by adding the ginseng polysaccharide extract GPS in a liquid culture medium containing Lachnospiraceae bacteria in step (3) at a concentration of 5 to 10 mg / ml.

[0019] The ginseng-derived probiotic fermented polysaccharide GPS-F comprises the following raw materials per liter of liquid culture medium: 10g of tryptone, 17.5g of beef heart extract powder, 5g of sodium chloride, 2.5g of sodium dihydrogen phosphate, and the balance is distilled water; the pH value is 7.2-7.6.

[0020] The invention relates to an application of the ginseng-derived probiotic fermentation polysaccharide GPS-F in the preparation of a medicine for treating infections caused by Shigella flexneri.

[0021] The invention relates to an application of the ginseng-derived probiotic fermentation polysaccharide GPS-F in the preparation of a medicine for treating bacillary dysentery.

[0022] The present invention obtains a polysaccharide fermented by probiotics through the above-mentioned method and purifies it to obtain the ginseng-derived probiotic fermented polysaccharide GPS-F. The polysaccharide was then characterized by molecular weight, monosaccharide composition, scanning electron microscopy, methylation, nuclear magnetic resonance, and structural elucidation of its sugar chain units. Shigella flexneri is one of the most common pathogens of bacillary dysentery. It is highly contagious and can be spread through contaminated food, water, and other sources. Following infection, patients experience typical symptoms such as fever, abdominal pain, and diarrhea, often accompanied by mucus, pus, and blood in the stool. Shigella flexneri is prone to causing chronic bacillary dysentery, which can be protracted and severely impact the patient's health. The ginseng-derived probiotic fermented polysaccharide GPS-F can be used to prepare pharmaceuticals, foods, and health products, including those for inhibiting Shigella flexneri infection. Furthermore, the ginseng-derived probiotic fermented polysaccharide GPS-F can be used alone or in combination with other active ingredients or probiotics, demonstrating promising application prospects and commercial value. Its smaller molecular weight and well-defined sugar chain units also facilitate subsequent synthetic synthesis, potentially alleviating the burden of ginseng consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Shown is the gel chromatography spectrum of GPS-F, a probiotic fermentation polysaccharide derived from ginseng.

[0024] Figure 2 Shown is the Fourier transform infrared spectrum of ginseng-derived probiotic fermentation polysaccharide GPS-F.

[0025] Figure 3 Shown is the monosaccharide composition profile of ginseng-derived probiotic fermentation polysaccharide GPS-F.

[0026] Figure 4 Shown is the molecular weight profile of GPS-F, a probiotic fermentation polysaccharide derived from ginseng.

[0027] Figure 5 Shown is a scanning electron micrograph of GPS-F, a probiotic fermented polysaccharide derived from ginseng.

[0028] Figure 6 Shown is the methylated total ion current of GPS-F, a probiotic fermented polysaccharide derived from ginseng.

[0029] Figure 7 Shown is the 1H NMR spectrum of GPS-F, a probiotic fermentation polysaccharide derived from ginseng.

[0030] Figure 8 Shown is the 13C NMR spectrum of ginseng probiotic fermentation polysaccharide GPS-F.

[0031] Figure 9 Shown is the 1H-1H COSY spectrum of ginseng probiotic fermentation polysaccharide GPS-F.

[0032] Figure 10 Shown is the HMBC spectrum of GPS-F, a polysaccharide fermented from ginseng probiotics

[0033] Figure 11 Shown is the HSQC spectrum of ginseng probiotic fermentation polysaccharide GPS-F.

[0034] Figure 12 Shown is the NOESY spectrum of ginseng probiotic fermentation polysaccharide GPS-F

[0035] Figure 13 Shown is the sugar chain unit structure of GPS-F.

[0036] Figure 14 Shown are the results of the inhibition of Shigella flexneri growth by ginseng probiotic fermented polysaccharide GPS-F. DETAILED DESCRIPTION

[0037] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention record, those skilled in the art can make various changes or modifications to the present invention, but these equivalent forms fall within the scope limited by the application's appended claims equally.

[0038] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention relates. In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by those of ordinary skill in the art without making any creative work shall fall within the scope of protection of the present invention.

[0039] Example 1: Preparation of Ginseng-derived Probiotic Fermented Polysaccharide GPS-F

[0040] (1) Take 500g of ginseng slices, grind them into powder and place them in a round-bottom flask. After reflux and boiling, extract with 5L pure water for 2h and filter with a vacuum pump. Add 4L pure water to the residue and continue reflux extraction for 2h. Filter and repeat once. Combine the filtrates, concentrate under reduced pressure to a volume of 500ml, add 2L of anhydrous ethanol, and let it settle overnight.

[0041] (2) Separate the precipitate and dissolve it in water;

[0042] (3) Chloroform and n-butanol were mixed in a volume ratio of 5:1 to form Sevage reagent. Sevage reagent was added to the precipitate, shaken thoroughly for 3 minutes, vortexed, and centrifuged at 3000 rpm for 15 minutes. The mixture was allowed to stand and separated into three layers. The top layer was taken and the above process was repeated 8 times until no protein layer was precipitated. The mixture was then freeze-dried under reduced pressure to obtain 36.5 g of ginseng polysaccharide extract GPS with a yield of 7.31%.

[0043] (4) The activation and culture method of Lachnospiraceae bacteria is as follows: dip the first generation bacterial solution of Lachnospiraceae bacteria in the preservation tube, streak the plate and then culture it overnight at 37°C in an anaerobic workstation; pick a single colony on the plate and inoculate it in liquid culture medium for 24 hours to obtain a second generation bacterial solution, wherein each liter of liquid culture medium is composed of the following raw materials: 10g of tryptone, 17.5g of ox heart extract powder, 5g of sodium chloride, 2.5g of sodium dihydrogen phosphate, and the balance is distilled water; the pH value is 7.4, and 1ml of the second generation bacterial solution is added to 9ml of liquid culture medium (the same as above) for passage, and 200μl of bacterial solution is taken every 2 hours, and its absorbance value is detected at OD600, and a 24-hour bacterial growth curve is drawn to determine the bacterial logarithmic growth phase and plateau phase.

[0044] (5) When the concentration of Lachnospiraceae bacteria is 1.5x10 8 CFU / ml LB bacterial solution was added with pre-fermentation ginseng polysaccharide GPS (concentration of 5-10 mg / ml), fermented in an anaerobic chamber at 37°C for 12 hours, centrifuged at 12000 rpm for 15 minutes, and the supernatant was collected, extracted with water and precipitated with alcohol, and freeze-dried under reduced pressure to obtain the crude polysaccharide after fermentation.

[0045] (6) The crude polysaccharide obtained in step (5) is eluted by ion exchange column chromatography, and the eluate is collected, concentrated, dialyzed and freeze-dried to obtain a white powder; and then purified by gel chromatography column to obtain ginseng-derived probiotic fermentation polysaccharide GPS-F;

[0046] The specific steps are:

[0047] Ion exchange column chromatography: 1 mg / ml of the crude polysaccharide obtained after fermentation in step (5) above was taken and eluted using an ion exchange chromatography column filled with DEAE-52 cellulose. The eluted components were concentrated, dialyzed and freeze-dried to obtain a white powder.

[0048] Gel column purification: Take approximately 1g of the ion-purified polysaccharide sample and add it to 20ml of pure water. Centrifuge at 10,000g for 10 minutes. Pass the supernatant through a gel chromatography column at a flow rate of 1ml / min. Elute 1.5 times the column volume with pure water, collecting 10ml into a tube and collecting all the eluate.

[0049] The total sugar content of the eluate in each collection tube was determined using the sulfuric acid-phenol method. The specific procedure was as follows: 100 μl of the diluted polysaccharide supernatant was added to 600 μl of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v), the same below), mixed, and reacted in the dark for 10 minutes. The absorbance was measured at 490 nm. The gel purification elution curve was then plotted.

[0050] (7) subjecting the product obtained in step (6) to a sulfuric acid-phenol method to determine the total sugar content of the eluate in each collection tube;

[0051] (8) Determine the main elution peak (the default is to select the main peak, such as Figure 1 The same elution peaks were merged;

[0052] (9) Rotary evaporation to 1 / 5 of the original volume;

[0053] (10) Dialyze for 24-48 hours using a 3 kDa dialysis bag to remove small molecule components.

[0054] (11) The mixture was freeze-dried and the purity of the polysaccharide after gel purification was determined by the sulfuric acid-phenol method, thereby obtaining 17.5 g of ginseng-derived probiotic fermented polysaccharide GPS-F.

[0055] Example 2: Infrared Detection Analysis of Ginseng-derived Probiotic Fermented Polysaccharide GPS-F

[0056] The ginseng-derived probiotic fermented polysaccharide GPS-F prepared by the method of the above-mentioned Example 1 was subjected to infrared detection and analysis using the potassium bromide tableting method. First, 5 g of potassium bromide was dried in an oven at 105°C for 4 hours and stored in a desiccator for later use. 200 mg of potassium bromide powder was placed in an agate mortar and ground under an infrared lamp. After grinding until completely dry and without lumps, an appropriate amount of GPS-F polysaccharide sample prepared by the method of the above-mentioned Example 1 was added and continued to grind to mix the sample evenly in potassium bromide. During the grinding process, it was ensured that the moisture in the GPS-F polysaccharide was completely removed and no lumps were formed. Wipe the tableting mold with anhydrous ethanol. After it was completely dry, an appropriate amount of the mixed polysaccharide sample was added to the mold, spread evenly, and pressed into a transparent thin sheet by a tablet press. The sheet was placed in a Fourier transform infrared spectrometer for infrared spectroscopy, and the scanning range was set to 4000 cm -1 -400cm -1 .

[0057] like Figure 2The infrared scanning spectrum of GPS-F is shown in the figure. -1 There is a broad and blunt strong absorption peak around 2900cm, which is caused by the stretching vibration of -OH in the polysaccharide structure. -1 The absorption peaks on the left and right are caused by the stretching vibration of CH in the polysaccharide structure. These are typical absorption peaks of sugar molecules.

[0058] Example 3: Detection of Monosaccharide Composition of Ginseng-derived Probiotic Fermented Polysaccharide GPS-F

[0059] In a clean chromatographic bottle, weigh an appropriate amount of ginseng-derived probiotic fermented polysaccharide GPS-F prepared by the method of Example 1 above, add 1 ml of 2M TFA acid solution, and heat at 121°C for 2 hours. Pass nitrogen and blow dry. Add 99.99% methanol to wash, blow dry again, and repeat the methanol washing 2-3 times. Add sterile water to dissolve, and transfer to a chromatographic bottle for testing. Take an appropriate amount of supernatant, rotary concentrate or blow dry with nitrogen. Add 1 ml of 2M TFA solution, and heat at 121°C for 2 hours. Pass nitrogen and blow dry. Add 99.99% methanol to wash, blow dry again, and repeat the methanol washing 2-3 times. Add sterile water to dissolve, and transfer to a chromatographic bottle for testing.

[0060] The chromatographic system used was the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), and the monosaccharide components were analyzed and detected using an electrochemical detector. TM CarboPac TM PA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume is 5μl. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 ml / min; column temperature 30℃; elution gradient: 0 min phase A / phase B / phase C (95:5:0, V / V), 26 min phase A / phase B / phase C (85:5:10, V / V), 42 min phase A / phase B / phase C (85:5:10, V / V), 42.1 min phase A / phase B / phase C (60:0:40, V / V), 52 min phase A / phase B / phase C (60:40:0, V / V), 52.1 min phase A / phase B / phase C (95:5:0, V / V), 60 min phase A / phase B / phase C (95:5:0, V / V).

[0061] like Figure 3The results showed that GPS-F is composed of four monosaccharides: rhamnose (Rha), arabinose (Ara), galactose (Gal), and glucose (Glc), with a monosaccharide concentration ratio of 0.4394:2.9619:5.1779:50.6018.

[0062] Example 4: Detection of polysaccharide molecular weight of ginseng-derived probiotic fermented polysaccharide GPS-F

[0063] The ginseng-derived probiotic fermented polysaccharide GPS-F prepared by the method of Example 1 was dissolved in a 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL and filtered through a 0.45 μm pore size filter before analysis. The chromatography system used was a gel chromatography-differential refractive index-multi-angle laser light scattering system, a liquid phase system of U3000 (Thermo, USA), an Optilab T-rEX differential detector (Wyatt Technology, CA, USA), and a laser light scattering detector of DAWN HELEOS II (Wyatt Technology, CA, USA). The specific chromatographic column and elution conditions were as follows: an Ohpak SB-805HQ (300×8 mm) gel exclusion chromatography column and an Ohpak SB-803HQ (300×8 mm) gel exclusion chromatography column were connected in series. The column temperature was 45° C., the injection volume was 100 μL, the mobile phase was A (0.02% NaN 3 , 0.1 M NaNO 3 ), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min.

[0064] like Figure 4 The results showed that the weight-average molecular weight of GPS-F was 358.238 kDa, and the number-average molecular weight was 337.065. This is approximately one-tenth of the weight of the polysaccharide before fermentation. The red line represents the multi-angle laser light scattering signal (LS, in V). The scattered light intensity is proportional to the molecular size and molecular weight of the substance. The blue line represents the difference signal (RI, in RIU). The response value depends on the change in the refractive index of the post-column effluent and is related to the type, concentration, and molecular weight of the substance. The black line represents the molecular weight (Molar Mass (g / mol)) fitted from the two signals.

[0065] Example 5: Scanning electron microscopy examination of the polysaccharide surface physical structure of ginseng-derived probiotic fermented polysaccharide GPS-F

[0066] The polysaccharide GPS-F prepared by the method of Example 1 was passed through a 100-mesh sieve, and a small amount was placed on a conductive carbon tape. After gold spraying, it was photographed using an electron microscope with a magnification of 500-10,000 times.

[0067] Technical parameters:

[0068] Resolution: 1.0nm@15kV; 1.9nm@1kV; 0.8nm@30kV (STEM mode); Accelerating voltage: 0.02–30kV; Beam current: 12pA to 100nA; Magnification: 500-10,000; Electron gun: Thermal field emission Schottky electron gun, beam stability better than 0.2% / h; Detector: High-efficiency in-lens Duo secondary electron detector, Everhart Thornley secondary electron detector; Sample chamber: 330mm(ф)×270mm(h); Image processing: Storage resolution up to 32768×24576 pixels; Multiple integration and averaging modes.

[0069] like Figure 5 Electron microscopy revealed that the surface structure of GPS-F was circular and mainly fluffy and porous, which was similar to the characterization of polysaccharides with immunomodulatory and pathogenic microorganism inhibitory activities reported in many literatures.

[0070] Example 6: Polysaccharide Structure Analysis of Ginseng-derived Probiotic Fermented Polysaccharide GPS-F

[0071] Polysaccharide methylation

[0072] (1) Take a small amount of ginseng probiotic fermented polysaccharide GPS-F (2-3 mg) prepared by the method of Example 1 above, add 500 μl DMSO and dissolve it.

[0073] (2) Add 1 mg of NaOH and incubate for 30 minutes.

[0074] (3) Add 50 μl of iodomethane solution and react for 1 hour.

[0075] (4) Add 1 ml of water and 2 ml of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times.

[0076] (5) Aspirate the lower dichloromethane phase and blow dry with nitrogen.

[0077] (6) Add 100 μl of 2 M TFA and react at 121°C for 90 min.

[0078] (7) Evaporate to dryness at 0℃.

[0079] (8) Add 50 μl of 2 M ammonia water and 50 μl of 1 M NaBD4, mix well, and react at room temperature for 2.5 h.

[0080] (9) Add 20 μl of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μl of methanol, and blow dry with nitrogen.

[0081] (10) Add 250 μl of acetic anhydride, vortex mix, and react at 100 °C for 2.5 h.

[0082] (11) Add 1 ml of water and let it stand for 10 minutes.

[0083] (12) Add 500 μl of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and repeat the water washing three times.

[0084] (13) The lower dichloromethane phase was removed and tested by GC-MS.

[0085] Chromatographic parameters: The chromatographic system used was an Agilent gas chromatograph (Agilent 6890A; Agilent Technologies, USA), using a TG-200 column (30 m × 0.25 mm × 0.25 μm, SGE, Australia). The injection volume was 1 μl, the split ratio was 10:1, and the carrier gas was high-purity helium at a flow rate of 1.5 ml / min. The column oven temperature was initially set at 150°C for 2.0 min, then programmed to 210°C at a rate of 2°C / min, held for 3 min, and then programmed to 240°C at a rate of 2°C / min, held for 5 min. The total ion profile is shown in Figure 1. Figure 6 shown.

[0086] Polysaccharide NMR scanning

[0087] (1) Take an appropriate amount of purified polysaccharide and fully dissolve it in D2O to prepare a polysaccharide solution with a concentration greater than or equal to 40 mg / mL.

[0088] (2) Transfer the dissolved solution to an NMR tube, adding 0.5 mL.

[0089] (3) Place the NMR tube into the NMR spectrometer to scan the one-dimensional 1H spectrum, 13C spectrum, and the two-dimensional COSY, HSQC, HMBC, and NOESY spectra.

[0090] Quantitative analysis of the target compounds was performed using a Bruker (Germany) 600 MHz NMR spectrometer at a scanning temperature of 25°C. The liquid probe used was a QXI 1H / 31P / 13C / 15N 5mm quadruple-resonance reverse detection probe (Z-gradient, ATM Acc). Specifications included: signal-to-noise ratio (1H): 888; resolution (Hz): 0.32 (rotating). The BBFO 1H-19F, 31P-15N, 1H decoupling / observe multinuclear forward detection probe (Z-gradient, ATM) had the following specifications: signal-to-noise ratio (1H): 798; resolution (Hz): 0.26 (rotating); signal-to-noise ratio (13C): 328; resolution (Hz): 0.1.

[0091] Polysaccharide structure analysis process

[0092] like Figure 7As shown, the sample's proton spectrum signals are primarily concentrated between δ3.0 and 5.5 ppm, with multiple coupled signal peaks identified in the anomeric signal region of δ4.3-5.4 ppm, indicating the presence of multiple sugar residues. The corresponding anomeric hydrogen chemical shifts are δ4.54, 4.86, 5.05, 5.25, and 5.3, respectively. Non-anomeric hydrogen signals are primarily concentrated in the δ3.1-4.2 ppm region. Due to significant overlap in some individual signals, the H2-H6 chemical shifts of the sugar residues must be assigned using COSY and HSQC spectra. The strong signal peak near δ4.71 ppm is a solvent peak.

[0093] like Figure 8 As shown, multiple signal peaks were identified in the anomeric carbon region of the sample, and combined with the 13C NMR spectrum and HSQC spectrum ( Figure 11 ) cross-peaks in the anomeric region, and the anomeric signals present in the sample were determined to be: δ5.3 / 99.57, 4.86 / 98.55, 5.25 / 99.67, 4.54 / 104.31, 5.05 / 107.11 ppm, which were recorded as sugar residues A, B, C, D, and E, respectively. Combining the sample bonding structure (methylation) information, anomeric signals, and comprehensive literature reports, it was speculated that sugar residue A was →4)-α-D-Glcp-(1→, sugar residue B was α-D-Glcp-(1→[6,7], sugar residue C was →4,6)-α-D-Glcp-(1→, sugar residue D was →4)-β-D-Galp-(1→, and sugar residue E was α-L-Araf-(1→[8].

[0094] The process of assigning the NMR signals of the main sugar residues is as follows:

[0095] Sugar residue A: The anomeric signal δ5.3 / 99.57 ppm (H1 / C1) indicates that residue A may be an α-configuration glucose residue, such as Figure 9 As shown, in the COSY spectrum, H2 (3.53 ppm) of residue A was determined based on the cross peak δ5.3 / 3.53 ppm, H3 (3.86 ppm) of residue A was determined based on the cross peak δ3.53 / 3.86 ppm, H4 (3.56 ppm) of residue A was determined based on the cross peak δ3.86 / 3.56 ppm, H5 (3.73 ppm) of residue A was determined based on the cross peak δ3.56 / 3.73 ppm, and H6 (3.75 ppm) of residue A was determined based on the cross peak δ3.73 / 3.75 ppm, thereby completing the chemical shifts of hydrogen on the sugar ring. Then, as Figure 11The chemical shifts of C on the sugar ring were assigned by HSQC signals. The chemical shift of C1 of residue A was δ99.57ppm, the chemical shift of C2 of residue A was δ71.45ppm, the chemical shift of C3 of residue A was δ73.28ppm, the chemical shift of C4 of residue A was δ76.57ppm, the chemical shift of C5 of residue A was δ71.09ppm, and the chemical shift of C6 of residue A was δ60.32ppm. The chemical shifts of C1 and C4 shifted to the downfield, indicating that the residue was substituted at the O-1 and O-4 positions of the sugar ring. Combined with the methylation analysis results and literature reports, it was inferred that the sugar residue A might be →4)-α-D-Glcp-(1→

[0096] Sugar residue B: The anomeric signal δ4.86 / 98.55ppm (H1 / C1) indicates that residue B may be an α-configuration glucose residue. In the COSY spectrum, H2 (3.49ppm) of residue B was determined according to the cross peak δ4.86 / 3.49ppm, H3 (3.6ppm) of residue B was determined according to the cross peak δ3.49 / 3.6ppm, H4 (3.31ppm) of residue B was determined according to the cross peak δ3.6 / 3.31ppm, H5 (3.57ppm) of residue B was determined according to the cross peak δ3.31 / 3.57ppm, and H6 (3.73ppm) of residue B was determined according to the cross peak δ3.57 / 3.73ppm, so that the chemical shifts of hydrogen on the sugar ring can be assigned. The chemical shifts of C on the sugar ring were then assigned by HSQC signals. The chemical shift of C1 of residue B was δ98.55 ppm, the chemical shift of C2 of residue B was δ71.65 ppm, the chemical shift of C3 of residue B was δ72.8 ppm, the chemical shift of C4 of residue B was δ69.23 ppm, the chemical shift of C5 of residue B was δ72.62 ppm, and the chemical shift of C6 of residue B was δ60.39 ppm. The chemical shift of C1 shifted to the downfield, indicating that the residue was substituted at the O-1 position of the sugar ring. Combined with the methylation analysis results and literature reports, it was inferred that sugar residue B might be α-D-Glcp-(1→

[0097] Sugar residue C: The anomeric signal δ5.25 / 99.67ppm (H1 / C1) indicates that residue C may be an α-configuration glucose residue. In the COSY spectrum, H2 (3.52ppm) of residue C was determined according to the cross peak δ5.25 / 3.52ppm, H3 (3.84ppm) of residue C was determined according to the cross peak δ3.52 / 3.84ppm, H4 (3.57ppm) of residue C was determined according to the cross peak δ3.84 / 3.57ppm, H5 (3.66ppm) of residue C was determined according to the cross peak δ3.57 / 3.66ppm, and H6 (3.74ppm) of residue C was determined according to the cross peak δ3.66 / 3.74ppm, so that the chemical shifts of hydrogen on the sugar ring can be assigned. The chemical shifts of C on the sugar ring were then assigned by HSQC signals. The C1 chemical shift of residue C was δ99.67 ppm, the C2 chemical shift of residue C was δ71.57 ppm, the C3 chemical shift of residue C was δ73.09 ppm, the C4 chemical shift of residue C was δ76.63 ppm, the C5 chemical shift of residue C was δ72.94 ppm, and the C6 chemical shift of residue C was δ67.8 ppm. The chemical shifts of C1, C4, and C6 shifted to the downfield, indicating that the residue was substituted at the O-1, O-4, and O-6 positions of the sugar ring. Combined with the methylation analysis results and literature reports, it was inferred that the sugar residue C might be →4,6)-α-D-Glcp-(1→

[0098] Sugar residue D: The anomeric signal δ4.54 / 104.31ppm (H1 / C1) indicates that residue D may be a β-configuration galactose residue. In the COSY spectrum, H2 (3.57ppm) of residue D was determined according to the cross peak δ4.54 / 3.57ppm, H3 (3.66ppm) of residue D was determined according to the cross peak δ3.57 / 3.66ppm, H4 (4.08ppm) of residue D was determined according to the cross peak δ3.66 / 4.08ppm, H5 (3.68ppm) of residue D was determined according to the cross peak δ4.08 / 3.68ppm, and H6 (3.65ppm) of residue D was determined according to the cross peak δ3.68 / 3.65ppm, so that the chemical shifts of hydrogen on the sugar ring can be assigned. The chemical shifts of C on the sugar ring were then assigned by HSQC signals. The C1 chemical shift of residue D was δ104.31 ppm, the C2 chemical shift of residue D was δ71.74 ppm, the C3 chemical shift of residue D was δ71.33 ppm, the C4 chemical shift of residue D was δ77.83 ppm, the C5 chemical shift of residue D was δ71.24 ppm, and the C6 chemical shift of residue D was δ60.48 ppm. The chemical shifts of C1 and C4 shifted to the downfield, indicating that the residue was substituted at the O-1 and O-4 positions of the sugar ring. Combined with the methylation analysis results and literature reports, it was inferred that the sugar residue D might be →4)-β-D-Galp-(1→

[0099] Sugar residue E: The anomeric signal δ5.05 / 107.11ppm (H1 / C1) indicates that residue E may be an α-configuration arabinose residue. In the COSY spectrum, H2 (4.04ppm) of residue E was determined according to the cross peak δ5.05 / 4.04ppm, H3 (3.84ppm) of residue E was determined according to the cross peak δ4.04 / 3.84ppm, H4 (3.94ppm) of residue E was determined according to the cross peak δ3.84 / 3.94ppm, and H5 (3.62ppm) of residue E was determined according to the cross peak δ3.94 / 3.62ppm, thereby completing the chemical shifts of hydrogen on the sugar ring. The chemical shifts of C on the sugar ring were then assigned by HSQC signals. The chemical shift of C1 of residue E was δ107.11 ppm, the chemical shift of C2 of residue E was δ81.19 ppm, the chemical shift of C3 of residue E was δ76.72 ppm, the chemical shift of C4 of residue E was δ83.77 ppm, and the chemical shift of C5 of residue E was δ60.66 ppm. The chemical shift of C1 shifted to the downfield, indicating that the residue was substituted at the O-1 position of the sugar ring. Combined with the methylation analysis results and literature reports, it was inferred that the sugar residue E might be α-L-Araf-(1→

[0100] According to the chemical shift of 13C and 1H of each sugar residue in the sample, the structure and connection mode of the polysaccharide were analyzed by combining HMBC and NOESY spectra. Figure 10 and Figure 12 As shown, the structure and connection mode of the polysaccharide were analyzed by combining HMBC and NOESY spectra. According to the HMBC spectrum, there is a cross peak between H1 of sugar residue A and C4 of sugar residue A at δ5.3 / 76.57 ppm, there is a cross peak between H1 of sugar residue A and C4 of sugar residue C at δ5.3 / 76.63 ppm, there is a cross peak between C1 of sugar residue A and H4 of sugar residue A at δ99.57 / 3.56 ppm, and there is a cross peak between C1 of sugar residue A and H4 of sugar residue C at δ99.57 / 3.57 ppm. According to the NOESY spectrum, H1 of sugar residue A and H4 of sugar residue A have cross peaks at δ5.3 / 3.56 ppm, H1 of sugar residue A and H4 of sugar residue C have cross peaks at δ5.3 / 3.57 ppm, H1 of sugar residue B and H6 of sugar residue C have cross peaks at δ4.86 / 3.74 ppm, and H1 of sugar residue C and H4 of sugar residue A have cross peaks at δ5.25 / 3.56 ppm. Due to the low content of sugar residues D and E, the signals in the HMBC and NOESY spectra are weak and therefore not reflected in the sugar chain connection.

[0101] Therefore, based on the analysis of one-dimensional and two-dimensional nuclear magnetic resonance information and methylation results, it was inferred that the polysaccharide is mainly composed of →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→, etc., which are connected to form the main chain. The main branch chain is composed of α-D-Glcp-(1→ connected to the O-6 position of the sugar residue →4,6)-α-D-Glcp-(1→, etc. The sugar chain structure is as follows Figure 13 shown.

[0102] Example 7: Ginseng probiotic fermentation polysaccharide GPS-F inhibits the growth of Shigella flexneri

[0103] Take Shigella flexneri grown to OD = 600 of 0.9-1, dilute it to OD = 600 of 0.1 with sterilized culture medium, and divide it into 9 equal parts, numbered by group and time point. Except for the control group (Control), ginseng polysaccharide GPS before fermentation and ginseng polysaccharide fermentation product GPS-F (ginseng probiotic fermentation polysaccharide GPS-F prepared by the method of Example 1) were added to make the final concentration of polysaccharide 10mg / ml, and the OD = 600 of the corresponding numbered samples was measured at 4h, 8h and 12h, and the bacterial proliferation curve was drawn. At 12h, 1ml of each of the three groups of bacterial liquid was taken, diluted 6 times with sterile PBS at a ratio of 1:10, and 200μl of each bacterial liquid was evenly spread on Columbia blood plates. Bacterial proliferation was observed after 24h.

[0104] like Figure 14 The results showed that GPS-F could inhibit the proliferation of pathogenic bacteria Shigella flexneri in vitro, while the polysaccharide GPS before fermentation had no obvious inhibitory effect on pathogenic bacteria.

[0105] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A ginseng-derived probiotic fermented polysaccharide GPS-F, characterized in that: The ginseng-derived probiotic fermented polysaccharide GPS-F is prepared by fermenting ginseng polysaccharide extract GPS with Lachnospiraceae bacterium. The Lachnospiraceae bacterium is from the German National Culture Collection DSMZ, with the collection number DMS24404.

2. The ginseng-derived probiotic fermented polysaccharide GPS-F according to claim 1, characterized in that: The preparation steps are: (1) Preparation of ginseng polysaccharide extract GPS; (2) Activation and cultivation of Lachnospiraceae bacteria; (3) adding ginseng polysaccharide extract GPS to the bacterial liquid of Lachnospiraceae bacterium for fermentation, centrifuging, taking the supernatant, extracting with water and precipitating with alcohol, and freeze-drying under reduced pressure to obtain crude polysaccharide after fermentation; (4) The crude polysaccharide obtained in step (3) is eluted by ion exchange column chromatography, and the eluate is collected; and then purified by gel chromatography column to obtain ginseng-derived probiotic fermentation polysaccharide GPS-F.

3. The ginseng-derived probiotic fermented polysaccharide GPS-F according to claim 2, characterized in that: The ginseng polysaccharide extract in the preparation step (1) is prepared by the following steps: (1) Ginseng slices were ground into powder, and then refluxed with water for extraction, and filtered; the residue was further refluxed with water for extraction, filtered, and the filtrates were combined, concentrated, and ethanol was added, and the mixture was allowed to settle overnight; (2) Separate the precipitate and dissolve it in water; (3) Chloroform and n-butanol were mixed in a volume ratio of 5:1 to form Sevage reagent, and Sevage reagent was added to the precipitate. The mixture was shaken thoroughly, vortexed, and centrifuged. The mixture was allowed to stand and separated into three layers. The top layer was taken and the above process was repeated 5-10 times until no protein layer was precipitated. The mixture was then freeze-dried under reduced pressure to obtain the ginseng polysaccharide extract GPS.

4. The ginseng-derived probiotic fermented polysaccharide GPS-F according to claim 2, characterized in that: The activation and culture method of the Lachnospiraceae bacterium in the preparation step (2) is as follows: dip the first-generation bacterial solution of the Lachnospiraceae bacterium in the storage tube, streak the plate, and then culture it in an anaerobic workstation at 37°C overnight; pick a single colony on the plate and inoculate it in a liquid culture medium for 24 hours to obtain a second-generation bacterial solution, take the second-generation bacterial solution and add it to the liquid culture medium for passage, aspirate the bacterial solution every 2 hours, detect its absorbance value at OD600, draw a 24-hour bacterial growth curve, determine the bacterial logarithmic growth phase and plateau phase, and finally the concentration of the Lachnospiraceae bacterium in the liquid culture medium is 1.5×10 8 CFU / ml.

5. The ginseng-derived probiotic fermented polysaccharide GPS-F according to claim 2, characterized in that: In the preparation step (3), the concentration of the ginseng polysaccharide extract GPS in the liquid culture medium containing Lachnospiraceae bacteria is 5-10 mg / ml.

6. The ginseng-derived probiotic fermented polysaccharide GPS-F according to claim 4, characterized in that: The liquid culture medium is composed of the following raw materials per liter: 10g of tryptone, 17.5g of beef heart extract powder, 5g of sodium chloride, 2.5g of sodium dihydrogen phosphate, and the balance is distilled water; the pH value is 7.2-7.

6.

7. Use of the ginseng-derived probiotic fermentation polysaccharide GPS-F according to claim 1 in the preparation of a medicament for treating infections caused by Shigella flexneri.

8. Use of the ginseng-derived probiotic fermentation polysaccharide GPS-F according to claim 1 in the preparation of a drug for treating bacillary dysentery.

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