Lilium lanceolatum polysaccharide as well as preparation method and application of lilium lanceolatum polysaccharide

Abstract

The invention discloses lilium lancifolium polysaccharide as well as a preparation method and application thereof. The lilium lancifolium polysaccharide L01-B1 is extracted from lilies through a process of combining water extraction and alcohol precipitation and a column separation and purification technology. The activity screening research finds that the polysaccharide L01-B1 can selectively enrich the potential probiotic Bifidobacterium longum subsp.suillum in the human intestinal tract, and the polysaccharide L01-B1 can be used for improving the composition of the intestinal tract florae, so that the polysaccharide L01-B1 can be used for selectively enriching the potential probiotic Bifidobacterium longum subsp.suillum in the human intestinal tract.

Description

Technical Field

[0001] The invention belongs to the field of polysaccharides, and in particular relates to a polysaccharide extracted from Lilium lancifolium Thunb., a preparation method and application of the polysaccharide. Background Art

[0002] Lily is the flower bud of Lilium lancifolium Thunb., Lilium brownie F.E. Brown var. viridulum Baker or Liliumpumilum DC. of the Liliaceae family. It has the functions of moistening the lungs, clearing away heat and calming the mind. It is used to treat cough, dizziness, restless sleep and pemphigus. In China, many species belonging to the genus Lilium have been widely used as traditional medicine or dietary resources. Lilies contain a variety of compounds, including polysaccharides, saponins, phenols, alkaloids and flavonoids. Research on lily plants mainly focuses on bulbs. The polysaccharides in the bulbs have pharmacological effects such as antioxidant, immunomodulatory, anti-tumor and antibacterial, but there is little research on the polysaccharides in lily buds. Summary of the invention

[0003] The invention adopts a simple and effective polysaccharide extraction method, and obtains a uniform polysaccharide L01-B1 by using Lilium lily as raw material. Activity screening finds that L01-B1 can selectively enrich Bifidobacterium longum (Bifidobacterium. Longum subsp. suillum) in the human intestine.

[0004] Based on this, the purpose of the present invention is to provide a Lilium lily polysaccharide L01-B1, a preparation method thereof, and a method for selectively enriching Bifidobacterium longum (Bifidobacterium. Longum subsp. suillum) thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] In the first aspect, the present invention provides a Lilium lily polysaccharide L01-B1, whose monosaccharide composition includes 35-40% galactose, 35-40% arabinose, 10-15% rhamnose, 10-15% galacturonic acid, and 1-3% glucuronic acid, based on the molar number of all monosaccharides contained in the polysaccharide as 100%.

[0007] In a specific embodiment, based on the molar number of all monosaccharides contained in the polysaccharide as 100%, the monosaccharide composition of the Lilium lily polysaccharide L01-B1 includes 37.2% galactose, 36.5% arabinose, 13.4% rhamnose, 11.1% galacturonic acid and 1.8% glucuronic acid.

[0008] The structural unit of the polysaccharide is a main chain of 1→4-linked α-galactopyranose uronic acid (GalpA), 1→2-linked α-rhamnose pyranose (Rhap) and 1→6-linked α-galactopyranose (Galp), which has the following structure:

[0009]

[0010] Among them, 1≤n≤11, for example, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11; preferably, 5≤n≤6.

[0011] In a specific embodiment, the weight average molecular weight of the polysaccharide L01-B1 is in the range of 3-300 kDa, preferably 4-60 kDa, more preferably 6-50 kDa, and more preferably 43.9 kDa.

[0012] In a second aspect, a method for preparing the Lilium lily polysaccharide L01-B1 according to the first aspect is provided, comprising the following steps:

[0013] (1) Polysaccharide extraction: The flowers of Lilium lily are extracted with boiling water, the obtained extract is concentrated and dialyzed, the dialyzate is centrifuged to remove the precipitate, and the obtained supernatant is precipitated with ethanol aqueous solution and separated to obtain the precipitate, i.e., the crude polysaccharide LLP of Lilium lily;

[0014] (2) Polysaccharide purification:

[0015] (i) dissolving the crude polysaccharide LLP prepared in step (1) in water and centrifuging the solution, taking the supernatant, and performing preliminary purification using an anion exchange column, sequentially using water and 0.05-0.1 M NaCl aqueous solution for gradient elution, collecting the elution fraction of 0.1 M NaCl aqueous solution, and obtaining a preliminary purified secondary crude polysaccharide LLP01;

[0016] (ii) The secondary crude polysaccharide LLP01 prepared in step (i) is dissolved in water and then centrifuged. The supernatant is purified by gel column to obtain Lilium lily polysaccharide L01-B1.

[0017] Preferably, in step (1), before the boiling water extraction, the Lilium lily flowers are first soaked in an ethanol aqueous solution with a volume fraction of 80 to 98%, such as 95%, for 5 to 14 days, such as 14 days, and then dried (such as air-dried).

[0018] Preferably, in step (1), in the boiling water extraction of Lilium lily flowers, the dosage ratio of Lilium lily flowers to water is 3:30-50, kg / L, preferably 3:30, unit: kg / L; the number of boiling water extractions is 4-10 times, preferably 8 times, each time for 3-5 hours, preferably 4 hours.

[0019] Preferably, in step (1), the extract is concentrated to 1 / 40 to 1 / 25 of the original volume, preferably 1 / 30.

[0020] Preferably, in step (1), the dialysis is performed by dialyzing the concentrated extract in water for 24 to 72 hours. Preferably, in step (1), the volume fraction of the ethanol aqueous solution used for the alcohol precipitation is 80 to 98%, preferably 95%; the volume of the ethanol aqueous solution used for precipitation is 3 to 6 times, for example 4 times, that of the concentrated solution.

[0021] Preferably, in step (1), the alcohol precipitation is performed by adding the ethanol aqueous solution to the supernatant and allowing the supernatant to stand for 6 to 24 hours, for example, 12 hours.

[0022] Preferably, in step (1), after obtaining the precipitate, the precipitate may be washed alternately with ethanol and acetone, each washing 3 to 4 times. Further, the washed precipitate may be dried (eg, freeze-dried) to obtain the crude polysaccharide LLP of Lilium lily.

[0023] Preferably, in step (i), during the dissolution process, the ratio of the crude polysaccharide LLP from Lilium lily flowers to water is 1:2 to 20, for example 1:10, unit: g / mL.

[0024] Preferably, in step (i), the anion exchange column may be DEAE Sepharose TM Fast Flow or DEAEcellucose-52, preferably DEAE Sepharose TM Fast Flow.

[0025] Preferably, in step (i), when the anion exchange column is used for preliminary purification, deionized water, 0.05 M and 0.1 M NaCl aqueous solutions are used in sequence for elution, and the elution components of the 0.1 M NaCl eluate are collected.

[0026] Preferably, step (i) further comprises concentrating the collected elution fraction of the 0.1 M NaCl aqueous solution, dialyzing it in water, and drying (eg freeze-drying) to obtain the secondary crude polysaccharide LLP01.

[0027] Preferably, in step (ii), during the dissolution process, the ratio of the crude polysaccharide of Lilium lily LLP01 to water is 1:5-40, for example 1:20, unit: g / mL.

[0028] Preferably, in step (ii), the gel column can be made of Sephacryl S-200HR or Sephacryl S-300HR, preferably Sephacryl S-300HR; and the eluent used for purifying the supernatant by the gel column is a 0.2M NaCl aqueous solution.

[0029] Preferably, step (ii) further comprises drying (eg freeze-drying) the collected elution fractions to obtain Lilium lily polysaccharide L01-B1.

[0030] In a third aspect, the present invention provides a pharmaceutical composition comprising the Lilium lily flower polysaccharide L01-B1 described in the first aspect or the Lilium lily flower polysaccharide L01-B1 prepared by the preparation method described in the second aspect, and pharmaceutically acceptable excipients.

[0031] In a fourth aspect, the present invention provides the use of the Lilium lily polysaccharide L01-B1 described in the first aspect, the Lilium lily polysaccharide L01-B1 prepared by the preparation method described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of a drug, reagent, diet or nutritional supplement having one or more of the following effects:

[0032] a. Improve the composition of human intestinal flora;

[0033] b. Enrichment of Bifidobacterium longum subsp. suillum in the human intestine;

[0034] c. Enrichment and cultivation of Bifidobacterium longum subsp. suillum from human fecal culture in vitro.

[0035] In a fifth aspect, the present invention provides a method for enriching and culturing Bifidobacterium longum subsp. suillum, which comprises co-culturing the lily lily polysaccharide L01-B1 described in the first aspect or the lily lily polysaccharide L01-B1 obtained by the preparation method described in the second aspect with human intestinal flora.

[0036] In a specific embodiment, the method for enriching and culturing Bifidobacterium longum subsp. suillum comprises the following steps:

[0037] S1: Cultivation of human intestinal flora: Fecal samples collected from healthy individuals were initially cultured in vitro using GB liquid medium, the culture was collected, centrifuged, and the bacteria were collected;

[0038] S2: Co-culture of Lilium lily polysaccharide L01-B1 and human intestinal flora: The human intestinal flora obtained by preliminary in vitro culture in step S1 is enriched and cultured in a GB liquid medium containing Lilium lily polysaccharide L01-B1 at a final concentration of 5 to 20 mg / mL (e.g., 10 mg / mL), and then the culture is collected, and the bacteria are collected by centrifugation, part of the bacteria are stored at -80°C for metagenomic sequencing, and part of the bacteria are stored in glycerol to obtain glycerol-preserved bacteria;

[0039] S3: performing metagenomic sequencing on part of the bacteria collected by centrifugation in step S2 to determine that Bifidobacterium longum subsp. suillum is enriched;

[0040] S4: Isolation, culture, purification and identification of bacteria: The glycerol-preserved bacteria obtained in step S2 are isolated, cultured and purified to obtain Bifidobacterium longum subsp. suillum.

[0041] In a specific embodiment, in steps S1, S2, and S4, the culture conditions are 37°C, anaerobic, and the culture time is 1 to 72 hours, such as 24 hours or 48 hours.

[0042] In a specific embodiment, after the bacterial cells are collected in step S1, glycerol is also used to preserve the bacterial cells.

[0043] In a specific embodiment, the step of using glycerol to preserve bacteria in steps S1 and S2 includes: resuspending the bacteria collected by centrifugation in fresh GB liquid culture medium, adding 80% glycerol in half according to the resuspended volume, and storing at -80°C for later use, that is, glycerol-preserved bacteria.

[0044] In a specific embodiment, in step S2, when step S1 uses glycerol to preserve the bacteria, it also includes a step of reviving the glycerol-preserved bacteria, including: mixing the glycerol-preserved bacteria with GB liquid culture medium for culturing and reviving, the volume ratio of the glycerol-preserved bacteria to the GB liquid culture medium is 1:1000, the culture conditions are 37°C, strictly anaerobic, and the culture time is 24 to 48 hours.

[0045] In a specific embodiment, in step S2, the step of enriching and culturing the human intestinal flora in a GB liquid medium containing Lilium lily polysaccharide L01-B1 at a final concentration of 5 to 20 mg / mL comprises:

[0046] The revived bacterial solution was mixed with GB liquid culture medium, wherein the volume ratio of the bacterial solution to the GB liquid culture medium was 1:1000, and 20-100 mg / mL of Lilium lily polysaccharide L01-B1 aqueous solution was added to make the final concentration of polysaccharide L01-B1 5-20 mg / mL for enrichment culture.

[0047] In a specific embodiment, in step S4, the culture medium used for the isolation and culture of bacteria can be GB solid culture medium, which contains mupirocin with a final concentration of 50 mg / L; the isolation and culture of bacteria can be carried out by dilution plate method, streak method, spread plate method, enrichment culture method, etc., preferably streak method.

[0048] In a specific embodiment, in step S4, after the bacteria are separated and cultured by the streaking method, the method for purifying the bacteria may include the following steps:

[0049] A single colony on the GB solid culture medium was picked and re-inoculated on the GB solid culture medium containing 50 mg / L mupirocin for subculture and purification. After two consecutive subcultures, a single colony was picked for expansion culture using GB liquid culture medium.

[0050] In a specific embodiment, in step S4, 16S rDNA sequencing is performed by PCR, and the obtained sequence is compared with SEQ ID NO: 5. If the similarity is above 98%, it can be determined that Bifidobacterium longum subsp. suillum is obtained by enrichment culture.

[0051] Beneficial effects achieved by the technical solution of the present invention:

[0052] The present invention uses Lilium lily as raw material to obtain a uniform Lilium lily polysaccharide L01-B1. Activity screening found that the polysaccharide L01-B1 can selectively enrich Bifidobacterium longum (Bifidobacterium. Longum subsp. suillum) in the human intestine, and can be further used to improve the composition of intestinal flora. Furthermore, the present invention also confirms that Bifidobacterium longum (Bifidobacterium. Longum subsp. suillum) can produce short-chain fatty acids that are beneficial to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the characteristic HPLC chromatogram of Lilium lily polysaccharide L01-B1 prepared in Example 1.

[0054] Figure 2 This is the characteristic infrared spectrum of the Lilium lily polysaccharide L01-B1 prepared in Example 1.

[0055] Figure 3 The polysaccharide L01-B1 prepared in Example 1 is 1 H NMR(a) and 13 C NMR (b) spectrum and 2D NMR spectra HSQC and HMBC (c).

[0056] Figure 4 It is a spectrum of the metagenomic sequencing results of the intestinal flora in Example 3; wherein, a is a bar graph showing the flora composition analyzed at the phylum level, b is a heat map showing the flora composition analyzed at the species level, c is the relative abundance of Bifidobacterium.longumsubsp.suillum, d is a comparative analysis of two groups at the species level, and e is a LEfSe analysis at the species level.

[0057] Figure 5 Schematic diagram of the isolation and culture of bacteria in Example 3; wherein a is a schematic diagram of the isolation and culture of bacteria, and b is an enlarged display of the colony in a.

[0058] Figure 6 This is the spectrum of the PCR amplification results of the bacteria in Example 3.

[0059] Figure 7 The electron microscopy results of the bacteria in Example 3, wherein a is a transmission electron microscopy image of the bacteria, and b is an enlarged image of a.

[0060] Figure 8 The graphs of short-chain fatty acids produced by the bacteria in Example 3 are as follows: (a) Standard; (b) SCFA graph of Bifidobacterium.longum subsp.suillum; (c) SCFA graph of L01-B1 co-cultured with Bifidobacterium.longum subsp.suillum. DETAILED DESCRIPTION

[0061] The present invention is described in detail below by examples. It should be noted that the examples are intended to better explain the present invention, rather than to limit its scope. Various variations and modifications are permitted without departing from the spirit and scope of the present invention. Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present invention are all conventional raw materials and reagents in this area, and can be obtained by purchasing commercially available products.

[0062] Example 1 Preparation of Lilium lily polysaccharide L01-B1

[0063] (1) Extraction and separation of polysaccharides

[0064] 3kg of Lilium lily (origin in Zhejiang) was soaked in 95% ethanol by volume for 14 days and then air-dried. 30L of water was added to the medicinal material, and the extract was extracted by boiling water extraction for 4 hours each time. The sugar content of the extract was detected by sulfuric acid-phenol method until the color reaction was not obvious. Boiling water extraction was used for 8 times. The extract was concentrated to 1 liter and dialyzed in convection water for 48 hours, and then the dialyzate was concentrated and centrifuged to remove the precipitate. A 95% ethanol aqueous solution was added to the supernatant for alcohol precipitation. While adding ethanol, it was stirred in the same direction. The volume ratio of the extract to ethanol was 1:4 (v / v), and it was allowed to stand overnight. The upper ethanol was discarded, the precipitate was collected by centrifugation, and then the precipitate was washed alternately with ethanol and acetone three times, the precipitate was redissolved in water, and freeze-dried to obtain 132g of Lilium lily crude polysaccharide LLP (relative to the weight of the dry medicinal material, the yield was 4.4%).

[0065] (2) Purification of polysaccharides

[0066] Dissolve 50 g of crude polysaccharide LLP in 500 mL of deionized water, heat and stir to dissolve overnight. Centrifuge to obtain the supernatant and pass it through DEAE Sepharose TM Fast Flow anion exchange column was used for separation. Deionized water, 0.05M NaCl solution and 0.1M NaCl solution were used for elution in turn, the flow rate was set to 100mL / min, and the eluate was collected in a 500mL conical flask. 100μL of solution was taken from each bottle, the sulfuric acid-phenol method was used for color development and the absorbance was detected at 490nm, and the elution curve of absorbance and elution volume was drawn. The components were collected according to the elution curve, concentrated under reduced pressure, dialyzed in water and freeze-dried, and finally the component of 0.1M NaCl eluate was obtained, named secondary crude polysaccharide LLP01 (6.1g).

[0067] 200 mg of secondary crude polysaccharide LLP01 was dissolved in 4 mL of deionized water, centrifuged (4000 r / min), the supernatant was taken, and loaded onto a Sephacryl S-300HR gel column, eluted with 0.2 M NaCl solution, the flow rate was controlled to be 5 mL / 15 min, and the eluate was collected by an automatic collector. The sulfuric acid-phenol method was used to develop the color and detect the absorbance, the elution curve was drawn, the desired separation components were collected, concentrated, dialyzed, and freeze-dried to obtain a uniform polysaccharide L01-B1 (1.04 g).

[0068] Example 2 Structural Identification of Polysaccharides

[0069] The uniform polysaccharide L01-B1 from Lilium lily flowers obtained according to the method of Example 1 was identified. The specific analysis method and results are as follows.

[0070] (1) Polysaccharide homogeneity and molecular weight determination

[0071] The average molecular weight of the polysaccharide sample L01-B1 from Lilium lily was determined by high performance gel permeation chromatography (HPGPC). 4 Da) and KS-804 (exclusion limit 4x10 5 Da) analytical gel column for analysis. The chromatographic conditions are as follows: mobile phase is 0.1M NaNO3, flow rate is 0.5mL / min, column temperature is 40°C, Agilent 1260 liquid chromatograph is used, and the detectors are differential detector and ultraviolet detector (detection wavelength is 280nm). Figure 1 The characteristic high performance liquid chromatography spectrum shown indicates that the average relative molecular mass of the Lilium lily flower polysaccharide sample L01-B1 is 43.9 kDa.

[0072] (2) Determination of monosaccharide composition by PMP-HPLC derivatization

[0073] Preparation of monosaccharide standard solutions: The nine monosaccharide standards are D-mannose, L-rhamnose, D-glucuronic acid, D-galacturonic acid, D-glucose, D-galactose, L-xylose, L-arabinose and L-fucose. Dissolve 9 mg of each of the nine monosaccharides in 1 mL of deionized water, then take 100 μL of each of the above monosaccharide solutions and mix them to obtain standard solutions with a concentration of 1 mg / mL for each of the nine monosaccharides.

[0074] Preparation of monosaccharide standard: Take 50 μL of the above standard solution in a 2 mL EP tube, add 50 μL of 0.6 M NaOH solution and 100 μL of 0.5 M PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution, react for 100 min in a 70 ° C water bath, add 100 μL of 0.3 M hydrochloric acid solution to the solution to neutralize and end the reaction, then add 700 μL of deionized water and 1 mL of chloroform, vortex for 30 min and let stand for 30 min, then centrifuge and draw 800 μL of the upper aqueous phase solution into a new 2 mL EP tube. Add chloroform again, extract twice, centrifuge and take the upper aqueous phase, filter through a 0.22 μm microporous membrane and put into a liquid phase vial to obtain the monosaccharide standard.

[0075] Sample preparation: Take 2-3 mg of dried Lilium polysaccharide L01-B1 sample into a pear-shaped flask, add 2 mL of deionized water and 2 ml of 4 M trifluoroacetic acid, seal it and completely acid hydrolyze it at 110 ° C for 4 hours, cool the sample to room temperature, repeatedly add methanol and rotary evaporation to remove excess trifluoroacetic acid, add 200 μL of deionized water to dissolve the sample, take out 50 μL and add it to a 2 mL EP tube, and the subsequent steps (derivation and extraction) are the same as the preparation steps of monosaccharide standards.

[0076] Preparation of mobile phase: Weigh 13.6 g of potassium dihydrogen phosphate and 1.8 g of sodium hydroxide and dissolve them in 2 L of milli Q water. Pass the aqueous phase through a filter membrane to obtain solution ①. Measure acetonitrile (HPLC grade) and mix it with solution ① in a ratio of 17:83 (v / v). Ultrasonicate for 30 min to mix them evenly to obtain the mobile phase.

[0077] The monosaccharide composition results showed that in the monosaccharide composition of L01-B1, the molar ratio of galactose, arabinose, rhamnose, galacturonic acid and glucuronic acid was 37.2:36.5:13.4:11.1:1.8.

[0078] (3) Infrared spectroscopy analysis

[0079] About 2 mg of Lilium lily polysaccharide L01-B1 sample was taken and the infrared spectrum of the polysaccharide sample was measured by potassium bromide (KBr) tablet method. Figure 2 ), at 3394.67cm -1 、2936.13cm -1 、1417.85-1041.73cm -1 、1728.96cm -1 There is an absorption peak.

[0080] (4) Nuclear Magnetic Resonance Analysis

[0081] Take 35 mg of Lilium lily polysaccharide L01-B1 sample, dissolve it in 0.5 mL D2O, and add 1.5 μL acetone as internal standard (δ H =2.29ppm,δ C =31.5ppm), and the one-dimensional 1 H NMR, 13 C NMR and 2D NMR spectra HSQC and HMBC, the results are as follows Figure 3 shown.

[0082] Lilium lily polysaccharide L01-B1 1 H NMR spectrum ( Figure 3In a), in the anomeric hydrogen region, 5.30ppm, 5.22ppm and 5.16ppm are the H-1 of T-α-Araf, 1,3,5-α-Araf and 1,5-α-Araf respectively; 4.70ppm, 4.55ppm and 4.71ppm are the H-1 of T-β-Galp, 1,3,6-β-Galp and 1,4-β-Galp respectively; the H-1 signals of 1,3-β-Galp and 1,6-β-Galp are Overlap at 4.59ppm; the H-1 signals of 1,2-α-Rhap and 1,2,4-α-Rhap overlap at 5.30ppm; the signal peak at 5.08ppm is attributed to the H-1 of 1,4-α-GalpA; the signal peak at 4.69ppm is attributed to the H-1 of T-β-GlcpA, and the signals at 1.32ppm and 1.37ppm are attributed to the H signals on the methyl carbon of 1,2-α-Rhap and 1,2,4-α-Rhap, respectively.

[0083] Lilium lily polysaccharide L01-B1 13 C NMR spectrum ( Figure 3 b), in the anomeric carbon region, 110.54ppm, 108.75ppm and 108.34ppm are attributed to the C-1 signals of T-α-Araf, 1,5-α-Araf and 1,3,5-α-Araf, respectively; the C-1 signals of T-β-Galp, 1,3-β-Galp and 1,4-β-Galp are at 104.62ppm, 103.99ppm and 105.59ppm, respectively; the C-1 signals of 1,3,6-β-Galp and 1,6-β-Galp overlap at 104.84ppm; 1, The C-1 signals of 2-α-Rhap and 1,2,4-aα-Rhap overlap at 99.96ppm; the C-1 signal peak of 1,4-α-GalpA is at 98.84ppm; the C-1 signal peak of T-β-GlcpA is at 102.65ppm; the carboxyl carbon signal peaks of 1,4-α-GalpA and T-β-GlcpA are at 175.29ppm and 174.62ppm respectively; the methyl carbon signal peaks of 1,2-α-Rhap and 1,2,4-α-Rhap are at 17.84ppm and 18.09ppm respectively.

[0084] Through two-dimensional NMR HSQC ( Figure 3 The chemical shifts of sugar residues C and H were assigned in HMBC ( Figure 3The connection mode was determined on the black (black) in middle c): the long-range correlation peak δ105.59 / 4.23 indicated that C-1 of 1,4-Galp(H) and H-4 of 1,4-Galp(H) were long-rangely correlated, the long-range correlation peak 681.41 / 4.70 indicated that C-4 of 1,4-Galp(H) and H-1 of 1,4-Galp(H) were long-rangely correlated, indicating that there were continuous 1,4-Galp residues in the side chain, and the long-range correlation peak δ105.59 / 3.78 indicated that C-1 of 1,4-Galp(H) and H-4 of 1,2,4-Rhap(K) were long-rangely correlated, indicating that the 1,4-Galp residue was connected to C-4 of 1,2,4-Rhap. The long-range correlation peak δ103.99 / 3.71 indicates that C-1 of 1,3-Galp (E) is long-rangely correlated with H-3 of 1,3,6-Galp (F), indicating that the 1,3-Galp residue is connected to C-3 of 1,3,6-Galp. The long-range correlation peak δ81.46 / 4.70 indicates that C-3 of 1,3,6-Galp (F) is long-rangely correlated with H-1 of T-Galp (D), indicating that a T-Galp residue is connected to C-3 of 1,3,6-Galp. The long-range correlation peak δ83.65 / 5.30 indicates that C-3 of 1,3-Galp (E) is long-rangely correlated with H-1 of T-Araf (B), indicating that a T-Araf residue is connected to 1,3-Galp. The long-range correlation peak δ108.75 / 3.95 (δ108.75 / 3.85) indicates that C-1 of 1,5-Araf(B) is long-rangely correlated with H-5 of 1,5-Araf(B), and the long-range correlation peak δ68.05 / 5.16 indicates that C-5 of 1,5-Araf(B) is long-rangely correlated with H-1 of 1,5-Araf(B), indicating that there are continuous 1,5-Araf residues in the side chain. The long-range correlation peak δ68.09 / 5.30 indicates that C-5 of 1,5-Araf(B) is long-rangely correlated with H-1 of T-Araf(A), indicating that there are T-Araf residues connected to 1,5-Araf. The long-range correlation peak δ85.17 / 5.30 indicates that C-3 of 1,3,5-Araf(C) is long-rangely correlated with H-1 of T-Araf(A). The long-range correlation peak δ108.75 / 4.01 indicates that C-1 of 1,5-Araf (B) is long-rangely correlated with H-3 of 1,3,5-Araf (C), indicating that a 1,5-Araf residue is attached to C-3 of 1,3,5-Araf. The long-range correlation peak δ77.85 / 5.16 indicates that H-1 of 1,5-Araf (B) is long-rangely correlated with C-4 of 1,2,4-Rhap (K), indicating that a 1,5-Araf residue is attached to C-4 of 1,2,4-Rhap.The long-range correlation peak δ108.75 / 3.85 indicates that C-1 of 1,5-Araf (B) is long-rangely correlated with H-5 of 1,3,5-Araf (C), indicating that there is a 1,5-Araf residue attached to the C-5 position of 1,3,5-Araf. The long-range correlation peak δ77.56 / 4.69 indicates that H-1 of T-GlcpA (L) is long-rangely correlated with C-4 of 1,2,4-Rhap (K).

[0085] In summary, the structural unit of Lilium lily polysaccharide L01-B1 is based on 1→4 linked α-galactopyranose uronic acid (GalpA), 1→2 linked α-rhamnose pyranose (Rhap) and 1→6 linked α-galactopyranose (Galp) as the main chain, and has the following structure:

[0086]

[0087] Among them, 5≤n≤6.

[0088] Example 3 Isolation, culture, purification and identification of bacteria

[0089] (1) Cultivation of human intestinal flora

[0090] GB liquid medium was used to culture the stool samples of 10 healthy people (the ethical approval number for the collection of stool samples of healthy people is: 2022ZSZY-LLK-266) in vitro. The culture conditions were strictly anaerobic at 37°C for 24 hours. After 24 hours, the pure culture was centrifuged at low speed (5000rpm / min) for 3 minutes, the supernatant was discarded, and the cells were resuspended in fresh GB liquid medium. 80% glycerol was added in half according to the resuspended volume, and stored at -80°C for later use.

[0091] (2) Co-culture of L01-B1 with human intestinal flora

[0092] The glycerol-preserved bacteria prepared above were revived, and the culture medium was GB liquid culture medium (the volume ratio of glycerol-preserved bacteria to culture medium was 1:1000), the culture conditions were strictly anaerobic at 37°C, and the culture time was 24h. The revived bacterial solution was added to the fresh GB culture medium at a volume ratio of 1:1000, and a 100mg / mL polysaccharide L01-B1 aqueous solution was added at the same time, so that the final concentration contained 10mg / mL polysaccharide L01-B1, and the culture conditions were strictly anaerobic at 37°C, and the culture time was 48h; at the same time, a control group was set up, the difference was that polysaccharide L01-B1 was not added during the culture process. After 48h, the pure culture was divided into two parts, one of which was centrifuged at low speed (5000rpm / min) for 3min, and the bacterial precipitate and the culture supernatant of the culture were collected respectively, wherein the bacterial precipitate was washed once with 1×PBS and stored at -80°C for standby use. The other part was preserved in glycerol for bacterial isolation.

[0093] (3) Metagenomic sequencing of intestinal flora

[0094] The prepared bacteria were sent to Shanghai Meiji Biotech Co., Ltd. for metagenomic sequencing, including the extraction and quality identification of bacterial genomic DNA. The metagenomic sequencing result spectrum is shown in Figure 4 The results of the bar chart of the community composition at the phylum level showed that polysaccharide L01-B1 significantly improved the composition of the bacterial community, and the proportion of Actinobacteria increased significantly ( Figure 4 a); at the species level, we found that L01-B1 was significantly enriched with Bifidobacterium longum ( Figure 4 b and c); at the species level, two groups were compared and LEfSe was analyzed, and it was also found that polysaccharide L01-B1 was significantly enriched in Bifidobacterium longum ( Figure 4 d and e).

[0095] (4) Isolation, culture, purification and identification of bacteria

[0096] According to the results of metagenomic sequencing, the dominant bacteria enriched in L01-B1 were found, and the bacteria were isolated, purified and identified. GB solid culture medium was used for bacterial isolation, but mupirocin antibiotics with a final concentration of 50 mg / L needed to be added. The glycerol bacteria preserved in step 2 were inoculated on a plate supplemented with mupirocin antibiotics using a bacterial inoculation loop. Incubate at 37°C for 24 hours under strict anaerobism. Pick a single colony on the plate and re-inoculate it on a plate containing mupirocin for subculture purification (such as Figure 5As shown), the colonies were milky white and grew in convex shapes on the plate. After two consecutive passages, a single colony was picked and expanded with 3 mL of GB liquid medium. The bacteria were collected by centrifugation, and bacterial genomic DNA was extracted using a bacterial genome extraction kit (Tiangen Biochemical Technology, Cat# DP302). The extracted bacterial genomic DNA was used as a template for PCR amplification. The primers used for PCR amplification are shown in Table 1 below, and the PCR system and amplification conditions are shown in Table 2 below:

[0097] Table 1 Bacterial identification primers

[0098]

[0099] Table 2 Bacterial identification amplification system and amplification procedure

[0100]

[0101]

[0102] Using the genome of DK001 as a template, a band of about 1500 bp was obtained by electrophoresis after PCR amplification.

[0103] After PCR amplification, 5 μL was taken for agarose gel electrophoresis, and the remaining sample was sent to Sangon Biotech Co., Ltd. for sequencing. The full-length sequencing results of 16S rRNA are as follows:

[0104]

[0105] Pick a single colony on GB solid medium, fix it with electron microscopy fixative at 4℃ for ~24h, and then send it to Wuhan Seville for transmission electron microscopy embedding and sectioning. Figure 7 As shown, the results of transmission electron microscopy showed that the isolated strain DK001 was oval in shape, without flagella, and had a distinct cell wall structure.

[0106] Example 4 Production of short-chain fatty acids by Bifidobacterium longum subsp. suillum

[0107] Bifidobacterium longum subsp. suillum (DK001) was cultured in MRS medium alone, and in MRS medium containing Lilium lily polysaccharide L01-B1 (final concentration of 2-10 mg / mL), and the culture conditions were anaerobic culture at 37°C for 24 hours.

[0108] The culture supernatant was collected and freeze-dried. After freeze-drying, the culture supernatant was suspended with 200 μL of sterile water, 100 μL of 50% sulfuric acid and 1 mL of ether were added, and the mixture was shaken for 30 seconds at 10,000 rpm, 4°C, and 5 minutes. 200 μL of the supernatant was taken and analyzed by GC-MS. The method of GC-MS is as follows:

[0109] Preparation of standards:

[0110] A standard solution containing acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid (using sterile water as solvent) was serially diluted with sterile water at concentrations of 25, 50, 100, 200, 400 and 800 μg / mL to serve as a standard sample for preparing a standard curve.

[0111] Conditions for chromatography and mass spectrometry:

[0112] The gas chromatography-mass spectrometer is composed of a TRACE 1300 gas chromatograph and an ISQ series mass spectrometer;

[0113] Analytical column: Thermo TraceGOLD TG-WaxMS 30m×0.25mm×0.25μm column;

[0114] Chromatographic conditions: injection port temperature: 250°C; split ratio: 50:1; column flow rate: 1 mL / min; program temperature rise: 50°C for 3 min, 15°C / min to 240°C, hold for 3 min;

[0115] Mass spectrometry conditions: scanning time: 20.067 min; ion source type: EI; detector temperature: 250°C; transfer line temperature: 250°C; ion source temperature: 300°C; scanning range: 30-300 Da; scanning mode: SCAN.

[0116] Results Figure 8 , showing that Bifidobacterium longum subsp. suillum can produce a large amount of acetic acid with or without the addition of L01-B1.

Claims

1. A polysaccharide from Lilium lily L01-B1, characterized in that: Taking the molar number of all monosaccharides contained in the polysaccharide as 100%, the monosaccharide composition of the Lilium lily polysaccharide L01-B1 includes 35-40% galactose, 35-40% arabinose, 10-15% rhamnose, 10-15% galacturonic acid, and 1-3% glucuronic acid.

2. The Lilium lily polysaccharide L01-B1 according to claim 1, characterized in that Taking the molar number of all monosaccharides contained in the polysaccharide as 100%, the monosaccharide composition of the Lilium lily polysaccharide L01-B1 includes 37.2% galactose, 36.5% arabinose, 13.4% rhamnose, 11.1% galacturonic acid and 1.8% glucuronic acid.

3. The Lilium lily polysaccharide L01-B1 according to claim 1 or 2, characterized in that: The structural unit of the polysaccharide is a main chain of α-galactopyranose uronic acid linked in 1→4, α-rhamnose linked in 1→2 and α-galactopyranose linked in 1→6, and has the following structure: Wherein, 1≤n≤11, preferably, 5≤n≤6; Preferably, the weight average molecular weight of the polysaccharide L01-B1 is in the range of 3-300 kDa, preferably 4-60 kDa, more preferably 6-50 kDa, and further preferably 43.9 kDa.

4. The method for preparing the Lilium lily polysaccharide L01-B1 according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) Polysaccharide extraction: The flowers of Lilium lily are extracted with boiling water, the obtained extract is concentrated and dialyzed, the dialyzate is centrifuged to remove the precipitate, and the obtained supernatant is precipitated with ethanol aqueous solution and separated to obtain the precipitate, i.e., the crude polysaccharide LLP of Lilium lily; (2) Polysaccharide purification: (i) dissolving the crude polysaccharide LLP prepared in step (1) in water and centrifuging the solution, taking the supernatant, and performing preliminary purification using an anion exchange column, sequentially using water and 0.05-0.1 M NaCl aqueous solution for gradient elution, collecting the elution fraction of 0.1 M NaCl aqueous solution, and obtaining a preliminary purified secondary crude polysaccharide LLP01; (ii) The secondary crude polysaccharide LLP01 prepared in step (i) is dissolved in water and then centrifuged. The supernatant is purified by gel column to obtain Lilium lily polysaccharide L01-B1.

5. The method for preparing Lilium lily polysaccharide L01-B1 according to claim 4, characterized in that: In step (1): Before the boiling water extraction, soak the Lilium lily in an ethanol aqueous solution with a volume fraction of 80 to 98%, for example, 95%, for 5 to 14 days and then dry; and / or In the boiling water extraction of Lilium lily, the ratio of Lilium lily to water is 3:30-50, kg / L, preferably 3:30, unit: kg / L; the number of boiling water extractions is 4-10 times, preferably 8 times, each time for 3-5 hours, preferably 4 hours; and / or The extract is concentrated to 1 / 40 to 1 / 25 of the original volume, preferably 1 / 30; and / or The dialysis is to dialyze the concentrated extract in water for 24 to 72 hours; and / or The volume fraction of the ethanol aqueous solution used for the alcohol precipitation is 80-98%, preferably 95%; the volume of the ethanol aqueous solution used for precipitation is 3-6 times that of the concentrated solution; and / or The alcohol precipitation is performed by adding the ethanol aqueous solution to the supernatant and allowing the solution to stand for 6 to 24 hours; and / or After the precipitate is obtained, it is washed alternately with ethanol and acetone, each washing 3 to 4 times; further, the washed precipitate is dried to obtain the crude polysaccharide LLP of Lilium lily flower.

6. The method for preparing the Lilium lily polysaccharide L01-B1 according to claim 4, characterized in that: In step (i), during the dissolution process, the ratio of the crude polysaccharide LLP from Lilium lily flower to water is 1:2 to 20, for example 1:10, unit: g / mL; In step (i), the anion exchange column is selected from DEAE Sepharose TM Fast Flow or DEAE cellucose-52, preferably DEAE Sepharose TM Fast Flow; and / or In step (i), when the anion exchange column is used for preliminary purification, deionized water, 0.05 M and 0.1 M NaCl aqueous solutions are used for elution in sequence, and the elution fraction of the 0.1 M NaCl eluate is collected; and / or Step (i) further comprises concentrating the collected elution fraction of the 0.1M NaCl aqueous solution, dialyzing it in water, and drying it to obtain the secondary crude polysaccharide LLP01; and / or In step (ii), during the dissolution process, the ratio of the crude polysaccharide of Lilium lily LLP01 to water is 1:5-40, for example 1:20, unit: g / mL; and / or In step (ii), the gel column is selected from Sephacryl S-200HR or Sephacryl S-300HR, preferably Sephacryl S-300HR; the eluent used for purifying the supernatant by the gel column is a 0.2M NaCl aqueous solution; and / or Step (ii) also includes drying the collected elution fractions to obtain Lilium lily polysaccharide L01-B1.

7. A pharmaceutical composition comprising the Lilium lily flower polysaccharide L01-B1 according to any one of claims 1 to 3 or the Lilium lily flower polysaccharide L01-B1 prepared by the preparation method according to any one of claims 4 to 6, and pharmaceutically acceptable excipients.

8. Use of the Lilium lily polysaccharide L01-B1 according to any one of claims 1 to 3, the Lilium lily polysaccharide L01-B1 prepared by the preparation method according to any one of claims 4 to 6, or the pharmaceutical composition according to claim 7 in the preparation of drugs, reagents, diets or nutritional supplements having one or more of the following effects: a. Improve the composition of human intestinal flora; b. Enrichment of Bifidobacterium longum subsp. suillum in the human intestine; c. Enrichment and cultivation of Bifidobacterium longum subsp. suillum from human fecal culture in vitro.

9. A method for enriching and culturing Bifidobacterium longum subsp. suillum, characterized in that: The lily lily flower polysaccharide L01-B1 described in claims 1-3 or the lily lily flower polysaccharide L01-B1 obtained by the preparation method described in claims 4-6 is co-cultured with human intestinal flora.

10. The method for enriching and culturing Bifidobacterium longum subsp. suillum according to claim 9, characterized in that: The method comprises the following steps: S1: Cultivation of human intestinal flora: Fecal samples collected from healthy individuals were initially cultured in vitro using GB liquid medium, the culture was collected, centrifuged, and the bacteria were collected; S2: Co-culture of Lilium lily polysaccharide L01-B1 and human intestinal flora: The human intestinal flora obtained by preliminary in vitro culture in step S1 is enriched and cultured in a GB liquid medium containing Lilium lily polysaccharide L01-B1 at a final concentration of 5 to 20 mg / mL (e.g., 10 mg / mL), and then the culture is collected, and the bacteria are collected by centrifugation, and part of the bacteria are stored at -80°C for metagenomic sequencing, and part of the bacteria are stored in glycerol to obtain glycerol-preserved bacteria; S3: performing metagenomic sequencing on part of the bacteria collected by centrifugation in step S2 to determine that Bifidobacterium longum subsp. suillum is enriched; S4: Isolation, culture, purification and identification of bacteria: The glycerol-preserved bacteria obtained in step S2 are isolated, cultured and purified to obtain Bifidobacterium longum subsp. suillum. Preferably, in steps S1, S2 and S4, the culture conditions are 37°C, anaerobic, and the culture time is 1 to 72 hours, such as 24 hours or 48 hours; and / or After collecting the bacterial cells in step S1, glycerol is used to preserve the bacterial cells; The step of using glycerol to preserve the bacteria in steps S1 and S2 comprises: resuspending the bacteria collected by centrifugation in fresh GB liquid culture medium, adding 80% glycerol in half according to the resuspended volume, and storing at -80°C for later use, i.e., glycerol-preserved bacteria; and / or In step S2, when glycerol is used to preserve the bacteria in step S1, the step of resuscitating the glycerol-preserved bacteria is further included, including: mixing the glycerol-preserved bacteria with GB liquid culture medium for culturing and resuscitating, wherein the volume ratio of the glycerol-preserved bacteria to the GB liquid culture medium is 1:1000, the culture conditions are 37°C, strictly anaerobic, and the culture time is 24 to 48 hours; and / or In step S2, the step of enriching and culturing the human intestinal flora in a GB liquid culture medium containing Lilium lily polysaccharide L01-B1 at a final concentration of 5 to 20 mg / mL comprises: The revived bacterial solution is mixed with GB liquid culture medium, wherein the volume ratio of the bacterial solution to the GB liquid culture medium is 1:1000, and a 20-100 mg / mL aqueous solution of Lilium lily polysaccharide L01-B1 is added to make the final concentration of the polysaccharide L01-B1 5-20 mg / mL, and enrichment culture is performed; and / or In step S4, the culture medium used for the isolation and culture of bacteria is GB solid culture medium, which contains mupirocin with a final concentration of 50 mg / L; the isolation and culture of bacteria is carried out by dilution plate method, streak method, spread plate method or enrichment culture method, preferably streak method; and / or In step S4, after the bacteria are separated and cultured by the streaking method, the method for purifying the bacteria may include the following steps: Pick a single colony on the GB solid medium and re-inoculate it on the GB solid medium containing 50 mg / L mupirocin for subculture and purification. After two consecutive subcultures, pick a single colony and use GB liquid medium for expansion culture; and / or In step S4, 16S rDNA sequencing is performed using PCR, and the obtained sequence is compared with SEQ ID NO:

5. If the similarity is above 98%, it can be determined that Bifidobacterium longum subsp. suillum is obtained by enrichment culture.

Images