Dendrobium huoshanense neutral polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses dendrobium huoshanense neutral polysaccharide as well as a preparation method and application thereof, and relates to the technical field of traditional Chinese medicinal material processing, the dendrobium huoshanense neutral polysaccharide has Mn of 5-15 * 10 < 3 > Da and MW of 5-15 * 10 < 3 > Da, monosaccharide comprises arabinose (Ara), glucose (Glc) and mannose (Man) in a molar mass ratio of (0.5-1.0%): (25-30%): (69-74.5%), and the purity is greater than 90%; the determination of molecular weight and the analysis of monosaccharide composition of the dendrobium huoshanense neutral polysaccharide show that the dendrobium huoshanense neutral polysaccharide has an obvious promotion effect on the proliferation of animal bifidobacterium in a specific concentration range (0.25%-0.5%), is added into food or developed into health-care food for regulating the intestinal flora function, and has an obvious promotion effect on the proliferation of animal bifidobacterium. The proliferation of bifidobacteria can be effectively promoted, the micro-ecological balance of intestinal tracts is improved, the health of human bodies is maintained, and wide development potential and wide market prospects are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese medicinal material processing, and particularly relates to a Huoshan dendrobium neutral polysaccharide and a preparation method thereof. Background Art

[0002] Bifidobacterium animalis is an important probiotic in the intestines of humans and animals. In recent years, more and more studies have shown that it is closely related to the health of humans and animals.

[0003] Studies have shown that animal Bifidobacterium is closely related to intestinal inflammation, intestinal barrier, and lipid metabolism. As an inherent flora in the intestine, animal Bifidobacterium can inhibit inflammation or activate innate immunity, adhere to and colonize in the intestine, restore the host intestinal microbiota, and significantly alleviate acute colitis caused by dextran sulfate sodium (DSS). Animal Bifidobacterium effectively enhances the immune function of cyclophosphamide-induced immunosuppressive mice by regulating the intestinal microbiota. In addition, animal Bifidobacterium reduces lipid accumulation in atherosclerosis model mice by regulating bile acid metabolites and downregulating intestinal FXR. Animal Bifidobacterium inhibits the occurrence of non-alcoholic fatty liver by restoring intestinal barrier function, inhibiting inflammation and lipid accumulation.

[0004] Huoshan Dendrobium (Dendrobium huoshanense) belongs to the genus Dendrobium of the Orchidaceae family, commonly known as rice dendrobium. It is a perennial herb with a creeping or upright growth habit. It likes cool and humid conditions and avoids high temperature, dryness, strong light and long daylight. Its natural distribution area is narrow, and it is mostly distributed in the Dabie Mountains centered on Huoshan, Anhui. Huoshan Dendrobium is sweet and slightly cold. It enters the stomach and kidney meridians, benefits the stomach and produces fluid, nourishes yin and clears heat, and long-term use thickens the stomach and intestines, lightens the body and prolongs life. Huoshan Dendrobium has been included in the 2020 edition of the "Chinese Pharmacopoeia", in which polysaccharide content is one of its quality control indicators. At the same time, Huoshan Dendrobium polysaccharide is also the main active ingredient of Huoshan Dendrobium. In recent years, there have been many studies on the biological activities of crude polysaccharides from Dendrobium huoshanense, including anti-inflammatory, antioxidant, anti-tumor, and lipid metabolism regulation, but there are almost no reports on the activity of purified uniform polysaccharides from Dendrobium huoshanense, especially its effect on the proliferation of probiotics.

[0005] The invention uses Huoshan dendrobium as raw material, extracts and purifies a Huoshan dendrobium neutral polysaccharide, and proves that it has an obvious effect of promoting the proliferation of animal bifidobacteria within a certain concentration. Summary of the invention

[0006] The purpose of the present invention is to provide a neutral polysaccharide from Dendrobium huoshanense and a method for separating and purifying the same in order to solve the above problems.

[0007] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0008] The present invention provides a neutral polysaccharide of Dendrobium huoshanense, wherein the Mn of the polysaccharide is 5-15×10 3 Da, M W 5-15×10 3 Da, monosaccharide composition is arabinose (Ara), glucose (Glc) and mannose (Man) with a molar mass ratio of 0.5-1.0%: 25-30%: 69-74.5%, and the purity is greater than 90%.

[0009] The second aspect of the present invention provides a method for preparing neutral polysaccharides from Dendrobium huoshanense, which specifically comprises the following steps:

[0010] S1. Extracting the powder of Dendrobium huoshanense with anhydrous ethanol, centrifuging and collecting the precipitate;

[0011] S101. Pretreatment of Huoshan Dendrobium: Crush the dried strips of Huoshan Dendrobium, pass through a 60-mesh sieve, and store in a dry and cool place;

[0012] S102, defatting of Huoshan Dendrobium: adding anhydrous ethanol to Huoshan Dendrobium powder at a mass volume ratio of 1:10-1:20, stirring to extract fat-soluble components, collecting the precipitate and centrifuging at 6000r / min for 10min;

[0013] S2, extract the precipitate twice with purified water, centrifuge, combine the supernatants of the two extractions, and vacuum concentrate the supernatant to 1 / 10 of the original volume; add 3 times anhydrous ethanol to the concentrate, precipitate overnight, and collect the precipitate by centrifugation to obtain a crude polysaccharide extract;

[0014] S201, initial extraction: add purified water to the precipitate at a mass volume ratio of 1:10-1:30, and extract in a 60°C water bath for 2 h;

[0015] Centrifugation: The extract was centrifuged at 6000r / min for 10min to separate the supernatant and the precipitate;

[0016] S202, re-extraction: extract the precipitate after centrifugation in S201, add purified water to the precipitate at a mass volume ratio of 1:10-1:30, and extract in a 60°C water bath for 2h;

[0017] Centrifuge again: centrifuge the extract at 6000r / min for 10min to separate the supernatant and the precipitate; combine the supernatants of the two extractions;

[0018] S203, impurity removal: the supernatant is filtered under reduced pressure to remove tiny particles, and a polysaccharide extraction clear solution is prepared;

[0019] S204, precipitating polysaccharides: the clear solution is concentrated to 1 / 10 of the original volume, anhydrous ethanol is added to the solution at a volume ratio of 1:3, and the precipitated solid is collected by alcohol precipitation at 8000r / min for 10min to obtain a crude polysaccharide extract;

[0020] S205, determining the purity of polysaccharide by phenol-sulfuric acid method;

[0021] S3, dissolving the crude polysaccharide extract in water, removing protein, decolorizing, dialyzing, and freeze-drying the polysaccharide solution to obtain refined polysaccharide;

[0022] S301, adding pure water to the solid of the crude polysaccharide extract of Dendrobium huoshanense to fully dissolve the crude polysaccharide, adding papain, and performing enzymolysis overnight;

[0023] S302, add 1 / 4 savage reagent (chloroform and n-butanol (5:1)) to the aqueous phase, mix thoroughly, collect the upper aqueous phase, and repeat 4 or 5 times;

[0024] S303, adding petroleum ether to the liquid, mixing thoroughly, and collecting the lower aqueous phase;

[0025] S304, adding macroporous resin AB-8 to the aqueous phase, mixing thoroughly, and adsorbing overnight;

[0026] S305, collecting the liquid, dialyzing it with a 3000Da dialysis bag for 48 hours to remove small molecular components, and freeze-drying the polysaccharide liquid;

[0027] S306, the purity of polysaccharide was determined by phenol-sulfuric acid method;

[0028] S4, the Huoshan Dendrobium polysaccharide was separated by anion cellulose exchange column, and purified by dextran gel column chromatography to obtain Huoshan Dendrobium neutral polysaccharide;

[0029] S401, taking an appropriate amount of crude polysaccharide sample and dissolving it in pure water to prepare a polysaccharide mother solution of a certain concentration;

[0030] S402, centrifuge at 10000r / min for 10min, take the supernatant and purify it through an ion exchange column at a flow rate of 4mL / min; use pure water, 0.1M, 0.2M and 0.3M NaCl solution in turn for gradient elution, collect one tube for every 15mL, and collect all the eluate;

[0031] S403, combining the eluates from the collection tubes corresponding to the same elution peak, and concentrating by rotary evaporation to 1 / 5 of the original volume;

[0032] S404, 3000Da dialysis bag dialysis for 48-72h desalination;

[0033] S405, determining the purity of polysaccharide by phenol-sulfuric acid method;

[0034] S406, taking the polysaccharide sample purified by anion column chromatography (the sample collected by elution with pure water) and adding pure water to prepare a polysaccharide mother solution of a certain concentration;

[0035] S407, centrifuge at 10000r / min for 10min; take the supernatant and pass it through a gel chromatography column for separation and purification at a flow rate of 1mL / min, use pure water to select 1.5 times the column volume, collect one tube for every 12mL, and collect all the eluate;

[0036] S408, combining the eluates from the collection tubes corresponding to the same elution peak, and concentrating by rotary evaporation to 1 / 5 of the original volume;

[0037] S409, freeze-drying to obtain neutral polysaccharides from Dendrobium huoshanense;

[0038] S410. The purity of polysaccharides was determined by phenol-sulfuric acid method.

[0039] The third aspect of the present invention also provides a use of neutral polysaccharides from Dendrobium huoshanense in the preparation of health food or medicine having the effect of promoting the proliferation of bifidobacteria. The polysaccharide has a promoting effect on the growth of animal Bifidobacterium GDMCC1.4430 within a concentration range of 0.25%-0.5%.

[0040] The present invention has at least the following beneficial effects: the present invention uses Huoshan Dendrobium as raw material, extracts, and successfully separates and purifies Huoshan Dendrobium neutral polysaccharides through sophisticated separation and purification technology, and the neutral polysaccharides have a specific molecular weight, monosaccharide composition and high purity; and the Huoshan Dendrobium neutral polysaccharides prepared by the present invention have an obvious effect of promoting the proliferation of animal bifidobacteria, which provides a new idea for developing foods or health foods with the function of regulating intestinal flora. Adding to foods or developing into health foods with the function of regulating intestinal flora can effectively promote the proliferation of bifidobacteria, improve the balance of intestinal microecology, and maintain human health, and has broad development potential and broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The present invention is a preparation process of the neutral polysaccharide of Dendrobium huoshanense;

[0042] Figure 2 This is the elution curve of the Huoshan Dendrobium polysaccharide DEAE-anion column separation of the present invention;

[0043] Figure 3 This is the elution curve of the Huoshan Dendrobium polysaccharide gel column purification of the present invention;

[0044] Figure 4 The present invention is to determine the molecular weight of neutral polysaccharides from Dendrobium huoshanense;

[0045] Figure 5 The present invention is the analysis of the monosaccharide composition of the neutral polysaccharide of Dendrobium huoshanense-monosaccharide standard ion chromatography;

[0046] Figure 6 The invention relates to the analysis of the monosaccharide composition of neutral polysaccharides of Dendrobium huoshanense-ion chromatography of polysaccharide hydrolysis samples. DETAILED DESCRIPTION

[0047] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0048] like Figure 1 As shown, a method for preparing neutral polysaccharides from Dendrobium huoshanense comprises the following specific steps:

[0049] 1. Preparation of materials and reagents:

[0050] Huoshan Dendrobium officinale dried strips were purchased from Huoshan Tianxia Zeyu Biotechnology Development Co., Ltd.; Table 1 shows the main experimental reagents:

[0051] Table 1

[0052]

[0053]

[0054] Table 2 shows the main experimental instruments:

[0055] Table 2

[0056]

[0057]

[0058] 2. Preparation process:

[0059] 2.1. Pretreatment of Huoshan Dendrobium: The dried strips of Huoshan Dendrobium were crushed, passed through a 60-mesh sieve, and stored in a cool and dry place.

[0060] 2.2. Defatting of Huoshan Dendrobium: Anhydrous ethanol was added to Huoshan Dendrobium powder at a mass volume ratio of 1:10-1:20, and the fat-soluble components were extracted by stirring. The precipitate was collected and centrifuged at 6000r / min for 10min.

[0061] 2.3 Polysaccharide Extraction

[0062] Initial extraction: add pure water to the precipitate at a ratio of 1:20 by volume, and extract in a 60℃ water bath for 2h;

[0063] Centrifugation: The extract was centrifuged at 6000r / min for 10min to separate the supernatant and the precipitate;

[0064] Re-extraction: extract the precipitate, add pure water to the precipitate at a mass volume ratio of 1:20, and extract in a 60°C water bath for 2 hours; centrifuge the extract at 6000r / min for 10 minutes to separate the supernatant and the precipitate; combine the supernatants of the two extractions;

[0065] Impurity removal: the supernatant is filtered under reduced pressure to remove tiny particles, and a polysaccharide extraction clear solution is prepared;

[0066] Precipitated polysaccharides: The clear liquid was concentrated to 1 / 20 of the original volume, anhydrous ethanol was added to the solution at a volume ratio of 1:3, and the precipitated solid was collected by centrifugation at 8000r / min for 10min to obtain a crude polysaccharide extract.

[0067] 2.4. Determination of polysaccharide purity by phenol-sulfuric acid method

[0068] 2.4.1. Method for drawing glucose standard curve: After drying to constant mass, accurately weigh 25.00 mg of glucose standard to prepare a glucose reference solution with a concentration of 0.10 mg / mL. As shown in Table 3 below, add sample and react for 30 minutes, measure the absorbance OD value at a wavelength of 490 nm, and draw the standard curve; Table 3 shows the sample addition amount for glucose standard curve:

[0069] Table 3

[0070]

[0071] 2.4.2. Determination of polysaccharide content: accurately weigh 2.00g of the extracted polysaccharide powder, add an appropriate amount of water to fully dissolve it, transfer it to a 10.00mL volumetric flask, add water to the scale line and shake well, determine the absorbance according to the operating method of the standard product and substitute it into the drawn standard curve equation to calculate the concentration of the polysaccharide solution;

[0072] The polysaccharide content was calculated as follows: mg = C × V / M;

[0073] Wherein, C is the sample concentration, which is calculated by substituting the measured absorbance value into the standard curve; V is the actual dissolved volume of the sample; M is the actual weighed mass.

[0074] 2.5. Deproteinization

[0075] Adding pure water to the solid of the crude polysaccharide extract of Dendrobium huoshanense to fully dissolve the crude polysaccharide, adding papain, and performing enzymolysis overnight;

[0076] Add 1 / 4 chloroform and n-butanol (5:1) to the aqueous phase and mix thoroughly, collect the upper aqueous phase, and repeat 5 times.

[0077] 2.6. Degreasing

[0078] Add petroleum ether to the liquid, mix thoroughly, and collect the lower aqueous phase.

[0079] 2.7. Depigmentation

[0080] Add macroporous resin AB-8 to the aqueous phase, mix thoroughly, and adsorb overnight.

[0081] 2.8 Dialysis

[0082] The liquid was collected and dialyzed for 48 h using a 3000Da dialysis bag to remove small molecule components, and the polysaccharide liquid was freeze-dried.

[0083] 2.9. Determination of polysaccharide purity: the method is the same as 2.4 above.

[0084] 2.10. Separation of polysaccharides by ion exchange column chromatography

[0085] Take an appropriate amount of crude polysaccharide sample and dissolve it in pure water to prepare a polysaccharide mother solution of a certain concentration;

[0086] Centrifuge at 10000r / min for 10min, take the supernatant and purify it through an ion exchange column at a flow rate of 4mL / min; use pure water, 0.1M, 0.2M and 0.3M NaCl solutions in turn for gradient elution, collect one tube for every 15ml, and collect all the eluent. Draw the ion purification elution curve of the sample with the number of eluent tubes as the horizontal axis and the total sugar content (blue) and the NaCl concentration in the eluent (green) as the vertical axis, as shown in the figure below: Figure 2 As shown. Ion purification mainly uses the fact that charged polysaccharides can bind to column chromatography media, while the interaction between neutral polysaccharides and fillers is very weak and cannot be adsorbed on the chromatography column media. Neutral polysaccharides and acidic polysaccharides with different charges can be separated by pure water and eluents with different ionic strengths, thereby achieving the purpose of separation and purification.

[0087] Combine the eluates from each collection tube corresponding to the same elution peak and concentrate by rotary evaporation to 1 / 5 of the original volume;

[0088] Use 3000Da dialysis bag to dialyze for 48-72h to remove salt.

[0089] 2.11. Determination of polysaccharide purity: the method is the same as 2.4 above.

[0090] 2.12. Purification of polysaccharides by gel chromatography

[0091] Take the polysaccharide sample after ion purification (the sample collected by elution with pure water) and add pure water to prepare a polysaccharide mother solution of a certain concentration;

[0092] Centrifuge at 10000r / min for 10min; take the supernatant and pass it through a gel chromatography column for separation and purification at a flow rate of 1mL / min. Use pure water to elute 1.5 times the column volume. Collect one tube for every 12ml and collect all the eluent. Draw the gel purification elution curve of the sample with the number of eluent tubes as the horizontal axis and the total sugar content (blue) as the vertical axis, such as Figure 3 As shown;

[0093] This gel purification mainly obtained an elution peak component, and the eluates from each collection tube corresponding to the elution peak (tubes 33-51) were combined and concentrated by rotary evaporation to 1 / 5 of the original volume;

[0094] Freeze drying can obtain neutral polysaccharides from Dendrobium huoshanense.

[0095] 2.13. Determination of polysaccharide purity: the method is the same as 2.4 above.

[0096] 3. Determination of the properties of Huoshan neutral polysaccharides

[0097] 3.1 Molecular weight determination of neutral polysaccharides from Dendrobium huoshanense

[0098] The polysaccharide sample was dissolved in 0.1M NaNO3 aqueous solution at a concentration of 1.00mg / mL, passed through a filter with a pore size of 0.45μm, and tested on a gel exclusion chromatography column. The injection volume was 100μL using an automatic sampler, the column temperature was 45°C, the mobile phase was A (0.02% NaN3; 0.1M NaNO3), the flow rate was 0.60mL / min, and the distilled water was isocratic eluted for 75min. The chromatographic data were processed using the software ASTRA6.1.

[0099] like Figure 4 As shown, the horizontal axis represents the retention time (Time, min) of the detection, the vertical axis represents the molar mass (g / mol), the red line represents the multi-angle laser light scattering signal; the blue line represents the difference signal, and the black line is the molecular weight fitted by the two signals, among which the inorganic salt phase near 37min is the solvent peak of the mobile phase. The spectrum was processed by software to obtain the number average molecular weight (Mn) of the neutral polysaccharide of 8.437×10 3 Da; weight average molecular weight (Mw) is 9.624×10 3 Da; Z average molecular weight (Mz) is 10.824×10 3 Da; polydispersity Mw / Mn is 1.141.

[0100] 3.2 Analysis of monosaccharide composition of neutral polysaccharides from Dendrobium huoshanense

[0101] Preparation of standard products: Accurately weigh the required standard products and use distilled water to prepare a 10mg / mL standard product stock solution. Take an appropriate amount of the stock solution mixture to prepare a variety of standard product mixed solutions with different concentrations. The concentration range is 60ug / mL, 40ug / mL and 20μg / mL. Prepare different concentration gradients according to experimental requirements;

[0102] Analysis of monosaccharide composition of polysaccharide samples: Select a clean chromatographic bottle, weigh an appropriate amount of sample, add 1mL2M TFA solution, and heat at 21℃ for 2h. After heating, use nitrogen to blow dry, and use 99.99% pure methanol to wash the sample 2 to 3 times. Add ultrapure water to dissolve the remaining sample, transfer it to a chromatographic bottle, and test it. Use a liquid chromatography column, injection volume 5μL, column temperature 30℃, flow rate 0.5mL / min; elution gradient is shown in Table 4 below: mobile phase A is H2O; mobile phase B is 0.1M NaOH; mobile phase C is 0.1M NaOH and 0.2M NaAc; use Chromeleon software to process chromatographic data.

[0103] Table 4

[0104]

[0105] The content of each component in the liquid sample was calculated according to the following formula:

[0106] Content of each component in the liquid sample (μg / mL) = C×F×V / M

[0107] Where, C is the concentration read by the instrument (μg / mL); F is the dilution factor; V is the volume of the sample extract (mL); M is the total weight of the sample;

[0108] The polysaccharide was hydrolyzed into monosaccharides by adding acid, and its monosaccharide composition was measured by ion exchange chromatography column separation and serial electrochemical detection. The percentages of arabinose, glucose and mannose were 0.66%, 27.50% and 71.84%, respectively.

[0109] Monosaccharide composition molar ratio (%) = monosaccharide composition content percentage / M0

[0110] Among them, M0 is the molecular mass of the corresponding monosaccharide;

[0111] The molar mass ratio of monosaccharide composition is shown in Table 5: the molar mass ratio of monosaccharide composition is 0.79%: 27.46%: 71.75%;

[0112] Table 5

[0113]

[0114]

[0115] like Figure 5-Figure 6 As shown in the figure, the horizontal axis represents the retention time (Time, min) of the detection, and the vertical axis represents the response value (Response, nC) of the ion detection; the monosaccharide composition of the obtained DHP-1 was qualitatively analyzed to be mannose, glucose and arabinose.

[0116] Application of neutral polysaccharide of Dendrobium huoshanense in preparing health food or medicine having the effect of promoting the proliferation of bifidobacteria;

[0117] Testing the effect of neutral polysaccharides from Dendrobium huoshanense on the proliferation of animal Bifidobacterium

[0118] 1. Preparation of materials and reagents:

[0119] Dried Dendrobium huoshanense strips: purchased from Tianxia Zeyu Biotechnology Development Co., Ltd., Huoshan County; Dendrobium huoshanense polysaccharides: homemade in the laboratory of the School of Biological and Pharmaceutical Engineering, Wanxi University; Bifidobacterium animalis GDMCC 1.4430, preserved in the Institute of Microbiology, Guangdong Academy of Sciences; sugar-free MRS medium.

[0120] Table 6 shows the main experimental equipment

[0121] Table 6

[0122]

[0123]

[0124] 2. Experimental steps

[0125] 2.1. Activation of Bifidobacterium animalis

[0126] In a sterile and anaerobic environment, animal Bifidobacterium was inoculated onto a basic culture medium and cultured under anaerobic conditions at (37±1)°C for 48 hours. 5% of the inoculum was taken for continued subculture. After 3 subcultures and activation, it was set aside.

[0127] 2.2 Preparation of sugar-free MRS medium

[0128] Weigh 10 g of peptone, 5 g of yeast extract, 0.1 g of magnesium sulfate, 0.05 g of citric acid, 2 g of potassium dihydrogen phosphate, 5 g of sodium chloride, and 0.0025 g of ammonium ferric citrate (III), put them into a container, dissolve the reagents with 1 L of sterile water, and adjust the pH to about 6.2-6.8;

[0129] Fill the culture medium with nitrogen to drive away the oxygen in the culture medium and cover it;

[0130] Heat the culture medium to boiling for sterilization, then cool to room temperature for later use.

[0131] 2.3. Polysaccharide mother solution preparation

[0132] Weigh 0.2 g of neutral polysaccharide of Dendrobium huoshanense prepared by the method of Example 1, and add 5 mL of sterilized sugar-free MRS medium;

[0133] Dissolving while stirring, filtering and sterilizing, thus preparing a 4% Huoshan Dendrobium neutral polysaccharide mother solution;

[0134] In the anaerobic workstation, sugar-free MRS medium was used to prepare a certain initial concentration of bacterial solution, with a bacterial concentration of about 10 6 / mL, and use the blood cell counting method to determine the specific value for later use.

[0135] 2.4 Experimental plan on the effect of polysaccharides on the proliferation of animal bifidobacteria

[0136] In the anaerobic workstation, add the sample to a 24-well plate, perform three parallel independent experiments for each concentration, and culture at 37°C for 48 hours under anaerobic conditions. Table 7 shows the sample addition scheme for Bifidobacterium animalis culture:

[0137] Table 7

[0138]

[0139] The bacterial counts at each polysaccharide concentration were determined 48 h later, strictly following aseptic operation specifications.

[0140] Calculation of Bifidobacterium animalis proliferation:

[0141] Logarithmic increase = logB-logA

[0142] Where A is the bacterial count at 0 h (cfu / mL), and B is the bacterial count after 48 h (cfu / mL).

[0143] 2.5 Experimental results on the effect of polysaccharides on the proliferation of animal bifidobacteria

[0144] After 48 hours of cultivation, the culture fluid of animal Bifidobacterium in each sample concentration and the negative control wells all showed visible turbidity, indicating that the bacteria had grown. Then the specific value of the number of bacteria was obtained by blood cell counting; the blank control wells were always clear and turbid before and after cultivation, indicating that there was no bacterial growth. Table 8 shows the changes in the number of bacteria before and after the cultivation of animal Bifidobacterium GDMCC1.4430:

[0145] Table 8

[0146]

[0147] Note: A is the number of bacteria at 0h (cells / mL), B is the average number of bacteria at 48h

[0148] From the data in Table 8, it can be seen that in the range of sample concentration of 0.25%-1%, the increase in the number of bacteria of animal Bifidobacterium GDMCC1.4430 is greater than the logarithmic increase value (1.5) of the negative control, indicating that Dendrobium polysaccharide has a certain promoting effect on the growth of animal Bifidobacterium; and in the concentration range of 0.25%-0.5%, the higher the concentration of added Dendrobium polysaccharide, the greater the logarithmic change value of bacteria, indicating that it has a significant promoting effect on the proliferation of animal Bifidobacterium. This promoting effect is not only reflected in the positive correlation between concentration and bacterial count, but also in its clear optimal concentration. When the concentration of Dendrobium polysaccharide is 0.5%, the logarithmic change value of bacteria reaches the maximum value (1 .8), that is, when the concentration of Dendrobium polysaccharide is 0.5%, the effect of promoting the growth of animal Bifidobacterium is the best. At this time, the logarithmic change value of bacterial growth is increased by 12.5% ​​compared with the sugar-free MRS culture medium without added Dendrobium polysaccharide, which provides a clear concentration guide for the application of polysaccharides, reveals the potential of Huoshan Dendrobium neutral polysaccharide as an animal Bifidobacterium proliferation promoter, and provides a new idea for the development of food or health food with the function of regulating intestinal flora. Adding it to food or developing it into a health food with the function of regulating intestinal flora can effectively promote the proliferation of Bifidobacterium, improve the balance of intestinal microecology, and maintain human health. It has broad development potential and broad market prospects.

[0149] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A neutral polysaccharide from Dendrobium huoshanense, characterized in that: The polysaccharide Mn is 5-15×10 3 Da, M W 5-15×10 3 Da, monosaccharide composition is arabinose (Ara), glucose (Glc) and mannose (Man) with a molar mass ratio of 0.5-1.0%: 25-30%: 69-74.5%, and the purity is greater than 90%.

2. A method for preparing the neutral polysaccharide from Dendrobium huoshanense as claimed in claim 1, characterized in that: The method comprises the following steps: S1. Extracting the powder of Dendrobium huoshanense with anhydrous ethanol, centrifuging and collecting the precipitate; S2, extract the precipitate twice with purified water, centrifuge, combine the supernatants of the two extractions, and vacuum concentrate the supernatant to 1 / 10 of the original volume; add 3 times anhydrous ethanol to the concentrate, precipitate overnight, and collect the precipitate by centrifugation to obtain a crude polysaccharide extract; S3. The crude polysaccharide extract is dissolved in water, deproteinized, decolorized, dialyzed, and the polysaccharide solution is freeze-dried to obtain refined polysaccharide; the Huoshan Dendrobium officinale refined polysaccharide is separated by anion cellulose exchange column, and purified by dextran gel column chromatography to obtain Huoshan Dendrobium officinale neutral polysaccharide.

3. The method for preparing neutral polysaccharides from Dendrobium huoshanense according to claim 2, characterized in that: In step S1, the Huoshan Dendrobium powder is obtained by crushing Huoshan Dendrobium dried strips and passing the powder through a 60-mesh sieve.

4. The method for preparing neutral polysaccharides from Dendrobium huoshanense according to claim 2, characterized in that: In step S1, the mass volume ratio of the Huoshan Dendrobium powder and anhydrous ethanol is 1:10-1:20, and the centrifugation conditions are precipitation at 6000r / min and centrifugation for 10min.

5. The method for preparing neutral polysaccharides from Dendrobium huoshanense according to claim 2, characterized in that: The warm immersion extraction twice described in step S2 comprises: First, add purified water to the precipitate at a mass volume ratio of 1:10-1:30, extract in a 60°C water bath for 2 h, centrifuge, and centrifuge the extract at 6000 r / min for 10 min to separate the supernatant and precipitate; The second time, the precipitate obtained in the first time was extracted, purified water was added to the precipitate at a mass volume ratio of 1:10-1:30, and the extraction was carried out in a water bath at 60°C for 2 hours; centrifugation, the extract was centrifuged at 6000r / min for 10 minutes, and the supernatant and the precipitate were separated.

6. The method for preparing neutral polysaccharides from Dendrobium huoshanense according to claim 2, characterized in that: In step S3, the ion exchange column was purified at a flow rate of 4 mL / min; pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions were used in sequence for gradient elution, and all the eluates were collected and concentrated in vacuum by rotary evaporation to 1 / 5 of the original volume.

7. The method for preparing neutral polysaccharides from Dendrobium huoshanense according to claim 2, characterized in that: In step S3, the dialysis condition is to use a 3000Da dialysis bag for 48-72 hours to remove salt.

8. The method for preparing neutral polysaccharides from Dendrobium huoshanense according to claim 2, characterized in that In step S3, the flow rate of the dextran gel chromatography purification is 1 mL / min, and 1.5 column volumes of pure water are used for elution.

9. The use of the neutral polysaccharide of Dendrobium huoshanense according to claim 1 in the preparation of a health food or medicine having the effect of promoting the proliferation of bifidobacteria, characterized in that: The bifidobacterium is animal bifidobacterium.

10. The use of the neutral polysaccharide of Dendrobium huoshanense according to claim 9 in the preparation of a health food or medicine having the effect of promoting the proliferation of bifidobacteria, characterized in that: The polysaccharide concentration is within the range of 0.25%-0.5%, which has a promoting effect on the growth of animal bifidobacterium GDMCC1.4430.

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