CN106868072B - Preparation method of small-molecular-weight dendrobium officinale polysaccharide - Google Patents
Preparation method of small-molecular-weight dendrobium officinale polysaccharide Download PDFInfo
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Abstract
The invention relates to a preparation method of dendrobium officinale polysaccharide with small molecular weight, which is characterized by comprising the following steps: A. strain activation and expanding culture: under the aseptic condition, taking a proper amount of liquid culture medium to prepare fusarium avenae bacterial suspension; B. repeatedly homogenizing at high pressure for 5-10 times by using a high-pressure micro-jet homogenizer to obtain cell-broken homogenate of Dendrobium officinale; C. fermentation culture: injecting a proper amount of liquid culture medium into a fermentation tank, weighing the dendrobium officinale wall-breaking homogenate and the fusarium avenaceum seed liquid, adding into the fermentation tank, and fermenting to obtain fermentation liquid; D. polysaccharide purification: after fermentation, decolorizing active carbon is used, standing and adsorbing for 1h, filtering to remove residues to obtain filtrate, vacuum-filtering to obtain precipitate, washing with ethanol for 3 times, and vacuum-drying to obtain small molecular weight Dendrobium officinale polysaccharide. The small-molecular-weight dendrobium officinale polysaccharide prepared by the invention has the average molecular weight of less than 300kDa, can effectively promote the proliferation of skin probiotics, can improve the Langerhans cell density on the surface of the skin, and can improve the immunity and the defense of the skin.
Description
Technical Field
The invention relates to the technical field of extraction of active ingredients of natural products, and particularly relates to a preparation method of dendrobium officinale polysaccharide with small molecular weight.
Background
Dendrobium officinale (Dendrobium candidum wall. ex Lindl) is a perennial epiphytic herbaceous plant of the genus Dendrobium (Dendrobium) of the family Orchidaceae, is rich in dendrobii polysaccharide, dendrobine, amino acid, phenanthrene compounds and various trace elements, and is a traditional and rare Chinese medicinal material in China. The dendrobium officinale is mainly used in the health-care product industry, and the dendrobium officinale polysaccharide is the main active ingredient, so that the dendrobium officinale polysaccharide oral liquid has the effects of enhancing the immunity of the organism, resisting tumors, resisting oxidation, delaying senescence and the like. At present, the dendrobium officinale polysaccharide is also used as a cosmetic raw material in the cosmetic industry, but the dendrobium officinale polysaccharide has a lasting moisturizing effect of large molecular weight (> 1000 kDa), and the research on the effect of the dendrobium officinale polysaccharide with small molecular weight (the average molecular weight is less than 300 kDa) on the aspect of enhancing skin immunity is not reported in documents.
The skin is used as an ecological system, is a habitat where microbial flora depends to live, and about billions of bacteria are mutually beneficial and symbiotic on the surface of the skin per square centimeter to form a micro-ecological protection shield. On the surface of human skin, there exist probiotics, mainly including bifidobacteria, lactobacillus species such as lactobacillus acidophilus, and harmful bacteria including streptococcus and staphylococcus aureus. The skin health condition is good, the normal flora is balanced, the probiotics account for the population dominance, most metabolites (sebum and sweat) on the skin are utilized by the beneficial flora, the pH value is reduced, the skin barrier is formed, harmful bacteria can be inhibited, and the skin state is normal; however, when the skin flora is disturbed and harmful bacteria are dominant, the pH value of the skin rises, the skin barrier function disappears, and harmful microorganisms can erode the skin and cause inflammation.
Langerhans Cells (LC) are important antigen presenting cells and mononuclear phagocytes in immune response, can capture and process antigens invaded in the skin, form antigen peptide-MHC molecule complexes, and transfer the complexes to T cells, so that specific T cells can be proliferated and activated to trigger immune response.
Therefore, as long as the dendrobium polysaccharide with the small molecular weight prepared by the invention can be proved to be capable of promoting the growth of probiotics on the surface of the skin and improving the density of Langerhans cells in the skin, the effect of the dendrobium polysaccharide on improving the immunity of the skin can be represented.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of the dendrobium officinale polysaccharide with the small molecular weight, wherein the average molecular weight of the dendrobium officinale polysaccharide is less than 300kDa, and the dendrobium officinale polysaccharide with the small molecular weight prepared by the method can promote the proliferation of skin probiotics and improve the skin immunity.
In order to solve the technical problems, the invention adopts the following technical scheme: a preparation method of dendrobium officinale polysaccharide with small molecular weight is characterized by comprising the following steps:
A. strain activation and expanding culture: under aseptic condition, taking appropriate amount of liquid culture medium to prepare strain with concentration of 1 × 105-1×106CFU/mL bacterial suspension, wherein the strain is fusarium avenae with the strain preservation number CICC 14038, and then 0.2-0.5mL bacterial suspension is streaked on the surface of a PDA solid culture medium and cultured for 24-48h at the temperature of 20-30 ℃; selecting well dispersed single colony, inoculating to a fermentation shake flask containing liquid culture medium components, performing propagation at 20-30 deg.C and 200r/min, performing oxygen supply culture for 24-72 hr at oxygen concentration of 0.010-0.030mol/L to obtain Fusarium avenaceum fermentation seed liquid with Fusarium avenaceum concentration of 1 × 104-1×105CFU/mL; the liquid culture medium is an MS liquid culture medium containing 15.0-30.0g/L of sucrose and 5.0-10.0g/L of yeast powder;
B. and (3) performing cell wall breaking treatment on the dendrobium officinale: pre-mixing the dendrobium officinale powder and distilled water according to a mass ratio of 1:20 at 50-52 ℃, pouring the mixture into a stainless steel container, stirring the mixture for 6-8h at a speed of 15rpm while scraping, and then repeatedly homogenizing the mixture for 5-10 times at high pressure by using a high-pressure micro-jet homogenizer, wherein the homogenization pressure is 90-130MPa, and the homogenization temperature is 50-52 ℃ to obtain the dendrobium officinale wall-broken homogenate;
C. fermentation culture: injecting a proper amount of liquid culture medium into a fermentation tank, weighing the wall-broken homogenate of the dendrobium officinale obtained in the step B, putting the wall-broken homogenate of the dendrobium officinale into the fermentation tank, weighing fusarium avenae seed liquid with the volume of 5.0-10.0% of that of the wall-broken homogenate of the dendrobium officinale, adding the seed liquid into the fermentation tank, and adding KH2PO4And Na2HPO4Adjusting pH of the feed liquid in the fermentation tank to 5.5-7.0 with buffer solution, fermenting at 25-35 deg.C for 3-5d with oxygen supply,controlling the oxygen concentration to be 0.020-0.040mol/L, and obtaining fermentation liquor after fermentation is completed;
D. polysaccharide purification: cooling to room temperature after fermentation, adding a diatomite filter aid accounting for 0.5-1.0% of the mass of the fermentation liquor and decolorizing active carbon accounting for 5.0-8.0% of the mass of the fermentation liquor into a fermentation tank, standing and adsorbing for 1h, filtering and removing residues by using a plate-and-frame filter press to obtain a filtrate, mixing the filtrate with a Sevage reagent according to the volume ratio of 4.0-6.0:1.0, fully oscillating for 30min, centrifuging at the rotating speed of 3000r/min for 10min, and taking a supernatant; mixing the supernatant with 95% ethanol at a mass ratio of 1.0:1.0-3.0, standing for 12-36h, vacuum filtering to obtain precipitate, washing with 80% ethanol for 3 times, and vacuum drying at 60 deg.C for 24h to obtain small molecular weight Dendrobium officinale polysaccharide.
The content of the small molecular weight dendrobium officinale polysaccharide prepared by the invention is measured as follows: reference is made to the phenol-sulfuric acid process; and (3) measuring the molecular weight of the small molecular weight dendrobium officinale polysaccharide: sephadex G-200 gel exclusion chromatography; the structural characterization of the dendrobium officinale polysaccharide with the small molecular weight needs to utilize a high performance liquid chromatograph to measure the polysaccharide after acidolysis. The results show that: the yield of the dendrobium officinale polysaccharide with the small molecular weight prepared by the invention is higher than 32.50%, the average molecular weight is less than 300KDa, and the main chain of the dendrobium officinale polysaccharide consists of D-glucose, D-galactose, L-rhamnose, L-arabinose and D-mannose.
Fusarium avenae (Fusarium avenaceum) used in the invention is purchased from China center for culture collection of industrial microorganisms, and the number of the strain is as follows: CICC 14038; the PDA culture medium is purchased from Hangzhou Baisi biotechnology limited; the sucrose (CAS 57-50-1) is purchased from Aladdin reagent (Shanghai) Limited company, and the purity is more than or equal to 99.5 percent; the yeast powder (CAS 119-44-8) is purchased from Beijing Hongrunbaoshu science and technology Limited, and has a purity of more than or equal to 99.0%; the MS liquid culture medium is produced by SIGMA company, and is available under the brand number M5519; the Dendrobium officinale powder is 2-year-old fresh Dendrobium officinale branches purchased from Wenzhou Leqing city of Zhejiang, and is automatically cleaned, air-dried and crushed into 50-100 meshes; the high-pressure micro-jet homogenizer is purchased from Shanghai carp leap precision machinery trade company, model M-110 EH; the fermentation tank is purchased from Nanjing Runzi bioengineering equipment Co., Ltd, model RZY-XGX; the diatomite is purchased in ShengzhouCommercially available diatomaceous earth products, Inc., under the designation CD06, SiO2(%) is more than or equal to 85 percent; the used decolorizing active carbon is purchased from Huzhou Sensheng active carbon Co., Ltd, and has the brand number of ZL-786 and the methylene blue adsorption capacity of 12mL/0.1 g; the plate-and-frame filter press is purchased from Kunshan environmental protection machinery Co., Ltd, and has the model of XAMGZ/30U; the gas bath constant temperature oscillator is purchased from Jiangsu Jierel electric appliances Co., Ltd, model number SHZ-82; the centrifuge used was purchased from machinery limited, Zhang hong City, model number PSF-1000; the vacuum suction filter is purchased from Shanghai Kesheng instruments Co., Ltd, and has the model of ZF-30L; the vacuum dryer was purchased from Changzhou day flight drying equipment, Inc., model SZG-350.
The dendrobium officinale polysaccharide with small molecular weight prepared in the invention has a promoting effect on the growth and proliferation of bifidobacterium adolescentis, bifidobacterium infantis, bifidobacterium bifidum and lactobacillus acidophilus, and the experiment is as follows by taking the product prepared in the embodiment 1 as an example:
the activated 4 strains were inoculated at 1.0% (10 concentration)6CFU/mL) was inoculated into MRS medium supplemented with small molecular weight Dendrobium officinale polysaccharides at mass fractions of 0, 0.5%, 1.0%, 1.5%, 2.0%, and 2.5%, each group was subjected to 3 parallel experiments, the culture was incubated at 38 deg.C for 24h, and the absorbance value (OD 600) of each culture at 600nm was measured using an ultraviolet-visible spectrophotometernm). The number of the used Bifidobacterium adolescentis (Bifidobacterium adolesentis) is CICC 6070, the number of the used Bifidobacterium infantis (Bifidobacterium infantis) is CICC 6069, the number of the used Bifidobacterium bifidum (Bifidobacterium bifidum) is CICC 6071, and the number of the used Lactobacillus acidophilus (Lactobacillus acidophilus) is CICC 6096, which are purchased from China center for the preservation and management of industrial microbial strains; the MRS culture medium is purchased from Qingdao Haibo biotechnology limited; the UV-visible spectrophotometer used was purchased from Youngco (Shanghai) instruments Inc., model UV-2100 PC.
OD of bifidobacterium and lactobacillus acidophilus in dendrobium officinale polysaccharide culture solutions with different concentrations and small molecular weights600nmThe values are shown in table 1, and the effect of different amounts of small molecular weight dendrobium officinale polysaccharide on the growth of bifidobacteria and lactobacillus acidophilus is shown in fig. 1.
TABLE 1 Bifidobacterium and Lactobacillus acidophilus OD in different concentrations of low molecular weight Dendrobium officinale polysaccharide culture600nmValue of
As can be seen from Table 1 and FIG. 1, with the increase of the addition amount of the small molecular weight Dendrobium officinale polysaccharide, the growth of typical skin probiotics Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacterium bifidum and Lactobacillus acidophilus shows a tendency to increase, and the OD is 2.0% (mass percent) of the addition amount600nmThe value reaches the maximum, when the addition amount of the dendrobium officinale polysaccharide with the small molecular weight reaches 2.5 percent (mass percentage), the growth promotion effect is weakened, which is probably caused by the fact that the osmotic pressure and the pH value of the culture solution are changed and the proliferation of probiotics is limited by the accumulation of metabolites due to the high concentration of the polysaccharide. The experimental result can show that when the concentration is less than 2 percent (mass percentage), the dendrobium officinale polysaccharide with small molecular weight has good promotion effect on the growth and proliferation of probiotics and is an effective prebiotic.
The effect of the dendrobium officinale polysaccharide with small molecular weight on the cell density of the spontaneous SLE model female mouse NZBHF 1/J epidermal Langerhans cell, which is the animal model closest to human SLE, is further used for representing the effect of the dendrobium officinale polysaccharide on the aspect of improving the skin immunity, and the experiment is as follows by taking the product prepared in the example 2 as an example:
dividing 12 NZBHF 1/J female mice with age of 2 months into 6 Dexamethasone (Dexamethasone) groups, and injecting Dexamethasone 1.0mg/kg intraperitoneally daily; 6 dendrobium officinale polysaccharide groups with small molecular weight are injected into the dendrobium officinale polysaccharide groups with small molecular weight of 50mg/kg every day in the abdominal cavity, and the dendrobium officinale polysaccharide groups are continuously taken from 2 months of age of a mouse for 6 months to 8 months of age of the mouse; SPF grade C57BL/6 inbred female mice were 6, 8 months old.
Shearing the ear of the same mouse under the condition of full-anesthesia of mouse ether, separating the skin on both sides of the ear by using a dissecting microscope, scraping the cartilage on both sides, putting into a biological EDTA separating solution with pH7.2, soaking for 2h at the constant temperature of 38 ℃, separating the true epidermis under the microscope, fixing the epidermis in acetone at 4 ℃ for 20min, and washing for 3 times with PBS phosphate buffer solution for 3min each time. Adding anti-mouse Ia antigen OX3 with the concentration of 1:50, anti-mouse Ia antigen OX4 with the concentration of 1:200 and mouse epidermis into eppendorf microtubes respectively, overnight at 4 ℃, washing with PBS, adding biotinylated goat anti-mouse IgG serum, incubating at 38 ℃ for 2h, washing with PBS, adding ABC (ovalbumin/biotin/enzyme) complex, incubating for 1h, and washing with PBS. Under a dissecting microscope, the epidermis is laid on a glass slide, the substrate 3-amino-9-ethyl carbazol (AEC) is added for color development and drying at room temperature, and then the glycerol gelatin is used for sealing.
The cells were observed under a microscope, and the cells were positive if they appeared in orange color. Epidermal Langerhans cell density (LC/mm)2) = average LC number per field/area of epidermis, area of epidermis determined by ocular grid calibrated with micrometer. The epidermal LC density data for each experimental group of mice is presented in table 2. The spontaneous SLE model female mouse NZBWF1/J used was purchased from Nanjing university-Nanjing biomedical research institute; the SPF grade C57BL/6 pure line mouse is female, 8 months old, and purchased from the center of Guangdong province medical experimental animals; the dissecting microscope was purchased from Shenzhen Bo Shida optical instruments, Inc., model number BD-60T.
TABLE 2 average value of epidermal LC density (LC/mm) of mice in each experimental group2）
As can be seen from Table 2, compared with the SLE type mouse NZBWF1/J in the dexamethasone group, the density of epidermal LC in SLE type mouse NZBWF1/J in the dendrobium officinale polysaccharide group with small molecular weight is obviously increased, probably because the dendrobium officinale polysaccharide with small molecular weight promotes the generation of GM-CSF, simultaneously stimulates immature granulocytes and macrophages to differentiate and mature, improves the immune function of an organism, and dexamethasone serving as an immunosuppressant is widely applied to the treatment and research of autoimmune diseases of human beings and animals; compared with C57BL/6 pure line mice, the density of epidermal LC of the SLE type mice NZBHF 1/J of the dendrobium officinale polysaccharide group is also remarkably increased.
Experiments prove that the dendrobium officinale polysaccharide with the small molecular weight can promote the increase of the Langerhans cell density of the skin, so that the immunity of the skin can be improved, and the physique of the skin can be improved.
Control test:
preparing the high molecular weight dendrobium officinale polysaccharide according to the following steps: putting the dendrobium officinale powder and distilled water into a reaction container according to the mass ratio of 1:20, heating to 85 ℃, leaching for 3 hours, performing suction filtration after completion, repeating leaching for 3 times, collecting filtrate, performing vacuum drying and concentration to 1/5 of the original volume, and then performing vacuum drying and concentration according to the following steps: mixing Sevage reagent =6.0:1.0 (volume ratio), fully shaking for 30min, centrifuging for 10min at 3000r/min, taking supernatant, and then: adding 95% ethanol =1.0:2.0 (mass ratio) into ethanol, standing for 24h, filtering by a vacuum filter to obtain precipitate, washing for 3 times by 80% ethanol, and vacuum drying at 60 ℃ for 24h to obtain the high molecular weight dendrobium officinale polysaccharide product.
The obtained high molecular weight Dendrobium officinale polysaccharide has a molecular weight of 730-1180kDa, and its main chain comprises D-glucose, D-galactose, L-rhamnose, L-arabinose and D-mannose.
The growth and proliferation effects of the high molecular weight dendrobium officinale polysaccharide on bifidobacterium adolescentis, bifidobacterium infantis, bifidobacterium bifidum and lactobacillus acidophilus are researched, the experimental method is the same as the above, and the experimental results are shown in table 3.
TABLE 3 OD of Bifidobacterium and Lactobacillus acidophilus in Dendrobium officinale polysaccharide culture solution with different concentrations and large molecular weight600nmValue of
As can be seen from Table 3 and FIG. 2, with the increase of the addition amount of the high molecular weight Dendrobium officinale polysaccharide, the growth of the typical skin probiotics Bifidobacterium adolescentis, Bifidobacterium infantis, Bifidobacterium bifidum and Lactobacillus acidophilus does not show a significant growth trend, and the experimental results show that the high molecular weight Dendrobium polysaccharide (730-1180kDa) does not have a significant promotion effect on the growth and proliferation of the probiotics.
The effect of the dendrobium officinale polysaccharide on the skin immunity is characterized by the influence of the dendrobium officinale polysaccharide with large molecular weight on the density of NZBHWF 1/J epidermal Langerhans cells of a spontaneous SLE model female mouse, the experimental method is the same as that of the dendrobium officinale polysaccharide, and the experimental result is shown in Table 4.
TABLE 4 average value of epidermal LC density (LC/mm) of mice in each experimental group2）
As can be seen from Table 4, compared with the C57BL/6 pure line mice, the density of the epidermal LC of the SLE type mice NZBHWF 1/J of the high molecular weight dendrobium officinale polysaccharide group is not obviously increased, and experiments show that the high molecular weight dendrobium polysaccharide (730-1180kDa) can not obviously promote the increase of the skin Langerhans cell density and hardly plays a role in improving the skin immunity.
In conclusion, a contrast test shows that the high molecular weight dendrobium polysaccharide has no obvious promotion effect on growth and proliferation of probiotics and improvement of skin Langerhans cell density, and cannot effectively improve skin immunity.
The method comprises the steps of firstly performing wall breaking treatment on the dendrobium officinale powder by using a high-pressure micro-jet homogenization method, and then fermenting and preparing the dendrobium officinale polysaccharide with small molecular weight, wherein the average molecular weight of the dendrobium officinale polysaccharide is less than 300KDa, and the fusarium avenaceum is selected from endophytic fungi of the dendrobium officinale. Through research on the influence of the dendrobium officinale polysaccharide with the small molecular weight on the growth of probiotics and the density of mouse Langerhans Cells (LC), the result shows that the dendrobium officinale polysaccharide with the small molecular weight can effectively promote the proliferation of skin probiotics and improve the density of the Langerhans cells on the surface of the skin, so that the immunity and the defensive power of the skin are improved, and the healthy state of the skin is maintained.
The preparation method of the small-molecular-weight dendrobium officinale polysaccharide provided by the invention mainly utilizes fusarium avenaceum to ferment so as to prepare the small-molecular-weight dendrobium officinale polysaccharide, and the average yield is higher than 32.50%.
In conclusion, the dendrobium officinale polysaccharide with the small molecular weight prepared by the invention has the average molecular weight of less than 300kDa, can effectively promote the proliferation of skin probiotics, and can improve the Langerhans cell density on the surface of the skin, so that the immunity and the defensive power of the skin are improved, and the healthy state of the skin is maintained.
Drawings
FIG. 1 shows the effect of different amounts of small molecular weight Dendrobium officinale polysaccharide on the growth of Bifidobacterium and Lactobacillus acidophilus;
FIG. 2 shows the effect of different amounts of high molecular weight Dendrobium officinale polysaccharide on the growth of Bifidobacterium and Lactobacillus acidophilus.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to these examples.
Example 1:
A. strain activation and expanding culture: under aseptic condition, using aseptic dropper to suck appropriate amount of liquid culture medium and drip it into strain tube containing freeze-dried powder of fusarium avenae (L.) danielli (endophytic fungus of Dendrobium officinale) to prepare strain with concentration of 1 × 106CFU/mL bacterial suspension, streaking 0.2mL bacterial suspension on the surface of PDA solid medium, culturing at 25 deg.C for 48h, selecting well dispersed single colony, inoculating into a fermentation shake flask containing liquid medium component, culturing at 25 deg.C under oxygen supply of 200r/min for 48h with oxygen concentration of 0.030mol/L, and making into fermentation seed liquid, wherein the concentration of Fusarium avenae is 1 × 105CFU/mL, wherein the used liquid culture medium is an MS liquid culture medium containing 30.0g/L of sucrose and 8.0g/L of yeast powder;
B. and (3) performing cell wall breaking treatment on the dendrobium officinale: pre-mixing 3kg of dendrobium officinale powder with 60L of distilled water at 50 ℃, pouring the mixture into a stainless steel container, stirring for 6 hours at a speed of 15rpm while scraping, and then repeatedly homogenizing for 8 times at high pressure by using a high-pressure micro-jet homogenizer at the pressure of 90MPa and the temperature of 50 ℃ to obtain the dendrobium officinale wall-breaking homogenate;
C. fermentation culture: putting the wall-broken homogenate of the dendrobium officinale obtained in the step B into a fermentation tank, inoculating fusarium avenae seed liquid according to the inoculation amount of 7.0% of the volume of the homogenate, and adding KH into the liquid culture medium in the fermentation tank, wherein the components of the liquid culture medium in the step A are the same as those in the step A2PO4And Na2HPO4Adjusting the pH of the feed liquid to be 7.0 by using a buffer solution, and performing oxygen supply fermentation for 3d at the temperature of 35 ℃ and the oxygen concentration to be 0.040 mol/L;
D. polysaccharide purification: cooling to room temperature after fermentation, adding diatomite filter aid accounting for 0.5% of the mass of the fermentation liquor and decolorizing active carbon accounting for 7.0% of the mass of the fermentation liquor into a fermentation tank, standing for adsorption for 1h, and filtering with a plate-and-frame filter press to remove residues to obtain filtrate; then according to the filtrate: mixing Sevage reagent =4.0:1.0 (volume ratio), fully shaking for 30min, centrifuging for 10min at 3000r/min, taking supernatant, and then: adding 95% ethanol =1.0:2.0 (mass ratio) into ethanol, standing for 24h, filtering by a vacuum filter to obtain precipitate, washing for 3 times by 80% ethanol, and vacuum drying at 60 ℃ for 24h to obtain 1005.6g of the small-molecular-weight dendrobium officinale polysaccharide product.
The yield of the dendrobium officinale polysaccharide with the small molecular weight prepared by the embodiment is 33.52%, the average molecular weight is 221.60kDa, the main chain of the dendrobium officinale polysaccharide is composed of D-glucose, D-galactose, L-rhamnose, L-arabinose and D-mannose, and the molar ratio is 3.112: 2.013: 1.561: 1.034: 2.487.
Example 2:
A. strain activation and expanding culture: under aseptic condition, using aseptic dropper to suck appropriate amount of liquid culture medium and drip it into strain tube containing freeze-dried powder of fusarium avenae (L.) baill as endophytic fungus of dendrobium officinale to prepare strain with concentration of 8.5 × 105CFU/mL bacterial suspension, streaking 0.4mL bacterial suspension on the surface of PDA solid medium, culturing at 20 deg.C for 36h, selecting well dispersed single colony, inoculating into a fermentation shake flask containing liquid medium component, culturing at 20 deg.C under 200r/min with oxygen supply of 0.010mol/L for 72h, and making into fermented seed liquid, wherein the concentration of Fusarium avenae is 6.5 × 104CFU/mL, wherein the used liquid culture medium is an MS liquid culture medium containing 18.0g/L of sucrose and 10.0g/L of yeast powder;
B. and (3) performing cell wall breaking treatment on the dendrobium officinale: pre-mixing 3kg of dendrobium officinale powder with 60L of distilled water at 52 ℃, pouring the mixture into a stainless steel container, stirring the mixture for 8 hours at a speed of 15rpm while scraping, and then repeatedly homogenizing the mixture for 10 times at a high pressure and a temperature of 52 ℃ by using a high-pressure micro-jet homogenizer at a pressure of 130MPa to obtain the dendrobium officinale wall-breaking homogenate;
C. fermentation culture: c, putting the dendrobium officinale wall-broken homogenate obtained in the step B into a fermentation tank, inoculating fusarium avenae seed liquid according to the inoculation amount of 10.0% of the volume of the homogenate, and synchronously performing liquid culture medium components in the fermentation tankA, and adding KH2PO4And Na2HPO4Adjusting pH of the feed liquid to be 5.5 by using buffer solution, and performing oxygen supply fermentation for 5d at 25 ℃ with oxygen concentration of 0.030 mol/L;
D. polysaccharide purification: cooling to room temperature after fermentation, adding diatomite filter aid accounting for 0.8% of the mass of the fermentation liquor and decolorizing active carbon accounting for 8.0% of the mass of the fermentation liquor into a fermentation tank, standing for adsorption for 1h, and filtering with a plate-and-frame filter press to remove residues to obtain filtrate; then according to the filtrate: mixing Sevage reagent =5.0:1.0 (volume ratio), fully shaking for 30min, centrifuging for 10min at 3000r/min, taking supernatant, and then: adding 95% ethanol =1.0: 3.0 (mass ratio) into ethanol, standing for 36h, filtering by a vacuum filter to obtain precipitate, washing for 3 times by 80% ethanol, and vacuum drying at 60 ℃ for 24h to obtain 1035.6g of the small-molecular-weight dendrobium officinale polysaccharide product.
The yield of the dendrobium officinale polysaccharide with the small molecular weight prepared by the embodiment is 34.52%, the average molecular weight is 1.5kDa, the main chain of the dendrobium officinale polysaccharide consists of D-glucose, D-galactose, L-rhamnose, L-arabinose and D-mannose, and the molar ratio is 2.452:1.025:2.231:3.154: 1.562.
Example 3:
A. strain activation and expanding culture: under aseptic condition, using aseptic dropper to suck appropriate amount of liquid culture medium and drip it into strain tube containing freeze-dried powder of fusarium avenae (L.) danielli (endophytic fungus of Dendrobium officinale) to prepare strain with concentration of 1 × 105CFU/mL bacterial suspension, streaking 0.5mL bacterial suspension on the surface of PDA solid medium, culturing at 30 deg.C for 24h, selecting well dispersed single colony, inoculating into a fermentation shake flask containing liquid medium component, culturing at 30 deg.C under 200r/min with oxygen supply of 0.020mol/L for 24h, and making into fermentation seed liquid, wherein the concentration of Fusarium avenae is 1 × 104CFU/mL, wherein the used liquid culture medium is an MS liquid culture medium containing 15.0g/L of sucrose and 10.0g/L of yeast powder;
B. and (3) performing cell wall breaking treatment on the dendrobium officinale: pre-mixing 3kg of dendrobium officinale powder with 60L of distilled water at 51 ℃, pouring the mixture into a stainless steel container, stirring the mixture for 6.5 hours at a speed of 15rpm while scraping, and then repeatedly homogenizing the mixture for 5 times at a pressure of 100MPa and a temperature of 51 ℃ by using a high-pressure micro-jet homogenizer to obtain the dendrobium officinale wall-breaking homogenate;
C. fermentation culture: putting the wall-broken homogenate of the dendrobium officinale obtained in the step B into a fermentation tank, inoculating fusarium avenae seed liquid according to the inoculation amount of 5.0% of the volume of the homogenate, and adding KH into the fermentation tank, wherein the components of a liquid culture medium in the fermentation tank are the same as those in the step A2PO4And Na2HPO4Adjusting the pH of the feed liquid to be 6.0 by using a buffer solution, and performing oxygen supply fermentation for 4d at the temperature of 30 ℃ and the oxygen concentration of 0.020 mol/L;
D. polysaccharide purification: cooling to room temperature after fermentation, adding diatomite filter aid accounting for 0.7% of the mass of the fermentation liquor and decolorizing active carbon accounting for 5.0% of the mass of the fermentation liquor into a fermentation tank, standing for adsorption for 1h, and filtering with a plate-and-frame filter press to remove residues to obtain filtrate; then according to the filtrate: mixing Sevage reagent =6.0:1.0 (volume ratio), fully shaking for 30min, centrifuging for 10min at 3000r/min, taking supernatant, and then: adding 95% ethanol =1.0:1.0 (mass ratio) into ethanol, standing for 24h, filtering by a vacuum filter to obtain precipitate, washing for 3 times by 80% ethanol, and vacuum drying at 60 ℃ for 24h to obtain 975.0g of the small-molecular-weight dendrobium officinale polysaccharide product.
The yield of the dendrobium officinale polysaccharide with the small molecular weight prepared by the embodiment is 32.50%, the average molecular weight is 110.50kDa, the main chain of the dendrobium officinale polysaccharide consists of D-glucose, D-galactose, L-rhamnose, L-arabinose and D-mannose, and the molar ratio is 1.547: 1.751:2.323:2.872: 1.310.
Example 4:
A. strain activation and expanding culture: under aseptic condition, using aseptic dropper to suck appropriate amount of liquid culture medium and drip it into strain tube containing freeze-dried powder of fusarium avenae (L.) baill as endophytic fungus of dendrobium officinale, the concentration of strain is 4.2X 105CFU/mL bacterial suspension, streaking 0.3mL bacterial suspension on the surface of PDA solid medium, culturing at 28 deg.C for 24h, selecting well dispersed single colony, inoculating into a fermentation shake flask containing liquid medium component, culturing at 28 deg.C under oxygen supply of 200r/min for 30h with oxygen concentration of 0.020mol/L, and making into fermentation seed liquid, wherein the concentration of Fusarium avenae is 3 × 104CFU/mL, wherein the used liquid culture medium is an MS liquid culture medium containing 18.0g/L of sucrose and 5.0g/L of yeast powder;
B. and (3) performing cell wall breaking treatment on the dendrobium officinale: pre-mixing 3kg of dendrobium officinale powder with 60L of distilled water at 50 ℃, pouring the mixture into a stainless steel container, stirring the mixture for 6.5 hours at a speed of 15rpm while scraping, and then repeatedly homogenizing the mixture for 7 times at a high pressure of 110MPa and a temperature of 50 ℃ by using a high-pressure micro-jet homogenizer to obtain the dendrobium officinale wall-breaking homogenate;
C. fermentation culture: putting the wall-broken homogenate of the dendrobium officinale obtained in the step B into a fermentation tank, inoculating fusarium avenae seed liquid according to the inoculation amount of 8.5% of the volume of the homogenate, and adding KH into the fermentation tank, wherein the components of a liquid culture medium in the fermentation tank are the same as those in the step A2PO4And Na2HPO4Adjusting the pH of the feed liquid to be 6.8 by using a buffer solution, and performing oxygen supply fermentation for 4d at the temperature of 28 ℃ and the oxygen concentration to be 0.030 mol/L;
D. polysaccharide purification: cooling to room temperature after fermentation, adding diatomite filter aid accounting for 0.6% of the mass of the fermentation liquor and decolorizing active carbon accounting for 6.5% of the mass of the fermentation liquor into a fermentation tank, standing for adsorption for 1h, and filtering with a plate-and-frame filter press to remove residues to obtain filtrate; then according to the filtrate: mixing Sevage reagent =4.5:1.0 (volume ratio), fully shaking for 30min, centrifuging for 10min at 3000r/min, taking supernatant, and then: adding 95% ethanol =1.0:2.0 (mass ratio) into ethanol, standing for 12h, filtering by a vacuum filter to obtain precipitate, washing for 3 times by 80% ethanol, and vacuum drying at 60 ℃ for 24h to obtain 988.5g of the small-molecular-weight dendrobium officinale polysaccharide product.
The yield of the dendrobium officinale polysaccharide with the small molecular weight prepared by the embodiment is 32.95%, the average molecular weight is 300kDa, the main chain of the dendrobium officinale polysaccharide consists of D-glucose, D-galactose, L-rhamnose, L-arabinose and D-mannose, and the molar ratio is 3.005:2.645:1.006:2.336: 1.496.
Example 5:
A. strain activation and expanding culture: under aseptic condition, using aseptic dropper to suck appropriate amount of liquid culture medium and drip it into strain tube containing freeze-dried powder of fusarium avenae (L.) baill as endophytic fungus of dendrobium officinale, the concentration of strain is 7.8X 105CFU/mL bacterial suspension, streaking 0.25mL bacterial suspension on the surface of PDA solid culture medium, culturing at 23 deg.C for 30h, selecting single colony with good dispersion, inoculating into a fermentation shake flask containing liquid culture medium component, performing propagation, and supplying oxygen at 23 deg.C and 200r/minCulturing for 72h with oxygen concentration of 0.030mol/L to obtain fermented seed liquid, wherein the concentration of Fusarium avenaceum is 5.3 × 104CFU/mL, wherein the used liquid culture medium is an MS liquid culture medium containing 28.0g/L of sucrose and 6.0g/L of yeast powder;
B. and (3) performing cell wall breaking treatment on the dendrobium officinale: pre-mixing 3kg of dendrobium officinale powder with 60L of distilled water at 52 ℃, pouring the mixture into a stainless steel container, stirring the mixture for 6 to 8 hours at a speed of 15rpm while scraping, and then repeatedly homogenizing the mixture for 8 times at high pressure and a pressure of 120MPa and a temperature of 52 ℃ by using a high-pressure micro-jet homogenizer to obtain the dendrobium officinale wall-breaking homogenate;
C. fermentation culture: putting the wall-broken homogenate of the dendrobium officinale obtained in the step B into a fermentation tank, inoculating fusarium avenae seed liquid according to the inoculation amount of 9.0% of the volume of the homogenate, and adding KH into the liquid culture medium in the fermentation tank, wherein the components of the liquid culture medium in the step A are the same as those in the step A2PO4And Na2HPO4Adjusting the pH of the feed liquid to be 6.0 by using a buffer solution, and performing oxygen supply fermentation for 4d at the temperature of 28 ℃ and the oxygen concentration to be 0.035 mol/L;
D. polysaccharide purification: cooling to room temperature after fermentation, adding diatomite filter aid accounting for 1.0% of the mass of the fermentation liquor and decolorizing active carbon accounting for 7.5% of the mass of the fermentation liquor into a fermentation tank, standing for adsorption for 1h, and filtering with a plate-and-frame filter press to remove residues to obtain filtrate; then according to the filtrate: mixing Sevage reagent =5.5:1.0 (volume ratio), fully shaking for 30min, centrifuging for 10min at 3000r/min, taking supernatant, and then: adding 95% ethanol =1.0:2.5 (mass ratio) into ethanol, standing for 30h, filtering by a vacuum filter to obtain precipitate, washing for 3 times by 80% ethanol, and vacuum drying at 60 ℃ for 24h to obtain 1026.0g of the small-molecular-weight dendrobium officinale polysaccharide product.
The yield of the dendrobium officinale polysaccharide with the small molecular weight prepared by the embodiment is 34.20%, the weight average molecular weight is 98.74kDa, the main chain of the dendrobium officinale polysaccharide consists of D-glucose, D-galactose, L-rhamnose, L-arabinose and D-mannose, and the molar ratio is 1.894:2.620:3.128:1.772: 2.315.
Claims (1)
1. A preparation method of dendrobium officinale polysaccharide with an average molecular weight of less than 300kDa is characterized by comprising the following steps:
A. strain activation and expanding culture: is free ofUnder the condition of bacteria, taking a proper amount of liquid culture medium to prepare a strain with the concentration of 1 multiplied by 105-1×106CFU/mL bacterial suspension, wherein the strain is Fusarium avenae (Fusarium avenaceum), the strain preservation number is CICC 14038, then 0.2-0.5mL bacterial suspension is taken to be streaked on the surface of a PDA solid culture medium, and the culture is carried out for 24-48h at the temperature of 20-30 ℃; selecting well dispersed single colony, inoculating to a fermentation shake flask containing liquid culture medium components, performing propagation at 20-30 deg.C and 200r/min, performing oxygen supply culture for 24-72 hr at oxygen concentration of 0.010-0.030mol/L to obtain Fusarium avenaceum fermentation seed liquid with Fusarium avenaceum concentration of 1 × 104-1×105CFU/mL; the liquid culture medium is an MS liquid culture medium containing 15.0-30.0g/L of sucrose and 5.0-10.0g/L of yeast powder;
B. and (3) performing cell wall breaking treatment on the dendrobium officinale: pre-mixing the dendrobium officinale powder and distilled water according to a mass ratio of 1:20 at 50-52 ℃, pouring the mixture into a stainless steel container, stirring the mixture for 6-8h at a speed of 15rpm while scraping, and then repeatedly homogenizing the mixture for 5-10 times at high pressure by using a high-pressure micro-jet homogenizer, wherein the homogenization pressure is 90-130MPa, and the homogenization temperature is 50-52 ℃ to obtain the dendrobium officinale wall-broken homogenate;
C. fermentation culture: injecting a proper amount of liquid culture medium into a fermentation tank, weighing the wall-broken homogenate of the dendrobium officinale obtained in the step B, putting the wall-broken homogenate of the dendrobium officinale into the fermentation tank, weighing fusarium avenae seed liquid with the volume of 5.0-10.0% of that of the wall-broken homogenate of the dendrobium officinale, adding the seed liquid into the fermentation tank, and adding KH2PO4And Na2HPO4Adjusting pH of the feed liquid in the fermentation tank to 5.5-7.0 with buffer solution, performing oxygen supply fermentation at 25-35 deg.C for 3-5d, controlling oxygen concentration at 0.020-0.040mol/L, and fermenting to obtain fermentation liquid;
D. polysaccharide purification: cooling to room temperature after fermentation, adding a diatomite filter aid accounting for 0.5-1.0% of the mass of the fermentation liquor and decolorizing active carbon accounting for 5.0-8.0% of the mass of the fermentation liquor into a fermentation tank, standing and adsorbing for 1h, filtering and removing residues by using a plate-and-frame filter press to obtain a filtrate, mixing the filtrate with a Sevage reagent according to the volume ratio of 4.0-6.0:1.0, fully oscillating for 30min, centrifuging at the rotating speed of 3000r/min for 10min, and taking a supernatant; mixing the supernatant with 95% ethanol according to the mass ratio of 1.0:1.0-3.0, standing for 12-36h, vacuum filtering to obtain precipitate, washing with 80% ethanol for 3 times, and vacuum drying at 60 deg.C for 24h to obtain Dendrobium officinale polysaccharide with average molecular weight less than 300 kDa.
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