CN102559802A - Method for preparing high-yield Tremella polysaccharide by adopting culture medium for fermenting - Google Patents
Method for preparing high-yield Tremella polysaccharide by adopting culture medium for fermenting Download PDFInfo
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Abstract
The invention relates to a method for preparing high-yield Tremella polysaccharide by taking a culture medium for fermenting and belongs to the technical field of microbial fermentation culture. The method comprises the following steps of: based on Tremellafuciformis CGMCCC (China General Microbiological Culture Collection Center) 5.466 as a strain, firstly, inoculating the strains into a seed culture medium to culture for one day; inoculating cultured seeds into a fermentation culture medium to culture, wherein the fermentation liquid loading amount is 5L, and the fermentation period is 72 hours; centrifuging fermentation broth, collecting, concentrating, carrying out alcohol precipitation and diethyl ether washing, and drying to obtain 9.5g/L high-yield Tremella polysaccharide. By using the method, the fermentation period is effectively shortened; and the method has the characteristics that process is simple and easy to operate, environment pollution is little, high-yield polysaccharide can be obtained, and industrialization is easy to realize.
Description
Technical field
The invention belongs to microbial fermentation culture technique field, relate to a kind of method of substratum fermentation high yield tremella polysaccharide.
Background technology
White fungus is claimed Tremella again, is a kind of higher fungi, is the important medicinal material of generally acknowledging in the world.A kind of acidic polysaccharose class material that contains in the white fungus can improve body's immunological function, has antiviral effect, promotes the mobility of T cell, so can prevent effectively that curing cancers is in interior multiple disease.Tremella polysaccharide can also improve the protective capacities of body to atomic radiation, reduces the sedimentary material of body lipofuscin, has anti-ageing effect of prolonging life etc.
Will obtain tremella polysaccharide at present on the market in a large number and mainly adopt conidium is carried out liquid submerged fermentation, fermentative prodn is with low cost, and the high yield polysaccharide can be realized industrialization, can obtain remarkable economic efficiency to manufacturing enterprise.The research great majority of high yield tremella polysaccharide concentrate on fermentation condition are optimized, and the optimization of fermention medium is very important in the production of mikrobe industry, is to industrial necessary link from the laboratory.Can design a good fermention medium, be a very important step in the leavened prod industriallization success.
The present invention is a tremella polysaccharide liquid submerged fermentation medium optimization; Through a large amount of experimental study screenings; Found a kind of substratum of the most suitable its growth yeast shape conidium bacterium fermentation, produced on the market at present about polysaccharide 4 g/L, method of the present invention is produced polysaccharide up to 9.5 g/L; On original basis, improve the output of fermentation polysaccharide, in the hope of reaching the purpose of the highest fermentation polysaccharide of production.
Summary of the invention
The objective of the invention is deficiency to prior art; A kind of method of medium liquid submerged fermentation high yield tremella polysaccharide is provided; Be characterized in supplying aborning growth breeding and accumulation tunning; Keep tremella polysaccharide thalline good cell form, highdensity growth obtains a large amount of high-purity tremella polysaccharide products.
Technical scheme of the present invention is following:
A kind of method of substratum fermentation high yield tremella polysaccharide is characterized in that being undertaken by following step:
(1) bacterial classification:
White fungus gemma Tremella fuciformis CGMCCC5.466 purchases in China Committee for Culture Collection of Microorganisms common micro-organisms center;
(2) substratum preparation:
White fungus gemma glycerine storage medium: glycerine 300g/L;
White fungus gemma slant preservation substratum: potato 200g/L, glucose 20g/L, peptone 5g/L, sal epsom 0.3g/L, potassium primary phosphate 2g/L, agar 20g/L;
White fungus gemma seed culture medium: glucose 20g/L, peptone 8 g/L, sal epsom 2.5 g/L, YNB (not having amino yeast nitrogen) 1.5g/L, potassium primary phosphate 0.5g/L, PH requires 6.0;
White fungus gemma fermention medium: glucose 30g/L, peptone 8g/L, sal epsom 2.5 g/L, YNB (not having amino yeast nitrogen) 2g/L, potassium primary phosphate 0.5g/L, PH requires 6.0;
(3) preparation technology:
Culture presevation: use the glycerine storage medium, preserved 1 year for-70 ℃;
The seed shake-flask culture: inoculum size is linked in the seed culture medium with 5～10% bacterium liquid and cultivated 25 ℃ of culture temperature 1 day;
Fermentation culture: cultured seed is linked in the fermentor tank with 8～10% inoculum sizes, fermentation liquid amount 5L, 25 ℃ of leavening temperatures, mixing speed 200rpm controls PH6.0 with ammoniacal liquor; Fermentation time transfers mixing speed to 300rpm to 48h, and the fermentation culture cycle finishes to 72h, and centrifugal collection supernatant concentrates, and alcohol precipitation obtains tremella polysaccharide.
The more detailed method of the present invention is following:
(1) bacterial classification: Tremella fuciformis CGMCCC5.466 purchases in China Committee for Culture Collection of Microorganisms common micro-organisms center.
(2) substratum preparation:
White fungus gemma glycerine storage medium: glycerine 300g/L.
White fungus gemma slant preservation substratum: potato 200g/L, glucose 20g/L, peptone 5g/L, sal epsom 0.3g/L, potassium primary phosphate 2g/L, agar 20g/L.
White fungus gemma seed culture medium: glucose 20g/L, peptone 8 g/L, sal epsom 2.5 g/L, YNB (not having amino yeast nitrogen) 1.5g/L, potassium primary phosphate 0.5g/L, PH requires 6.0.
White fungus gemma fermention medium: glucose 30g/L, peptone 8g/L, sal epsom 2.5 g/L, YNB (not having amino yeast nitrogen) 2g/L, potassium primary phosphate 0.5g/L, PH requires 6.0.
(3) preparation technology:
Culture presevation: use the glycerine storage medium, preserved 1 year for-70 ℃.
The seed shake-flask culture: inoculum size is linked in the seed culture medium with 5～10% bacterium liquid and cultivated 25 ℃ of culture temperature 1 day.
Fermentation culture: cultured seed is linked in the fermentor tank with 10% inoculum size, fermentation liquid amount 5L, 25 ℃ of leavening temperatures, mixing speed 200rpm controls PH6.0 with ammoniacal liquor; Fermentation time transfers mixing speed to 300rpm to 48h, and the fermentation culture cycle finishes to 72h, and centrifugal collection supernatant concentrates the alcohol precipitation polysaccharide.Fermention medium is: (glucose 30g/L, peptone 8g/L, sal epsom 2.5g/L, potassium primary phosphate 0.5g/L, YNB (not having amino yeast nitrogen) 2g/L, PH requires 6.0).
The physicochemical property of the fermentation polysaccharide of the present invention's preparation is following
Method: adopt deproteinated (Sevage method) earlier, take off small molecules (dialysis), gel chromatography purifying (EDAE post), during column purification, adopt the monitoring of sulfuric acid phynol method, carry out the map analysis of DEAE column chromatography then and obtain two kinds of equal one polysaccharide: BN516 and BN608.
The biological activity of the equal one polysaccharide of BN516 and BN608
Suppress the lymphoma experiment
Get BN516 and BN608 polysaccharide, be configured to concentration 5mg/kg, 10mg/kg with water for injection.The IRM-2 mouse inbred lines, male and female half and half.Inguinal region place's subcutaneous injection in IRM-2 mouse inbred lines left side cultivations of going down to posterity of IRM-2 mouse inbred lines spontaneous lymphoma, is put to death the IRM-2 mouse inbred lines and is peeled off the knurl piece, claims the knurl weight, shows that through experiment tremella polysaccharide is inhibited to lymphoma.
Beneficial effect of the present invention: through the substratum that is designed in the fermentation culture; Make the growth of favourable conidium thalline in the fermenting process; The title product that metabolism is a large amount of; Obtained High-efficient Production polysaccharide 9.5g/L, helped shortening fermentation period, with low cost, technology is simple to operation, environmental pollution is little, can obtain the polysaccharide and the easy realization of industrialization of high yield.
Embodiment
Below in conjunction with embodiment the present invention is described; The scheme of embodiment described here; Do not limit the present invention; One of skill in the art can make improvements and change according to spirit of the present invention, and described these improvement and variation all should be regarded as within the scope of the invention, and scope of the present invention and essence are limited claim.A wherein used substratum all ingredients all has commercially available.
Embodiment 1
The bacterial classification that the present invention uses is purchased in China Committee for Culture Collection of Microorganisms common micro-organisms center as Tremella fuciformis CGMCCC5.466.Be transferred to bacterial classification in the white fungus gemma slant preservation substratum earlier and cultivated 4 days, 25 ℃ of temperature requirements, the slant preservation substratum is: (potato 200g/L, glucose 20g/L, peptone 5g/L, sal epsom 0.3g/L, potassium primary phosphate 2g/L, agar 20g/L); Be transferred to cultured slant strains then and carry out-70 ℃ of preservations in the glycerine storage medium, the shelf time is 1 year, and the glycerine storage medium is: (glycerine 300g/L);
Be transferred in the shake-flask seed substratum with 10% inoculum size at the cultured bacterial classification of glycerine preservation and cultivated 25 ℃ of culture temperature, shaking speed 160rpm 1 day; Seed culture medium is: (glucose 20g/L; Peptone 8g/L, sal epsom 2.5g/L, YNB (not having amino yeast nitrogen) 1.5 g/L; Potassium primary phosphate 0.5g/L, PH requires 6.0); Cultured seed is linked in the fermentor tank with 10% inoculum size, fermentation liquid amount 5L, 25 ℃ of leavening temperatures, mixing speed 200rpm is controlled at 50% to dissolved oxygen according to the growing state of cell, and Ventilation Rate 3vvm controls PH6.0 with ammoniacal liquor; When fermentation time transfers mixing speed to 300rpm to 48h, the fermentation culture cycle finishes to 72h, and fermention medium is: (glucose 30g/L; Peptone 8g/L, sal epsom 2.5g/L, potassium primary phosphate 2.5g/L; YNB (not having amino yeast nitrogen) 2g/L, PH requires 6.0);
Carry out centrifugal collecting cell and supernatant to fermented liquid, centrifugal rotational speed is 3000 rpm, and the time is 20min; The fermentation supernatant that collection is good is rotated evaporimeter and concentrates 75 ℃ of thickening temperatures; Add the long-pending ethanol of triploid and carry out polysaccharide precipitation, leave standstill centrifugal collecting precipitation behind the 10min, centrifugal rotational speed is 3000 rpm, and the time is 20min, and deposition is washed with a little ether and decoloured, and with 50 ℃ of oven dry of incubator, collection obtains high yield polysaccharide 9.5g/L.
Embodiment 2
(1) bacterial classification: white fungus gemma Tremella fuciformis CGMCCC5.466 purchases in China Committee for Culture Collection of Microorganisms common micro-organisms center;
(2) substratum preparation:
White fungus gemma glycerine storage medium: glycerine 300g/L;
White fungus gemma slant preservation substratum: potato 200g/L, glucose 20g/L, peptone 5g/L, sal epsom 0.3g/L, potassium primary phosphate 2g/L, agar 20g/L;
White fungus gemma seed culture medium: glucose 20g/L, peptone 8 g/L, sal epsom 2.5 g/L, YNB (not having amino yeast nitrogen) 1.5g/L, potassium primary phosphate 0.5g/L, PH requires 6.0;
White fungus gemma fermention medium: glucose 30g/L, peptone 8g/L, sal epsom 2.5 g/L, YNB (not having amino yeast nitrogen) 2g/L, potassium primary phosphate 0.5g/L, PH requires 6.0;
(3) preparation technology:
Culture presevation: use the glycerine storage medium, preserved 1 year for-70 ℃;
The seed shake-flask culture: inoculum size is linked in the seed culture medium with 5% bacterium liquid and cultivated 25 ℃ of culture temperature 1 day;
Fermentation culture: cultured seed is linked in the fermentor tank with 10% inoculum size, fermentation liquid amount 5L, 25 ℃ of leavening temperatures, mixing speed 200rpm, dissolved oxygen control 50% is controlled PH6.0 with ammoniacal liquor; Fermentation time transfers mixing speed to 300rpm to 48h, and the fermentation culture cycle finishes to 72h, carries out centrifugal collecting cell and supernatant to fermented liquid, and centrifugal rotational speed is 3000 rpm, and the time is 20min; The fermentation supernatant that collection is good is rotated evaporimeter and concentrates 75 ℃ of thickening temperatures; Add the long-pending ethanol of triploid and carry out polysaccharide precipitation, leave standstill centrifugal collecting precipitation behind the 10min, centrifugal rotational speed is 3000 rpm, and the time is 20min, and deposition is washed with a little ether and decoloured, and with 50 ℃ of oven dry of incubator, collection obtains high yield polysaccharide 9.5g/L.
Embodiment 3 comparison tests
Ordinary method:
(1) method: liquid submerged fermentation
(2) step: cultivate seed with seed culture medium earlier, seed culture medium is: SANMALT-S 10g/L, glucose 10g/L, peptone 4g/L, potassium primary phosphate 0.46g/L, potassium hydrogenphosphate 1g/L, sal epsom 1g/L, PH nature; Cultured seed is cultivated in being transferred to fermention medium, and fermention medium is: SANMALT-S 10g/L, glucose 20g/L, dregs of beans 20g/L, potassium primary phosphate 0.46g/L, potassium hydrogenphosphate 1g/L, sal epsom 1g/L, PH nature; Cultivate 120h collection fermented liquid and concentrate, alcohol precipitation is collected polysaccharide.
(3) result: collection obtains about polysaccharide 4 g/L.
Method of the present invention:
(1) method: liquid submerged fermentation
(2) step: cultivate seed with seed culture medium earlier, seed culture medium is: glucose 20g/L, and peptone 8 g/L, sal epsom 2.5 g/L, YNB (not having amino yeast nitrogen) 1.5g/L, potassium primary phosphate 0.5g/L, PH requires 6.0; Cultured seed is cultivated in being transferred to fermention medium, and fermention medium is: glucose 30g/L, and peptone 8g/L, sal epsom 2.5 g/L, YNB (not having amino yeast nitrogen) 2g/L, potassium primary phosphate 0.5g/L, PH requires 6.0; Cultivate 72h collection fermented liquid and concentrate, alcohol precipitation is collected polysaccharide.
(3) result: collect and obtain high yield polysaccharide 9.5 g/L.
Claims (1)
1. the method for substratum fermentation high yield tremella polysaccharide is characterized in that being undertaken by following step:
(1) bacterial classification:
White fungus gemma Tremella fuciformis CGMCCC5.466 purchases in China Committee for Culture Collection of Microorganisms common micro-organisms center;
(2) substratum preparation:
White fungus gemma glycerine storage medium: glycerine 300g/L;
White fungus gemma slant preservation substratum: potato 200g/L, glucose 20g/L, peptone 5g/L, sal epsom 0.3g/L, potassium primary phosphate 2g/L, agar 20g/L;
White fungus gemma seed culture medium: glucose 20g/L, peptone 8 g/L, sal epsom 2.5 g/L, YNB (not having amino yeast nitrogen) 1.5g/L, potassium primary phosphate 0.5g/L, PH requires 6.0;
White fungus gemma fermention medium: glucose 30g/L, peptone 8g/L, sal epsom 2.5 g/L, YNB (not having amino yeast nitrogen) 2g/L, potassium primary phosphate 0.5g/L, PH requires 6.0;
(3) preparation technology:
Culture presevation: use the glycerine storage medium, preserved 1 year for-70 ℃;
The seed shake-flask culture: inoculum size is linked in the seed culture medium with 5～10% bacterium liquid and cultivated 25 ℃ of culture temperature 1 day;
Fermentation culture: cultured seed is linked in the fermentor tank with 8～10% inoculum sizes, fermentation liquid amount 5L, 25 ℃ of leavening temperatures, mixing speed 200rpm controls PH6.0 with ammoniacal liquor; Fermentation time transfers mixing speed to 300rpm to 48h, and the fermentation culture cycle finishes to 72h, and centrifugal collection supernatant concentrates, and alcohol precipitation obtains tremella polysaccharide.
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Cited By (6)
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| CN106135903A (en) * | 2015-03-31 | 2016-11-23 | 北京九和药业有限公司 | A kind of for functional food alleviating irritable bowel syndrome and preparation method thereof |
| CN107446825A (en) * | 2017-09-11 | 2017-12-08 | 华熙福瑞达生物医药有限公司 | One plant of white fungus bacterial strain and its application |
| CN108342429A (en) * | 2018-05-04 | 2018-07-31 | 宁德师范学院 | A kind of preparation method of tremella spore fermentation high yield tremella polysaccharides |
| CN108441431A (en) * | 2018-03-27 | 2018-08-24 | 南京工业大学 | One plant height resistance Yarrowia lipolytica and its application |
| CN110964761A (en) * | 2019-12-13 | 2020-04-07 | 江苏远大仙乐药业有限公司 | Tremella and application thereof |
| CN111920779A (en) * | 2020-07-20 | 2020-11-13 | 源合盛(吉林)药业有限公司 | Tremella fuciformis polysaccharide capsule medicament and preparation method thereof |
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| WO2001032830A2 (en) * | 1999-10-15 | 2001-05-10 | Medmyco, Inc. | Process for producing, methods and compositions of glucuronoxylomannan as nutriceutical agent from higher basidiomycetes mushroom |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106135903A (en) * | 2015-03-31 | 2016-11-23 | 北京九和药业有限公司 | A kind of for functional food alleviating irritable bowel syndrome and preparation method thereof |
| CN107446825A (en) * | 2017-09-11 | 2017-12-08 | 华熙福瑞达生物医药有限公司 | One plant of white fungus bacterial strain and its application |
| CN107446825B (en) * | 2017-09-11 | 2019-12-06 | 华熙生物科技股份有限公司 | Tremella fuciformis strain and application thereof |
| CN108441431A (en) * | 2018-03-27 | 2018-08-24 | 南京工业大学 | One plant height resistance Yarrowia lipolytica and its application |
| CN108342429A (en) * | 2018-05-04 | 2018-07-31 | 宁德师范学院 | A kind of preparation method of tremella spore fermentation high yield tremella polysaccharides |
| CN110964761A (en) * | 2019-12-13 | 2020-04-07 | 江苏远大仙乐药业有限公司 | Tremella and application thereof |
| CN110964761B (en) * | 2019-12-13 | 2021-07-30 | 江苏远大仙乐药业有限公司 | Tremella and application thereof |
| CN111920779A (en) * | 2020-07-20 | 2020-11-13 | 源合盛(吉林)药业有限公司 | Tremella fuciformis polysaccharide capsule medicament and preparation method thereof |
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