CN1483827A - Method for preparing active pachyman - Google Patents
Method for preparing active pachyman Download PDFInfo
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- CN1483827A CN1483827A CNA021370060A CN02137006A CN1483827A CN 1483827 A CN1483827 A CN 1483827A CN A021370060 A CNA021370060 A CN A021370060A CN 02137006 A CN02137006 A CN 02137006A CN 1483827 A CN1483827 A CN 1483827A
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Abstract
The present invention relates to a method for preparing active poria polysaccharide. It adopts the submerged fermentation process, and makes the poria strain undergo the processes of breeding, fermentation, extraction and refining so as to obtain the invented poria polysaccharide. SAid invention has no need of making chemical treatment of oxidation and carboxy methylation, and said product not only has the action of resisting tumor, but also can have the functions of resisting pollen hypersensitivity and improving renal function.
Description
Technical field
The present invention relates to microbial fermentation and separating and purifying technology field, relate in particular to a kind of preparation method of active pachyman.
Background technology
Pachymose promptly was proved as far back as the seventies has antineoplastic action, and it also is proved to be a kind of very strong immunostimulant material simultaneously.But extract this Pachymose with antitumor action from natural Poria cocos but is not easily, because the Pachymose in the natural Poria cocos is to form at interval with β 1 → 6 key bonded dextran base with 1～2 with β 1 → 3 key bonded dextran by 50.Anti-tumor experiment shows that this polysaccharide does not have anti-tumor activity, needs to remove with β 1 → 6 key bonded dextran, just can obtain having the Pachymose of anti-tumor activity.At present, the preparation Pachymose generally is to adopt chemical semi-synthesis method, this method is to be raw material with the Indian Bread, obtains the Pachymose highly finished product by alkaline solvent extraction, precipitation Crude polysaccharides, process for refining, obtains having the Pachymose of antitumor action again by oxidation, carboxymethylation.Therefore this method technology more complicated for preparing Pachymose, tooling cost, have limited the production of Pachymose to a certain extent than higher.
Summary of the invention
Purpose of the present invention is exactly the preparation method that the active pachyman that a kind of technology is simple, tooling cost is lower is provided for the defective that overcomes above-mentioned prior art existence.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of preparation method of active pachyman is characterized in that, this method adopts bio-fermentation process, and its processing step is as follows:
A. spawn culture:
Take the Poria cocos bacterial classification that liquid nitrogen is preserved, be implanted on the PDA slant medium, under 28 ℃～32 ℃ temperature, cultivated 10～15 days, obtain cultured Poria cocos bacterial classification;
B. mycelial fermentative preparation:
Adopt three grades of aerobic fermentation methods, 60～72 hours production cycles, concrete technical process is as follows: the mycelium of getting Poria cocos bacterial classification among the step a, immigration has in the Erlenmeyer flask of liquid nutrient medium, reciprocating vibration is 5～7 days under 20～30 ℃ of temperature, stroke 4～the 10cm of reciprocating vibration, oscillation frequency 100～120rpm, inoculum size 5% (v/v); The shake-flask culture thing is merged, move in the seeding tank, charging 70% (v/v) in jar, inoculum size 10% (v/v), stirring velocity 200～250rpm, air flow 1: 0.4v/vmin, tank pressure 0.4 * 10
5Pa, aerated culture moves into the breeding jar under 28～30 ℃ of temperature after 36～60 hours; Breeding jar mycelium long dense after, again with 1: 10 volume ratio expanding propagation to fermentor tank; Fermentor tank jar temperature is 28～32 ℃, air flow 1: 1 (v/v), and stirring velocity 100～200rpm, the fermentor tank breeding is after 60～72 hours, and ferment liquid gets the mycelium filter cake through filter press;
C. the extraction of Pachymose:
The filter cake that step b is made is smashed cell with the cell stamp mill after adding 10 times in water, adds the boiling water leaching 3～5 hours that refluxes then, obtains leaching liquid; Leaching liquid is concentrated through film under vacuum again, get 1 portion of concentrated solution and add 3 times of volumes 95% (weight %) ethanol, alcohol was analysed 8 hours under 0～4 ℃ of condition, got alcohol and analysed the thing spraying drying, promptly got the active pachyman extract of brown powder shape; Perhaps leaching liquid is crossed ultra-filtration membrane, collect filtrate, spray-dried, promptly get the active pachyman extract of brown powder shape.
Among the described step a, the prescription of PDA slant medium is as follows: potato juice 960ml, glucose 20 grams, agar 20 grams, PH5.5～6.5.
Described potato juice adopts following method to obtain: peeling potato 200 grams, added water boil 5 minutes, and get juice, add water to 1000ml, at 1.2kg/cm
2Under sterilized 30 minutes.
Among the described step b, the prescription of liquid nutrient medium is: soybean sprout liquor 1000ml, glucose 10 grams, dipotassium hydrogen phosphate 0.5 gram, potassium primary phosphate 0.5 gram, sal epsom 0.1 gram, PH5.5～6.5.
Described soybean sprout liquor adopts following method to obtain: add water 1000ml with 200 gram soybean sprout and boil and get juice after 10 minutes.
Among the described step b, (weight %) is as follows for the fermentative medium formula of fermentor tank: soybean cake powder 4%, sucrose 2%, peptone 0.2%, yeast powder 0.5%, potassium primary phosphate 0.1%, sal epsom 0.06%, add water to 100%, PH5.5～6.5.
Being compared as follows of Pachymose extract of Sheng Chaning and existing prepared product according to the method described above:
The present invention has technology now
Effective constituent polysaccharide polypeptide carboxymethyl polysaccharide
The raw materials for production deep layer is cultivated mycelium seed of Poria cocos entity
Heavy metal≤1mg/Kg≤10mg/Kg
Antitumor, the anti-pollen hypersensitivity of indication, to improve renal function antitumor
Preparation method's submerged fermentation chemistry is semi-synthetic
Tooling cost is 30% of existing processes cost
Extract the production technique for preparing active pachyman with biological fermentation process, can be without oxidation and the carboxymethylation process in the existing technology, the product of making are the active pachyman extract of forming with β 1 → 3 key bonded dextran.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1
Take the Poria cocos bacterial classification (Poria cocos) that liquid nitrogen is preserved, be implanted on PDA (potato-glucose-agar) slant medium, cultivated 10 days under 28～32 ℃ of temperature, the PDA prescription is as follows:
Potato juice * 960ml, glucose 20 grams, agar 20 grams, PH5.5～6.5
* remove the peel potato 200 grams, added water boil 5 minutes, get juice, add water to 1000ml, sterilization (1.2Kg/cm
2) 30 minutes.
Get the mycelium of slant strains, moving into has in 500 milliliters of Erlenmeyer flasks of 100 milliliters of liquid substratum, 25 ℃ of reciprocating vibrations 5 days.Stroke 4cm, oscillation frequency 100rpm, inoculum size 5%.The shake-flask culture thing is merged, move in 50～100 milliliters of seeding tanks, charging 70% (V/V) in jar, inoculum size 10% (V/V), stirring velocity 200rpm, air flow 1: 0.4V/Vmin, tank pressure 0.4 * 10 with pressure differential method
5Pa.28 ℃ of aerated culture move into 500 liters breeding jar after 48 hours.Breeding jar mycelium long dense after, again with 1: 10 volume ratio expanding propagation to fermentor tank.28～32 ℃ of jar temperature, air flow 1: 1 (V/V), stirring velocity 100～200rpm.
Fermentative medium formula:
Soybean cake powder 4%, sucrose 2%, peptone 0.2%, yeast powder 0.5%, potassium primary phosphate 0.1%, sal epsom 0.06% adds water to 100%, PH5.5～6.5.
The fermentor tank breeding is after 60～72 hours, and ferment liquid gets the mycelium filter cake through filter press.After filter cake added 10 times in water, smash cell with the cell stamp mill, add boiling water backflow leaching 3～5 hours then, leaching liquid is concentrated through film under vacuum again, get 1 portion of concentrated solution and add 3 part of 95% ethanol, alcohol was analysed 8 hours under 0～4 ℃ of condition, got alcohol and analysed the thing spraying drying, can obtain the Pachymose extract of brown powder shape.
Embodiment 2
Get one of Poria cocos (Poria cocos) slant strains of cryopreservation, move into and be equipped with in 1500 milliliters of culturing bottles of 50 milliliters of soybean sprout juice substratum, aerated culture 5 days, its culture medium prescription is:
Soybean sprout liquor * 1000ml glucose 10g dipotassium hydrogen phosphate 0.5g potassium primary phosphate 0.5g sal epsom 0.1g PH5.5～6.5
* adding water 1000ml with 200 gram soybean sprout boils and gets juice after 10 minutes.
Add 50 milliliters of seeding tanks then successively, 500 milliliters of breedings jar, and aerated culture 2～3 days in 5 tons of fermentor tanks, with fermentor tank after press filtration, receive wet mycelium with boiling water leaching 3 hours, with leaching liquid by ultra-filtration membrane (10,000 molecular weight), at 4KG/CM
2Filtering is less than the material of 10,000 molecular weight under the nitrogen pressure.Use the membrane filtration of 150,000 molecular weight then, collect filtrate, promptly spray-dried.
Embodiment 3
A kind of preparation method of active pachyman, its processing step a. spawn culture, the mycelial fermentative preparation of b. are identical with embodiment 1, different is, in the extraction of processing step c. Pachymose, after filter cake adds 10 times in water, smash cell with the cell stamp mill, add boiling water backflow leaching 3～5 hours then, obtain leaching liquid; Leaching liquid is crossed ultra-filtration membrane, collect filtrate, spray-dried, promptly get the active pachyman extract of brown powder shape.
Claims (6)
1. the preparation method of an active pachyman is characterized in that, this method adopts bio-fermentation process, and its processing step is as follows:
A. spawn culture:
Take the Poria cocos bacterial classification that liquid nitrogen is preserved, be implanted on the PDA slant medium, under 28 ℃～32 ℃ temperature, cultivated 10～15 days, obtain cultured Poria cocos bacterial classification;
B. mycelial fermentative preparation:
Adopt three grades of aerobic fermentation methods, 60～72 hours production cycles, concrete technical process is as follows: the mycelium of getting Poria cocos bacterial classification among the step a, immigration has in the Erlenmeyer flask of liquid nutrient medium, reciprocating vibration is 5～7 days under 20～30 ℃ of temperature, stroke 4～the 10cm of reciprocating vibration, oscillation frequency 100～120rpm, inoculum size 5% (v/v); The shake-flask culture thing is merged, move in the seeding tank, charging 70% (v/v) in jar, inoculum size 10% (v/v), stirring velocity 200～250rpm, air flow 1: 0.4v/vmin, tank pressure 0.4 * 10
5Pa, aerated culture moves into the breeding jar under 28～30 ℃ of temperature after 36～60 hours; Breeding jar mycelium long dense after, again with 1: 10 volume ratio expanding propagation to fermentor tank; Fermentor tank jar temperature is 28～32 ℃, air flow 1: 1 (v/v), and stirring velocity 100～200rpm, the fermentor tank breeding is after 60～72 hours, and ferment liquid gets the mycelium filter cake through filter press;
C. the extraction of Pachymose:
The filter cake that step b is made is smashed cell with the cell stamp mill after adding 10 times in water, adds the boiling water leaching 3～5 hours that refluxes then, obtains leaching liquid; Leaching liquid is concentrated through film under vacuum again, get 1 portion of concentrated solution and add 3 times of volumes 95% (weight %) ethanol, alcohol was analysed 8 hours under 0～4 ℃ of condition, got alcohol and analysed the thing spraying drying, promptly got the active pachyman extract of brown powder shape; Perhaps leaching liquid is crossed ultra-filtration membrane, collect filtrate, spray-dried, promptly get the active pachyman extract of brown powder shape.
2. the preparation method of active pachyman according to claim 1 is characterized in that, among the described step a, the prescription of PDA slant medium is as follows: potato juice 960ml, glucose 20 grams, agar 20 grams, PH5.5～6.5.
3. the preparation method of active pachyman according to claim 2 is characterized in that, described potato juice adopts following method to obtain: peeling potato 200 grams, added water boil 5 minutes, and get juice, add water to 1000ml, at 1.2kg/cm
2Under sterilized 30 minutes.
4. the preparation method of active pachyman according to claim 1, it is characterized in that, among the described step b, the prescription of liquid nutrient medium is: soybean sprout liquor 1000ml, glucose 10 grams, dipotassium hydrogen phosphate 0.5 gram, potassium primary phosphate 0.5 gram, sal epsom 0.1 gram, PH5.5～6.5.
5. the preparation method of active pachyman according to claim 4 is characterized in that, described soybean sprout liquor adopts following method to obtain: add water 1000ml with 200 gram soybean sprout and boil and get juice after 10 minutes.
6. the preparation method of active pachyman according to claim 1, it is characterized in that, among the described step b, (weight %) is as follows for the fermentative medium formula of fermentor tank: soybean cake powder 4%, sucrose 2%, peptone 0.2%, yeast powder 0.5%, potassium primary phosphate 0.1%, sal epsom 0.06%, add water to 100%, PH5.5～6.5.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021370060A CN1483827A (en) | 2002-09-16 | 2002-09-16 | Method for preparing active pachyman |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021370060A CN1483827A (en) | 2002-09-16 | 2002-09-16 | Method for preparing active pachyman |
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| CN1483827A true CN1483827A (en) | 2004-03-24 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308350C (en) * | 2004-07-28 | 2007-04-04 | 武汉大学 | Process for preparing tuckahoe mycelium extracellular polysacchride with anti-tumor activity |
| CN101711778B (en) * | 2008-10-07 | 2011-12-07 | 天津中医药大学 | Pachymaran composition used for suppressing tumours and preventing and treating metastasis and relapse and application |
| CN104351750A (en) * | 2014-10-31 | 2015-02-18 | 颍上县永祥旱粮研究所 | Method for treating poria cocos before peeling |
| CN105585638A (en) * | 2014-10-22 | 2016-05-18 | 北京中安佐际生物科技有限公司 | Pachymaran active component and composition, preparation method and application thereof |
| CN109876005A (en) * | 2019-03-15 | 2019-06-14 | 湖南补天药业股份有限公司 | A kind of tuckahoe effervescence tablet and preparation method thereof |
| CN114886834A (en) * | 2022-02-08 | 2022-08-12 | 杭州梵歌生物科技有限公司 | Whitening essence and preparation method thereof |
-
2002
- 2002-09-16 CN CNA021370060A patent/CN1483827A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308350C (en) * | 2004-07-28 | 2007-04-04 | 武汉大学 | Process for preparing tuckahoe mycelium extracellular polysacchride with anti-tumor activity |
| CN101711778B (en) * | 2008-10-07 | 2011-12-07 | 天津中医药大学 | Pachymaran composition used for suppressing tumours and preventing and treating metastasis and relapse and application |
| CN105585638A (en) * | 2014-10-22 | 2016-05-18 | 北京中安佐际生物科技有限公司 | Pachymaran active component and composition, preparation method and application thereof |
| CN105585638B (en) * | 2014-10-22 | 2017-12-15 | 北京中安佐际生物科技有限公司 | Pachymaran active component and composition, preparation method and the usage |
| CN104351750A (en) * | 2014-10-31 | 2015-02-18 | 颍上县永祥旱粮研究所 | Method for treating poria cocos before peeling |
| CN104351750B (en) * | 2014-10-31 | 2017-01-11 | 颍上县永祥旱粮研究所 | Method for treating poria cocos before peeling |
| CN109876005A (en) * | 2019-03-15 | 2019-06-14 | 湖南补天药业股份有限公司 | A kind of tuckahoe effervescence tablet and preparation method thereof |
| CN114886834A (en) * | 2022-02-08 | 2022-08-12 | 杭州梵歌生物科技有限公司 | Whitening essence and preparation method thereof |
| CN114886834B (en) * | 2022-02-08 | 2023-10-20 | 王梅枝 | Whitening essence and preparation method thereof |
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Owner name: YANG QINGLONG Free format text: FORMER OWNER: SHANGHAI YISI IMPORT AND EXPORT CO., LTD. Effective date: 20050121 |
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Effective date of registration: 20050121 Address after: Room 2, No. 111, Lane 501, happy road, Shanghai, China Applicant after: Yang Qinglong Address before: 3 floor, No. 749, Lane 38, Yuyuan Road, Shanghai Applicant before: Shanghai ever Import & Export Co., Ltd. |
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