CN1556212A - Technology of producing morin aqueous extract and polysaccharide using large scale liquid submerged fermentation process - Google Patents
Technology of producing morin aqueous extract and polysaccharide using large scale liquid submerged fermentation process Download PDFInfo
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- CN1556212A CN1556212A CNA2004100138696A CN200410013869A CN1556212A CN 1556212 A CN1556212 A CN 1556212A CN A2004100138696 A CNA2004100138696 A CN A2004100138696A CN 200410013869 A CN200410013869 A CN 200410013869A CN 1556212 A CN1556212 A CN 1556212A
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Abstract
A process for preparing the aqueous extract and polyose from the Sanghuang fungus in large scale by deep liquid fermenting includes slo slant shake-flask culture, enlarge culture, class-one seeding culture, fermenting culture, heating while stirring for 2-4 hr, press fitlering, concentrating, spray drying to obtain its aqueous extract or depositing in alcohol, dissolving deposit in water, and spray drying to obtain its polyose.
Description
Technical field
The present invention relates to a kind of large-scale deep liquid fermentation production Phellinus water extract and polysaccharide process.
Technical background
The modern science name of Phellinus (Phellinus igniarius) is called the Bao Shi shelf fungus; also be called mulberry ear, pin cracked feet, P .linteus etc.; be a kind of fungi that parasitizes on the mulberry tree of rotting, just Phellinus recorded as medicinal material simply in the Compendium of Material Medica of LI Shi-Zhen.After the mid-90 in 20th century, the researchist of Korea S and Japan finds: the extract of Phellinus has outstanding restraining effect to vegetations such as tumours.Experiment in vitro confirms, but Phellinus water extract inducing cancer cell enters program death.In addition, Phellinus also has very strong anticancer transferance.More stem-winding is that phellinus linteus extract is harmless, even long-term heavy dose is thrown gives, and is also without any side effects to people or laboratory animal.The main active ingredient of the water extract of Phellinus is a polysaccharose substance.
Because the Phellinus quantity of natural origin is very rare, be difficult to become stable Industrial products source.Korea S one drugmaker has taken the lead in developing the phellinus igniarius mycelium culture technique, is that Phellinus is fermented in special culture tank, at last its extract is processed into powder with lyophilization.Because anticancer effect is remarkable, phellinus linteus extract dry powder in the price in Korea S market up to 2000 dollars/g.According to statistics, the sale of Korea S in 2002 and Japanese two countries market Phellinus preparation adds up to and crosses 10,000,000,000 Yen.And the U.S. in 2000 just from Japanese import Phellinus preparation as dietary supplements.Have not yet to see the report of large-scale deep liquid fermentation production Phellinus water extract and polysaccharide.
Summary of the invention
The objective of the invention is with production Phellinus water extract and polysaccharide is target, invents a kind of Phellinus water extract of Cheap highly effective and the large-scale deep liquid fermentation process of polysaccharide, for from now on commercial application lays the first stone.
Realize technical scheme of the present invention: with the Phellinus fungi is starting strain, adopts slant strains to carry out liquid shaking bottle cultivation, liquid shaking bottle enlarged culturing, first order seed cultivation and fermentation culture.After fermentation ends; the end of a period fermented liquid is continued to be heated to certain temperature; certain hour is kept in stirring; fermented liquid is through filter press; clear liquid concentrates, and spraying drying obtains the Phellinus water extract, or clear liquid is concentrated an amount of ethanol sedimentation in back; taking precipitate adds the suitable quantity of water dissolving, and spraying drying can get the Phellinus polysaccharide.
This bacterial strain is numbered AS5.95 available from the bacterial classification at Chinese microorganism strain preservation center.
Realize that concrete steps of the present invention are as follows:
(1) takes out slant strains and insert in the fresh slant medium of preparing, culture temperature 25-33 ℃, cultivated 150-250 hour.
(2) slant strains in the step 1 is transferred the shaking in the bottle of shake-flask culture base is housed, 25-33 ℃ of cultivation, rotating speed 120-180rpm/min cultivates the enlarged culturing of shaking bottle after 80-120 hour.
(3) bottle bacterial classification that shakes in the step 2 is transferred and carried out enlarged culturing again into the bottle that shakes that shakes bottle enlarged culturing base is housed, 25-33 ℃ of cultivation, rotating speed 120-180rpm/min cultivates after 35-60 hour the access first class seed pot and cultivates.
(4) bottle bacterial classification that shakes in the step 3 is transferred one and is equipped with in the first class seed pot of seed culture medium, inoculum size is 4-10%, keep warm 25-33 ℃ in jar, tank pressure 0.5-0.9kPa, stir speed (S.S.) 80-150rpm/min, air flow 1: 0.3-0.5v/v.m fermented 30-55 hour, and ferment tank is gone in switching then.
(5) bacterial classification in the first class seed pot is transferred one be equipped with in the fermentor tank of fermention medium, inoculum size is 4-10%, keeps jar warm 25-33 ℃, tank pressure 0.5-0.9kPa, stir speed (S.S.) 80-150rpm/min, air flow 1: 0.3-0.5v/v.m fermented 120-200 hour.When being reduced to the 1.0-2.0% left and right sides, the fermented liquid reducing sugar content, puts jar for fermentation ends.
The prescription of slant medium is (g/100ml) among the present invention: potato 10-20, glucose 1-3, agar 1.5-2.5, add water be settled to volume required, pH value nature.
The shake-flask culture base is (g/100ml) with shaking bottle prescription of enlarged culturing base among the present invention: glucose 2-4, peptone 0.05-0.5, wheat bran juice 1.0-2.5, KH
2PO
40.1-0.5, MgSO
40.01-0.2, adding water and be settled to volume requiredly, the pH value is 4.5-5.0.
The culture medium prescription of first order seed cultivation and fermentation culture is (g/100ml) among the present invention: glucose 2-4, peptone 0.03-0.7, corn steep liquor 0.5-1.5, Semen Maydis powder 0.5-2, wheat bran juice 1-3, KH
2PO
40.1-0.5, MgSO
40.01-0.2, adding water and be settled to volume requiredly, the pH value is 4.0-5.0.
The shake-flask culture base is 1: 5 with shaking bottle volumetric ratio in the step 2 of the present invention and 3.
The volume of first class seed pot is 100-300L in the step 4 of the present invention, and the first order seed substratum that is equipped with in this first class seed pot should be 50-180L mutually; The volume of fermentor tank is 1000-5000L in the step 5, and the fermention medium that is equipped with in this fermentor tank should be 500-3500L mutually.
Jar standard of putting of the present invention is that fermented liquid begins the thickness that becomes, and reducing sugar content is reduced to about 1.0-2.0%, and microscopy finds that mycelium senesces.
Obtain its water extract and polysaccharide as need, then after fermentation of the present invention stopped, the end of a period liquid temp that will ferment was heated to 60-100 ℃, stirring velocity is 150-250rpm/min, keeps 2-4 hour, with pump fermented liquid is transported in the basin, through filter press, get clear liquid.Concentrating filter liquor, making last volume is the 1/4-1/10 of stoste, gets the concentrated solution spraying drying and obtains the Phellinus water extract.Or clear liquid concentrates the back and adds an amount of ethanol and make alcohol concn reach 75%, and alcohol was analysed 20-30 hour, got the precipitate with deionized water dissolving, and spraying drying promptly obtains the Phellinus polysaccharide.
Beneficial effect of the present invention: the present invention is applicable to large-scale deep liquid fermentation production Phellinus water extract and polysaccharide, and is significant for the development of from now on suitability for industrialized production and relevant application industry.Gained Phellinus water extract of the present invention and polysaccharide color are brown.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment.
Embodiment 1:
Inoculation is available from Chinese microorganism strain preservation center (bacterium numbering: phellinus igniarius mycelium 5.95), 28 ℃ of culture temperature, incubation time 180 hours in slant medium.Fresh slant strains access is equipped with in the 500ml triangular flask of 100ml substratum, and 28 ℃ of shaking tables were cultivated shaking speed 150rpm/min 100 hours.Then gained is shaken transfer 500ml triangle that the 100ml substratum is housed of bottle bacterial classification and shake and carry out enlarged culturing in the bottle, 28 ℃ of shaking tables were cultivated shaking speed 150rpm/min 45 hours.During the bacterial classification 2200ml that triangular flask is enlarged transfers the 100L first class seed pot that the 50L seed culture medium is housed then, keep 28 ℃ of jar temperature, tank pressure 0.7kPa, stir speed (S.S.) 100rpm/min, air flow 1: 0.5v/v.m, fermentation time 45 hours.The first class seed pot bacterial classification is transferred in the 1500L fermentor tank that the 800L fermention medium is housed, keep 28 ℃ of jar temperature, tank pressure 0.7kPa, stir speed (S.S.) 100rpm/min, air flow 1: 0.5v/v.m, fermentation time 144 hours, mycelium counts 2.9% with dry weight in the fermented liquid, and polysaccharide content is 30%.
To form be (g/100ml) to slant medium in the present embodiment 1: potato 15, and glucose 2, agar 2 adds water and is settled to volume requiredly, and the pH value is a nature.
The shake-flask culture base is (g/100ml) with shaking a bottle enlarged culturing base composition in the present embodiment 1: glucose 3, peptone 0.2, wheat bran juice 2, KH
2PO
40.2, MgSO
40.1, adding water and be settled to volume requiredly, the pH value is a nature.
The substratum of 100L first class seed pot composition is (g/100ml) in the present embodiment 1: glucose 3, peptone 0.2, corn steep liquor 1, Semen Maydis powder 1.5, wheat bran juice 2, KH
2PO
40.2, MgSO
40.1, adding water and be settled to 50L, the pH value is 4.5.
The culture medium prescription of 1500L fermentor tank is (g/100ml) in the present embodiment 1: glucose 3, peptone 0.2, corn steep liquor 1.0, Semen Maydis powder 1.5, wheat bran juice 2, KH
2PO
40.2, MgSO
40.1, adding water and be settled to 800L, the pH value is 4.5.
The fermentation ends standard is in the present embodiment: fermented liquid begins the thickness that becomes, and reducing sugar content is reduced to 1.5%, and microscopy finds that mycelium senesces.
The method of obtaining Phellinus water extract and polysaccharide is: after fermentation of the present invention stops; temperature is heated to 90 ℃; stirring velocity is 220rpm/min; kept 3 hours; with pump fermented liquid is transported in the basin, through filter press, concentrating filter liquor; making last volume is 1/5 of stoste, gets the concentrated solution spraying drying and obtains the Phellinus water extract.Obtain polysaccharide as need, then concentrated solution is added an amount of ethanol and make alcohol concn reach 75%, alcohol was analysed 24 hours, got the precipitate with deionized water dissolving, and spraying drying obtains the Phellinus polysaccharide.
Claims (9)
1; the technology of a kind of liquid submerged fermentation production Phellinus water extract and polysaccharide; it is characterized in that with Phellinus fungi (Phellinus igniarius) be starting strain; adopting slant strains to carry out liquid shaking bottle cultivates; the liquid shaking bottle enlarged culturing; first order seed is cultivated and fermentation culture gets phellinus igniarius mycelium fermentation liquid; after fermentation ends; the end of a period fermented liquid is continued to be heated to certain temperature; certain hour is kept in stirring; fermented liquid is through filter press; clear liquid concentrates; spraying drying obtains the Phellinus water extract; or clear liquid is concentrated the back use an amount of ethanol sedimentation, taking precipitate to add suitable quantity of water to dissolve, spraying drying can get the Phellinus polysaccharide.
2, the technology of liquid submerged fermentation production Phellinus water extract according to claim 1 and polysaccharide is characterized in that with the Phellinus fungi AS5.95 available from Chinese microorganism strain preservation center be starting strain.
3, the technology of liquid submerged fermentation production Phellinus water extract according to claim 1 and polysaccharide; it is characterized in that described slant strains cultivation; it is (g/100ml) that substratum is formed: potato 10-20; glucose 1-3; agar 1.5-2.5 adds water and is settled to volume requiredly, and the pH value is a nature; 25-33 ℃ of slant strains culture temperature cultivated 150-250 hour.
4, the technology of liquid submerged fermentation production Phellinus water extract according to claim 1 and polysaccharide is characterized in that described shake-flask culture, and it is (g/100ml) that substratum is formed: glucose 2-4, peptone 0.05-0.5, wheat bran juice 1.0-2.5, KH
2PO
40.1-0.5, MgSO
40.01-0.2, adding water and be settled to volume requiredly, the pH value is 4.5-5.0, and culture condition is: culture temperature 25-33 ℃, rotating speed 120-180rpm/min cultivated 80-120 hour, and switching is gone into to shake bottle and is carried out enlarged culturing then.
5, the technology of liquid submerged fermentation production Phellinus water extract according to claim 1 and polysaccharide is characterized in that the described bottle enlarged culturing of shaking, and it is (g/100ml) that substratum is formed: glucose 2-4, peptone 0.05-0.5, wheat bran juice 1.0-2.5, KH
2PO
40.1-0.5, MgSO
40.01-0.2, adding water and be settled to volume requiredly, the pH value is 4.5-5.0, and culture condition is: culture temperature 25-33 ℃, rotating speed 120-180rpm/min cultivated 35-60 hour, and switching is gone into first order seed and is cultivated then.
6, the technology of liquid submerged fermentation production Phellinus water extract according to claim 1 and polysaccharide is characterized in that described first order seed cultivation, and it is (g/100ml) that substratum is formed: glucose 2-4; peptone 0.03-0.7, corn steep liquor 0.5-1.5, Semen Maydis powder 0.5-2; wheat bran juice 1-3, KH
2PO
40.1-0.5, MgSO
40.01-0.2, adding water is settled to volume required, the pH value is 4.0-5.0, inoculum size is 4-10%, and culture condition is: culture temperature 25-33 ℃, and tank pressure 0.5-0.9kPa, stir speed (S.S.) 80-150rpm/min, air flow 1: 0.3-0.5v/v.m fermented 30-55 hour, and ferment tank is gone in switching then.
7, the technology of liquid submerged fermentation production Phellinus water extract according to claim 1 and polysaccharide is characterized in that described fermentation culture, and it is (g/100ml) that fermention medium is formed: glucose 2-4; peptone 0.3-0.7, corn steep liquor 0.5-1.5, Semen Maydis powder 0.5-2; wheat bran juice 1-3, KH
2PO
40.1-0.5, MgSO
40.01-0.2, adding water is settled to volume required, the pH value is 4.0-5.0, and inoculum size is 4-10%, and culture condition is: culture temperature 25-33 ℃, tank pressure 0.5-0.9kPa, stir speed (S.S.) 80-150rpm/min, air flow 1: 0.3-0.5v/v.m fermented 120-200 hour, when the fermented liquid reducing sugar content drops to 1.0-2.0%, for fermentation ends.
8, the technology of liquid submerged fermentation production Phellinus water extract according to claim 1 and polysaccharide is characterized in that described fermentation culture, can select the fermentor tank of 1000-5000L to carry out fermentation culture.
9, the technology of liquid submerged fermentation production Phellinus water extract according to claim 1 and polysaccharide, it is characterized in that end of a period fermented liquid temperature is heated to 60-100 ℃, stirring velocity is 150-250rpm/min, kept 2-4 hour, with pump fermented liquid is transported in the basin, obtain clear liquid through filter press, it is original volume 1/4-1/10 that clear liquid is concentrated into volume, and spraying drying obtains the Phellinus water extract; Or clear liquid is concentrated the back, and use an amount of ethanol sedimentation, alcohol to analyse the liquid alcohol concn be 75%, and alcohol was analysed 20-30 hour, and taking precipitate adds suitable quantity of water and dissolves, and spraying drying can get the Phellinus polysaccharide.
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|---|---|---|---|
| CN 200410013869 CN1259423C (en) | 2004-01-07 | 2004-01-07 | Technology of producing morin aqueous extract and polysaccharide using large scale liquid submerged fermentation process |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410013869 CN1259423C (en) | 2004-01-07 | 2004-01-07 | Technology of producing morin aqueous extract and polysaccharide using large scale liquid submerged fermentation process |
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|---|---|
| CN1556212A true CN1556212A (en) | 2004-12-22 |
| CN1259423C CN1259423C (en) | 2006-06-14 |
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Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100438882C (en) * | 2005-08-02 | 2008-12-03 | 福建农林大学 | Health care oral liquid of phellinus igniarius and its preparing method |
| CN101928173A (en) * | 2010-08-26 | 2010-12-29 | 陕西省微生物研究所 | Foliage fertilizer containing phellinus igniarius mycelium fermentation liquid and preparation method thereof |
| CN101933439A (en) * | 2010-07-15 | 2011-01-05 | 西南大学 | Method for improving phellinus igniarius hypha amount of submerged culture by utilizing plant oil |
| CN101323646B (en) * | 2008-06-23 | 2011-01-19 | 广东省微生物研究所 | Phellinus polysaccharide II having antineoplastic activity, extraction and separation method thereof |
| CN101297821B (en) * | 2007-09-18 | 2011-01-26 | 江苏大学 | Phellinus linteus mycelia active glucoprotein and use thereof and preparation |
| CN102491827A (en) * | 2011-11-14 | 2012-06-13 | 江苏省农业科学院 | Solid fermentation culture medium for phellinus igniarius mycelium and solid fermentation cultural method for phellinus igniarius mycelium |
| CN102771760A (en) * | 2012-07-20 | 2012-11-14 | 黄晓青 | Phellinus igniarius health care product manufactured through liquid submerged fermentation and manufacturing method thereof |
| CN102875225A (en) * | 2012-09-20 | 2013-01-16 | 福建农林大学 | Phellinus igniarius bacterial strain liquid fermenting culture medium and method for fermenting and producing phellinus linteus polysaccharides |
| CN103750029A (en) * | 2013-12-31 | 2014-04-30 | 西南大学 | Feed additive with immune regulation effect, feed and preparation method and application of feed additive |
| CN104397699A (en) * | 2014-12-04 | 2015-03-11 | 郭士平 | Health care product for conditioning syndromes before and after menstruation and dysmenorrhea and preparation technology of health care product |
| CN104892793A (en) * | 2015-05-25 | 2015-09-09 | 江苏大学 | Male agaric mycelium polysaccharides as well as preparation method and application thereof in resisting tumors |
| CN105524846A (en) * | 2016-01-14 | 2016-04-27 | 山东鲁北药业有限公司 | Phellinus igniarius strain and application thereof |
| CN105533715A (en) * | 2015-12-31 | 2016-05-04 | 天津天林欣然生物科技有限公司 | Phellinus igniarius and germinated brown rice health-care food and preparation method thereof |
| CN106070879A (en) * | 2016-06-21 | 2016-11-09 | 福建农林大学 | A kind of Phellinus igniarius (L. ex Fr.) Quel. kudzu root tea and preparation method thereof |
| CN106701598A (en) * | 2016-12-22 | 2017-05-24 | 菏泽学院 | Culture medium applicable to liquid fermentation production of phellinus linteus |
| CN106811420A (en) * | 2016-12-22 | 2017-06-09 | 菏泽学院 | A kind of Phellinus liquid fermentation production method |
| CN111172202A (en) * | 2018-11-09 | 2020-05-19 | 财团法人食品工业发展研究所 | Phellinus strain of phellinus, and product, extract and application thereof |
-
2004
- 2004-01-07 CN CN 200410013869 patent/CN1259423C/en not_active Expired - Fee Related
Cited By (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100438882C (en) * | 2005-08-02 | 2008-12-03 | 福建农林大学 | Health care oral liquid of phellinus igniarius and its preparing method |
| CN101297821B (en) * | 2007-09-18 | 2011-01-26 | 江苏大学 | Phellinus linteus mycelia active glucoprotein and use thereof and preparation |
| CN101323646B (en) * | 2008-06-23 | 2011-01-19 | 广东省微生物研究所 | Phellinus polysaccharide II having antineoplastic activity, extraction and separation method thereof |
| CN101933439A (en) * | 2010-07-15 | 2011-01-05 | 西南大学 | Method for improving phellinus igniarius hypha amount of submerged culture by utilizing plant oil |
| CN101928173A (en) * | 2010-08-26 | 2010-12-29 | 陕西省微生物研究所 | Foliage fertilizer containing phellinus igniarius mycelium fermentation liquid and preparation method thereof |
| CN102491827A (en) * | 2011-11-14 | 2012-06-13 | 江苏省农业科学院 | Solid fermentation culture medium for phellinus igniarius mycelium and solid fermentation cultural method for phellinus igniarius mycelium |
| CN102771760A (en) * | 2012-07-20 | 2012-11-14 | 黄晓青 | Phellinus igniarius health care product manufactured through liquid submerged fermentation and manufacturing method thereof |
| CN102875225A (en) * | 2012-09-20 | 2013-01-16 | 福建农林大学 | Phellinus igniarius bacterial strain liquid fermenting culture medium and method for fermenting and producing phellinus linteus polysaccharides |
| CN103750029B (en) * | 2013-12-31 | 2015-07-01 | 西南大学 | Feed additive with immune regulation effect, feed and preparation method and application of feed additive |
| CN103750029A (en) * | 2013-12-31 | 2014-04-30 | 西南大学 | Feed additive with immune regulation effect, feed and preparation method and application of feed additive |
| CN104397699A (en) * | 2014-12-04 | 2015-03-11 | 郭士平 | Health care product for conditioning syndromes before and after menstruation and dysmenorrhea and preparation technology of health care product |
| CN104892793A (en) * | 2015-05-25 | 2015-09-09 | 江苏大学 | Male agaric mycelium polysaccharides as well as preparation method and application thereof in resisting tumors |
| CN104892793B (en) * | 2015-05-25 | 2017-11-17 | 江苏大学 | Phellinus igniarius mycelium polysaccharide, preparation method and its in antitumor application |
| CN105533715A (en) * | 2015-12-31 | 2016-05-04 | 天津天林欣然生物科技有限公司 | Phellinus igniarius and germinated brown rice health-care food and preparation method thereof |
| CN105524846A (en) * | 2016-01-14 | 2016-04-27 | 山东鲁北药业有限公司 | Phellinus igniarius strain and application thereof |
| CN106070879A (en) * | 2016-06-21 | 2016-11-09 | 福建农林大学 | A kind of Phellinus igniarius (L. ex Fr.) Quel. kudzu root tea and preparation method thereof |
| CN106701598A (en) * | 2016-12-22 | 2017-05-24 | 菏泽学院 | Culture medium applicable to liquid fermentation production of phellinus linteus |
| CN106811420A (en) * | 2016-12-22 | 2017-06-09 | 菏泽学院 | A kind of Phellinus liquid fermentation production method |
| CN111172202A (en) * | 2018-11-09 | 2020-05-19 | 财团法人食品工业发展研究所 | Phellinus strain of phellinus, and product, extract and application thereof |
| CN111172202B (en) * | 2018-11-09 | 2021-12-31 | 财团法人食品工业发展研究所 | Phellinus strain of phellinus, and product, extract and application thereof |
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| Publication number | Publication date |
|---|---|
| CN1259423C (en) | 2006-06-14 |
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