CN101348803A - Artificial nutrient medium for Phellinus linteus fluid culture and method for fermentation of Phellinus linteus polysaccharide - Google Patents
Artificial nutrient medium for Phellinus linteus fluid culture and method for fermentation of Phellinus linteus polysaccharide Download PDFInfo
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- CN101348803A CN101348803A CNA2008100702244A CN200810070224A CN101348803A CN 101348803 A CN101348803 A CN 101348803A CN A2008100702244 A CNA2008100702244 A CN A2008100702244A CN 200810070224 A CN200810070224 A CN 200810070224A CN 101348803 A CN101348803 A CN 101348803A
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Abstract
The invention provides a synthetic medium for culturing a phellinus igniarius liquid. The synthetic medium takes glucose as a carbon source and 20 kinds of amino acid as a nitrogen source, and consists of inorganic salt and vitamin. The ratio of carbon to nitrogen is designed as 50 to 1; and the specific concentration g/l is as follows: the glucose: 30 to 80 grams; glutamic acid: 0.1 to 1.5 grams; glutamine: 0.1 to 1.5 grams; aspartic acid: 0.1 to 1.5 grams; asparagines: 0.1 to 1.5 grams; glutamic acid: 0.01 to 0.1 gram; tryptophan: 0.01 to 0.1 gram; valine: 0.01 to 0.1 gram; alanine: 0.01 to 0.1 gram; etc. During the whole culture process of the synthetic medium, phellinus igniarius mycelium is quick in growth; the yield of the phellinus igniarius mycelium can reach 20 grams per liter to the highest degree; simultaneously no agricultural and sideline product is introduced as the carbon source and the nitrogen source; substances such as proteins in the medium, medium compositions and so on are not carried; phellinus igniarius exopolysaccharide produced through fermentation has high purity and stable quality; and the yield of phellinus igniarius polysaccharide can reach 0.198 gram per liter to the highest degree. The synthetic medium has the advantages of clear compositions, stable quality and so on, and can be used for mass culture of the phellinus igniarius liquid and research of the regulation and control rule of polysaccharide anabolism.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method that is used for Phellinus bacterium fermentative production Phellinus polysaccharide full-synthetic culture medium and that adopt described substratum of medicinal fungi Phellinus bacteria liquid cultivation.
Background technology
Phellinus (Phellinus igniarius) claim phelliuns igniarius again; belong to Basidiomycetes; shelf fungus belongs to; parasitize on the mulberry tree trees; be a kind of rare edible and medicinal fungi, the Phellinus main active ingredient is the Phellinus polysaccharide, energy enhance immunity power; have notable antitumor activity, have exploitation and be worth.
Since complicacy, the singularity of Phellinus bacterium physiological ecological, Phellinus sporophore quantity rareness.Adopt deep liquid culture technique scale operation phellinus igniarius mycelium with and the active polysaccharide material be the effective way that satisfies the domestic and international market needs.In existing production technology and bibliographical information, it is carbon nitrogen source that the fermention medium that the Phellinus liquid culture adopts mainly adopts natural agricultural byproducts, as murphy juice, molasses, soybean cake powder, corn steep liquor etc.Because the complicated component of composite carbon nitrogenous source exists the difference on the composition often to cause the Phellinus bacteria liquid to cultivate production mycelium production and polysaccharide molecular weight instability between batch, also separate simultaneously and bring difficulty to the downstream extraction of Phellinus polysaccharide.
Advantages such as full-synthetic culture medium has to become to distinguish one from the other, steady quality, impurity are few can be used for directed regulation and control of Phellinus polysaccharide synthetic and metabolic flux analysis and the needs of large-scale production process quality control; Helping the downstream separation of polysaccharides simultaneously extracts.At present, the document and the patent report that also do not have the Phellinus full-synthetic culture medium both at home and abroad.
Summary of the invention
The object of the present invention is to provide to be used for the synthetic medium prescription that Phellinus is cultivated, and the method for utilizing this substratum production Phellinus polysaccharide.
The concrete technical solution that realizes the object of the invention is as follows:
Be carbon source with glucose in the synthetic medium, 20 seed amino acids are nitrogenous source, and inorganic salt and VITAMIN are formed, carbon-nitrogen ratio is approximately by design in 50: 1, concrete concentration following (g/L): glucose 30～80g, L-glutamic acid 0.1～1.5g, glutamine 0.1～1.5g, aspartic acid 0.1～1.5g, l-asparagine 0.1～1.5g L-glutamic acid 0.01～0.1g, tryptophane 0.01～0.1g, Xie Ansuan 0.01～0.1g, L-Ala 0.01～0.1g, leucine 0.01～0.1g, Isoleucine 0.01～0.1g, phenylalanine 0.001～0.1g, methionine(Met) 0.01～0.1g, proline(Pro) 0.01～0.1g, glycine 0.01～0.1g, Serine 0.01～0.1g, Methionin 0.01～0.1g, halfcystine 0.01～0.1g, tyrosine 0.01～0.1g, arginine 0.01～0.1g, Histidine 0.01～0.1g, MgSO
40.5～2g, KH
2PO
40.5～2g, NaCl0.3～1.5g, FeSO
40.01～0.15g, MnSO
47H
2O0.01～0.07g, ZnSO
4.7H2O 0.01～0.1g, CoCl
20.0001～0.05g, CuSO
40.0001～0.03g, CaCl
20.05～0.5g, VB
150～500ug, H
2O 1.0L.
The initial pH of substratum is controlled at 5.5～7.5,115～121 ℃ of sterilising temps, sterilization time 30min.
Utilize this synthetic medium shake flask fermentation to produce the zymotechnique of phellinus igniarius mycelium and Phellinus polysaccharide: inoculum size 5～15%, 24～28 ℃ of culture temperature, reciprocating type shaking table revolution 160～220rpm.
The advantage that the present invention had: adopt the growth of full-synthetic culture medium culturing process phellinus liteus rapidly, phellinus igniarius mycelium output reaches as high as 20g/L, is that present deep layer is cultivated the highest report of phellinus igniarius mycelium output; Not introducing agricultural byproducts in the substratum simultaneously is carbon nitrogen source, does not carry material such as protein, medium component in the substratum in the Phellinus polysaccharide secretly, the Phellinus exocellular polysaccharide purity height of fermentative production, steady quality, and the Phellinus polysaccharide yield is up to 0.198g/L.The present invention has advantages such as composition is clear, steady quality, can be used for the research of Phellinus bacteria liquid large scale culturing and polysaccharide anabolism regulation rule.
Specific implementation method
The bacterial classification that present embodiment uses is to purchase in Chinese common micro-organisms culture presevation administrative center (CGMCC) Phellinus bacterial classification (phelliuns igniarius, numbering 5.95),
Embodiment 1:
Fermention medium 1L described in preparation the present invention, its each component concentrations (g/L) is: glucose 50g, L-glutamic acid 0.8g, glutamine 0.8g, aspartic acid 0.8g, l-asparagine 0.8g L-glutamic acid 0.04g, tryptophane 0.04g, Xie Ansuan 0.04g, L-Ala 0.04g, leucine 0.04g, Isoleucine 0.04g, phenylalanine 0.04g, methionine(Met) 0.04g, proline(Pro) 0.04g, glycine 0.04g, Serine 0.04g, Methionin 0.04g, halfcystine 0.04g, tyrosine 0.04g, arginine 0.04g, Histidine 0.04g, MgSO
41.0g, KH
2PO
41.0g, NaCl 0.85g, FeSO
40.05g, MnSO
47H
2O 0.0383g, ZnSO
47H2O 0.0383g, CoCl
20.0043g, CuSO
40.0096g, CaCl
20.25g, VB
1200ug, H
2O 1.0L regulates pH to 6.0, the substratum branch is installed in the triangular flask of 250mL, and liquid amount is 50mL, 115 ℃ of sterilising temps, time 30min.
Phellinus liteus is cultivated: inoculation 10% (V/V) Phellinus seed liquor, 25 ℃ of culture temperature, rotating speed 180rpm/min, every 24h sampling, the centrifugal 10min of 3500rpm.With distilled water wash mycelium 3 times, to dry to constant weight, the 264 hours dry cell weights of weighing reach 17.67g/l.Supernatant concentration, 1: 4 by volume ratio adds alcohol, 4 ℃ of refrigerator overnight, centrifugation with 1: 4 alcohol wash 3 times, is water-solublely surveyed polysaccharide content with the phenol sulfuric acid process, and the mensuration polysaccharide content was 0.198g/l in 264 hours.
Embodiment 2:
Fermention medium 1L described in preparation the present invention, its each component concentrations (g/L) is: glucose 70g, L-glutamic acid 0.5g, glutamine 0.5g, aspartic acid 0.5g, l-asparagine 0.5g, L-glutamic acid 0.04g, tryptophane 0.04g, Xie Ansuan 0.04g, L-Ala 0.04g, leucine 0.04g, Isoleucine 0.04g, phenylalanine 0.04g, methionine(Met) 0.04g, proline(Pro) 0.04g, glycine 0.04g, Serine 0.04g, Methionin 0.04g, halfcystine 0.04g, tyrosine 0.04g, arginine 0.04g, Histidine 0.04g, MgSO
41.0g, KH
2PO
41.0g, NaCl 0.85g, FeSO
40.05g, MnSO
47H
2O 0.0383g, ZnSO
47H2O 0.0383g, CoCl
20.0043g, CuSO
40.0096g, CaCl
20.25g, VB
1200ug, H
2O 1.0L regulates pH to 6.0, the substratum branch is installed in the triangular flask of 250mL, and liquid amount is 50mL, 115 ℃ of sterilising temps, time 30min.
Phellinus liteus is cultivated: inoculation 10% (V/V) Phellinus seed liquor, 25 ℃ of culture temperature, rotating speed 180rpm/min, every 24h sampling, the centrifugal 10min of 3500rpm.With distilled water wash mycelium 3 times, dry to constant weight, dry cell weight reached 19.2g/l in 192 hours.Supernatant concentration, 1: 4 by volume ratio adds alcohol, 4 ℃ of refrigerator overnight, centrifugation with 1: 4 alcohol wash 3 times, is water-solublely surveyed polysaccharide content with the phenol sulfuric acid process, and the mensuration polysaccharide content was 0.08g/l in 192 hours.
Embodiment 3:
Fermention medium 1L described in preparation the present invention, its each component concentrations (g/L) is: glucose 50g, L-glutamic acid 0.8g, glutamine 0.8g, aspartic acid 0.8g, l-asparagine 0.8g, L-glutamic acid 0.04g, tryptophane 0.04g, Xie Ansuan 0.04g, L-Ala 0.04g, leucine 0.04g, Isoleucine 0.04g, phenylalanine 0.04g, methionine(Met) 0.04g, proline(Pro) 0.04g, glycine 0.04g, Serine 0.04g, Methionin 0.04g, halfcystine 0.04g, tyrosine 0.04g, arginine 0.04g, Histidine 0.04g, MgSO
41.0g, KH
2PO
41.0g, NaCl 0.85g, FeSO
40.05g, MnSO
47H
2O0.0383g, ZnSO
47H2O 0.0383g, CoCl
20.0043g, CuSO
40.0096g, CaCl
20.25g, VB
1200ug, H
2O1.0L regulates pH to 6.0, the substratum branch is installed in the triangular flask of 250mL, and liquid amount is 50mL, 115 ℃ of sterilising temps, time 30min.
Phellinus liteus is cultivated: inoculation 10% (V/V) Phellinus seed liquor, 25 ℃ of culture temperature, rotating speed 180rpm/min, every 24h sampling, the centrifugal 10min of 3500rpm.With distilled water wash mycelium 3 times, dry to constant weight, dry cell weight reached 20.63g/l in 240 hours.Supernatant concentration, 1: 4 by volume ratio adds alcohol, 4 ℃ of refrigerator overnight, centrifugation with 1: 4 alcohol wash 3 times, is water-solublely surveyed polysaccharide content with the phenol sulfuric acid process, and the mensuration polysaccharide content was 0.141g/l in 240 hours.
Claims (2)
1. one kind is used for the synthetic medium that the Phellinus bacteria liquid is cultivated; be carbon source with glucose in the described synthetic medium; 20 seed amino acids are nitrogenous source; and inorganic salt and VITAMIN composition, carbon-nitrogen ratio was by design in 50: 1, and concrete concentration g/L is as follows: glucose 30～80g; L-glutamic acid 0.1～1.5g; glutamine 0.1～1.5g, aspartic acid 0.1～1.5g, l-asparagine 0.1～1.5g L-glutamic acid 0.01～0.1g; tryptophane 0.01～0.1g; Xie Ansuan 0.01～0.1g, L-Ala 0.01～0.1g, leucine 0.01～0.1g; Isoleucine 0.01～0.1g; phenylalanine 0.001～0.1g, methionine(Met) 0.01～0.1g, proline(Pro) 0.01～0.1g; glycine 0.01～0.1g; Serine 0.01～0.1g, Methionin 0.01～0.1g, halfcystine 0.01～0.1g; tyrosine 0.01～0.1g; arginine 0.01～0.1g, Histidine 0.01～0.1g, MgSO
40.5～2g, KH
2PO
40.5～2g, NaCl0.3～1.5g, FeSO
40.01～0.15g, MnSO
47H
2O0.01～0.07g, ZnSO
4.7H2O 0.01～0.1g, CoCl
20.0001～0.05g, CuSO
40.0001～0.03g, CaCl
20.05～0.5g, VB
150～500ug, H
2O 1.0L; The initial pH of substratum is controlled at 5.5～7.5,115～121 ℃ of sterilising temps, sterilization time 30min.
2, utilize the described synthetic medium shake flask fermentation of claim 1 to produce the zymotechnique of phellinus igniarius mycelium and Phellinus polysaccharide, process is as follows: inoculation Phellinus seed liquor, inoculum size V/V 5～15%, 24～28 ℃ of culture temperature, reciprocating type shaking table revolution 160～220rpm, 24h sampling at interval, the centrifugal 10min of 3500rpm; With distilled water wash centrifugation 3 times, the mycelium that obtains; Supernatant concentration to original volume 1/3rd after, 1: 4 by volume ratio adds alcohol, 4 ℃ of refrigerator overnight, centrifugation after 1: 4 alcohol wash of volume ratio precipitation 3 times, is water-solublely surveyed polysaccharide content with the phenol sulfuric acid process.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN101983955A (en) * | 2010-08-13 | 2011-03-09 | 浙江省农业科学院 | Phellinus igniarius mycelium culture medium and process for fermenting phellinus igniarius using same |
| 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 |
| CN103044228A (en) * | 2012-12-15 | 2013-04-17 | 青岛农业大学 | Technology for separating acetophenone from Phellinus |
| CN104087632A (en) * | 2014-07-15 | 2014-10-08 | 江苏神华药业有限公司 | Method for producing phellinus igniarius extracellular polysaccharides by deep liquid fermentation |
| CN104278070A (en) * | 2014-10-21 | 2015-01-14 | 浙江省林业科学研究院 | Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius |
| CN108103122A (en) * | 2018-02-01 | 2018-06-01 | 江南大学 | A kind of method added phenylalanine and improve edible and medicinal fungi yield of extracellular polysaccharide |
| WO2019148419A1 (en) * | 2018-02-01 | 2019-08-08 | 江南大学 | Method for improving yield of edible and medicinal fungus exopolysaccharide by adding tyrosine |
| CN112322572A (en) * | 2020-11-19 | 2021-02-05 | 陕西省微生物研究所 | Liquid fermentation method for increasing yield of phellinus igniarius mycelium |
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| CN102766663B (en) * | 2012-05-30 | 2014-11-05 | 陕西师范大学 | Preparation method of active polysaccharides from phellinus linteus |
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- 2008-09-03 CN CN2008100702244A patent/CN101348803B/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN101983955A (en) * | 2010-08-13 | 2011-03-09 | 浙江省农业科学院 | Phellinus igniarius mycelium culture medium and process for fermenting phellinus igniarius using same |
| 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 |
| CN103044228A (en) * | 2012-12-15 | 2013-04-17 | 青岛农业大学 | Technology for separating acetophenone from Phellinus |
| CN104087632A (en) * | 2014-07-15 | 2014-10-08 | 江苏神华药业有限公司 | Method for producing phellinus igniarius extracellular polysaccharides by deep liquid fermentation |
| CN104087632B (en) * | 2014-07-15 | 2016-08-24 | 江苏神华药业有限公司 | A kind of deep layer liquid fermentation produces the method for Phellinus igniarius (L. ex Fr.) Quel. extracellular polysaccharide |
| CN104278070A (en) * | 2014-10-21 | 2015-01-14 | 浙江省林业科学研究院 | Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius |
| CN108103122A (en) * | 2018-02-01 | 2018-06-01 | 江南大学 | A kind of method added phenylalanine and improve edible and medicinal fungi yield of extracellular polysaccharide |
| WO2019148419A1 (en) * | 2018-02-01 | 2019-08-08 | 江南大学 | Method for improving yield of edible and medicinal fungus exopolysaccharide by adding tyrosine |
| CN108103122B (en) * | 2018-02-01 | 2020-06-09 | 江南大学 | Method for increasing yield of edible and medicinal fungus exopolysaccharide by adding phenylalanine |
| CN112322572A (en) * | 2020-11-19 | 2021-02-05 | 陕西省微生物研究所 | Liquid fermentation method for increasing yield of phellinus igniarius mycelium |
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