CN102465154B - Method for improving hypocrellin yield in shiraia bambusicola - Google Patents
Method for improving hypocrellin yield in shiraia bambusicola Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a technology for improving hypocrellin yield in shiraia bambusicola by using a polystyrene type nonpolar adsorption resin, wherein the technology comprises: carrying out activating culture for a strain, carrying out liquid seed culture, and carrying out co-culture fermentation for the strain and the adsorption resin. The method is characterized in that: with the co-culture fermentation, the resin can absorb the hypocrellin secreted by the shiraia bambusicola, and the hypocrellin content in the shiraia bambusicola can be improved so as to improve the hypocrellin yield. Therefore, with the present invention, the synthesis of the hypocrellin in the shiraia bambusicola can be effectively stimulated, the hypocrellin production in the culture system can be improved, the yield is high, the cost is low, the steps are simple, and the method can be used for the industrial production, and has a good industrial application prospect.
Description
Technical field
The invention belongs to microbial fermentation engineering, be specifically related to a kind of method of utilizing the two-phase culture technique to improve hypocrellin yield in shiraia bambusicola.
Background technology
Bamboo parasitic fungus (Shiraia bambusicola P.Hennigs) has another name called bamboo pseudo-ginseng, bamboo cocoon, the red dumpling of bamboo etc., it is a kind of special fungi parasitized on some bamboo spray, its stroma claims tabasheer, is under the jurisdiction of Hypocreaceae (Hypocreaceae) tabasheer and belongs to (Shiraia).Contain multiple physiologically active ingredient in tabasheer, that the most noticeable is hypocrellin (Hypocrellin).
Hypocrellin Shu Yu perylene quinone compound mainly comprises two kinds of compositions: Hypocrellin A (HypocrellinA is called for short HA) and Hypocrellin B (Hypocrellin B is called for short HB) are wherein the first element more than 95%, and HA can dewater and be converted into HB.Hypocrellin is a kind of novel photosensitive agent of finding the earliest in China, is current known photosensitizers good in visible region.This compounds can produce through common biosynthetic pathway, and their structure has stereochemical feature, shows the photodynamics activity, has significant Phototherapy, has been used for treating clinically tetter, as leukoplakia vulvae and softening keloid.In recent years, research finds that it has the effect of good photosensitive killing tumor cell and inhibition hiv virus HIV-1, and can be used as novel photo-activation pesticide and potential photoelectric conversion material, research and application prospect very wide (referring to: China is practical medical, 2008,3 (11): 147-149, hypocrellin progress of research).
At present, the method for production hypocrellin mainly contains following several:
(1) directly extract hypocrellin (referring to Shandong Technology Univ's journal from natural tabasheer stroma, 2004,18 (2): 91-94, the isolation and purification of Hypocrellin A in tabasheer), but regional distribution condition and temporal restriction due to bamboo parasitic fungus, seriously restricted the source on the resource, and the field acquisition tabasheer also easily causes decaying of bamboo grove.(referring to: Agriculture of Anhui science, 2009,37 (28): 13589-13590, the pharmaceutical use of medicinal fungus tabasheer and conservation of resources).
(2) utilize and to separate the bacterial strain obtained carry out solid culture (referring to biotechnology from tabasheer, 2004,14 (4): 46-47, the research of bamboo parasitic fungus solid state fermentation hypocrellin condition) and liquid culture (referring to the Zhejiang edible mushrooms, 2008,16 (5): 24-25, the liquid fermenting of bamboo parasitic fungus is studied) thus obtain hypocrellin and precursor species compound thereof.Solid culture or liquid culture are inoculating strains in solid or liquid nutrient medium, and standing or shaking culture, after for some time, is collected mycelium, then from mycelium the separation and Extraction hypocrellin.But in the method, tunning is stored in born of the same parents, all unfavorable for collecting product and process product outside born of the same parents, and have the step complexity from extraction separation and purification hypocrellin in born of the same parents, the problem that cost is high, hindered the development of using bamboo parasitic fungus fermentation production commercial applications.
In recent years, utilize the in situ product removal technology, the method for especially in substratum, adding solid adsorbent absorption promotes the synthetic research of secondary metabolite more and more.The in situ product removal technology refers to that the meta-bolites that thalline is produced is transferred to other position or other material from a certain position, is a kind of mode that increases output, belongs to a kind of of two-phase culture technique.In fermenting process, the transfer of the original position of product is comprehensive a production and the bioprocess separated, and for the efficiency that overcomes feedback inhibition and the whole process of raising, good effect is arranged.Yan Q. etc. by adding resin X-5 in the Hairy Root Cultures of Salvia miltiorrhiza culturing process, unit content and the volumetric production of total tanshinone have not only been improved, its remarkable effect is to have adsorbed from root most TANSHINONES (referring to Yan Q.et al.2005.Efficient production and recovery of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures with in situ adsorption, elicitation and semi-continuous operation.Journal of Biotechnology, 119:416～424).Xu L-J etc. add resin X-5 in fragrant fusarium (Fusarium redolens) culturing process, make the output of beauvericin be increased to 265mg/L from 194mg/L, and 65% beauvericin by solid phase adsorption (referring to: Xu Li-Jian et al.2009.Enhanced beauvericin production with in situ adsorption in mycelial liquid culture of Fusarium redolens Dzf2.Process Biochemistry 44:1063-1067).But about utilizing the in situ product removal technology to increase hypocrellin output, and the technology that is beneficial to product separation is without relevant report.
Summary of the invention
The object of the invention is to provide a kind of method that improves hypocrellin yield in shiraia bambusicola, to improve hypocrellin yield in shiraia bambusicola, simplifies collection and the treatment step of tunning hypocrellin simultaneously.
For achieving the above object, the technical solution used in the present invention is: a kind of method that improves hypocrellin yield in shiraia bambusicola, take the tabasheer bacterial strain as starting strain, carry out seed culture and fermentation culture after activation, fermentation culture is collected mycelium after at least 4 days, obtain hypocrellin after separation and purification, wherein, in the fermentation culture process, in fermention medium, add the macroporous adsorbent resin co-cultivation.
In technique scheme, described tabasheer bacterial strain refers to all fungies that can produce hypocrellin under state of nature, can be selected from tabasheer bacterial strain or its mutant strain that any strain library can openly be buied, be preferably bamboo parasitic fungus (Shiraia bambusicola) S8, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number is: CGMCC No.3984, preservation date is: on July 7th, 2010.
In technique scheme, described macroporous adsorbent resin is the polystyrene type nonpolar adsorption resin, in preferred technical scheme, described macroporous adsorbent resin particle diameter is 0.3-1.25mm, mean pore size is 29-30nm, and pore volume is 1.20-1.24, for example macroporous adsorption resin X-5.
Technique scheme specifically comprises the following steps:
(1) by potato dextrose agar (PDA) substratum of the new preparation of bamboo parasitic fungus bacterial strain access, 25～28 ℃ of culture temperature, incubation time 9～10 days;
The moiety of described potato dextrose agar (PDA) substratum is: each rise in distilled water containing following ingredients (unit: gram): potato 200 (liquor), glucose 20, agar 15, the pH value is nature pH; The prior art that the preparation method of described potato dextrose agar (PDA) substratum is known to the skilled person;
(2) bacterial classification in step (1) is equipped with in the triangular flask of seed culture medium with access after the punch tool punching, inoculum size is 4 holes/200mL nutrient solution, 25～28 ℃ of culture temperature, and shaking speed 150～160rpm, cultivate after 5 days and obtain seed liquor;
The moiety of described seed culture medium is that each rises in distilled water containing following ingredients (unit: gram): potato 200 (liquor), glucose 20, KH
2PO
43, MgSO
41.5, vitamins B
10.01, yeast extract paste 5, liquid amount is dress 200mL seed culture medium in every 500mL triangular flask;
(3) seed liquor of gained in step (2) is transferred in the triangular flask that fermention medium is housed and carry out fermentation culture, inoculum size 10% (v/v), 25～28 ℃ of culture temperature, shaking speed 150～160rpm, within 1st～7 days in fermentation culture, in fermention medium, add macroporous adsorbent resin, gathering in the crops mycelium and taking out macroporous adsorbent resin in 5th～8 days in fermentation culture;
The moiety of described fermention medium is identical with the moiety of seed culture medium in step (2), and liquid amount is dress 50mL fermention medium in every 150mL triangular flask, and the add-on of macroporous adsorbent resin is 0.5～3g/50mL fermention medium;
After macroporous adsorbent resin wraps up also sterilizing with 200 order nylon cloths, then add in fermention medium;
(4) by weighing after the mycelium oven dry of results in step (3), extract the hypocrellin in mycelium and macroporous adsorbent resin.
In technique scheme, in step (1), the medium optimization that the activation bacterial strain is used is potato dextrose agar (PDA) substratum, and culture temperature is preferably 28 ℃.
In technique scheme, in step (2), in the seed culture process, culture temperature is preferably 28 ℃; Shaking speed is preferably 150rpm; Incubation time is preferably 5 days.
In technique scheme, in step (3), in the fermentation culture process, culture temperature is preferably 28 ℃, and shaking speed is preferably 150rpm; The add-on of macroporous adsorbent resin is preferably every 100mL fermention medium 1g, and the joining day is preferably the 4th day of fermentation culture; The time of fermentation culture is preferably 8 days, gathering in the crops mycelium and taking out macroporous adsorbent resin in the 8th day in fermentation culture.
Because technique scheme is used, the present invention compared with prior art has following advantages:
(1) due to using polystyrol type nonpolar adsorption resin of the present invention and bamboo parasitic fungus, cultivate altogether, promote bamboo parasitic fungus to produce hypocrellin, improve the output of hypocrellin, compared with the control group that does not add the polystyrene type nonpolar adsorption resin, improved 3～4 times;
(2) the polystyrene type nonpolar adsorption resin to the growth of bamboo parasitic fungus without obvious inhibition; In common cultivation and fermentation process, the hypocrellin of absorption bamboo parasitic fungus secretion, can effectively overcome the feedback inhibition of product, and be conducive to the separation and Extraction of later stage hypocrellin, thereby reduce the cost of the method.
Preservation information
Bamboo parasitic fungus (Shiraia bambusicola) S8, the preservation address: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is: CGMCC No.3984, preservation date is: on July 7th, 2010.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
The extraction and determination of hypocrellin is with reference to the extraction and determination method of hypocrellin in hypocrellin ointment in Chinese Pharmacopoeia (2010 editions).
Embodiment mono-: using bamboo parasitic fungus fermentation produces hypocrellin
1, activated spawn: take out in the new PDA substratum of preparing of bamboo parasitic fungus bacterial strain S8 access of preservation, consisting of of PDA substratum, each rises in distilled water containing following ingredients (unit: gram): potato 200 (liquor), glucose 20, agar 15, the pH value is nature pH, 25～28 ℃ of culture temperature, incubation time 9～10 days;
2, the bacterial classification after activation is equipped with in the triangular flask of seed culture medium with access after punch tool punching, the consisting of of seed culture medium, each rises in distilled water and contains following ingredients (unit: gram): potato 200 (liquor), glucose 20, KH
2PO
43, MgSO
41.5, vitamins B
10.01, yeast extract paste 5, liquid amount is dress 200mL seed culture medium in every 500mL triangular flask, inoculum size is 4 holes/200mL nutrient solution, 25～28 ℃ of culture temperature, shaking speed 150～160rpm, cultivate after 5 days and obtain seed liquor;
3, seed liquor is transferred in the triangular flask that fermention medium is housed and carry out fermentation culture, the moiety of fermention medium is identical with the moiety of seed culture medium, liquid amount is dress 50mL fermention medium in every 150mL triangular flask, inoculum size 10% (v/v), 25～28 ℃ of culture temperature, shaking speed 150～160rpm;
4, fermentation culture was gathered in the crops mycelium after 8 days, with vacuum filtration isolation medium and mycelium, after mycelium is clean with distilled water flushing, in 40 ℃ of oven dry.
5, the bamboo parasitic fungus mycelium of drying is ground, get the 0.1g powder, add 5ml dehydrated alcohol lixiviate 24 hours, this step repeats once, merges extracted twice liquid, and 4000r is centrifugal, and 10min removes mycelium, extracting solution is settled to 10ml, measures the OD value under the 465nm wavelength, presses Hypocrellin A (C
30H
20O
10) the optical density coefficient be 459 and formula
A＝C＊E＊b
A is absorbancy, and C is concentration (g/L), and E is optical density coefficient (L/g*cm), and b is that cuvette thickness (cm) calculates.
Do not adding under macroporous adsorbent resin X-5 condition after using bamboo parasitic fungus fermentation, the output of hypocrellin is 0.423mg/L.
Embodiment bis-:
1, activated spawn: take out in the new PDA substratum of preparing of bamboo parasitic fungus bacterial strain S8 access of preservation, consisting of of PDA substratum, each rises in distilled water containing following ingredients (unit: gram): potato 200 (liquor), glucose 20, agar 15, the pH value is nature pH, 25～28 ℃ of culture temperature, incubation time 9～10 days;
2, the bacterial classification after activation is equipped with in the triangular flask of seed culture medium with access after punch tool punching, the consisting of of seed culture medium, each rises in distilled water and contains following ingredients (unit: gram): potato 200 (liquor), glucose 20, KH
2PO
43, MgSO
41.5, vitamins B
10.01, yeast extract paste 5, liquid amount is dress 200mL seed culture medium in every 500mL triangular flask, inoculum size is 4 holes/200mL nutrient solution, 25～28 ℃ of culture temperature, shaking speed 150～160rpm, cultivate after 5 days and obtain seed liquor;
3, seed liquor is transferred in the triangular flask that fermention medium is housed and carry out fermentation culture, the moiety of fermention medium is identical with the moiety of seed culture medium, liquid amount is dress 50mL fermention medium in every 150mL triangular flask, inoculum size 10% (v/v), 25～28 ℃ of culture temperature, shaking speed 150～160rpm added the macroporous adsorption resin X-5 of 0.5～3g with 200 order nylon cloths parcels sterilizing at the 4th day of fermentation culture in fermention medium;
4, fermentation culture is gathered in the crops mycelium and resin pocket after 7 days, with vacuum filtration isolation medium and mycelium, after mycelium is clean with distilled water flushing, in 40 ℃ of oven dry.
5, the bamboo parasitic fungus mycelium of drying is ground, get the 0.1g powder, add 5ml dehydrated alcohol lixiviate 24 hours, this step repeats once, merges extracted twice liquid, and 4000r is centrifugal, and 10min removes mycelium, extracting solution is settled to 10ml, measures the OD value under the 465nm wavelength, presses Hypocrellin A (C
30H
20O
10) the optical density coefficient be 459 and formula
A＝C＊E＊b
A is absorbancy, and C is concentration (g/L), and E is optical density coefficient (L/g*cm), and b is that cuvette thickness (cm) calculates.
6,25ml dehydrated alcohol lixiviate 24 hours for resin pocket, this step repeats once, merges extracted twice liquid, be concentrated into 10ml, concentrated solution, in the centrifugal 10min of 4000r, is measured the OD value under the 465nm wavelength, then calculates the content of hypocrellin according to the method in above-mentioned steps 5.
In this embodiment, when the macroporous adsorption resin X-5 add-on is 0.5g, the output of hypocrellin is the highest, is 0.795mg/L, is 1.88 times of output that do not add resin.
Embodiment tri-:
1, activated spawn: take out in the new PDA substratum of preparing of bamboo parasitic fungus bacterial strain S8 access of preservation, consisting of of PDA substratum, each rises in distilled water containing following ingredients (unit: gram): potato 200 (liquor), glucose 20, agar 15, the pH value is nature pH, 25～28 ℃ of culture temperature, incubation time 9～10 days;
2, the bacterial classification after activation is equipped with in the triangular flask of seed culture medium with access after punch tool punching, the consisting of of seed culture medium, each rises in distilled water and contains following ingredients (unit: gram): potato 200 (liquor), glucose 20, KH
2PO
43, MgSO
41.5, vitamins B
10.01, yeast extract paste 5, liquid amount is dress 200mL seed culture medium in every 500mL triangular flask, inoculum size is 4 holes/200mL nutrient solution, 25～28 ℃ of culture temperature, shaking speed 150～160rpm, cultivate after 5 days and obtain seed liquor;
3, seed liquor is transferred in the triangular flask that fermention medium is housed and carry out fermentation culture, the moiety of fermention medium is identical with the moiety of seed culture medium, liquid amount is dress 50mL fermention medium in every 150mL triangular flask, inoculum size 10% (v/v), 25～28 ℃ of culture temperature, shaking speed 150～160rpm added the macroporous adsorption resin X-5 of 0.5g with 200 order nylon cloths parcels sterilizing at 1st～7 days of fermentation culture in fermention medium;
4, fermentation culture is gathered in the crops mycelium and resin pocket after 7 days, with vacuum filtration isolation medium and mycelium, after mycelium is clean with distilled water flushing, in 40 ℃ of oven dry.
5, the bamboo parasitic fungus mycelium of drying is ground, get the 0.1g powder, add 5ml dehydrated alcohol lixiviate 24 hours, this step repeats once, merges extracted twice liquid, and 4000r is centrifugal, and 10min removes mycelium, extracting solution is settled to 10ml, measures the OD value under the 465nm wavelength, presses Hypocrellin A (C
30H
20O
10) the optical density coefficient be 459 and formula
A＝C＊E＊b
A is absorbancy, and C is concentration (g/L), and E is optical density coefficient (L/g*cm), and b is that cuvette thickness (cm) calculates.
6,25ml dehydrated alcohol lixiviate 24 hours for resin pocket, this step repeats once, merges extracted twice liquid, be concentrated into 10ml, concentrated solution, in the centrifugal 10min of 4000r, is measured the OD value under the 465nm wavelength, then calculates the content of hypocrellin according to the method in above-mentioned steps 5.
In this embodiment, when the joining day of macroporous adsorption resin X-5 is the 4th day, the output of hypocrellin is the highest, is 1.108mg/L, is 2.62 times of output that do not add resin.
Embodiment tetra-:
1, activated spawn: take out in the new PDA substratum of preparing of bamboo parasitic fungus bacterial strain S8 access of preservation, consisting of of PDA substratum, each rises in distilled water containing following ingredients (unit: gram): potato 200 (liquor), glucose 20, agar 15, the pH value is nature pH, 25～28 ℃ of culture temperature, incubation time 9～10 days;
2, the bacterial classification after activation is equipped with in the triangular flask of seed culture medium with access after punch tool punching, the consisting of of seed culture medium, each rises in distilled water and contains following ingredients (unit: gram): potato 200 (liquor), glucose 20, KH
2PO
43, MgSO
41.5, vitamins B
10.01, yeast extract paste 5, liquid amount is dress 200mL seed culture medium in every 500mL triangular flask, inoculum size is 4 holes/200mL nutrient solution, 25～28 ℃ of culture temperature, shaking speed 150～160rpm, cultivate after 5 days and obtain seed liquor;
3, seed liquor is transferred in the triangular flask that fermention medium is housed and carry out fermentation culture, the moiety of fermention medium is identical with the moiety of seed culture medium, liquid amount is dress 50mL fermention medium in every 150mL triangular flask, inoculum size 10% (v/v), 25～28 ℃ of culture temperature, shaking speed 150～160rpm added the macroporous adsorption resin X-5 of 0.5g with 200 order nylon cloths parcels sterilizing at the 4th day of fermentation culture in fermention medium;
4, fermentation culture is gathered in the crops mycelium and resin pocket after 5～8 days, with vacuum filtration isolation medium and mycelium, after mycelium is clean with distilled water flushing, in 40 ℃ of oven dry.
5, the bamboo parasitic fungus mycelium of drying is ground, get the 0.1g powder, add 5ml dehydrated alcohol lixiviate 24 hours, this step repeats once, merges extracted twice liquid, and 4000r is centrifugal, and 10min removes mycelium, extracting solution is settled to 10ml, measures the OD value under the 465nm wavelength, presses Hypocrellin A (C
30H
20O
10) the optical density coefficient be 459 and formula
A＝C＊E＊b
A is absorbancy, and C is concentration (g/L), and E is optical density coefficient (L/g*cm), and b is that cuvette thickness (cm) calculates.
6,25ml dehydrated alcohol lixiviate 24 hours for resin pocket, this step repeats once, merges extracted twice liquid, be concentrated into 10ml, concentrated solution, in the centrifugal 10min of 4000r, is measured the OD value under the 465nm wavelength, then calculates the content of hypocrellin according to the method in above-mentioned steps 5.
In this embodiment, when the time that macroporous adsorption resin X-5 and using bamboo parasitic fungus fermentation are cultivated altogether is 8 days, the output of hypocrellin is the highest, is 1.483mg/L, is 3.51 times of output that do not add resin.
Embodiment five:
1, activated spawn: take out in the new PDA substratum of preparing of bamboo parasitic fungus bacterial strain S8 access of preservation, consisting of of PDA substratum, each rises in distilled water containing following ingredients (unit: gram): potato 200 (liquor), glucose 20, agar 15, the pH value is nature pH, 28 ℃ of culture temperature, incubation time 9 days;
2, the bacterial classification after activation is equipped with in the triangular flask of seed culture medium with access after punch tool punching, the consisting of of seed culture medium, each rises in distilled water and contains following ingredients (unit: gram): potato 200 (liquor), glucose 20, KH
2PO
43, MgSO
41.5, vitamins B
10.01, yeast extract paste 5, liquid amount is dress 200mL seed culture medium in every 500mL triangular flask, inoculum size is 4 holes/200mL nutrient solution, 28 ℃ of culture temperature, shaking speed 150rpm, cultivate after 5 days and obtain seed liquor;
3, seed liquor is transferred in the triangular flask that fermention medium is housed and carry out fermentation culture, the moiety of fermention medium is identical with the moiety of seed culture medium, liquid amount is dress 50mL fermention medium in every 150mL triangular flask, inoculum size 10% (v/v), 28 ℃ of culture temperature, shaking speed 150rpm added the macroporous adsorption resin X-5 of 0.5g with 200 order nylon cloths parcels sterilizing at the 4th day of fermentation culture in fermention medium;
4, fermentation culture is gathered in the crops mycelium and resin pocket after 8 days, with vacuum filtration isolation medium and mycelium, after mycelium is clean with distilled water flushing, in 40 ℃ of oven dry.
5, the bamboo parasitic fungus mycelium of drying is ground, get the 0.1g powder, add 5ml dehydrated alcohol lixiviate 24 hours, this step repeats once, merges extracted twice liquid, and 4000r is centrifugal, and 10min removes mycelium, extracting solution is settled to 10ml, measures the OD value under the 465nm wavelength, presses Hypocrellin A (C
30H
20O
10) the optical density coefficient be 459 and formula
A＝C＊E＊b
A is absorbancy, and C is concentration (g/L), and E is optical density coefficient (L/g*cm), and b is that cuvette thickness (cm) calculates.
6,25ml dehydrated alcohol lixiviate 24 hours for resin pocket, this step repeats once, merges extracted twice liquid, be concentrated into 10ml, concentrated solution, in the centrifugal 10min of 4000r, is measured the OD value under the 465nm wavelength, then calculates the content of hypocrellin according to the method in above-mentioned steps 5.
At bamboo parasitic fungus and macroporous adsorption resin X-5, altogether under the cultivation and fermentation optimal conditions, the output of hypocrellin is 1.622mg/L, is 3.83 times of output that do not add resin.
Claims (6)
1. a method that improves hypocrellin yield in shiraia bambusicola, the tabasheer bacterial strain of take carries out seed culture and fermentation culture as starting strain, collect mycelium, obtain hypocrellin after separation and purification, it is characterized in that, in the fermentation culture process, in fermention medium, add the macroporous adsorbent resin co-cultivation; Described tabasheer bacterial strain be bamboo parasitic fungus (
Shiraia bambusicola) S8, the preserving number that provide at China Committee for Culture Collection of Microorganisms's common micro-organisms center is: CGMCC No. 3984, and preservation date is: on July 7th, 2010; Described macroporous adsorbent resin is the polystyrene type nonpolar adsorption resin; Described macroporous adsorbent resin particle diameter is 0.3-1.25mm, and mean pore size is 29-30nm, and pore volume is 1.20-1.24;
Specifically comprise the following steps:
(1) by the potato dextrose agar of the new preparation of bamboo parasitic fungus bacterial strain access, 25 ~ 28 ℃ of culture temperature, incubation time 9 ~ 10 days;
(2) bacterial classification in step (1) is equipped with in the triangular flask of seed culture medium with access after the punch tool punching, inoculum size is 4 holes/200 mL nutrient solutions, 25 ~ 28 ℃ of culture temperature, and shaking speed 150 ~ 160 rpm, cultivate after 5 days and obtain seed liquor;
The moiety of described seed culture medium is that each rises in distilled water and contains following ingredients: potato 200g is cooked into juice, glucose 20g, KH
2PO
43g, MgSO
41.5g, vitamins B
10.01g, yeast extract paste 5g, liquid amount is to fill 200 mL seed culture mediums in every 500 mL triangular flasks;
(3) seed liquor of gained in step (2) is transferred in the triangular flask that fermention medium is housed and carry out fermentation culture, inoculum size counts 10% with v/v, 25 ~ 28 ℃ of culture temperature, shaking speed 150 ~ 160 rpm, within 1st ~ 7 days in fermentation culture, in fermention medium, add macroporous adsorbent resin, gathering in the crops mycelium and taking out macroporous adsorbent resin in 5th ~ 8 days in fermentation culture;
The moiety of described fermention medium is identical with the moiety of seed culture medium in step (2), and liquid amount is to fill 50 mL fermention mediums in every 150 mL triangular flasks, and the add-on of polymeric adsorbent is 0.5 ~ 3 g/50 mL fermention medium;
After the polystyrene type nonpolar adsorption resin wraps up also sterilizing with 200 order nylon cloths, then add in fermention medium;
(4) mycelium of results in step (3) is weighed after 40 ℃ of oven dry, extract the hypocrellin in mycelium and macroporous adsorbent resin.
2. improve according to claim 1 the method for hypocrellin yield in shiraia bambusicola, it is characterized in that, in step (1), culture temperature is 28 ℃.
3. improve according to claim 1 the method for hypocrellin yield in shiraia bambusicola, it is characterized in that, in step (2), in the seed culture process, culture temperature is 28 ℃; Shaking speed is 150 rpm; Incubation time is 5 days.
4. improve according to claim 1 the method for hypocrellin yield in shiraia bambusicola, it is characterized in that, in step (3), in the fermentation culture process, culture temperature is 28 ℃, and shaking speed is 150 rpm.
5. improve according to claim 1 the method for hypocrellin yield in shiraia bambusicola, it is characterized in that, in step (3), in the fermentation culture process, the add-on of macroporous adsorbent resin is every 100mL fermention medium 1g, and the joining day is fermentation culture the 4th day.
6. improve according to claim 1 the method for hypocrellin yield in shiraia bambusicola, it is characterized in that, in step (3), in the fermentation culture process, the time of fermentation culture is 8 days, gathering in the crops mycelium and taking out macroporous adsorbent resin in the 8th day in fermentation culture.
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| CN103070021B (en) * | 2013-02-22 | 2014-05-14 | 贵州省生物技术研究所 | Method for artificially culturing fungus tabasheer |
| CN104846044B (en) * | 2014-02-17 | 2019-02-01 | 上海医药工业研究院 | A kind of fermentation medium improving feldamycin yield |
| CN104480224B (en) * | 2014-12-13 | 2016-08-24 | 河南天冠企业集团有限公司 | A kind of control method of fermentation process pH value |
| CN107099489B (en) * | 2017-06-30 | 2020-10-09 | 苏州大学 | Associated bacterial strain for improving hypocrellin fermentation yield and application thereof |
| CN113149819B (en) * | 2021-05-18 | 2022-04-15 | 山东国力生物科技有限公司 | Method for extracting hypocrellin from solid-state fermentation material of tabasheer fungus |
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| CN101113130A (en) * | 2006-07-24 | 2008-01-30 | 昆明振华制药厂有限公司 | Method for preparing high-purity hypocrellin A |
| CN101113132A (en) * | 2006-07-24 | 2008-01-30 | 昆明振华制药厂有限公司 | Method for preparing high-purity hypocrellin B |
| CN101186932A (en) * | 2007-11-11 | 2008-05-28 | 杨海龙 | Method for synchronously producing hypocrellin and shiraia bambusicola polysaccharides |
| CN101875905A (en) * | 2009-11-06 | 2010-11-03 | 首都师范大学 | Mao bamboo seed endophytic fungi strain for efficiently producing hypocrellin and application thereof |
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| CN101113130A (en) * | 2006-07-24 | 2008-01-30 | 昆明振华制药厂有限公司 | Method for preparing high-purity hypocrellin A |
| CN101113132A (en) * | 2006-07-24 | 2008-01-30 | 昆明振华制药厂有限公司 | Method for preparing high-purity hypocrellin B |
| CN101186932A (en) * | 2007-11-11 | 2008-05-28 | 杨海龙 | Method for synchronously producing hypocrellin and shiraia bambusicola polysaccharides |
| CN101875905A (en) * | 2009-11-06 | 2010-11-03 | 首都师范大学 | Mao bamboo seed endophytic fungi strain for efficiently producing hypocrellin and application thereof |
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