CN105925493B - A strain of tabasheer and its application in fermentation production of hypocrellin - Google Patents
A strain of tabasheer and its application in fermentation production of hypocrellin Download PDFInfo
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- CN105925493B CN105925493B CN201610496366.1A CN201610496366A CN105925493B CN 105925493 B CN105925493 B CN 105925493B CN 201610496366 A CN201610496366 A CN 201610496366A CN 105925493 B CN105925493 B CN 105925493B
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- VANSZAOQCMTTPB-SETSBSEESA-N hypocrellin Chemical compound C1[C@@](C)(O)[C@@H](C(C)=O)C2=C(OC)C(O)=C3C(=O)C=C(OC)C4=C3C2=C2C3=C4C(OC)=CC(=O)C3=C(O)C(OC)=C21 VANSZAOQCMTTPB-SETSBSEESA-N 0.000 title claims abstract description 44
- BQJKVFXDDMQLBE-UHFFFAOYSA-N shiraiachrome A Natural products COC1=C2C3=C(OC)C=C(O)C4=C3C3=C5C(CC(C)(O)C(C(C)=O)C3=C(OC)C4=O)=C(OC)C(=O)C(C(O)=C1)=C25 BQJKVFXDDMQLBE-UHFFFAOYSA-N 0.000 title claims abstract description 44
- 238000000855 fermentation Methods 0.000 title claims abstract description 38
- 230000004151 fermentation Effects 0.000 title claims abstract description 38
- KGHNSNSWRMJVND-UHFFFAOYSA-N Hypocrellin Natural products COC1=CC(=O)C2=C3C4C(C(C(=O)C)C(C)(O)Cc5c(OC)c(O)c6C(=O)C=C(OC)C(=C13)c6c45)C(=C2O)OC KGHNSNSWRMJVND-UHFFFAOYSA-N 0.000 title claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 title abstract description 9
- 241000233866 Fungi Species 0.000 claims abstract description 25
- 239000001963 growth medium Substances 0.000 claims abstract description 24
- 241000277045 Shiraia bambusicola Species 0.000 claims abstract description 16
- 238000009629 microbiological culture Methods 0.000 claims abstract description 3
- 241001330002 Bambuseae Species 0.000 claims description 28
- 235000017166 Bambusa arundinacea Nutrition 0.000 claims description 27
- 235000017491 Bambusa tulda Nutrition 0.000 claims description 27
- 235000015334 Phyllostachys viridis Nutrition 0.000 claims description 27
- 239000011425 bamboo Substances 0.000 claims description 27
- 230000003071 parasitic effect Effects 0.000 claims description 24
- 240000008042 Zea mays Species 0.000 claims description 21
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 21
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 19
- 235000005822 corn Nutrition 0.000 claims description 19
- 239000002609 medium Substances 0.000 claims description 19
- 238000011218 seed culture Methods 0.000 claims description 19
- 239000007787 solid Substances 0.000 claims description 19
- 241000196324 Embryophyta Species 0.000 claims description 18
- 239000000463 material Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 17
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 11
- 241001465754 Metazoa Species 0.000 claims description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 244000046052 Phaseolus vulgaris Species 0.000 claims description 9
- 235000010627 Phaseolus vulgaris Nutrition 0.000 claims description 9
- 229910052602 gypsum Inorganic materials 0.000 claims description 9
- 239000010440 gypsum Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 235000015278 beef Nutrition 0.000 claims description 8
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 8
- 239000002504 physiological saline solution Substances 0.000 claims description 8
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- 239000002994 raw material Substances 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- 235000007164 Oryza sativa Nutrition 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 235000013312 flour Nutrition 0.000 claims description 6
- 235000009566 rice Nutrition 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 235000015099 wheat brans Nutrition 0.000 claims description 5
- 229930091371 Fructose Natural products 0.000 claims description 4
- 239000005715 Fructose Substances 0.000 claims description 4
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 4
- 239000007836 KH2PO4 Substances 0.000 claims description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 229910052564 epsomite Inorganic materials 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- GVALZJMUIHGIMD-UHFFFAOYSA-H magnesium phosphate Chemical compound [Mg+2].[Mg+2].[Mg+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GVALZJMUIHGIMD-UHFFFAOYSA-H 0.000 claims description 4
- 239000004137 magnesium phosphate Substances 0.000 claims description 4
- 229960002261 magnesium phosphate Drugs 0.000 claims description 4
- 229910000157 magnesium phosphate Inorganic materials 0.000 claims description 4
- 235000010994 magnesium phosphates Nutrition 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 4
- 239000004317 sodium nitrate Substances 0.000 claims description 4
- 235000010344 sodium nitrate Nutrition 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 229940100486 rice starch Drugs 0.000 claims description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims 1
- 235000009754 Vitis X bourquina Nutrition 0.000 claims 1
- 235000012333 Vitis X labruscana Nutrition 0.000 claims 1
- 240000006365 Vitis vinifera Species 0.000 claims 1
- 235000014787 Vitis vinifera Nutrition 0.000 claims 1
- 238000010884 ion-beam technique Methods 0.000 abstract description 8
- 239000010902 straw Substances 0.000 abstract description 7
- 239000010871 livestock manure Substances 0.000 abstract description 5
- 239000000758 substrate Substances 0.000 abstract description 5
- 238000012258 culturing Methods 0.000 abstract description 3
- 239000002699 waste material Substances 0.000 abstract description 3
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- 230000002503 metabolic effect Effects 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract 1
- 239000008675 zhuhuang Substances 0.000 abstract 1
- 238000000034 method Methods 0.000 description 16
- YDLBDQPPRTYAIG-UHFFFAOYSA-N hypocrellin A Natural products COC1C2CC(C)(O)C(C(=O)C)C3=C(OC)C(=O)c4c(O)cc(OC)c5c6c(OC)cc(O)c(C1=O)c6c2c3c45 YDLBDQPPRTYAIG-UHFFFAOYSA-N 0.000 description 13
- 239000002361 compost Substances 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 11
- 210000003608 fece Anatomy 0.000 description 8
- 239000010907 stover Substances 0.000 description 8
- 230000035772 mutation Effects 0.000 description 6
- 241000209094 Oryza Species 0.000 description 5
- 239000000049 pigment Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 4
- 239000012084 conversion product Substances 0.000 description 4
- 238000003113 dilution method Methods 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 229960000935 dehydrated alcohol Drugs 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- UYEMGAFJOZZIFP-UHFFFAOYSA-N 3,5-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC(O)=CC(O)=C1 UYEMGAFJOZZIFP-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 235000009973 maize Nutrition 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001373 regressive effect Effects 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
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- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 230000008821 health effect Effects 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 238000011090 industrial biotechnology method and process Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
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- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000518 rheometry Methods 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 210000003497 sciatic nerve Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The invention discloses a strain of tabasheer and its application in fermentation production of hypocrellin, which is prepared by separating tabasheer collected in Anhui Huangshan area, Zhejiang Quzhou area and Sichuan Yibin area to obtain three strains of tabasheer, culturing in basic culture medium, treating with ion beam to obtain a strain with exuberant activity, which is classified and named as tabasheer (Zhuhuang)Shiraia bambusicola) NJTECH-1, which is preserved in China general microbiological culture collection center with the preservation number of CGMCC NO. 11617. The obtained tabasheer fungus has high metabolic activity, and the yield of the fermentation production of the hypocrellin is high. The waste straws and the livestock manure in the field can be used as fermentation substrates, the number of straw burning can be reduced, and the environmental pollution is reduced while the cost is saved.
Description
Technical field
The invention belongs to technical field of biological fermentation, and in particular to one plant of bamboo parasitic fungus and its in fermenting and producing hypocrellin
Application.
Background technique
Tabasheer is distributed mainly on Southeast China side, province, part, in addition, the Japan in Asia is also distributed, other countries are then
It has not been reported.The hypocrellin that bamboo parasitic fungus generates has good scattered silt promoting blood circulation, expelling wind and removing dampness, tonifying middle-Jiao and Qi and promotes new old generation
Thank, build up health and other effects, clinically it is chiefly used in treating peratodynia, rheumatic arthritis, traumatic injury and sciatic nerve
The diseases such as pain.And this pigment is safe and non-toxic, color is scarlet, strong coloring force and rich in healthcare function, as edible pigment
There is wide prospect.
Traditional hypocrellin production method, is generally divided into biological synthesis process and chemical synthesis, and biological synthesis process is main
It is using corn flour, wheat bran, the cereal crops such as wheat are raw material, produce hypocrellin using bamboo parasitic fungus fermentation, then separated
Purifying, although yield increased for other production methods, in the epoch that provision price gradually rises, this side
Method is unfavorable for the large-scale application of hypocrellin but also the cost of final product improves many.Another is closed for chemistry
Cheng Fa is initial reaction object using 3,5- dihydroxy-benzoic acid, chemically reacts by 12 steps, has synthesized hypocrellin, but originally
Method reaction step is more and complicated, and side reaction is more, and agents useful for same is more toxic, and cost is also high, is not production hypocrellin
Good method.
2005, Chinese maize stalk just had reached about 20207 × 104T, and the ratio of straw-returning is very low, though
Right country prohibites crop straw burning, but still cannot eradicate the excessive problem of stalk completely.Seek rationally to utilize discarded straw
The method of stalk is particularly important.
Summary of the invention
The object of the present invention is to provide one plant of bamboo parasitic fungus and its application in fermenting and producing hypocrellin, the bamboo parasitic fungus
Hypocrellin yield is high, and can efficiently use the fermentation of materials such as waste straw, discarded fowl and animal excrement and produce hypocrellin, reduces life
Produce cost and safety and environmental protection.
To achieve the above object, The technical solution adopted by the invention is as follows:
The present invention will be adopted respectively in Mt. Huang in Anhui area, the tabasheer in Zhejiang Quzhou area and Sichuan Yibin Prefecture, through separating
Three plants of bamboo parasitic fungus are obtained, base culture base is carried out and obtains one plant of vigor tabasheer the most vigorous after ion beam mutagenesis processing
Bacterium, classification naming be tabasheer (Shiraia bambusicola) NJTECH-1, it has been preserved on November 30th, 2015
State's General Microbiological Culture preservation administrative center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro- life of the Chinese Academy of Sciences
Object research institute, deposit number are CGMCC NO.11617.
The bacterial strain screens obtain by the following method: adopt respectively in Mt. Huang in Anhui area, Zhejiang Quzhou area and
The tabasheer of Sichuan Yibin Prefecture, isolated three plants of bamboo parasitic fungus carry out basic culture, 26 DEG C of trainings using liquid murphy juice culture medium
After supporting five days, while biomass and hypocrellin secretory volume are measured, obtaining one plant of vigor bacterial strain the most vigorous is to acquire
Bacterial strain from Sichuan Yibin, is named as Super-3, as dominant strain.
The spore of Super-3 is washed down with physiological saline, is connected in liquid seed culture medium and cultivates.By cultured tabasheer
It is 10 that bacterium spore suspension, which is diluted to cell number by 10 times of dilution methods,7/ mL takes 0.1 mL of bacterium solution to be uniformly applied to sterile empty plate
In, N is carried out after air drying+Ion beam mutation, N+Ion beam mutation dosage is (90,135,180,225,270) × 2.6 × 1013N+/cm2, N+Ion beam mutation energy is 15keV.With the sterile water washing cell of 1 mL after irradiation, diluted by 10 times of dilution methods
After be painted into plating medium, 45 d are cultivated in 26 DEG C of inversions, and to picking single colonie, shaking flask detection filters out mycelial growth rate
The fast and highest bacterial strain of hypocrellin secretion rate, be named as tabasheer (Shiraia bambusicola) NJTECH-1。
The tabasheer (Shiraia bambusicola) NJTECH-1 condition of culture are as follows:
The strain is by the way of aerobic culture.Carbon source for cultivating the bacterial strain can be glucose, sucrose, fructose,
The materials such as lactose, soluble starch, rice, corn；Nitrogen source for cultivating the bacterial strain can be beef extract, peptone, yeast
The materials such as cream, sodium nitrate, ammonium sulfate.The optimum temperature range of the strain growth is 24-28 DEG C, pH range 4-9, optimum PH range
For 5-7.Dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, the inorganic salts such as magnesium phosphate can be also added during strain culturing.
The tabasheer (Shiraia bambusicola) NJTECH-1 physiology and appearance feature are as follows:
Plate culture form: the well-grown on PDA plate, culture to third day bacterium colony start with radial growth；Bacterium
Silk is white fluffy, and middle section is compared with the prosperity of both sides mycelia；Bacterium colony central part culture medium is red, is the color of thallus secretion
Element, pigment at kermesinus, form annular concentric circle in the medium, and intermediate color is deep, and ambient color is shallow.Culture five days or so,
Culture medium all becomes red, and the back side obviously can see the agglomerating spore of a large amount of black, visible tubulose mycelia under microscope, spore
About 1.1 μm or so.
The tabasheer (Shiraia bambusicola) application of the NJTECH-1 in fermenting and producing hypocrellin.
Include the following steps:
1) seed culture: being washed down the spore of bamboo parasitic fungus with physiological saline, is accessed in liquid seed culture medium, 24 ~ 30 DEG C
Cultivate 40 ~ 48h；
2) it takes the seed liquor 5mL of step 1) to access in solid medium and carries out fermented and cultured, cultivation temperature is 26 DEG C ~ 30
DEG C, produce hypocrellin.
Liquid seed culture medium described in step 1) includes the component of following mass percent: carbon source 2% ~ 3%, nitrogen source 0.2%
~ 0.4%, inorganic salts 0.05% ~ 0.1%, pH is naturally, wherein the carbon source is selected from glucose, sucrose, fructose, lactose, soluble shallow lake
At least one of powder, rice, corn；The nitrogen source in beef extract, peptone, yeast extract, sodium nitrate, ammonium sulfate extremely
Few one kind, the inorganic salts are selected from least one of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, magnesium phosphate.
Solid medium described in step 2 can be to use grain as the culture medium of configuration, it may for example comprise following quality
The component of percentage: corn flour 68.9%-75%, wheat bran 5%-10%, rice bran 15%-20%, glucose 1%-3%, KH2PO4
0.1%-0.2% ,MgSO4·7H2O 0.05%-0.07%。
Or it is with weeds, corn stover, fowl and animal excrement (based on chicken manure and cow dung), gypsum and a small amount of dregs of beans
Solid medium, specific fermentation process is prepared in fermenting raw materials are as follows: first weeds and corn stover are mixed with fowl and animal excrement,
Pre-wetted treatment adds the auxiliary materials such as gypsum, dregs of beans later again, and one time fermentation is carried out in fermentation tunnel, rises to 70-80 DEG C to temperature
Shi Baowen 4-5 days, then cools down, and turning several times, obtains the compost that water content is 70%-80% during cooling.After carry out two
Compost obtained by one time fermentation is transferred in secondary fermentation tunnel, using the microbial fermentation in compost, to temperature by secondary fermentation
When reaching 55-60 DEG C, 9-11h is kept the temperature, then compost is carried out to 4-5 days by a definite date decomposed fermentation stages, finally obtains water content
For the culture base-material of 65%-70%, the solid state substrate of culture bamboo parasitic fungus can be used as.
The proportionate relationship of above-mentioned each material can be adjusted according to the actual situation.Such as first by weeds and corn stover with
Fowl and animal excrement is mixed in 1:1:0.7 or so ratio, and pre-wetted treatment is later again in mixed material and auxiliary material with the ratio of 1:2.5 or so
Add the auxiliary materials such as gypsum, dregs of beans.
Weeds in the culture medium prescription, corn stover, fowl and animal excrement (based on chicken manure and cow dung), gypsum and a small amount of beans
The dregs of rice are that the raw material being easy to get at a low price not only substantially reduces the production of hypocrellin using them as the fermentation substrate of bamboo parasitic fungus
Cost, and advantageous secondary use can be carried out to stalk, protect environment.
There is very big market potential for bamboo parasitic fungus solid state fermentation after being handled with stalk fermentation to produce hypocrellin,
Be mainly manifested in: 2005, Chinese maize stalk just had reached about 20207*104T, and the ratio of straw-returning is very low,
This measure can make full use of waste straw, not only from a wealth of sources, but also can also effective protection environment.Since hypocrellin is in medicine
It learns, has very big application prospect in biology, but price is costly, causes to be widely used, such method is significantly
Production cost is reduced, provides possibility for a wide range of use of hypocrellin.
The utility model has the advantages that the present invention has the advantage that compared with prior art
1. bamboo parasitic fungus of the invention has very high metabolic activity, fermenting and producing hypocrellin compared with other strains
Yield is high.
2. the raw material of culture is become what field was discarded from cereal crops (corn flour etc.) compared with traditional bioanalysis
Stalk and fowl and animal excrement, from cost for, obtained significantly reducing very much, and the quantity of crop straw burning can be reduced,
While save the cost, environmental pollution is reduced.
3. the method for the present invention is easy compared with conventional chemical methods, further reduces the cost, bring huge economic benefit.
Specific embodiment
By following embodiments, the present invention may be better understood.Then, as it will be easily appreciated by one skilled in the art that it is real
Specific material ratio described in example is applied, process conditions and its result are merely to illustrate the present invention, without that should will not limit
Claims say the present invention described in detail.
1 tabasheer of embodiment (Shiraia bambusicola) NJTECH-1 mutagenesis screening
To adopt respectively in Mt. Huang in Anhui area, the tabasheer in Zhejiang Quzhou area and Sichuan Yibin Prefecture, isolated three plants
Bamboo parasitic fungus carries out basic culture using liquid murphy juice culture medium, and 26 DEG C after culture five days, while measuring biomass and bamboo is red
Rhzomorph secretory volume, obtaining one plant of vigor bacterial strain the most vigorous is the bacterial strain acquired from Sichuan Yibin, is named as Super-3,
As dominant strain.
The spore of Super-3 is washed down with physiological saline, is connected in liquid seed culture medium, 26 DEG C are cultivated 12 hours, will
It is 10 that cultured bamboo parasitic fungus spore suspension, which is diluted to cell number by 10 times of dilution methods,7/ mL takes 0.1 mL of bacterium solution to be uniformly applied to
In sterile empty plate, N is carried out after air drying+Ion beam mutation, N+Ion beam mutation dosage is (90,135,180,225,270)
×2.6×1013N+/cm2, N+Ion beam mutation energy is 15keV.With the sterile water washing cell of 1 mL after irradiation, by 10
Plating medium is painted into after the dilution of times dilution method, 4-5 d are cultivated in 26 DEG C of inversion, and to picking single colonie, shaking flask is detected, filtered out
Mycelial growth rate is fast and the highest bacterial strain of hypocrellin secretion rate, be named as tabasheer (Shiraia bambusicola)
NJTECH-1。
2 tabasheer of embodiment (Shiraia bambusicola) NJTECH-1 culture and physiologic character
The tabasheer (Shiraia bambusicola) NJTECH-1 condition of culture are as follows:
The strain by the way of aerobic culture, the carbon source for cultivating the bacterial strain can be glucose, sucrose, fructose,
The materials such as lactose, soluble starch, rice, corn；Nitrogen source for cultivating the bacterial strain can be beef extract, peptone, yeast
The materials such as cream, sodium nitrate, ammonium sulfate；The optimum temperature range of the strain growth is 24-28 DEG C, pH range 4-9, optimum PH range
For 5-7, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, the inorganic salts such as magnesium phosphate can be also added during strain culturing.
The tabasheer (Shiraia bambusicola) NJTECH-1 physiology and appearance feature are as follows:
Plate culture form: the well-grown on PDA plate, culture to third day bacterium colony start with radial growth；Bacterium
Silk is white fluffy, and middle section is compared with the prosperity of both sides mycelia；Bacterium colony central part culture medium is red, is the color of thallus secretion
Element, pigment at kermesinus, form annular concentric circle in the medium, and intermediate color is deep, and ambient color is shallow.Culture five days or so,
Culture medium all becomes red, and the back side obviously can see the agglomerating spore of a large amount of black, visible tubulose mycelia under microscope, spore
About 1.1 μm or so.
3 tabasheer of embodiment (Shiraia bambusicola) (LBR-SB6) fermenting and producing hypocrellin A
Bamboo parasitic fungus (LBR-SB6) (is protected by key lab, the industrial biotechnology Ministry of Education, Southern Yangtze University with physiological saline
Hiding) spore wash down, access liquid seed culture medium in, 26 DEG C, 120rpm cultivate 40h, take 5mL seed liquor access solid state rheology
In base, temperature control is cultivated at 26 DEG C.Liquid seed culture medium includes following component: glucose 2g/L, dipotassium hydrogen phosphate 0.1g/
L, anhydrous magnesium sulfate 0.05g/L, beef extract 0.3g/L, pH are natural.
Fermented and cultured is carried out in seed liquor access solid medium, cultivation temperature is 26 DEG C ~ 30 DEG C, produces hypocrellin.
Solid medium includes the ingredient (%) of following mass ratio: the corn flour 68.9 of 60 mesh, wheat bran 9.85, rice bran 20, glucose
1, KH2PO4 0.2 ,MgSO4·7H2O 0.05。
Fermentation results, main conversion product are hypocrellin A, and yield is about 40mg/kg.
4 tabasheer of embodiment (Shiraia bambusicola) NJTECH-1 fermenting and producing hypocrellin A
Seed culture: being washed down the spore of bamboo parasitic fungus with physiological saline, is accessed in liquid seed culture medium, 26 DEG C of cultures
40h；Liquid seed culture medium includes following component: glucose 2g/L, dipotassium hydrogen phosphate 0.1g/L, anhydrous magnesium sulfate 0.05g/
L, beef extract 0.3g/L, pH are natural.
Fermented and cultured is carried out in seed liquor access solid medium, cultivation temperature is 26 DEG C ~ 30 DEG C, produces hypocrellin.
Solid medium includes the ingredient (%) of following mass ratio: the corn flour 68.9 of 60 mesh, wheat bran 9.85, rice bran 20, glucose
1, KH2PO4 0.2 ,MgSO4·7H2O 0.05。
Fermentation results, main conversion product are hypocrellin A, yield 78.1mg/kg.
5 tabasheer of embodiment (Shiraia bambusicola) NJTECH-1 with stalk fermentation produce hypocrellin A
The preparation of solid medium: with weeds, corn stover, fowl and animal excrement (based on chicken manure and cow dung), gypsum and
A small amount of dregs of beans is raw material, is first mixed weeds and corn stover and fowl and animal excrement in 1:1:0.7 or so ratio, after pre-wetted treatment
Again in auxiliary materials such as the ratio addition gypsum of 1:2.5 or so, dregs of beans, one time fermentation is carried out in fermentation tunnel, temperature rises to 70-80
DEG C when keep the temperature 4-5 days, then cool down, during cooling turning several times, obtain water content for 70%-80% compost.After carry out
Gained compost is transferred in secondary fermentation tunnel by secondary fermentation, using the microbial fermentation in compost, is reached to temperature
At 55-60 DEG C, 9-11h is kept the temperature, then compost is carried out to 4-5 days by a definite date decomposed fermentation stages, finally obtaining water content is
The culture base-material of 65%-70%, the solid state substrate as culture bamboo parasitic fungus.
Seed culture: being washed down the spore of bamboo parasitic fungus with physiological saline, is accessed in liquid seed culture medium, 26 DEG C of cultures
40h；Liquid seed culture medium includes following component: glucose 2g/L, dipotassium hydrogen phosphate 0.1g/L, anhydrous magnesium sulfate 0.05g/
L, beef extract 0.3g/L, pH are natural.
It takes and carries out fermented and cultured in seed liquor 5mL access solid medium, cultivation temperature is 26 DEG C ~ 30 DEG C, and production bamboo is red
Rhzomorph measures the content of hypocrellin A, fermentation ends after 168h.Fermentation results, main conversion product are hypocrellin A, and yield is
4mg/kg。
6 tabasheer of embodiment (Shiraia bambusicola) NJTECH-1 with stalk fermentation produce hypocrellin A
The preparation of solid medium: use weeds, corn stover, fowl and animal excrement (based on chicken manure and cow dung), gypsum and
A small amount of dregs of beans is raw material, is first mixed weeds and corn stover and fowl and animal excrement in 1:1:1 or so ratio, after pre-wetted treatment again
Gypsum is added in the ratio of 1:3 or so, the auxiliary materials such as dregs of beans carry out one time fermentation in fermentation tunnel, rise to 70-80 DEG C to temperature
Shi Baowen 4-5 days, then cools down, and turning several times, obtains the compost that water content is 70%-80% during cooling.After carry out two
Gained compost is transferred in secondary fermentation tunnel by secondary fermentation, using the microbial fermentation in compost, reaches 55- to temperature
At 60 DEG C, 9-11h is kept the temperature, then compost is carried out to 4-5 days by a definite date decomposed fermentation stages, finally obtaining water content is 65%-
70% culture base-material, the solid state substrate as culture bamboo parasitic fungus.
Seed culture: being washed down the spore of bamboo parasitic fungus with physiological saline, is accessed in liquid seed culture medium, 26 DEG C of cultures
40h；Liquid seed culture medium includes following component: glucose 2g/L, dipotassium hydrogen phosphate 0.1g/L, anhydrous magnesium sulfate 0.05g/
L, beef extract 0.3g/L, pH are natural.
Fermented and cultured is carried out in seed liquor access solid medium, cultivation temperature is 26 DEG C ~ 30 DEG C, hypocrellin is produced,
The content of hypocrellin A is measured after 168h, fermentation ends, main conversion product is hypocrellin A, yield 8.5mg/ in fermentation liquid
kg。
Hypocrellin A is measured with the following method in above embodiments:
It first determines the standard curve of hypocrellin A: accurately weighing hypocrellin A mark product 3.6mg, be placed in 10ml volumetric flask
In, scale is dissolved and is settled to dehydrated alcohol, and obtaining standard solution concentration is 0.36mg/ml, is diluted to one with dehydrated alcohol
Series of concentrations (10,20,30,50,100,120,180,240, unit: μ g/ml), a series of mark product solution of various concentrations is existed
Absorbance is surveyed at 464nm, using hypocrellin concentration as abscissa, absorbance is ordinate, carries out recurrence calculating, obtains the red bacterium of bamboo
Plain standard regressive method is y=0.0403x-0.0283, R2=0.9997.
The measurement of hypocrellin content: solid medium is put into drying box drying, is crushed with food processor, takes 0.1g
Crushed material mixed with 50ml dehydrated alcohol, stirring and leaching 2h takes leaching liquor high speed centrifugation, takes supernatant after extraction, obtain clear
Clear pigment solution measures trap using spectrophotometer method after diluting several times with dehydrated alcohol at 464nm wavelength,
The content of hypocrellin is calculated according to standard regressive method.
Claims (6)
1. one plant of bamboo parasitic fungus, classification naming be tabasheer (Shiraia bambusicola) NJTECH-1, it has been preserved in China
Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC NO.11617.
2. application of the bamboo parasitic fungus described in claim 1 in fermenting and producing hypocrellin.
3. application according to claim 2, it is characterised in that include the following steps:
1) seed culture: being washed down the spore of bamboo parasitic fungus with physiological saline, is accessed in liquid seed culture medium, 24 ~ 30 DEG C of cultures
40~48h；
2) it takes the seed liquor of step 1) to access in solid medium and carries out fermented and cultured, cultivation temperature is 26 DEG C ~ 30 DEG C, produces bamboo
Red rhzomorph.
4. application according to claim 3, which is characterized in that liquid seed culture medium described in step 1) includes following matter
The component of percentage: carbon source 2% ~ 3%, nitrogen source 0.2% ~ 0.4%, inorganic salts 0.05% ~ 0.1% are measured, wherein the carbon source is selected from grape
At least one of sugar, sucrose, fructose, lactose, soluble starch, rice, corn；The nitrogen source be selected from beef extract, peptone,
At least one of yeast extract, sodium nitrate, ammonium sulfate, the inorganic salts be selected from dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate,
At least one of magnesium phosphate.
5. application according to claim 3, which is characterized in that solid medium described in step 2 is with weeds, corn
At least one of stalk, fowl and animal excrement, gypsum or dregs of beans are raw material, and fermentation obtains the culture medium that water content is 65%-70%
Material.
6. application according to claim 3, which is characterized in that solid medium described in step 2 includes following quality hundred
Divide the component of ratio: corn flour 68.9% ~ 75%, wheat bran 5% ~ 10%, rice bran 15% ~ 20%, glucose 1% ~ 3%, KH2PO4 0.1%~
0.2% ,MgSO4·7H2O 0.05%~0.07%。
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| CN110172409B (en) * | 2019-04-26 | 2020-04-28 | 华南理工大学 | Shiraia bambusicola strain capable of highly producing hypocrellin A and application thereof |
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| CN101168728A (en) * | 2007-09-30 | 2008-04-30 | 江南大学 | Shiraia strain for perylene producing quinone compound |
| CN102703328A (en) * | 2012-04-27 | 2012-10-03 | 苏州维因生物科技有限公司 | Hypocrellin high-yielding Shiraia bambusicola strain |
| CN104277990A (en) * | 2014-09-25 | 2015-01-14 | 南京工业大学 | Saccharomyces cerevisiae and application thereof in fermentation production of fuel ethanol |
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| CN101168728A (en) * | 2007-09-30 | 2008-04-30 | 江南大学 | Shiraia strain for perylene producing quinone compound |
| CN102703328A (en) * | 2012-04-27 | 2012-10-03 | 苏州维因生物科技有限公司 | Hypocrellin high-yielding Shiraia bambusicola strain |
| CN104277990A (en) * | 2014-09-25 | 2015-01-14 | 南京工业大学 | Saccharomyces cerevisiae and application thereof in fermentation production of fuel ethanol |
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