CN102181488A - Method for preparing active phenolic compounds through rosin-induced bioconversion - Google Patents
Method for preparing active phenolic compounds through rosin-induced bioconversion Download PDFInfo
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- CN102181488A CN102181488A CN2011100597216A CN201110059721A CN102181488A CN 102181488 A CN102181488 A CN 102181488A CN 2011100597216 A CN2011100597216 A CN 2011100597216A CN 201110059721 A CN201110059721 A CN 201110059721A CN 102181488 A CN102181488 A CN 102181488A
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- KHPCPRHQVVSZAH-UHFFFAOYSA-N trans-cinnamyl beta-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OCC=CC1=CC=CC=C1 KHPCPRHQVVSZAH-UHFFFAOYSA-N 0.000 title claims abstract description 37
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Abstract
The invention provides a method for preparing active phenolic compounds through rosin-induced bioconversion. The method comprises the following steps: performing the routine culture of the microbial strain A5<+> in potato dextrose agar (PDA) culture medium to obtain the activated strain, inoculating the activated strain in the PDA culture medium to ferment for 48-72 hours; adding the inducer in the fermented strain to perform bioconversion; filtering and concentrating the conversion fluid to obtain conversion product crude extract; performing gradient alcohol precipitation on the conversion product crude extract, extracting with petroleum ether and ethyl acetate, finally separating and purifying the product through thin layer chromatography, silica gel column chromatography LH-20 gel chromatography and high performance liquid chromatography to obtain O,O-1,1'-di(2-hydroxyl-3,4,5-trimethyl)phenyl oxo bridge and 4,5,6-trimethyl-1,2-pyrocatechol. The bioconversion method has the advantages of high efficiency and selectivity and mild reaction conditions, and can hardly cause environmental pollution; the cost of the method is lower than the extraction method, thus the cost can be saved; and the obtained phenolic compounds have the functions of antioxidant property, antibacterial property, antiviral property, anticancer property, radiation resistance and antiallergic property.
Description
Affiliated field
The present invention relates to rosin and induce bio-transformation to prepare the method for active phenolic compound, belong to fine chemistry industry and pharmacy biological technical field.
Background technology
Rosin is the transparent solid of little Huang to yellowish red color, and is water insoluble, but be soluble in organic solvent, as acetone, ether, ethanol, vinyl acetic monomer, Virahol, turps, benzene and dimethylbenzene etc.The rosin chemical property: rosin acid contains double-stranded and carboxyl active gene, has conjugated double bond and typical carboxyl reaction.Rosin also has disproportionation, hydrogenation, addition, the two key reactions of polymeric except itself being easy to oxidation and isomerization reaction.Also have simultaneously esterification, alcoholization, salify, decarboxylation, ammonia and carboxyl reaction such as separate.Rosin secondary reprocessing just has the characteristic of two keys and carboxyl reaction based on rosin, with in addition modification of rosin, generate a series of modified rosins, has improved rosin use value.
Rosin is a kind of natural reproducible resource of important value, and world's annual production at present is more than 100 ten thousand tons, wherein, 600,000 tons of China's annual production, based on outlet raw material rosin, be the first in the world big rosin producing country and raw material rosin export State more than 70%, the rosiny utilization ratio only is 35%.And what beat up the outlet of developed country such as U.S. is the rosiny deep processed product mostly, and the rosin utilization ratio is almost near 100%.China's rosin resource faces serious waste and the backward situation of deep process technology, needs to be resolved hurrily.The rosin derivative of preparation high added value has become the research focus at present.
Yunnan Province has abundant rosin resource, is the second largest producing region of China's rosiny, and rosin output reaches 200,000 tons, and product mainly is rosin, 2 kinds of elementary processed goods of turps, and deep processed product does not have substantially.Output has got on, and the output value is still lower.Walk deep processing, prolong product chain, increasing value-added content of product is the only way that Yunnan rosin industry future realizes Sustainable development.
Bio-transformation (biotransformation) claims biocatalysis (biocatalysis) again, be meant that (enzyme and multienzyme system comprise microorganism to the applying biological reactor, animal and plant cells etc.) precursor compound is carried out structural modification and transformation, the organic compound of synthesizing new.Its essence is that the enzyme that utilizes living things system itself to be produced carries out enzymic catalytic reaction to xenobiontics; the catalyzed reaction type almost comprises all external organic chemical reactionses, as: hydroxylation, oxidation, dehydrogenation, hydrogenation, reduction, hydrolysis, hydration, esterification, transesterify, dehydration, decarboxylation, acidylate, amination, isomerization and aromizing etc.Bio-transformation is to carry out in room temperature or neutral environment mostly, has advantages such as nontoxic, pollution-free, less energy-consumption, high-level efficiency, highly selective.The living things system that is used for Study on Transformation mainly contains fungi, bacterium, algae, plant suspension cell, tissue or organ and zooblast, tissue etc., and wherein using maximum is vegetable cell suspension culture system and microorganism system.
Along with going deep into and expansion of bio-transformation research, the range of application of bio-transformation is also more and more wider, not only in medicine, foodstuffs industry, has also obtained using widely in departments such as fuel, makeup, Industrial Wastewater Treatment and agricultural chemical insecticides.What wherein research the widest maximum application were the most general is exactly the application of bio-transformation in pharmaceutical industry.Based on the characteristics of bio-transformation and the history of China's drug research, the bio-transformation research of aspect such as bio-transformation, active skull cap components and the lead compound of pharmaceutically active ingredient and chiral drug, chiral intermediate and medicine be synthetic in mainly concentrating on.
According to bibliographical information, at present, phenolic compound mainly is that separation and Extraction and chemical process are synthetic from natural plant, and the report of producing phenolic compound by biotransformation method seldom, because the plant extract method is subjected to season limit and the not high factor affecting of extracted amount, the chemical method step is various and can cause serious environmental to pollute, by comparison, biotransformation method is because of its high efficiency, the reaction that highly selective makes some chemical method be difficult to finish can realize with biotransformation method even once going on foot, the more important thing is, this method can not brought pollution to environment, and based on these advantages, biotransformation method more and more receives publicity.From
Eurotium chevalieriIsolate phenoloid flaveglaucin in the mycelium, be added to 0.05 addition and show excellent oxidation-resistance in the vegetables oil.People such as Ma Guilei have studied the drug metabolism study of 7 strain filamentous funguss conversion osalmides (diphenols), find, have only 4 strain bacterial strain (cunninghamella eleganses of Cunninghammella
Cunninghamella elegansAS 3. 156); Cunninghamella elegans
C. elegansAS3. 2028); Cunninghamella echinulata
C. echinulataAS3. 2004); Cunninghamella blakesleana C. blacksleana AS3. 153) osalmide is had very strong conversion capability, its conversion capability not only with the kind of bacterial strain even relevant with a kind of different strains, but also closely related with the structure of substrate.The medicine that has phenolic hydroxyl structure according to statistics can obtain two-phase drug metabolism binding substancess such as glucose binding substances and sulfuric acid fine wine binding substances by microbial transformation; Up to the present, related microorganism report 2 examples only that form glucuronide conjugate.
The activity of phenolic compound mainly contains oxidation-resistance, antibacterial, antiviral, anticancer, radioprotective and antianaphylaxis etc., and relevant study on mechanism is also more deep, and wherein the research of the biological activity of tea-polyphenol and the mechanism of action thereof is a lot.The anticancer research of phenolic compound is focus day by day.
Summary of the invention
The present invention relates to a strain and from the pine forest soil ulmin of Yunnan, screen the Mucoraceae that obtains
UmbelopsisBelong to A5+ bacterial strain and cultivation and fermentation optimal conditions thereof, and be used for rosin and induce bio-transformation to prepare active phenolic compound O, O-1,1 '-two (2-hydroxyl-3,4, the 5-trimethylammonium) phenyl oxo bridge and 4,5,6-trimethylammonium-1, the method for 2-pyrocatechol realizes by following technical proposal.
The object of the present invention is to provide a kind of rosin to induce the method for producing active phenolic compound, it is characterized in that through following each step:
A. microorganism strains A5+ is carried out routine and cultivate in the PDA substratum, obtain the activation bacterial strain, insert the 48～72h that ferments in the PDB substratum again;
B. prepare the inductor of converted product;
C. the inductor of being prepared among the step B is added the bacterial strain after the fermentation in the steps A, carry out bio-transformation;
D. the conversion fluid with C carries out filtering and concentrating, obtains converted product crude extract medicinal extract;
E. converted product crude extract medicinal extract carries out the gradient alcohol precipitation with 50～90% dehydrated alcohol, and ethanol concentrates mutually; Use petroleum ether extraction and then, water concentrates; Then use ethyl acetate multi-stage solvent extraction 3 times, ethyl acetate concentrates mutually; Obtaining powder compound SX-1 through thin-layer chromatography, silica gel column chromatography LH-20 gel chromatography and high performance liquid chromatography separation and purification at last is O, O-1,1 '-two (2-hydroxyl-3,4, the 5-trimethylammonium) phenyl oxo bridge and crystalline compounds SX-2 are 4,5,6-trimethylammonium-1,2-pyrocatechol.
O, O-1,1 '-two (2-hydroxyl-3,4,5-trimethylammonium) phenyl oxo bridge
4,5,6-trimethylammonium-1,2-pyrocatechol
The agar of glucose+1.5%～2% that described PDA substratum is potato+20g/L of 200g/L, pH value are 6, in 110～125 ℃ of following autoclaving 15～30min.
Described PDB substratum is the glucose of potato+20g/L of 200g/L, and the pH value is 6, in 110～125 ℃ of following autoclaving 15～30min.
Described inductor adds rosin 0.1～0.3g in the PDA substratum by every 200mL, ethanol 5～8mL sampling of solubility promoter 95%, and the solution that rosin fully is dissolved in gained in the ethanol is inductor.
The condition of described bio-transformation is reacted 72～96h for being under 120～128rpm in 26～30 ℃ of temperature, stir speed (S.S.).
The activation bacterial strain of described microorganism strains A5+, it is the bacterial strain that separation and purification obtains from the pine forest soil ulmin of Simao, Yunnan, earlier on potato culture, cultivate 2d with 28 ℃ of stationary temperatures, colony diameter 10mm can reach 23mm after cultivating 4d again, reach 42mm after cultivating 7d, bacterium colony is rounded gradually, neat in edge, concentric wheel stripe shape, mycelia elder generation white then turns greyish white to black gradually, fine and close fasciculation, the back side is colourless, by microscopic examination, the transparent nothing of mycelium every, longer, divide dendritic, many to life, single green-ball shape sporocyst is directly born on the branch top, produce a large amount of spherical in the capsule, wall is thin, slick sporangiospore, sporocyst promptly breaks and discharges spore after the spore maturation, belongs to Mucoraceae strain morphology feature.
Described microorganism strains A5+ passes through extracting genome DNA, pcr amplification and sequencing bacterial strain kind:
(1) extracting genome DNA:
1. mycelium is put into aseptic mortar, is poured into liquid nitrogen then, treat that moisture content loses fully after, add a small amount of quartz sand, be ground to powder rapidly, rapidly pulverous material is changed in the centrifuge tube of 2 mL then.2. in centrifuge tube, add rapidly 600 μ L FG1, violent mixing, water-bath 10 min then, twice of vibration mixing.3. add 140 μ L FG2, vibration mixing, centrifugal 10min(revolution 12000 rpm).4. suck supernatant to new centrifuge tube, add 490 μ L Virahol mixings, be settled out DNA, rapidly centrifugal 2 min(revolutions, 10000 rpm).5. abandon supernatant, and centrifuge tube is inverted on the paper, the sucking-off residual liquid.6. add 300 μ L aseptic deionized waters (being heated to 65 in advance), resuspended precipitation adds 4 μ L RNaseA, and mixing adds 150 μ L FG3 then, and 300 uL dehydrated alcohols are inverted.Centrifugal 1 min abandons supernatant, and all samples are transferred in the connecting pipe.7. pillar is changed in another collection tube, adds 700 μ L rinsing liquids, centrifugal 1 min(revolution, 10000 rpm), abandon liquid.8. adding 700 μ L rinsing liquids, centrifugal 2 min(revolutions are 12000 rpm).9. pillar changes 1.5 mL centrifuge tubes over to, adds 50～100 μ L dissolving damping fluid (water-bath to 65 in advance ℃) room temperature and places 3～5 min, centrifugal 3～5 min.10. 50 μ L dissolve damping fluid wash-out dissolving DNA once more, and repeat 10 step.
(2) pcr amplification and sequencing:
With the amplification ITS of fungi ribosomal gene transcribed spacer universal primer ITS 5(5 '-GGAAGTAAAAGTCGTAACAAGG) and ITS 4(5 '-TCCTCCGCTTATTGATATGC).Be reflected in the 50 μ L systems and carry out, system contains: 2 μ L dNTP(2.5 mmol/L), 0.28 μ L Taq polysaccharase (5 U/ μ L), 30 ng genomic dnas, 1 μ L primer ITS 5(28 μ M/L), 1 μ L primer ITS 4(28 μ M/L), 5 μ L Ta buffer(* 10 contain Mg
2+) and dd H
2O.Response procedures is: 94
oPre-sex change 4 min of C; Enter circulation, 94
oC sex change 1 min, 58
oThe C 1min that anneals, 72
OCExtend 1.5 min, 35 circulations; Last 72
oC extends 7 min, 4
OCPreserve.Get 8 μ L reaction product electrophoresis in the gel electrophoresis of 1% agarose sugar, under ultraviolet transilluminator, detect PCR product size.After observing the ITS band that is fit to size, reclaim test kit (Beijing hundred Tyke Bioisystech Co., Ltd) the remaining PCR product of purifying, submit the order-checking of Beijing three rich Bioisystech Co., Ltd to absorption pillar PCR product.The sequence results that order-checking obtains.Carry out Blast on software NCBI, ITS (intervening sequence district) homology of not naming the 23 S rDNA of kind of (retrieve sequence GQ241270) that the sequence of this bacterial strain and bacterial strain Umbelopsis belong to is up to 99%.
Combining form is identified, strains A 5+ is accredited as mucormycosis subphylum (Mucoromycotina), mucorales (Mucorales), Mucoraceae (Mucorales incertae sedis).
The advantage that the present invention possesses: 1) from the source of phenolic compound preparation, compare with synthetic the sending out of chemical process with traditional separation and Extraction from natural phant, the biotransformation method that the present invention uses can be realized with biotransformation method even once going on foot because of the reaction that its high efficiency, highly selective make some chemical method be difficult to finish, the more important thing is, this method reaction conditions gentleness can be brought pollution to environment hardly; 2) can carry out scale operation, not be subjected to seasonal effect; 3) lower than extraction method expense, save cost; 4) the selected strain of the present invention can bio-transformation prepares active phenolic compound
UmbelopsisThe A5+ bacterial strain, and be under the condition that inductor rosin exists, just to take place, this not only can be for realizing that the suitability for industrialized production of this type of active compound provides feasible theoretical foundation in the future, the exploitation that also can help to realize the rosin deep process technology provides new thinking, and the activity of gained phenolic compound has effects such as oxidation-resistance, antibacterial, antiviral, anticancer, radioprotective and antianaphylaxis.
Specific embodiment
Embodiment 1
A. microorganism strains A5+ is carried out routine and cultivate in the PDA substratum, obtain the activation bacterial strain, insert the 48h that ferments in the PDB substratum again; The PDA substratum is the agar of glucose+2% of potato+20g/L of 200g/L, and the pH value is 6, in 121 ℃ of following autoclaving 15min; The PDB substratum is the glucose of potato+20g/L of 200g/L, and the pH value is 6, in 121 ℃ of following autoclaving 15min;
B. prepare the inductor of converted product; Inductor adds rosin 0.1g in the PDA substratum by every 200mL, the ethanol 5mL sampling of solubility promoter 95%, and the solution that rosin fully is dissolved in gained in the ethanol is inductor;
C. the inductor of being prepared among the step B being added the bacterial strain after the fermentation in the steps A, is under the 120rpm in 30 ℃ of temperature, stir speed (S.S.), and reaction 96h carries out bio-transformation;
D. the conversion fluid with C carries out filtering and concentrating, obtains converted product crude extract medicinal extract;
E. converted product crude extract medicinal extract carries out the gradient alcohol precipitation with 50% dehydrated alcohol, and ethanol concentrates mutually; Use petroleum ether extraction and then, water concentrates; Then use ethyl acetate multi-stage solvent extraction 3 times, ethyl acetate concentrates mutually; Obtaining powder compound SX-1 through thin-layer chromatography, silica gel column chromatography LH-20 gel chromatography and high performance liquid chromatography separation and purification at last is O, O-1,1 '-two (2-hydroxyl-3,4, the 5-trimethylammonium) phenyl oxo bridge and crystalline compounds SX-2 are 4,5,6-trimethylammonium-1,2-pyrocatechol.
Embodiment 2
A. microorganism strains A5+ is carried out routine and cultivate in the PDA substratum, obtain the activation bacterial strain, insert the 72h that ferments in the PDB substratum again; The PDA substratum is the agar of glucose+1.5% of potato+20g/L of 200g/L, and the pH value is 6, in 110 ℃ of following autoclaving 20min; The PDB substratum is the glucose of potato+20g/L of 200g/L, and the pH value is 6, in 110 ℃ of following autoclaving 30min;
B. prepare the inductor of converted product; Inductor adds rosin 0.2g in the PDA substratum by every 200mL, the ethanol 8mL sampling of solubility promoter 95%, and the solution that rosin fully is dissolved in gained in the ethanol is inductor;
C. the inductor of being prepared among the step B being added the bacterial strain after the fermentation in the steps A, is under the 128rpm in 26 ℃ of temperature, stir speed (S.S.), and reaction 72h carries out bio-transformation;
D. the conversion fluid with C carries out filtering and concentrating, obtains converted product crude extract medicinal extract;
E. converted product crude extract medicinal extract carries out the gradient alcohol precipitation with 70% dehydrated alcohol, and ethanol concentrates mutually; Use petroleum ether extraction and then, water concentrates; Then use ethyl acetate multi-stage solvent extraction 3 times, ethyl acetate concentrates mutually; Obtaining powder compound SX-1 through thin-layer chromatography, silica gel column chromatography LH-20 gel chromatography and high performance liquid chromatography separation and purification at last is O, O-1,1 '-two (2-hydroxyl-3,4, the 5-trimethylammonium) phenyl oxo bridge and crystalline compounds SX-2 are 4,5,6-trimethylammonium-1,2-pyrocatechol.
Embodiment 3
A. microorganism strains A5+ is carried out routine and cultivate in the PDA substratum, obtain the activation bacterial strain, insert the 60h that ferments in the PDB substratum again; The PDA substratum is the agar of glucose+1.8% of potato+20g/L of 200g/L, and the pH value is 6, in 125 ℃ of following autoclaving 30min; The PDB substratum is the glucose of potato+20g/L of 200g/L, and the pH value is 6, in 125 ℃ of following autoclaving 20min;
B. prepare the inductor of converted product; Inductor adds rosin 0.3g in the PDA substratum by every 200mL, the ethanol 6mL sampling of solubility promoter 95%, and the solution that rosin fully is dissolved in gained in the ethanol is inductor;
C. the inductor of being prepared among the step B being added the bacterial strain after the fermentation in the steps A, is under the 125rpm in 29 ℃ of temperature, stir speed (S.S.), and reaction 80h carries out bio-transformation;
D. the conversion fluid with C carries out filtering and concentrating, obtains converted product crude extract medicinal extract;
E. converted product crude extract medicinal extract carries out the gradient alcohol precipitation with 90% dehydrated alcohol, and ethanol concentrates mutually; Use petroleum ether extraction and then, water concentrates; Then use ethyl acetate multi-stage solvent extraction 3 times, ethyl acetate concentrates mutually; Obtaining powder compound SX-1 through thin-layer chromatography, silica gel column chromatography LH-20 gel chromatography and high performance liquid chromatography separation and purification at last is O, O-1,1 '-two (2-hydroxyl-3,4, the 5-trimethylammonium) phenyl oxo bridge and crystalline compounds SX-2 are 4,5,6-trimethylammonium-1,2-pyrocatechol.
The bacteriostatic activity of two compounds detects: select the phenolic compound of embodiment 1 gained for use, respectively intestinal bacteria, subtilis, Bacillus cereus, bacillus megaterium and streptococcus aureus are suppressed experiment.
Adopt K-B(filter paper diffusion process) method.Prepare the aseptic filter paper sheet with punch tool, diameter is 6 mm.1.5 the mL centrifuge tube is soaked overnight in 75% alcohol, takes out oven dry.20 filter papers are placed centrifuge tube, add the compound S X-1 be dissolved in the methyl alcohol and the solution of SX-2, concentration is 1 mg/mL, soak 1 h after, take out piecewise and place sterile petri dish, in 40 ° of baking ovens, dry.
Indicator is cultivated (microbial culture 8 h) behind the certain hour, adjust bacterial concentration with sterilized water and reach standard reduced turbidity 0.5 Maxwell (BaSO4 solution is at 625 nm places, and light absorption value is 0.08～0.1), obtain normal concentration indicator suspension.
Aseptic cotton carrier dips in indicator suspension evenly coating on flat board of getting a certain amount of normal concentration.Rotating Plates 60 degree are constantly proceeded coating during operation, and it is even to guarantee to be coated with bacterium.The filter paper dispersion of oven dry is affixed on the plate that is coated with indicator, places incubator to cultivate 24 h, observe, measure and the record antibacterial circle diameter, the result is as showing:
Claims (6)
1. a rosin is induced the method for producing active phenolic compound, it is characterized in that through following each step:
A. microorganism strains A5+ is carried out routine and cultivate in the PDA substratum, obtain the activation bacterial strain, insert the 48～72h that ferments in the PDB substratum again;
B. prepare the inductor of converted product;
C. the inductor of being prepared among the step B is added the bacterial strain after the fermentation in the steps A, carry out bio-transformation;
D. the conversion fluid with C carries out filtering and concentrating, obtains converted product crude extract medicinal extract;
E. converted product crude extract medicinal extract carries out the gradient alcohol precipitation with 50～90% dehydrated alcohol, and ethanol concentrates mutually; Use petroleum ether extraction and then, water concentrates; Then use ethyl acetate multi-stage solvent extraction 3 times, ethyl acetate concentrates mutually; Obtaining powder compound SX-1 through thin-layer chromatography, silica gel column chromatography LH-20 gel chromatography and high performance liquid chromatography separation and purification at last is O, O-1,1 '-two (2-hydroxyl-3,4, the 5-trimethylammonium) phenyl oxo bridge and crystalline compounds SX-2 are 4,5,6-trimethylammonium-1,2-pyrocatechol.
2. method according to claim 1 is characterized in that: the agar of glucose+1.5%～2% that described PDA substratum is potato+20g/L of 200g/L, pH value are 6, in 110～125 ℃ of following autoclaving 15～30min.
3. method according to claim 1 is characterized in that: described PDB substratum is the glucose of potato+20g/L of 200g/L, and the pH value is 6, in 110～125 ℃ of following autoclaving 15～30min.
4. method according to claim 1 is characterized in that: described inductor adds rosin 0.1～0.3g in the PDA substratum by every 200mL, ethanol 5～8mL sampling of solubility promoter 95%, and the solution that rosin fully is dissolved in gained in the ethanol is inductor.
5. method according to claim 1 is characterized in that: the condition of described bio-transformation is reacted 72～96h for being under 120～128rpm in 26～30 ℃ of temperature, stir speed (S.S.).
6. method according to claim 1 is characterized in that: described microorganism strains A5+ is mucormycosis subphylum (Mucoromycotina), mucorales (Mucorales), Mucoraceae (Mucorales incertae sedis).
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105076220A (en) * | 2014-05-15 | 2015-11-25 | 钦州学院 | Mycelium extract of strain culture of marine fungus umbelopsis sp. |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105076220A (en) * | 2014-05-15 | 2015-11-25 | 钦州学院 | Mycelium extract of strain culture of marine fungus umbelopsis sp. |
| CN105087397A (en) * | 2014-05-15 | 2015-11-25 | 钦州学院 | Application of mycelium extract of strain culture of marine fungus umbelopsis sp. in preparation of antibacterial drugs |
| CN105087396A (en) * | 2014-05-15 | 2015-11-25 | 钦州学院 | Preparation method of mycelium extract of strain culture of marine fungus umbelopsis sp. |
| CN105168265A (en) * | 2014-05-15 | 2015-12-23 | 钦州学院 | Application of mycelium extract of umbelopsis sp. strain culture in preparing antifungal drugs |
| CN105076220B (en) * | 2014-05-15 | 2017-11-03 | 钦州学院 | A kind of hypha extract of marine fungi umbrella branch trichoderma strain culture |
| CN105087396B (en) * | 2014-05-15 | 2018-02-02 | 钦州学院 | A kind of preparation method of the hypha extract of marine fungi umbrella branch trichoderma strain culture |
| CN105087397B (en) * | 2014-05-15 | 2018-02-02 | 钦州学院 | A kind of application of hypha extract of marine fungi umbrella branch trichoderma strain culture in anti-bacterial drug is prepared |
| CN105168265B (en) * | 2014-05-15 | 2019-02-15 | 钦州学院 | A kind of hypha extract of marine fungi umbrella branch trichoderma strain culture is preparing the application in antifungal drug |
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