CN104087514B - Excellent aspergillosis and the application thereof of feruloyl esterase is produced in one strain - Google Patents
Excellent aspergillosis and the application thereof of feruloyl esterase is produced in one strain Download PDFInfo
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- CN104087514B CN104087514B CN201410299853.XA CN201410299853A CN104087514B CN 104087514 B CN104087514 B CN 104087514B CN 201410299853 A CN201410299853 A CN 201410299853A CN 104087514 B CN104087514 B CN 104087514B
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- aspergillosis
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- feruloyl esterase
- ferulic acid
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- 201000002909 Aspergillosis Diseases 0.000 title claims abstract description 52
- 208000036641 Aspergillus infections Diseases 0.000 title claims abstract description 52
- 101001065065 Aspergillus awamori Feruloyl esterase A Proteins 0.000 title claims abstract description 19
- 238000000855 fermentation Methods 0.000 claims abstract description 36
- 230000004151 fermentation Effects 0.000 claims abstract description 36
- 235000001785 ferulic acid Nutrition 0.000 claims abstract description 36
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 claims abstract description 35
- 229940114124 ferulic acid Drugs 0.000 claims abstract description 35
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 claims abstract description 35
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 claims abstract description 35
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims abstract description 32
- 235000019504 cigarettes Nutrition 0.000 claims abstract description 22
- 241000228193 Aspergillus clavatus Species 0.000 claims abstract description 17
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 12
- 239000000284 extract Substances 0.000 claims description 5
- 239000002054 inoculum Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 2
- 239000012879 subculture medium Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 14
- 230000001580 bacterial effect Effects 0.000 abstract description 13
- 150000007965 phenolic acids Chemical class 0.000 abstract description 7
- 238000010170 biological method Methods 0.000 abstract description 4
- 210000002421 cell wall Anatomy 0.000 abstract description 4
- 150000004676 glycans Chemical class 0.000 abstract description 4
- 229920001282 polysaccharide Polymers 0.000 abstract description 4
- 239000005017 polysaccharide Substances 0.000 abstract description 4
- 239000000758 substrate Substances 0.000 abstract description 4
- -1 ferulic acid ester Chemical class 0.000 abstract description 3
- 229920005610 lignin Polymers 0.000 abstract description 3
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract description 2
- 230000002255 enzymatic effect Effects 0.000 abstract 1
- 235000009048 phenolic acids Nutrition 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 20
- 230000001954 sterilising effect Effects 0.000 description 11
- 235000015099 wheat brans Nutrition 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 229920001817 Agar Polymers 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- 150000001299 aldehydes Chemical class 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
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- 102000004190 Enzymes Human genes 0.000 description 7
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 239000002253 acid Substances 0.000 description 6
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- 238000012216 screening Methods 0.000 description 5
- 238000011218 seed culture Methods 0.000 description 5
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- 239000002775 capsule Substances 0.000 description 4
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
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- ATJVZXXHKSYELS-FNORWQNLSA-N ethyl (e)-3-(4-hydroxy-3-methoxyphenyl)prop-2-enoate Chemical compound CCOC(=O)\C=C\C1=CC=C(O)C(OC)=C1 ATJVZXXHKSYELS-FNORWQNLSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- ATJVZXXHKSYELS-UHFFFAOYSA-N ferulic acid ethyl ester Natural products CCOC(=O)C=CC1=CC=C(O)C(OC)=C1 ATJVZXXHKSYELS-UHFFFAOYSA-N 0.000 description 3
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- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 description 2
- NGSWKAQJJWESNS-ZZXKWVIFSA-M 4-Hydroxycinnamate Natural products OC1=CC=C(\C=C\C([O-])=O)C=C1 NGSWKAQJJWESNS-ZZXKWVIFSA-M 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
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- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
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- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 description 2
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- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
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Abstract
The present invention provides a strain to produce the excellent aspergillosis of feruloyl esterase and application thereof, the Classification And Nomenclature of described rod aspergillosis be excellent aspergillosis (Aspergillus clavatus) cigarette is blue or green, by China typical culture collection center preservation, is called for short CCTCC, deposit number is CCTCC NO:M2013641, and preservation date is December in 2013 8, and rod aspergillosis is applied to produce feruloyl esterase.Bacterial strain of the present invention carrys out fermenting and producing feruloyl esterase with Testa Tritici for fermentation substrate, and with the ester bond between ferulic acid ester enzymatic breaking plant cell wall polysaccharides and lignin and ferulic acid, the method obtaining bound form phenolic acid ferulic acid, and utilize the growth of microorganism cycle short, climate does not limits, it is easily achieved large-scale production, provides new biological method for bound form phenolic acids such as ferulic acids.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a strain and produce excellent aspergillosis and the application thereof of feruloyl esterase.
Background technology
Semen Tritici aestivi is one of most commonly used crop of cultivated area in the world, accounts for the 26% of plant of grain crops area, China
Semen Tritici aestivi annual production is high, the annual production of Testa Tritici up to more than 20,000,000 tons, but all the time for the exploitation of wheat bran
Less, the most only to be used as feedstuff.Testa Tritici mainly contains non-starch polysaccharides(nsp), starch, fat,
The Multiple components such as lignin, protein, vitamin and aldehydes matter, wherein the non-starch polysaccharides(nsp) based on araboxylan contains
46%, it is the main component of Wheat cell wall.Possibly together with a number of phenolic acid in wheat bran, such as ferulic acid (ferulic
Acid), P-coumaric acid (p-coumaricacid), caffeic acid (caffeicacid), sinapic acid (sinapicacid) and Flos Caryophylli
The compositions such as acid (syringicacid), wherein with ferulic acid as main component, about the 0.4%～0.7% of wheat bran quality.Ferulic acid
Mainly by ester bond and cell wall polysaccharides and lignin necklace, highly basic typically can be used to be disconnected by ester bond and discharge.
In phenolic acid group Testa Tritici to be present in cortex, it has stronger antioxidant activity and antitumor action, and to environment
In noxious substance such as polycyclic aromatic hydrocarbon and inferior ammonium nitrate and mycotoxin have antimutagenesis.Studies have reported that discovery, in wheat
In the isolated fermentation test of bran dietary fiber, faecal flora can be incorporated on wheat-bran dietary fiber by Fermentation Ah
Wei, acid discharged.And ferulic acid can significantly reduce the survival ability of human cervical carcinoma cell, survival and antioxidant levels, simultaneously
Improve the reactive oxygen species in cancerous cell is protected and increase lipid peroxidation and the DNA damage effect of cancerous cell.Separately have been reported that and think
Ferulic acid can be by VEGF (VEGF), platelet derived growth factor (PDGF) and hypoxia inducible factor
(HIF) effect regulates the formation of blood vessel.
At present, research shows that aldehydes matter is mainly present in plant with several forms such as sequestered, conjunction type and bound forms
In body, in general, free property and conjunction type aldehydes matter are solubility phenols；Bound form is then insoluble aldehydes matter,
Combine with other materials (including protein, saccharide, organic acid etc.) with ester bond, glycosidic bond and ether glycosidic bond form.
For the extraction of aldehydes matter, general to use organic solvent be medium, is aided with ultrasonic, microwave, the method such as subcritical
Carry out.Owing to bound form aldehydes matter is typically connected with different of bondings from other materials, traditional extracting method is not enough to
By ester bond, glycosidic bond or ether bond rupture, therefore the extraction to bound form aldehydes matter mainly uses strong acid and strong base or biological method
(enzyme process and fermentation method), its main purpose is all to destroy the structure that cell wall fiber composition is fine and close, so that constraint row aldehydes matter
Discharge.
Although chemical method is simple to operate, efficiency is high, but follow-up needs process and extract waste liquid, are unfavorable for that sustainability is sent out
Exhibition.Therefore compare, use biological method more to meet the demand for development of green, environmental protection.Fermentation aspect is carried out for Testa Tritici
Focus mostly in the field such as xylooligosaccharides production and inulinase-producing activity, the most lot of documents report Testa Tritici fermented after wait until meals
Food fiber functional character greatly improves, such as retentiveness, holding oiliness and the binding ability etc. to harmful substance than before fermentation.
Ferulic acid, as the main phenolic acid in Testa Tritici, is mainly enzyme process to the mode that it obtains, at present with fermentation mode
Report less, it is accordingly required in particular to it is noted that what the form of the substantially suitable bound form of ferulic acid in Testa Tritici existed, its sequestered:
Conjunction type: the ratio of bound form is 0.1:1:100.In recent years, the research to aldehydes matter existence form is increasingly becoming new grinding
Study carefully focus.At present to some sequestereds, esterification type, glucoside type, the extraction of bound form aldehydes matter, mensuration and antioxidant activity
Etc. the report of aspect.The existence shape of aldehydes matter has important relationship with its content and antioxidant activity, and bound form aldehydes matter is not
Only content is significantly larger than sequestered and conjunction type, and antioxidant activity and suppression DNA damage aspect are higher than other two kinds
Type.
Need it is contemplated that utilize microbial resources to use fermentation engineering that agricultural resource converts formation human society
The material asked, the problem solving antioxidant shortage of resources.
Summary of the invention
The technical problem to be solved is: the new raw material of offer supplied for existing antioxidation and method, and
Large plants such as Semen Tritici aestivi are recycled, it is provided that excellent aspergillosis and application, its available Semen Tritici aestivi of feruloyl esterase is produced in a strain
Wheat bran fermenting and producing ferulic acid, owing to the growth of microorganism cycle is short, not climate restriction, it is easy to accomplish large-scale production, thus
Solve the problems such as ferulic acid is not enough.
The excellent aspergillosis of feruloyl esterase is produced in the strain that the present invention provides, and derives from South China Botanical Garden soil, through screening, point
Obtain from purification.This its Classification And Nomenclature of rod aspergillosis is that rod aspergillosis (Aspergillus clavatus) cigarette is blue or green, by Chinese Typical Representative
The preservation of culture collection center, is called for short CCTCC, and deposit number is CCTCC NO:M2013641, its ITS sequence table such as sequence table 1
Shown in, preservation date is December in 2013 8, and preservation address is Wuhan, China Wuhan University, China typical culture collection
Center.
The flat board of gained rod aspergillosis (Aspergillus clavatus) cigarette of the present invention green grass or young crops observes feature and microscopical character
As follows: rod aspergillosis (Aspergillus clavatus) cigarette green grass or young crops colony growth rate on Czapek's medium is relatively slow, cultivates 7d straight
Footpath 30mm；Quality velvet shape is that dark blue is green, without transudate to granular, white mycelium, conidium structure.At beerwort
The agar colony speed of growth is very fast, cultivates 7d diameter 45mm, smooth, quality granular, and conidium structure is many, celadon,
Bacterium colony reverse side bronzing.On Shi yeast extract agar, bacterium colony cultivates 7d diameter 40mm, and quality velvet shape, bottle is the thickest；Mitogenetic spore
Minor structure is many, and in dusty blue, edge is diluter, white, without transudate.It is observed that during conidial head children be under microscope
Rod, conidiophore betides substrate, and falx stem is different in size；Top capsule is gradually expanded by falx top and becomes club shape, directly
Footpath 50～60mm, produce spore structure monolayer, bottle stalk intensive this be born in top capsule all surfaces, bottle stalk size be 8～12 μ m 2～
3μm;Conidium is oval, and conidium size is 4～5 μ m 3 μm, and wall is smooth.
ITS and the rod aspergillosis (Aspergillus that described rod aspergillosis (Aspergillus clavatus) cigarette is blue or green
Clavatus) homology reaches 100%.
The preparation method of a kind of excellent aspergillosis producing feruloyl esterase, the excellent aspergillosis of the present invention derives from South China Botanical Garden soil
Earth, uses different culture medium to screen, and screens multiple bacterial strain, carries out ferulaic acid content one by one and Oxford cup transparent circle is surveyed
Fixed experiment, excellent aspergillosis (Aspergillus clavatus) cigarette obtaining high yield feruloyl esterase is blue or green, comprises the steps: to weigh
Pedotheque 10g puts into and sways 18 in the 300mL triangular flask of the normal saline containing bead aseptic equipped with 90 ~ 100 mL ~
20min, makees 10 times of ascending series dilute samples even liquid 1:10,1:100,1:1000 and 1:10000, takes the even liquid of dilute sample
The each 1.0mL of 1:100,1:1000 and 1:10000 is added separately to sterilized petri dishes；Add 10 again～the mixing of 15mL PDA culture medium is equal
Even, stand 1 ~ 2 min, be inverted cultivation 70 ~ 72h in 28 DEG C ± 1 DEG C, bacterium colony single in PDA culture medium is transferred to inclined-plane
Cultivate 70 ~ 72h for 28 DEG C ± 1 DEG C in PDA culture medium, then proceed to primary dcreening operation culture medium, cultivate 58 ~ 60h in 28 DEG C ± 1 DEG C, use cattle
Tianjin cup transparent circle experiment, excellent aspergillosis (Aspergillus clavatus) cigarette that screening obtains producing feruloyl esterase is blue or green；Described soil
Earth sample gathers from South China Botanical Garden.
In said method, it is inoculated into the training of triangular flask seed by obtaining the excellent aspergillosis producing feruloyl esterase in primary dcreening operation culture medium
Support in base, cultivate 58 ~ 60h in 28 DEG C ± 1 DEG C, be rod aspergillosis seed；Again rod aspergillosis seed is inoculated in fermentation medium
Cultivate, for producing the extract of the excellent aspergillosis of ferulic acid.
Primary dcreening operation culture medium: FeSO4·7H2O 0.01g, NaNO32.0 g, KCl 0.5 g, MgSO4·7H2O 0.5 g,
K2HPO41.0g, sucrose 30 g, agar 20 g, distilled water 1000m L, pH value is natural；121 DEG C of sterilizing 20 min.Cultivate after sterilizing
Base is as cold as about the 60 DEG C dimethyl formamide solution 100mL adding 10% aseptic ferulic acid ethylester, shakes up to the milkyst white
Color is down flat plate.
Seed culture medium: FeSO4·7H2O 0.01g, NaNO32.0 g, KCl 0.5 g, MgSO4·7H2O 0.5
G, K2HPO41.0g, wheat bran 1000 g, distilled water 1000m L, pH value is natural.121 DEG C of sterilizing 20 min.
PDA culture medium: PDA(potato agar) 40g, it is settled to 1L, pH natural.121 DEG C of insulated sterilizing 20min.
Fermentation medium: Testa Tritici: water is weight ratio 1:1, pH value is natural.
It is a further object to provide the fermentation application producing ferulic acid that described rod aspergillosis cigarette is blue or green.Described rod is bent
Mould cigarette green grass or young crops is applied to produce feruloyl esterase.
Above-mentioned application, comprises the steps: to utilize Testa Tritici direct inoculation rod aspergillosis (Aspergillus clavatus) cigarette
Green grass or young crops, carries out koji plate fermentation, it is thus achieved that fermented product extract in fermentation medium, utilizes in high-efficient liquid phase color spectrometry fermentation medium
The ferulic acid burst size of Testa Tritici.
In above-mentioned application, the condition of described fermentation is: carry out solid fermentation on koji tray with Testa Tritici, and Testa Tritici THICKNESS CONTROL exists
In 2 ~ 3cm, rod aspergillosis seed inoculum concentration is that 0.5% (bacteria containing amount is 1～2 × 109Individual/g), cultivation temperature is 25～28 DEG C, enters
Row aerlbic culture, fermentation time is 48～55h；PH is natural.Described Testa Tritici is natural Testa Tritici.Described rod aspergillosis seed is bent by rod
Mould it is inoculated in triangular flask seed culture medium, cultivates 58 ~ 72h in 28 DEG C ± 1 DEG C and obtain.
Compared with prior art, the invention has the beneficial effects as follows:
The invention provides a kind of new excellent aspergillosis (Aspergillus clavatus) cigarette blue or green, described bacterial strain have product Ah
The ability of acid Wei esterase, supplements the information bank extracting bound form ferulic acid bacterium greatly；Described rod aspergillosis (Aspergillus
Clavatus) cigarette green grass or young crops is isolated and purified from biosphere out and is prone to cultivate and preserves, and rapidly, energy for growth is strong in breeding.
Rod aspergillosis of the present invention fermentation is produced the enzyme of feruloyl esterase and is lived the highest, can be as extracting bound form phenolic acid from plant
Biological bacteria.
The invention provides the blue or green bound form ferulic acid biology in plant of rod aspergillosis (Aspergillus clavatus) cigarette
The application of method, for release and the follow-up antioxidation engineering of bound form phenolic acid, it is provided that new biological method and strong skill
Art is supported.
Accompanying drawing explanation
Fig. 1 is the transparent loop graph of bacterial strain of the present invention and the ferulic acid ester enzyme activity of other bacterial strain fermentation liquors.
Fig. 2 is the blue or green 400 times of podocyte aspect graphs observed under the microscope of rod aspergillosis cigarette.
Fig. 3 is the blue or green 400 times of conidial head aspect graphs observed under the microscope of rod aspergillosis cigarette.
Fig. 4 is the blue or green bacterium colony figure in PDA cultivation of rod aspergillosis cigarette.
Fig. 5 is the chromatogram of the ferulic acid standard substance that HPLC measures.
Fig. 6 is the standard curve of the ferulic acid standard substance that HPLC measures.
Fig. 7 is rod aspergillosis fermentation chromatogram.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further described.
The culture medium prescription used in following example:
Primary dcreening operation culture medium: FeSO4·7H2O 0.01g, NaNO32.0 g, KCl 0.5 g, MgSO4·7H2O 0.5 g,
K2HPO41.0g, sucrose 30 g, agar 20 g, distilled water 1000m L, pH value is natural；121 DEG C of sterilizing 20 min.Cultivate after sterilizing
Base is as cold as about the 60 DEG C dimethyl formamide solution 100mL adding 10% aseptic ferulic acid ethylester, shakes up to the milkyst white
Color is down flat plate.
Seed culture medium: FeSO4·7H20 0.01g, NaNO32.0 g, KCl 0.5 g, MgSO4·7H20 0.5 g,
K2HPO41.0g, wheat bran 1000 g, distilled water 1000m L, pH value is natural.121 DEG C of insulated sterilizing 20min.
PDA culture medium: PDA(potato agar) 40g, it is settled to 1L, pH natural.121 DEG C of insulated sterilizing 20min.
Fermentation medium: Testa Tritici: water weight ratio is that 1:1, pH are natural.121 DEG C of insulated sterilizing 20min.
Embodiment 1
The primary dcreening operation of rod aspergillosis, Oxford cup transparent circle sieve bacterium experiment screening superior strain.
Gather pedotheque from South China Botanical Garden, weigh sample 10g and put into the physiology containing bead aseptic equipped with 90mL
The 300mL triangular flask of saline sways 20 min, make 10 times of ascending series dilute sample even liquid 1:10,1:100,1:1000 and
1:10000, takes the dilute sample even liquid each 1.0mL of 1:100,1:1000 and 1:10000 and is separately added into sterilized petri dishes；Add 15mL again
PDA culture medium mix homogeneously, stands 1min, is inverted in 28 DEG C and cultivates 72h, bacterium colony single in culture medium is transferred to inclined-plane
Cultivating 72h for 28 DEG C in PDA culture medium, then proceed to the primary dcreening operation culture medium 28 DEG C cultivation 60h containing ferulic acid ethylester, ferulic acid is produced in screening
The bacterial strain of ester, standby；
Make of the experiment of plate Oxford cup transparent circle and again sieve bacterium, use liquid by separating the bacterial strain producing ferulic acid ester obtained
Fermentation process in the fermentation medium 28 DEG C cultivate 2 days, it is thus achieved that fermentation liquid, take on 200uL after centrifugal for fermentation liquid 6000r/min
Clear liquid, is put in primary dcreening operation culture medium in the cup of Oxford, is placed in 37 DEG C of incubators cultivation 5-27h and observes transparent circle, fermentation liquid upper
Clear liquid is cleared up substrate and is produced transparent circle, can illustrate to produce the height of ferulaic acid esterase activity, produce Resina Ferulae in this approach
Contrast screening transparent circle in the bacterial strain of acid esters enzyme, what transparent circle was big is the bacterial strain of high yield feruloyl esterase.As shown in accompanying drawing 1,
Wherein a strain is gained rod aspergillosis (Aspergillus clavatus) cigarette of the present invention green grass or young crops, and its transparent circle is compared to other bacterium
Strain is big.
Embodiment 2
The Microbiological Characteristics of bacterial strain and qualification
Rod aspergillosis colony growth rate on Czapek's medium is relatively slow, cultivates 7d diameter 30mm；Quality velvet shape is to powder
Shape, white mycelium, conidium structure is that dark blue is green, without transudate.On wort agar, colony growth rate is very fast,
Cultivating 7d diameter 45mm, smooth, quality granular, conidium structure is many, celadon, bacterium colony reverse side bronzing.Shi ferment
Female cream agar colony cultivates 7d diameter 40mm, and quality velvet shape, bottle is the thickest；Conidium structure is many, and in dusty blue, edge is relatively
Dilute, white, without transudate.
It is observed that be rod during conidial head children under microscope, conidiophore betides substrate, and falx stem is long
Short differ；Top capsule is gradually expanded by falx top and becomes club shape, diameter 50～60mm, produces spore structure monolayer, and bottle stalk is intensive
This is born in all surfaces of top capsule, bottle stalk size 8～12 μ m 2～3 μm;Conidium is oval, spore size 4～5 μm
× 3 μm, wall smooth (shown in accompanying drawing 2-4).
ITS and the rod aspergillosis (Aspergillus that described rod aspergillosis (Aspergillus clavatus) cigarette is blue or green
Clavatus) homology reaches 100%.
Embodiment 3
Solid fermentation measures bound form ferulic acid burst size
By seed culture based formulas, claiming the 10g wheat bran 10g that adds water to be mixed into 500mL triangular flask, seed culture medium sterilizing is followed by
Planting rod aspergillosis (Aspergillus clavatus) cigarette green grass or young crops inclined-plane bacterial strain, inoculum concentration is 0.5%, after 28 DEG C are cultivated 3d, obtains rod bent
Mould seed；
By fermentative medium formula, claim 200g wheat bran to add water sterilizing after 200g mixing, put porcelain dish cooling, Testa Tritici THICKNESS CONTROL
In 2.5cm, rod aspergillosis seed inoculum concentration is that 0.5% (bacteria containing amount is 1～2 × 109Individual/g), cultivation temperature is 28 DEG C, carries out
Aerlbic culture, fermentation time is 48h.By solid fermentation wheat bran, add 90mL water ratio in 15g and extract in 40 DEG C of water-baths
After 60min, in 4 DEG C, 12 000 r/min are centrifuged 20 min, take supernatant enzyme liquid and are placed in refrigerator preservation.
Take supernatant 3mL to enter 10mL centrifuge tube and add 3mL methanol mixed, 10000 r/min centrifuging and taking supernatant 0.45 μm
Membrane filtration enters sample injection bottle Refrigerator store, measures as liquid phase and uses.It is azymous by this that sample does blank i.e. bran mass
Method processes and get final product.HPLC uses Venusil XBP C18(L) (4.6mm × 250mm, 5 μm), guard column is C18(4.6×
7.5 mm, 5 μm) condition is: 10 μ L sample sizes, flowing is 1% glacial acetic acid mutually: acetonitrile (4:1), wavelength 320nm, column temperature 20 DEG C,
Flow velocity is 0.9mL min-1.Take 1.0mg ferulic acid standard substance, through methanol constant volume to 10mL, be configured to respectively concentration be 40,60,
80, the solution of 100,120,140 μ g/mL, is measured (being certain concentration ferulic acid standard substance color see Fig. 5 through above-mentioned chromatographic condition
Spectrogram), and be abscissa (x) by ferulic acid concentration, peak area is vertical coordinate (Y), finally obtains regression equation and is: Y=311134x
+ 202717,R2=0.9969 (standard curve see Fig. 6 is ferulic acid standard substance), further according to the Y of the ferulic acid of sample determination
Value (see figure 7), substitutes into Y value regression equation Y=311134x+202717, obtains x value, be sample ferulic acid concentration.Utilize
The bound form ferulic acid burst size of liquid chromatogram measuring reaches 0.3-0.5%, and in pertinent literature, the content of bound form ferulic acid is
0.3%-0.7%.Prove to have obtained great release by bound form ferulic acid after fermentation.This bacterial strain be high yield feruloyl esterase and
Related enzyme systems the method that can pass through to ferment obtain the bacterial strain of bound form ferulic acid from plant.
Sequence table 1
SEQUENCE LISTING
<110>South China Science & Engineering University
Excellent aspergillosis and the application thereof of feruloyl esterase is produced in<120>one strains
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 594
<212> DNA
<213>rod aspergillosis (Aspergillus clavatus) cigarette is blue or green
<400> 1
tttccgtagg tgaacctgcg gaaggatcat taccgagtgc gggccctctg ggtccaacct 60
cccacccgtg tttatcgtac cttgttgctt cggcgggccc gccgtcttcg gacggccgcc 120
ggggaggcct ccgcgccccc gggcccgcgc ccgccgaaga ccacaacatg aactctgttc 180
tgaagttttg cagtctgagt tgattatcat aatcagttaa aactttcaac aacggatctc 240
ttggttccgg catcgatgaa gaacgcagcg aaatgcgata actaatgtga attgcagaat 300
tcagtgaatc atcgagtctt tgaacgcaca ttgcgccccc tggtattccg gggggcatgc 360
ctgtccgagc gtcattgctg ccctcaagca cggcttgtgt gttgggcccc cgtccccggt 420
ttcccgggga cgggcccgaa aggcagcggc ggcaccgcgt ccggtcctcg agcgtatggg 480
gctttgtcac ccgctcttgt agggccggcc ggcgcctgtc gacaccaacc caatttttct 540
aaggttgacc tcggatcagg tagggatacc cgctgaactt aagcatatca ataa 594
Claims (5)
1. the excellent aspergillosis of feruloyl esterase is produced in a strain, it is characterised in that the Classification And Nomenclature of described rod aspergillosis is rod aspergillosis
(Aspergillus clavatus) cigarette is blue or green, and by China typical culture collection center preservation, deposit number is CCTCC NO:
M2013641, preservation date is December in 2013 8, and its ITS sequence is as shown in sequence table 1.
2. rod aspergillosis application in producing feruloyl esterase described in claim 1.
Application the most according to claim 2, it is characterised in that comprise the steps: to utilize Testa Tritici direct inoculation rod aspergillosis
(Aspergillus clavatus) cigarette is blue or green, carries out koji plate fermentation, it is thus achieved that fermented product extract in fermentation medium, utilizes efficiently
Liquid chromatograph measures the ferulic acid burst size of Testa Tritici in fermentation medium.
Application the most according to claim 3, it is characterised in that the condition of described koji plate fermentation is: with Testa Tritici on koji tray
Carry out solid fermentation, Testa Tritici THICKNESS CONTROL in 2 ~ 3cm, rod aspergillosis seed inoculum concentration be 0.5% i.e. bacteria containing amount be 1～2 ×
109Individual/g, cultivation temperature is 25～28 DEG C, is aerated cultivating, and fermentation time is 48～55h；PH is natural.
Application the most according to claim 4, it is characterised in that described rod aspergillosis seed is inoculated into triangular flask kind by rod aspergillosis
In sub-culture medium, cultivate 58 ~ 60h in 28 DEG C ± 1 DEG C and obtain.
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