CN105018352A - Fungal strain capable of producing kojic acid and preparation method of fungal strain - Google Patents
Fungal strain capable of producing kojic acid and preparation method of fungal strain Download PDFInfo
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Abstract
The invention discloses a fungal strain capable of producing kojic acid and a preparation method of the fungal strain, and belongs to microbial fungus isolation and culture, and particularly relates to isolation and culture of strains capable of producing kojic acid. The fungal strain refers to aspergillus nomius FECH-998, and the collection number is CGMCC NO. 10987. The preparation method includes steps such as strain selection, fermentation of liquid culture medium, pre-processing of fermentation liquid, isolation and purification of chemical compound and chemical compound structural identification. The strain is capable of producing kojic acid effectively, optimization feasibility of strain fermentation condition and strain breeding in later period is high, and the strain can be a candidate with high production of kojic acid.
Description
Technical field
The invention belongs to microbial fungi separation and Culture, be specifically related to the separation and Culture of producing kojic acid bacterial strain.
Background technology
Kojic acid is a kind of acidic metabolite produced by the microorganism such as aspergillus oryzae, can water-soluble, ethanol or acetic ester etc.The microorganism majority that can produce kojic acid is fungi, as aspergillus oryzae, cream-coloured aspergillus, flavus, Aspergillus candidus, excellent aspergillus, huge aspergillus, Aspergillus fumigatus, Aspergillus tamarii, Aspergillus wentti and Aspergillus amstelodami.Some bacterium as rose gluconobacter sp, wax-like gluconobacter sp and some pseudomonass also can utilize carbohydrate generate kojic acid (
rosfarizan Mohamad, Mohd Shamzi Mohamed, Arbakariya B. Ariff et al., Kojic acid:Applications and development of fermentation process for production.
biotechnology and Molecular Biology Reviewsvol. 5 (2), pp. 24-37,2010).
Continue to carry out high yield kojic acid microorganism strains and be separated and cultivate that to make it have industrial use necessary.This is because kojic acid is of many uses, act on collect antibacterial, anticorrosion, fresh-keeping, protect look color development, anti-oxidant etc. all over the body.As foodstuff additive, the Performance Ratio Sorbic Acid of its food preservatives is better; Also can be used as the fresh-keeping of raw vegetable, fruit, sea-food etc., and partly can replace Sodium Nitrite protecting on look color development, and Nitrite transformation can be suppressed to be carcinogenic nitrosamine.Kojic acid, as tyrosinase inhibitor, is tried to be the first and the chelating copper ions in tyrosine oxidase, is made cupric ion ineffective, suppresses the synthesis of dopachrome.Kojic acid can suppress 5,6-dihydroxy indole, i.e. the polymerization of DHI, suppresses the oxidasic activity of DHICA.
Add the function that the makeup of kojic acid have skin care, moisturizing, whitening, light, sun-proof, nti-freckle.Although the biological safety of kojic acid in makeup causes more arguement within for some time, up-to-date evidence shows: the interpolation concentration of kojic acid in makeup within 1% be safe (
christina L. Burnett, Wilma F. Bergfeld, F. Alan Andersen, et al.final report of the safety assessment of kojic acid as used in cosmetics.
international Journal of Toxicology.29 (Supplement 4), 244S-273S, 2010).Can predict, along with the support of more safety evaluation evidences, kojic acid also has very large wider space in the application of household chemicals field.
Summary of the invention
The present invention be intended to be provided by Isolation and screening a kind of kojic acid synthesized micro-organism-collection peak aspergillus (
aspergillus nomius) FECH-998 bacterial strain, and with this bacterial strain for producing the candidate strain of kojic acid, provide fermentation condition and bacterial strain breeding optimization method.
The present invention is achieved through the following technical solutions above-mentioned technological invention object.
(1) a kind of fungal bacterial strain producing kojic acid compound
This bacterial strain for collection peak aspergillus (
aspergillus nomius) FECH-998, deposit number is CGMCC No:10987, and the iron chelating compounds chemical structural formula that described bacterial strain FECH-998 produces is as shown in Figure 3.
(2) method of the fungal bacterial strain of kojic acid compound is produced in preparation
Comprise the following steps:
(1) spore suspension and liquid medium is prepared
Bacterial strain FECH-998 is inoculated on the agar slant of de-iron Cha Shi nutrient agar, slant tube specification is 15 × 150 mm, this nutrient agar volume 5 mL, cultivate 4 ~ 7 d for 28 DEG C, formed after a large amount of spore until mycelium, add 5 mL aseptic deionized waters to inclined-plane, sterilizing bamboo let gently scrapes lawn and makes spore suspension;
Being inoculated into respectively by bacterial strain FECH-998 spore suspension is equipped with in 3 test tubes of de-iron Cha Shi basic medium, test tube specification is 15 × 150 mm, this basic medium volume 5 mL, 28 DEG C cultivate 7 d, until basic medium surface occur a large amount of mycelial growth and spore time based on it nutrient solution;
Described de-iron Cha Shi nutrient agar (g/L): NaNO
32.0 g, K
2hPO
41.0 g, KCl 0.5 g, MgSO
40.5 g, sucrose 30.0 g, agar 20.0 g, deionized water 1 L, pH nature, prepare rear 121 DEG C of sterilizing 20 min;
The composition of described de-iron Cha Shi basic medium and de-iron Cha Shi nutrient agar is distinguished and is: the Cha Shi basic medium of de-iron does not add agar, and all the other compositions are identical;
(2) screen
With the liquid medium adding 20 microlitre CAS dye liquors and isopyknic step (1) in the 96 every holes of orifice plate, abundant mixing, take color reaction as red or orange or brown color or purple fungi alternatively bacterial strain, it is prepared spore suspension by step 1 method, be inoculated in 100mL de-iron Cha Shi basic medium, 28 DEG C, 180rpm cultivates 5d;
Add 40 microlitre CAS dye liquors and isopyknic candidate strain nutrient solution with in the 96 every holes of orifice plate, fully mix, the record colour-change time, the shortest for the reaction used time, reaction color are changed difference bacterial strain FECH-998 greatly as preferred strain;
(3) preparation fermentation crude extract
Get step (2) 10 FECH-998 bacterial strains to prepare spore suspension and be inoculated in 10 × 200 mL sterilising liq de-iron Cha Shi substratum, 28 DEG C, 180 rpm cultivate 5 d, treat that having a large amount of mycelium pellet to grow in nutrient solution stops cultivating, with this nutrient solution for seed liquor;
(4) step (3) seed liquor is got, by 10% inoculative proportion aseptic straw, it is on average inoculated in 50 bottles of 500 mL triangular flasks that 200 mL sterilizing de-iron Cha Shi basic mediums are respectively housed, 28 DEG C ~ 32 DEG C, 180 rpm cultivate 3 d ~ 8 d, in triangular flask, throw in particle diameter 60 ~ 16 object macroporous adsorbent resin in 2% ratio when having a large amount of mycelium pellet to grow in nutrient solution, 28 DEG C ~ 32 DEG C, 180 rpm original positions stop cultivating after adsorbing 24 h, collect nutrient solution, filter; Bacterium slag and resin methanol extraction, filtrate is extracted with ethyl acetate, and obtains methanol extract and acetic acid ethyl ester extract;
(5) by methanol extract concentrating under reduced pressure, drying, yellow oil 23.8 g being mingled with a large amount of white fine needle crystal is obtained; By acetic acid ethyl ester extract concentrating under reduced pressure, drying, obtain brown light brown solid 11.1 g being mingled with a large amount of white fine needle crystal, both merging, as crude extract.
Further, described preparation method is: described step (4) is got with high glucose medium
For de-iron Cha Shi basic medium, this high sugar-fermenting substratum (g/L) is: sucrose 100.0 g, yeast extract paste 6.0 g, NaNO
32.0 g, K
2hPO
41.0 g, KCl 0.5 g, MgSO
40.5 g, deionized water 1 L, pH nature; Prepare rear 121 DEG C of sterilizing 20 min; In the fermented liquid of described high glucose medium, kojic acid content is 15.65g/L, and kojic acid conversion ratio is 0.157g kojic acid/g sucrose.
Further, described preparation method is purification procedures (5) crude extract, comprising:
(1) normal phase silicagel column chromatographic grade wash-out, this chromatographic system gradient is: chloroform, chloroform: methyl alcohol=9:1 and methyl alcohol; CAS liquid development process follows the trail of the cut that each section of elutriant contains iron sequestering activity composition;
(2) cut evaporated under reduced pressure activeconstituents, removes oily matter by petroleum ether, dissolve with methanol, and under room temperature, periodic crystallisation and recrystallization obtain white fine acicular compound, and this compound has very strong iron sequestering activity.
The present invention has following substantive distinguishing features and progress:
The present invention limits 100 fungus strains of iron hoop border pedotheque for material to be separated from alkalescence, developing the color principle for instructing the screening model of screening different levels, obtaining the collection peak aspergillus FECH-998 microorganism strains that in model, iron chelate matter activity is strong with CAS.Through molecular biology identification, FECH-998 be accredited as collection peak aspergillus (
aspergillus nomius) bacterial strain.This bacterial strain on June 17th, 2015 " (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; postcode: 100101) carried out effective preservation, deposit number is CGMCC No:10987 to China Committee for Culture Collection of Microorganisms's common micro-organisms " center ".
The present invention carries out liquid culture to integrate peak aspergillus FECH-998 as starting strain further, draw Duan Chufen crude extract, and by investigating bacterial strain, to produce kojic acid ability be that the liquid nutrient medium of seed liquor makes kojic acid content in fermented liquid bring up to 15.65g/L with high glucose medium, and kojic acid conversion ratio is 0.157g kojic acid/g sucrose.
In addition, present invention also offers reactive site purification refine, point out the step that active substance chemical structure etc. obtains target compound.
The present invention shows: bacterial strain FECH-998 wild strain synthesis kojic acid nutritional requirement is simple, growth is rapid, sporulation quantity is large, later stage bacterial strain breeding space is large, has the good potentiality as producing kojic acid superior strain.
Accompanying drawing explanation
Fig. 1 is bacterial strain FECH-998 and reference culture
aspergillus nomiusthe systematic evolution tree of NRRL13137 ITS sequence.
Fig. 2 is bacterial strain FECH-998 iron sequestering activity crystalline material HPLC analysis chart.
Fig. 3 is FECH-998 iron sequestering activity matter chemistry structural formula.
Fig. 4 is " concentration-light absorption value " curve of kojic acid standard substance.
Embodiment
example 1.
Bacterial strain primary dcreening operation
Early stage is separated and is inoculated on iron Cha Shi medium agar inclined-plane from 100 fungal strains of Yunnan Cheng Hai bed mud sample and Geju City great Tun alkalescence tailing soil, slant tube specification is 15 × 150 mm, volume 5 mL, every strain bacterium inoculates 2 inclined-planes, and wherein 1 is inclined-plane for subsequent use, cultivate 4 ~ 7 d for 28 DEG C, formed after a large amount of spore until mycelium, except inclined-plane for subsequent use, in inclined-plane, add 5 mL aseptic deionized waters, gently scrape lawn with sterilizing bamboo let and make spore suspension, for subsequent use.
The spore suspension of every strain bacterium is inoculated in the test tube that de-iron Cha Shi basic medium is housed respectively, test tube specification is 15 × 150 mm, culture volume 5 mL, every strain bacterium inoculates 3 test tubes, arrange 3 test tubes not connecing bacterium is negative control simultaneously, cultivate 7 d, can screen when Tube propagation primary surface has a large amount of mycelial growth and spore to occur for 28 DEG C.
Wherein, the Cha Shi nutrient agar (g/L) of de-iron comprises: NaNO
32.0 g, K
2hPO
41.0 g, KCl 0.5 g, MgSO
40.5 g, sucrose 30.0 g, agar 20.0 g, deionized water 1 L, pH nature.Prepare rear 121 DEG C of sterilizing 20 min.
Distinguishing with nutrient agar is do not add agar in basic media components, and all the other compositions are identical.
In screening link, in 96 orifice plates, add 20 microlitre CAS dye liquors in every hole, add isopyknic above-mentioned bacterial strains test tube liquid medium afterwards respectively.Often add a kind of nutrient solution all slight wobble nutrient solution is fully mixed with CAS dye liquor, after record mixing, color becomes the strain number corresponding to nutrient solution sample of redness, purple or yellow, using the multiple sieve bacterial strain of corresponding bacterial strain as next step.There is no the nutrient solution sample of inoculated fungi as negative control.Ethylenediamine tetraacetic acid (EDTA) (EDTA) saturated deionized water solution is set to positive control.
Wherein, preparation reference literature (the Brian C. Louden of CAS blue detection liquid; Daniel Haarmann; Aaron Lynne.Use of Blue Agar CAS Assay for Siderophore Detection.
jM & BE., 2011,12 (1): 51-53.) carry out.CAS colour developing principle is: iron ion and HTDMA complexing can form blue complex (FeDye
3-x), when one is to Fe
3+there is the part (L) of more high-affinity, as ethylenediamine tetraacetic acid (EDTA) (EDTA), add iron-dyestuff (FeDye to
3-x) in mixture, the iron in mixture is transferred on part, and discharges dyestuff freely, the color of solution becomes yellow, purple or redness from blueness simultaneously.Process color useful chemical formula is simply expressed as: FeDye
3-x+ Ly-→ FeL
3-y+ Dye
x-.
Detect liquid reaction according to CAS, from 100 fungal strains, filter out 36 strains has the fungi of colour-change as the aimed strain of multiple sieve, and wherein 24 strain color reactions are red or orange, and 7 strains are brown color, and 5 strains are purple.
CAS liquid detecting method combines as to the screening of producing iron chelate matter fungi by primary dcreening operation program of the present invention with 96 well plate method, this method is simple to operate, and fast, test-results is explicit, and is easy to record and preserves, and is suitable for large batch of screening.
Example 2. bacterial strain sieves again
1. nutrient solution 96 orifice plate replies immediately sieve soon
36 fungal strains just screened are prepared respectively spore suspension (example 1), be inoculated in 100 mL liquid de-iron Cha Shi substratum respectively, every inoculation to 250 mL triangular flask 3 bottles, spore suspension inoculum size is 5 mL/100 mL, does not have the nutrient solution sample of inoculated fungi to be set to negative control.Each shaking flask 28 DEG C, 5 d cultivated by 180 rpm shaking tables, treat in substratum, have a large amount of mycelium pellet to occur carrying out multiple screening procedure.
In multiple sieve link, in 96 orifice plates, add 40 microlitre CAS dye liquors in every hole, add isopyknic above-mentioned bacterial strains test tube liquid medium afterwards respectively.Often add a kind of nutrient solution all slight wobble nutrient solution is fully mixed with CAS dye liquor, with stopwatch measure mixing after the colour-change time, and record corresponding to strain number.In reaction process, the depth of color and the length of colour-change required time reflect the ability of bacterial strain chelated iron, and colour-change difference is larger, and the time is shorter, and the sequestering power producing iron chelate matter is stronger.The shortest for the reaction used time, reaction color change difference bacterial strain are greatly selected bacterial strain as being sieved into again.There is no the nutrient solution sample of inoculated fungi in the process as negative control.Ethylenediamine tetraacetic acid (EDTA) (EDTA) saturated deionized water solution is set to positive control.The depth of colour-change and the length of colour-change required time after mixing with CAS dye liquor according to each strain cultured solution, we find to sieve again in bacterial strain in 36 strains, and the nutrient solution of FECH-998 bacterial strain and the developing time of CAS nitrite ion are 4s, and color is pale brown.The developing time of positive control-ethylenediamine tetraacetic acid (EDTA) (EDTA) saturated deionized water solution is 2 ± 0.12 seconds, and color is purplish red.
2. CAS double-layer plate contrast screening verification
The bacterial strain selecting CAS double-layer agar technique to verify that above-mentioned screening procedure obtains further is the fungi of high yield iron chelate matter.The every strain bacterium of 7 fungal strain that multiple for early stage sieve obtains is inoculated 2 inclined-planes, 1 is inclined-plane for subsequent use, cultivates 4 ~ 7 d, is formed after a large amount of spore until mycelium for 28 DEG C, in inclined-plane, add 5 mL aseptic deionized waters (inclined-plane for subsequent use does not process), gently scraping lawn with sterilizing bamboo let, to prepare spore suspension for subsequent use.
Aseptically, every bacterial strain spore suspension is drawn 10 microlitre bacteria suspensions instillations with aseptic dropper and is placed on 5 mm sterilizing filter papers on the double-deck agar plate of CAS.The flat board sealed membrane handled well is sealed, observations after 28 DEG C of cultivation 3 ~ 5 d.Do not connect the blank cultures of bacterium as negative control, the saturated deionized water solution of EDTA that 0.2 mm filter filters is as positive control.
Wherein, CAS double-layer plate compound method is: when 1% agar temperature drops to 65 DEG C, in 5 mL CAS blue detection liquid: the ratio of 100 mL 1% agar, the CAS blue detection liquid of 65 DEG C of water-bath 30 min is mixed with it, pour 105 mm into by 20 mL/ ware amounts dull and stereotyped, be positioned under room temperature dull and stereotyped as lower floor after natural coagulation.Afterwards the de-iron Cha Shi substratum after sterilizing is inverted on lower floor CAS flat board by 15 mL/ ware amounts, under room temperature after natural coagulation as upper panel.After double-layer plate prepares, put 5mm sterilizing filter paper by aseptic technique in its centre for subsequent use.
De-iron Cha Shi culture medium prescription is (g/L): NaNO
32.0 g, K
2hPO
41.0 g, KCl 0.5 g, MgSO
40.5 g, sucrose 30.0 g, agar 20.0 g, deionized water 1 L, pH nature, prepare rear 121 DEG C of sterilizing 20 min.
The blank cultures formula not connecing bacterium is (g/L): NaNO
32.0 g, K
2hPO
41.0 g, KCl 0.5 g, MgSO
40.5 g, sucrose 30.0 g, deionized water 1 L, pH nature, prepare rear 121 DEG C of sterilizing 20 min.
Compared with negative blank, after 7 strain bacterial strain 3 d, on CAS double-layer plate, all there is obvious colony growth, the below of bacterium colony or around all there is obvious colour-change.Wherein, FECH-998 periphery of bacterial colonies colour-change is obvious, and in brown color, and along with the continuity of colony growth, rapidly by whole dull and stereotyped color change, diffusion tendency is strong.Illustrate: the activity that bacterial strain FECH-998 produces iron chelate matter is very strong, good hydrophilic property, and is constantly secreted into extracellular with mycelial growth, with it for selected bacterial strain.
The molecular biology identification of example 3. object bacterial strain FECH-998
1, extracting genome DNA, amplification and order-checking
Be seeded in sterilized 50 mLPDA liquid nutrient mediums by high yield iron chelate matter bacterial strain FECH-998,180 rpm, cultivate 3 d at 28 DEG C, carry out the extraction of genomic dna, amplification and order-checking.
Wherein, the composition of PDA substratum is (g/L): potato 200.0 g, sucrose 20.0 g, water 1 L, pH nature.Peeling potatoes, be cut into block and boil 30 min, filtered through gauze, sugaring are dissolved, moisturizing to 1 L, 121 DEG C of sterilizing 30 min.
Wherein, the extracting method of genomic dna is: get appropriate 3 ~ 5 mL cultures, 13000 rpm, centrifugal 1 min, abandons substratum.Add fungal DNA and extract lysate 300 microlitre, vortex precipitates.65 DEG C of water-bath 30 min.Add isopyknic chloroform: primary isoamyl alcohol (24:1), vortex mixes, centrifugal 5 min of 13000 rpm.Get supernatant, add isopyknic Virahol, mixing, 13000 rpm, centrifugal 5 min.Abandon supernatant, add 70% ethanol mixing, centrifugal 1 min of 13000 rpm.Abandon ethanol, be inverted centrifuge tube and dry.Add 30 microlitre ddH
2o, dissolving DNA, carries out downstream PCR amplification or-20 DEG C of preservations.
Wherein, ITS rDNA amplification PCR reaction system consists of: 5 microlitre 10 × KOD buffer; 5 microlitre 2 mm dNTPs; 1 microlitre Genomic DNA; 1 microlitre Forward Primer (10 micromole); 1 microlitre Reverse Primer (10 micromole); 1 microlitre KOD DNA Polymerase; 36 microlitre ddH
2o.Amplification program is: 94 DEG C of 3 min; 94 DEG C of 30 s, 58 DEG C of 30 s, 72 DEG C of 3 min, 35 circulations; 72 DEG C of 10 min.PCR product 1% agarose gel electrophoresis detects, then after the PCR of 1 the 50 microlitre system that increases, product reclaims.PCR primer reclaimer is: add the 4 times of volumes i.e. Buffer CP of 800 microlitres to the 1.5mL centrifuge tube containing PCR reaction volume 200 microlitre; Concussion, of short duration centrifugal.
Adsorption column is placed in collection tube, and the mixture getting above-mentioned PCR primer proceeds to adsorption column with each 750 microlitres, centrifugal 1 min of 13000 rpm.Abandon waste liquid; Add 700 microlitre elutriants, centrifugal 1 min of 13000 rpm, abandons waste liquid; Add 500 microlitre elutriants, centrifugal 1 min of 13000 rpm, abandons waste liquid; Adsorption column is put into collection tube, and centrifugal 2 min of blank pipe 13000 rpm, to remove the ethanol on adsorption column; Adsorption column is forwarded to new 1.5 mL centrifuge tubes, adds 30 microlitre ddH at adsorption column center
2o, room temperature places 1 min, centrifugal 1 min of 13000 rpm, and namely filtrate be the DNA reclaimed.
PCR primer connects carrier T.PCR primer carrier T linked system is: 4 microlitre 5 × Reaction Buffer; 10 microlitre PCR Product; 2 microlitre pTZ57R/T Vector; 1 microlitre T4 DNA Ligase; 3 microlitre ddH
2o.PCR primer carrier T linker: shake up, centrifugal, place 1 h, be converted into connection product for 22 DEG C.Take out Competent cell TOP10, thaw, add connection product, place 30 min on ice, 42 DEG C of water-bath 90 s, ice bath 2 min, room temperature 5 min.Often pipe adds the LB liquid nutrient medium of 800 microlitre antibiotic-frees, and 37 ° of C vibrate 45 min recoveries, and the centrifugal 5min of 8000 rpm, abandons supernatant, and resuspended rear even spread arrives
ampin resistant panel, room temperature dries up, and is inverted in 37 DEG C of incubators, cultivates 12 ~ 16 h and grows bacterium colony, PCR qualification and order-checking bacterium colony.
By to FECH-998 bacterial strain ITS sequence through pcr amplification and order-checking, obtaining length is the ITS sequence of 606 effective bases, for subsequent analysis.
2. the analysis of ITS sequence
The DNA sequence dna warp recorded
nCBI(National Center for Biotechnology Information)
bLASTobtain the ITS gene order of related species after engine search, arrange with ClustalX 1.8 software.Base deletion site is got rid of, with adjacent method (Neighbor-joining analysis) phylogenetic tree construction during Phylogenetic Analysis.Distance matrix according to
kimura' s two-parameter model calculates,
bootstrap1000 sub-samplings are carried out in inspection.Result shows, the position of bacterial strain FECH-998 on evolutionary tree reliable (
bootstrapvalue >70).Line segment scale (0.01) represents the branch length of 1% sequence difference.Bacterial strain FECH-998 and reference culture can be learnt by systematic evolution tree
aspergillus nomiusnRRL13137 ITS sequence maximum comparability reaches 100%(and sees Fig. 1).
From microscopy results, bacterial strain FECH-998 is Aspergillus microorganism characteristic feature, and conidiophore is formed by a upright mycelia, and the end of mycelia forms spherical expansion.Bacterial strain conidium bunchy and is born in top capsule.Observe under natural light, this bacterial strain conidium is breen.
Comprehensive above information, infer bacterial strain FECH-998 bacterial strain belong to collect peak aspergillus (
aspergillus nomius).
The preparation of example 4. bacterial strain FECH-998 iron chelate matter, purifying and structure are pointed out
1. prepared by the crude extract that ferments
Spore suspension (example 1) prepared by 1 FECH-998 bacterial strain and be inoculated in (example 1 in 200 mL sterilising liq de-iron Cha Shi substratum.Inoculation shaking flask specification is 500 mL).28 DEG C, 180 rpm cultivate 5 d.Treat that having a large amount of mycelium pellet to grow in nutrient solution can stop cultivating.With this nutrient solution for seed liquor, by 5% inoculative proportion, it be on average inoculated in 20 bottles of 500 mL triangular flasks that 200 mL sterilising liq de-iron Cha Shi substratum are respectively housed with aseptic straw, 28 DEG C, 180 rpm cultivate 5 d.Treat that having a large amount of mycelium pellet to grow in nutrient solution can throw in particle diameter 60 ~ 16 order macroporous adsorbent resin in 2% ratio to triangular flask, 28 DEG C, 180 rpm original positions stop cultivating after adsorbing 24 h, collect nutrient solution, filter, and bacterium slag and resin adopt methanol extraction; Filtrate adopts extraction into ethyl acetate.Merge after methyl alcohol and ethyl acetate extract concentrating under reduced pressure, obtain fermentation crude extract.
2. the separation and purification of target iron chelate matter
First crude extract is carried out purification on normal-phase silica gel column chromatography, and chromatographic system is: chloroform, chloroform: methyl alcohol=9:1 and methyl alcohol follow the trail of the cut containing iron sequestering activity composition to each section of elutriant CAS liquid development process.Remove oily matter by petroleum ether after reactive site cut evaporated under reduced pressure, then use dissolve with methanol, put and at room temperature repeatedly carry out crystallization and re-crystallization step obtains white fine acicular compound crystal.
This crystalline material adopts the detection of CAS liquid development process to have very strong iron sequestering activity; This crystallization detects through HPLC chromatography, this chromatographic system: water: methyl alcohol=8:2, shows its purity and has reached compound structure analysis and require (see figure 2).
3. the structure of bacterial strain FECH-998 iron chelate matter is pointed out
Point out in work at compound structure, according to two characteristic ion peaks [M+Na] that matter ESI (+) mass spectroscopy of FECH-998 iron chelate occurs
+=165 and [M+K]
+=181 signals, infer that the molecular weight of compound is 142.Combine afterwards
1h-NMR spectrogram,
13c-NMR spectrogram and
dEPTthe hydrogen carbon information that spectrogram provides, infers that the molecular formula of compound is C
6h
6o
4.Afterwards, with this molecular formula for searched targets,
reaxys(Beilstein/Gmelin) and
sciFinderretrieve in database.Result for retrieval shows, deriving from FECH-998 iron sequestering activity material is kojic acid (kojic acid), and its structural formula is shown in Fig. 3, and concrete nuclear magnetic data is as following table 1:
the NMR data of table 1 compound F 17-hydroxy-corticosterone S-03 (solvent: deuterated dimethyl sulfoxide,
1
h:400 MHz,
13
c:100 MHz)
| Position | δ C (ppm， muLt.) | δ H (ppm， muLt.， J in Hz) |
| 2 | 168.3， C | - |
| 3 | 110.0， CH | 6.34，s |
| 4 | 174.1， C | - |
| 5 | 145.8， C | - |
| 6 | 139.4， CH | 8.02， s |
| 7 | 59.6， CH 2 | 4.28， d， 1.0 |
| 5-OH | - | 9.09， br s |
| 7-OH | - | 5.71， t， 5.6′(2) |
Wherein, the analysis that compound structure is pointed out measures entrusts Kunming Inst. of Botany, Chinese Academy of Sciences to complete.
reaxys(Beilstein/Gmelin) and
sciFinderdatabase is provided by Library of Yunnan University.
Example 4. bacterial strain FECH-998 iron chelate matter simple culture media expands preparation and kojic acid Production rate
1, kojic acid typical curve is set up
The establishment method of kojic acid typical curve is: accurately take 100.0mg kojic acid standard substance with analytical balance, distilled water dissolves, constant volume 100mL is standard sample liquid, with transfer pipet accurately draw 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL totally 6 standard sample liquids in 6 brown volumetric flasks of 100mL, 2 mL developers are added after respectively adding 50 mL distilled water, last constant volume 100 mL, under wavelength is 500 nm, measures its light absorption value, asks equation of linear regression.Wherein, the assay of kojic acid carries out with reference to GB/T 23534-2009 standard.
The process for preparation of above developer is: accurately take the anhydrous FeCl of 10g
3, add the dense HCl of 22.5 mL, distilled water dissolves, constant volume 1000 mL.
From the typical curve set up, its coefficient R
2=0.9999(is shown in Fig. 4), curve quality meets analyzes requirement.
2. bacterial strain FECH-998 iron chelate expands preparation
Spore suspension (example 1) prepared by 10 FECH-998 bacterial strains to be inoculated in 10 × 200 mL sterilising liq de-iron Cha Shi substratum (example 1), 28 DEG C, 180 rpm cultivate 5 d.Treat that having a large amount of mycelium pellet to grow in nutrient solution stops cultivating.With this nutrient solution for seed liquor, it be on average inoculated in 50 bottles of 500 mL triangular flasks that 200 mL sterilising liq de-iron Cha Shi substratum are respectively housed by 10% inoculative proportion aseptic straw, 28 DEG C, 180 rpm cultivate 3 d.Treat that having a large amount of mycelium pellet to grow in nutrient solution can throw in particle size range in 2% ratio to often propping up in triangular flask: 60 ~ 16 order macroporous adsorbent resins, 28 DEG C, 180 rpm original positions stop cultivating after adsorbing 24 h.The each shake flask culture of unified collection, filters with filter cloth, and bacterium slag and resin adopt methanol extraction, and filtrate adopts extraction into ethyl acetate.Wherein, methanol extract part obtains yellow oily crude extract 23.8 g being mingled with a large amount of white fine needle crystal after concentrating under reduced pressure also abundant drying; Filtrate acetic acid ethyl ester extract obtains brown light brown curable type crude extract 11.1 g being mingled with a large amount of white fine needle crystal after concentrating under reduced pressure also abundant drying.Merge two kinds of crude extracts, obtain 34.9 g and expand tunning sample.
3. bacterial strain FECH-998 kojic acid Production rate
Expand the kojic acid assay reference GB/T 23534-2009 standard in tunning sample.Accurately take 100.0 mg and expand tunning sample, 100 mL are settled to distilled water after a small amount of dissolve with methanol, get 10 mL, 0.45 mm syringe needle membrane filtration, then get 2 mL filtered liquids in the brown volumetric flask of 100 mL, add distilled water constant volume 100 mL after 2 mL developers.Under wavelength is 500 nm, measure its light absorption value respectively, gained light absorption value substitutes into equation of linear regression y=0.0087x+0.0021(Fig. 4), determine kojic acid content.
Pass through measure and calculation, it is 0.1642 that said determination sample absorbancy measures mean value three times, being calculated as kojic acid concentration is 18.63 mg/L, therefore to get the kojic acid content that 100.0 mg expand tunning samples be 93.15 mg, this content accounts for and expands 93.15% of tunning, and kojic acid content reaches 3.25g/L in bacterial strain FECH-998 fermented liquid, kojic acid conversion ratio is: 0.108g kojic acid/g sucrose.
Example 5. bacterial strain FECH-998 kojic acid high glucose medium ferments
High density carbon source material can improve or affect the output of bacterial strain FECH-998 kojic acid.The high sugar-fermenting substratum of this example carries out, and its component is (g/L): sucrose 100.0 g, yeast extract paste 6.0 g, NaNO
32.0 g, K
2hPO
41.0 g, KCl 0.5 g, MgSO
40.5 g, deionized water 1 L, pH nature; Prepare rear 121 DEG C of sterilizing 20 min.
Spore suspension (example 1) prepared by 10 FECH-998 bacterial strains to be inoculated in 10 × 200 mL sterilizing basis de-iron Cha Shi substratum (example 1), 28 DEG C, 180 rpm cultivate 5 d.Treat that having a large amount of mycelium pellet to grow in nutrient solution stops cultivating.With this nutrient solution for seed liquor, by 10% inoculative proportion, with aseptic straw, it is on average inoculated in 100 bottles of 500 mL triangular flasks that 200 mL sterilising liq high glucose mediums are respectively housed, 32 DEG C, after 180 rpm cultivate 8 d, particle size range 60 ~ 16 order macroporous adsorbent resin is thrown in often propping up in triangular flask in 5% ratio, 32 DEG C, 180 rpm original positions stop cultivating after adsorbing 24 h, collect nutrient solution, filter, bacterium slag and resin adopt methanol extraction, and methanol extract obtains pale brown curable type crude extract 362.2 g through concentrating under reduced pressure, drying; Filtrate is very low through detecting kojic acid content, is abandoned.
Crude extract kojic acid content assaying method is with example 4.By determination and analysis, get in 100.0 mg crude extract samples, the content of kojic acid is 86.42 mg, accounts for 86.42% of whole crude extract; In bacterial strain FECH-998 fermented liquid, kojic acid content reaches 15.65 g/L.Kojic acid conversion ratio is: 0.157 g kojic acid/g sucrose.
Claims (4)
1. produce a kojic acid fungal bacterial strain, it is characterized in that: described bacterial strain for collection peak aspergillus (
aspergillus nomius) FECH-998, deposit number is CGMCC:10987, and the iron chelating compounds chemical structural formula that described bacterial strain FECH-998 produces is:
。
2. preparation method of producing kojic acid fungal bacterial strain as claimed in claim 1, comprises the following steps:
(1) spore suspension and liquid medium is prepared
Bacterial strain FECH-998 is inoculated on the agar slant of de-iron Cha Shi nutrient agar, slant tube specification is 15 × 150 mm, this nutrient agar volume 5 mL, cultivate 4 ~ 7 d for 28 DEG C, formed after a large amount of spore until mycelium, add 5 mL aseptic deionized waters to inclined-plane, sterilizing bamboo let gently scrapes lawn and makes spore suspension;
Being inoculated into respectively by bacterial strain FECH-998 spore suspension is equipped with in 3 test tubes of de-iron Cha Shi basic medium, test tube specification is 15 × 150 mm, this basic medium volume 5 mL, 28 DEG C cultivate 7 d, until basic medium surface occur a large amount of mycelial growth and spore time based on it nutrient solution;
Described de-iron Cha Shi nutrient agar (g/L): NaNO
32.0 g, K
2hPO
41.0 g, KCl 0.5 g, MgSO
40.5 g, sucrose 30.0 g, agar 20.0 g, deionized water 1 L, pH nature, prepare rear 121 DEG C of sterilizing 20 min;
The composition of described de-iron Cha Shi basic medium and de-iron Cha Shi nutrient agar is distinguished and is: the Cha Shi basic medium of de-iron does not add agar, and all the other compositions are identical;
(2) screen
With the liquid medium adding 20 microlitre CAS dye liquors and isopyknic step (1) in the 96 every holes of orifice plate, abundant mixing, take color reaction as red or orange or brown color or purple fungi alternatively bacterial strain, it is prepared spore suspension by step 1 method, be inoculated in 100mL de-iron Cha Shi basic medium, 28 DEG C, 180rpm cultivates 5d;
Add 40 microlitre CAS dye liquors and isopyknic candidate strain nutrient solution with in the 96 every holes of orifice plate, fully mix, the record colour-change time, the shortest for the reaction used time, reaction color are changed difference bacterial strain FECH-998 greatly as preferred strain;
(3) preparation fermentation crude extract
Get step (2) 10 FECH-998 bacterial strains to prepare spore suspension and be inoculated in 10 × 200 mL sterilising liq de-iron Cha Shi substratum, 28 DEG C, 180 rpm cultivate 5 d, treat that having a large amount of mycelium pellet to grow in nutrient solution stops cultivating, with this nutrient solution for seed liquor;
(4) step (3) seed liquor is got, by 10% inoculative proportion aseptic straw, it is on average inoculated in 50 bottles of 500 mL triangular flasks that 200 mL sterilizing de-iron Cha Shi basic mediums are respectively housed, 28 DEG C ~ 32 DEG C, 180 rpm cultivate 3 d ~ 8 d, in triangular flask, throw in particle diameter 60 ~ 16 object macroporous adsorbent resin in 2% ratio when having a large amount of mycelium pellet to grow in nutrient solution, 28 DEG C ~ 32 DEG C, 180 rpm original positions stop cultivating after adsorbing 24 h, collect nutrient solution, filter; Bacterium slag and resin methanol extraction, filtrate is extracted with ethyl acetate, and obtains methanol extract and acetic acid ethyl ester extract;
(5) by methanol extract concentrating under reduced pressure, drying, yellow oil 23.8 g being mingled with a large amount of white fine needle crystal is obtained; By acetic acid ethyl ester extract concentrating under reduced pressure, drying, obtain brown light brown solid 11.1 g being mingled with a large amount of white fine needle crystal, both merging, as crude extract.
3. preparation method according to claim 2, is characterized in that: described step (4) replaces de-iron Cha Shi basic medium with high glucose medium, and this high sugar-fermenting substratum (g/L) is: sucrose 100.0 g, yeast extract paste 6.0 g, NaNO
32.0 g, K
2hPO
41.0 g, KCl 0.5 g, MgSO
40.5 g, deionized water 1 L, pH nature; Prepare rear 121 DEG C of sterilizing 20 min; In the fermented liquid of described high glucose medium, kojic acid content is 15.65g/L, and kojic acid conversion ratio is 0.157g kojic acid/g sucrose.
4. the preparation method according to Claims 2 or 3, is characterized in that further separation
Purification step (5) crude extract, comprising:
(1) normal phase silicagel column chromatographic grade wash-out, this chromatographic system gradient is: chloroform, methyl alcohol; CAS liquid development process follows the trail of the cut that each section of elutriant contains iron sequestering activity composition;
(2) cut evaporated under reduced pressure activeconstituents, removes oily matter by petroleum ether, dissolve with methanol, and under room temperature, periodic crystallisation and recrystallization obtain white fine acicular compound, and this compound has very strong iron sequestering activity.
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| CN114032178A (en) * | 2021-07-08 | 2022-02-11 | 西安文理学院 | Acid-producing bacterium JC-H, application thereof and culture and identification method of acid-producing bacterium JC-H |
| CN114032178B (en) * | 2021-07-08 | 2023-11-17 | 西安文理学院 | Acid-producing bacteria JC-H and application thereof, and culture and identification method of acid-producing bacteria JC-H |
| CN114181964A (en) * | 2021-11-02 | 2022-03-15 | 云南大学 | Expression cassette combination, recombinant vector, recombinant saccharomyces cerevisiae and application thereof |
| CN114181964B (en) * | 2021-11-02 | 2023-06-09 | 云南大学 | Expression cassette combination, recombinant vector, recombinant saccharomyces cerevisiae and application of recombinant saccharomyces cerevisiae |
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