CN104726347A - Penicillium vulpinum fungi strain and method for preparing levo 7-hydroxyl butylphthalide employing fungi strain - Google Patents
Penicillium vulpinum fungi strain and method for preparing levo 7-hydroxyl butylphthalide employing fungi strain Download PDFInfo
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Abstract
The invention discloses a penicillium vulpinum fungi strain and a method for preparing levo 7-hydroxyl butylphthalide ((S)-(-)-butyl-7-hydroxyl phthalide) employing the fungi strain. The fungi strain disclosed by the invention is a penicillium vulpinum fungi strain NCC3421 (penicillium vulpinum) and is preserved at the China General Microbiological Culture Collection Center (CGMCC for short); and the preservation number is CGMCC No.9094. Under the culture condition provided by the method, the fermentation unit of the levo 7-hydroxyl butylphthalide reaches over 1600mg/L; the fermented product is relatively single in components, and easy to separate and purify, and can be applied to industrialized production of the levo 7-hydroxyl butylphthalide.
Description
Technical field
The present invention relates to microbial technology field, specifically a strain fox excrement mould fungal bacterial strain and utilize this bacterial strain to prepare the method for left-handed 7-hydroxyl NBP ((S)-(-)-3-butyl-7-hydroxyphthalide).
Background technology
Left-handed 7-hydroxyl NBP is separated by Japanese scholars Makino M etc. the earliest and obtains [Heterocycles from fox excrement mould, 1998,48 (9): 1931-1933], other phthalide analog compounds application alone or in combination of this compound, has the illness (US20100184852A1) relevant with impaired neurotransmission and the diseases (US20060246157A1) relevant with vasculogenesis such as prevention and therapy diabetes (US20090192218Al), depression and anxiety.The 7-hydroxyl NBP access approaches reported in the world at present has: extraction in Ligusticum wallichii, chemosynthesis, be separated from fox excrement mould nutrient solution.
Open in patent US20060246157A1, extract from the active compound of Ligusticum wallichii left-handed 7-hydroxyl NBP can separately or with Vanillin, coniferyl ferulate and falcarindiol arbitrary combination, be used for the treatment of and prevent the medicine based on the disease of vasculogenesis and nutritive food.It is obtained by solvent extraction-silica gel column chromatography-HPLC wet chemical methods that this patent is separated left-handed 7-hydroxyl NBP, its complex steps.Left-handed 7-hydroxyl NBP content in Ligusticum wallichii is extremely low, and Ligusticum wallichii resource-constrained, be unsuitable for being that left-handed 7-hydroxyl NBP prepared on a large scale by raw material with Ligusticum wallichii.
Document Biosci Biotechnol Biochem, 2003,67 (10): the 2240-2244 synthetic methods having delivered 7-hydroxyl NBP two kinds of enantiomers, utilize Sharpless bishydroxy reagent A D-mix-α and AD-mix-β to carry out asymmetric synthesis respectively and obtain two kinds of optically pure enantiomorphs, and to prove that Makino M in 1998 etc. are separated the 7-hydroxyl NBP obtained from fox excrement mould be S configuration.
Document Tetrahedron Letters, 2014,55:1303 – 1305 reports, the propargyl alcohol utilizing lipase Novozym-435 to split racemization obtains key intermediate (S)-propargyl alcohol acetic ester (optical purity 95%), then by Alder – Rickert Reactive Synthesis left-handed 7-hydroxyl NBP.Key intermediate building-up reactions comprises 5 steps, yield 21.2%; Also need 3 step reactions from obtaining key intermediate to synthesis finished product, yield 35.8%, therefore total recovery only has 7.6%, and whole process relates to pyroreaction and uses multiple organic solvent, is not suitable for scale operation.
Document Heterocycles, 1998,48 (9): 1931-1934 report the left-handed 7-hydroxyl NBP of Late Cambrian, and source is fox excrement mould nutrient solution.In nutrient solution, left-handed 7-hydroxyl butylphthalide content is very low, and containing other components multiple, separation and purification process have employed the absorption of HP-20 post, silica gel column chromatography and HPLC preparation, complex process, product yield low (16mg just won by 10L fermented liquid), commercial application difficulty.
This patent discloses a strain fox excrement mould fungal bacterial strain and utilizes this bacterial strain to prepare the method for left-handed 7-hydroxyl NBP, under bacterial strain provided by the invention and culture condition, the fermentation unit of left-handed 7-hydroxyl NBP reaches more than 1600mg/L, and fermentation component is comparatively single, separation purifying technique is simple, may be used for the suitability for industrialized production of left-handed 7-hydroxyl NBP.
Summary of the invention
The object of this invention is to provide the superior strain of a strain left-handed 7-hydroxyl NBP, a kind of method utilizing this bacterial strain to prepare left-handed 7-hydroxyl NBP is provided simultaneously.
Left-handed 7-hydroxyl NBP producing strains provided by the present invention is fox excrement mould fungal bacterial strain, and its preservation name is called fox excrement mould
( penicillium vulpinum )nCC3421, depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date on April 25th, 2014, and deposit number is CGMCC No.9094, and preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Fungal bacterial strain NCC3421 provided by the present invention, through macroscopic view, microscopic morphology is observed and Molecular Identification is accredited as fox excrement mould
( penicillium vulpinum ).
Authentication method of the present invention is morphological specificity and Molecular Identification.The spore liquid of bacterial strain NCC3421 is inoculated in PDA substratum, and 25 DEG C of cultivations carry out microscopic morphology observation in 7 days under scanning electron microscope.By the spore liquid dibbling of bacterial strain NCC3421 in 4 kinds of general qualification substratum: CYA(Cha Shi yeast agar), MEA(malt meal agar), G25N(25% glycerophosphate agar), PDA(potato dextrose agar) in, cultivate 7 days at 25 DEG C, carry out macroscopic form observation.Carry out ITS sequence mensuration to NCC3421, submit to NCBI to compare, phylogenetic tree construction, with type strain comparison.Observe and Molecular Identification through macroscopic view, microscopic morphology, NCC3421 is accredited as fox excrement mould
( penicillium vulpinum ).
Fox excrement mould used in the present invention
( penicillium vulpinum )fungal bacterial strain NCC3421 is separated the strain wild strain obtained from the mountain soil of Baoxing County, Ya'an, Sichuan Province.
Collecting soil sample method of the present invention is scalp vegetable mould near plant root soil top layer with aseptic shovel, and then getting the distance earth's surface degree of depth is that the soil sample about 30 grams of 5 ~ 10cm is placed in sterilized paper bag and preserves.
The separation method of pedotheque of the present invention joins in 10mL sterilized water fully vibrate for getting 1 gram of this sample, and get 1mL suspension subsequently and join another and be equipped with in the test tube of 9mL sterilized water and fully vibrate, the rest may be inferred carries out gradient dilution value to 10
-10concentration.Each concentration is got 0.2mL and is coated with PDA two dish, subsequently quiescent culture at 26 DEG C, and two dish culture cycle is 3 ~ 10 days, and the colony inoculation that picking form is different from two dish is during this period in PDA(Beijing bispin) to cultivate in inclined-plane, the slant culture time is 14 days.A fungal strain is obtained, by its called after NCC3421 by aforesaid method.
Adopt preparation method of the present invention, the fermentation unit of its left-handed 7-hydroxyl NBP reaches more than 1600mg/L, and meta-bolites is single, therefore it can be applied in the left-handed 7-hydroxyl NBP of preparation.
The method utilizing fox excrement mould fungal bacterial strain to prepare left-handed 7-hydroxyl NBP provided by the present invention, comprises the following steps:
The seed liquor of a, preparation fox excrement mould fungal bacterial strain NCC3421:
The slant culture of bacterial strain NCC3421 or spore liquid are inoculated in seed culture medium, 22 ~ 30 DEG C, 100 ~ 220 rpm, cultivate 48 ~ 72 h obtain seed liquor;
Wherein said seed culture medium obtains by the following method: W-Gum 10.0 ~ 40.0 grams, glucose 10.0 ~ 40.0 grams, hot moulding soybean cake powder 2.0 ~ 10.0 grams, malt meal 1.0 ~ 6.0 grams, yeast powder 1.0 ~ 3.0 grams, 0.5 ~ 2.0 gram, sodium-chlor, magnesium sulfate heptahydrate 0.5 ~ 2.0 gram, add water and be settled to 1000mL, pH6.5 ~ 7.0,121 DEG C of sterilizing 30min.
The fermented liquid of b, preparation fox excrement mould fungal bacterial strain NCC3421:
Above-mentioned seed liquor is inoculated in fermention medium with the inoculum size of volume percent 3 ~ 10%, in shaking flask or fermentation cylinder for fermentation, leavening temperature 22 ~ 30 DEG C, 100 ~ 220 rpm, fermentation time 120 ~ 180 h, obtains fermented liquid;
Wherein said fermention medium obtains by the following method: glucose 10.0 ~ 40.0 grams, Zulkovsky starch 10.0 ~ 40.0 grams, glycerine 10.0 ~ 40.0 grams, hot moulding soybean cake powder 4.0 ~ 20.0 grams, cottonseed meal 4.0 ~ 20.0 grams, corn steep liquor 4.0 ~ 15.0 grams, potassium primary phosphate 1.0 ~ 5.0 grams, 1.0 ~ 5.0 grams, ammonium sulfate, 0.5 ~ 2.0 gram, sodium-chlor, magnesium sulfate heptahydrate 0.2 ~ 2.0 gram, add water and be settled to 1000mL, pH6.5 ~ 7.0,121 DEG C of sterilizing 30min.
C, by above-mentioned fermented liquid centrifugation, supernatant discarded obtains mycelium, adds methyl alcohol, ethanol or acetone extraction in mycelium, stirs, and filters, and filter residue repeats lixiviate once, and merge twice lixiviate filtrate, concentrating under reduced pressure obtains medicinal extract.
D, medicinal extract is added ethyl acetate, chloroform or normal hexane lixiviate, stir, leave standstill, be separated to obtain supernatant liquor, repeat lixiviate once, merge twice supernatant liquor, add anhydrous sodium sulfate dehydration, filter, filtrate decompression is dense dryly obtains oily crude extract.
E, by after oily crude extract methyl alcohol or dissolve with ethanol, be added on HZ816, D312 or AB-8 chromatographic resin post capital, with the polar organic solvent aqueous solution for eluent carries out wash-out, HPLC detects effluent liquid, collects containing left-handed 7-hydroxyl NBP part.Concentrating under reduced pressure evaporate to dryness, obtains left-handed 7-hydroxyl NBP crude product.
F, left-handed 7-hydroxyl NBP crude product is dissolved in crystallization in the polar organic solvent aqueous solution, filter, vacuum-drying obtains left-handed 7-hydroxyl NBP fine work.
Shake flask fermentation wherein described in step b, its rotating speed is 100 ~ 220rpm.
Ferment tank wherein described in step b, its tank pressure is 0.05 ± 0.01Mpa, air flow is 10 ~ 30L/min, and stirring velocity is 400 ~ 500rpm.
Wherein add alcohol steep in step c, the volume added is 1 ~ 3 times of mycelium volume, preferably adds the industrial alcohol of mycelium 2 times of volumes.
Wherein add ethyl acetate lixiviate in steps d, the volume added is 3 ~ 5 times of medicinal extract volume, preferably 5 times.
Wherein oily crude extract dissolve with methanol in step e, consumption is 2 times of oily crude extract volume, and eluent is methanol aqueous solution, aqueous ethanolic solution or aqueous acetone solution, and the preferred alcohol aqueous solution carries out three grades of gradient elutions, and gradient is 30%, 50%, 70%.Post blade diameter length ratio is the preferred 1:5 of 1:3 ~ 1:10, every grade of wash-out 5 ~ 10 times of column volumes, flow velocity 2 ~ 4 times of column volumes/hour, preferable flow rate 2 times of column volumes/hour.Collect the above elution fractions of left-handed 7-hydroxyl butylphthalide content 10mg/ml.
Wherein the crystallization of the step f polar organic solvent aqueous solution used is methanol aqueous solution, aqueous ethanolic solution or aqueous acetone solution, and the concentration expressed in percentage by volume of polar organic solvent is 70 ~ 80%, preferably carries out crystallization with methanol aqueous solution.
The wherein crystallisation process of step f, every gram of dissolving crude product crystallization in 20 ~ 30 milliliters of polar organic solvent aqueous solution, the time is between 2 ~ 24h.
Above-mentioned optimum condition, it is higher that it obtains left-handed 7-hydroxyl NBP output, better quality.
Fox excrement mould fungal bacterial strain NCC3421 used in the present invention is wild strain, and its left-handed 7-hydroxyl NBP fermentation unit reaches 1600 more than mg/L, and fermentation component is comparatively single, is easy to separation and purification.In the present invention left-handed 7-hydroxyl NBP preparation technology easy, be suitable for suitability for industrialized production, product total recovery reaches 60 more than %.
Accompanying drawing explanation
Fig. 1 is the coremium of bacterial strain.
Fig. 2 is the conidiophore of bacterial strain.
Fig. 3 is the conidia chain of bacterial strain.
Fig. 4 is the colonial morphology that bacterial strain is cultivated 7 days on CYA.
Fig. 5 is the colonial morphology that bacterial strain is cultivated 7 days on MEA.
Fig. 6 is the colonial morphology that bacterial strain is cultivated 7 days on G25N.
Fig. 7 is the colonial morphology that bacterial strain is cultivated 7 days on PDA.
Fig. 8 is the phylogenetic tree built according to the ITS sequence of NCC3421.
Fig. 9 is that NCC3421 fermented liquid thallus extract HPLC detects collection of illustrative plates.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.Experiment material used in following embodiment is industrial raw material if no special instructions; Analyze chromatographic column Inertsil ODS-SP 5 μm of C18 4.6 × 250 mm purchased from Japanese Shimadzu, leaching process organic solvent used is purchased from Beijing Chemical Plant, and chromatographic solvent is purchased from Honeywell trade (Shanghai) Co., Ltd..
the Isolation and Identification of embodiment 1, fox excrement mould NCC3421
The separation of (a) bacterial strain NCC3421
The separation substratum of fungal bacterial strain NCC3421 is PDA(Beijing bispin);
Get 1 gram of pedotheque (picking up from Baoxing County, Sichuan Province China province Ya'an mountain soil), put into the 50mL centrifuge tube that 9mL sterilized water is housed, fully vibrate.Get 1mL suspension liquid and put into the test tube that 9mL sterilized water is housed, mixing.Carry out serial dilutions until 10 successively
-10.Getting 0.2mL under each gradient is coated with dull and stereotyped.Be coated with flat board and be placed in 26 DEG C of quiescent culture, therefrom obtain a fungal strain, by its called after NCC3421.
The qualification of (b) bacterial strain NCC3421---morphological specificity and Molecular Identification
The spore liquid of bacterial strain NCC3421 is inoculated in PDA substratum, cultivates after 7 days for 25 DEG C and carry out microscopic morphology observation under scanning electron microscope.By the spore liquid dibbling of bacterial strain NCC3421 in 4 kinds of general qualification substratum: CYA(Cha Shi yeast agar), MEA(malt meal agar), G25N(25% glycerophosphate agar), PDA(potato dextrose agar) in, cultivate 7 days, carry out macroscopic form observation for 25 DEG C.
Carry out ITS sequence mensuration to NCC3421, submit to NCBI to compare, phylogenetic tree construction, with type strain comparison.
1) morphological specificity
The Photomicrograph of bacterial strain NCC3421, the conidiophore that the coremium that Fig. 1 is bacterial strain, Fig. 2 are bacterial strain, Fig. 3 are the conidia chain of bacterial strain, and conidiophore is combined closely the coremium in handle as seen from the figure; Falx stem is thin and bend, smooth surface; The irregular branch of penicillus, is entangled with into thalamium mutually; Metulae 8-12 μm; Each metulae has 3-5 bottle stalk, 10-12 μm, bottle stalk neck is short and attenuate suddenly; Conidium is oval, smooth, general 3.5-4.0 μm, have long and narrow spore every, illumination long-chain, covers coremium top and forms a column.
The morphological specificity of bacterial strain NCC3421 in 4 kinds of general qualification substratum is as follows:
NCC3421 is at CYA(Cha Shi yeast agar) upper 7 day, colony diameter reaches 30-34mm; Bacterium colony superficial white is velvet-like; There is the radial lines compared with regular distribution; Centre produces large-scale coremium, the tight and irregular tufted growth of handle, and top covers abundanter greyish-green spore; The coremium comparatively dense tufted growth of proximal edge position presents endless belt; Part coremium there is drop; The radial lines in bacterium colony back is clear in comparatively regular distribution; Coremium back is brown.The form that NCC3421 cultivates 7 days bacterium colonies on CYA is shown in Fig. 4.
NCC3421 is at MEA(malt meal agar) upper 7 day, colony diameter reaches 24-26mm; Bacterium colony superficial white is velvet-like; Nearly central part coremium tufted dense growth, present endless belt, coremium is more elongated, is upwards that rhodo is to white gradual change by base portion; The extremely short and small coremium of edge adularescent; Surface is without obvious radial lines; The radial lines in bacterium colony back is clear in comparatively regular distribution; Coremium back is brown.The form that NCC3421 cultivates 7 days bacterium colonies on MEA is shown in Fig. 5.
NCC3421 is at G25N(25% glycerophosphate agar) upper 7 day, colony diameter reaches 16-18mm; Bacterium colony superficial white is velvet-like; Central part is protuberance slightly, and comparatively dense tufted is diametrically 4-6mm greyish-green subcircular; There is the radial lines compared with regular distribution; Bacterium colony back is light yellow; Radial lines is clear, in brown color.The form that NCC3421 cultivates 7 days bacterium colonies on G25N is shown in Fig. 6.
NCC3421 is at PDA(potato dextrose agar) upper 7 day, colony diameter reaches 24-26mm; Bacterium colony superficial white staple; Bacterium colony central authorities have 1-2 white short and small coremium once in a while, and top covers greyish-green spore; The extremely short and small coremium of edge adularescent; Bacterium colony back white is to light yellow; Coremium back is brown.The form that NCC3421 cultivates 7 days bacterium colonies on PDA is shown in Fig. 7.
2) Molecular Identification
The ITS sequence of NCC3421 after measured length is 582bp:
Tccgtaggtgaacctgcggaaggatcattaccgagctgacggcctctgggtccacctcccacccgtgtttatttaccttgttgcttcggcgggcccgccttaactggccgccggggggcttccgcccccgggcccgcgcccgccgaagacacccccgaactctgtctgaagattgcagtctgagtgaaaatataaattatttaaaactttcaacaacggatctcttggttccggcatcgatgaagaacgcagcgaaatgcgatacgtaatgtgaattgcaaattcagtgaatcatcgagtctttgaacgcacattgcgccccctggtattccggggggcatgcctgtccgagcgtcatttctgccctcaagcccggcttgtgtgttgggccccgtcccccgatcccgggggacgggcccgaaaggcagcggcggcaccgcgtccggtcctcgagcgtatggggctttgtcacccgctctgtaggcccggccggcgcctgccaccaacccaaatttttatccaggttgacctcggatcaggtagggatacccgctgaacttaagcatatcaataagcggagga
Submit to NCBI to compare sequence, and phylogenetic tree construction, according to the phylogenetic tree (see figure 8) that the ITS sequence of NCC3421 builds, can find out, typical strain fox excrement mould in this bacterium and Penicillium mould subgenus (
penicillium vulpinumnRRL 1002) be in a branch, illustrate that the sibship between them is nearer.
According to macroscopic view, micro-morphology and Molecular Identification, NCC3421 is accredited as fox excrement mould (
penicillium vulpinum).
the preparation of embodiment 2,5L tank fermentation 7-hydroxyl NBP
A () prepares fox excrement penicillium bacterial strain NCC3421 seed liquor
Fox excrement penicillium bacterial strain NCC3421 is inoculated in seed culture medium, 26 DEG C, rotating speed 200 rpm cultivate 72 h obtain seed liquor.The preparation method of seed culture medium is: W-Gum 20.0 grams, glucose 10.0 grams, hot moulding soybean cake powder 2.0 grams, malt meal 6.0 grams, yeast powder 3.0 grams, 2.0 grams, sodium-chlor, magnesium sulfate heptahydrate 1.0 grams, add tap water dissolving and be settled to 1000 ml, pH value 6.8,121 DEG C of sterilizing 30 min.
B () prepares the fermented liquid of fox excrement penicillium bacterial strain NCC3421
Seed liquor is inoculated in the fermentor tank that 5 L fermention mediums are housed with the inoculum size of volume percent 5%, tank temperature 26 DEG C, tank pressure 0.05 ± 0.01 Mpa, air flow 30 L/min, 220 rpm stir, and ferment 168 h.The preparation method of fermention medium is: glucose 14.5 grams, Zulkovsky starch 18.0 grams, glycerine 21.5 grams, hot moulding soybean cake powder 15.5 grams, cottonseed meal 8.5 grams, corn steep liquor 8.5 grams, potassium primary phosphate 2.5 grams, 2.5 grams, ammonium sulfate, 1.2 grams, sodium-chlor, magnesium sulfate heptahydrate 0.4 gram, add tap water and be settled to 1000mL, pH6.5,121 DEG C of sterilizing 30min.After fermentation, left-handed 7-hydroxyl NBP fermentation unit is 1650mg/L after testing.
C () gets the fermented liquid 2.0 L(left-handed 7-hydroxyl butylphthalide content 1650mg/L of fox excrement penicillium bacterial strain NCC3421) centrifugal 20min in the whizzer of 4000r/min, collect mycelia and be about 500ml, add the lixiviate of 1000ml industrial acetone, stir 1 hour, filter, collect filtrate.Repeat lixiviate once.Merge twice filtrate and be about 2.2L.Concentrating under reduced pressure reclaims acetone and removes large portion moisture, obtains medicinal extract and is about 80g.
D () 80g medicinal extract adds 320ml chloroform, stir 30min, leaves standstill 1 hour, separates supernatant.Repeat once.Merge supernatant liquor, evaporated under reduced pressure reclaims chloroform, obtains about 3.9ml oily crude extract.
E (), with AB-8 resin dress post, column internal diameter 3.2cm, post bed height is about 16cm.Post bed is rinsed with 500ml salt-free water, for subsequent use.3.9ml crude extract is dissolved in 8ml methyl alcohol, and filter, filtrate is added on the AB-8 chromatography column capital handled well at twice, and all use 200ml salt-free water to rinse resin column after each application of sample, teeter column top board knot is divided a little if desired.Then carry out gradient elution with each 1200ml of 30%, 50%, 70% ethanol water to resin column respectively, flow velocity 8.5ml/min, HPLC detect elutriant.When 70% ethanol water section wash-out, collect the above elution fractions of left-handed 7-hydroxyl butylphthalide content 10mg/ml, pressure reducing and steaming ethanol, filter and drain to obtain left-handed 7-hydroxyl NBP crude product 2.5g.
F () left-handed 7-hydroxyl NBP crude product is about 2.5g, with 52ml dissolve with methanol, drip about 22ml distilled water, micro-aobvious muddiness.The warm solution that makes is clarified, and crystal is slowly separated out, crystallization 2 hours, filters.Vacuum-drying, obtains left-handed 7-hydroxyl NBP fine work 2.1g, content 99.5%, total recovery 63.3%.
(g) left-handed 7-hydroxyl NBP, water white transparency sheet or styloid; Be soluble in methyl alcohol, ethanol, chloroform, ethyl acetate, be dissolved in normal hexane, be slightly soluble in water; Maximum absorption band is had at 234 nm, 298 nm places; HRMS shows its molecular ion peak [M+H]
+be 207.1015; Actual measurement [α]=-48.5
o(c, 0.8, CHCl
3).Pure state room-temperature stable, stable under solution state, more stable under direct sunlight, less stable when soda acid exists.
What the hydrocarbon signals assignment of left-handed 7-hydroxyl NBP was reported with document * compares as table 1.Nucleus magnetic resonance testing tool be Varian Inova 500 MHz (
1h), 125MHz (
13c).
Table 1 is made by oneself and reported in literature left-handed 7-hydroxyl NBP
13c-NMR and
1h-NMR attribution data table
Document * is reported as HETEROCYCLES, Vol.48, NO.9,1998, P1931-1934
The liquid chromatographic detection condition of (h) left-handed 7-hydroxyl NBP
Chromatographic column: Inertsil ODS-SP 5 μm of C18 4.6 × 250 mm
Moving phase: A acetonitrile; B water, containing 0.05% phosphoric acid.Flow velocity 1ml/min.Gradient is arranged as following table.
Detector: UV, 210nm.
NCC3421 fermented liquid thallus extract HPLC detects collection of illustrative plates (see figure 9).From the UV 3D collection of illustrative plates gathered, apart from outside some strong extreme type-I in NCC3421 fermented liquid thalline ethanol extract, primary product is left-handed 7-hydroxyl NBP (retention time is about 17.2min).
(I) the liquid chromatography rapid detection condition of left-handed 7-hydroxyl NBP
Chromatographic column: Inertsil ODS-SP 5 μm of C18 4.6 × 250 mm
Moving phase: acetonitrile/water=60/40, containing 0.05 phosphoric acid; Flow velocity 1ml/min
Detector: UV, 210nm.
Under above-mentioned condition, the retention time of left-handed 7-hydroxyl NBP is about 5.9min.
the preparation of embodiment 3, shake flask fermentation 7-hydroxyl NBP
A () prepares the seed liquor of fox excrement penicillium bacterial strain NCC3421
Fox excrement penicillium bacterial strain NCC3421 is inoculated in seed culture medium, 26 DEG C, rotating speed 200 rpm cultivate 72 h obtain seed liquor.The preparation method of seed culture medium is: W-Gum 20.0 grams, glucose 10.0 grams, hot moulding soybean cake powder 2.0 grams, malt meal 6.0 grams, yeast powder 3.0 grams, 2.0 grams, sodium-chlor, magnesium sulfate heptahydrate 1.0 grams, add tap water dissolving and be settled to 1000 ml, pH value 6.5,121 DEG C of sterilizing 30 min.
B () prepares the fermented liquid of fox excrement penicillium bacterial strain NCC3421
Seed liquor is inoculated in the 250mL shaking flask that 40mL fermention medium is housed with the inoculum size of volume percent 5%, 26 DEG C, rotating speed 200 rpm, ferment 144 h.The preparation method of fermention medium is: glucose 20.5 grams, Zulkovsky starch 18.0 grams, glycerine 21.5 grams, hot moulding soybean cake powder 15.5 grams, cottonseed meal 15.5 grams, corn steep liquor 8.5 grams, potassium primary phosphate 2.5 grams, 2.5 grams, ammonium sulfate, 0.5 gram, sodium-chlor, magnesium sulfate heptahydrate 0.2 gram, add tap water and be settled to 1000mL, pH6.7,121 DEG C of sterilizing 30min.After fermentation, left-handed 7-hydroxyl NBP fermentation unit is 1638mg/L after testing.
C () gets the fermented liquid 2.0 L(left-handed 7-hydroxyl butylphthalide content 1638mg/L of fox excrement penicillium bacterial strain NCC3421) centrifugal 20min in the whizzer of 4000r/min, collect mycelia and be about 500ml, add the lixiviate of 1000ml industrial alcohol, stir 1 hour, filter, collect filtrate.Repeat lixiviate once.Merge twice filtrate and be about 2.2L.Concentrating under reduced pressure reclaims ethanol and removes large portion moisture, obtains medicinal extract and is about 77g.
D () 77g medicinal extract adds 250ml ethyl acetate, stir 30min, leaves standstill 1 hour, separates supernatant.Repeat once.Merge supernatant liquor, evaporated under reduced pressure reclaims ethyl acetate, obtains about 3.7ml oily crude extract.
E (), with HZ816 resin dress post, column internal diameter 3.2cm, post bed height is about 16cm.Post bed is rinsed with 500ml salt-free water, for subsequent use.3.7ml crude extract is dissolved in 8ml methyl alcohol, and filter, filtrate is added on the HZ816 chromatography column capital handled well at twice, and all use 200ml salt-free water to rinse resin column after each application of sample, teeter column top board knot is divided a little if desired.Then carry out gradient elution with each 650ml of 30%, 50%, 70% ethanol water to resin column respectively, flow velocity 4.5ml/min, HPLC detect elutriant.When 70% ethanol water section wash-out, collect the above elution fractions of left-handed 7-hydroxyl butylphthalide content 10mg/ml, pressure reducing and steaming ethanol, filter and drain to obtain left-handed 7-hydroxyl NBP crude product 2.4g.
F () left-handed 7-hydroxyl NBP crude product is about 2.4g, with 56ml dissolve with methanol, drip about 14ml distilled water, micro-aobvious muddiness.The warm solution that makes is clarified, and crystal is slowly separated out, crystallization 10 hours, suction filtration, vacuum-drying, obtains left-handed 7-hydroxyl NBP fine work 2.0g, content 99.4%, total recovery 60.7%.
the preparation of embodiment 4, shake flask fermentation 7-hydroxyl NBP
A () prepares the seed liquor of fox excrement penicillium bacterial strain NCC3421
Fox excrement penicillium bacterial strain NCC3421 is inoculated in seed culture medium, 22 DEG C, rotating speed 100 rpm cultivate 66 h obtain seed liquor.The preparation method of seed culture medium is: W-Gum 40.0 grams, glucose 20.0 grams, hot moulding soybean cake powder 5.5 grams, malt meal 4.0 grams, yeast powder 3.0 grams, 0.5 gram, sodium-chlor, magnesium sulfate heptahydrate 0.5 gram, add tap water dissolving and be settled to 1000 ml, pH value 6.5,121 DEG C of sterilizing 30 min.
B () prepares the fermented liquid of fox excrement penicillium bacterial strain NCC3421
Seed liquor is inoculated in the 250mL shaking flask that 40mL fermention medium is housed with the inoculum size of volume percent 10%, 22 DEG C, rotating speed 100 rpm, ferment 180 h.The preparation method of fermention medium is: glucose 10.0 grams, Zulkovsky starch 40.0 grams, glycerine 10.0 grams, hot moulding soybean cake powder 20.0 grams, cottonseed meal 4.0 grams, corn steep liquor 4.0 grams, potassium primary phosphate 1.0 grams, 1.0 grams, ammonium sulfate, 2.0 grams, sodium-chlor, magnesium sulfate heptahydrate 2.0 grams, add tap water and be settled to 1000mL, pH6.5,121 DEG C of sterilizing 30min.Must about 2L fermented liquid after fermentation, 7-hydroxyl NBP fermentation unit is 1696mg/L after testing.
C () gets the fermented liquid 2.0 L(left-handed 7-hydroxyl butylphthalide content 1696mg/L of fox excrement penicillium bacterial strain NCC3421) centrifugal 20min in the whizzer of 4000r/min, collect mycelia and be about 500ml, add the lixiviate of 1000ml methyl alcohol, stir 1 hour, filter, collect filtrate.Repeat lixiviate once.Merge twice filtrate and be about 2.2L.Concentrating under reduced pressure reclaims methyl alcohol and removes large portion moisture, obtains medicinal extract and is about 83g.
D () 83g medicinal extract adds 400ml normal hexane, stir 30min, leaves standstill 1 hour, separates supernatant.Repeat once.Merge supernatant liquor, evaporated under reduced pressure reclaims normal hexane, obtains about 3.5ml oily crude extract.
E (), with D312 resin dress post, column internal diameter 3.2cm, post bed height is about 16cm.Post bed is rinsed with 500ml salt-free water, for subsequent use.3.5ml crude extract is dissolved in 7ml methyl alcohol, and filter, filtrate is added on the D312 chromatography column capital handled well at twice, and all use 200ml salt-free water to rinse resin column after each application of sample, teeter column top board knot is divided a little if desired.Then carry out gradient elution with each 1000ml of 30%, 50%, 70% ethanol water to resin column respectively, flow velocity 6ml/min, HPLC detect elutriant.When 70% ethanolic moiety wash-out, collect the above elution fractions of left-handed 7-hydroxyl butylphthalide content 10mg/ml, pressure reducing and steaming ethanol, filter and drain to obtain left-handed 7-hydroxyl NBP crude product 2.5g.
F () left-handed 7-hydroxyl NBP crude product is about 2.5g, with 37ml dissolve with methanol, drip about 13ml distilled water, micro-aobvious muddiness.The warm solution that makes is clarified, and crystal is slowly separated out, crystallization 20 hours, suction filtration, filter cake vacuum-drying, obtains left-handed 7-hydroxyl NBP fine work 2.2g, content 99.5%, total recovery 64.5%.
the preparation of embodiment 5, shake flask fermentation 7-hydroxyl NBP
A () prepares the seed liquor of fox excrement penicillium bacterial strain NCC3421
Fox excrement penicillium bacterial strain NCC3421 is inoculated in seed culture medium, 30 DEG C, rotating speed 220 rpm cultivate 48 h obtain seed liquor.The preparation method of seed culture medium is: W-Gum 10.0 grams, glucose 40.0 grams, hot moulding soybean cake powder 10.0 grams, malt meal 1.5 grams, yeast powder 1.0 grams, 1.0 grams, sodium-chlor, magnesium sulfate heptahydrate 2.0 grams, add tap water dissolving and be settled to 1000 ml, pH value 7.0,121 DEG C of sterilizing 30 min.
B () prepares the fermented liquid of fox excrement penicillium bacterial strain NCC3421
Seed liquor is inoculated in the 250mL shaking flask that 40mL fermention medium is housed with the inoculum size of volume percent 3%, 30 DEG C, rotating speed 220 rpm, ferment 120 h.The preparation method of fermention medium is: glucose 40.0 grams, Zulkovsky starch 10.0 grams, glycerine 40.0 grams, hot moulding soybean cake powder 4.0 grams, cottonseed meal 20.0 grams, corn steep liquor 15.0 grams, potassium primary phosphate 5.0 grams, 5.0 grams, ammonium sulfate, 0.5 gram, sodium-chlor, magnesium sulfate heptahydrate 1.0 grams, add tap water and be settled to 1000mL, pH value 7.0,121 DEG C of sterilizing 30min.Obtain fermented liquid after fermentation and be about 2L, 7-hydroxyl NBP fermentation unit is 1739mg/L after testing.
C () gets the fermented liquid 2.0 L(left-handed 7-hydroxyl butylphthalide content 1739mg/L of fox excrement penicillium bacterial strain NCC3421) centrifugal 20min in the whizzer of 4000r/min, collect mycelia and be about 500ml, add the lixiviate of 1000ml industrial acetone, stir 1 hour, filter, collect filtrate.Repeat lixiviate once.Merge twice filtrate and be about 2.2L.Concentrating under reduced pressure reclaims acetone and removes large portion moisture, obtains medicinal extract and is about 85g.
D () 85g medicinal extract adds 300ml ethyl acetate, stir 30min, leaves standstill 1 hour, separates supernatant.Repeat once.Merge supernatant liquor, evaporated under reduced pressure reclaims ethyl acetate, obtains about 4.1ml oily crude extract.
E (), with D312 resin dress post, column internal diameter 3.2cm, post bed height is about 16cm.Post bed is rinsed with 500ml salt-free water, for subsequent use.4.1ml crude extract is dissolved in 8ml methyl alcohol, and filter, filtrate is added on the D312 chromatography column capital handled well at twice, and all use 200ml salt-free water to rinse resin column after each application of sample, teeter column top board knot is divided a little if desired.Then carry out gradient elution with each 800ml of 30%, 50%, 70% ethanol water to resin column respectively, flow velocity 5ml/min, HPLC detect elutriant.When 70% ethanolic moiety wash-out, collect the above elution fractions of left-handed 7-hydroxyl butylphthalide content 10mg/ml, pressure reducing and steaming ethanol, filter and drain to obtain left-handed 7-hydroxyl NBP crude product 2.7g.
F () left-handed 7-hydroxyl NBP crude product is about 2.7g, with 60ml dissolve with ethanol, drip about 20ml distilled water, micro-aobvious muddiness.The warm solution that makes is clarified, and crystal is slowly separated out, crystallization 24 hours, suction filtration, vacuum-drying, obtains left-handed 7-hydroxyl NBP fine work 2.3g, content 99.3%, total recovery 65.7%.
Claims (10)
1. a strain fox excrement mould fungal bacterial strain NCC3421(
penicillium vulpinum), its deposit number is CGMCC No.9094.
2. utilize the bacterial strain described in claim 1 to prepare a method for left-handed 7-hydroxyl NBP, comprise the following steps:
The seed liquor of a, preparation fox excrement mould fungal bacterial strain NCC3421:
The slant culture of bacterial strain NCC3421 or spore liquid are inoculated in seed culture medium, 22 ~ 30 DEG C, 100 ~ 220 rpm, cultivate 48 ~ 72 h obtain seed liquor;
Wherein said seed culture medium obtains by the following method: W-Gum 10.0 ~ 40.0 grams, glucose 10.0 ~ 40.0 grams, hot moulding soybean cake powder 2.0 ~ 10.0 grams, malt meal 1.0 ~ 6.0 grams, yeast powder 1.0 ~ 3.0 grams, 0.5 ~ 2.0 gram, sodium-chlor, magnesium sulfate heptahydrate 0.5 ~ 2.0 gram, add water and be settled to 1000mL, pH6.5 ~ 7.0,121 DEG C of sterilizing 30min;
The fermented liquid of b, preparation fox excrement mould fungal bacterial strain NCC3421:
Above-mentioned seed liquor is inoculated in fermention medium with the inoculum size of volume percent 3 ~ 10%, and in shaking flask or fermentation cylinder for fermentation, leavening temperature 22 ~ 30 DEG C, 100 ~ 220 rpm, fermentation time 120 ~ 180 h, obtain fermented liquid;
Wherein said fermention medium obtains by the following method: glucose 10.0 ~ 40.0 grams, Zulkovsky starch 10.0 ~ 40.0 grams, glycerine 10.0 ~ 40.0 grams, hot moulding soybean cake powder 4.0 ~ 20.0 grams, cottonseed meal 4.0 ~ 20.0 grams, corn steep liquor 4.0 ~ 15.0 grams, potassium primary phosphate 1.0 ~ 5.0 grams, 1.0 ~ 5.0 grams, ammonium sulfate, 0.5 ~ 2.0 gram, sodium-chlor, magnesium sulfate heptahydrate 0.2 ~ 2.0 gram, add water and be settled to 1000mL, pH6.5 ~ 7.0,121 DEG C of sterilizing 30min;
C, by above-mentioned fermented liquid centrifugation, supernatant discarded obtains mycelium, adds methyl alcohol, ethanol or acetone extraction in mycelium, stirs, and filters, and filter residue repeats lixiviate once, and merge twice lixiviate filtrate, concentrating under reduced pressure obtains medicinal extract;
D, medicinal extract is added ethyl acetate, chloroform or normal hexane lixiviate, stir, leave standstill, be separated to obtain supernatant liquor, repeat once, merge twice supernatant liquor, add anhydrous sodium sulfate dehydration, filter, filtrate decompression is dense dryly obtains oily crude extract;
E, by after oily crude extract methyl alcohol or dissolve with ethanol, be added on HZ816, D312 or AB-8 chromatographic resin post capital, with the polar organic solvent aqueous solution for eluent carries out wash-out, HPLC detects effluent liquid, collects containing left-handed 7-hydroxyl NBP part,
Concentrating under reduced pressure evaporate to dryness, obtains left-handed 7-hydroxyl NBP crude product;
F, above-mentioned left-handed 7-hydroxyl NBP crude product is dissolved in crystallization in the polar organic solvent aqueous solution, filter, vacuum-drying obtains left-handed 7-hydroxyl NBP fine work.
3. method according to claim 2, wherein adds alcohol steep in step c.
4. method according to claim 2, wherein adds ethyl acetate lixiviate in steps d.
5. method according to claim 2, wherein oily crude extract dissolve with methanol in step e, chromatographic resin is HZ816, and eluent is methanol aqueous solution, aqueous ethanolic solution or aqueous acetone solution.
6. method according to claim 5, wherein step e eluent methanol aqueous solution, aqueous ethanolic solution or aqueous acetone solution are three grades of gradients 30%, 50%, 70% wash-outs, every grade of wash-out 5 ~ 10 times of column volumes, flow velocity 2 ~ 4 times of column volumes/hour.
7. method according to claim 2, wherein the crystallization of the step f polar organic solvent aqueous solution used is methanol aqueous solution, aqueous ethanolic solution or aqueous acetone solution, and the concentration expressed in percentage by volume of polar organic solvent is 70 ~ 80%.
8. method according to claim 7, the wherein crystallisation process of step f, every gram of dissolving crude product crystallization in 20 ~ 30 milliliters of polar organic solvent aqueous solution, the time is between 2 ~ 24h.
9. method according to claim 2, the shake flask fermentation wherein described in step b, its rotating speed is 100 ~ 220rpm.
10. method according to claim 2, the ferment tank wherein described in step b, its tank pressure is 0.05 ± 0.01Mpa, air flow is 10 ~ 30L/min, and stirring velocity is 400 ~ 500rpm.
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| CN112226370A (en) * | 2019-07-15 | 2021-01-15 | 浙江泛亚生物医药股份有限公司 | New strain of penicillium fox dung and application thereof |
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| CN114762699A (en) * | 2021-01-13 | 2022-07-19 | 浙江泛亚生物医药股份有限公司 | External preparation of penicillium fox dung culture |
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| WO2024027251A1 (en) * | 2022-08-03 | 2024-02-08 | 华北制药集团新药研究开发有限责任公司 | Method for producing 7-hydroxybutylphthalide by means of fermentation of penicillium vulpinum |
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