CN109468233A - A kind of Fusidic Acid superior strain and its selection and application - Google Patents

A kind of Fusidic Acid superior strain and its selection and application Download PDF

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CN109468233A
CN109468233A CN201811242444.0A CN201811242444A CN109468233A CN 109468233 A CN109468233 A CN 109468233A CN 201811242444 A CN201811242444 A CN 201811242444A CN 109468233 A CN109468233 A CN 109468233A
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高健
黄巍巍
徐虹
李军
张丽
薛锋
倪浩
熊璐璐
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Yancheng Institute of Technology
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Abstract

The invention discloses a kind of Fusidic Acid superior strain and its selection and applications.The entitled fusidinic acid rouge coccus NJWW-0520 (Fusidium coccineum NJWW-0520) of the Fusidic Acid superior strain, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, the deposit date is on March 21st, 2018, deposit number was CGMCC No.15473.The present invention selects the Fusidic Acid superior strain by that stress orient domestication to Fusidic Acid production bacterial strain progress long lasting for using DMSO (dimethyl sulfoxide).The yield that naturalized strain fermentation produces Fusidic Acid is up to 4932 μ g/ml, improves 53.88% than starting strain yield (3205 μ g/ml), efficiently prepares in Fusidic Acid production in practical bioanalysis and is sufficiently applied.

Description

A kind of Fusidic Acid superior strain and its selection and application
Technical field
The invention belongs to the technical field of Fermentation Engineering and microorganism fungus kind breeding, in particular to a kind of Fusidic Acid high yield Bacterial strain and its selection and application.
Background technique
Fusidic Acid, molecular formula C31H48O6, molecular weight 516.69, chemical name are as follows: trans- -3 β of 16 α-carboxyl, 11 β - - 5 β of dihydroxy -4 β, 8 β, 14 α-trimethyl -18- demethyl, 10 α-cholesteric-(17Z) -17 (20), sour half water of 24- diene -21- Object is closed, there is steroidal spline structure, molecular structure and cigarette song spore acid, cephalosporin suffer from higher similarity, small part base Group has differences, and the presence of difference group enhances its bacteriostasis.It is demonstrated experimentally that Fusidic Acid water solubility is atomic, fat-soluble Preferably, and sodium fusidate is soluble easily in water.
Fusidic Acid is a kind of important narrow-spectrum antibiotic, to G+Bacterium has very strong inhibitory activity.Drug commodity mainly have The types such as sodium fusidate ointment, sodium fusidate injection and dry suspensoid agent, these different dosage forms are applicable to by various quick Feel the illness that the infection of bacterium causes, as sodium fusidafe as injection can be used for: extremities joint infection, septicemia (are drawn by bacterium Rise), cardiac intima inflammation, cystic fibrosis, osteomyelitis, skin structure infections, pneumonia and body traumatic infection etc. everywhere； Sodium fusidate ointment is applicable to treatment due to staphylococcus, streptococcus, minimum corynebacteria and other pairs of sodium fusidates Infection etc. caused by sensitive bacterial.Fusidic Acid belongs to steroidal compounds, due to the presence of asymmetric center in its molecule, chemistry Synthesis step is cumbersome, at high cost, yield is low, and chemical method is difficult to apply in the production of actual industrial, main both at home and abroad at present It to be prepared using Production by Microorganism Fermentation.Fusidic Acid is isolated from the secondary metabolite of fungi fermentation, wherein with Acremonium mold fermentation produces Fusidic Acid yield highest.
Liposoluble sex steroid sample Fusidic Acid is primarily involved in the formation of cell membrane in bacterial strain, and chemical structure is unique, can mention The toughness of high cell membrane.When, there are when organic solvent, to avoid cell itself from coming to harm, thallus can be generated in bacterial culture fluid Stress reaction synthesizes more Fusidic Acids, and cell membrane is promoted to enhance stability, forms barrier and organic solvent is prevented to enter thallus Cell causes to damage to it.
Response is quickly generated to the variation of external environmental condition, be the microorganism advantage that can adapt to and survive as early as possible it One.The variation of living environment promotes micropopulation bulk properties that corresponding adjustment occurs.It is micro- in the adverse circumstance existing for organic solvent Biological cell choosing multiple mode is adapted, the main change for including cell membrane component feature and accordingly forming: for example, cell Membrane permeability variation, long-chain unsaturated fatty acid, phosphoethanolamine and phosphoinositide, ergosterol, Fusidic Acid, trehalose etc. Constituent content increases, impact albumen synthesis, mitochondria activity and plasma membrane ATPase increased activity etc., and above-mentioned It influences each other, interact again between factor, therefore, cell is complicated to the acknowledgement mechanism of organic solvent, and reaction system is related to multilayer It is secondary, many-sided.And the changes of contents plays of the steroidals such as cell membrane itself permeability and steroid, lipid.
Long lasting for ground using the highly polar organic solvent of high concentration to Fusidic Acid production bacterial strain progress stress orient it is tame and docile Change, change the physiological status of production strain cell film, enhances it and resist organic solvent toxic action, and then it is high to obtain Fusidic Acid Bacterial strain is produced, this method is a kind of effective technological means, is conducive to strain improvement improvement.
Summary of the invention
The shortcomings that in order to overcome present strain and deficiency, the purpose of the present invention is to provide a kind of Fusidic Acid superior strains And its selection and application, the yield that naturalized strain fermentation produces Fusidic Acid is up to 4932 μ g/ml, than starting strain yield (3205 μ g/ml) improves 53.88%, efficiently prepares in Fusidic Acid production in practical bioanalysis and is sufficiently applied.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of Fusidic Acid superior strain, the entitled spherical shape shuttle pink mold NJWW-0520 (Fusidium of bacterial strain Coccineum NJWW-0520), it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation Location is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, and the deposit date is March 21 in 2018 Day, deposit number is CGMCC No.15473.
The Fusidic Acid superior strain is grown in PDA culture medium, 27 DEG C of culture 6d, colony diameter 4-6mm, bacterium Colours white is fallen to canescence, occasionally has pale pink, aerial hyphae is enriched, substrate mycelium and spore are less, and there is sporogenesis at center Small particles sample protrusion, have a slight diffusate, no soluble pigment, bacterium colony integrality is more stiff.
A kind of selection of Fusidic Acid superior strain, comprising the following steps:
(1) preliminary screening of resistance to highly polar organic solvent Fusidic Acid production bacterial strain
The starting strain of Fusidic Acid production bacterial strain after solid medium culture 5-6d is accessed into first order seed culture In base, expand in culture medium after 27 DEG C of 72 ± 5h of culture by 1% inoculum concentration second level of transferring, 27 DEG C of 25 ± 5h of culture, then press 10% inoculum concentration is transferred in fermentation medium, and DMSO, the final concentration of percent by volume of DMSO is added after 27 DEG C of culture 3d 12%, pressure stress dispose 6h, be coated on PDA solid plate after shaken cultivation on constant-temperature table；After strong point bacterium colony, choose Choosing is grown fine, bacterium colony is biggish accesses primary-seed medium again, carries out another wheel screening；Wherein, the starting of DMSO is dense Degree is percent by volume 12%, and every wheel screening DMSO concentration improves percent by volume 1% than last round of concentration, until culture medium Several single colonies can only be grown on plate；
(2) the highly polar organic solvent bacterial strain acclimating of enduring high-concentration and plate separation secondary screening choosing
In the resistant strains access seed culture medium that primary dcreening operation is obtained, transfer after 27 DEG C of 72 ± 5h of culture by 1% inoculum concentration Enter second level to expand in culture medium, 27 DEG C of 25 ± 5h of culture, then transfer in fermentation medium by 10% inoculum concentration, 27 DEG C are cultivated DMSO is added after 3d, the DMSO concentration is the maximum concentration of DMSO used in step (1), in constant-temperature shaking culture on shaking table After be coated on PDA solid plate, select the best bacterium colony of growing way and carry out next-generation domestication；Step (1) institute is pressed by five generations The DMSO acclimating culture of the maximum concentration used, obtains Fusidic Acid superior strain.
Preferably, the composition of the primary-seed medium are as follows: seed culture medium contains glucose 2.5%, fish meal protein peptone 1.0%, yeast powder 0.2%, KH2PO4 0.1%, MgSO4 0.05%, corn pulp 4.0%, precipitated calcium carbonate 0.2%, pH 5.9±0.1。
Preferably, the second level expands the composition of culture medium are as follows: glucose 2.5%, fish meal protein peptone 1.0%, yeast powder 0.2%, KH2PO4 0.1%, MgSO4 0.05%, corn pulp 4.0%, precipitated calcium carbonate 0.2%, pH 5.9 ± 0.1.
Preferably, the composition of the fermentation medium are as follows: sucrose 10.0%, seitan powder 0.5%, yeast powder 2.0%, corn Slurry 1.0%, soybean cake powder 0.5%, KH2PO4 0.1%, MgSO4 0.05%, precipitated calcium carbonate 0.3%, pH 5.9 ± 0.1.
Preferably, the PAD culture medium is glucose agar medium；The glucose agar medium composition are as follows: Potato 20.0% (peeling stripping and slicing is boiled to pureed, and six layers of filtered through gauze remove slag), glucose 2.0%, agar 2.0% are natural pH。
The strain that sets out is spherical shuttle pink mold (Fusidium coccineum).
The Fusidic Acid superior strain is for producing Fusidic Acid.
The following steps are included: by the Fusidic Acid superior strain access primary-seed medium, 27 DEG C of cultures 72 After ± 5h by 1% inoculum concentration transfer second level expand culture medium in, 27 DEG C of 25 ± 5h of culture, then by 10% inoculum concentration transfer into Fermentation medium after 27 DEG C of culture 7-8d, obtains target product Fusidic Acid fermentation liquid, isolates and purifies, obtain Fusidic Acid.
Compared with prior art, the present invention has the advantages that:
(1) Fusidic Acid superior strain provided by the invention, fermentation yield are up to 4932 μ g/ml, mention than starting strain It is high by 53.88%.
(2) selection and improvement strain method breeding condition provided by the invention is mild, easy to operate, ratio for input and output is high, holds Easily implement.
Detailed description of the invention:
Fig. 1 is the colonial morphology figure of spherical shuttle pink mold NJWW-0520 (Fusidium coccineum NJWW-0520) With mycelia, spore microscopy figure；Wherein, figure (a) is bacterium colony picture；Scheming (b) is the micro- sem observation figure of mycelium；Scheming (c) is spore Micro- sem observation figure；
Fig. 2 is the standard curve of HPLC method measurement Fusidic Acid potency；
Fig. 3 is that Fusidic Acid output condition and biomass show during the domestication of DMSO tolerance spherical shape shuttle pink mold bacterial strain It is intended to；
Fig. 4 is height endurability spherical shape shuttle pink mold bacterial strain Fusidic Acid output condition and corresponding life during different domestications The schematic diagram of object amount.
Specific embodiment
Below by specific embodiment, invention is further described in detail.But those skilled in the art will manage Solution, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Specific skill is not specified in embodiment Art or condition person, described technology or conditions carry out to specifications according to the literature in the art.Agents useful for same or instrument Production firm person is not specified, being can be with conventional products that are commercially available.
Embodiment 1
(1) preliminary screening of resistance to highly polar organic solvent Fusidic Acid production bacterial strain
Starting strain spherical shape shuttle pink mold (Fusidium coccineum, screened from Nanjing Laoshan area scenic spot soil) is connect Enter primary-seed medium (glucose 2.5%, fish meal protein peptone 1.0%, yeast powder 0.2%, KH2PO40.1%, MgSO4 0.05%, corn pulp 4.0%, precipitated calcium carbonate 0.2%, pH5.9 ± 0.1) in, 1% inoculum concentration is pressed after 27 DEG C of 72 ± 5h of culture Second level of transferring expands in culture medium (being formulated same primary-seed medium), 27 DEG C of 25 ± 5h of culture, then presses 10% inoculum concentration It transfers into fermentation medium (sucrose 10.0%, seitan powder 0.5%, yeast powder 2.0%, corn pulp 1.0%, soybean cake powder 0.5%, KH2PO40.1%, MgSO40.05%, precipitated calcium carbonate 0.3%, pH 5.9 ± 0.1) in, it is inhaled after 27 DEG C of culture 3d It takes 1ml bacteria suspension into 2ml sterile centrifugation tube, is added DMSO (dimethyl sulfoxide), the final concentration of percent by volume of DMSO 12%, pressure stress dispose 6h, and PDA solid plate (potato 20.0%, Portugal are coated on after shaken cultivation on constant-temperature table Grape sugar 2.0%, agar 2.0%, natural pH) on.After strong point bacterium colony, select grow fine, bacterium colony is biggish accesses one again Grade seed culture medium carries out another wheel screening；Wherein, the initial concentration of DMSO is percent by volume 12%, and every wheel screens DMSO Concentration improves percent by volume 1% than last round of concentration, until can only grow several single colonies on culture medium flat plate, passes through primary dcreening operation It is finally obtained DMSO resistant strains 0328FA and 0416FA.
(2) the highly polar organic solvent bacterial strain acclimating of enduring high-concentration and plate separation secondary screening choosing
Using the highest organic solvent concentration (DMSO concentration is percent by volume 18%) of primary dcreening operation to primary dcreening operation bacterial strain obtained Carry out acclimating culture.In the resistant strain access seed culture medium that primary dcreening operation is obtained, 1% is pressed after 27 DEG C of 72 ± 5h of culture Inoculum concentration is transferred in second level expansion culture medium, 27 DEG C of 25 ± 5h of culture, then is transferred by 10% inoculum concentration into fermentation medium In, 1ml bacteria suspension is drawn after 27 DEG C of culture 3d into 2ml sterile centrifugation tube, and the final concentration of volume of highest that primary dcreening operation determines is added The DMSO of percentage 18% is coated on PDA solid plate after shaken cultivation on constant-temperature table, selects the best bacterium colony of growing way Carry out next-generation domestication.By the volume ratio 18%DMSO acclimating culture in five generations, it is stable that finishing screen selects heritability DMSO resistant strain NJWW-0520 and NJWW-0627, colonial morphology is rounded, central uplift, more stiff, and color is white Or canescence, as shown in Fig. 1 (a)；Hypha form is elongated, has a diaphragm, and about 15 μm of width, as shown in Fig. 1 (b)；Its spore is aobvious It is in shuttle shape or ellipse under micro mirror, as shown in Fig. 1 (c).
(3) organic solvent acclimating height endurability spherical shape shuttle pink mold produces the measurement of Fusidic Acid ability
In the DMSO high resistant strains NJWW-0520 access primary-seed medium that secondary screening domestication is obtained, 27 DEG C of cultures After 72 ± 5h by 1% inoculum concentration transfer second level expand culture medium in, 27 DEG C of 25 ± 5h of culture, then by 10% inoculum concentration switching Enter in 100ml fermentation medium, 27 DEG C of culture 8d, then sampling carries out biomass (see measuring method II) and Fusidic Acid yield The measurement of (potency).
I, the measurement of Fusidic Acid potency is measured using HPLC method:
1. the measurement of Fusidic Acid standard curve:
Precise Fusidic Acid standard items 25mg is poured into 25mL volumetric flask, uses CH3OH dilutes and determines molten, turns upside down Mixing to get concentration is 1.0mg/mL Fusidic Acid standard solution, distinguishes the gradient dilution solution with dilution, obtains concentration point Not Wei 1.0mg/mL, 0.8mg/mL, 0.6mg/mL, 0.4mg/mL, 0.2mg/mL, 0.0mg/mL Fusidic Acid solution, utilize The standard curve of Fusidic Acid standard items is drawn out after HPLC detection, as shown in Figure 2.
2. the preparation of Fusidic Acid fermentation broth sample
It takes sample 2ml in the sterilized EP pipe of 10ml, adjusts pH to 3.5~4.0 with dilute hydrochloric acid, after standing 0.5h, take 8ml dehydrated alcohol is mixed with sample liquid, is shaken up, and 0.5h is placed, and constantly shakes centrifuge tube therebetween.EP pipe is put into supercentrifuge It is centrifuged 15min in (10000rpm), takes supernatant to cross disposable aspiration needle filter membrane with syringe spare in HPLC sample injection bottle.
II, the measuring method of biomass:
Take fermentation liquid 20ml in 50ml centrifuge tube, 8000rpm is centrifuged 10min, abandons supernatant, is precipitated with RO water washing And it is centrifuged, 3 times repeatedly.Then it is filtered with the filter paper dried and weighed, gained spherical shape shuttle pink mold NJWW-0520 bacterium Drying to constant weight in 60 DEG C of oven overnights for body, then weighs, the biomass being converted into every 100ml fermentation liquid, gained knot Fruit is the biomass estimation result (mg/100ml) of fermentation later period (8d).
As a result as shown in Figures 3 and 4: with the increase for disposing stress domestication number with DMSO, spherical shuttle pink mold strain is right The tolerance of DMSO increases, and Fusidic Acid fermentation yield is also in the trend being gradually increasing.The spherical shuttle of high tolerance obtained The Fusidic Acid fermentation yield (potency) of pink mold bacterial strain NJWW-0520 has reached 4932 μ g/ml, improves compared with starting strain 53.88%.And after five repetitions domestication, the biomass of resistant strain spherical shape shuttle pink mold NJWW-0520 relative to Bacterial strain NJWW-0627 has obtained largely restoring, and maximum cell biomass is up to 1665mg/100ml (dry weight), unit The production capacity of the fermentation liquid of volume is 1.54 times of starting strain up to 4932 μ g/ml, is spherical shuttle chain by the Strain Designation Spore bacterium Fusidium coccineum NJWW-0520, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms Center, the deposit date is on March 21st, 2018, deposit number was CGMCC No.15473.
The above is only preferred embodiments of the present invention, is not intended to limit the scope of the present invention, Therefore any trickle amendment, equivalent variations and modification made to the above embodiment according to the technical essence of the invention, belong to In the range of technical solution of the present invention.

Claims (8)

1. a kind of Fusidic Acid superior strain, which is characterized in that the entitled spherical shuttle pink mold NJWW-0520 of the bacterial strain (Fusidium coccineum NJWW-0520), is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms The heart, the deposit date is on March 21st, 2018, deposit number was CGMCC No.15473.
2. a kind of selection of Fusidic Acid superior strain described in claim 1, which comprises the following steps:
(1) preliminary screening of resistance to highly polar organic solvent Fusidic Acid production bacterial strain
The starting strain of Fusidic Acid production bacterial strain after solid medium culture 5-6d is accessed in primary-seed medium, Expand in culture medium after 27 DEG C of 72 ± 5h of culture by 1% inoculum concentration second level of transferring, 27 DEG C of 25 ± 5h of culture, then is connect by 10% Kind amount is transferred in fermentation medium, and DMSO is added after 27 DEG C of culture 3d, and the final concentration of percent by volume 12% of DMSO is forced 6h stress be disposed, is coated on after shaken cultivation on constant-temperature table on PDA solid plate；After strong point bacterium colony, it is good to select growing way Well, bacterium colony is biggish accesses primary-seed medium again, carries out another wheel screening；Wherein, the initial concentration of DMSO is volume Percentage 12%, every wheel screening DMSO concentration improves percent by volume 1% than last round of concentration, until can only on culture medium flat plate Grow several single colonies；
(2) the highly polar organic solvent bacterial strain acclimating of enduring high-concentration and plate separation secondary screening choosing
In the resistant strains access seed culture medium that primary dcreening operation is obtained, transfer by 1% inoculum concentration into two after 27 DEG C of 72 ± 5h of culture Grade expands in culture medium, 27 DEG C of 25 ± 5h of culture, then transfers in fermentation medium by 10% inoculum concentration, after 27 DEG C of culture 3d DMSO is added, the DMSO concentration is the maximum concentration of DMSO used in step (1), is coated with after constant-temperature shaking culture on shaking table On PDA solid plate, selects the best bacterium colony of growing way and carry out next-generation domestication；By five generations by used in step (1) The DMSO acclimating culture of maximum concentration, obtains Fusidic Acid superior strain.
3. a kind of selection of Fusidic Acid superior strain according to claim 2, which is characterized in that the level-one kind The composition of sub- culture medium are as follows: seed culture medium contains glucose 2.5%, fish meal protein peptone 1.0%, yeast powder 0.2%, KH2PO4 0.1%, MgSO4 0.05%, corn pulp 4.0%, precipitated calcium carbonate 0.2%, pH 5.9 ± 0.1.
4. a kind of selection of Fusidic Acid superior strain according to claim 2, which is characterized in that the second level expands The composition of big culture medium are as follows: glucose 2.5%, fish meal protein peptone 1.0%, yeast powder 0.2%, KH2PO4 0.1%, MgSO4 0.05%, corn pulp 4.0%, precipitated calcium carbonate 0.2%, pH 5.9 ± 0.1.
5. a kind of selection of Fusidic Acid superior strain according to claim 2, which is characterized in that the fermentation training Support the composition of base are as follows: sucrose 10.0%, seitan powder 0.5%, yeast powder 2.0%, corn pulp 1.0%, soybean cake powder 0.5%, KH2PO4 0.1%, MgSO4 0.05%, precipitated calcium carbonate 0.3%, pH 5.9 ± 0.1.
6. a kind of selection of Fusidic Acid superior strain according to claim 2, which is characterized in that the PAD Culture medium is glucose agar medium；The glucose agar medium composition are as follows: potato 20.0%, glucose 2.0%, Agar 2.0%, natural pH.
7. a kind of Fusidic Acid superior strain described in claim 1 is in the application of production Fusidic Acid.
8. a kind of Fusidic Acid superior strain according to claim 7 is in the application of production Fusidic Acid, which is characterized in that The following steps are included: being pressed in the Fusidic Acid superior strain access primary-seed medium after 27 DEG C of 72 ± 5h of culture 1% inoculum concentration is transferred in second level expansion culture medium, 27 DEG C of 25 ± 5h of culture, then is transferred by 10% inoculum concentration into fermented and cultured Base after 27 DEG C of culture 7-8d, obtains target product Fusidic Acid fermentation liquid, isolates and purifies, obtain Fusidic Acid.
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CN110016491A (en) * 2019-05-13 2019-07-16 福建康鸿生物科技有限公司 A kind of preparation method of Fusidic Acid

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