CN101974465A - Bacillus subtilis strain and application thereof - Google Patents

Abstract

The invention discloses a Bacillus subtilis strain and application thereof. The strain is named as Bacillus subtilis ZJU007 and preserved in the China general microbiological culture collection center (CGMCC) of Chinese Academy of Sciences on yard No.1 on Beichen West Road, in Chaoyang district in Beijing on September 3rd, 2010, and has the preservation number of CGMCC No.4140. A bactericidal substance generated by the strain of the invention has very strong inhibition effect on Candida albicans and has better bacteriostasis effect compared with common fungicidin. The bactericidal substance not only has very strong bacteriostasis effect on the Candida albicans and other yeast like fungi, and the like, but also has excellent effect on Aspergillus niger and other filamentous fungi, and meanwhile, has better bacteriostasis effect on escherichia coli and other bacteria.

Description

A kind of bacillus subtilis strain and application thereof

Technical field

The present invention relates to technical field of bioengineering, relate in particular to a kind of bacillus subtilis strain and application thereof.

Background technology

In recent years, raising along with the modern medical service level, patients' such as cancer, acquired immune deficiency syndrome (AIDS), organ and bone marrow transplantation survival rate rises greatly, but these patients are owing to often use medicines such as heavy dose of microbiotic, antitumor drug, hormone, immunosuppressor, and chemotherapy and interventional therapies such as venous cannula, hemodialysis, cause immunity degradation, very easily do not shown pathogenic condition pathomycete intrusion originally and cause deep mycosis (Systemic fungal infection).The fungi infestation of this in recent years serious threat life presents a rapidly rising trend, and patient's number constantly increases.

Clinically, along with the rising of fungi infestation rate, the rate of utilization of antifungal drug has been improved, following Resistant strain also is on the increase, and brings certain difficulty for clinical treatment.And antimycotic new drug is with respect to the bacterial-infection resisting medicine and the hysteresis that seems, the novel anti fungi material of therefore seeking safety, efficient, low toxicity, wide spectrum more and more causes people's attention.

Cause the pathogenic bacteria of deep mycosis to be mainly Candida and Aspergillus, wherein Candida albicans (Candida albicans) is the main pathogeny fungi of a class, can infect the many positions of human body, comprises internal organ and nervus centralis.It is the major cause that causes hospital's blood infection, have very strong lethality when infecting in wound and body fluid, and clinical study finds that its lethality rate does not descend with the use of conventional antifungal drug.

Although invasive fungi infestation phenomenon is comparatively rare, its total incidence climbs up and up in the past 20 years.According to the Datamonitor prediction,, in 9,000,000 high-risk disease patients, there are 1,200,000 patients will face the threat of fungi infestation by 2010.Past, fungi infestation generally only occurs among the specific subgroup patient, yet along with increasing of immunocompromised patient quantity such as chemotherapy, transplanting, HIV/ADIS infection or diabetes, especially concerning the patient who accepts long-term treatment, the danger occurred frequently of fungi infestation makes antifungal therapy receive concern more and more widely.Because to accepting crowd's expansions such as chemotherapy, transplanting and HIV/ADIS or diabetic subject, this has also indicated the huge of antifungal drug market potential just gradually for the prevention of invasive fungi infestation (IFIs) and treatment field.

Antifungal drug market is a sophisticated market, yet from the classification and the quantity of product, it is many like that and comprehensively that antifungal drug but can not show a candle to other antibacterials.First kind of antifungal drug came out the 1950's in the world, yet until 1978, the medicine that can be used for treating systemic fungal infection on the market only is 3 kinds of amphotericin B (amphotericins B, Am B), nystatin (nystatin) and flucytosines (fiucytosine) etc.In decades thereafter, though the listing of tens of kinds of antifungal drugs is also arranged, the broad-spectrum antifungal medicine that really accounts for the market dominant position but has only few in number several.Over nearly 10 years (1996～2005), the new antifungal antibiotic of various countries' report has 174 kinds approximately, chemical structure is a type surplus in the of 30 nearly, the special plant epiphyte resisting of some microbiotic wherein, and the microbiotic of most of anti-animal fungies or narrow because of anti-fungus spectra, activity in vivo is faint, or because of cytotoxicity stronger etc., natural goods itself with through the product of structural modification, it is few to have a development prospect person.

Antifungal drug commonly used clinically at present can be divided into three major types by its mechanism of action: 1) disturb the fungal nucleic acid synthetic drugs, as grisovin and flucytosine etc., this class medicine is because toxicity is big, and problems such as narrow antimicrobial spectrum, recurrence rate height are seldom used at present.2) disturb fungal cell membrane ergosterol synthetic medicine, as polyene antibiotics (amphotericin B, nystatin etc.), azole chemical synthetic drug (as itraconazole, fluconazole etc.) and third rare amine microbiotic (Terbinafine (Terbinafine) and naftifungin (Naftifine) etc.).Yet, the amphotericin B good anti-bacterial effect, has a broad antifungal spectrum is the most frequently used antifungal drug, but limits its application greatly owing to have renal toxicity; Azole drug also is effective antifungal drug, the exploitation of such medicine from the clotrimazole of the first-generation to the fluconazole of the third generation and itraconazole etc., toxicity is less, and the antimicrobial spectrum of broad is arranged, but its anti-microbial activity is starkly lower than amphotericin and easily produces resistance; Terbinafine is invalid to deep fungal infection.3) suppress β-(1,3)-medicine of D glucan synthase, as the new drug Caspofungin (Caspofungin) of Meck company calendar year 2001 listing and the Mi Kafen clean (Micafungin) of rattan pool medicine listing in 2002 etc., this class medicine can suppress the synthetic essential β-(1 of fungal cell wall, 3)-and the D glucan synthase, good application prospects is arranged.In addition, the external application therapy can have been cured most of superficial mycosis patients, but the medicine that can treat deep mycosis also seldom, and some disease does not still have effective methods of treatment.

Genus bacillus (Bacillus sp.) is a kind of non-pathogenic bacteria that occurring in nature extensively exists, can produce the metabolite of multiple antimycotic and bacterium, wherein lipopeptid class, peptide class, phospholipid are main type, and they all have the obvious suppression effect to the conditioned pathogen of pathogenic bacterium and autogenous infection.

Summary of the invention

The invention provides a kind of bacillus subtilis strain, can separate obtaining a kind of antimicrobial substance from this bacterial strain fermentation liquor, this material through ESI/MS, ¹H-NMR reaches ¹³Analyses such as C-NMR find that it is the Macrolide material of molecular weight 402.5Da.

A kind of bacillus subtilis strain, called after subtilis (Bacillus subtilis) ZJU007, be preserved in the Chinese common micro-organisms culture presevation administrative center (CGMCC) that is positioned at No. 3 institutes of microbiology of the Chinese Academy of Sciences in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preserving number is CGMCCNo.4140, September 03 2010 preservation time.

The main biological property of above-mentioned bacterial strains is: colony colour is a grey, and the edge is a white, and colonial morphology is than rounding, and there is the fold projection on the surface, and diameter is medium-sized (about about 2mm), and the sporiferous time of bacterial strain was peaked 4 days fully matureds in 3 days in cultivation.

The present invention also provides the application of above-mentioned subtilis in producing compound as the formula (1).

The antimicrobial substance that bacterial strain of the present invention produces is very strong to the oidiomycetic restraining effect of white, have better fungistatic effect than mildew making commonly used at present.This antimicrobial substance not only has the intensive fungistatic effect to yeast class fungies such as Candida albicanss, and filamentous funguss such as aspergillus niger are also had good effect, simultaneously bacteriums such as intestinal bacteria is also had fungistatic effect preferably.

Description of drawings

Fig. 1 a is the light microscopic figure of bacterial strain of the present invention;

Fig. 1 b is the Electronic Speculum figure of bacterial strain of the present invention;

Fig. 2 a is the influence curve figure that temperature is tired to fermented liquid meta-bolites anti-candida albicans;

Fig. 2 b is the influence curve figure that the pH value is tired to fermented liquid meta-bolites anti-candida albicans;

Fig. 2 c is the influence curve figure that liquid amount is tired to fermented liquid meta-bolites anti-candida albicans;

Fig. 2 d is the influence curve figure that inoculum size is tired to fermented liquid meta-bolites anti-candida albicans;

Fig. 2 e is the influence curve figure that shaking speed is tired to fermented liquid meta-bolites anti-candida albicans;

Fig. 2 f is the influence curve figure that fermentation time is tired to fermented liquid meta-bolites anti-candida albicans;

Fig. 3 is the antimycotic material HPLC of a present invention collection of illustrative plates;

Fig. 4 analyzes collection of illustrative plates for the antimycotic material EI/MS of the present invention;

Fig. 5 is the antimycotic material of the present invention ¹³The C-NMR collection of illustrative plates;

Fig. 6 is the antimycotic material of the present invention ¹The H-NMR collection of illustrative plates.

Embodiment

Substratum

(1) LB slant medium (%): yeast powder 0.5, peptone 1.0, NaCl 0.5, and agar 2.0 is transferred pH7.4～7.6.

(2) seed culture medium (%): glucose 1.5, yeast powder 1.0, K ₂HPO ₄0.1, MgSO ₄0.05, FeSO ₄0.001, add CaCO behind the accent pH7.0 ₃0.04.

(3) fermention medium (%): analysis for soybean powder 0.6, Zulkovsky starch 3.0, yeast powder 1.0, K ₂HPO ₄0.1 NaCl 0.2, MgSO ₄0.005, FeSO ₄0.002 pH 6.5.

(4) Candida albicans substratum (%): glucose 2.0, peptone 1.0, agar 2.0.

The strains separation purifying

Pedotheque carries out 5 ALTERNATE SAMPLING in the zone that the East Sea, Zhejiang delimited; take soil sample 10g at every; put into Erlenmeyer flask; earth sample 10g fetches earth behind the mixing; be added in the Erlenmeyer flask that the 90mL sterilized water is housed; place the 30min that vibrates on the shaking table, the thalline in the soil sample is well-dispersed in the water, be 10 ^-1Bacterium liquid.Dipping in after the cooling of transfering loop flame sterilization and getting a ring extent of dilution is 10 ^-1Bacterium liquid is done the inoculation of four rides on the LB culture medium flat plate, cultivate 48h for 30 ℃, and picking list bacterium colony line then is further purified.Single bacterium colony after cultivating is taken out with puncher together with fritter agar on every side, move into no substratum plate, move into the flat board that is coated with Candida albicans behind 30 ℃ of cultivation 4d, agar block centre compartment distance is 4cm, overnight incubation selects single bacterium colony to carry out multiple sieve according to suppressing the circle size.Multiple sieve adopts shaking culture to carry out, and gets primary dcreening operation and obtains 50 strain bacterial classifications, be linked in the seed bottle, and dress 25mL substratum in the 250mL Erlenmeyer flask, 7.0,30 ℃ of following 200r/min of initial pH cultivate 24h.Again the inoculum size of seed liquor with 8% (v/v) is linked in the fermention medium, dress 40mL substratum in the 250mL triangular flask, 200r/min cultivates 36h under 28 ℃, pH6.5, relatively 50 strain bacterial classifications produce the degree that antimycotic material suppresses Candida albicans, the final bacterial strain that obtains the most efficient inhibition Candida albicans of 1 strain is numbered ZJU007.LB slant medium preservation strain is in 4 ℃ of refrigerators, and long-term preservation adopts glycerine pipe preserving process frozen in-20 ℃.

Identification of strains

The main biological property of this bacterial strain is: colony colour is a grey, and the edge is a white, and colonial morphology is than rounding, and there is the fold projection on the surface, and diameter is medium-sized (about about 2mm), and the sporiferous time of bacterial strain was peaked 4 days fully matureds in 3 days in cultivation.After cultivating three days on the LB flat board, picking list bacterium colony is observed under opticmicroscope and scanning electron microscope, specifically shown in Fig. 1 a and Fig. 1 b.

Design following primer according to spore bacillus 16S rRNA conserved sequence, and give birth to worker bio-engineering corporation by Shanghai and synthesize:

Upstream primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 '

Downstream primer: 5 '-AAGGAGGTGATCCAGCCGCA-3 ',

Utilize above-mentioned primer that the 16S rDNA sequence of this bacterial strain is carried out pcr amplification, the PCR system is:

Pcr amplification reaction system 20 μ L

ddw 14.25μL

10×PCR?Buffer 2.0μL

dNTP?mixture 0.5μL

Taq polysaccharase 0.25 μ L

Primer 1 (16f) 1.0 μ L

Primer 2 (16R ₂) 1.0 μ L

Dna profiling 1.0 μ L (quantitative) according to concentration

The PCR reaction conditions is: the PCR cycling condition: 95 ℃ of pre-sex change 5min, 95 ℃ of sex change 1min of loop parameter, 52 ℃ of renaturation 45s, 72 ℃ are extended 1.5min, repeat 35 circulations after, 72 ℃ are extended 10min again, last 4 ℃ of insulations.Detect the PCR product with 0.8% agarose gel electrophoresis.Select for use band clearly product carry out purifying.

Amplified production reclaims through gel electrophoresis, and checks order after being connected to the T carrier, has obtained the 1513bp characteristic sequence (shown in SEQ ID NO.1) of this bacterial strain 16s rDNA primary structure.By in the Genebank database, carrying out similarity searching (blast), found that the 16s rDNA sequence homology of itself and withered grass subtilis Bacillus subtilis isolate C8-4 is 97% (registration number of Genebank is EU257436.1).

Naming this bacterial strain according to above-mentioned feature is subtilis (Bacillus subtilis) ZJU007, be preserved in the Chinese common micro-organisms culture presevation administrative center (CGMCC) that is positioned at No. 1 No. 3 institutes of microbiology of the Chinese Academy of Sciences of institute in BeiChen West Road, Chaoyang District, BeiJing City, preserving number is CGMCC No.4140, September 03 2010 preservation time.

Fermentation condition optimization

Get the activated above-mentioned bacterial classification of 1 ring, be linked in the seed bottle, dress 25mL substratum in the 250mL Erlenmeyer flask, 7.0,30 ℃ of following 200r/min of initial pH value cultivate 24h, make seed liquor.

Seed liquor is inoculated in the 250mL Erlenmeyer flask that contains fermention medium, the shaking table shaking culture, the content of measurement meta-bolites of anti-candida albicans in different fermentations culture temperature, medium pH value, liquid amount, inoculum size, shaking speed and fermentation time bottom fermentation liquid (with the expression of tiring of nystatin), its influence curve are respectively shown in Fig. 2 a, Fig. 2 b, Fig. 2 c, Fig. 2 d, Fig. 2 e and Fig. 2 f.

Concrete grammar is as follows:

(Candida albicans ATCC 64548) is indicator with Candida albicans, and cup-plate method is measured inhibition zone size under the different nystatin solution of tiring, and obtains typical curve equation r ²=1.233lg ^(U)-1.5926, R ²=0.9927.U wherein: nystatin is tired (U/mL), r: inhibition zone radius (cm).

Cup-plate method is measured the inhibition zone size of the fermented liquid that obtains under the different condition then, is converted into tiring of nystatin by typical curve.By analyzing each influence curve, optimal conditions of fermentation is: 28 ℃ of temperature, pH 6.5, shaking speed 200r/min, inoculum size 8%, liquid amount 40mL/250mL, fermentation time 36h.

Cup-plate method is specific as follows: with the Candida albicans inclined-plane of 37 ℃ of constant temperature culture 2d, wash with the 30mL stroke-physiological saline solution and to make bacteria suspension, get 2mL to 100mL in 50 ℃ Sha Shi solid medium, mix the back and draw 20mL to the culture dish of diameter 9cm, the Oxford cup is placed in media surface in the cooling back, get 150 μ L liquid to be measured and add in the cup of Oxford, cultivate 18h, measure transparent antibacterial circle diameter for 37 ℃.

It is as follows that the fermentation culture parameter is set: 28 ℃ of temperature, and pH 6.5, shaking speed 200r/min, inoculum size 8%, liquid amount 40mL/250mL, fermentation time 36h ferments 3 batches, obtains tiring of fermented liquid anti-candida albicans and is respectively:

First fermentation titer is 7060U/mL; Second batch fermentation is tired and is 7250U/mL; The 3rd batch fermentation is tired and is 7120U/mL, and the mean value of tiring of anti-candida albicans is 7143U/mL.

The separation and purification of antimicrobial substance and analysis

With the centrifugal 15min of first fermented liquid 5000r/min; Clear liquid is transferred pH3.0 with the HCl of 1N, uses the equal-volume ethyl acetate extraction then 3 times; Ethyl acetate partly adopt silica gel (Merck, USA) (Φ 2.5 * 20cm) separates column chromatography, and ethyl acetate/methanol is a moving phase, employing step increase ethyl acetate/methanol (100: 0,80: 20,60: 40,40: 60,20: 80,0: 100) the method wash-out of volume ratio; Collect 100% (v/v) eluent ethyl acetate liquid, last Sephadex LH20 (GE Healthcare Life Sciences) post (Φ 2 * 97cm, mobile phase methanol/chloroform (v/v=4: 1)), flow velocity is 1cm/min, and collected volume is than (elution volume/total moving phase) position between 0.65～0.8; The elutriant evaporated under reduced pressure adopts C ₁₈Preparative high-performance liquid chromatographic (OSD, Φ 8 * 250cm, 5um) carry out purifying (moving phase: methanol (70: 30, v/v); Flow velocity: 1.5mL/min, 254nm detects), obtain antimycotic material.

(2) antimycotic metabolite HPLC analyzes

Adopt Agilent-1100 (Agilent Technologies, USA) efficient liquid phase chromatographic analysis.Analytical column: Agilent ZORBAX SB-C ₁₈Post (5 μ m, Φ 4.6mm * 250mm); Moving phase: A: methyl alcohol; B: water; (0～30min), 35～45%A (30～60min) for gradient elution (v/v): 5～35%A; Flow: 1mL/min; Detect wavelength: 254nm; Column temperature: 25 ℃, the result as shown in Figure 3.

(3) EI-MS analyzes

Adopt APEX III fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, USA) electrospray ionization mass spectrum (ESI/MS) that carries out sample is analyzed, condition is: ionization voltage 32V; Spray voltage 5kV; 320 ℃ of sample temperatures; Scanning specific charge (m/z) scope: 150～600Da; Detection mode: positive ion, analytical results are as shown in Figure 4.

By atlas analysis as can be known, [M+NH ₄ ⁺]=420, [M+Na ⁺]=425 illustrate the molecular weight Mr=402.5 of compound.Nucleo plasmic relation (m/z) is that 385 quasi-molecular ions is its dewatered product.

(4) NMR ( ¹H and ¹³C) analyze

Adopt ^UnitINOVA 500 type nuclear magnetic resonance analyser (Varian, USA) carry out sample hydrogen spectrum ( ¹H-NMR) and carbon spectrum ( ¹³C-NMR) mensuration, condition: vibrational frequency: 500MHz; Solvent: deuterochloroform; Chemical shift (δ) calibration reagent: tetramethylsilane, analytical results are as shown in Figure 5 and Figure 6.

Cyclic ester base (R in this material contains is represented in chemical shift at the peak at 166.289 places ₁-COO-R ₂), 19.901 places represent to contain a CH ₃, the expression of 20-40 place contains 6 CH ₂, 120-140 contains at the place two keys.Amount to 24 carbon atoms, 2 quaternary carbons wherein, 16 CH, 6 CH ₂, 1 CH ₃34 of hydrogen atoms contain 3 OH.Hence one can see that, and this material is a Macrolide material, and molecular formula is C ₂₄H ₃₄O ₅, structure is as follows:

The antimicrobial spectrum of antimicrobial substance and effect

Strains tested: Candida albicans (Candida albicans) ATCC 64548, aspergillus niger (Aspergillus niger) ATCC20611; Intestinal bacteria (Escherichia coli) ATCC8739; Klebsiella Pneumoniae (Klebsiella pneumoniae) ATCC10031; Pseudomonas aeruginosa (Pseudomonsa aeruginosa) ATCC9027; Streptococcus aureus (Staphycococcus aureus) ATCC29213; Cereuisiae fermentum (Sacharomyces cerevisae) ATCC2601, above-mentioned bacterial strains is test strain, no specificity requirement, and can obtain by the ordinary method separation.

Adopt cup-plate method to test antimycotic material that above-mentioned separation the obtains bacteriostatic activity to above-mentioned strains tested, and make Mycinomycin II positive control and physiological saline blank, concrete test result is as shown in the table:

Claims (2)

1. subtilis, it is characterized in that: called after subtilis (Bacillus subtilis) ZJU007, preserving number is CGMCC No.4140.

2. the application of the described subtilis of claim 1 in producing compound as the formula (1).

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