CN102766592A - Bacillus amyloliquefaciens and application thereof - Google Patents
Bacillus amyloliquefaciens and application thereof Download PDFInfo
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Abstract
The invention relates to a strain of bacillus amyloliquefaciens and an application thereof and belongs to the field of microorganisms. According to the bacillus amyloliquefaciens JNC-002 and the application thereof, the conservation number of the strain is China general microbiological culture collection center (CGMCC) No.5740, and the strain is separated and obtained from distiller's yeast of Sichuan jiannanchun liquor (enterprise group) limited liability company. The capacity of producing tetramethylpyrazine by the bacillus amyloliquefaciens can reach to 2.4g/L, and the bacillus amyloliquefaciens can be applied to producing the tetramethylpyrazine.
Description
Technical field
The present invention relates to a kind of bacillus amyloliquefaciens and uses thereof, belong to microorganism field.
Background technology
TMPZ is Ligustrazine (Ligustrazine is abbreviated as TTMP) again, and its pharmacological action mainly contains vasodilation; Slight step-down; Improve and organize microcirculation, improve the tissue blood perfusion; Suppressing platelet adhesion assembles and thrombosis; Suppress smooth muscle cell and fibroblast proliferation; Regulate lipid metabolism, anti peroxidation of lipid; The effect of certain conciliation immunity is arranged; Anti-tissue fibrosis has certain therapeutic action to pulmonary fibrosis and hepatic fibrosis.And pyrazine compounds (content 3000 ~ 6000ug/L) in liquor is the distinctive flavor compound of Chinese conventional solid-state fermentation liquor, also is the health factor in the wine body.Therefore, the content of TMPZ can help HUMAN HEALTH in the raising liquor.If can from the wine brewing environment, select the function stem of metabolism TMPZ, concerning the liquor industry, be of great immediate significance.
Report shows that bacillus amyloliquefaciens can produce AMS, β-1 at present, 3-1, and multiple enzymes such as 4-LSD, proteolytic enzyme have been widely used in zymin production
[1-3]Some researchs show; Bacillus amyloliquefaciens can also produce antibacterial protein, iturin (iturin), surfactin (surfactivity is plain), fengycin (fragrant shepherd's purse is plain) and bacillomycin antimicrobial substances such as (bacillomycins); Has a broad antifungal spectrum particularly has good inhibitory effect for mould
[4-8], and also do not appear in the newspapers for the bacillus amyloliquefaciens that produces pyrazine compounds.
Summary of the invention
Technical problem to be solved by this invention provides a bacillus amyloliquefaciens Bacillus amyloliquefaciens JNC-002; This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on April 6th, 2012; Preserving number is CGMCC No.5740; Preservation address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode 100101.
Further, the 16S rDNA sequence of bacillus amyloliquefaciens is shown in SEQ ID No.3.
Further, the bacterial characteristics of this bacterial strain is following: thalline is shaft-like, length 2 ~ 3 μ m, diameter 0.6 ~ 0.8 μ m, no pod membrane, central spore; Cream-colored, opaque, the coarse diffusion of colony edge that bacterium colony is; Physiological and biochemical property is a Gram-positive, the hydrogen peroxide enzyme positive, and egg yolk reaction is negative; Indole reaction is positive, and hydrolyzable gelatin, starch, casein utilize Citrate trianion, ammonium salt, propanedioic acid; Nitrate reduction is positive, replaces glucose can produce acid with pectinose, wood sugar, N.F,USP MANNITOL.
The present invention also provides the cultural method of bacillus amyloliquefaciens; It is sucrose 80～120g/L that bacillus amyloliquefaciens is inoculated into composition; Soybean cake powder 25 ~ 40g/L, yeast extract paste 3 ~ 5g/L, Secondary ammonium phosphate 25 ~ 60g/L; In the substratum of pH7.0 ~ 7.5, under 35 ~ 37 ℃ of temperature, 180 ~ 200rpm cultivates 96～120h.
Further, said medium component is sucrose 100g/L, soybean cake powder 40g/L, yeast extract paste 5g/L, Secondary ammonium phosphate 40g/L, pH7.5.
The present invention also provides the purposes of bacillus amyloliquefaciens in producing TMPZ.
The present invention also provides the purposes of bacillus amyloliquefaciens in wine brewing.
The present invention also provides the purposes of cultural method in producing TMPZ of bacillus amyloliquefaciens.
The present invention also provides the purposes of cultural method in wine brewing of bacillus amyloliquefaciens.
Bacterial strain of the present invention is from the distiller's yeast of Sichuan Jian Nan Chun (group) Ltd, to separate to obtain.
Beneficial effect of the present invention is: the present invention finds that first a plant height produces the bacillus amyloliquefaciens of TMPZ.The ability that this bacterial strain produces TMPZ can reach about 2.4g/L, for the production of TMPZ provides new selection, for bio-pharmaceuticals provides valuable microorganism resource, has great applications value.Bacillus amyloliquefaciens of the present invention is separated from distiller's yeast, can be used for producing liquor, improves the content of TMPZ in the wine body, for strengthening wine body typical case flavor characteristic new way is provided.
Bacillus amyloliquefaciens Bacillus amyloliquefaciens provided by the invention is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on February 1st, 2012, and preserving number is CGMCC No.5740.
Description of drawings
Fig. 1, primary dcreening operation obtain the ability of the product TMPZ of each bacterial strain.
Fig. 2, nitrogenous source optimization experiment result.
The thalli morphology of Fig. 3, bacillus amyloliquefaciens of the present invention, microscope magnification are 100 times.
Embodiment
Bacillus amyloliquefaciens Bacillus amyloliquefaciens JNC-002 bacterial strain of the present invention is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on April 6th, 2012; Preserving number is CGMCC No.5740; Preservation address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode 100101.
Further, the 16S rDNA sequence of bacillus amyloliquefaciens is shown in SEQ ID No.3.
Further, the bacterial characteristics of this bacterial strain is following: thalline is shaft-like, length 2 ~ 3 μ m, diameter 0.6 ~ 0.8 μ m, no pod membrane, central spore; Cream-colored, opaque, the coarse diffusion of colony edge that bacterium colony is; Physiological and biochemical property is a Gram-positive, the hydrogen peroxide enzyme positive, and egg yolk reaction is negative; Indole reaction is positive, and hydrolyzable gelatin, starch, casein utilize Citrate trianion, ammonium salt, propanedioic acid; Nitrate reduction is positive, replaces glucose can produce acid with pectinose, wood sugar, N.F,USP MANNITOL.
Concrete; The cultural method of bacillus amyloliquefaciens: it is sucrose 80 ~ 120g/L that bacillus amyloliquefaciens is inoculated into composition; Soybean cake powder 25 ~ 40g/L, yeast extract paste 3 ~ 5g/L, Secondary ammonium phosphate 25 ~ 60g/L; In the substratum of pH7.0 ~ 7.5, under 35 ~ 37 ℃ of temperature, 180 ~ 200rpm cultivates 96 ~ 120h.
Further, said medium component is sucrose 100g/L, soybean cake powder 40g/L, yeast extract paste 5g/L, Secondary ammonium phosphate 40g/L, pH7.5.
The present invention also provides the purposes of bacillus amyloliquefaciens in producing TMPZ.
The present invention also provides the purposes of bacillus amyloliquefaciens in wine brewing.
The present invention also provides the purposes of cultural method in producing TMPZ of bacillus amyloliquefaciens.
The present invention also provides the purposes of cultural method in wine brewing of bacillus amyloliquefaciens.
Bacterial strain of the present invention is from the distiller's yeast of Sichuan Jian Nan Chun (group) Ltd, to separate to obtain.
Bacillus amyloliquefaciens of the present invention produces the TMPZ ability can reach 2.0 ~ 2.4g/L, is the new bacterial strain that a plant height produces TMPZ, can be used for the production of TMPZ.Bacillus amyloliquefaciens of the present invention is separated from distiller's yeast, can be used for producing liquor, improves the content of TMPZ in the wine body, for strengthening wine body typical case flavor characteristic new way is provided.Simultaneously, bacillus amyloliquefaciens of the present invention also provides valuable microorganism resource for bio-pharmaceuticals, has great using value.
1, the primary dcreening operation of bacterial strain
Material: the distiller's yeast that Sichuan Jian Nan Chun (group) Ltd uses
Substratum: casein 20.0g, ammonium chloride 1.0g, Carnis Bovis seu Bubali cream 1.0g, magnesium chloride 5.0g, agar 20.0g, zero(ppm) water is settled to 1000mL, and pH 7.0
Experimental technique:
Under sterile conditions at 10 g koji with 90mL sterile water and put a bead of 25mL flask, set Oscillating bed shaking at room temperature for 30 minutes.Get bacterium liquid by 10 times gradient dilution to 10
-6, get each extent of dilution bacterium liquid 1mL and put into aseptic plate, pour the substratum of sterilization postcooling to 50 ℃ into, mixing is put into 37 ℃ of incubators and was cultivated 5～7 days after waiting to solidify, whether produce transparent circle and transparent circle inner and outer diameter ratio carries out primary dcreening operation to bacterial strain by it.Microbiology is thought can produce pyrazine class material by the caseic bacterium of can degrading.During thalline degraded casein, can around thalline, form a transparent circle because of utilizing casein.Because microbe colony size itself is variant, so screen, the caseic ability of microbiological deterioration can be described more intuitively, and then reflects the ability of production by biological pyrazine class material according to the ratio of transparent circle outside diameter and interior diameter.The bacterial strain that primary dcreening operation obtains finds that through morphologic observation bacterial strain basically all is a bacillus.
Table 1 primary dcreening operation obtains the transparent circle ratio of bacterial strain
| Strain number | Ratio | Strain number | Ratio |
| TTMP-1 | 2.3 | TTMP-13 | 1.1 |
| TTMP-2 | 3.1 | TTMP-14 | 1.7 |
| TTMP-3 | 1.7 | TTMP-15 | 2.3 |
| TTMP-4 | 2.6 | TTMP-16 | 4.1 |
| TTMP-5 | 3.7 | TTMP-17 | 2.0 |
| TTMP-6 | 2.0 | TTMP-18 | 1.4 |
| TTMP-7 | 1.4 | TTMP-19 | 2.3 |
| TTMP-8 | 3.1 | TTMP-20 | 2.0 |
| TTMP-9 | 2.3 | TTMP-21 | 3.8 |
| TTMP-10 | 1.1 | TTMP-22 | 4.0 |
| TTMP-11 | 2.6 | TTMP-23 | 2.6 |
| TTMP-12 | 2.6 | TTMP-24 | 1.7 |
2, the multiple sieve of bacterial strain
The primary dcreening operation bacterial strain is inoculated in liquid state fermentation substratum (sucrose 100g/L, soybean cake powder 40g/L, yeast extract paste 5g/L with 5% inoculum size respectively after activated; Secondary ammonium phosphate 30g/L, pH7.5), 37 ℃ of 180rpm cultivate 120h; Content (see figure 1) with TMPZ in each bacterial strain fermentation liquor of gas chromatographic detection; Screen the bacterial strain of high yield TMPZ with this, higher with TTMP-16, TTMP-22, TTMP-21, TTMP-5, TTMP-8, TTMP-2, the result is consistent with primary dcreening operation.
3, bacterial strain produces the TMPZ fermentation condition optimization
Nitrogenous source produces in the TTMP culturing process at bacterial strain has two-layer significance, and the one, satisfy the needs of cell growth, the 2nd, stable and regulate the pH of fermenting process.Bibliographical information shows to the metabolism of TTMP whether the situation of utilizing of nitrogenous source directly influences bacterial strain
[9], therefore, the optimization of nitrogenous source condition is even more important, and this experiment is optimized nitrogen concentration, optimum result such as Fig. 2.
Nitrogenous source optimization experiment culture medium prescription: sucrose 100g/L, soybean cake powder 40g/L, yeast extract paste 5g/L, pH7.5; Adopt different phosphoric acid hydrogen two ammonium usage quantitys to be optimized, concentration is respectively 1%, 2%, 3%, 4%, 5%, 6%.
Can know that by Fig. 2 the trend that each bacterial strain produces pyrazine all is the increase increase earlier along with nitrogen concentration, arrives one and reduces just when the back.Find that through the bacterial strain optimization experiment under optimal conditions, the ability that each bacterial strain produces TTMP all promotes to some extent, especially the TTMP-16 bacterial strain produces TTMP at nitrogen concentration during for 40g/L and is largely increased, and before optimizing, has improved 33%, reaches 2398mg/L.
In six strain bacterial strains of multiple sieve, it is the strongest that the TTMP-16 bacterial strain produces the TMPZ ability, called after JNC-002, and it is identified.
The evaluation of embodiment 2TTMP-16 bacterial strain
1,16S rDNA sequential analysis
(1) DNA extraction
Collect thalline, be dissolved in 5mL and extract damping fluid (100mMTrisCl, 100mM EDTA-Na
2, 200mM NaCl, 2%CTAB, pH8.0) in, 37 ℃ the vibration 45min.Add 0.75mL 20%SDS, 65 ℃ of water-bath 1h.12,000rpm, centrifugal 10min collects supernatant.Supernatant adds the NaAC (pH5.2) of final concentration 0.3M and the absolute ethyl alcohol of 2 times of volumes, precipitation at room temperature 1h with isopyknic Fen ︰ Lv Fang ︰ primary isoamyl alcohol (25 ︰, 24 ︰ 1) extracting 2 times.4 ℃, 12,000rpm, centrifugal 20min, collecting precipitation with 70% ethanol rinsing 2 times, is dissolved in 50 μ L TE (10mmTris-Hcl, 1mmNa after drying
2EDTA) in, promptly get total DNA.
(2) amplification 16S rDNA
With total DNA is template, and bacterial 16 S rDNA universal primer EU27 (shown in SEQ ID No.1) and 1492R (shown in SEQ ID No.2) are respectively forward primer and reverse primer amplification 16S rDNA.Amplification reaction system is 50 μ L:10 * Buffer, 5 μ L, dNTP 1 μ L, Taq enzyme 0.5 μ L (2U), each 1 μ L of forward and reverse primer, template DNA 1 μ L, ddH
2O40.5 μ L.Amplification reaction condition: 94 ℃ of preparatory sex change of 4min; 94 ℃ of 0.5min, 56 ℃ of 1min, 72 ℃ of 0.5min, 30 circulations; 72 ℃ are extended 7min.
(3) 16S rDNA sequential analysis
The 16S rDNA fragment of amplification is served Hai Shenggong order-checking, and the blast search program through American National biotechnology information center compares and obtains homology analysis result (seeing table 2).
Shown in the 16S rDNA amplification sequencing result SEQ ID No.3, as follows:
TGGTTACCTTGTTACGACTTCACCCCAATCATCTGTCCCACCTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGTACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCTTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTCTGAACCATGCGGTTCAGACAACCGTCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGGATCAAACTCTA
The homology compare of analysis of table 2 amplified fragments
| Similar bacterial strain | Homology |
| Bacillus subtilis (subtilis) | 99% |
| Bacillus amyloliquefaciens (bacillus amyloliquefaciens) | 99% |
| Uncultured Bacillus (not cultivating genus bacillus) | 99% |
| Bacillus sp (genus bacillus) | 99% |
2, Biolog microorganism identification appearance is identified
Through 16S rDNA sequential analysis; Find bacterial strain and subtilis, bacillus amyloliquefaciens that multiple sieve obtains, do not cultivate genus bacillus, the equal homology of genus bacillus all reaches 99%; In order further to identify this bacterial strain, the contriver determines to adopt Biolog GENIII full automatic microorganism assessing instrument (U.S. BIOLOG company) to identify.
On the substratum BUG+B that inoculation to be identified is recommended to Biolog, 33 ℃ of cultivations, incubation time should be 16 hours.
With nonvaccinated inoculation liquid serve as blank adjustment turbidimeter to make reading be 100% to be designated as T, prepare the inoculum of expection turbidity.There is the place of cell growth to pick the bacterium colony of diameter 3mm with aseptic cotton carrier from culture plate.For the bacterium of quick growth, pick a single bacterium colony and get final product; Bacterium for moderate speed's growth then picks a tuftlet bacterium colony; And, then pick the cross zone of growth of first bacterium colony for the bacterium of slow growth.The bottom that the end of cotton swab deeply is equipped with the inoculated tube of inoculation liquid, and teetertotter back and forth, stir, so that bacterium fully is discharged in the inoculation liquid.Detect with turbidimeter, the adjustment cell concn is 90 ~ 98%T, obtains bacteria suspension.
Bacteria suspension is poured in the V-type application of sample tank, used 8 road pipettors that bacteria suspension is sucked in the pipettor suction nozzle.By the amount of every hole 100 μ L with bacteria suspension be sequentially added into the institute of microwell plate porose in.Build the lid of microwell plate, eject the rifle head.Microwell plate is directly put into readout instrument, perhaps put into 33 ℃ of incubators and cultivated 24 ~ 48 hours.Read plate, annotate result's (seeing table 3).Through identifying, the bacterial strain that screening obtains the high yield TMPZ is a bacillus amyloliquefaciens.
Table 3Biolog microorganism identification appearance qualification result
| English name | With reference to Chinese name |
| Bacillus?amyloliquefaciens | Bacillus amyloliquefaciens |
The morphological specificity of embodiment 3 bacillus amyloliquefaciens strains of the present invention and biological characteristics are identified
All will use the activatory thalline in following each experiment, the LB plate culture medium is adopted in activation, and streak inoculation is inverted for 37 ℃ and was cultivated 1 ~ 2 day.
1, bacterium colony and thalli morphology are observed
Streak inoculation activatory thalline is on the LB plate culture medium, and colonial morphology is observed in 37 ℃ of cultivations; With a little thalline of inoculating needle picking, observe the thalli morphology (see figure 3) in microscopically.
Observations is following: thalline is shaft-like, no pod membrane, central spore; Cream-colored, opaque, the coarse diffusion of colony edge that bacterium colony is.
2, gramstaining
A little is fixed on the clean slide to get new activatory thalline, dye 1min with the mixed solution of Viola crystallina after, washing; Use iodine liquid effect 1min again, washing is blotted; With 95% ethanol or acetone soln decolouring 30s, dye 2 ~ 3min with sarranine liquid, washing again; Air-dry back is observed in microscopically, observes and is found to be the Gram-positive rod bacterium, produces gemma.
3, katalase experiment
[10]
New activatory bacterium one ring of transfering loop picking, being applied in to drip has on the slide of 3% hydrogen peroxide, and then positive if any the bubble generation in half a minute, no bubble then is a property, and the result sees table 5.
4, starch hydrolysising experiment
[10]
Get the activatory thalline and select and be connected on the bouillon media flat board that contains 0.2% Zulkovsky starch, cultivated 4 days for 37 ℃, form obvious bacterium colony after; On flat board, drip Lu Ge Shi allusion quotation liquid, periphery of bacterial colonies is if any the water white transparency circle, and the hydrolysis of expression starch is positive; Be still black-and-blue negatively, the result sees table 5.
5, nitrate reduction experiment
[10]
The activatory thalline is inoculated in the nitrate salt liquid nutrient medium, cultivated 5 days for 37 ℃, get one of nutrient solution, add accord with regular rules in one of Si Shi reagent; Become redness like solution, positive for nitrate reduction, occur like redfree; Adding two pentanoic reagent again, as not being blue reaction, still is that nitrate reduction is positive; As be blue reaction, negative for nitrate reduction, the result sees table 5.
6, indole reaction experiment
[10]
The 1% tryptone aqueous solution is transferred pH7.2 ~ 7.6, divides to install in the test tube 115 ℃ of sterilization 30min.Insert new activatory thalline after the cooling, cultivated 5 days for 37 ℃, slowly add the high Paradimethylaminobenzaldehyde reagent of 5mm in the nutrient solution surface along tube wall, at the liquid layer interface redness takes place, be positive reaction, the result sees table 5.
7, gelatin puncture experiment
[10]
Peptone gelatine culture branch is installed in the test tube 115 ℃ of sterilization 20min.Whether puncture inserts new activatory thalline, cultivates for 3 weeks for 25 ℃, observe growing state and gelatin and liquefy, and the result sees table 5.
8, lecithinase experiment
[10]
Get the thalline point of cultivating 24h and be connected on the 20% egg yolk agar culture medium flat plate, cultivate 20h for 37 ℃ and observe, form the muddy ring of oyster white like periphery of bacterial colonies, the expression lecithinase is positive; Otherwise negative, the result sees table 5.
9, litmus milk experiment
[10]
Skimmed milk 100mL is mixed the back divide and to install to test tube with 2.5% reindeer moss aqueous solution 4mL, 112 ℃ of sterilization 25min, the activatory thalline is inserted in the cooling back, cultivates for 3 weeks for 37 ℃ and observes acid-base reactions, and acid cure, enzyme peptonize with fixed attention, reduction etc., and the result sees table 5.
10, glycitols fermenting experiment
[10]
Get new activatory thalline percutaneous puncture-inoculation in stop with the gloomy Er Shi substratum of sharp husband in (glucose replaces with 1% sugar alcohol), cultivate after 4 days for 37 ℃ and observe, like the indicator flavescence, acid is produced in expression, produces sour positively, otherwise negative, the result sees table 5.
11, Citrate trianion utilization experiment
[10]
Get the streak inoculation on Xi Mengsishi Citrate trianion slant medium of new activatory thalline, 37 ℃ are cultured to 5 days, and indicator shows blue expression can utilize Citrate trianion, and positive, otherwise negative, the result sees table 5.
12, malonate utilization experiment
[10]
Get new activatory thalline and be inoculated in propanedioic acid-dibromothymolsulfonphthalein substratum, cultivated 2 days for 37 ℃, to turn blue person positive by green for substratum, and substratum nondiscoloration person is negative, and the result sees table 5.
13, nitrogenous source utilization experiment
[10]
Press auxanographic method, fall plate with the substratum of no nitrogenous source, after media surface is solidified; Inoculation activatory thalline; With optionally sprinkling nitrate salt and ammonium salt at last in certain zone, cultivated 2 days for 37 ℃, observe bacterial strain and whether grow; All ability assimilation persons enclose in the growth that forms on every side of adding nitrogenous source, and the result sees table 5.
The physiological and biochemical property of table 5 bacterial strain
Annotate: "+" is positive, and "-" is negative.
14, utilization of carbon source situation
Adopt Biolog GP Microplate to carry out the utilization of carbon source experiment, microbial bacteria suspension cell concn is adjusted to 28%T, 100 μ L are inserted in every hole, cultivate 24 hours for 35 ℃, by the readout instrument sense data, obtain the utilization of carbon source situation and see table 6:
The utilization of carbon source situation of table 6 bacillus amyloliquefaciens of the present invention
| The carbon source title | Utilize situation | The carbon source title | Utilize situation | The carbon source title | Utilize situation |
| Dextrin | + | α-D-glucose | + | The D-sorbyl alcohol | + |
| D-SANMALT-S | - | The D-seminose | + | D-N.F,USP MANNITOL | + |
| The D-trehalose | - | D-fructose | + | The D-arabitol | - |
| The D-cellobiose | + | The D-semi-lactosi | - | Inositol | - |
| Gentiobiose | + | 3-formyl glucose | - | Glycerine | + |
| Sucrose | + | D-fructose | - | The D-Robison ester | - |
| The D-turanose | + | L-fructose | - | The D-fructose-6-phosphate | - |
| Stachyose | - | The L-rhamnosyl | - | The D-aspartic acid | + |
| Raffinose | - | Gelatin | + | Pectin | + |
| α-D-lactose | + | Melibiose | - | The D-galacturonic acid | - |
| Beta-hydroxy-D, the L butyric acid | - | The L-L-Ala | + | L-lactic acid | + |
| β-formyl-D-glucoside | + | The L-l-arginine | - | The D-glyconic acid | + |
| The D-saligenin | - | The L-aspartic acid | + | The D-glucuronic acid | - |
| N-acetyl-D-glycosamine | + | L-L-glutamic acid | + | Glucuronamide | - |
| N-acetyl-β-D-reveals osamine | - | L-histamine propylhomoserin | + | Glactaric acid | - |
| N-acetyl-D-galactosamine | - | The L-Pyrrolidonecarboxylic acid | - | Quininic acid | - |
| P-hydroxyl-toluylic acid | - | Polysorbate40 | - | L MALIC ACID | - |
| Pyruvic Acid Methyl ester | - | Gamma-amino-butyric acid | - | Acetate | + |
| The D-methyl lactate | - | Alpha-hydroxy-butyric acid | - | The D-oxysuccinic acid | - |
| Glycyl-L-proline(Pro) | + | Hydrocerol A | - | Propionic acid | - |
| L-galacturonic acid lactone | - | α-ketone-butyric acid | - | Etheric acid | + |
| α-ketone-pentanedioic acid | - |
Annotate: "+" is positive, and "-" is negative.
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Claims (9)
1. bacillus amyloliquefaciens Bacillus amyloliquefaciens JNC-002, preserving number is CGMCC No.5740.
2. bacillus amyloliquefaciens according to claim 1 is characterized in that: its 16S rDNA sequence is shown in SEQ ID No.3.
3. bacillus amyloliquefaciens according to claim 1 and 2 is characterized in that: the bacterial characteristics of this bacterial strain is following: thalline is shaft-like, length 2 ~ 3 μ m, diameter 0.6 ~ 0.8 μ m, no pod membrane, central spore; Cream-colored, opaque, the coarse diffusion of colony edge that bacterium colony is; Physiological and biochemical property is a Gram-positive, the hydrogen peroxide enzyme positive, and egg yolk reaction is negative; Indole reaction is positive, and hydrolyzable gelatin, starch, casein utilize Citrate trianion, ammonium salt, propanedioic acid; Nitrate reduction is positive, replaces glucose can produce acid with pectinose, wood sugar, N.F,USP MANNITOL.
4. the cultural method of bacillus amyloliquefaciens; It is characterized in that: it is sucrose 80 ~ 120g/L that bacillus amyloliquefaciens is inoculated into composition; Soybean cake powder 25 ~ 40g/L, yeast extract paste 3 ~ 5g/L, Secondary ammonium phosphate 25 ~ 60g/L; In the substratum of pH7.0 ~ 7.5, under 35 ~ 37 ℃ of temperature, 180 ~ 200rpm cultivates 96 ~ 120h.
5. the cultural method of bacillus amyloliquefaciens according to claim 4, it is characterized in that: said medium component is sucrose 100g/L, soybean cake powder 40g/L, yeast extract paste 5g/L, Secondary ammonium phosphate 40g/L, pH7.5.
6. the purposes of each described bacillus amyloliquefaciens of claim 1 ~ 3 in producing TMPZ.
7. the purposes of each described bacillus amyloliquefaciens of claim 1 ~ 3 in wine brewing.
8. the purposes of the cultural method of claim 4 or 5 described bacillus amyloliquefaciens in producing TMPZ.
9. the purposes of cultural method in wine brewing of claim 4 or 5 described bacillus amyloliquefaciens.
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