CN103789239A - Bacillus strain, and screening method and application thereof - Google Patents
Bacillus strain, and screening method and application thereof Download PDFInfo
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- CN103789239A CN103789239A CN201410040021.6A CN201410040021A CN103789239A CN 103789239 A CN103789239 A CN 103789239A CN 201410040021 A CN201410040021 A CN 201410040021A CN 103789239 A CN103789239 A CN 103789239A
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- mesh
- food grade
- vanillin food
- genus bacillus
- bacillus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The invention belongs to the technical field of microorganism and particularly discloses a bacillus strain, and screening method and an application thereof. The bacillus strain is preserved in China General Microbiological Culture Collection Center, with a preservation number of CGMCC No.8629. The bacillus capable of generating beta-D-glucosidase is screened from vanilla, and has a relatively strong capacity of hydrolyzing vanillin glucoside, so as to form vanillin. The bacillus stain does not pollute the environment and is ecological and safe, and can be widely applied to preparation of vanillin.
Description
Technical field
The present invention relates to microorganism field, relate in particular a kind of genus bacillus and screening method thereof and application.
Background technology
Herba vanillae Planifoliae originates in Mexico, belongs to the orchid family climbing vine.Herba vanillae Planifoliae is senior flavouring agent, has the title of " king of flavouring agent ", and beanpod contains 2～3% vanillin food grade,1000.000000ine mesh, is widely used in foodstuffs industry.
The main flavour substances of Herba vanillae Planifoliae fermentation beanpod is vanillin food grade,1000.000000ine mesh, and wherein vanillin food grade,1000.000000ine mesh is formed through the self-contained endogenous β-D-Glucose glycosides enzymic hydrolysis of beanpod by its precursor vanillin food grade,1000.000000ine mesh glucoside.β-D-Glucose glycosides enzyme belongs to Mierocrystalline cellulose enzyme, can be hydrolyzed β-D-Glucose glycosidic bond, discharges β-D-Glucose and corresponding aglucon simultaneously, distributes very extensive at occurring in nature.β-D-Glucose glycosides enzyme has participated in the carbohydrate metabolism of organism, plays an important role to maintaining organism normal physiological function.Research before finds that β-D-Glucose glycosides enzyme can be hydrolyzed to the flavor precursors in plant materials to have strong natural flavour mountaineous aroma substance, therefore has important use value at field of food.
At present, be separated to the bacterium of multiple product β-D-Glucose glycosides enzyme, but about the genus bacillus that produces β-D-Glucose glycosides enzyme, particularly separate product β-D-Glucose glycosides enzyme genus bacillus from Herba vanillae Planifoliae there are no report, and this bacterial strain can be applicable to the hydrolysis of vanillin food grade,1000.000000ine mesh glucoside and forms vanillin food grade,1000.000000ine mesh.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of genus bacillus and screening method thereof and application, make this bacterial strain can be hydrolyzed vanillin food grade,1000.000000ine mesh glucoside and form vanillin food grade,1000.000000ine mesh, be applied in the preparation of vanillin food grade,1000.000000ine mesh.
Another object of the present invention is providing a kind of stronger genus bacillus of vanillin food grade,1000.000000ine mesh glucoside ability that is hydrolyzed.
For realizing the object of the invention, the invention provides a kind of genus bacillus obtaining that separates from bean pod of Herba vanillae Planifoliae, called after strain X Y18, strain X Y18 bacterium colony on LB substratum is rounded, oyster white, opaque, arch upward slightly in centre.Microscopic examination thalline is unit cell, shaft-like, chaining sometimes, amphitrichous.Gram-positive, gemma ellipse or oval.Can 20-45 ℃ of growth on the LB substratum containing 0-8%NaCl and pH4.0-8.0.
Carry out this bacterial strain of pcr amplification 16S rDNA sequence with bacterial 16 S rDNA universal primer A8-27f and B1523-1504r, sequence length is that 1403bp(sequence is referring to nucleotide sequence shown in SEQ ID NO:1).Submit GenBank to, the number of logging in is KF986320.Through GenBank database BLAST comparison, result shows, the 100 strain bacterial strains high with strain X Y18 homology are all bacillus (Bacillus sp.), and its homology reaches more than 99%.In conjunction with physiological and biochemical property, identify that it is bacillus (Bacillus sp.).
Genus bacillus of the present invention has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 23rd, 2013, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.8629.
Genus bacillus of the present invention take fermentation after bean pod of Herba vanillae Planifoliae as material, screen purifying, finally obtained object bacterial strain, step is as follows:
Step 1, get the bean pod of Herba vanillae Planifoliae having fermented, be placed in sterilized water, fully, after concussion, mixed solution is coated to 28 ℃ of cultivations on LB plate culture medium, grow after bacterium colony on plate culture medium, picking list bacterium colony line purifying is cultivated and is obtained single bacterial strain;
Inoculation 28 ℃ of cultivations on the LB solid medium containing Vitamin C2 and citric acid high ferro ammonia after step 2, purifying that step 1 is obtained, produce the bacterium colony of black hydrolysis circle on picking flat board, obtain described genus bacillus.
Wherein, as preferably, LB plate culture medium pH is 7.0 described in step 1, and every liter comprises 10g NaCl, 10g Tryptones, 5g yeast extract and 15g agar.
As preferably, be 7.0 containing the LB solid medium pH of Vitamin C2 and citric acid high ferro ammonia described in step 2, every liter comprises 10g NaCl, 10g Tryptones, 5g yeast extract, 15g agar, 1g Vitamin C2 and 2.5g citric acid high ferro ammonia.
As preferably, the concrete steps of fermenting described in step 1 are:
Get 70 ℃ of 5min that complete of bean pod of Herba vanillae Planifoliae hot water, 50 ℃ of hot blast sweating 25h of beanpod after completing, and then through drying at room temperature 1 month, last room temperature Chen Xiang completes fermentative processing half a year.
Genus bacillus of the present invention is inoculated into the inorganic salt liquid substratum take vanillin food grade,1000.000000ine mesh glucoside as sole carbon source, vanillin food grade,1000.000000ine mesh glucoside concentration is 255mg/L, through the cultivation of 20h, in substratum, vanillin food grade,1000.000000ine mesh glucoside concentration is 0, by complete hydrolysis, in substratum, detect that vanillin food grade,1000.000000ine mesh concentration is 42.8mg/L simultaneously, shown the vanillin food grade,1000.000000ine mesh glucoside complete hydrolysis that genus bacillus XY18 of the present invention can be 255mg/L by concentration in 20h, and generated vanillin food grade,1000.000000ine mesh.The invention provides thus described genus bacillus at product β-D-Glucose glycosides enzyme, hydrolysis vanillin food grade,1000.000000ine mesh glucoside and/or prepare the application in vanillin food grade,1000.000000ine mesh.
Meanwhile, the present invention also provides a kind of method of preparing vanillin food grade,1000.000000ine mesh, comprises the following steps:
Being configured to vanillin food grade,1000.000000ine mesh glucoside is the inorganic salt liquid substratum of sole carbon source, and genus bacillus described in claim 1 is inoculated in inorganic salt liquid substratum, and 28 ℃ of 170rpm shaking tables are cultivated, and can obtain vanillin food grade,1000.000000ine mesh.
Wherein, as preferably, described inorganic salt liquid substratum is that pH7.0 comprises NH
4nO
31.5g/L, K
2hPO
41.5g/L, KH
2pO
40.5g/L, MgSO
40.2g/L, NaCl1g/L, the inorganic salt liquid substratum of vanillin food grade,1000.000000ine mesh glucoside 255mg/L.
From above technical scheme, the present invention filters out the genus bacillus of strain product β-D-Glucose glycosides enzyme from Herba vanillae Planifoliae, and its hydrolysis vanillin food grade,1000.000000ine mesh glucoside ability is stronger, can form vanillin food grade,1000.000000ine mesh, and free from environmental pollution, ecological, safety, can be widely used in the preparation of vanillin food grade,1000.000000ine mesh.
Biological preservation explanation
Classification And Nomenclature: genus bacillus, Bacillus sp. is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 23rd, 2013, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, and deposit number is CGMCC No.8629.
Embodiment
The invention discloses a kind of genus bacillus and screening method thereof and application, those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Genus bacillus of the present invention is described by preferred embodiment, related personnel obviously can change methods and applications as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: the screening method of genus bacillus of the present invention
Get the bean pod of Herba vanillae Planifoliae 0.2-0.5cm having fermented, be placed in sterilized water 10mL, fully after concussion, 0.2mL mixed solution is coated to LB plate culture medium (1L:10g NaCl, 10g Tryptones, 5g yeast extract, 15g agar, pH is 7.0) upper, cultivate after 24h, on solid plate, grow bacterium colony for 28 ℃.Picking list bacterium colony is cultivated through 3 line purifying, obtains single bacterial strain.
Respectively by purified inoculation in LB solid medium (the 1L substratum: 10g NaCl containing Vitamin C2 and citric acid high ferro ammonia, 10g Tryptones, 5g yeast extract, 15g agar, 1g Vitamin C2,2.5g citric acid high ferro ammonia, pH is 7.0) upper 28 ℃ of cultivation 24h, on picking flat board, produce the bacterium colony of black hydrolysis circle, called after strain X Y18.
Embodiment 2: the mensuration of genus bacillus Physiology and biochemistry of the present invention and 16S DNA sequence dna
1, Morphology and physiology biochemistry detection
Detect growing state, colonial morphology and color, gramstaining, spore staining and the flagella staining etc. of strain X Y18 on LB flat board, method is carried out with reference to the method for Shen Ping chief editor " Microbiology Experiment (third edition) ".Obtained strains XY18 is on LB culture medium flat plate, and colony colour is opaque oyster white, in the middle of circular colony edge is neat, slightly arches upward.Microscopic examination thalline is unicellular, shaft-like.Gramstaining is positive, gemma ellipse or oval.
Detect strain X Y18 respectively under differing temps, under different pH condition, carry out LB liquid culture under different N aCl concentration, the temperature optimal selection of cultivation is 28-35 ℃; The pH value optimal selection of cultivating is 5.0-7.0; The NaCl optimal concentration of cultivating is chosen as 1%.
Adopting Biolog system to carry out physiological and biochemical analysis and Sherlock MIDI(Microbial Identification to strain X Y18) system carries out determination of fatty acid, result shows, strain X Y18 can utilize the multiple kinds of carbohydrate such as gentiobiose, xylan, and in strain cell, main fatty acid is anteiso-C
15:0, iso-C
15:0, anteiso-C
17:0and iso-C
17:0.
2, the mensuration of 16S rDNA sequence
With reference to bacterial genomes DNA extraction method (the Tian Gen biochemical technology DP302-02 of company limited), carry out pcr amplification with bacterial 16 S rDNA universal primer A8-27f and B1523-1504r.
The reaction system of 25 μ L: 2.5 μ L10 × PCR buffer, 200 μ M dNTP (each), 80nM A8-27f, 80nM B1523-1504r, 1.25U Taq polymerase(Takara, Dalian, and PRC) and 1 μ L genomic dna (approx.10ng).
Pcr amplification reaction condition: 94 ℃ of 3min; 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 2min, totally 30 circulations; Then 72 ℃ keep 10min.PCR product entrusts Hua Da gene company limited to carry out direct Sequencing (sequence is referring to nucleotide sequence shown in SEQ ID NO:1).The sequence recording is carried out sequence alignment analysis with BLAST software, and with GenBank in known 16S rDNA carry out homology comparison.
Detected result is as follows:
By this pcr amplification product, in GenBank, (accession number: KF986320) carries out BLAST comparison.Result shows, the 100 strain bacterial strains high with strain X Y18 homology are all bacillus (Bacillus sp.), and its homology reaches more than 99%, and the Y18 of deducibility strain X thus belongs to bacillus (Bacillus sp.).
Adopt various biological software to analyze the sequence recording, phylogenetic tree construction.For other all bacterial strain sequences of Phylogenetic Analysis and related data all from GenBank database.First download Bacillus in this database and belong to the kind type strain sequence nearer with XY18 sibship, be organized into the file of FASTA form.According to the result of comparing with Clustalx1.81 software, to selected go out sequence length arrange, the sequence length that is used in Phylogenetic Analysis is basically identical.Adopt MEGA5.05 software building phylogenetic tree.
Phylogeny Epidemiological Analysis shows, strain X Y18 and B.amyloliquefaciens NBRC15535
twith B.siamensis PD-A10
tgather for independent one.16S rDNA sequence homology analysis is found strain X Y18 and B.amyloliquefaciens NBRC15535
twith B.siamensis PD-A10
tsequence similarity be 99.1% and 99.2%.
In conjunction with Physiology and biochemistry and 16S rDNA analytical results, strain X Y18 is accredited as to the bacterial strain (Bacillus sp.) that a bacillus belongs to.
Embodiment 3: the ability test of genus bacillus hydrolysis vanillin food grade,1000.000000ine mesh glucoside of the present invention
Being configured to vanillin food grade,1000.000000ine mesh glucoside is the inorganic salt liquid substratum (MM) of sole carbon source: NH
4nO
31.5g/L, K
2hPO
41.5g/L, KH
2pO
40.5g/L, MgSO
40.2g/L, NaCl1g/L, pH7.0, wherein vanillin food grade,1000.000000ine mesh glucoside concentration is 255mg/L.
Genus bacillus XY18 is inoculated in above-mentioned substratum, 28 ℃ of 170rpm shaking tables are cultivated after 20h, in substratum, vanillin food grade,1000.000000ine mesh glucoside concentration is 0, by complete hydrolysis, in substratum, detect that vanillin food grade,1000.000000ine mesh concentration is 42.8mg/L simultaneously, show the vanillin food grade,1000.000000ine mesh glucoside complete hydrolysis that genus bacillus XY18 of the present invention can be 255mg/L by concentration in 20h, and generate vanillin food grade,1000.000000ine mesh, illustrated that strain X Y18 can be applicable to the hydrolysis of vanillin food grade,1000.000000ine mesh glucoside to generate vanillin food grade,1000.000000ine mesh, and hydrolysis ability is stronger.
Embodiment 4: the method for preparing vanillin food grade,1000.000000ine mesh of the present invention
Being configured to vanillin food grade,1000.000000ine mesh glucoside is the inorganic salt liquid substratum (MM) of sole carbon source: NH
4nO
31.5g/L, K
2hPO
41.5g/L, KH
2pO
40.5g/L, MgSO
40.2g/L, NaCl1g/L, pH7.0, wherein vanillin food grade,1000.000000ine mesh glucoside concentration is 255mg/L.
Genus bacillus XY18 is inoculated in above-mentioned substratum, after 28 ℃ of 170rpm shaking tables are cultivated 20h, can obtains vanillin food grade,1000.000000ine mesh.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. a genus bacillus (Bacillus sp.), is characterized in that, deposit number is CGMCC No.8629.
2. genus bacillus according to claim 1, is characterized in that, 16S rDNA sequence is as shown in SEQ ID NO:1.
Described in claim 1 genus bacillus producing β-D-Glucose glycosides enzyme, hydrolysis vanillin food grade,1000.000000ine mesh glucoside and/or prepare the application in vanillin food grade,1000.000000ine mesh.
4. the screening method of genus bacillus described in claim 1, is characterized in that, comprises the following steps:
Step 1, get the bean pod of Herba vanillae Planifoliae having fermented, be placed in sterilized water, fully, after concussion, mixed solution is coated to 28 ℃ of cultivations on LB plate culture medium, grow after bacterium colony on plate culture medium, picking list bacterium colony line purifying is cultivated and is obtained single bacterial strain;
Inoculation 28 ℃ of cultivations on the LB solid medium containing Vitamin C2 and citric acid high ferro ammonia after step 2, purifying that step 1 is obtained, produce the bacterium colony of black hydrolysis circle on picking flat board, obtain described genus bacillus.
5. screening method according to claim 4, is characterized in that, LB plate culture medium pH is 7.0 described in step 1, and every liter comprises 10g NaCl, 10g Tryptones, 5g yeast extract and 15g agar.
6. screening method according to claim 4, it is characterized in that, described in step 2, be 7.0 containing the LB solid medium pH of Vitamin C2 and citric acid high ferro ammonia, every liter comprises 10g NaCl, 10g Tryptones, 5g yeast extract, 15g agar, 1g Vitamin C2 and 2.5g citric acid high ferro ammonia.
7. screening method according to claim 4, is characterized in that, the concrete steps of fermenting described in step 1 are:
Get 70 ℃ of 5min that complete of bean pod of Herba vanillae Planifoliae hot water, 50 ℃ of hot blast sweating 25h of beanpod after completing, and then through drying at room temperature 1 month, last room temperature Chen Xiang completes fermentative processing half a year.
8. a method of preparing vanillin food grade,1000.000000ine mesh, is characterized in that, comprises the following steps:
Being configured to vanillin food grade,1000.000000ine mesh glucoside is the inorganic salt liquid substratum of sole carbon source, and genus bacillus described in claim 1 is inoculated in inorganic salt liquid substratum, and 28 ℃ of 170rpm shaking tables are cultivated, and can obtain vanillin food grade,1000.000000ine mesh.
9. method according to claim 8, is characterized in that, described inorganic salt liquid substratum is that pH7.0 comprises NH
4nO
31.5g/L, K
2hPO
41.5g/L, KH
2pO
40.5g/L, MgSO
40.2g/L, NaCl1g/L, the inorganic salt liquid substratum of vanillin food grade,1000.000000ine mesh glucoside 255mg/L.
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Cited By (2)
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Cited By (3)
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| CN104630288A (en) * | 2015-02-02 | 2015-05-20 | 中国热带农业科学院香料饮料研究所 | Method for fermenting vanilla pods |
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| CN107201324A (en) * | 2017-04-25 | 2017-09-26 | 贵州大学 | The β glucuroides producing strains and its screening technique of one plant of production hydrolysis Gingko yellow ketoside |
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Application publication date: 20140514 |