CN105925511A - Bacillus and application thereof - Google Patents
Bacillus and application thereof Download PDFInfo
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- CN105925511A CN105925511A CN201610518822.8A CN201610518822A CN105925511A CN 105925511 A CN105925511 A CN 105925511A CN 201610518822 A CN201610518822 A CN 201610518822A CN 105925511 A CN105925511 A CN 105925511A
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- bacillus
- chondroitin sulfate
- bacillus cereus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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Abstract
The invention belongs to the field of microbiology, and provides a bacillus with a collection number of CCTCC NO: M2016263. The invention also provides an application of the bacillus in fermentation production of chondroitin sulfate. The strain of the bacillus can synthesize and excrete chondroitin sulfate. The bacillus provided by the invention is not restricted by the short of animal cartilage resources, and is beneficial for mass production; the production process is simple, and the conditions are mild, so that the cost is relatively low; and moreover, the environmental pollution is small. The chondroitin sulfate produced by using the strain of the bacillus provided by the invention can be used as a raw material of medicines, health products and the like.
Description
Technical field
The invention belongs to microbiological art, relate to a kind of bacillus cereus, specifically a kind of bacillus cereus and system thereof
Preparation Method and application.
Background technology
Chondroitin sulfate (ChondroitinSulfate, CS) is that the class being distributed widely in animal cartilage tissue is acid
Mucopolysaccharide.Within 2010, many fermentable are analyzed by Jolly, are found to have some microorganisms and also can produce chondroitin sulfate
Element.Such as p pestic (Pasteurellamultocida), escherichia coli (Escherichiacoli), bacillus cereus
(Bacillus), etc. minority antibacterial.Chondroitin sulfate molecular structure is by D-Glucose aldehydic acid and N-acetylamino galactosamine structure
The polysaccharide of the repetition disaccharide unit composition become, chondroitin sulfate typically contains 50 ~ 70 diglycosyl our units, relative molecular mass
Between 10000 ~ 50000.Different by the sulfate group position of esterification, chondroitin sulfate is divided into the multiple types such as A, B, C, D, E, F
Type.Being mainly used in the auxiliary treatments such as arthritis, omarthralgia, coronary heart diseases and angina pectoris clinically, in recent years, chondroitin sulfate exists
The fields such as pharmaceutical preparation, medical material, cosmetics, health product are more widely available application, and the market demand becomes the biggest.China is raw
Producing and export volume increases substantially year by year, development prospect is wide.
At present, China mainly use enzymatic isolation method or alkali carry-enzymatic isolation method technique prepares chondroitin sulfate from animal bone.Seldom
There is the screening microorganism report for fermenting and producing chondroitin sulfate.And animal bone resource is poorer, anxious new life to be developed
Goods and materials source and new technical matters produce chondroitin sulfate.
Summary of the invention
For above-mentioned technical problem of the prior art, the invention provides a kind of bacillus cereus and preparation method thereof and answer
With, described this kind of bacillus cereus and its preparation method and application prior art to be solved produces sulphuric acid from animal bone
In the method for chrondroitin, there is lack of raw materials is difficult to scale up production-scale technical problem for bone.
The present invention improves a kind of bacillus cereus, its deposit number CCTCC NO:M2016263.
Present invention also offers the application in fermenting and producing chondroitin sulfate of the above-mentioned a kind of bacillus cereus.
A kind of bacillus cereus of the present invention, its Classification And Nomenclature is bacillus cereus G-09 Bacillussp.G-09, this bacterial strain
Being preserved in China typical culture collection center (CCTCC), the address of China typical culture collection center is: Chinese, military
The Chinese, Wuhan University, preservation date is on May 16th, 2016, and preserving number is CCTCC NO:M201623.
The present invention carries out strains separation, purification, cultivation by conventional method from nature, it is thus achieved that this bacillus cereus
(Bacillus.sp) G-09 bacterial strain.The present invention applies this bacillus cereus to produce chondroitin sulfate by fermentation culture.
China mainly use enzymatic isolation method or alkali carry-enzymatic isolation method technique prepares chondroitin sulfate from animal bone.And animal bone
Resource is poorer, and anxious new living resources to be developed and new technical matters produce chondroitin sulfate.Fermentable is raw
Product chondroitin sulfate extracts the advantage of chondroitin sulfate than from animal cartilage: do not retrained by animal cartilage scarcity of resources,
Be conducive to a large amount of production；Production technology is simple, mild condition, thus cost is relatively low；In addition environmental pollution is little.Produce with this bacterial strain
Chondroitin sulfate can be as pharmaceutical raw material and the raw material of health product.
The present invention compares with prior art, and its technological progress is significant.The bacterial strain of the present invention can synthesize and secrete sulphuric acid
Chrondroitin, uses the bacillus cereus of the present invention, can produce chondroitin sulfate on a large scale, and cost ratio is relatively low.
Accompanying drawing illustrates:
Fig. 1 is chondroitin sulfate standard sample HPLC analysis chart.
Fig. 2 is that G-09 bacterial strain produces chondroitin sulfate HPLC figure
Fig. 3 is the photo of Bacterial stain.
Fig. 4 is the photo of the spore staining of G-09 bacterial strain.
Detailed description of the invention:
By embodiment, the present invention is described in detail below, and embodiment does not constitute any restriction to the present invention.
Embodiment 1: the screening technique of bacillus cereus.
Bacillus cereus is isolated and purified: from the soil being collected in countryside deadwood and rotten leaf, weighs 3-5g soil sample, after air-drying, puts
Enter in the triangular flask of 30-50ml sterile purified water (bead 5), shaking flask 30-60min, then it is placed on 85 DEG C of water-bath 30min,
After cooling, it is diluted to 10-3, 10-4, 10-5Diluent, takes diluent 0.1-0.2ml respectively and coats beef extract-peptone flat board,
It is placed in 35 DEG C, is inverted and cultivates 32-48h, observe the morphological characteristic of bacterium colony.And transfer single bacterium colony, with Malachite green stain method dye into
One step microscopy is observed.Malachite green stain method step is as follows:
(1) choose bacterium colony by Gram staining method smear after, with 6% malachite green solution dye 10min.
(2) rinse with tap water.(3) 2min is redyed with 0.5% sarranine liquid, washing, blot.In basis of microscopic observation, spore
In green, thalline takes on a red color.Choose sporiferous bacterium colony numbering to preserve, as the strain of screening further.
Produce the screening of chondroitin sulfate bacillus cereus: the above-mentioned isolated and purified strain obtained is inoculated in LB broth bouillon
(peptone 10g, Carnis Bovis seu Bubali cream 15g, NaCl15g, distilled water 1000ml, regulate PH7.2) is centrifugal after cultivating 24h-48h collects fermentation
Liquid, fermentation liquid uses in high-efficient liquid phase chromatogram technique analysis (Fig. 1, Fig. 2), Fig. 1, and peak 2 is chondroitin sulfate standard specimen, retention time
It is 2.04 min.In Fig. 2, peak 1 is chondroitin sulfate, and retention time is 2.12 min.Select and fermentation liquid produces aching and limp ossein relatively
High bacterial strain, is the bacillus cereus producing chondroitin sulfate.This bacterial strain is preserved in China typical culture collection center (CCTCC)
In, its preserving number is CCTCCNo:M201623.
The bacillus cereus producing chondroitin sulfate screening obtained is accredited as bacillus cereus through morphology, Physiology and biochemistry, and
Named Bacillus.sp G-09.
The qualification of embodiment 2:Bacillus.sp G-09 bacterial strain
(1) morphological characteristic is observed
By G-09 inoculation in LB solid medium, under 35 DEG C of constant temperatures, cultivate 48h, form bacterium colony, colony characteristics
Opaque, moistening, the thickness for milky；It is shaft-like that microscope observes thalline, end circle, single or in short catenation.Thalline
Having sporulation, brood cell's diameter is more than 1.0 μm, and Gram’s staining is purple, for Gram-positive bacillus.(see Fig. 3, Fig. 4)
(2) Physiology and biochemistry is identified
With reference to RE. uncommon Kan Nan. the method in " primary Jie Shi Bacteria Identification handbook " (Beijing: Science Press, 1989), to G-09
Bacterial strain carries out physio-biochemical characteristics qualification, and qualification result is the VP test positive, and MR tests the positive, the catalase experiment positive, gelatin
The liquefaction positive, indole test is positive, the citrate experiment positive, the Starch Hydrolysis experiment positive, the salt tolerant experiment positive.
(3) 16S rDNA molecular biology identification
The amplification of 16S rDNA:
Amplimer: P1:5-AGAGTTTGATCCTGGCTCAG-3,
P2 :5-AAGGAGGTGATCCAGCCGCA-3 。
PCR reaction system: 0.20 μ l Taq DNA polymerase；2.5 μ l 10 × PCR reaction buffers；1μl dNTP
(each 2.5mM)；0.5μl P1(10 uM)；0.5μl P2(10 uM)；0.5 μ l genomic DNA (20-50ng/ μ l), adds double steaming
H2O to 25 μ l.
PCR reaction condition: 94 DEG C of 5 min；94 DEG C of 30 S, 55 DEG C of 40 S, 72 DEG C of 90 s, 30 circulations；72 DEG C of ends
Extend 10 min.
The detection of PCR product and the mensuration of 16S rDNA sequence: PCR product is through 1% sepharose electrophoresis (150V, 100mA
After 20min), pcr amplification product is checked order, then the 16 S rDNA sequences obtained are submitted to NCBI nucleic acid database
In carry out BLAST on-line analysis.Gained 16S rDNA sequence carries out homology with GenBank data base's nucleotide sequence and divides
Analysis, obtains the strain Bacillus sp 0711P6-2 that sibship therewith is nearest;HM226670, homology reaches 99%.
According to the form of bacterium colony, physio-biochemical characteristics, according to morphological characteristic, physiological and biochemical property and 16S rDNA sequence
Analyze, identify that G-09 bacterial strain is bacillus cereus (Bacillus.sp), it is named: bacillus cereus G-09(Bacillus.sp
G-09).
Embodiment 3: bacillus cereus is application experiment in producing chondroitin sulfate
Take the above-mentioned isolated and purified G-09 bacterial strain 1mL obtained, be inoculated in 50mL nutrient broth medium (peptone 10g, Carnis Bovis seu Bubali cream
10g, NaCl 1g, distilled water 1000ml, regulate PH7.2) at rotating speed 120r/m, temperature is at 28 DEG C, cultivates 18h.Again by this bacterium
Plant culture fluid to be transferred in 1000mL nutrient broth medium, be: rotating speed 120r/m that temperature is 30 DEG C at condition of culture, cultivate
35h.Discard precipitation by centrifugal for fermentation liquid, collect supernatant.It is purified process again through ethanol precipitation commonly used in the art
I.e. can get chondroitin sulfate, the yield of polysaccharide about 105mg/L.
Claims (2)
1. a bacillus cereus, it is characterised in that: deposit number CCTCC NO:M2016263.
2. a kind of bacillus cereus application in fermenting and producing chondroitin sulfate described in claim 1.
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| CN201610518822.8A CN105925511A (en) | 2016-07-04 | 2016-07-04 | Bacillus and application thereof |
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| CN201610518822.8A CN105925511A (en) | 2016-07-04 | 2016-07-04 | Bacillus and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708576A (en) * | 2020-12-28 | 2021-04-27 | 华东师范大学 | Bacterial strain for producing sulfated polysaccharide by fermentation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220270A (en) * | 2011-05-18 | 2011-10-19 | 江南大学 | Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of chondroitin sulfate |
| CN102337312A (en) * | 2011-10-19 | 2012-02-01 | 江南大学 | Method for increasing yield of chondroitin sulfate produced by fermentation method |
| CN102965414A (en) * | 2012-11-27 | 2013-03-13 | 江南大学 | Method for extracting chondroitin sulfate from fermentation broth |
-
2016
- 2016-07-04 CN CN201610518822.8A patent/CN105925511A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220270A (en) * | 2011-05-18 | 2011-10-19 | 江南大学 | Screening method for producing chondroitin sulfate bacterial strain and application of bacterial strain fermentation method in production of chondroitin sulfate |
| CN102337312A (en) * | 2011-10-19 | 2012-02-01 | 江南大学 | Method for increasing yield of chondroitin sulfate produced by fermentation method |
| CN102965414A (en) * | 2012-11-27 | 2013-03-13 | 江南大学 | Method for extracting chondroitin sulfate from fermentation broth |
Non-Patent Citations (1)
| Title |
|---|
| 石玉刚等: "生物法与化学法制备硫酸软骨素", 《化学进展》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708576A (en) * | 2020-12-28 | 2021-04-27 | 华东师范大学 | Bacterial strain for producing sulfated polysaccharide by fermentation method and application thereof |
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