CN101935623B - Agrobacterium ZX09, water-soluble beta-glucan prepared from Agrobacterium ZX09 and preparation method thereof and application on reducing blood sugar - Google Patents
Agrobacterium ZX09, water-soluble beta-glucan prepared from Agrobacterium ZX09 and preparation method thereof and application on reducing blood sugar Download PDFInfo
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- 229920002498 Beta-glucan Polymers 0.000 title claims abstract description 39
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 title claims abstract description 37
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- 239000011780 sodium chloride Substances 0.000 claims abstract description 12
- 150000003839 salts Chemical class 0.000 claims abstract description 7
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- 230000028327 secretion Effects 0.000 claims abstract description 3
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- 150000004676 glycans Chemical class 0.000 claims description 34
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- 239000005017 polysaccharide Substances 0.000 claims description 34
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- 238000000855 fermentation Methods 0.000 claims description 11
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- 230000000877 morphologic effect Effects 0.000 claims description 2
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- 239000008103 glucose Substances 0.000 description 9
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 9
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
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Images
Abstract
The invention discloses Agrobacterium ZX09, water-soluble beta-glucan prepared from the Agrobacterium ZX09 and the preparation method thereof and the application on reducing blood sugar, wherein the bacterial strain of the Agrobacterium ZX09 is obtained by separating saline soil from Dongying city in Shandong province; the bacterial strain is Agrobacterium sp. and is collected in China Center for Type Culture Collection (CCTCC) on January 21st, 2010; and the collection number is CCTCC NO:M2010020. The structure general formula of the water-soluble beta-glucan prepared from the Agrobacterium ZX09 is as follows: ->(3)-beta-D-Glup-(1->3)-[beta-D-Glup-(1->3)-beta-D-Glup-]3-alpha-D-Glup-(1->3)-alpha-D-Glup-(1)n->, and n in the structural formula is from 100 to 200. The bacterial strain has the advantages of novelty, special properties, salt resistant growth, no pigment secretion in the process of growth, simple and convenient cultivation operation, simple technology, total synthetic media, definite components and low cost, and is suitable for industrial production.
Description
Technical field
The invention belongs to mikrobe and Microbial exopolysaccharides technical field, particularly a kind of edaphic bacillus Agrobacteriumsp.ZX09, new type water-solubility beta-glucan molecule that this bacterium produces and preparation method thereof and the application on lowering blood glucose.
Background technology
Beta-glucan is distributed in fungi, bacterium and the plant materials widely; Medicinal at present, edible fungus beta-dextran is mainly derived from the cell walls of edible, medicinal basidiomycete and blastomycetes; Beta-glucan in the cereal mainly is present in the cell walls of oat and barley endosperm; The technology of from these materials, extracting beta-glucan is complicated, and output is lower.
Beta-glucan not only in various organisms the performance various biological active, and in the various interbiotic processes that influence each other, also have multiple function, be biological respinse regulatory factor (BRM) efficiently.It is reported; Beta-glucan has functions such as antitumor, anti-oxidant; The long-time edible food that contains beta-glucan can reduce the SUV in the human blood of hypercholesterolemia, and the beta-glucan that extracts in the oat can reduce diabetics's postprandial blood sugar (Jenkins, A.L.; D.J.A.Jenkins; U.Zdravkovic, P.W ü rsch and V.Vuksan.2002.Depression of the glycemic index byhigh levels of β-glucan fiber in two functional foods tested in type 2diabetes.EuropeanJournal of Clinical Nutrition 56,622-628).Yet the performance of beta-glucan effect needs the support of certain solubility, space structure and suitable molecular weight.Much beta-glucans is water-soluble lower; For example, curdlan is a kind of water-insoluble curdlan type VISOSE that is produced by edaphic bacillus or Alcaligenes, it by the D-glucosyl residue through the beta-glucoside key at C1; C3 linearity (the Kim that is formed by connecting; M.K., K.E.Ryu, W.A.Choi; Y.H.Rhee, andI.Y.Lee.2003.Enhanced production of (1 → 3)-β-D-glucan by a mutant strain ofAgrobacterium species.Biochem.Eng.J.16:163-168); Lentinan has restraining effect to tumour, but its solubleness in water is not high yet.And non-water-soluble VISOSE is difficult to directly absorbed after getting into human body, makes its range of application receive great restriction.
For solving this difficult problem, a lot of scholar's research the derivatize of beta-glucan improve its water-soluble and biological activity, in studying like the variation of anti-tumor activity before and after the beta-glucan that from pleurotus tuberregium (Franch.) Singer. (Pleurotus tuber-regium), extracts being carried out sulfation; Polysaccharide solubleness increases after finding sulfation; Anti-tumor capacity also increases (Lina Zhang, Mei Zhang, Jing Dong accordingly simultaneously; Ji Guo; Yinyin Song, Peter Chi keung Cheung.2001.Chemical structure and chain conformation of the water-insoluble glucan isolated fromPleurotus tuber-regium.Biopolymers 59:457-464. opens clever Na, Zhang Mei; Zhang Zhiqiang, Huang Rongchun.Chain conformation and the structure activity relationship of Pleurotus tuber-regium dextran sulfate in the aqueous solution.The academic free paper session of national polymer in 2003).In addition, in Chinese patent (CN:1583802A), water-insoluble yeast β-(1,3)-VISOSE is degraded through formic acid, obtain water-soluble β-(1, the 3)-VISOSE of different molecular weight.But all these solutions have all increased the difficulty that obtains required beta-glucan, and preparation cost is improved greatly, have limited the extensive use in production field.
Summary of the invention
The object of the present invention is to provide a kind of edaphic bacillus (Agrobacterium sp.ZX09) of producing water-soluble beta-glucan.
Another object of the present invention provides a kind of water-soluble high viscosity beta-glucan of producing with edaphic bacillus ZX09 and preparation method thereof.
The present invention also aims to provide a kind of application of water-soluble high viscosity beta-glucan on lowering blood glucose of producing with edaphic bacillus ZX09.
The technical solution that realizes the object of the invention is: a kind of edaphic bacillus ZX09; In the solonchak of Shandong Dongying, separate and obtain bacterial strain; This bacterial strain is Agrobacterium bacterial strain (Agrobacterium sp.); Bacterial strain on January 21st, 2010 in China typical culture collection center (CCTCC) preservation, deposit number is CCTCC NO:M2010020.
A kind of water-soluble beta-glucan of producing with edaphic bacillus ZX09, general structure is as follows:
→{3)-beta-D-Glup-(1→3)-[beta-D-Glup-(1→3)-beta-D-Glup-]
3-alpha-D-Glup-(1→3)-alpha-D-Glup-(1}
n→
N in the said structure is 100～200.
A kind of preparation method of the water-soluble beta-glucan of producing with edaphic bacillus ZX09, step is following:
(1) configuration nutrient solution: with NaH
2PO
40.5 ‰-2.5 ‰, KNO
32 ‰-4 ‰, CaCl
20.07 ‰-0.15 ‰, MgCl
20.2 ‰-0.5 ‰, FeSO
47H
2O 0.005 ‰-0.0125 ‰, MnSO
40.001 ‰-0.003 ‰, ZnCl
20.005 ‰-0.0075 ‰, NaCl 0-7%, sucrose 2%-4% is dissolved in H
2Among the O, pH is 5-11, and above salt concn is a mass ratio;
(2) nutrient solution is heated 110-121 ℃ of sterilization 15-30 minute, cooling then;
(3) in step (2), be cooled in the nutrient solution after 30-37 ℃ the sterilization and add plate bacterium colony liquid, form seed culture fluids at 28 ℃～30 ℃ fermentation 24-48h;
(4) in step (2), be cooled in the nutrient solution after 30-37 ℃ the sterilization and add the seed culture fluid that obtains in the step (3), at 28 ℃～30 ℃ fermentation 48～72h;
(5) in (4) fermented liquid that obtains of step, inject ethanol, acetone or the primary isoamyl alcohol that surpasses the fermented liquid two volumes, precipitate and obtain Crude polysaccharides;
(6) to obtaining pure article behind the Crude polysaccharides Deproteinization.
A kind of application of the water-soluble beta-glucan of producing with edaphic bacillus ZX09 is applied to lowering blood glucose with the beta-glucan that utilizes edaphic bacillus ZX09 to produce.
The present invention compared with prior art, its remarkable advantage: (1) bacterial strain is novel, and is unique, but pigment is not secreted in the salt tolerant growth in the process of growth; (2) culture of strains is easy and simple to handle, and technology is simple, and substratum is a full-synthetic culture medium, and composition is clear and definite, and cost is low, is suitable for suitability for industrialized production; (3) beta-glucan of gained is the new texture compound of polysaccharide; (4) beta-glucan simple and convenient extraction, the beta-glucan of gained can directly be dissolved in cold water; (5) mouse feeding is mixed with the polysaccharide of starch, can effectively controls the rising of postprandial blood sugar.
Below in conjunction with accompanying drawing the present invention is described in further detail.
Description of drawings
Fig. 1 is flat-plate bacterial colony, Photomicrograph and the salt tolerant growing state of bacterial strain.
Fig. 2 is the gas chromatogram of VISOSE compound mensuration.
Fig. 3 is the periodate oxidation result of VISOSE.
Fig. 4 is the ir spectra of VISOSE.
Fig. 5 is the nuclear magnetic resonance spectrum of VISOSE.
Fig. 6 is the change of blood sugar figure after mouse is fed polysaccharide.
The bacterial strain of edaphic bacillus is preserved (CCTCC) on January 21st, 2010 at China typical culture collection center; The address of depositary institution is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University preservation center; Deposit number is CCTCC NO:M2010020, the classification called after of its biomaterial (Agrobacterium sp.ZX09).
Embodiment
Edaphic bacillus of the present invention (Agrobacterium sp.ZX09) is in the solonchak of Shandong Dongying, to separate to obtain.This bacterial strain has following characteristics:
A. morphological specificity: on the inorganic salt Agar Plating 28 ℃ cultivate these bacterial strains and observe after 3-5 days, bacterium colony is white in color, circle, smooth surface, neat in edge, the exocellular polysaccharide that the bacterial strain secretion is a large amount of.
B. physiological and biochemical property: can effectively reduce nitrate salt, can not degraded cellulose, do not secrete pigment.Can in 7% NaCl fermented liquid, grow, the speed of growth is the fastest in containing the fermented liquid of 2%NaCl.With sucrose or lactose is that carbon source can be grown well, and the suspicious carbon source of utilizing is D-glucose, D-fructose, D-seminose or D-wood sugar, can not utilize carbon source to be SANMALT-S.The HV of fermented liquid of fermenting after 48 hours can reach 20000mPas (NDJ-1 RotationalViscometer, Rotor are 3, and RPM is 6, Shanghai balance equipment factory).Among Fig. 1, A is the bacterium colony on the plate culture medium, and B is the microphotograph of bacterium, and C is that bacterial strain shakes the relative growth rate after cultivating in the nutrient solution of different salt concentration.
C. the 16S dna sequence dna of edaphic bacillus is seen sequence table.
D. this bacterial strain is the Agrobacterium bacterial strain.
The present invention extracts water-soluble beta-glucan outside the macromolecular born of the same parents from the bacterial strain of edaphic bacillus ZX09, general structure is as follows:
→{3)-beta-D-Glup-(1→3)-[beta-D-Glup-(1→3)-beta-D-Glup-]
3-alpha-D-Glup-(1→3)-alpha-D-Glup-(1}
n→
N in the said structure is 100～200.
The characteristic of above-mentioned beta-glucan is following
A. polysaccharide fraction is single glucose.Fig. 2 is a gas chromatogram.Same component goes out the peak at same position.A figure be the ZX09 after the hydrolysis produce polysaccharide go out the peak situation, single peak explanation polysaccharide is made up of one-component.Among the B figure Polysaccharides with two peak has appearred after seminose mixes, and C scheme in Polysaccharides with have only a peak after glucose mixes, this one-component that composition polysaccharide just has been described is exactly a glucose.This polysaccharide is a VISOSE.
B. Periodic acid 99 can not oxidation of polysaccharides, sees Fig. 3.VISOSE has different mode of connection.When reacting with periodate, with the glycosyl of 1 → 2 or 1 → 4 bonding, on average each glycosyl consumes a part Periodic acid 99; Glycosyl or non-reduced terminal group with 1 → 6 bonding consume two molecule Periodic acid 99, and the glycosyl of 1 → 3 bonding do not consume Periodic acid 99.Zulkovsky starch is the VISOSE with 1 → 4 bonding; Do reference with Zulkovsky starch; The VISOSE that ZX09 is produced carries out periodate oxidation; Find that Zulkovsky starch has consumed periodate, and the VISOSE that ZX09 produces does not consume periodate, the glycosyl of this this VISOSE of explanation is all with 1 → 3 bonding.
C. the ir spectra of polysaccharide is seen Fig. 4, and this superpolymer does not have deriveding group and exists.
D. the nuclear magnetic resonance spectrum of polysaccharide is seen Fig. 5.
1Among the H NMR, two signals are arranged, show to have alpha conformation (δ 5.1-5.3ppm) and beta conformation (δ 4.92-4.96ppm) in the VISOSE;
13In the C NMR spectrum, a low δ 106.4,105.75,105.69,105.51,105.37,105.31,105.14,99.59, the peak at 99.01ppm place has characterized the beta conformation of C-1, and High-Field δ 98, and the peak at 96ppm place has characterized the alpha conformation of C-1
The present invention utilizes edaphic bacillus ZX09 to produce water-soluble beta-glucan, and its step comprises as follows:
A. dispose nutrient solution: with NaH
2PO
40.5 ‰-2.5 ‰, KNO
32 ‰-4 ‰, CaCl
20.07 ‰-0.15 ‰, MgCl
20.2 ‰-0.5 ‰, FeSO
47H
2O 0.005 ‰-0.0125 ‰, MnSO
40.001 ‰-0.003 ‰, ZnCl
20.005 ‰-0.0075 ‰, NaCl 0-7%, sucrose 2%-4% is dissolved in H
2Among the O, pH 5-11, above salt concn is a mass ratio.
B. nutrient solution is heated 110-121 ℃ of sterilization 15-30 minute, cooling then;
C. in step b, be cooled to and add plate bacterium colony liquid in 30-37 ℃ the nutrient solution;
D. vaccinated nutrient solution 24-48h forms seed culture fluid in 28 ℃～30 ℃ fermentation step c;
E. in step b, be cooled in 30-37 ℃ the nutrient solution and add seed culture fluid;
F. with vaccinated nutrient solution among the step e 28 ℃～30 ℃ the fermentation 48～72h;
H. inject ethanol, acetone or the primary isoamyl alcohol that surpasses the fermented liquid two volumes in the fermented liquid that in step f, obtains, obtain Crude polysaccharides;
I. obtain pure article behind the Crude polysaccharides Deproteinization that obtains.
The aqueous solution of the beta-glucan through method for preparing has very high viscosity, and it is with a wide range of applications.The beta-glucan that utilizes edaphic bacillus ZX09 to produce is the application of formulations of active ingredients on food, beverage and reduction level of postprandial blood sugar., the mouse after the fasting irritated feed with after starch mixes like this beta-glucan or VISOSE, detect the postprandial plasma glucose level of different time.The mouse postprandial plasma glucose level of a feeding starch contains the mouse of the food of beta-glucan of the present invention among Fig. 6 apparently higher than feeding.
The configuration nutrient solution, NaH
2PO
40.5g, KNO
32g, CaCl
20.07g, MgCl
20.2g, FeSO
47H
2O 0.005g, MnSO
40.001g, ZnCl
20.005g, sucrose 20g, H
2O1000mL, pH 5.0.With 121 ℃ of sterilizations of nutrient solution heating 15 minutes, be cooled to 30 ℃ and insert plate bacterium colony liquid 200 μ L, cultivate 24h, be seed liquor for 28 ℃.To the bacterium of going out (121 ℃ sterilization 15 minutes) and be cooled to and add 2% seed culture fluid in 30 ℃ the nutrient solution, 28 ℃ of fermentation 48h.In this fermented liquid, add the ethanol of two volumes, obtain the beta-glucan bullion.
15 hours mouse of fasting, directly feeding polysaccharide 200mg/kg
-1Or feeding is mixed with the polysaccharide 200mg/kg of starch
-1, can effectively control the rising of postprandial blood sugar.
The configuration nutrient solution, NaH
2PO
40.5g, KNO
32g, CaCl
20.07g, MgCl
20.2g, FeSO
47H
2O 0.0075g, MnSO
40.002g, ZnCl
20.005g, NaCl 20g, sucrose 20g, H
2O1000mL, pH 7.2.With 121 ℃ of sterilizations of nutrient solution heating 15 minutes, be cooled to 30 ℃ and insert plate bacterium colony liquid 100 μ L, cultivate 24h, be seed liquor for 28 ℃.To the bacterium of going out (121 ℃ sterilization 15 minutes) and be cooled to and add 10% seed culture fluid in 30 ℃ the nutrient solution, 28 ℃ of fermentation 72h.95% ethanol that in fermented liquid, adds two volumes must precipitate and is Crude polysaccharides, will precipitate taking-up, is dissolved in the water again, utilizes sevag method Deproteinization 3 times.Add 2 times of volume ethanol in the supernatant of Deproteinization again, the deposition polysaccharide, and should precipitate and be dissolved in water once more.The polysaccharide solution that obtains is through the DEAE-32 Mierocrystalline cellulose with after sepharose Sepharose CL-4B separates, ethanol sedimentation, and drying promptly gets the polysaccharide of purifying.
The configuration nutrient solution, NaH
2PO
41.0g, KNO
32.5g, CaCl
20.07g, MgCl
20.2g, FeSO
47H
2O 0.0075g, MnSO
40.002g, ZnCl
20.005g, NaCl 20g, sucrose 30g, H
2O1000mL, pH 7.2, with 121 ℃ of sterilizations of nutrient solution heating 15 minutes, are cooled to 30 ℃ and insert plate bacterium colony liquid 200 μ L, cultivate 24h, are seed liquor for 30 ℃.To the bacterium of going out (121 ℃ sterilization 15 minutes) and be cooled to and add 4% seed culture fluid in 30 ℃ the nutrient solution, 28 ℃ of fermentation 48h.95% ethanol that in fermented liquid, adds two volumes must precipitate and is bullion, will precipitate taking-up, is dissolved in the water again, utilizes sevag method Deproteinization 3 times.Add 2 times of volume ethanol in the supernatant of Deproteinization again, the deposition polysaccharide, and should precipitate and be dissolved in water once more.The polysaccharide solution that obtains is through the DEAE-32 Mierocrystalline cellulose with after sepharose Sepharose CL-4B separates, ethanol sedimentation, and drying promptly gets the polysaccharide of purifying.
The configuration nutrient solution, NaH
2PO
41.5g, KNO
33g, CaCl
20.07g, MgCl
20.4g, FeSO
47H
2O 0.0075g, MnSO
40.002g, ZnCl
20.005g, NaCl 20g, sucrose 40g, H
2O1000mL, pH11 with 121 ℃ of sterilizations of nutrient solution heating 15 minutes, is cooled to 30 ℃ and inserts plate bacterium colony liquid 100 μ L, cultivates 24h, is seed liquor for 28 ℃.To the bacterium of going out (121 ℃ sterilization 15 minutes) and be cooled to and add 10% seed culture fluid in 30 ℃ the nutrient solution, 30 ℃ of fermentation 48h.95% ethanol that in fermented liquid, adds two volumes must precipitate and is bullion, will precipitate taking-up, is dissolved in the water again, utilizes cetyl trimethylammonium bromide (CTAB) method Deproteinization.Add 2 times of volume ethanol in the supernatant of Deproteinization again, the deposition polysaccharide, and should precipitate and be dissolved in water once more.The polysaccharide solution that obtains is through the DEAE-32 Mierocrystalline cellulose with after sepharose Sepharose CL-4B separates, ethanol sedimentation, and drying promptly gets the polysaccharide of purifying.
The configuration nutrient solution, NaH
2PO
42.5g, KNO
34g, CaCl
20.15g, MgCl
20.5g, FeSO
47H
2O 0.0125g, MnSO
40.003g, ZnCl
20.0075g, NaCl 70g, sucrose 40g, H
2O1000mL, pH 7.2, with 110 ℃ of sterilizations of nutrient solution heating 30 minutes, are cooled to 30 ℃ and insert plate bacterium colony liquid 100 μ L, cultivate 24h, are seed liquor for 28 ℃.To the bacterium of going out (121 ℃ sterilization 15 minutes) and be cooled to 30 ℃ nutrient solution in add 5% seed culture fluid, 30 ℃ of fermentation 72h add 95% ethanol of two volumes in this fermented liquid, must precipitate to be bullion.
Mouse feeding is with embodiment 1.
Sequence table
< 110>Institutes Of Technology Of Nanjing
< 120>edaphic bacillus ZX09, water-soluble beta-glucan of producing with edaphic bacillus ZX09 and preparation method thereof and falling
Application on the hypoglycemia
<130>5
<160>1
<170>PatentIn?version?3.5
<210>1
<211>1357
<212>DNA
<213>Agrobacterium?sp.
<220>
<221>misc_feature
<222>(1)..(1357)
< 223>edaphic bacillus ZX09 16SrDNA sequence
<400>1
atgcagtcga?ccgccccggg?aggggagtgg?cagacgggtg?agtaacgggt?ggcaacctac 60
cctttcctgc?ggaatacctc?cggaaaactg?gaattaacac?cgcatccccc?ctagggggga 120
aatatttatc?ggggaaggat?tgccccgcgt?tgtattagct?agttgggggg?gaaacggcct 180
accaaggcga?ccatccatag?ctgggctgag?aggatgatcc?gcctcgttgg?gactgacccc 240
ccccccaccc?tcctacggga?ggcagcgggg?gaaaatattg?aacgatgggc?gcacgcctga 300
tcccgccctg?ccgggtgagt?gaagaaggcc?ttagggttgt?aatctttttt?caccgatgaa 360
aataatgacg?gtagtcaaaa?aagacccccc?ggctaacttc?ctgccagccg?ccgcgataat 420
acgaaggggg?ctagtgttgt?tcagaattac?tgggcgtaaa?gcgcacgtag?gcgtttattt 480
aagtggggga?agaaatcccg?cagctcaact?gcggaactgt?ctttgatgct?gggtatgttg 540
aggatggaag?aggtaagtgg?aattccgagt?gtagaggaga?aattcgtata?tattcggagg 600
accaccagtg?gcgaaggctt?cttactggtc?cattactgac?gctgaggtgc?gaaagcgtgg 660
ggagcaaaca?ggattagata?ccctggtagt?ccacgccgta?aacgatgaat?gttagccgtc 720
gggcagtata?ctgttcggtg?gcgcagctaa?cgcattaaac?attccgcctg?gggagtacgg 780
tcgcaagatt?aaaactcaaa?ggaattgacg?ggggcccgca?caagcggtgg?agcatgtggt 840
ttaattcgaa?gcaacgcgca?gaaccttacc?agctcttgac?attcggggta?tgggcattgg 900
agacgatgtc?cttcagttag?gctggcccca?gaacaggtgc?tgcatggctg?tcgtcagctc 960
gtgtcgtgag?atgttgggtt?aagtcccgca?acgagcgcaa?ccctcgccct?tagttgccag 1020
catttagttg?ggcactctaa?ggggactgcc?ggtgataagc?cgagaggaag?gtggggatga 1080
cgtcaagtcc?tcatggccct?tacgggctgg?gctacacacg?tgctacaatg?gtggtgacag 1140
tgggcagcga?gacagcgatg?tcgagctaat?ctccaaaagc?catctcagtt?cggattgcac 1200
tctgcaactc?gagtgcatga?agttggaatc?gctagtaatc?gcagatcagc?atgctgcggt 1260
gaatacgttc?ccgggccttg?tacacaccgc?ccgtcacacc?atgggagttg?gttttacccg 1320
aaggtagtgc?gctaaccgca?aggaggcagc?taaccac 1357
Claims (3)
1. edaphic bacillus ZX09 is characterized in that: in the solonchak of Shandong Dongying, separate obtaining bacterial strain, this bacterial strain is the Agrobacterium bacterial strain
(Agrobacterium sp.
), bacterial strain on January 21st, 2010 in China typical culture collection center (CCTCC) preservation, deposit number is CCTCC NO:M2010020, this bacterial strain has following characteristics:
A. morphological specificity
Observe after 3-5 days at 28 ℃ of these bacterial strains of cultivation on the inorganic salt Agar Plating, bacterium colony is rounded, the exocellular polysaccharide that the bacterial strain secretion is a large amount of;
B. physiological and biochemical property
Can effectively reduce nitrate salt, can not degraded cellulose, do not secrete pigment, can in 7% NaCl fermented liquid, grow, the speed of growth is the fastest in containing the fermented liquid of 2%NaCl; With sucrose or lactose is that carbon source can be grown well, and the suspicious carbon source of utilizing is D-glucose, D-fructose, D-seminose or D-wood sugar, can not utilize carbon source to be SANMALT-S.
2. edaphic bacillus ZX09 according to claim 1 is characterized in that: edaphic bacillus (Agrobacteriumsp.ZX09) 16S rDNA sequence is:
atgcagtcga?ccgccccggg?aggggagtgg?cagacgggtg?agtaacgggt?ggcaacctac 60
cctttcctgc?ggaatacctc?cggaaaactg?gaattaacac?cgcatccccc?ctagggggga 120
aatatttatc?ggggaaggat?tgccccgcgt?tgtattagct?agttgggggg?gaaacggcct 180
accaaggcga?ccatccatag?ctgggctgag?aggatgatcc?gcctcgttgg?gactgacccc 240
ccccccaccc?tcctacggga?ggcagcgggg?gaaaatattg?aacgatgggc?gcacgcctga 300
tcccgccctg?ccgggtgagt?gaagaaggcc?ttagggttgt?aatctttttt?caccgatgaa 360
aataatgacg?gtagtcaaaa?aagacccccc?ggctaacttc?ctgccagccg?ccgcgataat 420
acgaaggggg?ctagtgttgt?tcagaattac?tgggcgtaaa?gcgcacgtag?gcgtttattt 480
aagtggggga?agaaatcccg?cagctcaact?gcggaactgt?ctttgatgct?gggtatgttg 540
aggatggaag?aggtaagtgg?aattccgagt?gtagaggaga?aattcgtata?tattcggagg 600
accaccagtg?gcgaaggctt?cttactggtc?cattactgac?gctgaggtgc?gaaagcgtgg 660
ggagcaaaca?ggattagata?ccctggtagt?ccacgccgta?aacgatgaat?gttagccgtc 720
gggcagtata?ctgttcggtg?gcgcagctaa?cgcattaaac?attccgcctg?gggagtacgg 780
tcgcaagatt?aaaactcaaa?ggaattgacg?ggggcccgca?caagcggtgg?agcatgtggt 840
ttaattcgaa?gcaacgcgca?gaaccttacc?agctcttgac?attcggggta?tgggcattgg 900
agacgatgtc?cttcagttag?gctggcccca?gaacaggtgc?tgcatggctg?tcgtcagctc 960
gtgtcgtgag?atgttgggtt?aagtcccgca?acgagcgcaa?ccctcgccct?tagttgccag 1020
catttagttg?ggcactctaa?ggggactgcc?ggtgataagc?cgagaggaag?gtggggatga 1080
cgtcaagtcc?tcatggccct?tacgggctgg?gctacacacg?tgctacaatg?gtggtgacag 1140
tgggcagcga?gacagcgatg?tcgagctaat?ctccaaaagc?catctcagtt?cggattgcac 1200
tctgcaactc?gagtgcatga?agttggaatc?gctagtaatc?gcagatcagc?atgctgcggt 1260
gaatacgttc?ccgggccttg?tacacaccgc?ccgtcacacc?atgggagttg?gttttacccg 1320
aaggtagtgc?gctaaccgca?aggaggcagc?taaccac 1357
3. a kind of edaphic bacillus ZX09 according to claim 1, with the preparation method of the water-soluble beta-glucan of its production, step is following:
(1) configuration nutrient solution: with NaH
2PO
40.5 ‰-2.5 ‰, KNO
32 ‰-4 ‰, CaCl
20.07 ‰-0.15 ‰, MgCl
20.2 ‰-0.5 ‰, FeSO
47H
2O 0.005 ‰-0.0125 ‰, MnSO
40.001 ‰-0.003 ‰, ZnCl
20.005 ‰-0.0075 ‰, NaCl 0-7%, sucrose 2%-4% is dissolved in H
2Among the O, pH is 5-11, and above salt concn is a mass ratio;
(2) nutrient solution is heated 110-121 ℃ of sterilization 15-30 minute, cooling then;
(3) in step (2), be cooled in the nutrient solution after 30-37 ℃ the sterilization and add plate bacterium colony liquid, form seed culture fluids at 28 ℃～30 ℃ fermentation 24-48h;
(4) in step (2), be cooled in the nutrient solution after 30-37 ℃ the sterilization and add the seed culture fluid that obtains in the step (3), at 28 ℃～30 ℃ fermentation 48～72h;
(5) in (4) fermented liquid that obtains of step, inject ethanol, acetone or the primary isoamyl alcohol that surpasses the fermented liquid two volumes, precipitate and obtain Crude polysaccharides;
(6) to obtaining pure article behind the Crude polysaccharides Deproteinization.
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| CN103540628A (en) * | 2012-07-09 | 2014-01-29 | 南京理工大学 | Method for preparing Soraean gum and application thereof in water treatment |
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| CN109517838B (en) * | 2018-12-18 | 2020-07-10 | 中国海洋大学 | Method for agrobacterium-mediated molecular breeding of kelp |
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| CN111778300A (en) * | 2020-07-31 | 2020-10-16 | 四川合泰新光生物科技有限公司 | Beta-1, 3-glucan and preparation method and application thereof |
| CN111773239A (en) * | 2020-07-31 | 2020-10-16 | 四川合泰新光生物科技有限公司 | Application of beta-1, 3-glucan in preparation of medicines and daily chemical products for repairing skin injuries |
| CN112625960B (en) * | 2020-12-28 | 2022-10-25 | 山东大学 | Agrobacterium DP3, microbial inoculum thereof and application thereof in field of biological fertilizer preparation |
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