CN101935623A - 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 42
- 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 40
- 239000008280 blood Substances 0.000 title claims abstract description 13
- 210000004369 blood Anatomy 0.000 title claims abstract description 13
- 241000589158 Agrobacterium Species 0.000 title claims abstract description 10
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 230000001580 bacterial effect Effects 0.000 claims abstract description 25
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 21
- 239000011780 sodium chloride Substances 0.000 claims abstract description 11
- 150000003839 salts Chemical class 0.000 claims abstract description 7
- 239000000049 pigment Substances 0.000 claims abstract description 4
- 241000589159 Agrobacterium sp. Species 0.000 claims abstract description 3
- 230000028327 secretion Effects 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 42
- 150000004676 glycans Chemical class 0.000 claims description 34
- 229920001282 polysaccharide Polymers 0.000 claims description 34
- 239000005017 polysaccharide Substances 0.000 claims description 34
- 235000015097 nutrients Nutrition 0.000 claims description 30
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 24
- 241000894006 Bacteria Species 0.000 claims description 19
- 230000001954 sterilising effect Effects 0.000 claims description 17
- 238000004659 sterilization and disinfection Methods 0.000 claims description 17
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 13
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- 239000002244 precipitate Substances 0.000 claims description 12
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- 238000000855 fermentation Methods 0.000 claims description 11
- 230000004151 fermentation Effects 0.000 claims description 11
- 238000011218 seed culture Methods 0.000 claims description 11
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- 229930006000 Sucrose Natural products 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 239000005720 sucrose Substances 0.000 claims description 10
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- 238000004519 manufacturing process Methods 0.000 claims description 8
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- 238000001816 cooling Methods 0.000 claims description 3
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- 239000000047 product Substances 0.000 claims description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 2
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- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 claims description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 2
- 229910002651 NO3 Inorganic materials 0.000 claims description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 2
- 239000008272 agar Substances 0.000 claims description 2
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
- 230000000877 morphologic effect Effects 0.000 claims description 2
- 238000007747 plating Methods 0.000 claims description 2
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- 108020004465 16S ribosomal RNA Proteins 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 7
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- 229960002086 dextran Drugs 0.000 description 17
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 229920002472 Starch Polymers 0.000 description 7
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- 125000003147 glycosyl group Chemical group 0.000 description 5
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- 241001365490 Agrobacterium sp. ZX09 Species 0.000 description 3
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- 239000006228 supernatant Substances 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 229920002558 Curdlan Polymers 0.000 description 2
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
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- 125000002353 D-glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
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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 microorganism 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 extracting beta-glucan from these materials is complicated, and output is lower.
Beta-glucan is performance various biological activity in various organisms not only, and also has multiple function in the various interbiotic processes that influence each other, and is biological respinse regulatory factor (BRM) efficiently.It is reported, beta-glucan has antitumor, function such as anti-oxidant, the long-time edible food that contains beta-glucan can reduce the cholesterol in the human blood of hypercholesterolemia, 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 dextran 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 dextran is difficult to directly be absorbed after entering 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 the variation research as anti-tumor activity the beta-glucan that extracts is carried out sulfation from pleurotus tuberregium (Franch.) Singer. (Pleurotus tuber-regium) before and after, polysaccharide solubleness increases after finding sulfation, anti-tumor capacity also increases (Lina Zhang accordingly simultaneously, Mei Zhang, Jing Dong, 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)-dextran is degraded by formic acid, obtain water-soluble β-(1, the 3)-dextran 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 using edaphic bacillus ZX09 production 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 who uses the water-soluble beta-glucan of edaphic bacillus ZX09 production, step is as follows:
(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 product behind the Crude polysaccharides Deproteinization.
A kind of application of using the water-soluble beta-glucan of edaphic bacillus ZX09 production, the beta-glucan that will utilize edaphic bacillus ZX09 to produce is applied to lowering blood glucose.
The present invention compared with prior art, its remarkable advantage: (1) bacterial strain novelty, unique, but pigment is not secreted in 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 dextran compound mensuration.
Fig. 3 is the periodate oxidation result of dextran.
Fig. 4 is the infrared spectra of dextran.
Fig. 5 is the nuclear magnetic resonance spectrum of dextran.
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 to separate to obtain in the solonchak of Shandong Dongying.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, can not degraded cellulose, do not secrete pigment.Can grow in 7% NaCl fermented liquid, 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 maltose.The high viscosity 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 concn.
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 as follows
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 among the C figure Polysaccharides with have only a peak after glucose mixes, this has just illustrated that one-component of forming polysaccharide is exactly a glucose.This polysaccharide is a dextran.
B. Periodic acid can not oxidation of polysaccharides, sees Fig. 3.Dextran 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; Glycosyl or non-reduced terminal group with 1 → 6 bonding consume two molecule Periodic acid, and the glycosyl of 1 → 3 bonding do not consume Periodic acid.Zulkovsky starch is the dextran with 1 → 4 bonding, do reference with Zulkovsky starch, the dextran that ZX09 produces is carried out periodate oxidation, find that Zulkovsky starch has consumed periodate, and the dextran that ZX09 produces does not consume periodate, and this glycosyl that this dextran is described is all with 1 → 3 bonding.
C. the infrared spectra of polysaccharide is seen Fig. 4, and this superpolymer there is no 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 dextran;
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, then cooling;
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 product behind the Crude polysaccharides Deproteinization that obtains.
The aqueous solution of the beta-glucan by 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 as this beta-glucan or dextran, 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 crude product.
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 adds two volumes in fermented liquid 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 liquor of Deproteinization again, the precipitation 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 adds two volumes in fermented liquid must precipitate and is crude product, 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 liquor of Deproteinization again, the precipitation 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 adds two volumes in fermented liquid must precipitate and is crude product, 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 liquor of Deproteinization again, the precipitation 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 crude product.
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 (5)
1. edaphic bacillus ZX09 is characterized in that: separate obtaining bacterial strain in the solonchak of Shandong Dongying, 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, 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 maltose.
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. water-soluble beta-glucan of producing with edaphic bacillus ZX09 is characterized in that 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.
4. the preparation method of a water-soluble beta-glucan of producing with edaphic bacillus ZX09, step is as follows:
(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
2O0.005 ‰-0.0125 ‰, MnSO
40.001 ‰-0.003 ‰, ZnCl
20.005 ‰-0.0075 ‰, NaCl0-7%, 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 product behind the Crude polysaccharides Deproteinization.
5. application with the water-soluble beta-glucan of edaphic bacillus ZX09 production is characterized in that the beta-glucan that will utilize edaphic bacillus ZX09 to produce is applied to lowering blood glucose.
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