CN106520641A - Bacillus amyloliquefaciens and preparation method of exopolysaccharides thereof - Google Patents
Bacillus amyloliquefaciens and preparation method of exopolysaccharides thereof Download PDFInfo
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Abstract
The invention provides bacillus amyloliquefaciens EZ99. The bacillus amyloliquefaciens is gram positive bacteria, exists in plant rhizosphere soil, has no pathogenicity for people and plants, is collected from an orchard county, in the west of a Qilihe district in the Lanzhou city of the Gansu province, and has been identified, registered and preserved in the China common microorganism strain preservation center, and the preservation number is CGMCC No.13267. Component analysis is performed on bacterial colonies formed by the bacillus amyloliquefaciens on a PMA and MS culture medium, and it is found that the bacillus amyloliquefaciens has the characteristic of synthesizing and secreting exopolysaccharides. Optimal culture medium formulas and fermentation optimal conditions based on fermentation production of the exopolysaccharides of the strains are built, a fermentation solution is subjected to centrifuging, alcohol precipitation, dialysis and chromatographic separation, four purification polysaccharide components are obtained, and four-polysaccharide-component monosaccharide composition and monomer molecular weight are determined. The exopolysaccharides obtained through the strain and the method have the various applications of medicines, cosmetics, food additives, biological film raw materials and the like.
Description
Technical field
The present invention relates to microbial fermentation product field, especially, it is related to a kind of bacillus amyloliquefaciens and its extracellular many
The preparation method of sugar.
Background technology
Extracellular polysaccharide is a class by Microbe synthesis and the natural high molecular substance being secreted in environment, by multiple monosaccharide point
Sub- dehydration polymerization, glycosidic bond in a variety of manners are formed by connecting, and can form straight chain or have the backbone of branch.Outside extracellular microbial
The effect of polysaccharide itself is the suitable living environment for creating microorganism.It participates in forming the extracellular matrix of microorganism, is microorganism
Medium of the cell attachment on environmental interface (such as the surface of sandy soil, rock or plant), forms microorganism envelope (biofilm),
It is allowed to obtain a metastable living environment.Additionally, extracellular polysaccharide is also a kind of mode that microorganism stores nutrient substance.
With the progress of science and technology, the extracellular polysaccharide for having more and more species is found and is identified.Extracellular polysaccharide it is abundant many
Sample, is allowed to be provided with various purposes.As people constantly separate, identify and use extracellular polysaccharide, extracellular polysaccharide
The utilization of various modes has been obtained in fields such as medicine, food, material, cosmetics and agriculturals.It is particularly in field of medicaments, various
Extracellular polysaccharide is developed to the products such as medicine, vaccine, vaccine adjuvant.
Obtaining the extracellular polysaccharide of bacteria that extensive exploitation utilizes at present has：Xanthan gum (Xanthan gum), gellan gum
The extracellular polysaccharide of bacteria such as (Gellan gum), heat setting polysaccharide (Curdlan) extensively exist as industrial thickening agent and emulsifying agent
The industries such as food, feedstuff, chemical industry are used；Left-handed glucosides, because of its nontoxic, nonirritant, no antigen and metabolizability, has been used as
Plasma expander；Lactobacilluss extracellular polysaccharide is good food thickening agent and local flavor regulator, at the same time as prebioticses, adjust
Agent intestinal microbial population cosmetic field：Hyaluronic acid etc. with good moisturizing, antioxidation and membrane ability, as excellent cosmetic
Product additive, can be used in various hair-washing hair-care articles for use, cosmetics；In field of new：Some extracellular polysaccharide are in special process
Under the conditions of, degradable biological thin film and plastic can be prepared into, can be used as the environment-friend substitution product of plastics.
However, the Microbial exopolysaccharides kind that current China has put into industrialized production is not enriched, yield can not
Meet China market demand.Therefore urgently Efforts To Develop possesses the Screening and Identification work of the excellent bacterial strain for producing sugared performance, develops
More microorganism fungus kinds for possessing industrial production potential, accelerate the lifting and development of China's extracellular polysaccharide industry.
The present invention relates to one plant of solution starch spore bar (Bacillus for possessing extracellular polysaccharide characteristic
amyloliquefaciens)EZ99.Found by studying, EZ99 bacterial strains can on the solid medium containing special component
Colloidal substance, the bacterium colony of translucent quality is produced, and then produces closely knit, opaque milky bacterium on general culture medium
Fall.Inventor has further carried out chemical analyses to the chemical composition of this kind of colloid semipermeable membrane bacterium colony, contains a large amount of in finding bacterium colony
Polysaccharide.After being cultivated in polysaccharide fluid medium is produced, a large amount of polysaccharide in bacterium solution, are also enriched.Take centrifugation, precipitate with ethanol etc.
After step, the crude extract of the polysaccharide is obtained.Through being further analyzed to fluid medium drive member, in finding thalline
Without identical composition, it is inferred that it is the bacteriogenic extracellular polysaccharide to go out polysaccharide component in culture fluid, and non-bacterial itself
Structural polysaccharide.The separated purification of the extracellular polysaccharide can be split as four polysaccharide components, respectively with different qualities and purposes.
The content of the invention
It is an object of the invention to provide one plant of bacillus amyloliquefaciens with higher yield of extracellular polysaccharide.
Bacillus amyloliquefaciens EZ99 bacterial strains provided by the present invention, are preserved in Chinese micro- life on October 12nd, 2015
Thing culture presevation administration committee common micro-organisms center (abbreviation CGMCC), preserving number are CGMCC No.13267.Solution starch bud
Spore bacillus (Bacillus amyloliquefaciens) EZ99CGMCC No.13267 are isolated from Lanzhou Qi Lihe district west fruit
In garden township lanzhou lily planting site rhizosphere soil.In MS culture medium, bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) bacterium colony of EZ99CGMCC No.13267 is the translucent moistening shape of light oyster white, has radial wrinkle
Pleat, diameter about 0.5-1.5mm.Individual morphology is irregular G+ bacillus cereuss, is presented shaft-like, is that bacillus amyloliquefaciens are peculiar
Form.
Second object of the present invention is to provide a kind of bacillus amyloliquefaciens extracellular polysaccharide and its production, separation and purification
Method.The method of production bacillus amyloliquefaciens extracellular polysaccharide provided by the present invention, is fermentation bacillus amyloliquefaciens
The extracellular polysaccharide that (Bacillus amyloliquefaciens) EZ99CGMCC No.13267 are obtained.
A kind of preparation method of bacillus amyloliquefaciens extracellular polysaccharide, including following methods：
A, strain separating identification and preservation；
B, the culture medium of extracellular polysaccharide are prepared and are fermented；
The preparation of C, rough extracellular polysaccharide；
The preparation of D, refined extracellular polysaccharide；
E, extracellular polysaccharide each component are isolated and purified；
The molecular weight determination of F, polysaccharide each component；
The monosaccharide components identification of G, polysaccharide each component.
For cultivating bacillus amyloliquefaciens (Bacillus amyloliquefaciens) EZ99CGMCC No.13267
Culture medium in carbon source and nitrogen source be respectively sucrose and yeast extract, for cultivating bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) inorganic salt in the culture medium of EZ99CGMCC No.13267 contains MnSO4。
For cultivating bacillus amyloliquefaciens (Bacillus amyloliquefaciens) EZ99CGMCC No.13267
Culture medium preferably constitute (g/L) by following compositions：Sucrose 80-125, yeast extract 3.5-6, peptone 8-12, (NH4)2SO4
2.5-4, NaCl 2-4, MgSO40.03-0.08, CaCl20.03-0.08, K2HPO4 0.8-1.2。
Preferably, the fermentation condition of described extracellular polysaccharide is:Liquid amount is the bottled 30mL culture medium of every 100mL triangles,
Inoculum concentration is 7.2-7.7% (v/v), and original ph is 5.8-6.2, and 30-35 DEG C, 160pm cultivates 30-40h.
Preferably, the preparation of described rough extracellular polysaccharide, comprises the following steps：
(1) actication of culture:Strain will be preserved to be connected in beef extract-peptone slant medium, in 30-35 DEG C of constant temperature culture
After 20-26h, 4 DEG C of refrigerators are stored in standby；
(2) seed culture medium is prepared and inoculated and cultured:Seed liquid culture medium adopts LB culture medium, and liquid amount is per 100mL tri-
The bottled 30mL culture medium in angle；Beef extract-peptone slant strains in step (1) are inoculated in into seed culture medium, in cultivation temperature
28-32 DEG C, cultivate 10-15h under conditions of rotating speed 175-220rpm, obtain seed culture fluid；
(3) fermentation is produced sugar culture-medium and is prepared and fermentation culture:Fermentation produces sugar culture-medium component as above, and pH value is 5.8-6.2；
The seed liquor that step (2) is fermented is inoculated in fermentation medium with 4-6% (v/v) inoculum concentration, cultivation temperature 28-32 DEG C,
The condition of rotating speed 175-220rpm obtains EPS fermentation liquids in culture 32-40h；
(4) preparation of polysaccharide:The fermentation culture medium 5500-6500rpm centrifugation 8-12min centrifugations that step (3) is obtained are gone
After thalline, supernatant pH is adjusted to 6.8-7.2, adds 3 times of 95% ethanol of volume to stand 10-15h, 3500-4500rpm at 4 DEG C
Centrifugation 8-15min obtains white precipitate, after washed twice by precipitation, obtains final product rough extracellular many after 45-55 DEG C of drying
Sugared total sugar.
Preferably, the preparation of described refined extracellular polysaccharide, comprises the following steps：
Rough polysaccharide is added into deionized water dissolving, the polysaccharide solution that concentration is 10% is configured to.By polysaccharide solution totality
Long-pending 1/2 addition Sevag reagents (V chloroforms: V n-butyl alcohol=4: 1), stand after fully vibrating, centrifugation, take supernatant repetition, until
Without floating preteinses；Repeat with Sevag reagents extracted protein two to three times, to White Flocculus naked eyes between water phase and organic faciess
It is invisible.Supernatant A is collected, plus dehydrated alcohol stands 10-15 hours to 75% concentration at 4 DEG C, 75% ethanol of precipitate Jing is washed
Rear vacuum lyophilization is washed, refined extracellular polysaccharide is obtained.
The extracellular polysaccharide obtained in methods described can be followed the steps below and be isolated and purified：
Using the supernatant A obtained during ion exchange chromatography purification refine extracellular polysaccharide, the ion exchange layer
The separation condition of analysis method is：With DEAE-Sepharose Fast Flow as filler, 5mL is loaded, sample concentration is 10mg/mL,
Difference deionized water, 50mM, the NaCl solution gradient elution of 100mM, 200mM and 800mM, with the flow velocity eluting of 120mL/h,
Sample at 490nm absworption peaks is collected, and molecular weight is respectively obtained for 175766Da, the polysaccharide of 24806Da, 62375Da and 17691Da
Component.
Jing 1H- nuclear magnetic resonance spectroscopies, wherein component 1 are for β-(2,6) glycosidic bond levan (Levan), account for total sugar composition
90% or so；Component 2-4 is the heteropolysaccharide containing various monosaccharide.Based on the performance of the extracellular polysaccharide of above-mentioned bacterial strains, the present invention builds
The complete skill technique of its fermentation, extraction and purification is found, yield of extracellular polysaccharide reaches more than 13.9g/L, possesses excellent life
Produce Development volue.
In methods described, step 1) in the dehydrated alcohol volume that added for fermented supernatant fluid 3 times of volume；Step 3) in
Extracted 4 times with Sevag reagents, vibrate 20min every time；Step 4) in the column length of used chromatographic column be 300mm, column internal diameter is
2.5mm。
The bacillus amyloliquefaciens extracellular polysaccharide that said method is obtained falls within protection scope of the present invention.The present invention is extracellular
Polyose has extensive use：Field of medicaments：Act on immunomodulating, antitumor, antioxidation etc., can develop as new
Pharmaceutical products；The wherein maximum composition β of content-(2,6) glycosidic bond levan have nontoxic, nonirritant, no antigen and point
The characteristics of son amount is moderate.The polysaccharide dissolubility is high, and viscosity is extremely low in aqueous, only has 0.14dl/g, can be prepared as blood
Slurry expander；Field of health care products：This product is good prebioticses, with promote intestinal beneficial flora growth, strengthen it is prebiotic because
The function of son synthesis, can develop as health food or food additive.Cosmetic field：This product molecule particles compared with
Little, its diameter only has 50-100 nanometers, just can form hard glittering protecting film after this polysaccharide of a thin layer is dry, and this causes product to touch
Sense more silk is slided.This product has good moisturizing, antioxidation and membrane ability, as excellent cosmetics additive, can use
In various hair-washing hair-care articles for use, cosmetics.Technical field of biological material：This product can be prepared as thickness under the conditions of special process
Degree only has the transparent organism thin film of 9nm, and the thin film has excellent oxygen barrier ability, is the environmental protection package of good medicine, food
Material.
The invention has the advantages that：Bacillus amyloliquefaciens (the Bacillus of the present invention
Amyloliquefaciens) EZ99, is one plant of gram-positive bacterium, is present in plant rhizosphere soil, to people and animals and plants
Equal no pathogenicity.Bacillus amyloliquefaciens EZ99 bacterial strains system inventor is gathered from Lanzhou City, Gansu Province Qi Lihe district Xi Guoyuanxiang,
Preservation is identified and registered to China General Microbiological strain collections, and preserving number is CGMCC No.13267.Jing pair
The bacterium forms bacterium colony in PDA and MS culture medium and carries out component analyses, it is found which has the characteristic of synthesis and secretion extracellular polysaccharide.
The present invention establishes the liquid of the preferred culture medium formula and above-mentioned extracellular polysaccharide produced based on above-mentioned bacterial strains Exopolysaccharide Production From The Fermentation
Fermentation optimum condition.Fermentation liquid Jing centrifugations, precipitate with ethanol, dialysis and chromatography, obtain four purified polysaccharide components, Jing nuclear magnetic resonance, NMR
And efficient liquid phase chromatographic analysis, it is determined that the monosaccharide composition of four polysaccharide components and monomer molecule amount.Using bacterial strain of the present invention and
Extracellular polysaccharide prepared by method has the multiple uses such as medicine, cosmetics, food addition and biofilm raw material.
In addition to objects, features and advantages described above, the present invention also has other objects, features and advantages.
The present invention is further detailed explanation below.
Description of the drawings
Fig. 1 is：The molecular size range and scattergram of extracellular polysaccharide EPS.
Fig. 2 is：The 1H-NMR spectrograms of extracellular polysaccharide EPS-0, EPS-200 and EPS-800.
Fig. 3 is：The PMP column front derivation HPLC chromatograms of extracellular polysaccharide EPS each components.
Specific embodiment
Hereinafter embodiments of the invention are described in detail, but the present invention can be limited according to claim and be covered
Multitude of different ways implement.
Embodiment one：Strain separating is identified
By lanzhou lily rhizosphere soil sample, in adding the triangular flask equipped with 25ml enrichment mediums, 30 DEG C, 160r/min
Shake-flask culture 48h.Then by enrichment culture liquid with the dilution of 10 times of physiological saline solution after, take 0.2ml coating isolation mediums and put down
Plate.28 DEG C are inverted culture 48h, and acquisition has the bacterium colony of colloidal substance transparent appearance on flat board, obtains producing extracellular polysaccharide
Antibacterial.Enrichment medium and isolation medium are all PDA plate culture medium.Carry out 16S rDNA genes according to a conventional method to bacterial strain
Sequencing, the sequence of bacillus (Bacillus) in sequencing result and Genebank is compared similarity highest, it is determined that
Experimental strain is bacillus amyloliquefaciens.Bacillus amyloliquefaciens are confirmed as in multiple Jing China typical culture collection centers identification,
Name the bacterial strain to be Bacillus amyloliquefaciens EZ99 strains, and carry out preservation, deposit number CGMCC
No.13267。
Embodiment two：The nutrient media components of extracellular polysaccharide, fermentation condition
Seed culture medium adopts LB culture medium；It is (g/L) that sugar culture-medium component is produced in fermentation：Sucrose 50.0.Yeast extract 5.0,
Peptone 10.0, (NH4)2SO43.0, NaCl 3.0, MgSO40.05, CaCl20.05, K2HPO4 1.0。
Fermentation condition:Liquid amount is the bottled 30mL culture medium of every 100mL triangles, and inoculum concentration is 7.5% (v/v), initial pH
Be worth 12h is cultivated for 7.0,32C, 160pm.
Embodiment three：Extracellular polysaccharide it is rough and refined
(1) actication of culture:Strain will be preserved to be connected in beef extract-peptone slant medium, in 32 DEG C of constant temperature culture 24h
Afterwards, 4 DEG C of refrigerators are stored in standby.
(2) seed culture medium is prepared and inoculated and cultured:Seed liquid culture medium adopts LB culture medium, and liquid amount is per 100mL tri-
The bottled 30mL culture medium in angle.Beef extract-peptone slant strains in step (1) are inoculated in into seed culture medium, in cultivation temperature
30 DEG C, cultivate 12h under conditions of rotating speed 200rpm, obtain seed culture fluid.
(3) fermentation is produced sugar culture-medium and is prepared and fermentation culture:Fermentation produces sugar culture-medium component as above, and pH value is 6.0.Will step
Suddenly the seed liquor that (2) ferment is inoculated in fermentation medium with 5% (v/v) inoculum concentration, in 30 DEG C of cultivation temperature, rotating speed 200rpm
Condition culture 36h, obtain EPS fermentation liquids.
(4) preparation of polysaccharide:The fermentation culture medium 6000rpm centrifugation 10min centrifugations that step (3) is obtained remove thalline
Afterwards, supernatant pH is adjusted to 7.0, adds 3 times of 95% ethanol of volume to stand 12h at 4 DEG C, and it is heavy that 4000rpm centrifugation 10min obtain white
Form sediment, after washed precipitation twice, after 50 DEG C of drying, obtain final product rough extracellular polysaccharide total sugar.
(5) polysaccharide is refined：Rough polysaccharide is added into deionized water dissolving, the polysaccharide solution that concentration is 10% is configured to.
By 1/2 addition Sevag reagents of polysaccharide solution cumulative volume, (V chloroforms: V n-butyl alcohol=4: 1), stand after fully vibrating, centrifugation takes
Supernatant repeats, until without floating preteinses；Repeat with Sevag reagents extracted protein two to three times, between water phase and organic faciess
White Flocculus naked eyes are invisible.Supernatant is collected, plus dehydrated alcohol stands 12 hours to 75% concentration at 4 DEG C, precipitate Jing
Vacuum lyophilization after 75% washing with alcohol, obtains refined extracellular polysaccharide.
Example IV：Extracellular polysaccharide each component is isolated and purified
Ion-exchange chromatography：Rough extracellular polysaccharide 100mg is dissolved in 5ml deionized waters, the polysaccharide of 20mg/mL is obtained
Solution, Jing millipore0.45 μm membrane filtrations are carried out with DEAE-sepharose Fast Flow anion chromatographies exchange columns
Separate.Difference deionized water, 0.05M, 0.2M, 0.8MNaCl solution gradient eluting, elution speed are 2mL/min, and branch receives
Collection eluent, while detect polyoses content with phend-sulphuric acid.All eluents are concentrated respectively, molecular cut off 8000- is used
14000 bag filter flowing water dialysis 48h, vacuum lyophilization obtains polysaccharide products.Wherein water elution is neutral polysaccharide, eluting salt
For acidic polysaccharose.
Embodiment five：The molecular weight determination of polysaccharide component
Weight average molecular weight is determined using High Performance Gel Permeation Chromatography.Glucosan serial standards Dextran (is divided equally again
Son amount for 2000.0kDa, 133.8kDa, 84.4kDa, 41.1kDa, 21.4kDa, 10.0kDa, 7.1kDa, 4.6kDa,
2.5kDa, Sigma) and extracellular polysaccharide sample be made into 4mg/mL, gpc analysis software is with the logarithm pair of dextran standards molecular weight
Retention time is mapped, and draws standard curve and calculates each component molecular size range.HPGPC operating conditions：Instrument：High-efficient liquid phase color
1100 types of spectrometer Agilent；Gel column：TSKgel GMPffXLGPC column (Japanese Tosoh companies)；Mobile phase：
0.1MNaNO3 and carbamide；Flow velocity：1mL/min；Detector：Differential refraction detector (RI)；Temperature：30℃；Sample size：20μL.
Polysaccharide molecular weight is determined using GPC, component 1-4 molecular weight is respectively 175766Da, 24806Da, 62375Da and 17691Da.
Embodiment six：The monosaccharide components identification of polysaccharide component
(1) polysaccharide acid hydrolysis：It is 4～5mg/mL that each component polysaccharide for obtaining is made into concentration, draws 100 μ L in 3mL water
In solution bottle, the 2mol/L trifluoroacetic acids (TFA) of 100 μ L are added, hydrolyze 6h in putting baking oven at 105 DEG C or in 110 DEG C of oil baths
Hydrolysis 2h；Lid is opened after cooling, is dried up with nitrogen after adding 200 μ L methanol, so repeat to add methanol and blown 3 times with nitrogen,
TFA is removed, its residue deionized water is dissolved and is settled to 5mL.
(2) polysaccharide PMP derives：Precision weighs 8 kinds of monosaccharide such as Man, GlcA, Gal, Glc, GalA, Xyl, Arb, Rha respectively
The each 10.0mg of standard substance, the constant volume that adds water fully are dissolved in the volumetric flask of 10mL.Take the mixing monosaccharide titer of 100 μ L respectively with
And the polysaccharide after step (1) acid hydrolysis is placed in the tool plug test tube of 1mL, add the NaOH solution mixing of 100 μ L0.3mol/L equal
It is even, the PMP- methanol solutions of 120 μ L0.5mol/L are added, whirlpool is mixed, and is wrapped at 70 DEG C with masking foil and reacts after sealing
60min, taking-up add the HCl solution of 100 μ L0.3mol/L to neutralize after being cooled to room temperature, and add water to 1mL.It is eventually adding 700 μ
L dichloromethane solutions, repeat extraction 3 times, combining extraction liquid.It is insoluble that sample after derivative needs Jing 3500r/min to be centrifuged off
Precipitation.
(3) efficient liquid phase HPLC chromatogram analysis monosaccharide composition：0.45 μm of micropore of each component polysaccharide Jing that step (2) is obtained
The laggard Agilent1200 high performance liquid chromatographs of membrane filtration are analyzed.Chromatographic column：ZORBAX Eclipse XDB-C18
(250mm×4.6mm)；Mobile phase：0.1mol/L phosphate (pH 6.7) buffer-acetonitrile (volume ratio is 83: 17)；Column temperature：
30℃；Ultraviolet detection wavelength：254nm；Flow velocity：1mL/min；Sampling volume：20μL.Each component monosaccharide composition is as follows：Component 1：
Fructose (Fru)；Component 2:Mannose (Man), rhamnose (Rha), glucose (Glc), galactose (Gal)；Component 3：Mannose
(Man), rhamnose (Rha), glucose (Glc), galactose (Gal), xylose (Xyl)；Component 4:Mannose (Man), rhamnose
(Rha), glucose (Glc), galactose (Gal).
The preferred embodiments of the present invention are the foregoing is only, the present invention is not limited to, for the skill of this area
For art personnel, the present invention can have various modifications and variations.It is all within the spirit and principles in the present invention, made any repair
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (7)
1. a kind of bacillus amyloliquefaciens EZ99 bacterial strains, it is characterised in that during the bacterial strain is preserved on November 14th, 2016
State's Microbiological Culture Collection administration committee common micro-organisms center, preserving number are CGMCC No.13267.
2. the preparation method of a kind of bacillus amyloliquefaciens and its extracellular polysaccharide, it is characterised in that comprise the following steps：
A, strain separating identification and preservation；
B, the culture medium of extracellular polysaccharide are prepared and are fermented；
The preparation of C, rough extracellular polysaccharide；
The preparation of D, refined extracellular polysaccharide；
E, extracellular polysaccharide each component are isolated and purified；
The molecular weight determination of F, polysaccharide each component；
The monosaccharide components identification of G, polysaccharide each component；
In described step A, the bacterial strain of preservation is bacillus amyloliquefaciens EZ99, and preserving number is CGMCC No.13267.
3. the preparation method of bacillus amyloliquefaciens as claimed in claim 1 and its extracellular polysaccharide, it is characterised in that described
Culture medium in step B constitutes (g/L) by following compositions：Sucrose 80-125, yeast extract 3.5-6, peptone 8-12, (NH4)2SO42.5-4, NaCl 2-4, MgSO40.03-0.08, CaCl20.03-0.08, K2HPO4 0.8-1.2。
4. the preparation method of bacillus amyloliquefaciens as claimed in claim 1 and its extracellular polysaccharide, it is characterised in that described
The fermentation condition of the extracellular polysaccharide in step B is:Liquid amount is the bottled 30mL culture medium of every 100mL triangles, and inoculum concentration is 7.2-
7.7% (v/v), original ph are 5.8-6.2, and 30-35 DEG C, 160pm cultivates 30-40h.
5. the preparation method of bacillus amyloliquefaciens as claimed in claim 1 and its extracellular polysaccharide, it is characterised in that described
The preparation of rough extracellular polysaccharide, comprises the following steps：
(1) actication of culture:Strain will be preserved to be connected in beef extract-peptone slant medium, in 30-35 DEG C of constant temperature culture 20-
After 26h, 4 DEG C of refrigerators are stored in standby；
(2) seed culture medium is prepared and inoculated and cultured:Seed liquid culture medium adopts LB culture medium, and liquid amount is per 100mL triangular flasks
Dress 30mL culture medium；Beef extract-peptone slant strains in step (1) are inoculated in into seed culture medium, in cultivation temperature 28-
32 DEG C, cultivate 10-15h under conditions of rotating speed 175-220rpm, obtain seed culture fluid；
(3) fermentation is produced sugar culture-medium and is prepared and fermentation culture:Fermentation produces sugar culture-medium component as above, and pH value is 5.8-6.2；Will step
Suddenly the seed liquor that (2) ferment is inoculated in fermentation medium with 4-6% (v/v) inoculum concentration, in cultivation temperature 28-32 DEG C, rotating speed
The condition of 175-220rpm obtains EPS fermentation liquids in culture 32-40h；
(4) preparation of polysaccharide:The fermentation culture medium 5500-6500rpm centrifugation 8-12min centrifugations that step (3) is obtained are gone degerming
After body, supernatant pH is adjusted to 6.8-7.2, adds 3 times of 95% ethanol of volume to stand 10-15h, 3500-4500rpm centrifugations at 4 DEG C
8-15min obtains white precipitate, after washed twice by precipitation, obtains final product rough extracellular polysaccharide total after 45-55 DEG C of drying
Sugar.
6. the preparation method of bacillus amyloliquefaciens as claimed in claim 1 and its extracellular polysaccharide, it is characterised in that described
The preparation of refined extracellular polysaccharide, comprises the following steps：Rough polysaccharide is added into deionized water dissolving, it is 10% to be configured to concentration
Polysaccharide solution；By 1/2 addition Sevag reagents of polysaccharide solution cumulative volume, stand after fully vibrating, centrifugation, take supernatant repetition, directly
To without floating preteinses；Repeat with Sevag reagents extracted protein two to three times, to White Flocculus meat between water phase and organic faciess
Eye is invisible；Supernatant A is collected, plus dehydrated alcohol stands 10-15 hours, 75% ethanol of precipitate Jing to 75% concentration at 4 DEG C
Vacuum lyophilization after washing, obtains refined extracellular polysaccharide.
7. the preparation method of bacillus amyloliquefaciens as claimed in claim 6 and its extracellular polysaccharide, it is characterised in that described
Isolating and purifying for extracellular polysaccharide each component is comprised the following steps：During ion exchange chromatography purification refine extracellular polysaccharide
The supernatant A for obtaining, the separation condition of the ion exchange chromatography is：With DEAE-Sepharose Fast Flow as filling out
Material, is loaded 5mL, and sample concentration is 10mg/mL, respectively deionized water, 50mM, the NaCl solution of 100mM, 200mM and 800mM
Gradient elution, with the flow velocity eluting of 120mL/h, collects sample at 490nm absworption peaks, and it is 175766Da to respectively obtain molecular weight,
The polysaccharide component of 24806Da, 62375Da and 17691Da.
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| CN110607254A (en) * | 2019-08-27 | 2019-12-24 | 华南理工大学 | Bacillus amyloliquefaciens and preparation method of extracellular polysaccharide thereof |
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| CN110607254B (en) * | 2019-08-27 | 2022-06-14 | 华南理工大学 | Bacillus amyloliquefaciens and preparation method of extracellular polysaccharide thereof |
| CN113755372A (en) * | 2021-08-31 | 2021-12-07 | 中国科学院天津工业生物技术研究所 | Corynebacterium glutamicum producing exopolysaccharides and application thereof |
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| CN113755403A (en) * | 2021-10-12 | 2021-12-07 | 浙江省农业科学院 | Exopolysaccharide-producing lactobacillus pentosus, and fermentation process and application thereof |
| CN114163542A (en) * | 2021-11-26 | 2022-03-11 | 中国农业科学院植物保护研究所 | Extract containing exopolysaccharide, exopolysaccharide and composition |
| CN116925962A (en) * | 2023-06-19 | 2023-10-24 | 东北农业大学 | Bacillus amyloliquefaciens JM033 exopolysaccharide with intestinal flora and immunoregulatory function and application thereof |
| CN116925962B (en) * | 2023-06-19 | 2024-03-29 | 东北农业大学 | Bacillus amyloliquefaciens JM033 exopolysaccharide with intestinal flora and immunoregulatory function and application thereof |
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