CN104231106A - Paenibacillus exopolysaccharide and preparation method and application of paenibacillus exopolysaccharide - Google Patents
Paenibacillus exopolysaccharide and preparation method and application of paenibacillus exopolysaccharide Download PDFInfo
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Abstract
The invention discloses a paenibacillus exopolysaccharide and a preparation method and application of the paenibacillus exopolysaccharide. The structural formula of the paenibacillus exopolysaccharide is shown in a formula (1), wherein n=15-30; the average molecular weight distribution of the paenibacillus exopolysaccharide is 2500-5000 Da, the degree of polymerization of the paenibacillus exopolysaccharide is 15-30, and the monosaccharide component of the paenibacillus exopolysaccharide is fructose. The paenibacillus exopolysaccharide is single in the monosaccharide component and can be prepared by fermenting a paenibacillus BD3526 with a preservation number of CGMCC (China General Microbiological Culture Collection Center) NO. 8333. The fermentation operations of the preparation method of the paenibacillus exopolysaccharide are simple and easy to implement. The invention also discloses a purification method of the paenibacillus exopolysaccharide and application of the paenibacillus exopolysaccharide to externally promoting of proliferation of bifidobacterium infantis and adjusting adult intestinal flora in vitro.
Description
Technical field
The invention belongs to microorganism field, be specifically related to the exocellular polysaccharide of a Bacillus species, preparation method and application thereof.
Background technology
Certain micro-organisms (as milk-acid bacteria, series bacillus, root nodule bacterium etc.), in growth metabolism process, can secrete a class saccharide compound to outside cell walls.Wherein, the glycocalix depended on outside microorganism wall is called capsular polysaccharide, with in the growing environment form of permeating exist for glue polysaccharide.
Microbial exopolysaccharides (be called for short EPS) not only can have special local flavor, also has certain reducing blood-fat, immunomodulatory, the function such as antitumor, therefore can as foodstuff additive.And, along with food-safety problem is more and more subject to the attention of human consumer, how to obtain clear and definite, stable yield, diverse in function new food additive (as thickening material, emulsifying agent, stablizer etc.) of originating, more and more cause the attention of investigator.
Polylevulosan (Levan), connects and composes the side chain of glycan main chain also containing the connection of a small amount of β (2 → 1) fructose glycosidic bond by a large amount of fructose units with β (2 → 6) fructose glycosidic bond and forms.The Polylevulosan of the polymerization degree (being called for short DP) lower (DP=2 ~ 9) is commonly referred to oligofructose, and the Polylevulosan of the polymerization degree 10 ~ 30 is commonly referred to polyfructosan, and the polymerization degree is commonly referred to high levan higher than the Polylevulosan of 40.The microbe-derived Levan of part has the important biological activity such as antitumor, antiviral, hypoglycemic, reducing blood-fat, immunostimulant, in medicine and functional foodstuff, have huge application potential.
A large amount of Levan of acquisition has three kinds of methods at present: chemical synthesis, microbial fermentation solution extract and enzymatic clarification.But the Levan of current chemical synthesis process synthesis is only the trisaccharide structure that β-glycosidic link is formed by connecting.Although plant and microorganism can synthesize Levan, the Levan that fermentable produces at present is high molecular, high-polymerization degree, is generally 2 × 10
6~ 100 × 10
6da (DP is far above 40).And, extract Levan output and Sucrose conversion from microbial fermentation solution at present general lower, and other products such as other superpolymer simultaneous and glucose, fructose, oligofructose in fermented liquid, be thus unfavorable for the mass-producing purifying of Levan.In addition, the enzymatic clarification of Levan, needs to react under the conditions such as certain pH, temperature, and reaction conditions is complicated, is difficult to control.
Summary of the invention
Technical problem to be solved by this invention be overcome Levan high molecular that fermentable produces, defect that high-polymerization degree and tunning are unfavorable for purifying, the exocellular polysaccharide of a Bacillus species, preparation method and application thereof are provided.The described exocellular polysaccharide polymerization degree is moderate, and monosaccharide components is single; Described preparation method is simple, is beneficial to and carries out purifying to described exocellular polysaccharide.
The invention provides the exocellular polysaccharide of a Bacillus species, the structural formula of described exocellular polysaccharide is such as formula shown in (1):
Wherein, n=15 ~ 30.
Preferably, the molecular-weight average of described exocellular polysaccharide is distributed as 2500 ~ 5000Da; And/or there is following outward appearance: pure white filament or powder.
Described exocellular polysaccharide is produced by the series bacillus bacterial strain of the product exocellular polysaccharide of this area routine.Preferably, series bacillus (the Paenibacillus damxungensis sp.nov.) BD3526 being CGMCC NO.8333 by preserving number produces, and is produced by the mutant strain obtained for starting strain with series bacillus BD3526 or derivative.
Described series bacillus preserving number is that the series bacillus BD3526 of CGMCC NO.8333 has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and open in Chinese patent application CN103740618A, the full text of this patent application is quoted by the present invention.
The invention provides a kind of preparation method preparing the exocellular polysaccharide of described series bacillus, it comprises the following steps:
(1) ferment series bacillus CGMCC NO.8333 to obtain fermented liquid;
(2) fermented liquid of step (1) gained is heated 10 ~ 30 minutes at 95 ~ 100 DEG C, after being cooled to 15 ~ 25 DEG C, regulate pH to 4.4 ~ 4.8, leave standstill 3 ~ 5 hours, centrifuging and taking supernatant liquor, adds 80 ~ 100% ethanolic solns of described supernatant volume 2 ~ 4 times, hold over night, centrifugal collecting precipitate, described per-cent is the mass percent accounting for ethanolic soln;
(3) with the precipitate dissolves of 50 ~ 80 DEG C of distilled water by step (2) gained, obtain the throw out solution that concentration is 0.5 ~ 1.0%, described per-cent is the quality volume percent accounting for described throw out solution, trichoroacetic acid(TCA) is added when described solution is cooled to 20 ~ 25 DEG C, trichoroacetic acid(TCA) is made to be finally 4% ~ 10%, described per-cent is the quality volume percent accounting for described solution, leave standstill centrifugal acquisition supernatant liquor again, described supernatant liquor is obtained after the film dialysis that molecular weight cut-off is 1000Da the aqueous solution containing exocellular polysaccharide;
(4) by step (3) gained containing exocellular polysaccharide aqueous solution drying after namely obtain exocellular polysaccharide crude product.
Step (1) is: ferment series bacillus CGMCC NO.8333 to obtain fermented liquid.Fermentation described in step (1) is the fermentation of this area routine; Preferably, described fermentation is 30 DEG C of fermentations 72 hours.Preferably, described fermentation is produced in sugar culture-medium at liquid and is carried out, and described liquid produces sugar culture-medium by 10% sucrose, 1% pancreas casein peptone, 0.5% yeast extract paste, 0.5%K
2hPO
4, 0.034%CaCl
2form with distilled water, described per-cent is account for the mass percent that described liquid produces sugar culture-medium.The inoculum size of described fermentation is the inoculum size of this area routine; Be preferably 1%, described per-cent is account for the mass percent that described liquid produces sugar culture-medium.
Step (2) is: by the fermented liquid of step (1) gained 95 ~ 100 DEG C of heating 10 ~ 30 minutes, after being cooled to 15 ~ 25 DEG C, regulate pH to 4.4 ~ 4.8, leave standstill 3 ~ 5 hours, centrifuging and taking supernatant liquor, adds 80 ~ 100% ethanolic solns of described supernatant volume 2 ~ 4 times, hold over night, centrifugal collecting precipitate, described per-cent is the mass percent accounting for ethanolic soln.Centrifugal condition described in step (2) is that this area is conventional; Be preferably centrifugal 10 minutes of 14000g.Preferably, described adjust ph is for being adjusted to 4.6.Preferably, the ethanolic soln described in step (2) is 95% ethanolic soln, and described per-cent is the mass percent that ethanol accounts for ethanolic soln.Preferably, the add-on of described ethanolic soln is 3 times of described supernatant volume.
Step (3) is: with the precipitate dissolves of 50 ~ 80 DEG C of distilled water by step (2) gained, obtain the throw out solution that concentration is 0.5 ~ 1.0%, described per-cent is the quality volume percent accounting for described throw out solution, trichoroacetic acid(TCA) is added when described solution is cooled to 20 ~ 25 DEG C, trichoroacetic acid(TCA) is made to be finally 4% ~ 10%, described per-cent is the quality volume percent accounting for described solution, leave standstill centrifugal acquisition supernatant liquor again, described supernatant liquor is obtained after the film dialysis that molecular weight cut-off is 1000Da the aqueous solution containing exocellular polysaccharide.Dissolving described in step (3), be preferably with the precipitate dissolves of 60 DEG C of distilled water by step (2) gained, obtain the throw out solution that concentration is 0.8%, described per-cent is the quality volume percent accounting for described throw out solution, trichoroacetic acid(TCA) is added when described solution is cooled to 25 DEG C, make trichoroacetic acid(TCA) be finally 4%, described per-cent is the quality volume percent accounting for described solution.
Step (4) is: by step (3) gained containing exocellular polysaccharide aqueous solution drying after namely obtain exocellular polysaccharide crude product.Drying described in step (4) is the drying of this area routine; It is preferably vacuum lyophilization; More preferably, be 0.160mBar, vacuum lyophilization 72 hours under-30 DEG C of conditions.
Preferably, the preparation method of the exocellular polysaccharide of described series bacillus, also comprises the following steps:
(5) the exocellular polysaccharide crude product that step (4) is obtained is dissolved in 0.05mol/L, in the Tris-HCl damping fluid of pH7.60, wiring solution-forming, through DEAE-Sepharose FF pillar chromatography; Successively with described Tris-HCl damping fluid and the described Tris-HCl damping fluid linear gradient elution containing 0.2 ~ 1.2mol/L NaCl, flow velocity is 2 ~ 6mL/min, surveys absorbancy in 490nm wavelength place, with the pipe number mapping that absorbancy is corresponding, obtains elution curve A;
(6) in elution curve A step (5) obtained, the aqueous solution of unimodal corresponding dialysis is collected, and deionized water is dialysed, and vacuum lyophilization, obtains exocellular polysaccharide component B;
(7) exocellular polysaccharide component B step (6) obtained, is dissolved in described Tris-HCl damping fluid, wiring solution-forming, and upper DEAE-Sepharose CL-4B ion exchange column carries out chromatography; By the described Tris-HCl buffer solution elution containing 0.2 ~ 1.2mol/L NaCl, flow velocity is 2 ~ 6mL/min, surveys absorbancy in 490nm wavelength place, with the pipe number mapping that absorbancy is corresponding, obtains elution curve B;
(8) in elution curve B step (7) obtained, the aqueous solution of unimodal corresponding dialysis is collected, with water dialysis, dry.
Preferably, in step (5), obtained exocellular polysaccharide crude product 50 ~ 200mg is dissolved in 0.05mol/L, in the Tris-HCl damping fluid of pH7.60, is made into the solution of 5 ~ 20mg/mL.Preferably, the flow velocity described in step (5) is 3mL/min.DEAE-Sepharose FF pillar described in step (5) is that this area is conventional; Be preferably D1.6 × 100cm.
Preferably, the flow velocity described in step (7) is 3mL/min.In step (7), described DEAE-Sepharose CL-4B ion exchange column is that this area is conventional; Preferably, be D1.6 × 100cm.
Of the present inventionly spending the night for this area general term, preferably, is 4 ~ 24 hours.
The invention provides described exocellular polysaccharide and promote the application in bifidus bacillus propagation.
Described exocellular polysaccharide is produced by series bacillus, and preferably, series bacillus (the Paenibacillus damxungensis sp.nov.) BD3526 that described exocellular polysaccharide is CGMCC NO.8333 by preserving number produces.Described exocellular polysaccharide is obtained by the method for this area routine, preferably, is obtained by described preparation method.
Described bifidus bacillus is intestinal microflora bifidus bacillus preferably.Described intestinal microflora from the faecal samples of healthy population, preferably, from the faecal samples of 3 ~ 6 years old healthy children.Described bifidus bacillus is preferably bifidobacterium breve (B.breve), long bifidus bacillus (B.longum) or bifidobacterium infantis (B.infantis); More preferably bifidobacterium breve (B.breve).
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: the invention discloses a Bacillus species exocellular polysaccharide, preparation method and application thereof.Exocellular polysaccharide composition of the present invention is single, has the moderate polymerization degree (DP=15 ~ 30), can be fermented and obtain, have the effect similar to commercialization oligofructose by series bacillus BD3526.Described preparation method is easy and simple to handle, is beneficial to and carries out purifying to described exocellular polysaccharide.The present inventor is the physico-chemical property of described exocellular polysaccharide through tight experimental analysis, analyzes its structure composition, and bifidobacteria infantis is bred, the external application regulating adult's intestinal microflora to provide it to promote in vitro.
Accompanying drawing explanation
Fig. 1 is that series bacillus BD3526 exocellular polysaccharide crude product of the present invention surveys absorbance OD at A490nm and A280nm place.
Fig. 2 is the elution curve of series bacillus BD3526 exocellular polysaccharide crude product of the present invention on DEAE-Sepharose ion exchange column; Wherein, ordinate zou be elutriant through the absorbance value OD of phenol-sulfuric acid colometry at n=490nm place, X-coordinate is test tube number.
Fig. 3 is the elution curve of the electrically charged part peak (F1) of series bacillus BD3526 exocellular polysaccharide of the present invention on Sepharose CL-4B molecular sieve gel post; Wherein, ordinate zou be elutriant through the absorbance value OD of phenol-sulfuric acid colometry at n=490nm place, X-coordinate is test tube number.
Fig. 4 is the infared spectrum of series bacillus BD3526 exocellular polysaccharide after purifying of the present invention.
Fig. 5 is series bacillus BD3526 exocellular polysaccharide after purifying of the present invention
13c-NMR composes.
Fig. 6 is the liquid chromatogram measuring molecular weight of series bacillus BD3526 exocellular polysaccharide after purifying of the present invention; Wherein, ordinate zou is retention time, and X-coordinate is peak area.
Fig. 7 is the monose composition of series bacillus BD3526 exocellular polysaccharide after purifying of the present invention; Wherein, ordinate zou is retention time, and X-coordinate is peak area.A () is fructose collection of illustrative plates (object of reference), (b) is series bacillus BD3526 exocellular polysaccharide sample after purifying.
Fig. 8 is series bacillus BD3526 exocellular polysaccharide external promotion bifidobacteria infantis propagation collection of illustrative plates after purifying of the present invention.Wherein, blank group is blank (namely not adding the vitro culture liquid phase of the infant faeces of polysaccharide to micro-flora content); BD3526 exocellular polysaccharide is for adding the relative increase of the vitro culture liquid micro-flora content of the infant faeces of series bacillus BD3526 exocellular polysaccharide after 0.4% (w/v) purifying of the present invention; FOS is for adding the relative increase of the vitro culture liquid micro-flora content of infant faeces after 0.4% (w/v) business oligofructose.
Fig. 9 is the PCR-DGGE collection of illustrative plates in stool culture total thalline V3 district.Wherein, blank group is the coproculture matter sample not adding polysaccharide, and FOS is the coproculture matter sample adding business oligofructose, and BD3526 EPS is the coproculture matter sample adding series bacillus BD3526 exocellular polysaccharide after purifying of the present invention.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Of the present inventionly spending the night for this area general term, preferably, is 4 ~ 24 hours.
The preparation of embodiment 1 BD3526 polysaccharide
(1) preparation of series bacillus fermentation and fermented liquid
Dissolved with 0.1mL sterile distilled water by the lyophilized powder 100 milligrams of series bacillus BD3526, (described product sugar solid medium is by 1.2% agar, 10% sucrose, 1% pancreas casein peptone, 5% yeast extract paste, 5%K to line the sugared solid medium of product with transfering loop picking one ring
2hPO
4, 0.034%CaCl
2form with distilled water, described per-cent is the mass percent accounting for the sugared solid medium of described product) on, 30 DEG C of aerobic cultivations are taken out for 24 hours.
Picking produces the single bacterium colony in sugared solid medium, and (described liquid produces sugar culture-medium by 10% sucrose, 1% pancreas casein peptone, 0.5% yeast extract paste, 0.5%K to be transferred to 20mL liquid product sugar culture-medium
2hPO
4, 0.034%CaCl
2form with distilled water, described per-cent is account for the mass percent that described liquid produces sugar culture-medium) in, cultivate 24 hours, obtain seed liquor for 30 DEG C.Seed liquor is transferred to new liquid in the ratio of inoculum size 1% (v/v) produces in sugared nutrient solution, cultivates 72 hours, obtains fermented liquid for 30 DEG C.
(2) preparation of exocellular polysaccharide crude product in fermented liquid
(2.1) fermented liquid step (1) obtained is at 100 DEG C of heating 30min, after being cooled to 25 DEG C, with lactic acid or the salt acid for adjusting pH to 4.60 of food grade, leave standstill 4 hours, the centrifugal 10min of 14000g, get supernatant liquor, 95% ethanolic soln adding described supernatant liquor 3 times of volumes in above-mentioned centrifugal after supernatant liquor in, hold over night, the centrifugal 10min of 14000g, collecting precipitation thing, described per-cent is the mass percent that ethanol accounts for ethanolic soln.
(2.2) with the precipitate dissolves that step 2.1 obtains by 60 DEG C of distilled water, its final concentration is made to be 0.8%, described per-cent is the quality volume percent accounting for described throw out solution, trichoroacetic acid(TCA) is added when solution is cooled to 25 DEG C, make trichoroacetic acid(TCA) be finally 4%, described per-cent is the quality volume percent accounting for described solution.Again described solution is left standstill 16 hours in 4 DEG C, throw out is removed in centrifugal or filtration, obtains supernatant liquor.Be that the dialysis tubing (U.S.'s spectrum medical science (spectrumlabs)) of 1000Da is dialysed 72 hours in distilled water by described supernatant liquor molecular weight cut-off, within every 12 hours, change water once, the aqueous solution that must dialyse.
(2.3) the direct vacuum lyophilization of the aqueous solution of dialysis step 2.2 obtained: 0.160mBar, under-30 DEG C of conditions, namely vacuum lyophilization obtains exocellular polysaccharide crude product in 72 hours.And survey absorbancy at A490nm and A280nm place, judge purity.Result is as shown in Figure 1: A490nm is polysaccharide characteristic peak, and A280nm is protein characteristic peak, and result display only has a single symmetrical peak of A490nm, illustrates that exocellular polysaccharide raw protein content can be ignored; Learn according to A490nm place peak area and A280nm place calculated by peak area, exocellular polysaccharide crude product purity is higher, and polysaccharide content is 95.7%, and described per-cent is the mass percent accounting for exocellular polysaccharide crude product simultaneously.
The purifying of embodiment 2 exocellular polysaccharide crude product
(1) the exocellular polysaccharide crude product 100mg obtained by embodiment 1 is dissolved in Tris-HCl damping fluid (0.05mol/L, pH7.60) in, be made into the solution of 10.0mg/mL, through DEAE-Sepharose FF pillar (D1.6 × 100cm) (GE Medical Group) chromatography.Successively with Tris-HCl damping fluid and the 0.05mol/L containing 0.2 ~ 1.2mol/L NaCl, the Tris-HCl damping fluid linear gradient elution of pH7.60, flow velocity is 3mL/min, and every 6mL collects 1 pipe.With sulfuric acid-phynol method (Dubois, M., K.A.Gilles, J.K.Hamilton, P.A.Rebers, and F.Smith, 1956.Colorimeteric method for determination of sugars and related substances.Anal.Chem.28:350-356) tracking monitor, survey absorbancy in 490nm wavelength (i.e. polysaccharide characteristic wavelength) place, with the pipe number mapping that absorbancy is corresponding, obtain elution curve A, the data of elution curve are in table 1.Obtain the polysaccharide component of a preliminary purification thus, namely peak F1 (see Fig. 2) may contain two or more different polysaccharide component, needs next step purifying to verify.
Table 1 elution curve A data
| Test tube number | OD 490nm |
| 2 | 0.0711 |
| 6 | 0.0597 |
| 10 | 0.0539 |
| 14 | 0.0758 |
| 18 | 0.062 |
| 22 | 0.06 |
| 26 | 0.063 |
| 28 | 0.083 |
| 29 | 0.093 |
| 30 | 0.112 |
| 31 | 0.1845 |
| 32 | 0.3545 |
| 33 | 0.7526 |
| 34 | 1.0895 |
| 35 | 1.1524 |
| 36 | 1.0752 |
| 37 | 0.8605 |
| 38 | 0.4123 |
| 39 | 0.2715 |
| 40 | 0.1628 |
| 41 | 0.098 |
| 42 | 0.085 |
| 43 | 0.078 |
| 47 | 0.068 |
| 51 | 0.07 |
| 52 | 0.0656 |
| 55 | 0.0731 |
| 58 | 0.0664 |
| 63 | 0.0671 |
| 67 | 0.0723 |
| 71 | 0.0689 |
| 75 | 0.0742 |
| 79 | 0.0853 |
| 81 | 0.0725 |
| 82 | 0.0834 |
| 83 | 0.0923 |
| 84 | 0.0808 |
| 85 | 0.0946 |
| 86 | 0.0989 |
| 87 | 0.0873 |
| 88 | 0.0622 |
| 89 | 0.0676 |
| 90 | 0.0648 |
(2) aqueous solution that in Fig. 2 step (1) obtained, in unimodal (F1) corresponding pipe number, wash-out obtains is collected, dialyse 72 hours with the dialysis tubing deionized water of 1000Da, 0.160mBar vacuum ,-30 DEG C are freezing, after dry 72 hours, obtain exocellular polysaccharide component B.
(3) exocellular polysaccharide component B step (2) obtained, be dissolved in Tris-HCl damping fluid, be made into the solution of 10.0mg/mL, (filler is DEAE-Sepharose CL-4B to upper ion exchange column, and pillar: D1.6 × 100cm (GE Medical Group) carries out chromatography.By the Tris-HCl buffer solution elution containing 0.2 ~ 1.2mol/L NaCl, flow velocity is 3mL/min, and every 6mL collects 1 pipe.Use sulfuric acid-phynol method tracking monitor, survey absorbancy in 490nm wavelength place, with the pipe number mapping that absorbancy is corresponding, obtain elution curve B.Experimental result, see only there being a unimodal F2 in Fig. 3, Fig. 3, illustrate only containing a kind of polysaccharide component, and F2 is the polysaccharide component obtained be further purified the polysaccharide component F1 of preliminary purification.
(4) aqueous solution that the chromatography in Fig. 3 step (3) obtained in unimodal (F2) respective tube number obtains is collected, dialyse 72 hours with the dialysis tubing deionized water of 1000Da, 0.160mBar, under-30 DEG C of conditions, namely vacuum lyophilization obtains the exocellular polysaccharide of purifying after 72 hours, polysaccharide content is 100%, not containing protein, described per-cent is the mass percent accounting for exocellular polysaccharide.
The exocellular polysaccharide of the purifying prepared by embodiment 1 ~ 2 will be used in embodiment 5 ~ 9.
Embodiment 3
One, the preparation of BD3526 polysaccharide
(1) preparation of series bacillus fermentation and fermented liquid
Dissolved with 0.1mL sterile distilled water by the lyophilized powder 100 milligrams of series bacillus BD3526, (described product sugar solid medium is by 1.2% agar, 10% sucrose, 1% pancreas casein peptone, 0.5% yeast extract paste, 0.5%K to line the sugared solid medium of product with transfering loop picking one ring
2hPO
4, 0.034%CaCl
2form with distilled water, described per-cent is the mass percent accounting for the sugared solid medium of described product) on, 30 DEG C of aerobic cultivations are taken out for 24 hours.
Picking produces the single bacterium colony in sugared solid medium, and picking produces the single bacterium colony in sugared solid medium, and (described liquid produces sugar culture-medium by 10% sucrose, 1% pancreas casein peptone, 0.5% yeast extract paste, 0.5%K to be transferred to 20mL liquid product sugar culture-medium
2hPO
4, 0.034%CaCl
2form with distilled water, described per-cent is account for the mass percent that described liquid produces sugar culture-medium) in, cultivate 24 hours, obtain seed liquor for 30 DEG C.Seed liquor is transferred to new liquid in the ratio of inoculum size 1% (v/v) produces in sugared nutrient solution, cultivates 72 hours, obtains fermented liquid for 30 DEG C.
(2) preparation of exocellular polysaccharide crude product in fermented liquid
(2.1) fermented liquid step (1) obtained is at 95 DEG C of heating 10min, after being cooled to 15 DEG C, with lactic acid or the salt acid for adjusting pH to 4.4 of food grade, leave standstill 3 hours, the centrifugal 10min of 14000g, get supernatant liquor, 80% ethanolic soln adding described supernatant liquor 4 times of volumes in above-mentioned centrifugal after supernatant liquor in, hold over night, the centrifugal 10min of 14000g, collecting precipitation thing, described per-cent is the mass percent that ethanol accounts for ethanolic soln.
(2.2) with the precipitate dissolves that step 2.1 obtains by 50 DEG C of distilled water, its final concentration is made to be 0.5%, described per-cent is the quality volume percent accounting for described throw out solution, trichoroacetic acid(TCA) is added when solution is cooled to 25 DEG C, make trichoroacetic acid(TCA) be finally 10%, described per-cent is the quality volume percent accounting for described solution.Again by described solution in 4 DEG C of standing 16h, centrifugal or filter remove throw out, obtain supernatant liquor.Be that the dialysis tubing (U.S.'s spectrum medical science (spectrumlabs)) of 1000Da is dialysed 72 hours in distilled water by described supernatant liquor molecular weight cut-off, within every 12 hours, change water once, the aqueous solution that must dialyse.
(2.3) the direct vacuum lyophilization of the aqueous solution of dialysis step 2.2 obtained: 0.160mBar, under-30 DEG C of conditions, namely vacuum lyophilization obtains exocellular polysaccharide crude product in 72 hours.And survey absorbancy at A490nm and A280nm place, judge purity.
Two, the purifying of exocellular polysaccharide crude product
(1) the exocellular polysaccharide crude product 100mg obtained by embodiment 1 is dissolved in Tris-HCl damping fluid (0.05mol/L, pH7.60) in, be made into the solution of 5mg/mL, through DEAE-Sepharose FF pillar (D1.6 × 100cm) (GE Medical Group) chromatography.Successively with Tris-HCl damping fluid and the 0.05mol/L containing 0.2 ~ 1.2mol/L NaCl, the Tris-HCl damping fluid linear gradient elution of pH7.60, flow velocity is 2mL/min, and every 6mL collects 1 pipe.Survey absorbancy with sulfuric acid-phynol method in 490nm wavelength (i.e. polysaccharide characteristic wavelength) place, with the pipe number mapping that absorbancy is corresponding, obtain elution curve A.Obtain the polysaccharide component of a preliminary purification thus, i.e. unimodal F1.
(2) aqueous solution that in unimodal (F1) that step (1) obtained corresponding pipe number, wash-out obtains is collected, dialyse 72 hours with the dialysis tubing deionized water of 1000Da, 0.160mBar vacuum ,-30 DEG C of lyophilizes, after 72 hours, obtain exocellular polysaccharide component B.
(3) exocellular polysaccharide component B step (2) obtained, be dissolved in Tris-HCl damping fluid, be made into the solution of 5mg/mL, (filler is DEAE-Sepharose CL-4B to upper ion exchange column, and pillar: D1.6 × 100cm (GE Medical Group) carries out chromatography.By the Tris-HCl buffer solution elution containing 0.2 ~ 1.2mol/L NaCl, flow velocity is 2mL/min, and every 6mL collects 1 pipe.Use sulfuric acid-phynol method tracking monitor, survey absorbancy in 490nm wavelength place, with the pipe number mapping that absorbancy is corresponding, obtain elution curve B.Obtain the polysaccharide component that a component F1 is further purified thus, i.e. unimodal F2.
(4) aqueous solution that the chromatography in unimodal (F2) respective tube of step (3) being obtained number obtains is collected, dialyse 72 hours with the dialysis tubing deionized water of 1000Da, 0.160mBar, under-30 DEG C of conditions, namely vacuum lyophilization obtains the exocellular polysaccharide of purifying after 72 hours.
Embodiment 4
One, the preparation of BD3526 polysaccharide
(1) preparation of series bacillus fermentation and fermented liquid
Dissolved with 0.1mL sterile distilled water by the lyophilized powder 100 milligrams of series bacillus BD3526, (described product sugar solid medium is by 1.2% agar, 10% sucrose, 1% pancreas casein peptone, 0.5% yeast extract paste, 0.5%K to line the sugared solid medium of product with transfering loop picking one ring
2hPO
4, 0.034%CaCl
2form with distilled water, described per-cent is the mass percent accounting for the sugared solid medium of described product) on, 30 DEG C of aerobic cultivations are taken out for 24 hours.
Picking produces the single bacterium colony in sugared solid medium, and picking produces the single bacterium colony in sugared solid medium, and (described liquid produces sugar culture-medium by 10% sucrose, 1% pancreas casein peptone, 0.5% yeast extract paste, 0.5%K to be transferred to 20mL liquid product sugar culture-medium
2hPO
4, 0.034%CaCl
2form with distilled water, cultivate 24 hours, obtain seed liquor for 30 DEG C.Seed liquor is transferred to new liquid in the ratio of inoculum size 1% (v/v) produces in sugared nutrient solution, cultivates 72 hours, obtains fermented liquid for 30 DEG C.
(2) preparation of exocellular polysaccharide crude product in fermented liquid
(2.1) fermented liquid step (1) obtained is at 100 DEG C of heating 30min, after being cooled to 25 DEG C, with lactic acid or the salt acid for adjusting pH to 4.8 of food grade, leave standstill 5 hours, centrifugal 10 minutes of 14000g, get supernatant liquor, 80% ethanolic soln adding described supernatant liquor 2 times of volumes in above-mentioned centrifugal after supernatant liquor in, hold over night, centrifugal 10 minutes of 14000g, collecting precipitation thing, described per-cent is the mass percent that ethanol accounts for ethanolic soln.
(2.2) with the precipitate dissolves that step 2.1 obtains by 80 DEG C of distilled water, its final concentration is made to be 1%, described per-cent is the quality volume percent accounting for described throw out solution, trichoroacetic acid(TCA) is added when solution is cooled to 20 DEG C, make trichoroacetic acid(TCA) be finally 6%, described per-cent is the quality volume percent accounting for described solution.Again described solution is left standstill 16 hours in 4 DEG C, throw out is removed in centrifugal or filtration, obtains supernatant liquor.Be that the dialysis tubing (U.S.'s spectrum medical science (spectrumlabs)) of 1000Da is dialysed 72 hours in distilled water by described supernatant liquor molecular weight cut-off, within every 12 hours, change water once, the aqueous solution that must dialyse.
(2.3) the direct vacuum lyophilization of the aqueous solution of dialysis step 2.2 obtained: 0.160mBar, under-30 DEG C of conditions, namely vacuum lyophilization obtains exocellular polysaccharide crude product in 72 hours.And survey absorbancy at A490nm and A280nm place, judge purity.
Two, the purifying of exocellular polysaccharide crude product
(1) the exocellular polysaccharide crude product 100mg obtained by embodiment 1 is dissolved in Tris-HCl damping fluid (0.05mol/L, pH7.60) in, be made into the solution of 20mg/mL, through DEAE-Sepharose FF pillar (D1.6 × 100cm) (GE Medical Group) chromatography.Successively with Tris-HCl damping fluid and the 0.05mol/L containing 0.2 ~ 1.2mol/L NaCl, the Tris-HCl damping fluid linear gradient elution of pH7.60, flow velocity is 6mL/min, and every 6mL collects 1 pipe.Survey absorbancy with sulfuric acid-phynol method in 490nm wavelength (i.e. polysaccharide characteristic wavelength) place, with the pipe number mapping that absorbancy is corresponding, obtain elution curve A.Obtain the polysaccharide component of a preliminary purification thus, i.e. unimodal F1.
(2) aqueous solution that in unimodal (F1) that step (1) obtained corresponding pipe number, wash-out obtains is collected, dialyse 72 hours with the dialysis tubing deionized water of 1000Da, 0.160mBar vacuum ,-30 DEG C of lyophilizes, after 72 hours, obtain exocellular polysaccharide component B.
(3) exocellular polysaccharide component B step (2) obtained, be dissolved in Tris-HCl damping fluid, be made into the solution of 20mg/mL, (filler is DEAE-Sepharose CL-4B to upper ion exchange column, and pillar: D1.6 × 100cm (GE Medical Group) carries out chromatography.By the Tris-HCl buffer solution elution containing 0.2 ~ 1.2mol/L NaCl, flow velocity is 6mL/min, and every 6mL collects 1 pipe.Use sulfuric acid-phynol method tracking monitor, survey absorbancy in 490nm wavelength place, with the pipe number mapping that absorbancy is corresponding, obtain elution curve B.Obtain the polysaccharide component that a component F1 is further purified thus, i.e. unimodal F2.
(4) aqueous solution that the chromatography in unimodal (F2) respective tube of step (3) being obtained number obtains is collected, dialyse 72 hours with the dialysis tubing deionized water of 1000Da, 0.160mBar, under-30 DEG C of conditions, namely vacuum lyophilization obtains the exocellular polysaccharide of purifying after 72 hours.
The structure elucidation of embodiment 5 exocellular polysaccharide
(1) infrared (FI-IR) analyzes
Exocellular polysaccharide 2.0mg and the KBr of purifying embodiment 2 obtained grinds compressing tablet, at 4000 ~ 500cm
-1infrared spectrum scanning (Xu Guangtong is carried out in region, Yuan Hongfu, Lu Wanzhen etc. Modern Near-Infrared Spectroscopic Techniques and application progress. spectroscopy and spectroscopic analysis, 2000,02:134-142.) (infrared spectrometer: Thermo Fischer Scient Inc.).As shown in Figure 4, detailed data is in table 1 for infrared spectra collection of illustrative plates.
Table 1 infrared spectra spectrum data
(2) nucleus magnetic resonance (NMR) is analyzed
The exocellular polysaccharide 10 milligrams of purifying embodiment 2 obtained is dissolved in 1mL heavy water.JNM-A500 (being manufactured by Nippon Denshi) is used to measure
1h-NMR spectrum and
13c-NMR composes.
13c-NMR spectrum represents in Figure 5.
1it is as follows that H-NMR composes concrete data: δ (3.538) ~ (3.585) ppm is H-6b, δ (3.661) ~ (3.692) ppm is H-1b, δ (3.748) ~ (3.793) ppm is H-1a, δ (3.894) ppm is H-6a, δ (3.935) ppm is H-5, δ (4.082) ~ (4.132) ppm is H-4, δ (4.082) ~ (4.132) ppm is H-4, δ (4.184) ~ (4.205) ppm is H-3.In Fig. 5, δ (62.67) is C-1, δ (66.46) ~ δ (66.25) is C-6, δ (77.38) ~ δ (77.98) is C-4, δ (79.07) is C-3, δ (83.07) ~ δ (83.92) for C-5, δ (79.07) be C-2.Thus with the glycosidic link standard diagram comparison of fructose, show that described exocellular polysaccharide is the Polylevulosan that β (2 → 6) glycosidic link connects.Meanwhile, the data results of NMR also shows, the exocellular polysaccharide of the purifying that embodiment 2 obtains, not containing the characteristic peak of other non-saccharide structures of albumen, sulfuration, amino etc., namely not containing impurity such as protein, is pure Polylevulosan.
The molecular weight distribution of the exocellular polysaccharide after embodiment 6 purifying
(1) by the standard substance of different molecular weight sample introduction in succession, record retention time TR, take TR as X-coordinate, lgM is ordinate zou drawing standard curve, draws the regression equation of molecular weight and retention time TR.
(2) after purifying to be measured, exocellular polysaccharide is dissolved in ddH
2in O, concentration is 10g/L.Sample introduction, obtains TR, by the relative molecular weight of regression equation calculation sample.
Chromatographic condition is as follows:
Chromatographic instrument: Viscotek TDAmax (waters China)
Detector: Composition distribution (waters China)
Chromatographic column: GPCmaxTM scope 2000-20000000 (waters China)
Moving phase: 0.1mol/L NaNO
3
Column temperature: 30 DEG C; Flow velocity: 1mL/min; Sample concentration: 10mg/mL
The molecular weight distribution result of polysaccharide described above as shown in Figure 6, result shows: at retention time 9.7min place appearance absorption peak, with the polysaccharide Chromatographic Comparison of standard molecular weight, this polysaccharide is made up of single polysaccharide, the average molecular weight range utilizing TR to calculate polysaccharide described above is 2500 ~ 5000Da, the exopolysaccharide molecule of about 90% is in the scope of 2500 ~ 5000Da, and described per-cent is the molar percentage accounting for exopolysaccharide molecule.
The monosaccharide composition analysis of the exocellular polysaccharide after embodiment 7 purifying
The exocellular polysaccharide 10mg of purifying embodiment 2 obtained, is dissolved in the H of 2mL 0.05mol/L
2sO
4in solution, 80 DEG C, hydrolysis 2h obtains hydrolyzed solution A, hydrolyzed solution A is diluted 100 times and obtains hydrolyzed solution B.Hydrolyzed solution B by high-efficient ion chromatogram (HPAEC-PAD) analyze its monose composition (Suo Hui. the primary structure research (D) of garlic fructosan. Guangdong: Ji'nan University, 2010).Ion chromatography condition is: chromatographic column (Carbopac China): Carb1,4 × 250mm; Flow velocity: 1.0mL/min; Sample size: 20 μ L; Detector: pulsed amperometry (Carbopac China), gold electrode; Probe temperature: 30 DEG C.With 15mM NaOH solution isocratic elution.Experimental result is shown in Fig. 7, and result shows, and fructose, when 14:36min, produces the peak of an about 4200nA.Exocellular polysaccharide sample after purifying produces a peak when 14:54min, identical with fructose appearance time, illustrates, the monose of the exocellular polysaccharide after purifying is made up of single fructose.
Result according to embodiment 5 ~ 7 illustrates, the repeated structural unit of described exocellular polysaccharide is fructose, and the mode of connection of fructose is → 2,6-fructose → 2,6-fructose, and described exocellular polysaccharide is Polylevulosan, the structural formula of described exocellular polysaccharide such as formula shown in (1),
Wherein, n=15 ~ 30, and described exocellular polysaccharide has the outward appearance of pure white filament or powder.
The external promotion bifidobacteria infantis propagation of embodiment 8 exocellular polysaccharide
(1) (age is 6 ~ 24 months to gather the ight soil of 30 babies, recent without intestinal tract disease and take antibiotics), according to method preparation standard faecal microbiota nutrient solution (the Models of the gastrointestinal tract to study microbial interactions Original Research Article Biology of Growing Animals of Minekus etc., 2005,2:142-154).
(2) by a certain amount of standard ileal jet substratum (SIEM, pectin 0.047g/L, xyloglucan 0.047g/L, arabogalactan 0.047g/L, amylose starch 0.047g/L, starch 0.392g/L, casein 24.0g/L, tween 80 17.0g/L, bacto peptone 24.0g/L, oxgall 0.4g/L, halfcystine 0.2g/L) join 96 hole titer plate after, add the standard faecal microbiota nutrient solution of 0.1%, described per-cent is the volume ratio accounting for described standard ileal jet substratum, then in 96 different holes, add the exocellular polysaccharide (being called for short BD3526 exocellular polysaccharide) of the purifying that embodiment 2 obtains by 0.4% respectively, 0.4% business oligofructose (being called for short FOS), in each hole, the final volume of liquid is 1.5mL, make blank simultaneously, 37 DEG C, Anaerobic culturel 8h, described per-cent is the quality volume percent of the final volume accounting for liquid in a hole in described 96 hole titer plate.
(3) after cultivation terminates, fermented liquid is mixed with 250 μ L lysis buffers, 250 μ L zirconiums pearl liquid (0.1mm) and 200 μ L phenol solution, then the upper homogenate of pearl grinder (BIO SPEC company of the U.S.) 2 times, each 2 minutes, with DNA test kit (Tian Gen biochemical technology company limited) extracting DNA.Then PCR-enteric microorganism microarray assay (SALAZAR N is adopted, GUEIMONDEM, HERNANDEZ-BARRANCO A M, et al.Exopolysaccharides produced by intestinal Bifidobacterium strains act as fermentable substrates for human intestinal bacteria.Applied and environmental microbiology, 2008, 74 (15): 4737-45.) in analytic sample, the relative blank of flora (does not add polysaccharide, the standard ight soil bacterium liquid of direct cultivation) quantity, experimental result as Suo Shi Fig. 8 and table 2 (herein, log (ORF/mL) the value numerical value of all floras of blank is defined as 0).As shown in Figure 8, compared with blank group, series bacillus BD3526 EPS can promote the propagation of bifidus bacillus in infant faeces flora particularly bifidobacterium breve (B.breve) as FOS.As shown in Table 2, relative to blank, the quantity of adding bifidus bacillus in the sample flora of FOS and BD3526 exocellular polysaccharide both increases.Especially bifidobacterium breve (B.breve), that adds its quantitative value (log (ORF/mL) value) in the sample of BD3526 exocellular polysaccharide increases to 0.81, almost will reach the numerical value (1.18) adding FOS.And bifidus longum bb (B.Longum), add in the sample of BD3526 exocellular polysaccharide, its quantitative value (log (ORF/mL) value) increase to 0.36, close to the value 0.37 of adding FOS sample.
The quantity of bacterial classification in rear flora cultivated for 8 hours by table 2
The external effect regulating adult's intestinal microflora composition of embodiment 9 exocellular polysaccharide
(1) gather 1 normal adults ight soil (age be 23-25 year sex, recent without intestinal tract disease and take antibiotics), be dissolved in pH 7.3 phosphate buffered saline buffer (8g/L NaCl, 0.2g/L KCl, 1.15g/L NaHPO
4, 0.2g/L KH
2pO
4) mix afterwards, volume ratio with 1: 10 adds in culture system, exocellular polysaccharide (being called for short BD3526EPS), the 0.4% business oligofructose (being called for short FOS) of the purifying then embodiment 2 obtained add in culture system respectively, do blank simultaneously, 37 DEG C, Anaerobic culturel 24h, described per-cent is the quality volume percent accounting for culture system.
(2) after cultivation terminates, adopt phenol chloroform method (ERCOLINI D, HILL P J, DODD C E.Bacterial community structure and location in Stilton cheese.Applied and environmental microbiology, 2003, 69 (6): 3540-8.) DNA of culture and faecal microbiota is extracted, then PCR-DGGE (denaturing gradient gel electrophoresis) method (Analysis of inherited and acquired mutations using PCR and denaturing gradient gel electrophoresis (DGGE) Mutation Research/Environmental Mutagenesis and Related Subjects is adopted, Volume 252, Issue 2, 1991, 175-176.A.-L.
e.Hovig, B.Smith-
s.Lystad, A.
) composition of analytic sample flora.Namely universal primer 357F (as shown in SEQ ID NO.1) (5 '-TACGGGAGGCAGCAG-3 '), 518R (as shown in SEQ ID NO.2) (5 '-ATTACCGCGGCTGCTGG-3 ') and the GC clip (as shown in SEQ ID NO.3) of bacterial 16 S rDNA is adopted.(5 '-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCC-3 ') PCR amplification system is 25 μ L (each 1 μ L of aseptic double-distilled water 8.5 μ L, primer 518R and GC-357F, template 2 μ L, 2X Plus-Mix 12.5 μ L).Amplification program is: 94 DEG C, 5min; Then 94 DEG C of 30s, 56 DEG C of 30s, 68 DEG C of 40s, 35 circulations; Last 72 DEG C extend 10min.It is 8% (wt) that amplified production joins concentration of polyacrylamide, and denatured gradient is on the glue of 40% (wt) ~ 60% (wt), with 1X TAE for electrophoretic buffer, at 60 DEG C, runs 13 hours with the voltage of 75V.After electrophoresis completes, dye with ethidium bromide, take pictures in gel imaging instrument, the results are shown in Figure 9.In Fig. 9, the band of arrow indication is obviously bright in FOS group and blank group, illustrates that BD3526 EPS can promote the propagation of this bacterioid in people's intestinal microflora, has the ability regulating human intestinal microflora composition.
Claims (10)
1. the exocellular polysaccharide of a Bacillus species, is characterized in that, the structural formula of described exocellular polysaccharide such as formula shown in (1),
Wherein, n=15 ~ 30.
2. exocellular polysaccharide as claimed in claim 1, it is characterized in that, the molecular-weight average of described exocellular polysaccharide is distributed as 2500 ~ 5000Da; And/or there is following outward appearance: pure white filament or powder.
3. exocellular polysaccharide as claimed in claim 1, it is characterized in that, series bacillus (the Paenibacillus damxungensis sp.nov.) BD3526 that described exocellular polysaccharide is CGMCC NO.8333 by preserving number produces.
4. prepare a preparation method for the exocellular polysaccharide of the series bacillus as described in claims 1 to 3 any one, it comprises the following steps:
(1) ferment series bacillus CGMCC NO.8333 to obtain fermented liquid;
(2) fermented liquid of step (1) gained is heated 10 ~ 30 minutes at 95 ~ 100 DEG C, after being cooled to 15 ~ 25 DEG C, adjust ph to 4.4 ~ 4.8, leave standstill 3 ~ 5 hours, centrifuging and taking supernatant liquor, adds 80 ~ 100% ethanolic solns of described supernatant volume 2 ~ 4 times, hold over night, centrifugal collecting precipitate, described per-cent is the mass percent that ethanol accounts for ethanolic soln;
(3) with the precipitate dissolves of 50 ~ 80 DEG C of distilled water by step (2) gained, obtain the throw out solution that concentration is 0.5 ~ 1.0%, described per-cent is the quality volume percent accounting for described throw out solution, trichoroacetic acid(TCA) is added when described solution is cooled to 20 ~ 25 DEG C, trichoroacetic acid(TCA) is made to be finally 4% ~ 10%, described per-cent is the quality volume percent accounting for described solution, leave standstill centrifugal acquisition supernatant liquor again, described supernatant liquor is obtained after the film dialysis that molecular weight cut-off is 1000Da the aqueous solution containing exocellular polysaccharide;
(4) by step (3) gained containing exocellular polysaccharide aqueous solution drying after namely obtain exocellular polysaccharide crude product.
5. preparation method as claimed in claim 4, is characterized in that, the fermentation described in step (1) is 30 DEG C of fermentations 72 hours; Described fermentation is produced in sugar culture-medium at liquid and is carried out, and described liquid produces sugar culture-medium by 10% sucrose, 1% pancreas casein peptone, 0.5% yeast extract paste, 0.5%K
2hPO
4, 0.034%CaCl
2form with distilled water, described per-cent is account for the mass percent that described liquid produces sugar culture-medium;
Centrifugal described in step (2) is centrifugal 10 minutes of 14000g; Described adjust ph is for being adjusted to 4.6; Described ethanolic soln is 95% ethanolic soln, and described per-cent is the mass percent accounting for ethanolic soln; The add-on of described ethanolic soln is 3 times of described supernatant volume;
Be dissolved as with the precipitate dissolves of 60 DEG C of distilled water by step (2) gained described in step (3), obtain the throw out solution that concentration is 0.8%, described per-cent is the quality volume percent accounting for described throw out solution, trichoroacetic acid(TCA) is added when described solution is cooled to 25 DEG C, make trichoroacetic acid(TCA) be finally 4%, described per-cent is the quality volume percent accounting for described solution; Or,
Drying described in step (4) is vacuum lyophilization; Preferably, be 0.160mBar, vacuum lyophilization 72 hours under-30 DEG C of conditions.
6. preparation method as claimed in claim 4, it is characterized in that, described preparation method also comprises the following steps:
(5) the exocellular polysaccharide crude product that step (4) is obtained is dissolved in 0.05mol/L, in the Tris-HCl damping fluid of pH7.60, wiring solution-forming, through DEAE-Sepharose FF pillar chromatography; Successively with described Tris-HCl damping fluid and the described Tris-HCl damping fluid linear gradient elution containing 0.2 ~ 1.2mol/L NaCl, flow velocity is 2 ~ 6mL/min, surveys absorbancy in 490nm wavelength place, with the pipe number mapping that absorbancy is corresponding, obtains elution curve A;
(6) in elution curve A step (5) obtained, the aqueous solution of unimodal corresponding dialysis is collected, with water dialysis, dry, obtains exocellular polysaccharide component B;
(7) exocellular polysaccharide component B step (6) obtained, is dissolved in described Tris-HCl damping fluid, wiring solution-forming, and upper DEAE-Sepharose CL-4B ion exchange column carries out chromatography; By the described Tris-HCl buffer solution elution containing 0.2 ~ 1.2mol/L NaCl, flow velocity is 2 ~ 6mL/min, surveys absorbancy in 490nm wavelength place, with the pipe number mapping that absorbancy is corresponding, obtains elution curve B;
(8) in elution curve B step (7) obtained, the aqueous solution of unimodal corresponding dialysis is collected, with water dialysis, dry.
7. preparation method as claimed in claim 6, is characterized in that, in step (5), obtained exocellular polysaccharide crude product 50 ~ 200mg is dissolved in 0.05mol/L, in the Tris-HCl damping fluid of pH7.60, is made into the solution of 5 ~ 20mg/mL; Flow velocity described in step (5) is 3mL/min; DEAE-Sepharose FF pillar described in step (5) is D1.6 × 100cm; In step (7), obtained exocellular polysaccharide crude product 50 ~ 200mg is dissolved in described Tris-HCl damping fluid, is made into the solution of 5 ~ 20mg/mL; Flow velocity described in step (7) is 3mL/min; Or the DEAE-Sepharose FF pillar described in step (7) is D1.6 × 100cm.
8. the exocellular polysaccharide as described in any one of claims 1 to 3 is promoting the application in bifidus bacillus propagation.
9. apply as claimed in claim 8, it is characterized in that, described bifidus bacillus is bifidobacterium breve (B.breve).
10. apply as claimed in claim 8, it is characterized in that, described bifidus bacillus is the bifidus bacillus in human intestinal microflora.
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