CN104403981A - Novel exopolysaccharide as well as production strain and preparation method thereof - Google Patents

Novel exopolysaccharide as well as production strain and preparation method thereof Download PDF

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CN104403981A
CN104403981A CN201410786092.0A CN201410786092A CN104403981A CN 104403981 A CN104403981 A CN 104403981A CN 201410786092 A CN201410786092 A CN 201410786092A CN 104403981 A CN104403981 A CN 104403981A
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exocellular polysaccharide
novel
polysaccharide
exocellular
strain
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方亚鹏
崔娜娜
赵萌
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Hubei University of Technology
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Hubei University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/22—Klebsiella

Abstract

The invention discloses a novel exopolysaccharide as well as a production strain and a preparation method thereof and belongs to the technical field of microorganisms. The novel exopolysaccharide is an acidic polysaccharide, exists in an aqueous phase in the form of nanoparticles and is completely dissolved in dimethyl sulfoxide; the structure is stable when the pH is in a range of 3-11, and gel is formed during neutralization after alkali treatment. The strain capable of being used for producing the exopolysaccharide through fermentation is Klebsiella pneumoniae PHRC1.001 with the preservation number of CCTCC NO:M2014427. The strain is used as an original strain, a carbohydrate is used as a main carbon source, nitrate is used as a main nitrogen source, and the novel exopolysaccharide can be produced through fermentation. An obtained novel exopolysaccharide product is a hydrophilic colloid additive which has the great market potential, can be applied to fields of flocculation, cement, ceramic, food additives and the like, and can be used for preparing metal nanoparticles.

Description

A kind of novel exocellular polysaccharide and produce bacterial strain and preparation method
Technical field
The present invention relates to microbial technology field, particularly relate to a kind of novel exocellular polysaccharide and produce bacterial strain and preparation method.
Background technology
After xanthan gum first time realizes suitability for industrialized production and realizes huge business success, the exploitation of microbial polysaccharide causes the great interest of scientific circles and industry member, and numerous and confused input human and material resources and financial resources carry out the new microbial polysaccharide research with property.
Microbial polysaccharide comprises intracellular polyse, cell wall polysaccharide and exocellular polysaccharide.Microbial exopolysaccharides (Exopolysaccharide) refers in the vegetative activity of many microorganisms under different external conditions and is secreted into polysaccharide outside cell walls or polysaccharide mixture.On the one hand, exocellular polysaccharide is easily separated with thalline, can realize suitability for industrialized production by submerged fermentation.Compared with animals and plants polysaccharide, the impact of the factor such as production climate, geographical environment, natural disaster of microbial polysaccharide is less, with short production cycle, output and steady quality, and microbial polysaccharide safety non-toxic.On the other hand, microbial polysaccharide has unique physico-chemical property, can be used as emulsifying agent, lubricant, jelling agent, membrane-forming agent, suspension agent, thickening material and stablizer and is widely used in multiple fields such as chemical industry, agricultural, oil, pharmacy and food.
Up to the present, maturation has been studied and the microbial polysaccharide put into production mainly contains xanthan gum (Xanthan gum), Welan gum (Welan gum), gelling gum (Gellan gum), scleroglycan (Scleeroglucan), Pul (Pullulan), heat setting polysaccharide (Curdlan) etc.The output of microbial polysaccharide and annual increment are all significantly going up in the world, and the annual increment of some novel polysaccharides also reaches more than 30%, and according to estimates, the year industrial output value of whole world microbial polysaccharide can reach hundreds of hundred million dollars.But the research of domestic microbiological polysaccharide and produce innovative research and have much room for improvement, domestic Late Cambrian have the novel microorganism polysaccharide of industrialization potential few, and relevant patent report is also little, so be worth the research of Efforts To Develop novel microorganism polysaccharide.
Klebsiella ( kleibsiela pneumoniae) be a kind of microorganism that can produce exocellular polysaccharide, the patent that domestic and international citric acid granulose is relevant is little, and in some articles of non-patentability, some investigators report citric acid granulose and can be applicable to immunocompetence adjustment, Adsorption of Heavy Metal Ions, flocculation etc.; But in bibliographical information, citric acid granulose unit output low (<15g/L), carbon source low conversion rate, is difficult to realize large-scale commercial production, and does not have special rheological properties, and application has certain limitation.
Summary of the invention
The object of the invention is to: provide a kind of novel exocellular polysaccharide, this polysaccharide has good ph stability and special alkaline purification gel property; Thering is provided can the microorganism strains of this novel exocellular polysaccharide of fermentative production, and uses this bacterial strain to prepare the method for novel exocellular polysaccharide.
Object of the present invention is achieved through the following technical solutions:
The bacterial strain of novel exocellular polysaccharide is produced in one strain, screen from the active sludge of Wuhan City, Hubei Province sewage work of weaving university and obtain, this bacterial strain is preserved in China typical culture collection center (China, Wuhan) on September 18th, 2014, its Classification And Nomenclature be Klebsiella pneumonia ( kleibsiella pneumoniae) PHRC1.001, deposit number is CCTCC NO:M 2014427.
A kind of novel exocellular polysaccharide, is obtained by above-mentioned bacterial strains Klebsiella pneumonia PHRC1.001 fermentative production.Show through chemical composition analysis and physico-chemical property research, this exocellular polysaccharide is a kind of acidic polysaccharose, exists in aqueous phase with nanoparticle morphology, and dissolves completely in dimethyl sulfoxide (DMSO), stablize in pH 3-11 scope inner structure, the exocellular polysaccharide after alkaline purification forms gel in N-process.
Use Klebsiella pneumonia PHRC1.001 to prepare a method for novel exocellular polysaccharide, comprise the steps:
(1) seed culture
Seed liquid nutrient medium: glucose 10-30g/L, Repone K 5-20g/L, peptone 5-10g/L, pH 7.0;
By Klebsiella pneumonia PHRC1.001 strain inoculation in seed liquid nutrient medium, 30-35 DEG C, hunting speed cultivates 10-24h to obtain seed culture fluid under being the condition of 150-300rpm;
(2) fermentation culture
Fermention medium: sucrose 10-50g/L, saltpetre 1-10g/L, SODIUM PHOSPHATE, MONOBASIC 1-10g/L, bitter salt 1-10g/L, calcium chloride 0.5-2g/L, pH 7.0;
Seed culture fluid is inoculated in fermention medium with the inoculum size of 2.5-10%, 25-30 DEG C, hunting speed is 200-500rpm, air flow quantity is 5-30m 3/ (L .h), under condition, fermentation 48-96h produces exocellular polysaccharide.
The method of the novel exocellular polysaccharide of described preparation also comprises following treatment step: by fermented liquid prior to 80-100 DEG C of water-bath deactivation thalline, then through centrifugal removing thalline; Add the ethanol of 2-5 times of volume in fermented supernatant fluid, leave standstill 12-24h, make exocellular polysaccharide become threadiness to separate out, collect exocellular polysaccharide precipitation with 300-500 object filter-cloth filtering; By multiple soluble in water for exocellular polysaccharide precipitation, be made into the polysaccharide solution of 0.5-5%, with polysaccharide solution: Sevag reagent=(2 ~ 4): 1(v/v) ratio add Sevag reagent (propyl carbinol: chloroform=1:(3 ~ 5) (v/v)) precipitation remove albumen, centrifugal acquisition supernatant liquor, repeats 2-5 time; By polysaccharide solution (supernatant liquor) vacuum concentration to concentration 10-30%, adopt vacuum-drying or lyophilize, the obtained exocellular polysaccharide powder of grinding.The exocellular polysaccharide powder obtained can be soluble in waterly again further purified by dialysis.
Beneficial effect of the present invention: the bacterial strain providing the novel exocellular polysaccharide of a kind of fermentative production kleibsiella pneumoniaepHRC1.001, and determine optimum fermention medium, fermentation condition and fermentation aftertreatment preparation method, obtain this microbial polysaccharide powder.This novel exocellular polysaccharide exists with nanoparticle morphology in aqueous phase, and dissolve completely in dimethyl sulfoxide (DMSO), there is good ph stability and special alkaline purification gel property simultaneously, it is a kind of hydrophilic colloid additive having very much market potential, can be applicable to, in the fields such as flocculation, cement, pottery, foodstuff additive, also can be used for preparing metal nanoparticle.
Accompanying drawing explanation
Fig. 1 be embodiment 1 screen the colonial morphology figure producing sugared bacterial strain.
Fig. 2 is Klebsiella pneumonia PHRC1.001 aspect graph under the microscope.
Fig. 3 is the OD(600nm of Klebsiella pneumonia PHRC1.001 suspension) and dry cell weight DCW(g/L) between the canonical plotting of relation.
Fig. 4 is each parametric line of Klebsiella pneumonia PHRC1.001 in 7L fermentation cylinder for fermentation process.
Fig. 5 is the grain size distribution that Klebsiella pneumonia PHRC1.001 exocellular polysaccharide is dissolved in different solvents.
Fig. 6 is the form of the Klebsiella pneumonia PHRC1.001 exocellular polysaccharide aqueous solution under transmission electron microscope.
Fig. 7 is pH on the impact of Klebsiella pneumonia PHRC1.001 exocellular polysaccharide viscosity and structure.
Fig. 8 to be pH be 14 impacts Klebsiella pneumonia PHRC1.001 exocellular polysaccharide being formed to gel.
Embodiment
Following examples are used for further illustrating content of the present invention, but should not be construed as limitation of the present invention, and without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement all belong to scope of the present invention.
The screening of embodiment 1 exocellular polysaccharide producing strains and qualification
(1) sample collecting and pre-treatment
Sample from the active sludge of Wuhan City, Hubei Province sewage work of weaving university.The sample of collection is put into the other refrigeration of ice bag, take back laboratory under remaining on the environment of lesser temps and process as early as possible.Get solid-liquid biased sample 10g and put into the 250mL Erlenmeyer flask that 150mL, 0.9% stroke-physiological saline solution are housed, for subsequent use after shaken well.
(2) initial gross separation of sugared bacterial strain is produced
Use 0.9% stroke-physiological saline solution by volume according to 10 -1-10 -8gradient serial dilution is carried out to above-mentioned pretreatment sample, 0.1mL dilute sample is got at each extent of dilution, spread plate respectively, constant temperature culture 36-72h under 30 DEG C of conditions, comparatively large and single bacterium colony of thickness with aseptic inoculation ring picking, colonial morphology as shown in Figure 1, is surrounded by a circle viscous polysaccharide around the bacterial strain of thickness.Then be separated at corresponding flat lining out, obtain pure single bacterium colony, isolate 40 strain bacterium altogether.The bacterial strain of purifying is deposited on slant medium, the preservation of 4 DEG C, refrigerator.
(3) secondary separation of sugared bacterial strain is produced
40 strain bacterium are carried out fermentative production exocellular polysaccharide by the following method:
1) seed culture
Seed liquid nutrient medium: glucose 15g/L, Repone K 5g/L, peptone 7g/L, pH 7.0, tap water is prepared;
By the strain inoculation of slant preservation in seed liquid nutrient medium, 30 DEG C, hunting speed cultivates 12h to obtain seed culture fluid under being the condition of 220rpm.
2) fermentation culture
Fermention medium: sucrose 40g/L, yeast extract paste 3g/L, potassium primary phosphate 2.6g/L, bitter salt 2g/L, calcium chloride 0.5g/L, pH 7.0, tap water is prepared;
Seed culture fluid is inoculated in fermention medium with the inoculum size of 2.5%, 30 DEG C, hunting speed is 220rpm, air flow quantity is 60m 3/ (L .h), under condition, fermentation 72h produces exocellular polysaccharide.
Measure viscosity and the thick output of exocellular polysaccharide of fermented liquid, output and the fermented liquid apparent viscosity of final acquisition one strain bacterium Crude polysaccharides are all relatively high, in conjunction with the viscosity of bacterium colony, choose this strain bacterium, called after PHRC1.001.
Wherein, the method measuring fermented liquid apparent viscosity is: 25 DEG C time, adopts vertebral plate rotor (C60/1 °, Til, gap 0.052mm), under steady state shearing pattern, when shearing rate is 0.01-500 1/s, measure the apparent viscosity of fermented liquid with Haake Rheostress 6000 rotational rheometer.
The method measuring the thick output of exocellular polysaccharide is: get 20mL fermented liquid prior to 80-100 DEG C of water-bath deactivation thalline, removes thalline through the centrifugal 20min of 10000r/min; Add the ethanol of 2-5 times of volume in fermented supernatant fluid, leave standstill 12-24h, make exocellular polysaccharide become threadiness to separate out, collect exocellular polysaccharide precipitation with 300-500 object filter-cloth filtering; Polysaccharide precipitation is put in 35-45 DEG C of vacuum-drying 3-4h in vacuum drying oven, the numerical value after the total mass numerical value claimed deducts filter cloth quality and the ratio of fermentating liquid volume are the thick output of exocellular polysaccharide, and unit is g/L.
The bacterial strain PHRC1.001 of screening is carried out gramstaining, the form of basis of microscopic observation bacterial strain, as shown in Figure 2, this strain bacterium is Gram-negative bacteria, in rod-short.
(4) qualification of bacterium
Sangon Biotech (Shanghai) Co., Ltd. is sent by bacterial strain PHRC1.001 to carry out 16S rDNA molecular biology identification.This strain bacterium sequencing result is carried out Blast in GenBank database, according to the sequential analysis comparison of 16S rDNA, bacterium is accredited as Klebsiella pneumonia ( kleibsiella pneumoniae), this bacterial strain is preserved in China typical culture collection center (China, Wuhan) on September 18th, 2014, and its deposit number is CCTCC NO:M 2014427.
Embodiment 2 sucrose concentration produces the impact of exocellular polysaccharide to Klebsiella pneumonia PHRC1.001
Using sucrose as carbon source, according to yeast extract paste 3g/L, potassium primary phosphate 2.6g/L, bitter salt 2g/L, calcium chloride 0.5g/L, pH 7.0, be mixed with the fermention medium of different sucrose, fermentation culture is carried out by the method described in embodiment 1, found that when sucrose concentration is lower, thalline mainly carries out self growth, it is slow that sugared Comparison of Gardening Activities is produced in metabolism, along with the increase of sucrose concentration, biomass and output of sugar are all in increase, but reach maximum when 40g/L, and increase along with the continuation of sucrose concentration, the growth of thalline and the accumulation of sugar will be suppressed, therefore determine that sucrose concentration is 40g/L.The impact that sucrose concentration produces exocellular polysaccharide to fermentation is as shown in table 1.
 
Table 1 sucrose concentration pair kleibsiella pneumoniaepHRC1.001 produces the impact of exocellular polysaccharide
Sucrose concentration (g/L) The thick output of exocellular polysaccharide (g/L) Dry cell weight DCW(g/L)
20 7.9 1.5
40 10.2 1.7
60 8.4 1.5
80 7.9 1.5
100 6.6 1.4
The measuring method of somatic cells dry weight is: get appropriate fermented liquid, after 20min centrifugal under 10000r/min, with distilled water wash precipitation and centrifugal, after repeating 3 times, gained cell redissolves the bacteria suspension being made into serial different concns in distilled water, the OD value (control OD value is within the scope of 0.2-08) of bacteria suspension is measured under 600nm, 0.5cm light path, correspondence gets 50mL bacteria suspension in aluminium box simultaneously, at 105 DEG C constant temperature deposit and spend the night after constant weight weigh, obtain dry cell weight (dry cell weight, DCW, g/L).Do typical curve with the OD value of bacteria suspension and dry cell weight, relevant criterion curve as shown in Figure 3.Fermented liquid is cooked suitable dilution, does blank reference with the fermented supernatant fluid after centrifugal, the method repeating above-mentioned survey OD value records the OD value of dilution secondary fermentation liquid, and calculates dry cell weight with this reference standard opisometer.
Embodiment 3 nitrogenous source kind pair kleibsiella pneumoniaepHRC1.001 produces the impact of exocellular polysaccharide
Using ammonium chloride, saltpetre, ammonium nitrate, ammonium sulfate, peptone, extractum carnis, yeast extract paste and urea as different nitrogenous sources, according to sucrose 40g/L, nitrogenous source 2g/L, potassium primary phosphate 2.6g/L, bitter salt 2g/L, calcium chloride 0.5g/L, pH 7.0, the fermention medium of preparation different nitrogen sources, fermentation culture is carried out by the method described in embodiment 1, found that and be inorganic nitrogen-sourcedly better than organic nitrogen source, be wherein all maximum using saltpetre as exopolysaccharides in inorganic nitrogen-sourced fermented liquid and thalline content, therefore determine using saltpetre as nitrogenous source.Fermention medium component after optimization is sucrose 40g/L, saltpetre 2g/L, SODIUM PHOSPHATE, MONOBASIC 3.5g/L, bitter salt 7g/L, calcium chloride 0.8g/L, pH 7.0.The impact that nitrogenous source kind produces exocellular polysaccharide to fermentation is as shown in table 2.
Table 2 nitrogenous source kind pair kleibsiella pneumoniaepHRC1.001 produces the impact of exocellular polysaccharide
Nitrogenous source kind The thick output of exocellular polysaccharide (g/L) Dry cell weight DCW(g/L)
Ammonium chloride 7.4 3.1
Saltpetre 15.0 4.5
Ammonium nitrate 8.0 3.4
Ammonium sulfate 9.1 2.1
Urea 8.7 2.5
Extractum carnis 4.3 0.8
Yeast extract paste 7.8 1.0
Peptone 7.8 1.9
The fermenting process research of embodiment 4 7L fermentor tank
Adopt the fermention medium after optimizing and envrionment conditions, in 7L rotor stirred pot, observe fermentation situation, liquid amount 4L, stirring velocity 300r/min, air flow 4 × 10 -3m 3/ min, result as shown in Figure 4, as can be seen from the fermentation diagram of parameter each in figure, thalline is after the lag phase of 4h, enter logarithmic phase rapidly, from about 6h, synthesize polysaccharide in a large number, to about 48h, polysaccharide yield reaches maximum, from 60h, polysaccharide accumulation starts to decline, and therefore stops fermentation at 72h, fermented liquid Crude polysaccharides output is up to about 21g/L, and output reaches a reasonable level.
The preparation and purification of embodiment 5 exocellular polysaccharide
By fermented liquid prior to 100 DEG C of water-bath deactivation thalline, then through centrifugal removing thalline; Add the ethanol of 3 times of volumes in fermented supernatant fluid, leave standstill 24h, make exocellular polysaccharide become threadiness to separate out, collect exocellular polysaccharide precipitation with 500 object filter-cloth filterings; By multiple soluble in water for exocellular polysaccharide precipitation, be made into the polysaccharide solution of 2%, with polysaccharide solution: Sevag reagent=4:1(v/v) ratio add Sevag reagent (propyl carbinol: chloroform=1:4(v/v)) precipitation remove albumen, centrifugal acquisition supernatant liquor, repeats twice; By supernatant liquor and polysaccharide solution vacuum concentration to concentration 10-30%, adopt vacuum-drying or spraying dry, the obtained exocellular polysaccharide powder of grinding.
Exocellular polysaccharide powder is multiple soluble in water, and be made into the polysaccharide solution of 1.0%, dialyse with the dialysis tubing of 14KDa, every 2h changes a water, dialysis 48h; Polysaccharide solution after dialysis is adopted lyophilize, obtained exocellular polysaccharide purified powder.
Embodiment 6 kleibsiela pneumoniaethe character of PHRC1.001 exocellular polysaccharide
(1) chemical constitution
Adopt the moisture content in hyperthermia drying weight difference subtraction mensuration exocellular polysaccharide, Folin-phenol method measures the protein content in exocellular polysaccharide, sulfuric acid-phynol method measures the total sugar content in exocellular polysaccharide, between xenol method measure glucuronic acid content in exocellular polysaccharide, ash oontent in calcination gravimetric determination exocellular polysaccharide, the results are shown in Table 3.
The basic chemical constitution of table 3 exocellular polysaccharide
Composition Moisture Protein Total reducing sugar Uronic acid Ash content
Content (%) 8.37 2.16 83.69 8.54 1.05
As can be seen from Table 3, in the polysaccharide sample after purifying, protein content is lower, and glucuronic acid content is 8.54%, illustrates that this polysaccharide is a kind of acidic polysaccharose.
(2) solvability in different solvents
Exocellular polysaccharide after purifying is dissolved in respectively in dimethyl sulfoxide (DMSO) (DMSO) and water, be made into the solution of 0.1%, the distribution of its grain size is measured with nano particle size instrument, concrete grammar is: 25 DEG C time, and measure the size distribution situation of exocellular polysaccharide solution with Nano ZS nano particle size instrument, laser detection angle is 173 °, polysaccharide material refractive index is 0.147, the refractive index of water is 1.330, and the refractive index of dimethyl sulfoxide (DMSO) is 1.477, and sample pool is glass PCS1115 type.
Result as shown in Figure 5, can find out in aqueous, the nano particle that polysaccharide becomes mainly with molecular aggregates exists, and in DMSO, polysaccharide molecule is not assembled, may be very strong chemical bond rupture agent due to DMSO, destroy the chemical bond between polysaccharide molecule, polysaccharide molecule is existed with individual molecule.
(3) molecular conformation under transmission electron microscope
Exocellular polysaccharide after purifying is dissolved in the water, is made into the solution of 10 μ g/mL concentration, the pattern under transmission electron microscope as shown in Figure 6, can find out the molecule of polysaccharide be conglobulation together, corresponding with the size distribution in water.
Embodiment 7 pH couple kleibsiella pneumoniaethe impact of PHRC1.001 exocellular polysaccharide rheological characteristics
(1) ph stability
Prepare with HCl and NaOH the aqueous solution that pH is 1,2,3,4,6,8,10,11,12,13,14, dissolve exocellular polysaccharide powder, make exocellular polysaccharide final concentration be 1.0%, normal temperature places 6h.Exocellular polysaccharide solution apparent viscosity under different pH is measured according to the method for said determination fermented liquid apparent viscosity.As shown in Figure 7, within the scope of pH 3-11, the change of exocellular polysaccharide solution viscosity is little, illustrates that exocellular polysaccharide is stablized in this pH scope inner structure; But under pH 1,2,12,13,14 condition, exocellular polysaccharide viscosity obviously declines, illustrate that strong acid (pH≤2) and highly basic (pH >=12) can damage exocellular polysaccharide structure.
(2) in alkaline purification and gel property
With the exocellular polysaccharide solution of deionized water preparation 1.0%, normal temperature places 6h, and this is control sample; With the exocellular polysaccharide solution (pH 14) of 1M NaOH solution preparation 1.0%, normal temperature places 6h, then with 2M HCl, polysaccharide soln is adjusted to pH 7, and this is in alkaline purification and sample.Measure in control sample and alkaline purification and the apparent viscosity of sample according to the method for said determination fermented liquid apparent viscosity.As shown in Figure 8, in alkaline purification and the viscosity of sample apparently higher than control sample, illustrate that the exocellular polysaccharide after highly basic process defines gel in N-process, this may be that in N-process, exopolysaccharide molecule occurs to reset and gelation in order because alkaline purification destroys the nanoparticle structure that exopolysaccharide molecule formed.

Claims (10)

1. the bacterial strain of exocellular polysaccharide is produced in a strain, it is characterized in that: Classification And Nomenclature be Klebsiella pneumonia ( kleibsiella pneumoniae) PHRC1.001, deposit number is CCTCC NO:M 2014427.
2. use the bacterial strain described in claim 1 to prepare the method for novel exocellular polysaccharide, it is characterized in that comprising the steps:
(1) seed culture
By bacterial strain strain inoculation according to claim 1 in seed liquid nutrient medium, 30-35 DEG C, hunting speed cultivates 10-24h to obtain seed culture fluid under being the condition of 150-300rpm; Described seed liquid nutrient medium is: glucose 10-30g/L, Repone K 5-20g/L, peptone 5-10g/L, pH 7.0;
(2) fermentation culture
Seed culture fluid is inoculated in fermention medium with the inoculum size of 2.5-10%, 25-30 DEG C, hunting speed is 200-500rpm, air flow quantity is 30-60m 3/ (L .h), under condition, fermentation 48-96h produces exocellular polysaccharide; Described fermention medium is: sucrose 10-50g/L, saltpetre 1-10g/L, SODIUM PHOSPHATE, MONOBASIC 1-10g/L, bitter salt 1-10g/L, calcium chloride 0.5-2g/L, pH 7.0.
3. the method for the novel exocellular polysaccharide of preparation according to claim 2, is characterized in that: the fermention medium described in step (2) is: sucrose 40g/L, saltpetre 2g/L, SODIUM PHOSPHATE, MONOBASIC 3.5g/L, bitter salt 7g/L, calcium chloride 0.8g/L, pH 7.0.
4. the method for the novel exocellular polysaccharide of preparation according to claim 2, characterized by further comprising following treatment step: by fermented liquid prior to 80-100 DEG C of water-bath deactivation thalline, then through centrifugal removing thalline; Add the ethanol of 2-5 times of volume in fermented supernatant fluid, leave standstill 12-24h, make exocellular polysaccharide become threadiness to separate out, collect exocellular polysaccharide precipitation with 300-500 object filter-cloth filtering; By multiple soluble in water for exocellular polysaccharide precipitation, be made into the polysaccharide solution of 0.5-5%, with polysaccharide solution: Sevag reagent=(2 ~ 4): the ratio of 1 adds Sevag agent precipitate and removes albumen, and centrifugal acquisition supernatant liquor, repeats 2-5 time; By polysaccharide solution vacuum concentration to concentration 10-30%, vacuum-drying or lyophilize, the obtained exocellular polysaccharide powder of grinding.
5. the method for the novel exocellular polysaccharide of preparation according to claim 4, is characterized in that: exocellular polysaccharide powder is soluble in water by dialysis purifying again.
6. a novel exocellular polysaccharide, is characterized in that: prepared by the method described in any one of claim 2-5.
7. novel exocellular polysaccharide according to claim 6, is characterized in that: described novel exocellular polysaccharide is acidic polysaccharose.
8. novel exocellular polysaccharide according to claim 6, is characterized in that: described novel exocellular polysaccharide exists with nanoparticle morphology in aqueous phase, dissolves completely in dimethyl sulfoxide (DMSO).
9. novel exocellular polysaccharide according to claim 6, is characterized in that: described novel exocellular polysaccharide is stablized in pH 3-11 scope inner structure.
10. novel exocellular polysaccharide according to claim 6, is characterized in that: in N-process, form gel after described novel exocellular polysaccharide alkaline purification.
CN201410786092.0A 2014-12-18 2014-12-18 Novel exopolysaccharide as well as production strain and preparation method thereof Pending CN104403981A (en)

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