CN104372051B - The method for producing ethyl pyruvate Glucomannan - Google Patents
The method for producing ethyl pyruvate Glucomannan Download PDFInfo
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- CN104372051B CN104372051B CN201410462307.3A CN201410462307A CN104372051B CN 104372051 B CN104372051 B CN 104372051B CN 201410462307 A CN201410462307 A CN 201410462307A CN 104372051 B CN104372051 B CN 104372051B
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- glucomannan
- ethyl pyruvate
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Abstract
The present invention is to utilize bacterial strainPantoea agglomeransKFS 9 ferments in liquid medium, obtain the zymotic fluid containing ethyl pyruvate Glucomannan, the tunning is precipitated through certain density ethyl alcohol, it can obtain using ethyl pyruvate Glucomannan as the sediment of primary product, the sediment through further isolating and purifying obtains single symmetrical peak in gel column.It is made of two kinds of monosaccharide of mannose and glucose through analyzing the glycopolymers, molar ratio 4:3, cellular construction is the recurring unit of seven sugar, and the both ends of molecular weight 1251.12, this repeated heptasaccharide uint are β D mannopyranoses, and alternate links in a like fashion are adopted between mannose and glucose.There are an ethyl pyruvate bases to be substituted on end group mannose in EPS.
Description
Technical field
The present invention relates to biotechnology, in particular to a kind of utilizationsPantoea agglomeransKFS-9 bacterial strains
The method of fermentation production of acetone acetoacetic ester Glucomannan.
Background technology
Ethyl pyruvate Glucomannan is the macromolecular by being connected with ethyl pyruvate using Glucomannan as the polysaccharide of straight chain
Compound has various biological activity, can be obtained by the chemical modification of Glucomannan.Currently, utilizing micro- life not yet
Object produces the report of ethyl pyruvate Glucomannan.
It has been reported：Pantoea agglomeransKFS-9 can generate solubility by being metabolized under given conditions
Exocellular polysaccharide, to obtain bacterial strainPantoea agglomeransThe exocellular polysaccharide of KFS-9 centrifuges thalline zymotic fluid, removal
Graininess impurity and precipitation somatic cells in fluid nutrient medium, at 70 DEG C rotation be concentrated by evaporation 7 times of volumes, 3 times of volumes are added afterwards
95% ethyl alcohol, precipitate polysaccharides.Precipitation is dissolved in water, is centrifuged off insoluble polysaccharide, supernatant adds 95% ethyl alcohol, and 3000
Rpm centrifuges 5 min, collects precipitation.By the precipitation of acquisition, redissolves in suitable water, isometric 5% will be added in polysaccharide solution
Solution of trichloroacetic acid stands 4 h, and 95% ethanol precipitation is added in centrifuging and taking supernatant, after redissolved with water, repeat above operation,
TCA method removing proteins are used again.As clear as crystal liquid glucose is finally obtained, 95% ethyl alcohol is added, 3000 rpm centrifuge 5 min, precipitation
It is washed twice with absolute ethyl alcohol, vacuum drying, obtains water-soluble general bacterium exopolysaccharide（PAEPS）, standard monosaccharide and general mycetocyte are more outside
Sugared monosaccharide component gas-chromatography comparison shows that general bacterium exopolysaccharide is a kind of more complicated polysaccharide, mainly by arabinose,
Galactolipin, glucose group are at molar ratio L-Ara: D-Gal: D-Glc=3.10：8.42：6.68=1：2.71：2.15.
It has been reported：General bacterium can generate lipopolysaccharides.It is by polysaccharide O antigens, core polysaccharide and lipoid A (Lipid
A) three parts form, and are located at the outermost layer of bacterium, polysaccharide O antigens are outside, are made of the recurring unit of several oligosaccharide
Polysaccharide chain has specificity;Core polysaccharide is divided into inner core and outer core, and outer core has several hexoses, including glucose, gala
The compositions such as sugar, acetylglucosamine.
The present invention is to be related toPantoea agglomeransKFS-9 strain fermentations produce ethyl pyruvate Glucomannan and
Its method.
Invention content
The present invention is to provide a kind of microorganism that can generate ethyl pyruvate GlucomannanPantoea agglomerans
KFS-9 and the method for producing ethyl pyruvate Glucomannan using the bacterial strain.
The present invention provides one plant of bacteriumPantoea agglomeransKFS-9 has generation ethyl pyruvate Portugal sweet
The characteristic of glycan.Using Gram's staining, pod membrane, flagellum, motility observation etc. Preliminary Identification of the morphological observations as strain,
Then basis《Primary Jie Shi Bacteria Identifications handbook the 9th edition》With《Common bacteria system identification handbook》Bio-chemical characteristics are carried out to do
Further identification.By 16SrDNA gene sequencing and Phylogenetic analysis, qualification result isPantoeaBelong to.But its form and
Physiological and biochemical property withagglomeransIt is identical.The bacterial strain is named to bePantoea agglomeransKFS-9.Culture presevation
Unit：China typical culture collection center.Address：Wuhan, China, Wuhan University.The deposit date is Augusts in 2005 3 days, protect
Tibetan number is CCTCC NO:M205086.
The present invention is fermented in liquid medium using the strain, obtains the hair containing ethyl pyruvate Glucomannan
Zymotic fluid, the tunning are precipitated through certain density ethyl alcohol, and it is main production that can obtain with ethyl pyruvate Glucomannan
The sediment of object, the sediment through further isolating and purifying obtain single symmetrical peak in S100 gel columns.It is poly- through analyzing the sugar
It closes object to be made of two kinds of monosaccharide of mannose and glucose, molar ratio 4:3.Cellular construction is the recurring unit of seven sugar, molecule
Amount is 1251.12, and the both ends of this repeated heptasaccharide uint are β-D- mannopyranoses, are used between mannose and glucose
Identical mode alternately connects.There are an ethyl pyruvate bases to be substituted on end group mannose in EPS.Basic structural unit is such as
Shown in lower：
Description of the drawings
Fig. 1：The monosaccharide of gas chromatography analysis EPS forms
Fig. 2：Bglii fragment mass spectrogram
Specific implementation mode
1, fermented and cultured obtains the zymotic fluid containing ethyl pyruvate Glucomannan：It willPantoea agglomerans
KFS-9 bacterial strains, sterile working are linked into the fermentation medium of sterilizing, and 48h is cultivated under 26 °C, 190 rpm.By zymotic fluid
10min is centrifuged under conditions of 4000 r/min, obtains the supernatant containing ethyl pyruvate Glucomannan.
Culture medium for above-mentioned fermented and cultured forms：20 g/L of sucrose, peptone 3.5 g/L, ZnSO4 0. 2
G/L, KH2PO41 g/L, (NH4)2SO43 g/L, pH 8.0.
2, the extracting method of ethyl pyruvate Glucomannan：
It by 3 times of ethanol precipitations of above-mentioned fermented supernatant fluid, is stood overnight in 4 DEG C of refrigerators, 3600 rpm centrifuge 10 min
White precipitate is obtained, precipitation is dissolved in water, and obtained 3600 rpm of redissolution liquid is centrifuged 10 min, removes insoluble matter.Redissolve liquid again
3 times of ethanol precipitation polysaccharide of secondary addition, precipitation are washed twice with absolute ethyl alcohol, dry, obtain ethyl pyruvate Glucomannan white powder
End.
3, ethyl pyruvate Glucomannan monosaccharide composition analysis：
Using monosaccharide composition analysis-alditol acetate Derivative gas chromatography of polysaccharide
(l) standard items measure
Standard monosaccharide (glucose, galactolipin, mannose, xylose, arabinose, rhamnose) each 5mg is weighed, is added
30mg NaBH4With 5mg internal standard compounds inositol in ampere bottle, 2ml water is added, places 2h after oscillation at room temperature, makes sugared reduction
At corresponding sugar alcohol, instills acetic acid and decompose excessive NaBH4, until bubble-free generates.Solution is concentrated under reduced pressure at 60 DEG C or less
To doing, 0.1% (v/v) methanol hydrochloride solution 2ml is added, is evaporated to dryness under reduced pressure after oscillation, repeats 4~5 times to remove boric acid
Root, residue heat 15min in 105 DEG C of baking ovens and remove moisture, and 0.5ml pyridines and 0.5ml acetic anhydrides is then added,Envelope PipeAfterwards at 100 DEG C（Boiling water bath）Lower reaction lh, reaction product can directly carry out gas chromatographic analysis.Each standard monosaccharide can be measured
Retention time.
(2) sample measures
It weighs about 20mg samples and 3mL 2mol/L trifluoroacetic acids (TFA) is added, be placed in 110 DEG C of hydrolysis 0.5h in baking oven,
Cooling, decompression eliminates TFA, adds 50mg NaBH4With 5mg internal standard compound inositols, remaining operation is same as above.When according to opposite reservation
Between speculate monosaccharide composition and each monosaccharide content.
(3) operating condition of GC
Chromatograph:Aglient6890N gas chromatographs;Chromatographic column:DB-225 capillary columns;
Detector:Flame ionization detector (FID);Flow velocity:l.0ml/min;Column temperature：220℃;Sample size：1μL.
Standard monosaccharide and the monosaccharide gas chromatogram that ethyl pyruvate Glucomannan hydrolyzes are as shown in Figure 1.Six kinds of marks
The gas chromatogram of quasi- monosaccharide, be followed successively by from left to right rhamnose, arabinose, xylose, mannose, galactolipin, glucose and
Internal standard inositol.The retention time for the monosaccharide (Fig. 1) that polysaccharide hydrolysis obtains is compared with the retention time of standard monosaccharide can be with
Find out that EPS is made of two kinds of monosaccharide of mannose and glucose, the peak area ratio of the two is about 4：3.
4, the structural unit detection method of ethyl pyruvate Glucomannan：
Using ESI-LC-MS analytic approach
It is prepared by the unit polysaccharide of ethyl pyruvate Glucomannan：The ethyl pyruvate Glucomannan for taking 0.1g, with 0.5M's
Hydrogen peroxide 5ml dissolvings, closed 50 DEG C keep the temperature 3 hours, are cooled to room temperature, go to precipitate with the absolute ethyl alcohol of 30ml, be contained
The supernatant of unit segment, the supernatant are washed 2 times with 90% ethyl alcohol until doing in 70 DEG C of revolvings, obtain pure ethyl pyruvate
Glucomannan unit segment, the sample carry out ESI-LC-MS analyses, and mass spectrogram is as shown in Figure 2.Show that the ethyl pyruvate Portugal is sweet
The structural unit fragments molecules amount of glycan is 1251.12, is a seven sugared units by mannose and glucose alternate links.This
The both ends of a repeated heptasaccharide uint are mannose, and alternate links in a like fashion are adopted between mannose and glucose.Often
There are an ethyl pyruvates to be substituted on end group mannose for a cellular construction middle.
According to the above analysis, bacterial strain is utilizedPantoea agglomeransThe pyruvic acid second of KFS-9 fermenting and producings
Ester Glucomannan is constituted using repeated heptasaccharide uint as basic structural unit, molecular weight 1251.12, this seven sugar repeats
Unit is with mannose and glucose again with 4:3 ratio alternate links form, and both ends are mannose.In addition, each unit
There are an ethyl pyruvates to be substituted on end group mannose for structure middle.
Claims (2)
1. a kind of utilizationPantoea agglomeransThe method that KFS-9 produces ethyl pyruvate Glucomannan, feature exist
It is in production bacteriumPantoea agglomeransKFS-9 bacterial strains are preserved in China typical culture collection center (CCTCC),
Preserving number is CCTCC NO:M205086, the deposit date is Augusts in 2005 3 days.
2. a kind of sweet poly- using Pantoea agglomerans KFS-9 production ethyl pyruvates Portugal according to claim 1
The method of sugar, it is describedPantoea agglomeransThe ethyl pyruvate Glucomannan of KFS-9 productions, it is characterized in that with sweet dew
Sugar and two kinds of monosaccharide of glucose press 4:3 ratios, seven sugar that alternately connection forms are recurring unit, and recurring unit's molecular weight is
1251.12, and there are an ethyl pyruvate base substituent groups on the end group mannose of repetitive unit.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1958785A (en) * | 2005-11-05 | 2007-05-09 | 中国海洋大学 | Using Pantoea agglomcrans KFS-9 fungus to produce 2, 3 butanediol, and application in cosmetic |
| CN103484392A (en) * | 2012-06-13 | 2014-01-01 | 中国海洋大学 | Application of pseudomonas fluorescens PGM37 strain to produce glucomannan |
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2014
- 2014-09-12 CN CN201410462307.3A patent/CN104372051B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1958785A (en) * | 2005-11-05 | 2007-05-09 | 中国海洋大学 | Using Pantoea agglomcrans KFS-9 fungus to produce 2, 3 butanediol, and application in cosmetic |
| CN103484392A (en) * | 2012-06-13 | 2014-01-01 | 中国海洋大学 | Application of pseudomonas fluorescens PGM37 strain to produce glucomannan |
Non-Patent Citations (2)
| Title |
|---|
| Pantoea agglomerans KFS胞外多糖抗辐射性能及其结构分析;臧聚玲;《CNKI中国知网》;20140527;摘要1-2,8,16-20行 * |
| 抗UV-B辐射菌紫外吸收代谢产物分析及其抗紫外辐射活性研究;王洪媛等;《中国海洋药物杂志》;20060831;第25卷(第4期);1-5 * |
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