CN100335614C - Production of fructosan by Cellulomonas sp.nov.GJT07 strain - Google Patents
Production of fructosan by Cellulomonas sp.nov.GJT07 strain Download PDFInfo
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- CN100335614C CN100335614C CNB2005101065343A CN200510106534A CN100335614C CN 100335614 C CN100335614 C CN 100335614C CN B2005101065343 A CNB2005101065343 A CN B2005101065343A CN 200510106534 A CN200510106534 A CN 200510106534A CN 100335614 C CN100335614 C CN 100335614C
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Abstract
The present invention relates to a microbe, namely Cellulomonas sp. nov. GJT07 capable of generating fructan and a method for producing fructan by using a Cellulomonas sp. nov. GJT07 strain. The present invention aims to provide a Cellulomonas sp. nov. GJT07 strain from nature and a method for producing fructan by using the Cellulomonas sp. nov. GJT07 strain. A Cellulomonas sp. nov. GJT07 strain is separated and purified from orchard soil and has the characteristic of generating fructan. On the basis of the ninth edition of the Bergey's Manual of Systematic Bacteriology, the morph and the physiological and biochemical property of the Cellulomonas sp. nov. GJT07 strain is coincident with that of cellulomonas. The Cellulomonas sp. nov. GJT07 strain has the characteristics of wide range of nutritional requirements and easy culture. When used as a production bacterial strain of fructan, the Cellulomonas sp. nov. GJT07 strain of the present invention has the characteristics of high stability, easy production and operation, low cost, etc., and industrialized production can be realized.
Description
Invention field
The present invention relates to a kind of method that can produce the microorganism Cellulomonas sp.nov.GJT07 of Polylevulosan and utilize this bacterial strain production Polylevulosan.
Background of invention
Polylevulosan is the occurring in nature biopolymer the most widely that distributes, and is by with β-2, and 6-key and β-2, the homopolysaccharide that the fructofuranose base that the 1-key connects is formed.Polylevulosan produces as the carbohydrate stored substance in plant.By known to inspection information, the literature search, some microorganism also can produce Polylevulosan, comprises bacterium, yeast and fungi according to the inventor.As: genus acetobacter, actinomyces, Aspergillus, Bacillus, Corynebacterium, erwinia, Rhodopseudomonas and streptococcus etc.But at home and abroad there is no the report of Cellulomonas (Cellulomonas) production by biological Polylevulosan.
Qualitative and the quantitative analytical procedure of Polylevulosan mainly adopts: spectrophotometry and chromatography.Wherein, chromatography is the most commonly used, comprising: ply of paper is analysed, thin-layer chromatography and high performance liquid chromatography.
Summary of the invention
The purpose of this invention is to provide a kind of method that can produce the microorganism Cellulomonas sp.nov.GJT07 of Polylevulosan and utilize this bacterial strain production Polylevulosan.
Separation and purification of the present invention goes out the microorganism Cellulomonas sp.nov.GJT07 that a strain derives from soil, and it has the characteristic that produces Polylevulosan, and depositary institution is called for short CCTCC, and preserving number is M205085.
Characteristics of the present invention are: separate the Cellulomonas sp.nov.GJT07 bacterial strain of producing Polylevulosan first.This bacterial strain is irregular shaft-like, Gram-positive, and 05～0.6 μ m * 1.8～2.1 μ m, single or be V word shape and arrange, motion can not form gemma, and is not antiacid.This bacterial strain 37 ℃ of cultivation 24h on nutrient broth medium form single bacterium colony, the faint yellow projection of bacterium colony, circle, neat in edge.The catalase positive, the energy decomposition of cellulose, reduction nitrate is to nitrite.According to uncle Jie Shi handbook the 9th edition, determine that it is Cellulomonas, but belonging to known bacterium with physiological and biochemical property and this, its form compares different (the feature contrast of this bacterial strain and other bacterial classifications of Cellulomonas sees Table 1).
Cellulomonas sp.nov.GJT07 inoculation of the present invention in fermention medium, behind 30 ℃ of shake-flask culture 48h, is collected fermented liquid, and frozen centrifugation removal thalline, acquisition contains the supernatant liquor of polysaccharide product.Then, extract the purified product polysaccharide with ethanol precipitation and sevag method, lyophilize obtains the polysaccharide finished product.
Description of drawings
Fig. 1: the efficient liquid phase chromatographic analysis figure of fructose standard substance.
Fig. 2: the efficient liquid phase chromatographic analysis figure of Cellulomonas sp.nov.GJT07 bacterial strain exocellular polysaccharide hydrolyzed solution.
Embodiment
The acquisition of Cellulomonas sp.nov.GJT07 bacterial strain of the present invention: gather pedotheque from the orchard, join in the triangular flask that the 25ml enrichment medium is housed, 30 ℃, 160r/min shake-flask culture 48h.After then enrichment culture liquid being used 10 times of dilutions of stroke-physiological saline solution, get 0.2ml coating isolation medium flat board.Be inverted for 30 ℃ and cultivate 48h, obtain on flat board, to have the bacterium colony of thickness transparent appearance, obtain the bacterium that a strain can be produced exocellular polysaccharide.Observe and the Physiology and biochemistry evaluation by strain morphology, determine that it is Cellulomonas according to uncle Jie Shi handbook the 9th edition.Enrichment medium is by sucrose 2g, yeast extract paste 3g, NaNO
31g, K
2HPO
40.5g, MgSO
40.5g, NH
4Cl 0.5g, FeSO
40.01g form, adding distil water 1000ml transfers pH7.5; Isolation medium is by sucrose 10g, yeast extract paste 3g, NaNO
31g, K
2HPO
40.5g, MgSO
40.5g, NH
4Cl0.5g, FeSO
40.01g agar 15g forms, adding distil water 1000ml transfers pH7.5.
The production and the preparation of Cellulomomnas sp.nov.GJT07 bacterial strain exocellular polysaccharide: with bacterial classification inoculation in 100ml fermention medium (sucrose 40g, peptone 7g, ammonium sulfate 1.4g, K are housed
2HPO
42g, MgSO
40.25g, FeSO
40.01g, H
2O 1000ml, pH 8) the 250ml triangular flask in, behind 30 ℃ of shake-flask culture 48h, 4 ℃ of following 4000rpm of nutrient solution were removed thalline in centrifugal 10 minutes, obtain to contain the supernatant liquor of polysaccharide product.Then, 95% ethanol that adds 2.5 times of volumes is in supernatant liquor, and glass stick stirs, leave standstill to flocks occurring, after the centrifugal supernatant liquor of removing, precipitation redissolved with distilled water, (chloroform-propyl carbinol volume ratio that adds 1/4 times of volume was 4: 1 a mixed solution with the sevag method, jolting 30min, the centrifuging and taking water) remove albumen, use 95% ethanol sedimentation polysaccharide of 2.5 times of volumes again, centrifugal, lyophilize obtains the exocellular polysaccharide finished product.
The compositional analysis of Cellulomonas sp.nov.GJT07 bacterial strain exocellular polysaccharide: with exocellular polysaccharide dilute acid hydrolysis (1mol/L sulfuric acid, 90 ℃, 4h) afterwards, BaCO
3Neutralizing hydrolysis liquid, centrifugal, get supernatant liquor and analyse with high performance liquid chromatography by ply of paper and analyze.The supernatant liquor point sample is carried out ply of paper in Xinhua's middling speed chromatography filter paper analyse analysis, developping agent is a propyl carbinol: acetate: water=4: 1: 5 (solvent layer), developer is aniline-pentanoic, and standard monose is glucose, semi-lactosi, wood sugar, seminose, pectinose, rhamnosyl, fructose.Find that by paper chromatographic analysis the result is shown as single spot, its Rf value and color reaction are all consistent with the fructose standard substance.Again supernatant liquor and fructose standard substance are analyzed chromatographic column Inertsil NH through the high performance liquid chromatograph of precision
2(4.6 * 250mm, 5 μ m), moving phase is acetonitrile: water=75: 25, flow are 0.8mL/min, differential detects.The result shows that this polysaccharide hydrolysis liquid chromatography honeybee only contains a fructose main peak (Fig. 1, Fig. 2), shows in this polysaccharide hydrolysis liquid and has only fructose.Prove that thus the exocellular polysaccharide by the production of Cellulomonas sp.nov.GJT07 bacterial strain is a Polylevulosan.
Table 1 cellulomonas cartae belongs to the discriminating between planting
| Feature | C. biazotes | C. celasea | C. cellulans | C. fimi | C. flavigena | C. gelida | C. uda | C. sp.nov |
| Motility is produced acid with sole carbon utilization: D-ribose raffinose L (+)-lactate proline from dextrin | + - + + - - | - - - + + - | ND + - + + ND | + - - + - + | - + - - - + | + - - - - + | - - - - - + | + - + + + - |
Claims (2)
1. a cellulomonas cartae (Cellulomonas sp.nov.GJT07), it is characterized in that: can produce Polylevulosan, and carry out preservation by China typical culture collection center, deposit number is CCTCC NO:M205085.
2. the described cellulomonas cartae of claim 1 purposes that is used to produce Polylevulosan.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005101065343A CN100335614C (en) | 2005-09-29 | 2005-09-29 | Production of fructosan by Cellulomonas sp.nov.GJT07 strain |
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|---|---|---|---|
| CNB2005101065343A CN100335614C (en) | 2005-09-29 | 2005-09-29 | Production of fructosan by Cellulomonas sp.nov.GJT07 strain |
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| CN1778896A CN1778896A (en) | 2006-05-31 |
| CN100335614C true CN100335614C (en) | 2007-09-05 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5926277B2 (en) * | 1976-06-07 | 1984-06-26 | 武田薬品工業株式会社 | Method for producing D-fructose |
| US4908310A (en) * | 1987-12-23 | 1990-03-13 | University Of Kansas | Water insoluble polysaccharide polymer and method thereof |
| CN1533430A (en) * | 2000-12-21 | 2004-09-29 | �Ʒ� | Lactobacillus strain producing levan and its use in human or pet food products |
-
2005
- 2005-09-29 CN CNB2005101065343A patent/CN100335614C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5926277B2 (en) * | 1976-06-07 | 1984-06-26 | 武田薬品工業株式会社 | Method for producing D-fructose |
| US4908310A (en) * | 1987-12-23 | 1990-03-13 | University Of Kansas | Water insoluble polysaccharide polymer and method thereof |
| CN1533430A (en) * | 2000-12-21 | 2004-09-29 | �Ʒ� | Lactobacillus strain producing levan and its use in human or pet food products |
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Granted publication date: 20070905 Termination date: 20091029 |