CN104164385A - Sphingomonas paucimobilis strain gxas-815 and applications thereof - Google Patents
Sphingomonas paucimobilis strain gxas-815 and applications thereof Download PDFInfo
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- CN104164385A CN104164385A CN201410309093.6A CN201410309093A CN104164385A CN 104164385 A CN104164385 A CN 104164385A CN 201410309093 A CN201410309093 A CN 201410309093A CN 104164385 A CN104164385 A CN 104164385A
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Abstract
The invention discloses a sphingomonas paucimobilis strain gxas-815 and applications thereof. The strain is classified and named as sphingomonas paucimobilis gxas-815, and is deposited in the China Center for Type Culture Collection with an accession number of CCTCC M2013327. Gellan gum is produced by fermentation by utilizing the sphingomonas paucimobilis and by adopting cane sugar as a raw material. The output of the gellan gum reaches 13.75 g/L.
Description
Technical field
The present invention relates to technical field of bioengineering, specifically a strain sphingomonas paucimobilis bacterial strain gxas-815, obtains from the screening of Nanning collected specimens, and this bacterial strain is applied to take sucrose as fermenting raw materials production gelling gum.
Background technology
Gelling gum is a kind of novel microorganism exocellular polysaccharide, is the high molecular polymer consisting of D-Glucose, D-Glucose aldehydic acid, D-Glucose and L-rhamnosyl or 4 kinds of sugar units of mannosans.Gelling gum purposes is very extensive, at field of food, mainly as thickening material, is to collect in the world at present thickening, suspension, emulsification, be stable at one, one of foodstuff glue of superior performance.As novel microbiological gum, in industries such as food, medicine, chemical industry, gelling gum will progressively replace vegetable jelly, as agar, carrageenin etc., have good market outlook.(Wei Chun, Zhang Donghui, Ying Xiangxian etc. produce the yellow pigment defective type resistance Sphingol single-cell WZ013 fermentation condition optimization of gelling gum. Chinese food journal .2013,23 (2): 101-107).Gelling gum is stable to enzyme, is good, the easy natural degradation of flocculating effect, can not causes the novel flocculant of the features such as secondary pollution.
Sphingomonas paucimobilis again translated name moves sphingolipid Zymomonas mobilis less, is the fermentative production bacterial strain of gelling gum.In the active sludge of sewage disposal, once separating for several times goes out sphingomonas paucimobilis, it has flocculation activity, the gelling gum of producing can be dissolved in cold water, there is satisfactory stability, acidproof, high temperature resistant, the characteristics such as heat is reversible, the effect that can also resist microorganism and enzyme, can exercise the biological restoration functions such as wastewater treatment as the integral part of biological flocculant.The industrial scale gelling gum specification little, that produce of domestic gelling gum manufacturer is less, in suitability for industrialized production, still exist fermentation production rate low, in fermenting process, aeration-agitation energy consumption is high, product extracts loaded down with trivial details, the problems such as product clarification difficulty, directly cause production cost higher, limited the development of domestic gelling gum industry.In the fermenting process of microbial polysaccharide, it is extremely important selecting suitable fermentation condition.Gelling gum is synthetic through carbohydrate metabolism in suitable substratum by sphingomonas paucimobilis, in building-up process, carbohydrate is that main carbon source, protein is the largest portion (Hu Guiping that main nitrogenous source forms fermentation raw material cost, Liu Bo, Zhu Yujing etc. moving sphingolipid Zymomonas mobilis produces the response surface method optimization of gellan gum fermentation substratum less. biomathematics journal .2012,27 (3): 507-517).
Sphingomonas paucimobilis is a kind of aerobic gram negative bacillus, can make carbon source with glucose, starch, sucrose etc., and ammonium nitrate, yeast extract paste, peptone etc. are grown and produce glue for nitrogenous source and containing in other micro-substratum.Lot of domestic and foreign scholar's research finds, carbon source, nitrogenous source, kind age, inoculum size, temperature, the initial pH of fermention medium produces gelling gum to its fermentation larger impact.
Take sphingomonas paucimobilis or less moving sphingolipid Zymomonas mobilis as retrieval key element, in China Intellectual Property Office, retrieve altogether 44 patents.These patent coverages mainly contain: production method and the extracting method that the discovery of the new bacterial strain of sphingomonas paucimobilis, the excavation of gene and gene knockout recombinant bacterium build, gelling gum is produced in the production of relevant enzyme preparation, fermentation, as microbe additive, for flavour of food products, improve and the application such as wastewater treatment.
Summary of the invention
The object of this invention is to provide strain sphingomonas paucimobilis bacterial strain and an application thereof.This bacterial strain has good fermentation optimization and genetic modification prospect, also has important industrial application value simultaneously.
Sphingomonas paucimobilis provided by the present invention (Sphingomonas paucimobilis) gxas-815 obtains from Nanning sampling separation screening.
Described sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815CCTCC M2013327 separating screening method:
A) sampling: from Nanning collected specimens,
B) enrichment: each sample respectively takes 1g and is placed in and take the substratum that sucrose is main carbon source, is placed in 30 ℃ of fermentation 72h of 220r/min shaking table, gets the karusen that viscosity is high and carries out primary dcreening operation,
C) primary dcreening operation: by the high karusen dilution spread screening plate culture medium of viscosity, be placed in 30 ℃ of incubators and cultivate 24h, the bacterium colony of selecting yellow, circle, smooth, glossy, low projection, neat in edge from flat board carries out multiple sieve,
D) multiple sieve: select single bacterium colony in sucrose fermention medium from screening flat board, be placed in 30 ℃ of fermentation 72h of 220r/min shaking table, 80 ℃ of water-bath 30min of karusen, centrifugal thalline, the ethanol of going precipitate, dry, through Fourier transformation infrared spectrometer Nicoletis10 (Thermo), detect, the higher bacterial strain of screening gelling gum output is optimized
E) identify: through the automatic assessing instrument of biochemistry and 16S rDNA, identify that this bacterial strain is sphingomonas paucimobilis (Sphingomonaspaucimobilis).
Sphingomonas paucimobilis of the present invention (Sphingomonas paucimobilis) gxas-815, can be used for fermentative production gelling gum.
The concrete grammar of described fermentative production gelling gum is: sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815 is inoculated in fermention medium, be placed in 220r/min shaking table, leavening temperature is 30 ℃, and fermentation 72h obtains gellan gum fermentation wine with dregs.
Described fermention medium is to take the fermention medium that sucrose is main carbon source, and the quality percentage composition of sucrose is 3～4%, and optimal dose is 3%; The quality percentage composition of peptone is 0.2～0.4%, and optimal dose is 0.3%; The quality percentage composition of dipotassium hydrogen phosphate is 0.05～0.2%, and optimal dose is 0.15%; The quality percentage composition of potassium sulfate is 0.1～0.3%, and optimal dose is 0.15%; Potassium primary phosphate quality percentage composition is 0.3～0.5%, and optimal dose is 0.25%; The quality percentage composition of anhydrous magnesium sulfate is 0.2～0.4%, and optimal dose is 0.3%.
Described inoculation is that the inoculum size that is 10% according to volumn concentration by the sphingomonas paucimobilis that grows to logarithmic phase (Sphingomonas paucimobilis) gxas-815 is inoculated in fermention medium.
Sphingomonas paucimobilis of the present invention (Sphingomonas paucimobilis) gxas-815 be take sucrose as fermenting raw materials production gelling gum, and gelling gum output reaches as high as 13.75g/L.
The explanation of preservation information
Sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815, has been deposited in Chinese Typical Representative culture collection center, and deposit number is: CCTCC No:M 2013327.Preservation date is on July 10th, 2013, and preservation address is wuchang, wuhan road Jia Shan Wuhan University.
Embodiment
Method in the following example, if no special instructions, is ordinary method.
Embodiment 1
The separation of sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815, screening and evaluation
The first step: sampling
In Weizhou Island, Beihai, Guangxi collected specimens.
Second step: enrichment
Enrichment medium component: sucrose 5g, peptone 10g, extractum carnis 3g, sodium-chlor 5g, uses distilled water constant volume to 1000mL, pH6.0～7.2.Above composition is mixed, be sub-packed in the triangular flask of 250mL volume, every bottled 50ml, 115 ℃ of high pressure steam sterilization 20min.
Each sample respectively takes 1g and is placed in respectively the triangular flask that enrichment medium is housed, and is placed in 30 ℃ of shaking tables, and 220r/min cultivates 72h, selects higher the screening of karusen viscosity.
The 3rd step: primary dcreening operation
Primary dcreening operation plate culture medium: sucrose 5g, peptone 10g, extractum carnis 3g, sodium-chlor 5g, agar 20g, distilled water 1000mL, pH7.0～7.2,115 ℃ of high pressure steam sterilization 20min.
By the higher diluted sample coating primary dcreening operation plate culture medium of karusen viscosity, be placed in 30 ℃ of incubators and cultivate 24h, the bacterium colony of selecting yellow, circle, smooth, glossy, low projection, neat in edge carries out multiple sieve.
The 4th step: multiple sieve
Sucrose fermention medium: sucrose 30g, peptone 3g, dipotassium hydrogen phosphate 1.5g, potassium sulfate 1.5g, potassium primary phosphate 2.5 g, anhydrous magnesium sulfate 3g, uses distilled water constant volume to 1000mL, pH7.0.Above composition is mixed, be sub-packed in the triangular flask of 250mL volume, every bottled 50ml, 115 ℃ of high pressure steam sterilization 20min.
From primary dcreening operation culture medium flat plate, select single bacterium colony in fermention medium, 30 ℃ of shaking table 220r/min fermentation 72h, karusen is carried out centrifugally going ethanol precipitation after thalline, drying, with infrared spectrometer, detect and analyze, a final selected strain utilizes the higher bacterial strain of sucrose fermentative production gelling gum output, called after gxas-815.
The 5th step: identify
Physiological and biochemical property:
(note: in table, "+" is positive, "-" is negative)
16S rDNA identifies:
To bacterial strain sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-81516S rDNA sequence amplification, after sequencing analysis, the sequence obtaining is compared in GenBank database (www.ncbi.nlm.nih.gov).The sequencing results shows: sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815 and sphingomonas paucimobilis (Sphingomonas paucimobilis) homology are more than 99%.
This bacterial strain sphingomonas paucimobilis morphology, physiological and biochemical property meet sphingomonas paucimobilis (Sphingomonas paucimobilis) feature, approach most sphingomonas paucimobilis, and sequential analysis shows, bacterial strain gxas-815 and sphingomonas paucimobilis (Sphingomonas paucimobilis) homology is more than 99%.Identify thus, bacterial strain gxas-815 belongs to sphingomonas paucimobilis (Sphingomonas paucimobilis).
From occurring in nature, successfully separate a kind of sucrose that can utilize for the new bacterial strain of fermenting raw materials high yield gelling gum, widened the bacterial strain that utilizes microorganism fermentative production gelling gum.
Embodiment 2
Sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815 fermentative production gelling gum
Plate culture medium: sucrose 5g, peptone 10g, extractum carnis 3g, sodium-chlor 5g, agar 20g, distilled water 1000mL, pH7.0～7.2,115 ℃ of high pressure steam sterilization 20min.
Seed culture medium: sucrose 5g, peptone 10g, extractum carnis 3g, sodium-chlor 5g, uses distilled water constant volume to 1000mL, pH7.0～7.2,115 ℃ of high pressure steam sterilization 20min.
Sucrose fermention medium: sucrose 30g, peptone 3g, dipotassium hydrogen phosphate 1.5g, potassium sulfate 1.5g, potassium primary phosphate 2.5g, anhydrous magnesium sulfate 3g, pH7.0, uses distilled water constant volume to 1000mL.Above composition is mixed, be sub-packed in the triangular flask of 250mL volume, every bottled 50ml, 115 ℃ of high pressure steam sterilization 20min.
Get sphingomonas paucimobilis (Sphingomonas paucimobilis) the gxas-815 spread plate substratum of glycerine preservation, be placed in 30 ℃ of incubators and cultivate 20h, choose single bacterium colony in seed culture medium, at 30 ℃ of shaking speed 220r/min, being cultured to OD600 is 1.0 left and right, the inoculum size that is 10% according to volumn concentration is inoculated in the 250ml triangular flask of dress 50ml fermention medium, 30 ℃ of shaking speed 220r/min fermentation 72h, precipitate 80 ℃ of water-bath 30min of karusen, centrifugal thalline, the ethanol of going, dry and detect with infrared spectrometer.Sucrose fermention medium used is through optimizing, and gelling gum output reaches as high as 13.75g/L.
Embodiment 3
The detection analysis of gelling gum
Infrared spectrometer detects to be analyzed: karusen adds the water of 2 times of volumes, 80 ℃ of water-bath 30min, 12000r/min is centrifugal, and 30min removes thalline, get the dehydrated alcohol that supernatant liquor adds 2 times of volumes, 4 ℃ of precipitations are spent the night, and the centrifugal 30min of 12000r/min will precipitate 60 ℃ of oven dry, grind loading infrared spectrometer and detect, analyze peak shape.
By above example, can find out that sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815 has good leavening property during the fermentation, the optimization gelling gum output of condition reaches as high as 13.75g/L by fermentation.
Claims (7)
1. a strain sphingomonas paucimobilis bacterial strain, its Classification And Nomenclature is sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815, on July 10th, 2013, be preserved in Chinese Typical Representative culture collection center, deposit number is: CCTCC M2013327.
2. the application of sphingomonas paucimobilis claimed in claim 1 (Sphingomonas paucimobilis) gxas-815 in fermentative production gelling gum.
3. application according to claim 2, it is characterized in that, the method of described fermentation gelling gum is that sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815 is inoculated in fermention medium, be placed in 220r/min shaking table, leavening temperature is 30 ℃, and fermentation 72h obtains gellan gum fermentation wine with dregs.
4. according to the application described in claim 2-3, it is characterized in that, described inoculation is that the inoculum size that is 5～10% according to volumn concentration by the sphingomonas paucimobilis that grows to logarithmic phase (Sphingomonas paucimobilis) gxas-815 is inoculated in fermention medium.
5. application according to claim 2, is characterized in that: described fermention medium is to take the fermention medium that sucrose is main carbon source, and the quality percentage composition of sucrose is 3～4%.
6. application according to claim 5, it is characterized in that: in described fermention medium, also contain peptone, quality percentage composition is 0.2～0.4%, the quality percentage composition of dipotassium hydrogen phosphate is 0.05～0.2%, the quality percentage composition of potassium sulfate is 0.1～0.3%, potassium primary phosphate quality percentage composition is 0.3～0.5%, and the quality percentage composition of anhydrous magnesium sulfate is 0.2～0.4%.
7. application according to claim 5, is characterized in that: in described fermention medium, the quality percentage composition of each composition is peptone 0.3%, dipotassium hydrogen phosphate 0.15%, potassium sulfate 0.15%, potassium primary phosphate 0.25%, anhydrous magnesium sulfate 0.3%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106011012A (en) * | 2016-06-22 | 2016-10-12 | 中国人民解放军总医院 | Space Sphingomonas sp. LCT-S10-1 (sphingomonas paucimobilis LCT-S10-1) |
| CN112159775A (en) * | 2020-09-27 | 2021-01-01 | 浙江工业大学 | Sphingomonas zeae and application thereof in biological deodorization |
| CN113151050A (en) * | 2021-03-10 | 2021-07-23 | 南京工业大学 | Sphingomonas and application thereof |
| CN117286081A (en) * | 2023-11-24 | 2023-12-26 | 中国农业科学院草原研究所 | Microorganism strain combination for promoting growth of elymus chinensis and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011012A (en) * | 2016-06-22 | 2016-10-12 | 中国人民解放军总医院 | Space Sphingomonas sp. LCT-S10-1 (sphingomonas paucimobilis LCT-S10-1) |
| CN106011012B (en) * | 2016-06-22 | 2021-07-23 | 中国人民解放军总医院 | Space sphingomonas paucimobilis LCT-S10-1 |
| CN112159775A (en) * | 2020-09-27 | 2021-01-01 | 浙江工业大学 | Sphingomonas zeae and application thereof in biological deodorization |
| CN112159775B (en) * | 2020-09-27 | 2022-03-08 | 浙江工业大学 | Sphingomonas zeae and application thereof in biological deodorization |
| CN113151050A (en) * | 2021-03-10 | 2021-07-23 | 南京工业大学 | Sphingomonas and application thereof |
| CN117286081A (en) * | 2023-11-24 | 2023-12-26 | 中国农业科学院草原研究所 | Microorganism strain combination for promoting growth of elymus chinensis and application |
| CN117286081B (en) * | 2023-11-24 | 2024-01-26 | 中国农业科学院草原研究所 | Microorganism strain combination for promoting growth of elymus chinensis and application |
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