CN104164385A - Sphingomonas paucimobilis strain gxas-815 and applications thereof - Google Patents

Abstract

The invention discloses a strain of Sphingomonas paucimobilis and its application, which is classified as Sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815, and is preserved in the China Center for Type Culture Collection with a preservation number For: CCTCC M2013327. The invention utilizes Sphingomonas paucimobilis to ferment and produce gellan gum with sucrose as raw material, and the yield of gellan gum reaches 13.75g/L.

Description

A strain of Sphingomonas paucimobilis gxas-815 and its application

technical field

The invention relates to the technical field of bioengineering, in particular to a sphingomonas paucimobilis strain gxas-815, which is obtained by screening samples collected from Nanning, Guangxi. The strain is used to ferment and produce gellan gum with sucrose as a raw material. the

Background technique

Gellan gum is a new type of microbial exopolysaccharide, which is a polymer composed of four sugar units: D-glucose, D-glucuronic acid, D-glucose, and L-rhamnose or mannan. Gellan gum has a wide range of uses and is mainly used as a thickener in the food field. It is currently one of the food gums with superior performance in the world that integrates thickening, suspension, emulsification, and stabilization. As a new type of microbial glue, gellan gum will gradually replace vegetable gums, such as agar and carrageenan, in the food, pharmaceutical, and chemical industries, and has a good market prospect. (Wei Chun, Zhang Donghui, Ying Xiangxian et al. Optimization of fermentation conditions for yellow pigment-deficient drug-resistant Sphingomonas WZ013 producing gellan gum. Chinese Journal of Food Science. 2013,23(2):101-107). Gellan gum is stable to enzymes, and is a new type of flocculant with good flocculation effect, easy natural degradation, and no secondary pollution. the

Sphingomonas paucimobilis, also translated as Sphingomonas paucimobilis, is a fermentation-producing strain of gellan gum. In the activated sludge of sewage treatment, Sphingomonas paucimobilis has been isolated many times, which has flocculation activity, and the gellan gum produced can be dissolved in cold water, has good stability, acid resistance, high temperature resistance, and heat reversibility It can also resist the action of microorganisms and enzymes, and can be used as a component of bioflocculants to perform bioremediation functions such as wastewater treatment. The production scale of domestic gellan gum manufacturers is small, the specifications of gellan gum produced are less, and there are still problems such as low fermentation yield in industrial production, high energy consumption for aeration and stirring during fermentation, cumbersome product extraction, and difficult product clarification. Lead to higher production costs, which limits the development of the domestic gellan gum industry. In the fermentation process of microbial polysaccharides, it is extremely important to choose suitable fermentation conditions. Gellan gum is synthesized by Sphingomonas paucimobilis through sugar metabolism in a suitable medium. During the synthesis process, the carbohydrate-based carbon source and the protein-based nitrogen source constitute the largest part of the cost of fermentation raw materials (Hu Guiping, Liu Bo, Zhu Yujing, etc. Optimization of Gellan Gum Fermentation Medium by Sphingomonas paucimobilis by Response Surface Method. Acta Biomathematica Sinica. 2012,27(3):507-517). the

Sphingomonas paucimobilis is an aerobic Gram-negative bacillus that can be cultured in cultures that use glucose, starch, sucrose, etc. as carbon sources, ammonium nitrate, yeast extract, peptone, etc. as nitrogen sources, and other trace elements. Grow and produce glue in the base. Many scholars at home and abroad have found that carbon source, nitrogen source, seed age, inoculum size, temperature, and initial pH of the fermentation medium all have a greater impact on the production of gellan gum by fermentation. the

Using Sphingomonas paucimobilis or Sphingomonas paucimobilis as the search element, a total of 44 patents were retrieved in the China Intellectual Property Office. The scope of these patents mainly includes: the discovery of new strains of Sphingomonas paucimobilis, the discovery of genes and the construction of gene knockout recombinant bacteria, the production of related enzyme preparations, the production method and extraction method of gellan gum produced by fermentation, as Microbial additives are used in applications such as food flavor improvement and wastewater treatment. the

Contents of the invention

The purpose of the present invention is to provide a Sphingomonas paucimobilis bacterial strain and application thereof. This strain has good prospects for fermentation optimization and genetic transformation, and also has important industrial application value. the

The Sphingomonas paucimobilis gxas-815 provided by the present invention is obtained by sampling, separating and screening from Nanning, Guangxi. the

Said Sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815CCTCC M2013327 isolation and screening method:

a) Sampling: samples were collected from Nanning, Guangxi,

b) Enrichment: 1g of each sample was weighed and placed in a medium with sucrose as the main carbon source, placed in a 220r/min shaker at 30°C for 72h fermentation, and the fermented mash with high viscosity was taken for preliminary screening.

c) Preliminary screening: Dilute the high-viscosity fermented mash and spread the screening plate medium, place it in an incubator at 30°C for 24 hours, and select yellow, round, smooth, shiny, low convex, and neat edges from the plate. Bacterial colonies were re-screened,

d) Re-screening: select a single colony from the screening plate in the sucrose fermentation medium, place it on a 220r/min shaker at 30°C for 72h, ferment the fermented mash in a water bath at 80°C for 30min, centrifuge to remove bacteria, ethanol precipitate, dry, and pass through Fourier transform infrared spectrometer Nicoletis10 (Thermo) detection, screening of strains with higher yield of gellan gum for optimization,

e) Identification: The bacterial strain was identified as Sphingomonas paucimobilis by a biochemical automatic identification instrument and 16S rDNA. the

The Sphingomonas paucimobilis gxas-815 of the present invention can be used to ferment and produce gellan gum. the

The specific method for producing gellan gum by fermentation is as follows: inoculate Sphingomonas paucimobilis gxas-815 into the fermentation medium, place it on a shaker at 220r/min, and ferment at a temperature of 30°C. 72h to obtain gellan gum fermented mash. the

The fermentation medium is a fermentation medium with sucrose as the main carbon source, the mass percentage of sucrose is 3-4%, and the optimum amount is 3%; the mass percentage of peptone is 0.2-0.4%, and the optimum amount is 0.3%; the mass percentage of dipotassium hydrogen phosphate is 0.05-0.2%, the optimum amount is 0.15%; the mass percentage of potassium sulfate is 0.1-0.3%, the optimum amount is 0.15%; the mass percentage of potassium dihydrogen phosphate The content of magnesium sulfate is 0.3-0.5%, the optimal amount is 0.25%; the mass percentage of anhydrous magnesium sulfate is 0.2-0.4%, the optimal amount is 0.3%. the

The inoculation is to inoculate the Sphingomonas paucimobilis gxas-815 grown to the logarithmic phase into the fermentation medium according to the inoculum amount of 10% by volume. the

The Sphingomonas paucimobilis gxas-815 of the present invention uses sucrose as a raw material to ferment and produce gellan gum, and the yield of gellan gum can reach up to 13.75g/L. the

Preservation Information Description

Sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815 has been preserved in the China Center for Type Culture Collection, and the preservation number is: CCTCC No: M 2013327. The preservation date is July 10, 2013, and the preservation address is Wuhan University, Jiashan, Wuchang Road, Wuhan. the

Detailed ways

The methods in the following examples are conventional methods unless otherwise specified. the

Example 1

Isolation, Screening and Identification of Sphingomonas paucimobilis gxas-815

Step 1: Sampling

Samples were collected in Weizhou Island, Beihai, Guangxi. the

Step Two: Enrichment

Enrichment medium components: 5g sucrose, 10g peptone, 3g beef extract, 5g sodium chloride, dilute to 1000mL with distilled water, pH 6.0-7.2. Mix the above ingredients evenly, divide them into 250mL Erlenmeyer flasks, fill each bottle with 50ml, and sterilize under high pressure steam at 115°C for 20min. the

Weigh 1 g of each sample and place them in Erlenmeyer flasks equipped with enrichment medium, place them on a shaker at 30°C, culture them at 220r/min for 72 hours, and select those with higher viscosity of fermented mash for screening. the

Step Three: Preliminary Screening

Primary screening plate medium: 5g sucrose, 10g peptone, 3g beef extract, 5g sodium chloride, 20g agar, 1000mL distilled water, pH7.0～7.2, sterilized by high pressure steam at 115℃ for 20min. the

The samples with higher viscosity of fermented mash were diluted and coated with the primary screening plate medium, placed in a 30°C incubator for 24 hours, and yellow, round, smooth, shiny, low convex, and neatly edged colonies were selected for secondary screening. the

Step Four: Rescreening

Sucrose fermentation medium: 30 g of sucrose, 3 g of peptone, 1.5 g of dipotassium hydrogen phosphate, 1.5 g of potassium sulfate, 2.5 g of potassium dihydrogen phosphate, 3 g of anhydrous magnesium sulfate, dilute to 1000 mL with distilled water, pH 7.0. Mix the above ingredients evenly, divide them into 250mL Erlenmeyer flasks, fill each bottle with 50ml, and sterilize under high pressure steam at 115°C for 20min. the

Select a single colony from the primary screening medium plate in the fermentation medium, and ferment on a shaker at 30°C at 220r/min for 72 hours. After centrifuging the fermented mash to remove bacteria, ethanol precipitation, drying, and infrared spectrometer detection and analysis, the final selection A strain with high yield of gellan gum produced by sucrose fermentation is named gxas-815. the

Step Five: Identification

Physiological and biochemical characteristics:

(Note: "+" in the table is positive, "-" is negative)

16S rDNA identification:

The gxas-81516S rDNA sequence of the strain Sphingomonas paucimobilis (Sphingomonas paucimobilis) was amplified, and after sequencing analysis, the obtained sequences were compared in the GenBank database (www.ncbi.nlm.nih.gov). Sequence analysis results showed that the homology between Sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815 and Sphingomonas paucimobilis was more than 99%. the

The morphological, physiological and biochemical characteristics of the strain Sphingomonas paucimobilis conformed to the characteristics of Sphingomonas paucimobilis, and was closest to Sphingomonas paucimobilis, and sequence analysis showed that the strain gxas- 815 has more than 99% homology with Sphingomonas paucimobilis. Thus identified, the strain gxas-815 belongs to Sphingomonas paucimobilis (Sphingomonas paucimobilis). the

A new strain that can use sucrose as raw material to ferment high-yield gellan gum was successfully isolated and screened from nature, which broadened the strains that can produce gellan gum by microbial fermentation. the

Example 2

Production of Gellan Gum by Fermentation of Sphingomonas paucimobilis gxas-815

Plate medium: 5g sucrose, 10g peptone, 3g beef extract, 5g sodium chloride, 20g agar, 1000mL distilled water, pH7.0-7.2, sterilized by high pressure steam at 115°C for 20min. the

Seed medium: 5g sucrose, 10g peptone, 3g beef extract, 5g sodium chloride, dilute to 1000mL with distilled water, pH7.0-7.2, sterilize by high pressure steam at 115℃ for 20min. the

Sucrose fermentation medium: 30 g sucrose, 3 g peptone, 1.5 g dipotassium hydrogen phosphate, 1.5 g potassium sulfate, 2.5 g potassium dihydrogen phosphate, 3 g anhydrous magnesium sulfate, pH 7.0, and dilute to 1000 mL with distilled water. Mix the above ingredients evenly, divide them into 250mL Erlenmeyer flasks, fill each bottle with 50ml, and sterilize under high pressure steam at 115°C for 20min. the

Take the Sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815 coated plate medium preserved in glycerol, place it in a 30°C incubator and cultivate it for 20h, pick a single colony in the seed medium, shake it at 30°C at a speed of 220r Cultivate at OD600 of about 1.0 per minute, inoculate 10% inoculum into a 250ml Erlenmeyer flask containing 50ml of fermentation medium, ferment at 30°C at a shaker speed of 220r/min for 72h, place the fermented mash in a water bath at 80°C After 30 minutes, centrifuge to remove bacteria, precipitate with ethanol, dry and detect with infrared spectrometer. The sucrose fermentation medium used is optimized, and the yield of gellan gum can reach up to 13.75g/L. the

Example 3

Detection and Analysis of Gellan Gum

Infrared spectrometer detection and analysis: add 2 times the volume of water to the fermented mash, bathe in 80°C for 30 minutes, centrifuge at 12000r/min for 30min to remove bacteria, take the supernatant and add 2 times the volume of absolute ethanol, settle overnight at 4°C, and centrifuge at 12000r/min After 30 minutes, the precipitate was dried at 60°C, ground and loaded to an infrared spectrometer for detection, and the peak shape was analyzed. the

It can be seen from the above examples that Sphingomonas paucimobilis (Sphingomonas paucimobilis) gxas-815 has good fermentation performance during the fermentation process, and the gellan gum yield can reach up to 13.75g/L after optimization of fermentation conditions. the

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%.