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Screening method for 2-keto-D-gluconic acid high-yield bacterial strain, and fermentation method of such bacterial strain

A technology of gluconic acid and screening methods, applied in the direction of microorganism-based methods, fermentation, biochemical equipment and methods, etc., which can solve the problems of long fermentation cycle, high energy consumption, low fermentation production efficiency, etc.

Inactive Publication Date: 2012-02-29
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At the same time, there are also some problems in my country's domestic fermentation production of 2KGA: serious bacteriophage pollution; long fermentation cycle; low fermentation production efficiency, and energy consumption is higher than that of similar foreign enterprises

Method used

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  • Screening method for 2-keto-D-gluconic acid high-yield bacterial strain, and fermentation method of such bacterial strain
  • Screening method for 2-keto-D-gluconic acid high-yield bacterial strain, and fermentation method of such bacterial strain
  • Screening method for 2-keto-D-gluconic acid high-yield bacterial strain, and fermentation method of such bacterial strain

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Experimental program
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Effect test

Embodiment 1

[0044] Weigh about 0.2g of soil into 20ml of 0.85% sterile normal saline, shake and mix well, spread the bacterial suspension onto beef extract peptone medium with 5% salt concentration and incubate at 30°C for 24h, and inoculate the purple pigment-producing colonies Put it into the fermentation medium for re-screening, culture at 30°C for 24 hours, and collect the fermentation broth by centrifugation. The fermentation broth is analyzed by the internal standard method in the high performance liquid phase method, and the 2-keto-D-gluconate calcium standard is used as the internal standard , The strain that increases the peak height of the 2-keto-D-gluconate standard product is the 2-keto-D-gluconate-producing strain.

Embodiment 2

[0046] The morphological characteristics of the screened strains were identified according to Microbial Taxonomy. The strains were short rod-shaped, Gram-negative bacteria, without spores, with perinatal flagella, and could move. The colony is round, with smooth edges, moist surface, slightly raised, producing purple-red pigment, and has a foul smell. Physiological and biochemical characteristics (see Table 1), and extract genomic DNA according to the extraction method of the bacterial genome extraction kit, with P1 and P2 as forward and reverse primers:

[0047] P1: 5'-AGAGTTTGATCCTGGCTCAG-3',

[0048] P2: 5'-GGCTACCTTGTTACGACTT-3'.

[0049] The 16S rDNA gene was amplified by PCR, and the BGI Research Center was commissioned to perform 16S rDNA sequencing. After obtaining the partial 16S rDNA sequence of this strain (GenBank accession number: JF794580), the homology comparison analysis was performed on the NCBI website using the BLAST search tool. . Based on 16S rDNA seque...

Embodiment 3

[0054] The strain was preserved in a glycerol tube with a final concentration of 15%. Take 200μl of the preserved bacteria liquid and insert it into 50ml of beef extract peptone medium with 5% salt concentration and cultivate it at 30°C for 12h, and insert it into a 70ml fermentation culture medium with a 10% inoculum size. The base 750mL two-thorn Erlenmeyer flask was used for fermentation. See claim 2 for the fermentation medium, and the initial glucose is 140g / L. Fermentation was carried out at 30° C. and 200 rpm for 24 hours, and the content of 2-keto-D-gluconic acid in the fermentation liquid was determined by HPLC. The output was 119.7g / L, the yield was 0.84g / g, and the production intensity was 3.74g / (L·h).

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Abstract

The invention discloses a screening method for a high-yield 2-keto-D-gluconic acid Serratieae bacterial strain, and the application of the bacterial strain for producing 2-keto-D-gluconic acid, belonging to the field of biological engineering. The screening method comprises the following steps of: weighing about 0.2 g of soil, adding the soil in 20 ml 0.85% of sterile normal saline, mixing uniformly via shaking, coating the bacterial suspension on a beef extract peptone culture medium with salt concentration of 5%, culturing for 36 hours at 30 DEG C, culturing the bacterial colony of the purplish red pigment in a fermentation medium, then, executing qualitative analysis via a high performance liquid chromatograph by an internal standard method, and identifying the screened bacterial strain on the aspects of morphology, physiology and biochemistry, and molecular biology, so as to get one high-yield 2-keto-D-gluconic acid Serratieae bacterial strain; the invention also discloses a method for producing the 2-keto-D-gluconic acid by the bacterial strain, and yield of the 2-keto-D-gluconic acid produced by fermentation according to the method is 181.2g / L.

Description

technical field [0001] The invention relates to a screening method for a high-yield 2-keto-D-gluconic acid strain and a method for fermenting and producing 2-keto-D-gluconic acid with the strain. Background technique [0002] 2-keto-D-gluconic acid (2-keto-D-gluconic acid, 2KGA), is one of the organic acids produced on a large scale at present, mainly used in food antioxidant D-sodium erythorbate (sodium erthorbate, EN) And the synthesis of D-erythorbic acid (EA), also known as isovitamin C, is widely used as a food antioxidant in the food industry. The production methods of 2KGA mainly include enzymatic method, chemical synthesis method and fermentation method. At present, bacterial fermentation method is mainly used in industry. [0003] Bacteria of the genus Pseudomonas (Pseudomonas), especially Pseudomonas fluorescens (Pseudomonas fluorescens) are usually used for 2KGA fermentation at home and abroad. Usually starch hydrolyzed sugar is used as the carbon source, corn s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P7/58C12R1/425
Inventor 刘立明牛盼清杨爱华杨松鑫
Owner JIANGNAN UNIV
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