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Strain for producing 2-keto-L-gulonic acid and production method thereof

A technology of keto-gulonic acid bacteria and gulonic acid, which is applied in the field of bacterial strains in which the substrate L-sorbose is transformed into 2-keto-L-gulonic acid, can solve the problems of large pollution, high material consumption, complex process control, etc., and achieve The effect of shortening the fermentation period

Inactive Publication Date: 2014-11-19
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The purpose of the present invention is to overcome the deficiencies such as high material consumption, heavy pollution and complex process control in the original two-step fermentation process, and to provide a method for converting L-sorbose into 2-KLG by pure bacteria fermentation. strains and methods

Method used

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  • Strain for producing 2-keto-L-gulonic acid and production method thereof
  • Strain for producing 2-keto-L-gulonic acid and production method thereof
  • Strain for producing 2-keto-L-gulonic acid and production method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Implementation example 1: biological characteristics of ketoglobulina SPUB-003

[0035] After Gram staining, ketoglobulina SPUB-003 was a Gram-negative brevibacterium, which could form short chains or filaments, but could not form spores. The bacterium was grown on the slant medium for 4 days, and its colony form was point-like, raised, complete, smooth round colony, and the surface of the colony was oily, transparent, and almost no pigment was produced. Its physiological and biochemical indicators are shown in Table 1.

[0036]Genomic DNA of strain SPUB-003 was extracted and PCR amplified with Primer(S): 5'AGA GTT TGA TCC TGG CTC AG 3'; and Primer(A): 5'AAG GAG GTG ATC CAG CCG CA 3' The 16s rRNA gene was purified and sequenced, and the measured sequence was compared with the rRNA homologous sequence in Genbank, and 7 strains with high homology were selected, and the phylogenetic tree of the strain was constructed using the software Mega5. Strain SPUB-003 and Ketogul...

Embodiment 2

[0040] Implementation example 2: strain SPUB-003 shake flask fermentation characteristics

[0041] The purely cultured strain SPUB-003 was inoculated in the basic seed medium, and after 24 hours of shaking flask culture at 30°C and 220rpm, it was inoculated in the basic fermentation medium with a 10% transplantation amount, and the fermentation medium contained 7% of Sorbus Sugar, cultured at 25~34°C, shaking at 220r / min for 72h. Samples were taken every 4 hours, and the strain concentration (OD650 value), pH value, 2-KLG yield and conversion rate were measured at 650nm. The experimental results are shown in Table 2. It can be seen that the conversion rate of strain SPUB-003 was the highest at 32°C for 68 hours, reaching 73.32%.

[0042]

[0043]

[0044]

[0045]

[0046]

Embodiment 3

[0047] Implementation Example 3: Determination of the optimum sorbose concentration fermented by bacterial strain SPUB-003

[0048] The purely cultured strain SPUB-003 was inoculated into the basic seed medium, and after 24 hours of shaking flask culture at 32°C and 220rpm, it was inoculated into the basic fermentation medium with a 10% transfer amount, and the fermentation medium contained 1%~13 % sorbose, cultivated at 32°C with shaking at 220r / min for 68 hours, and controlled the pH to 6.8~7.0 during the fermentation process. After the fermentation, the output and conversion rate of 2-KLG were measured. As shown in Table 3, it can be seen that the optimum sorbose substrate concentration of bacterial strain SPUB-003 is no more than 7%, and the conversion rate can reach 76.20% under this condition; preferred 1 -5%, its conversion rate is more than 85%; more preferably 1-3%, its conversion rate is more than 95%.

[0049]

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Abstract

The invention belongs to the field of microbial fermentation, relates to a strain for producing 2-keto-L-gulonic acid and a production method of the strain, and in particular relates to a strain and process for converting a substrate namely L-sorbose into 2-keto-L-gulonic acid (2-KLG) by using a biological fermentation method. According to the strain disclosed by the invention, a new strain SPUB-003 which is stable in genetic character, can grow independently, and can be used for producing 2-KLG is obtained by separating and screening from soil and further performing artificial domestication. SPUB-003 is authenticated to be Ketogulonicigenium sp, and has been sent to General microorganism center of China Committee for Culture Collection of Microorganisms (CGMCC) for collection, wherein the collection date is July 3, 2013, and the collection number is CGMCC No.7876. SPUB-003 can perform independent growth and acid production at 20-34 DEG C; the strain can be used for independently producing 2-KLG by virtue of liquid state fermentation; after performing shake-flask fermentation for 24-96 hours, the conversion rate of obtained 2-KLG can be more than 70%; after fermenting for 24-68 hours by using a 5L fermentation tank, the conversion rate of 2-KLG can reach more than 80%. In a fermentation process, by adding micro physiological active substances, the conversion rate can be increased to more than 90%, and the fermentation period can be shortened.

Description

Technical field: [0001] The invention belongs to the field of microbial fermentation, and relates to a strain producing 2-keto-L-gulonic acid (2-KLG) and a production method thereof, in particular to converting substrate L-sorbose into 2-KLG by means of microbial fermentation. The strain and process of keto-L-gulonic acid. Background technique: [0002] VC is an essential vitamin for the human body. It has a wide range of physiological functions in anti-oxidation and maintaining metabolic balance. It has important uses in the pharmaceutical industry, food industry, feed industry and cosmetics industry. It has a wide range of applications and a huge market. [0003] At present, more than 90% of the world's VC production uses the "two-step fermentation method" invented by my country. The first step of fermentation is to oxidize D-sorbitol to L-sorbose under the action of Gluconobacter oxidans. The second step is to further oxidize L-sorbose to 2-keto-L-gulonic acid (2-KLG),...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P7/60C12P17/04C12R1/01
Inventor 张怡轩吴春福
Owner SHENYANG PHARMA UNIVERSITY
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