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Agarase generating vibrio alginolyticus and application thereof

A technology of Vibrio alginolyticus and agar oligosaccharides, applied in the direction of bacteria, microorganisms, biochemical equipment and methods, etc., can solve the problems of long production cycle, unfavorable recovery and utilization of agar oligosaccharides, complex degradation components, etc. To achieve the effect of short production cycle

Active Publication Date: 2016-02-24
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Our country is rich in seaweed resources, which has laid a material foundation for the development and production of agar oligosaccharides. Now the traditional industry mainly uses acid hydrolysis to produce agar oligosaccharides. There are relatively few industries that produce agar oligosaccharides by enzymatic methods. There are many deficiencies in the production of agar oligosaccharides, including long production cycle, cumbersome production operations, complex degradation components, unstable hydrolyzed products, and difficult purification, which is not conducive to the recovery and utilization of agar oligosaccharides

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Take the sample liquid from the intestinal tract of marine shellfish, draw 1ml for dilution (10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -5 , 10 -6 , 10 -7 , 10 -8 ), draw 0.2ml and spread it on the agar screening medium, and after culturing for 48 hours at 28°C, select colonies with obvious depressions, and after separation and purification, transfer the strains to the liquid medium at 28°C, 150rpm, Shake the flask for 32 hours, centrifuge to take the supernatant (the supernatant is the crude enzyme solution), and use spectrophotometry to detect the agarase activity in the medium. After preliminary screening, a total of 6 strains of bacteria capable of growing agarase were isolated and obtained. , combined with the production capacity of agarase, the final selection of Vibrio alginolyticus ( Vibrio alginolyticus ) A-001 as the starting strain.

Embodiment 2

[0025] Through the study of morphology and physiological and biochemical characteristics, the characteristics of the agarase-producing strain are as follows:

[0026] Colony morphology: the colony is moist, milky white, with neat edges and round shape.

[0027] Cell morphology: curved short rod.

[0028] Physiological and biochemical characteristics: Gram-negative bacteria with motility and positive oxidase, can use glucose and starch as carbon sources, and can also use peptone, yeast extract, ammonium nitrate, ammonium sulfate, ammonium chloride, etc. as growth needs nitrogen source.

[0029] PCR amplification and sequence determination were performed on the 16SrDNA gene of the strain with the preservation number CCTCCM2015555, and it was found that the length of the 16SrDNA gene part was 1420bp. After comparison between NCBI and the ribosome database, it was identified as Vibrio alginolyticus ( Vibrio alginolyticus ) A-001, to be named Vibrio alginolyticus ( Vibrio algino...

Embodiment 3

[0031] (1) Vibrio alginolyticus ( Vibrio alginolyticus ) A-001 in the seed medium, use 250mL Erlenmeyer flask to fill 62mL seed medium, sterilize according to the conventional method, cool down, and inoculate the colony, after inoculation, culture at 25°C, 140rpm shaker for 24h to obtain the seed fermentation liquid. Said seed culture medium is: sodium chloride 35g / L, peptone 5g / L, yeast powder 10g / L, agar 2.0g / L, ferrous sulfate heptahydrate 0.1g / L, adjust pH to be 7, tap water preparation.

[0032] (2) Pack 90mL fermentation medium in a 250mL Erlenmeyer flask, sterilize according to the conventional method, cool down, and insert the seed fermentation liquid into the fermentation medium according to the inoculation amount of 11%, and cultivate it on a shaker at 28°C and 150rpm after inoculation After 48h, the fermented liquid containing agarase was obtained, and the seed culture medium was: sodium chloride 35g / L, peptone 5g / L, yeast powder 10g / L, agar 2.0g / L, ferrous sulfate...

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Abstract

The invention relates to vibrio alginolyticus A-001 and a method for preparing agaro-oligosaccharide with agarase generated by the vibrio alginolyticus A-001. The vibrio alginolyticus A-001 is preserved in CCTCC (China Center for Type Culture Collection) in September 18, 2015, and a preservation number of the vibrio alginolyticus A-001 is CCTCC M2015555. The method includes: taking strains for culture, using a fermentation medium composed of a carbon source, a nitrogen source and inorganic salt to generate enzymes, salting out fermentation liquor through ammonium sulfate, centrifuging, dialyzing, passing through iron exchange columns and gel columns to obtain dry enzyme powder, allowing reaction between agarase and 0.5% agar substrate, filtering through an ultrafiltration membrane, collecting filtrate and performing freeze drying to obtain the agaro-oligosaccharide.

Description

technical field [0001] The invention belongs to the field of food biotechnology and relates to screening an agarase-producing Vibrio alginolyticus and a method for preparing agar oligosaccharides. Background technique [0002] Agar, also known as agar, is water soluble and is a complex mixture, one of the three polysaccharides from seaweed. Because agar has good stability and gelling properties, it is widely used in food, medicine and other industries. With the sustained and healthy development of the seaweed polysaccharide industry, the depth and breadth of research on agar polysaccharide degrading enzymes are also increasing. According to the difference of the action point of agarase on the glycosidic bond, it can be divided into α-agarase and β-agarase. 4 glycosidic linkages, and obtain 3,6-endether-α-L-galactose and D-galactose. This type of enzyme uses agar as a reaction substrate, and can use its functional specificity to obtain agar-oligosaccharides with different ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P19/14C12R1/63
Inventor 郑宝东张林林江玉姬张龙涛张怡郭娟娟王培森李庆旺
Owner FUJIAN AGRI & FORESTRY UNIV
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