Roseobactersp.zjut and application thereof in preparation of agaro-oligosaccharide

A technology of rose and bacillus, applied to rose bacillus and its application field in the preparation of agar oligosaccharide, can solve the problems of high medium cost, low enzyme activity, long fermentation period and the like

Active Publication Date: 2011-01-26
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The present invention provides a new strain of agarase-producing bacteria——Roseobacter sp. zjut, and its application in the preparation of agar oligosaccharides, which can better overcome the cultivation of agarase produced by fermentation of existing microorganisms. Disadvantages of high base cost, long fermentation cycle and low enzyme activity

Method used

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  • Roseobactersp.zjut and application thereof in preparation of agaro-oligosaccharide

Examples

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Embodiment 1

[0035] Embodiment 1: Enrichment, separation and screening of microorganisms producing agarase

[0036] Add 50mL of natural seawater and 5g of strip agar to a 250mL Erlenmeyer flask, culture the flask at 28°C and 200r / min in a vibrating shaker, and pipette 5mL of the culture solution to another In the Erlenmeyer flask with 5g of agar and 46mL of seawater, the above culture process was repeated, so that the number of microorganisms that could grow with agar as the sole carbon source in seawater increased. Dilute the enriched culture solution with sterile water and spread it on the plate medium, and cultivate the culture plate in a biochemical incubator at 28°C until the number of colonies no longer increases. Observe the colony shape on the plate, pick the colonies with obvious depressions around them to the slant medium, mark the bottom of the petri dish, and add Lugol's iodine solution dropwise for staining. Measure the diameter of the transparent circle around the bacterium ...

Embodiment 2

[0040] Embodiment 2: Mutation breeding of high-yield agarase strain

[0041] The strain HS0326-601 screened in Example 1 was irradiated with ultraviolet rays and gamma rays for mutagenesis, and the mutant strains with improved agarase-producing activity were screened.

[0042] Ultraviolet radiation mutagenesis method: After activating the slant strain HS0326-601, pick 6 rings of bacteria into a conical flask containing 50mL of sterile artificial seawater, shake at room temperature for 20-30min, and make a bacterial suspension. Count the bacteria with a hemocytometer under a microscope, and control the number of cells in the bacterial suspension to 1×10 8 A / mL or so. Under red light, take 2 mL of the above-mentioned bacterial suspension and a sterile paper clip into 6 petri dishes with a diameter of 6 cm, place the petri dishes on a magnetic stirrer, and irradiate them under a preheated ultraviolet lamp for about 20 minutes for 1 ~5min. Take 0.5mL of the above-mentioned irra...

Embodiment 3

[0048] Embodiment 3: the method for preparing agarase by Roseobacter zjut fermentation

[0049] Using roseobacter zjut as the strain, after optimizing the medium composition and fermentation conditions, the preparation method of agarase is as follows:

[0050] (1) Inoculate the strain of Roseobacter zjut that was freeze-dried or preserved on the slant of the test tube into the slant medium, and the slant was cultured in a biochemical incubator at 28°C for 36 hours. The slant medium consists of: agar 20g / L, yeast extract powder 5g / L, NaCl 20g / L, MgSO 4 ·7H 2 O 5g / L; KCl1g / L, FeSO 4 ·7H 2 O 0.02g / L, CaCl 2 0.2g / L, NaH 2 PO 4 0.6g / L, solvent is water, pH 7.0.

[0051] (2) Use an inoculation loop to pick out the roseobacter zjut cells after activation in step (1) into the liquid seed medium, and the liquid seed culture is based on 28°C and 200r / min shaking conditions for 24 hours to obtain seed liquid (diluted 5 times OD 620 =0.882); The liquid seed medium consists of: ag...

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Abstract

The invention provides a novel agarase generating strain (roseobactersp.zjut) and application thereof in the preparation of agaro-oligosaccharide. The roseobactersp.zjut is preserved in the China Center for Type Culture Collection, the collection number is CCTCC No. M 209292, and the preservation date is December 3, 2009. The roseobactersp.zjut and the application thereof in the preparation of the agaro-oligosaccharide have the advantages that: (1) the roseobactersp.zjut of the invention has low requirements on nutrition and can be easily cultured; (2) agarase generated by the roseobactersp.zjut is an extracellular enzyme, separation and purification are convenient, and cell breakage is not required; (3) the roseobactersp.zjut has high agarase generating activity, and the activity of crude agarase liquid which is not separated and purified can reach 215.8U/ml under optimized conditions; (4) the agarase generated by the roseobactersp.zjut degrades agar under mild reaction conditions, and the reaction time is short; and (5) the agarase in the invention has high stability and basically has no enzyme activity loss (inactivation efficiency is less than or equal to 5 percent) after being stored in a refrigerator for 7 days at the temperature of less than 4 DEG C.

Description

(1) Technical field [0001] The invention relates to a new agarase-producing bacterial strain—Roseobacter sp. zjut, and its application in preparing agar oligosaccharides. (2) Background technology [0002] Agar (agar) is a polysaccharide extracted from marine red algae. It has a long history of application in food, medical and health industries, but due to its high viscosity and low water solubility, it is not easy to be absorbed. Applications in the pharmaceutical industry are very limited. Agaro-oligosaccharide (agaro-oligosaccharide) is an oligosaccharide with a degree of polymerization of 2-10 after degradation of agar polysaccharide, also known as agaro-oligosaccharide, which is mainly composed of repeating units of agarobiose. Well, it is beneficial to human body absorption, and its application value in the field of medicine will be significantly improved. Studies have shown that agar oligosaccharides have good physiological activities such as anti-cancer, anti-oxida...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/01C12N9/24C12P19/00C12R1/01
Inventor 梅建凤应国清刘美英王鸿易喻陈建澍
Owner ZHEJIANG UNIV OF TECH
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