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Alcaligenes faecalis mutant strain and method for preparing curdlan by using alcaligenes faecalis mutant strain

A technology of Alcaligenes faecalis and strains, which is applied in the field of preparation of curdlan gum, can solve the problems of curdlan gum strength damage, easy-to-break gel strength, and low curdlan gum strength, and achieve simple post-processing technology Easy to operate, good mass transfer and oxygen transfer effect, simple and easy extraction process

Active Publication Date: 2014-10-08
JIANGSU YIMING BIOLOGICAL SCI & TECH +1
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AI Technical Summary

Problems solved by technology

[0003] At present, curdlan gum is mainly prepared by fermenting sucrose or glucose with Agrobacterium biovarl or Alcaligenes faecalis, and the nitrogen source used is yeast extract, which leads to a darker color of the fermented product and lower fermentation efficiency. Low and high dosage of calcium carbonate lead to a large amount of calcium carbonate remaining after fermentation, resulting in problems such as low product purity, low transparency and low strength of curdlan
Due to the above problems, cumbersome and complicated extraction processes are required to obtain high-quality products. At the same time, the strength of curdlan gum is easily damaged during the extraction process, resulting in a large amount of waste products.
Such as the Chinese patent whose application number is 200410041271.8 (invention name is: a kind of microbial polysaccharide-thermogel extraction process), the extraction process of its fine-quality thermogel needs to go through high-concentration alkali solution, high-speed centrifugation, a large amount of hydrochloric acid callback and a large amount of Processes such as water washing are not only complicated in process, easy to destroy the gel strength, but also seriously polluted, not suitable for mass production, and the purity of the final product is only 83-86%; the product obtained by direct centrifugal washing without acid and alkali treatment has a purity Only 66.7%
The Chinese patent with the application number 201110218879.3 (invention name: post-extraction method of curdlan gum), although the entire extraction process uses enzyme treatment to remove impurities, it also requires a lot of acid-base treatment and a lot of water washing

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Mutation screening of embodiment 1 Alcaligenes faecalis bacterial strain

[0023] The present invention takes CICC20602 Alcaligenes faecalis as starting bacterial strain, after ultraviolet ray, nitrosoguanidine mutagenesis treatment, coat high-sugar aniline blue separation and screening culture medium, culture medium is composed as follows (wt%): sucrose 10%, (NH 4 ) 2 HPO 4 0.20%, KH 2 PO 4 0.20%, MgSO 4 ·7H 2 O 0.10%, CaCO 3 0.2%, corn syrup powder 0.08%, the amount of aniline blue added , pH7.0, aniline blue can be complexed with curdlan gum to form a blue complex, the formation rate of the blue complex depends on the concentration and degree of polymerization of curdlan gum, the darker the blue color of the colony, it means curdlan gum The higher the concentration and the degree of polymerization, the larger the colony, indicating the higher growth activity of the strain. Based on this, the strains with high degree of polymerization and good growth activi...

Embodiment 2

[0026] Slant culture medium (wt%): glucose or sucrose 1.00%, yeast extract 0.30%, peptone 0.50%, NaCl, 0.50%, agar 1.50%, pH adjusted to 7.0.

[0027] Seed medium (wt%): glucose 2.00%, (NH 4 ) 2 HPO 4 0.50%, KH 2 PO 4 0.15%, MgSO 4 ·7H 2 O0.10%, corn syrup powder 0.05%, CaCO 3 0.30%, pH7.2, sterilized at 121°C for 20min.

[0028] The fermentation medium (wt%): glucose 3.00%, sucrose 4%, (NH 4 ) 2 HPO4 0.20%, KH 2 PO 4 0.20%, MgSO 4 ·7H 2 O 0.10%, CaCO 3 0.05%, corn syrup powder 0.08%, pH 7.0, sterilized at 121°C for 20min.

[0029] (1) Incline cultivation:

[0030] Take an inoculation loop of the Alcaligenes faecalis mutant strain, inoculate it on a slant medium by streaking, culture it statically at 28-30°C for 50 hours, store it in a refrigerator at 4°C for one month and use it;

[0031] (2) Seed cultivation:

[0032] Take 1 branch of the culture of step (1), inoculate a ring and connect a ring of seeds in a 500ml Erlenmeyer flask equipped with 100ml of s...

Embodiment 3

[0039] The composition of the culture medium is the same as above, and the preparation method is as follows:

[0040] (1) Incline cultivation:

[0041] Take an inoculation loop of the Alcaligenes faecalis mutant strain, inoculate it on a slant medium by streaking, culture it statically at 28-30°C for 60 hours, store it in a refrigerator at 4°C for one month and use it;

[0042] (2) Seed cultivation:

[0043] Take 1 branch of the culture of step (1), inoculate a ring and connect a ring of seeds in a 500ml Erlenmeyer flask equipped with 100ml of seed medium, and cultivate it for 24hr at 28-30°C with shaking on a shaker at 180 rpm to obtain a seed solution. The effective number of viable bacteria in the seed liquid is 10 12 -10 14 / ml;

[0044] (3) Fermentation:

[0045] The seed solution in step (2) is inoculated into the fermentation medium with an inoculum size of 5%, and cultured with aeration and stirring at 28-30° C. for 92 hours; the fermentation yield is as high as 4...

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Abstract

The invention discloses an alcaligenes faecalis mutant strain and a method for preparing curdlan by using the alcaligenes faecalis mutant strain. The alcaligenes faecalis mutant strain has the collection number of CGMCC No.9291. The method for preparing curdlan by using the alcaligenes faecalis mutant strain comprises slant culture, seed culture, fermenting and post-extraction process, wherein the post-extraction process comprises the following steps: uniformly stirring lysozyme in fermentation liquor, performing centrifugal separation, adding solid matter into water, stirring and performing centrifugal separation again; taking out the solid matter, adding ethanol, uniformly stirring, performing plate-frame pressure filtration, and drying to obtain the product. The sugar concentration in the early stage of fermentation is 7 percent, the final curdlan yield is 4.8 percent during batch fermentation, the fermentation liquor is not viscous in the fermentation process, the mass transfer and oxygen transfer effects are good, the amount of calcium carbonate used in the strain fermentation process is only 0.05 percent, almost no calcium carbonate is remained after fermentation is ended, and the purity of the extracted product can be over 90 percent. The obtained colloid is semi-transparent and high in toughness, and the after-treatment process is simple and feasible.

Description

technical field [0001] The invention relates to a method for preparing curdlan, in particular to a method for fermenting a mutant strain of Alcaligenes faecalis to produce curdlan and a method for extracting the product afterward. Background technique [0002] Curdlan gum (Curdlan) is a new type of microbial exopolysaccharide. Because the polysaccharide has the unique property of forming a gel under heating conditions, it is also called thermal gel. Both chemical and enzymatic analysis have confirmed that curdlan gum is an unbranched homogeneous polysaccharide polymer formed by a single D-glucose linked by β-1,3-glycosidic bonds at the C1 and C3 positions. In general, each curdlan molecule is composed of 300-500 glucose residues, the average degree of polymerization is 450, the relative molecular weight is about 74000, and the molecular formula is (C 6 h 10 o 5 )n, n>250. According to the analysis, the difference in the degree of molecular polymerization may be caused...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P19/04C12R1/05
Inventor 周站平袁方郭宏明
Owner JIANGSU YIMING BIOLOGICAL SCI & TECH
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