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Short bacillus and method for preparing trehalose by virtue of fermentation

A technology of trehalose and short, which is applied in Brevibacterium and its application in the fermentation and preparation of trehalose, in the field of microbial fermentation, can solve the problems of complex preparation process, high cost, and difficult separation, so as to achieve simple preparation process and increase yield , the effect of a wide range of sources

Inactive Publication Date: 2013-06-26
SUZHOU UNIV
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Problems solved by technology

Chinese patent application CN101481719 discloses a method for simultaneously preparing zymosan, trehalose and yeast extract by using waste beer yeast. The method includes debittering and impurity removal of waste beer yeast, autolysis, enzymolysis and inactivation of waste beer yeast , the separation and drying of zymosan, the membrane separation, concentration, crystallization and drying of trehalose, the concentration and drying of yeast extract and other steps; however, the yield of trehalose prepared by microbial extraction is not high, and the production cost is high, which is not conducive to Industrial application
The advantage of this method is that starch is used as raw material, and the production cost is relatively low. The disadvantage is that the production process is complex, and the product trehalose is difficult to separate from other sugars in the conversion system, such as maltose and maltotriose, and it is difficult to obtain high-purity trehalose. Using maltose as raw material, one-step synthesis of trehalose through microbial fermentation or microbial enzyme conversion has the advantage of simple process, but the disadvantage is high cost. In addition, similar to the enzyme conversion starch method, there is also the problem that it is difficult to obtain high-purity trehalose products; Glucose is used as a substrate, and trehalose can be efficiently generated by using highly specific trehalose-6-phosphate synthase and trehalose-6-phosphatase to jointly catalyze glucose, but the high-energy substance UDP needs to be consumed during the entire reaction process Glucose, GDP glucose, and ADP glucose are difficult to achieve large-scale industrial production
At the same time, the enzyme conversion method also needs multiple steps such as enzyme preparation and enzyme conversion reaction, which leads to the disadvantage of complex preparation process.

Method used

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  • Short bacillus and method for preparing trehalose by virtue of fermentation
  • Short bacillus and method for preparing trehalose by virtue of fermentation
  • Short bacillus and method for preparing trehalose by virtue of fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Mutagenesis Treatment Screening of Brevibacterium Mutants and Performance Testing

[0043] The strain was originally isolated from the soil, and it is a high-yielding strain of trehalose obtained through primary screening, re-screening and ultraviolet mutagenesis. Curtobacterium sp. SY311, with the preservation number CGMCC No.7181, was preserved in the General Microbiology Center of the China Microbiological Culture Collection Management Committee, and the preservation date was January 21, 2013. It specifically includes the following steps:

[0044] 1) Primary screening

[0045] From the candidate strains, select the strain that can decompose trehalose, that is, produce trehalase. The specific method is to plant the various strains preserved on the slant on the screening plate with trehalose as the only carbon source (the composition of the medium (mass volume ratio): 2% trehalose; 0.5% (NH 4 ) 2 SO 4 ;0.05% yeast extract; 0.1%KH 2 PO 4 ;0.06%Na 2 HPO...

Embodiment 2

[0051] Example 2 Preparation of Trehalose by Shake Flask Fermentation

[0052] The high-yield trehalose strains screened by the present invention can be prepared by optimizing the fermentation process to obtain high-yield trehalose, which specifically includes the following steps:

[0053] The Brevibacterium with the preservation number CGMCC No.7181 Curtobacterium sp. SY311 was transferred to the slant, 30°C, cultured for 48 hours, took 1 ring, inoculated in a 300ml Erlenmeyer flask with 30ml seed medium, 30°C, 200rpm, shaken and cultured for 20h, as the seed liquid; then, the seed liquid was pressed 2- 4% seed quantity is inserted in the 500ml Erlenmeyer flask that 50ml fermented liquid is housed.

[0054] According to the mass volume ratio, the composition of the slant medium: 1% peptone, 0.5% yeast extract, 0.5% wort juice, 0.5% casein, 0.2% beef extract, 0.2% glycerol, 0.005% Toween80, 0.1% MgSO 4 ·7H 2 O, 1.5% agar, the rest is water; pH is 7.0-7.2.

[0055] Accordi...

Embodiment 3

[0061] The Brevibacterium CGMCC No.7181 prepared in Example 1 was transferred to the slant, 30 ° C, cultured for 48 hours, took 1 ring, inoculated in a 300 ml Erlenmeyer flask with 30 ml of seed medium, 30 ° C, 200 rpm, shaking culture for 24 hours, as Seed liquid; then, the seed liquid was inserted into a 2-liter bench-top fermenter equipped with 1.2 liters of fermentation liquid by 3% seed amount and fermented for 72 hours.

[0062] Put 1370ml of the fermentation supernatant (containing 13.8g of trehalose) in a boiling water bath for 10 minutes, and filter to remove the coagulated protein. The clear night flows into the 732 cation exchange column (H + Type, 25×265mm), eluted with deionized water, collected trehalose fraction, flowed into 717 anion exchange column (OH - type, 39×370mm), eluted with deionized water, collected the trehalose fraction, then added 2% activated carbon to decolorize while stirring, and microfiltered the filter membrane (pore size: 0.45 μm) to obtai...

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Abstract

The invention discloses a short bacillus and an application thereof. The short bacillus is a curtobacterium sp. SY311 bacterial strain and is preserved in China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.7181. The method for preparing trehalose by fermenting the short bacillus concretely comprises the following steps of: (1) activating the strain; (2) preparing a seed solution; (3) fermenting; (4) deproteinizing; (5) collecting a trehalose component; and (6) carrying out posttreatment, namely carrying out decoloration, filtering, low pressure concentration, crystallization and drying on the trehalose component, so that the trehalose product is obtained. According to the invention, the adopted strain is mutated by ultraviolet, trehalose enzymatic activity is low, and accumulative increase of trehalose in fermentation liquor can be facilitated, so that the yield of the final trehalose product is increased; and the preparation technology disclosed by the invention is simple, trehalose is directly synthesized by adopting a one-step method, raw materials are available, cost is low, less impurities are contained in the trehalose product, purification is easy, and the method provided by the invention is applicable to industrial production.

Description

technical field [0001] The invention relates to microbial fermentation technology, in particular to a Brevibacterium with low trehalase activity and its application in fermenting and preparing trehalose, belonging to the field of biotechnology. Background technique [0002] Trehalose is a non-reducing disaccharide formed by connecting two glucose groups through a-a-1?1 bonds, and its molecular formula is C 12 h 22 o 11 ?2H 2 O, was first isolated from a beetle pupa living in the desert, and later found to be widely present in plants, algae, bacteria, molds, yeasts, insects and invertebrates. It is not only a kind of energy stored by organisms, but also has an important anti-stress and fresh-keeping effect on biologically active substances. When organisms are in adverse environments such as drying, freezing, high osmotic pressure, and strong radiation, they can increase the content of trehalose through in vivo regulation. content to resist external adverse damage, in addi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P19/12C12R1/01
Inventor 王蕾黄瑞顾冠彬
Owner SUZHOU UNIV
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