A method and application of immobilizing Phaffia rhodozyme and preparing Xinkesi sugar

A technology of Phaffia rhodozyma and Xinkesi sugar, which is applied in the field of bioengineering, can solve the problems of easy inactivation of free cells, easy inactivation of batches, and low number of repeated uses, so as to improve mechanical strength and stability, and effectively It is beneficial to industrial production and overcomes the effect of easy inactivation

Active Publication Date: 2017-12-01
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the separation and purification of enzymes limit the application of this method
Another method is the whole cell transformation method, that is, the cells are directly acted on sucrose to produce oligosaccharides. This method avoids the complicated steps of separation and purification of enzymes, but free cells are easily inactivated in extreme environments, and the number of reuses is low. The disadvantages limit the industrial application of this method
[0004] Phaffia rhodozyme can use sucrose as a substrate to synthesize new cosiose, but the conversion of free cells to sucrose to produce new cosiose has problems such as easy inactivation and few batches

Method used

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  • A method and application of immobilizing Phaffia rhodozyme and preparing Xinkesi sugar
  • A method and application of immobilizing Phaffia rhodozyme and preparing Xinkesi sugar
  • A method and application of immobilizing Phaffia rhodozyme and preparing Xinkesi sugar

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] (1) Preparation of the activated Phaffia rhodozyme strain: inoculate the cryopreserved Phaffia rhodozyme As 2.1557 (China Ordinary Microorganism Culture Collection and Management Center) in YPS medium, and cultivate it to logarithmic at 20°C and 220rpm period, to obtain first-class seeds; the first-class seed solution diluted 10 5After doubling, take 200 μL and spread it on YPSA medium, and culture it upside down at 20°C until a single red colony grows to obtain secondary seeds; streak the secondary seeds on YPSA medium, culture upside down at 20°C for 48 hours, and obtain activated Phaffia rhodozyme slant; use an inoculation loop to pick 1 to 2 rings of slant cells and inoculate them in 25ml of YPS medium, culture at 20°C and 220rpm for 36h to obtain activated Phaffia rhodozyme liquid. The composition of YPS (yeast extract powder peptone sucrose) medium is as follows: 10g / L sucrose, 3g / L yeast extract powder, 5g / L peptone, 22g / L wort, and the initial pH of the medium i...

Embodiment 2

[0057] (1) Preparation of seed solution: same as step (1) of Example 1.

[0058] (2) Proliferation culture of cells: same as step (2) of Example 1.

[0059] (3) Preparation of bacterial suspension: same as step (3) of Example 1.

[0060] (4) Preparation of chitosan-coated immobilized cells after calcium alginate embedding: same as step (4) of Example 1.

[0061] (5) Transformation of immobilized cells: get five 50ml Erlenmeyer flasks with a volume, add immobilized cells in each Erlenmeyer flask (the corresponding wet cell concentration is 115.9g / L, and the corresponding dry cell concentration is 16g / L L); Then, 10ml of sucrose solution with a concentration of 400g / L was added to the Erlenmeyer flask, and cultured with shaking at 220rpm in a shaker at 20°C, 30°C, 40°C, and 50°C for 24h, and samples were taken every 2h. Sampling method: 50 μL for each sample. After being diluted 20 times, the content of substrate and product in the reaction solution was detected by high perfo...

Embodiment 3

[0064] (1) Preparation of seed solution: same as step (1) of Example 1.

[0065] (2) Proliferation culture of cells: same as step (2) of Example 1.

[0066] (3) Preparation of bacterial suspension: same as step (3) of Example 1.

[0067] (4) Preparation of chitosan-coated immobilized cells after calcium alginate embedding: same as step (4) of Example 1.

[0068] (5) Syncosiose produced by catalyzing sucrose in a stacked bed reactor: the immobilized cells were filled in a chromatographic column (φ1.5cm×20cm), and the filling height was 16.5cm. The 400g / L sucrose solution stored in the feeding bottle that has been sterilized flows through the reactor at a certain flow rate through the pump, so that the residence time of the sucrose solution (residence time=filling volume / flow rate of the pump, the calculation of filling volume The formula is (1.5 / 2) 2 ×16.5×3.14) were 1.1h, 1.2h, 1.4h, 1.7h, 2.0h, 2.5h, 3.3h, respectively, and the reactor was placed in a 20°C thermostat. Sam...

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Abstract

The invention discloses a method and application of fixing Phaffia rhodozyme and preparing Xinkesi sugar. The present invention mixes evenly dispersed Phaffia rhodozyme suspension with sodium alginate solution in physiological saline, and then adds it dropwise into calcium chloride solution to stand for calcification, separates solid from liquid, washes the solid, and then transfers the solid to the shell The polysaccharide solution is covered with a membrane, washed to obtain immobilized cells; finally, the immobilized cells are placed in a shaker flask or a reactor for transformation to obtain Neocosiose. This method has the following advantages: it overcomes the disadvantage that free cells are easily inactivated; on the basis of the traditional calcium alginate embedding method, chitosan coating is introduced to improve the mechanical strength and stability of immobilized particles; The chemical cells convert sucrose to produce Xinkesi sugar, which runs continuously for 30 days and 360 batches. The yield of Xinkesi sugar is maintained at 50.7g / L, and the yield of Xinkesi sugar does not decrease significantly. Therefore, the method provided by the invention is beneficial to industrial production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method and application of immobilizing Phaffia rhodozyme and preparing Xinkesi sugar. Background technique [0002] Neo-kestose (neo-kestose) is a kind of sucrose trisaccharide oligosaccharides, which is an oligomer formed by combining a fructosyl group with D-fructose in sucrose molecules through a β(2-6) glycosidic bond. Not only does it have the physiological functions of ordinary fructooligosaccharides: it lowers the pH of the intestinal tract, regulates the flora in the body in two directions, prevents constipation and diarrhea; lowers blood lipids and blood pressure, prevents cardiovascular diseases; promotes vitamin synthesis and improves immunity; absorption; prevention of dental caries, etc. Moreover, it also has the ability to promote the proliferation of bifidobacteria that is superior to ordinary fructooligosaccharides. Therefore, it is called a high-effici...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/10C12N11/04C12P19/00C12R1/645
Inventor 朱明军别晓颖
Owner SOUTH CHINA UNIV OF TECH
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