Method for producing lactulose through whole-cell catalysis

A technology of lactulose and lactulose, which is applied in the field of whole-cell catalysis to produce lactulose, can solve the problems of food safety hazards and increase the cost of lactulose, and achieve the effects of clean and non-toxic products, promotion of cleanliness, and simple separation and purification process

Inactive Publication Date: 2014-08-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

When the pure enzyme of cellobiose epimerase is used for the enzymatic production of lactulose, a large amount of boric acid needs to be added to the reaction system. Boric acid is a harmful substance to the human body. The removal of boric acid requires membrane separation technology combined with expensive deboronation resin, which will increase the cost of lactulose industrial production, while bringing food safety hazards

Method used

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  • Method for producing lactulose through whole-cell catalysis
  • Method for producing lactulose through whole-cell catalysis

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Experimental program
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Effect test

Embodiment 1

[0026] The construction conditions of recombinant Escherichia coli E.coli BL21 (DE3) are as follows: the cellobiose epimerase gene is derived from Caldicellulosiruptor saccharolyticus DSM8903, its nucleotide sequence is shown in SEQ ID NO.1, and the host cell is E.coli BL21(DE3), the carrier is pET-28a(+), using IPTG and lactose as inducers to induce enzyme production.

[0027] The recombinant Escherichia coli E.coli BL21 (DE3) producing cellobiose epimerase constructed was fermented and cultivated as a production strain, and the fermentation and cultivation steps were as follows:

[0028] (1) Take the strains in 30% (v / v) glycerol at -80°C, use 1% inoculation amount, 37°C, 200r / min shaker for 12h as the seed culture solution;

[0029](2) Select LB liquid medium for fermentation medium, add 50mL LB liquid medium to a 250mL Erlenmeyer flask, sterilize at 121°C for 20 minutes, add seed culture medium according to 1% inoculation amount after cooling, and add kalamycin to a final ...

Embodiment 2

[0032] In Example 1, the bacterial cells fermented with lactose as an inducer were subjected to vacuum freeze-drying to obtain bacterial powder, and the freeze-dried bacterial powder was directly used as a cell biocatalyst to catalyze the transformation of lactose into lactulose, and the substrate lactose was dissolved in Tris-HCl (pH7. 5) In the buffer solution, the concentration is 600g / L, the amount of bacteria powder added is 12.5U / mL, the reaction temperature is 80°C, and the reaction time is 3h. The lactulose content in the final reaction solution is 251.4g / L, and the lactulose conversion rate reaches 41.9%. .

Embodiment 3

[0034] In Example 1, the bacterial cells fermented with lactose as an inducer were permeabilized with 40% (v / v) ethanol at 4°C for 15 minutes, vacuum freeze-dried to obtain a permeabilized bacterial powder, and the obtained permeable bacterial powder was directly used as a cell The biocatalyst converts lactose to produce lactulose, the substrate lactose concentration is 600g / L, the buffer is Tris-HCl buffer (pH7.5), the reaction temperature is 80°C, the amount of bacteria powder added is 12.5U / mL, the reaction time is 3h, and the final reaction The content of lactulose in the liquid is 310g / L, the conversion rate of lactulose reaches 51.8%, the content of ipilactose is 49g / L, and the conversion rate of ipilactose reaches 8.2%.

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Abstract

The invention discloses a method for producing lactulose through whole-cell catalysis, and belongs to the field of the food biotechnology. According to the method, recombinant escherichia coli E. coli BL 21 (DE3) for producing cellobiose epimerase is taken as production bacterial strains, lactose is used for replacing isopropyl-beta-D-sulfo-galactoside (IPTG) and taken as an inductive agent for fermental cultivation, and thalluses obtained through centrifugation are subjected to ethyl alcohol permeabilization and vacuum freeze drying to serve as a cell biocatalyst for directly converting the lactose to produce the lactulose. The maximum percent conversion of the lactulose can reach 65.1%, the concentration of the lactulose reaches 390.6 g/L, the production rate of the lactulose reaches 195.3 g/(L*h), and the production quantity of by-product epidepride lactulose is smaller than 2%(w/w). According to the method, microbial cells are directly used for converting the lactose to produce the lactulose, and the method is simple and easy to implement, avoids the loss of enzyme activity in the separation and purification process, greatly reduces the cost of producing the lactulose through an enzymic method and is beneficial for achieving industrialization of lactulose production through the enzymic method early.

Description

technical field [0001] The invention relates to a method for catalyzing the production of lactulose by whole cells, which belongs to the field of food biotechnology. Background technique [0002] In recent years, the population of obesity, diabetes and cardiovascular disease has shown a trend of increasing year by year and younger age, making functional sweeteners with low calories and more functional properties become the focus of research and development and attention of food practitioners. 70% of people in the world have lactose intolerance. After eating lactose, they will cause serious gastrointestinal problems. Every year, millions of tons of lactose in the world are wasted because they cannot be further utilized and cause environmental pollution. How to efficiently utilize lactose has become an urgent problem to be solved. Lactulose obtained through lactose isomerization is becoming a new focus of attention of consumers and food practitioners due to its superior funct...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/90C12P19/24C12P19/12C12R1/19
Inventor 杨瑞金汪明明华霄张文斌沈秋云
Owner JIANGNAN UNIV
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