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Bacterial strain producing beta-galactosidase and method for preparing high-purity galactooligosaccharide

A technology of galactooligosaccharides and galactosidase, which is applied in the field of preparation of galactooligosaccharides, can solve the problems that galactose and lactose are not well separated, GOS has not realized industrial production, etc., and achieve the purity and yield of GOS. High efficiency, optimized production process, and simple process

Inactive Publication Date: 2015-07-29
SHANDONG FREDA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the monosaccharide components in the GOS mixture, there is a patent to use yeast and other fermentation to consume the glucose, but the galactose and lactose are not well separated
At the same time, my country began to conduct industrial research on GOS in the 1990s, but high-purity GOS has not yet achieved industrial production.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction and preservation of Pichia pastoris strain SMD1168H-pGAZaA-lac

[0025] The genome was extracted from Aspergillus leucobacterium, and then the β-galactosidase gene was obtained, which was verified by electrophoresis; the target gene fragment was amplified by PCR, connected with the plasmid vector pGAPZaA after double enzyme digestion, and transformed into Escherichia coli, and the transformation was successful Afterwards, single clones were picked, and plasmids were extracted for verification. Cultivate the successfully transformed Escherichia coli, extract the plasmid, and perform linearization, then electrotransform the linearized plasmid containing the target gene into Pichia pastoris SMD1168H, add X-gal to the medium, and use the principle of color development to detect β - Screen the strains with galactosidase activity, and select the dark-colored bacteria for secondary screening; express the recombinant Pichia pastoris with dark color, measu...

Embodiment 2

[0028] (1) 10L fermenter expands the cultivation of Pichia pastoris strain SMD1168H-pGAZaA-lac to prepare β-galactosidase: insert the glycerol tube of Pichia pastoris strain SMD1168H-pGAZaA-lac stored at -80°C into the seed medium, After culturing at 30°C for 16 hours, the seed solution was obtained; then, the inoculum was inserted into a 10L fermenter with a mass ratio of 10%, and the pH was adjusted to 7.0, and cultivated at 30°C, pressure 0.15Mpa, and dissolved oxygen 5-10% After 72 hours, the fermentation broth was obtained; the fermentation broth was centrifuged at 10,000 r / min for 10 minutes, and the supernatant was collected to be the crude enzyme solution of β-galactosidase. The crude β-galactosidase enzyme solution was concentrated 10 times through a 10kDa ultrafiltration membrane at 4°C, and the concentrated solution was freeze-dried to obtain 75000U / g β-galactosidase powder.

[0029] Wherein, the above-mentioned seed medium composition is as follows: yeast powder 5g...

Embodiment 3

[0034] (1) 10L fermenter expanded culture Pichia pastoris strain SMD1168H-pGAZaA-lac to prepare β-galactosidase, the fermentation supernatant was concentrated and then freeze-dried to obtain β-galactosidase solid powder, the method was the same as in Example 2 Step (1) described.

[0035] (2) Using 4 L of lactose solution with a concentration of 350 g / L as the substrate, adding 40 U / g of lactose β-galactosidase powder to catalyze the formation of GOS, the reaction temperature was 37 ° C, and the reaction time was 36 h to obtain GOS crude liquid; TLC and HPLC detection, its composition is a small amount of glucose, galactose, a large amount of unreacted lactose and the GOS of generation, and its GOS content is 32%.

[0036] (3) 10L fermenter expanded culture K.marxianus, culture method is described in step (3) with embodiment 2;

[0037] (4) Pour the treated GOS mixture into a yeast fermenter for purification. The inoculation amount of K. marxianus wet bacteria is 7%, and cult...

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Abstract

The invention discloses a bacterial strain producing beta-galactosidase and a method for preparing high-purity galactooligosaccharide. The bacterial strain producing beta-galactosidase is pichia pastoris SMD1168H-pGAZaA-lac, and the preservation number is CGMCC No. 8349. The invention also discloses the method for preparing high-purity galactooligosaccharide. The method includes the following steps: using pichia pastoris strain SMD1168H-pGAZaA-lac to prepare beta-galactosidase; using beta-galactosidase to catalyze lactose, so as to synthesize galactooligosaccharide; purifying galactooligosaccharide mixed liquid through Kluyveromyces marxianus; refining to obtain galactooligosaccharide with high purity. The purity of galactooligosaccharide obtained through the method is equal to or greater than 90%, and the yield is equal to or greater than 30%.

Description

technical field [0001] The invention relates to a preparation method of galactooligosaccharides, in particular to a production method for catalyzing the synthesis of galactooligosaccharides by using the β-galactosidase produced by the recombinant Aspergillus leucobacterium β-galactosidase gene, It belongs to the technical field of microorganisms. Background technique [0002] Galactooligosaccharides (Galactooligosaccharides, GOS) have special properties such as low sweetness, low calorie, low cariogenicity, heat and acid stability, and food safety. Good source of nutrients and potent proliferative factors. It can improve the digestion and absorption function of the human intestinal tract, and is often used as a new type of functional food additive, known as the "bifidus factor" with the best therapeutic effect. In September 2008, according to the provisions of the "Food Sanitation Law of the People's Republic of China" and the "Administrative Measures for New Resource Food...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N9/38C12P19/14C07H3/06C07H1/06C12R1/84
CPCC07H1/06C07H3/06C12N9/2471C12P19/14C12Y302/01023C12N1/165C12R2001/84
Inventor 李珍爱苏移山刘英梅朱云峰刘飞宗工理张林军马双双钊倩倩赵文刚
Owner SHANDONG FREDA BIOTECH
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