Method for producing high-activity beta-galactosidase bacillus circulans strain and breeding method of strain

Abstract

The invention discloses a method for producing high-activity beta-galactosidase bacillus circulans strain and a breeding method of the strain. The produced high-activity beta-galactosidase bacillus circulans strain is bacillus circulans QHT-310-M481 is preserved in the China center for type culture collection on October 22nd, 2018. The preservation number is CCTCC NO:M 2018699, and the preservation address is Wuhan university, Wuhan, China. The produced high-activity beta-galactosidase bacillus circulans strain is obtained from a bacillus circulans original strain through mutagenizing by meansof 1-methyl-3-nitro-1-nitrosoguanidine, and a beta-galactosidase solution high in fermentation activity can be produced and used for an industrial catalytic galactosyl transfer reaction to produce galactooligosaccharide.

Description

technical field

[0001] The invention belongs to the fields of fermentation engineering and microbial breeding, and in particular relates to a bacillus circulus strain producing high-activity β-galactosidase and a breeding method thereof. Background technique

[0002] Galactooligosaccharides (GOS) is a functional oligosaccharide with natural properties. Galacto-oligosaccharides are low-energy sugars that occur naturally in animal and human milk. Galacto-oligosaccharide is an excellent nutrient source and effective proliferation factor for beneficial bacteria such as Bifidobacterium and Lactobacillus acidophilus in the human intestinal tract, which can improve the digestion and absorption function of the human intestinal tract. In addition, galacto-oligosaccharides can also improve lipid metabolism, reduce serum cholesterol concentration, promote the absorption of mineral elements, improve lactose intolerance, improve the body's immunity and other functions.

[0003] Galacto...

Examples

preparation example Construction

[0068] The seed medium used in the examples is prepared by the following method: add 1000 mL of distilled water to 15-20 g of prefabricated nutrient broth dry powder, heat to dissolve, the pH value does not need to be adjusted separately, and cool to obtain the seed medium.

[0069] The sterile phosphate buffer solution is a 0.01mol / L phosphate buffer solution with a pH value of 6.

[0070] The solid medium plate is prepared by the following method: add 1000mL distilled water to 30-35g prefabricated nutrient agar dry powder, heat to dissolve, the pH value does not need to be adjusted separately, pour it into a petri dish, cool down, and obtain a solid medium plate.

[0071] The fermentation medium comprises the following components by weight percentage: 0.6-1.6% soybean peptone, 0.35-0.65% yeast extract, 0.2-0.5% disodium hydrogen phosphate, 0.12-0.25% sodium carbonate, 0.12-0.55% magnesium sulfate, 1.0-1.5% lactose and 95.5-97.5% water, the pH value of the fermentation medium...

Embodiment 1

[0073] 1. The preliminary screening of the circular bacillus producing highly active β-galactosidase comprises the following steps:

[0074] 1) Activation of the starting strain: Bacillus circulars (Bacillus circulars, purchased from the American Type Culture Collection (ATCC, No. 31382)) was used as the starting strain for streak culture. , inoculated in 100mL fermentation medium (the composition of fermentation medium is: the soybean peptone of 1.2wt%, the yeast extract of 0.65wt%, the disodium hydrogen phosphate of 0.35wt%, the sodium carbonate of 0.25wt%, the sodium carbonate of 0.55wt% Magnesium sulfate, 1.5wt% lactose and 95.50wt% water, pH 6.0-6.5), 37 ° C, shake the flask after 48 hours of centrifugation, get the supernatant, and carry out the enzyme activity assay of β-galactosidase, Its measurement result is shown in table 1:

[0075] Table 1 β-galactosidase enzyme activity of the starting strain of Bacillus circulans

[0076]

[0077] 2) Pick a ring of slant st...

technical field

[0001] The invention belongs to the fields of fermentation engineering and microbial breeding, and in particular relates to a bacillus circulus strain producing high-activity β-galactosidase and a breeding method thereof. Background technique

[0002] Galactooligosaccharides (GOS) is a functional oligosaccharide with natural properties. Galacto-oligosaccharides are low-energy sugars that occur naturally in animal and human milk. Galacto-oligosaccharide is an excellent nutrient source and effective proliferation factor for beneficial bacteria such as Bifidobacterium and Lactobacillus acidophilus in the human intestinal tract, which can improve the digestion and absorption function of the human intestinal tract. In addition, galacto-oligosaccharides can also improve lipid metabolism, reduce serum cholesterol concentration, promote the absorption of mineral elements, improve lactose intolerance, improve the body's immunity and other functions.

[0003] Galacto...

Examples

preparation example Construction

[0068] The seed medium used in the examples is prepared by the following method: add 1000 mL of distilled water to 15-20 g of prefabricated nutrient broth dry powder, heat to dissolve, the pH value does not need to be adjusted separately, and cool to obtain the seed medium.

[0069] The sterile phosphate buffer solution is a 0.01mol / L phosphate buffer solution with a pH value of 6.

[0070] The solid medium plate is prepared by the following method: add 1000mL distilled water to 30-35g prefabricated nutrient agar dry powder, heat to dissolve, the pH value does not need to be adjusted separately, pour it into a petri dish, cool down, and obtain a solid medium plate.

[0071] The fermentation medium comprises the following components by weight percentage: 0.6-1.6% soybean peptone, 0.35-0.65% yeast extract, 0.2-0.5% disodium hydrogen phosphate, 0.12-0.25% sodium carbonate, 0.12-0.55% magnesium sulfate, 1.0-1.5% lactose and 95.5-97.5% water, the pH value of the fermentation medium...

Embodiment 1

[0073] 1. The preliminary screening of the circular bacillus producing highly active β-galactosidase comprises the following steps:

[0074] 1) Activation of the starting strain: Bacillus circulars (Bacillus circulars, purchased from the American Type Culture Collection (ATCC, No. 31382)) was used as the starting strain for streak culture. , inoculated in 100mL fermentation medium (the composition of fermentation medium is: the soybean peptone of 1.2wt%, the yeast extract of 0.65wt%, the disodium hydrogen phosphate of 0.35wt%, the sodium carbonate of 0.25wt%, the sodium carbonate of 0.55wt% Magnesium sulfate, 1.5wt% lactose and 95.50wt% water, pH 6.0-6.5), 37 ° C, shake the flask after 48 hours of centrifugation, get the supernatant, and carry out the enzyme activity assay of β-galactosidase, Its measurement result is shown in table 1:

[0075] Table 1 β-galactosidase enzyme activity of the starting strain of Bacillus circulans

[0076]

[0077] 2) Pick a ring of slant st...

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