Fermentation preparation method, and separation and purification method of heat-resistant and acid-resistant glucose oxidase

Abstract

The invention discloses a fermentation preparation method, and a separation and purification method of heat-resistant and acid-resistant glucose oxidase, and belongs to the technical field of microorganisms. The glucose oxidase prepared by fermentation has high heat resistance and acid resistance, and the defect of low enzyme activity caused by use in food and feed in a high temperature and acidic environment is overcome. The fermentation preparation method comprises the following specific steps: culturing a bacterial strain into a spore, cleaning the spore and then preparing a mono-spore suspension; introducing the mono-spore suspension into a seed culture medium for fermentation culture; introducing seed culture fluid into a shaking flask fermentation medium for fermentation culture, adding a fermentation supplementary nutrient solution into fermentation liquid; adding dextrin, soluble starch and potassium sorbate into the fermentation liquid, and uniformly stirring; freezing, drying and crushing the fermentation liquid to obtain the glucose oxidase. After crude enzyme fluid of the glucose oxidase obtained by the fermentation is subjected to super-heating treatment, ammonium sulfate-ethanol-compound precipitation, ion exchange chromatography and molecular sieve gel filtration chromatography, the glucose oxidase with high recovery rate and high purity is obtained.

Description

technical field

[0001] The invention relates to the technical field of microorganisms, in particular to a fermentation preparation and separation and purification method of heat-resistant and acid-resistant glucose oxidase. Background technique

[0002] Glucose oxidase is a very important enzyme in the field of enzyme technology. It can use molecular oxygen as an electron acceptor to specifically catalyze the oxidation of β-D-glucose into gluconic acid and hydrogen peroxide. Because of its close relationship with the vital substances glucose and oxygen, it has been widely used in scientific research, medicine and food industry production. In the food industry, glucose oxidase can be used to remove glucose in food or medicine, thereby preventing browning of food or obtaining oligosaccharides with higher purity; at the same time, glucose oxidase can be used as a relatively An ideal potassium bromate substitute, used as a safer flour improver; in the fermentation industry, the...

Examples

Embodiment 1

[0047] A fermentation preparation method for heat-resistant and acid-resistant glucose oxidase produced by Paecilomyces thermophila, the specific steps are as follows:

[0048] (1) Streak and activate Paecilomyces thermophila species on the PDA activation medium plate, and place the PDA activation medium plate in a constant temperature incubator at 35° C. for 50 h to obtain Paecilomyces thermophila filaments;

[0049] (2) Scrape the Paecilomyces thermophila filaments prepared in step (1) with an inoculation loop and inoculate them on the Paecilomyces thermophila spore medium, and place the Paecilomyces thermophila spore medium at 35°C Cultivate for 3 days to obtain spores;

[0050] (3) The prepared spores in the step (2) are cleaned with 0.75% sterile saline containing 0.01% (v / v) Tween 80, and the cleaned spores are put into 50mL sterile saline and Place in a conical flask with sterile glass beads and fully shake for 40 minutes. After the spores are fully dispersed, prepare ...

Embodiment 2

[0063] A fermentation preparation method for heat-resistant and acid-resistant glucose oxidase produced by Paecilomyces thermophila, the specific steps are as follows:

[0064] (1) Streak and activate Paecilomyces thermophila species on the PDA activation medium plate, and place the PDA activation medium plate in a constant temperature incubator at 30°C for 48 hours of activation culture to obtain Paecilomyces thermophila filaments;

[0065] (2) Use the inoculation loop to scrape the prepared Paecilomyces thermophila filaments in step (1) and inoculate them on the Paecilomyces thermophila spore medium, and place the Paecilomyces thermophila spore medium at 30°C Under culture for 2 days, the spores are obtained;

[0066](3) The prepared spores in the step (2) are cleaned with 0.75% sterile saline containing 0.01% (v / v) Tween 80, and the cleaned spores are put into 50mL sterile saline and Place in a conical flask with sterile glass beads and fully shake for 30 minutes. After th...

Embodiment 3

[0079] A fermentation preparation method for heat-resistant and acid-resistant glucose oxidase produced by Paecilomyces thermophila, the specific steps are as follows:

[0080] (1) Streak and activate Paecilomyces thermophila species on a PDA activation medium plate, and place the PDA activation medium plate in a constant temperature incubator at 40°C for 72 hours of activation culture to obtain Paecilomyces thermophila filaments;

[0081] (2) Use the inoculation loop to scrape the prepared Paecilomyces thermophila filaments in step (1) and inoculate them on the Paecilomyces thermophila spore medium, and place the Paecilomyces thermophila spore medium at 40°C Under culture for 4 days, the spores are obtained;

[0082] (3) The prepared spores in the step (2) are cleaned with 0.75% sterile saline containing 0.01% (v / v) Tween 80, and the cleaned spores are put into 55mL sterile saline and Place in a conical flask with sterile glass beads and fully shake for 45 minutes. After the...

technical field

[0001] The invention relates to the technical field of microorganisms, in particular to a fermentation preparation and separation and purification method of heat-resistant and acid-resistant glucose oxidase. Background technique

[0002] Glucose oxidase is a very important enzyme in the field of enzyme technology. It can use molecular oxygen as an electron acceptor to specifically catalyze the oxidation of β-D-glucose into gluconic acid and hydrogen peroxide. Because of its close relationship with the vital substances glucose and oxygen, it has been widely used in scientific research, medicine and food industry production. In the food industry, glucose oxidase can be used to remove glucose in food or medicine, thereby preventing browning of food or obtaining oligosaccharides with higher purity; at the same time, glucose oxidase can be used as a relatively An ideal potassium bromate substitute, used as a safer flour improver; in the fermentation industry, the...

Examples

Embodiment 1

[0047] A fermentation preparation method for heat-resistant and acid-resistant glucose oxidase produced by Paecilomyces thermophila, the specific steps are as follows:

[0048] (1) Streak and activate Paecilomyces thermophila species on the PDA activation medium plate, and place the PDA activation medium plate in a constant temperature incubator at 35° C. for 50 h to obtain Paecilomyces thermophila filaments;

[0049] (2) Scrape the Paecilomyces thermophila filaments prepared in step (1) with an inoculation loop and inoculate them on the Paecilomyces thermophila spore medium, and place the Paecilomyces thermophila spore medium at 35°C Cultivate for 3 days to obtain spores;

[0050] (3) The prepared spores in the step (2) are cleaned with 0.75% sterile saline containing 0.01% (v / v) Tween 80, and the cleaned spores are put into 50mL sterile saline and Place in a conical flask with sterile glass beads and fully shake for 40 minutes. After the spores are fully dispersed, prepare ...

Embodiment 2

[0063] A fermentation preparation method for heat-resistant and acid-resistant glucose oxidase produced by Paecilomyces thermophila, the specific steps are as follows:

[0064] (1) Streak and activate Paecilomyces thermophila species on the PDA activation medium plate, and place the PDA activation medium plate in a constant temperature incubator at 30°C for 48 hours of activation culture to obtain Paecilomyces thermophila filaments;

[0065] (2) Use the inoculation loop to scrape the prepared Paecilomyces thermophila filaments in step (1) and inoculate them on the Paecilomyces thermophila spore medium, and place the Paecilomyces thermophila spore medium at 30°C Under culture for 2 days, the spores are obtained;

[0066](3) The prepared spores in the step (2) are cleaned with 0.75% sterile saline containing 0.01% (v / v) Tween 80, and the cleaned spores are put into 50mL sterile saline and Place in a conical flask with sterile glass beads and fully shake for 30 minutes. After th...

Embodiment 3

[0079] A fermentation preparation method for heat-resistant and acid-resistant glucose oxidase produced by Paecilomyces thermophila, the specific steps are as follows:

[0080] (1) Streak and activate Paecilomyces thermophila species on a PDA activation medium plate, and place the PDA activation medium plate in a constant temperature incubator at 40°C for 72 hours of activation culture to obtain Paecilomyces thermophila filaments;

[0081] (2) Use the inoculation loop to scrape the prepared Paecilomyces thermophila filaments in step (1) and inoculate them on the Paecilomyces thermophila spore medium, and place the Paecilomyces thermophila spore medium at 40°C Under culture for 4 days, the spores are obtained;

[0082] (3) The prepared spores in the step (2) are cleaned with 0.75% sterile saline containing 0.01% (v / v) Tween 80, and the cleaned spores are put into 55mL sterile saline and Place in a conical flask with sterile glass beads and fully shake for 45 minutes. After the...

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