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A kind of aspergillus oryzae and application thereof

A kind of Aspergillus oryzae, application technology, applied in Aspergillus oryzae and its application field, can solve the problems of lack of high-yield strains, poor stability, high production cost, etc., achieve good application prospects, stable enzyme activity, and cost-saving effects

Active Publication Date: 2020-08-21
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the research on naringinase is getting more and more in-depth, only a small number of naringinases have been industrialized at present, and there are two problems restricting the large-scale industrial application of naringinase: the first major problem is the lack of High-yield enzyme strains suitable for industrial production greatly limit the application; the difficulty in cultivating high-yield naringinase strains is how to efficiently select excellent mutants from a large number of mutagenized progeny, so far there is no A better way is that the breeding of strains is progressing slowly, and it is necessary to develop new and efficient screening methods, so the cultivation of high-yielding strains is an urgent task at present
The second problem is that there are still many defects in the immobilization technology of naringinase in China. The reagents or carriers used in the immobilization have high cost, low immobilization efficiency, poor stability, and equipment used for continuous operation. It is more complex, etc., which requires our country to further develop a simpler and more applicable immobilization method. There have been relevant reports abroad that naringinase has been used in the debittering and processing of citrus and other products, but naringinase has not been used in China. There is no large-scale application in production. The main reason is that there is no commercial naringinase prepared on a large scale in China. In the future, we should strengthen the method of breeding high-yield naringinase strains and prepare low-cost pure naringinase , has important practical significance
However, the naringinase activity produced by mold has the following disadvantages: it has been found that there are fewer types of naringinase-producing bacteria and lack of high-yielding strains; the fermentation efficiency of mold-producing naringinase is low and the production cost is high; there are not many domestically available for industrial production. commercial enzyme preparations; less research on the structure of naringinase

Method used

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  • A kind of aspergillus oryzae and application thereof
  • A kind of aspergillus oryzae and application thereof
  • A kind of aspergillus oryzae and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Aspergillus oryzae 11250 screening isolation and identification

[0056] 1. Aspergillus oryzae ( Aspergillus oryzae ) 11250 screening isolate

[0057] Weigh 2g of soil samples collected from all over the country and shake them for 30min in a 100mL Erlenmeyer flask filled with 18mL of sterilized normal saline. It is best to put a little glass beads in the Erlenmeyer flask. Then diluted with sterile water to a volume concentration of 1 × 10 -3 , 1×10 -4 For two gradient dilutions, pipette 100 μL dilutions with sterilized pipette tips and apply to the pre-prepared and sterilized selective plate medium (the composition of the selective plate medium is (g / L): MgSO 4 0.5, KH 2 PO 4 1.5, CaCl 2 0.1, (NH 4 ) 2 SO 4 1.5, KCl 0.5, KNO 3 1.5, yeast extract 2, orange peel powder 7.5, agar 20, water balance), cultivated at 30°C for 48-96h. Separate the single colony by streaking from the selective plate medium, and check the purity of the strain by microscopi...

Embodiment 2

[0083] Embodiment 2 The preparation of the liquid fermented liquid of Aspergillus oryzae 11250 containing naringinase

[0084] (1) Slant culture: Aspergillus oryzae 11250 was inoculated on the slant medium and cultured at 40°C for 48 hours to obtain the slant strain. The slant medium used is: MgSO 4 0.5, KH 2 PO 4 1.5, CaCl 2 0.1, (NH 4 ) 2 SO 4 1.5, KCl 0.5, KNO31.5, yeast extract 2, orange peel powder 15, agar 20, water balance, (g / L); pH 6.0, 1×10 5 Pa sterilized for 20 min.

[0085] (2) Liquid shake flask fermentation: Inoculate the obtained slant strain into the liquid shake flask medium with an inoculation amount of 0.2% (V / V), culture at 30°C, shaker speed 180r / min, and culture keep The time is 96h; the fermentation broth is obtained. Liquid shake flask medium is: maltose concentration 2, beef extract peptone 3, MgSO 4 0.5, KH 2 PO 4 1.5, CaCl 2 0.1, (NH 4 ) 2 SO 4 1.5, KCl 0.5, KNO 3 1.5, yeast extract 2, orange peel powder 7.5, water balance...

Embodiment 3

[0090] Embodiment 3 Aspergillus oryzae 11250 liquid fermentation produces naringinase

[0091] Centrifugal separation: centrifuge the fermentation broth, and take the supernatant; the centrifugation speed is 8000rpm, and the centrifugation time is 10min.

[0092] The supernatant ammonium sulfate is graded and precipitated: add ammonium sulfate to the supernatant (ammonium sulfate is an analytically pure ammonium sulfate reagent) to make the mass volume concentration 50%, filter to remove the resulting precipitate, and then continue to add ammonium sulfate to the filtrate , until its mass volume concentration is 50%-70%, and the precipitate containing naringinase is obtained by filtration. The mass volume concentration means: the mass (g) of ammonium sulfate contained in each mL supernatant.

[0093] Elution: After the naringinase-containing precipitate is eluted with 0~1mol / L NaCl (the optimal concentration is 0.6mol / L), the naringinase eluate is collected.

[0094] Q Sephar...

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Abstract

The invention relates to aspergillus oryzae and application thereof, belonging to the technical field of microbial fermentation. The Aspergillus oryzae of the present invention is named as Aspergillus oryzae 11250, and its preservation number is CGMCC NO.11320. The activity of naringinase in its liquid fermentation broth can reach 251 U / mL, the total enzyme activity of purified naringinase can reach 12052U, and the specific enzyme activity can reach 2198U / mg; its fermentation broth also contains naringin and naringin hydrolysis substrates Naringenin: Naringenin is a flavonoid substance, which has health effects such as lowering blood pressure, detoxification, diuresis, and antibacterial. In addition, the aspergillus oryzae of the present invention uses orange peel as a fermentation substrate, fully utilizes domestic waste, and saves costs. The naringinase produced by Aspergillus oryzae of the present invention has a molecular weight of 48 kDa, and the optimum action temperature and pH are respectively 45° C. and 5.0. In addition, the Aspergillus oryzae 11250 of the present invention has "high enzyme production level, and the produced naringinase is an extracellular enzyme, and it is not necessary to use methods such as ultrasonic wall breaking to release intracellular enzymes, which reduces the loss of enzyme activity, thereby greatly reducing industrial production. cost in application".

Description

technical field [0001] The invention relates to aspergillus oryzae and application thereof, belonging to the technical field of microbial fermentation. Background technique [0002] Naringinase is an important enzyme used in the hydrolysis of bitter substances in fruit juice. It is composed of α-L-rhamnosidase (α-L-rhamnosidase, EC 3.2.1.40) and β-D-glucosidase (β -D-glucosidase, EC3.2.1.21) is a glycoside hydrolysis complex enzyme system, which has the activities of both α-L-rhamnosidase and β-D-glucosidase, and naringinase can act on The bitter naringin is hydrolyzed to produce flavonoids. The process is: naringin first generates rhamnose and prunin under the action of α-L-rhamnosidase (Prunin, also translated as prunin) , the bitterness of Pruning is about one-third of that of naringin, so the bitterness is reduced, and then Pruning can be decomposed into non-bitter naringin flavonoids (mainly for naringin). The microbial sources of naringinase are mainly concentrated ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12N9/24C12N9/42C12R1/69
CPCC12N9/2402C12N9/2445C12Y302/01021C12Y302/0104C12N1/145C12R2001/69
Inventor 李秀婷朱运平郦金龙郗梦露
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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