Aspergillus oryzae and application thereof

A technology of Aspergillus oryzae and seeds, applied in the direction of enzymes, biochemical equipment and methods, microorganisms, etc., can solve the problems of lack of high-yield strains, poor stability, high production costs, etc., and achieve good application prospects, stable enzyme activity, and cost-saving effects

Active Publication Date: 2016-06-22
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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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 com

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  • Aspergillus oryzae and application thereof
  • Aspergillus oryzae and application thereof
  • 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. Screening and isolation of Aspergillus oryzae 11250

[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, (NH4) 2 SO 4 1.5, KCl0.5, 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 microscopic inspection of some o...

Embodiment 2

[0082] The preparation of the liquid fermented liquid of embodiment 2 containing naringinase, Aspergillus oryzae 11250

[0083] (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, KCl0.5, KNO31.5, yeast extract 2, orange peel powder 15, agar 20, water balance, (g / L); pH6.0, 1×10 5 Pa sterilization for 20min.

[0084] (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: 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, KCl0.5, KNO 3 1.5, yeast extract 2, orange peel powder 7.5, water balance, (g / L); pH5...

Embodiment 3

[0089] Embodiment 3 Aspergillus oryzae 11250 liquid fermentation produces naringinase

[0090] Centrifugal separation: centrifuge the fermentation broth, and take the supernatant; the centrifugation speed is 8000rpm, and the centrifugation time is 10min.

[0091] 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.

[0092] 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.

[0093] QSepharo...

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Abstract

The invention relates to aspergillus oryzae and application thereof and belongs to the technical field of microbial fermentation. The aspergillus oryzae is named aspergillus oryzae 11250, and the preservation number is CGMCC NO.11320. The activity of naringinase in liquid fermentation broth of the aspergillus oryzae reaches 251 U/mL, the activity of total naringinase after purification can reach 12,052 U, and the specific enzymatic activity reaches 2,198 U/mg; the fermentation broth of the aspergillus oryzae also contains naringin and naringenin which is a naringin hydrolysis substrate; naringenin is a flavonoid substance and has the healthcare effects of reducing blood pressure, removing toxicity, promoting diuresis, resisting bacteria and the like. Besides, orange peel serves as a fermentation substrate of the aspergillus oryzae, household refuse waste is fully utilized, and cost is reduced. The molecular weight of naringinase produced by the aspergillus oryzae is 48 kDa, the optimum action temperature is 45 DEG C, and the optimum pH is 5.0. Furthermore, the aspergillus oryzae 11250 has the advantages that the enzyme output is high, produced naringinase is an extracellular enzyme, there is no need to adopt an ultrasonic wall-breaking mode and the like to release an intracellular enzyme, enzyme activity losses are reduced, and therefore cost in industrial application is greatly reduced.

Description

technical field [0001] The invention relates to aspergillus oryzae and application thereof, belonging to the technical field of microbial fermentation. technical background [0002] Naringinase is an important enzyme for the hydrolysis of bitter substances in fruit juice. It is composed of α-L-rhamnosidase (α-L-rhamnosidase, EC3.2.1.40) and β-D-glucosidase ( β-D-glucosidase, EC3.2.1.21) is a glycoside hydrolysis compound enzyme system, which has the activities of both α-L-rhamnosidase and β-D-glucosidase, and naringinase can It acts on bitter naringin and hydrolyzes it to produce flavonoids. The process is: naringin first generates rhamnose and prunin (Prunin, also translated as prunin) under the action of α-L-rhamnosidase ), 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 under the action of β-D-glucosidase ( mainly naringin). The microbial sources of na...

Claims

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Application Information

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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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