A kind of aspergillus oryzae and application thereof

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 ...

Examples

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...

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 ...

Examples

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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