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A kind of bacillus amyloliquefaciens and application thereof

A technology for dissolving amyloid spores and bacilli, applied in the directions of bacteria, hydrolase, enzymes, etc., can solve the reports that Bacillus amyloliquefaciens produce naringinase, the difficulty in cultivating high-yielding naringinase strains, the lack of large-scale naringinase, etc. problems, to achieve mild conditions, easy separation, and cost savings

Inactive Publication Date: 2017-04-05
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0005] 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 lies in how to efficiently select excellent mutants from a large number of mutagenized offspring. 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, compared with mold, the activity of naringinase produced by bacteria still has a certain gap; the reported level of enzyme production by bacterial fermentation does not exceed 1000 U units per milliliter of fermentation broth; the optimum temperature for bacterial fermentation to produce naringinase is moderate Strong environment may limit its application in acidic environment
[0007] The Bacillus amyloliquefaciens reported in the prior art (before the present invention) is used more in the biological control of plant pathogenic fungi, but there is no report on the production of naringinase by Bacillus amyloliquefaciens

Method used

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  • A kind of bacillus amyloliquefaciens and application thereof
  • A kind of bacillus amyloliquefaciens and application thereof
  • A kind of bacillus amyloliquefaciens and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Screening, isolation and identification of Bacillus amyloliquefaciens 11568

[0055] 1. Screening and isolation of Bacillus amyloliquefacines JNU002 (Bacillus amyloliquefacines) 11568

[0056] Weigh 2g of soil samples or moldy orange peel or / and pomelo peel collected from all over the country, and shake it in a 100mL conical flask filled with 18mL of sterilized normal saline for 30min. It is best to put a little glass beads in the conical flask . Then diluted with sterile water to a volume concentration of 1 × 10 -3 , 1×10 -4 For the two gradient dilutions, 100 μL of the dilutions were pipetted with a sterilized pipette tip and spread on the pre-prepared and sterilized selective plate medium, and incubated 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 examination of part of the single colonies picked (microscopic observation of the bacteria shows the same morpho...

Embodiment 2

[0076] Example 2 Preparation of liquid fermentation broth of Bacillus amyloliquefaciens 11568 containing naringinase

[0077] (1) Slant culture: Bacillus amyloliquefaciens 11568 was inoculated on the slant medium and cultured at 40°C for 48 hours to obtain the slant strain. The slant medium used (g / L) 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, KNO3 1.5, yeast extract 2, orange peel powder 15, agar 20, pH 6.0, 1×10 5 Pa sterilized for 20 min.

[0078] (2) Liquid shake flask fermentation: Inoculate the obtained slant strain into the liquid shake flask culture medium with an inoculum amount of 0.2% (v / V), culture at 40.9°C, shaker speed 180r / min, and culture for 48 hours; the obtained liquid fermentation broth. Liquid shake flask medium (g / L) is: MgSO 4 0.5, KH 2PO 4 1.5, CaCl 2 0.1, (NH 4 ) 2 SO 4 1.5, KCl 0.5, KNO 3 1.5. Yeast paste 2. Orange peel powder 7.5, pH 6.0, 1×10 5 Pa sterilized for 20 min.

[0079] (3) Determi...

Embodiment 3

[0081] Example 3 Preparation of liquid fermentation broth of Bacillus amyloliquefaciens 11568 containing naringinase

[0082] Inoculate the slant strain cultivated in step (1) of Example 2 into a 250ml Erlenmeyer flask containing liquid medium at an inoculum size of 0.2% (v / V), and carry out liquid shake flask fermentation at a fermentation temperature of 40.9°C. The bed speed was 180 r / min, and the fermentation time was 48 h; a liquid fermentation liquid was obtained. Liquid medium composition (g / L): naringin 10, tryptone 10, (NH 4 ) 2 HPO 4 20, NaCl10, yeast extract 5; pH 7.51, 1×10 5 Pa sterilized for 20 min.

[0083] (3) Determine the composition of the liquid fermentation broth by high performance liquid chromatography (water: methanol = 1:1), and the test results are as follows: figure 1 : Contains naringin and naringenin in liquid fermented liquid.

[0084] (4) The naringinase activity of the liquid fermentation broth prepared in this example was measured accord...

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Abstract

The invention relates to a bacillus amyloliquefaciens and application thereof, belonging to the technical field of microbial fermentation. The invention provides a new bacterial strain; a Bacillus amyloliquefaciens, its preservation number is CGMCCNo.9928, the preservation date is November 4, 2014, and the preservation unit is the General Microbiology Center (CGMCC ). The Bacillus amyloliquefaciens of the present invention whose preservation number is CGMCC No. 9928 can produce naringinase. The present invention isolates and cultivates the amylase bacillus capable of producing naringinase for the first time; the activity of naringinase in the liquid fermentation broth can be as high as 201.12 U / mL.

Description

technical field [0001] The invention relates to a bacillus amyloliquefaciens and application thereof, belonging to the technical field of microbial fermentation. [0002] technical background [0003] 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 that 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 (mainly naringin). The microbial sources of naringinase are mai...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/24C12R1/07
CPCC12N9/2402C12N9/2445C12Y302/01021C12Y302/0104C12N1/205C12R2001/07
Inventor 李秀婷朱运平伍少明郗梦露
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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