Bacillus amyloliquefaciens separated from fermented soya beans and used for producing protease

A technology for decomposing starch spores and producing protease, which is applied in the field of bioengineering, and can solve the problems of uneven fermentation ability of wild strains, unstable product quality of fermented soy beans, product transportation and storage restrictions, etc., and achieve high production safety and high enzyme activity. High, easy separation and purification effect

Inactive Publication Date: 2015-06-10
JIANGXI NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in my country, due to the traditional natural fermentation method used in the industrial fermentation of bacterial fermented soy beans, pure fermentation has not been realized. The microorganisms in the traditional natural fermentation process mainly come from the microorganisms in the air and on the utensils, resulting in unstable quality of fermented soy bean products , the transportation and storage of products are also limited and it is difficult to achieve large-scale production. Usually, wild strains in the natural environment have uneven fermentation ability and low safety; There are likely to be some harmful bacteria in the uncontrolled fermentation system, how to avoid harmful bacteria contamination is a crucial public health issue

Method used

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  • Bacillus amyloliquefaciens separated from fermented soya beans and used for producing protease
  • Bacillus amyloliquefaciens separated from fermented soya beans and used for producing protease
  • Bacillus amyloliquefaciens separated from fermented soya beans and used for producing protease

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Embodiment 1

[0029] Embodiment 1: Preparation or screening of Bacillus amyloliquefaciens strain of the present invention

[0030] Step 1. Sample collection: collect fermented soybeans during the koji making process in the fermented soybean production factory, then seal them in sample bags and store them at low temperature.

[0031]Step 2. Enrichment culture: Take 1g of the sample and put it into a 10mL test tube, shake it with a vortex shaker for 5min, draw 1mL of the bacterial solution and place it in a 250mL Erlenmeyer flask with 50mL broth medium, and then place it in a 37±0.1 ℃, 72h in a shaker at 170r / min.

[0032] Step 3. Primary screening of strains: using the plate dilution method, take 200 μL of the enriched and cultured bacterial solution and spread it on the casein screening medium, cultivate it at 37±0.1°C for 48 hours, and pick out the strains that can produce transparent circles, among which , the casein screening medium of protease-producing Bacillus amyloliquefaciens is: c...

Embodiment 2

[0035] Embodiment 2: Microscopic morphology and molecular biological identification of Bacillus amyloliquefaciens strain of the present invention

[0036] The strain with the highest transparent circle was taken for microscopic morphology and molecular biology identification, and the specific process was as follows:

[0037] (1) Adopt solid plate culture method: solid culture is broth medium, cultivate at 37±1°C for 24 hours, and the colony diameter is 8-12mm. White opaque colonies, rough surface, irregular colony edges, no pigmentation on various media.

[0038] The microscopic morphological characteristics of Bacillus amyloliquefaciens are as follows: the bacterium is short rod-shaped, uniformly stained, motile, facultatively anaerobic, can form endophytic spores, the cysts are enlarged and oval, and the surface of free spores is weakly colored. Gram stain positive. Colony morphology of Bacillus amyloliquefaciens figure 1 , the micrographs of the spore morphology observe...

Embodiment 3

[0041] Embodiment 3: The Bacillus amyloliquefaciens strain 16S rDNA sequence analysis implementation process for protease production according to the present invention is as follows:

[0042] Primers were designed according to the most conserved sequence in bacterial 16S rDNA, in which the forward primer was F: 5'-GAGAGTTTGATCCTGGCTCAG-3', and the reverse primer was R: 5'-AAGGAGGTGATCCAGCCGCA-3'. PCR reaction system 20μL. Thermal cycle parameters Pre-denaturation at 94°C for 5min, denaturation at 94°C for 30s, annealing at 55°C for 40s, extension at 72°C for 1.5min, 35 cycles, and extension at 72°C for 7min. The PCR product was detected by agarose gel electrophoresis, and the amplified product was a specific band with a size of about 1.4kb. The amplified sequence was sequenced, and the 16S rDNA nucleotide sequence of the strain Jxnuwy-1 was 1457bp in size. The sequence was compared with the 16S rDNA nucleotide sequences of 10 strains in the GenBank database, and the strain wa...

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Abstract

The invention discloses bacillus amyloliquefaciens separated from fermented soya beans and used for producing a protease and belongs to the technical field of biological engineering. The bacillus amyloliquefaciens is separated from traditional fermented soya beans by adopting a separation and purification technique. The bacillus amyloliquefaciens which is identified and named according to the microbial taxonomy is collected in the China Center for Type Culture Collection (CCTCC) on December 10. 2014 with the collection number of CCTCC M 2014639. The bacillus amyloliquefaciens used for producing protease is easy to culture and is highly safe during production, the screened strain for liquid fermenting and culturing has the enzyme activity of 148-2,231U/mL, has good genetic stability; the metabolite is easy to separate and purify; a foundation is laid for the scientific pure culture in the traditional fermented soya bean industry.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a strain of Bacillus amyloliquefaciens isolated and purified from traditional fermented soya bean, which is of great significance for the scientific and pure cultivation of the traditional fermented soya bean industry. Background technique [0002] Tempeh has a history of more than 2,000 years. It has high nutritional value and the effects of dispelling cold and strengthening food. In the past, Western scholars believed that Bacillus subtilis was a very harmful spoilage bacteria. The ancestors of our country were able to turn decay into magic, and they were first used in food brewing. Since bacterial tempeh is mostly made and eaten by families, there are few records in ancient documents, and few modern scholars have dabbled in it. On the contrary, there are more researches abroad. According to modern research, Bacillus subtilis can form a large amount of amylase and prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/54C12R1/07
CPCC12N9/54C12N1/205C12R2001/07
Inventor 王筱兰李保根涂宗财杨林杨慧林
Owner JIANGXI NORMAL UNIV
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