Bacillus subtilis with phosphate-solubilizing effect and application thereof

A technology of Bacillus subtilis and phosphorus solubilization, which is applied in the direction of bacteria, hydrolytic enzymes, and microorganism-based methods to achieve the effects of improving quality, improving soil microenvironment, and reducing phosphorus pollution

Active Publication Date: 2011-05-18
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
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Problems solved by technology

On the other hand, in the aquaculture industry, 40-70% of the phosphorus in fish feed also exists in the form of phytic acid. Because the fish digestive tract lacks enzymes to hydrolyze phytate phosphorus, most of the phosphorus is excreted with feces. Make full use of the phosphorus source in the feed, and cause serious water phosphorus pollution

Method used

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  • Bacillus subtilis with phosphate-solubilizing effect and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1. Isolation and identification of Bacillus subtilis XF-8, CCTCC NO: M2010182

[0027] The experimental materials come from the field soil of Jining City, Shandong Province.

[0028] The specific implementation steps are as follows: under aseptic operation, weigh 1.0g soil sample and dissolve it in 9ml sterile physiological saline. on the screening plate. Place it upside down in an incubator at 28-30°C for 2-3 days, use an inoculation needle to pick up the colony with a transparent circle, and further separate and purify it. The strains were then placed in the enzyme-producing medium for re-screening to determine the strains producing phytase. After Gram staining, 16S rDNA gene sequence analysis and BIOLOG identification, it was confirmed that the strain was Bacillus subtilis, named as Bacillus subtilis (Bacillus subtilis) XF-8, and the deposit number was: CCTCC NO: M 2010182.

Embodiment 2

[0029] Example 2, Bacillus subtilis (Bacillus subtilis) XF-8, CCTCC NO: M 2010182 enzyme production culture experiment 1

[0030] The enzyme-producing medium was used, and the specific steps were as follows: the strains were inoculated into LB medium, shaken and cultured overnight at 30°C, inoculated in the enzyme-producing medium with 1.0% inoculum, and cultured at 25°C, pH 5.5, 160rpm shaking for 2 days. 4°C, 5000rpm, centrifuge for 15min, take the supernatant to obtain the neutral phytase crude enzyme; after diluting 10 times, measure the enzyme activity of the fermentation broth.

[0031] Drawing of Phosphorus Standard Curve

[0032] Use solutions 4 and 5 to make a phosphorus standard curve. The specific steps are as follows: add 1ml of substrate solution to 250μl of phosphorus standard solutions of different concentrations, and add 1.25ml of stop solution after 30min in water bath at 37℃; Add 2.5ml of chromogenic solution to 1ml of substrate solution in both the experim...

Embodiment 3

[0042] Example 3, Bacillus subtilis (Bacillus subtilis) XF-8, CCTCC NO: M2010182 enzyme production culture experiment 1

[0043] The enzyme-producing medium was used, and the specific steps were as follows: the strains were inoculated into LB medium, shaken at 30°C overnight, inoculated in the enzyme-producing medium with 2.5% inoculum, 30°C, pH 6.5, 160rpm, shaken for 3 days, 4 Centrifuge at 5000rpm for 15min, take the supernatant to obtain the neutral phytase crude enzyme; after diluting 10 times, measure the enzyme activity of the fermentation broth.

[0044] The drawing of phosphorus standard curve and neutral phytase enzyme activity determination method are the same as embodiment 2.

[0045] The neutral phytase activity of Bacillus subtilis XF-8 calculated according to the equation obtained from the standard curve and the definition of enzyme activity was 0.51 U / ml.

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Abstract

The invention discloses bacillus subtilis with phosphate-solubilizing effect and application thereof, and relates to bacillus subtilis XF-8 (CCTCC No: M2010182) and a method for producing neutral phytase from the bacillus subtilis XF-8. The application comprises the following steps: inoculating a strain in an LB culture medium, and carrying out shake culture overnight at the temperature of 30 DEG C; inoculating the strain in an enzyme-producing culture medium with 1-5% of inoculation amount, and carrying out shake culture at the temperature of 25-37 DEG C for 2-4 days, wherein, the pH value is 5.5-7.5; centrifuging at the speed of 5000rpm at the temperature of 4 DEG for 15 minutes, and taking supernatant to obtain crude neutral phytase; and diluting the crude neutral phytase by 10 folds, and then measuring enzyme activity of fermentation liquor, wherein, the carbon source of the enzyme-producing culture medium is wheat bran, and the nitrogen source of the enzyme-producing culture medium comprises peptone from casein and (NH4)2SO4. The neutral phytase produced by the bacillus subtilis has high enzyme activity (pH=7.0), so the neutral phytase can be applied to development of fish feed for lowering phosphorus pollution in water, and can be also applied to development of biological bacterial fertilizer for effectively utilizing organic phosphorus in soil, reducing application amount of phosphate fertilizer, improving soil microenvironment and enhancing the quality of agricultural products.

Description

technical field [0001] The invention relates to a new bacterial strain of bacillus subtilis and its application, in particular to a strain of bacillus subtilis (Bacillus subtilis) XF-8 with phosphorus-solubilizing effect and a method for producing neutral phytase by using the bacteria. Background technique [0002] Phosphorus is an essential element for the growth of animals and plants, and plays an important role in the process of life activities. Most of the phosphorus in the soil exists in the form of organic phosphorus such as phytic acid, nucleic acid, and phospholipids, which cannot be directly absorbed and utilized by plants, and the underground storage of phosphate rock as a raw material source for phosphate fertilizers is very limited, which will make phosphorus a constraint in modern times. An important factor for sustainable agricultural development. On the other hand, in the aquaculture industry, 40-70% of the phosphorus in fish feed also exists in the form of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N9/16C12R1/125
CPCC12N1/20C12N9/16
Inventor 宋存江李保宾王淑芳李强耿伟涛曹名锋孙秀梅苏文萍张伟
Owner NANKAI UNIV
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