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A kind of lysine high-yield bacterial strain and application

A high-yield strain and lysine technology, applied in the direction of application, bacteria, additional food elements, etc., can solve the problems of reverse mutation and insufficient increase of lysine level, so as to inhibit growth and reproduction, enhance animal immunity, and improve the ability Effect

Active Publication Date: 2017-08-18
HUNAN SHENGSHI FENGHUA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the lysine production level of the obtained mutant strains is not improved enough by the method of ultraviolet mutagenesis, and it is easy to produce back mutations.

Method used

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  • A kind of lysine high-yield bacterial strain and application
  • A kind of lysine high-yield bacterial strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1 The preparation of bacterial strain Bacillus subtilis PL13-hom::Km of the present invention

[0020] Isolation of the original strain: Take chyme 10g from the pig intestine of the Experimental Animal Breeding Center of the Institute of Subtropical Agroecology, Chinese Academy of Sciences (Changsha, Hunan Province), add 10 times sterile water to vortex for 5 minutes, stand still for 5 minutes, and take 1ml for 10-fold dilution , at 10 6 、10 7 、10 8 For each dilution, take 100 microliters of the supernatant and coat the LB solid plate. For each dilution, carry out three repetitions, incubate at 37°C for 24 hours, and pick a single colony in a 10ml EP tube containing 3ml LB liquid medium. place in a shaker at 37°C for 24 hours, centrifuge at 12000rpm for 5 minutes, take the supernatant to analyze the effect of inhibiting E. The original strain of Bacillus subtilis, preserved with 25% glycerol.

[0021] Homoserine gene knockout of the original strain:

[0...

Embodiment 2

[0040] Embodiment 2 The preparation of bacterial strain Bacillus subtilis PL13-hom::Km fermentation broth of the present invention

[0041] The preparation of shake flask fermentation seed liquid: get bacterial strain Bacillus subtilis PL13-hom::Km 2ml of the present invention (concentration of living bacteria is 10 8 -10 10 CFU / ml), inoculated in 100ml LB liquid medium for shake flask fermentation culture, the fermentation temperature is 37°C, the pH value is 7.2, the rotation speed is 200r / min, and the fermentation time is 24h. Among them, the composition of LB liquid medium is: tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water 1000ml, pH value is 7.2.

[0042] Carry out fermentor pilot test after shake bottle fermentation finishes, get 100ml shake bottle fermentation seed liquid (the concentration of seed liquid is 10 8 CFU / ml) was inoculated into a 10L fermenter with a liquid volume of 5L, the fermentation temperature was 37°C, the pH value was 7.2, th...

Embodiment 3

[0043] Example 3 Analysis of lysine yield in bacterial strain Bacillus subtilis PL13-hom::Km fermentation broth of the present invention

[0044] Take 5ml of the fermented liquid obtained in Example 2, centrifuge at 12000r / min for 10min, take 1ml of supernatant, add 2.5ml of 7.5% (mass ratio) trichloroacetic acid per ml, mix well, centrifuge at 18000r / min at 4°C for 15min, draw 1ml of the supernatant was transferred to an amino acid analysis vial, and the contents of arginine and other amino acids were determined with a Hitachi L-8800 automatic amino acid analyzer. The results showed that the content of lysine in the fermentation broth of the bacterial strain Bacillus subtilis PL13-hom::Km of the present invention was 63g / L.

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Abstract

A lysine high-yielding bacterial strain, which is classified as Bacillus subtilis PL13‑hom::Km (Bacillus subtilis PL13‑hom::Km), which has been preserved in the China Center for Type Culture Collection on January 5, 2015 , the deposit number is CCTCC: M 2015002. The concentration of lysine in the bacterial strain Bacillus subtilis PL13-hom::Km fermentation liquid of the present invention is as high as 50-100g / L, and the fermentation liquid can inhibit the growth of Escherichia coli in vitro, and the fermentation liquid is added to animal feed or animal drinking water, It can enhance animal immunity, promote animal growth, and reduce the amount of lysine added in animal feed, thereby effectively reducing feed production costs.

Description

technical field [0001] The invention relates to the field of feed additives, more specifically to a high-lysine-producing Bacillus subtilis PL13-hom::Km (Bacillus subtilis PL13-hom::Km), and also relates to the application of the strain in the preparation of animal feed additives. Background technique [0002] Lysine can regulate the body's metabolic balance, increase calcium absorption and accumulation in the body, increase appetite, thereby promoting growth and development, reducing diseases and enhancing immune function. Therefore, in feed production, the proportion of lysine added in the complete feed is about 0.7%. However, after 30 years of development in my country's feed industry, the profit rate has been declining, and it has entered the era of meager profits. At present, the profit of full-price materials in my country is about 20 yuan / ton. Especially now that various natural disasters, inflation and other factors have caused the prices of bulk raw materials, suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/08A23K20/142C12R1/125
CPCA01N63/00C12P13/08C12N1/205C12R2001/125
Inventor 李丽立王升平张彬范觉鑫王来强蒋国礼刘志强
Owner HUNAN SHENGSHI FENGHUA BIOTECH CO LTD
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