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Halobacillus and method for industrially producing Ectoin by using halobacillus

A technology of bacillus and ectoine, applied in the field of microorganisms, can solve the problems of limited scope of use, high cost and high price, and achieve the effect of reducing corrosion and environmental pollution and reducing salt concentration

Active Publication Date: 2021-02-02
SHANDONG FREDA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Based on its special physical and chemical characteristics, most of the high-yield ectoine-producing strains are screened from extreme environments such as high salt. Moreover, the yield of ectoine prepared by wild fungus reported at home and abroad is low, the extraction process is complicated in the later stage, the required equipment and operation steps are strict, and the cost is high, which leads to the high price of ectoine sold in the market, which limits its use. The scope of use also makes consumers discouraged from it, and has to find alternatives with good effects and low prices.

Method used

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  • Halobacillus and method for industrially producing Ectoin by using halobacillus
  • Halobacillus and method for industrially producing Ectoin by using halobacillus
  • Halobacillus and method for industrially producing Ectoin by using halobacillus

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1: the acquisition of bacterial strain

[0024] Dilute the sea mud samples collected in the coastal waters of Rizhao in sterile water, spread the diluted solution on medium plates containing different NaCl concentrations, and incubate at 28°C. After the colonies grow out, select a single colony and continue to separate and purify until The pure bacteria were obtained, the strains with fast growth rate and wide range of salt concentration were first screened, and the HPLC method was used to determine the yield of ectoine in the second screening, and the strain with ectoine yield of 2.4g / L was obtained through laboratory screening.

[0025] The strain was bred through chemical and physical compound mutagenesis, and was screened in a large number in the laboratory to obtain a mutant strain FL-2423 with high yield of ectoine, which has the ability of industrial production of ectoine and stable genetic characteristics. Growth curve such as figure 1 As shown, the...

Embodiment 2

[0026] Embodiment 2: Amplification, sequencing and sequence comparison of 16SrDNA of bacterial strain

[0027] Collect fresh bacteria, use the Ezup column bacterial genome DNA extraction kit to extract the total DNA template, and use universal primers to amplify the 16S rDNA gene. After the PCR product is tested and purified, it is directly sequenced. Technology Co., Ltd.

[0028] The primer sequence is as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0029] 1495R: 5'-GGTTACCTTGTTACGACTT-3';

[0030] The 10 μL PCR reaction system is as follows: template 1 μL; upstream primer, downstream primer 1 μL; Dream Taq GreenPCR Master Mix 5 μL; ddH 2 O 2 μL.

[0031] PCR conditions: 95°C for 5min, 94°C for 30s, 52°C for 2min, 72°C for 1min; cycle 30 times.

[0032] The measured full sequence results are as follows: the 16S rDNA sequence of the strain is shown in the sequence table SEQ No.1, and the colony characteristics of the mutant strain are as follows: figure 2 shown. And the o...

Embodiment 3

[0033] Embodiment 3: the culture method of halobacillus sp. FL-2423

[0034] 1) Inoculate the seed medium with Halobacillus sp. FL-2423 and culture it at 28°C for 24 hours to obtain the seed solution.

[0035] Seed medium components: monosodium glutamate 25g / L, dipotassium hydrogen phosphate 5g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 0.4g / L, manganese sulfate 0.01g / L, yeast powder 1g / L, sodium chloride 30g / L, pH 7.0.

[0036] 2) The above seed solution was inoculated in a shake flask with an inoculation amount of 5%, and cultured at 28° C. for 72 hours, and the yield of ectoine in the fermentation broth was 12.5 g / L.

[0037] Fermentation medium components: sodium glutamate 40g / L, dipotassium hydrogen phosphate 8g / L, potassium dihydrogen phosphate 4g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.05g / L, yeast powder 10g / L, Sodium chloride 60g / L, ammonium sulfate 10g / L, pH 7.0.

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Abstract

The invention discloses a halobacillus and a method for industrially producing Ectoin by using the halobacillus. According to the invention, the method comprises the following steps: firstly, separating a bacterial strain with the Ectoin-secretion characteristic from coastal mud of Rizhao, and then carrying out mutation screening to obtain the Halobacillu sp. FL-2423 with high Ectoin yield and stable hereditary characteristics. The preservation number of the bacterial strain is CGMCC No.18783. The above bacterial strain is moderate halophilic bacteria and is subjected to fermentation culture in a culture medium with a NaCl concentration of 50-100g / L, Ectoin is synthesized in cells, and the fermentation yield reaches 10-16g / L.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a halo-loving bacillus and a method for industrialized production of ectoine. Background technique [0002] Ecdoine, the chemical name is 1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid, also known as tetrahydropyrimidine, is a cyclic amino acid derivative, which is the response of certain microorganisms An osmotic pressure compensating solute synthesized by environmental osmotic stress. Ectoin is widely used, not only for moisturizing, water-locking and anti-wrinkle in cosmetics, but also for stabilizing the activity of cells and macromolecules, such as enzymes and nucleic acid stabilizers. At the same time, ectoine has broad application prospects and development value in medicine. It can not only be used as a protective agent for healthy cells during chemotherapy, but also have a certain preventive effect on Alzheimer's disease and Parkinson's disease. [0003...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P17/12C12R1/01
CPCC12N1/20C12P17/12C12R2001/01C12N1/205
Inventor 李珍爱李海军苏移山张英华马双双王兆兰王庆波周济源刘芳李松
Owner SHANDONG FREDA BIOTECH
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