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Method of producing lipopolysaccharide having antibacterial activity by means of Serratia marcescens NS-17 bacterial strain

A technology of Serratia marcescens and antibacterial activity, applied in the field of microbial preparations, can solve the problems of high cost, low yield, complicated production process, etc., and achieve the effect of low cost, good effect and simplified extraction process

Active Publication Date: 2017-05-31
XIAMEN HUAXIA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, studies have shown that most of the clinically isolated strains do not produce pigment, and most of the pigment-producing strains are isolated from the environment, indicating that the pathogenicity of Serratia marcescens has a certain relationship with whether it produces pigment. Serratia producing prodigal polysaccharides has high biological safety and less threat of environmental pollution, but there are disadvantages such as complicated production process, low yield and high cost

Method used

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  • Method of producing lipopolysaccharide having antibacterial activity by means of Serratia marcescens NS-17 bacterial strain
  • Method of producing lipopolysaccharide having antibacterial activity by means of Serratia marcescens NS-17 bacterial strain

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1: 5L fermenter produces prodigy polysaccharide.

[0023] The slant strains stored at 4°C were inoculated on the slant of nutrient agar, cultured at 37°C for 24 hours and then quickly inoculated into the liquid seed medium. Before inoculation, the seed culture was kept at a 40°C incubator. Use a 250mL Erlenmeyer flask to fill 50mL. After inoculation, culture at 37°C and 200rpm for 16h. The qualified seed solution was inoculated into a 5L fermenter. Before sterilization, the liquid volume in the fermentation tank was 3.2L, sterilized by steam at 121°C for 20 minutes, cooled to 37°C and inoculated with 100mL seed liquid, cultivated at 37°C, and the initial ventilation volume was 100L / h, the initial stirring speed is 200rpm, and the ventilation volume and stirring speed are increased alternately with the decrease of dissolved oxygen, the maximum ventilation volume is 210L / h, and the maximum stirring speed is 700rpm. Control the pH of the fermentation broth ...

Embodiment 2

[0026] Embodiment 2: 50L fermentation tank produces prodigal polysaccharide.

[0027] The slant strains preserved at low temperature were inoculated on the slant of nutrient agar, cultured at 37°C for 24 hours and then quickly inoculated into the liquid seed medium. Before inoculation, the seed culture was kept at 40°C in an incubator. Use a 1L Erlenmeyer flask to fill 200mL of liquid. After inoculation, culture at 37°C and 200rpm for 16h. The qualified seed solution was inoculated into a 50L fermenter. Before sterilization, the liquid volume in the fermentation tank was 33L, steam sterilized at 121°C for 20 minutes, cooled to 37°C and inoculated with 1.6L seed liquid, cultivated at 37°C, and the initial ventilation volume was 18L / min, the initial stirring speed is 100rpm, and the ventilation volume and stirring speed are increased alternately with the decrease of dissolved oxygen, the maximum ventilation volume is 35L / min, and the maximum stirring speed is 300rpm. Contro...

Embodiment 3

[0030] Embodiment three: antibacterial experiment.

[0031] Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Aspergillus niger, Saccharomyces cerevisiae and Candida albicans were selected as the test bacteria. Serratia marcescens Minimum Inhibitory Concentration (MIC) of the prodigy polysaccharides produced by NS-17 against different strains, among which Escherichia coli, Staphylococcus aureus and Bacillus subtilis use commercially available MH broth medium, pH 7.2-7.4; Aspergillus niger , Saccharomyces cerevisiae and Candida albicans use commercially available liquid Sabouraud medium. After the slant surface of the tested bacteria was activated, the bacterial lawn was scraped and diluted with two culture media to about 1×10 6 CFU / mL, where Aspergillus niger was diluted with spores.

[0032] Take the prodigal polysaccharides obtained in Example 2, prepare 3200 μg / mL solutions with the above two mediums, and dilute to 640, 320, 160, 80, 40, 20, 10 μg / mL, and 1 ...

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Abstract

The invention discloses a method of producing lipopolysaccharide having antibacterial activity by means of a Serratia marcescens NS-17 bacterial strain. The method includes the steps of activation of the bacterial strain, seed culture, fermenting culture, cell collection, cell wall disruption, and extraction of the lipopolysaccharide. The Serratia marcescens NS-17, which is screened and obtained from soil, can generate a pigment prodigiosin under culture at 30 DEG C, but not generates the prodigiosin under culture at 37 DEG C. In the method, culture temperature is strictly controlled and a simple culture medium is employed; after culture for 32 h, thalluses are collected through centrifugation; polysaccharides are extracted through a phenol-water method after repeated freezing-thawing cell wall disruption; alcohol precipitation is carried out and then the precipitate is washed with ethanol and acetone in gradient concentrations; and vacuum freeze drying is carried out to prepare the lipopolysaccharide having significant antibacterial activity. The prodigiosin polysaccharide has significant inhibition effect on various microorganisms, especially staphylococcus aureus.

Description

technical field [0001] The invention relates to the technical field of microbial preparations, in particular to a production process for producing lipopolysaccharide with antibacterial activity by using a conditionally pigmented Serratia marcescens NS-17. Background technique [0002] Serratia marcescens ( Serratia marcescens ) are Gram-negative bacteria that can be used to produce two important bioactive substances: prodigiosin and prodigiosan. Prodigiosin is a new type of anti-tumor drug that is in the clinical trial stage; and prodigiosan is a lipopolysaccharide that has biological activities such as increasing white blood cells, improving the body's specific immunity, suppressing tumors, and anti-infection. It is used for the adjuvant treatment of diseases such as leukopenia, hepatitis B and acute and chronic pelvic inflammatory disease caused by various reasons. [0003] At present, the Prodigobacterium polysaccharide products on the market are all injections for inje...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/04C12R1/43
Inventor 傅奇肖玉娟庄峙厦黄华斌郝春丽
Owner XIAMEN HUAXIA UNIV
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