Serratia marcescen capable of producing red pigment through fermentation and method for producing red pigment by fermentation

A technology of Serratia marcescens and Serratia marcescens, which is applied in the field of fermentation engineering and can solve the problems that the fermentation amplification experiment cannot be realized.

Inactive Publication Date: 2012-02-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The prodigiosin-producing bacteria are mostly isolated from seawater or soil, and the research on the prodigiosin-producing system of endophytic fungi is yet to be carried out; the yield of prodigiosin is not very satisfactory, and it is screened from natural resources. The pathway of microorganisms with high prodigiosin production is very promising; the production of prodigiosin is related to the production of other phenotypes (such as caseinase activity, hemagglutinin activity), and these phenomena can be explained from a genetic perspective Further research is needed; at present, the shake bottle production of prodigiosin has a considerable output, but the fermentation scale-up experiment has not been realized, and there is a distance from industrialized large-scale production; prodigiosin is a natural pigment and can be used in large quantities in the food industry and medical services etc. The current research mainly focuses on the effect of prodigiosin in inducing tumor cell apoptosis. At present, the pharmacological efficacy, pharmacological mechanism, and toxic and side effects of prodigiosin have not yet been resolved, and these aspects need further in-depth research.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: Pigment related assay method and extraction and purification of strain red pigment

[0012] Determination of the relative content of the pigment: take 1 mL of the fermentation broth and centrifuge at 12000 rpm for 10 min, wash the bacteria twice with distilled water, add 5 mL of methanol, and repeatedly shake and extract twice, combine the two extracts, let stand for 10 min, and then centrifuge at 12000 rpm for 10 min The supernatant was diluted 10 times, and its absorbance value at 535 nm was measured with a UV spectrophotometer, and the blank was methanol.

[0013] Pigment extraction and purification: extract the pigment with acidic methanol (pH 3.0), extract with chloroform-water system, and obtain the pigment concentrate by rotary evaporation of the chloroform phase. The concentrate was separated and purified by silica gel column chromatography to obtain pigment components. The diameter-to-length ratio of the chromatographic column is 1:40, the stationar...

Embodiment 2

[0014] Embodiment two: the identification of the structure of pigment

[0015] For pigment analysis and UV-Vis analysis, the purified pigment was dissolved in methanol, the pH value was adjusted to 3.0 / 9.0 (adjusted by HCl / NaOH), and the absorbance of each component at the wavelength range of 300-700nm was traced at these two pH values. Under acidic conditions, there is a maximum absorption peak at 534nm, and under alkaline conditions, the maximum absorption peak moves to 465nm, which is consistent with the reported absorption spectrum of prodigiosin Figure 1 Sincerely.

[0016] LC / MS analysis, instrument: Waters ZMD 4000 liquid-mass spectrometer; Chromatographic column: LichrospherC 18column (4.6mm×250mm), mobile phase: 80% methanol in water, flow rate: 1mL / min, column temperature: 30°C; MS Conditions: ESI(+) ionization mode, ESI-MS cone voltage 60V, capillary voltage 3.88KV, ion source temperature 120°C, desolvation temperature 300°C. The pure pigment is analyzed by time-o...

Embodiment 3

[0017] Embodiment three: the preparation method of prodigiosin

[0018] The present invention also provides a method for producing prodigiosin, which is characterized in that Serratia marcescens is used as a production strain to produce prodigiosin through fermentation and culture, and the present invention specifically provides Serratia marcescens producing prodigiosin The optimum fermentation conditions of red pigment are as follows:

[0019] Seed medium (g / L): peptone 3, beef extract 3, sodium chloride 1, magnesium sulfate 0.5, pH7.2~7.5; fermentation medium (g / L): glycerin 4, peptone 16, sodium chloride 3 , Potassium chloride 4, pH7.2~7.5.

[0020] Strains were fermented, using liquid fermentation medium, 500mL Erlenmeyer flask, containing 50mL of culture solution, sterilized at 115°C for 15min, and shaken at 30°C at 200r / min.

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Abstract

The invention discloses Serratia marcescen capable of producing red pigment through fermentation and a method for producing red pigment by fermentation, belonging to the field of fermentation engineering. The method comprises the following steps: carrying out lixiviating, extraction and rotary evaporation so as to obtain a pigment concentrated liquid; separating and purifying through silicagel column chromatography to obtain a pigment component; carrying out UV-Vis (ultraviolet-visible) analysis so as to determine that the maximum absorption peaks are respectively located at the wavelengths of 530nm and 465nm under the condition that the pH value is 3.0 and 9.0; and measuring the accurate relative molecular mass of the pigment component through LC/MS (liquid chromatography-mass spectrometry) analysis so as to determine that the pigment is prodigiosin. In the method provided by the invention, an LB (load balance) liquid cultural medium is adopted for fermentation; and 50 mL of culture solution is contained in a 500mL triangular flask; sterilization is carried out for 15 minutes at the temperature of 115 DEG C; shaking culture is carried out at the temperature of 30 DEG C at the speed of 200r/min, and then centrifuging operation is carried out for 10 minutes at the speed of 8000r/min to remove impurities; and through extraction with acidic methanol, the prodigiosin Serratia marcescen strain pigment is produced and the yield is 150mg/L. Prodigiosin has important application prospect in the fields of tumor treatment, foods, cosmetics and the like.

Description

technical field [0001] The invention relates to a fermented Serratia neomarcescens strain producing red pigment, which belongs to the technical field of fermentation engineering. The invention also relates to the identification of the red pigment produced by the above method in a fermentation method, which belongs to the field of fermentation engineering. Background technique [0002] Prodigiosin (PG) is a general term for a family of natural red pigments. It has a methoxypyrrole skeleton structure composed of three pyrrole rings. It is often dark red and has a melting point of 151-152°C. It is insoluble in water and belongs to Fat-soluble pigment, easily soluble in methanol and benzene. In methanol solution, there is a characteristic absorption at 540nm under acidic conditions, and a characteristic absorption at 466nm under alkaline conditions. The pigment is stable to temperature and is greatly affected by pH. The molecular formula is C 20 h 25 N 3 O. Prodigiosin is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P17/16C07D207/36C09B23/04G01N27/62C12R1/425
Inventor 刘立明张传亮王鸿江牛盼清
Owner JIANGNAN UNIV
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