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Method for Catalyzed Synthesis of Conjugated α-Linolenic Acid Isomers by Surfactant Coated Bacteria

A technology of surfactant and coating bacteria, which is applied in the field of biomedicine, can solve the problems of bacteria can not be reused, the amount of addition is limited, and the fermentation cycle is long, so as to improve permeability, reduce inhibition, and high catalytic activity Effect

Inactive Publication Date: 2018-04-17
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, microbial fermentation is carried out in aqueous solution. Since the added fermentation substrate - α-linolenic acid is a fat-soluble substance, α-linolenic acid needs to be emulsified before being added to the fermentation broth, and the amount of addition is limited. From the fermentation broth The product of conjugated α-linolenic acid needs to be extracted by organic solvent, and the whole production process has the disadvantages of long fermentation period, high production cost and low yield.
Invention patent CN200410060670.9 reports specific biosynthesis by Lactobacillus casei CGMCC 1.574 c 9, t 11, c 15-conjugated α-linolenic acid isomer method, the substrate addition amount is 1mg / mL, the fermentation time is as long as 42h, the conversion rate of the substrate is less than 50%, and the bacteria cannot be reused

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The activated Lactobacillus casei ( Lactobacillus casei ) CGMCC 1.574 strains were inoculated into MRS medium, cultured at 30°C for 20 hours, centrifuged at 5000g for 10 minutes to collect the bacteria, and 10 grams of wet bacteria were resuspended in 50 mL of Triton X-100 containing 1.5% by volume and 50 mM phosphoric acid In the permeabilization treatment solution of salt buffer solution (pH5.8), treat in a water bath at 37°C for 20 minutes, and centrifuge at 5000g for 10 minutes to obtain the permeabilized bacteria.

[0027]After the permeabilized bacteria were washed with 50mL of 60mM phosphate buffer (pH5.8), the bacteria were collected by centrifugation at 6000g for 10min, and the wet bacteria were resuspended in 200mL of 60mM phosphate buffer (pH5.8), and 40mL of Acetone solution of 1% glutamic acid dioleyl alcohol ester ribitol (Song Baodong et al., Petrochemical Industry, 2001, Volume 30 Supplement: 376-378), mix well, stir at 4°C for 4 hours, centrifuge at 60...

Embodiment 2

[0030] The activated Lactobacillus casei ( Lactobacillus casei ) CGMCC 1.574 strains were inoculated into MRS medium, cultured at 30°C for 18 hours, centrifuged at 4000g for 5 minutes to collect the bacteria, and 10 grams of wet bacteria were resuspended in 50 mL of Triton X-100 containing 3.0% by volume and 200 mM phosphoric acid In the permeabilization treatment solution of salt buffer solution (pH5.8), treat in a water bath at 37°C for 20 minutes, and centrifuge at 5000g for 10 minutes to obtain the permeabilized bacteria.

[0031] After the permeabilized bacteria were washed with 40mL of 60mM phosphate buffer (pH5.8), the bacteria were collected by centrifugation at 5000g for 10min, and the wet bacteria were resuspended in 200mL of 60mM phosphate buffer (pH5.8), and 40mL of The acetone solution of dioleyl glutamate ribitol with a mass percentage of 1% was mixed evenly, stirred at 4°C for 2 hours, centrifuged at 6000g for 5 minutes to collect the bacteria, and the bacteria...

Embodiment 3

[0034] The activated Lactobacillus casei ( Lactobacillus casei ) CGMCC 1.574 strains were inoculated into MRS medium, cultured at 30°C for 18 hours, centrifuged at 4000g for 5 minutes to collect the bacteria, and 10 grams of wet bacteria were resuspended in 50 mL of Triton X-100 containing 0.5% by volume and 20 mM phosphoric acid In the permeabilization treatment solution of salt buffer solution (pH5.8), treat in a water bath at 37°C for 20 minutes, and centrifuge at 5000g for 10 minutes to obtain the permeabilized bacteria.

[0035] After the permeabilized bacteria were washed with 50mL of 60mM phosphate buffer (pH5.8), the bacteria were collected by centrifugation at 4000g for 10min, and the wet bacteria were resuspended in 200mL of 60mM phosphate buffer (pH5.8), and 40mL of The acetone solution of dioleyl glutamate ribitol with a mass percentage of 1% was mixed evenly, stirred at 4°C for 6h, centrifuged at 5000g for 5min to collect the bacteria, and the bacteria were pre-f...

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Abstract

The method for catalyzing the synthesis of conjugated α-linolenic acid isomers by surfactant-coated bacteria comprises the following steps: (1) Resuspending each gram of Lactobacillus casei CGMCC 1.574 bacteria in 5 mL of permeabilization treatment solution, 37 ° C Treat for 20 minutes, and collect the permeabilized bacteria by centrifugation; (2) After washing with 60mM, pH5.8 phosphate buffer, centrifuge to collect the bacteria, and suspend each gram of wet bacteria in 20mL of 60mM, pH5.8 phosphate buffer Then, dropwise add acetone solution containing 1% dioleyl glutamic acid ester ribitol, mix well while adding, treat at 4-40°C for 2-6h, collect the cells by centrifugation, and obtain the coated cells after freeze-drying (3) Add each gram of freeze-dried coated bacteria into 500mL of n-hexane, stir evenly, add α-linolenic acid with a volume percentage of n-hexane of 0.1-0.3%, and react at 4-40°C for 1-12h; (4 ) Centrifuge the coated bacteria, recover n-hexane, and the products are c9, t11, c15-conjugated α-linolenic acid isomers. The invention has short reaction time, the coated bacterium can be reused for many times, the output is significantly improved, no environmental pollution, and the production cost is significantly reduced.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. Background technique [0002] Conjugated α-linolenic acid is a conjugated isomer of α-linolenic acid, which is a general term for a group of octadecyl-conjugated trienoic acids. There are various positional isomers and geometric isomers, such as: c 9, t 11, c 15-conjugated alpha-linolenic acid and t 10, c 12, c 15-conjugated α-linolenic acid isomers, etc. Studies have shown that conjugated α-linolenic acid has physiological functions such as anti-cancer, prevention of atherosclerosis, and weight loss, and its physiological activities are isomer-specific, such as: c 9, t 11, c 15-conjugated α-linolenic acid isomers have anticancer effects. In nature, conjugated α-linolenic acid mainly exists in the milk and meat fat of ruminants, mainly composed of c 9, t 11, c 15-conjugated α-linolenic acid and other isomers, but its content is usually very low, which cannot meet the development a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/64C12R1/245
Inventor 刘晓华李加肖李海星
Owner NANCHANG UNIV