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Non-aqueous enzymatic synthesis of conjugated γ-linolenic acid isomers

A synthesis method and technology of linolenic acid, applied in the field of biomedicine, can solve the problems of long fermentation period, high production cost, and limited addition amount, and achieve the effects of reducing inhibition, improving permeability, and improving thermal stability

Active Publication Date: 2018-05-22
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 fermentation substrate - γ-linolenic acid is a fat-soluble substance, γ-linolenic acid needs to be emulsified before being added to the fermentation broth, and the amount of addition is limited. The product of conjugated γ-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

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 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 cells, and 40 mL of 50 mM phosphate buffer (pH 6.8), 5 mg / mL lysozyme was added to 10 grams of wet cells and 2mM sodium ethylenediaminetetraacetate, shake in a water bath at 37°C for 20 minutes, take it out, and put it in an ice bath, and ultrasonicate it with a power of 200W, with an interval of 5s for 30s, sonicate twice, and centrifuge at 5000g for 10min to obtain permeabilized bacteria.

[0026] After the permeabilized bacteria were washed with 40mL of 50mM phosphate buffer (pH5.8), the bacteria were collected by centrifugation at 6000g for 10min, and the wet bacteria were resuspended in 200mL of 50mM phosphate buffer (pH5.8), and 2mL of Tween-80 was added and mixed evenly, stirred at 4°C for 4 hours, centrifuged at 6000g for 10 minutes to collect the bacterial cells, and the...

Embodiment 2

[0029] 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 40mL of 50mM phosphate buffer (pH6.8) and 5mg / mL lysozyme were added to 10 grams of wet bacteria and 2mM sodium ethylenediaminetetraacetate, shake in a water bath at 37°C for 20 minutes, take it out, and put it in an ice bath, ultrasonic treatment with a power of 200W, every ultrasonic treatment for 5s and a 30s pause, ultrasonic treatment twice, and centrifugation at 5000g for 5min to obtain permeabilized bacteria.

[0030] After the permeabilized bacteria were washed with 30mL of 50mM phosphate buffer (pH5.8), the bacteria were collected by centrifugation at 5000g for 10min, the wet bacteria were resuspended in 200mL of 50mM phosphate buffer (pH5.8), and 4mL of Tween-80 was added and mixed evenly, stirred at 4°C for 2 hours, centrifuged at 5000g for 10 minutes to c...

Embodiment 3

[0033] 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 40mL of 50mM phosphate buffer (pH6.8) and 5mg / mL lysozyme were added to 10 grams of wet bacteria and 2mM sodium ethylenediaminetetraacetate, shake in a water bath at 37°C for 20 minutes, take it out, and put it in an ice bath, ultrasonic treatment with a power of 200W, every ultrasonic treatment for 5s and a 30s pause, ultrasonic treatment twice, and centrifugation at 5000g for 5min to obtain permeabilized bacteria.

[0034] After the permeabilized bacteria were washed with 40mL of 50mM phosphate buffer (pH5.8), the bacteria were collected by centrifugation at 5000g for 10min, and the wet bacteria were resuspended in 200mL of 50mM phosphate buffer (pH5.8), and 1mL of Tween-80 was added. and mixed evenly, stirred at 4°C for 6 hours, centrifuged at 5000g for 10 minutes...

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PUM

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Abstract

A non-aqueous Enzymological synthesis method of a conjugated Nu-linolenic acid isomer includes: 1, adding 4mL of 50mM pH6.8 phosphate buffer, 5mg / L lysozyme and 2mM sodium ethylene diamine tetracetate per gram of Lactobacillus casei CGMCC 1.574, and performing processing at 37 DEG C for 20 min; under ultrasonic power 200W, at 30s intervals per 5s of ultrasonic processing, performing ultrasonic processing twice, and performing centrifuging to obtain permeabilized bacteria; 2, performing washing with 50mM pH5.8 phosphate buffer, collecting the bacteria, re-suspending each gram of permeabilized bacteria in 20mL of 50mM pH5.8 phosphate buffer, adding tween 80 accounting for 0.5 to 2.0% of volume of bacterial suspension, performing mixing, performing processing at 4 to 40 DEG C for 2 to 6 hours, performing centrifuging to collect bacteria, and performing freeze drying to obtain coated bacteria; 3, adding each gram of coated bacteria into 600mL of n-hexane, performing mixing, adding Nu-linolenic acid accounting for 0.1 to 0.3% of the volume of the n-hexane, and allowing reacting at 4 to 40 DEG C for 1 to 12 hours; 4, performing centrifugal separating on the coated bacteria, recovering the n-hexane to obtain a product, the c6, c9 and t11-conjugatedNu-linolenic acid isomer. The method has the advantages that reaction time is short, the coated bacteria are reusable, the yield is evidently increased, no environmental pollution is caused, and production cost is significantly lowered.

Description

technical field [0001] The invention belongs to the technical field of biomedicine. Background technique [0002] Conjugated γ-linolenic acid is a conjugated isomer of γ-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 6, c 9, t 11-conjugated γ-linolenic acid and c 6, t 10, c 12-conjugated γ-linolenic acid isomers, etc. Studies have shown that conjugated γ-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 6, c 9, t 11-conjugated γ-linolenic acid isomers have anticancer effects. In nature, conjugated γ-linolenic acid mainly exists in the milk and meat fat of ruminants, mainly composed of c 6, c 9, t 11-conjugated γ-linolenic acid and other isomers, but its content is usually very low, which cannot meet the development and appli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/64C12R1/245
Inventor 刘晓华魏华
Owner NANCHANG UNIV