Method for synthesizing conjugated gamma-linolenic acid isomers catalyzed by surfactant-coated bacteria

Abstract

The invention discloses a method for catalytic synthesis of a conjugated gamma-linolenic acid isomer by virtue of a surfactant-coated bacterium, wherein the method comprises the following steps (1) re-suspending each gram of lactobacillus casei CGMCC 1.574 bacterium in 5ml of a permeabilization treatment solution, treating at 37 DEG C for 20min, centrifuging and collecting a permeabilized bacterium; (2) washing the permeabilized bacterium with 60mM of a phosphate buffer solution which is at 5.8 in pH value; centrifuging and collecting the bacterium; re-suspending each gram of the wet bacterium in 20mL of the 60mM of phosphate buffer solution which is at 5.8 in pH value, dropping an acetone solution which contains 1% of dioleyl glutamate ribitol and uniformly mixing at the same time; processing at 4-40 DEG C for 2-6h, centrifuging and collecting the bacterium, and freeze-drying so as to obtain a coated bacterium; (3) adding each gram of the freeze-dried coated bacterium to 500ml of normal hexane, uniformly stirring, adding 0.1-0.3% of gamma-linolenic acid in terms of the volume percentage of the normal hexane and reacting at 4-40 DEG C for 1-12h; and (4) centrifuging and isolating the coated bacterium and recovering the normal hexane, so as to obtain a finished product, namely c6,c9,t11-conjugated gamma-linolenic acid isomer. The method disclosed by the invention is short in reaction time; the coated bacterium can be recycled for several times, so that yield is significantly improved; and the method is free from environmental pollution and is capable of remarkably reducing production cost.

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...

Examples

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...

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...

Examples

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...