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Method for screening microbe producing polyunsaturated fatty acid

A technology of unsaturated fatty acids and microorganisms, which is applied in the field of screening microorganisms producing polyunsaturated fatty acids, can solve the problems of cumbersome operation, cumbersome, and inability to directional, rapid and high-throughput screening of microorganisms producing polyunsaturated fatty acids, so as to improve screening efficiency, The effect of simplifying the screening process

Inactive Publication Date: 2012-07-04
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The carbon starvation detection method has high accuracy, but it is too cumbersome; the fat granule counting method is simple, but lacks accuracy, and the size of the fat granule must be considered when using it; although the Sudan III sludge staining method has high accuracy, However, microscopic observation is required, and the operation is still cumbersome. It will be a very time-consuming task to detect and screen dozens, hundreds, or even thousands of microorganisms
Moreover, a common shortcoming of the above-mentioned methods is that they cannot be targeted for rapid and high-throughput screening of polyunsaturated fatty acid-producing microorganisms. It is not clear whether the oil-containing strains screened by these methods contain the required target polyunsaturated fatty acids.

Method used

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  • Method for screening microbe producing polyunsaturated fatty acid
  • Method for screening microbe producing polyunsaturated fatty acid

Examples

Experimental program
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Effect test

Embodiment 1

[0063] The soil was collected from Yujiashan, Huazhong University of Science and Technology, and the topsoil was shoveled off with a cleaning tool, and about 150 grams of soil at a distance of 5-15 cm was taken and packed in a sterilized bottle. DNA was extracted from the collected soil samples using a soil microbial genome DNA extraction kit, and the D6 gene fragment was amplified using the extracted DNA as a template. The PCR reaction system consists of extracted microbial genomic DNA, 50 μM D6-F and 50 μM D6-R, 100 μM dNTPs, 5 μL 10×PCR reaction buffer, 1units Taq DNA polymerase, and sterilized ddH 2 O to make up to 50 μL. The PCR amplification conditions were: 94°C, 6min; 94°C, 1min, 50°C, 45sec, 72°C, 1min for a total of 32 cycles; after that, 72°C for an additional 8min. The results of agarose gel electrophoresis showed that there was D6 gene in the soil sample DNA.

[0064] Microorganisms in the collected soil samples were isolated using conventional microbial isolati...

Embodiment 2

[0078] Soil was collected from Huazhong Agricultural University campus flower garden. Microorganisms in the soil were isolated using conventional microbial isolation methods. Put the soil sample in a sterile empty petri dish, put in the lure, see that mycelium grows on the lure, and immediately transplant it to a PDA plate added with streptomycin and penicillin for cultivation. They were cultured statically in an incubator at 22°C. According to the growth of microorganisms, 35 strains of microorganisms were obtained by separating and purifying the top of mycelia.

[0079] 35 strains of microorganisms were respectively inoculated into 40 mL of PDA liquid medium, cultured at 25°C and 160 rpm for 5 days, the genomic DNA of these microorganisms was extracted, and 0.3 μL was taken for group screening. Every 10 strains of microbial genome DNA constituted a screening group, with A, Group B, and another screening group C containing 15 strains of microbial genomic DNA. The D6 gene fr...

Embodiment 3

[0088] Soil was collected from the vegetable garden of Huashan Town, Hongshan District, Wuhan City, and microorganisms were isolated by conventional microbial separation methods. Under aseptic conditions, pick 0.1-0.25mg of soil particles and place them on the surface of the PDA plate added with antibiotics, and place 3 places on each plate in a triangular shape. Cultivate at 30° C. and 220 rpm for 5 days, and if mycelium grows, it is immediately transplanted to a PDA plate added with streptomycin and penicillin for cultivation. According to the growth of microorganisms, 42 strains of microorganisms were obtained by separating and purifying the top of mycelia.

[0089] 42 strains of microorganisms were inoculated into 60mL of PDA liquid medium, cultured at 30°C and 220rpm for 5 days, the genomic DNA of these microorganisms was extracted, and 0.5μL was taken for group screening. Every 5 strains of microbial genome DNA constituted a screening group, with A~ There were 8 groups ...

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Abstract

The invention discloses a method for rapidly screening produced polyunsaturated fatty acid. In the method, by taking a key enzyme gene as a molecular marker, microbes producing the polyunsaturated fatty acid can be rapidly and directionally screened on the basis of a PCR amplification technology. The method comprises the following steps of: designing a primer according to conserved regions of reported delta 6 fatty acid desaturase (D6) and delta 5 fatty acid desaturase gene (D5), amplifying and screening microbes obtained by separating soil or a water body in sequence to produce GLA by amplifying the gene of D6 and to produce AA and EPA by amplifying the genes of D6 and D5 simultaneously; then fermenting and culturing the microbes capable of producing the gene of D6 and simultaneously producing the genes of D6 and D5 by amplifying and extracting fatty acid; analyzing the fatty acid synthesized by the microbes by using GC and MS; and finally determining the microbe producing the polyunsaturated fatty acid. The invention provides an effective strain or algae seed separating and screening method and a batch of effective starting strains or algae seeds for producing the polyunsaturated fatty acid by using micro to ferment.

Description

technical field [0001] The invention belongs to the technical fields of microbial biotechnology and biopharmaceuticals, and in particular relates to a method for screening microorganisms producing polyunsaturated fatty acids. Background technique [0002] Polyunsaturated fatty acids have a wide range of physiological functions and biological effects in the body, and the more double bonds, the higher the degree of unsaturation and the higher the nutritional value. Polyunsaturated fatty acids have physiological functions such as maintaining the structure and function of biological membranes, treating cardiovascular diseases, anti-inflammation, anti-cancer, promoting brain development, losing weight, and increasing animal litter rate and survival rate. Polyunsaturated fatty acids are usually derived from higher plants, animal offal and fish oil. However, the above-mentioned sources all have the disadvantages that the output cannot meet the market demand and the price is expens...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 余龙江李运涛栗茂腾张鹏
Owner HUAZHONG UNIV OF SCI & TECH