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Method capable of simply and efficiently screening chlorogenic acid producing strains on culture medium

A culture medium and chlorogenic acid technology, applied to the separation of microorganisms, etc., can solve the problems of cumbersome operation, high cost, unsuitable screening work, etc., and achieve the effect of wide application prospect and low cost

Active Publication Date: 2018-02-02
GUANGXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the qualitative and quantitative techniques of chlorogenic acid mainly include spectrophotometry, which is easy to operate, has low requirements for instruments, is easily interfered by other structural analogues, and the detection results are not accurate enough; thin-layer-ultraviolet spectrophotometry, combined with color reagents Chlorogenic acid has fluorescence absorption at a specific wavelength, so as to qualitatively and quantitatively eliminate the interference of impurities, and the detection result is more accurate; high performance liquid chromatography has remarkable separation effect, good linear relationship, low detection limit and high recovery rate. High precision and high cost; gas chromatography has high sensitivity and good reproducibility, but the esterification temperature is too high, the operation is cumbersome, and the cost is high; the above method is suitable for the verification of a small amount of chlorogenic acid strains, not suitable for A lot of screening work, but the screening work involved in excavating chlorogenic acid-producing strains from the environment is large, time-consuming, and expensive. Therefore, it is urgent to develop a simple, fast, and low-cost screening method for chlorogenic acid-producing strains. method

Method used

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  • Method capable of simply and efficiently screening chlorogenic acid producing strains on culture medium
  • Method capable of simply and efficiently screening chlorogenic acid producing strains on culture medium
  • Method capable of simply and efficiently screening chlorogenic acid producing strains on culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 : Isolation of chlorogenic acid (Chlorogenic acid, CGA) strain

[0036] The isolation of chlorogenic acid (CGA) strains includes medium preparation, sampling, primary screening and re-screening in four steps, as follows:

[0037] 1.1 Culture medium preparation

[0038] Beef extract peptone medium and No. ① and No. ③ medium formulations (Table 1) of beef extract peptone medium and improved beef extract peptone medium, and No. ② and No. ④ medium formulations of potato dextrose agar medium and improved potato medium (Table 2) As follows, and the medium used in the experiment was prepared with distilled water, and sterilized by high-pressure damp heat at 121°C for 20min.

[0039]Table 1 Beef extract peptone medium and improved No. ① and No. ③ medium formulations

[0040]

[0041] Table 2 Potato dextrose agar medium and improved No. ② and No. ④ medium formulations

[0042]

[0043] 1.2 Sampling

[0044] The plant samples used for separating chlorogenic ...

Embodiment 2

[0050] Example 2 : Comparison of methods for efficient screening of chlorogenic acid (CGA) strains

[0051] 2.1 Establishment of efficient screening method

[0052] Chlorogenic acid belongs to organic carboxylic acid substances, and under alkaline conditions, it can form a stable purple-red complex with aluminum ions. Utilize the special group reaction in the chemical structure of chlorogenic acid to improve the screening medium and screen the strains producing chlorogenic acid through the special color reaction on the medium. The present invention designs a new type of screening medium to screen and produce chlorogenic acid through color reaction. Acidic strains; at the same time, thin layer chromatography (TLC) preliminary qualitative, high performance liquid chromatography precise qualitative and quantitative.

[0053] The present invention explores the addition amount of aluminum ions and the solidification state of the culture medium, and the results show that Al 3+ T...

Embodiment 3

[0077] Example 3 : Determination of the content of chlorogenic acid produced by microorganisms

[0078] 3.1 Quantification by HPLC

[0079] According to the screening scheme established above, the standard curve of chlorogenic acid was established by high performance liquid chromatography (Fig. 7), and then some bacterial strains obtained by screening were quantitatively analyzed, and the results found that the amount of chlorogenic acid produced by the obtained bacterial strains was 0.70 mg / L, 0.67mg / L, 1.87mg / L, 2.16mg / L, 5.43mg / L, 13.04mg / L, etc. The amount of chlorogenic acid produced by these strains is relatively higher than that of the previously reported chlorogenic acid-producing strains. Through the optimization of fermentation conditions and product leaching methods, the yield can be increased, which provides the possibility for later industrial production.

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Abstract

The invention belongs to the technical field of secondary metabolite-chlorogenic acid research, and particularly relates to a method capable of simply, rapidly and efficiently screening chlorogenic acid producing strains on a culture medium. Plants such as sweet potatoes, eucommia barks, immature flowers of wild honeysuckle, Japanese camellia flowers, lonicera hypoglauca miq, wild mint herbs and Laurocerasus phaeosticta are selected to serve as main research objects, different plants are screened by the method to totally obtain 11 endophytic bacteria, 10 secondary metabolites such as chlorogenic acid can be produced, and screening efficiency is 91%. Compared with high-performance liquid chromatography, the method has the advantages of time and labor saving properties, simplicity, rapidness, low cost, high efficiency and the like. The principle can be analogized to screening of other secondary metabolite producing strains, and is wide in application prospect.

Description

technical field [0001] The invention belongs to the technical field of secondary metabolite-chlorogenic acid research, and relates to a method capable of simply and efficiently screening chlorogenic acid-producing strains on a culture medium. Background technique [0002] In 1891, Kossel clearly proposed the concept of plant secondary metabolites, using intermediate products of primary metabolism as starting materials to release energy. There are many kinds of plant secondary metabolites with different structures. According to the structural formula and their properties, they are mainly divided into phenols, terpenes and nitrogen-containing compounds. Although secondary metabolites are not necessary for cell life activities or plant growth and development, they help improve the ability of plants to adapt to the external environment during plant growth, such as plant resistance to disease, stress and animal hazards. play a key role. [0003] Chlorogenic acid (CGA) is an imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/02
CPCC12N1/02
Inventor 樊宪伟王晓陈鑫鑫李有志
Owner GUANGXI UNIV
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