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Method for separating microorganism capable of producing ferulic acid esterase

A technology of ferulic acid esterase and ethyl ferulate, applied in microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve problems such as inability to intuitively judge, cumbersome steps, heavy workload, etc. High practical value, simplified screening process, shortened time effect

Inactive Publication Date: 2008-07-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the research aimed at screening out anaerobic fungi that produce ferulic acid esterase, this process is a lot of work, the purpose is not clear enough, lack of pertinence, cumbersome steps, time-consuming and labor-intensive
The most prominent performance is: in the process of screening single bacteria using the in vitro culture medium of common anaerobic fungi, it is necessary to go through a series of enzyme activity tests and it is impossible to intuitively judge whether the screened colonies have the ability to produce ferulic acid esterase

Method used

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  • Method for separating microorganism capable of producing ferulic acid esterase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Preparation of Liquid Screening Medium

[0028] Add ethyl ferulate to conventional liquid selection medium without major carbon source (such as: glucose) to make the concentration of ethyl ferulate reach 2 μM. For example, the liquid screening medium with the following configuration is used, and each 1000ml contains: peptone 1.0g, yeast extract 1.0g, NaHCO 3 7.0g, crushed wheat straw 7g, solution I 165ml, solution II 165ml, cell-free rumen fluid 170ml, L-cysteine ​​hydrochloride 1.7g, resazurin (0.1% w / v) 1.0ml, distilled water 500ml ; Solution I 1000ml contains: KH 2 PO 4 3g, (NH 4 ) 2 SO 4 3g, NaCl 6g, CaCl 2 2H 2 O 0.4g, MgSO 4 ·7H 2 O 0.6 g; solution II 1000ml contains: K 2 HPO 4 4g. Add penicillin (1600IU / mL) and streptomycin (2000IU / mL) before inoculation to inhibit bacteria.

Embodiment 2

[0029] Example 2 Preparation of solid screening medium

[0030] Add ethyl ferulate to conventional solid selection medium without major carbon source (such as: glucose) to make the concentration of ethyl ferulate reach 9 μM. For example, the solid screening medium with the following configuration is used, and each 1000ml contains: peptone 1.0g, yeast extract 1.0g, NaHCO 3 7.0g, 15g of agar, 165ml of solution I, 165ml of solution II, 170ml of cell-free rumen fluid, 1.7g of L-cysteine ​​hydrochloride, 1.0ml of resazurin (0.1% w / v), 500ml of distilled water; solution I Contains in 1000ml: KH 2 PO 4 3g, (NH 4 ) 2 SO 4 3g, NaCl 6g, CaCl 2 2H 2 O 0.4g, MgSO 4 ·7H 2 O 0.6g; solution II 1000ml contains: K 2 HPO 4 4g. Add penicillin (1600IU / mL) and streptomycin (2000IU / mL) before inoculation to inhibit bacteria.

Embodiment 3

[0031] Example 3 Screening and isolation of anaerobic fungi producing ferulic acid esterase in bovine rumen microorganisms

[0032] 1) Collect fresh rumen fluid samples through ruminal fistula of cows, quickly inoculate 1 mL of it into a Heinz tube containing 9 mL of conventional liquid in vitro medium with a syringe, make gradient dilutions, and incubate at 39°C for 4 days;

[0033] 2) Inoculate the bacteria surviving in the Heinz tube with the highest dilution in a conventional liquid in vitro medium at a volume ratio of 1:9, and culture at 39°C for 4 days;

[0034] 3) Inoculate the bacterium into the liquid screening medium prepared in Example 1, and cultivate it at 39° C. for 3 days;

[0035] 4) Inoculate it into the solid screening medium prepared in Example 2, roll the tube on ice, and culture at 39°C for 2 days;

[0036] 5) Inoculate a single colony in the liquid screening medium prepared in Example 1, and culture at 39° C. for 3 days;

[0037]6) Repeat 4) and 5). Aft...

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Abstract

The invention provides a separation method used for producing ferulate enzyme microorganism, which cultures the sample with ferulate as culture medium of carbon source, wherein the microorganism producing ferulate enzyme can utilize ferulate as carbon source so as to form bacterial colony, nevertheless the microorganism which can't utilize ferulate can't grow so as to achieve the aim of separating the microorganism producing ferulate enzyme. The invention completes separation of microorganism and qualitative determination of ferulate enzyme activity in one step, which shortens the screening time, and has high operability. The invention has highly actual application value in the production of fodder industry, paper making industry, spice industry and pharmacy material industry.

Description

technical field [0001] The invention relates to a method for isolating microorganisms, in particular to a method for isolating microorganisms producing ferulic acid esterase. The invention also relates to a special selection medium for use in the method. Background technique [0002] About 30% to 80% of the dry matter of straw and forage grass is cell wall components, however, the digestion and utilization of these cell walls by ruminants does not exceed 50%. Therefore, the cell wall becomes the bottleneck factor for straw and forage utilization. Studies have shown that molecules such as ferulic acid, p-coumaric acid, and dimerized ferulic acid in the lignin of the feed cell wall form a dense network cross-linked structure with ester bonds and hemicellulose branches, which limits the ability of rumen microorganisms to interact spatially. For the effective degradation of cellulose and hemicellulose in the cell wall, ferulic acid esterase has been speculated to be one of the...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N9/16C12R1/645
Inventor 杨红建岳群
Owner CHINA AGRI UNIV
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