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, time-consuming and labor-intensive, etc. Practical application value, simplified screening process, strong operability effect
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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). Afte...
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