Method for preparing microbial extracellular enzyme dielectrophoresis sample in finished wheat koji used for making Shao-hsing rice wine

Abstract

The invention discloses a method for preparing a microbial extracellular enzyme dielectrophoresis sample in finished wheat starter used for making Shao-hsing rice wine, which comprises the following steps of: lixiviating microbial extracellular enzyme in the wheat starter at the low temperature by selecting buffer solution of acetic acid-sodium acetate, precipitating albumen by using pre-cooled acetone solution I containing dithiothreitol and trichloroacetic acid and rinsing a precipitate by using pre-cooled acetone solution containing the dithiothreitol, and finally adding cracking liquid toprepare a high-quality sample which is suitable for dimensional electrophoresis. The method for preparing the microbial extracellular enzyme dielectrophoresis sample in the Shao-hsing rice wine finished wheat starter is quick, reliable, simple and easy to implement, can prepare a dielectrophoresis map with high quality and repeatability for facilitating subsequent analytical identification of albumen by bio-mass spectra, and has a high practical application value.

Description

technical field

[0001] The invention relates to a method for preparing Shaoxing rice wine finished wheat koji containing various microbial extracellular enzyme proteins, in particular to a method for preparing Shaoxing yellow rice wine finished wheat koji containing various microbial extracellular enzyme proteins by two-dimensional electrophoresis. Background technique

[0002] Yellow rice wine is one of the three major brewed wines in the world. "Using wheat to make koji and using koji to make wine" is the characteristic of Chinese rice wine. The traditional production of wheat koji is made from crushed wheat, which is prepared by natural enrichment and natural cultivation of microorganisms. Wheat koji plays an extremely important role in the brewing of yellow rice wine, and is known as the "bone of wine". It is not only the "crude enzyme preparation" for rice wine brewing, but also the metabolites of various microorganisms are accumulated in the wheat koji, which endows ye...

Examples

Embodiment 1

[0025] 1. Sample preparation

[0026] 1) Weigh 25g of wheat koji, the finished product of a rice wine factory in Shaoxing City, Zhejiang, add 50mL of acetic acid-sodium acetate buffer solution with a concentration of 0.09mol / L and pH5.0, and add 1 protease inhibitor mixed tablet at the same time, at 4°C Under leaching for 12h. Then filter with 4 layers of gauze and filter paper successively to obtain the filtrate. Centrifuge the filtrate at 4° C. and 10,000 r / min for 30 min with a refrigerated high-speed centrifuge, and collect the supernatant, which is the crude enzyme extract.

[0027] 2) Add 4 times the volume of pre-cooled acetone solution I (which contains 20mmol / L dithiothreitol and 0.1g / mL trichloroacetic acid) to the crude enzyme extract obtained in step 1), invert several times to mix After homogenization, place at -20°C for 1 hour to precipitate protein; then centrifuge at 12000r / min for 30min at 4°C, discard the supernatant, and collect the precipitate. Add 2 mL ...

Embodiment 2

[0039] 1) Weigh 30g of wheat koji, the finished product of a rice wine factory in Shaoxing City, Zhejiang, add 50mL of acetic acid-sodium acetate buffer solution with a concentration of 0.15mol / L and pH5.0, and add 1 protease inhibitor mixed tablet at the same time, at 4°C Under leaching for 4h. Then filter with 4 layers of gauze and filter paper successively to obtain the filtrate. Centrifuge the filtrate at 4° C. and 10,000 r / min for 30 min with a refrigerated high-speed centrifuge, and collect the supernatant, which is the crude enzyme extract.

[0040] 2) Add 6 times the volume of pre-cooled acetone solution I (which contains 10mmol / L dithiothreitol and 0.2g / mL trichloroacetic acid) to the crude enzyme extract obtained in step 1), and mix it upside down several times. After homogenization, place at -20°C for 1 hour to precipitate protein; then centrifuge at 12000r / min for 30min at 4°C, discard the supernatant, and collect the precipitate. Add 2 mL of pre-cooled acetone s...

Embodiment 3

[0052] 1) Weigh 20g of wheat koji, a finished product of a rice wine factory in Shaoxing City, Zhejiang, add 50mL of acetic acid-sodium acetate buffer solution with a concentration of 0.10mol / L and pH5.0, and add 1 protease inhibitor mixed tablet at the same time, at 4°C Under leaching for 10h. Then filter with 4 layers of gauze and filter paper successively to obtain the filtrate. Centrifuge the filtrate at 4° C. and 10,000 r / min for 30 min with a refrigerated high-speed centrifuge, and collect the supernatant, which is the crude enzyme extract.

[0053] 2) Add 4 times the volume of pre-cooled acetone solution I (which contains 30mmol / L dithiothreitol and 0.15g / mL trichloroacetic acid) to the crude enzyme extract obtained in step 1), and mix it upside down several times. After homogenization, place at -20°C for 1 hour to precipitate protein; then centrifuge at 12000r / min for 30min at 4°C, discard the supernatant, and collect the precipitate. Add 2 mL of pre-cooled acetone s...

technical field

[0001] The invention relates to a method for preparing Shaoxing rice wine finished wheat koji containing various microbial extracellular enzyme proteins, in particular to a method for preparing Shaoxing yellow rice wine finished wheat koji containing various microbial extracellular enzyme proteins by two-dimensional electrophoresis. Background technique

[0002] Yellow rice wine is one of the three major brewed wines in the world. "Using wheat to make koji and using koji to make wine" is the characteristic of Chinese rice wine. The traditional production of wheat koji is made from crushed wheat, which is prepared by natural enrichment and natural cultivation of microorganisms. Wheat koji plays an extremely important role in the brewing of yellow rice wine, and is known as the "bone of wine". It is not only the "crude enzyme preparation" for rice wine brewing, but also the metabolites of various microorganisms are accumulated in the wheat koji, which endows ye...

Examples

Embodiment 1

[0025] 1. Sample preparation

[0026] 1) Weigh 25g of wheat koji, the finished product of a rice wine factory in Shaoxing City, Zhejiang, add 50mL of acetic acid-sodium acetate buffer solution with a concentration of 0.09mol / L and pH5.0, and add 1 protease inhibitor mixed tablet at the same time, at 4°C Under leaching for 12h. Then filter with 4 layers of gauze and filter paper successively to obtain the filtrate. Centrifuge the filtrate at 4° C. and 10,000 r / min for 30 min with a refrigerated high-speed centrifuge, and collect the supernatant, which is the crude enzyme extract.

[0027] 2) Add 4 times the volume of pre-cooled acetone solution I (which contains 20mmol / L dithiothreitol and 0.1g / mL trichloroacetic acid) to the crude enzyme extract obtained in step 1), invert several times to mix After homogenization, place at -20°C for 1 hour to precipitate protein; then centrifuge at 12000r / min for 30min at 4°C, discard the supernatant, and collect the precipitate. Add 2 mL ...

Embodiment 2

[0039] 1) Weigh 30g of wheat koji, the finished product of a rice wine factory in Shaoxing City, Zhejiang, add 50mL of acetic acid-sodium acetate buffer solution with a concentration of 0.15mol / L and pH5.0, and add 1 protease inhibitor mixed tablet at the same time, at 4°C Under leaching for 4h. Then filter with 4 layers of gauze and filter paper successively to obtain the filtrate. Centrifuge the filtrate at 4° C. and 10,000 r / min for 30 min with a refrigerated high-speed centrifuge, and collect the supernatant, which is the crude enzyme extract.

[0040] 2) Add 6 times the volume of pre-cooled acetone solution I (which contains 10mmol / L dithiothreitol and 0.2g / mL trichloroacetic acid) to the crude enzyme extract obtained in step 1), and mix it upside down several times. After homogenization, place at -20°C for 1 hour to precipitate protein; then centrifuge at 12000r / min for 30min at 4°C, discard the supernatant, and collect the precipitate. Add 2 mL of pre-cooled acetone s...

Embodiment 3

[0052] 1) Weigh 20g of wheat koji, a finished product of a rice wine factory in Shaoxing City, Zhejiang, add 50mL of acetic acid-sodium acetate buffer solution with a concentration of 0.10mol / L and pH5.0, and add 1 protease inhibitor mixed tablet at the same time, at 4°C Under leaching for 10h. Then filter with 4 layers of gauze and filter paper successively to obtain the filtrate. Centrifuge the filtrate at 4° C. and 10,000 r / min for 30 min with a refrigerated high-speed centrifuge, and collect the supernatant, which is the crude enzyme extract.

[0053] 2) Add 4 times the volume of pre-cooled acetone solution I (which contains 30mmol / L dithiothreitol and 0.15g / mL trichloroacetic acid) to the crude enzyme extract obtained in step 1), and mix it upside down several times. After homogenization, place at -20°C for 1 hour to precipitate protein; then centrifuge at 12000r / min for 30min at 4°C, discard the supernatant, and collect the precipitate. Add 2 mL of pre-cooled acetone s...

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