- ENZYME EXTRACTION:
A. Wheat and Barley Flours:
1. Mill wheat, barley or other grain (approx. 10-50 g sample) to pass a 0.5 mm screen (e.g. with a Fritsch centrifugal mill).
2. Accurately weigh 3.0 g of flour into a flask of 50 mL capacity.
3. To each flask add 20.0 mL of Extraction Buffer solution (pH 5.4) and stir the flask contents vigorously.
4. Allow the enzyme to extract over 15-20 min at 40°C with occasional mixing.
5. Filter an aliquot of the solution through a Whatman GF/A glass fibre filter paper, or centrifuge an aliquot at 1,000 g for 10 min. Assay enzyme activity within 2 h.
B. Malt Flours:
1. Mill malt (20 g sample) to pass a 0.5 mm screen.
2. Accurately weigh 0.5 g malt flour into a 100 mL volumetric flask.
3. To the volumetric flask add a solution of 1% sodium chloride plus 0.02% calcium chloride plus 0.02% sodium azide; adjust to volume.
4. Allow the enzyme to extract for 15-20 min at room temperature, with occasional stirring.
5. Filter an aliquot of the solution through a Whatman GF/A glass fibre filter paper or centrifuge at 1,000 g for 10 min.
6. Dilute 0.5 mL of the filtrate with 9.5 mL of Extraction Buffer Solution. Assay enzyme activity (see page 6, section A, point 5) within 2 h.
C. Microbial Preparations:
Liquid preparations:
1. Add 1 mL of liquid enzyme preparation (using a positive displacement dispenser) to Buffer A or B (49 mL, pH 5.4 or 6.5) and mix thoroughly. This is termed the Original Extract.
2. Dilute 1.0 mL of Original Extract 10-fold by addition to 9.0 mL of appropriate Buffer (A or B) and mix thoroughly. Repeat this step until a dilution suitable for assay is obtained. For example, for the industrial enzyme preparation, Bacterial Alpha-Amylase (from Kerry Ingredients, Ireland) a dilution of the Original Extract of approx. 4,000-fold is required.
Powder preparations:
1. Add 1 g of enzyme powder preparation to 50 mL of Buffer A or B (pH 5.4 or 6.5) and gently stir the slurry over a period of about 15 min or until the sample is completely dispersed or dissolved.
2. Clarify this solution (the Original Extract) by centrifugation (1,000 g, 10 min) or filtration through Whatman No. 1 (9 cm) filter circles.
3. Dilute 1.0 mL of this solution 10-fold by addition to 9.0 mL of appropriate Extraction/Dilution buffer and mix thoroughly. Repeat this step until a dilution suitable for assay is obtained.
- ASSAY PROCEDURE:
A. Wheat and barley flours:
1. Dispense 0.2 mL aliquots of Amylase HR Reagent solution (unbuffered) into test tubes and pre-incubate the tubes and contents at 40°C for 5 min.
2. Pre-incubate cereal extract at 40°C for 5 min.
3. To each tube containing Amylase HR Reagent solution (0.2 mL), add 0.2 mL of pre-equilibrated wheat or barley extract directly to the bottom of the tube. Incubate at 40°C for exactly 20 min (from time of addition).
4. At the end of the 20 min incubation period, add exactly 3.0 mL of Stopping Reagent and stir the tube contents vigorously.
5. Read the absorbance of the solutions and the reaction blank at 400 nm against distilled water.
B. Malt and microbial preparations:
1. Dispense 0.2 mL aliquots of Amylase HR Reagent solution (unbuffered) into test tubes and pre-incubate the tubes and contents at 40°C for 5 min.
2. Pre-incubate buffered malt or microbial preparation extract at 40°C for 5 min.
3. To each tube containing Amylase HR Reagent solution (0.2 mL), add 0.2 mL of pre-equilibrated (and suitably diluted) microbial enzyme or malt extract directly to the bottom of the tube. Incubate at 40°C for exactly 10 min (from time of addition).
4. At the end of the 10 min incubation period, add exactly 3.0 mL of Stopping Reagent and stir the tube contents vigorously.
5. Read the absorbance of the solutions and the reaction blank at 400 nm against distilled water.
莆崧實業股份有限公司版權所有 © copyright Reserved.