GB 5521-1989 Determination of α-amylase activity in cereals and cereal products - Colorimetric method
Some standard content:
National Standard of the People's Republic of China
Determination of alpha-amylase activity in cereals and cereal products
Colorimetric method
Method for determination of alpha-amylase activity in cereals and cereal products-Colorimetric method
GB 5521-89
Replaces GB5521-85
This standard is equivalent to the international standard ISO 3983-1977 "Colorimetric method for determination of alpha-amylase activity in cereals and cereal products". 1 Subject content and scope of application
This standard specifies the instruments, reagents, analytical steps and result calculation used in the colorimetric method for determining the alpha-amylase activity in cereals and cereal products.
This standard is applicable to the determination of alpha-amylase activity in cereals and cereal products, and can also be used to determine the activity of alpha-amylase dry powder derived from fungi or bacteria.
2 Definition
The enzyme extracted from 1g of sample by 1L solvent degrades 1.024×10-5 units of β-limit dextrin substrate solution per second under specified conditions. The α-amylase activity of the sample is equal to one unit. Limit dextrin is the starch product completely degraded by β-amylase.
3 Principle
Enzyme degrades β-limit dextrin substrate solution. After different reaction times, the reaction mixture is equally added to the iodine solution. As the reaction time increases, the color intensity decreases to determine the enzyme activity. ,. Reagents
Iodine (GB 678-77);
Potassium iodide (GB1272-77);
4.3Glacial acetic acid (GB 676-78);bZxz.net
Sulfuric acid (GB 625-78);
4.5Anhydrous sodium acetate (GB694-81),4.6Calcium chloride,
4.7Soluble starch\
Note: 1) Liatner starch or domestic soluble starch of equivalent quality can be used. Shijiang Linghu Chemical Reagent Products meet the requirements. 4.8Pumice powder,
.4.9Iodine stock solution:
Weigh 11.0g potassium iodide and dissolve it in a small amount of water, add 5.50g crystal iodine, and stir until the iodine is completely dissolved. Then make up to 250mL, place in a brown bottle, store in a dark place, this solution can be stored for one month; 4.10 Iodine diluent:
Weigh 40.0g potassium iodide and dissolve it in water, add 4.00mL iodine stock solution, and dilute to 1L, prepare it when needed, do not keep it overnight, approved by the Ministry of Commerce of the People's Republic of China on December 31, 1988 284
Implementation on September 1, 1989
GB5521-—89
4.11 Buffer solution:
Weigh 164g anhydrous sodium acetate and dissolve it in water, add 120mL glacial acetic acid and make up to 1L with water 4.120.2% (m/V) calcium chloride solution,
4.130.05mol /L sulfuric acid solution
4.14β-amylase solution:
Weigh 10g of soybean powder with enzyme activity (the coarseness is to pass through a 1.5mm sieve), add 85mL of water and 15mL of 0.05mol/L sulfuric acid, stir thoroughly, let stand for 15min, filter, and the filtrate is used to prepare β-limit dextrin substrate solution. 4.15B-limit dextrin substrate solution:
Weigh 5.00g (thousand base) soluble starch in a small beaker, add about 20mL of water to mix, slowly pour it into a 500mL tall beaker containing 100mL of boiling water while stirring, and use a wash bottle to transfer all the starch in the small beaker to the tall beaker, continue stirring and heating, and slowly boil for 2min. Cover the surface blood, cool to below 30℃ in tap water, add 25mL buffer solution and 50mL β-amylase solution, place at room temperature for 20h, add 1 spoon of pumice powder, slowly boil the solution within 10min, and then continue to boil for more than 5min. Cool to below 30℃, dilute to 250mL with water, shake well, and filter. Add a few drops of toluene to the filtrate. The pH value of this solution should be 4.7±0.1, and it can be used continuously for five days below 25℃. 5 Instruments
Constant temperature water bath, 30±0.1℃,
Constant temperature water bath, 20±0.1℃
Spectrophotometer or colorimeter,
Stopwatch,
Sieve: aperture is 1.0mm, 1.5mm,
5.6 pH meter.
6 Analysis steps
6.1 Spectrophotometer and blank adjustment:
Connect the spectrophotometer to a regulated power supply, preheat for 20 minutes, and adjust the wavelength to 575nm. Add 2.0mL of calcium chloride solution to 10.0mL of dilute iodine solution, and then dilute with appropriate water V. (the amount of water added is generally 20-40mL, V. See 6.2 for details), mix well and reach 20℃, test with a 1cm cuvette, and adjust the instrument slit width to make the absorbance zero. 6.2 Calibration of substrate solution:
Put 5.0mL of β-limit dextrin solution and 15.0mL of calcium chloride solution in a 100mL beaker, mix and take out 2.0mL of the mixture into another 100mL beaker, add 10.0mL of dilute iodine solution and appropriate amount of water, mix well and place in a 20℃ water bath, use 1cm colorimetric III at a wavelength of 575nm, and use the solution used in 6.1 as a reference to measure its absorbance. Adjust the amount of water added so that the measured absorbance is between 0.55 and 0.60. If the measured absorbance is greater than 0.60, increase the amount of water added. If the measured value is less than 0.55, reduce the amount of water added. Repeat the test until the measured absorbance value is between 0.55 and 0.60, record the amount of water added Vo at this time, and use it to adjust the amount of water added V of the blank solution. This is the amount of water added during the β-limit dextrin test. 6.3 Extraction of α-amylase:
Weigh about 5g of grain sample (accurate to 0.05g), pour it into a 300mL conical flask, add 100±0.5mL of calcium chloride solution preheated to 30℃, shake well, and place it in a constant temperature water bath at 30℃. Take it out of the water bath at 15, 30, and 45min, turn the conical flask upside down 10 times, put it back in the water bath, and extract for a total of 60min. Take it out and filter it, and the filtrate is the enzyme extract. 6.4 Determination of α-amylase activity:
Put the enzyme extract and β-limit dextrin solution in a 30℃ water bath. After 10min, use a fast pipette to pipette 5.0mL of β-limit dextrin solution into the conical flask containing 15.0mL of enzyme extract, add a stopper and spread it evenly. While adding liquid, use a stopwatch to time. Pipette 10.0mL of dilute iodine solution into each 50mL volumetric flask, add VmL of water, mix well, stopper, and place in a 20℃ water bath. Perform the following operations every 5min or 10min:
GB 5521-89
6.4.1 Pipette 2.0mL of enzyme and β-limit dextrin mixture into a volumetric flask containing dilute iodine solution and V.mL of water, shake well, and place in a water bath to make the solution temperature reach 20℃.
6.4.2 Pour into a colorimetric dish to measure its absorbance.
7 Calculation of results
7.1 Calculation formula:
α-amylase activity is obtained by the following formula:
500×f
In the formula, A——α-amylase activity, unit, m
100mL calcium chloride extracted sample mass, g, h——water content in the sample, %,
f-dilution multiple of enzyme extract;
t1, t2—different reaction times, min, D, Dz—absorbance at time t1, 2, b——absolute value of the slope of the curve of lg D against t, 500——coefficient.
Ig Di - Ig D2
7.2 Calculation example:
5.20g of sample containing 14.6% water is extracted with 100mL calcium chloride solution. Since the reaction rate of enzyme extract and limit dextrin is too fast, calcium chloride solution is used to dilute the enzyme extract, and 1.5 parts of calcium chloride solution are added to 1 part of enzyme extract. Therefore, f=1+1.5=2.5. After β~limit dextrin is mixed with enzyme extract, the absorbance after intervals of 5, 10 and 20 minutes are: time, min
According to the observation results after 5 minutes and 10 minutes: b=
Absorbance D
0.697 -0.628
Use the three observation results to draw a curve of lgD versus t (see figure). Substituting b=0.01391 into the formula, we get:
4= 500×2.5×0.01391
IgD+ 1
=0.0138min-1
3.9 units
7.3 Repeatability of results,
GB 5521-89
b = I&D -ig D,
Time, min
=0.01391min
Absorbance D
The difference between two simultaneous or consecutive determination results in the same laboratory shall not exceed 10% of the average value. If the two determination results meet the requirements, the average value of the results shall be taken. Rounding according to the following table: Activity, unit
50~500
500~5000
5000~50000
Rounding to integer range, unit
Note: ① The concentration of the enzyme should be reasonably adjusted during the determination process so that 35%~60% of β-limit dextrin is degraded within 15~40min, that is, the absorbance measured at the end is 40%~65% of the absorbance during substrate calibration. If the absorbance drops too quickly, the enzyme solution can be diluted with 0.2% calcium chloride solution. If the activity of the enzyme is too low, the reaction time can be extended to more than 60 minutes to obtain correct results. ② When measuring absorbance with a spectrophotometer, the temperature of the solution will affect the result and must be controlled at 20°C. In the operation of 6.4, the interval from color development to absorbance determination generally has no effect on the result. If a series of samples are measured, the absorbance can be measured together after all samples have completed color development. However, the long interval time shall not exceed 1 hour. 287
Additional Notes:
GB5521—89
This standard was proposed and managed by the Grain Storage and Transportation Bureau of the Ministry of Commerce of the People's Republic of China. This standard was drafted by the Institute of Cereal Oil Chemistry of the Ministry of Commerce. The main drafter of this standard is Wang Weiguang.
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