The Entry-Exit Inspection and Quarantine Industry Standard SN/T 1113-2002 in the People's Republic of China has specified the Sampling, sample preparation and spectrophotometric methods for the determination of phycocyanin content in imported and exported spirulina powder . This standard is applicable to the determination of phycocyanin content of import and export spirulina powder in China.
2. Normative document of reference
3. Sampling and sample preparation
According to the method specifed in GB/T 16919
4.1 Determination of phycocyanin content
4.1.1 Method and principle
Dissolve the sample into a phosphate buffer solution, frozen in a refrigerator and melted, to separate out the pigment proteins, and measure the separated liquid extract by spectrophotometry.
Phosphate buffer solution: Mix 0.1 mol/L potassium dihydrogen phosphate solution with 0.1 mol/L dipotassium hydrogen phosphate solution (45 + 55, V/V), and the pH value of the solution is 7. 0 .
4. 1. 3. 1 Spectrophotometer
4. 1. 3. 2 Ultrasonic oscillator
4. 1. 3. 3 Centrifuge ：3000 r /min
4. 1. 3. 4 Cryogenic refrigerator：-20 ℃
4. 1. 3. 5 Centrifuge tube：50 mL
4.1. 4 Sample determination
Take a sample 0. 25 g ~ 0. 5 g (accurate to 0.0001 g), dissolve in buffer solution (4.1. 2), oscillate for 5 minutes by ultrasonic oscillator , dilute to volume in a 250 mL volumetric flask, shake well . Transfer all the solution into a 250 mL wide-mouth plastic bottle, freeze it in a refrigerator at -20 ℃ for 12 hours (or overnight) , then thaw it and shake well. Take a portion of solution in a centrifuge tube and centrifuge at 3 000 r/min for 15 min. Take the supernatant, 1 cm cuvette, and measure the absorbance at 620 nm, 652 nm, and 562 nm by spectrophotometer, and use the buffer solution (4.1. 2) as a blank.
5. 1. 5 Calculate the results
Calculate the mass fraction content of phycocyanin of the sample according to below equations (1),(2),(3),(4).
In these equations:
X1—— the content of phycocyanin in the test solution, in milligrams per milliliter (mg/mL);
X2 —— the content of allophycocyanin in the test solution, in milligrams per milliliter (mg/mL);
X3 —— the content of phycoerythrin in the test solution, in milligrams per milliliter (mg / mL);
A —— the absorbance measured at the corresponding wavelength (620 nm, 652 nm, 562 nm).
X4 —— the mass fraction of phycocyanin in the sample, in grams per 100 grams (g/100 g);
V —— the constant volume of sample, in milliliters (mL).
m —— the mass of the sample, in grams (g).
Take the arithmetic mean of the parallel test results as the measurement results, and retain two numbers after the decimal point.
The parallel test errors (relative) are allowed no more than 4%.
Note: The whole process should be protected from light, and the spectrophotometry determination should be completed within 15 minutes.