Test Method of Yeast Beta Glucan

A.4.1 Acid hydrolysis method (arbitration method) 

A.4.1.1 Principle 

After acid hydrolysis, the sample was analyzed using high-performance liquid chromatography. The glucose and other components in the sample were separated by a chromatographic column, detected by a differential refractive detector, and quantitatively determined using an external standard method. 

(Note: During the hydrolysis process of yeast beta glucan, there may be incomplete hydrolysis and other side reactions of the hydrolysis product glucose due to high temperature, resulting in a low content of yeast beta glucan in the test results.)

A.4.1.2 Instruments and Equipment

A. 4.1.2.1 Vortex mixer.

A. 4.1.2.2 High performance liquid chromatography: equipped with a differential detector and a sugar column (6.5 mmx300 mm), or a separation column with equivalent analytical performance. A. 4.1.2.3 Chromatographic conditions: Use pure water as the mobile phase, with a flow rate of 0.5mL/min and a column temperature of 80 ° C. Inject the sample after the instrument baseline is stable.

A.4.1.3 Reagents and Solutions

A. 4.1.3.1 Water: GB/T 6682, Grade 1 water. 

A. 4.1.3.2 Hydrochloric acid: 37% 

A. 4.1.3.3 Anhydrous glucose

A. 4.1.3.4 Glucose standard solution (2.0 g/L): Weigh 0.20g (accurate to 0.001 g) of glucose and dry it at 98 ° C to 100 ° C for 2 hours (A.4.1.3.3). Dissolve it in water (A.4.1.3.1) and bring to a constant volume of 100 mL, shake well.

A. 4.1.3.5 Yeast beta glucan reference substance: Known purity, and purity ≥ 70%. 

A. 4.1.3.6 Sodium hydroxide solution (300 g/L): Weigh 300.0 g of sodium oxide, dilute to 1000 mL with water (A.4.1.3.1), and shake well

A.4.1.4 Sample processing 

Weigh 0.40 g (accurate to 0.001 g) of the sample or yeast beta glucan reference substance A.4.1.3.5 to 20mL in a nut capped test tube, add 6.0mL of hydrochloric acid (A.4.1.3.2), cover tightly, and shake to obtain a uniform suspension. Place the test tube in a 30 ° C water bath for 45 minutes (mix with a vortex mixer every 15 minutes). Then transfer the floating substance to a 200 mL heat-resistant spiral cap bottle, wash the test tube several times with 100mL-120mL of water, and merge the washing solution into the heat-resistant spiral cap bottle. Place the heat-resistant bottle with a screw cap into a high-pressure sterilization pot at 121 ° C for 60 minutes. After removal, cool to room temperature, adjust the pH of the solution to 6-7 using sodium hydroxide solution (A.4.1.3.6), and then transfer to a 200mL volumetric flask. Bring to volume with water (A.4.1.3.1) and mix well. Use 0.22 μ Filter with m pore size cellulose alginate membrane for backup.

A.4.1.5 Drawing of standard curves 

Take 2.0mL, 4.0mL, 6.0mL, 8.0mL, and 10.0mL of glucose standard solution (A.4.1.3.4) into a 10mL volumetric flask, dilute with water (A.4.1.3.1) and shake well to obtain a series of standard solutions with glucose concentrations of 400 mg/L, 800 mg/L, 1200 mg/L, 1600 mg/L, and 2000 mg/L. Inject 20 samples under chromatographic conditions in A.4.1.2.3 μ L. Draw a standard curve based on the chromatographic peak area and glucose concentration.

A.4.1.6 Determination of samples and reference standard 

Under the same chromatographic conditions, inject the processed sample and yeast beta glucan reference solution into the chromatograph respectively, and record the retention time and peak area of each chromatographic peak. Qualitative analysis is performed using the retention time of the chromatographic peak of glucose standard solution, and quantitative analysis is performed using the peak area of the chromatographic peak of glucose standard solution.

A.4.1.7 Result calculation 

The content of yeast beta glucan is calculated according to formula (1):

In the equation:

X₁ -------- The content of yeast beta glucan in the sample, in grams per hundred grams (g/100 g);

C₁ -------- Calculate the glucose content of the sample solution based on the peak area of the sample solution and the standard curve, in milligrams per liter (mg/L);

0.2-------- The volume of the sample or standard sample treated with yeast beta glucan, expressed in liters (L);

100 ------ Conversion coefficient of percentage content;

M₁ ------- Weigh the mass of the sample, in grams (g);

1000------ Conversion coefficient between milligrams and grams;

0.9 ------- Coefficient for converting glucose to yeast beta glucan;

F₁ -------- Empirical compensation coefficient for low results caused by the destruction of glucose during sample acid hydrolysis.

Calculate the F₁ value according to formula (2):

In the equation:

P₁ --------The purity of yeast beta glucan reference substance (based on the test report provided by the reagent manufacturer), in grams per hundred grams (g/100 g);

W ------- Moisture content of yeast beta glucan reference substance (according to the test report provided by the reagent manufacturer), in grams per hundred grams (g/100 g);

M₂ ------ Weigh the mass of yeast beta glucan reference substance, in grams (g);

1000----- Conversion coefficient between milligrams and grams;

C₂ ------ --Calculate the glucose content of the reference solution based on the peak area of the yeast beta glucan reference solution and the standard curve, in milligrams per liter (mg/L);

0.2------- The volume of the sample or yeast beta glucan reference substance after treatment, expressed in liters (L);

100 ------ Conversion coefficient of percentage content;

0.9-------- Conversion coefficient between glucose and yeast beta glucan

The calculation results are represented as the arithmetic mean of two independent measurement results obtained under repeatability conditions, rounded to an integer.

A.4.1.8 Precision

The absolute difference between two independent measurement results obtained under repeatability conditions should not exceed 5% of the arithmetic mean.