GB/T 18246-2019Determination of amino acids in feeds (English PDF)

3.2.4 Sodium citrate buffer solution [pH = 2.2, c(Na+) =

0.2 mol/L]: Weigh

19.6 g of trisodium citrate into a 1 000 mL volumetric flask; add water to dissolve; then, add

16.5 mL of hydrochloric acid (3.2.2),

5.0 mL of thiodiglycol, and 1 g of phenol; use water to fix the volume and filter.

3.2.5 Amino acid standard stock solution: Containing 17 kinds of amino acids for routine protein hydrolysate analysis (aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine and arginine). The concentration of each component is 2.50 µmol/mL. Certified standard solutions shall be used.

3.2.6 Amino acid standard working solution: Accurately transfer 2 mL of amino acid standard stock solution (3.2.5) into a 50 mL volumetric flask; use sodium citrate buffer solution (3.2.4) to fix the volume, with a concentration of 100 nmol/mL for each amino acid component; package and seal in ampoules, approximately 1 mL each; store at 2 °C ~ 8 °C, valid for 3 months.

3.2.7 Sodium citrate buffer solutions and ninhydrin solutions of different pH values and ionic strengths: Prepare or purchase according to the instrument manual.

3.2.8 Liquid nitrogen.

3.3 Instruments and equipment

3.3.1 Automatic amino acid analyzer: equipped with cation exchange column, ninhydrin post-column derivatization device and 570 nm and 440 nm photometric detectors.

3.3.2 Balance: sensitivity

0.1 mg and

0.01 mg.

3.3.3 Vacuum pump.

3.3.4 Blowtorch.

3.3.5 Constant temperature drying oven: The temperature can reach 110 °C ± 2 °C.

3.3.6 Centrifuge: speed not less than 4 000 r/min.

3.3.7 Rotary evaporator or concentrator: The temperature can be adjusted between room temperature and 65 °C, with a temperature control accuracy of ±1 °C and a vacuum degree as low as 3.3×103 Pa (25 mmHg).

3.4 Test sample Prepare the sample according to GB/T 20195; crush it through a

0.25 mm sieve; mix it evenly; store it in a sealed container for later use. For samples with crude fat content greater than or equal to 5%, the defatted samples need to be air-dried, mixed, and placed in a sealed container for later use. For samples with crude fat content less than 5%, they can be weighed directly.

3.5 Test procedure

3.5.1 Sample pretreatment Perform two tests in parallel. Weigh 50 mg ~ 100 mg of the sample (containing about

7.5 mg ~ 25 mg of protein, accurate to

0.1 mg) into a 20 mL ampoule or hydrolysis tube; accurately add 10 mL of hydrochloric acid hydrolysis solution (3.2.3); freeze in liquid nitrogen (3.2.8); use a vacuum pump to evacuate to

7 Pa (<=5×10-2 mmHg); then use a blowtorch to seal the mouth or fill with nitrogen for 1 min and tighten the tube cap. Place the ampoule or hydrolysis tube in a constant temperature drying oven at 110 °C ± 2 °C and hydrolyze for 22 h ~ 24 h. Cool; mix well; open the tube; filter; use a pipette to accurately draw an appropriate amount of filtrate; place it in a rotary evaporator or concentrator; evaporate to dryness under vacuum at 60 °C. If necessary, add a little water and repeat the evaporation 1 ~ 2 times. Add 3 mL ~ 5 mL of sodium citrate buffer solution (3.2.4) to re-dissolve, so that the amino acid concentration in the sample solution reaches 50 nmol/mL ~ 250 nmol/mL; shake well; filter or centrifuge; take the supernatant for determination.

3.5.2 Determination Vi - injection volume of sample solution, in microliters (µL); F - crude fat content, %. Report the result as the arithmetic mean of the results of two parallel samples, retaining two decimal places.

3.7 Precision Under repeatability conditions, the absolute difference between two independent measurement results and their arithmetic mean shall not exceed 5% of the arithmetic mean of the two measurement values when the content is not greater than 0.5%; and shall not exceed 4% when the content is greater than 0.5%.

4.2 Determination of other amino acids

4.2.1 Principle When sodium metabisulfite is used as the oxidation terminator, the content of other protein hydrolyzed amino acids except tyrosine and tryptophan can be determined; when hydrobromic acid is used as the terminator, the content of other amino acids including sulfur-containing amino acids except tyrosine, phenylalanine, histidine and tryptophan can be determined.

4.2.2 Sample pretreatment Carry out sample pretreatment in accordance with the provisions of GB/T 15399.

4.2.3 Determination Inject the standard solution containing cysteine, methionine sulfone and amino acids into the automatic amino acid analyzer; use sodium citrate buffer solution and ninhydrin solution (3.2.7) of different pH and ionic strength as mobile phase and derivatization agent; adjust the instrument operation procedures and parameters, and the ratio of buffer solution reagents for elution, which shall comply with the requirements for the verification procedures of JJG 1064 Automatic amino acid analyzer; ensure that the separation degrees of threonine-serine, glycine-alanine, leucine-isoleucine and methionine sulfone-aspartic acid are not less than 85%, 90%, 80% and 85% respectively. The chromatogram of the standard solution is shown in Figure B.3 of Appendix B. In the determination, every 5 samples (i.e., 10 single samples) form a group, and cysteine, methionine sulfone and amino acid standard solutions are inserted between the groups

6.2.6 Sodium citrate buffer solution [pH = 2.2, c(Na+) =

0.2 mol/L]: same as 3.2.4.

6.2.7 Amino acid standard stock solution: same as 3.2.5.

6.2.8 Amino acid standard working solution: same as 3.2.6.

6.2.9 Sodium citrate buffer solutions and ninhydrin solutions of different pH and ionic strength: same as 3.2.7.

6.3 Instruments and equipment

6.3.1 Automatic amino acid analyzer: same as 3.3.1.

6.3.2 Balance: same as 3.3.2.

6.3.3 Centrifuge: same as 3.3.6.

6.3.4 Ultrasonic oscillator.

6.4 Test sample Prepare the sample according to GB/T 20195; crush it through a

0.25 mm sieve; mix it evenly; store it in a sealed container for later use.

6.5.1 Sample pretreatment

6.5.1.1 Additive premix feed Perform two tests in parallel. Weigh 1 g ~ 2 g of sample (accurate to

0.2 mg); add 30 mL of hydrochloric acid extraction solution (6.2.3); use ultrasonic oscillator to extract for 15 min; let it stand for a while; filter the supernatant into a 100 mL volumetric flask; repeat the extraction twice with 15 mL ~ 20 mL of water; filter the supernatant into the above volumetric flask; use water to rinse the residue on the extraction flask and filter paper; fix the volume. If the filtration is too slow during the sample extraction process, centrifuge for 10 minutes (4 000 r/min), combine the supernatant and add water to fix the volume. Shake well and use the filtrate for determination.

6.5.1.2 Compound feeds, concentrated feeds and concentrate supplements Perform two tests in parallel. Weigh 2 g ~ 5 g (accurate to