T/CBFIA 02002-2020 in English

Technical background of the standard

This standard was jointly formulated by the China Bio-fermentation Industry Association and 8 other units including Wuhan Xinhua Yang Biology. Aiming at the industry pain point of phytase activity detection in feed, a phosphorus release rate determination system based on spectrophotometry was established. Compared with GB/T 18634-2005, this method innovatively adopts two-stage simulated digestion (stomach/intestinal environment) to more realistically reflect the effect of phytase under actual feeding conditions.

Core principle analysis

Phytase catalyzes the hydrolysis of phytic acid phosphorus to generate inorganic phosphorus under pH2.5-5.5 conditions, and the absorbance at 415nm is determined by the vanadium molybdenum yellow color reaction. Key technological breakthroughs include:

Key operating points

1. Establishment of standard curve

Prepare 1.5625-25.000μmol/mL gradient standard solution according to Table 1. After color development, centrifuge at 4000r/min for 10min to eliminate turbidity interference.

2. Sample pretreatment

The corn and soybean meal mixture (7:3) needs to be crushed and passed through a 0.42mm sieve. For total phosphorus determination, high-temperature ashing treatment is performed according to GB/T 6437.

3. Enzymatic process control

• The phytase solution needs to be diluted to 50U/mL with a buffer containing Triton X-100
• Add 49mL of pH2.5 hydrochloric acid solution in the stomach stage
• Add 50mL of pH5.5 acetic acid buffer in the intestine stage

Result calculation specification

Phosphorus release rate calculation formula:
R=(P₂-P₁)/P₀×100%
Wherein P₂ is the inorganic phosphorus content of the enzymatic group, P₁ is the blank group, and P₀ is the total phosphorus content. The allowable deviation of parallel determination is ≤10%.

Implementation Suggestions

1. Key points of quality control

① The colorimetric solution must be prepared and used immediately; ② The pH meter must be calibrated daily; ③ If the supernatant is turbid after centrifugation, it must be filtered

2. Troubleshooting of Common Problems