- 1 -  
Phytase No.2(7) 
Specifications for method of ma nufacture of feed in general 
 Phytase No.2(7) is allowed to be used as feed additives for pigs, broilers, layers and quails  
only.  Specifications for feed addtives  
Active Substance 
Compositional specification 
Unit ≥150,000 phytic acid degradation/g.  
Physical and Chemical Properties  
(1) It comes in greenish brown liquid.  
(2) pH of aqueous solution (1 part  solute/100 parts solution) is 4.0–5.0.  
(3) It has the best enzyme activ ity when pH is between 2.5 and 5.5. 
Purity test  
(1) Lead:  Weigh 1.0 g (0.95–1.04 g) of this product and prepare a test solution according to 
the Lead Test (Atomic Absorption Spectropho tometry Method I). The concentration of 
lead must be 5 μg/g or below. To prepare a Standa rd Lead Solution, use a volumetric 
pipette to pipet and transfer 0.5 mL into a 10- mL volumetric flask and add nitric acid (1 
in 150) to the 10-mL mark.  
(2) Arsenic:  Purity test No.2 for Active Substance of Phytase No.1.  
(3) Antimicrobial activity:  Purity test No.3 for Active Substance of Phytase No.1. 
Ignition residue: Not more than 5.0% (0.5 g).  
Strength test of enzyme: Method No.4 of Strength test of decomposing phytic acid.  
Preparation of sample solution:  Weigh 0.5 g of this product to  three significant digits and 
record the weight. Transfer to a 100-mL volumetric flask, and add 80 mL of 0.25 mol/L 
sodium acetate-hydrochloric acid buffer solution (pH5.5) containing polysorbate 20 
(0.01%). Using a stirrer, stir for 30 minutes at room temperat ure, remove the stirrer, and 
add the same buffer to the 100-mL mark. Use this solution as a stock solution. Take a 
sufficient amount of the stoc k solution using a micropipette. Add th e same buffer to 
accurately dilute and make a test solution containing approximately 0.1 unit of phytic acid 
degradation per mL.  
Standard for manufacture 
Culture a Komagataella phaffii strain containing a phytase-pr oducing recombinant. At the 
end of the culture, filter the cult ure to remove bacterial cells. C oncentrate the filtrate to obtain 
Phytase No.2(7). Standard for method of storage  - 2 - Store in an air-tight cont ainer protected from light.  
Standard for method of labelling  
Containers or packaging of th e products must be labeled that  pH value (up to the first 
decimal place) showing the maximum enzyme activity.  Product No.1 
Compositional specification 
This product comes in the form of liquid and is produced by adding sodium benzoate 
followed by pH adjustment with  hydrochloric acid and mixing with water and glycerin as 
necessary.  
Unit: This product contains the amount of P hytase corresponding to  85 to 170% of the 
phytic acid degradation unit of  activity shown on the label. 
Strength test of enzyme: Test according to Method 4 of the Phytase Activity Test.  
Preparation of sample solution:  Weigh 1 g of this product to three significant digits and 
record the weight. Transfer to a 100-mL volumetric flask, and add 80 mL of 0.25 mol/L 
sodium acetate-hydrochloric acid buffer solution (pH5.5) containing polysorbate 20 
(0.01%). Using a stirrer, stir for 30 minutes at room temperat ure, remove the stirrer, and 
add the same buffer to the 100-mL mark. Use this solution as a stock solution. Take a 
sufficient amount of the stoc k solution using a micropipette. Add th e same buffer to 
accurately dilute and make a test solution containing approximately 0.1 unit of phytic acid 
degradation per mL.  
Standard for method of storage  
Same as the standard for method of Active Substance of Phytase No.2(7). 
Standard for method of labelling  
Same as the standard for method of Active Substance of Phytase No.2(7). 
 Product No.2 
Compositional specification 
This product comes in the form of powder or  granules and is produced by adding wheat or 
α-starch to spray-dried Phytase No.2(7). 
Unit: This product contains the amount of P hytase corresponding to  85 to 170% of the 
phytic acid degradation unit of  activity shown on the label. 
Enzyme Activity Test: Test according to Method 4 of the Phytase Activity Test.  
Preparation of sample solution:  Weigh 1 g of this product to three significant digits and 
record the weight. Transfer to a 250-mL conical flask, and add 100 mL of 0.25 mol/L 
sodium acetate-hydrochloric acid buffer solution (pH5.5) containing polysorbate 20 
(0.01%). Using a stirrer, stir fo r 30 minutes at room temperature. Centrifuge about 2 mL at 
11,000 × g for 3 minutes. Use the obtained supern atant as a stock solution. Take a 
sufficient amount of the stoc k solution using a micropipette. Add th e same buffer to - 3 - accurately dilute and make a test solution containing approximately 0.1 unit of phytic acid 
digestive activity per mL.  
Standard for method of storage  
Same as the standard for method of Active Substance of Phytase No.2(7). 
Standard for method of labelling  
Same as the standard for method of Active Substance of Phytase No.2(7). 
 Product No.3 
Compositional specification 
This product comes in the form of small piec es or granules is produced by adding wheat  to 
Phytase No.2(7) followed by drying, coating with vegetable oil and glycerin fatty acid ester. 
If necessary, it is mixed with chaff.  
Unit: This product contains the amount of P hytase corresponding to  85 to 170% of the 
phytic acid degradation unit of  activity shown on the label. 
Enzyme Activity Test: Test according to Method 4 of the Phytase Activity Test.  
Preparation of test solution:  Strength test of enzyme for Active Substance of Phytase 
No.2(7).  
Standard for method of storage  
Same as the standard for method of Active Substance of Phytase No.2(7). 
Standard for method of labelling  
Same as the standard for method of Active Substance of Phytase No.2(7). 
 Analytical method 
Strength test of enzyme 
Method No.4 of Strength test of decomposing phytic acid  (See Japanese attachment.)