Amendments of standards and specifications of Phytase   
 
Ministry of Agriculture, Forestry and Fisheries (MAFF)  will add standards and specifications to 
“Ministerial Ordinance on the Specifications and Standards of Feeds and Feed Additives ” 
(Ordinance No. 35 of July 24th, 1976 of the Ministry of Agriculture and Forestry).   
Major additions are  as follows.  
 
 
Phytase  No.2（6） 
 
Specifications for method  of manufacture  of feed in general  
Phytase No.2 （6）is allowed to be used as feed additives for pigs, broilers, layers, quails, fish and 
crustaceans only.  
 
Specifications for feed additives  
Active Substance  
Compositional specifications  
Unit    ≥ 200,000 phytic acid degradation /g 
Physical and chemical properties  
(1) It comes in  yellow -brown powde r or particles . 
(2) pH of aqueous solution (1 part solute/100 parts solution ) is 4.0 to 5.0. 
(3) It has the best enzyme  activity when pH is between 2.5 and 5.0. 
Purity test:  
(1) Lead: 0.1 g (0.95~1.04 g) of this product is weighed. When lead is tested using this sample by 
the lead test method (Method No. 1 of atomic absorption spectrophotometry test method), the 
concentration of the solution shall not exceed  5 µg/g.  
(2) Arsenic: Purity test No.2 for Active Substance of Phytase No.1.  
(3) Antibacterial activity:  Purity test No.3 for Active Substance of Phytase No.1.  
Loss on drying: ≤ 12.0% (1  g, 105 °C, 3 hours)  
Ignition residue : ≤ 25.0% ( 0.5 g) 
Strength test of enzyme:  Method No.3 of Strength test of decomposing phytic acid.  
 Preparation of Sample solution:  2.00 g (1.995 ~ 2.004 g) of this product is diluted  with 0.2 
mol/L citric acid /sodium citrate buffer (pH 5.5)  to total 100mL . This solution is stirred at room 
temper ature for 1 hour and centrifuged (3,400 × g for 10 minutes), and the supernatant is diluted 
with the same buffer to 0.1 phytic acid degradation /mL as sample solution.  
 
Standard  for method  of manufacture  
Genetically modified Komagataella  pastoris  should be cultured. T he cultured solution  should be 2 
 
filtered. The bacteria in the cultured solution has to be  removed by filtration, and the filtrate  must 
be spray -dried. 
Standard  for method  of storage  
The products  must be stored in light-shielding  and airtight  containers.  
 Standard for method of labelling  
  Containers or packaging of t he products must be labeled that pH  value  (up to the first decimal 
place)  showing the maximum  enzyme  activity.  
 
Product  No. 1 
Compositional specifications  
This product comes  in the form of liquid  and is produced by  adding  aqueous solution containing 
sucrose to the Active Substance of Phytase  No.2(6). 
Unit    This product contains the amount of Phytase  corresponding to 85 to 17 0% of the phytic acid 
degradation  unit of activity shown  on the label.  
Strength test of enzyme: Method No.3 of Strength test of decomposing phytic acid . 
Preparation of Sample solution:  5 mL of this product is  diluted with 0.2 mol/L citric acid/sodium 
citrate buffer (pH 5.5) to total 50mL. This solution is diluted with the same buffer to 0.1 phytic acid 
degradation  /mL as sample solution.  
Standard for method of storage   
Same as the standard for method of  Active Substance  of Phytase  No.2(6). 
Standard for method of labelling  
Containers or packages of the products must be labeled pH value (up to the first decimal place) that 
shows the maximum enzyme activity.   
 
Product  No. 2 
Compositional specifications  
This product comes  in the form of pieces , particles  or powder  and is produced by  adding flour , 
mixing with aqueous solution  containing α-starch  to the Active Substance of Phytase No.2( 6), 
granulating  and drying . If necessary, it is mixed with excipient substances.  
Unit   This product contains the amount of Phytase  corresponding to 85 to 17 0% of the phytic acid 
degradation  unit of activity shown on the label.  
Strength test of enzyme: Method No.3 of Strength test of decomposing phytic acid . 
Preparation of Sample solution:  Strength test of enzyme  for Active Substance of Phytase No.2 
(6). 
Standard for method of storage  
Same as the standard for method of Active Substance  of Phytase  No.2(6). 
Standard for method of labelling  
Containers or packag es of the products must be labeled pH value (up to the first decimal place) that 3 
 
show s the maximum  enzyme activity . 
 
Analytical method  
 Strength test of enzyme  
  Method No.3 of Strength test of decomposing phytic acid  
    (See Japanese  attachment .)