Amendment to the Enforcement Ordinance of the Food Sanitation Law and the 
Standards and Specifications for Foods and Food Additives 
 
The government of Japan will designate Aspara ginase as an authorized food additive. 
 Summary 
Under Article 10 of the Food Sanitation Law (hereinafter referred to as the “Law”), 
food additives shall not be used or marketed  without authorization by the Minister of 
Health, Labour and Welfare (hereinafter referred to as “the Minister”). In addition, when 
specifications or standards are established for f ood additives based on Article 11 of the Law and 
stipulated in the Ministry of Health, Labour a nd Welfare Notification (Ministry of Health and 
Welfare Notification No. 370, 1959), those additiv es shall not be used or marketed unless they 
meet the standards or specifications. 
 
In response to a request from the Minister, the Committee on Food Additives of the 
Food Sanitation Council that is establishe d under the Pharmaceutical Affairs and Food 
Sanitation Council has discussed the adequacy of the designation of Asparaginase as a 
food additive. The conclusion of the committee is outlined below.  Outline of conclusion  
The Minister, based on Article 10 of the Law, should designate Asparaginase, as a 
food additive unlikely to harm human health, and establish standard for use and compositional specifications, based on Ar ticle 11 of the law (see Attachment). 
    Attachment 
Asparaginase 
アスパラギナーゼ  
 
 
 Standards for use 
Not established.  Compositional specifications 
Substance name  Asparaginase  
Definition  Asparaginase is obtained from the filamentous fungi,  A. niger  ASP-72, in 
which asparaginase productivity is improv ed by amplifying the asparaginase gene 
intrinsically occurring in Aspergillus niger. It is an enzyme that hydrolyzes asparagine 
into aspartic acid and ammonia. It may cont ain glycerine, maltodextrin, or wheat flour. 
Enzyme Activity  Asparaginase contains the enzyme activity equivalent to not less 
than 2375 units per gram or milliliter.    
Description   Asparaginase occurs as a clear, yell ow to brown liquid or as pale gray or 
slightly yellowish white granules.   
Identification  When tested by the enzyme activity determination, Asparaginase 
exhibits activity.   
Purity  
(1) Lead  Not more than 5.0 μg/g as Pb. 
Test Solution  Weigh 0.8 g of Asparaginase into a platinum, quartz, or porcelain 
crucible or a quartz beaker, and moisten it wit h a small amount of diluted sulfuric acid 
(1 in 4). Heat it by increasing the temper ature gradually until the sample is carbonized 
and the white fumes of sulfuric acid are no longer evolved. If necessary, add diluted sulfuric acid (1 in 4) again, and heat until the sample is almost carbonized. For a liquid 
s a m p l e  o r  a  s a m p l e  t h a t  i s  h a r d  t o  b e  c a rbonized, concentrated sulfuric acid may be 
used, instead of diluted sulfuric acid (1 in 4).  After the sample is carbonized, lid the 
crucible or beaker loosely if necessary, heat in an electric furnace by increasing the 
temperature gradually, and ignite at 450–600 C to incinerate. If any carbonized residue 
is present, crush the residue with a glass ro d if necessary, moisten with 1 ml of diluted sulfuric acid (1 in 4) and 1 ml of nitric ac id, heat until the white fumes of sulfuric acid 
are no longer evolved, and ignite in the elec tric furnace to completely incinerate it. To 
residue, add 10 ml of diluted hydrochloric acid (1 in 4), heat on a water bath, and 
evaporate to dryness. To the residue, add a sm all amount of diluted nitric acid (1 in 100), 
and warm to dissolve it.  After cooling, add d iluted nitric acid (1 in 100) to make exactly 
10 ml.  
When incineration is done at 500 C or below, a heat-resistant glass beaker can be 
used.     
Control Solution   To 1 ml of Lead Standard Stock Solution, exactly measured, add 
water to make exactly 100 ml. To exactly me asured 4 ml of this solution, add diluted 
nitric acid (1 in 100) to make 10 ml.  
Procedure   Proceed as directed in Method 1 of the Lead Limit Test.  
(2) Arsenic  Not more than 4.0 μg/g as As 2O3 (0.50 g, Method 3, Apparatus B). 
Microbial Limits  Proceed as directed in the Microbial  Limit Tests. The total bacterial 
count is not more than 50,000/g. Escerichia coli and Salmonella are negative. The 
Salmonella  tests should be conducted in the same  manner as for the microbial limit test 
f o r  N i s i n .      Enzyme Activity Determination  
(i) Substrate Solution  Weigh 1.50 g of 
L-asparagine monohydrate, add citric acid–
sodium hydroxide buffer (pH 5.0), and dissolve it  completely by stirring. Add citric acid–
sodium hydroxide buffer (pH 5.0) again to make exactly 100 ml. Prepare fresh before 
use.   
(ii) Sample Solution  Weigh accurately about 2.5 g of Asparaginase, dissolve in 20 
ml of citric acid–sodium hydroxide buffer (p H 5.0) , and then add citric acid–sodium 
hydroxide buffer (pH 5.0) to make exactly 25 ml. Dilute this solution with citric acid–
sodium hydroxide buffer (pH 5.0) to prep are a solution containing 6 units/ml.  
(iii) Control Stock Solution  Weigh an amo unt of asparaginase for enzyme activity 
determination equivalent to 4000 units, disso lve in 20 ml of citric acid–sodium 
hydroxide buffer (pH 5.0) , and then add citr ic acid–sodium hydroxide buffer (pH 5.0) to 
make exactly 25 ml. Dilute this solution wit h citric acid–sodium hydroxide buffer (pH 
5.0) to prepare a solution containing 6 units/ml. 
(iv) Ammonium Sulfate Standard Soluti ons  Weigh accurately about 3.9 g of 
ammonium sulfate, add 40 ml of citric acid–sodium hydroxide buffer (pH 5.0), and 
shake for 15 minutes. Again add citric acid–s odium hydroxide buffer (pH 5.0) to make 
50 ml. Dilute this solution with citric acid–s odium hydroxide buffer (pH 5.0) to prepare 
4-fold, 6-fold, 10-fold, 30-fold, and 60- fold dilutions, respectively.   
(v) Procedure   Test Solution and Control Solution   Place 2.0 ml portions of the substrate solution 
into two separate test tu bes, and warm them at 37 C for 10 minutes. Add 0.100 ml of 
the sample solution to one test tube and 0. 100 ml of the control st ock solution to the 
other, and stir. Warm them at 37 C exactly for 30 minutes, add 0.400 ml portions of 
trichloroacetic acid solution (1  in 4) to them, stir, and add 2.5 ml of water to each, and 
again stir. Take 0.100 ml from each test tube , and add 4.0 ml of water and 0.850 ml of  
phenol-nitroprusside (basic), and stir . Add 0.850 ml of sodium hypochlorite −sodium 
hydroxide TS (for asparaginase activity test ) to each, and allow them to stand at 37 C 
for 10 minutes. Use them as the test solution and the control solution, respectively.        
Reference Solutions  Place 2.0 ml portions of the substrate solution into two 
separate test tubes, an d add 0.400 ml portions of trichloroa cetic acid solution (1 in 4) to 
them, and stir. Add 0.100 ml of the sample solu tion to one test tube and 0.100 ml of the 
control stock solution to the other, stir, and warm them at 37 C for 30 minutes. Add 2.5 
ml of water to each, and stir again. Take 0. 100 ml from each test tu be, and add 4.0 ml of 
water and 0.850 ml of phenol-nitroprusside (basic), and stir. Add 0.850 ml of sodium 
hypochlorite −sodium hydroxide TS (for asparaginase activity test) to each, allow them 
stand at 37 C for 10 minutes. Use them as the reference solutions for the test solution 
and the control solution, respectively.  
Calibration Curve   Place 2.0 ml portions of the subs trate solution into five separate 
test tubes, and warm them at 37 C for 10 minutes. To them, add 0.100 ml portions of the 
ammonium sulfate standard solutions with different concentrations instead of the 
sample solution, and proceed as directed in the preparation of the test solution.  Measure the absorbances of them against wa ter at 600 nm. Prepare a calibration curve 
using the absorbance values obtained and the ammonium sulfate concentrations in the ammonium sulfate standard solutions.   
Determination  Measure  the absorbances (A T and  AC) of the test solution and the 
control solution against wa ter at 600 nm. Also measure  the absorbances (A BT and  ABC) of 
the reference solutions for the test solution  and the control solution against water at 
600 nm. Measure the gradient, a (ml/mg),  of the calibration curve. Calculate the enzyme 
activity of asparaginase for enzyme activity  determination used for the preparation of 
the control solution by the formula given belo w. When the obtained activity is in the 
range of 91 to 109% of the labeled value, de termine the enzyme activity of the sample 
also by the formula. One unit of enzyme ac tivity corresponds to the amount of the 
enzyme required to liberate 1 μmol of ammonia per minute from L-asparagine when the 
enzyme activity is determined as directed in the Procedure.            
௙	ൈ25	ൈ2	ൈ	10ଷ
ൈ132.14	ൈ30 A = the value obtained by deducting the absorbance (A BT or ABC) of the 
corresponding reference solution from the absorbance (A T or AC) of the 
test solution or the control solu tion, whichever is appropriate, 
Df = the dilution ratio of the sample solution or the control stock solution,  
W = the weight (g) of the sample or asparaginase for enzyme activity 
determination.  
 Solutions and Reagents  
Asparaginase for Enzyme Activity Determination   Obtained from the filamentous 
fungi,  A. niger  ASP-72, in which asparaginase produc tivity is improved by amplifying 
the asparaginase gene intrinsically occurring in Aspergillus niger.  Occurs as a clear, 
yellow to brown liquid or as pa le gray or slightly yellowish white granules.  One unit of 
Asparaginase for Enzyme Activity Determin ation corresponds to the amount of the 
enzyme required to liberate 1 μmol of ammonia in one minute at pH 5.0 and at 37 C 
when L-asparagine as the substrate is used.   
L-Asparagine Monohydrate   C 4H8N2O3･H2O [K8021] 
Citric acid–Sodium Hydroxide Buffer (pH 5.0)   Dissolve 21 g of citric acid 
monohydratein 500 ml of water, adjust the pH to 5.0 with sodium hydroxide TS (2 
mol/L), and add water to make 1000 ml.       
Phenol-Nitroprusside TS (Basic)   To 8 to 10 ml of sodium hydroxide solution (13 in 50), 
add 0.1 ml of sodium nitroprusside solution  (1 in 100), stir, and add 10 ml of phenol 
solution in ethanol (5 in 8) and water to  make 50 ml. Prepare fresh before use.  
Sodium Hydroxide TS, 2 mol/L  Dissolve 80 g of sodium hydroxide in water to make 
100 mL.   
Sodium Hypochlorite −Sodium Hydroxide TS (for Asparaginase Activity Test)  To 2.5 ml 
of sodium hypochlorite TS, add water to make  10 ml. Standardize the resulting solution 
using 3 ml of it , as directed in Assay fo r “Sodium Hypochlorite” in the Monographs a nd 
adjust the solution so that it is a solution  of 0.32 to 0.38 mol/L sodium hypochlorite. 
Adjust its pH to 12.5 with sodium hydroxide solution with an appropriate concentration. 
To 3 ml of this solution, add 85 ml of wa ter, and adjust its pH to 12.5 with sodium 
hypochlorite solution with an appropriate concentration.  Add water to make 100 ml. 
Store in a cool, dark place.