Amendment to the Ordinance for Enforcement of the Food Sanitation Act and the 
Specifications and Standards for Foods, Food Additives, Etc. 
 
The government of Japan will designate Psicose Epimerase as an authorized food 
additive and establish the standards for use and the compositional specifications. 
 
Summary 
Japan prohibits the sale etc. of food additives which are not designated by the 
Minister of Health, Labour and Welfare (hereinafter referred to as “the Minister”) 
under Article 10 of the Food Sanitation Act (Act No. 233 of 1947; hereinafter referred 
to as “the Act”). In addition, when specifications or standards for food additives are 
stipulated in the Specifications and Standards for Foods, Food Additives, Etc. 
(Ministry of Health and Welfare Notification No. 370, 1959) pursuant to Article 11 of 
the Act, Japan prohibits the sale etc. of those additives unless they meet the 
specifications or the standards. 
 
In response to a request from the Minister, the Committee on Food Additives of the 
Food Sanitation Council under the Pharmaceutical Affairs and Food Sanitation 
Council (hereinafter referred to as “the Committee”) has discussed the adequacy of 
the designation of Psicose Epimerase as a food additive. The conclusion of the 
Committee is outlined below. 
 
Outline of conclusion 
The Minister, pursuant to Article 10 of the Act, should designate Psicose Epimerase 
as a food additive having no risk to harm human health and establish the standards 
for use and the compositional specifications pursuant to Article 11 of the Act (see 
Attachment for the details). 
   
 
Psicose Epimerase 
プシコースエピメラーゼ 
 
Standard for Use (draft) 
 
Not established. 
 
 
Compositional Specifications (draft) 
 
Substance Name  Psicose Epimerase, Allulose Epimerase 
CAS Number  [1618683-38-7] 
Definition  Psicose Epimerase is an enzyme that mutually isomerizes fructose and 
psicose to each other. It is derived from the culture of Escherichia coli (limited to E. coli 
K12 W3110 strain) in which the psicose epimerase gene, intrinsically occurring in the 
bacterium (limited to Arthrobacter globiformis), is introduced. 
It may contain foods used exclusively for bulking, powdering, diluting, stabilizing, or 
preserving it or for adjusting its activity. It may also contain food additives used for 
bulking, powdering, diluting, stabilizing, or preserving it or for adjusting its pH or 
activity. 
Enzyme Activity  Psicose Epimerase has an enzyme activity of not less than 230 units 
per gram. 
Description  Psicose Epimerase occurs as a light to dark brown liquid or as a gray 
powder. 
Identification  When tested by the enzyme activity determination specified below, 
Psicose Epimerase shows activity. 
Purity  
(1) Lead  Not more than 5 μg/g as Pb (0.80 g, Method 1, Control Solution: Lead 
Standard Solution 4.0 mL, Flame Method). If the residue does not dissolve in 5 mL of 
diluted nitric acid (1 in 100) in the preparation of the test solution specified in Method 1, 
proceed as directed in Method 3. 
(2) Arsenic  Not more than 3 μg/g as As (0.50 g, Method 5, Standard Color: Arsenic 
Standard Solution 3.0 mL, Apparatus B). 
Microbial Limits  Proceed as directed under Microbial Limit Tests. 
Total plate count: Not more than 50,000 per gram.  
 
Escherichia coli: Negative per test. 
Salmonella: Negative per test. 
Sample Fluid  Prepare as directed in Method 3 for total plate count. 
Pre-enrichment Culture  Prepare as directed in Method 3 for the Escherichia coli 
test and Method 2 for the Salmonella test. 
Enzyme Activity Determination 
(i) Substrate Solution  Weigh 0.18 g of D(+)-psicose, dissolve it in water, and add 
water to make exactly 5 mL. Prepare fresh before use. 
(ii) Sample Solution  Weigh accurately about 1.0 g of Psicose Epimerase, and 
dissolve it in the diluent prepared as directed below to make a constant volume so that 
the resulting solution has 4−10 units per mL.  The diluent: Mix phosphate buffer (0.05 
mol/L) at pH 8.0 and magnesium chloride TS (1 mol/L) at a rate of 199 : 1. 
(iii) D(−)-Fructose Standard Solutions  Weigh accurately about 0.27 g of D(−)-
fructose for enzyme activity determination, dissolve it in water to make exactly 100 mL. 
Use this solution as the standard stock solution. Prepare four standard solutions with 
different concentrations—10 mol, 5 mol, 3 mol, and 1 mol of D(−)-fructose (C6H12O6 
= 180.16) per mL—by exactly diluting the standard stock solution with water to 1.5, 3, 
5, and 15 times, respectively. 
(iv) Procedure 
Test Solution  Place 0.100 mL of the sample solution into a test tube, mix it with 
0.400 mL of the diluent prepared in (ii), lid the test tube, and equilibrate at 50°C ± 0.5°C 
for 5 minutes. Add 0.500 mL of the substrate solution to the test tube, mix, incubate at 
50°C ± 0.5°C for exactly 10 minutes, and then heat in a water bath for 2 minutes. After 
cooling, add about 100 mg of strongly acidic cation-exchange resin and about 100 mg of 
weakly basic anion-exchange resin (free-form), both whose surface water was previously 
removed with a filter paper, shake for 15 minutes, and filter through a membrane filter 
(pore size: 0.2 m). 
For strongly acidic cation-exchange resin, wash with water before use as directed in 
the Strongly Acidic Cation-exchange Resin section under C. REAGENTS, SOLUTIONS, 
AND OTHER REFERENCE MATERIALS, beginning with “Weigh about 50 g of strongly 
acidic cation-exchange resin,” and confirm that the pH of the effluent is 5.0−6.5. 
Reference Solution  Place 0.100 mL of the diluent into a test tube instead of the 
sample solution, and proceed as directed for the test solution. 
Determination  Analyze 10 L portions of the test solution, the reference solution, 
and the four D(−)-fructose standard solutions by liquid chromatography using the 
operating conditions given below. Prepare a calibration curve from the peak area and 
the concentration (mol/mL) of each D(−)-fructose standard solution. Measure the peak 
areas of D(−)-fructose in the test solution and the reference solution, and then determine 
the concentration (mol/mL) of D(−)-fructose in each solution from the calibration curve. 
Calculate the enzyme activity by the following formula. One unit of the enzyme activity  
 
is equivalent to the amount of the enzyme required to liberate 1 mol of D(−)-fructose 
per minute when determined as directed in the Procedure. 
Enzyme activity (unitg⁄)  = (C − CB)× V
 M  
CT = concentration (mol/mL) of D(−)-fructose in the test solution, 
CB = concentration (mol/mL) of D(−)-fructose in the reference solution, 
VT = whole volume (mL) of the sample solution prepared, 
M = weight (g) of the sample. 
 
Operating conditions 
Detector: Differential refractometer. 
Column: A stainless steel tube (8 mm internal diameter and 30 cm length). 
Column packing material: About 9-m cation-exchange resin for liquid 
chromatography (Ca-form). 
Column temperature: 80°C. 
Mobile phase: Water. 
Flow rate: 0.4 mL/min. 
 
 
Reagents, Solutions, and Other Reference Materials 
D(−)-Fructose for Enzyme Activity Determination  C6H12O6  [57-48-7]  Colorless to 
white crystals or powder. 
Specific rotation  [α]: −90 to −94°  Weigh accurately about 4 g of D(−)-fructose 
for enzyme activity determination, add 0.2 mL of ammonia TS and 80 mL of water to 
dissolve it, allow to stand for 30 minutes, and add water to make exactly 100 mL. 
Measure the optical rotation of the resulting solution. 
Purity (1) Clarity of solution  Clear (1.0 g, water 20 mL). 
(2) Loss of drying  Not more than 2.0% (reduced pressure, 18 hours). 
(3) Related substances Prepare a test solution by dissolving 20 mg of D(−)-fructose 
for enzyme activity determination in 2 mL of water. Prepare a control solution by 
diluting exactly measured 1 mL of the test solution with water to exactly 50 mL. Analyze 
10 L portions of the test solution and the control solution by liquid chromatography 
using the operating conditions given below. Continue the chromatography for three 
times the retention time of the main peak, and measure the peak areas. The sum of the 
areas of all peaks, other than the main peak and the solvent peak, from the test solution 
is not greater than the area of main peak from the control solution.    
Operating conditions 
Detector: Differential refractometer.  
 
Column: A stainless steel tube (3−8 mm internal diameter and 15−30 cm length). 
Column packing material: 5−10 m aminopropyl-bonded silica gel for liquid 
chromatography.   
Column temperature: A constant temperature of 35−40°C. 
Mobile phase: A 7 : 3 mixture of acetonitrile/water. 
Flow rate: Adjust the retention time of D(−)-fructose to 4−7 minutes. 
Magnesium Chloride TS (1 mol/L)  Dissolve 203 g of magnesium chloride hexahydrate 
in water to make 1000 mL. 
D(+)-Psicose  C6H12O6  [551-68-8]  A white to slightly pale yellow crystalized powder 
or powder.  
Specific rotation  [α]: +2.0 to +6.0° (0.1 g, water, 10 mL). 
Purity  Related substances  Prepare a test solution by dissolving 20 mg of D(+)-
psicose in 2 mL of water. Prepare a control solution by diluting exactly measured 1 mL 
of the test solution with water to exactly 50 mL. Analyze 10 L portions of the test 
solution and the control solution by liquid chromatography using the operating 
conditions given below. Continue the chromatography for three times the retention time 
of the main peak, and measure the peak areas. The sum of the areas of all peaks, other 
than the main peak and the solvent peak, from the test solution is not greater than the 
area of main peak from the control solution. 
Operating conditions 
Detector: Differential refractometer. 
Column: A stainless steel tube (3−8 mm internal diameter and 15−30 cm length). 
Column packing material: 5−10 m aminopropyl-bonded silica gel for liquid 
chromatography.   
Column temperature: A constant temperature of 35−40°C. 
Mobile phase: A 7 : 3 mixture of acetonitrile/water. 
Flow rate: Adjust the retention time of D(+)-psicose to 6−9 minutes.