Attachment 
 
Isomaltodextranase 
イソマルトデキストラナーゼ 
 
 
Compositional Specifications 
Definition  Isomaltodextranase includes dextran-degrading enzymes derived from 
the culture of bacteria limited to the genus Arthrobacter. 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 exclusively for bulking, 
powdering, diluting, stabilizing, or preserving it or for adjusting its pH or activity. 
Description  Isomaltodextranase occurs as white to dark brown granules, powder, 
or paste, or as a colorless to dark brown liquid. It is odorless or has a characteristic 
odor. 
Identification test  Isomaltodextranase complies with the Isomaltodextranase 
Activity Test. 
Purity test 
(1) Lead  Not more than 5 g/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 g/g as As (0.50 g, Method 5, Standard Color: 
Arsenic Standard Solution 3.0 mL, Apparatus B). 
Microbial Limits  Proceed as directed under the 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. 
The requirement of total plate count can be disregarded if unsterilized 
Isomaltodextranase is used for captive consumption in the manufacture of food that 
will be sterilized or pasteurized before the completion of the final product. Isomaltodextranase Activity Test  Perform the test using the method given below. 
If the activity test cannot be performed by the given method, appropriate 
replacement of the sample dilution factor, buffer solution, and temperature for 
reaction is acceptable where this is scientifically justifiable. 
Method 1 
Sample Solution  Weigh 1.0 g of Isomaltodextranase, add water or acetate buffer 
(0.05 mol/L) at pH 4.5 to dissolve it or disperse it uniformly, and make 10 mL. As 
necessary, prepare a 10-fold, 100-fold, or 1000-fold dilution by adding water or the 
same buffer to the resulting solution. 
Substrate Solution  Weigh 1.25 g of dextran (molecular weight: 150,000), and 
dissolve it in acetate buffer (0.05 mol/L) at pH 4.5 to make 100 mL. 
Test Solution  To 5 mL of the substrate solution, equilibrated at 40C, add 0.2 
mL of the sample solution, and mix. Incubate the mixture at 40C for 20 minutes. 
Transfer 1 mL of this solution into a test tube containing 2 mL of Somogyi copper TS. 
Cover the mouth of the test tube with a glass bead, heat in a water bath for 10 minutes, 
and cool to room temperature. Add 2 mL of Nelson TS, mix, allow to stand for 30 
minutes, and add 5 mL of water. 
Control Solution  To 5 mL of the substrate solution, equilibrated at 40C, add 0.2 
mL of the sample solution and mix. Transfer 1 mL of this solution into a test tube 
containing 2 mL of Somogyi copper TS, and mix immediately. Cover the mouth of the 
test tube with a glass bead, heat in a water bath for 10 minutes, and cool to room 
temperature. Add 2 mL of Nelson TS, mix, allow to stand for 30 minutes, and add 5 
mL of water. 
Procedure  Measure the absorbance of the test solution and the control solution 
at a wavelength of 520 nm. The absorbance value of the test solution is higher than 
that of the control solution. If the test solution and the control solution are turbid, 
centrifuge them, and use the supernatants. 
Method 2 
Sample Solution  Weigh 1.0 g of Isomaltodextranase, add water or acetate buffer 
(0.05 mol/L) at pH 4.5 to dissolve it or disperse it uniformly, and make 10 mL. As 
necessary, prepare a 10-fold, 100-fold, 1000-fold, or 10,000-fold dilution by adding 
water or the same buffer to the resulting solution. 
Substrate Solution  Weigh 1.25 g of dextran (molecular weight: 150,000), and 
dissolve it in acetate buffer (0.05 mol/L) at pH 4.5 to make 50 mL. 
Test Solution  Add 500 μL of the sample solution to 500 μL of the substrate 
solution and mix. Incubate the mixture at 40C for 4 hours, heat in a water bath for 
10 minutes, and cool to room temperature. Standard Solution  Dissolve 0.13 g of isomaltose in 10 mL of water. 
Control Solution  Add 500 μL of the sample solution to 500 μL of the substrate 
solution, and mix. Heat immediately in a water bath for 10 minutes and cool. 
Procedure  Analyze 2-μL portions of the test solution, standard solution, and 
control solution by thin-layer chromatography using a 6 : 4 : 1 mixture of 1-
butanol/pyridine/ water as the developing solvent. Use a thin-layer plate coated with 
silica gel for thin-layer chromatography as the solid support and then dried at 110C 
for 1 hour. Stop the development when the solvent front has ascended to a point 
about 15 cm above the original line, and air-dry the plate. Spray with 15% sulfuric 
acid–methanol TS, heat at 100C for 10 minutes, and examine. One of the spots from 
the test solution has the same Rf value as that from the standard solution and is 
darker in color than the spot with the same Rf value from the control solution. 
 
Reagents, Solutions, and Other Reference Materials 
Isomaltose  C12H22O11  Use a product suitable for the corresponding enzyme activity 
tests. 
 
Dextran (molecular weight: 150,000)  (C6H10O5)n  Use a product suitable for the 
corresponding enzyme activity tests. 
  Japanese Persimmon Color 
カキ色素 
 
 
Compositional Specifications 
Definition 
Japanese Persimmon Color is obtained from the fruits of the Diospyros kaki Thunb. 
It is produced from fermented and roasted fruits through extraction with hydrous 
ethanol or through extraction with an alkaline solution and neutralization. It may 
contain dextrin or lactose. 
Color value  The Color Value E1cm10% of Japanese Persimmon Color is not less 
than 20 and is in the range of 90-110% of the labeled value. 
Description  Japanese Persimmon Color occurs as red-brown to dark brown lumps, 
powder, paste, or liquid. It has a slight characteristic odor.   
Identification test 
(1) Weigh an amount of Japanese Persimmon Color equivalent to 2.5 g of Japanese 
persimmon color with a Color Value 20, and dissolve it in 100 mL of citrate buffer 
(pH 7.0). The resulting solution is red-brown to dark brown. 
(2) To 5 mL of the solution prepared in Identification (1), add 2–3 drops of 
hydrochloric acid and allow to stand. A red-brown to dark brown precipitate is 
produced. 
(3) To 5 mL of the solution prepared in Identification (1), add 2 mL of iron(III) 
chloride hexahydrate solution (1 in 50). A gray to dark brown precipitate is produced. 
(4) Weigh an amount of Japanese Persimmon Color equivalent to 1 g of Japanese 
persimmon color with a Color Value 20, and dissolve it in 100 mL of sodium 
hydroxide solution (1 in 250). To 5 mL of this solution, add 10 mL of diluted 
hydrochloric acid (9 in 1000) and 0.1 mL of zinc chloride TS (pH 3.0), and stir. Stopper 
it, and warm at 50C for 20 minutes. If necessary, centrifuge at 3000 rpm for 10 
minutes. A yellow-brown to dark brown precipitate is produced. 
Purity test 
(1) Lead  Not more than 2 μg/g as Pb (2.0 g, Method 1, Control Solution: Lead 
Standard Solution 4.0 mL, Flame Method). 
(2) Arsenic  Not more than 3 μg/g as As (0.50 g, Method 3, Standard Color: 
Arsenic Standard Solution 3.0 mL, Apparatus B). Color value determination  Proceed as directed under Color Value Determination, 
according to the following operating conditions: 
Operating Conditions 
Solvent: Citrate buffer (pH 7.0). 
Wavelength: 500 nm. 
 
  Enju Extract 
Japanese Pagoda Tree Extract 
エンジュ抽出物 
 
 
The compositional specifications for Enju extract were revised as follows: 
 
 
 
 
 
dl-α-Tocopherol 
dl-α-トコフェロール 
 
The compositional specifications of dl-α-Tocopherol were revised as follows: 
 
 
 After the revision Before the revision 
Identification 
(3) Dissolve 20 mg of Enju Extract in 100 
mL of ethanol (95) and to 2 mL of this 
solution, add ethanol (95) to make 20 mL. 
The solution exhibits absorption maxima 
at wavelengths of approximately 257 nm 
and 361 nm. Identification 
(3) Dissolve 10 mg of Enju Extract in 
100 mL of ethanol (95). The solution 
exhibits absorption maxima at 
wavelengths of approximately 257 nm 
and 361 nm. 
After the revision Before the revision 
Description 
dl--Tocopherol is a light yellow to red-
brown, clear, viscous liquid. It is odorless. Description 
dl--Tocopherol is a light yellow to 
yellow-brown, viscous liquid. It is 
odorless.