Amendment to the Ordinance for Enforcement of the Food Sanitation Act  and the 
Specifications and Standards for Food s, Food Additives , Etc . 
 
The government of Japan will designate chitin -glucan  as an authorized food additive  
and establish compositional specifications  and use standards for this additive . 
 
Background  
Japan prohibits the sale  of food additives that are not designated by the Minister of 
Health, Labour and Welfare (hereinafter referred to as “the Minister”) under Article 
12 of the Food Sanitation Act (Act No. 233 of 1947; hereinafter referred to as “the 
Act”). In addition, when specifications or stand ards for food additives are stipulated 
in the Specifications and Standards for Foods , Food Additives , Etc. (Public Notice of 
the Ministry of Health and Welfare No. 370, 1959) , Japan prohibits the sale  of those 
additives unless they meet the specifications or the standards  pursuant to Article 13 
of the Act . 
 
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 referre d to as “the Committee”)  has discussed the adequacy of 
the designation of chitin -glucan  as a food additive. The conclusion of the Committee 
is outlined below.  
 
Outline of conclusion  
The Minister  should designate chitin -glucan  as a food additive unlikely to cause 
harm to human health pursuant to Article 12 of the Ac t and should establish 
compositional specificatio ns and use standards for this additive pursuant to  Article 
13 of the Act (see Attachment  for the details ). 
 
  Chitin -Glucan  
キチングルカン  
 
Standards for Use (draft)  
Permitted for use in grape juice used for wine production and grape wine only . 
Shall be used at  not more than  5 g/L in grape wine  as chitin -glucan.  When Chitin -
Glucan is used in grape juice for wine production, this additive is consid ered to be 
used in grape wine.  
The Chitin -Glucan used shall be removed before the completion of the final product.  
 
Compositional Specifications (draft)  
Substance Name  Chitin -Glucan  
Definition  
Chitin -Glucan is a copolymer composed of chitin and β-1,3-glucan that  are derived 
from the culture of filamentous fungi ( limited to  Aspergillus niger ). 
Content  
Chitin -Glucan  contains not less than 95% of chitin -glucan . 
Description   Chitin -Glucan  occurs as a white to light yellow -brown powder. It is 
odorless . 
Identification  
Chitin/ glucan ratio   25 : 75 to 60 : 40. 
Sample   Place 2.0 g of Ch itin-Glucan in a centrifuge tube, and add 40 mL of 
hydrochloric acid TS (1 mol/L). After shaking it for 30 minutes, centrifuge at 3000 
rpm for 10 minutes, and remove the supernatant. Again  add 40 mL of hydrochloric 
acid TS (1 mol/L), and perform the same st ep. Then, add 40 mL of water to the 
residue, agitate , centrifuge at 3000 rpm for 10 minutes, and remove the supernatant. 
Repeat the same step with 40 mL of water at each time until the electric conductivity 
of the supernatant is below 100 μS/cm . Then, add  40 mL of ethanol (99.5) to the 
residue, agitate , centrifuge at 3000 rpm for 10 minutes, and remove the supernatant. 
Again add 40 mL of ethanol (99.5) to the residue, and perform the same step . Then, 
add 40 mL of a 1 : 1 mixture of chloroform/methanol  to th e residue,  shake for 30 
minutes, centrifuge at 3000 rpm for 10 minutes, and remove the supernatant. Again Attachment  add 40 mL of a 1  : 1 mixture of chloroform/methanol to the residue, and perform the 
same step. Then, add 40 mL of acetone to the residue, shake for 30  minutes, and 
centrifuge at 3000 rpm for 10 minutes.  Filter the supernatant through a filter paper 
(30 μm pore size) , and discard the filtrate. Again add acetone t o the residue in the 
centrifuge tube, shake, filter the whole content in the tube through the  filter paper, 
and remove the  filtrate . Place the residue with the filter  paper  on a watch glass, and 
dry it at room temperature  in a draft chamber. U se the resulting residue on the filter 
paper as the sample.  
Procedure   Put the sample  in a solid -state NMR tube (3 –4 mm external 
diameter),  and seal tightly . Measure the CP/MAS 13C NMR spect rum using a n NMR 
spectrometer — conditioned so that the carbon signal from adamant ane in the high 
magnetic field is 
  29.5 ppm —with a proton resonance frequency of 400 MHz or more 
under the following operating conditions. Separately, measure the CP/MAS 13C NMR 
spectr um of chitin as directed for the sample.  For the obtained spectra, correct the 
baseline and conduct waveform separation treatment . Confirm that signals  are 
detected  in each  CP/MAS 13C NMR spect rum of  the sample and chitin  at SN ratio 50 
or more  in the regions of  around 
  23 ppm, 
  55 ppm,  
 61 ppm, and 
 104 ppm . 
Designate each signal area intensity as A 1, A2, A3, and A 4 for the sample;  and B 1, B2, 
B3, and B 4 for chitin, respectively . Determine the chitin composition  percentage  (%) 
and the glucan  composition  percentage  (%) by the following  formula e. The 
chitin/glucan ratio  is expressed  as the ratio of chitin composition percentage  (%) and 
the glucan composition percentage  (%). 
Chitin composition ratio (%) = C1 + C2 + C3 + C4
4 ×100 
Glucan composition ratio (%) = 100  − Chitin  composition   ratio  (%) 
A1 = signal area intensity of Chitin -Glucan at around 
  23 ppm,  
A2 = signal area intensity of Chitin -Glucan at around 
  55 ppm,  
A3 = signal area intensity of Chitin -Glucan at around 
  61 ppm,  
A4 = signal area intensity of Chitin -Glucan at around 
  104 ppm,  
B1 = signal area intensity of Chitin at around 
  23 ppm,  
B2 = signal area intensity of Chitin at around 
  55 ppm,  
B3 = signal area intensity of Chitin -Glucan at around 
  61 ppm,  
B4 = signal area intensity of Chitin -Glucan at around 
  104 ppm , 
C1 = (B3/B1)/(A 3/A1), 
C2 = (B3/B2)/(A 3/A2), 
C3 = (B4/B1)/(A 4/A1), C4 = (B4/B2)/(A 4/A2). 
Operating conditions  
Spinning  rate: Not less than 7 kHz . 
Contact time: Constant time at around 2 mill isecond s. 
Delay time: Not less than 5  seconds.  
Number of accumulation: Not less than 3000 . 
Purity  
(1) Lead   Not more than1  μg/g as Pb ( an amount equivalent to 4.0 g on the dried 
basis, Method 1 , Control Solution: Lead  Standard Solution 4.0 mL, Flame 
Method).  
(2) Arsenic   Not more than 1  μg/g as As (an amount equivalent to 1.0 g on the 
dried basis , Method 3 , Standard Color: Arsenic Standard Solution 2.0 mL, 
Apparatus B).  
Loss on Drying   Not more than 10 % (105C, 3 hours) . 
Ash  Not more than 3% (600 C, 6 hours, on the dried basis ). 
Microbial Limits  Proceed as directed in the Microbial Limit Tests.  
Total plate count: Not more than 1000 per gram.  
Yeasts and molds: Not more than 200 per gram.  
Escherichia coli : Negative per test.  
Salmonella : Negative per test.  
Sample Fluid   Prepare as directed in Method 1 for  the total plate count and the  
enumeration of yeasts and molds.  
Pre-enrichment Culture  Prepare as directed in Method 1 for the Escherichia coli 
test and the Salmonella  test. 
Assay  Weigh accurately about 5 g of Chitin -Glucan, transfer it into a flask, add 100 
mL of water, and stir for 2 minutes. Filter the resulting suspension by suction 
through a membrane filter (1 μm pore size ). Place the filtrate in an evaporating dish, 
previously dried at 1 05C for 30 minutes, cooled in a desiccator, and accurately 
weighed (the mass of the dish  = m g) , and evaporate it to dryness. Then, dry it at 
105C for 4 hours, leave to cool in the desiccator, accurately weigh  the mass (M g) , 
and calculate the content.  
Content (%) of chitin -glucan = Weight  (g) of the sample  −(M (g) −m (g))
Weight  (g) of the sample ×100 Reagents, Solutions, and Other Reference Materials  
Adamantane  C10H16  [281-23-2]  White to light brown crystals or powder.  
Purity   Related  substances   Prepare a test solution by dissolving 0.5 g of 
adamantine in 10 mL of toluene . Prepare a control solution by diluting exactly 
measured 1.5 mL of the test solution with toluene  to exactly 50 mL. Analyze 1.0 L-
portions of the test solution and the control solution by gas chromatography using 
the operating conditions given below. Con tinue the ch romatography for two  times 
the retention time of the main peak, and measure the peak areas. The sum of the 
areas of all peaks, excluding  the main peak  and solvent peaks , from the test solution 
is not greater than the area of main peak from the control solution.  
Operating conditions  
Detector: Frame -ionization detector . 
Column: A fused silica tube ( 0.53 mm internal diameter and 15–30 m length)  
coated with a 5.0 μm thick layer of dimethylpolysiloxane for gas 
chromatography.  
Column temperature: Raise the temperature at 10 °C/minute from 100 °C to 
250 °C and m aintain the temperature at 250°C for 5 minutes . 
Injection port temperature : 250°C . 
Detector temperature: 250°C.  
Carrier gas: Helium . 
Flow rate: Adjust so that the peak of adamantine appears about 6–12 minutes 
after  injection.  
Injection method: Split.  
Split ratio: 1 : 2 0. 
Chitin   (C8H13NO 5 )n  [1398 -61-4]  A white to light brown powder or scale -like 
substance.  
Identification  Add 1 g of chitin to 200 mL of diluted acetic acid (1 in 100). It 
does not solve.  
Loss on drying  Not more than 15.0% ( 1g, 105 °C, 2 hours) .