Chitin -Glucan  
キチングルカン  
 
Standards for Use  
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 Specifi cations  
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 u ntil 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 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.  Attachment  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 adamantane in the high magnetic field is 
  
29.5 ppm —with a proton resonance fr equency 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 formulae. The chitin/glucan ratio is expressed as the ratio of chitin composition 
percentage  (%) and the g lucan 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 = sig nal 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 = (B 3/B1)/(A 3/A1), 
C2 = (B 3/B2)/(A 3/A2), 
C3 = (B 4/B1)/(A 4/A1), 
C4 = (B 4/B2)/(A 4/A2). 
Operating conditions  
Spinning  rate: Not less than 7 kHz . 
Contact time: Constant time at around 2 millisecond 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 each  of the test 
solution and the control solu tion by gas chromatography using the operating conditions 
given below. Continue 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 the 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) .