Grape sugar
RESOLUTION OENO 47/2000
INTERNATIONAL OENOLOGICAL CODEX
GRAPE SUGAR
1. Objective, Origin and Scope of Application
Grape sugar is obtained exclusively from grape musts. The addition of grape sugar to wine is subject to regulation.
The label, or, when this is absent, the documentation accompanying the containers of grape sugar, must cite the sugar percentage.
2. PROPERTIES
Syrupy, milk-white or slightly yellowish liquid with a sugary flavor.
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Refraction index at 20 °C |
1.42410-1.46663 |
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Total sugar in terms of invert sugar |
63% (m/m) minimum |
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Absorbance at 425 nm under 1 cm at 25° Brix |
maximum 0.100 |
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pH at 25° Brix |
maximum 5 * |
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Titration acidity in mEq/kg of sugar |
maximum 15 * |
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Sucrose |
negative byrecommended method |
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Sulfur dioxide in mg/kg of sugar |
maximum 25 |
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Folin-Ciocalteu index at 25° Brix |
maximum 6 |
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Total cations in mEq/kg of sugar |
maximum 8 |
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Conductivity at 25° Brix in |
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Micro-Siemens/cm (μScm-1) |
maximum 120 |
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5-(hydroxymethyl)furfural in mg/kg sugar |
maximum 25 |
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Residual ethanol in g/kg sugar |
maximum 8 |
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Heavy metals in mg/kg grape sugar expressed in terms of lead |
less than 10 |
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No antiseptics and anti-fermenting agents |
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1° Brix = 1 g of sugar in 100 g of solution
* after vacuum removal of the carbon dioxide
3. TESTS
3.1. Preparing the Sample
Drawing samples for the various different analyses is difficult; therefore. the following two dilutions are recommended:
3.1.1. Principal Solution I
For the following tests: titration acidity, total sulfur dioxide and total cations
Weigh exactly 200 g of grape sugar. Fill to 500 ml with water.
3.1.2. Principal Solution II
Necessary for the following tests: Folin-Ciocalteu index, pH, conductivity, sucrose test and absorbance at 425 nm.
Dilute the grape sugar with water until it has a concentration of 25° ± 0.5° Brix (25 g of sugar in 100 g of solution).
3.2. Refraction Index at 20 °C (total sugars)
3.2.1. Equipment:
The refractometer used gives the following, based on type of graduation:
- 0.1% by mass of sucrose (or dry matter or Brix degrees)
- the 5th decimal of the index of refraction
The refractometer used should be equipped with a thermometer (+ 10 °C at + 30 °C).
3.2.2. Procedure Method:
Place two drops of grape sugar on the surface of the fixed prism. Lower the moving prism and point the instrument toward a light source that illuminates the graduated scale. Observe the line of separation on this scale between a lower clear zone and an upper dark. Read the graduation line at which this line of separation occurs and record the temperature in °C.
3.2.3. Calculation:
If the device is graduated in percentage (m/m) of sucrose (or dry matter or Brix degrees), the measurement converted to 20 °C using Table 2 is recorded in Table 1 which provides (Column 3) total sugar content in percent (m/m) expressed in terms of sugar.
If the device is graduated by refraction index, the index measured at t °C is used to obtain the corresponding value in percent of sucrose (m/m) at t °C in Table 1 (Column 1). This value as expressed at 20 °C using the temperature correction table N° 2, transferred to Table 1, which, in Column 3, gives the total sugar number in percent (m/m) of invert sugar.
To obtain the refraction index at 20 °C, refer to the total sugar content expressed in terms of invert sugar in Table 1.
3.2.4. Recording the Findings:
Total sugar content is expressed parts per 100 by mass of sucrose and is recorded with a decimal.
The refraction index at 20 °C is expressed to 5 decimal places.
3.3. Absorbance of a 25° Brix Solution at 425 nm
Prior to taking the measurements, filter Principal Solution II using a membrane with a porosity of 0.45 µm.
The absorbance of Principal Solution II (25° Brix) is measured in a 1 cm vessel at 425 nm.
3.4. Measuring pH
Measure the pH of Principal Solution II (25° Brix) at 20 °C. Take at least two measurements on the same sample. Take as the finding the arithmetic mean of the two measurements, which should not differ by more than 0.05.
3.5. Titration Acidity
Place 10 ml of Principal Solution I in a cylindrical vessel (3.1.1). Add 0.1M (or 0.01M) sodium hydroxide solution until the pH, as measured with a glass electrode, equal s 7.0 at 20 °C.
The sodium hydroxide solution should be added slowly and the solution should be stirred constantly.
1 ml 0.1M NaOH = 7.5 mg of tartaric acid or 0.1 mEq
Expressing the findings:
In mEq per kilogram of total sugar, with one decimal place.
3.6. Sucrose Test by Thin Layer Chromatography
Equipment and reagents (R) (see Annex).
3.6.1. Preparing the Sample
Dilute Principal Solution II (25° Brix) to 1/4: 25 ml are topped off to 100 ml with water in a volumetric flask.
3.6.2. Obtaining the Chromatogram
Deposit 10 µl of the sample and 10 µl of the reference solution on the start line of the plate (R), this line being 2 cm wide.
After positioning the substances, place the plate in the developing chamber containing the solvent (R).
Let the solvent migrate to a height of 16 cm from the starting line.
Dry the plate in an air current after removing it from the vessel. Place it in an oven at 105 °C for 15 minutes, parallel to the air current, after grinding the developer reagent.
An orange-yellow colored stain appears in the presence of sucrose. Its Rf is identical to that of sucrose in the reference solution. Glucose and fructose produce yellow-orange stains whose Rf is greater than that of the sucrose stain.
If an orange-yellow stain appears, it should not be more intense that that obtained by the sucrose benchmark solution.
It is also possible to use high-performance liquid or gas chromatography techniques. The method thus implementend should allow detection of at least 2g sucrose per kg of grape sugar.
3.7. Sulfur Dioxide
Place 25.0 ml of Principal Solution I and 5 ml of orthophosphoric acid (25%) (R) in the distillation device and proceed as indicated in the reference method detailed in the Compendium.
3.8. Folin-Ciocalteu Index of the 25° Brix Solution
Place the following, in order, in a 100 ml volumetric flask:
- 5 ml of Principal Solution II
- 50 ml water
- 5 ml Folin-Ciocalteu reagent (R)
- 20 ml of sodium carbonate solution (R)
Fill to the 100 ml level with water. Stir to homogenize. Wait 30 minutes for the reaction to stabilize.
Determine absorbance at 750 nm in 1 cm as compared with a control prepared with water instead of Principal Solution II.
Expressing the results:
Express the results in the form of an index obtained by multiplying the absorbance by 16 in order to obtain a scale comparable to that used for wines.
3.9. Total Cations
Place approximately 10 ml of cation-exchange resin in acid form in a column with an inner diameter of 1 cm. Wash with water until the acidity disappears from the wash water, as indicated by pH test paper.
Pour 100 ml of Principal Solution I (3.1.1) (rate: one drop/second). Wash with 50 ml of water and titrate the acidity in the effluent using 0.1M NaOH solution until a pH of 7.0 is reached. Let n be the volume of sodium hydroxide, in ml, that has been poured.
Calculation:
Q mEq/kg of grape sugar = 2.5n
Total cations in mEq/kg of grape sugar = Q - titration acidity in mEq/kg of grape sugar.
Expressing the results:
Total cations are expressed in mEq/kg of total sugar to one decimal place.
3.10. Conductivity of the Solution at 25° Brix
Bring Principal Solution II to a temperature of 20 °C by immersing it in a water bath and proceed as indicated in Annex II (sugars).
Expressing the results:
Conductivity is expressed in micro-Siemens per cm (µS.cm-1) at 20 °C without a decimal place and is for a 25° Brix solution of grape sugar.
3.11. 5-(Hydroxymethyl)furfural(HMF)
Principle :
HMF is determined by HPLC (high-performance liquid chromatography).
Equipment (cited as an example)
Instrument specifications :
- chromatograph equpped with an isocratic pump
- UV/visible light detection apparatus
- Column : grafted silicon dioxide C 18 (20 cm ; 4.6 mm ; 5μm)
- Liquid phase : ultrafiltered demineralized water/methanol/acetic acid (80, 10, 3: v/v/v)
- Flow rate : 0.5 ml/mn
- Detection wavelength : 280 nm
- Volume injected : 20 μl
Preparation of the Reference Solution
In a 100 ml volumetric flask, add 20 mg HMF preliminarily weighed at approximately 0.1 mg, and fill to the gauge line with ultrafiltered demineralized water.
Place 10 of this solution in a 100 ml volumetric flask and fill with ultrafiltered demineralized water (or the equivalent having a resistivity of 18 MΩ, for example). The solution will have a concentration of 20 mg/l and must be prepared daily.
Preparation of the samples :
The samples and the reference solution are injected after filtration on a membrane (pore diameter : 0.45 μm).
Working Method :
The chromatographic column is filled with the liquid phase for approximately 30 minutes before injecting the samples.
3.12. Heavy Metals
Dissolve 12 g of grape sugar in 15 ml of water. Place 10 ml of this solution in a test tube with 2 ml of pH 3.5 buffer solution and 1.2 ml of thioacetamide reagent (R). No precipitate should form. If a brown coloration appears, it should be less intense than that obtained in a control prepared as indicated in the Annex.
(Heavy metal content, expressed in terms of lead, should be less than 10 mg/kg).
3.13. Lead
Using the method set forth in the Compendium, quantitatively analyze lead in the Principal Solution I (3.1.1). (Lead content should be less than 1 mg/kg.)
3.14. Mercury
Using the method set forth in the annex, quantitatively analyze mercury in the Principal Solution I (3.1.1). (Mercury content should be less than 0.3 mg/kg.)
3.15. Arsenic
Using the method described in the annex, quantitatively analyze arsenic in the Principal Solution I (3.1.1). (Arsenic concentration should be less than 0.5 mg/kg.)
3.16. Ethanol
3.16.1. Principle
Simple distillation of the alkalinized sample. The alcohol is oxidized in an acidic medium using a potassium dichromate solution in acetic acid.
The dichromate excess is titrated using an iron (III) solution and ammonium.
3.16.2. Method
Distillation: Separate out the alcohol by distillation using a device which does not result in a loss of alcohol greater than 0.02% by volume during distillation.
Place 100 g of grape sugar and 100 ml of water in the distillation vessel. Collect the distillate in a 100 ml volumetric flask and fill to the gauge line with water.
Oxidation: Take a 300 ml flask with an emery stopper with a wide neck ending in a flared portion allowing the neck to be washed without loss. Place 20.0 ml of titrated potassium dichromate solution (33.79 g/l), 20 ml of sulfuric acid diluted to 1/2 (v/v) in the flask and agitate. Add 20 ml of distillate. Place the stopper in the flask, shake, and wait at least 30 minutes while shaking from time to time.
Volumetric analysis: Add 4 drops of orthophenanthroline reagent (0.695 g iron (II) sulfate dissolved in 100 ml of water, to which 1.485 g of monohydrated orthophenantroline is added, then heat and agitate).
Titrate the excess dichromate by pouring the iron (II) sulfate and ammonium solution (R). Stop adding iron (II) solution as soon as the indicator turns from blue-green to chestnut brown.
If the indicator turns too far, return to the exact color by using a potassium permanganate solution (1.083 g/l). One-tenth of the volume of the solution used is subtracted from the volume of the iron (II) solution, where n is this difference.
Perform the same operation in a similar flask containing the same volumes of the same reagents, but in which the 20.0 ml of distillate are replaced with 20.0 ml of water. Let n be the volume of iron (II) solution used.
3.16.3. Calculations
A quantity of n' ml of iron (II) solution reduces 20 ml of dichromate solution, which oxidize 157.85 mg ethanol.
One milliliter of iron (II) solution has the same reducing power as
- 157.85/n’ mg ethanol
n' - n ml of iron (II) solution have the same reducing power as
- 157.85 n’-n/n’ mg ethanol
If 100 g of grape sugar have been distilled to 100 ml and 20 ml have been treated, the concentration of pure alcohol is:
Ethanol in g/kg grape sugar = 7.892 n-n’/n’
3.16.4. Expressing the results: in g per kilogram of grape sugar to 1 decimal place.
3.17. Meso-Inositol
Gas phase chromatography of a silyl-containing derivative.
N.B. : The information given above is provided for informational purposes. There are other techniques for deriving sugars and polyhydroxy alcohols, and chromatographic methods for determining meso-inositol concentrations
3.17.1. Preparing the sample:
Dilute 5 g of grape sugar in 50 ml of water. Dry 50 µl of the dilution and 50 µl of a methyl D-glucopyranoside solution in a concentration of 1 g/liter, (internal standard) under a vacuum in a small 2 ml flask.
Dissolve the residue with 100 µl of pyridine. Add 100 µl of trimethylchlorosilane. Seal the small flask with a teflon stopper and heat at 80 °C for 1 hour. Inject 1 µl with division of the injected volume to 1/60.
3.17.2. Separation
Column: apolar capillary type of fused silica 25 m long and inner diameter of 0.2 mm.
Supporting Gas: helium, 1 ml/minute
Injector and detector: 280 °C
Column temperature: 60-250 °C, at 4 °C per minute, then isothermal at 250 °C.
3.17.3. Expressing the results: g per kg of sugar
4. STORAGE
Grape sugar must be stored in impermeable containers and at ambient termperature from the time it is made.
ANNEX 1 (sugars)
TABLE 1
Sugar Content in Musts Using Refractometry
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% Succrose (m/m) |
Index of refraction at 20°C |
Density at 20°C |
Sugars in g/l |
Sugars in g/kg |
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TABLE 2
Correction of the Conventional Sugar Mass Titer as a Function of Temperature
Mass Titer Measured in %
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(N.B. : In the French original reproduced here, commas should be replaced with decimal points)
TABLE 3
Conductivity Corrections for Temperatures Other Than 20oC in μ siemens/cm-1
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(1) Subtract the correction
(2) Add the correction
(N.B. : In the French original reproduced here, commas should be read as decimal points.
ANNEX II (sugars)
CONDUCTIVITY
1. APPARATUS
Conductometer allowing measurement in the range of 1-1000 micro-Siemens per cm.
2. EQUIPMENT
200 ml volumetric flasks
Water bath to bring samples to a temperature of 20 °C
3. REAGENTS
Water: specific conductivity should be less than 2 micro-Siemens per cm
KCl reference solution (R)
4. WORKING METHOD
Bring the solution to be analyzed to a temperature of 20 °C by immersing it in the water bath. Wash the measurement cells of the conductometer twice with the solution to be tested. Measure conductivity expressed in micro-Siemens per cm at 20 ± 0.1 °C.
5. CALCULATION
5.1. Temperature Correction
If the apparatus is not equipped with a temperature compensator, correct conductivity using Table 3 (Annex 1 (sugars)).
If the temperature is below 20 °C, add the correction.
If the temperature is above 20 °C, subtract the correction.
5.2. Water Correction
Subtract one-half of the conductivity, as measured in the water used for the dilution at a temperature of 20°C, from the conductivity of the prepared sample.
ANNEX III (sugars)
SUCROSE DETERMINATION IN GRAPE SUGAR USING THIN LAYER CHROMATOGRAPHY
1. Principle Underlying the Method
Sucrose is separated out from the other sugars using thin layer chromatography and is detected using urea/hydrochloric acid reagent.
2. Equipment and Reagents
- Chromatographic plates covered with a thin layer of cellulose (thickness of 0.1 mm)
- Liquid phase: methylene chloride + acetic acid + ethanol + methanol + water (50 + 25 + 9 + 6 + 10)
- Developing agent: urea: 5 g + 20 ml of 2M HCl + 100 ml ethanol
- Reference solution which contains the following per liter:
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Glucose |
160 g |
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Fructose |
160 g |
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Sucrose |
0.8 g |
3. Preparing the Sample
Use Principal Solution I (3.1.1) (200 g of grape increased to a volume of 500 ml with water).
4. Chromatography
Place the following 2.5 cm from the lower edge the plate:
- 0.5 µl of the sample
- 0.5 µl of the reference solution
Place the plate in the chromatography tank saturated by steam from the liquid phase. Let the liquid travel to a level 1 cm from the upper edge. Remove the plate and dry under hot air. Repeat this migration procedure twice, drying the plate each time.
Uniformly spray 15 ml of the developing agent over the plate and maintain at 100 °C in the oven for 5 minutes.
The sucrose and fructose will appear as orange stains.