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COEI-1-ALGIAC Alginic acid

Sin no. 400

C.A.S. no.: 9005-32-7

1. Subject, origin and scope

Alginic acid is a colloidal polysaccharide extracted from various varieties of brown algae in particular from Laminaria. The monomers constituting the -L-glucuronic acid and - D-mannuronic acid are bound in pairs as connections of the type 1 4

-[ α-glu-14- α-glu-14- β-man-1- β-man-1- α-glu

A clarifying agent, which, after being neutralized before use by potassium chloride, or potassium carbonate or potassium hydrogenocarbonate can be added to the drawn-off liquid, designed to carry out the second fermentation of sparkling wines (foam formation).

Alginic acid is made up on average of 200 basic units of uronic acids.

Their molecular weight ranges between 10 000 and 600 000 U.

2. Labeling

The concentration of alginic acid must be indicated on the label, as well as the conditions of safety and conservation.

3. Characteristics

Alginic acid exists in powder or filament form, or as amorphous granules of a yellowish white to brown color, insoluble in pure water and the various organic solvents. It can dissolve in water alkalized by sodium carbonate, sodium hydroxide or trisodium phosphate.

 

4. Identifying characteristics

4.1. pH

A suspension of 3% alginic acid in water has a pH ranging between 2 and 3.5.

4.2. Differentiation with other polysaccharides

An alginic acid solution of 5 g/l in sodium hydroxide (dissolve 4.3 g of sodium hydroxide in water and complete to 100 ml) precipitate in gelatinous form by adding a fifth of volume of a 2.5% solution of calcium chloride.

Furthermore, an addition of a half volume of a solution saturated with ammonia sulfate to the solution previously described does not cause any turbidity.

These two tests can be used to differentiate alginic acid from other polysaccharides that may be used in foodstuffs or pharmaceuticals.

4.3. Organoleptic characteristics

Alginic acid must have no taste, or abnormal odor.

5. Tests

All the limits described below refer to the dry weight of alginic acid.

5.1. Insoluble in a solution of sodium hydroxide

Dissolve by prolonged magnetic agitation 1 g of alginic acid weighed with precision in 100 ml of a solution of sodium hydroxide (dissolve 4.3 g of sodium hydroxide in water and complete to 100 ml), centrifuge, decant, and wash the residue 5 times with distilled water, with centrifugation and drainage of the washwater each time. Transfer all the residue using distilled water to a Gooch filter that has been tared beforehand (filter made of sintered glass of low porosity), dry for 1 hour at 105°C and weigh again.

The rate of insoluble should not exceed 2% in relation to the dry weight of the alginic acid.

5.2. Loss on desiccation

Determine until constant weight, on a test specimen of 2 g, the loss of weight, at 100-105°C, the alginic acid must be lower than 15 p. 100

5.3. Sulfuric ash

Proceed as indicated in chapter II of the international oenological Codex. The sulfuric ash content should not be higher than 8 p 100 in weight of the alginic acid.

5.4. Preparation of the solution for tests

After weighing the ashes, dissolve them in 2 ml of concentrated hydrochloric acid (R) and 10 ml of water. Heat to activate the dissolution and add water until a volume equal to 25 times the weight of the dry alginic acid is obtained. 1 ml of this solution contains the mineral matter of 0.04 g of dry alginic acid.

5.5. Lead

On the solution prepared for tests (5.4), to carry out the dosage of lead according to the method described in chapter II of the international oenological Codex.

The lead content must be lower than 5 mg/kg.

5.6. Cadmium

On the solution prepared for the tests (5.4), determine the cadmium using the method described in chapter II of the international oenological Codex.

The cadmium content must be lower than 1 mg/kg.

5.7. Mercury

Determine the mercury using the method described in Chapter II of the international oenological Codex.

The mercury content must be lower than 1 mg/kg.

5.8. Arsenic

On the solution prepared for the tests (5.4), determine the arsenic using the method described in Chapter II of the international oenological Codex.

The arsenic content must be lower than 3 mg/kg.

5.9. Bacteriological control

Proceed as indicated in chapter II of the international Oenological Codex for each parameter.

Limit: total viable microorganisms: less than 5 x 10³ CFU/g.

5.10.      Coliforms

The number of coliforms must be lower than or equal to 1 per g.

5.11.      Staphilococca

The number of staphilococca (-haemolytics with positive coagulase) must be lower than or equal to 1 per g.

5.12.      Salmonella

The number of salmonella must be lower than 1 per 100 g.

5.13.      Yeast

Limit concentration: 5 x 10² CFU per g of preparation.

5.14.      Lactic bacteria

Limit concentration: 10² CFU per g of preparation.

5.15.      Lactobacillus sp.

Limit concentration: 10 CFU per g of preparation.

5.16.      Pediococcus sp.

Limit concentration: absence in a sample of 10 g of preparation.

5.17.      Acetic bacteria

Limit concentration: 10³ CFU per g of preparation.

5.18.      Moulds

Limit concentration: 5 x 10² CFU per g of preparation.

6. Storage

Alginic acid must be kept in sealed bags.

Calcium (Alignate)

SIN N°: 402

1. Object, origin and scope of application

Calcium alginate is obtained from a 1 % aqueous solution of potassium alginate or alginic acid placed in contact with a 20 % aqueous solution of calcium chloride. Beads of calcium alginate can be produced by dropping droplets of potassium alginate solution into a calcium chloride solution.

Beads of calcium alginate, dry or wet, can contain yeasts or lactic bacteria, dry or wet. They are used for foam forming purposes in the bottle for sparkling wine or to restart alcoholic fermentation in still wines or to start the malolactic fermentation.

These beads can be coated with a double layer of potassium or calcium alginate or with colloidal silica to prevent the precipitation of the yeasts or bacteria incorporated into the beads.

2. Labelling

The label should indicate the product’s purity and the safety and storage conditions for calcium alginate, the yeasts or bacteria incorporated into the beads, the expiration date and the lot number.

3. Characteristics

Calcium alginate is a translucent gel, which is insoluble in water and wine. It only dissolves in a sodium metaphosphate solution.

An alginic acid precipitate is also produced if 1 ml of sulfuric acid diluted to 10 % (R) is added to 10 ml of an aqueous 1 % (m/v) suspension of calcium alginate.

COEI-1-ACIGRA Anti-Foaming Agents (Fatty acid mono- and diglycerides)

SIN NO. 471

1. Objective, origin and scope of application

The mixture of fatty acid glyceric mono- and diesters (with a small quantity of tri-esters), with fatty oils and acids and alimentary fats are termed mono- and diglycerides.  The mixture of mono- and diglycerides used as anti-foaming agents are essentially constituted by oleic acid esters.

The product thus defined can contain small quantities of fatty acids and free glycerol. It is used under appropriate technological conditions and does not leave measurable traces in wine after filtering.

2. Labelling

The label must indicate the mono- and diglyceride content of the preparation, the storage and safety conditions, and the final date of use.

3. Properties

The product is usually found in the form of an oily liquid with a straw yellow color, a doughy product with an ivory color or a hard waxy solid with a white or off-white color.  All of the forms have a pleasant odor and taste.  The solid form can be found in flakes, powder or small granules.

The product used as an anti-foaming agent is liquid at normal temperatures, but can become cloudy at low temperatures.

4. Solubility

Insoluble in water.

Soluble in ethanol, chloroform and benzene.

5. Identifying characteristics

5.1.      Hydrolysis of the Sample

Treat 1 g of the sample by reflux using a 0.5 M potassium hydroxide solution for 1 hour.  Add 15 ml of water and acidify with hydrochloric acid diluted to 30 pp 100 (v/v) (R) (approximately 4-5 ml).  Oily drops or a white/yellowish white precipitate will form.  Extract the fatty acids released using 5 ml hexane, separating the solvent.  Repeat the extraction with 5 ml of hexane and reunite the two extracts.

Set aside the aqueous phase.

5.2.      Detection of the Fatty Acids in the Hexane Extract Using Gas Phase Chromatography

For the purpose of example, use may be made of a semi-polar column, e.g., Carbowax 20M ® measuring 25m x 0.32 mm x 0.25 m phase thickness.

5.3.      Detection of Glycerol

Place 5 ml of the aqueous phase in a test tube.  Add an excess amount of powdered calcium hydroxide and place the test tube in boiling water for five minutes, stirring from time to time.  Cool and filter.

Place one drop of the filtrate in a test tube and add approximately 50 mg of potassium hydrogen sulfate, At the end of the test tube, place a piece of  filter paper soaked in the reagent obtained by mixing extemporaneously equal volumes of a sodium nitrosopentacyanoferrate solution (R) and piperidine (F').  Heat using a small flame.  A blue coloring of the reactive paper indicates the presence of acrolein.

The color turns red by adding 1M sodium hydroxide solution.

6. Tests

6.1.      Drying Loss at 100 °C

Weigh exactly a quantity of about 5 g of the product to be analyzed in a glass crystallizing dish with a diameter of 70 mm, which has been preliminarily dried in  an oven, cooled in a desiccator and calibrated.  Place the crystallizing dish with the fatty material into a 103 °C oven and maintain this temperature for 30 minutes.  Remove the crystallizing dish, let cooll in the desiccator, then weigh.  Place the sample in the oven again for 30 minutes.  Weigh it again after cooling.  Drying loss in the oven is completed when weight loss does not exceed 0.05% per half-hour of heating.

Drying loss at 100 °C should be less than 2 pp 100.

6.2.      Sulfur Ash

Sulfur ash is quantified as indicated in the Annex using a test sample of 5 g.  The sulfur ash should weigh less than 0.2 g/kg.

6.3.      Arsenic

Determined as indicated in the Annex using a test sample of 5 g.  The arsenic should weigh less than 3 mg/kg.

6.4.      Heavy metals

Test for heavy metals either:

• After mineralization at 450 5 °C of the residue left by the drying loss test.  Take up the ash using 1 ml of diluted hydrochloric acid (R) and one drop of concentrated nitric acid (R) while heating in a 100 °C water bath to activate dissolution, then decant in a 25 ml volumetric flask, washing the cap with distilled water.  Fill up to gauge line.

Draw off a volume of v ml of solution corresponding to 2 g of the sample to be analyzed and proceed with the test for heavy metals as indicated in the Annex.

• or, after liquid mineralization of an sample weighed with precision to about 5 g using concentrated nitric acid (R), Perhydrol and a microwave digester to accelerate the operation.

Decant the liquid obtained in a 25 ml volumetric flask and fill to the line with the wash water.  Continue as indicated in the heavy metal tests.

Heavy metal content, expressed in terms of lead, should be less than 10 mg/kg.

6.5.      Lead

Using the technique set forth in the Compendium, determine the quantity of lead in one of the two aforementioned preparations (6.4). The lead content should be less than 5 mg/kg.

6.6.      Mercury

Using the technique described in the annex, determine the quantity of lead in one of the two aforementioned preparations (6.4). The lead content should be less than 1 mg/kg.

6.7.      Cadmium

Using the technique detailed in the annex, determine the quantity of cadmium in one of the two aforementioned preparations (6.4). The lead content should be less than 1 mg/kg.

6.8.      Free Fatty Acids

Prepare 125 ml of a mixture of equal volumes of isopropyl alcohol and toluene.  Add 2 ml of 1 pp 100 phenolphthalein solution (m/v) in isopropyl alcohol and neutralize using an alkaline solution until a persistent but weak pink coloring appears.

Weigh with precision an amount of approximately 5 g of the sample to be analyzed in a 500 ml conical flask.  Add the neutralized solvent mixture and dissolve the test sample, by heating if necessary, while stirring vigorously.  Pour the 0.1 M potassium hydroxide solution until a pink color identical to that obtained during the solvent neutralization process is obtained. Let n be the volume in ml poured:

Fatty acid content expressed in g of oleic acid pp 100 (m/m):

• 2.8n / test sample in g

The fatty acid content in terms of oleic acid should be less than 3 pp 100 (m/m).

6.9.      Soaps

Weigh precisely about 10 g of the product to be analyzed in a 250 ml conical flask.  Add a mixture of 60 ml of acetone and 0.15 ml of 0.5 pp 100 (m/v) bromophenol blue solution in 95% alcohol by volume which has first been neutralized with a 0.1 M hydrochloric acid solution or a 0.1 M sodium hydroxide solution.  Gently heat in a 70 °C water bath and titrate with a 0.1 M hydrochloric acid solution until the blue color disappears.  Let sit for 20 minutes.  Heat until the precipitate redissolves and, if the blue color reappears, continue titration.

1 ml 0.1 M hydrochloric acid solution corresponds to 0.0304 g of sodium oleate (NaC₁₈H₃₃O₂).

Soap content expressed in g of sodium oleate pp 100 (m/m):

• 3.04n / test sample in g

Soap content expressed in g of sodium oleate should be less than 6 pp 100 (m/m).

6.10.  Monoglycerides

6.10.1. Sample preparation

If the sample is in solid form, melt it by heating it to its melting point at a temperature of less than 10 °C. Liquid samples which are cloudy or have particles in them should also be heated. Mix vigorously.

6.10.2. Method

Weigh precisely a test sample, Q, of approximately 1 g to be analyzed in a 100 ml cylindrical flask.  Dissolve using 25 ml of chloroform.  Transfer this solution to a decanting glass. Wash the cylindrical flask with 25 ml of chloroform, then with 25 ml of water and add these liquids to the contents of the decanting glass.

Seal the decanting glass hermetically. Stir for 30-60 seconds. Let the two phases separate out (add 1-2 ml of crystallizable acetic acid (R) to break the emulsion). Collect the aqueous phase in a 500 ml conical flask with an emery stopper.  Extract the chloroform phase remaining in the decanting glass twice with 25 ml of water.  Separate the aqueous phase and place it in the 500 ml conical flask.  These aqueous extracts will be used for the free glycerol analysis.

Transfer the chloroform from the decanting glass to a 500 ml conical flask with an emery stopper.  Add 50 ml of periodic acetic acid solution (R) while stirring.

In the two other 500 ml conical flasks with emery stoppers to be used as "blanks", place 50 ml of chloroform and 10 ml of water.  Add 50 ml of periodic acetic acid solution (R) while stirring to each of the two flasks.  Let the three flasks sit at least 30 minutes, but no more than 90 minutes.

While gently stirring, add 20 ml of potassium iodide solution (R) to each of these containers.  Let sit at least 1 minute but no more than 5 minutes before volumetric analysis.

Add 100 ml water and titrate with a 0.05 M sodium thiosulfate solution using a magnetic stirrer until the brown color disappears from the aqueous phase.  Add 2 ml of starch solution (R) and continue to add the reagent until the iodine disappears from the chloroform layer and the blue color disappears from the aqueous phase.

6.10.3. Calculate the percentage of monoglycerides using the formula:

 

• B is the average volume in ml of the sodium thiosulfate solution used for analysis of the "blanks" containing chloroform.
• S is the amount of sodium thiosulfate solution in ml used to titrate the sample.
• M is the exact molarity of the sodium thiosulfate solution.
• P is the weight of the sample to be analyzed in the volume of chloroform used for the analysis.
• 17.927 is the molar mass of glycerol monostearate, divided by 20.

The monoglyceride content expressed in terms of glycerol monostearate should be greater than 30 pp 100 (m/m).

6.11.  Free glycerol

Add 50 ml of periodic acetic acid solution (R) to the aqueous extracts obtained during the monoglyceride-analysis process.  Simultaneously prepare a "blank" by adding to 75 ml of water in a 500 ml conical flask 50 ml of periodic acetic acid solution (R).  Continue the determination process as indicated in the method described for monoglycerides.

Calculate the percentage of glycerol using the following formula:

• b is the volume in ml of sodium thiosulfate solution used in the quantitative analysis  the "blank" containing 75 ml of water
• S is the volume in ml of sodium thiosulfate solution used in the quantitative analysis of the aqueous extracts
• M is the molarity of the sodium thiosulfate solution.
• Q is the weight of the first sample to to be analyzed (see monoglyceride determination).
• Glycerol content should be less than 7 pp 100 (m/m).

N.B. : Glycerol can also be disclosed and identified by high performance liquid chromatography (HPLC) (5.3).

7. Storage

Anti-foaming agents should be kept in completely water-tight containers and away from heat.

COEI-1-ACILAC L-Lactic acid, D-Lactic acid, D,L-Lactic acid2-hydroxypropanoic acid

N° SIN : 270

C.A.S.  number 50-21-5

(L-: 79-33-4;  D-: 10326-41-7;  DL-: 598-82-3)

chemical formula C3H6O3

Molecular mass: 90.08, density 1.20-1.21.

1. Object, origin and field of application

 

An acid of natural origin obtained by lactic fermentation of sugars or synthetically made; it may contain condensation products such as lactate from lactic acid and dilactide.

It is used for the acidification of musts and wines in the conditions set by the regulation.

 

2. Labelling

The label must mention particularly clearly that it concerns L-lactic or D-Lactic acids obtained by fermentation or D,L-Lactic obtained by a chemical process, the storage conditions and expiration date.

The common commercial products are solutions at 50%-90%.

Solid products containing about 100%-125% of titrable lactic acid also exist. (Note: Lactic acid is hygroscopic and once concentrated by boiling or distillation, it forms condensation products that hydrolyse into lactic acid by dilution and by heating in water).

Purity level: not less than 95.0% and not more than 105.0% of the concentration marked.

 

3. Characteristics

Colourless or slightly yellow and syrupy liquid with a clearly acid flavour to a slightly lactic taste.

4. Solubility

• Water at 20°C: very soluble
• Alcohol at 95% vol.: Very soluble
• Ether: very soluble
• Insoluble in chloroform.

5. Optical rotation

 

For L-lactic acid aqueous solution at 2.5 g for 100 ml.

is 2.6°

For D-lactic acid in aqueous solution at 8 g for 100 ml.

is -2.6°

6. Identity characters

6.1.         Characterisation of lactic acid

In a 100 ml conical flask, weigh 10 g of lactic acid, add 5 ml of sulphuric acid 0.5 M, shake, add 25 ml of potassium permanganate at 0.33% and place on a hot plate. Collect the vapour released on a filter paper soaked with a solution at 50% vol/vol of morpholine at 20% and potassium nitrocyanoferrate (II) at 5%.

The filter paper becomes blue.

6.2.         Determination of total lactic acid

Titrate the free lactic acid with sodium hydroxide 1 M then hydrolyse the polymerised lactic acid using an excess of sodium hydroxide and then determined by sulphuric acid 0.5 M.

6.3.         Colour

Compare the colour with the standards of the alpha scale (colour standards of platinum-cobalt).

6.4.         Stereochemical purity

The method is based on the separation by HPLC using a chiral phase of two enantiomers of lactic acid. The product is diluted in water beforehand. Enzymatic determinations can also be performed according to the methods in the Compendium of international methods of analysis of wines and musts.

7. Test trials

 

7.1.         Preparation of the test trial solution

For the purity test trials, prepare a solution containing 10% m/v of lactic acid by using the concentration marked.

7.2.         Sulphuric ashes

From a 2 g sample of lactic acid, determine the sulphuric ashes as indicated in chapter II of the International Oenological Codex.

The content must be less than or equal to 1 g/kg.

7.3.         Chlorides

To 0.5 ml of the test trial solution (7.1), add 14.5 ml of water, 5 ml of diluted nitric acid (R) and 0.5 ml of silver nitrate solution at 5% (R). The solution should satisfy for the test trial, the determination limit of chlorides described in chapter II of the International Oenological Codex.

The chloride content must be less than 1 g/kg expressed in hydrochloric acid.

7.4.         Iron

To 10 ml of the test trial solution (7.1), add 1 ml of concentrated hydrochloric acid (R) and 2 ml of the potassium thiocyanate solution at 5% (R). The red colouration obtained must not be darker than the control prepared with 1 ml of the iron salt solution (III) at 0.010 g of iron per litre (R), 9 ml of water and the same quantities of the same reagents.

The iron content must be less than 10 mg/kg.

Iron can also be determined by atomic absorption spectrometry according to the method described in chapter II of the International Oenological Codex.

7.5.         Lead

Using the test trial solution (7.1), apply the method described in chapter II of the International Oenological Codex.

Lead content should be less than 2 mg/kg.

7.6.         Mercury

Using the test trial solution (7.1), determine the mercury according to the method described in chapter II of the International Oenological Codex.

Mercury content should be less than 1 mg/kg.

7.7.         Cadmium

Using the test trial solution (7.1), determine the cadmium according to the method described in chapter II of the International Oenological Codex.

Cadmium content should be less than 1mg/kg.

7.8.         Arsenic

Using the test trial solution (7.1), determine the arsenic according to the method described in chapter II of the International Oenological Codex.

Arsenic content should be less than 3 mg/kg.

7.9.         Sulphates

To 1 ml of the test trial solution (7.1), add 18 ml of water, 1 ml of diluted hydrochloric acid at 10% (R) and 2 ml of barium chloride solution at 10% (R). The solution should satisfy for the test trial, the determination limit of sulphates described in chapter II of the International Oenological Codex.

Sulphate content should be less than 1 g/kg, expressed in sulphuric acid.

7.10.     Cyanides

In a 40 ml volumetric flask containing 25 ml of distilled water and 2.5 ml of buffer solution at pH 7.5 (R), introduce 0.4 ml of the test trial solution (7.1), add 0.3 ml of chloramine T solution at 0.1% (R). Wait 90 seconds and add 6 ml of pyridine-pyrazolone reagent (R). Complete to 40 ml with distilled water and mix. The colouration obtained must not be darker than that obtained by treating the same way 4 ml of a freshly prepared potassium cyanide solution titrating 1 mg of hydrocyanic acid per litre (R).

Free cyanide content expressed in hydrocyanic acid should be less than 1 mg/kg.

7.11.     Citric acid

To 5 ml of the test trial solution (7.1), add 5 ml of water, 2 ml of mercury sulphate solution (II) (R), bring to the boil and add a few drops to the potassium permanganate solution at 2% (R). No white precipitate should form.

7.12.     Citric, oxalic, tartaric and phosphoric acids

Dilute 1 ml of the test trial solution (7.1) in 10 ml of water, add 40 ml of the calcium hydroxide solution (R) bring to the boil for 2 minutes. No turbidity should form.

7.13.     Sugars

Add 2 ml of the test trial solution (7.1) to 10 ml of cupro-alkaline reagent (R). No red precipitate should form.

8. Storage

Lactic acid should be stored in hermetically sealed containers away from heat and light.

COEI-1-ACIMAL L-Malic acid , DL-Malic acid

2-hydroxybutanedioic acid

N° SIN: 296

C.A.S.  number 617-48-1

Chemical formula C4H6O5

Molecular mass: 134.09

1. Object, origin and field of application

 

An acid of natural origin contained in most fruit (L-malic acid) or synthetically made: DL-malic.

It is used for the acidification of musts and wines in the conditions set by the regulation.

 

2. Labelling

The label must mention particularly clearly that it is L-malic or D,L-malic acid, the storage conditions and date of expiry.

Malic acid content should be at least 99%.

3. Characteristics

 

White or off-white crystalline powder or granules with a clearly acid flavour.

Melting point of D,L-malic: 127°C-132°C

Melting point of L-malic: 100°C.

4. Solubility

 

• Water at 20°C: 55.8 g/100
• Alcohol at 95% vol.: 45.5 g/100.
• Ether: 0.84 g/ 100

5. Optical rotation

 

For the L-Malic acid in aqueous solution at 8.5 g for 100 ml.

is -2.3°

6. Identity characters

 

6.1. Characterisation of malic acid

Malic acid can be determined by an enzymatic process according to the methods in the Compendium of international methods of analysis of wines and musts (specifically L-malic and D-malic acids. Malic acid can also be determined by HPLC according to the method in the Compendium of international methods of analysis of wines and musts.

7. Test trials

 

7.1. Preparation of the test trial solution

For purity trials, prepare a solution containing 10% m/v of malic acid.

7.2. Sulphuric cinders

From a 2 g sample of malic acid, determine the sulphuric cinders as indicated in chapter II of the International Oenological Codex.

Content must be less than or equal to 1 g/kg.

7.3. Chlorides

To 0.5 ml of the test trial solution (7.1), add 14.5 ml of water, 5 ml of diluted nitric acid (R) and 0.5 ml of silver nitrate solution at 5% (R). The solution should satisfy for the test trial, the determination limit of chlorides described in chapter II of the International Oenological Codex.

Content must be less than 1 g/kg expressed in hydrochloric acid.

7.4. Iron

To 10 ml of the test trial solution (7.1), add 1 ml of concentrated hydrochloric acid (R) and 2 ml of potassium thiocyanate solution at 5% (R). The red colouration obtained should not be darker than that of the control prepared with 1 ml of an iron salt solution (III) at 0.010 g of iron per litre (R), 9 ml of water and the same quantities of the same reagents.

Content must be less than 10 mg/kg.

Iron can also be determined by atomic absorption spectrometry according to the method described in chapter II of the International Oenological Codex.

7.5. Lead

Using the test trial solution (7.1), apply the method described in chapter II of the International Oenological Codex.

Lead content must be less than 5 mg/kg.

7.6. Mercury

Using the test trial solution (7.1), determine the mercury according to the method described in chapter II of the International Oenological Codex.

Mercury content must be less than 1 mg/kg.

7.7. Cadmium

Using the test trial solution (7.1), determine the cadmium according to the method described in chapter II of the International Oenological Codex.

Cadmium content must be less than 1 mg/kg.

7.8. Arsenic

Using the test trial solution (7.1), determine the arsenic according to the method described in chapter II of the International Oenological Codex.

Arsenic content must be less than 3 mg/kg.

7.9. Sulphates

To 1 ml of the test trial solution (7.1), add 18 ml of water, 1 ml of diluted hydrochloric acid at 10% (R) and 2 ml of the barium chloride solution at 10% (R). The solution should satisfy for the test trial, the determination limit of sulphates described in chapter II of the International Oenological Codex.

Sulphates content must be less than 1 g/kg, expressed in sulphuric acid.

7.10.       Cyanides

In a 40 ml volumetric flask containing 25 ml of distilled water and 2.5 ml of buffer solution at pH 7.5 (R), introduce 0.4 ml of the test trial solution (7.1), add 0.3 ml of chloramine T solution at 0.1% (R). Wait 90 seconds and add 6 ml of pyridine-pyrazolone reagent (R). Complete to 40 ml with distilled water and mix. The colouration obtained must not be darker than that obtained by treating the same way 4 ml of a freshly prepared potassium cyanide solution titrating 1 mg of hydrocyanic acid per litre (R).

Free cyanide content expressed in hydrocyanic acid must be less than 1 mg/kg.

7.11.                                                                                                                                                                                                                                                                    Sugars

Add 2 ml of the test trial solution (7.1) to 10 ml of cupro-alkaline reagent (R). No red precipitate should form.

7.12.                                                                                                                                                                                                                                                                    Fumaric and maleic acids

Limit in fumaric acid: 1% in weight.

Limit in maleic acid: 0.05% in weight. These acids are determined by HPLC according to the method described in the Method of Analysis of Wines and Musts in the same way as malic and tartaric acids.

8. Storage

 

Malic acid should be stored in hermetically sealed containers away from heat and light.

COEI-1-ALCAGR Rectified alcohol of agricultural origin

1. Objective, Origin and Scope of Application

Rectified, or "neutral," alcohol obtained by distilling and rectifying alcohol from wine, wine sediments or alcoholic fermentation products derived from from grape or raisin marcs, and all other plant-based substances of agricultural origin.

Rectified alcohol of agricultural origin forms an ingredient of some spirits and special wines.

2. Composition

At a temperature of 20 °C, 100 parts of this alcohol contain at least 96 parts ethanol.

Note: The tests and controls described below in italics are not mandatory and are performed only upon request.

3. Properties

 

Colorless, clear, volatile liquid with a penetrating odor and fiery flavor. It is flammable and burns without smoke and with a blue flame.

It should be distilled completely at between 78 and 79 °C.

3.1.    Solubility

Neutral alcohol is miscible in water in all proportions with a notable release of heat and contraction of volume. It is also mixable with in acetone, chloroform, ethyl ether, glycerol, and an equal volume of castor oil.

3.2.    Characterization Procedure

• Slowly heat a mixture of 1 ml neutral alcohol, twenty drops of concentrated sulfuric acid (R) and 10 g of sodium acetate (R) in a test tube.  A strong, characteristic odor of ethyl acetate will be released.
• Mix several drops of alcohol and 1 ml of concentrated sulfuric acid (R), then add several drops of 10 pp 100  potassium dichromate solution.  The liquid will become green and emit the odor of ethanal.
• Dilute 0.5 ml of alcohol with 4.5 ml of water. Add 1 ml of 1M sodium hydroxide solution, then slowly add 2 ml of iodized potassium iodide (R). An odor of iodoform will be produced, following by the formation of a yellow precipitate.
  3.     Analysis of Agricultural Origin

This analysis is carried out by measuring the ethanol 14C/12C ratio (scintillation) in accordance with the method described in the Spirits Compendium.

4. Tests

4.1.    Appearance

Take two identical test tubes made of alkali-lime glass about 250 mm high, fill one with alcohol, the other with water, which will serve as a control. Examine the liquids along the cylinder's axis. The alcohol should not exhibit any noticeable coloration.

In one test tube about 250 mm high and 25 mm in diameter, pour 40 ml of alcohol, then dilute it with 80 ml of water. The mixture should not cloud nor present any odor or foreign taste.

4.2.    Foreign Odoriferous Substances

Let 10 ml of alcohol evaporate spontaneously on a strip of white filter paper. No foreign odor should be perceived during or after evaporation.

4.3.    Dry Extract or Non-Volatile Residue

In a 25 ml calibrated dish, heat to 100 °C in a water bath, then slowly evaporate 100 ml of alcohol.  Weigh.  The dry extract should be less than 1.5 g/hl 100% ethanol by volume.

4.4.    Heavy Metals

Take up, using 10 ml dilute chlorhydric acid (R), any residue left from the evaporation of 100 ml alcohol during the dry extract analysis. After heating for several minutes in a 100 °C water bath to stimulate dissolution of this residue, decant the acid solution in a 25 ml volumetric flask after the calibrated dish has been washed three times with 5 ml of water and the volume raised to 25 ml.  Take a 5 ml sample of this solution in a test tube.  Add 2 ml of a pH 3.5 (R) buffer solution, 7.5 ml of water and 1.2 ml of thioacetamide reagent (R). The solution should not produce any white or black precipitates nor any brown or other coloration. At the very least, any coloration produced should be no more intense than that obtained using the general method. (Heavy metal conent expressed in lead, after 50% concentration of the alcohol, should be 0.5 mg/l).

4.5.    Lead

Using the method set forth in the Compendium, perform the lead analysis lead in the solution obtained in the previous paragraph. (Lead content should be less than 0.5 mg/l).

4.6.    Mercury

Using the method described in the annex, carry out the mercury analysis in the solution obtained in Paragraph

(Mercury content should be less than 0.2 mg/l).

4.7.    Arsenic

Using the method described in the annex, carry ou the arsenic analysis in the solution obtained in Paragraph 4.4. (Arsenic content should be less than 0.5 mg/kg after 50% alcohol concentration).

4.8.    Ketones, propan-2-ol and 2-methylpropan-1-ol

Add 3 ml of water and 10 ml of mercury sulfate (II) solution (R) to 1 ml of alcohol, then heat in a 100 °C water bath.  No precipitate should form in the first three minutes.

4.9.    Permanganate Decolorization Time (Barbet Test)

Pour 50 ml of the alcohol sample into a flask. Add 2 ml of freshly prepared potassium permanganate solution to 0.20 g/l (R). Place the container in a 15 °C water bath and start a stopwatch.  Avoid directly exposing the sample to natural or artificial light during the test.

Simultaneously, place 50 ml of the comparison solution in the 15 °C water bath.  This solution is obtained by mixing 3 ml of 5 pp 100 cobalt chloride solution (R), 4.2 ml of 4 pp 100 uranyl nitrate solution (R) and filling to 50 ml with distilled water. Compare the test color to the standard.  Stop the timer when the colors are identical.  Note the amount of time elapsed.  The decolorization time of the permanganate should be at least 20 minutes.

4.10. Sulfured Derivatives

Add approximately 1 ml of mercury, then 20 ml of alcohol to a test tube.  Agitate for 1-2 minutes.  The surface of the mercury should remain brilliant with no black clouding.

4.11. Methanol

4.11.1.     Colorimetric Analysis

Standard solution: weigh 5 g of methanol in a 50 ml volumetric flask, then top off to the line with ethanol (free of methanol).

In a 1-liter volumetric flask, place 1 g of the preceding solution (i.e., 1.25 ml) containing 125 mg of methanol, 250 ml of pure alcohol (methanol free).  Top off with water to 1000 ml.

Test technique: place 1250/A ml of alcohol in a volumetric flask (A is the alcoholmetric titer of the alcohol to be tested.) and fill to the gauge line with water. Place 1 ml of alcohol, diluted to 25 pp 100 in a test tube. Add four drops of 50 pp 100 (m/m) phosphoric acid (R), four drops of 5 pp 100 (m/m) potassium permanganate solution (R), then stir and let sit 10 minutes.  Decolorize the permanganate with several (typically 8) drops of 2 pp 100 (m/v) of potassium anhydrous sulfite (metabisulfite) (R), avoiding any excess.  Add 5 ml of chromotropic sulfuric acid solution (R).  Place in a 70 °C water bath for 20 minutes.  No violet color should appear, or in the event it does appear, it should not be more intense than that of a control prepared using the same technique and the same reagents, with 1 ml of the aforementioned standard solution (maximum methanol content is 50 g/hl at 100% vol.).

4.11.2.     Gas phase chromatography Analysis

• Equipment (example):
• Gas phase chromatograph with a flame ionization detector
• Semi-polar capillary columns, for example Carbowax 20 M .

Test technique:

Prepare a water-alcohol solution using 1 g per liter of the internal standard (4-methylpentane-2-ol) in 50 pp 100 alcohol by volume.

Prepare the solution to be analyzed by adding 5 ml of this solution to 50 ml of alcohol reduced to 50 pp 100 by volume.

Prepare a reference solution of methanol at 100 mg per liter of alcohol at 50 pp 100 by volume.  Add 5 ml of the internal standard solution to 50 ml of this solution.

Inject 2 microliters of the solution to be analyzed added to the internal standard solution, into the chromatograph.

The oven temperature should be 90 °C and the supporting gas flow rate should be 25 ml per minute.  These settings are given as an example.

• S: surface of the methanol peak of the reference solution
• : surface of the methanol peak of the solution to be analyzed
• i: surface of the internal standard solution peak in the solution to be analyzed
• I: surface of the internal standard solution peak in the reference solution

The methanol content, expressed in milligrams per liter of alcohol at 50 pp 100 by volume, is given by the formula:

The content in grams per hectoliter of pure alcohol is 0.20C (maximum content in methanol 50 g/hl of ethanol at 100% by volume).

4.12. Ammonium Hydroxide and Nitrogenous Bases

Pour 50 ml of the alcohol to be examined into a 200 ml flask.  Add 40 ml of water and two drops of phosphoric acid (ρ 20 = 1.58). Distill and collect the 80 ml that are returned.  Add 2 ml of 10 pp 100 sodium hydroxide ® to the cooled residue. Distill again and collect approximately 7 ml of distillate in a test tube to which had previously been added 2 ml of water and one drop of methyl red solution ®.  The distillate should be drawn to the bottom of the tube using a slender tube.  Titrate using a solution of 0.01 M hydrochloric acid until the indicator turns to red. Let n be the number of milliliters of 0.01 M hydrochloric acid solution used.

1 ml of 0.01 M hydrochloric acid solution corresponds to 0.00014 g of nitrogen (ammoniacal or volatile nitrogen bases).

The quantity of ammoniacal nitrogen or nitrogenous bases expressed in milligrams of nitrogen per liter of ethanol is:

280n/A

Where A is the alcohometric titer by volume of the alcohol studied.

Neutral alcohol should not contain more than 1 mg of nitrogen (ammoniacal or of volatile nitrogenous bases) per liter of ethanol.

(Maximum ammonium hydroxide and nitrogenous base content is expressed in terms of nitrogen is 0.1 g/hl of ethanol at 100% by volume).

4.13. Acidity

Place 100 ml strengthened of 50 pp 100 by volume alcohol in a 250 ml conical flask. Add one drop of phenol red solution (R) and add 0.01 M sodium hydroxide, one drop at a time, until red, where n is the number of milliliters used.

1 ml of 0.01 M sodium hydroxide corresponds to 0.0006 g of acetic acid.

Acidity expressed in milligrams of acetic acid per liter of ethanol is equal to 12n.

This acidity should be less than 15 mg/l of ethanol (or 1.5 g/hl) at the time the alcohol is delivered.

(Maximum acidity expressed in terms of acetic acid is 1.5 g/hl of ethanol at 100% by volume).

Note: Indicator movement should be stable and clear cut during quantitative analysis of the acidity.  If it is not, and especially if the acidity exceeds 15 mg/l, a new test should be conducted after the sample is degassed using the following technique.

100 ml of alcohol at 50 pp 100 by volume is placed in a 250 ml flask whose stopper has two tubes through it.

One tube permits the flask to be kept under a vacuum using a glass filter pump.  Pressure is kept between 55 and 65 cm of mercury.

During the procedure, the other tube allows air bubbling from which carbon dioxide is removed by using a sode wash bottle. To accomplish this, the tube has a capillary portion which is submerged in the alcohol. The rate of air flow through the wash bottle is approximately 1 ml per second.

The procedure should last between 3 and five minutes. Titration is accomplished in the same flask.

4.14. Esters

Add 10 ml of 0.1 M sodium hydroxide solution measured with precision to the solution prepared to analyze acidity as detailed under 4.13 (or 100 ml of alcohol at 50% by volume). Cork the flask and stir while maintaining a temperature equal to or slightly higher than 20 °C. After 24 hours of contact, titrate the excess sodium hydroxide using a 0.1 M solution of hydrochloric acid, where n is the number of milliliters used.

To determine the quantity of 0.1 M hydrochloric acid solution which will neutralize 10 ml of 0.1 M sodium hydroxide solution in the presence of the same quantity of alcohol and of the same indicator movement obtained by decreased pH intervals, perform the following test: place 100 ml of degasified 50 pp 100 alcohol in a 250 ml conical flask.  Add one drop of phenol red solution (R) and n milliliters of 0.1 M sodium hydroxide. Which cuase the indicator to turn to red. Add 10 ml of the 0.1 M sodium hydroxide solution, and, immediately thereafter, add 0.1 M hydrochloric acid solution to obtain the same movement of the indicator, that is, nⁿof the volume used.

1 ml of 0.1 M sodium hydroxide solution corresponds to 0.0088 g of ethyl acetate.  The ester concentration, expressed in milligrams of ethyl acetate contained in 1 liter of ethanol is:

This content level should not exceed 13 mg for 1 liter of ethanol (or 1.3 g/hl) at the time the alcohol is delivered.

(Maximum ester content expressed in terms of ethyl acetate is 1.3 g/hl of ethanol at 100% of volume).

4.15. Aldehydes

Standard solution: Place 268.3 mg of pure acetal (boiling point: 102°C) in a 100 ml volumetric flask.  Top off to the line with 50 pp 100 alcohol by volume, free of aldehydes.

Dilute this solution to 1/10 in 50 pp 100 alcohol by volume, is free of aldehydes.  The solution obtained contains 100 mg of ethanal per liter of 50 pp 100 alcohol by volume, or 20 g in 100 liters of ethanol.

Test procedure: Place 10 ml of alcohol reduced to 50 pp 100 by volume in a test tube. In a second test tube, place 5 ml of the solution containing 100 mg of ethanal per liter of alcohol at 50 pp 100 and 5 ml of alcohol at 50 pp 100 by volume which is free of aldehydes.  Add to the two tubes 4 ml aniline red chlorhydrate solution decolorized by sulfuric acid (R), stir, and compare the colorations obtained after 20 minutes.

The alcohol to be tested should have a color approximately equal to that of the standard solution.

(Maximum aldehyde content expressed in ethanal is 0.5 g/hl at 100% of volume).

Note concerning 50 pp 100 alcohol by volume without aldehydes: Place 100 ml of alcohol diluted to 50 pp 100 by volume in a 250 ml flask with 2 g of metaphenylene diamine (R) and two pieces of pumice stone.  Connect the flask to a reflux condenser and maintain a gentle boil for one hour.  After cooling, connect the flask to the distilling apparatus and slowly distill without overheating the walls.  Collect 75 ml of distillate in a 100 ml volumetric flask.  Fill to the line with distilled water.

4.16. Superior Alcohols

Propan-1-ol, 2-methylpropan-1-ol, 2- and 3-methylbutan-1-ol.

Quantitative analysis by gas phase chromatography (see methanol).

Maximum content for the sum of each of the alcohols: 0.5 g/hl of ethanol at 100% of volume.

4.17. Furfural

Place 10 ml of alcohol reduced to 50% by volume in a test tube with an emery stopper.  Add 0.5 ml of aniline (R) and 2 ml of crystallizable acetic acid (R).  Stir.  No salmon pink coloration should be perceptible after 20 minutes.

5. Storage

The alcohol should be stored in inert containers which will not give off metals, ions or plastic constituents.

The containers, as well as storage methods,must be in compliance with safety standards.

COEI-1-ALCVIT Rectified alcohol of viti-vinicultural origin

1. Objective, Origin and Scope of Application

Alcohol obtained exclusively by distillation and rectification from wine, grape marcs, wine sediments, or fermented raisins.

Rectified alcohol of viti-vinicultural origin constitues a constituent of some spirits and special wines.

2. Composition

At a temperature of 20 °C, 100 parts of this alcohol contain at least 96 parts ethanol.

Note: The tests and controls described below in italics are not mandatory and are performed only upon request.

3. Properties

Colorless, clear, volatile liquid with a penetrating odor and fiery taste.  It is flammable and burns without smoke and with a blue flame.

It should be distilled completely at between 78 and 79 °C.

3.1.    Solubility

Neutral alcohol is miscible in water in all proportions with a notable release of heat and contraction of volume. It is also mixable with in acetone, chloroform, ethyl ether, glycerol, and an equal volume of castor oil.

3.2.    Characterization Procedure

• Slowly heat a mixture of 1 ml neutral alcohol, twenty drops of concentrated sulfuric acid (R) and 10 g of sodium acetate (R) in a test tube.  A strong, characteristic odor of ethyl acetate will be released.
• Mix several drops of alcohol and 1 ml of concentrated sulfuric acid (R), then add several drops of 10 pp 100 potassium dichromate solution.  The liquid will become green and emit the odor of ethanal.
• Dilute 0.5 ml of alcohol with 4.5 ml of water. Add 1 ml of 1M sodium hydroxide solution, then slowly add 2 ml of iodized potassium iodide (R). An odor of iodoform will be produced, following by the formation of a yellow precipitate.

3.3.    Determination of Viti-vinicultural Origin

This analysis is carried out by measuring the ethanol 14C/12C ratio (scintillation) in accordance with the method described in the Spirits Compendium.

3.4.    If necessary, the viti-vinicultural source of the alcohol can be determined using isotopic methods detailed in the Compendium of Wine and Must Analysis Methods.

4. Tests

Test are identical to those for rectified alcohol of agricultural origin , but with the following content limits:

4.1.    Methanol

Maximum content 50 g/hl of ethanol at 100% by volume.

4.2.    Acidity

Maximum acetic acid content 1.5 g/hl of ethanol at 100% by volume.

4.3.    Esters

Maximum content of ethyl acetate 1.3 g/hl of ethanol at 100% by volume (or 5 g/hl).

4.4.    Aldehydes

Maximum ethanal content 0.5 g/hl of ethanol at 100% by volume.

4.5.    Superior Alcohols

Maximum content 0.5 g/hl of ethanol at 100% by volume.

4.6.    Preparing the solution for tests

Using 10 ml of dilute hydrochloric acid (R), take up the residue left by evaporating 100 ml of alcohol during the dry extract analysis.  After heating for several minutes in a 100 °C water bath to stimulate dissolution of this residue, decant the acid solution in a 25 ml volumetric flask, and wash the dish three times with 5 ml of water and filled to 25 ml.

4.7.    Heavy metals

Place 5 ml of the prepared solution in a test tube in accordance with paragraph 4.6. Add 2 ml of pH 3.5 (R) buffer solution, 7.5 ml of water and 1.2 ml of thioacetamide reagent (R). The solution should not yield any white or black precipitate nor any brown or coloring.  At the very least, any coloring produced should be no more intense than that obtained using the general method (heavy metals content expressed in terms of lead, after 50% concentration of the alcohol, should be 0.5 mg/l).

4.8.    Lead

Using the method set forth in the Compendium, conduct the quantitative lead analysis on the solution prepared for testing (under paragraph 4.6) (lead content should be less than 0.5 mg/l).

4.9.    Mercury

Carry out the quantitative mercury analysis on the solution prepared for testing (under Paragraph 4.6), implementing the technique described in the annex (mercury content should be less than 0.2 mg/l).

4.10. Arsenic

Conduct the quantitative arsenic analysis on the solution prepared for testing (Paragraph 4.6), using the method described in the annex (Arsenic content should be less than 0.5 mg/kg).

5. Storage

 

Alcohol should be stored in inert containers which will not give off metals, ions or plastics constituents.

The containers as well as the storage methods must comply with safety standards.

COEI-1-AMMCHL Ammonium chloride

Ammonia Hydrochloride

Ammonii Chloridum

NCl=53.50

SIN NO.: 510

1. Objective, Origin and Domain of Application

This product is used as a fermentation activator and is reserved for fermentation operations.  It makes available ammonium ions which can be directly assimilated by the yeast.

Statutory limits regulate the amount of ammonium added.

2. Labeling

The concentration of this product should be indicated on the label, including cases in which it is mixed. In addition, safety and storage conditions should be stipulated.

3. Centesimal Composition

Cl	66.22
NH3	31.78

4. Properties

Colorless, odorless crystals with a fresh, salty and piquant taste.  It sublimes without decomposing and is stable in air.

5. Solubility

Water at 20 °C	350.8 g/l
Water at 100 °C	758 g/l
Alcohol, 95% by vol.	13.3 g/l

6. Identifying Characteristics

Aqueous solutions of ammonium chloride produce reactions of ammonium and those of chloride.

7. Testing

7.1.    Sulfur Ash

When quantitifed as indicated in the Annex, the sulfur ash content of the ammonium chloride should not be greater than 0.2 per 100.

7.2.    Preparing the solution for tests

Prepare an aqueous solution from NH₄Cl crystals at 10 per 100 (m/v).

7.3.    Sulfates

To 1 ml of solution prepared for tests under paragraph 7.2, add 2 ml of hydrochloric acid diluted to 10 pp 100 (m/v) (R), 17 ml of water and 2 ml of barium chloride solution (R).  The mixture should be clear, or else the opalescence observed after 15 minutes should be less than that of the control solution prepared as indicated in the Annex.  (Sulfate content expressed in terms of sulfuric acid should be less than 1 g/kg).

7.4.    Nitrates

Mix 5 ml of concentrated sulfuric acid (R) and 0.5 ml of an extemporaneously prepared iron (II) sulfate solution at 5 pp 100 in a test tube.  Without mixing, pour 5 ml of the solution prepared under paragraph 7.2.  No coloration should be observed at the surface line separating the two solutions.

7.5.    Phosphates

To 0.5 ml of the solution prepared for testing under Paragraph 7.2, add 5 ml of water and 10 ml of nitro-vanadomolybdic (R) reagent.  Leave in contact for 15 minutes at 20 °C.  If a yellow coloration appears, it should be less intense than that obtained by adding 0.5 ml of a solution of 0.05 g of phosphorous per liter (R), 5 ml of water and 10 ml of nitro-vanadomolybdic (R) reagent. (Phosphate content expressed in terms of phosphorous less than 500 mg/kg).

7.6.    Iron

To 5 ml of solution prepared under paragraph 7.2, add 1 ml of concentrated hydrochloric acid (R), one drop of 2 pp 100 potassium permanganate and 2 ml of 5 pp 100 potassium thiocyanate (R).

If a red coloration appears, it should be less intense than that of a control prepared with 2.5 ml of an iron (III) solution containing 0.01 g of iron per liter (R), 2.5 ml of water and the same quantities of the same reagents. (Iron content should be less than 50 mg/kg).

The iron may also be quantitatively analyzed using atomic absorption spectometry, in accordance with the method detailed in the Compendium.

7.7.    Arsenic

Using the method indicated in the annex, test for arsenic in the test solution prepared in accordance with Paragraph 7.2.  (Arsenic content should be less than 3 mg/kg.)

7.8.    Lead

Using the method described in the Compendium, quantify the lead in the solution obtained under Paragraph 7.2.  (Lead content should be less than 2 mg/kg.)

7.9.    Mercury

Using the method described in the annex, test for mercury in the solution prepared for testing under §7.2. (Mercury content should be less than 1 mg/kg.

7.10. Quantitative Ammonia Analysis

Dilute the solution prepared for testing under paragraph 7.2 to one-tenth strength, then place 10 ml of this dilute solution (i.e., 0.1 g of ammonium chloride) in a steam distillation device.  Add 10 ml of 30% sodium hydroxide (R) and distill 100 ml.  Quantify the distilled ammonia using 0.1 M hydrochloric acid.  Let n be the number of milliliters used:

100 g of ammonium chloride contains 1.7 n g of ammonia (NH₃).

(Ammonia content greater than 31.5 pp 100).

7.11. Quantitative Hydrochloric Acid Analysis

Take a 10 ml sample of the solution prepared for testing under paragraph 7.2, which has been diluted to one-tenth strength. Place the sample in a cylindrical flask.  Add 20 ml of 0.1 M silver nitrate solution, 1 ml of concentrated nitric acid (R), 5 ml of iron (III) sulfate solution and 10 pp 100 of ammonium (R). Titrate the excess silver nitrate with a 0.1 M potassium thiocyanate solution. Let n be the number of milliliters used:

100 g of ammonium chloride contains 3.65 (20-n) g of hydrochloric acid (HCl).  (Hydrochloric acid content greater than 67.5 pp 100).

8. Storage

Ammonium chloride must be stored in water-tight containers away from heat.

COEI-1-AMMHYD Ammonium hydrogen sulfite

Ammonium bisulfite

NH4HSO3 = 99.07

1. Objective, origin and scope of application

This product falls in the category of preservatives and is used exclusively for fermentation operations. It makes available sulfur dioxide and ammonium ions, which can be directly assimilated by the yeast. There are regulatory restrictions on the amount of ammonium that can be added and on sulfur dioxide content.

2. Labeling

The concentration of this product, as well as the safety and storage conditions, should be indicated on the label.

3. Centesimal composition

 

NH3	17.16
SO2	64.67

 

4. Properties

Ammonium hydrogen sulfite always takes an aqueous solution form. This solution emits a piquant sulfur dioxide odor.

 

5. Solubility

 

Water at 60 °C	847 g/l
Alcohol, 95% by vol.	Slightly soluble

6. Identifying characteristics

Aqueous solutions of ammonium hydrogen sulfite produce reactions of ammonium (release of ammonia in the presence of sodium hydroxide when heated) and sulfur dioxide (filter paper soaked in potassium iodate and starch turns blue).

7. Tests

 

7.1.     Sulfur Ash

As quantified as indicated in the Annex, the proportion of ammonium hydrogen sulfite ash should not be greater than 0.2 per 100.

7.2.     Preparing the Solution for Tests

Prepare a 10 pp 100 (m/v) solution.

 

7.3.     Iron

To 5 ml of the solution prepared for testing under paragraph 2, add 1 ml of concentrated hydrochloric acid (R), one drop of 2 pp 100 potassium permanganate (R) and 2 ml of 5 pp 100 potassium thiocyanate (R).

If a red colorating appears, it should be less intense than that of a control prepared with 2.5 ml of an iron (III) solution of 0.01 g of iron per liter (R), 2.5 ml of water and the same quantities of the same reagents.

(Iron content should be less than 50 mg/kg).

The iron may also be quantified by means of atomic absorption spectrometry, using the technique described in the Compendium.

7.4.     Lead

Use the method detailed in the Compendium on the solution in a concentration of 10 pp 100 prepared for testing (under 7.2) and diluted to one one-twentieth.

7.5.     Mercury

Test for mercury in the solution prepared for testing (under 7.2) using the technique detailed in the annex. (Mercury content should be less than 1 mg/kg.)

 

7.6.     Arsenic

Using the method indicated in the Annex, test for arsenic in 2 ml of the test solution prepared for testing in accordance with paragraph 7.2.  (Arsenic content should be less than 3 mg/kg).

7.7.     Quantitative Ammonia Analysis

Dilute the solution prepared for testing under paragraph 7.2 to one-tenth strength, then place 10 ml of this dilute solution (0.10 g of ammonium hydrogen sulfite) in a steam distillation device (described in the annex).  Add 10 ml of 30 pp 100 sodium hydroxide (R) and distill 100 ml. Quantify the distilled ammonia using 0.1 M hydrochloric acid. Let n be the number of milliliters used:

100 g of ammonium hydrogen sulfite contain 1.7 n g of ammonia (NH₃).  Ammonia content should be greater than 16.5 pp 100 (m/m).

7.8.     Quantitative Sulfur Dioxide Analysis

In a 200 ml conical flask, place 50 ml of cold water, then 5 ml of the freshly prepared ammonium hydrogen sulfite solution.  Titrate with 0.05 M iodine in the presence of starch.  Let n be the volume of iodine used.

SO₂ content per 100 g: 6.4n

Ammonium hydrogen sulfite should contain at least 62 pp 100 SO₂.

8. Storage

 

Ammonium hydrogen sulfite solutions should be stored in hermetically sealed containers away from heat and cold.

COEI-1-PHODIA Diammonium hydrogen phosphate

Ammonium hydrogen phosphate

Ammonii phosphas

SIN N°.342

1. Objective, Origin and Scope of Application

This product is used as a fermentation activator and is reserved for fermentation operations.  It makes available ammonium ions, which can be directly assimilated by the yeast. Excess phosphates can lead to iron breakdown.

Statutory provisions limit the amount of ammonium that can be added.

2. Labelling

The concentration of this product should be indicated on the label, including cases of mixtures. In addition, safety and storage conditions should also be stipulated.

3. Centesimal Composition

 

H3PO4	74.21
P2O5	53.75
NH3	25.79

4. Properties

Colorless, monoclinic crystals.  This salt slowly loses small quantities of ammonia in air.

5. Solubility

Water at 20 °C	689 g/l
Water at 100 °C	1060 g/l
Alcohol, 95% by vol.	insoluble

6. Identifying Characteristics

6.1.    Prepare a 1 pp 100 (m/v) solution in water.  The solution has a pH of approximately 8, and a slight pink color is produced with several drops of phenolphthalein (R). At 25 °C, the pH of this solution should be between 7.8 and 8.4.

6.2.    This solution produces a yellow precipitate with a nitro-molybdic reagent (R).

6.3.    When heated with several drops of 30% sodium hydroxide solution (R), this solution releases ammonia.

7. Tests

7.1.    Sulfur Ash

Quantified as indicated in the Annex, the proportion of diammonium phosphate ash should not be greater than 5 g/kg.

7.2.    Preparing the solution for tests

Prepare a 10 pp 100 (m/v) solution.

7.3.    Chlorides

To 0.5 ml of the solution prepared for testing under Paragraph 7.2, add 14.5 ml of water, 5 ml of nitric acid diluted to 10 pp 100 (R) and 0.5 ml of 5 pp 100 silver nitrate solution (R).  After 15 minutes at rest in the dark, there should be no clouding, or any clouding visible should be less intense than that observed in the control prepared as detailed in the annex.  (Hydrochloric acid content is less than 1 g/kg).

7.4.    Sulfates

To 1 ml of solution prepared for tests under paragraph 7.2, add 2 ml of dilute hydrochloric acid (R), 17 ml of water and 2 ml of barium chloride solution (R).  The mixture must not form any precipitate or any opalescence; or else, any opalescence that does occur should be less intense than that observed in the control prepared as indicated in the Annex.  (Sulfuric acid content should be less than 1 g/kg).

7.5.    Oxalic acid

To 5 ml of solution prepared for tests under paragraph 7.2, add 20 drops of acetic acid (R) and 5 ml of solution saturated with calcium sulfate (R). The solution should remain clear.

7.6.    Iron

To the 5 ml of solution prepared under paragraph 2, add 1 ml of concentrated hydrochloric acid (R) and 1 ml of 5 pp 100 potassium thiocyanate solution (R).

Coloring should be less intense than that of a control prepared with 2.5 ml of an iron solution in a concentration of 10 mg of iron per liter (R), 2.5 ml of water and the same quantities of the same reagents. (Iron content should be less than 50 mg/kg.)

Iron may also be analytically quantified by atomic absorption spectometry, according to the method specified in the Compendium.

7.7.    Lead

By implementing the method detailed in the Compendium, carry out quantitative analysis of the solution prepared for testing according to Paragraph 7.2.   (Lead content should be less than 5 mg/kg).

7.8.    Mercury

Test for mercury in the solution prepared for testing (Par. 7.2), in accordance with the method detailed in the Compendium. (Mercury content should be less than 1 mg/kg.)

7.9.    Arsenic

Using the method indicated in the Annex, test for arsenic in 2 ml of the test solution prepared in accordance with paragraph 7.2.  (Arsenic content should be less than 3 mg/kg.)

7.10. Quantitative Ammonia Analysis

Dilute the solution prepared under Paragraph 7.2 to one-tenth strength, then place 10 ml of this dilute solution (0.10 g of ammonium phosphate) in a steam distillation device (described in the Annex).  Add 10 ml of water, 10 ml of 30% sodium hydroxide (R) and distill 10 ml.  Analytically quantify the distilled ammonia using 0.1 M hydrochloric acid.  Let n be the number of milliliters used:

100 g of ammonium phosphate contains 1.7 n g of ammonia (NH₃). (Minimum content is 25 pp 100).

7.11. Quantitative Analysis of Phosphoric Acid

Place 25 ml of the solution prepared under paragraph 7.2 in a conical flask.  Add 5 drops of phenolphthalein (R).  The solution should have a pale pink color. If not, add just enough 0.1 M sodium hydroxide solution to cause incipient movement of the indicator. Add 10 drops of bromocresol green (R) and use a burette to pour 0.5 M sulfuric acid until the indicator turns green.

Let n be the volume in ml used:

One liter of 0.5 M solution corresponds to 71 g of phosphoric anhydride or 98 g of phosphoric acid.

Proportion of ammonium phosphate in g per 100 g:

in phosphoric anhydride	2.84 n
in phosphoric acid	3.92 n

The proportion of phosphoric anhydride must range between 51.6 and 55 pp 100, or between 71.5 and 76 pp 100 of phosphoric acid.

8. Storage

Ammonium phosphate must be stored away from moisture and heat, and in hermetically sealed containers.

COEI-1-AMMSUL Ammonium sulfate

Ammonium sulfuricum

(NH4)2SO4 = 132.10

SIN NO. 517

1. Objective, Origin and Scope of Application

This product is used as a fermentation activator and is reserved for fermentation operations.  It adds ammonium ions that can be directly assimilated by the yeast. The sulfates added are completely soluble in wine.

Statutory restrictions govern the addition of ammonium.

2. Labelling

The concentration of this product should be indicated on the label, including mixtures. In addition, safety and storage conditions should be noted.

3. Centesimal Composition

 

H2SO4	74.22
NH3	25.78
SO3	60.59
N	21.20

4. Properties

Transparent, anhydrous crystals with a bitter, pungent taste, which are similar to potassium sulfate crystals, with which this salt is isomorphous.

5. Solubility

Water at 20 °C	509 g/l
Water at 100 °C	1040 g/l
Alcohol, 90% by vol.	Insoluble
Acetone	Insoluble

6. Identifying Characteristics

 

Solutions of this salt in water in a concentration of 1 pp 100 (m/v) has a pH of approximately 5.5. This solution allows reactions of ammonium and those involving sulfates.

7. Tests

7.1.     Sulfur Ash

The concentration of sulfur ash of ammonium sulfate prepared as explained in the annex in a test sample of 1 g must not exceed 5 g/kg.

7.2.     Preparing the Solution for Tests

Prepare a 10 pp 100 (m/v) solution.

7.3.     Chlorides

To 0.5 ml of the solution prepared for testing under paragraph 7.2, add 14.5 ml of water, 5 ml of nitric acid (R) diluted to a concentration of 10 pp 100 and 0.5 ml of 5 pp 100 silver nitrate solution (R).  After 15 minutes at rest in the dark, there should be no clouding; or else, any clouding visible should be less intense than that observed in the control prepared as indicated in the annex.  (Hydrochloric acid content must be less than 1 g/kg).

7.4.     Phosphates

To 0.5 ml of the solution prepared for tests under paragraph 7.2, add 5 ml of water and 10 ml of nitro-vanadomolybdic reagent (R). Leave in contact for 15 minutes at 20 °C. If a yellow coloring appears, it should be less intense than that obtained by adding, to 0.5 g of a solution containing 0.05 g phosphorous per liter, 5 ml of water and 10 ml of nitro-vanadomolybdic reagent.  (Phosphate content expressed in terms of phosphorous should be less than 500 mg/kg).

7.5.     Nitrates

Mix 5 ml of concentrated sulfuric acid (R) and 0.5 ml of an previously prepared iron (II) sulfate solution in a concentration of 5 pp 100 (m/v) in a test tube. Without mixing, pour 5 ml of the solution obtained by dissolving 2 g of ammonium sulfate in 10 ml of water. No coloring should be observed at the surface separating the two solutions

7.6.     Iron

To 5 ml of the solution prepared for testing under paragraph 7.2, add 1 ml of concentrated hydrochloric acid (R), one drop of 2 pp 100 potassium permanganate (R) and 2 ml of 5 pp 100 potassium thiocyanate solution (R).

If a red coloring appears, it should be less intense than that of a control prepared with 2.5 ml of an iron (III) solution in a concentration of 0.01 g of iron per liter (R), 2.5 ml of water and the same quantities of the same reagents.  (Iron content should be less than 50 mg/kg).

The proportion of iron may also be quantified by atomic absorption spectometry, using the technique detailed in the Compendium.

7.7.     Lead

Use the quantitative analysis technique detailed in the Compendium on the solution prepared for testing under paragraph 7.2.

(Lead content should be less than 5 mg/kg.)

7.8.     Mercury

Test for mercury concentration in the solution prepared for testing (7.2), using the method explained in the annex. (Mercury content should be less than 1 mg/kg.)

7.9.     Arsenic

Using the method indicated in the Annex, test for arsenic concentration in the test solution prepared in accordance with paragraph 2.  (Arsenic content should be less than 3 mg/kg.)

7.10. Quantitative Analysis of Ammonia

Dilute the test solution prepared under paragraph 7.2 to one-tenth strength, then place 10 ml of this dilute solution (0.10 g of ammonium sulfate) in a steam distillation device (described in the Annex). Add 20 ml of 30% sodium hydroxide (R) and distill 100 ml. Quantitatively analyze the distilled ammonia using 0.1 M hydrochloric acid. Let n be the number of milliliters used:

100 g of ammonium sulfate contains 1.7 n g of ammonia (NH₃).

(Ammonia concentration greater than 25 pp 100.)

7.11. Quantitative Analysis of Sulfuric Acid

Dilute the test solution prepared for testing under paragraph 7.2 to one-tenth strength, then take 25 ml of this solution and add 75 ml of water and 1 ml concentrated hydrochloric acid (R).  Bring to a boil while slowly adding a small excess of barium chloride solution (R). Let the precipitate form for 30 minutes in a 100 °C water bath.  Collect the precipitate, then wash, calcine in an oven at 600 °C and weigh. Let p be the weight of the barium sulfate precipitate:

100 g of ammonium sulfate contains 16.80 p g of sulfuric acid (H₂SO₄).  (Sulfuric acid content greater than 73.5 pp 100.)

8. Storage

Ammonium sulfate should be stored in a dry place in hermetically sealed containers, away from heat.

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COEI-1-ARGON

Argon Ar = 40.0

N° SIN: 938

N°CAS = 7440-37-1

1. Object, origin and field of application

Neutral gas, used for operations of inerting or degassing, it is used in a mixture of nitrogen and/or of carbon dioxide.

2. Labelling

The label must mention the nature of the gas and refer to its composition and purity. The safety conditions must also be indicated on the packages.

3. Characteristics

Colourless and odourless gas without flavour. Non flammable, it does not support combustion.

The weight of a litre of argon under the pressure of 760 mm of mercury is 1.784 g at 0°C. A volume of water dissolves 0.0336 volume of argon at 20°C.

4. Test trials

The global purity of the argon used in oenology must not be less than 99% of argon in volume.

Before any measurement it is advisable to allow any gas to escape for a few minutes in order to purge the piping.

4.1. Chromatographic dosage

The search and determination of gases: Nitrogen, carbon monoxide (less than 10 µl/l), oxygen (10 ml/l), hydrogen, carbon dioxide (less than 300 µl/l), etc., are quickly obtained by chromatography in gaseous phase according to the method in chapter II of the International Oenological Codex.

The total surface are of hydrogen chromatographic peaks, of oxygen and nitrogen must not exceed 1% of gas surfaces to be examined.

The following chemical methods can also be used for oxygen.

4.2. Oxygen dosage by chemical method

Preparation of the flask for searching oxygen:

• Introduce in a 24 ml flask about two fragments of copper turnings of 2 cm, 16 ml of ammoniac solution of copper sulphate (R), then 2 ml of hydrazine dihydrochloride solution (R).
• Seal the flask with a rubber stopper that is easy to pierce with a needle for hypodermic injections. Crimp the neck with a metallic capsule. Then cover the capsule with wax in order to ensure perfect water tightness. Shake the flask and allow to stand away from light until complete discolouration is obtained after about eight days.

Conduct of the test trial:

• Pierce the flask’s stopper to search for oxygen with a needle of 8/10 millimetre for hypodermic injection (take care so as not to plunge it into the liquid) that then will be used for evacuating the gas after bubbling. Then introduce a second needle of the same diameter releasing the gas and plunging it into the liquid. After a minute of bubbling, a noticeable colouration should not be observed. In the presence of oxygen, the liquid quickly becomes blue and the colour darkens with time.

5. Packaging

The argon is supplied in highly resistant steel cylinders painted in white with needle valves. The resistance of these cylinders must be checked periodically.

COEI-1-ASCACI Ascorbic acid

2,3-didehydro-L-threohexano-4-lactone

Acidum ascorbicum

L-Ascorbic Acid

C6H8O6 = 176.1

SIN NO. 300

1. Objective, Origin and Scope of Application

Ascorbic acid is the enolic form of 3-oxo-L-gulofuranolactone (2,3-didehydro-L-threohexano-4-lactone).

This product falls into the category of antioxidants and is used as a reducing agent used to prevent oxidation.

Its use is subject to statutory regulations regarding limits.

 

2. Labelling

The concentration of this product should be indicated on the label, including cases in which it is used in mixtures, as should the safety and storage conditions.

3. Properties

Odorless white or very pale yellow crystalline powder with an acidic flavor. Aqueous solutions rapidly decay in air and light and have a maximum stability at pH 5.4.  Melting point in a capillary tube: approximately 190 °C with decomposition.

Ascorbic acid in aqueous solution has a pH of less than or equal to 3.

4. Solubility

Water at 20 °C	290 g/l
Alcohol, 95% by vol.	320 g/l
Methanol	125 g/l
Acetone	soluble
Benzene, chloroform, ethyl ether, petroleum ether	insoluble

5. Rotatory Power

 

In a 10 pp 100 (m/v) aqueous solution, ascorbic acid has a specific rotatory power

is between +20,5° et +21,5°

6. Absorption in Ultraviolet Light

Ascorbic acid in alcohol solutions in a concentration of 10 mg/l exihibits an absorption spectrum with a maximum of approximately 244 nm.

The solution has a specific extinction of:

 

approximately 560

7. Identifying Characteristics

7.1.     Preparation of the Solution for Testing

Dissolve 5 g ascorbic acid in water and fill to 100 ml using the same solvent.

7.2.     Add 0.5 g monosodium carbonate to 2 ml of the solution prepared for testing (Par. 7.1).

7.3.     Add several drops nitric acid diluted to 10 pp 100 (R) and several drops silver nitrate in a concentration of 1 pp 100 (R) to 1 ml of the solution prepared for tests (Par. 7.1). A gray precipitate will form.

7.4.     To 1 ml of the solution prepared for testing (Par. 7.1) add one drop of recently prepared sodium nitrohexacyanoferrate (III) Na₂[Fe(CN)₅NO], 2H₂O (sodium pentacyanonitrosylferrate) in a concentration of 5 pp 100 (m.v), and 2 ml of 10 pp 100 diluted sodium hydroxide solution (R).  Then, add 0.6-0.7 ml of concentrated hydrochloric acid (R) and stir.  The yellow color will turn to blue.

7.5.     Add drop by drop 2 ml of 2,6-dichlorophenolindophenol solution (R) to the solution prepared for testing (Par. 7.1). It will instantly become decolored.

8. Tests

8.1.     Sulfur Ash

As determined in 1.0 g ascorbic acid, the proportion of sulfur asheshould not be greater than 1 g/kg.

8.2.     Appearance of the Solution

The solution prepared for tests under paragraph 7.1 should be clear and colorless.

8.3.     Determining pH

The pH of the solution prepared for tests under paragraph 7.1 should be between 2.4 and 2.8.

8.4.     Heavy metals

10 ml of the solution prepared for tests under paragraph 7.1 should meet the heavy metal limit requirements described in the annex.  (The heavy metal concentration  expressed in terms of lead should be less than 10 mg/kg).

8.5.     Lead

Use the technique detailed in the Compendium to analyze the solution prepared for tests (Par. 7.1). (Lead concentration should be less than 2 mg/kg).

8.6.     Mercury

Use the technique described in the annex to analyze the solution prepared for tests (Par. 7.1). (Mercury concentration should be less than 1 mg/kg.)

8.7.     Arsenic

Using the method indicated in the Annex, test for arsenic in the test solution prepared in accordance with paragraph 7.1.  (Arsenic concentration should be less than 3 mg/kg).

8.8.     Iron

Implement the atomic absorption technique described in the Compendium to analyze the solution prepared for tests (Par. 7.1). (Iron concentration should be less than 5 mg/kg.)

8.9.     Copper

Implement the atomic absorption technique described in the Compendium to analyze the solution prepared for tests (Par. 7.1). (Copper concentration should be less than 2 mg/kg.)

8.10. Moisture

Dehydration loss after drying in a desiccator under a vacuum and in the presence of sulfuric acid for 24 hours must be less than 0.4%.

8.11. Quantitative Analysis

In 80 ml of recently boiled and cooled water to which 10 ml of sulfuric acid diluted to 10 pp 100 (R) has been added, dissolve a test sample weighed precisely at about 0.20 g. Add 1 ml of starch (R) and titrate using 0.05 M iodine until a persistent blue coloration appears.

1 ml of 0.05 M iodine corresponds to 8.81 mg ascorbic acid.

The product should contain at least 99 pp 100 ascorbic acid.

9. Storage

Ascorbic acid should be stored in tightly sealed non-metal containers in a dark place. Aqueous solutions decay rapidly in air and light.

 

 

Isoascorbic acid

Isoascorbic acid, or D-ascorbic acid or erythorbic acid has the same antioxidant power as ascorbic acid and can be used for the same oenological purpose.

This acid exhibits the same appearance and the same solubility properties as ascorbic acid.

It is, optically, the reverse of ascorbic acid and has, under the same conditions, a specific rotatory power of:

is between -20° et -21,5°

With the exception of rotatory power, this acid should exhibit the same properties as ascorbic acid, respond in the same way to the identifying reactions, pass the same tests and responds to the same quantitative analysis.

Note 1: The vitamin C efficacity of isoascorbic acid is approximately 1/20 of that of ascorbic acid.

Note 2: There is a preliminary draft resolution calling for registration of this product in the International Code of Oenological Practices.

COEI-1-AZOTE Nitrogen

Nitrogenum

N = 14.007

SIN NO. 941

1. Objective, Origin and Scope of Application

Neutral gas used to render inert or to degas.  It can be used pure or mixed with carbon dioxide.

2. Labelling

The label should mention the nature of this gas and reference its composition and purity, as well as its safety and storage conditions.

3. Properties

Colorless, odorless, flavorless gas.  It is not flammable and does not maintain combustion.

The weight of a liter of nitrogen under normal conditions is 1.250 g.

Under a pressure of 760 mm of mercury at 20 °C, a volume of water dissolves a 0.01507 volume of nitrogen, while a volume of alcohol dissolves a 0.1224 volume of nitrogen.

4. Tests

The purity of nitrogen used for oenological purposes should be 99 parts per 100 by volume.

Before undertaking any measurement, the gas should be allowed to escape for several moments in order to clean out the lines.

Gas detection and quantitative analysis: oxygen, carbon monoxide, argon, carbon dioxide, etc. are most rapidly detected using gas phase chromatography.  (See this method in the Annex.)

The following chemical methods can also be used.

4.1.     Phosphorous-containing Hydrogen, Arsenical Hydrogen and Reducing Substances

Let 1 liter of nitrogen to flow into a mixture of 10 ml of ammoniacal silver nitrate (R) and 15 ml of water.

Regulate the flow of gas so that the gas flows into the solution in approximately 15 minutes.

There should be no clouding or brown coloration when compared with an identical control solution through which no gas will flow.

4.2.     Oxygen

Prepare a flask to test for oxygen as follows:

Place 2 turned pieces of copper of approximately 2 cm², 16 ml of ammoniacal copper sulfate solution (R) and 2 ml of hydrazine dichlorhydrate in a 24 ml flask.

Stop the flask with a rubber stopper which can easily be pierced with a hypodermic needle.  Seal the collar with a metal cap, then cover the cap with wax to ensure a perfectly airtight seal.  Shake the flask, then let it sit in the dark until the color disppears completely, after approximately eight days.

Conducting the test:

Pierce the flask stopper with a 8/10 mm hypodermic needle (take care not to dip the needle into the liquid).  This will allow gas to escape after bubbling.  Next, insert a second hypodermic needle of the same diameter and plunge it into the liquid.  After a minute of bubbling, there should be no significant coloring.  In the presence of oxygen, the liquid will rapidly turn blue and the color becomes more intense over time.

The nitrogen must incorporate less than 10 ml/l oxygen.

5. Packing and Storage

 

Nitrogen is delivered in high-strength steel canisters which are painted black and equipped with a needle valve tap.  The strength of the canisters should be checked periodically.

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COEI-1-BENTON Bentonites

Bentonita

N° SIN: 558

1. Object, origin and field of application

Bentonites are hydrous aluminium silicates belonging to the montmorillonite group. The brute formula is:

Si4 (Al (2-x) Rx) (O10, H2O)(Cex, nH2O) or  Si4(Al(2-x)Rx)(H2O)n where:

R = Mg, Fe, M, Zn, Ni

Ce (exchangeable cations) = Ca, Na, Mg.

Bentonites are used for clarification operations or protein stabilisation in musts and wine. Bentonites fix to certain unstable proteins which allows them to be eliminated.

Bentonites are capable of fixing coloured matter.

2. Labelling

The nature of the bentonite (natural sodium, calcium, and activated calcium), batch number and the optimal expiration date for activated bentonites will be indicated on the label. The mention of risks and safety concerning the presence of crystalline silica should also be indicated.

2.1.    Natural Bentonites:

Depending on the nature of the of exchangeable cations present, there are 2 naturally occurring types of bentonite:

• Sodium bentonite, it swells and absorbs readily where sodium is the major exchangeable cation.
• Calcium bentonite, where calcium is the major exchangeable cation, it is lower swelling and lower absorbent than sodium bentonites.

These two types of betonites are simply grinded before their commercialisation after possibly being dried at 80°C to 90°C.

2.2.    Activated bentonites:

In order to improve the adsorption properties of calcium betonites, they are most often activated by sodium carbonate, then dried and grinded. This results in activated calcium bentonites with properties equal or superior to sodium bentonites.

The properties of these bentonites thus activated or permuted are less stable in time (3 to 18 months) and depend on the activation of magnesium, calcium, and sodium levels.

These different types of bentonites are in the form of powder, spherical or cylindrical granules. Colour can vary from white for the purest products to grey, beige or green for others.

3. Test trials

3.1.    Odour

Bentonite should not have any undesirable odour (e.g. no mould) and should not change the taste of wine.

3.2.    pH level

Shake 5g of bentonite with 100 ml of distilled water for 5 minutes. Allow to stand for 1 hour. Measure the pH level of the supernatant liquid. Natural calcium bentonites have a neutral pH (level around 6.5 to 8.5) whereas activated calcium bentonites have a much more alkaline pH (level around 8.5 to 10.0). Natural sodium bentonites have a wider range of pH (level around 4.7 to 10.0).

3.3.    Loss during desiccation

The desiccation of 5 g of bentonite at 105°C during 4 hours causes a weight loss of 5% to 15% of the initial weight (often around 10%).

3.4.    Preparation of the test trial solution

Weigh p g of bentonite containing 10 g of anhydrous bentonite.

In a 500 ml flask with a large opening which can be hermetically sealed, add 100 ml of tartaric acid solution to 5 g per litre until the solution has a pH level of 3 (R). Sprinkle the bentonite trial sample in the constantly shaken solution (for example using a magnetic stirrer) and a funnel. After this addition, shake vigorously for 5 minutes. Allow to stand for 24 to 48 hours. Decant, centrifuge or filter if necessary to obtain at least 100 ml of clear liquid.

All the following set limits for bentonite are for the weight of dried bentonite.

3.5.    Montmorillonite content

Minimum rate:

• Manufacturer indicates that the content should not be under 80% by x-ray diffraction analysis.

3.6.    Different forms of free silica content

• Crystal silica content must be less than 3% (quartz N° CAS 14080-60-7, cristobalite N° CAS 14464-46-1).
• Particle holdings under 10 microns must be less than 10%.
• Respirable crystal silica content must be under 0.3%.

These standards must be written on the security form supplied by the manufacturer.

3.7.    Lead

In the test trial solution (3.4) determine the lead content using the method described in Chapter II.

Lead content must be less than 5 mg/kg.

3.8.    Mercury

Determine the mercury content according to the method described in Chapter II with the test trial solution (3.4).

Mercury content should be less than 1 mg/kg.

3.9.    Arsenic

Determine the arsenic content of 5 ml of test trial solution (3.4) according to the method in Chapter II.

Soluble arsenic content should be less than 2 mg/kg.

3.10. Iron

Add 12.5 ml of water, 1 ml concentrated hydrochloric acid (R) and 2 ml of potassium thiocyanate at 5% (R) to 5 ml of the test trial solution (3.4). The red coloration should be lighter than what is obtained when using 2.5 ml citric acid at 5% at pH 3 (R), 1 ml concentrated hydrochloric acid (R), 15 ml of iron salt solution (III) at 0.010 g of iron per litre (R) and 2 ml of potassium thiocyanate solution at 5% (R).

Iron content should be less than 600 mg/kg).

Iron can also be determined by atomic absorption spectrometry according to the method in Chapter II.

3.11. Aluminium

On the test trial solution (3.4), find extractable aluminium according to the method described in Chapter II.

Extractable aluminium content should be less than 2.5 g/kg.

3.12. Calcium and magnesium

On the test trial solution (3.4), determine calcium and magnesium using the methods outlined in the Compendium of International Methods of Analysis of Wine and Musts.

Calcium and soluble magnesium combined should be less than 100 meq for 100 g.

3.13. Sodium

On the test trial solution (3.4), determine sodium using the method outlined in the Compendium of International Methods of Analysis of Wine and Musts.

Soluble sodium content should be less than 10 g/kg for natural bentonites and less than or equal to 35 g/kg for activated bentonites.

3.14. Presence of large particles

• Put 1 litre of water in a 1.5 litre long stem glass.
• Slowly add while shaking the liquid, a quantity of bentonite corresponding to 50 g of dried bentonite.
• Shake vigorously 2 to 3 minutes and allow to stand for 24 hours.
• Shake 2 to 3 minutes and allow to stand for 2 minutes.
• Using a siphon, take off 9/10 of the cloudy liquid exceeding 100 ml and leave the deposits at the bottom of the glass.
• Add 900 ml of water.
• Shake 1 minute.
• Allow to stand for 2 minutes and repeat to obtain 5 washings.
• Remove the deposit and put in a capsule.
• Dry and weigh.
• The residue must be less than 8 g for 100 g.

3.15. De-acidification tests trials

• Weigh (p) of bentonite containing 0.2 g of dried bentonite.
• Put this in a 125 ml flask containing 50 ml of citric acid 0.033 M solution (R).
• Shake vigorously for 5 minutes and allow to stand for 30 minutes.
• Either filter or centrifuge.
• Take 10 ml of filtrate and titrate with an acid solution of 0.1 M of sodium hydroxide with a drop of phenolphthalein solution (R), that is n ml the volume poured to obtain a colour change in the indicator: 250 (10 – n) is the number of milliequivalent of acids fixed or neutralised for 100 g of bentonite.
• The maximum limit is 2.5 eq/kg.

3.16. Rate of swelling

Swelling indicator: specific test is necessary.

2 g of bentonite is strewn over 100 ml of demineralised water and 100 ml of wine in a graduated test tube cylinder. After 24 hours, weigh the volume of bentonite. This will be expressed in ml/g of dried product.

3.17. Protein adsorption test trial (for bentonite to go through deproteinisation)

3.17.1.     Preparation of test trial solution:

Mix 5 g of egg white with a sufficient amount of citric acid solution of 5 g per litre (pH=3) to make 1 litre. Filter. Determine total nitrogen on 100 ml of this solution by using the procedure described in Chapter II. This solution contains approximately 90 mg of total nitrogen for 575 mg of proteins per litre.

3.17.2.     For each test trial using 100 ml of this solution, mix increasingly larger doses of bentonites prepared in a 5% suspension in order to process doses of 0.1 to 0.8 g/l. Shake vigorously and maintain at 15°C–20 °C for 6 hours. Centrifuge and proceed with determinations of nitrogen or residual proteins.

A de-proteinising bentonite should eliminate at least 50% of the proteins in a synthetic solution with a 0.4 g/l dose.

3.18. Determining the specific adsorption surface (or the adsorption indicator for methylene blue)

See method described in annex.

The accepted limit should be 300 mg/100g.

4. Storage

Bentonites must be stored in a ventilated area in watertight containers away from volatile objects that they could adsorb.

Annex: Determination of the specific surface of adsorption of bentonite

1. General information

1.1.    Aim of the test trial

This test trial enables to measure the capacity of bentonite to adsorb methylene blue.

Clays, organic matters, and iron hydroxide preferentially adsorb methylene blue. This capacity takes into account the activity on the surface of these elements. We call, “blue value” of bentonites, the quantity expressed in grams of methylene blue adsorbed per 100 g of bentonites.

1.2.    Principle of the test trial

Elemental doses of a methylene blue solution are injected successively into an aqueous solution containing the trial sample. The adsorption of blue is checked after each addition by making a spot on a paper filter (spot test, see paragraph 5).

For a simple conformity check, the specified quantity of blue is injected once.

2. Equipment and reagent

2.1.    A 25 ml burette graduated 1/10 ml.

2.2.    Paper filter: quantitative and without ashes (< 0.010); weight: 95 g/m²; thickness: 0.20 mm; filtration speed 75; retention: 8 micrometers.

2.3.    A glass rod: 300 mm length; 8 mm diameter.

2.4.    A magnetic stirrer and magnetic stirring bar.

2.5.    Methylene blue of medicinal quality at 10g/l 0.1 g/l.

The maximum duration for using the solution is one month. The solution must be stored away from light.

2.6.    Demineralised or distilled water.

3. Preparation of test trial samples

Add 10 g of bentonite in 200 ml of distilled water, allow to swell for 2 hours, then homogenise by shaking.

4. Carrying out test trial

4.1.    Definition of spot test

After each addition of blue (see paragraph 5.2), this test involves taking a drop of suspension that is placed on a paper filter using a glass rod. The spot that is formed is composed of a central deposit of matter, blue in colour surrounded by a humid colourless area.

The drop must be such that the diameter of the deposit is between 8 and 12 mm.

The test is positive if a persistent light blue ring appears around the middle deposit in the humid zone. The test is negative if the ring is colourless.

4.2.    Determination

Using a burette, pour 2 ml of blue solution in a container with 200 ml of suspension of bentonite maintained in agitation. After 2 minutes, add 1 ml of blue solution. This addition is followed by the spot test on filter paper.

Allow the asorption of blue to occur which is not instantaneous. Meanwhile tests should be conducted minute by minute.

If the light blue ring disappears at the fifth spot, proceed with elemental additions of 0.2 ml of blue and then 0.1 ml.

Each addition is followed by tests conducted minute by minute.

Renew these operations until the test remains positive for 5 consecutive minutes: the determination is considered as ended.

That is V ml poured.

5. Expression of results

5.1.    Blue value

The blue value expressed in grams of blue for 100 g of bentonite is shown in the following formula:

• V is the value of blue methylene poured in ml.

5.2.    Conformity check compared to a given specification

The specification is expressed in blue value for 100 g of bentonite, or s of this value.

The volume of blue solution to be added in one time to the preparation (3) is:

The spot test is done after eight minutes of shaking. If it is negative, the bentonite complies with the specification.

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COEI-1-WOOPIE Pieces of oak wood

1. Object, origin and field of application

Pieces of oak wood used for winemaking and for passing on certain constituents to the wine in conditions set by regulations.

The pieces of oak wood must come exclusively from the Quercus genus.

They can possibly be left in their natural state or they can be heated to a low, medium or high temperature but they must not be charred including on the surface, nor be carbonaceous, nor friable when touched.

No compound should be added to them for the purpose of increasing their natural aromatising capacity or their extractible phenolic compounds.

Likewise, they must not undergo any chemical, enzymatic or physical treatment other than heating.

2. Labelling

The label must mention the varietal origin of the oak and the intensity of any heating, the storage conditions and safety precautions.

3. Dimensions

The dimensions of these particles must be such that at least 95% in weight be retained by the screen of 2 mm mesh (9 mesh).

4. Purity

The pieces of oak wood must not release any substances in concentrations which may be harmful to health,

5. Storage conditions

The pieces of oak wood must be stored in sufficiently dry and odourless conditions free from substances liable to contaminate them.

6. Introduction in wine

Where bags or other containers are used as the means of introducing pieces of oak wood or related support system into wine, they must be made from materials that are approved for food contact in the country of use, and which do not release any substances into the wine in concentrations which may be harmful to health, or jeopardise to the quality of the final product.

Annex A: Determination of the size of pieces of oak wood by screening

 

1. Introduction

The use of pieces of oak wood, commonly called chips, to treat wine is authorised provided they comply with the specifications of the Oenological Codex (resolution OENO 3/2005). In particular, the pieces of oak wood used must meet a size requirement, and it is specified that "The dimensions of these particles must be such that at least 95% in weight be retained by the screen of 2 mm mesh (9 mesh)". The following operating procedure provides a method of sampling and then screening that can be used to verify this requirement.

2. Field of application

The method applies to oak wood test samples of more than 0.5 kg.

3. Principle

After dividing up the initial test sample, a known quantity of pieces of oak wood (approximately 200g) is placed on a vibrating screen. By weighing the pieces of oak wood remaining on the screen after shaking, it is possible to determine the percentage by weight of particles retained by the screen.

4. Equipment

• Standard laboratory equipment.
• Screen of 2 mm mesh (9 mesh), 30 cm in diameter, mounted on a vibrating plate provided with a recovery tray.
• Weighing machine capable of weighing to within 0.1 g.
• Slotted test specimen divider (see figure below as an example).

Slotted test sample divider (EN 1482-1: 2007 Scheme proposed as an example

5. Division of test sample

When the size of the test sample has to be reduced to obtain “sub-samples” of 200 g which retain a homogeneous nature representative of the initial test sample, a slotted test sample divider can be used which allows random separation of the test sample into 2 parts.

The test sample is poured entirely into the divider in order to separate it into two statistically equivalent parts. Half is put aside, while the other half is again split by means of the chip spreader. This operation is repeated as often as necessary, half being eliminated at each stage with the aim of obtaining 2 “sub-samples” of about 200 g each.

6. Operating procedure

• Weigh the empty screen (W_ES).
• Weigh the empty recovery tray (W_ET).
• Tare the screen + recovery tray unit and place on it about 200 g of pieces of oak wood weighed to within 0.1 g. Let W_OAK be the weight of the pieces of oak wood to be screened.
• Place the unit on the vibrating plate and close the cover with the clamping loops.
• Start up the device and allow it to vibrate for 15 minutes.
• Weigh the screen containing the remaining particles that have not passed through the 2mm meshes (W_PS).
• Weigh the recovery tray containing the particles that have passed through the screen (W_PT).

A second test is performed in these conditions on the second sub-sample of pieces of oak wood coming from the same initial test sample.

Comment: Weighing of the recovery tray before and after screening (W_RT and W_PT) serves to verify that there has been no loss of test sample during the operation.

One should have:

7. Calculation

The percentage (by weight) of particles retained by the screen of 2mm mesh is given by the following formula:

This calculation is performed for each of the 2 sub-samples coming from the initial test sample; the percentage of particles retained corresponds to the mean of the 2 results.

8. Bibliography

• Resolution OENO 3/2005 PIECES OF OAK WOOD
• EN1482-1 - Fertilizers and liming materials.  Sampling and sample preparation.  Part 1: Sampling.

COEI-1-WOOCON Wood for wine containers

1. Subject, origin and scope

 

The wood of containers used during the making, storage or transport of wines.

The pieces of wood must exclusively originate from species recognized as being suitable to store wine (oak, chestnut)

They can possibly be left in their natural state or they can be heated to a low, medium or high temperature, but they must not be charred, including on the surface, nor be carbonaceous, nor friable when touched.

No compound should be added to them for the purpose of increasing their natural aromatizing capacity or their extractable phenolic compounds.

They must not undergo any chemical, enzymatic or physical treatment other than heating when used for new containers.

If they have undergone chemical or physical treatment, in particular to clean containers having already been used, it is recommended to ensure the perfect harmlessness of any such treatment for materials in contact with foodstuffs, and in particular to ensure that sufficient rinsing has eliminated any trace of certain products that are not authorized in wine.

2. Container marking and/or accompanying document

Container markings or the accompanying document must indicate the origin of the botanical species of wood, the intensity of any heating and the safety instructions.

3. Purity

Wooden containers must not release substances in concentrations which may be harmful to health.

4. Storage

Wooden containers must be washed before first use and then stored under suitable conditions to prevent any development of undesirable micro-organisms when the containers are empty.

COEI-1-CALCAR Calcium carbonate

Calcii carbonas

CaCO3 = 100.1

SIN NO. 170

1. Objective, origin and scope of application

This product is used for deacidification.  The transport of calcium ions causes salification of free tartaric acid.  The use of calcium carbonate is also authorized when using the so-called "double salt" method of deacidification.  It may then contain small quantities of calcium tartromalate (double salt) and/or calcium tartrate There are regulations governing the use of this product..

2. Labelling

The label should indicate the proportion of pure ccalcium carbonate and the safety and storage requirements.

3. Centesimal composition

Carbon dioxide	43.97
Calcium	40.04

4. Properties

Calcium carbonate exists as a white powder with the reaction properties of carbonates.  In solution in a concentration of 5 pp 100 (m/v) in dilute acetic acid (R), it yields calcium reactions.

5. Solubility

Insoluble in water

Insoluble in alcohol at 95% by vol.

Soluble with effervescence in dilute acetic acid, hydrochloric acid and nitric acid solutions

6. Tests

6.1.     Desiccation loss

Weigh 2 g calcium carbonate in a dish. Place in an oven at 200 °C for 4 hours. Weight loss should not exceed 2 pp 100.

6.2.     Substances Soluble in Water

Mix 2 g of ground calcium carbonate with 20 ml of boiled water.  Filter. Collect 10 ml.  The solution should be neutral. Dry evaporate.  The residue should not be greater than 1 pp 100l.

6.3.     Ammoniacal Ions

Place 2 g of calcium carbonate, 25 ml of distilled water and 5 ml of 30 pp 100 sodium hydroxide solution (R) in the flask of a distillation device.

Distill and collect 20 ml distillate in 40 ml 4 pp 100 boric acid (R) in the presence of methyl red (R).  Two drops of 0.1 M hydrochloric acid solution should be sufficient to cause the indicator to turn color.

6.4.     Barium

Dissolve 0.50 g of calcium carbonate in 10 ml of nitric acid diluted to 10 pp 100 (R). Add 10 ml of saturated calcium sulfate solution (R). The mixture should remain llear.

6.5.     Preparing the Solution for Tests

Dissolve 10 g of calcium carbonate in 100 ml of 10 pp 100 dilute acetic acid (m/v) (take care as there will be effervescence due to the release of carbon dioxide).

6.6.     Magnesium

Use the method described in the Compendium on the solution prepared for testing under paragraph 6.5. (Content should be less than 1 pp 100 by weight).

6.7.     Iron

Use the atomic absorption spectrometry method described in the Compendium on the solution prepared under paragraph 6.5. (Iron content should be less than 300 mg/kg).

6.8.     Lead

Using the technique described in the annex to quantitatively analyze the lead in the solution prepared for testing (Par. 6.5). (Lead content should be less than 2 mg/kg).

6.9.     Mercury

Implement the technique described in the annex to quantitatively analyze the mercury in the solution prepared for testing (Par. 6.5). (Mercury content should be less than 1 mg/kg).

6.10. Arsenic

Using the method described in the annex, test for arsenic in the solution prepared for testing (Par. 6.5). (Arsenic content should be less than 3 mg/kg).

6.11. Sodium

In accordance with the method described in the Compendium, quantitatively determine sodium content by flame photometry in the solution prepared for testing (Par. 6.5). (Sodium content should be less than 500 mg/kg).

6.12. Quantitative Analysis

Dissolve a precisely weighed sample p of about 2 g in 50 ml of a 1 M hydrochloric acid solution.  Bring to a boil.  Allow to cool and and titrate the excess hydrochloric acid solution using 1 M sodium hydroxide solution and methyl red (R). Let n be the amount in ml of 1 M sodium hydroxide solution used:

1 ml of 1 M hydrochloric acid corresponds to 0.05005 g calcium carbonate.  Parts per 100 of calcium carbonate in the product tested:

The wine-making product must contain a minimum of 98 pp 100 calcium carbonate.

7. Storage

Calcium carbonate should be stored in a dry place in hermetically sealed containers away from volatile elements it could adsorb.

COEI-1-POTCAR Potassium carbonate

Potassium carbonate anhydrous (K₂CO₃, CAS No. 584-08-7)

Potassium carbonate hydrate (2K₂CO₃ · 3H₂O, CAS No.: 6381-79-9)

 

1. Objective, origin and scope of application

The addition of potassium carbonate can be used to deacidify musts and wines.

2. Labelling

The label should indicate the product's purity, lot code, date of manufacture, storage conditions and expiration date.

3. Characteristics

Anhydrous potassium carbonate (K₂CO₃) is the potassium salt of carbonic acid and occurs as a white, odourless, hygroscopic powder. The hydrate form (2K₂CO₃ · 3H₂O) occurs as small, white, translucent crystals or granules.

 

4. Identifying characteristics

 

• Solubility: Very soluble in water, insoluble in ethanol (95% by vol).
• Carbonate: Potassium carbonate is soluble with effervescence in dilute acetic acid or hydrochloric acid solutions, evolving a colourless gas (CO₂) that, when passed into calcium hydroxide solution, produces a white precipitate immediately.
• Potassium: The presence of potassium imparts a violet colour to a non-luminous flame if not masked by the presence of small quantities of sodium.

5. Tests

The limits are determined according to the values observed during production in line with the good manufacturing practices.

5.1.  Desiccation Loss

Through the desiccation of 3 g of potassium carbonate for 4 hours at 180°C, for the anhydrous form, the loss of weight must be lower than 1%, for the hydrate form, the loss of weight must be between 10,0% and 16,5%.

 

5.2.  Preparing the Solution for Tests

Dissolve 1 g of potassium carbonate in 20 mL water.

5.3.  Substances Insoluble in Water

Filter the solution prepared for testing under Paragraph 5.2. on a membrane of cellulose ester with a diameter of the pore lower or equal to 0,5 μm, no residue can be detected.

5.4.  Iron

Using the atomic absorption spectrometry technique detailed in chapter II of the International Oenological Codex, determine the iron content in the test solution (5.2); the content should be less than 10 mg/kg.

5.5.  Lead

Using the technique set forth in chapter II of the International Oenological Codex, determine the lead content in the test solution (5.2); the content should be less than 5 mg/kg.

5.6.  Mercury

Using the technique described in chapter II of the International Oenological Codex, determine the mercury content in the test solution (5.2); the content should be less than 1 mg/kg.

5.7.  Arsenic

Using the technique described in chapter II of the International Oenological Codex, determine the arsenic content in the test solution (5.2); the content should be less than 3 mg/kg.

5.8.  Sodium

Determine the sodium content in the test solution (5.2) using flame photometry described in chapter II of the International Oenological Codex; the content should be less than 1%.

5.9.  Cadmium

Using the technique described in chapter II of the International Oenological Codex, determine the cadmium content in the test solution (5.2); the content should be less than 1 mg/kg.

5.10.        Potassium Carbonate Content

Sample: 1 g previously dried.

Analysis: Transfer sample to a beaker and dissolve it in 50 mL water. Add 2 drops of methyl red TS and, while constantly stirring, slowly titrate with 1 N hydrochloric acid until the solution becomes faintly pink. Heat the solution to boiling, cool, and continue titration until the faint pink colour no longer fades after boiling.  The product intended for wine-making should contain a minimum of 98% potassium carbonate.

6. Storage

 

Potassium carbonate should be stored in airtight containers.

COEI-1-CALPHY Calcium phytate

Calcium inositol hexaphosphate

Calcii phytas

C6H6Ca6O24P6,3H20= 942.11

1. Objective, Origin and Scope of Application

Calcium phytate is the salt of the inositol hexaphosphoric ester, or inositohexaphosphoric or phytic acid.

In its calcium and magnesium double salt forms, phytic acid composes phytin, a reserve form of phosphorous in plants.

Since it is an iron (III) complexing agent approved for removal of excess iron in wines, its use must be strictly monitored.

Any excess phytate with respect to the iron (III) content causes deposits to build up when the slightest oxidation occurs.

2. Labelling

The label should indicate product concentration even when used in mixtures, as well as its safety and storage conditions.

3. Properties

White powder with an acidulous taste, which is minimally soluble in water, soluble in dilute strong acids, and difficult to dissolve in wine, in which solubility is incomplete.

Aqueous calcium phytate solution possess an acidic nature, which is disclosed by movement of the indicator to litmus.  It yields calcium reactions.

4. Tests

4.1.     Desiccation Loss

Dry a 1 g sample of calcium phytate in an oven at 105 °C until a constant weight is obtained.  Weight loss should be less than 12 pp 100.

Limits indicated below are for dry product.

4.2.     Ash

Incinerate a 0.250 g test sample of calcium phytate at 550 °C.  The residue should not be less than 65 pp 100 nor greater than 72 pp 100 of the dry product contained in the test sample.

4.3.     Insoluble Substances

Prepare a first solution contaning 1 g of calcium phytate, 7 ml of 1M hydrochloric acid solution, and 93 ml of distilled water.  Separately, prepare a solution of 1 g of calcium phytate with 50 ml of distilled water and 1.5 ml pure phosphoric acid (R). Filter each of the solutions separately and collect the deposit. Wash and dry the deposit at 100 °C. Each residue should be less than 1 part per 100 (10g/kg) of dried product at 105 °C.

4.4.     Starch

Add several drops of iodinated water (R) to the residues obtained under Paragraph 4.3; no blue coloration should develop.

4.5.     Sugars

• Stir 3 g of calcium phytate with 15 ml of distilled water.
• Filter.
• The filtrate should not reduce the cupro-alkaline reagent (R) before or after the sucrose inversion.

4.6.     Albumin

• Dissolve 1 g of the product in a mixture of 1 ml of concentrated hydrochloric acid (R) and 3 ml of distilled water.
• Add 3 ml of 30% sodium hydroxide solution (R).
• Filter.
• When one drop of 4 pp 100 (m/v) copper (II) sulfate solution is added to the filtrate, no violet color should appear.

4.7.     Preparing the Solution for Tests

Macerate a quantity of calcium phytate containing 5 g dry product with 100 ml of 10 g per liter citric acid (R) for 24 hours while agitating from time to time. Filter.

4.8.     Iron

• Add 1 ml of concentrated hydrochloric acid (R) and 2 ml of 5 pp 100 potassium thiocyanate to 10 ml of test solution prepared under paragraph 4.7.

• The resulting colorating should be less intense than that produced by a control tube prepared with 2.5 ml solution in a concentration of of 0.010 g of iron per liter (R), 7.5 ml of distilled water, 1 ml of concentrated hydrochloric acid (R) and 2 ml of 5 pp 100 thiocyanate (R).  (Iron content should be less than 50 mg/kg).

4.9.     Lead

Using the method described in the Compendium, quantify lead analytically in the test solution prepared according to Par. 4.7.  (Lead content should be less than 5 mg/kg).

4.10. Mercury

Using the method described in the annex, quantify mercury analytically in the test solution prepared according to Par. 4.7.  (Mercury content should be less than 1 mg/kg).

4.11. Arsenic

Using the method described in the annex, quantify arsenic analytically in the test solution prepared according to Par. 4.7.  (Arsenic content should be less than 3 mg/kg).

4.12. Mineral phosphates

• Place 0.50 g calcium phytate in a 200 ml volumetric flask.
• Add 100 ml of distilled water and 5 ml of concentrated nitric acid (R).
• Agitate for 15 minutes at 20 °C and top off to 200 ml with distilled water.
• To 10 ml of this solution, add 10 ml of nitro-vanadomolybdic reagent (R).
• Leave in contact for 15 minutes at 20 °C.  The resulting color should be less intense than that produced by adding 5 ml distilled water and 10 ml nitro-vanadomolybdic reagent (R) to 5 ml of a monopotassic phosphate solution containing 0.05 g phosphorous per liter (R).
• (Mineral phosphate content, expressed in terms of phosphorous, should be less than 1 pp 100).

4.13. Glycerophosphates

• Heat 0.50 g of calcium phytate in the presence of monopotassic sulfate.
• No acrolein fumes (odor of burnt horn) should be released.

4.14. Total Phosphorous Determination

• Weigh precisely a 0.25 g sample of calcium phytate which has already been dried at 105 °C.
• Place it in a flask which is ground and polished so it can be fitted with a tube 8 mm in diameter and 1 m long which will serve as a reflux condenser.
• Add 5 ml of concentrated sulfuric acid (R) and 0.5 ml concentrated nitric acid (R).
• Bring to boiling under reflux for approximately 15 minutes.
• After cooling, decant the contents of the flask diluted with water in a 1 liter volumetric flask.
• Wash the condenser and flask with water by pouring these liquids in the volumetric flask, and fill to gauge line after bringing the temperature to 20 °C. Agitate.
• Add 10 ml of nitro-vanadomolybdic reagent (R) to 10 ml of this solution.  Agitate in a 20 °C water bath and let sit in the water bath for 15 minutes.  The intensity of the resulting color should be equal to or greater than that of a control prepared under the same conditions using 8 ml of monopotassic phosphate solution in a concentration of 0.05 g of

phosphorous per liter (R), 2 ml of water and 10 ml of nitro-vanadomolybdic reagent (R).

Total phosphorous analysis can also be determined using a spectrophotometer with a wavelength of 425 nm whose calibration curve was obtained based on 4-6-8-10 ml of solution in a concentration of 0.05 mg phosphorous per liter (R).

Calcium phytate should contain at least 15 parts of phosphorous per 100 , as compared with a product dried at 105 °C.

5. Storage

Calcium phytate should be stored in a dry place in hermetically sealed containers.

COEI-1-CALTAR Calcium tartrate

Dextrorotatory Calcium Tartrate

Calcium tartaricum

(OOC-CHOH-CHOH-COO) Ca, 4H2O

Tetrahydric L(+)-2,3- calcium dihydoxybutanedioate

(OOC-CHOH-CHOH-COO) Ca, H2O

C4H12CaO10 = 260.13

SIN No. 354

1. Objective, Origin and Scope of Application

A natural wine salt primarily originating from wine residues.  It is therefore typically found in L(+) form. It usually crystallizes in tetrahydrated form.

This product promotes triggering of the precipitation of the natural calcium tartrate in wine by means of a seeding technique.

2. Labelling

The label should indicate product concentration, even when used in mixtures, as well as its safety and storage conditions.

3. Centesimal Composition

Tartaric acid	57.7
Calcium	15.4
Water	27.9

4. Properties

Fine, crystalline powder with a white or off-white color. Tasteless.  Melting point is 270 °C.

 

5. Solubility

 

Water at 20 °C	0.525 g/l
Alcohol, 95% by vol.	0.15 g/l
Ethyl ether	0.01 g/l

6. Tests

6.1.     Rotatory Power

Dissolve 1 g of the substance in 1l of 1 M hydrochloric acid.  After it has completely dissolved, it gives:

Rotatory power is sensitive to slight variations in pH.

6.2.     pH in Saturate Solution

• Add 1 g of the product to 100 ml of distilled water.
• After shaking for one hour and allowing the precipitate to resettle (15 minutes), an increase in pH of between 1.5 and 2.5 pH units should be observed.

6.3.     Desiccation Loss

• Desiccation loss is determined up to constant weight in precisely-weighed sample of about 1 g.
• At a temperature of between 100 and 105 °C, weight loss should be less than or equal to 2.5 pp 100.

6.4.     Preparing the Solution for Tests

Dissolve a sample precisely weight to about 1 g in 100 ml of 1 M hydrochloric acid.

6.5.     Sulfates

• Take 10 ml of the test solution (Par. 6.4) and add to it 1 ml of 10 pp 100 barium chloride solution (R).
• After homogenization, let sit after 15 minutes.
• No clouding should occur.
• If clouding does occur, it should be less intense than that in a control prepared using the method indicated in the Annex.  (Sulfate content, expressed in terms of sulfuric acid, should be less than 1 g/kg).

6.6.     Heavy Metals

Add 0.5 ml of concentrated ammonium hydroxide (R), 2 ml of pH 3.5 buffer solution (R) and 1.2 ml of thioacetamide reagent (R) to 10 ml of the test solution prepared under paragraph 6.4.  (Heavy metal content, expressed in terms of lead, should be less than 10 mg/kg).

6.7.     Lead

Using the method described in the Compendium, quantify lead analytically in the test solution prepared according to Par. 6.4. (Lead content should be less than 5 mg/kg).

6.8.     Mercury

Using the method described in the Annex, quantify mercury analytically in the test solution prepared according to Par. 6.4. (Mercury content should be less than 1 mg/kg).

6.9.     Arsenic

Using the method described in the Annex, quantify arsenic analytically in the test solution prepared according to Par. 6.4.

(Arsenic content should be less than 3 mg/kg).

6.10. Basic Residue Determination

• Dissolve a sample, p, of tetrahydric calcium tartrate weighed precisely at about 0.5 g in 25 ml of 1 M hydrochloric acid solution (R).
• Bring to boiling under reflux and allow to cool.
• Titrate the excess acid  using 1 M sodium hydroxide solution (R) and in the presence of methyl red (R). Let  n be the quantity in millimeters of  the 1 M sodium hydroxide solution used. 1 ml of 1 M hydrochloric acid corresponds to 0.05005 g of calcium carbonate.
• The content in parts per 100 of calcium carbonate is:

The products used in winemaking should contain a maximum of 3 pp 100 basic residues expressed in terms of calcium carbonate.

7. Storage

Calcium tartrate should be stored away from moisture in hermetically-sealed containers.

COEI-1-CALSUL Calcium sulfate

Ca x 2 O (Dihydrate)

CAS NUMBER 10101-41-4

1. Objective and scope of application

This product is used for must acidification in the production of liqueur wines. Calcium sulfate added reacts with tartrate ions of the must producing insoluble calcium tartrate and releasing ion sulfate in the must. These facts originate modifications in ions equilibria that liberates proton ions and reduces the pH without increasing the titratable acidity.

2. Labelling

The label should indicate the nature of calcium sulfate, batch number and the storage and safety requirements.

 

3. Stoichiometric composition

CaSO4	79.1 %
H2O	20.9 %

4. Properties

Calcium sulfate dihydrate exists as a white amorphous powder. Not to be confused with the anhydrous form which is very hygroscopic and sets in contact with must.

 

5. Solubility

Slightly soluble in water and soluble in hydrochloric, sulphuric and nitric acid solutions.

 

6. Tests

 

6.1. Desiccation losses

Free water: Weigh 50 g of calcium sulphate in a dish. Place it in an oven at 40 ºC until constant weight. Weight loss should not exceed 2 %.

Free and bonded water: Place another sample in an oven at 200 º C during 4 h. Total weight loss should not exceed 23 %.

 

6.2. Preparing the Solution for Tests

Weigh 10 g of calcium sulphate. In a 500 ml erlenmeyer flask which can be hermetically sealed, add 200 ml of tartaric acid solution at 5 g/L per litre and bring to pH 3 with HCl 0.1 N. Put this in a magnetic mixer, sprinkle gently the calcium sulphate and mix for 1 hour at a temperature of 20 2°C. Allow to settle and filter by eliminating the first 50 ml of filtrate. Collect at least 100 ml of clear liquid.

 

6.3. Lead

Using the technique described in the Compendium, analyse quantitatively the lead in the solution prepared for testing (Par. 6.2). Lead content in calcium sulfate should be less than 2 mg/kg.

 

6.4. Mercury

Using the technique described in the Compendium, analyse quantitatively the mercury in the solution prepared for testing (Par. 6.2). Mercury content in calcium sulfate should be less than 1 mg/kg.

 

6.5. Arsenic

Using the technique described in the Compendium, analyse quantitatively the arsenic in the solution prepared for testing (Par. 6.2). Arsenic content in calcium sulfate should be less than 3 mg/kg.

 

6.6. Iron

Using the technique described in the Compendium, analyse quantitatively the iron in the solution prepared for testing (Par. 6.2). Iron content should be less than 200 mg/kg.

 

6.7. Quantitative Analysis

• Any method of analysis included in the Compendium could be used. In the case of using the gravimetric method OIV-MA-AS321-05A, use the following procedure.
• Weigh 250 milligrams of the sample dried at 40 ºC with a precision of 1 mg and dissolve it in 10 mL HCl 1M.
• Take 5 mL of this solution and add 0,5 mL of HCl 2 M and 1,5 mL of a solution of BaCl₂ 400 g/L. Stir with a glass stirrer; rinse the stirrer with a little distilled water and leave to stand for 5 min.
• Centrifuge for 5 min at 3.000 rpm, then carefully decant the supernatant liquid.
• Wash the barium sulfate precipitate as follows: add 10 mL hydrochloric acid 2 M, place the precipitate in suspension and centrifuge for 5 min at 3.000 rpm, then carefully decant the supernatant liquid.
• Repeat the washing procedure twice as before using 15 mL distilled water each time.
• Quantitatively transfer the precipitate, with distilled water, into a tared platinum capsule and place over a water bath at 100°C until fully evaporated.
• The dried precipitate is calcined several times briefly over a flame until a white residue is obtained. Leave to cool in a desiccator and weigh:

 

7. Calculations

Content of calcium sulfate dihydrate in the product (%)=

• Where p is the measured weight of BaSO₄ in mg.

If other method of analysis of sulphates included in the Compendium is used to analyse the initial solution of calcium sulfate prepared for quantitative analysis:

Content of calcium sulfate dihydrate in the product (%)=

• Where c is the concentration of sulfates in mg/L of K₂SO₄

The wine-making product must contain a minimum of 90 pp 100 calcium sulfate.

8. Storage

Calcium sulfate should be stored in a dry place in hermetically sealed containers away from volatile elements it could adsorb.

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COEI-1-CELLUL Cellulose

(

INS N°: 460

1. Object, origin and field of application

Cellulose is obtained from mechanical processing and purification from an alpha-cellulose, which comes directly from vegetable fibres. Its molecular weight is 1.5. Dalton. Cellulose fibre is used for its absorbency traits, mainly for the filtration of wine.

2. Labelling

The concentration of the product and whether it was mixed, must be indicated on the label in addition to the change.

3. Characteristics

Cellulose is a white odourless, flavourless, fibre. It is insoluble in water.

4. Test trials

4.1.     pH

• Mix 5g of cellulose in 40 ml of water free of carbon dioxide, for 20 minutes.
• Centrifuge.
• The pH of the supernatant will be between 5.0 and 7.5.

4.2.     Humidity and volatile matter

• Put 5 g of cellulose in an incubator at 105°C for 3 hours.
• Mass loss must not exceed 8%.

 

All of the maximum limits set below refer to the dried product.

4.3.     Starch

• Add 90 ml of water (R) to 10 g of microcrystalline cellulose and boil for 5 minutes.
• Filter when hot. Cool and add 0.1 ml of 0.05 Miodine to the filtrate.
• A blue colour should not appear.

 

4.4.     Ashes

• Incinerate at 600 25°C the residue obtained according to point 4.2, for 4 hours.
• The weight of the ashes should not exceed 2%.

4.5.     Preparation of the test solution

• After weighing, dissolve ashes in 2 ml of concentrated hydrochloric acid (R) and 10 ml of water (R).
• Heat in order to dissolve and fill the water up to 50 ml. (R).

4.6.     Iron

Determine iron using an atomic absorption spectrophotometer (following the method described in Chapter II on the test solution (4.5).

Iron content must be less than 100 mg/kg.

4.7.     Lead

Measure out lead following the method described in Chapter II on the test solution (4.5). Lead content must be less than 5 mg/kg.

4.8.     Mercury

Measure out mercury following the method described in Chapter II on the test solution (4.5).

Mercury content must be less than 1 mg/kg.

4.9.     Cadmium

Measure out cadmium as described in Chapter II on the test solution (4.5).

Cadmium content must be less than 1 mg/kg.

4.10. Arsenic

Measure out arsenic following the method described in Chapter II on the test solution (4.5).

Arsenic content must be less than 2 mg/kg.

4.11. Calcium

Determine calcium using an atomic absorption spectrophotometer (see method described in Chapter II on the test solution (4.5). Calcium content must be less than 500 mg/kg.

4.12. Water soluble substances

Evaporate the aliquot part of the supernatant obtained when measuring the pH level at point 4.1, in an incubator at 105°C for 3 hours. The soluble substance content should not exceed 0.25%.

5. Storing conditions

Cellulose should be kept in a well-ventilated place in sealed packages away from volatile substances susceptible of being adsorbed.

POWDERED CELLULOSE

(C₁₂H₂₀O₁₀)_n
INS No.: 460 (ii)

CAS No.: 9004-34-6

OIV-OENO 681-2022

1. Object, origin and scope of application

Powdered cellulose is cellulose of food-grade quality. Cellulose is a linear glucose homopolymer composed of glucopyranose units linked by β-1,4-glycosidic bonds; its degree of polymerisation (DP) is dependent on the origin of the cellulosic material.

Powdered cellulose is a non-modified, purified cellulose, obtained by the mechanical disintegration of alpha-cellulose procured in pulp form from fibrous plant material.

Powdered cellulose plays a “support” role in clarified fermentation media; it allows for improved “degassing” of carbon dioxide at the start of alcoholic fermentation and thus shortens the latency phase.  It increases the fermentability of must.

2. Labelling

The following should be mentioned on the label:

- the identification of the cellulose and its use in food,

- the concentration of the product, including in the case of a mixture,

- safety and storage conditions

- the batch no.,

- the expiry date.

3. Characteristics

Powdered cellulose comes in the form of flakes or very fine fibres, and is whitish in colour, odourless and flavourless.

4. Test limits and methods

4.1 Identification

Place approx. 10 mg powdered cellulose on a watch glass and disperse into 2 mL iodinated zinc chloride solution (R). The solution will turn a blue-purple colour.

4.2 Solubility

Powdered cellulose is insoluble in water, ethanol, ether and diluted acids, and slightly soluble in sodium hydroxide solution.

4.3 Purity

The powdered cellulose content should not be less than 92%.

4.4 Size of fine particles

The size of fine particles should not be less than 5 µm; the number of particles smaller than 5µm should not exceed 10%.

4.5 pH

Mix approx. 10 g dry cellulose in 90 mL water free of carbon dioxide for 60 min. Centrifuge.
The pH of the supernatant liquid should be between 5.0 and 7.5.

4.6 Water-soluble substances

Mix around 6 g of sample, dried beforehand, with 90 mL water that has recently been boiled then cooled. Leave to rest for 10 min. Filter using a membrane with porosity of 3 µm, throw away the first 10 mL of filtrate and pass the filtrate through the same filter a second time if necessary to obtain a clear filtrate. Evaporate a 15-mL portion of filtrate to dryness in a tared evaporating dish over a water bath, and dry to 105 °C for 1 h. Weigh the dish containing the dry residue. Less than 15 mg of residue should be obtained.

4.7 Detection of starch

Add 90 mL demineralised water (R) to 10 g powdered cellulose and boil for 5 min. Filter while hot with a 25-µm membrane filter. Cool and add 0.1 mL 0.05 M iodine solution to the filtrate. No blue colouration should appear. With very fine grains, a light blue colour may be observed after the addition of the iodine solution but will disappear after 30 minutes.

4.8 Loss on drying

Place 1 g powdered cellulose in a tared dish in an oven at 100-105 °C for 3 hours. The loss on drying should not exceed 7.0%.

4.9 Ashes

Incinerate the residue obtained in point 4.8 at 800 ± 25 °C for 4 hours.
The weight of the ashes should not be greater than 0.3%.

All of the limits set below relate to dry products.

5. Preparation of the test solution

After weighing, dissolve the ashes in 2 mL concentrated hydrochloric acid (R) and 10 mL water (R). Heat to activate dissolution and make up to 50 mL with water.

5.1 Iron

Determine the iron content of the solution prepared for testing purposes (5) by atomic absorption spectrophotometry according to the method described in Chapter II.
The iron content should be less than or equal to 10 mg/kg.

5.2 Lead

Determine the lead content of the solution prepared for testing purposes (5) according to the method described in Chapter II.
The lead content should be less than 2 mg/kg.

5.3 Mercury

Determine the mercury content of the solution prepared for testing purposes (5) according to the method described in Chapter II.
The mercury content should be less than 1 mg/kg.

5.4 Cadmium

Determine the cadmium content of the solution prepared for testing purposes (5) according to the method described in Chapter II.
The cadmium content should be less than 1 mg/kg.

5.5 Arsenic

Determine the arsenic content of the solution prepared for testing purposes (5) according to the method described in Chapter II.
The arsenic content should be less than 1 mg/kg.

6. Storage

Powdered cellulose should be stored in a well-ventilated place in airtight packaging, away from any volatile substances that it could adsorb.

COEI-1-CELMIC Microcristlaline cellulose

INS N°: 460

1. Object, origin and field of application

Microcrystalline cellulose is purified cellulose and is partially depolymerised. It comes from the treatment of alpha-cellulose mineral acids from plant fibres. Its molecular weight is approximately 36 000

Microcrystalline cellulose plays an important role in “supporting” very clarified fermentation as it increases the fermentability of the juices.

2. Labelling

The concentration of the product must be mentioned on the label and if there is mixing as well as the method of preservation.

3. Characteristics

 

Cellulose is found in microcrystalline powder form. white. odourless and tasteless. It is almost insoluble in water. acetone. ethanol. toluene. diluted acids and in 50 g/l sodium hydroxide solutions.

4. Identification

4.1.    In a watch glass. put approximately 10 mg of microcrystalline cellulose and add 2 ml of zinc chloride iodated solution (R). The solution turns bluish purple.

4.2.    Degree of polymerisation

• Put 1.300 g of  microcrystalline cellulose in a 125 ml conical flask. Add 25 ml of water (R) and 25 ml of 1M cupriethylenediamine hydroxide.
• Immediately pass a nitrogen current. Close the flask and mix until completely dissolved.
• For 7 ml of the solution into an appropriate glass capillary viscosimetric tube.

Time how long it takes between two lines on the viscosimeter and express the time measured in (t₁). Calculate the kinematic viscosity V₁ of the solution using the following formula:

In which k₁ is the viscosimeter constant.

Take out an appropriate volume of 1M cupriethylenediamine hydroxide and dilute with the same volume of water. (R). Using an appropriate capillary viscosimeter. determine the time flow of this solution.

Calculate the kinematics viscosity V₂ of the solvent using the following formula.

In which k₂ is the viscosimeter constant.

Determine the relative viscosity η_rel  of the microcrystalline cellulose sample. using the following formula:

Determine the intrinsic viscosity []c by extrapolation. using the intrinsic viscosity table in Annex.

Calculate the degree of polymerisation P. using the formula:

In which m is the mass. in grams of the trial and b is the value obtained in the test trial " loss through drying " in %.

The degree of polymerisation is not over 350.

4.3.    pH

• Shake for 20 minutes about 5 g of cellulose in 40 ml of water free of carbon dioxide.
• Centrifuge.
• The pH of the supernatant liquid must be between 5.0 and 7.5.

4.4.    Soluble substances in ether

• Prepare a column of 10.0 g of microcrystalline cellulose in a glass tube with an inside diameter of approximately 20 mm.
• Put 50 ml of ether free of peroxides (R). through the column and evaporate the eluate until bone dry.
• The residue should not be above 5.0 mg (0.05%).

4.5.    Soluble substances in water

• Mix 5.0 g of microcrystalline cellulose with 80 ml of water (R) for 10 mn.
• Filter in a vacuum and collect the filtrate in a weighed vase.
• Evaporate over a bath of 100° C water until bone dry and dry at 100-105°C for 1 hour.
• The residue is not above 12.5 mg (0.25%).

4.6.    Starch

• Add 90 ml of water (R) to 10 g microcrystalline cellulose. and boil for 5 mn.
• Filter when hot.
• Let cool and add 0.1 ml iodine 0.05 M to filtrate.
• There is no blue colouring.

4.7.    Loss through drying

• Put 1 g of cellulose in a mass capsule for 3 hours in an incubator at 100-105°C.
• Loss through drying should not be more than 6.0%.

All limits set below refer to the dried product.

4.8.    Ashes

• Incinerate at 600 ± 25°C the residue obtained in point 4.7. for 4 hours.
• The mass of the ashes should not be more than 0.1%.

4.9.    Preparation of test solution

• After weighing. dissolve the ashes in 2 ml of concentrated hydrochloric acid (R) and 10 ml of water (R).
• Heat to activate the dissolution and fill up to 50 ml with water.

4.10. Iron

Determine iron with an atomic absorption spectrophotometer following the method described in Chapter II into the test solution (4.9).

Iron content must be less than less or equal to 10 mg/kg.

4.11. Lead

Determine the lead according to the method described in Chapter II. into the test solution (4.9).

Lead content must be less than 5 mg/kg.

4.12. Mercury

Determine the mercury according to the method described in Chapter II

Mercury content must be less than 1 mg/kg.

4.13. Cadmium

Determine the cadmium according to the method described in Chapter II. into the test solution (4.9).

Cadmium content must be less than 1 mg/kg.

4.14. Arsenic

Determine the arsenic according to the method described in Chapter II.

Arsenic content must be less than 1 mg/kg.

4.15. Calcium

Determine the calcium with an atomic absorption spectrophotometer. following the method described in Chapter II. into the test solution (4.9).

Calcium content must be less than 500 mg/kg.

5. Storing conditions

Cellulose must be stored in a well-ventilated place in sealed packages away from volatile substances which it might adsorb.

Table of instrinsic viscosity
Intrinsic viscosity. [η]c. according to value of relative viscosity.. ηrel
[η]c
rel	0.00	0.01	0.02	0.03	0.04	0.05	0.06	0.07	0.08	0.09
1.1	0.098	0.106	0.115	0.125	0.134	0.143	0.152	0.161	0.170	0.180
1.2	0.189	0.198	0.207	0.216	0.225	0.233	0.242	0.250	0.259	0.268
1.3	0.276	0.285	0.293	0.302	0.310	0.318	0.326	0.334	0.342	0.350
1.4	0.358	0.367	0.375	0.383	0.391	0.399	0.407	0.414	0.422	0.430
1.5	0.437	0.445	0.453	0.460	0.468	0.476	0.484	0.491	0.499	0.507
1.6	0.515	0.522	0.529	0.536	0.544	0.551	0.558	0.566	0.573	0.580
1.7	0.587	0.595	0.602	0.608	0.615	0.622	0.629	0.636	0.642	0.649
1.8	0.656	0.663	0.670	0.677	0.683	0.690	0.697	0.704	0.710	0.717
1.9	0.723	0.730	0.736	0.743	0.749	0.756	0.762	0.769	0.775	0.782
2.0	0.788	0.795	0.802	0.809	0.815	0.821	0.827	0.833	0.840	0.846
2.1	0.852	0.858	0.864	0.870	0.876	0.882	0.888	0.894	0.900	0.906
2.2	0.912	0.918	0.924	0.929	0.935	0.94 1	0.948	0.953	0.959	0.965
2.3	0.971	0.976	0.983	0.988	0.994	1.000	1.006	1.011	1.017	1.022
2.4	1.028	1.033	1.039	1.044	1.050	1.056	1.061	1.067	1.072	1.078
2.5	1.083	1.089	1.094	1.100	1.105	1.111	1.116	1.121	1.126	1.131
2.6	1.137	1.142	1.147	1.153	1.158	1.163	1.169	1.174	1.179	1.184
2.7	1.190	1.195	1.200	1.205	1.210	1.215	1.220	1.225	1.230	1.235
2.8	1.240	1.245	1.250	1.255	1.260	1.265	1.270	1.275	1.280	1.285
2.9	1.290	1.295	1.300	1.305	1.310	1.314	1.319	1.324	1.329	1.333
3.0	1.338	1.343	1.348	1.352	1.357	1.362	1.367	1.371	1.376	1.381
3.1	1.386	1.390	1.395	1.400	1.405	1.409	1.414	1.418	1.423	1.427
3.2	1.432	1.436	1.441	1.446	1.450	1.455	1.459	1.464	1.468	1.473
3.3	1.477	1.482	1.486	1.491	1.496	1.500	1.504	1.508	1.513	1.517
3.4	1.521	1.525	1.529	1.533	1.537	1.542	1.546	1.550	1.554	1.558
3.5	1.562	1.566	1.570	1.575	1.579	1.583	1.587	1.591	1.595	1.600
3.6	1;604	1.608	1.612	1.617	1.621	1.625	1.629	1.633	1.637	1.642
3.7	1.646	1.650	1.654	1.658	1.662	1.666	1.671	1.675	1.679	1.683
3.8	1.687	1.691	1.695	1.700	1.704	1.708	1.712	1.715	1.719	1.723
3.9	1.727	1.731	1.735	1.739	1.742	1.746	1.750	1.754	1.758	1.762
4.0	1.765	1.769	1.773	1.777	1.781	1.785	1.789	1.792	1.796	1.800
4.1	1.804	1.808	1.811	1.815	1.819	1.822	1.826	1.830	1.833	1.837
4.2	1.841	1.845	1.848	1.852	1.856	1.859	1.863	1.867	1.870	1.874
4.3	1.878	1.882	1.885	1.889	1.893	1.896	1.900	1.904	1.907	1.911
4.4	1.914	1.918	1.921	1.925	1.929	1.932	1.936	1.939	1.943	1.946
4.5	1.950	1.954	1.957	1.961	1.964	1.968	1.971	1.975	1.979	1.982
4.6	1.986	1.989	1.993	1.996	2.000	2.003	2.007	2.010	2.013	2.017
4.7	2.020	2.023	2.027	2.030	2.033	2.037	2.040	2.043	2.047	2.050
4.8	2.053	2.057	2.060	2.063	2.067	2.070	2.073	2.077	2.080	2.083
4.9	2.087	2.090	2.093	2.097	2.100	2.103	2.107	2.110	2.113	2.116
5.0	2.119	2.122	2.125	2.129	2.132	2.135	2.139	2.142	2.145	2.148
5.1	2.151	2.154	2.158	2.160	2.164	2.167	2.170	2.173	2.176	2.180
5.2	2.183	2.186	2.190	2.192	2.195	2.197	2.200	2.203	2.206	2.209
5.3	2.212	2.215	2.218	2.221	2.224	2.227	2.230	2.233	2.236	2.240
5.4	2.243	2.246	2.249	2.252	2.255	2.258	2.261	2.264	2.267	2.270
5.5	2.273	2.276	2.279	2.282	2.285	2.288	2.291	2.294	2.297	2.300
5.6	2.303	2.306	2.309	2.312	2.315	2.318	2.320	2.324	2.326	2.329
5.7	2.332	2.335	2.338	2.341	2.344	2.347	2.350	2.353	2.355	2.358
5.8	2.361	2.364	2.367	2.370	2.373	2.376	2.379	2.382	2.384	2.387
5.9	2.390	2.393	2.396	2.400	2.403	2.405	2.408	2.411	2.414	2.417
6.0	2.419	2.422	2.425	2.428	2.431	2.433	2.436	2.439	2.442	2.444
6.1	2.447	2.450	2.453	2.456	2.458	2.461	2.464	2.467	2.470	2.472
6.2	2.475	2.478	2.481	2.483	2.486	2.489	2.492	2.494	2.497	2.500
6.3	2.503	2.505	2.508	2.511	2.513	2.516	2.518	2.52 1	2.524	2.526
6.4	2.529	2.532	2.534	2.537	2.540	2.542	2.545	2.547	2.550	2.553
6.5	2.555	2.558	2.561	2.563	2.566	2.568	2.571	2.574	2.576	2.579
6.6	2.581	2.584	2.587	2.590	2.592	2.595	2.597	2.600	2.603	2.605
6.7	2.608	2.610	2.613	2.615	2.618	2.620	2.623	2.625	2.627	2.630
6.8	2.633	2.635	2.637	2.640	2.643	2.645	2.648	2.650	2.653	2.655
6.9	2.658	2.660	2.663	2.665	2.668	2.670	2.673	2.675	2.678	2.680
7.0	2.683	2.685	2.687	2.690	2.693	2.695	2.698	2.700	2.702	2.705
7.1	2.707	2.710	2.712	2.714	2.717	2.719	2.721	2.724	2.726	2.729
7.2	2.731	2.733	2.736	2.738	2.740	2.743	2.745	2.748	2.750	2.752
7.3	2.755	2.757	2.760	2.762	2.764	2.767	2.769	2.771	2.774	2.776
7.4	2.779	2.781	2.783	2.786	2.788	2.790	2.793	2.795	2.798	2.800
7.5	2.802	2.805	2.807	2.809	2.812	2.814	2.816	2.819	2.821	2.823
7.6	2.826	2.828	2.830	2.833	2.835	2.837	2.840	2.842	2.844	2.847
7.7	2.849	2.851	2.854	2.856	2.858	2.860	2.863	2.865	2.868	2.870
7.8	2.873	2.875	2.877	2.879	2.881	2.884	2.887	2.889	2.891	2.893
7.9	2.895	2.898	2.900	2.902	2.905	2.907	2.909	2.911	2.913	2.915
8.0	2.918	2.920	2.922	2.924	2.926	2.928	2.931	2.933	2.935	2.937
8.1	2.939	2.942	2.944	2.946	2.948	2.950	2.952	2.955	2.957	2.959
8.2	2.961	2.963	2.966	2.968	2.970	2.972	2.974	2.976	2.979	2.981
8.3	2.983	2.985	2.987	2.990	2.992	2.994	2.996	2.998	3.000	3.002
8.4	3.004	3.006	3.008	3.010	3.012	3.015	3.017	3.019	3.021	3.023
8.5	3.025	3.027	3.029	3.031	3.033	3.035	3.037	3.040	3.042	3.044
8.6	3.046	3.048	3.050	3.052	3.054	3.056	3.058	3.060	3.062	3.064
8.7	3.067	3.069	3.071	3.073	3.075	3.077	3.079	3.081	3.083	3.085
8.8	3.087	3.089	3.092	3.094	3.096	3.098	3.100	3.102	3.104	3.106
8.9	3.108	3.110	3.112	3.114	3.116	3.118	3.120	3.122	3.124	3.126
9.0	3.128	3.130	3.132	3.134	3.136	3.138	3.140	3.142	3.144	3.146
9.1	3.148	3.150	3.152	3.154	3.156	3.158	3.160	3.162	3.164	3.166
9.2	3.168	3.170	3.172	3.174	3.176	3.178	3.180	3.182	3.184	3.186
9.3	3.188	3.190	3.192	3.194	3.196	3.198	3.200	3.202	3.204	3.206
9.4	3.208	3.210	3.212	3.214	3.215	3.217	3.219	3.221	3.223	3.225
9.5	3.227	3.229	3.231	3.233	3.235	3.237	3.239	3.241	3.242	3.244
9.6	3.246	3.248	3.250	3.252	3.254	3.256	3.258	3.260	3.262	3.264
9.7	3.266	3.268	3.269	3.271	3.273	3.275	3.277	3.279	3.281	3.283
9.8	3.285	3.287	3.289	3.291	3.293	3.295	3.297	3.298	3.300	3.302
9.9	3.304	3.305	3.307	3.309	3.311	3.313	3.316	3.318	3.320	3.321
10	3.32	3.34	3.36	3.37	3.39	3.41	3.43	3.45	3.46	3.48
11	3.50	3.52	3.53	3.55	3.56	3.58	3.60	3.61	3.63	3.64
12	3.66	3.68	3.69	3.71	3.72	3.74	3.76	3.77	3.79	3.80
13	3.80	3.83	3.85	3.86	3.88	3.89	3.90	3.92	3.93	3.95
14	3.96	3.97	3.99	4.00	4.02	4.03	4.04	4.06	4.07	4.09
15	4.10	4.11	4.13	4.14	4.15	4.17	4.18	4.19	4.20	4.22
16	4.23	4.24	4.25	4.27	4.28	4.29	4.30	4.31	4.33	4.34
17	4.35	4.36	4.37	4.38	4.39	4.41	4.42	4.43	4.44	4.45
18	4.46	4.47	4.48	4.49	4.50	4.52	4.53	4.54	4.55	4.56
19	4.57	4.58	4.59	4.60	4.61	4.62	4.63	4.64	4.65	4.66

COEI-1-CHARBO Oenological carbon

INS N° :153

1. Object, origin and field of application

Oenological carbon are of plant (generally wood) origin. In order to increase their adsorption properties, the carbon is subjected to activation either at a high temperature or a lower temperature in the presence of an acid, (generally phosphoric acid). Oenological carbon must not be activated with a metal-based catalyser like zinc chloride.

It is in the form of very fine and light black powder, or in granulated form.

There are humid preparations which can reduce dust accumulation. In this case, weight loss as described in 3.1 can reach 60%.

Carbon can be agglomerated with bentonite.

Carbon for oenological purposes is used to correct alterations due to fungus in addition to the colour of white musts from purple, spotted or oxidised grapes. The carbon can eliminate anthocyanins and oxidised or non-oxidised polyphenols in addition to polysaccharides. The carbon are used to correct the organoleptic characteristics of musts made from grapes altered by fungus.

Oenological carbon can also be used to reduce the presence of Ochratoxin A in musts, the musts during fermentation and in white wine.

Decolourising carbon has a relatively weak deodorising effect.

Absorption by carbon is not very selective and depends on its structure, porosity and specific surface area.

The limit concerning the use of carbons should be compliant with the prescriptions of the OIV International Code of Oenological Practices (expressed by weight of dry carbon).

2. Labelling

The label should indicate the storage conditions, the expiration date for humid solutions, and a mention of whether there are existing regulations regarding the usage of the product and specify if it concerns decolourising or deodorising carbon.

3. Test trials

3.1.    Loss with dessication

• Put 5g of carbon in a silica capsule and heat to 100°C in an incubator.
• After 3 hours of dessicating, weight loss should not be more than 20%.

All limits set for carbon refer to dry carbon weight.

3.2.    Ashes

• Incinerate the previously obtained dry residue at 500°C-600°C.
• These ashes should not be more than 10%.
• Carbon agglomerated with bentonite should have ashes more than 10% and less than 30%.

3.3.    Soluble matter in acids

• Boil 5 g of dried carbon with 20 ml of concentrated hydrochloric acid (R) and 100 ml of water.
• Once cooled, filter using a fine filter or membrane.

Evaporate the filtrate and dry at 100°C–105°C. The soluble matter content in acids should not be more than 5%.

3.4.    Chlorides

• Shake 0.067g of dried carbon and 20 ml of distilled water.
• Filter.
• Add 5 ml of diluted nitric acid (R) to 5 ml of filtrate.
• Fill up to 20 ml and add 0.5 ml of silver nitrate solution at 5% (R).
• Compare any opalescence or cloudiness to a prepared control sample as indicated in the annex. Other methods such as ionic chromatography can be used.
• Chloride content should not be more than 3g/kg.

3.5.    Cyanides

• Put a quantity of carbon containing 1 g of dried carbon with 10 ml of diluted sulphuric acid (R) in a 100 ml conical flask.
• Adapt to the conical flask a pressure relief tube plunged into approximately 2 ml of saturated borax solution (R) in a test tube.
• Distil and gather 2 to 3 ml of distillate.
• Add 5 drops of potassium anhydrosulphite solution at 2% (R) and leave for 5 minutes.
• Add 1 ml of iron sulphate solution (II) at 5% (R) and leave for 15 minutes.
• Then add 2 drops of phenolphthalein (R). Use a saturated borax solution (R) to make the solution a little more alkaline.
• Leave for 5 minutes.
• Add 2 drops of iron sulphate (III) and ammonia solution at 10% (R) and 1 ml of concentrated hydrochloric acid (R).
• No colouration nor blue precipitate should form.

3.6.    Polycyclic  aromatic hydrocarbons

Polycyclic aromatic hydrocarbons including benzoa]pyrene are extracted by hexane; the solvent is evaporated and the residue is taken by the methanoltetrahydrofuran mixture for HPLC analysis following the method described in chapter II.

Note: It is also possible to determine benzoa]pyrene by gas chromatography by using an apolar capillary column with detection by mass spectrometry following the method described in chapter II of the International Oenological Codex.

Benzoa]pyrene content should not be more than 10 μg/kg.

3.7.    Sulphides

• Put a quantity of carbon containing 1 g of dried carbon with 10 ml of diluted hydrochloric acid and 10 ml of water in a 50 ml flask.
• Distil and collect 5 ml of distillate in a test tube containing 5 ml of 1 M sodium hydroxide solution.
• 0.5 ml of lead nitrate solution at 1 g per litre (R) is added to 1 ml of test trial solution.
• There should be no brown colouring or black precipitate.
• Sulphide content expressed in sulphur should not be more than 20 mg/kg.

3.8.    Preparation of test trial solution

• Put a quantity of carbon corresponding to 2.5 g of dried carbon with 50 ml of a citric acid solution at 5 g a litre with a pH of 3 (R), in a conical flask with a wide opening that can be sealed.
• Shake vigorously for 5 minutes and allow to stand at least 12 hours.
• Filter through a fine filter or a membrane in order to obtain a clear solution.

3.9.    Iron

• Add 5 ml of water, 1 ml of concentrated hydrochloric acid, 2 ml of 5% potassium thiocyanate solution (R) to 5 ml of test trial solution as prepared in point 3.8.
• The colouration obtained should be lighter than the control sample prepared with 10 ml of iron salt solution (II) at 0.010 g of iron per litre (R), and 1 ml of concentrated hydrochloric acid (R), 2 ml of 5% potassium thiocyanate solution (R). Atomic absorption spectrophotometry can also be used.

Iron content should not be more than 200 mg/kg.

3.10. Lead

Determine the lead according to the method described in chapter II on the test trial solution prepared according to point 3.8.

Lead content should not be more than 2 mg/kg.

3.11. Mercury

Determine the mercury according to the method described in chapter II on the test trial solution prepared according to point 3.8.

Mercury content should not be more than 1 mg/kg.

3.12. Arsenic

Determine the arsenic according to the method described in chapter II on the test trial solution prepared according to point 3.8.

Arsenic content should not be more than 3 mg/kg.

3.13. Calcium

Determine the calcium according to the method described in chapter II on the test trial solution prepared according to point 3.8.

Calcium content should not be more than 10 g/kg.

3.14. Cadmium

Determine cadmium according to the method described in chapter II on the test trial solution prepared according to point 3.8.

Cadmium content should not be more than 1 mg/kg.

3.15. Zinc

Determine the zinc according to the method described in chapter II on the test trial solution prepared according to point 3.8.

Zinc content should not be more than 25 mg/kg.

3.16. Specific surface area

The specific surface area of a decolourising carbon must be between 600 and 2000 m²/g.

Methylene blue decolourisation is the method used. (Methylene blue indicator).

3.17. Methylene blue indicator

• Prepare 4 conical flasks and place 0.1 g of carbon.
• Add 10, 15, 17 and 20 ml of methylene blue solution at 1.2 g/l (absorbance at 620 nm is between 0.830 and 0.850).
• After shaking for 5 minutes, filter through a slow filter and note the volume of the solution in the conical flask which underwent decolourisation.

Depending on the results, repeat this experiment with different volumes of solution.

Put the solution in a spectrophotometer at 664 nm with the absorbance value of 0.08 with an optical path of 1 cm.

The volume of the methylene blue test solution in ml just discoloured, represents the methylene blue indicator.

4. Phenol index

 

4.1.    Introduction

When activated carbon is applied in the treatment of wine, the phenol index can be used to define a limit value over which the carbon is considered as a decolouriser and under which it is regarded as a deodoriser. The phenol index selected is the AWWA B600-90 index

4.2.    Principle:

AWWA phenol index: this index, expressed in g of carbon scaled to the dry weight per l of solution represents the carbon powder concentration required to decrease the phenol concentration of a solution from 200 mg/l to 20 mg/l.

 

4.3.    Description of the AWWA method: 

This index is determined using an adsorption isotherm based on at least 4 different weights of carbon put in contact with a phenol solution.

This isotherm represents the weight of phenol adsorbed in mg/l/g carbon, in relation to the residual phenol concentration in the solution, expressed in mg/l.

4.4.    Reagents

4.4.1.  Pure disodic hydrogenophosphate Na₂HPO₄ for analysis

4.4.2.  Distilled water

4.4.3.  Pure phosphoric Acid (H₃PO₄)

4.4.4.  Pure phenol

4.4.5.  Buffer solution A of disodic hydrogenophosphate with a pH of 6.5 at 104 g/l

In a 1-litre graduated flask, dissolve 104 g of disodic hydrogenophosphate (4.4.1) in 300 ml of hot water (4.4.2), add 14 ml of phosphoric acid (4.4.3) and make up to one litre. Homogenise. Check that the pH is 6.5 0.1

4.4.6.  Buffer solution B of disodic hydrogenophosphate with a pH of 6.5 at 10.4 g/l

In a 1-litre graduated flask, place 100 ml of buffer solution A at 104 g/l (4.4.5) and make up with water (4.4.2). Homogenise.

4.4.7.  Phenol solution with 1 g/l

In a 100-ml graduated flask, place 100 mg of phenol (4.4.4) and make up to 100 ml with water (4.4.2). Obtain complete dissolution by stirring.

4.4.8.  Calibration solutions of phenol with 20, 40, 60, 80, 100, and 120 mg/l

In a series of 100-ml graduated flasks, respectively place 2 ml, 4 ml, 6 ml, 8 ml, 10 ml, and 12 ml of the phenol solution with 1 g/l (4.4.7). Make up to 100 ml using buffer solution B (4.4.6).

4.4.9.  Phenol solutions with 200 mg/l

In a 1-litre flask, place 200 ml of the phenol solution at 1 g/l (4.4.7), add 100 ml of buffer solution A (4.4.5), make up to 1 l with water (4.4.2). Homogenise.

4.4.10.     Measuring the phenol index of oenological carbon powder

Note: The water content of the carbon must be known in order to scale the index to the dry carbon weight.

4.5.    Apparatus

4.5.1.  Laboratory glassware i.e.: graduated precision pipettes to measure small volumes, 100-ml and 1-l graduated flasks, funnels, and 300-ml conical bottles

4.5.2.  Filter paper

4.5.3.  Laboratory balance, precision to within 0.10 mg

4.5.4.  Spectrometer capable of operating in the ultraviolet spectrum and housing quartz tanks with an optical thickness of 1 cm.

4.5.5.  Laboratory shaker (it is not recommended to use a magnetic bar)

4.6.    Procedure

4.6.1.  Phenol calibration curve.

Measure the absorbance at 270 nm in tanks with an optical thickness of 1 cm (4.5.4) of each phenol solution with 20, 40, 60, 80, 100, and 120 mg/l (4.4.8). Calculate the straight regression line of the absorbance in relation to the phenol concentration.

Note:The blank is based on buffer solution B (4.4.6).

4.6.2.  Determine the residual phenol for each carbon (4.4.10)

In a series of 300-ml conical flasks, place 200 ml of phenol solution at 200 mg/l (4.4.9), then respectively 0.4, 0.5, 0.6 and 0.7 g of carbon; close the bottle.

For these 4 preparations, stir for 30 minutes (4.5.5) so that the carbon remains in suspension.

Filter on paper (4.5.2) the 4 samples containing the carbon and a blank (phenol solution with 200 mg/l (4.4.8) without carbon).

Measure the absorbance at 270 nm in tanks with an optical thickness of 1 cm (4.5.4) of each one of the filtered solutions.

Note 1 The blank is based on buffer solution B (4.4.6).

Note 2 At least one of the quantities of carbon must adsorb 90% of the phenol in the solution; if not, widen the carbon weight range.

4.7.    Calculations

4.7.1.  Determine the percentage of residual phenol in each filtrate for each activated carbon:

residual % = milligram per litre of residual phenol filtrate * 100/200 (milligram per litre of phenol in the test solution).  i.e. a = % residual phenol

4.7.2.  Determine the percentage of X (adsorbed phenol):

% of X = 100 -% residual in the filtrate. i.e. X = 100 – a

4.7.3.  The quantities of activated carbon for 200 ml of phenol solution are multiplied by 5 to obtain the quantities of activated carbon, i.e. M in grams per litre.

4.7.4.  Calculate the percentage of the value of X/M for each activated carbon.

4.7.5.  Plot the isotherm: percentage of residual normality of the filtrate on the X-axis (a) and the percentage of X/M on the Y-axis using 2x2 logarithmic paper; establish the straight regression line and determine the regression equation. It is also possible to calculate the regression using the logarithm for the values of a and X/M.

4.7.6.  Determine X/M at 10%; i.e. C (when the residual phenol concentration of the filtrate is 10%).

4.7.7.  Phenol index in grams per litre = 90/C * (100 -% of humidity/100); i.e. P

This formula refers to activated carbon without humidity.

4.7.8.  Limit values

A carbon is regarded as a deodouriser if its phenol index is lower than 3.5

4.7.9.  Examples

Standard cuve
Abs at 270 nm	Phenol mg/l
0.303	20
0.603	40
0.8777	60
1.3443	100
1.53	120

Calibration straight line for phenol titration

A1	g of carbon	% humidity	Dry weight	Abs	C phenol
	0,25	1.31	0.2467	0.309	23
	0,6255	1.31	0.6173	0.065	5
	0,7136	1.31	0.7043	0.0454	3
	0,7829	1.31	0.7726	0.0367	3

A1 Calculations
a	X	M	a	X/M	Log a	Log X/M
11.53	88.47	1.23	11.53	71.72	1.061829	1.855636
2.42	97.58	3.09	2.42	31.61	0.384605	1.499871
1.69	98.31	3.52	1.69	27.92	0.228747	1.445885
1.37	98.63	3.86	1.37	25.53	0.136357	1.407065
				c			P
			10.00	66.16	1	1.8206	1.3

Adsorption isotherm of carbon A1

A2	g of carbon	% Humidity	Dry weight	Abs	C phenol
	0.4054	1.60	0.3989	0.9969	74
	0.6012	1.60	0.5916	0.679	51
	0.7914	1.60	0.7787	0.4972	37
	0.9032	1.60	0.8887	0.4126	31
	1.4040	1.60	1.3815	0.2654	20

A2 Calculations
a	X	M	a	X/M	Log a	Log X/M
37.18	62.82	1.99	37.18	31.49	1.570343	1.498241
25.33	74.67	2.96	25.33	25.25	1.403561	1.402199
18.54	81.46	3.89	18.54	20.92	1.268222	1.320569
15.39	84.61	4.44	15.39	19.04	1.187221	1.279687
9.90	90.10	6.91	9.90	13.04	0.995635	1.115409
				c			P
			10	13.70	1	1.1367	6.5

Adsorption isotherm of carbon A2

4.7.10.     Collaborative analysis: AWWA phenol indices in g/l

	Lab 1	Lab 2	Lab 3	Lab 4
A1	1.7	1.53	1.8	1.3
A2	5.1	4.56	6.2	6.5
A3	1.3	1.29	1.8	1.4
A4	5.8	4.95	10.0	7
B1	11.4	7.18	10.6	7.6
B2	1.8	1.47	2.3	1.4
B3	49.4	21.97	18.0	17.5
B4	2.9	2.80	3.6	2.6
C1	1.9	1.69	2.3	1.8
C2	1.7	1.56	2.0	1.5
C3	5.4	4.71	6.2	4.9
C4	5.4	4.55	6.0	4.7

Reproducibility: 2.88 for the 5.86 general average SR = 1,03

5. Determination of the decolourisation capacity of carbon

5.1.    Principle

Measuring the decolourisation of an oenocyanin solution with a precise amount of carbon under defined conditions.

5.2.    Apparatus:

Equipment:

5.2.1.  Precision balance in mg

5.2.2.  5.2.2 Magnetic stirrer

5.2.3.  Absorption spectrophotometer for OD to 420, 520 and 620 nm measures Glassware:

5.2.4.  250 ml cylindrical flask

5.2.5.  250 ml conical flask

5.2.6.  200 ml volumetric flask

5.2.7.  Chamber with a 1 mm optical path for an absorption spectrophotometer.

5.3.    Reagents

5.3.1.  Very pure demineralised water

5.3.2.  Crystallised acetic acid

5.3.3.  Tartaric acid

5.3.4.  Crystallised sodium acetate

5.3.5.  96% volume ethanol

5.3.6.  Oenocyanin powder

5.4.    Preparation of oenocyanin solution

5.4.1.  Pour approximately 150 ml of demineralised water (5.3.1) in a 250 ml cylindrical flask (5.2.4).

5.4.2.  Shake (5.2.2).

5.4.3.  Weigh 0.900 g 0.001 g of oenocynanin (5.3.6) and dissolve by adding small amounts while stirring in a vortex mixer.

5.4.4.  Weigh 1.400 g 0.01 g of tartaric acid (5.3.3) and pour into the cylindrical flask (5.2.4).

5.4.5.  Pour 0.8 ml of crystallised acetic acid (5.3.2) and 1.4 g of crystallised sodium acetate (5.3.4).

5.4.6.  Shake continuously until completely dissolved (5.2.2)

5.4.7.  Transfer to a 200 ml volumetric flask (5.2.6).

5.4.8.  Adjust to 200 ml with the rinsing water from the cylindrical flask of the 5.4 preparation.

5.4.9.  Transfer again into a 250 ml cylindrical flask (5.2.4).

5.4.10.     Shake (5.2.2).

5.4.11.     Centrifuge 150 ml of the solution for 10 minutes at 10,000 g place the supernatant in a cuvette with 1 mm optical path

5.4.12.     Measure the colour intensity of the spectrometer (5.2.3)

CI1=OD 420+ OD 520+ OD 620

CI1=

5.5.    Decolourisation by carbon

5.5.1.  Weigh 100 mg of dried carbon.

(Measure the humidity in order to define the exact dose of humid carbon to be used).

5.5.2.  Put the carbon in 100 ml of oenocyanin solution with colour intensity

CI1= 4

5.5.3.  Shake for 30 minutes (5.2.2).

5.5.4.  Allow to stand for 10 minutes and centrifuge 10 ml of this mixture for 10 minutes at 10,000 g.

5.5.5.  Measure the colour intensity with a spectrometer (5.2.3) under 1 mm of optical path:

CI2=OD 420+ OD 520+ OD 620

5.6.    Calculation of decolourisation capacity

The decolourisation capacity (DC):

DC=100 (CI1-CI2)/CI1

Carbon is considered as a ‘decolourising agent’ when DC is more than or equal to 40.

6. Storage

Carbon cannot be stored in open bags because of its adsorption capacities. Oenological carbon must be stored in sealed packages away from volatile substances that it could adsorb.

COEI-1-CHITGL Chitin-glucan

[]m - []n

CAS number Chitin: 1398-61-4

CAS number β-glucan: 9041-22-9

1. Purpose, origin and scope

Chitin-glucan is of fungus origin and is a natural polymer, the main component of the cellular walls of Aspergillus niger. It is initially extracted and purified from the mycelium of Aspergillus niger. This fungal resource is a by-product of the citric acid produced for the food and pharmaceutical markets.

Chitin-glucan is composed of polysaccharides chitin (repeat units N-acetyl-D-glucosamine) and 1,3-ß-glucan (repeat unit D-glucose). The two polymers are covalently connected and form a three-dimensional network. The chitin/glucan ratio ranges from 25:75 to 60:40 (m/m).

It is used as a fining agent of musts during racking in order to reduce the colloid content and cloudiness.

It is also used for stabilising wines prior to bottling after alcoholic fermentation. This polymer has a stabilising capacity with respect to ferric breakages. It also helps eliminate undesirable compounds such as heavy metals (lead, cadmium), mycotoxins, etc.

2. Synonyms

Poly(N-acetyl-D-glucosamine)-poly(D-glucose) and 1,3- β-glucan

3. Labelling

The following information must be stated on the packaging label: fungal origin, product for oenological use, use and conservation conditions and use-by date.

4. Characters

4.1.   Aspect

Chitin-glucan comes in the form of a white, odourless and flavourless powder. Chitin-glucan is almost completely insoluble in aqueous or organic medium.

4.2.   Purity and soluble residues

 

The purity of the product must be equal to or higher than 95 %.

Dissolve 5 g of chitin-glucan in 100 ml of bidistilled water and agitate for 2 minutes. Filter after cooling on a fine mesh filter or membrane.

Evaporate the filtrate and dry at 100-105 °C. The content of solubles should not be higher than 5 %.

5. Tests

5.1.   Identification and chitin-glucan ratio

5.1.1.       Determination of the chitin-glucan ratio

The chitin/glucan ratio is determined using the ¹³C NMR spectrum in solid phase, by comparison with the spectrum of a pure chitin reference sample.

This method is detailed in appendix I.

 

5.2.   Loss during desiccation

In a glass cup, previously dried for 1 hour in an oven at 100-105 °C and cooled in a desiccator, place 10 g of the analyte. Allow to desiccate in the drying oven at 100-105 °C to constant mass. Weigh the dry residue amount after cooling in the desiccator.

The weight loss must be lower than 10 %.

 

Note: all the limits stated below are reported in dry weight except for the microbiological analyses

 

5.3.   Ashes

Incinerate without exceeding 600°C the residue left from the determination of the loss during desiccation as described in 5.2. Allow to calcine for 6 hours. Allow the crucible to cool in a desiccator and weigh.

The total ash content should not be higher than 3 %.

 

5.4.   Preparation of the test solution

Before determining the metals, the sample is dissolved by acid digestion (HNO₃, H₂O₂ and HCl). Mineralisation is performed in a closed microwave system. The sample undergoes neither crushing nor drying before mineralisation.

The reagents used for the mineralisation of chitin-glucan are as follows: HNO₃ (65 %) (Suprapur), HCl (37 %) (Suprapur), H₂O₂ (35 %). The 0.5 to 2 g sample of chitin-glucan is placed in a flask to which are added 25 ml of HNO₃, 2 ml of HCl and 3 ml of H₂O₂. This is submitted to microwave digestion (Power of 60 % for 1 min, 30 % for 10 min, 15 % for 3 min, and 40 % for 15 min). The solution is diluted in a volumetric flask with bidistilled water to a final volume of 25.0 ml.

The metal contents can then be determined.

 

5.5.   Lead

Lead is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The lead content must be lower than 1 mg/kg.

It is also possible to achieve lead determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.6.   Mercury

Mercury is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The mercury content must be lower than 0.1 mg/kg.

It is also possible to achieve mercury determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

 

5.7.   Arsenic

Arsenic is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The arsenic content must be lower than 1 mg/kg.

It is also possible to achieve arsenic determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.8.   Cadmium

Cadmium is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The cadmium content must be lower than 1 mg/kg.

It is also possible to achieve cadmium determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.9.   Chromium

Chromium is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The chromium content must be lower than 10 mg/kg.

It is also possible to achieve chromium determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.10.         Zinc

Zinc is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The zinc content must be lower than 50 mg/kg.

It is also possible to achieve zinc determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.11.         Iron

Iron is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The iron content must be lower than 100 mg/kg.

It is also possible to achieve iron determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.12.         Copper

Copper is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The copper content must be lower than 30 mg/kg.

It is also possible to achieve copper determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

 

5.13.         Microbiological control

5.13.1.   Total bacteria count

The total bacteria count is performed according to the horizontal method by means of the colony count technique at 30 °C on the PCA medium described in appendix III.

Less than 1000 CFU/g of preparation.

It is also possible to carry out the enumeration as described in chapter II of the International Oenological Codex.

5.13.2.   Enterobacteria

The enumeration of Enterobacteria is carried out according to the horizontal method by means of the colony count technique at 30 °C described in appendix IV.

Less than 10 CFU/g of preparation.

5.13.3.   Salmonella

Carry out the enumeration as described in chapter II of the International Oenological Codex.

Absence checked on a 25 g sample.

5.13.4.   Coliform bacteria

Carry out the enumeration as described in chapter II of the International Oenological Codex.

Less than 100 CFU/g of preparation.

5.13.5.   Yeasts

The enumeration of yeasts is carried out according to the horizontal method by means of the colony count technique at 25 °C on the YGC medium described in appendix V.

Less than 100 CFU/g of preparation.

It is also possible to carry out the enumeration as described in chapter II of the International Oenological Codex.

5.13.6.   Moulds

The enumeration of moulds is carried out according to the horizontal method by means of the colony count technique at 25 °C on the YGC medium described in appendix VI.

Less than 100 CFU/g of preparation.

It is also possible to carry out the enumeration as described in chapter II of the International Oenological Codex.

6. Ochratoxin A testing

Prepare an aqueous solution (distilled water) of chitin-glucan at 1 % and agitate for 1 hour, then carry out determination using the method described in the Compendium of International Methods of Analysis of Wine and Musts.

Less than 5 µg/kg.

7. Storage

Keep container closed and store in a cool and dry place.

Appendix I: Determination of the chitin/glucan ratio

 

1. Principle

 

This method consists in determining the chitin/glucan ratio using the ¹³C RMN spectrum in solid phase.

2. Reagents and materials
   1.    Chitin glucan sample
   2.    Osmosis purified water
   3.    Hydrochloric acid 1 M
   4.    Pure ethanol
   5.    Pure chloroform
   6.    Pure methanol
   7.    Acetone
   8.    Standard laboratory material, pipettes, cylindrical glass vases, porosity filters 30 μm…
   9.    Rotary shaker
   10.          Laboratory centifuge
   11.          Conductimeter
   12.          Nuclear Magnetic resonance apparatus

3. Sample preparation

 

Before the determination, samples are prepared according to a precise protocol as described below:

3.1.   Washing with HCl 1 M (2.3)

This step consists in mixing 2 g of chitin-glucan (2.1) and 40 ml of HCl 1 M in a tube flask.

This mixture is agitated for 30 min at 320 rpm then centrifuged at 4000 rpm for 10 min. The supernatant is eliminated.

This step is repeated once.

3.2.   Washing with osmosis purified water

This step consists in mixing the sediment from the previous step with 40 ml of osmosis purified water (2.2).

This mixture is centrifuged for 10 min at 4000 rpm. The supernatant is eliminated.

This step is repeated until the supernatant conductivity is lower than 100 μS/cm.

3.3.   Washing with ethanol

This step consists in mixing the sediment from the previous step with 40 ml of ethanol (2.4).

This mixture is centrifuged for 10 min at 4000 rpm. The supernatant is eliminated.

This step is repeated once.

3.4.   Washing with chloroform/methanol

This step consists in mixing the sediment from the previous step with 40 ml of a of 50/50, v/v of chloroform (2.5) and methanol (2.6) mixture.

This mixture is agitated for 30 min at 320 rpm then centrifuged at 4000 rpm for 10 min. The supernatant is eliminated.

This step is repeated once.

3.5.   Washing with acetone and drying

This step consists in mixing the sediment from the previous step with 40 ml of acetone (2.7).

This mixture is agitated for 30 min at 320 rpm then centrifuged at 4000 rpm for 10 min.

After centrifugation, pour the supernatant on a 30 μm filter, rinse the tube flask with acetone (2.7) and pour everything on the filter.

Place the material located on the filter in a crystallising dish and allow to dry.

After drying, the product is ready to be analysed by NMR.

4. Procedure

 

The prepared samples are then analysed on the Brücker Avance DSX 400WB nuclear magnetic resonance instrument (or the equivalent).

The analysis conditions are as follows:

• Magnetic field: 9.04 Tesla
• Larmor frequency: 83 kHz
• Time interval between 2 magnetic pulses: 5s
• Time period during which the magnetic pulse is applied: 5,5ms
• Number of magnetic pulse sequences: 3000

5. Expression of the results

5.1.   The beta-glucan content is determined from the area of the four resonance bands.

5.2.   The results are expressed in mol %.

 

Appendix II : Metal determination by atomic emission spectroscopy

 

1. Principle

 

This method consists in measuring atomic emission by an optical spectroscopy technique.

 

2. Sample preparation

Before the determination of metals, the sample is dissolved by acid digestion (HNO₃, H₂O₂ and HCl). Mineralisation takes place in closed microwave system. The sample undergoes neither crushing nor drying before mineralisation.

The reagents used for the mineralisation of chitosan are as follows: HNO₃ (65 %) (Suprapur), HCl (37 %) (Suprapur), H₂O₂ (35 %). The 0.5 to 2 g sample of chitin-glucan is placed in a flask to which are added 25 ml of HNO₃, 2 ml of HCl and 3 ml of H₂O₂. The whole is then submitted to microwave digestion (Power of 60 % for 1 min, 30 % for 10 min, 15 % for 3 min, and 40 % for 15 min). The solution is then diluted in a volumetric flask with bidistilled water to a final volume of 25.0 ml.

The metal contents can then be determined.

 

3. Procedure

 

The dissolved samples are nebulised and the resulting aerosol is transported in a plasma torch induced by a high frequency electric field. The emission spectra are dispersed by a grating spectrometer and the line intensity is evaluated by a detector (photomultiplier).  The detector signals are processed and controlled by a computer system. A background noise correction is applied to compensate for the background noise variations.

 

4. Expression of the results

The metal concentrations in the oenological products are expressed in mg/kg

Appendix III : Total bacteria count by counting the colonies obtained at 30 °C

 

PCA medium

Composition:

Peptone	5.0 g
Yeast extract	2.5 g
Glucose	1.0 g
Agar-agar	15 g
Adjusted to	pH 7.0
Water	complete to 1000 ml

The medium is sterilised before use in an autoclave at 120 °C for 20 min.

The Petri dishes are inoculated by pour plate method and spiral plating method.

After inoculation, they are incubated at 30 °C in aerobiosis for 48 to 72 hours.

Count the CFU number.

Appendix IV : Enumeration of Enterobacteria is carried out according to the horizontal method by means of the colony count technique

 at 30 °C

 

VRBG medium

Composition:

Peptone	7 g
Yeast extract	3 g
Glucose	10 g
Sodium Chloride	5 g
Crystal Violet	0.002 g
Neutral Red	0.03 g
Agar-agar	13 g
Bile salts	1.5 g
Adjusted to	pH 7.4
Water	complete to 1000 ml

The medium is sterilised before use in an autoclave at 120 °C for 20 min.  

The Petri dishes are inoculated by pour plate method and spiral plating method.

After inoculation, they are incubated at 30 °C in aerobiosis for 18 to 24 hours.

Count the CFU number.

Appendix V : Enumeration of yeasts by counting

 

YGC medium

Composition:

Yeast extract	5.0 g
D-glucose	20 g
Agar-agar	14.9 g
Choramphenicol	0.1 g
Adjusted to	pH 6.6
Water	complete to 1000 ml

The medium is sterilised before use in an autoclave at 120 °C for 20 min.

The Petri dishes are inoculated by pour plate method and spiral plating method.

After inoculation, they are incubated at 25 °C in aerobiosis for 3 to 5 days without being turned over.

Count the number of yeasts.

 

Appendix VI :Enumeration of the moulds by counting

YGC medium

Composition:

Yeast extract	5.0 g
D-glucose	20 g
Agar-agar	14.9 g
Choramphenicol	0.1 g
Adjusted to	pH 6.6
Water	complete to 1000 ml

The medium is sterilised before use in an autoclave at 120 °C for 20 min.

The Petri dishes are inoculated by pour plate method and spiral plating method.

After inoculation, they are incubated at 25 °C in aerobiosis for 3 to 5 days without being turned over.

Count the number of moulds.

COEI-1-CHITOS Chitosan

[]n

CAS number Chitosan : [9012-76-4]

Chitosan

1. Purpose, origin and applicability

Chitosan, a natural polysaccharide prepared of fungal origin, is initially extracted and purified from reliable and abundant food or biotechnological fungal sources such as Agaricus bisporus or Aspergillus niger.

Chitosan is obtained by hydrolysis of a chitin-rich extract. Chitin is a polysaccharide composed of several N-acetyl-D-glucosamine units interconnected by β (1.4) type linkages.

Chitosan is composed of glucosamine sugar units (deacetylated units) and N-acetyl-D-glucosamine units (acetylated units) interconnected by β (1.4) type linkages.

It is used as a fining agent in the treatment of musts for flotation clarification to reduce cloudiness and the content of unstable colloids.

It is also used for stabilising wines. This polymer actually helps eliminate undesirable micro-organisms such as Brettanomyces.

2. Synonyms

Poly(N-acetyl-D-glucosamine)-poly(D-glucose).

3. Labelling

The following information must be stated on the packaging label: exclusively fungal origin, product for oenological use, use and conservation conditions and use-by date.

4. Characters

4.1.    Aspect and solubility

Chitosan comes in the form of a white, odourless and flavourless powder. Chitin-glucan is almost completely insoluble in aqueous or organic medium.

4.2.    Purity and soluble residues

 

The purity of the product must be equal to or higher than 95 %.

Dissolve 5 g of chitin-glucan in 100 ml of bidistilled water and agitate for 2 minutes. Filter after cooling on a fine mesh filter or membrane.

Evaporate the filtrate and dry at 100-105 °C. The content of solubles should not be higher than 5 %.

5. Tests

5.1.    Determination of the acetylation degree and chitosan origin

5.1.1.  Determination of the acetylation degree

The acetylation degree is determined by potentiometric titration, using the method described in Appendix I.

5.1.2.  Determination of the source

Chitosan, as a natural polymer, is extracted and purified from fungal sources; it is obtained by hydrolysis of a chitin-rich extract. This chitosan is considered identical to chitosan from shellfish in terms of structures and properties.

An identification of the origin of chitosan is made based on 3 characteristics: content of residual glucans (refer to method in annex II), viscosity of chitosan in solution 1 % and settled density (following settlement).

Only fungal origin chitosan has both contents of residual glucan > at 2 %, a settled density ≥ at 0,7 g/cm³ and viscosity in solution 1 % in acetic acid 1 % < at 15 cPs

 

5.2.    Loss during desiccation

In a glass cup, previously dried for 1 hour in an oven at 100-105 °C and cooled in a desiccator, place 10 g of the analyte. Allow to desiccate in

the drying oven at 100-105 °C to constant mass. Weigh the dry residue amount after cooling in the desiccator.

The weight loss must be lower than 10 %.

Note: all the limits stated below are reported in dry weight except for the microbiological analyses

 

5.3.    Ashes

Incinerate without exceeding 600 °C the residue left from the determination of the loss during desiccation as described in 5.2. Allow to calcine for 6 hours. Allow the crucible to cool in a desiccator and weigh.

The total ash content should not be higher than 3 %.

5.4.    Preparation of the test solution

Before determining the metals, the sample is dissolved by acid digestion (HNO₃, H₂O₂ and HCl). Mineralisation is performed in a closed microwave system. The sample undergoes neither crushing nor drying before mineralisation.

The reagents used for the mineralisation of chitosan are as follows: HNO₃ (65 %) (Suprapur), HCl (37 %) (Suprapur), H₂O₂ (35 %). The 0.5 to 2 g sample of chitosan is placed in a flask to which are added 25 ml of HNO₃, 2 ml of HCl and 3 ml of H₂O₂. This is submitted to microwave digestion with a maximum power of 1200 watts; Power of 60 % for 1 min, 30 % for 10 min, 15 % for 3 min, and 40 % for 15 min). The solution is diluted in a volumetric flask with bidistilled water to a final volume of 25.0 ml.

The metal contents can then be determined.

 

5.5.    Lead

Lead is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The lead content must be lower than 1 mg/kg.

It is also possible to achieve lead determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

 

5.6.    Mercury

Mercury is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The mercury content must be lower than 0.1 mg/kg.

It is also possible to achieve mercury determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

 

5.7.    Arsenic

Arsenic is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The arsenic content must be lower than 1 mg/kg.

It is also possible to achieve arsenic determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.8.    Cadmium

Cadmium is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The cadmium content must be lower than 1 mg/kg.

It is also possible to achieve cadmium determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.9.    Chromium

Chromium is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The chromium content must be lower than 10 mg/kg.

It is also possible to achieve chromium determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.10. Zinc

Zinc is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The zinc content must be lower than 50 mg/kg.

It is also possible to achieve zinc determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.11. Iron

Iron is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The iron content must be lower than 100 mg/kg.

It is also possible to achieve iron determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

5.12. Copper

Copper is determined by atomic absorption spectrophotometry, using the method described in appendix II.

The copper content must be lower than 30 mg/kg.

 

It is also possible to achieve copper determination by atomic absorption, using the method described in chapter II of the International Oenological Codex.

 

5.13. Microbiological control

5.13.1.     Total bacteria count

The total bacteria count is performed according to the horizontal method by means of the colony count technique at 30 °C on the PCA medium described in appendix III.

Less than 1000 CFU/g of preparation.

It is also possible to carry out the enumeration as described in chapter II of the International Oenological Codex.

5.13.2.     Enterobacteria

The enumeration of Enterobacteria is carried out according to the horizontal method by means of the colony count technique at 30 °C described in appendix IV.

Less than 10 CFU/g of preparation.

5.13.3.     Salmonella

Carry out the enumeration as described in chapter II of the International Oenological Codex.

Absence checked on a 25 g sample.

5.13.4.     Coliform bacteria

Carry out the enumeration as described in chapter II of the International Oenological Codex.

Less than 100 CFU/g of preparation.

5.13.5.     Yeasts

The enumeration of yeasts is carried out according to the horizontal method by means of the colony count technique at 25 °C on the YGC medium described in appendix VI.

Less than 100 CFU/g of preparation.

It is also possible to carry out the enumeration as described in chapter II of the International Oenological Codex.

5.13.6.     Moulds

The enumeration of moulds is carried out according to the horizontal method by means of the colony count technique at 25 °C on the YGC medium described in appendix VII.

Less than 100 CFU/g of preparation.

It is also possible to carry out the enumeration as described in chapter II of the International Oenological Codex.

6. Ochratoxin A testing

Prepare an aqueous solution (distilled water) of chitosan at 1 % and agitate for 1 hour, then carry out determination using the method described in the Compendium of International Methods of Analysis of Wine and Musts.

Less than 5 μg/kg.

6.1.    Storage

Keep container closed and store in a cool and dry place.

 

Appendix I: Determination of the acetylation degree

 

1. Principle

 

This method consists in determining the acetylation degree of chitosan by titration of the amino groups. The acetylation degree is the ratio of the number of N-acetyl-glucosamine units to the number of total monomers.

This method is based on the method described by Rinaudo et al., (1999).

The titration of a chitosan solution by means of NaOH at 0.1 M must be performed in order to identify two pH jumps from 0 to 14.

Chitosan is dissolved in 0.1M HCl, the amino groups (on the deacetylated glucosamine units (G)) are positively charged (HCl in excess)).

The chitosan solution (of known quantity) is titrated by NaOH of known concentration.  In the first part of the reaction, the excess quantity of HCl is determined:

1.1.    HCl (excess)+NaOH+ NH₃⁺Cl^-  -->  NaCl + H₂O + NH₃⁺Cl^-

After the first pH jump, the quantity of charged amino groups is determined:

1.2.    HCl +H₂O + NH₃⁺Cl^-+ NaOH --> NH₂ + 2H₂O + 2NaCl

1.3.    After the second pH jump, the excess quantity of NaOH is measured.

The determination of the NaOH volume between the two jumps makes it possible to identify the quantity of charged amines.

2. Reagents and materials

2.1.    Commercial preparation of chitosan

2.2.    Distilled or deionised water

2.3.    Chlorhydric acid 0,3 M

2.4.    Sodium Hydroxide 0,1M

2.5.    Glass cylindrical flasks, pipettes, burettes…

2.6.    Magnetic mixer and stir bar

2.7.    pH-meter with temperature sensor.

3. Samples preparation

 

Before determination, the samples are prepared according to the protocol described hereafter:

100 mg of chitosan are placed into a cylindrical flask to which 3 ml of 0.3 M HCl and 40 ml of water are added. Agitate for 12 hours.

 

4. Procedure

 

First introduce the pH electrode of the pH-meter as well as the temperature sensor into the cylindrical flask. Check that the pH value is lower than 3.

To bring to pH = 1, add a V1 volume (ml) of HCl 0.3 M and agitate.

Then to bring to pH = 7 with a V2 volume (ml) of 0.1 M NaOH 

These operations can be carried out using an automatic titrator.

5. Expression of results

 

The acetylation degree of chitosan is expressed in %. This formula is the ratio of the mass of acetylated glucosamine (aG) units in g actually present in the sample, to the mass in g that would be present if all the groups were acetylated, where:

• Mcs: dry weight of chitosan in g

For a 1 g sample

With G = Glucosamine part; a = acetylated part

aG weight actually present (in g) =

aG weight if all the deacetylated groups were acetylated (in g) =

The acetylation degree will be equal to DA, where:

Bibliography

• Rinaudo, M., G. Pavlov and J. Desbrieres. 1999. Influenced of acetic acid concentration on the solubilization of chitosan. Polym. 40, 7029-7032.

 

Appendix II: Determination of the residual glucan content

 

1. Principle

 

This method consists in determining the content of residual glucans in chitosan by means of spectrophotometry.

This method is based on a colorimetric reaction with a response depending on the degradation of the starch hydrolysates by hot concentrated sulphuric acid.

This degradation gives a brown yellow compound with a colour intensity proportional to the content of residual glucans.

2. Reagents and materials

2.1.    Glucan 97% (Société Mégazyme)

2.2.    Commercial preparation of chitosan

2.3.    Distilled or deionised water

2.4.    Ethanol

2.5.    Acetic acid 1%

2.6.    Solution of phenol 5%

2.7.    Glacial acetic acid 100%

2.8.    Glass cylindrical flasks, pipettes, volumetric flasks,…

2.9.    Magnetic mixer and stir bar

2.10. Chronometer

 

3. Preparation of the standard range

A stock solution of glucan (glucan with a purity of 97 % is provided by the company Megazyme) is prepared according to the precise protocol described hereafter:

500 mg of glucan are introduced into a volumetric flask of 100 ml into which 6 ml of ethanol and 80 ml of distilled water are added.

Agitate and boil out to allow glucan dissolution

Allow to cool, adjust to the filling mark with water

Agitate for 30 minutes.

Pour 1 ml of this solution into a 50 ml volumetric flask and adjust to the filling mark with 1 % acetic acid.

The solution is ready to use to produce the standard range according to the protocol hereafter.

Stock solution V (ml)	Water V (ml)	Glucan M (μg)
0	1	0
0.1	0.9	10
0.3	0.7	30
0.5	0.5	50
0.7	0.3	70

4. Samples preparation

Before determination, the samples are prepared according to the protocol described hereafter:

100 mg of chitosan are placed into a 50 ml volumetric flask to which 25 ml of 1 % acetic acid are added.

Agitate for 12 hours then adjust to the filling mark.

5. Procedure

In a test tube, add 1 ml of the analyte solution, 1 ml of phenol at 5 % and 5 ml of concentrated sulphuric acid.

Agitate this mixture using a vortex for 10 s, then allow to cool for 1 hour.

The absorbance A is measured at 490 nm.

6. Expression of the results

Determine the glucan content in µg/g from the calibration curve (0-70 μg). This content is expressed in μg/g of chitosan.

Appendix III: Metal determination by atomic emission spectroscopy

 

1. Principle

 

This method consists in measuring atomic emission by an optical spectroscopy technique.

 

2. Sample preparation

Before the determination of metals, the sample is dissolved by acid digestion (HNO₃, H₂O₂ and HCl). Mineralisation takes place in closed microwave system. The sample undergoes neither crushing nor drying before mineralisation.

The reagents used for the mineralisation of chitosan are as follows: HNO₃ (65 %) (Suprapur), HCl (37 %) (Suprapur), H₂O₂ (35 %). The 0.5 to 2 g sample of chitosan is placed in a flask to which are added 25 ml of HNO₃, 2 ml of HCl and 3 ml of H₂O₂. The whole is then submitted to microwave digestion (Power of 60 % for 1 min, 30 % for 10 min, 15 % for 3 min, and 40 % for 15 min). The solution is then diluted in a volumetric flask with bidistilled water to a final volume of 25.0 ml.

The metal contents can then be determined.

3. Procedure

 

The dissolved samples are nebulised and the resulting aerosol is transported in a plasma torch induced by a high frequency electric field. The emission spectra are dispersed by a grating spectrometer and the line intensity is evaluated by a detector (photomultiplier).  The detector signals are processed and controlled by a computer system. A background noise correction is applied to compensate for the background noise variations.

 

4. Expression of the results

The metal concentrations in chitosan are expressed in mg/kg.

 

Appendix IV: Total bacteria count by counting the colonies obtained at 30 °C

 

PCA medium

Composition:

Peptone	5.0 g
Yeast extract	2.5 g
Glucose	1.0 g
Agar-agar	15 g
Adjusted to	pH 7.0
Water	complete to 1000 ml

The medium is sterilised before use in an autoclave at 120 °C for 20 min.

The Petri dishes are inoculated by pour plate method and spiral plating method.

After inoculation, they are incubated at 30 °C in aerobiosis for 48 to 72 hours.

Count the CFU number.

Appendix V: Enumeration of Enterobacteria is carried out according to the horizontal method by means of the colony count technique at 30 °C

 

VRBG medium

Composition:

Peptone	7 g
Yeast extract	3 g
Glucose	10 g
Sodium Chloride	5 g
Crystal Violet	0.002 g
Neutral Red	0.03 g
Agar-agar	13 g
Bile salts	1.5 g
Adjusted to	pH 7.4
Water	complete to 1000 ml

The medium is sterilised before use in an autoclave at 120 °C for 20 min.

The Petri dishes are inoculated by pour plate method and spiral plating method.

After inoculation, they are incubated at 30  °C in aerobiosis for 18 to 24 hours.

Count the CFU number.

Appendix VI : Enumeration of yeasts by counting

 

YGC medium

Composition:

Yeast extract	5.0 g
D-glucose	20 g
Agar-agar	14.9 g
Choramphenicol	0.1 g
Adjusted to	pH 6.6
Water	complete to 1000 ml

The medium is sterilised before use in an autoclave at 120 °C for 20 min.

The Petri dishes are inoculated by pour plate method and spiral plating method.

After inoculation, they are incubated at 25 °C in aerobiosis for 3 to 5 days without being turned over.

Count the number of yeasts.

 

Appendix VII : Enumeration of the moulds by counting

YGC medium

Composition:

Yeast extract	5.0 g
D-glucose	20 g
Agar-agar	14.9 g
Choramphenicol	0.1 g
Adjusted to	pH 6.6
Water	complete to 1000 ml

The medium is sterilised before use in an autoclave at 120 °C for 20 min.

The Petri dishes are inoculated by pour plate method and spiral plating method.

After inoculation, they are incubated at 25 °C in aerobiosis for 3 to 5 days without being turned over.

Count the number of moulds.