1991R2568 — EN — 16.10.2015 — 028.001

This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents

Amended by:

Corrected by:

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COMMISSION REGULATION (EEC) No 2568/91

of 11 July 1991

on the characteristics of olive oil and olive-residue oil and on the relevant methods of analysis

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Article 1

1.  Oils, the characteristics of which comply with those set out in points 1 and 2 of Annex I to this Regulation, shall be deemed to be virgin olive oils within the meaning of point 1(a) and (b) of the Annex to Regulation No 136/66/EEC.

2.  Oil, the characteristics of which comply with those set out in point 3 of Annex I to this Regulation, shall be deemed to be lampante olive oil within the meaning of point 1(c) of the Annex to Regulation No 136/66/EEC.

3.  Oil, the characteristics of which comply with those set out in point 4 of Annex I to this Regulation, shall be deemed to be refined olive oil within the meaning of point 2 of the Annex to Regulation No 136/66/EEC.

4.  Oil, the characteristics of which comply with those set out in point 5 of Annex I to this Regulation, shall be deemed to be olive oil composed of refined olive oils and virgin olive oils within the meaning of point 3 of the Annex to Regulation No 136/66/EEC.

5.  Oil, the characteristics of which comply with those set out in point 6 of Annex I to this Regulation, shall be deemed to be crude olive-pomace oil within the meaning of point 4 of the Annex to Regulation No 136/66/EEC.

6.  Oil, the characteristics of which comply with those set out in point 7 of Annex I to this Regulation, shall be deemed to be refined olive-pomace oil within the meaning of point 5 of the Annex to Regulation No 136/66/EEC.

7.  Oil, the characteristics of which comply with those set out in point 8 of Annex I to this Regulation, shall be deemed to be olive-pomace oil within the meaning of point 6 of the Annex to Regulation No 136/66/EEC.

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Article 2

1.  The characteristics of oils laid down in Annex I shall be determined in accordance with the following methods of analysis:

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2.  Verification by national authorities or their representatives of the organoleptic characteristics of virgin oils shall be effected by tasting panels approved by the Member States.

The organoleptic characteristics of an oil as referred to in the first subparagraph shall be deemed consonant with the category declared if a panel approved by the Member State confirms the grading.

Should the panel not confirm the category declared as regards the organoleptic characteristics, at the interested party's request, the national authorities or their representatives shall have carried out without delay two counter-assessments by other approved panels, at least one by a panel approved by the producer Member State concerned. The characteristics concerned shall be deemed consonant with the characteristics declared if at least two of the counter-assessments confirm the declared grade. If that is not the case, the interested party shall be responsible for the cost of the counter-assessments.

3.  When the national authorities or their representatives verify the characteristics of the oil as provided for in paragraph 1, samples shall be taken in accordance with international standards EN ISO 661 on the preparation of test samples and EN ISO 5555 on sampling. However, notwithstanding point 6.8 of standard EN ISO 5555, in case of batches of such oils in immediate packaging, the sample shall be taken in accordance with Annex Ia to this Regulation. In case of bulk oils for which the sampling cannot be performed according to EN ISO 5555, the sampling shall be performed in accordance with instructions provided by the competent authority of the Member State.

Without prejudice to standard EN ISO 5555 and Chapter 6 of standard EN ISO 661, the samples taken shall be put in a dark place away from strong heat as quickly as possible and sent to the laboratory for analysis no later than the fifth working day after they are taken, otherwise the samples shall be kept in such a way that they will not be degraded or damaged during transport or storage before being sent to the laboratory.

4.  For the purposes of the verification provided for in paragraph 3, the analyses referred to in Annexes II, III, IX, XII and XX and, where applicable, any counter-analyses required under national law, shall be carried out before the minimum durability date in case of packaged products. In case of sampling of bulk oils, those analyses shall be carried out no later than the sixth month after the month in which the sample was taken.

No time limit shall apply to the other analyses provided for in this Regulation.

Unless the sample was taken less than two months before the minimum durability date, if the results of the analyses do not match the characteristics of the category of olive oil or olive-pomace oil declared, the party concerned shall be notified no later than one month before the end of the period laid down in the first subparagraph.

5.  For the purpose of determining the characteristics of olive oils by the methods provided for in the first subparagraph of paragraph 1, the analysis results shall be directly compared with the limits laid down in this Regulation.

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Article 2a

1.  For the purpose of this Article, ‘olive oil marketed’ means total quantity of olive oil and olive pomace oil of a relevant Member State that is consumed in that Member State or exported from that Member State.

2.  Member States shall ensure that conformity checks are carried out selectively, based on a risk analysis, and with appropriate frequency, so as to ensure that the olive oil marketed is consistent with the category declared.

3.  The criteria to assess the risk may include:

4.  Member States shall lay down in advance:

At least one conformity check per thousand tonnes of olive oil marketed in the Member State shall be carried out per year.

5.  Member States shall verify compliance by:

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Article 3

Where it is found that an oil does not correspond to its category description, the Member State concerned shall, without prejudice to any other penalties, apply effective, proportionate and dissuasive penalties to be determined in the light of the seriousness of the irregularity detected.

Where checks reveal significant irregularities, Member States shall increase the frequency of checks in relation to marketing stage, oil category, origin, or other criteria.

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Article 4

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1.  The Member States may approve assessment panels so that national authorities or their representatives can assess and verify organoleptic characteristics.

The terms of approval shall be set by Member States and ensure that:

Member States shall notify to the Commission a list of approved panels and the action taken under this paragraph.

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2.  Where Member States encounter difficulties in setting up tasting panels in their territory, they may call on a tasting panel approved in another Member State.

3.  Each Member State draw up a list of tasting panels set up by professional or inter-branch organizations in accordance with the conditions laid down in paragraph 1 and shall ensure that those conditions are complied with.

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Article 6

1.  The oil content of oil cake and other residues resulting from the extraction of olive oil (CN codes 2306 90 11 and 2306 90 19 ) shall be determined using the method set out in Annex XV.

2.  The oil content referred to in paragraph 1 shall be expressed as a percentage of the weight of oil to the weight of dry matter.

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Article 7

The Community provisions concerning the presence of contaminants shall apply.

As regards halogenated solvents, the limits for all categories of olive oils are as follows:

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Article 7a

Natural or legal persons and groups of persons who hold olive oil and olive pomace oil from the extraction at the mill up to the bottling stage included, for whatever professional or commercial purposes, shall be required to keep entry and withdrawal registers for each category of such oils.

Member State shall ensure that the obligation laid down in the first paragraph is duly complied with.

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Article 8

1.  Member States shall notify the Commission of the measures implementing this Regulation. They shall inform the Commission of any subsequent amendments.

2.  No later than 31 May of each year, Member States shall transmit to the Commission a report on the implementation of this Regulation during the previous calendar year. The report shall contain at least the results of the conformity checks carried out on olive oils as per the templates set out in Annex XXI.

3.  The notifications referred to in this Regulation shall be made in accordance with Commission Regulation (EC) No 792/2009 ( 5 ).

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Article 9

Regulation (EEC) No 1058/77 is hereby repealed.

Article 10

1.  This Regulation shall enter into force on the third day following its publication in the Official Journal of the European Communities.

However, the method set out in Annex XII shall apply from ►M1  1 November 1992 ◄ , except in so far as operations relating to the intervention system are concerned.

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That method shall not apply to virgin olive oil prepared for the market prior to 1 November 1992.

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2.  This Regulation shall not apply to olive oil and olive-residue oil packaged before the entry into force of this Regulation and marketed up to 31 October 1992.

This Regulation shall be binding in its entirety and directly applicable in all Member States.

ANNEXES

Summary

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ANNEX I

OLIVE OIL CHARACTERISTICS

Notes:

Appendix

DECISION TREE

Campesterol decision tree for virgin and extra virgin olive oils:

The other parameters shall comply with the limits fixed in this Regulation.

Delta-7-stigmastenol decision tree for:

The other parameters shall comply with the limits fixed in this Regulation.

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ANNEX Ia

SAMPLING OF OLIVE OIL OR OLIVE-POMACE OIL DELIVERED IN IMMEDIATE PACKAGING

This method of sampling is applied to batches of olive oil or olive-pomace oil put up in immediate packaging. Different sampling methods apply, depending on whether the immediate packaging exceeds 5 litres or not.

‘Batch’ shall mean a set of sales units which are produced, manufactured and packed in circumstances such that the oil contained in each sales unit is considered to be homogenous in terms of all analytical characteristics. The individuation of a batch must be done in accordance with Directive 2011/91/EU of the European Parliament and of the Council ( 6 ).

‘Increment’ shall mean the quantity of oil contained in an immediate package and taken from a random point of the batch.

1.   CONTENT OF PRIMARY SAMPLE

1.1.    Immediate packaging not exceeding 5 litres

‘Primary Sample’ for immediate packaging not exceeding 5 litres shall mean the number of increments taken from a batch and in agreement with Table 1.

The number of packs referred to in Table 1, which shall constitute a primary sample, can be increased by each Member State, according to their own needs (for example organoleptic assessment by a different laboratory from that which performed the chemical analyses, counter-analysis, etc.).

1.2.    Immediate packaging exceeding 5 litres

‘Primary Sample’ for immediate packaging exceeding 5 litres shall mean a representative part of the total increments, obtained by a process of reduction and in agreement with Table 2. The primary sample must be composed of various examples.

‘Example’ of a primary sample shall mean each of the packages making up the primary sample.

In order to reduce the volume of the sampling immediate packs, the content of the sampling increments is homogenised for the preparation of the primary sample. The portions of the different increments are poured into a common container for homogenisation by stirring, so that it will be best protected from air.

The content of the primary sample must be poured into a series of packages of the minimum capacity of 1,0 liter, each one of which constitutes an example of the primary sample.

The number of primary samples can be increased by each Member State, according to their own necessity (for example organoleptic assessment by a different laboratory from the one that performed the chemical analyses, counter-analysis, etc).

Each package must be filled in a way to minimise the air layer on top and then suitably closed and sealed to ensure the product is tamper-proof.

These examples must be labeled to ensure correct identification.

2.   ANALYSES AND RESULTS

3.   VERIFICATION OF THE CATEGORY OF BATCH

ANNEX Ib

DECISION TREE FOR VERIFYING WHETHER AN OLIVE OIL SAMPLE IS CONSISTENT WITH THE CATEGORY DECLARED

Table 1

Table 2

Table 3

Appendix 1

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ANNEX II

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DETERMINATION OF FREE FATTY ACIDS, COLD METHOD

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1.   DETERMINATION OF ACIDITY

The determination of free fatty acids in olive oils. The content of free fatty acids is expressed as acidity calculated conventionally.

1.1.   Principle

A sample is dissolved in a mixture of solvents and the free fatty acids present titrated using an ethanolic solution of potassium hydroxide.

1.2.   Reagents

All the reagents should be of recognized analytical quality and the water used either distilled or of equivalent purity.

1.3.   Apparatus

Usual laboratory equipment including:

1.4.   Procedure

1.5.   Acidity: expressed as percentage of oleic acid

Acidity as a percentage by weight is equal to:

where:

V = the volume of titrated potassium hydroxide solution used, in millilitres;

c = the exact concentration in moles per litre of the titrated solution of potassium hydroxide used;

M = the molar weight in grams per mole of the acid used to express the result (= 282);

m = the weight in grams of the sample.

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Take as the result, the arithmetic mean ►M6  of two calculations ◄ carried out.

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ANNEX III

DETERMINATION OF PEROXIDE VALUE

1.   SCOPE

This Standard describes a method for the determination of the peroxide value of oils and fats.

2.   FIELD OF APPLICATION

This Standard is applicable to animal and vegetable oils and fats.

3.   DEFINITION

The peroxide value is the quantity of those substances in the sample, expressed in terms of milliequivalents of active oxygen per kilogram, which oxidize potassium iodide under the operating conditions described.

4.   PRINCIPLE

Treatment of the test portion, in solution in acetic acid and chloroform, by a solution of potassium iodide. Titration of the liberated iodine with standardized sodium thiosulphate solution.

5.   APPARATUS

All the equipment used shall be free from reducing or oxidizing substances.

Note:

Do not grease ground surfaces.

6.   REAGENTS

7.   SAMPLE

Take care that the sample is taken and stored away from the light, kept cold and contained in completely filled glass containers, hermetically sealed with ground-glass or cork stoppers.

8.   PROCEDURE

The test shall be carried out in diffuse daylight or in artificial light. Weigh in a glass scoop (5.1) or, failing this, in a flask (5.2), to the nearest 0,001 g, a mass of the sample in accordance with the following table, according to the expected peroxide value:

Unstopper a flask (5.2) and introduce the glass scoop containing the test portion. Add 10 ml of chloroform (6.1). Dissolve the test portion rapidly by stirring. Add 15 ml of acetic acid (6.2), then 1 ml of potassium iodide solution (6.3). Insert the stopper quickly, shake for one minute, and leave for exactly five minutes away from the light at a temperature from 15 to 25 °C.

Add about 75 ml of distilled water. Titrate the liberated iodine with the sodium thiosulphate solution ►C1  (6.4) (0,002 Mol/L solution for expected values less than 12, and 0,01 Mol/L solution ◄ for expected values above 12) shaking vigorously, using starch solution (6.5) as indicator.

Carry out two determinations on the same test sample.

Carry out simultaneously a blank test. If the result of the blank exceeds 0,05 ml of ►C1  0,01 mol/L sodium ◄ thiosulphate solution (6.4), replace the impure reagents.

9.   EXPRESSION OF RESULTS

The peroxide value (PV), expressed in milliequivalents of active oxygen per kilogram, is given by the formula:

where:

V = the number of ml of the standardized sodium thiosulphate solution (6.4) used for the test, corrected to take into account the blank test;

T = ►C1  the exact molarity ◄ of the sodium thiosulphate solution (6.4) used;

m = the weight in g, of the test portion.

Take as the result the arithmetic mean of the two determinations carried out.

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ANNEX IV

DETERMINATION OF WAX CONTENT BY CAPILLARY COLUMN GAS CHROMATOGRAPHY

1.   SUBJECT

This method describes a process for determining the wax content of olive oils. Waxes are separated according to the number of their carbon atoms. The method may be used in particular to distinguish between olive oil obtained by pressing and that obtained by extraction (olive-residue oil).

2.   PRINCIPLE

Addition of a suitable internal standard to the fat or oil, then fractionation by chromatography on a hydrated silica gel column. Recovery under the test conditions of the fraction eluted first (the polarity of which is less than that of the triglycerides), then direct analysis by capillary column gas chromatography.

3.   EQUIPMENT

4.   REAGENTS

5.   PROCEDURE

5.1.   Preparation of the chromatographic column.

Suspend 15 g of silica gel (4.1) in the n-hexane (4.2) and introduce it into the column (3.2). Allow to settle spontaneously. Complete settling with the aid of an electrovibrator (3.5) to make the chromatographic layer more homogeneous. Percolate 30 ml of n-hexane to remove any impurities. Using the balance (3.8) weigh exactly 500 mg of the sample into the 25 ml Erlenmeyer flask (3.1), add the appropriate quantity of the internal standard (4.5) according to the presumed wax content. For example, add 0,1 mg of lauryl arachidate for olive oil, and 0,25 to 0,5 mg for olive-residue oil. Transfer the prepared sample to the chromotography column using two 2 ml portions of n-hexane (4.2).

Allow the solvent to flow away until it reaches 1 mm above the upper level of the absorbant then percolate a further 70 ml of n-hexane in order to eliminate the n-alkanes naturally present. Then start the chromatographic elution by collecting 180 ml of the mixture of n-hexane/ethyl ether (ratio 99:1), keeping a rate of flow of approximately 15 drops every 10 seconds. Elution of the sample must be carried out at a room temperature of 22 ± 4 °C.

NB:

Dry the fraction thus obtained in a rotary evaporator (3.6.) until virtually all the solvent has been eliminated. Eliminate the final 2 ml of solvent with the aid of a weak current of nitrogen; then add 2-4 ml n-heptane.

5.2.   Analysis by gas chromatography

5.2.1.   Preparatory work

Fit the column to the gas chromatograph (3.3) by connecting the inlet port to the on-column system and the outlet port to the detector. Perform a general check on the GC apparatus (operation of gas circuits, detector and recorder efficiency, etc.).

If the column is being used for the first time it should be conditioned first. Pass a little gas through the column, then turn on the GC apparatus. Heat gradually until 350 °C is reached after about four hours. Maintain that temperature for at least two hours then regulate the apparatus to operating conditions (set gas flow, light flame, connect to the electronic recorder (3.3.4), set temperature of column chamber, detector, etc.) and record the signal at a sensitivity at least twice as high as that required for the analysis. The baseline must be linear, with no peaks of any kind, and must not show any deviation.

A negative straight-line drift indicates that the column connections are not tight; a positive drift that the column has not been sufficiently conditioned.

5.2.2.   Choice of operating conditions

The operating conditions are generally as follows:

The conditions may be modified according to the characteristics of the column and the GC apparatus to obtain separation of all the waxes and a satisfactory peak resolution (see figure); the internal standard C32 retention time must be 18 ± 3 minutes. The most representative wax peak must be at least 60 % of the full scale.

The peak integration parameters must be established so as to obtain a correct evaluation of the areas of the peaks in question.

NB: Given the high final temperature, a positive drift of no more than 10 % of the full scale is permitted.

5.3.   Performance of the analysis

Sample 1 μl of the solution using the 10 μl microsyringe; withdraw the syringe plunger so that the needle is empty. Place the needle in the injector and after 1-2 seconds inject quickly; remove the needle slowly after about five seconds.

Record until the waxes are completely eluted.

The base line must always satisfy the required conditions.

5.4.   Identification of peaks

Identification of the different peaks should be based on retention time by comparison with wax mixtures of known retention times analysed under the same conditions.

The figure is a chromatogram of the waxes of a virgin olive oil.

5.5.   Evaluation of quantity

Calculate the areas of the peaks of the internal standard and the aliphatic esters of C40 to C46 using the integrator.

Calculate the wax content of each of the esters in mg/kg fat using the formula:

where:

6.   EXPRESSION OF RESULTS

Indicate the total of the contents of the various C40 to C46 waxes in mg/kg fat (ppm).

NB: The components to be quantified refer to the peaks with carbon pair numbers between esters C40 and C46, using the example of the olive oil wax chromatogram shown in the figure below. If ester C46 appears twice, it is recommended that to identify it the fraction of the waxes of an olive-residue oil should be analysed where the C46 peak is easy to identify because it is in the clear majority.

The results should be expressed to one decimal place.

Figure

Chromatogram of the waxes of an olive oil ( 7 )

Key:

Appendix

Determination of the linear velocity of the gas

Inject 1-3 μl methane (or propane) into the GC apparatus after it has been regulated to normal operating conditions. Measure the time it takes for the gas to flow through the column from the time it is injected to the time the peak appears (tM).

The linear velocity in cm/s is given by the formula L/tM, where L is the length of the column in cm and tM the time measured in seconds.

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ANNEX V

DETERMINATION OF THE COMPOSITION AND CONTENT OF STEROLS AND TRITERPENES DIALCOHOLS BY CAPILLARY-COLUMN GAS CHROMATOGRAPHY

1.   SCOPE

The method describes a procedure for determining the individual and total sterols and triterpene dialcohols content of olive oils and olive-pomace oils.

2.   PRINCIPLE

The oil, with added α-cholestanol as an internal standard, is saponified with potassium hydroxide in ethanolic solution and the unsaponifiable matter is then extracted with ethyl ether.

The sterols and triterpene dialcohols fraction is separated from the unsaponifiable matter by thin-layer chromatography on a basic silica gel plate. The fractions recovered from the silica gel are transformed into trimethylsilyl ethers and then analysed by capillary column gas chromatography.

3.   APPARATUS

The usual laboratory equipment and in particular the following:

4.   REAGENTS

5.   PROCEDURE

6.   EXPRESSION OF THE RESULTS

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Appendix

Determination of the linear speed of the gas

With the gas chromatograph set to normal operating conditions, inject 1 to 3 μl of methane (or propane) and measure the time taken by the gas to pass through the column, from the time of injection to the time at which the peak appears (tM).

The linear speed in cm/s is given by L/tM, where L is the length of the column in centimetres and tM is the measured time, in seconds.

Figure 1

Gas chromatogram of the sterol and triterpene dialchols fraction of a lampante olive oil (spiked with internal standard)

Figure 2

Gas chromatogram of the sterol and triterpene dialchols fraction of a refined olive oil (spiked with internal standard)

Figure 3

TLC plate olive-pomace oil with the zone that must be scraped for sterols and triterpenic dialcohols determination

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ANNEX VII

DETERMINATION OF THE PERCENTAGE OF 2-GLYCERYL MONOPALMITATE

1.   PURPOSE AND SCOPE

This method describes the analysis procedure for determining the percentage of palmitic acid in position 2 of the triglycerides by evaluating 2-glyceryl monopalmitate.

This method can be applied to liquid vegetable oils at ambient temperature (20 °C).

2.   PRINCIPLE

After preparation the oil sample is subjected to the action of pancreatic lipase: partial and specific hydrolysis in positions 1 and 3 of the triglyceride molecule causes monoglycerides to appear in position 2. The percentage of 2-glyceryl monopalmitate in the monoglyceride fraction is determined after silylation by capillary-column gas chromatography.

3.   APPARATUS AND MATERIALS

4.   REAGENTS

5.   METHOD

5.1.   Sample preparation

5.2.   Hydrolysis by pancreatic lipase

5.3.   Preparation of the silanised derivatives and gas chromatography

5.4.   Gas chromatography

Operating conditions:

5.4.1.   Identification of the peaks

The individual monoglycerides are identified from their retention times and by comparison with those obtained for standard monoglyceride mixtures under the same conditions.

5.4.2.   Quantitative evaluation

The area of each peak is calculated using an electronic integrator.

6.   EXPRESSION OF RESULTS

The percentage of glyceryl monopalmitate is calculated from the ratio between the area of the corresponding peak and the areas of the peaks of all the monoglycerides (see Figure 2) using the formula:

glyceryl monopalmitate (%):

where:

The result must be to one decimal place.

7.   ANALYSIS REPORT

The analysis report must specify:

Figure 1

Chromatogram of the products of the silanisation reaction obtained by the action of lipase on a refined olive oil with 20 % esterified oil added (100 %)

Key: ‘acides gras libres’ = free fatty acids; ‘Huile d’olive raffinée + 20 % huile estérifiée’ = refined olive oil + 20 % esterified oil; ‘1-2 monopalmitoléine’ = 1-2 monopalmitolein; ‘1-2 mono C18 insat.’ = unsaturated 1-2 mono C18

Figure 2

Chromatogram of:

(A)

unesterified olive oil, after lipase; after silanisation; under these conditions (8-12 m capillary column) the wax fraction is eluted at the same time as the diglyceride fraction or slightly afterwards. After lipase, the triglyceride content should not exceed 15 % Key: 1 = Free fatty acids 2 = Monoglycerides 3 = Diglycerides 4 = Triglycerides * = 2-monopalmitine ** = Triglyceride C54

Chromatogram of:

(B)

unesterified oil after lipase; after silanisation; under these conditions (8-12 m capillary column) the wax fraction is eluted at the same time as the diglyceride fraction or slightly afterwards. After lipase, the triglyceride content should not exceed 15 %. Key: 1 = Free fatty acids 2 = Monoglycerides 3 = Diglycerides 4 = Triglycerides * = 2-monopalmitine ** = C54 triglyceride

8.   NOTES

PREPARATION OF THE LIPASE

Lipases with satisfactory activity are commercially available. They can also be prepared in the laboratory in the following manner:

Cool to 0 °C 5 kg of fresh pig’s pancreas. Remove the surrounding solid fat and the connective tissue and grind to a liquid paste in a blender. Stir the paste with 2,5 litres of anhydrous acetone for 4-6 hours, then centrifuge. Extract the residue three more times with the same volume of anhydrous acetone, then twice with an acetone/diethyl ether mixture (1/1 v/v) and twice with diethyl ether.

Vacuum-dry the residue for 48 hours to obtain a stable powder which can be stored for a long time in a refrigerator away from moisture.

MONITORING LIPASE ACTIVITY

Prepare an olive oil emulsion as follows:

In a mixer stir for 10 minutes a mixture of 165 ml of a 100 g/l gum arabic solution, 15 g of crushed ice and 20 ml of a previously neutralised olive oil.

Pour 10 ml of the emulsion into a 50 ml beaker, then 0,3 ml of a 0,2 g/ml sodium cholate solution and then 20 ml of distilled water.

Put the beaker in a thermostat set at 37 °C; introduce the electrodes of the pH meter and the screw agitator.

Using a burette, add a 0,1 N sodium hydroxide solution drop by drop until a pH of 8,3 is obtained.

Add an aliquot of the lipase powder suspension in water (0,1 g/ml of lipase). As soon as the pH meter reads 8,3, start the chronometer and add the sodium hydroxide solution drop by drop at a rate which maintains the pH at 8,3. Note every minute the volume of solution consumed.

Record the data on an x/y graph with the time on the x-axis and millilitres of 0,1 N alkaline solution consumed to keep a constant pH on the y-axis. A linear graph should be obtained.

Lipase activity, expressed in lipase units per mg, is given by the following formula:

where:

A lipase unit is defined as the quantity of enzyme which releases 10 micro-equivalents of acid per minute.

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ANNEX IX

SPECTROPHOTOMETRIC INVESTIGATION IN THE ULTRAVIOLET

FOREWORD

Spectrophotometric examination in the ultraviolet can provide information on the quality of a fat, its state of preservation and changes brought about by technological processes. The absorption at the wavelengths specified in the method is due to the presence of conjugated diene and triene systems resulting from oxidation processes and/or refining practices. These absorptions are expressed as specific extinctions

(the extinction of 1 % w/v solution of the fat in the specified solvent, in a 10 mm cell) conventionally indicated by K (also referred to as ‘extinction coefficient’).

1.   SCOPE

This Annex describes the procedure for performing a spectrophotometric examination of olive oil in the ultraviolet region.

2.   PRINCIPLE OF THE METHOD

A sample is dissolved in the required solvent and the absorbance of the solution is measured at the specified wavelengths with reference to pure solvent.

The specific extinctions at 232 nm and 268 nm in iso-octane or 232 nm and 270 nm in cyclohexane are calculated for a concentration of 1 % w/v in a 10 mm cell.

3.   EQUIPMENT

4.   REAGENTS

During the analysis, unless otherwise stated, use only reagents of recognised analytical grade and distilled or demineralised water or water of equivalent purity.

Solvent: Iso-octane (2,2,4 trimethylpentane) for the measurements at 232 nm and 268 nm and cyclohexane for the measurements at 232 nm and 270 nm, having an absorbance less than 0,12 at 232 nm and less than 0,05 at 270 nm against distilled water, measured in a 10 mm cell.

5.   PROCEDURE

6.   EXPRESSION OF THE RESULTS

ANNEX X

DETERMINATION OF FATTY ACID METHYL ESTERS BY GAS CHROMATOGRAPHY

1.   SCOPE

This Annex gives guidance on the gas chromatographic determination of free and bound fatty acids in vegetable fats and oils following their conversion into fatty acid methyl esters (FAME).

The bound fatty acids of the triacylglycerols (TAGs) and, depending on the esterification method, the free fatty acids (FFA), are converted into fatty acid methyl esters (FAME), which are determined by capillary gas chromatography.

The method described in this Annex allows the determination of FAME from C12 to C24, including saturated, cis- and transmonounsaturated and cis- and trans-polyunsaturated fatty acid methyl esters.

2.   PRINCIPLE

Gas chromatography (GC) is used for the quantitative analysis of FAME. The FAME are prepared according to Part A. They are then injected into and vaporised within the injector. The separation of FAME is performed on analytical columns of specific polarity and length. A Flame Ionisation Detector (FID) is used for the detection of the FAME. The conditions of analysis are given in Part B.

Hydrogen or helium may be used as the carrier gas (mobile phase) in the gas chromatography of FAME with FID. Hydrogen speeds up separation and gives sharper peaks. The stationary phase is a microscopic layer of a thin liquid film on an inert solid surface made of fused silica.

As they pass through the capillary column the volatilised compounds being analysed interact with the stationary phase coating the inner surface of the column. Due to this different interaction of different compounds, they elute at a different time, which is called the retention time of the compound for a given set of analysis parameters. The comparison of the retention times is used for the identification of the different compounds.

PART A

PREPARATION OF THE FATTY ACID METHYL ESTERS FROM OLIVE OIL AND OLIVE-POMACE OIL

1.   SCOPE

This part specifies the preparation of the methyl esters of fatty acids. It includes methods for preparing fatty acid methyl esters from olive and olive-pomace oils.

2.   FIELD OF APPLICATION

The preparation of the fatty acid methyl esters from olive oils and olive-pomace oils are performed by transesterification with methanolic solution of potassium hydroxide at room temperature. The necessity of purification of the sample prior to the trans-esterification depends on the sample's free fatty acids content and the analytical parameter to be determined, it can be chosen according to the following table:

3.   METHODOLOGY

3.1.    Trans-esterification with methanolic solution of potassium hydroxide at room temperature

3.1.1.    Principle

Methyl esters are formed by trans-esterification with methanolic potassium hydroxide as an intermediate stage before saponification takes place.

3.1.2.    Reagents

3.1.3.    Apparatus

3.1.4.    Purification of oil samples

When necessary, the samples will be purified by passing the oil through a silica gel solid-phase extraction cartridge. A silica gel cartridge (3.1.2.8) is placed in a vacuum elution apparatus and washed with 6 ml of hexane (3.1.2.2); washing is performed without vacuum. Then a solution of the oil (0,12 g approximately) in 0,5 ml of hexane (3.1.2.2) is loaded onto the column. The solution is pulled down and then eluted with 10 ml of hexane/diethyl ether (87:13 v/v) (3.1.2.6). The combined eluates are homogenised and divided in two similar volumes. An aliquot is evaporated to dryness in a rotary evaporator under reduced pressure at room temperature. The residue is dissolved in 1 ml of heptane and the solution is ready for fatty acid analysis by GC. The second aliquot is evaporated and the residue is dissolved in 1 ml of acetone for triglyceride analysis by HPLC, if necessary.

3.1.5.    Procedure

In a 5 ml screw-top test tube (3.1.3.1) weigh approximately 0,1 g of the oil sample. Add 2 ml of heptane (3.1.2.2), and shake. Add 0,2 ml of the methanolic potassium hydroxide solution (3.1.2.7), put on the cap fitted with a PTFE joint, tighten the cap, and shake vigorously for 30 seconds. Leave to stratify until the upper solution becomes clear. Decant the upper layer containing the methyl esters. The heptane solution is ready for injection into the gas chromatograph. It is advisable to keep the solution in the refrigerator until gas chromatographic analysis. Storage of the solution for more than 12 hours is not recommended.

PART B

ANALYSIS OF FATTY ACID METHYL ESTERS BY GAS CHROMATOGRAPHY

1.   SCOPE

This part gives general guidance for the application of capillary column gas chromatography to determine the qualitative and quantitative composition of a mixture of fatty acid methyl esters obtained in accordance with the method specified in Part A.

The part is not applicable to polymerised fatty acids.

2.   REAGENTS

2.1.    Carrier gas

Inert gas (helium or hydrogen), thoroughly dried and with an oxygen content of less than 10 mg/kg.

2.2.    Auxiliary gases

2.3.    Reference standard

Mixture of methyl esters of pure fatty acids, or the methyl esters of a fat of known composition, preferably similar to that of the fatty matter to be analysed. Cis and trans isomers of octadecenoic, octadecadienoic and octadecatrienoic methyl esters are useful for the identification of trans isomers of unsaturated acids.

Care should be taken to prevent the oxidation of polyunsaturated fatty acids.

3.   APPARATUS

The instructions given are for the usual equipment used for gas chromatography, employing capillary columns and a flame-ionisation detector.

3.1.    Gas chromatograph

The gas chromatograph shall comprise the following elements.

3.1.1.    Injection system

Use an injection system with capillary columns, in which case the injection system should be specially designed for use with such columns. It may be of the split type or the splitless on-column injector type.

3.1.2.    Oven

The oven shall be capable of heating the capillary column to a temperature of at least 260 °C and of maintaining the desired temperature to within 0,1 °C. The last requirement is particularly important when a fused silica tube is used.

The use of temperature-programmed heating is recommended in all cases, and in particular for fatty acids with less than 16 carbon atoms.

3.1.3.    Capillary column

3.1.4.    Flame ionisation detector and converter-amplifier

3.2.    Syringe

The syringe shall have a maximum capacity of 10 μl, graduated in 0,1 μl divisions.

3.3.    Data acquisition system

Data acquisition system connected online with the detectors and employed with a software program suitable for peak integration and normalisation.

4.   PROCEDURE

The operations described in 4.1 to 4.3 are for the use of a flame-ionisation detector.

4.1.    Test conditions

4.1.1.    Selection of optimum operating conditions for capillary columns

Owing to the efficiency and permeability of capillary columns, the separation of the constituents and the duration of the analysis are largely dependent on the flow-rate of the carrier gas in the column. It will therefore be necessary to optimise the operating conditions by adjusting this parameter (or simply column head loss) depending on whether the aim is to improve separation or speed up analysis.

The following conditions have proved to be suitable for the separation of FAMEs (C4 to C26). Examples of chromatograms are shown in Appendix B:

4.1.2.    Determination of the resolution (see Appendix A)

Calculate the resolution, R, of two neighbouring peaks I and II, using the formula:

R = 2 × ((ddr(II) – dr(I) )/(ω(I) + ω(II))) or R = 2 × ((tr(II) – tr(I) )/(ω(I) + ω(II))) (USP) (United States Pharmacopeia),

or

R = 1,18 × ((tr(II) – tr(I))/(ω0,5(I) + ω0,5(II))) (EP, BP, JP, DAB), (JP (Japanese Pharmacopeia), EP (Pharmacopée Européenne), BP (British Pharmacopeia))

where:

If ω(I) ≈ ω(II), calculate R using the following formulas:

R = (dr(II) – d r(I))/ω = (dr(II) – d r(I))/4σ

where:

σ is the standard deviation (see Appendix A, Figure 1).

If the distance dr between the two peaks d r(II) - d r(I) is equal to 4σ, the resolution factor R = 1.

If two peaks are not separated completely, the tangents to the inflection points of the two peaks intersect at point C. In order to completely separate the two peaks, the distance between the two peaks must be equal to:

d r(II) - d r(I) = 6 σ from where R = 1,5 (see Appendix A, Figure 3).

5.   EXPRESSION OF RESULTS

5.1.    Qualitative analysis

Identify the methyl ester peaks of the sample from the chromatogram in Appendix B, figure 1, if necessary by interpolation, or by comparison with those of the methyl esters reference mixtures (as indicated at point 2.3).

5.2.    Quantitative analysis

5.2.1.    Determination of the composition

Calculate the mass fraction w i of the individual fatty acid methyl esters, expressed as a percentage by mass of methyl esters, as follows:

5.2.2.    Method of calculation

5.2.2.1.   General case

Calculate the content of a given component i, expressed as a percentage by mass of methyl esters, by determining the percentage represented by the area of the corresponding peak relative to the sum of the areas of all the peaks, using the following formula:

wi = (Ai/ΣA) × 100

where:

The results are expressed to two decimal places.

5.2.2.2.   Use of correction factors

In certain cases, for example in the presence of fatty acids with less than eight carbon atoms or of acids with secondary groups, the areas shall be corrected with specific correction factors (Fci). These factors shall be determined for each single instrument. For this purpose suitable reference materials with certified fatty acid composition in the corresponding range shall be used.

For this reference mixture, the mass percentage of the FAME i is given by the formula:

wi = (mi /Σm) × 100

where

From the chromatogram of the reference mixture, calculate the percentage by area for the FAME i as follows:

wi = (Ai/ΣA) × 100

where:

The correction factor Fc is then

Fc = (mi × ΣA)/(Ai/Σm)

For the sample, the percentage by mass of each FAME i is:

wi = (Fi × Ai)/Σ (Fi × Ai)

The results are expressed to two decimal places.

5.2.2.3.   Use of an internal standard

In certain analyses (for example where not all of the fatty acids are quantified, such as when acids with four and six carbons are present alongside acids with 16 and 18 carbons, or when it is necessary to determine the absolute amount of a fatty acid in a sample) it is necessary to use an Internal Standard. Fatty acids with 5, 15 or 17 carbons are frequently used. The correction factor (if any) for the Internal Standard should be determined.

The percentage by mass of component i, expressed as methyl esters, is then given by the formula:

wi = (mIS × Fi × Ai)/(m × FIS × AIS)

where:

The results are expressed to two decimal places.

6.   TEST REPORT

The test report shall specify the methods used for the preparation of the methyl esters and for the gas chromatographic analysis. It shall also mention all operating details not specified in this Standard Method, or regarded as optional, together with details of any incidents which may have influenced the results.

The test report shall include all the information necessary for complete identification of the sample.

7.   PRECISION

7.1.    Results of interlaboratory test

Details of an interlaboratory test on the precision of the method are set out in Annex C to standard IOC/T.20/Doc. No 33. The values derived from this interlaboratory test may not be applicable to concentration ranges and matrices other than those given.

7.2.    Repeatability

The absolute difference between two independent single test results, obtained using the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5 % of cases be greater than r given in tables 1 to 14 in Annex C to standard IOC/T.20/Doc. No 33.

7.3.    Reproducibility

The absolute difference between two single test results, obtained using the same method on identical test material in different laboratories with different operators using different equipment, will in not more than 5 % of cases be greater than R given in tables 1 to 14 in Annex C to standard IOC/T.20/Doc. No 33.

Appendix A

Figure 1

ω0,5 width at half height of the triangle (ABC) and b width at half height of the triangle (NPM).

Appendix B

Figure 1

Gas chromatographic profile obtained by the cold methylation method from olive-pomace oil

The chromatographic peaks correspond to the methyl and ethyl esters except where otherwise indicated.

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ANNEX XI

DETERMINATION OF VOLATILE HALOGENATED SOLVENTS CONTENT OF OLIVE OIL

1.   METHOD

Analysis by gas chromatography using the head space technique.

2.   EQUIPMENT

3.   REAGENTS

Standard: halogenated solvents of a degree of purity suitable for gas chromatography.

4.   PROCEDURE

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ANNEX XII

THE INTERNATIONAL OLIVE COUNCIL’S METHOD FOR THE ORGANOLEPTIC ASSESSMENT OF VIRGIN OLIVE OIL

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1.   PURPOSE AND SCOPE

The purpose of the international method described in this Annex is to determine the procedure for assessing the organoleptic characteristics of virgin olive oil within the meaning of point 1 of Part VIII of Annex VII to Regulation (EU) No 1308/2013 of the European Parliament and of the Council ( 8 ) and to establish the method for its classification on the basis of those characteristics. It also provides indications for optional labelling.

The method described is applicable only to virgin olive oils and to the classification or labelling of such oils according to the intensity of the defects perceived and of the fruitiness, as determined by a group of tasters selected, trained and monitored as a panel.

The IOC standards mentioned in this Annex are used in their last available version.

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2.   GENERAL BASIC VOCABULARY FOR SENSORY ANALYSIS

Refer to the standard IOC/T.20/Doc. No 4 "Sensory Analysis: General Basic Vocabulary"

3.   SPECIFIC VOCABULARY

3.1.    Negative attributes

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3.1.1.    Other negative attributes

3.2.    Positive attributes

3.3.    Optional terminology for labelling purposes

Upon request, the panel leader may certify that the oils which have been assessed comply with the definitions and ranges corresponding to the following adjectives according to the intensity and perception of the attributes.

Positive attributes (fruity, bitter and pungent): According to the intensity of perception:

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4.   GLASS FOR OIL TASTING

Refer to the standard IOC/T.20/Doc. No 5, "Glass for Oil Tasting".

5.   TEST ROOM

Refer to the standard IOC/T.20/Doc. No 6, "Guide for the Installation of a Test Room".

6.   ACCESSORIES

The following accessories, which are required by tasters to perform their task properly, must be supplied in each booth and must be within easy reach:

7.   PANEL LEADER AND TASTERS

7.1.    Panel leader

The panel leader must be a suitably trained person with an expert knowledge of the kinds of oils which he or she will come across in the course of their work. They are the key figure in the panel and responsible for its organisation and running.

The work of the panel leader calls for basic training in the tools of sensory analysis, sensory skill, meticulousness in the preparation, organisation and performance of the tests and skill and patience to plan and execute the tests in a scientific manner.

They are the sole person responsible for selecting, training and monitoring the tasters in order to ascertain their level of aptitude. They are thus responsible for the appraisal of the tasters, which must always be objective and for which they must develop specific procedures based on tests and solid acceptance and rejection criteria. See standard IOC/T.20/Doc. No 14, "Guide for the selection, training and monitoring of skilled virgin olive oil tasters".

Panel leaders are responsible for the performance of the panel and hence for its evaluation, of which they must give reliable, objective proof. In any case, they must demonstrate at all times that the method and tasters are under control. Periodic calibration of the panel is recommended (IOC/T.20/Doc. No 14, § 5).

They hold ultimate responsibility for keeping the records of the panel. These records must always be traceable. They must comply with the assurance and quality requirements laid down in international sensory analysis standards and ensure the anonymity of the samples at all times.

They shall be responsible for inventorying and ensuring that the apparatus and equipment needed to comply with the specifications of this method is properly cleaned and maintained and shall keep written proof thereof, as well as of the compliance with the test conditions.

They shall be in charge of the reception and storage of the samples upon their arrival at the laboratory as well as of their storage after being tested. When doing so, they shall ensure at all times that the samples remain anonymous and are properly stored, for which purpose they must develop written procedures in order to ensure that the entire process is traceable and affords guarantees.

In addition, they are responsible for preparing, coding and presenting the samples to the tasters according to an appropriate experimental design in line with pre-established protocols, as well as for assembling and statistically processing the data obtained by the tasters.

They shall be in charge of developing and drafting any other procedures that might be necessary to complement this standard and to ensure that the panel functions properly.

They must seek ways of comparing the results of the panel with those obtained by other panels undertaking the analysis of virgin olive oil in order to ascertain whether the panel is working properly.

It is the duty of the panel leader to motivate the panel members by encouraging interest, curiosity and a competitive spirit among them. To do so, they are strongly recommended to ensure a smooth two-way flow of information with the panel members by keeping them informed about all the tasks they carry out and the results obtained. In addition, they shall ensure that their opinion is not known and shall prevent possible leaders from asserting their criteria over the other tasters.

They shall summon the tasters sufficiently in advance and shall answer any queries regarding the performance of the tests, but shall refrain from suggesting any opinion to them on the sample.

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7.1.1.    Deputy panel leader

The panel leader may, on justified grounds, be replaced by a deputy panel leader who may stand in for duties regarding the performance of the tests. This substitute must have all the necessary skills required of a panel leader.

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7.2.    Tasters

The people acting as tasters in organoleptic tests carried out on olive oils must do so voluntarily. It is therefore advisable for candidates to submit an application in writing. Candidates shall be selected, trained and monitored by the panel leader in accordance with their skills in distinguishing between similar samples; it should be borne in mind that their accuracy will improve with training.

Tasters must act like real sensory observers, setting aside their personal tastes and solely reporting the sensations they perceive. To do so, they must always work in silence, in a relaxed, unhurried manner, paying the fullest possible sensory attention to the sample they are tasting.

Between 8 and 12 tasters are required for each test, although it is wise to keep some extra tasters in reserve to cover possible absences.

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8.   TEST CONDITIONS

8.1.    Presentation of the sample

The oil sample for analysis shall be presented in standardised tasting glasses conforming to the standard IOC/T.20/Doc. No 5 ‘Glass for oil tasting’.

The glass shall contain 14–16 ml of oil, or between 12,8 and 14,6 g if the samples are to be weighed, and shall be covered with a watch-glass.

Each glass shall be marked with a code made up of digits or a combination of letters and digits chosen at random. The code will be marked by means of an odourfree system.

8.2.    Test and sample temperature

The oil samples intended for tasting shall be kept in the glasses at 28 °C ± 2 °C throughout the test. This temperature has been chosen because it makes it easier to observe organoleptic differences than at ambient temperature and because at lower temperatures the aromatic compounds peculiar to these oils volatilise poorly while higher temperatures lead to the formation of volatile compounds peculiar to heated oils. See the standard IOC/T.20/Doc. No 5 ‘Glass for Oil Tasting’ for the method which has to be used for heating the samples when in the glass.

The test room must be at a temperature between 20 ° and 25 °C (see IOC/T.20/Doc. No 6).

8.3.    Test times

The morning is the best time for tasting oils. It has been proved that there are optimum perception periods as regards taste and smell during the day. Meals are preceded by a period in which olfactory–gustatory sensitivity increases, whereas afterwards this perception decreases.

However, this criterion should not be taken to the extreme where hunger may distract the tasters, thus decreasing their discriminatory capacity; therefore, it is recommended to hold the tasting sessions between 10.00 in the morning and 12 noon.

8.4.    Tasters: general rules of conduct

The following recommendations apply to the conduct of the tasters during their work.

When called by the panel leader to participate in an organoleptic test, tasters should be able to attend at the time set beforehand and shall observe the following:

9.   PROCEDURE FOR THE ORGANOLEPTIC ASSESSMENT AND CLASSIFICATION OF VIRGIN OLIVE OIL

9.1.    Tasting technique

9.2.    Use of the profile sheet by tasters

The profile sheet intended for use by tasters is detailed in Figure 1 of this Annex.

Each taster on the panel shall smell and then taste ( 9 ) the oil under consideration. They shall then enter the intensity with which they perceive each of the negative and positive attributes on the 10-cm scale shown in the profile sheet provided.

Should the tasters perceive any negative attributes not listed in section 4, they shall record them under the "others" heading, using the term or terms that most accurately describes the attributes.

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9.3.    Use of the data by the panel leaders

The panel leader shall collect the profile sheets completed by each taster and shall review the intensities assigned to the different attributes. Should they find any anomaly, they shall invite the taster to revise his or her profile sheet and, if necessary, to repeat the test.

The panel leader shall enter the assessment data of each panel member in a computer program like that provided by the standard IOC/T.20/Doc. No 15 with a view to statistically calculating the results of the analysis, based on the calculation of their median. See point 9.4 and the Appendix to this Annex. The data for a given sample shall be entered with the aid of a matrix comprising 9 columns representing the 9 sensory attributes and n lines representing the n panel members used.

When a defect is perceived and entered under the ‘others’ heading by at least 50 % of the panel, the panel leader shall calculate the median of the defect and shall arrive at the corresponding classification.

The value of the robust coefficient of variation which defines classification (defect with the strongest intensity and fruity attribute) must be no greater than 20 %.

If the opposite is the case, the panel leader must repeat the evaluation of the specific sample in another tasting session.

If this situation arises often, the panel leader is recommended to give the tasters specific additional training (IOC/T.20/Doc. No 14, § 5) and to use the repeatability index and deviation index to check taster performance (IOC/T.20/Doc. No 14, § 6).

9.4.    Classification of the oil

The oil is graded as follows in line with the median of the defects and the median for the fruity attribute. The median of the defects is defined as the median of the defect perceived with the greatest intensity. The median of the defects and the median of the fruity attribute are expressed to one decimal place.

The oil is graded by comparing the median value of the defects and the median for the fruity attribute with the reference ranges given below. The error of the method has been taken into account when establishing the limits of these ranges, which are therefore considered to be absolute. The software packages allow the grading to be displayed as a table of statistics or a graph.

For assessments intended to monitor compliance, one test shall be carried out. In the case of counter assessments, the panel leader must arrange for the assessment to be carried out in duplicate in different sessions; the median of the attributes will be calculated on the basis of all the profile sheet data for both tests.

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Appendix

Method for calculating the median and the confidence intervals

Median

The median is defined as the real number Xm characterised by the fact that the probability (p) that the distribution values (X) are below this number (Xm), is less than and equal to 0,5 and that simultaneously the probability (p) that the distribution values (X) are below or equal to Xm is greater than and equal to 0,5. A more practical definition is that the median is the 50th percentile of a distribution of numbers arranged in increasing order. In simpler terms, it is the midpoint of an ordered set of odd numbers, or the mean of two midpoints of an ordered set of even numbers.

Robust standard deviation

In order to arrive at a reliable estimate of the variability around the mean it is necessary to refer to the robust standard deviation as estimated according to Stuart and Kendall (4). The formula gives the asymptotic robust standard deviation, i.e. the robust estimate of the variability of the data considered where N is the number of observations and IQR is the interquartile range which encompasses exactly 50% of the cases of a given probability distribution:

The interquartile range is calculated by calculating the magnitude of the difference between the 75th and 25th percentile.

Where the percentile is the value Xpc characterised by the fact that the probability (p) that the distribution values are less than Xpc is less than and equal to a specific hundreth and that simultaneously the probability (p) that the distribution values are less than or equal to Xpc is greater than and equal to that specific hundredth. The hundredth indicates the distribution fractile chosen. In the case of the median it is equal to 50/100.

For practical purposes, the percentile is the distribution value corresponding to a specific area subtended from the distribution or density curve. To give an example, the 25th percentile represents the distribution value corresponding to an area equal to 0,25 or 25/100.

In this method percentiles are computed on the basis of the real values which appear in the data matrix (percentiles computing procedure).

Robust coefficient of variation (%)

The CVr% represents a pure number which indicates the percentage variability of the set of numbers analysed. For this reason it is very useful for checking the reliability of the panel assessors.

Confidence intervals of the median at 95%

The confidence intervals at 95% (value of the error of the first kind equal to 0,05 or 5%) represent the interval within which the value of the median could vary if it were possible to repeat an experiment an infinite number of times. In practice, it indicates the interval of variability of the test in the operating conditions adopted starting from the assumption that it is possible to repeat it many times. As with the CVr%, the interval helps to assess the reliability of the test.

where C = 1,96 for the confidence interval at the 95% level.

An example of the calculation sheet is presented in Annex I to the standard IOC/T 20/Doc. No 15.

References

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ANNEX XV

1.   OIL CONTENT OF OLIVE RESIDUE

1.1.   Apparatus

1.2.   Reagent

Normal hexane, technical grade, which must leave a residue of less than 0,002 g per 100 ml, on complete evaporation.

2.   PROCEDURE

2.1.   Preparation of the test sample

If necessary, use the mechanical mill, which has previously been properly cleaned, to grind the laboratory sample in order to reduce it to particles that can pass completely through the sieve.

Use about one twentieth of the sample to complete the process of cleaning the mill, discard the ground material, grind the remainder and collect, mix carefully and analyze without delay.

2.2.   Test portion

As soon as the grinding operation has been completed, weigh out about 10 g of the sample to the nearest 0,01 g for testing.

2.3.   Preparation of the extraction thimble

Place the test portion in the thimble and plug with cotton wool. If a filter paper is used, envelope the test portion in it.

2.4.   Peliminary drying

If the olive residues are very moist (i.e., moisture and volatile matter content more than 10 %), carry out preliminary drying by placing the loaded thimble (or filter paper) in the oven heated for an appropriate time at not more than 80° C in order to reduce the moisture and volatile matter content to less than 10 %.

2.5.   Preparation of the round-bottomed flask

Weigh to the nearest 1 mg the flask containing one or two particles of pumice stone, previously dried in the stove at 103 ± 2° C and then cooled in a dessicator for not less than one hour.

2.6.   Initial extraction

Into the extraction apparatus insert the thimble (or filter paper) containing the test portion. Pour into the flask the requisite quantity of hexane. Fit the flask to the extraction apparatus and place the whole on the electrically heated bath. Adjust the rate of heating in such a way that the reflux rate is not less than three drops per second (moderate, not violent boiling). After four hours extraction, allow to cool. Remove the thimble from the extraction apparatus and place it in a stream of air in order to drive off most of the impregnating solvent.

2.7.   Second extraction

Tip the contents of the thimble into the micro-grinder and grind as finely as possible. Return the ground mixture to the thimble without loss and place it back in the extraction apparatus.

Continue the extraction for a further two hours using the same round-bottomed flask containing the initial extract.

The resultant solution in the extraction flask must be clear. If not, filter it through a filter paper and wash the original flask and the filter paper several times with hexane. Collect the filtrate and the washing solvent in a second round-bottomed flask which has been dried and tared to the nearest 1 mg.

2.8.   Removal of solvent and weighing of extract

Remove the greater part of the solvent by distillation on an electrically heated bath. Remove the last traces of solvent by heating the flask in the oven at 103 ± 2° C for 20 minutes. Assist the elimination process either by blowing in air, or preferably an inert gas, at intervals or by using reduced pressure.

Leave the flask in a dessicator to cool for at least one hour and weigh to the nearest 1 mg.

Heat again for 10 minutes under the same conditions, cool in a dessicator and reweigh.

The difference between the two weighings shall not exceed 10 mg. If it does, heat again for periods of 10 minutes followed by cooling and weighing until the weight difference is 10 mg or less. Note the last weight of the flask.

Carry out duplicate determinations on the test sample.

3.   EXPRESSION OF RESULTS

3.1.   Method of calculation and formula

3.2.   Repeatability

The difference between the duplicate determinations carried out simultaneously or in rapid sucession by the same analyst shall not exceed 0,2 g of hexane extract per 100 g of sample.

If this condition is not satisfied, repeat the analysis on two other test portions. If, in this case too, the difference exceeds 0,2 g, take as the result the arithmetic mean of the four determinations.

ANNEX XVI

DETERMINATION OF IODINE VALUE

1.   SCOPE

This International Standard specifies a method for the determination of the iodine value of animal and vegetable fats and oils, referred to hereafter as fats.

2.   DEFINITION

For the purposes of this International Standard, the following definition applies:

3.   PRINCIPLE

Dissolution of a test portion in solvent and addition of Wijs reagent. After a specified time, addition of potassium iodide solution and water, and titration of the liberated iodine with sodium thiosulfate solution.

4.   REAGENTS

All reagents shall be of recognized analytical grade:

5.   APPARATUS

Usual laboratory apparatus and, in particular, the following:

6.   PREPARATION OF THE TEST SAMPLE

The homogenized sample is dried over sodium sulphate and filtered.

7.   PROCEDURE

8.   EXPRESSION OF RESULTS

The iodine value is given by the expression

where:

c = is the numerical value of the exact concentration, in moles per litre, of the standard volumetric sodium thiosulfate solution (4.4) used;

V1 = is the numerical value of the volume, in millilitres, of the standard volumetric sodium thiosulfate solution (4.4) used for the blank test;

V2 = is the numerical value of the volume, in millilitres, of the standard volumetric sodium thiosulfate solution (4.4) used for the determination;

m = is the numerical value of the mass, in grams, of the test portion (7.1).

Take as the result the arithmetic mean of the two determinations, provided that the requirement for repeatability (9.2) is satisfied.

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ANNEX XVII

METHOD FOR THE DETERMINATION OF STIGMASTADIENES IN VEGETABLE OILS

1.   PURPOSE

Determination of stigmastadienes in vegetable oils containing low concentrations of these hydrocarbons, particularly in virgin olive oil and crude olive-residue oil.

2.   SCOPE

The standard may be applied to all vegetable oils although measurements are reliable only where the content of these hydrocarbons lies between 0,01 and 4,0 mg/kg. The method is particularly suited to detecting the presence of refined vegetable oils (olive, olive residue, sunflower, palm, etc.) in virgin olive oil since refined oils contained stigmastadienes and virgin oils do not.

3.   PRINCIPLE

Isolation of unsaponifiable matter. Separation of steroidal hydrocarbon fraction by column chromatography on silica gel and analysis by capillary gas chromatography.

4.   APPARATUS

5.   REAGENTS

All reagents should be of analytical grade unless otherwise specified. The water used should be distilled water, or water of at least equivalent purity.

6.   PROCEDURE

6.1.   Preparation of unsaponifiable matter

6.2.   Separation of steroidal hydrocarbon fraction

6.3.   Gas chromatography

Figure 1

Gas chromatograms obtained from olive oil samples analysed on a fused silica capillary column (0,25 mm internal diameter by 25 m) coated with 5 %-phenylmethylsilicone, 0,25 μm film thickness.

Figure 2

Gas chromatogram obtained from a refined olive oil sample analysed on DB-5 column showing the isomer of 3,5-stigmastadiene.

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ANNEX XVIII

DETERMINATION OF THE DIFFERENCE BETWEEN ACTUAL AND THEORETICAL CONTENT OF TRIACYLGLYCEROLS WITH ECN 42

1.   SCOPE

Determination of the absolute difference between the experimental values of triacylglycerols (TAGs) with equivalent carbon number 42 (ECN42HPLC) obtained by determination in the oil by high performance liquid chromatography and the theoretical value of TAGs with an equivalent carbon number of 42 (ECN 42theoretical) calculated from the fatty acid composition.

2.   FIELD OF APPLICATION

The standard is applicable to olive oils. The method is applicable to the detection of the presence of small amounts of seed oils (rich in linoleic acid) in every class of olive oils.

3.   PRINCIPLE

The content of triacylglycerols with ECN 42 determined by HPLC analysis and the theoretical content of triacylglycerols with ECN 42 (calculated on the basis of GLC determination of fatty acid composition) correspond within a certain limit for genuine olive oils. A difference larger than the values adopted for each type of oil points out that the oil contains seed oils.

4.   METHOD

The method for the calculation of the theoretical content of triacylglycerols with ECN 42 and of the difference with respect to the HPLC data is essentially made by the coordination of analytical data obtained by means of other methods. It is possible to distinguish three phases: determination of fatty acid composition by capillary gas chromatography, calculation of theoretical composition of triacylglycerols with ECN 42, HPLC determination of ECN 42 triacylglycerols.

4.1.    Apparatus

4.2.    Reagents

The reagents should be of analytical purity. Elution solvents should be de-gassed, and may be recycled several times without effect on the separations.

4.3.    Sample preparation

As a number of interfering substances can give rise to false positive results, the sample must always be purified according to IUPAC method 2.507, used for the determination of polar compounds in frying fats.

4.3.1.    Chromatographic column preparation

Fill the column (4.1.3) with about 30 ml of elution solvent (4.2.3), then introduce inside the column some glass wool (4.2.5) pushing it to the bottom of the column by means of the glass rod (4.1.5).

In a 100 ml beaker, suspend 25 g of silica gel (4.2.4) in 80 ml of elution mixture (4.2.3), then transfer it to the column by means of a glass funnel (4.1.6).

To ensure the complete transfer of the silica gel to the column, wash the beaker with the elution mixture and transfer the washing portions to the column too.

Open the cock and let the solvent elute from the column until its level is about 1 cm over the silica gel.

4.3.2.    Column chromatography

Weigh with the accuracy of 0,001 g, 2,5 ± 0,1 g of oil, previously filtered, homogenised and anhydrified, if necessary, in a 50 ml volumetric flask (4.1.7).

Dissolve it in about 20 ml of elution solvent (4.2.3). If necessary, slightly heat it to make the dissolution easily. Cool at room temperature and adjust the volume with elution solvent.

By means of a volumetric pipette, introduce 20 ml of solution inside the column prepared according to 4.3.1, open the cock and let the solvent elute to the silica gel layer level.

Then elute with 150 ml of elution solvent (4.2.3), adjusting the solvent rate at about 2 ml/min (150 ml will take about 60-70 minutes to pass through the column).

The eluate is recovered in a 250 ml round-bottomed flask (4.1.1) previously tared in an oven and exactly weighed. Eliminate the solvent at reduced pressure in a rotary evaporator (4.1.9) and weigh the residue that will be used to prepare the solution for HPLC analysis and for methyl ester preparation.

The sample recovery from the column must be 90 % at least for the extra virgin, virgin, ordinary, refined and olive oil categories, and a minimum of 80 % for lampante and olive-pomace oils.

4.3.3.    SPE purification

Silica SPE column is activated by passing 6 ml of hexane (4.2.3) under vacuum, avoiding dryness.

Weigh to an accuracy of 0,001 g, 0,12 g in a 2 ml vial (4.1.15) and dissolve with 0,5 ml of hexane (4.2.3).

Load the SPE column with the solution and elute with 10 ml of hexane-diethyl ether (87:13 v/v) (4.2.3) under vacuum.

The collected fraction is evaporated to dryness in a rotary evaporator (4.1.9) under reduced pressure at room temperature. The residue is dissolved in 2 ml of acetone (4.2.6) for triacylglycerol (TAG) analysis.

4.4.    HPLC analysis

4.4.1.    Preparation of the samples for chromatographic analysis

A 5 % solution of the sample to be analysed is prepared by weighing 0,5 ± 0,001 g of the sample into a 10 ml graduated flask and making up to 10 ml with the solubilisation solvent (4.2.9).

4.4.2.    Procedure

Set up the chromatographic system. Pump elution solvent (4.2.8) at a rate of 1,5 ml/min to purge the entire system. Wait until a stable base line is obtained.

Inject 10 μl of the sample prepared as in point 4.3.

4.4.3.    Calculation and expression of results

Use the area normalisation method, i.e. assume that the sum of the areas of the peaks corresponding to TAGs from ECN 42 up to ECN 52 is equal to 100 %.

Calculate the relative percentage of each triglyceride using the formula:

.

The results should be given to at least two decimal places.

See notes 1 to 4.

4.5.    Calculation of triacylglycerols composition (moles %) from fatty acid composition data (area %)

4.5.1.    Determination of fatty acid composition

Fatty acid composition is determined by ISO 5508 by means of a capillary column. The methyl esters are prepared according to COI/T.20/Doc. No 24.

4.5.2.    Fatty acids for calculation

Glycerides are grouped by their Equivalent Carbon Number (ECN), taking into account the following equivalencies between ECN and fatty acids. Only fatty acids with 16 and 18 carbon atoms were taken into consideration, because only these are important for olive oil. The fatty acids should be normalised to 100 %.

4.5.3.    Conversion of area % into moles for all fatty acids (1)

4.5.4.    Normalisation of fatty acid moles to 100 % (2)

The result gives the percentage of each fatty acid in moles % in the overall (1, 2, 3–) position of the TAGs.

Then the sum of the saturated fatty acids P and S (SFA) and the unsaturated fatty acids Po, O, L and Ln (UFA) are calculated (3):

4.5.5.    Calculation of the fatty acid composition in 2- and 1, 3- positions of TAGs

The fatty acids are distributed to three pools as follows: one for 2- position and two identical for 1- and 3- positions, with different coefficients for the saturated (P and S) and unsaturated acids (Po, O, L and Ln).

4.5.5.1.   Saturated fatty acids in 2-position [P(2) and S(2)] (4):

4.5.5.2.   Unsaturated fatty acids in 2-position [Po(2), O(2), L(2) and Ln(2)] (5):

4.5.5.3.   Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3), O(1,3), L(1,3) and Ln(1,3)] (6):

4.5.6.    Calculation of triacylglycerols

4.5.6.1.   TAGs with one fatty acid (AAA, here LLL, PoPoPo) (7)

4.5.6.2.   TAGs with two fatty acids (AAB, here PoPoL, PoLL) (8)

4.5.6.3.   TAGs with three different fatty acids (ABC, here OLLn, PLLn, PoOLn, PPoLn) (9)

4.5.6.4.   Triacylglycerols with ECN42

The triacylglycerols with ECN42 are calculated according to equations 7, 8 and 9 and are then given in order of expected elution in HPLC (normally only three peaks).

The triacylglycerols with ECN42 are given by the sum of the nine triacylglycerols including their positional isomers. The results should be given to at least two decimal places.

5.   EVALUATION OF THE RESULTS

The calculated theoretical content and the content determined by the HPLC analysis are compared. If the difference in the absolute value of the HPLC data minus the theoretical data is greater than the values stated for the appropriate oil category in the standard, the sample contains seed oil.

Results are given to two decimal figures.

6.   EXAMPLE (THE NUMBERS REFER TO THE SECTIONS IN THE TEXT OF THE METHOD)

— 4.5.1.    Calculation of moles % fatty acids from GLC data (normalised area %)

The following data are obtained for the fatty acid composition by GLC:

— 4.5.3    Conversion of area % into moles for all fatty acids (see formula (1))

— 4.5.4    Normalisation of fatty acid moles to 100 % (see formula (2))

Sum of the saturated and unsaturated fatty acids in the 1,2,3-position of TAGs (see formula (3)):

— 4.5.5    Calculation of the fatty acid composition in 2- and 1,3-positions of the TAGs

— 4.5.5.1   Saturated fatty acids in 2-position [P(2) and S(2)] (see formula (4))

— 4.5.5.2   Unsaturated fatty acids in 2-position [Po(1,3), O(1,3), L(1,3) and Ln(1,3)] (see formula (5))

— 4.5.5.3   Fatty acids in 1,3-positions [P(1,3), S(1,3), Po(1,3), O(1,3), L(1,3) and Ln(1,3)] (see formula (6))

— 4.5.6.    Calculation of triacylglycerols

From the calculated fatty acid composition in sn-2- and sn-1,3-positions:

the following triacylglycerols are calculated:

— 4.5.6.1.   TAGs with one fatty acid (LLL, PoPoPo) (see formula (7))

, = 0,09706 mol LLL

— 4.5.6.2   TAGs with two fatty acids (PoLL, SLnLn, PoPoL) (see formula (8))

0,03210 mol PoLL

0,00094 mol SLnLn

0,00354 mol PoPoL

— 4.5.6.3   TAGs with three different fatty acids (PoPLn, OLLn, PLLn, PoOLn) See formula (9)