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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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Textual Amendments
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).
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.
Textual Amendments
After decanting, remove the lower layer containing the soaps. Remove any intermediate layers (mucilage and insoluble substances). Wash the hexane solution of the neutralised oil with successive portions of 50-60 ml of the 1/1 (v/v) isopropanol/water solution (4.4) until the pink colouration of the phenolphthalein disappears.
Remove most of the hexane by vacuum distillation (use a rotary evaporator, for example) and transfer the oil into a 100 ml flask (3.5). Dry the oil in vacuum until the solvent is completely removed.
After that procedure is completed, the acidity of the oil should be less than 0,5 %.
If the solution is cloudy centrifuge it to ensure optimum conditions for chromatography. (Ready-to-use 500 mg silica gel SPE cartridges can be used).
Pour about 30 ml of the developer solvent (4.10) into the column (3.3), insert a piece of cotton into the bottom part of the column using a glass rod; press to eliminate the air.
In a beaker prepare a suspension of 25 g of silica gel (4.1) in about 80 ml of developer solvent and pour it into the column using a funnel.
Check that all the silica gel is in the column; wash with developer solvent (4.10), open the stopcock and allow the liquid to reach a level about 2 mm above the level of the silica gel.
Weigh accurately 1,0 g of sample prepared as in point 5.1 into a 25 ml Erlenmeyer flask (3.1).
Dissolve the sample in 10 ml of developer solvent (4.10). Pour the solution into the chromatography column prepared as in point 5.1.3. Avoid disturbing the surface of the column.
Open the stopcock and pour the sample solution until it reaches the level of the silica gel. Develop with 150 ml of the developer solvent. Adjust the flow rate to 2 ml/min (so that 150 ml enters the column in about 60-70 minutes).
Recover the eluate in a previously weighed 250 ml flask. Evaporate the solvent under vacuum and remove the final traces of the solvent under a nitrogen current.
Weigh the flask and calculate the recovered extract.
(If ready-to-use silica gel SPE cartridges are used use the following method: Put 1 ml of solution (5.1.2) into the prepared cartridges with 3 ml of n-hexane.
After percolating the solution develop with 4 ml of n-hexane/diethyl ether 9/1 (v/v).
Recover the eluate in a 10 ml tube and evaporate to dry in a nitrogen current.
Expose the dry residue to pancreatic lipase (5.2). (It is essential to check the fatty acid composition before and after crossing the SPE cartridge.)
Operating conditions:
Injector temperature (on-column injector) lower than solvent boiling point (68 °C);
Detector temperature: 350 °C;
Column temperature: programming of furnace temperature: 60 °C for 1 minute, increasing by 15 °C per minute up to 180 °C, then by 5 °C per minute up to 340 °C, then 340 °C for 13 minutes;
Carrier gas: hydrogen or helium, set at a linear velocity sufficient to obtain the resolution reflected in Figure 1. The retention time of the C 54 triglyceride must be 40 ± 5 minutes (see Figure 2). (The operating conditions indicated above are indicative. Operators will have to optimise them to obtain the desired resolution. The peak corresponding to 2-glyceryl monopalmitate must have a minimum height equal to 10 % of the recorder scale.)
Quantity of substance injected: 0,5-1 μl of the n-hexane solution (5 ml) (5.3.3).
The individual monoglycerides are identified from their retention times and by comparison with those obtained for standard monoglyceride mixtures under the same conditions.
The area of each peak is calculated using an electronic integrator.
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:
where:
=
area of the peak corresponding to glyceryl monopalmitate
=
sum of the areas of all the monoglyceride peaks
The result must be to one decimal place.
The analysis report must specify:
reference to this method,
all the information needed for a full identification of the sample,
the analysis result,
any deviation from the method, whether as the result of a decision by the parties concerned or for another reason,
details to identify the laboratory, the date of the analysis and the signatures of those responsible for the analysis.
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 %)
Chromatogram of :
After lipase, the triglyceride content should not exceed 15 %
Chromatogram of :
After lipase, the triglyceride content should not exceed 15 % .
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.
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:
is activity in lipase units/mg
is the number of millilitres of 0,1 N sodium hydroxide solution per minute (calculated on the basis of the graph)
is the titre of the sodium hydroxide solution
is the mass in mg of the test lipase.
A lipase unit is defined as the quantity of enzyme which releases 10 micro-equivalents of acid per minute.]
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