Issue |
Lait
Volume 64, Number 640-642, 1984
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Page(s) | 340 - 355 | |
DOI | https://doi.org/10.1051/lait:1984640-64228 |
DOI: 10.1051/lait:1984640-64228
Dosage de l'activité lipasique et des acides gras libres du lait par titration automatique colorimétrique
P. CARTIERa, Y. CHILLIARDb, Marie-Paule CHAZALa and Jeanne FLECHETba Institut Technique de l'Elevage Bovin
b Laboratoire de la lactation, I.N.R.A.-Theix - 63122 Ceyrat
Abstract - Determination of milk lipase activity and milk free fatty acid content using a colorimetric automatic titration
The colorimetric automatic titration of free fatty acids (FFA) has been studied, with the Metrohm titration assembly, from Jellema's proposals (1981), to determine free fatty acids in raw milk using the BDI method. It has also been applied to the measurement of milk (lipoprotein) lipase activity.
1 Using standard solutions of palmitic acid, the assay was sensitive, precise, repeatable and rapid (20 to 45 sec. per measurement). During routine utilization, the rate of a series of measurements was limited by the number of successive determination that could be made without emptying the recipient. This number depended on the volume of each sample in relation to the volume of the titration vessel and to the amount of titrated FFA. Indeed, when this amount increased, turbidity occurred and disturbed the measurement. This occurred more quickly when the alkali was sodium hydroxide rather than potassium hydroxide and when the solvant was ether / ethanol (instead of heptan or butanol). It took place later with tetrabutyl ammonium hydroxide (TBAH) as titrant.
2 When applied to the determination of milk fat FFA (0.1 to 0.25 ml fat obtained by the miniaturized BDI extraction method), the automatic titration gave results comparable to those obtained by manual titration using an accurate microburette. The butanol / TBAH system was the one that allowed the greatest number of successive determinations to be made without any turbidity.
From a practical point of view, the precision of the automatic method depended on that of fat sampling with a pipette (whereas it was weighed in the manual method) especially with the miniaturized variants of the BDI method. Otherwise, its rapidity was limited by several precautions that had to be taken during calibration and during each series of determinations.
3 The lipoprotein lipase activity assay was based on the hydrolysis of a commercial triglyceride emulsion (Intralipid) containing long chain fatty acids, activated by bovine serum. Free fatty acids were extracted before and after enzymatic incubation by the method of Trout et al. (1960) and titrated on 3 ml of heptan phase. Although the automatic method overestimated the absolute values of free fatty acids, lipase activities (development of FFA during incubation) measured by the 2 methods were equal and correlated very well. During routine utilization, the speed of the method was decreased strongly on account of the volume of each sample, because of titration vessel emptying and recalibration frequency.
4. Finally, the automatic titration did not seem to allow any time to be saved for lipoprotein lipase measurement. With the determination of milk FFA (extracted by the BDI method) time can be saved, in addition to inherent advantages linked to the suppression of the visual judgment of the change in colour (removal of operator effect and decreased visual weariness). Further studies are needed to evaluate the suitability of this kind of method for large-scale routine assays
Résumé - Le dosage des acides gras libres (AGL) et de l'activité lipasique du lait par titration colorimétrique automatique a été étudié à l'aide de la chaîne de dosage Métrohm, à partir des propositions de Jellema (1981).
L'étalonnage de la méthode automatique montre qu'elle est sensible, précise, répétable et rapide. La rapidité d'une série de mesures est toutefois limitée par l'apparition d'un précipité, surtout lorsque le titrant est de la soude ou de la potasse et lorsque le solvant est le mélange éther / éthanol. L'emploi du tétra-butylammonium hydroxide comme titrant et du butanol ou de l'heptane comme solvant, permet de retarder l'apparition du précipité.
La méthode automatique est applicable au dosage de l'activité lipasique du lait, mais sans gain de temps par rapport à la méthode manuelle, en raison du volume des prises d'essai nécessaires au dosage. Par contre, l'automatisation de la titration des AGL du lait extraits par la méthode BDI peut permettre un gain de temps mais nécessite un pipettage volumétrique délicat de la matière grasse. Dans les 2 cas, l'automatisation permet de supprimer l'effet opérateur (appréciation visuelle du virage) et de diminuer la fatigue visuelle. L'intérêt de ce type de dosage en ateliers semi-industriels demande toutefois à être précisé
Key words: Milk / Fat-acidity / Lipase Activity / Free fatty acids / Colorimetric and automatic titration / Heptan / Butanol / Tetrabutyl ammonium hydroxide