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Determination of the Antihypertensive Peptide LHLPLP in Fermented Milk by High-Performance Liquid Chromatography–Mass Spectrometry

A. Quirós^*, M. Ramos^*, B. Muguerza, M. A. Delgado, P. J. Martín-Alvarez^*, A. Aleixandre and I. Recio^*,1

^* Instituto de Fermentaciones Industriales, Consejo Superior de Investigaciones Científicas (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
Department of Research and Development, Grupo Leche Pascual, Ctra. Palencia s/n, 09400 Aranda de Duero Burgos, Spain
Instituto de Farmacología y Toxicología (CSIC), Facultad de Medicina, Universidad Complutense de Madrid, Avda. Complutense s/n, 28040 Madrid, Spain

¹ Corresponding author: recio{at}ifi.csic.es

Among different lactic acid bacteria isolated from raw milk, 4 Enterococcus faecalis strains have stood out as producers of fermented milk with potent antihyperten-sive activity. The peptide ß-casein f(133–138), LHLPLP, was identified as one of the major peptides responsible for the activity of these fermented milk products. A simple method was developed to quantify this peptide in fermented milk using high-performance liquid chromatography coupled in line with mass spectrometry. This procedure does not require any previous sample fractionation or extraction, and direct analysis of the water-soluble extract obtained from the fermented milk can be performed. Validation studies showed sufficient speci-ficity, reproducibility, linearity, and recovery, demonstrating that this method can be used for the routine quantification of LHLPLP during the production of fermented milk products. The developed method was readily applied to quantify the peptide LHLPLP under different fermentation conditions and with different aromatized products.

Key Words: antihypertensive peptide • Enterococcus faecalis • quantification • mass spectrometry