Insights in the Peptidomics of buttermilk fermentation product by Lactobacillus delbrueckii subsp. lactis through MS screening focused on the ID of bioactive peptides as functional food additives.

KOCI, KAMILA; BINETTI, ANA G.; SOARES, RENATA; HAAG, ELIANA; VINDEROLA, GABRIEL; REINHEIMER, JORGE; COSTA, M. DO CÉU; VARELA COHELO, ANA

Lisboa (Portugal)

Congreso; 4th Portuguese Mass Spectrometry Meeting, Lisboa (Portugal); 2010

A spray-dried product of buttermilk fermentation naturally enriched of potentially bioactive peptides was analysed using LC-MS/MS and MALDI-MS/MS. In a previous study, mucosal immunomodulation was observed when the fermented product was administrated to BALB/c mice1. The aim of this work was to identify the peptides responsible to this immune activity. Due to high sample complexity (47% lactose, 29% protein, 12% fat, 7% ash) sample preparation optimization was carried out prior to MS analysis. Four sample treatments were tested in order to obtain peptide enriched extracts. The best results were achieved using 3 successive steps: 1) Extraction of lipids using chloroform; 2) Extraction of peptides using 50% MeOH; 3) Protein precipitation with 10% trichloroacetic acid. LC-MS/MS (Surveyor HPLC/LCQ-Thermo Finnigan) analysis was performed with the supernatant obtained from step 3. For the selective detection of peptides, BCA reaction was performed with 5 sub-fractions collected during the LC-MS run. The LC gradient was modified to improve the separation of the 4 peptide-containing fractions and 1 peptide (IPP) was identified. To overcome sensibility issues and improve fragmentation patterns MALDI-MS/MS (4800 MALDI TOF/TOF-ABI) were performed for 15 fractions using 2 approaches. First, MS/MS spectra for the 10 most intense peaks of each fraction were collected. Subsequently, a more selective MALDI-MS/MS analysis was performed focused on the peaks of interest. Obtained MS/MS patterns were compared with protein sequence databases using ProteinPilotTM search engine. This screening identified 9 proteolytic peptides originating from b-casein and a-lactalbumin bovine proteins. Some of the identified peptides are in good agreement with reported b-casein bioactive peptides released during dairy fermentation using Lactobacillus2. A guided immune active assay of prepared purified fractions is on its way. ACKNOWLEDGMENTS: The authors gratefully acknowledge support from CYTED-Project Novel ProBio. 1. P. Burns, F. Molinari, A. Beccaría, R. Páez, C. Meinardi, J. Reinheimer and G. Vinderola . J. Appl. Microbiol. Vol 109 (2010), 1370-1378. 2. E.M. Hebert, G. Mamone, G. Picariello, R.R. Raya, G. Savoy, P. Ferranti and F. Addeo Appl. Environ. Micorbiol. Vol 74 (2008), 3682–3689.