Discovering natural bioactive peptides in cheese with mass spectrometry

Randall Robinson, Dept. of Food Science and Technology, University of California, Davis, USA
Student Travel Award Recipient

Randall C. Robinson1, David C. Dallas2, and Daniela Barile1,3
1. Department of Food Science and Technology, University of California, Davis;
2. School of Biological and Population Health Sciences, Oregon State University, Corvallis;
3. Foods for Health Institute, University of California, Davis

Alterations in gastrointestinal microbiota are becoming increasingly common. Current anti-microbials are successful in treating overt infections, but are non-specific, cause many side effects, and can betoxic to the host’s cells. Non-selective interventions, notably broad-spectrum antibiotics, are proving highly deleterious to the healthy intestinal microbiota. There is a critical need for treatment options that will selectively eliminate pathogens, but at the same time improve or at least not impair the growth of commensal bacteria. Peptides can possess a variety of bioactivities, including antimicrobial functionality, and may be a favorable alternative to traditional antibiotics. Peptides occur naturally in dairy products as a result of microbial fermentation and proteolysis. Therefore, identification of bioactive sequences is important in understanding the physiological impact of these foods.

The objective of this study is to identify the sequences and potential functions of endogenous peptides from several cheeses by LC-Orbitrap MS/MS analysis, including Mimolette (France), Taleggio (Italy), and Stilton (UK), as well as traditional cheddar. Each of these cheeses contains a unique mix of micro-organisms that we hypothesized would contribute to proteolysis and bioactive peptide formation.

Water-soluble peptides were extracted from the rind and interior of each cheese and purified through C18 solid phase extraction. Each peptide extract was analyzed by LC-MS/MS using an Orbitrap Q Exactive Plus mass spectrometer in data-dependent mode. Peptides were then identified from the tandem spectra by an automated search, using the complete bovine proteome as a reference. The resulting peptide lists will be analyzed by an in-house homology search program that will identify peptide sequences from the cheeses which have previously been demonstrated to be bioactive.

The searches identified an outstandingly high number of peptides: 4,701 from Mimolette, 3,520 from Taleggio, 3,408 from Stilton, and 1827 from cheddar. Peptides ranged in length from 5 to 45 amino acid residues. The majority of peptides in each sample originated from caseins, although several other proteins, including β-lactoglobulin, lactoferrin, glycosylation-dependent cell adhesion molecule 1, butyrophilin subfamily 1 member A1, and xanthine dehydrogenase/oxidase, were also well represented in the peptide profiles. Mimolette was particularly notable in that it contained 117 lactoferrin derived peptide sequences in the rind and only 1 lactoferrin peptide in its interior, demonstrating the differential in proteolytic factors between the two regions. A functional homology search will be conducted in the near future to identify bioactive sequences in each cheese.

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