Editing of the Proteolytic System of Lactococcus lactis Increases Its Bioactive Potential

Abstract

Large-scale mass spectrometry-based peptidomics for bioactive peptide discovery is relatively unexplored because of challenges in intracellular peptide extraction and small peptide identification. Here we present an analytical pipeline for large-scale intracellular peptidomics of Lactococcus lactis. It entails an optimized sample preparation protocol for L. lactis, used as an “enzyme complex” to digest β-casein, an extraction method for its intracellular peptidome, and a peptidomics data analysis and visualization procedure. In addition, we proofread the publicly available bioactive peptide databases and obtained an optimized database of bioactive peptides derivable from bovine β-casein. We used the pipeline to examine cultures of L. lactis MG1363 and a set of 6 isogenic multiple peptidase mutants incubated with β-casein. We observed a clearly strain-dependent accumulation of peptides with several bioactivities, such as ACE inhibitory, DPP-IV inhibitory, immunoregulatory functions. The results suggest that both the number of different bioactive peptides and the bioactivity diversity can be increased by editing the proteolytic system of L. lactis. This comprehensive pipeline offers a model for bioactive peptide discovery in combination with other proteins and might be applicable to other bacteria. Importance Lactic acid bacteria (LAB) are very important for the production of safe and healthy human and animal fermented foods and feed and, increasingly more, in the functional food industry. The intracellular peptidomes of LAB are promising reservoirs of bioactive peptides. We show here that targeted genetic engineering of the peptide degradation pathway allows steering the composition of the peptide pool of the LAB Lactococcus lactis and producing peptides with interesting bioactivities. Our work could be used as a guideline for modifying proteolytic systems in other LAB to further explore their potential as cell peptide factories.

Positive Interactions between Lactic Acid Bacteria Promoted by Nitrogen-Based Nutritional Dependencies

“… Lactococcus lactis and Enterococcus faecalis were found to be adequate candidates. Both species possess a very efficient proteolytic system and can modify sensorial profiles in cheeses and other fermented dairy products ( 16 , 17 ). The E. faecalis proteolytic system was even shown to be responsible for a decrease of allergenicity of bovine milk proteins ( 18 ).…”

Lactococcus lactis in Dairy Fermentation—Health-Promoting and Probiotic Properties

“…Milk fermentation processes executed by LAB are preferable ways to generate peptides due to the GRAS status of these organisms. The proteolytic system of L. lactis has been comprehensively analyzed with respect to the genes and enzymes involved, as well as their regulation [261][262][263][264]. It was reported that during bovine milk fermentation with L. lactis, numerous casein-derived bioactive peptides can be generated, most of which have angiotensin-converting enzyme (ACE) inhibitory activity, leading to an antihypertensive effect [36,260,265].…”

Peptidomics: new trends in food science

“…The ability to preferentially cleave these regions could confer a selective growth advantage to L. helveticus. Huang and Kok [30 ] combined deletion of pep genes and nanoLC-MS/MS to identify novel bioactive peptides released from b-casein by Lactococcus lactis pep mutants.…”