Bitterness is a common flavor attribute of aged cheese associated with the peptide fraction, but excessive levels are a defect leading to consumer rejection. Bitterness in cheese has been primarily associated with peptides that arise from the breakdown of casein. The last review of bitter peptides was published in 1992. This updated review compiled information about the bitter peptides published up to 2022. Our comprehensive search of the literature compiled 226 peptides associated with bitterness and cheese protein origins into a database (Supplemental Materials). The influences of a peptide's physical properties, such as molecular weight, average hydrophobicity, peptide length, number of prolines and the presence of hydrophobic amino acids in the peptide's terminus, were assessed for correlation with bitterness threshold values this assessment found that, among variables considered, higher molecular weight had the strongest correlation with higher bitterness among known peptides. Heatmaps of bitter peptides and their bitterness threshold values highlight β-casein as the primary source of known bitter peptides in cheese. This comprehensive database of cheese protein-derived bitter peptides and this discovery of the correlation of a peptide's physical properties to bitterness will aid future researchers in the identification and discovery of contributors to cheese bitterness.

Despite bitterness being a common flavor attribute of aged cheese linked to casein-derived peptides, excessive bitterness is a sensory flaw that can lead to consumer rejection and economic loss for creameries. Our research employs a unique approach to identify bitter peptides in cheese samples using crossflow filtration-based fractionation, mass spectrometry-based peptidomics, statistics and sensory analysis. Applying peptidomics and statistical screening tools, rather than traditional chemical separation techniques, to identify bitter peptides allows for screening the whole peptide profile. Five peptides-YPFPGP (β-casein [60-65]), YPFPGPIPN (βA2-casein [60-68]), LSQSKVLPVPQKAVPYPQRDMPIQA (β-casein [165-189]), YPFPGPIHNS (βA1-casein [60-69]) and its serine phosphorylated version YPFPGPIHN[S] (βA1-casein [60-69])- demonstrated high levels of bitterness with mean bitterness intensity values above 7 on a 15-point scale. In the future, this data can be combined with the microbial and protease profile of the Cheddar samples to help understand how these factors contribute to bitter taste development.

Background/Objectives: Human milk is the optimal source of nutrition and protection against infection for infants. If breastfeeding is not possible, standard and hydrolyzed infant formulas (IF) are an alternative. Extensively hydrolyzed IFs (eHFs) contain bioactive peptides, but their activities have rarely been evaluated. The aim of this study was to characterize and compare the bioactive peptide profiles of different eHFs and standard IFs before and after in vitro digestion. Methods: Two forms, liquid and powder, of intact protein formula (iPF) and eHF were subjected to in vitro gastrointestinal digestion, mimicking a young infant’s gut (age 0–4 months) and an older infant’s gut (>6 months). Bioactive peptides of in vitro digested and undigested formulas were analysed with Liquid Chromatography–Mass Spectrometry (LC–MS). Results: In all samples, a variety of peptides with potential bioactive properties were found. Immuno-regulatory peptides, followed by antimicrobial and antioxidative peptides were most frequent, as were peptides promoting wound healing, increasing mucin secretion, regulating cholesterol metabolism, and preventing bacterial infection. Peptides typically found in yoghurt and colostrum were identified in some formula samples. Conclusions: The high amounts of bioactive peptides with various properties in eHFs and iPFs indicate a possible contribution to infection protection, healthy gut microbiomes, and immunological development of infants. eHFs showed similar compositions of bioactive peptides to iPFs, with intermittently increased peptide variety and quantity.

The PeptiLine peptidomics pipeline is a comprehensive software platform designed to transform peptidomic mass spectrometry output data into interactive, intuitive visualizations and statistical summaries. By integrating peptide-to-protein mapping, quantitative comparisons, bioactive annotations and descriptive and inferential analyses into a single user-friendly interface, PeptiLine addresses the bottleneck of timely and complex data exploration and visualization in bioactive peptide research. It enables visualization of peptidomic data through a series of modules allowing the user to create peptide sequence heatmaps, correlation plots and a series of categorical bar plots and pie plots to explore the absolute and relative bioactivity, protein origin, summed abundance and total peptide count. Here, we provide example uses of our Data Transformation, Descriptive Analysis and Heatmap Visualization tools to pinpoint functional peptide hotspots across protein sequences and compare samples across different protein variants. PeptiLine is freely available at https://mbpdb.nws.oregonstate.edu/peptiline/ and on GitHub ( https://github.com/Kuhfeldrf/peptiline/ ) under an MIT license. The software runs on Windows, macOS, and Linux systems with Python ≥3.10. Support and bug reports: contact- mbpdb@oregonstate.edu . Author summary: Bioactive peptides in food have health benefits ranging from antimicrobial activity to blood pressure regulation. We use mass spectrometry to identify these peptides, but analyzing the resulting datasets is challenging. The data comes out as massive spreadsheets that are difficult to interpret, and existing tools either require advanced programming skills, lack key analytical features, or are no longer maintained. We built PeptiLine to address these problems. It’s a user-friendly platform that transforms peptidomic data into interactive visualizations and statistical summaries. The key challenge we solved was handling peptides with multiple overlapping functions, for instance, a peptide that’s both antimicrobial and antihypertensive. Most tools would double-count such peptides, inflating the totals. We figured out how to accurately display both individual functional categories and true abundance totals without this double-counting bias. We demonstrated PeptiLine’s capabilities by analyzing bitter peptides in aged Cheddar cheese, successfully identifying specific protein regions responsible for bitterness and novel insight into Cheddars bioactive potential. The software is freely available as both a web application and for local installation, requiring no programming expertise.

Functional peptidomic studies produce data sets of thousands of peptides, which are assigned bioactivities through curated databases and prediction tools. Annotation, data preparation, and visualization are handled in different tools, fragmenting the workflow. Because peptides often have multiple bioactivities, grouping by function can double-count peptides and overstate total functional abundance within each sample. We developed PeptiLine, a browser-based platform for functional peptidomic analysis, whose integrated annotation tool searches bioactive databases. Automated column mapping and multifile merging accommodate diverse proteomic software exports, and analysis extends to noncanonical protein sequences. The Data Transformation module organizes data through an auditable workflow spanning functional annotation, column renaming, replicate and group assignment, and peptide-to-protein mapping. The Descriptive Analysis and Heatmap Visualization modules generate sequence heatmaps, correlation plots, categorical charts, and scaled abundance plots of bioactivity, protein origin, abundance, and peptide counts, with optional group-comparison statistics. Our scaled bar plots resolve double counting: bar heights derive from unique-peptide summation and stay comparable between samples, while a sample’s functional composition is proportionate to the unique-peptide summation within each sample. Case studies from food, clinical, and comparative-physiology peptidomes demonstrate the broad applicability of PeptiLine. PeptiLine is freely available at https://peptiline.nws.oregonstate.edu/ and on GitHub (https://github.com/kuhfeldrf/peptiline/) under an MIT license.