Oligopeptides from grape must represent a secondary source of nitrogen for yeasts to grow and carry out fermentation. Saccharomyces cerevisiae takes up oligopeptides from the environment through multiple oligopeptide transporters with different peptide length specificities. However, due to difficulties associated with the qualitative and quantitative measurement of peptides in natural matrices, peptide transporter specificities have been mostly researched in single peptide environments. Using a peptide mapping method, we monitored the relative consumption of peptides derived from a protein hydrolysate by a set of CRISPR-Cas9-engineered S. cerevisiae wine strains to study oligopeptide transporters from the Opt and Fot families. Results show that Opt2 can import peptides containing three to at least seven amino acid residues, which is a broader peptide length specificity than previously reported, while Opt1 activity was not sufficient to support growth on peptides as nitrogen source. Fot1, Fot2, and Fot3, previously referred to as di-tripeptide transporters in S. cerevisiae wine strains, could also import tetrapeptides. The consumption order of peptides was determined by the peptide length as higher chain length peptides were taken up by Opt2 only after most di-tetrapeptides were depleted from the media. Altogether, Fot and Opt2 activity assured completion of the fermentation process without necessarily requiring ammonia or free amino acids. Analysis of peptide transporter gene expression during fermentation showed an effect of SO ₄ ²⁻ not only on OPT1 but also on FOT and supported the assumption of a possible interplay between Fot and Opt2 activities. IMPORTANCE: Limited nitrogen supply can prevent the completion of alcoholic fermentation. Supplementation through peptides as an alternative, natural source of nitrogen for yeast offers an interesting solution for this issue. In this work, the S. cerevisiae peptide transporters of the Opt and Fot families were studied. We demonstrated that Fot and Opt2 have a broader peptide length preference than previously reported, enabling yeasts to acquire sufficient nitrogen from peptides without requiring additional ammonia or amino acids to complete fermentation. On the contrary, Opt1 was unable to consume any peptide in the given conditions, whereas it has been described elsewhere as the main peptide transporter for peptides longer than three amino acid residues in experiments in laboratory conditions. This controversy signifies the need in applied sciences for approaching experimental conditions to those prevalent in the industry for its more accurate characterization. Altogether, this work provides further evidence of the importance of peptides as a nitrogen source for yeast and their consequent positive impact on fermentation kinetics.

Several studies have shown the ability of yeast to consume peptides as a nitrogen source in single-peptide containing media. However, a suitable and cost-effective methodology to study the utilization of peptides by yeast and other microorganisms in a complex peptide mixture has yet to be put forward. This article addresses this issue by presenting a screening methodology for tracking the consumption of peptides by yeast during alcoholic fermentation. As a peptide source, the methodology makes use of an in-house prepared peptide-mapped bovine serum albumin (BSA) proteolytic digest, which was applied to a synthetic grape must. The peptide uptake was analyzed using high-throughput ultra-high-pressure liquid chromatography coupled to data-independent acquisition-based ion mobility separation-enabled high-resolution mass spectrometry (UPLC-DIA-IMS-HRMS) analysis. The relative changes of abundance of 123 di- to hexapeptides were monitored and reported during fermentations with three commercial wine strains, demonstrating different uptake kinetics for individual peptides. Using the same peptide-mapped BSA hydrolysate, the applicability of an untargeted workflow was additionally assessed for peptide profiling in unelucidated matrixes. The comparison of the results from peptide mapping and untargeted analysis experiments highlighted the ability of untargeted analysis to consistently identify small molecular weight peptides on the length and amino acid composition. The proposed method, in combination with other analytical techniques, such as gene or protein expression analysis, can be a useful tool for different metabolic studies related to the consumption of complex nitrogen sources by yeast or other microorganisms.

Scotch grain whisky is produced with a substantial proportion of unmalted grains, which can result in nitrogen deficiency for yeast in the fermentable grain mash. This study examined nitrogen source availability and utilisation by three commercial whisky strains ( Saccharomyces cerevisiae ) during Scotch grain whisky fermentation, focusing on oligopeptides. Peptide uptake kinetics in synthetic whisky mash with defined peptides showed that oligopeptides of up to nine amino acids were taken up by the strains, albeit with some variability between the strains. The study found that peptides with appropriate molecular weights could replace free amino acids without negatively affecting fermentation kinetics. Moreover, fermentation performance improved when additional nitrogen was provided via peptides rather than diammonium phosphate. Analysis of industrial grain mash indicated that despite low initial yeast assimilable nitrogen, residual proteolytic activity from malt increased nitrogen availability during fermentation. Approximately 30% of the nitrogen consumed by yeast during grain mash fermentation was derived from peptides. LC-HRMS peptide analysis revealed complex dynamics of peptide formation, degradation, and utilisation. This study highlights the importance of oligopeptides in ensuring optimal fermentation efficiency in Scotch grain mash and similar substrates.