Machine learning-assisted unveiling of aroma modulation by inorganic nitrogen during cider fermentation by Pichia kluyveri.

Non-Saccharomyces yeasts have been shown to enhance and diversify the aroma profile of cider. This study aimed to elucidate the regulatory role of inorganic nitrogen and to improve cider aroma quality by investigating how the type and concentration of nitrogen sources affect the fermentation performance and aroma production of Pichia kluyveri. The effects of three inorganic nitrogen sources-NH₄Cl, (NH₄)₂SO₄, and (NH₄)₂HPO₄-at two concentrations (140 and 200 mg YAN/L) on apple juice fermentation by Pichia kluyveri X31-10 were examined, with Saccharomyces cerevisiae WLS21 as a reference. Yeast growth was monitored, and volatile compounds were analyzed using GC-MS, GC-IMS, and machine learning to profile aroma composition and identify key differential compounds. Nitrogen strongly affected the growth and aroma production of both strains, especially P. kluyveri. Machine learning identified eight key compounds distinguishing the two strains and five nitrogen-source-dependent biomarkers in P. kluyveri, with 2-methylbutyl acetate, ethyl decanoate, and ethyl butyrate common to both sets, highlighting them as key differential compounds responsive to nitrogen regulation. Ester formation in P. kluyveri was strongly influenced by the type of nitrogen source: compared with S. cerevisiae, phosphate and sulfate supplementation markedly enhanced overall ester production, particularly affecting isoamyl acetate and 2-methylbutyl acetate. Nitrogen concentration further modulated the synthesis of key esters in P. kluyveri: elevated NH₄Cl and (NH₄)₂HPO₄ consistently increased key esters such as isoamyl acetate and hexyl acetate, whereas the effects of (NH₄)₂SO₄ were compound-specific. These findings demonstrate the potential of nitrogen management to optimize cider flavor using P. kluyveri.