Screening of aroma-producing Kluyveromyces marxianus in traditional fermented milk and its impact on flavor formation of fermented milk: Insights from genomics and metabolomics.

Kluyveromyces marxianus, as a dominant strain in traditional fermented milk, plays a significant role in the formation of its unique flavor. However, research on the aroma-producing mechanisms of K. marxianus in dairy products remains limited, which restricts its application in the food industry. To address this, in this study a total of 78 Kluyveromyces marxianus strains were isolated from traditional handmade dairy products in Xinjiang. Through a screening process, we finally obtained a K. marxianus strain, BSK1-4, with excellent aroma-producing characteristics. Compared with the other 77 K. marxianus strains, BSK1-4 showed a stronger ability to produce esters, particularly ethyl acetate, phenethyl acetate, isoamyl acetate, ethyl hexanoate and ethyl octanoate. These compounds can impart fruity notes such as pineapple and banana, as well as floral notes like rose, to fermented milk. A total of 35 characteristic volatile metabolites were identified across 6 fermentation stages of this strain. Among them, ethyl acetate (4.88 mg/L), isoamyl alcohol (2.25 mg/L), phenethyl acetate (1.16 mg/L), phenethyl alcohol (1.01 mg/L), isobutanol (0.31 mg/L), and isoamyl acetate (0.22 mg/L) serve as the primary flavor metabolites. Multi-omics analysis revealed that K. marxianus BSK1-4 contains various synthases and hydrolases (particularly pyruvate kinase, ethanol acetyltransferase 1 and medium - chain fatty acid ethyl ester synthase/esterase 2) that promote the involvement of the key intermediate pyruvate in the tricarboxylic acid cycle, as well as in the biosynthesis of amino acids such as phenylalanine, valine, leucine, and isoleucine. These pathways enhance the biosynthesis of flavor compounds such as ethyl acetate, phenethyl alcohol, phenethyl acetate, and isoamyl acetate, ultimately contributing to the unique flavor of the fermented milk.