Alcohol fermentation using yeast has been implicated in the production of important bioactive compounds. Both Saccharomyces and non-Saccharomyces strains of yeasts undertake alcohol fermentation and produce key bioactive compounds. Through the melatonin pathway synthesis, melatonin is the final product, while “tryptophan (L-TRP), 5-hydroxytryptophan (5-HTRP), serotonin (5-HT), and N-acetyl-5-hydroxytryptamine (N-acetyl-5-HT)” as its intermediate products in alcohol fermentation (Fernandez-Cruz 1554). Other indolic compounds, such as tryptophol (TOL), L-tryptophan ethyl ester (L-TRP EE), tryptamine (TRYP), and 3-indoleacetic acid (3IAA), originate from L-TRP.
The study used two strains of S. cerevisiae (QA23 and P24) and four strains of non-Saccharomyces (Hanseniaspora uvarum, Starmerella bacillaris Cz4, Metschnikowia pulcherrima Mpp, and Torulaspora delbrueckii Tdp in the fermentation process. Subsequently, yeast cells were grown in an enriched media overnight and sampled for analysis. The two strains of Saccharomyces and two of non-Saccharomyces strains (H. uvarum and T. delbrueckii) were utilized in the indolic synthesis via the fermentation process. The buffered ethanol approach was used to extract indolic metabolites and prepared for analysis by dilution. High-performance liquid chromatography and high-resolution mass spectrometry (HPLC-HRMS) were used in the analysis of samples and STATISTICA software was used in data analysis (Fernandez-Cruz 1555).
Results, Discussion, and Interpretation
The table shows the concentrations of both major and minor indolic compounds produced out of the fermentation process. It also shows the variation in indolic compounds during the initial, middle, and final exponential phases, as well as finalized fermentation stages of both strains of Saccharomyces (QA23 and P24) and non-Saccharomyces (Hu4 and Tdp) (Fernandez-Cruz 1557). As a reactant, the concentration of L-TRP decreased along with the fermentation points of the initial, middle, final, and finalized stages. QA23, P24, and Tdp produced quantifiable levels of 5-HTRP, while Hu4 did not synthesize quantifiable concentrations.
The levels of 5-TH were detectable in Saccharomyces strains but not detectable in non-Saccharomyces strains. N-acetyl-5-HT was not detectable in Saccharomyces strains but detectable in non-Saccharomyces strains. Likewise, MLT was not quantifiable in Saccharomyces but quantifiable in non-Saccharomyces with decreasing concentration along the fermentation stages. Both strains produced 3IAA and TOL with higher concentrations in Saccharomyces than in non-Saccharomyces strains. TRYP and L-TRP-EE were produced in detectable levels by Saccharomyces strains but in undetectable concentrations by non-Saccharomyces strains.
The heap map depicts the effects of L-TRP addition on the production of indolic compounds after 30 minutes on both cultured strains of Saccharomyces and non-Saccharomyces during the fermentation. All strains produced low concentrations of MLT, N-acetyl-5-HT, and L-TRP-EE, but mixed concentrations of 5-HTRP, 3IAA, L-TRP, TOL, TRYP, and 5-HT (Fernandez-Cruz 1557). QA23 strain produced high concentrations of TRYP and L-TRP, moderate concentrations of 3IAA, TOL, and 5-HT, and low concentrations of MLT, N-acetyl-5-HT, L-TRP-EE, and 5-HTRP. Comparatively, the P24 strain produced high concentrations of TOL, L-TRP, and TRYP, moderate concentrations of 3IAA, 5-HTRP, and 5-HT, and low levels of MLT, N-acetyl-5-HT, and L-TRP-EE. MPP strain had high levels of TRYP, 5-HT, moderate concentrations of 5-HTRP, 3IAA, L-TRP, and TRYP, and low levels of MLT, N-acetyl-5-HT, and L-TRP-EE.
Hu4 strain generated moderate levels of 3IAA, L-TRP, and TOL but low levels of 5-HTRP, TRYP, MLT, N-acetyl-5-HT, and L-TRP-EE. Cz strain produced high concentrations of 3IAA, moderate concentrations of 5-HTRP, L-TRP, TOL, and 5-HT, and low concentrations of TRYP, MLT, N-acetyl-5-HT, and L-TRP-EE. The top strain produced high levels of 5-HTRP, 3IAA, L-TRP, and TOL, a moderate concentration of 5-HT, and low levels of MLT, N-acetyl-5-HT, TRYP, and L-TRP-EE.
Bar graphs (A) show percent compositions of indolic compounds produced by different strains of yeasts in the fermentation process. L-TRP and TOL formed the major indolic compounds because they represented over 95% of the fermentation products. In various stages of fermentation, from initial, middle, final, and finalized phases, L-TRP formed larger proportions of indolic compounds than those of TOL.
Being a precursor of the fermentation process, L-TRP decreased in their proportions with time as the concentrations of TOL and other minor products increased. To allow comparisons of minor indolic compounds, major products (L-TRP and TOL) were removed from the overall proportions. Additionally, bar graphs (B) depict the percent distribution of the minor products with 3IAA being dominant while others are L-TRP EE, TRYP, MLT, N-acetyl-5-HT, 5-HT, and 5-HTRP were present at various proportions in phases of the fermentation process.
Principal component analysis indicates explained variance of indolic compounds and yeast strains. While component 1 accounts for 62.06% of the variation in indolic compounds, component 2 explains 13.84% of the variance. Moreover, component 1 has a negative relationship with L-TRP, TOL, TRYP, and 5-HTRP, and a positive relationship with MLT and N-acetyl-5-HT. Component 2 has a positive relationship with 31AA and a negative relationship with L-TRP-EE. The analysis of indolic profiles shows that Saccharomyces and non-Saccharomyces have unique variations (Fernandez-Cruz 1559).
The study demonstrated that both Saccharomyces and non-Saccharomyces can synthesize bioactive indolic compounds. However, the nature of indolic compounds produced via the fermentation process varies according to the strain of yeast and the metabolic state of cells. While some indolic compounds form the major products (L-TRP, 3IAA, and TOL), others constitute the minor products (5-HTRP, 5-HT, N-acetyl-5-HT, MLT, 3IAA, TRYP, TOL, and L-TRP-EE). The percent composition of indolic compounds shows that different strains exhibit unique variations as indicated by principal component analysis.
Fernandez‑Cruz, Edwin, et al. “Intracellular Biosynthesis of Melatonin and Other Indolic Compounds in Saccharomyces and Non-Saccharomyces Wine Yeasts.” European Food Research and Technology, vol. 245, no. 1, 2019, pp. 1553-1560.