Evaluation of the effect of Torulaspora delbrueckii on important volatile compounds in navel orange original brandy using E-nose combined with HS-SPME-GC-MS.

Simultaneous inoculation of non-Saccharomyces cerevisiae during the alcoholic fermentation process has been found to be an effective strategy for enhancing wine flavor. This study aimed to investigate the effect of Torulaspora delbrueckii NCUF305.2 on the flavor of navel orange original brandy (NOOB) using E-nose combined with HS-SPME-GC-MS. The results showed a significant increase (p < 0.05) in the sensitivity of NOOB to W5C, W3C, W1S, and W3S sensors by mixed fermentation (MF). Esters in NOOB increased by 4.13%, while higher alcohols increased by 21.93% (p < 0.001), terpenes and others increased by 52.07% and 40.99% (p < 0.01), respectively. Notably, several important volatile compounds with relative odor activity values above 10 showed an increase. Sensory analysis revealed that a more pronounced citrus-like flavor and higher overall appearance scores were found in MF than in pure fermentation (PF). These findings offer valuable theoretical guidance for enhancing the quality of fruit brandies.

Potential Use of Torulaspora delbrueckii As a New Source of Mannoproteins of Oenological Interest.

In this work, three MP extracts obtained from Torulaspora delbrueckii were added to red wine, and the changes in phenolic composition, color, and astringency were evaluated by HPLC-DAD-ESI-MS, tristimulus colorimetry, and sensory analysis, respectively. The MP extracts modified wine phenolic composition differently depending on the type of MP. Moreover, two MP extracts were able to reduce wine astringency. The fact that the MP-treated wines showed an increased flavanol content suggests the formation of MP-flavanol aggregates that remain in solution. Furthermore, the formation of these aggregates may hinder the interaction of flavanols with salivary proteins in the mouth. The effect of these MPs might be associated with their larger size, which could influence their ability to bind flavanols and salivary proteins. However, one of the astringent-modulating MPs also produced a loss of color, highlighting the importance of assessing the overall impact of MPs on the organoleptic properties of wine.

Effects of Torulaspora delbrueckii and Saccharomyces cerevisiae Co-Fermentation on the Physicochemical and Flavor Compounds of Huaniu Apple Cider.

The effects of different fermentation methods utilizing Torulaspora delbrueckii 1004 and Saccharomyces cerevisiae 32169 on the physicochemical properties, organic acid content, polyphenol and flavonoid concentrations, antioxidant activity, and volatile aroma compounds of Huaniu apple cider were investigated in this study. Employing methods of single inoculation, co-inoculation, and sequential inoculation, it was found that sequential fermentation exhibited strong fermentative power in the initial stages, effectively reducing the content of soluble solids and achieving a balanced composition of malic, succinic, and citric acids while maintaining a lower titratable acidity. Sequential inoculation was observed to significantly enhance the total polyphenols and flavonoids, as well as the antioxidant capacity (p < 0.05). Specifically, in the synthesis of volatile aroma compounds, sequential inoculation significantly enhanced the richness and diversity of the Huaniu apple cider's aromas, particularly in terms of the concentration of ester compounds (p < 0.05). Principal component analysis further confirmed the superiority of sequential inoculation in terms of aroma component diversity and richness. The findings of this study suggest that sequential inoculation of fermentation with non-Saccharomyces and S. cerevisiae is an effective strategy for optimizing the flavor characteristics of Huaniu apple cider, offering valuable theoretical support and practical guidance for enhancing cider quality and fostering the development of new products.

Enhancement of ester biosynthesis in blueberry wines through co-fermentation via cell-cell contact between Torulaspora delbrueckii and Saccharomyces cerevisiae.

This study investigated the effects of co-fermentation of T. delbrueckii and S. cerevisiae on the volatile composition and sensory characteristics of blueberry wines. Mixed fermentation led to higher levels of terpenes, higher alcohols, and esters compared to wines fermented with each yeast individually. Conversely, when T. delbrueckii were physically separated from S. cerevisiae in the double-compartment fermenter, contrasting outcomes emerged. The stronger fruity aroma induced by mixed fermentation were linked to higher ester concentrations, including isoamyl acetate, ethyl isovalerate, ethyl hexanoate, and diethyl succinate. The enhanced esters in mixed fermentation can be attributed to the upregulated alcohol acyltransferase activity and the expressions of ACC1, FAS2, ELO1 and ATF1 genes in late fermentation stage via the cell-cell contact between T. delbrueckii and S. cerevisiae. These findings can deepen the understanding of the interaction between non-Saccharomyces and S. cerevisiae in ester production, assisting wineries in effectively controlling wine aroma through mixed fermentations.

A comparative study to investigate the individual contribution of metabolic and physical interaction on volatiles formation in the mixed fermentation of Torulaspora delbrueckii and Saccharomyces cerevisiae.

It is well-known that the co-inoculation of Saccharomyces cerevisiae and non-Saccharomyces strains can modulate and improve the aromatic quality of wine through their multi-level interactions. However, the individual contribution of metabolic interaction (MI) and physical interaction (PI) on wine volatiles remains poorly understood. In this work, we utilized a double-compartment bioreactor to examine the aromatic effect of MI and PI by comparing the volatiles production in Torulaspora delbrueckii and Saccharomyces cerevisiae single fermentations to their mixed fermentations with or without physical separation. Results showed that the PI between T. delbrueckii and S. cerevisiae increased the production of most aroma compounds, especially for acetate esters and volatile fatty acids. In comparison, the MI only promoted a few volatile compounds, including ethyl decanoate, isoamyl acetate, and isobutanol. Noticeably, the MI significantly decreased the levels of ethyl dodecanoate, 2-phenylethyl alcohol, and decanoic acid, which exhibited opposite profiles in PI. Our results indicated that the PI was mainly responsible for the improved volatiles in T. delbrueckii/S. cerevisiae mixed fermentation, while the MI can be targeted to modulate the specific aroma compounds. A thorough understanding of the PI and MI aromatic effect will empower winemakers to accurately and directionally control the volatile profile of the wine, promoting the application of multi-starters to produce diverse styles of wines.

Genome-scale metabolic models reveal determinants of phenotypic differences in non-Saccharomyces yeasts.

BACKGROUND: Use of alternative non-Saccharomyces yeasts in wine and beer brewing has gained more attention the recent years. This is both due to the desire to obtain a wider variety of flavours in the product and to reduce the final alcohol content. Given the metabolic differences between the yeast species, we wanted to account for some of the differences by using in silico models. RESULTS: We created and studied genome-scale metabolic models of five different non-Saccharomyces species using an automated processes. These were: Metschnikowia pulcherrima, Lachancea thermotolerans, Hanseniaspora osmophila, Torulaspora delbrueckii and Kluyveromyces lactis. Using the models, we predicted that M. pulcherrima, when compared to the other species, conducts more respiration and thus produces less fermentation products, a finding which agrees with experimental data. Complex I of the electron transport chain was to be present in M. pulcherrima, but absent in the others. The predicted importance of Complex I was diminished when we incorporated constraints on the amount of enzymatic protein, as this shifts the metabolism towards fermentation. CONCLUSIONS: Our results suggest that Complex I in the electron transport chain is a key differentiator between Metschnikowia pulcherrima and the other yeasts considered. Yet, more annotations and experimental data have the potential to improve model quality in order to increase fidelity and confidence in these results. Further experiments should be conducted to confirm the in vivo effect of Complex I in M. pulcherrima and its respiratory metabolism.

Sequential inoculation of flocculent Torulaspora delbrueckii with Saccharomyces cerevisiae increases color density of Pinot Noir wines.

Pinot noir grapes require careful management in the winery to prevent loss of color density and promote aging stability. Winemaking with flocculent yeast has been shown to increase color density, which is desirable to consumers. This research explored interspecies sequential inoculation and co-flocculation of commercial yeast on Pinot noir wine color. Sedimentation rates of six non-Saccharomyces species and two Saccharomyces cerevisiae strains were assayed individually and in combination. The most flocculent pairings, Torulaspora delbrueckii BIODIVA with S. cerevisiae RC212 or VL3, were used to ferment 20 L Pinot noir must. Sequential fermentations produced wines with greater color density at 420 + 520 nm, confirmed by sensory panel. Total and monomeric anthocyanin concentrations were decreased in sequentially fermented wines, despite being the main source of red wine color. BIODIVA adsorbed more anthocyanins than S. cerevisiae, indicating a greater number of cell wall mannoproteins in flocculent yeast, that could then result in a later release of anthocyanins and enhance copigment formation in red wines.

Effects of Torulaspora delbrueckii co-fermented with Saccharomyces cerevisiae on physicochemical and aromatic profiles of blueberry fermented beverage.

To investigate the effects of mixed fermentation with T. delbrueckii on aroma profiles of blueberry fermented beverage, five fermentations were conducted: monoculture of T. delbrueckii and S. cerevisiae, respectively; co-inoculation of two strains; sequential inoculation of two strains at time intervals of 24 h and 48 h, respectively. Compared with pure S. cerevisiae fermentation, ethanol level was decreased by up to 1.1% vol., while total anthocyanins were increased by 27.7%-85.0% in mixed fermentations. Marker aroma compounds in different fermentations with relative odor activity values higher than 1were identified. T. delbrueckii significantly decreased volatile acid content (especially acetic acid) by 22.2%-83.3%. Ethyl 3-methylbutanoate, ethyl hexanoate and ethyl octanoate, in pure T. delbrueckii fermentation were significantly decreased, while their concentrations were increased by 1.6-4.4 folds in sequential fermentations. Besides, linalool, rose oxide, benzeneacetaldehyde were significantly increased by sequential fermentation, which was associated with the enhancement of fruity and sweet notes.

Importance of micronutrients and organic nitrogen in fermentations with Torulaspora delbrueckii and Saccharomyces cerevisiae.

The current use of non-Saccharomyces yeasts in mixed fermentations increases the relevance of the interactions between yeast species. In this work, the interactions between Saccharomyces cerevisiae and Torulaspora delbrueckii were analyzed. For this purpose, fermentations with and without contact between strains of those yeast species were performed in synthetic must. Fermentation kinetics, yeast growth and dynamics were measured over time. Additionally, the effects of nitrogen and other nutrient supplementations on the mixed fermentations were determined. Our results showed that S. cerevisiae did not always dominate the sequential fermentations, and experiments without yeast contact (in which T. delbrueckii cells were removed from the medium before inoculating S. cerevisiae at 48 h) resulted in stuck fermentations except when the inoculum size was increased (from 2 × 106 to 108 cells/mL) or there was a supplementation of thiamine, zinc and amino acids at the same concentration as initially found in the synthetic must. Our findings highlight the importance of inoculum size and ensuring the availability of enough micronutrients for all yeast species, especially in sequential fermentations.

The DNA replication protein Orc1 from the yeast Torulaspora delbrueckii is required for heterochromatin formation but not as a silencer-binding protein.

To understand the process by which new protein functions emerge, we examined how the yeast heterochromatin protein Sir3 arose through gene duplication from the conserved DNA replication protein Orc1. Orc1 is a subunit of the origin recognition complex (ORC), which marks origins of DNA replication. In Saccharomyces cerevisiae, Orc1 also promotes heterochromatin assembly by recruiting the structural proteins Sir1-4 to silencer DNA. In contrast, the paralog of Orc1, Sir3, is a nucleosome-binding protein that spreads across heterochromatic loci in conjunction with other Sir proteins. We previously found that a nonduplicated Orc1 from the yeast Kluyveromyces lactis behaved like ScSir3 but did not have a silencer-binding function like ScOrc1. Moreover, K. lactis lacks Sir1, the protein that interacts directly with ScOrc1 at the silencer. Here, we examined whether the emergence of Sir1 coincided with Orc1 acting as a silencer-binding protein. In the nonduplicated species Torulaspora delbrueckii, which has an ortholog of Sir1 (TdKos3), we found that TdOrc1 spreads across heterochromatic loci independently of ORC, as ScSir3 and KlOrc1 do. This spreading is dependent on the nucleosome binding BAH domain of Orc1 and on Sir2 and Kos3. However, TdOrc1 does not have a silencer-binding function: T. delbrueckii silencers do not require ORC-binding sites to function, and Orc1 and Kos3 do not appear to interact. Instead, Orc1 and Kos3 both spread across heterochromatic loci with other Sir proteins. Thus, Orc1 and Sir1/Kos3 originally had different roles in heterochromatin formation than they do now in S. cerevisiae.

Microencapsulation by spray drying of coffee epiphytic yeasts Saccharomyces cerevisiae CCMA 0543 and Torulaspora delbrueckii CCMA 0684.

The objective of this work was to evaluate the microencapsulation feasibility of Saccharomyces cerevisiae CCMA 0543 and Torulaspora delbrueckii CCMA 0684 in three different compositions of wall material by spray-dryer. The yeasts (109 CFU mL-1) were microencapsulated separately using maltodextrin (15%), maltodextrin (15%) with sucrose (2%), or maltose (2%) as wall material. The viability was evaluated for 6 months at two different temperatures (7 and 25 °C). The yield, cell viability after spray drying, and characterization of the microcapsules were performed. Results indicate that cell viability ranged between 94.06 and 97.97%. After 6 months, both yeasts stored at 7 °C and 25 °C presented 107 and 102 CFU mL-1, respectively. Regarding Fourier-transform infrared spectroscopy analysis, all microencapsulated yeasts presented typical spectra footprints of maltodextrin. After 6 months of storage, S. cerevisiae CCMA 0543 obtained a 10.8% increase in cell viability using maltodextrin with maltose as wall material compared to maltodextrin and maltodextrin with sucrose. However, T. delbrueckii CCMA 0684 obtained a 13.5% increase in cell viability using only maltodextrin. The study showed that maltodextrin as a wall material was efficient in the microencapsulation of yeasts. It is possible to assume that maltose incorporation increased the cell viability of S. cerevisiae CCMA 0543 during storage.

Identification of genes related to hydrolysis and assimilation of Agave fructans in Candida apicola NRRL Y-50540 and Torulaspora delbrueckii NRRL Y-50541 by denovo transcriptome analysis.

Fructans are the main sugar in agave pine used by yeasts during mezcal fermentation processes, from which Candida apicola NRRL Y-50540 and Torulaspora delbrueckii NRRL Y-50541 were isolated. De novo transcriptome analysis was carried out to identify genes involved in the hydrolysis and assimilation of Agave fructans (AF). We identified a transcript annotated as SUC2, which is related to ß-fructofuranosidase activity, and several differential expressed genes involved in the transcriptional regulation of SUC2 such as: MIG1, MTH1, SNF1, SNF5, REG1, SSN6, SIP1, SIP2, SIP5, GPR1, RAS2, and PKA. Some of these genes were specifically expressed in some of the yeasts according to their fructans assimilation metabolism. Different hexose transporters that could be related to the assimilation of fructose and glucose were found in both the transcriptomes. Our findings provide a better understanding of AF assimilation in these yeasts and provide resources for further metabolic engineering and biotechnology applications.

Co-inoculation of yeasts starters: A strategy to improve quality of low altitude Arabica coffee.

The study aimed to improve the quality of dry-processed coffee grown at low altitudes through yeast inoculation, using three species (Saccharomyces cerevisiae CCMA 0543, Torulaspora delbrueckii CCMA 0684, and Candida parapsilosis CCMA 0544) singly and with co-inoculation for fermentation. Important chemical compounds and groups were analyzed by liquid and gas chromatography and Fourier-transform infrared spectroscopy (FTIR). The inoculated coffees with yeast populations around 106 cell/g obtained the highest scores, and the co-inoculation with C. parapsilosis CCMA 0544 and T. delbrueckii CCMA 0684 had the highest score in the sensory analysis (85). Different descriptors were observed in each treatment, and body, flavor, balance, and aftertaste are strongly related to C. parapsilosis CCMA 0544. The fermentation process improved the quality of low-altitude coffees, and the combination of non-Saccharomyces yeasts (C. parapsilosis CCMA 0544 and T. delbrueckii CCMA 0684) was the most indicated as starter cultures.

Co-culture fermentation of Pediococcus acidilactici XZ31 and yeast for enhanced degradation of wheat allergens.

Previous researchers have shown the potential of sourdough and isolated lactic acid bacteria in reducing wheat allergens. As the interactions of lactic acid bacteria with yeast is a key event in sourdough fermentation, we wished to investigate how yeast affects metabolism of lactic acid bacteria, thereby affecting protein degradation and antigenic response. In this study, three strains isolated from sourdough were selected for dough fermentation, namely Pediococcus acidilactici XZ31, Saccharomyces cerevisiae JM1 and Torulaspora delbrueckii JM4. The changes in dough protein during the fermentation process were studied. Protein degradation and antigenic response in dough inoculated with Pediococcus acidilactici XZ31 monoculture and co-culture with yeast were mainly evaluated by SDS-PAGE, immunoblotting, ELISA and Liquid chromatography-tandem mass spectrometry assay. The whole-genome transcriptomic changes in Pediococcus acidilactici XZ31 were also investigated by RNA sequencing. The results showed that water/salt soluble protein and Tri a 28/19 allergens content significantly decreased after 24 h fermentation. Co-culture fermentation accelerated the degradation of protein, and reduced the allergen content to a greater extent. RNA-sequencing analysis further demonstrated that the presence of yeast could promote protein metabolism in Pediococcus acidilactici XZ31 for a certain period of time. These results revealed a synergistic effect between Pediococcus acidilactici XZ31 and yeast degrading wheat allergens, and suggested the potential use of the multi-strain leavening agent for producing hypoallergenic wheat products.

Impact of Sequential Inoculation with the Non-Saccharomyces T. delbrueckii and M. pulcherrima Combined with Saccharomyces cerevisiae Strains on Chemicals and Sensory Profile of Rosé Wines.

Controlled inoculations of non-Saccharomyces yeasts are becoming increasingly used to produce high-quality wines due to their enological potential. In this study, we evaluated the impact of sequential inoculation with the commercial non-Saccharomyces yeasts (Torulaspora delbrueckii and Metschnikowia pulcherrima) in combination with Saccharomyces cerevisiae on the chemical and sensory profile of rosé wines. Sequential inoculation with T. delbrueckii produced wines with an overall reduction in esters, mainly explained by the lower concentrations of ethyl esters of medium-chain fatty acids and isoamyl acetate. The lower ester concentrations of these wines were related to a reduction in fruity descriptors. An increase was observed, however, in other minor esters such as cinnamates and ethyl esters of branched acids. Zinc, ethyl isobutyrate, and ethyl dihydrocinnamate were selected as potential markers for this fermentation strategy. Sequential inoculation with M. pulcherrima resulted in rosé wines with an enhanced ester profile, reduced acetaldehyde, and increased anthocyans and tannins. Compared to the control wines fermented with S. cerevisiae, the changes observed in these wines were far subtler, especially for the volatile profile, sensory characteristics, and color parameters, with isobutyl hexanoate and isoamyl butyrate being selected as potential markers.

Influence of fermentation conditions on the secretion of seripauperin 5 (PAU5) by industrial sparkling wine strains of Saccharomyces cerevisiae.

The yeast mannoprotein seripauperin 5 (PAU5) from Saccharomyces cerevisiae is a negative gushing biomarker in sparkling wine with a direct gushing-reducing effect. The knowledge about the influence of the yeast strain and the fermentation conditions on the PAU5 content in the final product could reduce the gushing potential of sparkling wines and avoid economic losses for sparkling wine producers. The potential of 30 different commercially used (sparkling) wine S. cerevisiae strains to produce PAU5 was analyzed by RP-HPLC. The experiments revealed great differences between strains and identified three high-PAU5-producing yeast strains. The influence of different fermentation conditions was analyzed in two selected strains. Cultivation conditions of elevated temperature and lower inoculation density, as well as cultivation under diffuse daylight significantly enhanced PAU5 production by the tested S. cerevisiae strains as compared to standard conditions. However, cultivation as agitated cultures and co-cultivation with Metchnikowia pulcherrima or Torulaspora delbrueckii led to a reduction of PAU5 production as compared to standard conditions.

Impact of changes in wine composition produced by non-Saccharomyces on malolactic fermentation.

Non-Saccharomyces yeasts have increasingly been used in vinification recently. This is particularly true of Torulaspora delbrueckii and Metschnikowia pulcherrima, which are inoculated before S. cerevisiae, to complete a sequential alcoholic fermentation. This paper aims to study the effects of these two non-Saccharomyces yeasts on malolactic fermentation (MLF) carried out by two strains of Oenococcus oeni, under cellar conditions. Oenological parameters, and volatile and phenolic compounds were analysed in wines. The wines were tasted, and the microorganisms identified. In general, non-Saccharomyces created more MLF friendly conditions, largely because of lower concentrations of SO2 and medium chain fatty acids. The most favourable results were observed in wines inoculated with T. delbrueckii, that seemed to promote the development of O. oeni and improve MLF performance.

Giant GAL gene clusters for the melibiose-galactose pathway in Torulaspora.

In many yeast species, the three genes at the centre of the galactose catabolism pathway, GAL1, GAL10 and GAL7, are neighbours in the genome and form a metabolic gene cluster. We report here that some yeast strains in the genus Torulaspora have much larger GAL clusters that include genes for melibiase (MEL1), galactose permease (GAL2), glucose transporter (HGT1), phosphoglucomutase (PGM1) and the transcription factor GAL4, in addition to GAL1, GAL10, and GAL7. Together, these eight genes encode almost all the steps in the pathway for catabolism of extracellular melibiose (a disaccharide of galactose and glucose). We show that a progenitor 5-gene cluster containing GAL 7-1-10-4-2 was likely present in the common ancestor of Torulaspora and Zygotorulaspora. It added PGM1 and MEL1 in the ancestor of most Torulaspora species. It underwent further expansion in the T. pretoriensis clade, involving the fusion of three progenitor clusters in tandem and the gain of HGT1. These giant GAL clusters are highly polymorphic in structure, and subject to horizontal transfers, pseudogenization and gene losses. We identify recent horizontal transfers of complete GAL clusters from T. franciscae into one strain of T. delbrueckii, and from a relative of T. maleeae into one strain of T. globosa. The variability and dynamic evolution of GAL clusters in Torulaspora indicates that there is strong natural selection on the GAL pathway in this genus.

Fermentation characteristics of four non-Saccharomyces yeasts in green tea slurry.

The fermentation characteristics of non-Saccharomyces yeasts (Pichia kluyveri FrootZen, Torulaspora delbrueckii Prelude, Williopsis saturnus var. mrakii NCYC2251 and Torulaspora delbrueckii Biodiva) were evaluated in green tea slurry fermentation. Each yeast showed different fermentation performances: strains Prelude and Biodiva utilized sucrose faster than the other two yeasts; strain NCYC2251 was the only species that metabolized xylose. Strain FrootZen increased the caffeine content significantly and strain Prelude showed the opposite trend, both at a statistical level, while theanine contents in four samples were relatively stable. Biodiva and FrootZen significantly improved polyphenols content and the oxygen radical absorbance capacity of fermented teas. Some endogenous volatiles such as ketones, lactones and aldehydes decreased to lower or undetected levels, but one of the key tea aroma compounds methyl salicylate increased by 34-fold and 100-fold in P. kluyveri and W. saturnus samples respectively. Therefore, green tea fermentation by appropriate non-Saccharomyces yeasts can enhance its antioxidant capacity and alter the aroma compound profile.

Understanding the interaction of isoleucine paired with other amino acids in soy whey alcohol fermentation using Torulaspora delbrueckii.

Soy (tofu) whey is a liquid by-product generated from tofu (soybean curd) production and it is often discarded off as a waste liquid by the tofu manufacturers. Previous studies have demonstrated that soy whey can be biotransformed into a soy alcoholic beverage by using Saccharomyces and non-Saccharomyces yeasts even though soy whey is low in yeast assimilable nitrogen (YAN) content. In this study, the initial YAN of the soy whey was estimated to be 46.6 mg N/L and Torulaspora delbrueckii Biodiva was used to ferment soy whey supplemented with either isoleucine only or isoleucine paired with valine, leucine or phenylalanine (each amino acid supplemented at a dosage of 30 mg N/L). Amino acid supplementation was found to enhance sugar utilization by the yeast, which led to higher ethanol production (7.49% v/v in control versus 8.35-8.80% v/v in supplemented samples). Samples supplemented with isoleucine only experienced slower sugar utilization during the fermentation as compared to the paired amino acid samples, but the yeast was still able to utilize the sugar to low levels at the end of the fermentation. The presence of leucine supplementation counteracted the "inhibition" induced by the presence of isoleucine at the first day of the fermentation. Amino acid supplementation slowed down glutamic acid utilization and resulted in higher levels of residual glutamic acid and alanine. Amino acid supplementation increased the corresponding fusel alcohol production and the presence of other amino acids reduced the active amyl alcohol production. Therefore, interactions between amino acids can impact the metabolism of the yeast as well as the flavor modulation during soy whey fermentation.