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The yeast Lachancea thermotolerans has the ability to produce notable amounts of lactic acid and reduce alcoholic strength in fermentation, so it has a considerable potential for mitigating negative impacts of climate changes in winemaking. In this study, a treatment with L. thermotolerans and Saccharomyces cerevisiae in sequential inoculation was compared to a control S. cerevisiae monoculture fermentation of Malvazija istarska (aka Malvasia Istriana) white grape must. Standard physico-chemical parameters of the obtained wines were determined by the OIV methods. Targeted (GC/FID and GC/MS) and untargeted (GC×GC/TOF-MS) gas chromatographic techniques were combined for the analysis of volatile compounds. Phenolic compounds were analyzed by UPLC/QqQ-MS/MS, and proteins by RP-HPLC-DAD, while a sensory analysis of wines was performed by a panel of trained and certified tasters. L. thermotolerans co-fermentation treatment increased the concentration of lactic acid and decreased alcoholic strength. L. thermotolerans increased the concentrations of geraniol, β-ionone, isobutanol, isobutyric acid, ethyl isobutyrate, several major acetates, ethyl lactate, and diethyl succinate, followed by many minor compounds. This wine also contained more hydroxycinnamoyl tartrates, while control S. cerevisiae wine had higher levels of free hydroxycinnamates. The effects on PR proteins were minor. L. thermotolerans co-fermentation slightly enhanced the sensory perception of tropical fruit, herbaceous, tobacco, and buttery odor notes, as well as fullness of body. With the largest number of identified volatile compounds up to date and other results obtained, this study contributes to the better understanding of oenological and especially aromatic potential of L. thermotolerans in white wine production.

To date, there are no specific guidelines for the use of bioacidifying yeasts in winemaking. In this work, we aimed to characterize an oenological strain of Lachancea thermotolerans (Lt), a non-Saccharomyces lactic acid-producing yeast, and to test different sequential inoculation conditions with Saccharomyces cerevisiae (Sc). The results of bench scale vinifications showed that both the strategy of inoculating Sc 12 h after Lt and the mixing of Lt and Sc during fermentation delivered an acceptable increase in lactic acid (2 g/L) and a decrease in pH (about 0.15 units). Therefore, both strategies were implemented in winery experiments. Our results at the cellar scale showed no increase in acidity, which was likely due to the presence of indigenous yeasts. Overall, our experience shows the difficulty of translating laboratory protocols into cellar experiments and calls for further research into new strategies for implementing acidifying yeasts.

Increasingly high interest in yeast–yeast interactions in mixed-culture fermentation is seen along with beer consumers’ demands driving both market growth and requests for biotechnological solutions that can provide better sensory characteristics. In this study, Lachancea thermotolerans and Saccharomyces cerevisiae with a cell population ratio of 10:1 were inoculated for sour beer fermentation while the process conditions within the brewing industry remained unchanged. With L. thermotolerans producing lactic acid (1.5–1.8 g/L) and bringing down the pH to 3.3–3.4 whilst adding no foreign flavors herein, this study revealed a new natural, fruity sour beer with a soft, sour taste. In this study, the double-yeast mixed-culture fermentation produced more flavor substances than a single-culture process, and plenty of isobutyl acetate and isoamyl acetate enhanced the fruit aroma and balanced the sour beer with a refreshing taste. While playing a positive role in improving the beer’s quality, the double-yeast mixed-culture fermentation developed in this study helps to offer an alternative mass production solution for producing sour beer with the processes better controlled and the fermentation time reduced. The stress responses of the L. thermotolerans during the fermentation were revealed by integrating RNA sequencing (RNA-Seq) and metabolite data. Given that the metabolic flux distribution of the S. cerevisiae during the fermentation differed from that of the non-Saccharomyces yeasts, transcriptional analysis of non-Saccharomyces yeast and S. cerevisiae could be suitable in helping to develop strategies to modulate the transcriptional responses of specific genes that are associated with the aroma compounds released by S. cerevisiae and non-Saccharomyces yeasts. In the case of some non-Saccharomyces yeast species/strains, the diversion of alcoholic fermentation and the formation of a great number of secondary compounds may, in part, account for the low ethanol yield.

One of the most notable effects of climate change, especially in warm regions, is the decrease in acidity (i.e., increase in pH) of wines and a reduction in their aromatic profile. To address this issue, must from a white grape variety with low acidity were inoculated with two non-Saccharomyces yeasts (Lachancea thermotolerans and Torulaspora delbrueckii) to enhance the acidity of the resulting wines. Basic oenological variables and major volatile compounds and polyols of the wines were analyzed, and the results were compared with those obtained through a Saccharomyces cerevisiae strain. Through multiple regression analysis, we found relations between the production of lactic acid to compounds involved in yeast metabolism and redox balance, including glycerol, acetic acid, isobutanol, isoamyl alcohols, and 2-phenylethanol. By means of principal component analysis, we obtained three components that explain more than 89% of the observed variability. The first component differentiates wines produced by L. thermotolerans; the second differentiates wines obtained by S. cerevisiae from those obtained by T. delbrueckii; and the third component is related to the temperature of fermentation. Organoleptic wines produced with S. cerevisiae were the best valuated, but taste was a highlight of the wines produced with L. thermotolerans due to possessing the best acidity.

This work presents the attempt to develop a production technology for grape–plum low-alcohol beverages and enhance their chemical composition and flavor complexity through the non-Saccharomyces species. Saccharomyces cerevisiae (SC) pure cultures were used as reference beverages. Pure cultures of Lachancea thermotolerans (LT) and co-inoculated Lachancea thermotolerans with Saccharomyces cerevisiae (MIX) were included for grape–plum must fermentation at a pilot scale. The process involves two steps: a primary alcoholic fermentation in stainless steel tanks (F1) and a secondary fermentation in a bottle after dextrose syrup addition (F2). The chemical compositions of all beverages obtained in F1 and F2 were studied. Compared to SC, must inoculated with L. thermotolerans (LT and MIX) required four more days to complete the fermentation of sugars during F1. SC fermentation tended to have slightly higher pH and titratable acidity values and lower concentrations of total phenols. Final levels of aromatic precursor nitrogen and sulfur amino nitrogen were obtained more in SC than in LT and MIX. SC treatment had higher final levels of histidine, phenylalanine, isoleucine, lysine, methionine, threonine, valine, and cysteine. Related to individual amino acids, SC treatment had higher final levels of histidine, phenylalanine, isoleucine, lysine, methionine, threonine, valine, and cysteine. Analysis of the volatile composition showed that, compared with SC, MIX had the highest percentage of higher alcohols (3-methyl-1-butanol and 2-methyl-1-butanol) and acetates (isoamyl acetate and isobutyl acetate) which are associated with fruity and banana aromas. A decreasing trend in volatile fatty acids was observed in LT and MIX compared to SC. LT application, both in pure and mixed culture, significantly modified the values of the percentage of 5 of the 10 ethyl ester compounds analyzed. Finally, the sensory analysis showed that there were no significant differences, even though the non-Saccharomyces had a higher percentage of volatile metabolites. The results have shown that through this process an innovative and high-quality product was obtained: a low-alcohol beverage made from grapes and plums, which could be developed at an industrial level due to the increasing interest of consumers in this type of product.

The emerging low acidity in icewine grapes is becoming a major problem in producing quality icewine. Using non-Saccharomyces cerevisiae yeasts in fermentation can improve wine’s organoleptic characteristics and aromatic quality. This study evaluated two indigenous non-Saccharomyces cerevisiae yeasts, Lachancea thermotolerans (LT-2) and Torulaspora delbrueckii (TD-3), for their ability to improve the acidity and quality of ‘Semillon’ icewine. Five different inoculation schemes were implemented, including a single inoculation of S. cerevisiae (SC), L. thermotolerans (LT), and T. delbrueckii (TD); the sequential inoculation of L. thermotolerans, followed by S. cerevisiae after 6 days (L-S); and the sequential inoculation of L. thermotolerans, followed by T. delbrueckii after 6 days (L-D). The results showed that, during sequential fermentation (L-S and L-D), the presence of S. cerevisiae or T. delbrueckii slightly restrained the growth of L. thermotolerans. Single or sequential inoculation with L. thermotolerans and T. delbrueckii significantly reduced the amount of volatile acidity and increased the glycerol content. Furthermore, fermentations involving L. thermotolerans produced relevant amounts of lactic acid (2.04–2.2 g/L) without excessive deacidification of the icewines. Additionally, sequential fermentations increased the concentration of terpenes, C13-norisoprenoid compounds, and phenethyl compounds. A sensory analysis also revealed that sequentially fermented icewines (L-S and L-D) had more fruity and floral odors and aroma intensity. This study highlights the potential application of L. thermotolerans and T. delbrueckii in sequential fermentation to improve the icewine quality.

One of the alternatives to SO2 as an antimicrobial is the use of bioprotection yeasts, which colonize the medium preventing the proliferation of undesirable microorganisms. In this work, the bioprotective effect of a mixed inoculum formed by Torulaspora delbrueckii/Lachancea thermotolerans during fermentation was evaluated. For this purpose, fermentations were carried out using this mixed inoculum and the populations of yeasts, lactic bacteria and acetic bacteria, and the physical–chemical parameters of the wines obtained were studied. The results were compared with those obtained in spontaneous fermentation with and without SO2. The different fermentation strategies caused a differentiation in the yeast species present during fermentation. Regarding populations of lactic acid bacteria, results showed that the effect of the addition of the mixed inoculum was comparable to that exerted by SO2. On the other hand, due to the high sensitivity of acetic acid bacteria to SO2, the sulfite vinifications showed a lower population of acetic acid bacteria in the early stages of fermentation, followed by the vinifications with the mixed inoculum.

Climate change is affecting vineyards, resulting in grapes with a low acidity a high pH and sugar at harvest time. The most common procedure so far to improve the acidity and reduce the final pH of wines is to use tartaric acid, but wine can also be acidified microbiologically using Lachancea thermotolerans yeasts, a natural bio-tool that acidifies gradually during the first stage/days of fermentation. Two strains of L. thermotolerans were compared with one Saccharomyces cerevisiae at a pilot-scale under similar fermentation conditions and in duplicate. A sequential inoculation was performed on the third day for the non-Saccharomyces, producing only about 1 g/L of lactic acid, which was suitable for comparison with the Saccharomyces, to which 1.5 g/L of tartaric acid had been added to lower the final pH. The three fermentations ended with a total acidity without significant differences. A significant and normal feature of the L. thermotolerans yeasts is their higher propane-1,2,3-triol production, which was observed in the Laktia yeast, and the acetic acid was Saccharomyces and the increase was seen in carbonyl compounds, organic acids, lactones, fumaric compounds, and phenols. Finally, the sensory analysis showed that there were hardly any significant differences, even though the non-Saccharomyces had a higher quantity of volatile metabolites, which could lead to a good acceptance of the product, since biological acidification was used, generating a more natural product.

The use of non-Saccharomyces yeasts in sequential fermentations with S. cerevisiae has been proposed to improve the organoleptic characteristics involved in the quality of wine. The present study set out to select a non-Saccharomyces inoculum from the D.O.Ca. Rioja for use in winemaking. Strains included in the study belonged to Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, Zygosaccharomyces bailii, Williopsis pratensis, Debaryomyces hansenii, Pichia kluyveri, Sporidiobolus salmonicolor, Candida spp., Cryptococcus spp. and two mixed inocula of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio. In the first stage of the process, SO2 resistance and presence of enzymatic activities related to wine aroma and wine color and fining (esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, β-glucosidase, pectinase, cellulose, xylanase and glucanase) were studied. In the later stages, selection criteria such as fermentative behavior, aroma compound production or influence on phenolic compounds were studied in laboratory scale vinifications. Taking into account the results obtained in the different stages of the process, a mixed inoculum of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio was finally selected. This inoculum stood out for its high implantation capacity, the production of compounds of interest such as glycerol and lactic acid and the consequent modulation of wine acidity. Given these characteristics, the selected inoculum is suitable for the production of quality wines.

Since the beginning of enology and fermentation research, wine quality has been parametrized from a chemical and sensory point of view [...]

The present study aimed to investigate the oenological changes induced by non-Saccharomyces yeasts in three red grape varieties from the Rioja Qualified Designation of Origin. Pilot plants fermentation of three different varieties, were conducted following early inoculations with Metschnikowia pulcherrima and with mixed inoculum of Lachancea thermotolerans-Torulaspora delbrueckii from La Rioja and compared to a wine inoculated with Saccharomyces cerevisiae. The microbiological and physicochemical characteristics of vinifications were analysed. Results showed that most of the variations due to inoculation strategies were observed in Tempranillo just after the alcoholic fermentation, probably because of the better adaptation of the inocula to the must’s oenological properties. Finally, after the malolactic fermentation the inoculation with the mix of Lachancea thermotolerans and Torulaspora delbrueckii caused more changes in Tempranillo and Grenache wines while the early inoculation with Metschnikowia pulcherrima had more effects on Grenache wines. Therefore, the study was aimed to identify the fermentation effects of each inoculation strategy by using different non-Saccharomyces yeasts and different grape varieties.

In recent years, studies have reported the positive influence of non-Saccharomyces yeast on wine quality. Many grape varieties under mixed or sequential inoculation show an overall positive effect on aroma enhancement. A potential impact by non-Saccharomyces yeast on volatile and non-volatile compounds should benefit the flavor of Riesling wines. Following this trend, four separate sequential fermentations (using the non-Saccharomyces yeasts Torulaspora delbrueckii, Metschnikowia pulcherrima, Pichia kluyveri, and Lachancea thermotolerans with Saccharomyces cerevisiae) were carried out on Riesling must and compared to a pure culture of S. cerevisiae. Sequential fermentations influenced the final wine aroma. Significant differences were found in esters, acetates, higher alcohols, fatty acids, and low volatile sulfur compounds between the different trials. Other parameters, including the production of non-volatile compounds, showed significant differences. This fermentation process not only allows the modulation of wine aroma but also chemical parameters such as glycerol, ethanol, alcohol, acidity, or fermentation by-products. These potential benefits of wine diversity should be beneficial to the wine industry.

In the past, some microbiological studies have considered most non-Saccharomyces species to be undesirable spoilage microorganisms. For several decades, that belief made the Saccharomyces genus the only option considered by winemakers for achieving the best possible wine quality. Nevertheless, in recent decades, some strains of non-Saccharomyces species have been proven to improve the quality of wine. Non-Saccharomyces species can positively influence quality parameters such as aroma, acidity, color, and food safety. These quality improvements allow winemakers to produce innovative and differentiated wines. For that reason, the yeast strains Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, Schizosaccharomyces pombe, and Pichia kluyveri are now available on the market. Other interesting species, such as Starmerella bacillaris, Meyerozyma guilliermondii, Hanseniospora spp., and others, will probably be available in the near future.

In recent years, in line with consumer preferences and due to the effects of global climate change, new trends have emerged in wine fermentation and wine technology. Consumers are looking for wines with less ethanol and fruitier aromas, but also with a good balance in terms of acidity and mouthfeel. Nonconventional yeasts contain a wide range of different genera of non-Saccharomyces. If in the past they were considered spoilage yeasts, now they are used to enhance the aroma profile of wine or to modulate wine composition. Recent publications highlight the role of non-Saccharomyces as selected strains for controlling fermentations mostly in cofermentation with Saccharomyces. In this article, I have reviewed the ability of some bacteria and non-Saccharomyces strains to modulate wine acidity.

Lachancea (kluyveromyces) thermotolerans is a ubiquitous yeast that can be naturally found in grapes but also in other habitats as soil, insects and plants, extensively distributed around the world. In a 3-day culture, it shows spherical to ellipsoidal morphology appearing in single, paired cells or short clusters. It is a teleomorph yeast with 1–4 spherical ascospores and it is characterized by a low production of volatile acidity that helps to control global acetic acid levels in mixed or sequential inoculations with either S. cerevisiae or other non-Saccharomyces species. It has a medium fermentative power, so it must be used in sequential or mixed inoculations with S. cerevisiae to get dry wines. It shows a high production of lactic acid able to affect strongly wine pH, sometimes decreasing wine pH by 0.5 units or more during fermentation. Most of the acidification is produced at the beginning of fermentation facilitating the effect in sequential fermentations because it is more competitive at low alcoholic degree. This application is especially useful in warm areas affected by climatic change. pH reduction is produced in a natural way during fermentation and prevents the addition of tartaric acid, that produces tartrate precipitations, or the use of cation exchangers resins highly efficient reducing pH but with undesirable effects on wine quality. Production of lactic acid is done from sugars thus reducing slightly the alcoholic degree, especially in strains with high production of lactic acid. Also, an improvement in the production of 2-phenylethanol and glycerol has been described.