The Influence of Chitosan on the Chemical Composition of Wines Fermented with Lachancea thermotolerans.

Chitosan exerts a significant influence on various chemical parameters affecting the quality of wine produced using multiple strains of Lachancea thermotolerans. The impact of chitosan on these parameters varies depending on the specific strain studied. We observed that, under the influence of chitosan, the fermentation kinetics accelerated for all examined strains. The formation of lactic acid increased by 41% to 97% across the studied L. thermotolerans strains, depending on the specific strain. This effect also influenced acidity-related parameters such as total acidity, which increased by 28% to 60%, and pH, which experienced a decrease of over 0.5 units. The consumption of malic acid increased by 9% to 20% depending on the specific strain of L. thermotolerans. Nitrogen consumption also rose, as evidenced by all L. thermotolerans strains exhibiting a residual value of Primary Amino Nitrogen (PAN) of below the detection limit, and ammonia consumption increased by 90% to 100%, depending on the strain studied. However, certain parameters such as acetic acid, succinic acid, and glycerol showed contradictory results depending on the strain under investigation. In terms of volatile composition, chitosan supplementation led to increased production of i-butanol by 32% to 65%, 3-methylbutanol by 33% to 63%, and lactic acid ethyl ester by 58% to 91% across all studied strains of L. thermotolerans. Other analyzed aroma compounds exhibited varying changes depending on the specific strain of L. thermotolerans.

A comparative study of Lachancea thermotolerans fermentative performance under standardized wine production conditions.

The study explores diverse strains of Lachancea thermotolerans in single-inoculum wine fermentation conditions using synthetic grape must. It aims to analyze the role of the species without external influences like other microorganisms or natural grape must variability. Commercial strains and selected vineyard isolates, untested together previously, are assessed. The research evaluates volatile and non-volatile chemical compounds in final wine, revealing significant strain-based variations. L. thermotolerans notably produces lactic acid and consumes malic acid, exhibiting moderate ethanol levels. The volatile profile displays strain-specific impacts, affecting higher alcohol and ester concentrations compared to S. cerevisiae. These effects vary based on the specific compounds. Using a uniform synthetic must enables direct strain comparisons, eliminating grape-related, environmental, or timing variables in the experiment, facilitating clearer insights into the behavior of L. thermotolerans in wine fermentation. The study compares for the first time all available commercial strains of L. thermotolerans.

Microsatellite typing of Lachancea thermotolerans for wine fermentation monitoring.

Climate change is causing a lack of acidity during winemaking and oenologists use several solutions to cope with such a problem. Lachancea thermotolerans, which has the potential to tolerate the harsh physicochemical conditions of wine, has emerged as a promising alternative for pH management during winemaking and, currently, it is the most valuable yeast used for acidity control in wine. In this work a manageable method for L. thermotolerans genotyping based on a multiplexed microsatellite amplification in 6 different loci was developed. The proposed method was used to distinguish between 103 collection strains obtained from different geographical and isolation sources, and then challenged against a 429 L. thermotolerans isolates from several wineries and harvests. The procedure was also tested for fermentation monitoring and strain implantation. This approach was conceived to simplify the methodology available for L. thermotolerans genotyping, making it easy for applying in wine-related laboratories. This method can be applied to distinguish between L. thermotolerans strains in selection programs and to follow implantation of inoculated strains during winemaking with optimal results.

Impact of rare yeasts in Saccharomyces cerevisiae wine fermentation performance: Population prevalence and growth phenotype of Cyberlindnera fabianii, Kazachstania unispora, and Naganishia globosa.

Saccharomyces cerevisiae is a highly fermentative species able to complete the wine fermentation. However, the interaction with other non-Saccharomyces yeasts can determine the fermentation performance of S. cerevisiae. We have characterised three rare non-Saccharomyces yeasts (Cyberlindnera fabianii, Kazachstania unispora and Naganishia globosa), studying their impact on S. cerevisiae fitness and wine fermentation performance. Using a wide meta-taxonomic dataset of wine samples, analysed through ITS amplicon sequencing, we show that about a 65.07% of wine samples contains Naganishia spp., a 27.21% contains Kazachstania spp., and only a 4.41% contains Cyberlindnera spp; in all cases with average relative abundances lower than 1% of total fungal populations. Although the studied N. globosa strain showed a limited growth capacity in wine, both K. unispora and C. fabianii showed a similar growth phenotype to that of S. cerevisiae in different fermentation conditions, highlighting the outstanding growth rate values of K. unispora. In mixed fermentations with S. cerevisiae, the three yeast species affected co-culture growth parameters and wine chemical profile (volatile compounds, polysaccharides and proteins). K. unispora DN201 strain presents an outstanding capacity to compete with S. cerevisiae strains during the first stage of wine fermentation, causing stuck fermentations in both synthetic and natural grape musts.

Influence of different Lachancea thermotolerans strains in the wine profile in the era of climate challenge.

The study performed sequential fermentations of red grape juice using several strains of Lachancea thermotolerans and one strain of Saccharomyces cerevisiae. Due to the new conditions imposed by climate change, wine acidity must be affected as well as the volatile profile. Non-Saccharomyces yeasts such as L. thermotolerans are real alternatives to soften the impact of climate change in winemaking. The L. thermotolerans strains included three commercially available strains and two wine-related natural isolates. L. thermotolerans showed significant statistical differences in basic chemical parameters such as lactic acid, malic acid, or ethanol concentrations as well as in the volatile profile. S. cerevisiae clearly produced some volatile compounds in higher amounts than the studied L. thermotolerans strains while others showed the opposite effect. Sequential fermentations involving any of the studied strains of L. thermotolerans with S. cerevisiae showed an increased volatile profile compared to the S. ceresisiae single fermentation, highlighting the synergic effect between the studied species.

Expectable diversity patterns in wine yeast communities.

Wine fermentations are dominated by Saccharomyces yeast. However, dozens of non-Saccharomyces yeast genera can be found in grape musts and in the early and intermediate stages of wine fermentation, where they co-exist with S. cerevisiae. The diversity of non-Saccharomyces species is determinant for the sensorial attributes of the resulting wines, both directly (by producing aroma impact compounds) and indirectly (modulating the performance of Saccharomyces). Many research groups worldwide are exploring the great diversity of wine yeasts to exploit their metabolic potential to improve wine flavor or to prevent wine spoilage. In this work, we share a new data set from a wide ITS amplicon survey of 272 wine samples, and we perform a preliminary exploration to build a catalogue of 242 fungal and yeast genera detectable in wine samples, estimating global figures of their prevalence and relative abundance patterns across wine samples. Thus, our mycobiome survey provides a broad measure of the yeast diversity potentially found in wine fermentations; we hope that the wine yeast research community finds it useful, and we also want to encourage further discussion on the advantages and limitations that meta-taxonomic studies may have in wine research and industry.

Biological management of acidity in wine industry: A review.

Climate change is generating several problems in wine technology. One of the main ones is lack of acidity and difficulties performing malolactic fermentation to stabilize wines before bottling. Among the different available acidity management technologies, such as direct acid addition, ion exchange resins, electro-membrane treatments, or vineyard management, the microbiological option is reliable and deeply studied. The main approach is the increase in malic acid content because of the metabolism of specific Saccharomyces strains and to increase lactic acid because of the metabolism of Lachancea genus. Other non-Saccharomyces yeasts, such as Starmerella bacillaris or Candida stellata can also acidify significantly because of the production of pyruvic or succinic acid. Wine industry needs the removal of malic acid in most red wines before bottling to achieve wine stability. Oenococus oeni performs the malolactic fermentation of red wines on most conditions because of the metabolization of malic acid into lactic acid. However, modern oenology challenges such as high ethanol concentrations, high pH or low levels of malic acid have made researchers to look for other options to reduce potential risks of deviation. Other wine-related microorganisms able to de-acidify malic acid have appeared as interesting alternatives for specific difficult scenarios. Lactiplantibacillus plantarum and Schizosaccharomyces genus make up nowadays the main studied alternatives.

Niche differentiation drives microbial community assembly and succession in full-scale activated sludge bioreactors.

Network models and community phylogenetic analyses are applied to assess the composition, structure, and ecological assembly mechanisms of microbial communities. Here we combine both approaches to investigate the temporal dynamics of network properties in individual samples of two activated sludge systems at different adaptation stages. At initial assembly stages, we observed microbial communities adapting to activated sludge, with an increase in network modularity and co-exclusion proportion, and a decrease in network clustering, here interpreted as a consequence of niche specialization. The selective pressure of deterministic factors at wastewater treatment plants produces this trend and maintains the structure of highly functional and specialized communities responding to seasonal environmental changes.

An Integrative View of the Role of Lachancea thermotolerans in Wine Technology.

The interest in Lachancea thermotolerans, a yeast species with unusual characteristics, has notably increased in all ecological, evolutionary, and industrial aspects. One of the key characteristics of L. thermotolerans is the production of high quantities of lactic acid compared to other yeast species. Its evolution has mainly been driven by the influence of the environment and domestication, allowing several metabolic traits to arise. The molecular regulation of the fermentative process in L. thermotolerans shows interesting routes that play a complementary or protective role against fermentative stresses. One route that is activated under this condition is involved in the production of lactic acid, presenting a complete system for its production, showing the involvement of several enzymes and transporters. In winemaking, the use of L. thermotolerans is nowadays mostly focused in early-medium-maturity grape varieties, in which over-ripening can produce wines lacking acidity and with high concentrations of ethanol. Recent studies have reported new positive influences on quality apart from lactic acid acidification, such as improvements in color, glutathione production, aroma, malic acid, polysaccharides, or specific enzymatic activities that constitute interesting new criteria for selecting better strains. This positive influence on winemaking has increased the availability of commercial strains during recent years, allowing comparisons among some of those products. Initially, the management of L. thermotolerans was thought to be combined with Saccaharomyces cerevisiae to properly end alcoholic fermentation, but new studies are innovating and reporting combinations with other key enological microorganisms such as Schizosaccharomyces pombe, Oenocous oeni, Lactiplantibacillus plantarum, or other non-Saccharomyces.

Microbial Communities Present in Hydrothermal Sediments from Deception Island, Antarctica.

Deception Island is a geothermal location in Antarctica that presents active fumaroles, which confers unique characteristics to this habitat. Several studies about microbial communities in Antarctica have been carried out, nevertheless, Antarctic microbiota is still partially unknown. Here we present a multidisciplinary study about sediments obtained by deposition during 4 years in which several approaches have been considered for their characterization. First, a physicochemical characterization, using ionic chromatography and mass spectrometry for the determination of most abundant ions (chloride and sulphate) and elements (mainly silicon), was conducted. In addition, the total microbial community was studied using a metataxonomical approach, revealing a bacterial community dominated by Proteobacteria and Thaumarchaeota as the main archaeal genera and a fungal community mainly composed by Aspergillaceae. Culture-dependent studies showed low microbial diversity, only achieving the isolation of Bacillus-related species, some of them thermophilic, and the isolation of common fungi of Aspergillus or Penicillium spp. Furthermore, diatoms were detected in the sediment and characterized attending to their morphological characteristics using scanning electron microscopy. The study reveals a high influence of the physicochemical conditions in the microbial populations and their distribution, offering valuable data on the interaction between the island and water microbiota.

High Potential of Pichia kluyveri and Other Pichia Species in Wine Technology.

The surfaces of grapes are covered by different yeast species that are important in the first stages of the fermentation process. In recent years, non-Saccharomyces yeasts such as Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, and Pichia kluyveri have become popular with regard to winemaking and improved wine quality. For that reason, several manufacturers started to offer commercially available strains of these non-Saccharomyces species. P. kluyveri stands out, mainly due to its contribution to wine aroma, glycerol, ethanol yield, and killer factor. The metabolism of the yeast allows it to increase volatile molecules such as esters and varietal thiols (aroma-active compounds), which increase the quality of specific varietal wines or neutral ones. It is considered a low- or non-fermentative yeast, so subsequent inoculation of a more fermentative yeast such as Saccharomyces cerevisiae is indispensable to achieve a proper fermented alcohol. The impact of P. kluyveri is not limited to the grape wine industry; it has also been successfully employed in beer, cider, durian, and tequila fermentation, among others, acting as a promising tool in those fermentation processes. Although no Pichia species other than P. kluyveri is available in the regular market, several recent scientific studies show interesting improvements in some wine quality parameters such as aroma, polysaccharides, acid management, and color stability. This could motivate yeast manufacturers to develop products based on those species in the near future.

The Genus Metschnikowia in Enology.

Over the last decade, several non-Saccharomyces species have been used as an alternative yeast for producing wines with sensorial properties that are distinctive in comparison to those produced using only Saccharomyces cerevisiae as the classical inoculum. Among the non-Saccharomyces wine yeasts, Metschnikowia is one of the most investigated genera due to its widespread occurrence and its impact in winemaking, and it has been found in grapevine phyllospheres, fruit flies, grapes, and wine fermentations as being part of the resident microbiota of wineries and wine-making equipment. The versatility that allows some Metschnikowia species to be used for winemaking relies on an ability to grow in combination with other yeast species, such as S. cerevisiae, during the first stages of wine fermentation, thereby modulating the synthesis of secondary metabolites during fermentation in order to improve the sensory profile of the wine. Metschnikowia exerts a moderate fermentation power, some interesting enzymatic activities involving aromatic and color precursors, and potential antimicrobial activity against spoilage yeasts and fungi, resulting in this yeast being considered an interesting tool for use in the improvement of wine quality. The abovementioned properties have mostly been determined from studies on Metschnikowia pulcherrima wine strains. However, M. fructicola and M. viticola have also recently been studied for winemaking purposes.

Tuning up microbiome analysis to monitor WWTPs' biological reactors functioning.

Wastewater treatment plants (WWTPs) are necessary to protect ecosystems quality and human health. Their function relies on the degradation of organic matter and nutrients from a water influent, prior to the effluent release into the environment. In this work we studied the bacterial community dynamics of a municipal WWTP with a membrane bioreactor through 16S rRNA gene sequencing. The main phyla identified in the wastewater were Proteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Actinobacteria. The WWTP is located in Spain and, like other studied WWTP in temperate climate zones, the temperature played a major role in community assembly. Seasonal community succession is observed along the two years sampling period, in addition to a continual annual drift in the microbial populations. The core community of the WWTP bioreactor was also studied, where a small fraction of sequence variants constituted a large fraction of the total abundance. This core microbiome stability along the sampling period and the likewise dissimilarity patterns along the temperature gradient makes this feature a good candidate for a new process control in WWTPs.

Effects on varietal aromas during wine making: a review of the impact of varietal aromas on the flavor of wine.

Although there are many chemical compounds present in wines, only a few of these compounds contribute to the sensory perception of wine flavor. This review focuses on the knowledge regarding varietal aroma compounds, which are among the compounds that are the greatest contributors to the overall aroma. These aroma compounds are found in grapes in the form of nonodorant precursors that, due to the metabolic activity of yeasts during fermentation, are transformed to aromas that are of great relevance in the sensory perception of wines. Due to the multiple interactions of varietal aromas with other types of aromas and other nonodorant components of the complex wine matrix, knowledge regarding the varietal aroma composition alone cannot adequately explain the contribution of these compounds to the overall wine flavor. These interactions and the associated effects on aroma volatility are currently being investigated. This review also provides an overview of recent developments in analytical techniques for varietal aroma identification, including methods used to identify the precursor compounds of varietal aromas, which are the greatest contributors to the overall aroma after the aforementioned yeast-mediated odor release.

Occurrence and enological properties of two new non-conventional yeasts (Nakazawaea ishiwadae and Lodderomyces elongisporus) in wine fermentations.

The microbial diversity of wine alcoholic fermentation is not restricted to the presence and activity of Saccharomyces yeast strains. Some non-Saccharomyces species have been described as part of the fermentative microbiota, specially found in the initial steps of wine fermentations. These species may play roles from wine spoilage to flavor quality enhancement. From a large number of wine fermentations (429 wine samples), analyzed by ITS-amplicon sequencing to define their mycobiome, 2 non-conventional yeast species (Nakazawaea ishiwadae and Lodderomyces elongisporus) were detected, in a very limited number of samples but in significant levels of relative abundance. One strain of each species was isolated and their technological and enological potential have been characterized in this work. Compared with the Saccharomyces cerevisiae Viniferm Revelacion wine strain, the studied N. ishiwadae BMK17.1 and L. elongisporus BMK12.5 strains showed, as expected, a lower tolerance and growth fitness in high ethanol concentrations. However, N. ishiwadae BMK17.1 was able to grow also at 15% ethanol and L. elongisporus BMK12.5 at 10% reaching, in the latter case, slightly higher efficiency rates than S. cerevisiae at this level. Contrary to most non-Saccharomyces yeasts, these species were able to growth in presence of high doses of potassium-metabisulfite, reaching in both cases higher efficiency rates than S. cerevisiae. A notable affinity of L. elongisporus BMK12.5 for high pH values was clearly observed. Their fermentation kinetics and the final chemical-analytical characterization were studied in micro-fermentation assays, using synthetic grape must. L. elongisporus BMK12.5 was able to complete, in single inoculation, the sugar fermentation after 19 days, but, N. ishiwadae BMK17.1 left about 80 g/L sugars at this time. Co-inoculation assays (in a 1:100 proportion of S. cerevisiae:non-Saccharomyces strains) finished sugar consumption with similar kinetics than the S. cerevisiae single inoculation, in the case of L. elongisporus BMK12.5 co-inoculation, and with lower kinetics when using N. ishiwadae BMK17.1. A remarkable malic acid consumption and a low acetic acid production was associated with L. elongisporus BMK12.5 fermentations, together with a high production of 3-methyl-1-butanol and 2-phenylethanol, and the release of high amounts of proteins into the wines. N. ishiwadae BMK17.1, although unable to finish the fermentation itself, showed a high production of oligosaccharides and volatile compounds such as isobutanol or isobutyric acid. This work reports, for the first time, the occurrence and enological potential of two strains pertaining to the non-conventional yeast genera Lodderomyces and Nakazawaea.

Analytical impact of Metschnikowia pulcherrima in the volatile profile of Verdejo white wines.

Most wine aroma compounds, including the varietal fraction, are produced or released during wine production and derived from microbial activity. Varietal aromas, typically defined as terpenes and thiols, have been described as derived from their non-volatile precursors, released during wine fermentation by different yeast hydrolytic enzymes. The perception of these minority aroma compounds depends on the chemical matrix of the wine, especially on the presence of majority aroma compounds, such as esters or higher alcohols. Strategies aiming to reduce the production of these masking flavors are on the spotlight of enology research as a way to better define varietal standard profiles for the global market. Using a natural white must from Verdejo variety (defined as a thiol grape variety), here we describe the analytical and sensorial impact of using, in sequential inoculations, a selected strain of Metschnikowia pulcherrima, in combination with two different Saccharomyces cerevisiae strains. An increase in the levels of the thiol 4-MSP (4-methyl-4-sulfanylpentan-2-one) over its sensory threshold, together with a decrease in higher alcohol production, was observed when M. pulcherrima was used. This has an important impact on these wines, making them fruitier and fresher, always preferred by the sensory panel.

Quorum Sensing versus Quenching Bacterial Isolates Obtained from MBR Plants Treating Leachates from Municipal Solid Waste.

Quorum sensing (QS) is a mechanism dependent on bacterial density. This coordinated process is mediated by the synthesis and the secretion of signal molecules, called autoinducers (AIs). N-acyl-homoserine lactones (AHLs) are the most common AIs that are used by Gram-negative bacteria and are involved in biofilm formation. Quorum Quenching (QQ) is the interference of QS by producing hydrolyzing enzymes, among other strategies. The main objective of the present study was to identify QS and QQ strains from MBR wastewater treatment plants. A total of 99 strains were isolated from two Spanish plants that were intended to treat leachate from municipal solid waste. Five AHL producers were detected using AHL biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1). Fifteen strains of seventy-one Gram-positive were capable of eliminating or reducing at least one AHL activity. The analysis of 16S rRNA gene sequence showed the importance of the Pseudomonas genus in the production of biofilms and the relevance of the genus Bacillus in the disruption of the QS mechanism, in which the potential activity of lactonase or acylase enzymes was investigated with the aim to contribute to solve biofouling problems and to increase the useful lifespan of membranes.

Influence of Torulaspora delbrueckii in varietal thiol (3-SH and 4-MSP) release in wine sequential fermentations.

In last years, non-Saccharomyces yeasts have emerged as innovative tools to improve wine quality, being able to modify the concentration of sensory-impact compounds. Among them, varietal thiols released by yeasts, play a key role in the distinctive aroma of certain white wines. In this context, Torulaspora delbrueckii is in the spotlight because of its positive contribution to several wine quality parameters. This work studies the physiological properties of an industrial T. delbrueckii strain, for the production of wines with increased thiol concentrations. IRC7 gene, previously described in S. cerevisiae, has been identified in T. delbrueckii, establishing the genetics basis of its thiol-releasing capability. Fermentations involving T. delbrueckii showed improvements on several parameters (such as glycerol content, ethanol index, and major volatile compounds composition), but especially on thiols release. These results confirm the potential of T. delbrueckii on wine improvement, describing new metabolic features regarding the release of cysteinylated aroma precursors.

The Biology of Pichia membranifaciens Killer Toxins.

The killer phenomenon is defined as the ability of some yeast to secrete toxins that are lethal to other sensitive yeasts and filamentous fungi. Since the discovery of strains of Saccharomyces cerevisiae capable of secreting killer toxins, much information has been gained regarding killer toxins and this fact has substantially contributed knowledge on fundamental aspects of cell biology and yeast genetics. The killer phenomenon has been studied in Pichia membranifaciens for several years, during which two toxins have been described. PMKT and PMKT2 are proteins of low molecular mass that bind to primary receptors located in the cell wall structure of sensitive yeast cells, linear (1→6)-ß-d-glucans and mannoproteins for PMKT and PMKT2, respectively. Cwp2p also acts as a secondary receptor for PMKT. Killing of sensitive cells by PMKT is characterized by ionic movements across plasma membrane and an acidification of the intracellular pH triggering an activation of the High Osmolarity Glycerol (HOG) pathway. On the contrary, our investigations showed a mechanism of killing in which cells are arrested at an early S-phase by high concentrations of PMKT2. However, we concluded that induced mortality at low PMKT2 doses and also PMKT is indeed of an apoptotic nature. Killer yeasts and their toxins have found potential applications in several fields: in food and beverage production, as biocontrol agents, in yeast bio-typing, and as novel antimycotic agents. Accordingly, several applications have been found for P. membranifaciens killer toxins, ranging from pre- and post-harvest biocontrol of plant pathogens to applications during wine fermentation and ageing (inhibition of Botrytis cinerea, Brettanomyces bruxellensis, etc.).

Microbial Contribution to Wine Aroma and Its Intended Use for Wine Quality Improvement.

Wine is a complex matrix that includes components with different chemical natures, the volatile compounds being responsible for wine aroma quality. The microbial ecosystem of grapes and wine, including Saccharomyces and non-Saccharomyces yeasts, as well as lactic acid bacteria, is considered by winemakers and oenologists as a decisive factor influencing wine aroma and consumer's preferences. The challenges and opportunities emanating from the contribution of wine microbiome to the production of high quality wines are astounding. This review focuses on the current knowledge about the impact of microorganisms in wine aroma and flavour, and the biochemical reactions and pathways in which they participate, therefore contributing to both the quality and acceptability of wine. In this context, an overview of genetic and transcriptional studies to explain and interpret these effects is included, and new directions are proposed. It also considers the contribution of human oral microbiota to wine aroma conversion and perception during wine consumption. The potential use of wine yeasts and lactic acid bacteria as biological tools to enhance wine quality and the advent of promising advice allowed by pioneering -omics technologies on wine research are also discussed.