Functional analysis of lipid metabolism genes in wine yeasts during alcoholic fermentation at low temperature

11 pages, 1 table, 6 figures.Wine produced by low-temperature fermentation is mostly considered to have improved sensory qualities. However few commercial wine strains available on the market are well-adapted to ferment at low temperature (10 – 15°C). The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. One strategy to modify lipid composition is to alter transcriptional activity by deleting or overexpressing the key genes of lipid metabolism. In a previous study, we identified the genes of the phospholipid, sterol and sphingolipid pathways, which impacted on growth capacity at low temperature. In the present study, we aimed to determine the influence of these genes on fermentation performance and growth during low-temperature wine fermentations. We analyzed the phenotype during fermentation at the low and optimal temperature of the lipid mutant and overexpressing strains in the background of a derivative commercial wine strain. The increase in the gene dosage of some of these lipid genes, e.g., PSD1, LCB3, DPL1 and OLE1, improved fermentation activity during low-temperature fermentations, thus confirming their positive role during wine yeast adaptation to cold. Genes whose overexpression improved fermentation activity at 12°C were overexpressed by chromosomal integration into commercial wine yeast QA23. Fermentations in synthetic and natural grape must were carried out by this new set of overexpressing strains. The strains overexpressing OLE1 and DPL1 were able to finish fermentation before commercial wine yeast QA23. Only the OLE1 gene overexpression produced a specific aroma profile in the wines produced with natural grape must.This work has been financially supported by grants AGL2010-22001-C02-01 and PROMETEOII/2014/042 from the Spanish government and the Generalitat Valenciana, respectively, awarded to JMG. MLM wishes to thank the Spanish government for her FPI grant.Peer reviewe

Lactic Acid Bacteria Isolated from Fermented Doughs in Spain Produce Dextrans and Riboflavin

Many lactic acid bacteria (LAB) produce metabolites with applications in the food industry, such as dextran-type exopolysaccharides (EPS) and riboflavin (vitamin B2). Here, 72 bacteria were isolated from sourdoughs made by Spanish bread-makers. In the presence of sucrose, colonies of 22 isolates showed a ropy phenotype, and NMR analysis of their EPS supported that 21 of them were dextran producers. These isolates were identified by their random amplified polymorphic DNA (RAPD) patterns and their rrs and pheS gene sequences as LAB belonging to four species (Weissella cibaria, Leuconostoc citreum, Leuconostoc falkenbergense and Leuconostoc mesenteroides). Six selected strains from the Leuconostoc (3) and Weissella (3) genera grew in the absence of riboflavin and synthesized vitamin B2. The EPS produced by these strains were characterized as dextrans by physicochemical analysis, and the L. citreum polymer showed an unusually high degree of branching. Quantification of the riboflavin and the EPS productions showed that the W. cibaria strains produce the highest levels (585–685 μg/and 6.5–7.4 g/L, respectively). Therefore, these new LAB strains would be good candidates for the development of fermented foods bio-fortified with both dextrans and riboflavin. Moreover, this is the first report of riboflavin and dextran production by L. falkenbergense.This research was funded by the Spanish Ministry of Science, Innovation and Universities, (grants RTI2018-097114-B-I00 and PCIN-2017-075), by the Basque Government Industry and Education Department (grant PIBA_2020_1_0032) and by the University of the Basque Country (General Grant to Research Groups (GIU 19/014))

Lactic Acid Bacteria Isolated from Fermented Doughs in Spain Produce Dextrans and Riboflavin

Many lactic acid bacteria (LAB) produce metabolites with applications in the food industry, such as dextran-type exopolysaccharides (EPS) and riboflavin (vitamin B2). Here, 72 bacteria were isolated from sourdoughs made by Spanish bread-makers. In the presence of sucrose, colonies of 22 isolates showed a ropy phenotype, and NMR analysis of their EPS supported that 21 of them were dextran producers. These isolates were identified by their random amplified polymorphic DNA (RAPD) patterns and their rrs and pheS gene sequences as LAB belonging to four species (Weissella cibaria, Leuconostoc citreum, Leuconostoc falkenbergense and Leuconostoc mesenteroides). Six selected strains from the Leuconostoc (3) and Weissella (3) genera grew in the absence of riboflavin and synthesized vitamin B2. The EPS produced by these strains were characterized as dextrans by physicochemical analysis, and the L. citreum polymer showed an unusually high degree of branching. Quantification of the riboflavin and the EPS productions showed that the W. cibaria strains produce the highest levels (585–685 μg/and 6.5–7.4 g/L, respectively). Therefore, these new LAB strains would be good candidates for the development of fermented foods bio-fortified with both dextrans and riboflavin. Moreover, this is the first report of riboflavin and dextran production by L. falkenbergense.This research was funded by the Spanish Ministry of Science, Innovation and Universities, (grants RTI2018-097114-B-I00 and PCIN-2017-075), by the Basque Government Industry and Education Department (grant PIBA_2020_1_0032) and by the University of the Basque Country (General Grant to Research Groups (GIU 19/014))

Yeast Biodiversity in Fermented Doughs and Raw Cereal Matrices and the Study of Technological Traits of Selected Strains Isolated in Spain

[EN] Bakers use pure microorganisms and/or traditional sourdoughs as the leavening agent for making bread. The performance of each starter and the substances produced by the microorganisms greatly affect the dough rheology and features of breads. Modern sourdoughs inoculated with selected lactic acid bacteria and yeasts are microbiologically stable, safer than traditional sourdoughs, and easy to use. However, the commercial repertoire of baker’s yeasts is still limited. Therefore, there is a demand for new strains of yeast species, capable of conferring distinctive traits to breads made from a variety of agri-food matrices, in the design of innovative starters. In this context, we report the first comprehensive study on yeasts isolated from a wide range of fermented doughs, cereal flours, and grains of Spain. Nine yeast species were identified from 433 isolates, which were distributed among separate clades. Moreover, phenotypic traits of potential technological relevance were identified in selected yeast strains. Mother doughs (MDs) showed the greatest yeast biodiversity, whereas commercial Saccharomyces starters or related and wild strains often dominated the bakery doughs. A metataxonomic analysis of wheat and tritordeum MDs revealed a greater richness of yeast species and percentage variations related to the consistency, flour type, and fermentation time of MDs

Yeast Biodiversity in Fermented Doughs and Raw Cereal Matrices and the Study of Technological Traits of Selected Strains Isolated in Spain

Bakers use pure microorganisms and/or traditional sourdoughs as the leavening agent for making bread. The performance of each starter and the substances produced by the microorganisms greatly affect the dough rheology and features of breads. Modern sourdoughs inoculated with selected lactic acid bacteria and yeasts are microbiologically stable, safer than traditional sourdoughs, and easy to use. However, the commercial repertoire of baker’s yeasts is still limited. Therefore, there is a demand for new strains of yeast species, capable of conferring distinctive traits to breads made from a variety of agri-food matrices, in the design of innovative starters. In this context, we report the first comprehensive study on yeasts isolated from a wide range of fermented doughs, cereal flours, and grains of Spain. Nine yeast species were identified from 433 isolates, which were distributed among separate clades. Moreover, phenotypic traits of potential technological relevance were identified in selected yeast strains. Mother doughs (MDs) showed the greatest yeast biodiversity, whereas commercial Saccharomyces starters or related and wild strains often dominated the bakery doughs. A metataxonomic analysis of wheat and tritordeum MDs revealed a greater richness of yeast species and percentage variations related to the consistency, flour type, and fermentation time of MDs

Phenotypic analysis of mutant and overexpressing strains of lipid metabolism genes in Saccharomyces cerevisiae: implication in growth at low temperatures

11 pages, 6 figures, 1 table.-- Available online 31 December 2012.The growing demand for wines with a more pronounced aromatic profile calls for low temperature alcoholic fermentations (10–15 °C). However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase and sluggish or stuck fermentations. The lipid metabolism of Saccharomyces cerevisiae plays a central role in low temperature adaptation. The aim of this study was to detect lipid metabolism genes involved in cold adaptation. To do so, we analyzed the growth of knockouts in phospholipids, sterols and sphingolipids, from the EUROSCARF collection S. cerevisiae BY4742 strain at low and optimal temperatures. Growth rate of these knockouts, compared with the control, enabled us to identify the genes involved, which were also deleted or overexpressed in a derivative haploid of a commercial wine strain. We identified genes involved in the phospholipid (PSD1 and OPI3), sterol (ERG3 and IDI1) and sphingolipid (LCB3) pathways, whose deletion strongly impaired growth at low temperature and whose overexpression reduced generation or division time by almost half. Our study also reveals many phenotypic differences between the laboratory strain and the commercial wine yeast strain, showing the importance of constructing mutant and overexpressing strains in both genetic backgrounds. The phenotypic differences in the mutant and overexpressing strains were correlated with changes in their lipid composition.This work was financially supported by the grants from the Spanish government (projects AGL2010-22001-C02-02 and AGL2010-22001-C02-01, awarded to NR and JMG, respectively). MLM also wants to thank to Spanish government for her FPI grant.Peer reviewe

Analysis of low temperature-induced genes (LTIG) in wine yeast during alcoholic fermentation

Fermentations carried out at low temperatures, that is, 10-15 °C, not only enhance the production and retention of flavor volatiles, but also increase the chances of slowing or arresting the process. In this study, we determined the transcriptional activity of 10 genes that were previously reported as induced by low temperatures and involved in cold adaptation, during fermentation with the commercial wine yeast strain QA23. Mutant and overexpressing strains of these genes were constructed in a haploid derivative of this strain to determine the importance of these genes in growth and fermentation at low temperature. In general, the deletion and overexpression of these genes did affect fermentation performance at low temperature. Most of the mutants were unable to complete fermentation, while overexpression of CSF1, HSP104, and TIR2 decreased the lag phase, increased the fermentation rate, and reached higher populations than that of the control strain. Another set of overexpressing strains were constructed by integrating copies of these genes in the delta regions of the commercial wine strain QA23. These new stable overexpressing strains again showed improved fermentation performance at low temperature, especially during the lag and exponential phases. Our results demonstrate the convenience of carrying out functional analysis in commercial strains and in an experimental set-up close to industrial conditions. © 2012 Federation of European Microbiological Societies.This work was supported by a grant (AGL2010-22001-C02-01) from the Spanish government.Peer Reviewe

Chickpea flours as a high nutritional quality ingredient for healthy bakery innovation

Resumen del trabajo presentado al XXII EuroFoodChem Congress, celebrado en Belgrade, Serbia del 14 al 16 de junio de 2023.The growing demand from citizens, including those with gluten intolerance, for higher quality breads for daily consumption, justifies the industry's interest in developing new bakery products using flours obtained from vegetable matrices of geographical proximity, more sustainable from the environmental point of view, and with high nutritional quality and health-promoting properties. Pulses are edible seeds of plants belonging to the legume family that occupy an important place in the diet worldwide. Among pulses, chickpea flours are vegetable matrices that offer extraordinary opportunities to elaborate innovative breads made with mixes of flours, which would be healthier that those frequently consumed made exclusively with wheat flours. The objective of this study was the characterization of the nutritional and bioactive quality of five chickpea flours from Spain (Murcia, Cádiz, Zamora and Albacete) and Germany and the development of mother doughs with the aim of investigating their potential as a healthy an innovative ingredient for bread making. The protein content of chickpea flours was quantified by the Dumas method, and the total starch and phytic acid content were determined by using two commercial kits from Megazyme: Total starch assay kit, AA/AM (K-TSTA) and Phytic acid assay kit (K-PHYT), respectively, and the fatty acid profile was measured by gas chromatography with flame ionization detector. In addition, the content of free phenolic compounds was determined with the Fast Blue BB reagent, after extraction in acidified methanol and the antioxidant activity by the ORAC method. Finally, two types of spontaneously fermented mother doughs, daily back slopped until their fermentative maturity, were prepared with wheat flours combined with chickpea flours from Zamora at 30% (MDG1) and 50% (MDG2). The indigenous microbiota of each MDG was analysed with metagenetic techniques and breads were obtained with MDG1. Chickpea flours showed similar nutritional quality, regardless of their origin, although differences were observed in the content of certain nutrients and bioactive compounds. Protein (22-25%), starch (33-37%), and phytic acid (2.3-2.6%) contents were very similar in all flours, with the exception of flours from Murcia and Albacete, which had higher protein and phytic acid contents, respectively. The content of phenolic compounds varied between 27-51 mg gallic acid equivalents (GAE)/100 g, while the antioxidant activity ranged from 14 to 21 μmoles Trolox equivalents/g. Oleic and linoleic acids were the major fatty acids present in chickpea flours. The flours also proved to be suitable matrices for the growth of autochthonous LAB and yeast species. Breads developed with 30% of MDG1 showed good sensory properties. In conclusion, the good nutritional properties of chickpea flours, together with their good suitability for the production of mother doughs, make these flours a very attractive ingredient for the production of new bakery products.This research was funded by the Fundación General de la Universidad de Salamanca (Junta de Castilla y León/ FEDER) through the project PC_TCUE21-23_037 CICER4FOOD. F. Agahi thanks to Universidad de Valencia for the “Margarita Salas” contract.Peer reviewe

Comparative genomic analysis of Saccharomyces cerevisiae yeasts isolated from fermentations of traditional beverages unveils different adaptive strategies

Saccharomyces cerevisiae strains are the main responsible of most traditional alcohol fermentation processes performed around the world. The characteristics of the diverse traditional fermentations are very different according to their sugar composition, temperature, pH or nitrogen sources. During the adaptation of yeasts to these new environments provided by human activity, their different compositions likely imposed selective pressures that shaped the S. cerevisiae genome. In the present work we performed a comparative genomic hybridization analysis to explore the genome constitution of six S. cerevisiae strains isolated from different traditional fermentations (masato, mescal, cachaça, sake, wine, and sherry wine) and one natural strain. Our results indicate that gene copy numbers (GCN) are very variable among strains, and most of them were observed in subtelomeric and intrachromosomal gene families involved in metabolic functions related to cellular homeostasis, cell-to-cell interactions, and transport of solutes such as ions, sugars and metals. In many cases, these genes are not essential but they can play an important role in the adaptation to new environmental conditions. However, the most interesting result is the association observed between GCN changes in genes involved in the nitrogen metabolism and the availability of nitrogen sources in the different traditional fermentation processes. This is clearly illustrated by the differences in copy numbers not only in gene PUT1, the main player in the assimilation of proline as a nitrogen source, but also in CAR2, involved in arginine catabolism. Strains isolated from fermentations where proline is more abundant contain a higher number of PUT1 copies and are more efficient in assimilating this amino acid as a nitrogen source. A strain isolated from sugarcane juice fermentations, in which arginine is a rare amino acid, contains less copies of CAR2 and showed low efficiency in arginine assimilation. These results suggest that nitrogen metabolism has played an important role in the adaptive evolution of S. cerevisiae strains.C. Ibáñez was beneficiary of a FPI fellowship from the Spanish Government. R. Pérez-Torrado was supported from JAE-DOC postdoctoral program (IATA-CSIC) co-funded by FSE. This work was supported by CICYT grants (ref. AGL2012-39937-CO2-01 and AGL2012-39937-CO2-02) from the Ministerio de Educación y Ciencia and by grant PROMETEO (project PROMETEO/2009/019) from Generalitat Valenciana.Peer reviewe

Nitrogen requirements of commercial wine yeast strains during fermentation of a synthetic grape must

Nitrogen deficiencies in grape musts are one of the main causes of stuck or sluggish wine fermentations. Currently, the most common method for dealing with nitrogen-deficient fermentations is adding supplementary nitrogen (usually ammonium phosphate). However, it is important to know the specific nitrogen requirement of each strain, to avoid excessive addition that can lead to microbial instability and ethyl carbamate accumulation. In this study, we aimed to determine the effect of increasing nitrogen concentrations of three different nitrogen sources on growth and fermentation performance in four industrial wine yeast strains. This task was carried out using statistical modeling techniques. The strains PDM and RVA showed higher growth-rate and maximum population size and consumed nitrogen much more quickly than strains ARM and TTA. Likewise, the strains PDM and RVA were also the greatest nitrogen demanders. Thus, we can conclude that these differences in nitrogen demand positively correlated with higher growth rate and higher nitrogen uptake rate. The most direct effect of employing an adequate nitrogen concentration is the increase in biomass, which involves a higher fermentation rate. However, the impact of nitrogen on fermentation rate is not exclusively due to the increase in biomass because the strain TTA, which showed the worst growth behavior, had the best fermentation activity. Some strains may adapt a strategy whereby fewer cells with higher metabolic activity are produced. Regarding the nitrogen source used, all the strains showed the better and worse fermentation performance with arginine and ammonium, respectively. © 2012 Elsevier Ltd.This project was supported by AGROVIN Company and by the “Centro para el Desarrollo Tecnológico Industrial (CDTI)” (Project CENIT DEMETER) and the Spanish Government (project AGL2009-07331, awarded to GB).Peer Reviewe