Non-conventional Yeast Research Articles

The N.C.Yeastract and CommunityYeastract databases to study gene and genomic transcription regulation in non-conventional yeasts.

Responding to the recent interest of the yeast research community in non-Saccharomyces cerevisiae species of biotechnological relevance, the N.C.Yeastract (http://yeastract-plus.org/ncyeastract/) was associated to YEASTRACT + (http://yeastract-plus.org/). The YEASTRACT + portal is a curated repository of known regulatory associations between transcription factors (TFs) and target genes in yeasts. N.C.Yeastract gathers all published regulatory associations and TF-binding sites for Komagataellaphaffii (formerly Pichia pastoris), the oleaginous yeast Yarrowia lipolytica, the lactose fermenting species Kluyveromyces lactis and Kluyveromyces marxianus, and the remarkably weak acid-tolerant food spoilage yeast Zygosaccharomyces bailii. The objective of this review paper is to advertise the update of the existing information since the release of N.C.Yeastract in 2019, and to raise awareness in the community about its potential to help the day-to-day work on these species, exploring all the information available in the global YEASTRACT + portal. Using simple and widely used examples, a guided exploitation is offered for several tools: (i) inference of orthologous genes; (ii) search for putative TF binding sites and (iii) inter-species comparison of transcription regulatory networks and prediction of TF-regulated networks based on documented regulatory associations available in YEASTRACT + for well-studied species. The usage potentialities of the new CommunityYeastract platform by the yeast community are also discussed.

\u03b1-Farnesene production from lipid by engineered Yarrowia lipolytica

Producing high value-added products from waste lipid feedstock by microbial cell factory has great advantages to minimize the pollution as well as improve the economic value of wasted oils and fats. Yarrowia lipolytica is a non-conventional oleaginous yeast and can grow on a variety of hydrophobic substrates. In this study, we explored its ability to synthesize α-farnesene, an important sesquiterpene, using lipid feedstock. Based on the α-farnesene production strain, we constructed previously, we identified that Erg12 was the key limiting factor to further increase the α-farnesene production. The α-farnesene production was improved by 35.8% through increasing the copy number of ERG12 and FSERG20 on oleic acid substrate. Expression of heterologous VHb further improved α-farnesene production by 12.7%. Combining metabolic engineering with the optimization of fermentation conditions, the α-farnesene titer and yield reached 10.2 g/L and 0.1 g/g oleic acid, respectively, in fed-batch cultivation. The α-farnesene synthesis ability on waste cooking oil and other edible oils were also explored. Compared with using glucose as carbon source, using lipid substrates obtained higher α-farnesene yield and titer, but lower by-products accumulation, demonstrating the advantage of Y. lipolytica to synthesize high value-added products using lipid feedstock.

Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris.

The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has become a powerful eukaryotic expression platform for biopharmaceutical and biotechnological applications on both laboratory and industrial scales. Despite the fundamental role that artificial transcription factors (ATFs) play in the orthogonal control of gene expression in synthetic biology, a limited number of ATFs are available for P. pastoris. To establish orthogonal regulators for use in P. pastoris, we characterized ATFs derived from Arabidopsis TFs. The plant-derived ATFs contain the binding domain of TFs from the plant Arabidopsis thaliana, in combination with the activation domains of yeast GAL4 and plant EDLL and a synthetic promoter harboring the cognate cis-regulatory motifs. Chromosomally integrated ATFs and their binding sites (ATF/BSs) resulted in a wide spectrum of inducible transcriptional outputs in P. pastoris, ranging from as low as 1- to as high as ∼63-fold induction with only small growth defects. We demonstrated the application of ATF/BSs by generating P. pastoris cells that produce β-carotene. Notably, the productivity of β-carotene in P. pastoris was ∼4.8-fold higher than that in S. cerevisiae, reaching ∼59% of the β-carotene productivity obtained in a S. cerevisiae strain optimized for the production of the β–carotene precursor, farnesyl diphosphate, by rewiring the endogenous metabolic pathways using plant-derived ATF/BSs. Our data suggest that plant-derived regulators have a high degree of transferability from S. cerevisiae to P. pastoris. The plant-derived ATFs, together with their cognate binding sites, powerfully increase the repertoire of transcriptional regulatory modules for the tuning of protein expression levels required in metabolic engineering or synthetic biology in P. pastoris.

A Multiphase Multiobjective Dynamic Genome-Scale Model Shows Different Redox Balancing among Yeast Species of the Saccharomyces Genus in Fermentation.

ABSTRACTYeasts constitute over 1,500 species with great potential for biotechnology. Still, the yeast Saccharomyces cerevisiae dominates industrial applications, and many alternative physiological capabilities of lesser-known yeasts are not being fully exploited. While comparative genomics receives substantial attention, little is known about yeasts’ metabolic specificity in batch cultures. Here, we propose a multiphase multiobjective dynamic genome-scale model of yeast batch cultures that describes the uptake of carbon and nitrogen sources and the production of primary and secondary metabolites. The model integrates a specific metabolic reconstruction, based on the consensus Yeast8, and a kinetic model describing the time-varying culture environment. In addition, we proposed a multiphase multiobjective flux balance analysis to compute the dynamics of intracellular fluxes. We then compared the metabolism of S. cerevisiae and Saccharomyces uvarum strains in a rich medium fermentation. The model successfully explained the experimental data and brought novel insights into how cryotolerant strains achieve redox balance. The proposed model (along with the corresponding code) provides a comprehensive picture of the main steps occurring inside the cell during batch cultures and offers a systematic approach to prospect or metabolically engineering novel yeast cell factories.IMPORTANCE Nonconventional yeast species hold the promise to provide novel metabolic routes to produce industrially relevant compounds and tolerate specific stressors, such as cold temperatures. This work validated the first multiphase multiobjective genome-scale dynamic model to describe carbon and nitrogen metabolism throughout batch fermentation. To test and illustrate its performance, we considered the comparative metabolism of three yeast strains of the Saccharomyces genus in rich medium fermentation. The study revealed that cryotolerant Saccharomyces species might use the γ-aminobutyric acid (GABA) shunt and the production of reducing equivalents as alternative routes to achieve redox balance, a novel biological insight worth being explored further. The proposed model (along with the provided code) can be applied to a wide range of batch processes started with different yeast species and media, offering a systematic and rational approach to prospect nonconventional yeast species metabolism and engineering novel cell factories.

Revisiting the unique structure of autonomously replicating sequences in Yarrowia lipolytica and its role in pathway engineering.

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 2.2-fold increase in gene expression and 1.7-fold higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

Yarrowia lipolytica Strains and Their Biotechnological Applications: How Natural Biodiversity and Metabolic Engineering Could Contribute to Cell Factories Improvement.

Among non-conventional yeasts of industrial interest, the dimorphic oleaginous yeast Yarrowia lipolytica appears as one of the most attractive for a large range of white biotechnology applications, from heterologous proteins secretion to cell factories process development. The past, present and potential applications of wild-type, traditionally improved or genetically modified Yarrowia lipolytica strains will be resumed, together with the wide array of molecular tools now available to genetically engineer and metabolically remodel this yeast. The present review will also provide a detailed description of Yarrowia lipolytica strains and highlight the natural biodiversity of this yeast, a subject little touched upon in most previous reviews. This work intends to fill this gap by retracing the genealogy of the main Yarrowia lipolytica strains of industrial interest, by illustrating the search for new genetic backgrounds and by providing data about the main publicly available strains in yeast collections worldwide. At last, it will focus on exemplifying how advances in engineering tools can leverage a better biotechnological exploitation of the natural biodiversity of Yarrowia lipolytica and of other yeasts from the Yarrowia clade.

Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichiapastoris.

The industrial yeast Pichia pastoris has been harnessed extensively for production of proteins, and it is attracting attention as a chassis cell factory for production of chemicals. However, the lack of synthetic biology tools makes it challenging in rewiring P. pastoris metabolism. We here extensively engineered the recombination machinery by establishing a CRISPR-Cas9 based genome editing platform, which improved the homologous recombination (HR) efficiency by more than 54 times, in particular, enhanced the simultaneously assembly of multiple fragments by 13.5 times. We also found that the key HR-relating gene RAD52 of P. pastoris was largely repressed in compared to that of Saccharomyces cerevisiae. This gene editing system enabled efficient seamless gene disruption, genome integration and multiple gene assembly with positive rates of 68–90%. With this efficient genome editing platform, we characterized 46 potential genome integration sites and 18 promoters at different growth conditions. This library of neutral sites and promoters enabled two-factorial regulation of gene expression and metabolic pathways and resulted in a 30-fold range of fatty alcohol production (12.6–380 mg/l). The expanding genetic toolbox will facilitate extensive rewiring of P. pastoris for chemical production, and also shed light on engineering of other non-conventional yeasts.

Mitochondrial dynamics and degradation in the oleaginous yeast Lipomyces starkeyi.

Emerging evidence implicates the vital role of mitochondria in lipid consumption and storage, highlighting the intimate link between energy production and saving. Although formation of giant lipid droplets, which is the key hallmark of the oleaginous yeast Lipomyces starkeyi, appears to be regulated in response to changes in mitochondrial shape and metabolism, technical limitations of genetic manipulation have become an obstacle to uncover the mitochondrial behavior in this nonconventional yeast. Here, we established an L. starkeyi strain stably expressing a fluorescent marker for monitoring mitochondrial morphology and degradation and found that mitochondria are mostly fragmented in L. starkeyi cells under fermentable, nonfermentable, and nitrogen depletion conditions. Notably, a fraction of mitochondria-specific fluorescent signals was localized to the vacuole, a lytic organelle in yeast, indicating degradation of mitochondria in those cells. This possible catabolic event was more predominant in cells under nutrient-poor conditions than that in cells under nutrient-rich conditions, concomitantly with lipid droplet formation. Collectively, our studies provide a new tool to investigate mitochondrial dynamics in L. starkeyi and decipher the potential role of mitochondrial degradation in lipid metabolism.

Non-Conventional Yeasts as Alternatives in Modern Baking for Improved Performance and Aroma Enhancement

Saccharomyces cerevisiae remains the baker’s yeast of choice in the baking industry. However, its ability to ferment cereal flour sugars and accumulate CO2 as a principal role of yeast in baking is not as unique as previously thought decades ago. The widely conserved fermentative lifestyle among the Saccharomycotina has increased our interest in the search for non-conventional yeast strains to either augment conventional baker’s yeast or develop robust strains to cater for the now diverse consumer-driven markets. A decade of research on alternative baker’s yeasts has shown that non-conventional yeasts are increasingly becoming important due to their wide carbon fermentation ranges, their novel aromatic flavour generation, and their robust stress tolerance. This review presents the credentials of non-conventional yeasts as attractive yeasts for modern baking. The evolution of the fermentative trait and tolerance to baking-associated stresses as two important attributes of baker’s yeast are discussed besides their contribution to aroma enhancement. The review further discusses the approaches to obtain new strains suitable for baking applications.

Conferring thermotolerant phenotype to wild-type Yarrowia lipolytica improves cell growth and erythritol production.

In microbial engineering, heat stress is an important environmental factor modulating cell growth, metabolic flux distribution and the synthesis of target products. Yarrowia lipolytica, as a GARS (generally recognized as safe) nonconventional yeast, has been widely used in the food industry, especially as the host of erythritol production. Biomanufacturing economics is limited by the high operational cost of cooling energy in large-scale fermentation. It is of great significance to select thermotolerant Y. lipolytica to reduce the cooling cost and elucidate the heat-resistant mechanism at molecular level. For this purpose, we performed adaptive evolution and obtained a thermotolerant strain named Y. lipolytica BBE-18. Transcriptome analysis allows us to identify four genes in thiamine metabolism pathway that are responsible for the complicated thermotolerant phenotype. The heat-resistant phenotype was validated with the model strain Y. lipolytica Po1f by overexpression of single and combined genes. Then, conferring the thermotolerant phenotype to the wild-type Y. lipolytica BBE-17 enable the strain to produce three-times more erythritol of the control strain with 3°C higher than optimal cultivation temperature. To our knowledge, this is the first report on engineering heat-resistant phenotype to improve the erythritol production in Y. lipolytica. However, due to the increase of culture temperature, a large amount of adenosine triphosphate is consumed to ensure the life activities of Y. lipolytica which limits the potential of cell synthetic products to a certain extent. Even so, this study provides a reference for Y. lipolytica to produce other products under high temperature.

Advances in metabolic engineering of non-conventional yeasts

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.

The role of Mig1, Mig2, Tup1 and Hap4 transcription factors in regulation of xylose and glucose fermentation in the thermotolerant yeast Ogataea polymorpha.

Glucose is a preferred carbon source for most living organisms. The metabolism and regulation of glucose utilization are well studied mostly for Saccharomyces cerevisiae. Xylose is the main pentose sugar released from the lignocellulosic biomass, which has a high potential as a renewable feedstock for bioethanol production. The thermotolerant yeast Ogataea (Hansenula) polymorpha, in contrast to S. cerevisiae, is able to metabolize and ferment not only glucose but also xylose. However, in non-conventional yeasts, the regulation of glucose and xylose metabolism remains poorly understood. In this study, we characterize the role of transcriptional factors Mig1, Mig2, Tup1 and Hap4 in the natural xylose-fermenting yeast O. polymorpha. The deletion of MIG1 had no significant influence on ethanol production either from xylose or glucose, however the deletion of both MIG1 and MIG2 reduced the amount of ethanol produced from these sugars. The deletion of HAP4-A and TUP1 genes resulted in increased ethanol production from xylose. Inversely, the overexpression of HAP4-A and TUP1 genes reduced ethanol production during xylose alcoholic fermentation. Thus, HAP4-A and TUP1 are involved in repression of xylose metabolism and fermentation in yeast O. polymorpha and their deletion could be a viable strategy to improve ethanol production from this pentose.

The revenge of Zygosaccharomyces yeasts in food biotechnology and applied microbiology.

Non-conventional yeasts refer to a huge and still poorly explored group of species alternative to the well-known model organism Saccharomyces cerevisiae. Among them, Zygosaccharomyces rouxii and the sister species Zygosaccharomyces bailii are infamous for spoiling food and beverages even in presence of several food preservatives. On the other hand, their capability to cope with a wide range of process conditions makes these yeasts very attractive factories (the so-called "ZygoFactories") for bio-converting substrates poorly permissive for the growth of other species. In balsamic vinegar Z. rouxii is the main yeast responsible for converting highly concentrated sugars into ethanol, with a preference for fructose over glucose (a trait called fructophily). Z. rouxii has also attracted much attention for the ability to release important flavor compounds, such as fusel alcohols and the derivatives of 4-hydroxyfuranone, which markedly contribute to fragrant and smoky aroma in soy sauce. While Z. rouxii was successfully proposed in brewing for producing low ethanol beer, Z. bailii is promising for lactic acid and bioethanol production. Recently, several research efforts exploited omics tools to pinpoint the genetic bases of distinctive traits in "ZygoFactories", like fructophily, tolerance to high concentrations of sugars, lactic acid and salt. Here, I provided an overview of Zygosaccharomyces industrially relevant phenotypes and summarized the most recent findings in disclosing their genetic bases. I suggest that the increasing number of genomes available for Z. rouxii and other Zygosaccharomyces relatives, combined with recently developed genetic engineering toolkits, will boost the applications of these yeasts in biotechnology and applied microbiology.

Screening and Growth Characterization of Non-conventional Yeasts in a Hemicellulosic Hydrolysate.

Lignocellulosic biomass is an attractive raw material for the sustainable production of chemicals and materials using microbial cell factories. Most of the existing bioprocesses focus on second-generation ethanol production using genetically modified Saccharomyces cerevisiae, however, this microorganism is naturally unable to consume xylose. Moreover, extensive metabolic engineering has to be carried out to achieve high production levels of industrially relevant building blocks. Hence, the use of non-Saccharomyces species, or non-conventional yeasts, bearing native metabolic routes, allows conversion of a wide range of substrates into different products, and higher tolerance to inhibitors improves the efficiency of biorefineries. In this study, nine non-conventional yeast strains were selected and screened on a diluted hemicellulosic hydrolysate from Birch. Kluyveromyces marxianus CBS 6556, Scheffersomyces stipitis CBS 5773, Lipomyces starkeyi DSM 70295, and Rhodotorula toruloides CCT 7815 were selected for further characterization, where their growth and substrate consumption patterns were analyzed under industrially relevant substrate concentrations and controlled environmental conditions in bioreactors. K. marxianus CBS 6556 performed poorly under higher hydrolysate concentrations, although this yeast was determined among the fastest-growing yeasts on diluted hydrolysate. S. stipitis CBS 5773 demonstrated a low growth and biomass production while consuming glucose, while during the xylose-phase, the specific growth and sugar co-consumption rates were among the highest of this study (0.17 h–1 and 0.37 g/gdw*h, respectively). L. starkeyi DSM 70295 and R. toruloides CCT 7815 were the fastest to consume the provided sugars at high hydrolysate conditions, finishing them within 54 and 30 h, respectively. R. toruloides CCT 7815 performed the best of all four studied strains and tested conditions, showing the highest specific growth (0.23 h–1), substrate co-consumption (0.73 ± 0.02 g/gdw*h), and xylose consumption (0.22 g/gdw*h) rates. Furthermore, R. toruloides CCT 7815 was able to produce 10.95 ± 1.37 gL–1 and 1.72 ± 0.04 mgL–1 of lipids and carotenoids, respectively, under non-optimized cultivation conditions. The study provides novel information on selecting suitable host strains for biorefinery processes, provides detailed information on substrate consumption patterns, and pinpoints to bottlenecks possible to address using metabolic engineering or adaptive evolution experiments.

Physiological performance of Kazachstania unispora in sourdough environments

In this work we explored the potential of several strains of Kazachstania unispora to be used as non-conventional yeasts in sourdough fermentation. Properties such as carbohydrate source utilization, tolerance to different environmental factors and the performance in fermentation were evaluated. The K. unispora strains are characterized by rather restricted substrate utilization: only glucose and fructose supported the growth of the strains. However, the growth in presence of fructose was higher compared to a Saccharomyces cerevisiae commercial strain. Moreover, the inability to ferment maltose can be considered a positive characteristic in sourdoughs, where the yeasts can form a nutritional mutualism with maltose-positive Lactic Acid Bacteria. Tolerance assays showed that K. unispora strains are adapted to a sourdough environment: they were able to grow in conditions of high osmolarity, high acidity and in presence of organic acids, ethanol and salt. Finally, the performance in fermentation was comparable with the S. cerevisiae commercial strain. Moreover, the growth was more efficient, which is an advantage in obtaining the biomass in an industrial scale. Our data show that K. unispora strains have positive properties that should be explored further in bakery sector.Graphic abstract

Non-conventional yeasts from fermented honey by-products: Focus on Hanseniaspora uvarum strains for craft beer production

The increasing interest in novel beer productions focused on non-Saccharomyces yeasts in order to pursue their potential in generating groundbreaking sensory profiles. Traditional fermented beverages represent an important source of yeast strains which could express interesting features during brewing. A total of 404 yeasts were isolated from fermented honey by-products and identified as Saccharomyces cerevisiae, Wickerhamomyces anomalus, Zygosaccharomyces bailii, Zygosaccharomyces rouxii and Hanseniaspora uvarum. Five H. uvarum strains were screened for their brewing capability. Interestingly, Hanseniaspora uvarum strains showed growth in presence of ethanol and hop and a more rapid growth than the control strain S. cerevisiae US-05. Even though all strains showed a very low fermentation power, their concentrations ranged between 7 and 8 Log cycles during fermentation. The statistical analyses showed significant differences among the strains and underlined the ability of YGA2 and YGA34 to grow rapidly in presence of ethanol and hop. The strain YGA34 showed the best technological properties and was selected for beer production. Its presence in mixed- and sequential-culture fermentations with US-05 did not influence attenuation and ethanol concentration but had a significant impact on glycerol and acetic acid concentrations, with a higher sensory complexity and intensity, representing promising co-starters during craft beer production.

Non-<em>Saccharomyces</em> as a tool for modulating wine quality and stimulating malolactic fermentation

Recent research in non-Saccharomyces yeasts promotes their use as starter cultures in wine alcoholic fermentation together with S. cerevisiae. The use of these non-conventional yeasts can modulate the organoleptic profile of wines. However, it is unclear how they will modulate wines together with Oenococcus oeni after malolactic fermentation. In this article we discuss the main oenological consequences of these interactions and how malolactic fermentation can be stimulated using some of these non-Saccharomyces yeasts.

Evaluation of Bioethanol Production from a Mixed Fruit Waste by Wickerhamomyces sp. UFFS-CE-3.1.2

The use of fruit residues from domestic consumption for energy purposes is a perspective of innovation in the search for a more sustainable fuel, due to the low cost of this biomass, combined with its large amounts of generation, which has sugars readily available in its composition and does not require process pretreatment hydrolysis complexes of the material. This work evaluated residues of melon, pineapple, banana, apple, and mango, to compose a mixture of fruit waste (MFW), simulating a household waste for bioethanol production by a non-conventional yeast Wickerhamomyces sp. UFFS-CE-3.1.2. Sugar extraction was optimized by experimental design (MFW: 12% g dry mass v−1; 25 °C; 5 min) and resulted in the liberation of 36.32 ± 0.72 g L−1 fermentable sugar. MFW was used for alcoholic fermentation with and without nitrogen supplementation (urea and yeast extract). The results demonstrated that it is not necessary for the supplementation, making the process more economically viable. The maximum ethanol productivity (2.50±0.06 g L−1 h−1) was achieved in 9 h of the operation. The MFW extracted is an alternative for the bioethanol process as low cost and straightforward, adapted for different fruit residues, and used as a unique or diluent medium in the biorefinery context. Moreover, non-conventional yeast demonstrated the more new one in this study that explores the potential of yeast recently isolated.

Multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica.

Erythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica. The effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica. A combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol.

Assessment of non-conventional yeasts with potential probiotic for protein-fortified craft beer production

The use of non-conventional yeasts could be an interesting source of biodiversity for developing innovative fermented beverages. Here, 43 wild yeast strains belonging to different genera such as Lachancea, Kluyveromyces, Torulaspora, Metschnikowia, Kazachstania, Brettanomyces, Pichia, Candida, Hanseniaspora, Rhodotorula, Rodosporidobolus and Saccharomyces, previously evaluated for their probiotic traits, have been tested for craft beer production. Different experimental lines were carried out to develop a new beverage which could combine increased aromatic characteristics and improved nutritional properties: i) beers produced from pils wort (PW); ii) beers from pils wort enriched with lentil (PLW) or chickpea flour extracts (PCW).PW beer trials were characterized by a low ethanol content. The PCW results in beer with an unpleasant aromatic taste, while the presence of lentil (PLW) confers effective fermentative characters and pleasant aromatic notes to the final beers.The selected strains Lachancea thermotolerans, Kazachstania unispora and Saccharomyces cerevisiae determined a significantly increase in the main aromatic compounds such as ethyl acetate, isoamyl acetate and higher alcohols in PLW. The sensorial profile indicated that the beers were characterized with emphasized aromatic attributes. The yeasts selected here, could contribute to obtain a premium craft beer, with highly nutritional and functional characteristics, with a distinctive aromatic character.