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Beer brewing is a well-known process that still faces great challenges, such as the total consumption of sugars present in the fermentation media. Lager-style beer, a major worldwide beer type, is elaborated by Saccharomyces pastorianus (Sp) yeast, which must ferment high maltotriose content worts, but its consumption represents a notable problem, especially among Sp strains belonging to group I. Factors, such as fermentation conditions, presence of maltotriose transporters, transporter copy number variation, and genetic regulation variations contribute to this issue. We assess the factors affecting fermentation in two Sp yeast strains: SpIB1, with limited maltotriose uptake, and SpIB2, known for efficient maltotriose transport. Here, SpIB2 transported significantly more maltose (28%) and maltotriose (32%) compared with SpIB1. Furthermore, SpIB2 expressed all MAL transporters (ScMALx1, SeMALx1, ScAGT1, SeAGT1, MTT1, and MPHx) on the first day of fermentation, whereas SpIB1 only exhibited ScMalx1, ScAGT1, and MPH2/3 genes. Some SpIB2 transporters had polymorphic transmembrane domains (TMD) resembling MTT1, accompanied by higher expression of these transporters and its positive regulator genes, such as MAL63. These findings suggest that, in addition to the factors mentioned above, positive regulators of Mal transporters contribute significantly to phenotypic diversity in maltose and maltotriose consumption among the studied lager yeast strains.IMPORTANCEBeer, the third most popular beverage globally with a 90% market share in the alcoholic beverage industry, relies on Saccharomyces pastorianus (Sp) strains for lager beer production. These strains exhibit phenotypic diversity in maltotriose consumption, a crucial process for the acceptable organoleptic profile in lager beer. This diversity ranges from Sp group II strains with a notable maltotriose-consuming ability to Sp group I strains with limited capacity. Our study highlights that differential gene expression of maltose and maltotriose transporters and its upstream trans-elements, such as MAL gene-positive regulators, adds complexity to this variation. This insight can contribute to a more comprehensive analysis needed to the development of controlled and efficient biotechnological processes in the beer brewing industry.

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Lager yeasts are limited to a few strains worldwide, imposing restrictions on flavour and aroma diversity and hindering our understanding of the complex evolutionary mechanisms during yeast domestication. The recent finding of diverse S. eubayanus lineages from Patagonia offers potential for generating new lager yeasts with different flavour profiles. Here, we leverage the natural genetic diversity of S. eubayanus and expand the lager yeast repertoire by including three distinct Patagonian S. eubayanus lineages. We used experimental evolution and selection on desirable traits to enhance the fermentation profiles of novel S. cerevisiae x S. eubayanus hybrids. Our analyses reveal an intricate interplay of pre-existing diversity, selection on species-specific mitochondria, de-novo mutations, and gene copy variations in sugar metabolism genes, resulting in high ethanol production and unique aroma profiles. Hybrids with S. eubayanus mitochondria exhibited greater evolutionary potential and superior fitness post-evolution, analogous to commercial lager hybrids. Using genome-wide screens of the parental subgenomes, we identified genetic changes in IRA2, IMA1, and MALX genes that influence maltose metabolism, and increase glycolytic flux and sugar consumption in the evolved hybrids. Functional validation and transcriptome analyses confirmed increased maltose-related gene expression, influencing greater maltotriose consumption in evolved hybrids. This study demonstrates the potential for generating industrially viable lager yeast hybrids from wild Patagonian strains. Our hybridization, evolution, and mitochondrial selection approach produced hybrids with high fermentation capacity and expands lager beer brewing options.

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Kombucha is a fermented beverage derived from a sweetened tea fermentation inoculated with a bacteria-yeast consortium referred to as Symbiotic Culture of Bacteria and Yeast (SCOBY). Different SCOBY cultures can impact the beverage's quality and make the whole process highly variable. Adding Saccharomyces yeast cultures to the fermentation process can avoid stalled fermentations, providing a reproducible beverage. Here, we explored using different Saccharomyces eubayanus strains together with SCOBY in the context of kombucha fermentation. Our results show that yeast x SCOBY co-cultures exhibited a robust fermentation profile, providing ethanol and acetic acid levels ranging from 0,18-1,81 %v/v and 0,35-1,15 g/L, respectively. The kombucha volatile compound profile of co-cultures was unique, where compounds such as Isopentyl acetate where only found in yeast x SCOBY fermentations. Metabarcoding revealed that the SCOBY composition was also dependent on the S. eubayanus genotype, where besides Saccharomyces, amplicon sequence variants belonging to Brettanomyces and Starmerella were detected. These differences concomitated global changes in transcript levels in S. eubayanus related to the metabolism of organic molecules used in kombucha fermentation. This study highlights the potential for exploring different S. eubayanus strains for kombucha fermentation, and the significant yeast genotype effect in the profile differentiation in this process.

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BACKGROUND: Probiotics are effective for treating acute infectious diarrhoea caused by bacteria, but there are inconsistent results for the effectiveness of probiotics for diarrhoea caused by viruses. In this article we want to determine whether Sb supplementation has an effect on acute inflammatory viral diarrhoea diagnosed with the multiplex panel PCR test. The aim of this study was to evaluate the efficacy of Saccharomyces boulardii (Sb) as a treatment in patients diagnosed with viral acute diarrhoea. METHODS: From February 2021 to December 2021, 46 patients with a confirmed diagnosis of viral acute diarrhoea diagnosed with the polymerase chain reaction multiplex assay were enrolled in a double-blind, randomized placebo-controlled trial. Patients received paracetamol 500 mg as a standard analgesic and 200 mg of Trimebutine as an antispasmodic treatment plus 600 mg of Sb (n = 23, 1 × 109/100 mL Colony forming unit) or a placebo (n = 23) orally once daily for eight days. The improvement in and severity of symptoms were measured using a symptom diary, the Patient Global Impression and the Patient Global Impression of Change scales (days 4 and 8), both answered and recorded by the patient. RESULTS: Of the 46 patients who completed treatment, 24 (52%) were men and 22 (48%) were women. The average age was 35.6 ± 12.28 years (range 18 to 61 years). The average duration of the evolution of illness at the time of diagnosis was 0.85 ± 0.73 days (maximum 2 days). On day 4 after the diagnosis, 20% reported pain and 2% reported fever, but on day 8, no patient reported pain or fever. On day 4, 70% of patients in the Sb group and 26% in the placebo group reported improvement (P = 0.03), based on the Patients' Global Impression of Change scale, which assesses patient's rating of overall improvement. These findings suggest that 3 to 4 days of treatment with Sb helped to improve symptoms of diarrhoea caused by a virus. CONCLUSION: Treatment with Sb on acute inflammatory diarrhoea of viral aetiology shows no changes regarding the severity of the symptoms; nevertheless, it seems to impact improvement positively. TRIAL REGISTRATION: 22CEI00320171130 dated on 16/12/2020, NCT05226052 dated on 07/02/2022.

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Our objective was to evaluate the effect of supplementing ß-mannanase enzyme, with and without sugarcane yeast (Saccharomyces cerevisiae), on broiler chickens aged 1-21 days. The study used 720 one-day-old male Cobb chicks in a randomized design, with six treatments and six replications of 20 birds each. The treatments were: basal diet (BD), BD + ß-mannanase (100 g/t), 7% sugarcane yeast (SY), SY + ß-mannanase (80 g/t), SY + ß-mannanase (100 g/t), and SY + ß-mannanase (120 g/t). Zootechnical results (animal performance) were evaluated during the pre-starter (1 to 7 day-old) and starter (1 to 21 day-old) phases, as well as small intestine morphometry (duodenum, jejunum, and ileum) and diets' economic viability. Data were subjected to variance analysis using the SAS software, and the means were compared by the Student Newman Keuls (SNK) test. In the pre-starter phase, treatments with YD + ß-mannanase (80, 100, and 120 g/t) showed the best feed conversion averages. In the starter phase, chickens consuming the basal diet (BD) and BD + ß-mannanase showed better average weights, weight gains, and feed conversion rates. For intestinal morphometry, shallower ileal crypts were observed in the treatment with YD + ß-mannanase (120 g/t) compared to the basal diet, and wider ileal villi were observed in the treatment with YD + ß-mannanase (100 g/t) compared to the diet with YD + ß-mannanase (80 g/t). The thickness of the muscular wall in the duodenum was lower in chickens consuming BD compared to BD + ß-mannanase (100 g/t), higher in YD and supplementation with 100 g/t compared to 80 and 120 g/t in the jejunum, and higher in diets with ß-mannanase supplementation compared to BD and YD in the ileum. For economic viability, adding 7% sugar cane yeast, with or without enzyme, increased the average feed cost and cost index, and reduced the economic efficiency index. Based on the zootechnical results, YD + ß-mannanase (120 g/t) is recommended for the pre-starter and starter phases. However, using sugar cane yeast with or without ß-mannanase enzyme supplementation is not economically viable in the 1- to 21-day period.

Objetivou-se avaliar o efeito da suplementação da enzima ß-mananase com e sem levedura de cana de açúcar (Saccharomyces cerevisiae) para frangos de corte 1 a 21 dias de idade. Utilizou-se 720 pintos de corte de um dia de idade, machos, da linhagem Cobb, distribuídos em delineamento inteiramente casualizado com seis tratamentos e, seis repetições de 20 aves cada. Os tratamentos foram: Dieta basal (DB); DB + ß-mananase (100 g/t); DB + 7% de levedura de cana-de-açúcar (DL); DL + ß-mananase (80 g/t); DL + ß-mananase (100 g/t); DL + ß-mananase (120 g/t). Avaliou-se o desempenho zootécnico na fase pré-inicial (1 a 7) e na fase inicial (1 a 21 dias de idade), a morfometria do intestino delgado (duodeno, jejuno e íleo) e, a viabilidade econômica da ração. Os dados foram submetidos à análise da variância do programa SAS e as médias foram comparadas pelo teste de Student Newmann Keuls (SNK). Na fase pré-inicial, os tratamentos com DL + ß-mananase (80, 100 e 120 g/t) apresentaram as melhores médias de conversão alimentar. Na fase inicial, aos frangos que consumiram à dieta basal (DB) e DB + ß-mananase apresentaram peso médio, ganho de peso e conversão alimentar melhores. Para a morfometria intestinal, criptas ileais mais rasas foram observadas no tratamento com DL+ ß-mananase (120 g/t) em relação a dieta basal e vilos ileais mais largos foram observados no tratamento com DL+ ß-mananase (100 g/t) em relação a dieta com DL+ ß-mananase (80 g/t). A espessura da parede muscular, no duodeno, foi menor nos frangos que consumiram a DB em relação a DB + ß-mananase (100 g/t), no jejuno, foi maior na DL e na suplementação com 100 g/t em relação a 80 e 120 g/t e no íleo, foi maior nas dietas com suplementação de ß-mananase em relação a DB e DL. Para a viabilidade econômica, a adição de 7% de levedura de cana-de-açúcar, com ou sem enzima proporcionaram aumento do custo médio de ração e índice de custo, e redução do índice de eficiência econômica. Com base nos resultados zootécnicos recomenda-se DL + ß-mananase (120g/t) para a fase pré-inicial e inicial. Contudo, o uso de levedura de cana-de-açúcar com e sem suplementação da enzima ß-mananase não é economicamente viável no período de 1 a 21 dias.

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L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26-362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch® strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg-1. The resultant enzyme is 40% more stable than commercially available Escherichia coli's ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi.

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Since its identification, Saccharomyces eubayanus has been recognized as the missing parent of the lager hybrid, S. pastorianus. This wild yeast has never been isolated from fermentation environments, thus representing an interesting candidate for evolutionary, ecological and genetic studies. However, it is imperative to develop additional molecular genetics tools to ease manipulation and thus facilitate future studies. With this in mind, we generated a collection of stable haploid strains representative of three main lineages described in S. eubayanus (PB-1, PB-2 and PB-3), by deleting the HO gene using CRISPR-Cas9 and tetrad micromanipulation. Phenotypic characterization under different conditions demonstrated that the haploid derivates were extremely similar to their parental strains. Genomic analysis in three strains highlighted a likely low frequency of off-targets, and sequencing of a single tetrad evidenced no structural variants in any of the haploid spores. Finally, we demonstrate the utilization of the haploid set by challenging the strains under mass-mating conditions. In this way, we found that S. eubayanus under liquid conditions has a preference to remain in a haploid state, unlike S. cerevisiae that mates rapidly. This haploid resource is a novel set of strains for future yeast molecular genetics studies.

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A wide variety of microorganism species have been used as probiotics to improve the intestinal microbial balance, control and prevent the colonization of pathogenic bacteria and promote growth in animal production. Based on the various beneficial factors of probiotic agents, this study aimed to evaluate the effectiveness of three different formulations of probiotics strains through the analysis of antagonistic capacity against common pathogens from chickens gastrointestinal tract. Three formulations composed by Bacillus, Lactobacillus, Saccharomyces, Enterococcus, Bifidobacterium and Pediococcus were tested in vitro by the method of probiotic culture spot in plates seeded with inocula of Escherichia coli, Salmonella Heidelberg and Campylobacter jejuni. The inhibition halos were measured and classified according to the degree of pathogenic bacteria inhibition. The formulation containing an association among Bacillus, Lactobacillus and Saccharomyces presented better results when compared to the other formulations.

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The study's objective was to develop a co-fermentation process with appropriate fermentation parameters to produce a sour beer (similar to a Belgium sour beer) with an ethanol content of 6-8% (v/v) using a coculture of Saccharomyces pastorianus and Lactobacillus plantarum. Statistical optimization was conducted to determine fermentation conditions to produce a sour beer with ~ 3 mg/mL of lactic acid, similar to the traditional sour beer levels. Studies were conducted on the microbial dynamics and volatile compounds produced during this fermentation and aging process. GC-MS studies revealed the generation of novel bioactive compounds as well as the depletion of some volatile compounds during co-fermentation. The study detailed a 5-day co-fermentation process of S. pastorianus and L. plantarum and a 21-day aging process to prepare a sour beer with biochemical properties along the lines of traditional lambic beers. The interrelationship between the two microorganisms and the biochemical changes in the sour beer fermentation process was elucidated and the sensorial attributes have been described.

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Interest in the use of non-conventional yeasts in wine fermentation has been increased in the last years in the wine sector. The main objective of this manuscript was to explore the aromatic diversity produced by wild and non-wine strains of S. cerevisiae, S. eubayanus, S. kudriavzevii, and S. uvarum species in young and bottle-aged Tempranillo wines as well as evaluate their fermentation capacity and the yield on ethanol, glycerol, and organic acids, that can contribute to diminishing the effects of climate change on wines. S. uvarum strain U1 showed the highest ability to release or de novo produce monoterpenes, such as geraniol and citronellol, whose values were 1.5 and 3.5-fold higher than those of the wine S. cerevisiae strain. We found that compared to the normal values for red wines, ß-phenylethyl acetate was highly synthesized by U1 and E1 strains, achieving 1 mg/L. Additionally, after aging, wines of S. eubayanus strains contained the highest levels of this acetate. Malic acid was highly degraded by S. kudriavzevii yeasts, resulting in the highest yields of lactic acid (>5-fold) and ethyl lactate (>2.8-fold) in their wines. In aged wines, we observed that the modulating effects of yeast strain were very high in ß-ionone. S. uvarum strains U1 and BMV58 produced an important aging attribute, ethyl isobutyrate, which was highly enhanced during the aging. Also, the agave S. cerevisiae strain develops an essential aroma after aging, reaching the highest ethyl leucate contents. According to the results obtained, the use of wild non-wine strains of S. cerevisiae and strains of the cryotolerant species S. eubayanus, S. kudriavzevii, and S. uvarum in Tempranillo wine fermentation increase the aroma complexity. In addition, wines from S. kudriavzevii strains had twice additional glycerol, those from S. uvarum 4-fold more succinic acid, while wines from wild strains yielded 1% v/v less ethanol which may solve wine problems associated with climate change.

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