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The textile industry produces harmful effluents that are discharged into the environment, damaging the aquatic and other ecosystems. A yeast-based solution for decolorization of textile industrial wastewater was produced and evaluated. Three yeast strains, Candida parapsilosis (HOMOGS20B), Yarrowia lipolytica (HOMOGST27AB) and Candida pseudoglaebosa (LIIIS36B), isolated from a textile wastewater treatment plant and previously selected for their dye decolorization capacity, were freeze-dried. Additionally, Yarrowia lipolytica (HOMOGST27AB) was also spray-dried. Skim milk powder and maltodextrin were used as cell protectors, and the freeze-dried products were stored at cold (4 °C) and room temperature for 210 days. The viability of the yeast cells and their decolorization capacity over time were assessed. Dried yeast cells maintained their viability, and decolorization capacity for at least 90 days of storage after spray- and freeze-drying with both cell-protecting agents. The dried yeast-based solution for decolorizing textile industrial wastewater combines stability, efficiency, and convenience of production for application in real industrial facilities.

The purpose of this study is to evaluate the effect of high hydrostatic pressure (HHP) as a novel approach for yeast cell disruption and lipid extraction from Lipomyces starkeyi DSM 70295 grown in glucose medium (40 g/L and C/N:55/1) at initial pH of 5.0, 25°C, and 130 rpm for 8 days. HHP extraction conditions including pressure, time, and temperature were optimized by response surface methodology. The high speed homogenizer-assisted extraction (HSH) was also used for comparison. The biomass subjected to HHP was examined under scanning electron microscopy and light microscope. A maximal lipid yield of 45.8 ± 2.1% in dry cell basis (w/w) was achieved at 200 MPa, 40°C, and 15 min, while a minimum yield of 15.2 ± 0.9% was observed at 300 MPa, 40°C, and 10 min (p 0.05). The lipid yield decreased with increasing pressure. It was demonstrated that low pressure (200 MPa) collapsed the cells, while high pressure (400 MPa) created protrusions on the cell wall and cell fragments spread in the environment. This study favors HHP as a promising method for Lipomyces oil extraction. PRACTICAL APPLICATION: Single-cell oils are considered future alternatives to plant-based oils as food additives and dietary supplements. Oleaginous microorganisms accumulate oils in their cell plasma, which makes extraction essential. One of the main obstacles with existing methods is the utilization of strong acids to destroy cell walls. This study aims to demonstrate high hydrostatic pressure as a rapid method for lipid extraction from oleaginous yeast Lipomyces starkeyi.

Aims This study evaluated changes in epiphytic microbial population of alfalfa (Medicago sativa) during the growing season. First cut forage was harvested to study the effects of an inoculant combining two obligate heterofermentative lactic acid bacteria strains on the bacterial and fungal communities and the fermentation of alfalfa silage. Methods and Results The epiphytic microbiome of alfalfa was evaluated 10-times during the growing season. Alfalfa wilted to 395.0 g/kg was treated with water (Control) or with a combination of L. buchneri NCIMB 40788 and L. hilgardii CNCM-I-4785 (LBLH). Mini-silos were opened after 1, 4, 8, 16, 32, and 64 days of ensiling. The relative abundance (RA) of the epiphytic bacterial and fungal families varied during the growing season. After 1 day, Weissella was the most abundant genus and present at similar RA in the two treatments (average 80.4%). Compared with Control, LBLH had a higher RA of Lactobacillus at day 1, 16, 32, and 64, and a lower RA of Weissella from day 8 to 64. Control contained more bacteria belonging to the Enterobacteriales than LBLH up to day 16. Inoculated silage had more acetate than Control at day 32 and 64. The fungal population were similar between treatments. The enhanced development and dominance of Lactobacillus in inoculated silage led to greater accumulation of acetate and propionate, which reduced the numbers of culturable yeasts but did not markedly affect the fungal community structure. Conclusions The bacterial community composition of alfalfa stands in the filed changed over time and was affected by cutting. For the ensiling trial, inoculation modified the composition of the bacterial community of alfalfa, increasing the RA of Lactobacillus while reducing the RA of Weissella and of Enterobacteriaceae. Significance and Impact of Study Inoculation increased the RA of Lactobacillus, hampering the dominance of Weissella in the early stages of ensiling, improving antifungal compounds production and reducing the numbers of culturable yeasts.

Fungal agents emerged as post-pasteurization contamination are responsible for the spoilage in yogurt drink. In this work, the antifungal effects of some lactic acid bacteria (LAB) on the spoilage yeasts isolated from yogurt drink (Doogh) were evaluated. First, the microbial growth in the yogurt drink samples during the storage time was investigated, and the isolated microorganisms were identified using biochemical methods and sequencing of the specific amplicons. Yeasts (3-7 log CFU ml

Aims The objective of this study was to explore the potential of fermentation as a biovalorization strategy for spent tea leaves (STL), a major agrifood waste generated from the tea extraction industry. Fermentation by wine yeasts or lactic acid bacteria (LAB) has shown promising results in previous studies across various substrates. Methods and Results Konacha (green tea) STL slurries were inoculated with single strains of wine yeasts or LAB respectively. After a 48-h fermentation, changes in selected nonvolatile and volatile compositions were evaluated. Fermentation by LAB increased organic acid content by 5- to 7-fold (except Lactobacillus fermentum) and modulated the composition of major tea catechins, whereas wine yeast fermentation resulted in a 30% increase in amino acid content. Strain-specific production of specific volatile compounds was also observed such as butanoic acid (L. fermentum), isoamyl acetate (Pichia kluyveri) and 4-ethylphenol (L. plantarum). Conclusions Both volatile and nonvolatile compound compositions of Konacha STL were successfully modified via wine yeast and LAB fermentation. Significance and Impact of Study Our findings indicate that Konacha STL is a suitable medium for biovalorization by wine yeasts or LAB via the generation of commercially useful volatile and nonvolatile compounds. Future optimizations could further render fermentation an economically viable strategy for the upcycling of STL.

Aim We aim to explore the non-structural sugars from white wine grape pomace (WWGP) as the input carbon source for the co-production of multiple high-value products by the non-fastidious yeast Rhodotorula babjevae to create a sustainable and economically appealing process. Methods and Results Water extraction of unfermented, soluble sugars from WWGP yielded extracts with similar amounts of glucose and fructose, which were used to prepare a growth medium. Rhodorotula babjevae multiplied as fast on WWGP-based medium as on a reference medium but achieved higher cell dry weight (CDW) and lower intracellular triacylglycerol accumulation (22.5% vs. 28.6%) in WWGP-based medium. In addition, R. babjevae produced mannitol and arabitol and carotenoids and secreted polyol esters of fatty acids, a rare type of glycolipid as confirmed by Fourier transform-infrared, nuclear magnetic resonance and high-performance liquid chromatography analyses. Remarkably, R. babjevae consumed simultaneously both fructose and glucose when on WWGP-based medium and left glucose practically untouched in the reference medium, evidencing a fructophilic character. Conclusions Rhodorotula babjevae, a metabolic versatile yeast, proliferated on a minimally processed extract and successfully converted glucose and fructose into high-value products. Significance and Impact of Study Different chemicals with market potential can be produced through the valorization of abundant waste feedstocks generated by the wine industry to which R. babjevae can contribute.

Natural agents from microorganisms have emerged as suitable options to replace chemical preservatives in foods. In this study, the antibacterial activity of cell-free supernatant (CFS) from five native yeasts (Saccharomyces cerevisiae Lev6 and Lev30, C. pseudointermedia Lev8, Candida intermedia Lev9, C. parapsilosis Lev15) and the reference S. boulardi SSB, was evaluated against some indicator food pathogens. The generation of antimicrobials was reliant on strain-, and sugar-supplemented media, which supported yeast growth established at 30 °C and 200 rpm for 48 h. Treatment with proteinase K and catalase was unable to completely abolish the inhibitory effect, indicating that the active components are likely complex combinations of acids, proteins, hydrogen peroxide, and other metabolites. Although there was no impact on Listeria monocytogenes, exposure to CFS and extracellular fractions obtained through precipitation with methanol (PPm) at 120 °C for 60 min significantly (p < 0.05) increased the inhibitory activity against Escherichia coli, Salmonella enterica, Kosakonia cowanii, and Staphylococcus aureus, indicating that the inhibitory activity was stimulated by heat. Likewise, a synergistic inhibitory action against Listeria was obtained following the pretreatment of PPm with EDTA (ethylenediaminetetraacetic acid). These activities were yeast strain-dependent, with Lev6, Lev8, and Lev30 showing the highest activity. In addition, a heat-stable low-molecular-mass molecule under 5 kDa was detected in Lev30. Further research is required to evaluate the mode of action and characterize the composition of the released molecules in the CFS in order to develop a novel biocontrol agent based on yeasts.

Critically ill COVID-19 patients requiring mechanical ventilation in the intensive care unit are at risk of developing invasive candidiasis. In this study we aimed to (1) characterize oral cultivable mycobiota of mechanically ventilated adult COVID-19 patients in an ICU setting by sampling four distinct oral niches in two fixed time points with regards to oral health status, (2) investigate Candida spp. infections in this population, and (3) compare oral mycobiota with selected bacteriobiota strains during the observation in the ICU. We recruited 56 adult COVID-19 patients who qualified for mechanical ventilation. Patients received either standard or extended oral care procedures with tooth brushing. Oral samples were taken first within 36 h and after 7 days of intubation. Yeast-like fungi were identified by MALDI/TOF mass spectrometry. Yeast infection cases were retrospectively analyzed. Candida spp. in oral sampling was identified in 80.4% and 75.7%, C. albicans in 57.1% and 61.1%, and non-albicans Candida species in 48.2% and 47.2% patients at baseline and follow-up, respectively. There were no differences in the overall CFU counts of Candida spp. species and individual Candida species in oral samples, both at baseline and follow-up. At baseline, a higher prevalence of Candida spp. was associated with a higher identification rate of Lactobacillus spp. (64.4% vs. 27.3%, p = 0.041). At follow-up, there was a borderline lower prevalence of Candida spp. in patients with Lactobacillus spp. identified (57.1% vs. 87.0%, p = 0.057). The incidence rate of candidiasis was 5.4% and the incidence density was 3.1/1000 pds. In conclusion, non-albicans Candida species in oral samples were identified in nearly half of patients. Oral health was moderately impaired. A high incidence of yeast infections, including invasive cases, in patients hospitalized in the ICU due to COVID-19 and requiring mechanical ventilation was noted. Severe COVID-19 and disease-specific interventions within the ICU possibly played a major role promoting Candida spp. infections.

In order to address human zinc deficiency, fortifying staple foods with zinc is a safe and cost-effective solution. To ensure the nutritional properties and quality of a final product, zinc tolerance of the microorganisms involved in the fermentation is necessary. Bread, which is widely consumed, occupies a substantial place in many people’s diets, and is often based on a sourdough making process; thus, it might be an important headlining product. This study investigated the zinc tolerance of yeasts and lactic acid bacteria that are specifically suited to produce sourdough bread made with cricket powder hydrolysate and wheat flour. Amongst the yeasts, Kazachstania servazzii KAZ2 and Kazachstania unispora FM2 were only slightly affected in regard to cell growth and colony-forming ability when cultured in YPD broth spiked with 0.5 or 1 mM ZnSO4, respectively. Yarrowia lipolytica RO25 showed a higher tolerance for up to 2.5 mM zinc (ZnSO4). All the yeast strains were capable of accumulating zinc in the range between 200 and 400 fg/cell. The heterofermentative lactic acid bacterium Fructilactobacillus sanfranciscensis DG1 appeared to have a moderate zinc requirement and was homeostatically balanced, even under a high 20 mM extracellular ZnSO4 load. A better understanding of zinc homeostasis in yeast and lactic acid bacteria for food industry applications may lead to improvements in zinc fortification, which might contribute to diminishing Zn deficiencies, especially in vulnerable population groups.

Mexico is among the most important citrus fruit producers in the world. However, during storage, several problems related to fungi can arise. The most common fungal postharvest diseases detected on Citrus limon var Eureka (Italian lime) produced in the Tamaulipas state are green/blue mold (Penicillium spp.), fusarium rot (F. oxysporum, F. solani, F. proliferatum, among others), and anthracnose (Colletotrichum spp.). In this work, we selected yeasts, occurring as the natural epiphytic mycoflora of lemons or from fermented traditional products, to be tested as part of a formulation for protecting stored lemons against fungal diseases. The best-performing yeasts, labeled as LCBG-03 (Meyerozyma guilliermondii), LCBG-30 (Pseudozyma sp.), and LCBG-49 (Saccharomyces cerevisiae), were selected to test their compatibility and biocontrol performance against strains of Penicillium digitatum (AL-38), Fusarium sp. (AL-21), Colletotrichum gloeosporioides (AL-13), and Epicoccum sorghinum (H3A). Based on their in vitro performance regarding the percentage of radial growth inhibition, both applied individually or as two yeasts mixed at equal cellular concentrations, the best combinations (containing M. guilliermondii formulated with either Pseudozyma sp. or S. cerevisiae) were selected with efficacies higher than 95% in both in vitro fungal radial growth rate inhibition and on stored lemon fruits. This work contributes to the search for compatible yeast combinations with the aim to diminish the fungal losses of citrus fruits using biocontrol for citrus postharvest protection.

Pest resistance against fungicides is a widespread and increasing problem, with impact on crop production and public health, making the development of new fungicides an urgent need. Chemical analyses of a crude methanol extract (CME) of Guiera senegalensis leaves revealed the presence of sugars, phospholipids, phytosterols, guieranone A, porphyrin-containing compounds, and phenolics. To connect chemical composition with biological effects, solid-phase extraction was used to discard water-soluble compounds with low affinity for the C18 matrix and obtain an ethyl acetate fraction (EAF) that concentrates guieranone A and chlorophylls, and a methanol fraction (MF) dominated by phenolics. While the CME and MF exhibited poor antifungal activity against Aspergillus fumigatus, Fusarium oxysporum and Colletotrichum gloeosporioides, the EAF demonstrated antifungal activity against these filamentous fungi, particularly against C. gloeosporioides. Studies with yeasts revealed that the EAF has strong effectiveness against Saccharomyces cerevisiae, Cryptococcus neoformans and Candida krusei with MICs of 8, 8 and 16 μg/mL, respectively. A combination of in vivo and in vitro studies shows that the EAF can function as a mitochondrial toxin, compromising complexes I and II activities, and as a strong inhibitor of fungal tyrosinase (Ki = 14.40 ± 4.49 µg/mL). Thus, EAF appears to be a promising candidate for the development of new multi-target fungicides., This work received financial support from PT national funds (FCT/MCTES, Fundação para a Ciência e Tecnologia and Ministério da Ciência, Tecnologia e Ensino Superior) through the projects UIDB/50006/2020 of REQUIMTE/LAQV, and UIDB/04423/2020 and UIDP/04423/2020 of CIIMAR.

Efficient bioconversion of methanol, which can be generated from greenhouse gases, into valuable resources contributes to achieving climate goals and developing a sustainable economy. The methylotrophic yeast Ogataea methanolica is considered to be a suitable host for efficient methanol bioconversion because it has outstanding characteristics for the better adaptive potential to a high methanol environment (i.e., greater than 5%). This capacity represents a huge potential to construct an innovative carbon-neutral production system that converts methanol into value-added chemicals under the control of strong methanol-induced promoters. In this review, we discuss what is known about the regulation of methanol metabolism and adaptation mechanisms for 5% methanol conditions in O. methanolica in detail. We also discuss about the potential to breed "super methylotrophic yeast," which has potent growth characteristics under high methanol conditions.

There is a growing appreciation for the role that yeast play in biotransformation of flavour compounds during beverage fermentations. This is particularly the case for brewing due to the continued popularity of aromatic beers produced via the dry-hopping process. Here, we review the current literature pertaining to biotransformation reactions mediated by fermentative yeasts. These reactions are diverse and include the liberation of thiols from cysteine or glutathione-bound adducts, as well as the release of glycosidically bound terpene alcohols. These changes serve generally to increase the fruit and floral aromas in beverages. This is particularly the case for the thiol compounds released via yeast β-lyase activity due to their low flavour thresholds. The role of yeast β-glucosidases in increasing terpene alcohols is less clear, at least with respect to fermentation of brewer’s wort. Yeast acetyl transferase and acetate esterase also have an impact on the quality and perceptibility of flavour compounds. Isomerization and reduction reactions, e.g. the conversion of geraniol (rose) to β-citronellol (citrus), also have potential to alter significantly flavour profiles. A greater understanding of biotransformation reactions is expected to not only facilitate greater control of beverage flavour profiles, but also to allow for more efficient exploitation of raw materials and thereby greater process sustainability.Key points•Yeast can alter and boost grape- and hop-derived flavour compounds in wine and beer•β-lyase activity can release fruit-flavoured thiols with low flavour thresholds•Floral and citrus-flavoured terpene alcohols can be released or interconverted

Altres ajuts: acords transformatius de la UAB The function of the Saccharomyces cerevisiae Ppz1 phosphatase is controlled by its inhibitory subunit Hal3. Hal3 is a moonlighting protein, which associates with Cab3 to form a decarboxylase involved in the CoA biosynthetic pathway. Hal3 is composed by a conserved core PD region, required for both Ppz1 regulation and CoA biosynthesis, a long N-terminal extension, and an acidic C-terminal tail. Cab3 has a similar structure, but it is not a Ppz1 inhibitor. We show here that deletion or specific mutations in a short region of the N-terminal extension of Hal3 compromise its function as a Ppz1 inhibitor in vivo and in vitro without negatively affecting its ability to interact with the phosphatase. This study defines a R-K-X-VTFS- sequence whose presence explains the unexpected ability of Cab3 (but not Hal3) to regulate Ppz1 function in Candida albicans. This sequence is conserved in a subset of fungi and it could serve to estimate the relevance of Hal3 or Cab3 proteins in regulating fungal Ppz enzymes. We also show that the removal of the motif moderately affects both Ppz1 intracellular relocalization and counteraction of toxicity in cells overexpressing the phosphatase. Thus, our work contributes to our understanding of the regulation of Ppz phosphatases, which are determinants for virulence in some pathogenic fungi.

Onychomycosis was a common nail disease caused by dermatophytes, yeasts or molds. The prevalence of onychomycosis varied in different counties and it was necessary to understand the epidemiology in China.This study was conducted to investigate the epidemiology of onychomycosis in Chinese mainland in the past 30 years.A 30-year systemic review was carried out by searching publications investigating the prevalence of onychomycosis in Chinese mainland from 1991 to 2020.A total of 90 articles involving more than 40,000 onychomycosis patients were enrolled in this study. The ratio of males to females was 1:1.32. Fingernail onychomycosis was found in 36.12% cases, toenail onychomycosis in 48.31%, and both fingernail and toenail onychomycosis in 15.57%. The most common clinical type of onychomycosis was distal lateral subungual onychomycosis (60.99%), followed by total dystrophic onychomycosis (18.91%), proximal subungual onychomycosis (10.19%) and superficial white onychomycosis (9.92%). Dermatophytes (60.59%) were the most frequently isolated pathogens, followed by yeasts (30.09%), molds (7.91%) and mixed infection (1.41%). The primary pathogens in dermatophytes, yeasts and molds were Trichophyton rubrum (49.93%), Candida albicans (10.99%) and Aspergillus (3.11%), respectively. Additionally, dermatophytes were more commonly affected males than females (63.69% vs. 51.57%), and mostly involved in toenail onychomycosis (75.63%). The infection of yeasts was higher in females than males (40.97% vs. 29.52%), often causing onychomycosis in fingernail than toenail (41.03% vs. 17.08%), and it was more common in warm and humid southern regions than northern area (34.07% vs. 24.41%).The proportion of the causative agents changed over time, dermatophytes, especially T. rubrum had always been the predominant pathogen, followed by yeasts and molds. The distribution of fungal pathogens varied among clinical types, gender, infection sites and geography gender.

Oleaginous yeasts offer an interesting possibility for renewable lipid production, since the single cell oil accumulated can be based on a wide range of cheap, waste-derived carbon sources. Here, several short chain carboxylic acids and sugars commonly found in these substrates were assessed as carbon sources for Apiotrichum brassicae and Pichia kudriavzevii. While both strains were able to utilize all carbon sources employed, high volumetric lipid productivities (0.4 g/Lh) and lipid contents (68%) could be reached particularly with acetic acid as carbon source. Odd-numbered volatile fatty acids led to lower productivities and lipid contents, but the lipids contained unusually high proportions of odd-numbered fatty acids (up to 80% of total fatty acids). These fatty acids are rather uncommon in nature and might offer the possibility for various high value applications. In conclusion both strains are able to utilize a wide range of substrates potentially present in waste-derived substrates. Lipid content and volumetric lipid productivity strongly depend on the carbon source, with even-numbered volatile fatty acids resulting in the highest values. For volatile fatty acids in particular, the carbon source also strongly influences the composition of the lipids produced by the yeast strains.

Antibiotic resistance is a growing global health threat, demanding urgent responses. Tetracyclines, a widely used antibiotic class, are increasingly succumbing to antibiotic resistance; generating novel analogues is therefore a top priority for public health. Fungal tetracyclines provide structural and enzymatic diversity for novel tetracycline analogue production in tractable heterologous hosts, like yeasts, to combat antibiotic-resistant pathogens. Here, we successfully engineered

The plant phyllosphere is one of the largest sources of microorganisms, including yeasts. In bromeliads, the knowledge of yeasts is dispersed and still incipient. To understand the extent of our knowledge of the subject, this review proposes to compile and synthesize existing knowledge, elucidating possible patterns, biotechnological and taxonomic potentials, bringing to light new knowledge, and identifying information gaps. For such, we systematically review scientific production on yeasts in bromeliads using various databases. The results indicated that the plant compartments flowers, fruits, leaves, and water tank (phytotelma) have been studied when focusing on the yeast community in the bromeliad phyllosphere. More than 180 species of yeasts and yeast-like fungi were recorded from the phyllosphere, 70% were exclusively found in one of these four compartments and only 2% were shared among all. In addition, most of the community had a low frequency of occurrence, and approximately half of the species had a single record. Variables such as bromeliad subfamilies and functional types, as well as plant compartments, were statistically significant, though inconclusive and with low explanatory power. At least 50 yeast species with some biotechnological potentials have been isolated from bromeliads. More than 90% of these species were able to produce extracellular enzymes. In addition, other biotechnological applications have also been recorded. Moreover, new species have been described, though yeasts were only exploited in approximately 1% of the existing bromeliads species, which highlights that there is still much to be explored. Nevertheless, it appears that we are still far from recovering the completeness of the diversity of yeasts in this host. Furthermore, bromeliads proved to be a good ecological model for prospecting new yeasts and for studies on the interaction between plants and yeasts. In addition, the yeast community diverged among plant compartments, establishing bromeliads as a microbiologically complex and heterogeneous mosaic.

Trichomonascaceae is the largest family of ascomycetous yeast in the order Saccharomycetales. In spite of the extensive body of research on Trichomonascaceae in China, there remain new species to be discovered. Here, we describe four new species isolated from several rotting wood samples from Henan Province, Central China. Phylogenetic analysis of a combined ITS and nrLSU dataset with morphological studies revealed four new species in the Trichomonascaceae: Diddensiella luoyangensis, Sugiyamaella cylindrica, Su. robnettiae, and Zygoascus detingensis. Clustering in the Diddensiella clade, D. luoyangensis’ closest neighbour was D. transvaalensis. Meanwhile, Su. cylindrica clustered in the Sugiyamaella clade closest to Su. marilandica and Su. qingdaonensis. Also clustering in the Sugiyamaella clade, Su. robnettiae was most closely related to Su. chuxiongensis. Finally, Z. detingensis occupied a distinct and separated basal branch from the other species of the genus Zygoascus. These results indicate a high species diversity of Trichomonascaceae.

Fermentation of grape must to wine is carried out by a complex microbial mixture, which also involves spoilage yeasts of wine. The latter yeasts produce organoleptic changes that cause significant economic losses to the wine industry. SO2 is traditionally used to control this spoilage populations, but because of its harmful effects on human health, biocontrol has emerged as an alternative treatment. Although studies have been carried out to select biocontroller yeasts and examine their underlying mechanisms of action, reports on their application have not been published yet. To better understand the interaction and the successful application of biocontrol, the use of mathematical models, among other methods, is important, as they facilitate the prediction of success or failure of the antagonist. The objective of the present study was to use an existing mathematical model to obtain information about the yeast’s interaction assayed and to validate its predictive use under different physicochemical conditions during the wine fermentation, and eventually predict biocontrol kinetics. The mathematical model was applied to the fermentation conditions and provided information on the kinetic parameters of the biocontrol interaction and allowed interpretations about other parameters. The model was applied in the different physicochemical conditions for the biocontrol and did not fit correctly to experimental data, and therefore an improvement was proposed which was successful and presented new hypotheses.

Methylotrophic yeasts have been widely recognized as a promising host for production of recombinant proteins and value-added chemicals. Promoters for controlled gene expression are critical for construction of efficient methylotrophic yeasts cell factories. Here, we summarized recent advances in characterizing and engineering promoters in methylotrophic yeasts, such as Komagataella phaffii and Ogataea polymorpha. Constitutive and inducible promoters controlled by methanol or other inducers/repressors were introduced to demonstrate their applications in production of proteins and chemicals. Furthermore, efforts of promoter engineering, including site-directed mutagenesis, hybrid promoter, and transcription factor regulation to expand the promoter toolbox were also summarized. This mini-review also provides useful information on promoters for the application of metabolic engineering in methylotrophic yeasts. KEY POINTS: • The characteristics of six methylotrophic yeasts and their promoters are described. • The applications of Komagataella phaffii and Ogataea polymorpha in metabolic engineeringare expounded. • Three promoter engineering strategies are introduced in order to expand the promoter toolbox.

The yeast Komagataella phaffii (a.k.a. Pichia pastoris) harbours a unique glutamate utilization pathway in which the cytosolic enzymes glutamate dehydrogenase 2 (GDH2), aspartate aminotransferase 2 (AAT2) and phosphoenolpyruvate carboxykinase (PEPCK) catalyze the sequential conversion of glutamate to α-ketoglutarate, oxaloacetate and phosphoenolpyruvate respectively. GDH2 and PEPCK are essential for glutamate catabolism. Their synthesis is induced by autophagy during carbon starvation and are essential for cell survival. Here, we demonstrate that GDH2 and PEPCK reciprocally regulate each other's protein levels during glutamate catabolism such that GDH2 is downregulated in Δpepck and PEPCK is downregulated in Δgdh2. We further demonstrate that sequential conversion of glutamate to α-ketoglutarate and oxaloacetate by GDH2 and AAT2, respectively, is essential for PEPCK synthesis in cells metabolizing glutamate. Our studies indicate that translation of GDH2 mRNA is induced by glutamate while oxaloacetate derived from glutamate is likely to be the inducer of PEPCK mRNA translation during glutamate catabolism. Thus, GDH2- and PEPCK-catalyzed reactions are essential for ATP generation and gluconeogenesis respectively during carbon starvation and glutamate catabolism in K. phaffii. We conclude that K. phaffii harbours a unique translational regulatory circuit in which substrates of GDH2 and PEPCK act as inducers of their synthesis, a phenomenon not reported in any yeast species.

The yeasts involved in the ripening process of artisanal soft raw ewe milk Protected Designation of Origin (PDO) Torta del Casar and Queso de la Serena cheeses produced in Extremadura, Spain, were isolated throughout their ripening process, strain typed, and characterized for some important technological properties. A total of 508 yeast isolates were obtained and identified by inter-single sequence repeat anchored PCR amplification analysis and subsequent sequencing of the internal transcribed spacer ITS1/ITS2 5.8S rRNA. A total of 19 yeast species representing 8 genera were identified. Debaryomyces hansenii, Pichia kudriavzevii, Kluyveromyces lactis, and Yarrowia lipolytica were the predominant species. We selected 157 isolates, by genotyping and origin, for technological characterization. The evaluation of yeast isolates' growth under stress conditions of cheese ripening showed that 87 presented better performance. Among them, 71 isolates were not able to catabolize tyrosine to produce a brown pigment. Principal component analysis of the biochemical features of these isolates showed that 9 strains stood out, 3 K. lactis strains (2287, 2725, and 1507), 2 Pichia jadinii (1731 and 433), 2 Yarrowia alimentaria (1204 and 2150), Y. lipolytica 2495 and P. kudriavzevii 373. These strains displayed strong extracellular proteolytic activity on skim milk agar as well as an adequate enzymatic profile (strong aminopeptidase and weak protease activity), suggesting their great potential for cheese proteolysis. Extracellular lipolytic activity was mainly restricted to Yarrowia spp. isolates and weakly present in P. kudriavzevii 373 and K. lactis 2725, although enzymatic characterization by API-ZYM (bioMérieux SA) evidenced that all may contribute, at least in part, to the lipolysis process. Moreover, these strains were able to assimilate lactose, galactose, and glucose at NaCl concentrations higher than that usually found in cheese. However, lactate and citrate assimilation were limited to Y. lipolytica 2495, P. kudriavzevii 373, and P. jadinii 433, and may contribute to the alkalinizing process relevant to biochemical processes that take place in the last stages of ripening. By contrast, K. lactis strains showed acidifying capacity and β-galactosidase activity and may take part in the initial stages of ripening, together with lactic acid bacteria. Thus, considering the technological characteristics studied, the 9 selected strains presented biochemical features well suited to their potential use as adjunct cultures, alone or in combination with autochthonous starter bacteria in the cheesemaking process, to overcome the heterogeneity of these PDO cheeses, preserving their unique sensory characteristics.

This report is the first investigation of yeast biodiversity from the oligotrophic hypersaline coastal waters of the Arabian Gulf surrounding Qatar. Yeasts and yeast-like fungi, were cultured from seawater sampled at 13 coastal areas surrounding Qatar over a period of 2 years (December 2013-September 2015). Eight hundred and forty-two isolates belonging to 82 species representing two phyla viz., Ascomycota (23 genera) and Basidiomycota (16 genera) were identified by molecular sequencing. The results indicated that the coastal waters of the Qatari oligotrophic marine environment harbor a diverse pool of yeast species, most of which have been reported from terrestrial, clinical and aquatic sources in various parts of the world. Five species, i.e., Candida albicans, C. parapsilosis, C. tropicalis, Pichia kudriavzevii and Meyerozyma guilliermondii (n = 252/842; 30% isolates) are known as major opportunistic human pathogens. Fifteen species belonging to nine genera (n = 498/842; 59%) and 12 species belonging to seven genera (n = 459/842; 55%) are hydrocarbon degrading yeast and pollution indicator yeast species, respectively. Ascomycetous yeasts were predominant (66.38%; 559/842) as compared to their basidiomycetous counterparts (33.6%; 283/842). The most isolated yeast genera were Candida (28%; 236/842) (e.g., C. aaseri, C. boidinii, C. glabrata, C. intermedia, C. oleophila, C. orthopsilosis, C. palmioleophila, C. parapsilosis, C. pseudointermedia, C. rugopelliculosa, C. sake, C. tropicalis and C. zeylanoides), Rhodotorula (12.7%; 107/842), Naganishia (8.4%; 71/842), Aureobasidium (7.4%; 62/842), Pichia (7.3%; 62/842), and Debaryomyces (6.4%; 54/842). A total of eleven yeast species ( n = 38) isolated in this study are reported for the first time from the marine environment. Chemical testing demonstrated that seven out of the 13 sites had levels of total petroleum hydrocarbons (TPH) ranging from 200 to 900 µg/L, whereas 6 sites showed higher TPH levels (> 1000-21000 µg/L). The results suggest that the yeast community structure and density are impacted by various physico-chemical factors, namely total organic carbon, dissolved organic carbon and sulphur.

Yeasts are often present in floral nectar and can influence plant fitness directly (independently of pollinators) or indirectly by influencing pollinator visitation and behavior. However, few studies have assessed the effect of nectar yeasts on plant reproductive success or compared effects across different plant species, limiting our understanding of the relative impact of direct vs. indirect effects.We inoculated the nectar of six plant species in the field with the cosmopolitan yeast Metschnikowia reukaufii to analyze the direct and indirect effects on female reproductive success over 2 years. The pollinator assemblage for each species was recorded during both flowering years.Direct yeast effects on female fecundity were statistically nonsignificant for all plant species. There were significant indirect, pollinator-mediated effects on fruit production and seed mass for the two species pollinated almost exclusively by bumblebees or hawkmoths, with the direction of the effects differing for the quantity- and quality-related fitness components. There were no consistent effects of the yeast on maternal fecundity for any of the species with diverse pollinator assemblages.Effects of M. reukaufii on plant reproduction ranged from negative to neutral or positive depending on the plant species. The among-species variation in the indirect effects of nectar yeasts on plant pollination could reflect variation in the pollinator community, the specific microbes colonizing the nectar, and the order of microbial infection (priority effects), determining potential species interactions. Elucidating the nature of these multitrophic plant-pollinator-microbe interactions is important to understand complex processes underlying plant pollination.

The microbial conversion of pentoses to ethanol is one of the major drawbacks that limits the complete use of lignocellulosic sugars. In this study, we compared the yeast species Spathaspora arborariae, Spathaspora passalidarum, and Sheffersomyces stipitis regarding their potential use for xylose fermentation. Herein, we evaluated the effects of xylose concentration, presence of glucose, and temperature on ethanol production. The inhibitory effects of furfural, hydroxymethylfurfural (HMF), acetic acid, and ethanol were also determined. The highest ethanol yield (0.44 g/g) and productivity (1.02 g/L.h) were obtained using Sp. passalidarum grown in 100 g/L xylose at 32 °C. The rate of xylose consumption was reduced in the presence of glucose for the species tested. Hydroxymethylfurfural did not inhibit the growth of yeasts, whereas furfural extended their lag phase. Acetic acid inhibited the growth and fermentation of all yeasts. Furthermore, we showed that these xylose-fermenting yeasts do not produce ethanol concentrations greater than 4% (v/v), probably due to the inhibitory effects of ethanol on yeast physiology. Our data confirm that among the studied yeasts, Sp. passalidarum is the most promising for xylose fermentation, and the low tolerance to ethanol is an important aspect to be improved to increase its performance for second-generation (2G) ethanol production. Our molecular data showed that this yeast failed to induce the expression of some classical genes involved in ethanol tolerance. These findings suggest that Sp. passalidarum may have not activated a proper response to the stress, impacting its ability to overcome the negative effects of ethanol on the cells. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-022-00693-6.

Black yeasts can survive extreme conditions in food production owing to their polyextremotolerant character. However, significant strain-to-strain variation in black yeast thermoresistance has been observed. In this study, we assessed the variability in tolerance to nonthermal interventions among a collection of food-related black yeast strains. Variation in tolerance to UV light treatment, high pressure processing, sanitizers, and osmotic pressure was observed within each species. The two strains previously shown to possess high thermotolerance, Exophiala phaeomuriformis FSL-E2-0572 and Exophiala dermatitidis YB-734, were also the most HPP tolerant, but were the least halotolerant. Meanwhile, Aureobasidium pullulans FSL-E2-0290 was the most UV and sanitizer tolerant, but had been shown to have relatively low thermoresistance. Fisher's exact tests showed that thermoresistance in black yeasts was associated with HPP tolerance and inversely with halotolerance, but no association was found with UV tolerance or sanitizer tolerance. Collectively, the relative stress tolerance among strains varied across interventions. Given this variation, a broad range of different food products are susceptible to black yeast spoilage. Additionally, different strains should be selected in challenge studies specific to the intervention. (1312/2000 characters).

The diversity and ecological contribution of the fungus kingdom in the marine environment remain understudied. A recent survey in the Atlantic (Woods Hole, MA, USA) brought to light the diversity and unique biological features of marine fungi. The study revealed that black yeast species undergo an unconventional cell division cycle, which has not been documented in conventional model yeast species such as Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast). The prevalence of this unusual property is unknown. Here, I collected and identified 65 marine fungi species across 40 genera from the surface ocean water, sediment, and the surface of macroalgae (seaweeds) in the Pacific (Sugashima, Toba, Japan). The Sugashima collection largely did not overlap with the Woods Hole collection and included several unidentifiable species, further illustrating the diversity of marine fungi. Three black yeast species were isolated, two of which were commonly found in Woods Hole (Aureobasidium pullulans and Hortaea werneckii). Surprisingly, their cell division mode was dependent on cell density, and the previously reported unconventional division mode was reproduced only at a certain cell density. For all three black yeast species, cells underwent filamentous growth with septations at low cell density and immediately formed buds at high cell density. At intermediate cell density, two black yeasts (H. werneckii and an unidentifiable species) showed rod cells undergoing septation at the cell equator. In contrast, all eight budding yeast species showed a consistent division pattern regardless of cell density. This study suggests the plastic nature of the growth/division mode of marine-derived black yeast.

International audience; Mitochondria are essential to cell homeostasis, and alterations in mitochondrial distribution, segregation, and turnover have been linked to complex pathologies such as neurodegenerative diseases and cancer. Understanding how these functions are coordinated in specific cell types is a major challenge to discover how mitochondria globally shape cell functionality. In this review, we first describe how mitochondrial transport and dynamics are regulated throughout the cell cycle in yeast and in mammals. Second, we explore the functional consequences of mitochondrial transport and partitioning on cell proliferation, fate acquisition, and stemness and on the way cells adapt their metabolism. Finally, we focus on how mitochondrial clearance programs represent a further layer of complexity for cell differentiation or in the maintenance of stemness. Defining how mitochondrial transport, dynamics, and clearance are mutually orchestrated in specific cell types may help our understanding of how cells can transition from a physiological to a pathological state.

8 Páginas.-- 4 Figuras.-- 2 Tablas.-- Material suplementario, To turn table olives into appropriate carriers of beneficial bacteria and yeasts to consumers, it is essential to have reliable methods for analysing microorganisms in biofilms. This work validates the application of a non-destructive procedure to study the lactic acid bacteria and yeasts distribution in fruits during Spanish-style green table olive fermentations. Laboratory-scale fermentations were inoculated simultaneously with three Lactiplantibacillus pentosus strains (LPG1, 119, and 13B4) and two yeasts (Wickerhamomyces anomalus Y12 and Saccharomyces cerevisiae Y30), all of them natives of table olive fermentations. Data showed that L. pentosus LPG1 and yeasts W. anomalus Y12 were quite prone to colonise olive biofilms, but only the Lactiplantibacillus strain also can penetrate the epidermis of the fruit and colonise the flesh. Applying a non-destructive treatment consisting in shelling the fruits with glass beads led to obtaining similar lactic acid bacteria and yeast recovery than the classical stomacher destructive method. However, the glass bead procedure improved the quality of the metagenomics analysis (especially when using 16 S rRNA gene-based sequencing). Results show the great utility of procedures that do not destroy the fruit for studying fermented vegetable biofilms., This research was funded through the TOBE project (RTI 2018-100883-B-I00, MCIU/AEI/FEDER, UE). ELG thanks the Spanish Ministry of Science and Innovation for his FPI contract (PRE 2019-087812), while ABC thanks Junta de Andalucia for his postdoctoral contract PAIDI 2020-00162.

In recent years, research on yeasts as probiotics has gained more and more interest, which will allow the development of “new” products in the probiotics market. In this context, seventeen yeast strains isolated from Rabilé, a traditional beer produced in Burkina Faso, were assessed for their probiotic attributes. The yeast identification was performed by molecular methods, including PCR-RFLP and 5.8S-ITS region sequencing. Saccharomyces cerevisiae (14 strains) was the predominantly identified species, followed by Pichia kudriavzevii (2 strains) and Rhodotorula mucilaginosa (1 strain). Except for R. mucilaginosa, all yeast strains grew well at human temperature. The yeast strains showed high resistance when they were exposed to simulated gastrointestinal conditions. Auto-aggregation ability was between 70.20 ± 10.53% and 91.82 ± 1.96%, while co-aggregation with E. coli ranged from 24.92 ± 3.96% to 80.68 ± 9.53% and with S. enterica serovar Typhimurium from 40.89 ± 8.18% to 74.06 ± 7.94%. Furthermore, the hydrophobicity of isolated strains toward n-hexane was in the range from 43.17 ± 5.07% to 70.73 ± 2.42%. All yeast strains displayed high antioxidant capabilities, and the strains did not show hemolysis halos, such that they can be considered safe. Additionally, S. cerevisiae strains strongly inhibited the growth of foodborne pathogens. This is the first preliminary study to identify and characterize the yeast strains isolated from Rabilé with interesting probiotic properties.

Toxigenic members of Aspergillus flavus contaminate cereal grains, resulting in contamination by aflatoxin, a food safety hazard that causes hepatocellular carcinoma. This study identified probiotic strains as aflatoxin detoxifiers and investigated the changes to the grain amino acid concentrations during fermentation with probiotics in the presence of either A. flavus La 3228 (an aflatoxigenic strain) or A. flavus La 3279 (an atoxigenic strain). Generally, higher concentrations (p < 0.05) of amino acids were detected in the presence of toxigenic A. flavus La 3228 compared to the atoxigenic A. flavus La 3279. Compared to the control, 13/17 amino acids had elevated (p < 0.05) concentrations in the presence of the toxigenic A. flavus compared to the control, whereas in systems with the atoxigenic A. flavus 13/17 amino acids had similar (p > 0.05) concentrations to the control. There were interspecies and intraspecies differences in specific amino acid elevations or reductions among selected LAB and yeasts, respectively. Aflatoxins B1 and B2 were detoxified by Limosilactobacillus fermentum W310 (86% and 75%, respectively), Lactiplantibacillus plantarum M26 (62% and 63%, respectively), Candida tropicalis MY115 (60% and 77%, respectively), and Candida tropicalis YY25, (60% and 31%, respectively). Probiotics were useful detoxifiers; however, the extent of decontamination was species- and strain-dependent. Higher deviations in amino acid concentrations in the presence of toxigenic La 3228 compared to atoxigenic La 3279 suggests that the detoxifiers did not act by decreasing the metabolic activity of the toxigenic strain.

Fermentation is a promising solution to valorize cheese whey, the main by-product of the dairy industry. In Parmigiano Reggiano cheese production, natural whey starter (NWS), an undefined community of thermophilic lactic acid bacteria, is obtained from the previous day residual whey through incubation at gradually decreasing temperature after curd cooking. The aim of this study was to investigate the effect of fermentation regime (spontaneous (S) and NWS-inoculated (I-NWS)) on biofunctionalities and release of bioactive peptides during whey fermentation. In S and I-NWS trials proteolysis reached a peak after 24 h, which corresponded to the drop out in pH and the maximum increase in lactic acid. Biological activities increased as a function of fermentation time. NWS inoculum positively affected antioxidant activity, whilst S overcame I-NWS in angiotensin-converting enzyme (ACE) and DPP-IV (dipeptidyl peptidase IV) inhibitory activities. Peptidomics revealed more than 400 peptides, mainly derived from β-casein, κ-casein, and α-lactalbumin. Among them, 49 were bioactive and 21 were ACE-inhibitors. Semi-quantitative analysis strongly correlated ACE-inhibitory activity with the sum of the peptide abundance of ACE-inhibitory peptides. In both samples, lactotripeptide isoleucine-proline-proline (IPP) was higher than valine-proline-proline (VPP), with the highest content in S after 24 h of fermentation. In conclusion, we demonstrated the ability of whey endogenous microbiota and NWS to extensively hydrolyze whey proteins, promoting the release of bioactive peptides and improving protein digestibility.

[ES] El vino es una bebida fermentada que puede deteriorarse por el crecimiento de algunos microorganismos indeseables, produciendo un efecto irreversible en la calidad del vino y pérdidas económicas considerables. Por ello, el proceso de vinificación debe ser controlado a lo largo de los procesos de elaboración y almacenamiento. La estabilización microbiológica del vino es necesaria para controlar la actividad microbiana no deseada con el fin de mejorar la seguridad y calidad del vino. A día de hoy existen una multitud de soluciones y procesos tecnológicos disponibles para la estabilización microbiana del vino, pero en su mayoría afectan al perfil sensorial y calidad final del vino. El objetivo de este estudio fue la aplicación de partículas de óxido de silicio funcionalizadas con compuestos de aceites esenciales (CAE) con propiedades antimicrobianas como método de filtración para la estabilización microbiana de vino tinto. La capacidad de reducción microbiana del filtrado a través de las partículas funcionalizadas con vainillina se evaluó utilizando vino tinto inoculado con Acetobacter aceti, Lactobacillus plantarum, Dekkera bruxellensis, Zygosaccharomyces bailii y Saccharomyces cerevisiae. También se evaluó la influencia de la filtración sobre algunos parámetros fisicoquímicos del vino. Los resultados de capacidad de eliminación microbiana mostraron que los filtros funcionalizados con vainillina, no fueron capaces de eliminar los microrganismos Acetobacter aceti, Lactobacillus plantarum, y Saccharomyces cerevisiae en el vino tinto. Por otra parte, el filtrado fue capaz de reducir un orden logarítmico el microorganismo Dekkera bruxellensis y mostró una buena efectividad frente a Zygosaccharomyces bailii inoculado en vino tinto. Además, los resultados evidenciaron que, aunque tuvieron una baja efectividad sobre la mayoría de los microorganismos, los filtros no afectaban a las propiedades fisicoquímicas del vino. Nuevos CAE están siendo evaluados., [EN] Wine is a fermented beverage that can deteriorate due to the growth of some undesirable micro-organisms, producing an irreversible effect on the quality of the wine and considerable economic losses. Therefore, the winemaking process must be controlled throughout the winemaking and storage processes. Microbiological stabilization of wine is necessary to control undesirable microbial activity to improve the safety and quality of the wine. A multitude of technological solutions and processes are currently available for the microbial stabilization of wine, but most of them affect the sensory profile and final quality of the wine. The objective of this study was the application of silicon oxide particles functionalized with essential oil compounds (EOC) with antimicrobial properties as a filtration method for the microbial stabilization of red wine. The microbial reduction capacity of the filtrate through the vanillin-functionalized particles was evaluated using red wine inoculated with Acetobacter aceti, Lactobacillus plantarum, Dekkera bruxellensis, Zygosaccharomyces bailii and Saccharomyces cerevisiae. The influence of filtration on some physicochemical parameters of the wine was also evaluated. The results of microbial removal capacity showed that filters functionalized with vanillin were not able to remove the microorganisms Acetobacter aceti, Lactobacillus plantarum, and Saccharomyces cerevisiae in red wine. On the other hand, the filtrate was able to reduce by one logarithmic order the microorganism Dekkera bruxellensis and showed good effectiveness against Zygosaccharomyces bailii inoculated in red wine. Furthermore, the results showed that, although they had a low effectiveness on most of the microorganisms, the filters did not affect the physicochemical properties of the wine.

This work aimed to develop a non-dairy functional beverage fermented with probiotic strains and fortified with Brazilian red propolis (microencapsulated and extracted). The non-dairy matrix consisted of oats (75 g), sunflower seeds (175 g), and almonds (75 g). It was fermented by a starter co-culture composed of Lactiplantibacillus plantarum CCMA 0743 and Debaryomyces hansenii CCMA 176. Scanning electron microscopy analysis was initially performed to verify the integrity of the microcapsules. The viability of the microorganisms after fermentation and storage, chemical composition (high performance liquid chromatography (HPLC) and gas chromatography coupled to mass spectrometry (GC-MS) analyses), rheology, antioxidant activity, and sensory profile of the beverages were determined. After fermentation and storage, the starter cultures were well adapted to the substrate, reducing the pH (6.50 to 4) and cell count above 7.0 log CFU/mL. Lactic acid was the main organic acid produced during fermentation and storage. In addition, 39 volatile compounds were detected by gas chromatography coupled to mass spectrometry (GC-MS), including acids, alcohols, aldehydes, alkanes, alkenes, esters, ethers, phenols, terpenes, and others. The addition of propolis extract increased the antioxidant and phenolic activity and the presence of volatile esters but reduced the beverage’s acceptability. The addition of microencapsulated propolis was more associated with the presence of higher alcohols and had similar acceptance to the control beverage. The combination of a non-dairy substrate, a starter co-culture, and the addition of propolis led to the development of a probiotic beverage with great potential for health benefits.

Pigeon excreta can cause environmental and public health issues, particularly in urban and public areas. They are reservoirs of several human pathogens including fungi, bacteria, and viruses. Epidemiological data of pathogenic and opportunistic yeasts in pigeon droppings in Chon Buri, one of the most reputable tourist cities of Thailand, are scarce. The present study aimed to identify yeasts in pigeon droppings by MALDI-TOF mass spectrometry, and to study their prevalence in Chon Buri, Thailand. A total of 200 pigeon fecal samples were collected randomly from all 11 districts of Chon Buri. A sum of 393 yeast-like colonies were isolated on Sabourand’s dextrose agar and CHROMagar media. These isolates were further confirmed for their species by MALDI-TOF MS. Twenty-four yeast species belonging to 11 different genera were identified in pigeon fecal samples. Candida spp., predominantly C. krusei (14.32%), were the most prevalent yeast species. Other yeast species, including C. glabrata (12.73%), C. metapsilosis (11.93%), Lodderomyces elongisporus (10.87%), C. tropicalis (7.16%), C. albicans (5.83%), and Cryptococcus neoformans (4.77%) were identified. This study provides valuable epidemiological data and diversity of yeasts in pigeon droppings in Chon Buri, Thailand, and also supports the use of MALDI-TOF MS for yeast identification and epidemiological surveillance.

The aim of this study was to evaluate the chemical, microbiological and sensory characteristics of a Sangiovese wine aged in barrique with the addition of an unripe grape extract (UGE) as an alternative to sulfur dioxide. Three samples were considered: control wine (TQ) with free SO2 of approximately 15 mg/L; sample A with chitosan (100 mg/L) and UGE (200 mg/L); and sample B with UGE (400 mg/L). The results achieved in this work demonstrated that the UGE, either alone or in combination with chitosan, was able to maintain the color characteristics of the Sangiovese wine and its sensory quality. Moreover, the addition of UGE contributed to an early and better stabilization of the color through the formation of polymeric pigments. The microbiological stabilization was comparable to SO2 when UGE was used at 200 mg/L in combination with chitosan. The market survey conducted in the present study confirmed how the use of UGE as an alternative to sulfitation was positively accepted by consumers, who are increasingly attentive not only to the quality of the wines they select but also to the sustainability of the production processes from which they derive and to the fact that they are not harmful to human health.

Hanseniaspora opuntiae is a commonly found yeast species in naturally fermenting cocoa pulp-bean mass, which needed in-depth investigation. The present study aimed at examining effects of the cocoa isolate H. opuntiae IMDO 040108 as part of three different starter culture mixtures compared with spontaneous fermentation, regarding microbial community, substrate consumption, and metabolite production dynamics, including volatile organic compound (VOC) and phytochemical compositions, as well as compositions of the cocoa beans after fermentation, cocoa liquors, and chocolates. The inoculated H. opuntiae strain was unable to prevail over background yeasts present in the fermenting cocoa pulp-bean mass. It led to under-fermented cocoa beans after four days of fermentation, which was however reflected in higher levels of polyphenols. Cocoa fermentation processes inoculated with a Saccharomyces cerevisiae strain enhanced flavour production during the fermentation and drying steps, which was reflected in richer and more reproducible aroma profiles of the cocoa liquors and chocolates. Sensory analysis of the cocoa liquors and chocolates further demonstrated that S. cerevisiae led to more acidic notes compared to spontaneous fermentation, as a result of an advanced fermentation degree. Finally, different VOC profiles were found in the cocoa beans throughout the whole chocolate production chain, depending on the fermentation process.

In the last 20 years, a growing army of systems biologists has employed quantitative experimental methods and theoretical tools of data analysis and mathematical modeling to unravel the molecular details of biological control systems with novel studies of biochemical clocks, cellular decision-making, and signaling networks in time and space. Few people know that one of the roots of this new paradigm in cell biology can be traced to a serendipitous discovery by an obscure Russian biochemist, Boris Belousov, who was studying the oxidation of citric acid. The story is told here from an historical perspective, tracing its meandering path through glycolytic oscillations, cAMP signaling, and frog egg development. The connections among these diverse themes are drawn out by simple mathematical models (nonlinear differential equations) that share common structures and properties.

Kombucha has been gaining prominence around the world and becoming popular due to its good health benefits. This beverage is historically obtained by the tea fermentation of Camellia sinensis and by a biofilm of cellulose containing the symbiotic culture of bacteria and yeast (SCOBY). The other substrates added to the C. sinensis tea have also been reported to help kombucha production. The type as well as the amount of sugar substrate, which is the origin of SCOBY, in addition to time and temperature of fermentation influence the content of organic acids, vitamins, total phenolics, and alcoholic content of kombucha. The route involved in the metabolite biotransformation identified in kombucha so far and the microorganisms involved in the process need to be further studied. Some nutritional properties and benefits related to the beverage have already been reported. Antioxidant and antimicrobial activities and antidiabetic and anticarcinogenic effects are some of the beneficial effects attributed to kombucha. Nevertheless, scientific literature needs clinical studies to evaluate these benefits in human beings. The toxic effects associated with the consumption of kombucha are still unclear, but due to the possibility of adverse reactions occurring, its consumption is contraindicated in infants and pregnant women, children under 4-years-old, patients with kidney failure, and patients with HIV. The regulations in place for kombucha address a number of criteria, mainly for the pH and alcohol content, in order to guarantee the quality and safety of the beverage as well as to ensure transparency of information for consumers.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is widely used in clinical laboratories for routine identification of bacteria and yeasts. However, methodological difficulties are still apparent when applied to filamentous fungi. The liquid cultivation method recommended by Bruker Daltonics GmbH for identification of filamentous fungi by MALDI-TOF MS is labour intensive and time-consuming. In this study, growth of Aspergillus species on different (porous) surfaces was investigated with the aim to develop a more reliable, quicker and less laborious identification method using MALDI-TOF MS. Mycelial growth without sporulation mimicking liquid cultivation and reliable MALDI-TOF MS spectra were obtained when A. fumigatus strains were grown on and in between a polycarbonate membrane filter on Sabouraud dextrose agar. A database of in-house reference spectra was created by growing Aspergillus reference strains (mainly focusing on sections Fumigati and Flavi) under these selected conditions. A test set of 50 molecularly identified strains grown under different conditions was used to select the best growth condition for identification and to perform an initial validation of the in-house database. Based on these results, the cultivation method on top of a polycarbonate filter proved to be most successful for species identification. This method was therefore selected for the identification of two sets of clinical isolates that mainly consisted of Aspergilli (100 strains originating from Indonesia, 70 isolates from Qatar). The results showed that this cultivation method is reliable for identification of clinically relevant Aspergillus species, with 67% and 76% correct identification of strains from Indonesia and Qatar, respectively. In conclusion, cultivation of Aspergilli on top of a polycarbonate filter showed improved results compared to the liquid cultivation protocol recommended by Bruker in terms of percentage of correct identification, ease of MSP creation, time consumption, cost and labour intensity. This method can be reliably applied for identification of clinically important Aspergilli and has potential for identification of other filamentous fungi.

The rise of antibiotic resistance has increased the need for alternative ways of preventing and treating enteropathogenic bacterial infection. Various probiotic bacteria have been used in animal and human. However, Saccharomyces boulardii is the only yeast currently used in humans as probiotic. There is scarce research conducted on yeast species commonly found in kefir despite its claimed potential preventative and curative effects. This work focused on adhesion properties, and antibacterial metabolites produced by Kluyveromyces lactis and Saccharomyces unisporus isolated from traditional kefir grains compared to Saccharomyces boulardii strains. Adhesion and sedimentation assay, slide agglutination, microscopy and turbidimetry assay were used to analyze adhesion of Salmonella Arizonae and Salmonella Typhimurium onto yeast cells. Salmonella growth inhibition due to the antimicrobial metabolites produced by yeasts in killer toxin medium was analyzed by slab on the lawn, turbidimetry, tube dilution and solid agar plating assays. Alcohol and antimicrobial proteins production by yeasts in killer toxin medium were analyzed using gas chromatography and shotgun proteomics, respectively. Salmonella adhered onto viable and non-viable yeast isolates cell wall. Adhesion was visualized using scanning electron microscope. Yeasts-fermented killer toxin medium showed Salmonella growth inhibition. The highest alcohol concentration detected was 1.55%, and proteins with known antimicrobial properties including cathelicidin, xanthine dehydrogenase, mucin-1, lactadherin, lactoperoxidase, serum amyloid A protein and lactotransferrin were detected in yeasts fermented killer medium. These proteins are suggested to be responsible for the observed growth inhibition effect of yeasts-fermented killer toxin medium. Kluyveromyces lactis and Saccharomyces unisporus have anti-salmonella effect comparable to Saccharomyces boulardii strains, and therefore have potential to control Salmonella infection.

Yeasts are ubiquitous in temperate forests. While this broad habitat is well-defined, the yeasts inhabiting it and their life cycles, niches, and contributions to ecosystem functioning are less understood. Yeasts are present on nearly all sampled substrates in temperate forests worldwide. They associate with soils, macroorganisms, and other habitats and no doubt contribute to broader ecosystem-wide processes. Researchers have gathered information leading to hypotheses about yeasts' niches and their life cycles based on physiological observations in the laboratory as well as genomic analyses, but the challenge remains to test these hypotheses in the forests themselves. Here, we summarize the habitat and global patterns of yeast diversity, give some information on a handful of well-studied temperate forest yeast genera, discuss the various strategies to isolate forest yeasts, and explain temperate forest yeasts' contributions to biotechnology. We close with a summary of the many future directions and outstanding questions facing researchers in temperate forest yeast ecology. Yeasts present an exciting opportunity to better understand the hidden world of microbial ecology in this threatened and global habitat.

The 22 mitochondrial and ∼45 cytosolic tRNAs in human cells contain several dozen different post-transcriptional modified nucleotides such that each carries a unique constellation that complements its function. Many tRNA modifications are linked to altered gene expression, and deficiencies due to mutations in tRNA modification enzymes (TMEs) are responsible for numerous diseases. Easily accessible methods to detect tRNA hypomodifications can facilitate progress in advancing such molecular studies. Our laboratory developed a northern blot method that can quantify relative levels of base modifications on multiple specific tRNAs ∼10 yr ago, which has been used to characterize four different TME deficiencies and is likely further extendable. The assay method depends on differential annealing efficiency of a DNA-oligo probe to the modified versus unmodified tRNA. The signal of this probe is then normalized by a second probe elsewhere on the same tRNA. This positive hybridization in the absence of modification (PHAM) assay has proven useful for i6A37, t6A37, m3C32, and m2,2G26 in multiple laboratories. Yet, over the years we have observed idiosyncratic inconsistency and variability in the assay. Here we document these for some tRNAs and probes and illustrate principles and practices for improved reliability and uniformity in performance. We provide an overview of the method and illustrate benefits of the improved conditions. This is followed by data that demonstrate quantitative validation of PHAM using a TME deletion control, and that nearby modifications can falsely alter the calculated apparent modification efficiency. Finally, we include a calculator tool for matching probe and hybridization conditions.

Šljiva (Prunus domestica L.) je koštičava voćna vrsta vrlo cijenjena zbog poželjnih senzornih i nutritivnih svojstava plodova. Plodovi se konzumiraju svježi ili se prerađuju. Zbog relativno visokog sadržaja šećera i niže ukupne kiselosti plodovi sorte 'Stanley' predstavljaju dobar izbor za proizvodnju voćnih vina. Voćna vina proizvode se aktivnošću kvasaca u procesu alkoholne fermentacije. Obzirom da metabolizmom kvasaca nastaje čitav niz metabolita, izborom odgovarajuće vrste ili soja utječe se na tip vina koje se proizvodi. Cilj ovog rada bio je istražiti utjecaj različitih vrsta kvasaca na osnovni kemijski sastav, koncentraciju ukupnih fenola i ukupnih antocijana voćnih vina od šljive. Korištene su dvije različite vrste kvasaca Lalvin ICV D21 – Saccharomyces cerevisiae i Lalvin EC 1118 – Saccharomyces bayanus. Vina proizvedena aktivnošću kvasca ICV D21 imala su viši sadržaj alkohola, ekstrakta te ukupnih fenola i ukupnih antocijana. Rezultati ovog istraživanja ukazuju na značajan utjecaj vrste kvasca na kemijski sastav i kakvoću voćnih vina od šljive., Plum (Prunus domestica L.) is highly valued drupe because of its desirable sensory and nutritional properties. The fruits are consumed fresh or processed. The fruits of the ´Stanley´ variety are a good choice for the production of fruit wines due to their relatively high sugar content and lower total acidity. Fruit wines are produced by yeasts in the process of alcoholic fermentation. Yeast metabolism produces a number of metabolites, so we influence the type of the wine by choosing the appropriate strain. The aim of this study was to determine the influence of different yeast strains on the basic chemical composition, concentration of total phenols and total anthocyanins of plum fruit wines. Two different commercial yeasts were used, Lalvin ICV D21 - Saccharomyces cerevisiae and Lalvin EC 1118 - Saccharomyces bayanus. Wines produced under the influence of yeast ICV D21 had a higher content of alcohol, extract, total phenols and total anthocyanins. The obtained results of the research indicate a significant impact of yeast strains on the chemical composition and quality of plum fruit wines.

In this study, the effects of starch adjuncts with different fine molecular structures obtained by size-exclusion chromatography on the mashing and fermentation efficiencies of barley malts were investigated. Following fermentation, violate compounds of freshly-fermented beer samples were determined by headspace-solid-phase microextraction coupled with gas chromatography–mass spectrometry analysis (HS-SMPE-GC-MS). High performance liquid chromatography results showed that depending on their molecular structures, starch adjuncts addition significantly increased wort maltose and maltotriose content, whereas reducing the glucose content and thus both the ratios of glucose and maltotriose to that of the maltose. The whole fermentation by dry beer yeast was finished within the first 48 h and reached to equilibrium for the rest 72 h, represented by the stable soluble protein content. Results also showed that the addition of starch adjuncts resulted into increased alcohol content, which was mainly attributed to the altered glucose/maltose ratio. The HS-SPME-GC–MS results showed that whether or not with starch adjuncts addition, the composition of violate compounds were not significantly influenced, their content, on the contrary, were altered, represented by different peak heights. This study provides important information concerning the molecular effects of starch adjuncts on brewing performances of barley malts, and also provides a new pathway for choosing suitable types of adjuncts for making beer with better quality.

Apigenin is one of the most studied flavonoids and is widely distributed in the plant kingdom. Apigenin exerts important antioxidant, antibacterial, antifungal, antitumor activities, and anti-inflammatory effects in neurological or cardiovascular disease. Chalcone isomerase A (chiA) is an important enzyme of the flavonoid biosynthesis pathway. In order to enhance the apigenin production, the petunia chi A gene was transformed for Astragalus trigonus. Bialaphos survived plants were screened by PCR, dot blot hybridization and RT-PCR analysis. Also, jasmonic acid, salicylic acid, chitosan and yeast extract were tested to evaluate their capacity to work as elicitors for apigenin. Results showed that yeast extract was the best elicitor for induction of apigenin with an increase of 3.458 and 3.9 fold of the control for calli and cell suspension culture, respectively. Transformed cell suspension showed high apigenin content with a 20.17 fold increase compared to the control and 6.88 fold more than the yeast extract treatment. While, transformed T1 calli derived expressing chiA gene produced apigenin 4.2 fold more than the yeast extract treatment. It can be concluded that the highest accumulation of apigenin was obtained with chiA transgenic cell suspension system and it can be utilized to enhancement apigenin production in Astragalus trigonus.

Bloodstream infection survival is linked to timely administration of optimal antimicrobial therapy. Commercial multiplex polymerase chain reaction (PCR) assays, such as the BioFire Blood Culture Identification Panel (BCID) used for the rapid diagnosis of bloodstream infections, have significantly improved the turnaround time for optimisation of antimicrobial therapy. Reported concordance with culture-based methods and multiplex PCR analysis is high and only limited by (1) the range of targets available on the multiplex panel; and (2) the complexity of microorganisms present in the blood culture specimen. In this study, we evaluated the use of the BioFire Blood Culture Identification 2 panel (BCID2), including an expanded repertoire of targets for Gram-positive and Gram-negative bacteria, yeast and antimicrobial resistance genes compared to the BCID panel. The BCID2 panel identified microorganisms in 39/42 (92.9%) blood cultures where monomicrobial growth was detected; the three unidentified blood cultures contained organisms not included in the BCID2 panel. Polymicrobial blood culture analysis revealed a lower degree of concordance (28.6%); however, most disagreement was due to the culture-based identification of off-panel microorganisms of low clinical significance. Turnaround time, from blood culture collection to organism identification on the blood cultures correctly identified by BCID2, was 24.6 (±16.8) hours for the BCID2 panel versus 38.2 (±21.9) hours for conventional methods. Analysis of the theoretical impact of the BCID2 identification on clinical management found therapy would be altered in 45.1% (23/51) of patients. The BCID2 panel is anticipated to improve the diagnosis and antimicrobial management of patients with serious bloodstream infections.

© The Author(s) 2021., Temperature fluctuation is one of the most frequent threats to which organisms are exposed in nature. The activation of gene expression programs that trigger the transcription of heat stress-protective genes is the main cellular response to resist high temperatures. In addition, reversible accumulation and compartmentalization of thermosensitive proteins in high-order molecular assemblies are emerging as critical mechanisms to ensure cellular protection upon heat stress. Here, we summarize representative examples of membrane-less intracellular bodies formed upon heat stress in yeasts and human cells and highlight how protein aggregation can be turned into a cytoprotective mechanism., This work was supported by the Ministerio de Ciencia, Innovación y Universidades (Grant: PGC2018-099849-B-I00 to R.R. Daga) and Junta de Andalucía-FEDER-UPO (grant: UPO-1264663 to S.S.P.).

Efficient bioethanol production from lignocellulosic biomass requires thermotolerant yeasts capable of utilizing multiple sugars, tolerating inhibitors and fermenting at high temperatures. In this study, 98 thermotolerant yeasts were isolated from nine compost samples. We selected 37 yeasts that belonged to 11 species; 31 grew at 45°C; 6 strains grew at 47°C, while 9 yeasts could utilize multiple sugars. Many yeast isolates showed high ethanol production in the range of 12-24 g l-1 , with fermentation efficiencies of 47-94% at 40°C using 5% glucose. Kluyveromyces marxianus CSV3.1 and CSC4.1 (47°C), Pichia kudriavzevii CSUA9.3 (45°C) produced 21, 22 and 23 g l-1 of ethanol with efficiencies of 83, 87 and 90%, respectively, using 5% glucose. Among these yeasts, K. marxianus CSC4.1 and P. kudriavzevii CSUA9.3 exhibited high tolerance against furfural, 5-HMF, acetic acid and ethanol. These two strains produced high amounts of ethanol from alkali-treated RS, with 84 and 87% efficiency via separate hydrolysis and fermentation; 76 and 74% via simultaneous saccharification and fermentation at 47 and 45°C, respectively. Therefore, this study demonstrates compost as a potential anthropogenic niche for multiple sugar-utilizing, inhibitor-tolerant ethanologenic yeasts suitable for high-temperature ethanol production via SHF of rice straw.