Efficient PCR-based gene targeting in isolates of the nonconventional yeast Debaryomyces hansenii.

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates. It uses a simple polymerase chain reaction (PCR)-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology.

Beneficial microbes to suppress Vibrio and improve the culture performance of copepod Tigriopus japonicus Mori.

The use of beneficial microbes, i.e., probiotics, to reduce pathogens and promote the performance of the target species is an important management strategy in mariculture. This study aimed to investigate the potential of four microbes, Debaryomyces hansenii, Ruegeria mobilis, Lactobacillus plantarum, and Bacillus subtilis, to suppress Vibrio and increase survival, population growth and digestive enzyme activity (protease, lipase, and amylase) in the harpacticoid copepod, Tigriopus japonicus. Copepod, T. japonicus stock culture with an initial mean density of 50 individual/mL (25 adult male and 25 adult female) was distributed into five treatments (i.e., four experimental and a control, each with four replicates; repeated twice) using 20 beakers (100 mL capacity each). The copepods were fed a mixture of the dinoflagellate Alexandrium tamarense and the diatom Phyaeodactylum tricornutum (3 × 104 cells/mL-1). Each microbe's concentration was adjusted at 108 CFU/mL-1 and applied to the culture condition. D. hansenii, L. plantarum, and B. subtilis all improved the copepods' survival and population growth, likely by including a higher lipase activity (P < 0.05). In contrast, using R. mobilis did not improve the copepod's culture performance compared to control. B. subtilis was the most effective in decreasing the copepod's external and internal Vibrio loading. The probiotic concentrations in the copepod decreased within days during starvation, suggesting that routine re-application of the probiotics would be needed to sustain the microbial populations and the benefits they provide. Our results demonstrated that D. hansenii and B. subtilis are promising probiotics for mass copepod culture as live food for mariculture purposes.

Use of yeast isolates of cheese origin as adjunct culture in Beyaz cheese: Influence on sensorial, textural and quality characteristics.

The effects of various yeast species isolated from raw-milk cheese were evaluated in Beyaz cheese. Four batches of cheeses were produced, in which the control cheese involved only commercial starter culture while YL, DH and KL cheeses were produced with the incorporation of individual Yarrowia lipolytica, Debaryomyces hansenii and Kluyveromyces lactis, respectively. The chemical composition, microbial counts, sensory attributes, volatile compounds and textural properties of cheeses were determined on days 1, 30, and 60 during the ripening period. The results obtained demonstrated that chemical, microbial and sensory properties of cheese varied depending on yeast species. The cheese with Y. lipolytica was the most preferred and it contained more short chain fatty acids, particularly butyric acid. This result could be due to the higher fat content and advanced lipolytic activity. The ripening index of DH was found to be higher than the other cheeses, showing an advanced proteolytic activity in relation to lower hardness in the texture profile. K. lactis was associated with lactose metabolism and promoted the development of Lactococcus spp. The results highlighted a potential use of yeasts as adjunct cultures in Beyaz cheese to develop the sensory properties such as texture and flavor. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05791-3.

Selection of Debaryomyces hansenii isolates as starters in meat products based on phenotypic virulence factors, tolerance to abiotic stress conditions and aroma generation.

INTRODUCTION: Debaryomyces hansenii is a yeast widely used in meat fermentations as starter for the purpose of improving the aromatic quality of the final product. However, it has not been the subject of an extensive study regarding phenotypic characteristics important for starter selection, such as the capacity to grow at abiotic stress conditions occurring during fermentation, the ability to generate desirable aromas and the absence of virulence traits in yeasts. AIMS: The aim of this study was to screen 60 strains of D. hansenii isolated from assorted foods for their potential application as starters in dry-cured fermented sausages manufacture. METHODS: The abiotic stress factors tested were low aw and pH and high concentration of salt, acetic acid and lactic acid. The phenotypic virulence traits explored were growth at 37°C, pseudohyphal and biofilm generation, invasiveness and enzymatic activities present in virulent yeasts. The generation of desirable meat aromas was tested in models containing aroma precursors applying an olfactory analysis. A quantitative profiling of stress tolerance was used to test the potential performance of selected strains in meat fermentations. RESULTS: The results demonstrated that most strains displayed no virulence trait or were only positive for biofilm production. Moreover, the strains showed large heterogeneity regarding their tolerance to abiotic stress factors, although most of them could grow at intermediate to high levels of the traits. The sensory analysis was the criteria determining the selection of starter strains. CONCLUSIONS: The evaluation of the phenotypic traits demonstrates that D. hansenii is a safe yeast, it is able to tolerate the stress in meat fermentation and it is able to generate desirable aromas. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study confirm the adequacy of selected D. hansenii strains to be applied as starters in meat products.

Genomic features, aroma profiles, and probiotic potential of the Debaryomyces hansenii species complex strains isolated from Korean soybean fermented food.

Fermented soybean products are gaining attention in the food industry owing to their nutritive value and health benefits. In this study, we performed genomic analysis and physiological characterization of two Debaryomyces spp. yeast isolates obtained from a Korean traditional fermented soy sauce "ganjang". Both Debaryomyces hansenii ganjang isolates KD2 and C11 showed halotolerance to concentrations of up to 15% NaCl and improved growth in the presence of salt. Ploidy and whole-genome sequencing analyses indicated that the KD2 genome is haploid, whereas the C11 genome is heterozygous diploid with two distinctive subgenomes. Interestingly, phylogenetic analysis using intron sequences indicated that the C11 strain was generated via hybridization between D. hansenii and D. tyrocola ancestor strains. The D. hansenii KD2 and D. hansenii-hybrid C11 produced various volatile flavor compounds associated with butter, caramel, cheese, and fruits, and showed high bioconversion activity from ferulic acid to 4-vinylguaiacol, a characteristic flavor compound of soybean products. Both KD2 and C11 exhibited viability in the presence of bile salts and at low pH and showed immunomodulatory activity to induce high levels of the anti-inflammatory cytokine IL-10. The safety of the yeast isolates was confirmed by analyzing virulence and acute oral toxicity. Together, the D. hansenii ganjang isolates possess physiological properties beneficial for improving the flavor and nutritional value of fermented products.

Structural Characterization, Antioxidant and Antitumor Activities of the Two Novel Exopolysaccharides Produced by Debaryomyces hansenii DH-1.

Exopolysaccharides produced by edible microorganisms exhibit excellent constructive physicochemical and significant biological activity, which provide advantages for the food or pharmaceutical industries. Two novel exopolysaccharides produced by Debaryomyces hansenii DH-1 were characterized, named S1 and S2, respectively. S1, with a molecular weight of 34.594 kDa, primarily consisted of mannose and glucose in a molar ratio of 12.19:1.00, which contained a backbone fragment of α-D-Manp-(1→4)-α-D-Manp-(1→2)-α-D-Glcp-(1→3)-α-D-Manp-(1→3)-ß-D-Glcp-(1→4)-ß-D-Manp-(1→. S2, with a molecular weight of 24.657 kDa, was mainly composed of mannose and galactose in a molar ratio of 4.00:1.00, which had a backbone fragment of α-D-Manp-(1→6)-ß-D-Manp-(1→2)-α-D-Manp-(1→4)-α-D-Galp-(1→3)-ß-D-Manp-(1→6)-α-D-Manp-(1→. Both S1 and S2 exhibited good thermal stability and potent hydroxyl radical scavenging activity, with ~98%. Moreover, S1 possessed an additional strong iron-reducing capacity. In vitro antitumor assays showed that S1 and S2 significantly inhibited the proliferation of Hela, HepG2, and PC-9 cancer cells. Moreover, PC-9 was more sensitive to S1 compared with S2. The above results indicate that S1 and S2 have great potential to be utilized as natural antioxidants and candidates for cancer treatment in the food and pharmaceutical industries.

A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active ß-d-Galactosidase from Paracoccus sp. 32d.

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active ß-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.

Bioconversion study for xylitol and ethanol production by Debaryomyces hansenii: aeration, medium and substrate composition influence.

Debaryomyces hansenii has been employed to study, initially, the influence of the oxygen availability on D-xylose to xylitol fermentation, as this parameter is considered as one of the most critical variables for this bio alcohol accumulation. Apart from the air supplied in the fermentation process through the stirring vortex (0.0 v/v/min), additional aeration rates (0.1-2.0 v/v/min) effects were discussed. Furthermore, a change in the fermentative medium composition as well as a comparative analysis of D. hansenii behavior with respect to fermentation of D-glucose and D-xylose mixtures solutions, with the aim of producing both xylitol and ethanol bioproducts, were performed. For these purposes, specific growth rates, biomass productivities, specific substrate-uptake rates, overall biomass yields, specific xylitol formation rates and overall xylitol yields values have been calculated, applying a differential method to the kinetic data. Aeration influence was clearly evinced since a faster D-xylose metabolism, for aeration values close to 1.0 v/v/min, was noted. This yeast exhibited a sequential substrate consumption, firstly D-glucose and then D-xylose. The maximum xylitol yield (0.32 kg kg- 1) was obtained for 0.5 v/v/min airflow, remarking a significant reduction of this parameter for both above and below the quoted air supply value.

A physiological characterization in controlled bioreactors reveals a novel survival strategy for Debaryomyces hansenii at high salinity.

Debaryomyces hansenii is traditionally described as a halotolerant non-conventional yeast and has served as a model organism for the study of osmotolerance and salt tolerance mechanisms in eukaryotic systems for the past 30 years. However, unraveling of D. hansenii's biotechnological potential has always been difficult due to the persistent limitations in the availability of efficient molecular tools described for this yeast. Additionally, there is a lack of consensus and contradictory information along the recent years that limits a comprehensive understanding of its central carbon metabolism, mainly due to a lack of physiological studies in controlled and monitored environments. Moreover, there is little consistency in the culture conditions (media composition, temperature, and pH among others) used by different groups, which makes it complicated when trying to get prevalent conclusions on behavioral patterns. In this work, we present for the first time a characterization of D. hansenii in batch cultivations using highly controlled lab-scale bioreactors. Our findings contribute to a more complete picture of the central carbon metabolism and the external pH influence on the yeast's ability to tolerate high Na+ and K+ concentrations, pointing to a differential effect of both salts, as well as a positive effect in cell performance when low environmental pH values are combined with a high sodium concentration in the media. Finally, a novel survival strategy at very high salinity (2 M) is proposed for this yeast, as well as potential outcomes for its use in industrial biotechnology applications. TAKE AWAY: High salt concentrations stimulate respiration in Debaryomyces hansenii. Sodium exerts a stronger positive impact on cell performance than potassium. µmax is higher at a combination of low pH, high salt, and high temperature. Concentrations of 2 M salt result in slower growth but increased biomass yield. The positive effect of salts is enhanced at low glucose concentration.

Formation and characterization of biofilms formed by salt-tolerant yeast strains in seawater-based growth medium.

Yeast whole cells have been widely used in modern biotechnology as biocatalysts to generate numerous compounds of industrial, chemical, and pharmaceutical importance. Since many of the biocatalysis-utilizing manufactures have become more concerned about environmental issues, seawater is now considered a sustainable alternative to freshwater for biocatalytic processes. This approach plausibly commenced new research initiatives into exploration of salt-tolerant yeast strains. Recently, there has also been a growing interest in possible applications of microbial biofilms in the field of biocatalysis. In these complex communities, cells demonstrate higher resistance to adverse environmental conditions due to their embedment in an extracellular matrix, in which physical, chemical, and physiological gradients exist. Considering these two topics, seawater and biofilms, in this work, we characterized biofilm formation in seawater-based growth media by several salt-tolerant yeast strains with previously demonstrated biocatalytic capacities. The tested strains formed both air-liquid-like biofilms and biofilms on silicone surfaces, with Debaryomyces fabryi, Schwanniomyces etchellsii, Schwanniomyces polymorphus, and Kluyveromyces marxianus showing the highest biofilm formation. The extracted biofilm extracellular matrices mostly consisted of carbohydrates and proteins. The latter group was primarily represented by enzymes involved in metabolic processes, particularly the biosynthetic ones, and in the response to stimuli. Specific features were also found in the carbohydrate composition of the extracellular matrix, which were dependent both on the yeast isolate and the nature of formed biofilms. Overall, our findings presented herein provide a unique data resource for further development and optimization of biocatalytic processes and applications employing seawater and halotolerant yeast biofilms.Key points• Ability for biofilm formation of some yeast-halotolerant strains in seawater medium• ECM composition dependent on strain and biofilm-forming surface• Metabolic enzymes in the ECM with potential applications for biocatalysis.

Temporal differences in microbial composition of Époisses cheese rinds during ripening and storage.

Époisses is a protected designation of origin smear-ripened cheese from the Burgundy region in France. It has an orange color and a strong flavor, both of which are generated by surface microorganisms. The objective of the present study was to investigate the microbial dynamics at the surface of Époisses cheese during ripening and postmanufacturing storage at low temperatures. Rind samples were analyzed by enumeration on agar plates and by 16S rRNA gene and internal transcribed spacer amplicon sequencing. During most of the ripening process, the counts of yeasts, which corresponded to the species Debaryomyces hansenii and Geotrichum candidum, were higher than those of the aerobic acid-sensitive bacteria. Debaryomyces hansenii reached a level of about 3 × 108 cfu/cm2, and its viability strongly decreased in the late stage of ripening and during storage at 4°C. Two of the inoculated bacterial species, Brevibacterium aurantiacum and Staphylococcus xylosus, did not establish themselves at the cheese surface. At the end of ripening, among the 18 most abundant bacterial species detected by amplicon sequencing, 14 were gram-negative, mainly from genera Psychrobacter, Vibrio, Halomonas, and Mesonia. It was hypothesized that the high moisture level of the Époisses rinds, due the humid atmosphere of the ripening rooms and to the frequent washings of the curds, favored growth of these gram-negative species. These species may be of interest for the development of efficient ripening cultures. In addition, because the orange color of Époisses cheeses could not be attributed to the growth of Brevibacterium, it would be interesting to investigate the type and origin of the pigments that confer color to this cheese.

Counteraction of osmolytes on pH-induced unfolding of xylose reductase from Debaryomyces nepalensis NCYC 3413.

The stability of Debaryomyces nepalensis NCYC 3413 xylose reductase, a homodimeric enzyme recombinantly expressed and purified from E. coli Rosetta cells, was studied at different pH ranging from 5.0 to 10.0. Deactivation kinetics at different pH were studied by analyzing residual activity of the recombinant enzyme over time at 40 °C whereas conformational changes and stability dependence were investigated by using circular dichroism and differential scanning calorimetry. Four osmolytes viz. glycerol, sucrose, trehalose and sorbitol were explored for their effect on the deactivation and melting temperatures of the enzyme under neutral and extreme pH conditions. The enzyme was found to be catalytically and structurally stable at pH 7.0 with half-life of 250 min and a melting temperature of 50 °C. It was found that alteration in both secondary and tertiary structures caused enzyme deactivation in acidic pH while increased deactivation rates at alkaline pH was attributed to the variation of tertiary structure over time. Estimated thermodynamic parameters also showed that the enzyme stability was highest at neutral pH with ΔH of 348 kcal/mole and ΔG40 of 9.53 kcal/mole. All four osmolytes were effective in enhancing enzyme stability by several folds at extreme pH with sorbitol being the most efficient, which increased enzyme half-life by 11-fold at pH 10.0 and 8-fold at pH 5.0.

Tolerance to Oxidative Stress in Budding Yeast by Heterologous Expression of Catalases A and T from Debaryomyces hansenii.

The function of catalases A and T from the budding yeast Saccharomyces cerevisiae (ScCta1 and ScCtt1) is to decompose hydrogen peroxide (H2O2) to mitigate oxidative stress. Catalase orthologs are widely found in yeast, suggesting that scavenging H2O2 is crucial to avoid the oxidative damage caused by reactive oxygen species (ROS). However, the function of catalase orthologs has not yet been experimentally characterized in vivo. Here, we heterologously expressed Debaryomyces hansenii DhCTA1 and DhCTT1 genes, encoding ScCta1 and ScCtt1 orthologs, respectively, in a S. cerevisiae acatalasemic strain (cta1Δ ctt1Δ). We performed a physiological analysis evaluating growth, catalase activity, and H2O2 tolerance of the strains grown with glucose or ethanol as carbon source, as well as under NaCl stress. We found that both genes complement the catalase function in S. cerevisiae. Particularly, the strain harboring DhCTT1 showed improved growth when ethanol was used as carbon source both in the absence or presence of salt stress. This phenotype is attributed to the high catalase activity of DhCtt1 detected at the exponential growth phase, which prevents intracellular ROS accumulation and confers oxidative stress resistance.

Dynamics of microbial communities and metabolites in ganjang, a traditional Korean fermented soy sauce, during fermentation.

Six different ganjang batches were prepared and the microbial communities in the ganjang samples and raw materials (meju and solar salts), as well as the metabolites generated during fermentation were analyzed. The varying amounts of raw materials used differentially affected the microbial communities. Halophilic or halotolerant microbes derived from solar salts were abundant during middle or late fermentation. By contrast, non-halophilic microbes derived from meju were abundant mainly during early fermentation. Debaryomyces, Tetragenococcus, and Staphylococcus were abundant in all ganjang batches, which suggested that these may be the most common microbes involved in ganjang fermentation. The salt concentrations did not affect the abundance of Debaryomyces, which was abundant in all ganjang batches. Tetragenococcus was abundant in low salt ganjang, whereas Staphylococcus was abundant in high salt ganjang. Metabolite analysis revealed that carbohydrate concentrations were high in ganjang prepared using high amounts of meju. The level of lactate, which may be largely produced by Tetragenococcus, in low salt ganjang was high because of high microbial activity. The amino acid concentrations of the ganjang batches were mainly associated with meju quantity, but not salt concentration. These results indicated that the production of amino acids may be associated with indigenous proteases in meju, but not microbial activities during ganjang fermentation.

Yeast Microbiota during Sauerkraut Fermentation and Its Characteristics.

Sauerkraut is the most important fermented vegetable obtained in Europe. It is produced traditionally by spontaneous fermentation of cabbage. The aim of this study was to determine biodiversity of yeasts present during fermentation of eight varieties of cabbages (Ambrosia, Avak, Cabton, Galaxy, Jaguar, Kamienna Glowa, Manama and Ramco), as well as characterize obtained yeast isolates. WL Nutrient Agar with Chloramphenicol was used to enumerate yeast. Isolates were differentiated using RAPD-PCR and identified by sequencing of the 5.8S-ITS rRNA gene region. The volatiles production was analyzed using SPME-GC-TOFMS. Our research confirmed that during sauerkraut fermentation there is an active growth of the yeasts, which begins in the first phases. The maximal number of yeast cells from 1.82 to 4.46 log CFU g-1 occurred after 24 h of fermentation, then decrease in yeast counts was found in all samples. Among the isolates dominated the cultures Debaryomyces hansenii, Clavispora lusitaniae and Rhodotorula mucilaginosa. All isolates could grow at NaCl concentrations higher than 5%, were relatively resistant to low pH and the presence of lactic acid, and most of them were characterized by killer toxins activity. The highest concentration of volatiles (mainly esters and alcohols) were produced by Pichia fermentans and D. hansenii strains.

Cheese yeasts.

Numerous traditionally aged cheeses are surface ripened and develop a biofilm, known as the cheese rind, on their surfaces. The rind of such cheeses comprises a complex community of bacterial and fungal species that are jointly responsible for the typical characteristics of the various cheese varieties. Surface ripening starts directly after brining with the rapid colonization of the cheese surface by yeasts. The initially dominant yeasts are acid and salt-tolerant and are capable of metabolizing the lactate produced by the starter lactic acid bacteria and of producing NH3 from amino acids. Both processes cause the pH of the cheese surface to rise dramatically. This so-called deacidification process enables the establishment of a salt-tolerant, Gram-positive bacterial community that is less acid-tolerant. Over the past decade, knowledge of yeast diversity in cheeses has increased considerably. The yeast species with the highest prevalence on surface-ripened cheeses are Debaryomyces hansenii and Geotrichum candidum, but up to 30 species can be found. In the cheese core, only lactose-fermenting yeasts, such as Kluyveromyces marxianus, are expected to grow. Yeasts are recognized as having an indispensable impact on the development of cheese flavour and texture because of their deacidifying, proteolytic, and/or lipolytic activity. Yeasts are used not only in the production of surface-ripened cheeses but also as adjunct cultures in the vat milk in order to modify ripening behaviour and flavour of the cheese. However, yeasts may also be responsible for spoilage of cheese, causing early blowing, off-flavour, brown discolouration, and other visible alterations of cheese.

Reidentification and antifungal susceptibility profile of Candida guilliermondii and Candida famata clinical isolates from a culture collection in Argentina.

The aim of this work was to reidentify strains previously identified as Candida guilliermondii and Candida famata by conventional phenotypic methods conserved in a culture collection from Argentina using ribosomal DNA sequencing, ACT1 gene sequencing, and matrix-assisted laser desorption ionization - time of flight mass spectrometry (MALDI-TOF MS). In addition, we performed antifungal susceptibility tests of eight antifungal drugs commonly used in clinical treatment. We identified 68 isolates belonging to the Candida guilliermondii species complex (59 C. guilliermondii, 8 C. fermentati, and 1 Candida carpophila), 16 isolates belonging to the Candida famata species complex (8 C. famata, 6 Debaryomyces nepalensis, 1 Debaryomyces fabryi, and 1 Debaryomyces tyrocola). Although sequencing of ITS region was able to identify C. guilliermondii and D. nepalensis isolates, sequencing of ACT1 gene seems to be the most appropriate technique for differentiation between C. fermentati and C. carpophila and between members of the C. famata species complex others than D. nepalensis. MALDI-TOF MS has a good potential for the identification of these yeasts, particularly in clinical laboratories since is a rapid and easy to perform technique. Here, we report the first isolation of D. tyrocola from a human patient and the first isolation of D. nepalensis from lungs and blood of human patients. Finally, correct identification and determination of antifungal susceptibility of those closely related species could be a useful tool for clinicians to choose the most effective antifungal treatment.

Isolation, identification and screening of yeasts towards their ability to assimilate biodiesel-derived crude glycerol: microbial production of polyols, endopolysaccharides and lipid.

AIMS: To assess the ability of various newly isolated or belonging in official collections yeast strains to convert biodiesel-derived glycerol (Gly) into added-value compounds. METHODS AND RESULTS: Ten newly isolated yeast strains belonging to Debaryomyces sp., Naganishia uzbekistanensis, Rhodotorula sp. and Yarrowia lipolytica, isolated from fishes, metabolized Gly under nitrogen limitation. The aim of the study was to identify potential newly isolated microbial candidates that could produce single-cell oil (SCO), endopolysaccharides and polyols when these micro-organisms were grown on biodiesel-derived Gly. As controls producing SCO and endopolysaccharides were the strains Rhodotorula glutinis NRRL YB-252 and Cryptococcus curvatus NRRL Y-1511. At initial Gly (Gly0 ) ≈40 g l-1 , most strains presented remarkable dry cell weight (DCW) production, whereas Y. lipolytica and Debaryomyces sp. produced non-negligible quantities of mannitol and arabitol (Ara). Five strains were further cultivated at increasing Gly0 concentrations. Rhodotorula glutinis NRRL YB-252 produced 7·2 g l-1 of lipid (lipid in DCW value ≈38% w/w), whereas Debaryomyces sp. FMCC Y69 in batch-bioreactor experiment with Gly0 ≈80 g l-1 , produced 30-33 g l-1 of DCW and ~30 g l-1 of Ara. At shake-flasks with Gly0 ≈125 g l-1 , Ara of ~48 g l-1 (conversion yield of polyol on Gly consumed ≈0·62 g g-1 ) was achieved. Cellular lipids of all yeasts contained in variable concentrations oleic, palmitic, stearic and linoleic acids. CONCLUSIONS: Newly isolated, food-derived and non-previously studied yeast isolates converted biodiesel-derived Gly into several added-value metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Alternative ways of crude Gly valorization through yeast fermentations were provided and added-value compounds were synthesized.

Biocontrol of aflatoxigenic Aspergillus parasiticus by native Debaryomyces hansenii in dry-cured meat products.

Dry-cured meat products, such as dry-cured ham or dry-fermented sausages, are characterized by their particular ripening process, where a mould population grows on their surface. Some of these moulds are hazardous to the consumers because of their ability to produce mycotoxins including aflatoxins (AFs). The use of native yeasts could be considered a potential strategy for controlling the presence of AFs in dry-cured meat products. The aim of this work was to evaluate the antagonistic activity of two native Debaryomyces hansenii strains on the relative growth rate and the AFs production in Aspergillus parasiticus. Both D. hansenii strains significantly reduced the growth rates of A. parasiticus when grown in a meat-model system at different water activity (aw) conditions. The presence of D. hansenii strains caused a stimulation of AFs production by A. parasiticus at 0.99 aw. However, at 0.92 aw the yeasts significantly reduced the AFs concentration in the meat-model system. The relative expression levels of the aflR and aflS genes involved in the AFs biosynthetic pathway were also repressed at 0.92 aw in the presence of both D. hansenii strains. These satisfactory results were confirmed in dry-cured ham and dry-fermented sausage slices inoculated with A. parasiticus, since both D. hansenii strains significantly reduced AFs amounts in these matrices. Therefore, both tested D. hansenii strains could be proposed as biocontrol agents within a HACCP framework to minimize the hazard associated with the presence of AFs in dry-cured meat products.

Unveiling hákarl: A study of the microbiota of the traditional Icelandic fermented fish.

Hákarl is produced by curing of the Greenland shark (Somniosus microcephalus) flesh, which before fermentation is toxic due to the high content of trimethylamine (TMA) or trimethylamine N-oxide (TMAO). Despite its long history of consumption, little knowledge is available on the microbial consortia involved in the fermentation of this fish. In the present study, a polyphasic approach based on both culturing and DNA-based techniques was adopted to gain insight into the microbial species present in ready-to-eat hákarl. To this aim, samples of ready-to-eat hákarl were subjected to viable counting on different selective growth media. The DNA directly extracted from the samples was further subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) and 16S amplicon-based sequencing. Moreover, the presence of Shiga toxin-producing Escherichia coli (STEC) and Pseudomonas aeruginosa was assessed via qualitative real-time PCR assays. pH values measured in the analyzed samples ranged from between 8.07 ±â€¯0.06 and 8.76 ±â€¯0.00. Viable counts revealed the presence of total mesophilic aerobes, lactic acid bacteria and Pseudomonadaceae. Regarding bacteria, PCR-DGGE analysis highlighted the dominance of close relatives of Tissierella creatinophila. For amplicon sequencing, the main operational taxonomic units (OTUs) shared among the data set were Tissierella, Pseudomonas, Oceanobacillus, Abyssivirga and Lactococcus. The presence of Pseudomonas in the analyzed samples supports the hypothesis of a possible role of this microorganism on the detoxification of shark meat from TMAO or TMA during fermentation. Several minor OTUs (<1%) were also detected, including Alkalibacterium, Staphylococcus, Proteiniclasticum, Acinetobacter, Erysipelothrix, Anaerobacillus, Ochrobactrum, Listeria and Photobacterium. Analysis of the yeast and filamentous fungi community composition by PCR-DGGE revealed the presence of close relatives of Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida zeylanoides, Saccharomyces cerevisiae, Debaryomyces, Torulaspora, Yamadazyma, Sporobolomyces, Alternaria, Cladosporium tenuissimum, Moristroma quercinum and Phoma/Epicoccum, and some of these species probably play key roles in the development of the sensory qualities of the end product. Finally, qualitative real-time PCR assays revealed the absence of STEC and Pseudomonas aeruginosa in all of the analyzed samples.