The use of selected starter <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains to produce traditional and industrial cachaça: a comparative study

Six Saccharomyces cerevisiae strains from cachaça fermentation were characterized for biomass, ethanol, glycerol, and acetic acid yields, as well as productivity. Three strains presenting the best fermentation parameters were selected for cachaça production. The experiments were carried out in an industrial distillery that distills this beverage in a stainless steel column, and in a traditional distillery that uses copper alembic for distillation. The permanence of the selected strains was studied by restriction fragment analysis of mitochondrial DNA. Strains UFMG-A1007 and UFMG-A2097 were prevalent in the vats during the 5 days of the fermentation period. Non-Saccharomyces strains were isolated during the entire fermentation period. In general, the cachaças produced in the stainless steel column had the highest concentrations of volatile acidity, acetaldehyde, esters, and higher alcohols. Both cachaças did not differ statistically in aroma, taste, and overall impression. The use of these indigenous S. cerevisiae strains as starter ferment could improve the sensory attributes of both industrial and traditional cachaças.     

<Emphasis Type="Italic">Cachaça</Emphasis> yeast strains: alternative starters to produce beer and bioethanol

This work was performed to verify the potential of yeast strains isolated from cachaça distilleries for two specific biotechnological applications: beer and bioethanol production. In the beer production, the strains were tested for characteristics required in brewery practices, such as: capacity to ferment maltose and maltotriose, ability to grow at lowest temperatures, low H₂S production, and flocculation profile. Among the strains tested, two of them showed appropriate characteristics to produce two different beer styles: lager and ale. Moreover, both strains were tested for cachaça production and the results confirmed the capacity of these strains to improve the quality of cachaça. In the bioethanol production, the fermentation process was performed similarly to that used by bioethanol industries: recycling of yeast biomass in the fermentative process with sulfuric acid washings (pH 2.0). The production of ethanol, glycerol, organic acids, dry cell weight, carbohydrate consumption, and cellular viability were analyzed. One strain presented fermentative parameters similar to PE2, industrial/commercial strain, with equivalent ethanol yields and cellular viability during all fermentative cycles. This work demonstrates that cachaça distilleries seem to be an interesting environment to select new yeast strains to be used in biotechnology applications as beer and bioethanol production.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains associated with the production of cachaça: identification and characterization by traditional and molecular methods (PCR,PFGE and mtDNA-RFLP)

A study of the genetic diversity of populations of Saccharomyces cerevisiae was conducted in ten different cachaça producers (alambiques) in the southern state of Minas Gerais, Brazil. A total of 106 isolates were identified by PCR using the primer SCREC114, specific to S. cerevisiae, by pulsed-field gel electrophoresis (PFGE) and by restriction fragment polymorphism of mitochondrial DNA analysis (RFLP-mtDNA). PCR showed a product of amplification to 61 isolates, enabling a rapid identification of S. cerevisiae in different alambiques. Nine different profiles were found by PFGE; all the yeasts identified as S. cerevisiae by PCR had profiles similar to that of the marker S. cerevisiae, highlighting the specificity of primer SCREC114. RFLP-mtDNA, using four different enzymes, enabled the grouping of strains of S. cerevisiae, with 80%–100% similarity. Some alambiques that had a higher frequency of S. cerevisiae characterized by PCR and PFGE, had a lower level of genetic diversity determined by RFLP-mtDNA, indicating the ability of these strains to lead the fermentative process.     

Low-Input Fermentations of <Emphasis Type="Italic">Agave tequilana</Emphasis> Leaf Juice Generate High Returns on Ethanol Yields

During tequila production, up to 75 % w/w of the Agave plant is discarded when leaves are removed from the stem. The discarded leaves represent an extensive amount of unexploited biomass that was used here for bioethanol production in no-input fermentations, where no acid or enzymatic hydrolysis, supplementation of nutrients or standardization of carbohydrate content occur. Ethanol yield from Agave leaf juice is unaffected by sterilization but reduced if fermentation is reliant solely on endogenous microorganisms. Non-Saccharomyces yeasts, including Kluyveromyces marxianus and Candida akabanensis, proved to be more robust than standard Saccharomyces spp. and yielded up to 88 % of the theoretical maximum ethanol from leaf juice. Combining leaf and stem juice, as from a whole plant, was predicted to maximize yield at up to 19,439 L/ha of ethanol from mature plants.     

Yeasts in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> latex

Yeast abundance and species diversity in the latex of rubber tree Hevea brasiliensis (Willd. ex Juss.) Müll. Arg., on its green leaves, and in soil below the plant were studied. The yeasts present in the fresh latex in numbers of up to 5.5 log(CFU/g) were almost exclusively represented by the species Candida heveicola. This species was previously isolated from Hevea latex in China. In the course of natural modification of the latex (turned from liquid to solid form), yeast diversity increased, while yeast abundance decreased. The yeasts in thickened and solidified latex were represented by typical epiphytic and ubiquitous species: Kodamea ohmeri, Debaryomyces hansenii, Rhodotorula mucilaginosa, and synanthropic species Candida parapsilosis and Cutaneotrichosporon arboriformis. The role of yeasts in latex modification at the initial stages of succession and their probable role in development of antifungal activity in the latex are discussed.     

How much do we know about hemolytic capability of pathogenic <Emphasis Type="Italic">Candida</Emphasis> species?

Hemolytic factor production by pathogenic Candida species is considered an important attribute in promoting survival within the mammal host through the ability to assimilate iron from the hemoglobin-heme group. Hemolytic capability has been evaluated for Candida species based on hemolysis zones on plate assay, analysis of hemolytic activity in liquid culture medium, and hemolysis from cell-free culture broth. The production of hemolytic factor is variable among Candida species, where C. parapsilosis is the less hemolytic species. In general, no intraspecies differences in beta-hemolytic activities are found among isolates belonging to C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis. The production of hemolytic factor by Candida species is affected by several factors such as glucose supplementation in the culture medium, blood source, presence of erythrocytes and hemoglobin, and presence of electrolytes. On the basis of existing achievements, more researches are still needed in order to extend our knowledge about the biochemical nature of hemolytic molecules produced by distinct Candida species, the mechanism of hemolysis, and the molecular basis of the hemolytic factor expression.     

Yeast Communities of the Moscow City Soils

Yeast abundance and diversity were studied in the soils (topsoil) of Moscow city: urban soils under lawn vegetation and close to the areas of household waste disposal, as well as in zonal soddy-podzolic soils (retisols) in parks (Losiny Ostrov and Izmailovo). The numbers of soil yeasts were similar in all studied urban biocenoses (on average ~3.5 × 10³ CFU/g). From all studied soils, 54 yeast species were isolated. The highest yeast diversity was found in the soils adjacent to the areas of household waste storage. Soils from different urban sites were found to have different ratios of ascomycetous and basidiomycetous yeasts: basidiomycetes predominated in urban soils under lawn vegetation, while in the areas close to the waste disposal sites their share was considerably lower. The differences between the studied urban soils were also found in the structure of soil yeast complexes. In urban soils with high anthropogenic impact, the isolation frequency of clinically important yeast species (Candida parapsilosis, C. tropicalis, Diutina catenulata, and Pichia kudriavzevii) was as high as 35% of all studied samples, while its share in the community was 17%. The factors responsible for development of specific features of yeast communities in various urban soils are discussed in the paper.     

Identification and characterization of yeasts isolated from sedimentary rocks of Union Glacier at the Antarctica

The study of the yeasts that inhabit cold environments, such as Antarctica, is an active field of investigation oriented toward understanding their ecological roles in these ecosystems. In a great part, the interest in cold-adapted yeasts is due to several industrial and biotechnological applications that have been described for them. The aim of this work was to isolate and identify yeasts from sedimentary rock samples collected at the Union Glacier, Antarctica. Furthermore, the yeasts were physiologically characterized, including the production of metabolites of biotechnological interest. The yeasts isolated that were identified at the molecular level belonged to genera Collophora (1 isolate), Cryptococcus (2 isolates), Sporidiobolus (4 isolates), Sporobolomyces (1 isolate) and Torrubiella (2 isolates). The majority of yeasts were basidiomycetous and psychrotolerant. By cross-test assays for anti-yeast activity, it was determined that Collophora sp., Sporidiobolus salmonicolor, and Sporobolomyces roseus secreted a protein factor that kills Sporidiobolus metaroseus. The colored yeasts Sp. salmonicolor, Sp. metaroseus and Collophora sp. produced several carotenoid pigments that were identified as 2,3 dihydroxy-γ-carotene, -carotene, 4-ketotorulene, torulene β-cryptoxanthin and spirilloxanthin. Concerning analysis of mycosporines, these metabolites were only found in the yeasts Torrubiella sp. and Cryptococcus sp. T11-10-1. Furthermore, the yeasts were evaluated for the production of extracellular hydrolytic activities. Of the twelve activities analyzed, alkaline phosphatase, invertase, gelatinase, cellulase, amylase, and protease enzyme activities were detected. The yeasts Cryptococcus sp. T11-10-1 and Sporidiobolus metaroseus showed the highest number of different enzyme activities.     

Biogenic synthesis of multifunctional silver nanoparticles from <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> and <Emphasis Type="Italic">Rhodotorula mucilaginosa</Emphasis>: antifungal,catalytic and cytotoxicity activities

Silver nanoparticles (AgNPs) have several technological applications and may be synthetized by chemical, physical and biological methods. Biosynthesis using fungi has a wide enzymatic range and it is easy to handle. However, there are few reports of yeasts with biosynthetic ability to produce stable AgNPs. The purpose of this study was to isolate and identify soil yeasts (Rhodotorula glutinis and Rhodotorula mucilaginosa). After this step, the yeasts were used to obtain AgNPs with catalytic and antifungal activity evaluation. Silver Nanoparticles were characterized by UV–Vis, DLS, FTIR, XRD, EDX, SEM, TEM and AFM. The AgNPs produced by R. glutinis and R. mucilaginosa have 15.45?±?7.94 nm and 13.70?±?8.21 nm (average?±?SD), respectively, when analyzed by TEM. AgNPs showed high catalytic capacity in the degradation of 4-nitrophenol and methylene blue. In addition, AgNPs showed high antifungal activity against Candida parapsilosis and increase the activity of fluconazole (42.2% for R. glutinis and 29.7% for R. mucilaginosa), while the cytotoxicity of AgNPs was only observed at high concentrations. Finally, two yeasts with the ability to produce AgNPs were described and these particles showed multifunctionality and can represent a technological alternative in many different areas with potential applications.     

Endophytic yeasts in <Emphasis Type="Italic">Malus domestica</Emphasis> and <Emphasis Type="Italic">Pyrus communis</Emphasis> fruits under anthropogenic impact

Yeast abundance and species diversity on the surface and in inner tissues of Malus domestica and Pyrus communis fruits under high anthropogenic impact in the city of Moscow (Russia) were studied. Results demonstrated that abundance of epiphytic yeasts on the fruits increased gradually, reaching the maximum of 3.2 × 10⁴ CFU/g on mature fruits. During summer, abundance of endophytes did not change significantly (variation near 2.5 × 10³ CFU/g) until complete maturation, while in September their numbers increased to 10⁴ CFU/g. Basidiomycetous yeasts (Filobasidium wieringae, F. magnum, Rhodotorula glutinis, and Rhodosporidiobolus colostri) predominated on the fruit surface. Ascomycetous species were the most diverse group inside the fruits, which quantitatively increased through maturation. It was found that the share of opportunistic species Candida parapsilosis in internal tissues was significant during the entire period of fruit formation and development under anthropogenic impact in the city. Specific properties of epiphytic and endophytic yeast communities developing in natural ecological niches under synanthropic conditions and anthropogenic impact are discussed.     

Untangling the wine metabolome by combining untargeted SPME–GCxGC-TOF-MS and sensory analysis to profile Sauvignon blanc co-fermented with seven different yeasts

Saccharomyces cerevisiae (SC) is the main driver of alcoholic fermentation, however for aroma and flavor formation in wine, non-Saccharomyces species can have a powerful effect. This study aimed to compare untargeted volatile compound profiles from SPME–GCxGC-TOF-MS and sensory analysis data of Sauvignon blanc wine inoculated with six different non-Saccharomyces yeasts followed by SC. Torulaspora delbrueckii (TD), Lachancea thermotolerans (LT), Pichia kluyveri (PK) and Metschnikowia pulcherrima (MP) where commercial starter strains, while Candida zemplinina (CZ) and Kazachstania aerobia (KA), were isolated from wine grape environments. Each wine showed a distinct profile both sensorially and chemically. SC and CZ wines were the most distinct in both of these cases. SC wine had guava, grapefruit, banana, and pineapple aromas while CZ wine was driven by fermented apple, dried peach/apricot, and stewed fruit as well as sour flavor. Chemically over 300 unique features were identified as significantly different across the fermentations. SC wine had the highest number of esters in the highest relative concentration but all the yeasts had distinct ester profiles. CZ wine displayed the highest number of terpenes in high concentration but also produced a large amount of acetic acid. KA wine was high in ethyl acetate. TD wine had fewer esters but three distinctly higher thiol compounds. LT wine showed a relatively high number of increased acetate esters and certain terpenes. PK wine had some off odor compounds while the MP wine had high levels of methyl butyl-, methyl propyl-, and phenethyl esters. Overall, this study gives a more detailed profile of these yeasts contribution to Sauvignon blanc wine than previously reported.     

Invasive Fungal Infection in Romania: Changing Incidence and Epidemiology During Six Years of Surveillance in a Tertiary Hospital

The present study aimed to evaluate the incidence of candidemia, the trend of species distribution and the antifungal susceptibility patterns of all invasive strains of Candida spp. isolated over a 6-year period in an Eastern European University hospital. A total number of 156 isolates were reidentified by MALDI-TOF and tested for susceptibility by Sensititre^TM YeastOne?. Isolates were assigned as non-susceptible or belonging to the wild type according to the new CLSI (2017, 2018) break points and epidemiological cut-off values. C. parapsilosis (37.82%) was the most frequently isolated yeast, followed by C. albicans (26.28%). The general tendency of the species distribution during the surveyed period shifted towards an increase in C. parapsilosis and C. lusitaniae isolates. Fluconazole resistance was present in 23.52% of C. glabrata, 5.08% of C. parapsilosis and in none of the C. albicans, C. tropicalis and C. lusitaniae isolates. Echinocandin resistance was present only in 1(5.88%) C. glabrata isolate. The study emphasises the importance of monitoring local epidemiologic data and antifungal susceptibility trends due to the diversity of affected patient groups in our hospital.     

Molecular Genotyping of <Emphasis Type="Italic">Candida parapsilosis</Emphasis> Species Complex

Background

The Candida parapsilosis complex species has emerged as an important cause of human disease. The molecular identification of C. parapsilosis isolates at the species level can be helpful for epidemiological studies and then for the establishment of appropriate therapies and prophylactic measures.

Methods

The present study was undertaken to analyze 13 short tandem repeat (STR) markers (7 minisatellites and 6 microsatellites) in a global set of 182 C. parapsilosis complex isolates from different origins including invasive and superficial clinical sites.

Results

Upon the analysis of 182 strains of C. parapsilosis complex species, 10–17 haplotypes were detected for each minisatellite marker. The combination of 7 minisatellite markers yielded 121 different genotypes with a 0.995 D value. Upon the analysis of 114 isolates (68 from invasive infections and 46 from superficial infections), 21–32 genotypes were detected for each microsatellite marker. The combination of all 13 markers yielded 96 different genotypes among 114 isolates with a high degree of discrimination (0.997 D value).The same multilocus genotype was shared by isolates recovered from some patients and from the hand of theirs correspondent healthcare worker. For another patient, the same multilocus genotype of C. metapsilosis was detected in blood and skin confirming that candidemia usually arises as an endogenous infection following prior colonization.

Conclusions

These STR markers are a valuable tool for the differentiation of C. parapsilosis complex strains, to support epidemiological investigations especially studies of strain relatedness and pathways of transmission.

     

Molecular genetic polymorphism of soil yeasts of the genus <Emphasis Type="Italic">Williopsis</Emphasis> from Taiwan Island

Comparative molecular genetic study of Williopsis yeasts isolated in different world regions reveals some peculiarities of species content in Taiwan. Some Williopsis yeasts may represent novel species. In Taiwan, four of the five known Williopsis species are documented: W. saturnus, W. suaveolens, W. mrakii, and W. subsufficiens. The W. saturnus yeasts predominate in Taiwanese soils, while W. suaveolens is more frequently isolated in Europe.     

Yeast species diversity in apple juice for cider production evidenced by culture-based method

Identification of yeasts isolated from apple juices of two cider houses (one located in a plain area and one in an alpine area) was carried out by culture-based method. Wallerstein Laboratory Nutrient Agar was used as medium for isolation and preliminary yeasts identification. A total of 20 species of yeasts belonging to ten different genera were identified using both BLAST algorithm for pairwise sequence comparison and phylogenetic approaches. A wide variety of non-Saccharomyces species was found. Interestingly, Candida railenensis, Candida cylindracea, Hanseniaspora meyeri, Hanseniaspora pseudoguilliermondii, and Metschnikowia sinensis were recovered for the first time in the yeast community of an apple environment. Phylogenetic analysis revealed a better resolution in identifying Metschnikowia and Moesziomyces isolates than comparative analysis using the GenBank or YeastIP gene databases. This study provides important data on yeast microbiota of apple juice and evidenced differences between two geographical cider production areas in terms of species composition.     

Comparison of Killing Activity of Micafungin Against Six <Emphasis Type="Italic">Candida</Emphasis> Species Isolated from Peritoneal and Pleural Cavities in RPMI-1640, 10 and 30% Serum

Currently echinocandins are recommended in Candida peritonitis and pleuritis. We determined micafungin killing rates (k values) at therapeutic concentrations (0.25–2 mg/L) in RPMI-1640 with and without 10 and 30% serum mimicking in vivo conditions against six Candida species isolated from peritoneal and pleural fluid. In RPMI-1640, micafungin was fungicidal against C. glabrata, C. krusei and C. kefyr within 2.27?±?10.68, 2.69?±?10.29 and 3.10?±?4.41 h, respectively, while was fungistatic against C. albicans, C. tropicalis and C. parapsilosis. In 10% serum, ≥?0.25, ≥?0.5, ≥?0.5 and ≥?1 mg/L micafungin produced positive k values (killing) for all C. albicans, C. glabrata, C. kefyr and C. krusei, respectively. In 30% serum, 2 mg/L micafungin produced killing against all C. albicans, C. glabrata and C. kefyr isolates, but was ineffective against C. krusei, C. parapsilosis and 2 of 3 C. tropicalis. Micafungin exposure should be increased against non-albicans species to eradicate fungi from peritoneal and pleural cavities.     

Population dynamics and metabolite analysis of yeasts involved in a Chinese miscellaneous-flavor liquor fermentation

Baiyunbian liquor, the most popular miscellaneous-flavor liquor in China, is mainly produced in four successive fermentation batches every year. Sensory analysis based on the trained expert panel test revealed that the raw Baiyunbian liquor distilled from the last two fermentation batches always has higher quality than that distilled from the first two batches. In this study, the yeast composition, volatile compounds, and fermentation conditions associated with each fermentation batch were monitored. Significant differences were observed in the yeast community structure and fermentation parameters among different batches, which influenced the volatile profiles of the resulting liquors. Redundancy discriminate analysis and single-strain fermentation revealed that Pichia kudriavzevii and Candida humilis play important roles in enriching volatile compounds. Saccharomyces cerevisiae and Zygosaccharomyces bailii were the dominant yeasts in the last two fermentation batches. The sensorial characteristics of the liquors produced by single-strain fermentation were also analyzed. This study provides an in-depth understanding of the factors that influence liquor production and is beneficial in the development of new fermentation techniques with stable liquor quality.     

Comprehensive analysis of fungal diversity and enzyme activity in <Emphasis Type="Italic">nuruk</Emphasis>, a Korean fermenting starter,for acquiring useful fungi

Nuruk is a fermenting starter that is involved in the production of alcoholic beverages, and has been used in South Korea for a very long time. To analyze the fungal diversity, we collected a total of 59 nuruk samples from several companies and persons in 2013 to 2014, and obtained 364 isolates. All of the single isolated fungi were identified, both morphologically and molecularly, based on the sequences of ribosomal RNA gene [18S, ITS1-5.8S-ITS2, and 26S (D1/D2 region)]. In 46 nuruk samples out of 59 (78%), Saccharomycopsis fibuligera, a dimorphic yeast, was most frequently isolated. Among the filamentous fungi, Aspergillus and Lichtheimia were found in more than 50% of the samples with lower colony forming unit (CFU/g of sample) than those of yeasts. The yeasts S. fibuligera and Wickerhamomyces anomalus were counted with maximum 1.3–1.8 × 10⁸ CFU/g. Among Mucorales fungi, Lichtheimia and Mucor were isolated in much higher numbers than Rhizopus and Rhizomucor. Overall, the home-made nuruks tend to contain more diverse filamentous fungi than the commercial nuruks. To acquire industrially useful filamentous fungi and yeasts, we analyzed the enzyme activities of α-amylase, glucoamylase and acid protease associated with brewing properties for 131 strains. Aspergillus oryzae and S. fibuligera had high α- and glucoamylase activities and most isolates of Lichtheimia ramosa had high acid protease activity. For further applications, 27 fungal strains were chosen based on isolation frequencies from nuruk, and the ability to produce useful enzyme.