Zygosaccharomyces Bisporus Research Articles

Wine Barrel Biofilm as a Source of Yeasts with Non-Conventional Properties.

This study investigated the main microbial groups characterizing the interior surface of oak barrels from different years (1890, 1895, 1920, 1975, 2008) used in the production of vino cotto. The yeasts were characterized for the following properties: γ-aminobutyric acid (GABA) production, antioxidant activity, air-liquid interfacial biofilm formation, and anthocyanin adsorption capacity. Community-level physiological profile analysis revealed that the microbial communities inside the barrels used the tested carbon sources in different manners. The following yeast species were identified: Millerozyma farinosa, Zygosaccharomyces bisporus, Wickerhamiella versatilis, Zygosaccharomyces bailii, Starmerella lactis-condensi, and Zygosaccharomyces rouxii. All the strains were able to produce GABA, and S. lactis-condensi, Z. bisporus and Z. rouxii were the highest producers (more than 600 mg/L). The Z. rouxii and Z. bailii strains showed the highest antioxidant activity. Only seven strains out of ten M. farinosa formed air-liquid interfacial biofilm. None of the M. farinosa strains adsorbed anthocyanins on their cell wall. The other strains adsorbed anthocyanins in a strain-dependent way, and the highest adsorption was observed for the W. versatilis strains. The yeasts isolated in this study could be used to increase the functional properties and the quality of fermented foods and beverages.

Symbiosis of acetic acid bacteria and yeast isolated from black tea fungus mimicking the kombucha environment in bacterial cellulose synthesis

The symbiotic effect of acetic acid bacteria and yeast on bacterial cellulose (BC) synthesis in kombucha was explored. Firstly, the optimal culture ratio of acetic acid bacteria and yeast was optimised through single factor and orthogonal test. The results showed that when Komagataeibacter intermedius:Brettanomyces bruxellensis:Zygosaccharomyces bisporus ratio was 1:10:10, and the inoculation amounts of K. intermedius, B. bruxellensis, and Z. bisporus were 104, 105, and 105 CFU/mL, respectively, the yield of BC was the highest, and the dry basis was 5.51 g/L. It was determined that the metabolites of B. bruxellensis and Z. bisporus could promote the synthesis of BC by K. intermedius. In addition, the composition of yeast filtrate was analysed by amino acid analyser, gas chromatography-mass spectrometry (GC-MS), and high performance liquid chromatograph (HPLC). Results showed that 16 amino acids were detected in yeast filtrate, and cysteine was only detected in yeast filtrate. The increase in isoleucine before and after fermentation was the highest, which was 11.64 times that of the control group. The increase in aspartic acid and glycine were second and third, accounting for 60.00 and 41.67%, respectively. The main volatile substances were alcohols, accounting for 84.89%, of which the relative content of ethanol was the highest at 77.35%. The relative contents of 3-methyl-1-butanol and phenylethanol were also high, accounting for 4.13 and 3.14%, respectively. Tartaric, citric, and succinic acids were detected in the yeast filtrate. The chemical species did not change before and after fermentation, but the content decreased. Vitamins B2 and B6 were detected in yeast filtrate, and the species and content did not change significantly before and after fermentation. A theoretical basis for kombucha fermentation and BC synthesis was provided.

Identification and validation of core microbes associated with key aroma formation in fermented pepper paste (Capsicum annuum L.)

Fermented peppers are usually obtained by the spontaneous fermentation of microorganisms attached to fresh peppers, and the variable microbial composition would lead to inconsistencies in flavor between batches. To demonstrate the roles of microorganisms in flavor formation, the core microbes closely associated with the key aroma compounds of fermented pepper paste were screened and validated in this study. Lactobacillus was the dominant bacterial genus in fermented pepper paste, whereas the main fungal genera were Alternaria and Kazachstania. Nine strains of the genera Lactobacillus, Weissella, Bacillus, Zygosaccharomyces, Kazachstania, Debaryomyces, and Pichia were isolated from fermented pepper paste. Eleven key aroma compounds were identified using gas chromatography combined with olfactometry and relative odor activity values. Correlation analysis showed that Zygosaccharomyces and Kazachstania were positively correlated with the majority of the key aroma compounds, whereas Lactobacillus was negatively correlated with them. Thus, Zygosaccharomyces and Kazachstania were identified as core genera associated with the key odorants. Finally, Zygosaccharomyces bisporus, Kazachstania humilis, and Lactiplantibacillus plantarum were used as starter cultures for fermented peppers, confirming that Z. bisporus and K. humilis were more beneficial for the key aroma compounds (e.g., acetate, linalool, and phenyl ethanol) rather than L. plantarum. This study contributed to understanding the flavor formation mechanism and provided references for the quality control of food fermentation.

Microbial Diversity and Interaction Specificity in Kombucha Tea Fermentations.

ABSTRACTDespite the popularity of kombucha tea, the distribution of different microbes across kombucha ferments and how those microbes interact within communities are not well characterized. Using metagenomics, comparative genomics, synthetic community experiments, and metabolomics, we determined the taxonomic, ecological, and functional diversity of 23 distinct kombuchas from across the United States. Shotgun metagenomic sequencing demonstrated that the bacterium Komagataeibacter rhaeticus and the yeast Brettanomyces bruxellensis were the most common microbes in the sampled kombucha communities. To determine the specificity of bacterium-yeast interactions, we experimentally quantified microbial interactions within kombucha biofilms by measuring densities of interacting species and biofilm production. In pairwise combinations of bacteria and yeast, B. bruxellensis and individual strains of Komagataeibacter spp. were sufficient to form kombucha fermentations with robust biofilms, but Zygosaccharomyces bisporus, another yeast found in kombucha, did not stimulate bacteria to produce biofilms. Profiling the spent media of both yeast species using nuclear magnetic resonance spectroscopy suggested that the enhanced ability of B. bruxellensis to ferment and produce key metabolites in sucrose-sweetened tea may explain why it stimulates biofilm formation. Comparative genomics demonstrated that Komagataeibacter spp. with >99% genomic similarity can still have dramatic differences in biofilm production, with strong producers yielding five times more biofilm than the weakest producers.IMPORTANCE Through an integration of metagenomic and experimental approaches, our work reveals the diversity and nature of interactions among key taxa in kombucha microbiomes through the construction of synthetic microbial pairs. Manipulation of these microbes in kombucha has the potential to shape both the fermentation qualities of kombucha and the production of biofilms and is valuable for kombucha beverage producers, biofilm engineers, and synthetic ecologists.

Microbiological contamination profile in soft drinks.

Soft drinks are food matrices propitious to the growth of acidophilic bacteria, yeasts, and filamentous fungi due to their pH, water activity, and the presence of nutrients. Off-flavor, clouding, and package stuffing are the only parameters producers have to detect spoilage when it is often too late for the brand's reputation. In this work, microbiological analyses were performed on non-alcoholic beverages of Brazilian and Bolivian brands. As a result, Gram-positive, Gram-negative, yeast, and filamentous fungi were isolated. Zygosaccharomyces bisporus yeast was isolated from different flavored stuffed products, and Gluconacetobacter liquefaciens and Brevibacillus agri were isolated from packages without visible signs of deterioration. These microorganisms were identified by MALDI-TOF. For products with visible growth of filamentous fungi, microscopic identification keys identified Aspergillus flavus, Penicillium citrinum, Paecilomyces niveus, and Paecilomyces variotii. These work's findings reflect a failure to sanitize raw materials since the isolates' primary origin is the soil and the water, pointing to the lack of process control in soft drinks.

Functional Biodiversity of Yeasts Isolated from Colombian Fermented and Dry Cocoa Beans.

Yeasts play an important role in the cocoa fermentation process. Although the most relevant function is the degradation of sugars and the production of ethanol, there is little understanding of the enzyme activities and attributes that allow them to survive even after drying. The present study explored the functional biodiversity of yeasts associated with Criollo Colombian cocoa fermented beans, able to survive after drying. Twelve species belonging to 10 genera of osmo-, acid-, thermo-, and desiccation-tolerant yeasts were isolated and identified from fermented and dry cocoa beans, with Pichia kudriavzevii and Saccharomyces cerevisiae standing out as the most frequent. For the first time, we reported the presence of Zygosaccharomyces bisporus in cocoa fermented beans. It was found that resistance to desiccation is related to the different degradation capacities of fermentation substrates, which suggests that associative relationships may exist between the different yeast species and their degradation products. Besides, the increased thermotolerance of some species was related to the presence of polyphenols in the medium, which might play a fundamental role in shaping the microbial community composition.

Identification and Characterization of Non-Saccharomyces Species Isolated from Port Wine Spontaneous Fermentations.

In winemaking, non-Saccharomyces yeast species contribute important organoleptic complexity. Current interest focuses on abundant and dominant strains characteristically present in the early phase of spontaneous alcoholic fermentations. Non-Saccharomyces species are particularly relevant in Port wine production such that the fermentation is prematurely stopped, after the metabolism of only one half of the available sugar, through fortification with aguardente. This work aimed to isolate, identify and characterize non-Saccharomyces species present in spontaneously fermenting Port. To accomplish these goals, yeasts were isolated from a selection of frozen must samples (2012–2016 harvests), using a pre-screening process choosing only the best candidates based on the organoleptic quality of the corresponding fortified wine. From five hundred non-Saccharomyces isolates, twelve species were identified. The three most abundant species, Hanseniaspora uvarum, Lachancea thermotolerans, and Metschnikowia pulcherrima, representing 89% of the isolates, exhibited particularly high diversity with high growth performance variability when exposed to typical stress conditions associated with common enological parameters. Less abundant species included Issatchenkia orientalis, Torulaspora delbrueckii, Hanseniaspora vineae, Hanseniaspora osmophila, Candida zemplinina, Rhodotorula mucilaginosa, Hanseniaspora guilliermondii, Issatchenkia occidentalis, and Zygosaccharomyces bisporus. This is the first study providing insights into the identification and characterization of non-Saccharomyces species responsible for spontaneous Port wine production.

Modeling of yeast thermal resistance and optimization of the pasteurization treatment applied to soft drinks

Yeast are usually responsible for spoilage of soft drinks and fruit beverages, because of the particular characteristics of these products (low pH, high C/N ratio). The microbial stability is guaranteed by thermal treatments. However, excessive heat treatments can affect food sensorial quality. In this work the thermal resistance of different yeasts strains (seven belonging to the species Saccharomyces cerevisiae and six belonging to the species Kluyveromyces marxianus, Zygosaccharomyces bisporus, Z. mellis, Z. rouxii, Schizosaccharomyces pombe and Saccharomycodes ludwigii) was assessed in a model system. The results showed non-linear death curves and a high variability also within the same species. The most resistant strain, belonging to the species S. cerevisiae, was chosen for further experiments in orange juice based industrial beverages: first, death curves were performed; then, the probability of beverage spoilage in relation to process parameters (initial inoculum, temperature, treatment time) was evaluated using a logistic regression model. Finally, a cross-validation was performed to investigate the predictive capability of the fitted model. Pasteurization in the soft drink industry is commonly applied according to parameters defined several decades ago, which does not consider the successive findings concerning microbial physiology and stress response, the process improvement and the more recent tools provided by predictive microbiology. In this perspective, this study can fill a gap in the literature on this subject, going to be a basis for optimizing thermal processes. In fact, the data obtained indicated an interesting possibility for food industry to better modulated (and even reduce) thermal treatments, with the aim to guarantee microbial stability while reducing thermal damage and energy costs.

Effect of chlorine dioxide in reduction of harmful microbes on fermented hot pepper paste.

In this study, the in vitro effects of chlorine dioxide (ClO2) in growth reduction against Candia glaebosa, Zygosaccharomyces bisporus, Saccharomycopsis capsularis and Pichia pastoris involving in deterioration of fermented hot pepper paste were studied to assess the applicability of chlorine dioxide to preparation of fermented hot pepper paste, and the concentration of ClO2 required for destruction of harmful microorganisms through the fumigation of fermented hot pepper paste was evaluated. ClO2 was treated by using ClO2 generator for 15min. C. glaebosa, Z. bisporus and S. capsularis were reduced by ClO2 concentration dependent and not detected by ClO2 over 10ppmV, whereas the P. pastoris was significantly perished by the treatment of ClO2 over 30ppmV. We suggest that the ClO2 fumigation in stages of the preparation, disintegration, and fermentation of the paste made of fermented hot pepper might be useful for control of harmful microbes therein.

Fermentative capabilities of native yeast strains grown on juices from different Agave species used for tequila and mezcal production.

The Asparagaceae family is endemic from America, being the Agave genus the most important. The Agave species possess economic relevance and are use as raw material to produce several distilled alcoholic beverages, as bacanora, tequila, and mezcal. The fermentation process has been carry out either spontaneously or by adding a selected yeast strain. The latter is generally responsible for the production of ethanol and volatile compounds. This study comprised five Agave species (A. angustifolia, A. cupreata, A. durangensis, A. salmiana, and A. tequilana) and eight endogenous yeast strains: five of them were non-Saccharomyces (Torulaspora delbrueckii, Zygosaccharomyces bisporus, Candida ethanolica, and two Kluyveromyces marxianus) and three Saccharomyces cerevisiae strains. The results showed that the S. cerevisiae strains were not able to grow on A. durangensis and A. salmiana juices. The Kluyveromyces marxianus strains grew and fermented all the agave juices and displayed high ethanol production (48-52gL-1) and volatile compounds. The ethanol production was higher on A. angustifolia juice (1.1-2.8-fold), whereas the volatile compound was dependent on both yeast strain and the Agave species. The use of endogenous non-Saccharomyces yeast strains is feasible, as they may outperform S. cerevisiae regarding the production of fermented beverages from agave plants with a high content of ethanol and aromatic compounds. Graphical abstract.

English

English

The importance of antioxidants and plasma membrane ion transporters which play role in cellular response against salt induced oxidative stress

Abstract Salt-induced oxidative stress accounts for redox imbalance and cell death in the food spoilage yeasts. However, the physiological mechanisms underlying this imbalance and succeeding effective tolerance are still poorly understood. To elucidate this cellular protective mechanism governing in moderately halotolerant yeasts, Zygosaccharomyces bisporus was analyzed under sub-lethal conditions of sodium salt. For most stress intervals; high activity of enzymic antioxidants suggested their broad panorama in rescuing salt prompted apoptosis, attenuating intracellular degenerative reactive species with simultaneous reversal of characteristic apoptotic changes as nuclear fragmentation and chromatin condensation. In addition, the synergistic role of cell membrane cation transporters; plasma membrane ATPase and Na+/H+ like antiporter have been investigated by quantifying the external pH of the medium under stress, with the aid of specific ion transporter inhibitors like N,N'-Dicyclohexylcarbodiimide, sodium azide, and amiloride respectively. Taken together, the major outcomes from this study provide the evidence of salt imposed cell death by triggering unbalanced redox status via several key mitochondrial reactive species, allied with yeast defensive pleiotropic manipulations like antioxidants, expulsion of toxic Na+ by Nha transporters and re-establishing the optimal growth pH in alliance with PM-ATPase as a stress response resulting redox homeostasis restoration and osmoregulation. This study critically claims a proteostatic crosstalk mechanism between mitochondria and the cytosol, thus plays an important role in cell survival and advance halo-tolerance. Thereby, this inter-relationship can be further exploited for biotechnological and industrial applications in spoilage yeasts.

Physiological Basis for the Tolerance of Yeast Zygosaccharomyces bisporus to Salt Stress

Zygosaccharomyces bisporus is a moderately halotolerant yeast isolated from highly sugary and salty foods. We performed various evident biochemical and in vivo experiments as first of its kind to sketch out the possible overlay of salt tolerance mechanism in this model organism. The growth and survival curve analysis revealed that 1.0 M NaCl concentration (sublethal) enacts growth inhibitory effects with prompting immediate delay in cell division cycle; however, yeast cells adopted modified stress physiologically with further stretched stress spans which was accompanied by an upsurge in the level of cellular metabolites such as trehalose (reserve carbohydrate) and chiefly glycerol (polyols) as major compatible osmolytes, suggesting their role in defense mechanism against osmotic stress. To further elucidate the relation of osmotic stress cell physiology to salinity, thiobarbituric acid reactive substances, protein carbonyl, and reduced glutathione content were measured in salt-stressed cells demonstrating positive correlation of reactive oxygen species generation in Z. bisporus with an elevated concentration of lipid and protein oxidation, thereby damaging cell membrane and eventually causing cell death. We assessed NaCl exposure sourcing increased intracellular reactive oxygen species concentration, by an electron transfer-based colorimetric cupric-reducing antioxidant capacity assay justifying that cellular total antioxidant capacity which uses all the combined antioxidant activities present within vitamins, proteins, lipids, and glutathione reverses these deleterious stress effects. Henceforth, performance of Z. bisporus MTCC 4801 mounted because of stress regime seems to be multifactorial.

Fermentative aptitude of non-Saccharomyces wine yeast for reduction in the ethanol content in wine

Over the last few decades, there has been a progressive increase in the ethanol content in wines due to global climate change and to the new wine styles that are associated with increased grape maturity. This increased ethanol content can have negative consequences on the sensory properties of the wines, human health, and economic aspects. Several microbiological approaches for decreasing the ethanol content have been suggested, such as strategies based on genetically modified yeasts, the adaptive evolution of yeasts, and the use of non-Saccharomyces yeast. In the present study, we investigated the interspecies and intraspecies variability of some non-Saccharomyces wine yeast species under anaerobic fermentation conditions. Across different grape juices and fermentation trials, Hanseniaspora uvarum, Zygosaccharomyces sapae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus promoted significant reductions in ethanol yield and fermentation efficiency in comparison with Saccharomyces cerevisiae. The diversion of alcoholic fermentation and the abundant formation of secondary compounds might explain the marked reduction in ethanol yield, as determined through the segregation of the majority of the strains according to their species attributes observed using principal component analysis. These data suggest that careful evaluation of interspecies and intraspecies biodiversity can be carried out to select yeast that produces low-ethanol yields.

Evolution of microbiological and chemical parameters during red wine making with extended post-fermentation maceration

The aim of the present work was to investigate the microbiological, chemical, and sensory characteristics of red wine subjected to post-fermentation maceration that was extended to 90days. For this purpose, the ‘Aglianico di Taurasi’ grape was used as a case study. The total yeast concentration increased until day 40 of maceration and decreased thereafter, whereas the concentration of lactic acid bacteria slightly increased. Dekkera/Brettanomyces spp. and acetic acid bacteria were not detected. The yeast community was composed of Saccharomyces cerevisiae, Zygosaccharomyces bisporus, Metschnikowia pulcherrima, Hanseniaspora guilliermondii, Hanseniaspora uvarum, Pichia guilliermondii, Aureobasidium pullulans and Debaryomyces carsonii. Nine S. cerevisiae strains were detected at high levels at different times of maceration.The results of all the conventional chemical analyses of the wines were in agreement with the regulations of commercial production and, interestingly, the changes in terms of concentration demonstrated the presence of yeast and LAB populations that were not only alive but also in a metabolically active state until day 90 of maceration. The alcohol and glycerol contents slightly increased until day 90. The concentrations of malic acid decreased, whereas those of lactic acid slightly increased throughout the maceration process.Furthermore, different durations of maceration resulted in significant differences in the total polyphenol content, which was higher at 40–50days. The main phenolic compounds were benzoic and cinnamic acids and catechins. Interestingly, the highest ratio between (+)-catechin and (−)-epicatechin was found on day 40. In addition, the highest antioxidant activity was observed between days 40 and 50. The concentration of volatile organic compounds, which were mainly represented by alcohols, increased until the end of the maceration process. Sensory analysis revealed that samples that were subjected to maceration for a long period of time showed the highest odour and taste complexity and no off-odours and/or off-flavours were detected. These data confirmed that extending post-fermentation maceration to 90days has no negative impact on the microbiological, chemical and sensory composition of wines, but affects the polyphenol content and potential health benefits of the resulting wine.

Investigating of yeast species in wine fermentation using terminal restriction fragment length polymorphism method

The objective of this study was to examine the potential of terminal restriction fragment length polymorphism (T-RFLP) in monitoring yeast communities during wine fermentation and to reveal new information on yeast community of Chinese enology. Firstly, terminal restriction fragment (TRF) lengths database was constructed using 32 pure yeast species. Ten of these species were firstly documented. The species except for Candida vini, Issatchenkia orientalis/Candida krusei, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces cerevisiae, Saccharomyces kudriarzevii and Zygosaccharomyces bisporus could be distinguished by the T-RFLP targeting 5.8S-ITS rDNA. Moreover, the yeast communities in spontaneous fermentation of Chardonnay and Riesling were identified by T-RFLP and traditional methods, including colony morphology on Wallerstein Nutrient (WLN) medium and 5.8S-ITS–RFLP analysis. The result showed that T-RFLP profiles of the yeast community correlated well with that of the results identified by the traditional methods. The TRFs with the highest intensity and present in all the samples corresponded to Saccharomyces sp. Other species detected by both approaches were Hanseniaspora uvarum, Metschnikowia pulcherrima, Pichia minuta var. minuta, Saccharomycodes ludwigii/Torulaspora delbrueckii and Candida zemplinina. This study revealed that T-RFLP technique is a rapid and useful tool for monitoring the composition of yeast species during wine fermentation.

Selection of Phytase Producing Yeast Strains for Improved Mineral Mobilization and Dephytinization of Chickpea Flour

Among 600 yeast isolates screened for phytase production, five (Zygosaccharomyces bisporus NCIM 3265 and 3296, Williopsis saturnus NCIM 3298, Zygosaccharomyces priorionus NCIM 3299 and Schizosaccharomyces octosporus NCIM 3297) were selected as potential phytase producers. Phytase activity was found to be cell wall associated with temperature and pH optima of 50C and 4.0, respectively. Highest phytase production was attained in cane juice medium as compared with other media used. Addition of phytase to chickpea flour significantly enhanced mineral mobilization by approximately 20–28%, 26–37% and 24–42% for Zn2+, Fe2+ and Ca2+, respectively, and decreased phytic acid content by about 75–88%. Practical Applications Chick pea is one of the most popular vegetarian foods in most developing countries. It is a rich source of protein and minerals like Ca2+, Mn2+, Fe2+, Cu2+ and Mg2+; but these metal ions are bound to phytic acid, which acts as an anti-nutrient. Treatment of chickpea flour with yeast phytase hydrolyzed the phytic acid and released the bound metal ions. The ability of yeast phytase to improve mineral mobilization and dephytinization of chickpea flour suggests its potential application in food processing and feed industry.

Application of Proanthocyanidins from Peanut Skins as a Natural Yeast Inhibitory Agent

Proanthocyanidins were extracted from peanut skins and investigated for their antimicrobial activity against Saccharomyces cerevisiae, Zygosaccharomyces bailii, and Zygosaccharomyces bisporus in traditional growth media (Sabouraud Dextrose and Maltose broth) and a simulated apple juice beverage. Peanut skins extracts (PSE) were prepared through a multisolvent extraction procedure. The PSE extended the lag phase growth of the 3 yeasts studied at a concentration of 1 mg/mL and at 10 mg/mL yeast growth was totally inhibited for 120 h. PSE was fractionated by normal phase high performance liquid chromatography and the active components/fractions were determined. Compounds present in the fractions were identified by liquid chromatography-mass spectrometry to determine the compounds responsible for inhibition. Fractions consisting mostly of A-type proanthocyanidin dimers, trimers, and tetramers showed the highest percent inhibition toward the yeasts tested in this study. Both optical density (OD) and standard enumeration plating methods were performed in this study. The OD method led to an overestimation of the inhibitory effects of PSE, the 2 methods agreed in respect to treatment effects but not the severity of the inhibition. There is a growing consumer demand for "fresh like" products containing reduced amounts of chemical preservatives without compromising food safety and quality. Therefore, the goal of this study was to determine if an extract of peanut skins containing flavonoid rich compounds could function as a natural antimicrobial in a model beverage system. Proteins were removed through the process of producing the peanut skin extract, thus it is unlikely to contain peanut allergens. The antimicrobial compounds mentioned in this study were successfully integrated into a model beverage system, and were found to have antimicrobial effect. However, the incorporation of these compounds would likely lead to negative sensory attributes at the concentration needed to achieve an appreciable antimicrobial effect alone.

Fructanase and fructosyltransferase activity of non-Saccharomyces yeasts isolated from fermenting musts of Mezcal

Fructanase and fructosyltransferase are interesting for the tequila process and prebiotics production (functional food industry). In this study, one hundred thirty non-Saccharomyces yeasts isolated from “Mezcal de Oaxaca” were screened for fructanase and fructosyltransferase activity. On solid medium, fifty isolates grew on Agave tequilana fructans (ATF), inulin or levan. In liquid media, inulin and ATF induced fructanase activities of between 0.02 and 0.27U/ml depending of yeast isolate. High fructanase activity on sucrose was observed for Kluyveromyces marxianus and Torulaspora delbrueckii, while the highest fructanase activity on inulin and ATF was observed for Issatchenkia orientalis, Cryptococcus albidus, and Candida apicola. Zygosaccharomyces bisporus and Candida boidinii had a high hydrolytic activity on levan. Sixteen yeasts belonging to K. marxianus, T. delbrueckii and C. apicola species were positive for fructosyltransferase activity. Mezcal microbiota proved to showed to be a source for new fructanase and fructosyltransferases with potential application in the tequila and food industry.

Species-specific PCR primers for the rapid identification of yeasts of the genus Zygosaccharomyces

Species-specific primer pairs that produce a single band of known product size have been developed for members of the Zygosaccharomyces clade including Zygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces kombuchaensis, Zygosaccharomyces lentus, Zygosaccharomyces machadoi, Zygosaccharomyces mellis and Zygosaccharomyces rouxii. An existing primer pair for the provisional new species Zygosaccharomyces pseudorouxii has been confirmed as specific. The HIS3 gene, encoding imidazole-glycerolphosphate dehydratase, was used as the target gene. This housekeeping gene evolves slowly and is thus well conserved among different isolates, but shows a significant number of base pair changes between even closely related species, sufficient for species-specific primer design. The primers were tested on type and wild strains of the genus Zygosaccharomyces and on members of the Saccharomycetaceae. Sequencing of the D1/D2 region of rDNA was used to confirm the identification of all nonculture collection isolates. This approach used extracted genomic DNA, but in practice, it can be used efficiently with a rapid colony PCR protocol. The method also successfully detected known and new hybrid strains of Z. rouxii and Z. pseudorouxii. The method is rapid, robust and inexpensive. It requires little expertise by the user and is thus useful for preliminary, large-scale screens.