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Molecular characterization and oenological properties of wine yeasts isolated during spontaneous fermentation of six varieties of grape must
Abstract
Fermentation by naturally occurring yeasts may produce wines of complex oenological properties that are unique to a specific region. The present work analyses the population dynamics of the yeasts during spontaneous fermentation of six varieties of grape must from the "Valle del Andarax" area (Spain). In this study we identified members of the genera Candida, Hanseniaspora, Issatchenkia, Metschnikowia, Pichia and Saccharomyces by PCR-RFLP of the ITS region. The capability of these yeasts to ferment grape must of the Macabeo variety was studied, and the volatile profile of the wine from each microvinification has been determined. Of all the yeasts isolated Candida stellata and Saccharomyces cerevisiae were able to consume virtually all the initial glucose, producing ethanol contents typical of table wines. The best profile of higher alcohols was given by Saccharomyces cerevisiae followed by Hanseniaspora uvarum, Issatchenkia orientalis and Candida stellata. Metschnikowia pulcherrima and Pichia fermentans showed the highest production of ethyl caprilate and 2-phenyl ethanol, compounds associated with pleasant aromas.
... For example, the mono-culture fermentation of Torulaspora delbrueckii produces lower levels of volatile acids than Saccharomyces cerevisiae [41]. Metschnikowia pulcherrima generates more ethyl caprylate, providing the wine with pineapple or pear aromas [42]. Yeasts are in continuous and extensive competition during the alcoholic fermentation process. ...
... This study selected three mutants for alcoholic fermentation with commercial Saccharomyces cerevisiae S1 and strain H5 after analyzing their ethanol tolerance while evaluating the fermentation performance and volatile aromatic substances. The fermentation results showed that the three mutants and H5 could not complete alcoholic fermentation independently, while their fermentation ability was weaker than commercial Saccharomyces cerevisiae, which was consistent with the research results of Clemente-Jimenez et al. [42]. Although the residual sugar content exceeded 4 g/L when only Pichia terricola was used for alcoholic fermentation, the fermentation capacity of the UV5 mutant was improved. ...
... It is speculated that while the UV-irradiated yeast develops ethanol resistance, it may also affect the metabolic cell pathways related to citric and malic acid; however, the specific reasons for this require further analysis. Clemente-Jimenez et al. [42] indicated that Pichia terricola displayed low fermentation ability while producing high ethyl acetate levels, causing the wine to present an unpleasant odor. The H5 group did not exhibit high ethyl acetate content, while the sensory evaluation results did not indicate the presence of an unpleasant odor; however, high ethyl acetate levels were evident in the UV8 mutant group, displaying a severely unpleasant odor, which was consistent with the literature. ...
Although using non-Saccharomyces yeasts during alcoholic fermentation can improve the wine aroma, most of them are not ethanol tolerant; therefore, in 2017, this study screened 85 non-Saccharomyces yeasts isolated and identified from 24 vineyards in seven Chinese wine-producing regions, obtaining Pichia terricola strain H5, which displayed 8% ethanol tolerance. Strain H5 was subjected to ultraviolet (UV) irradiation and diethyl sulfate (DES) mutagenesis treatment to obtain mutant strains with different fermentation characteristics from the parental H5. Compared with strain H5, the UV-irradiated strains, UV5 and UV8, showed significantly higher ethanol tolerance and fermentation capacity. Modified aroma profiles were also evident in the fermentation samples exposed to the mutants. Increased ethyl caprate, ethyl caprylate, and ethyl dodecanoate content were apparent in the UV5 samples, providing the wine with a distinctly floral, fruity, and spicy profile. Fermentation with strain UV8 produced a high ethyl acetate concentration, causing the wine to present a highly unpleasant odor. To a certain extent, UV irradiation improved the ethanol tolerance and fermentation ability of strain H5, changing the wine aroma profile. This study provides a theoretical basis for the industrial application of non-Saccharomyces yeasts that can improve wine flavor.
... It has the ability to produce high concentrations of esters, especially isoamyl and isobutyl acetate, which contribute to the pleasant fruity aroma in wines [10,13]. Literature data showed that during the production of wine from different grape varieties, this yeast species produced higher amounts of glycerol compared to S. cerevisiae [18][19][20]. Although glycerol does not affect the aromatic profile of wine, it has a positive effect on the mouthfeel properties, providing the fullness and sweetness sensation [21,22]. ...
... The obtained results were in accordance with published results [19]. Lower amounts of glycerol than those achieved in this study were observed in the fermentation of the Macabeo grape variety [18] and the Trebbiano grape variety [11]. The difference in the results is due to the different compositions of the fermentation medium, especially the initial sugar content, as well as the fermentation conditions [61]. ...
... The ethanol content ranged between 3.13 and 4.68% v/v, which is consistent with the literature [14,33,62]. Jolly and coworkers showed that H. uvarum could produce 5.4-6.5% v/v of ethanol during the fermentation of two different Chardonnay grape must samples [21], while another research group reached a limit of 3.81% v/v of ethanol [18]. ...
The utilization of native yeast strains associated with a distinct terroir for autochthonous grape types represents a novel trend in winemaking, contributing to the production of unique wines with regional character. Hence, this study aimed to isolate native strains of the yeast H. uvarum from the surface of various fruits and to characterize its fermentation capability in Prokupac grape must. Out of 31 yeasts, 8 isolates were identified as H. uvarum. The isolates were able to grow at low (4 °C) temperatures, SO2 concentrations up to 300 ppm and ethanol concentrations up to 5%. Additionally, they provided a good profile of organic acids during the microvinification of sterile grape must. Although the content of acetic acid (0.54–0.63 g/L) was relatively high, the sniffing test proved that the yeast isolates developed a pleasant aroma characterized as fruity. All H. uvarum isolates produced twice the concentration of glycerol compared to commercial wine yeast Saccharomyces cerevisiae, contributing to the fullness and sweetness of the wine. The results for pure and sequential fermentation protocols confirmed that the selected S-2 isolate has good oenological characteristics, the capability to reduce the ethanol content (up to 1% v/v) and a potential to give a distinctive note to Prokupac-grape wines.
... As the spontaneous fermentation progressed, most of the non-Saccharomyces yeasts decreased their presence in the must, even disappearing on day 7. This might be explained by its low resistance to the harsh environmental conditions, characterized mostly by the lack of nutrients and high ethanol concentrations [23,24,26,27,49]. On the contrary, the Saccharomyces genera that showed a relative abundance from 10% to 40% at day 0 take advantage of these conditions, and therefore become dominant. ...
... The diversity observed in this study is in line with that previously reported for spontaneous grape juice fermentations [10,11,[25][26][27]49,87], in which a number of non-Saccharomyces yeast were detected and/or isolated primarily at the early stages of the fermentation process. In our study, non-Saccharomyces yeasts were also isolated after 7 days of fermentation, which indicates that the species have a certain tolerance to the ethanol concentration (13.08 ± 0.03 to 14.2 ± 0.11% v/v) and nutrient deprivation (glucose and fructose: 1.70 ± 0.05 to 2.12 ± 0.05 g/L, respectively) conditions reached in 7 days of the spontaneous fermentations (data not shown). ...
... The selected yeast strains used in this study showed ethanol yields of 0.18 and 0.40 for C. oleophila and C. stellata, respectively (Table 5), and therefore, the increased glycerol concentration might be attributed to the yeast s ability to canalize sugar consumption into glycerol production. C. stellata fermentative potential has been studied not only in wines, but also in beer and vinegar production [21,26,108]. The fructophilic yeast metabolism is characterized by the production of glycerol instead of ethanol, and thus produces wines with reduced alcohol content [20]. ...
Grapes are a source of native yeasts and lactic acid bacteria (LAB); however, the microbial make up is dependent on the grape cultivar and the regional growth conditions. Therefore, the aim of this study was to characterize the yeast and LAB in seven grape cultivars cultivated in Chile. Grape juices were fermented at 25 °C for 7 days. Samples were collected to analyze sugar, organic acids, and ethanol. Microbial evolution was measured with culture-dependent and molecular approaches. Then, a native isolated Candida oleophila was selected for further sequential fermentations with Saccharomyces cerevisiae. The grape cultivars in the Maule showed a diversity of non-Saccharomyces yeasts, with a greater diversity observed at the beginning of the fermentation. However, species from the Hansenasporia, Metschnikowia, Torulaspora, Lachancea, and Candida genera were detected after 7 days, suggesting tolerance to environments rich in ethanol, capability may be associated to the terroir studied, which is characterized by torrid weather and antique and traditional vineyards. The alcoholic fermentation negatively impacted the LAB population, and after 7 days only Leuconostoc mesenteroides was isolated. In the sequential fermentations, C. oleophila was able to produce fermented grape juices with <1.5 g/L glucose, 12.5% (v/v) alcohol, and low concentrations of malic (<1.00 g/L) and succinic (2.05 g/L) acids, while acetic acid reached values >0.3 (g/L). To our knowledge this is the first time C. oleophila has been reported as a potential starter culture for wine production. However, more studies are necessary to fully characterize the potential of C. oleophila on wine attributes.
... For this reason, it is not always possible to obtain the same end product from spontaneous fermentation. This issue is currently solved using commercial yeast strains in the fermentation process, however, at the expense of the autochthonous characteristics of wine (Clemente-Jimenez et al., 2004). This paper is focused on monitoring the effects of two consortia of wine microorganisms obtained from spontaneous fermentations in 2017 (variant b) and 2018 (variant c), compared with direct spontaneous fermentation without inoculation of any consortium (variant a). ...
... ght. This paper eliminates these effects by performing the experiment on one vintage wine, which further examines the influence of the consortia obtained in previous years. Some differences in the aromatic profile can be observed, which can be attributed to the individual yeast strains. This has been confirmed in many studies, such as the study by Clemente-Jimenez et. al. (2004), who state that it is not always possible to obtain the same product from spontaneous wine fermentation. This issue is currently solved by using commercial yeast strains in the fermentation process at the expense of the autochthonal character of wine, as a significant manifestation of yeast metabolism can cause some kind of wine uniform ...
... al. (2004), who state that it is not always possible to obtain the same product from spontaneous wine fermentation. This issue is currently solved by using commercial yeast strains in the fermentation process at the expense of the autochthonal character of wine, as a significant manifestation of yeast metabolism can cause some kind of wine uniformity (Chamberlain et al., 1997;Clemente-Jimenez et al., 2004;Barata et al., 2012). The last category of the sensory evaluation was the assessment of the aromatic and mightiness profiles. ...
In this paper we focus on possibilities of using natural microflora from vineyards to enhance spontaneous fermentation. Obtained yeast isolates, naturally occurring in vineyards, which were previously isolated in 2017 and 2018, were used for inoculation of Hibernal grape must. Spontaneously fermented musts and musts with the yeast isolates from 2017 and 2018 were compared. Basic parameters of the musts (sugar concentration, pH, concentration of titratable acids, concentration of assimilated nitrogen) were analysed studied, and a sensory analysis of the resultant wines was performed. The fermentation with the yeast isolates was quick and smooth. In the spontaneous fermentation lower ethanol production rate was observed at the end of the fermentation process. During the sensory evaluation, fruitiness of the spontaneously fermented batch was lower, but its vegetal characteristic was pronounced. The variant with the yeast isolates from 2017 was described as smooth, and the variant with yeast isolates from 2018 was evaluated as slightly vegetal.
... By HTS, Portillo and Mas [44] found yeast belonging to the genera Hanseniaspora and Issatchenkia in grape must, whereas Padilla, García-Fernández, González, Izidoro, Esteve-Zarzoso, Beltran, and Mas [45] observed H. uvarum and Issatchenkia terricola in some of the four analysed Grenache (Garnacha) grape musts. The presence of the Hanseniaspora species as H. uvarum or H. guilliermondii (or their teleomorphs Kloeckera apiculata and Kloeckera apis), as well as M. pulcherrima, is quite common in the grape must of many varieties [46][47][48][49]. The isolation of H. opuntiae, I. terricola, and W. anomalus (formerly Pichia anomala, Hansenula anomala, Candida pelliculosa) is less frequent. ...
... The isolation of H. opuntiae, I. terricola, and W. anomalus (formerly Pichia anomala, Hansenula anomala, Candida pelliculosa) is less frequent. I. terricola has been found in the grape must or at early fermentative times of Macabeo, Vermentino, and Viognier white wine fermentations [47]. ...
This work studies the variability of the Saccharomyces cerevisiae present during the spontaneous fermentation of Garnacha grapes’ musts from a “Pago” winery from the east of Spain. The parameters used to select yeast are those related to growth, fermentative behaviour, and the influence on the wine’s aroma and polyphenolic composition. Yeast identification was performed by ITS analysis and typed by Hinfl mDNA restriction profile analysis. Growth and metabolic characteristics of the isolates were determined by laboratory-scale fermentations of sterile Garnacha must, and the composition of the polyphenolic and the volatile compounds, and the sensory attributes of the small-scale produced red wines were determined. Ten S. cerevisiae strains were isolated and characterized. Overall, strain 22H quickly grew, produced wines with moderate ethanol concentrations and low volatile acidity, and obtained the highest colour and aroma scores, plus a high score for sensory attributes.
... Between veraison (color break) and grape harvest, a substantial increase in nutrients occurs within and at the surface of the berry, which significantly impacts yeast population size and diversity (Coombe and McCarthy 2000;Fleet 2003). Native yeasts can exert a positive or negative effect on any aspect of the vinification process (Fleet 2003;Fugelsang and Edwards 2007), and are responsible for some of the unique flavors, aromas, and textures of premium wines (Clemente-Jimenez et al. 2004;Ferreira et al. 2001; Romano et al. 2003). The mycobiomes of wine grapes or fermentation samples from California, Texas, Brazil, China, Germany, New Zealand and South Africa have been reported (Bokulich et al. 2014;Bougreau et al. 2019;Brysch-Herzberg and Seidel 2015;de Ponzzes-Gomes et al. 2014;Gayevskiy & Goddard 2012;Li et al. 2018;Setati et al. 2015;Vigentini et al. 2016). ...
... Cabernet Sauvignon berries grown in Washington State appeared to harbor a core group of yeasts that have been found on other cultivars throughout world (Bokulich et al. 2014;Clemente-Jimenez et al. 2004;Pinto et al. 2015). The yeast-like fungus A. pullulans was present at high populations on berries at both veraison and harvest. ...
Abstract:
To address a knowledge gap about the grape berry mycobiome from Washington State vineyards, next-generation sequencing of the internal transcribed spacer region (ITS1) was used to identify native yeast and fungal species on berries of cultivar ‘Cabernet Sauvignon’ from two vineyards at veraison and harvest in 2015 and 2016. Four hundred fifty-six different yeast amplicon sequence variants (ASV), representing 184 distinct taxa, and 2467 non-yeast fungal ASV (791 distinct taxa) were identified in this study. A set of 50 recurrent yeast taxa, including Phaeococcomyces, Vishniacozyma and Metschnikowia, were found at both locations and sampling years. These yeast species were monitored from the vineyard into laboratory-scale spontaneous fermentations. Taxa assignable to Metschnikowia and Saccharomyces persisted during fermentation, whereas Curvibasidium, which also has possible impact on biocontrol and wine quality, did not. Sulfite generally reduced yeast diversity and richness, but its effect on the abundance of specific yeasts during fermentation was negligible. Among the 106 recurring non-yeast fungal taxa, Alternaria, Cladosporium and Ulocladium were especially abundant in the vineyard. Vineyard location was the primary factor that accounted for the variation among both communities, followed by year and berry developmental stage. The Washington mycobiomes were compared to those from other parts of the world. Sixteen recurrent yeast species appeared to be unique to Washington State vineyards. This subset also contained a higher proportion of species associated with cold and extreme environments, relative to other localities. Certain yeast and non-yeast fungal species known to suppress diseases or modify wine sensory properties were present in Washington vineyards, and likely have consequences to vineyard health and wine quality.
... The remaining esters present in the wines occurred at a relatively low level (Table 5). According to the literature data, M. pulcherrima produces significant amounts of esters [16], especially ethyl octanoate [57,66]. The conducted research did not confirm this fact. ...
... In our research, the content of acetaldehyde in wines fermented with the monocultures of M. pulcherrima MG970690 and Z. bailli 749 did not exceed 1 mg/L (Table 5). Clemente-Jimenez et al. [66] demonstrated the synthesis of significant amounts of acetaldehyde in wine by strains of C. stellata, H. uvarum, I. orientalis, I. terricola and M. pulcherrima in relation to S. cerevisiae. Similar results were achieved by Mateos et al. [69], indicating higher levels of acetaldehyde in wines produced with native microbiota compared to Saccharomyces yeast. ...
Interest in the use of non-Saccharomyces yeast in mixed cultures is increasing due to the perceived improvement in the quality and complexity of the resulting wines. The aim of the study was to determine the ability of monocultures and mixed yeast cultures for deacidification and improvement of the composition of cold climate grape wines. Fermentation of grape musts with increased total acidity was carried out with the use of monocultures of Saccharomyces cerevisiae MH020215 (Sc), Zygosaccharomyces bailii 749 (Zb) and Metschnikowia pulcherrima MG970690 (Mp), and their mixed cultures, inoculated simultaneously and sequentially. Oenological parameters, organic acids and volatile compounds profiles of obtained wines were characterized. The fermentation kinetics and analytical profiles of the obtained wines showed that the use of mixed yeast cultures contributed to the reduction of volatile acidity and acetic acid content in the wines, as well as obtaining a favorable aromatic profile of the wines. The dominant higher alcohols in all wines were 2-methyl-1-propanol, 3-methyl-1-butanol and 2-methyl-1-butanol. Significantly higher amounts of the first two compounds were found in wines obtained with M. pulcherrima MG070690, both in monoculture and in mixed cultures. The monocultures of M. pulcherrima MG070690 (Mp) compared with Z. bailli 749 (Zb) synthesized higher levels of esters in wines, including ethyl acetate, ethyl propionate, isobutyl acetate, ethyl pyroracemate and isoamyl acetate.
... The PCR-RFLP results of ITS1-5.8S-ITS2 gene region are similar to previous studies which are especially used Hae III, Hinf I and Hha I (Cfo I) restriction endonucleases(Guillamon, Sabate, Barrio, Cano, & Querol, 1998;Rodriguez-Vico, Clemente-Jimenez, Mingorance-Cazorla, Martinez-Rodriguez, & Las Heras- Vazquez, 2003;Romano, Capece, Siesto, & Romaniello, 2009;Gibson et al., 2011). The Hae III, Hinf I, and Hha I restriction pattern of second group members was similar to M. pulcherrima yeast species recorded in previous studies(Esteve-Zarzoso, Belloch, Uruburu, & Querol, 1999;Rodriguez-Vico et al., 2003). ...
... gene region are similar to previous studies which are especially used Hae III, Hinf I and Hha I (Cfo I) restriction endonucleases(Guillamon, Sabate, Barrio, Cano, & Querol, 1998;Rodriguez-Vico, Clemente-Jimenez, Mingorance-Cazorla, Martinez-Rodriguez, & Las Heras- Vazquez, 2003;Romano, Capece, Siesto, & Romaniello, 2009;Gibson et al., 2011). The Hae III, Hinf I, and Hha I restriction pattern of second group members was similar to M. pulcherrima yeast species recorded in previous studies(Esteve-Zarzoso, Belloch, Uruburu, & Querol, 1999;Rodriguez-Vico et al., 2003). ...
Raspberry fruit contains phenolic compounds, flavones, flavonoids, vitamins, and antioxidant substances that are important properties for health and pharmacological sciences. Edible berries provide also a suitable habitat for the growth of various microorganisms. In this study, yeast biota associated with raspberry fruits was determined by molecular identification techniques. Raspberry fruits were collected from Çanakkale, Gelibolu (Gallipoli). Yeast strains were isolated and then identified by using the analysis of ITS1-5.8S-ITS2 rDNA gene sequences. The phylo-genetic analysis of all yeast strains was carried out by using the MEGA–X phylogenetic analysis tool. The extracel-lular enzyme profiles of identified yeast species were determined by the API-ZYM kit system. The distribution of yeast species on the raspberry fruits was determined as Hanseniaspora uvarum, Metschnikowia viticola, Aureo-basidium pullulans, and Metschnikowia pulcherrima. It was observed that yeast strains belong to Metschnikowia genus were dominant on raspberry fruits. All yeast strains in Metschnikowia genus showed different enzyme pro-files against seven extracellular enzymes. These enzymes may be the discriminatory enzymes for the yeast strains in the Metschnikowia genus. When the phylogenetic relationships among all yeast strains were investigated, all strains were divided into two main clades. While the first clade consists of only Metschnikowia genus, second clade includes H. uvarum and A. pullulans yeast species. Our results indicated that restriction patterns and also extracel-lular enzyme profiles could be utilized for differentiation of yeast strains within the genus. M. pulcherrima, H. uvarum, and A. pullulans can be used for industrial applications for future researches.
... While the HinfI restriction profile of H. uvarum strains was similar to previous results, HhaI restriction profile was different (Guillamon et al. 1998;Rodriguez-Vico et al. 2003;Romano et al. 2009;Baffi et al. 2010;Gibson et al. 2011). The HhaI, HaeIII and HinfI profiles of C. zeylanoides yeast strains showed similar restriction patterns with other researches (Guillamon et al. 1998;Esteve-Zarzoso et al. 1999). ...
... The HhaI, HaeIII and HinfI profiles of C. zeylanoides yeast strains showed similar restriction patterns with other researches (Guillamon et al. 1998;Esteve-Zarzoso et al. 1999). Similarly, HinfI, HaeIII and HhaI profiles of M. pulcherrima, A. pullulans and K. lactis yeast species revealed the same restriction pattern previously reported (Guillamon et al. 1998;Esteve-Zarzoso et al. 1999;Rodriguez-Vico et al. 2003;Romano et al. 2009;Baffi et al. 2010;Gibson et al. 2011). We also determined that the restriction profiles of both Kwoniella sp. and M. pulcherrima species members were different, but, they were determined as same species with respect to in the sequence analysis of ITS1-5.8S-ITS2 ...
Genc TT, Günay M. 2020. Internal transcribed spacer (ITS) sequence-based identification of yeast biota on pomegranate surface and determination of extracellular enzyme profile. Nusantara Bioscience 12: 59-67. Yeasts are the most significant organisms to produce fermented products from different types of fruits such as grape, strawberry and pomegranate. The native yeasts on these fruits contribute to beverages’ quality and aroma during fermentation. Pomegranate is used in fruit juice and wine production because of high antioxidant characteristic. In order to determine yeast microbiota on the pomegranate fruits collected from Gallipoli (Gelibolu), Çanakkale-Turkey, ITS-5.8S rDNA gene region have been utilized. Also, phylogenetic relationships among identified yeast species were assigned by using sequences of ITS-5.8S rDNA gene region. In addition, extracellular enzyme activity of identified yeast strains was detected by using API-ZYM. Kluyveromyces lactis, Aureobasidium pullulans, Hanseniaspora uvarum, Candida zeylanoides, Kwoniella sp., and Metschnikowia pulcherrima and Metschnikowia ziziphicola yeast species were identified on pomegranate surface. Phylogenetic analysis, carried out in ITS-5.8S rDNA gene region of identified yeast strains, revealed the presence of five clades. Kwoniella sp., H. uvarum, M. pulcherrima, and Kl. lactis yeast strains revealed high leucine arylamidase activity. Also valine arylamidase activity was determined in M. pulcherrima and Kl. lactis yeast species. Acid phosphatase activity was determined in H. uvarum and K. lactis yeast species. Uncultured Kwoniella sp. and H. uvarum yeast species displayed high β-galactosidase and β-glucosidase activities, respectively.
... Zhang et al. (2022) stated that isoamyl alcohol concentration increased constantly and reached the maximum level at the later stage of the fermentation with fluctuation similar to the findings of the present study. Phenylethyl alcohol is one of the main aroma compounds in wine fermentation and it is characterized by the fragrance of rose (Clemente-Jimenez et al., 2004). The concentration of phenylethyl alcohol also increased throughout the fermentation in all samples, except for BO20. ...
Spontaneous fermentation may produce complex and unique wines with geographical designations due to the region-specific yeast flora and variations in must characteristics. The current study focused on the yeast diversity and changes in volatile compounds during the spontaneous fermentation of ‘Karasakız’ grapes from three sub-regions of Çanakkale province, Turkey for two vintages (2019 and 2020). This is the first study on the diversity of autochthonous yeasts during wine fermentation of ‘Karasakız’ variety. In the present work, the strains belong to Hanseniaspora uvarum, Starmerella bacillaris, Pichia terricola, Metschnikowia pulcherrima and Zygoascus hellenicus by PCR-RFLP of the ITS region were identified . A total of 272 isolates were identified as S. cerevisiae. Yeast population and diversity changed related to the sub-region and the vintages. However, H. uvarum and S. bacillaris were common denominators of three regions evaluated. H. uvarum was dominant in the early stages of the fermentation, except for Bozcaada 2019 vintage. Bozcaada region also exhibited higher S. cerevisiae strain diversity compared to other regions. 1-Hexanol and 1-octanol gradually decreased during the fermentation of ‘Karasakız’ grapes, while the concentration of isoamyl alcohol, phenylethyl alcohol and ester compounds increased by the fermentation but with some fluctuation.
... W. anomalus and P. fermentans species were isolated in all the samples and at both temperatures but in low percentages (8% and 6%, respectively). W. anomalus has been isolated from natural habitats as soil, bark and tree leaves (Kurtzman, 2011;Martos et al., 2013) as well as from wines (Bagheri et al., 2015(Bagheri et al., , 2017Tofalo et al., 2016) and other fermented beverages (Lee et al., 2011), while P. fermentans species is strictly related to fermentative environments (Clemente-Jimenez et al., 2004;Li et al., 2010). ...
Patagonia (Argentina and Chile) harbors the highest Saccharomyces eubayanus genomic diversity and its widest predominance in natural environments. In this work, S. eubayanus was isolated for the first time from a fermentative environment. This species was found dominating both a traditional apple chicha fermentation as well as feral apple trees in the Andean region of Aluminé (Argentina). S. eubayanus was the only Saccharomyces species found in the isolation substrates, although it coexisted with other non‐Saccharomyces species. The absence of strong fermentative competitors of the Saccharomyces genus (like Saccharomyces uvarum or Saccharomyces cerevisiae) in the feral apples could promote the development and implantation of S. eubayanus in a spontaneous apple must fermentation. Phylogeographic analyses revealed a high intraspecific diversity in S. eubayanus, enabling the characterization of strains belonging to the genomic subpopulations PA1, PA2, and PB1 according to the sequences obtained for the intFR gene region. This result evidence that the studied sampling area represents a natural habitat for the species. Being a novel finding, studying the causes that allowed this species to prosper in a fermentative environment becomes essential. Hence, the physiological profile of the new isolates, including their ability to grow at different temperature, nitrogen, and ethanol concentrations was evaluated in comparison with a set of S. eubayanus strains previously isolated from natural environment and representing different genomic subpopulations. Greater physiological diversity was evidenced when strains isolated from both natural and fermentative environments were analyzed overall. Furthermore, no direct relationship between genomic population and physiological behavior was observed; on the opposite, strains appeared to exhibit similar behavior, primarily grouped by isolation origin.
... yeast isolated from Indian cashew apple were able to tolerate up to 10% (v/v) ethanol (Desai et al., 2012). Besides, a strain of Candida stellata was found to produce ethanol levels up to 13.48% (v/v) during the fermentation of Macabeo grape must, indicating a high tolerance to ethanol (Clemente-Jimenez et al., 2004). In this study, we obtained a strain of C. tropicalis that exhibited robust growth in YEPD broth containing 12% (v/v) of ethanol (Figure 1). ...
Ethanol tolerance is crucial for the oenological yeasts. Rosa roxburghii Tratt, a Rosaceae plant native to China, is rich in nutritional and medicinal ingredients. In this study, ethanol-tolerant non-Saccharomyces yeasts were screened, and their oenological properties were further evaluated. Three ethanol-tolerant yeast strains (designated as C6, F112, and F15), which could tolerate 12% (v/v) ethanol treatment, were isolated from R. roxburghii, and identified as Candida tropicalis, Pichia guilliermondii, and Wickerhamomyces anomalus, respectively. The winemaking condition tolerances of these ethanol-tolerant yeast strains were similar to those of Saccharomyces cerevisiae X16. However, their growth, sugar metabolic performance and sulphureted hydrogen activities, were different. The β-glucosidase production ability of strain W. anomalus F15 was lower than that of S. cerevisiae X16, and strains of C. tropicalis C6 and P. guilliermondii F112 were similar to S. cerevisiae X16. Electronic sensory properties of the R. roxburghii wines fermented using ethanol-tolerant yeasts together with S. cerevisiae showed no significant differences. However, the mixed inoculation of the ethanol-tolerant yeast strains with S. cerevisiae could regulate the volatile aroma characteristics of the fermented R. roxburghii wine, enriching and enhancing the aroma flavor. Therefore, the selected ethanol-tolerant yeasts have the potential for application in the production of unique R. roxburghii wine.
... 1 Two categories of microorganisms, fungi (predominantly yeasts) and bacteria, are recognised as the driving force of the primary and secondary fermentation processes, as well as the more recently recognised 'spontaneous fermentation' process performed by wild yeasts and bacteria, which originate from the plant's microbiome. [2][3][4][5][6] The grapevine microbiome, similar to other, complex microbiomes such as the human gut microbiome, is separated into distinct compartments with each hosting its own diversity of microbes. The main subdivisions of the grapevine microbiome are the rhizosphere microbiome (the area surrounding the roots), the endosphere microbiome (the area within the plant's tissues) and the phyllosphere microbiome (the surface of the aerial portion of the plant). ...
Grapevines that are used for winemaking host a diverse range of microorganisms that make up their microbiome. The microbes that inhabit the grapevine have been used by winemakers to produce wine for centuries, although modern wine producers often rely on inoculated microorganisms such as Saccharomyces cerevisiae. In the Australian wine industry, there is a movement towards returning to the utilisation of the microbiome for wine fermentation. With the recent increase in the understanding of the role of the grapevine microbiome in grapevine health, fermentation and subsequent wine sensory traits, the microbial world offers a new level of complexity that can be harnessed for winemaking. In order to develop and maintain a desired vineyard micro-biodiversity, extensive microbial monitoring is required. Here we discuss the utilisation of a viability selection dye in order to distinguish between microorganisms that are live and associated with the host, and relic signals generated from non-living sources.
... The majority of the higher alcohols in this study have significant differences between different regions (Table 1), which may have resulted from the vineyard and winemaking environment (Ciani et al., 2016;Clemente-Jimenez et al., 2004). Branched alcohols such as 2-methyl-1-propanol, 3-methyl-1-butanol, and aromatic alcohol phenylethyl alcohol were identified in this study, with concentrations ranging from 35.0-108.0 ...
HS-SPME/GC-MS and aroma descriptive analysis were used to gain insights into the volatile and sensory details of 99 red wine samples collected from four varieties in five regions. The general volatile fingerprints of Cabernet Sauvignon and Merlot wine samples in Xinjiang and Ningxia regions were similar, even though chemometric models could not discriminate between them. The main drivers of the diversity were secondary metabolites of grape such as terpenes, benzene-derivatives, and ketones. Fermentation-derivatives (esters and alcohols) were also responsible for region and variety-related differences in wines. Analysis of volatile compounds also showed that the primary factor accounting for diversity in wines in this study was region rather than variety. These results highlight the sensory attributes and volatiles of different regions and varieties, and provide a quantitative basis for screening for differential metabolites and potential markers in wines.
... on Day 1 to 0-8.0% at the end of the Trebbiano and Montepulciano fermentations, respectively (Day 30 in Figure 2B). Candida and Hanseniaspora genera have been frequently isolated on spontaneous fermentations [46] and these results agreed with previous studies on spontaneous fermentations, describing the abundance of Hanseniaspora at the beginning of the fermentation and Candida until the end of the alcoholic fermentation [47]. ...
Spontaneous wine fermentation is a meaningful topic that cannot be disregarded among winemakers and consumers due to the peculiarity of the organoleptic profile that it confers to the wine. Nevertheless, in this process the activity of indigenous microorganisms might be a threat. We studied the evolution of the spontaneous fermentation process in a traditional Italian winery in order to understand the origin of spoilage microorganisms, and to characterize the peculiarity of the microbiota associated with spontaneous fermentation. Six Trebbiano and Montepulciano wine production chains were monitored by plate counts made by OIV methods and by Illumina MiSeq technique. Despite some compositional deficiencies, all grape musts were characterized by a highly concentrated microbial population. Non-Saccharomyces yeasts revealed an unexpected tolerance to ethanol, which has contributed to the evolution of alcoholic fermentation. Lactic bacteria were detectable from the very first steps of the winemaking process, with a prevalence of Leuconostoc spp. which is nowadays, rarely isolated in wine. The combination between culture-dependent and high-throughput sequencing (HTS) approaches allowed to estimate microbial diversity and growth dynamics in wine fermentations of different grape varieties and under different treatments; these results could be used by winemakers as a starting point to drive a more mindful, accurate and, controlled fermentation process and to set up the most suitable environmental conditions to enhance wine singularities.
... Moreover, the sequential culture inoculation of Hanseniaspora guilliermondii and Saccharomyces cerevisiae gave a better aroma and quality in Campanino white grape wine (Lombardi et al., 2018). In addition, the metabolic products resulting from non-Saccharomyces yeast like higher alcohols (Heard, 1999;Zohre and Erten, 2002;Clemente-Jimenez et al., 2004) can affect the overall quality of the palm wine. In bacteria, fructophilic lactic acid bacteria (FLAB), mainly Fructobacillus and Leuconostoc, encountered during both types of palm wine tapping is due to their affinity to the fructose enriched habitat such as flowers, palm sap and honey bee gut (Endo et al., 2018). ...
Palm wine fermentation is a complex microbial process that evolves with tapping times. The dynamics in microbiota and metabolites throughout palm wine tapping days is still not established, which are critical for the distinctive characteristics of palm wine taste and quality, and thus the mastery of the daily quality fluctuation during tapping. We analyzed the changes in microbial community structure by amplicon sequencing of bacterial 16S rRNA gene and fungal internal transcribed spacer (ITS) region, and metabolite profile changes using mass spectrometry in palm wine collected over 25–30 days tapping of ron (Borassus aethiopum) and oil palms (Elaeis guineensis) from Côte d’Ivoire. The stage-wise collected palm wine samples showed distinct changes in microbial diversity and pH, supporting microbial community dynamics during palm wine tapping. Results highlighted the dominance of Saccharomyces cerevisiae in early stages and the emergence of non-Saccharomyces yeasts, particularly Hanseniaspora spp. in the later stages of oil palm wine tapping, vice versa in the case of ron palm wine tapping, with a unique presence of Saccharomycodes in the later stages (15–30 days). Fructophilic lactic acid bacteria (FLAB), mainly Fructobacillus and Leuconostoc, encountered in both types of palm wine tapping showed a decline at later stages of oil palm wine tapping. In this type of palm wine, acetic acid bacteria with genera Acetobacter and Glucanoacetobacter, by surpassing Lactobacillus in the last stage become dominant, whereas Lactobacillus remained dominant in ron palm wine throughout tapping days. The decline in the relative abundance of gevotroline and essential amino acids during the later stages of palm wine tapping (15–25 days) supports the difference in the health benefits of the palm wine obtained from different days of tapping, indicating that early stages of tapping is more nutritional and healthy than the later stages. The microbial dynamics may be a potential indicator of metabolite changes during palm sap fermentation, thus contributing to establish particular features of palm wines in different stages of tapping. This understanding of microbial ecology and chemical composition changes during palm wine tapping can be used as biomarkers to assess palm wine’s quality and help to design an optimum starter culture.
... Primers ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and NL4 (5 -GGTCCGTGTTTCAA GACGG-3 ) were used to amplify the conserved region of yeast DNA in order to identify the species of yeast [41]. Primers ITS1 and NL4 target ITS1 and D1/D2 region. ...
Cell-based agriculture is an emerging and attractive alternative to produce various food ingredients. In this study, five strains of marine yeast were isolated, molecularly identified and biochemically characterized. Molecular identification was realized by sequencing the DNA ITS1 and D1/D2 region, and sequences were registered in GenBank as Yarrowia lipolytica YlTun15, Rhodotorula mucilaginosa RmTun15, Candida tenuis CtTun15, Debaryomyces hansenii DhTun2015 and Trichosporon asahii TaTun15. Yeasts showed protein content varying from 26% (YlTun15) to 40% (CtTun15 and DhTun2015), and essential amino acids ranging from 38.1 to 64.4% of the total AAs (CtTun15-YlTun15, respectively). Lipid content varied from 11.15 to 37.57% with substantial amount of PUFA (>12% in RmTun15). All species had low levels of Na (<0.15 mg/100 g) but are a good source of Ca and K. Yeast cytotoxic effect was investigated against human embryonic kidney cells (HEK 293); results showed improved cell viability with all added strains, indicating safety of the strains used. Based on thorough literature investigation and yeast composition, the five identified strains could be classified not only as oleaginous yeasts but also as single cell protein (SCP) (DhTun2015 and CtTun15) and single cell oil (SCO) (RmTun15, YlTun15 and TaTun15) producers; and therefore, they represent a source of alternative ingredients for food, feed and other sectors.
... Despite the goaty aroma over the threshold of 15 mg/L [1], the high production of octanoic acid at both temperatures is a positive outcome, as it is the precursor of its ester, known for its fruity notes. According to Benito et al. [29] and Clemente-Jimenez et al. [30] M. pulcherrima is able to produce high amounts of this ester, surpassing S. cerevisiae by 25 %, thus enhancing the product with the aroma of sour apple [31]. Last of all, decanoic acid ethyl ester presented significantly higher amounts (p < 0.05) at lower temperature in mixed sugars fermentation (0.37 ± 0.09 mg/L), implying that in a wort fermented by pure culture of M. pulcherrima, the last would enrich the beer with apple-like flavour [1]. ...
Metschnikowia pulcherrima, also known as Candida pulcherrima (anamorph), is a non-Saccharomyces yeast, well known from the wine industry. It is currently used in sequential or co-fermentation with Saccharomyces cerevisiae to enrich the wine with more complex and varietal aromas. In this study, we examined the potential application of a commercial M. pulcherrima strain in the brewing industry. First, the fermentation dynamics in glucose, fructose, and maltose, at 13 and 20 °C, were studied, and GC-MS analysis of the volatile compounds related to beer flavour was performed. After being assured that M. pulcherrima is capable of metabolizing maltose, the main wort sugar, a Pale Ale beer was produced at 13 and 20 °C. A pure M. pulcherrima and two mixed cultures of S. cerevisiae with M. pulcherrima in ratios of 1:1 and 1:10 were used, and fermentation was monitored by specific gravity and free amino nitrogen measurements. The ethanol content at the end of fermentation varied within 4.0 – 5.5 % (v/v). The volatile profile analysis of the produced beers revealed the presence of higher alcohols, medium-chain fatty acids and their esters, with the obtained results presenting satisfactory complexity in flavour.
... In various investigations in countries like China (Sun et al. 2014), Czech-Republic, Slovakia, Hungary (Minarik 1964), Slovenia (Jemec et al. 2001), Austria (Lopandic et al. 2008), Germany (Sturm et al. 2006), Switzerland (Díaz et al. 2013;Schütz and Gafner 1993), Italy (Tofalo et al. 2009), France (Fleet et al. 1984;Zott et al. 2008), Spain (Beltran et al. 2002;Clemente-Jimenez et al. 2004;Hierro et al. 2006), Portugal (Barata et al. 2008;Couto et al. 2005), USA (Egli et al. 1998;van Keulen et al. 2003), Brazil (Bezerra-Bussoli et al. 2013) and Australia (Heard and Fleet 1985), S. pombe was not detected. Most likely this is due to the fact that S. pombe is a week competitor against other indigenous yeast in spontaneous grape must fermentations or against inoculated starter cultures (Brugirard and Roques 1972;Delteil 1988;Ribereau-Gayon and Peynaud 1962). ...
The fission yeast genus Schizosaccharomyces contains important model organisms for biological research. In particular, S. pombe is a widely used model eukaryote. So far little is known about the natural and artificial habitats of species in this genus. Finding out where S. pombe and other fission yeast species occur and how they live in their habitats can promote better understanding of their biology. Here we investigate in which substrates S. pombe, S. octosporus, S. osmophilus and S. japonicus are present. To this end about 2100 samples consisting of soil, tree sap fluxes, fresh fruit, dried fruit, honey, cacao beans, molasses and other substrates were analyzed. Effective isolation methods that allow efficient isolation of the above mentioned species were developed. Based on the frequency of isolating different fission yeast species in various substrates and on extensive literature survey, conclusions are drawn on their ecology. The results suggest that the primary habitat of S. pombe and S. octosporus is honeybee honey. Both species were also frequently detected on certain dried fruit like raisins, mango or pineapple to which they could be brought by the honey bees during ripening or during drying. While S. pombe was regularly isolated from grape mash and from fermented raw cacao beans S. octosporus was never isolated from fresh fruit. The main habitat of S. osmophilus seems to be solitary bee beebread. It was rarely isolated from raisins. S. japonicus was mainly found in forest substrates although it occurs on fruit and in fruit fermentations, too.
... Metschnikowia pulcherrima yeast species [31,32]. It was shown that the application of fungicides did not affect the diversity of the epiphytic yeast community on strawberries [12]. ...
Since yeasts can survive under variable environmental conditions using different food sources they have a wide distribution in nature. Fruits are suitable living spaces for yeasts and other microorganisms due to their high and different sugar contents. Strawberry fruit as well as other fruits are very sensitive to pathogenic fungi. Due to their residues on fruits, limitations on the use of fungicides have led to increased use of microorganisms with antagonistic effects as biological control agents. The biological agents to be used are selected mainly from the microorganisms found in the natural microbiota of the fruit. Therefore, in this study yeast biota on strawberry fruit collected from fungicide treated (Klorzon and Topas) and organic fields was determined using molecular identification methods. In addition, extracellular enzyme profiles of the identified yeast species were determined by the APIZYM-based system. There was no difference in the diversity of yeast species on strawberries collected from fungicide treated and organic fields, but the yeast density on organic strawberries was greater than fungicide treated fruits. The identified yeast species on fruits were determined as Metschnikowia pulcherrima (61.7%), Hanseniaspora uvarum (34.0%) and Wickerhamomyces pijperi (4.3%). W. pijperi yeast species was reported on strawberry fruit in our study first time. It was determined that H. uvarum and W. pijperi yeast species showed no α-glucosidase enzyme activity. All yeast strains showed industrially important β-glucosidase enzyme activity.
... Numerous studies have looked into different aspects of Saccharomyces cerevisiae's genetic diversity since the first application of molecular tools, some three decades ago, which made it possible to conduct such explorations, the scale of which grew over time. Many of these studies focusing on oenological S. cerevisiae communities have looked into vineyard floras [17,33,35,43,[75][76][77][78][79] collected to conduct fermentations in the lab, and only a few have explored the fermenting floras in their natural environment, i.e., the wineries [21][22][23], while some have looked at both populations [19,27,41,80]. Even if a constant circulation and exchange of strains is most probable between wineries and vineyards [19,27,43], wineries might also have their own implanted yeast lineages that conduct or participate in the natural fermentations and are different from the surviving ones in the vineyards [27,34,[80][81][82]. ...
A total of 296 isolates of Saccharomyces cerevisiae sampled from naturally fermenting grape musts from various locations in Lebanon were typed by interdelta fingerprinting. Of these, 88 isolates were compared with oenological strains originating from various countries, using microsatellite characterization at six polymorphic loci. These approaches evidenced a large diversity of the natural oenological Lebanese flora over the territory as well as in individual spontaneous fermentations. Several cases of dominance and perenniality of isolates were observed in the same wineries, where fermentations appeared to involve lineages of sibling isolates. Our work thus evidenced a “winery effect” on strains’ relatedness. Similarly, related or identical strains were also detected in vicinal wineries, suggesting strain circulation within small geographical areas and a further “vicinity effect”. Moreover, and despite its diversity, the Lebanese flora seemed interrelated, on the basis of microsatellite loci analysis, in comparison to worldwide communities. We finally tested the ability of 21 indigenous strains to act as potential starters for winemaking. Seven of them passed our pre-selection scheme and two of them at least may be good candidates for use provided pilot-scale assays confirm their suitability.
... M. pulcherrima also showed the highest concentrations of 2phenylethanol and 2-phenyl acetate (data not shown). High production of 2-phenylethanol was found in M. pulcherrima in studies carried out by other authors [72][73][74]. Regarding production of esters, Rodríguez et al. [75] observed high concentrations of these compounds in M. pulcherrima. On the other hand, M. pulcherrima showed the highest levels of ethyl acetate, a compound that can give rise to off odors. ...
The use of non-Saccharomyces yeasts in sequential fermentations with S. cerevisiae has been proposed to improve the organoleptic characteristics involved in the quality of wine. The present study set out to select a non-Saccharomyces inoculum from the D.O.Ca. Rioja for use in winemaking. Strains included in the study belonged to Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, Zygosaccharomyces bailii, Williopsis pratensis, Debaryomyces hansenii, Pichia kluyveri, Sporidiobolus salmonicolor, Candida spp., Cryptococcus spp. and two mixed inocula of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio. In the first stage of the process, SO2 resistance and presence of enzymatic activities related to wine aroma and wine color and fining (esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, β-glucosidase, pectinase, cellulose, xylanase and glucanase) were studied. In the later stages, selection criteria such as fermentative behavior, aroma compound production or influence on phenolic compounds were studied in laboratory scale vinifications. Taking into account the results obtained in the different stages of the process, a mixed inoculum of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio was finally selected. This inoculum stood out for its high implantation capacity, the production of compounds of interest such as glycerol and lactic acid and the consequent modulation of wine acidity. Given these characteristics, the selected inoculum is suitable for the production of quality wines.
... In a broad sense, the concentration observed for butanediol was in general higher in the fermentations carried out with PL-treated grapes, while 3-methyl-1-butanol and 2-methyl-1-butanol were generally in higher concentrations in the fermentations done with nontreated grapes. The species Saccharomyces cerevisiae produces high concentrations of butanediol in comparison to other non-Saccharomyces species [33]; thus, a better implantation in a fermentative must is expected to increase the amounts of this metabolite during fermentation. The concentration of higher alcohols has been reported to be directly proportional to the initial concentration of the inoculum [34], as was the case observed in treated grapes. ...
Pulsed light irradiation is a nonthermal technology currently used for the elimination of pathogens from a diverse range of food products. In the last two decades, the results obtained using PL at laboratory scale are encouraging wine experts to use it in the winemaking industry. PL can reduce native yeast counts significantly, which facilitates the use of starter cultures, reducing SO2 requirements at the same time. In this experimental set up, Tempranillo grapes were subjected to pulsed light treatment, and the fermentative performance of non-Saccharomyces yeasts belonging to the species Schizosaccharomyces pombe, Lachancea thermotolerans, Torulaspora delbrueckii, Metschnikowia pulcherrima and Hanseniaspora vineae was monitored in sequential fermentations against spontaneous fermentation and pure culture fermentation with the species Saccharomyces cerevisiae. The experimental analyses comprised the determination of anthocyanin (High performance liquid chromatography with photodiode array detector—HPLC-DAD), polyphenol index and colour (Ultraviolet-visible spectroscopy—UV-Vis spectrophotometer), fermentation-derived volatiles (Gas chromatography with flame ionization detector—GC-FID), oenological parameters (Fourier transform Infrared spectroscopy—FT-IR) and structural damage of the skin (atomic force microscopy—AFM). The results showed a decrease of 1.2 log CFU/mL yeast counts after pulsed light treatment and more rapid and controlled fermentation kinetics in musts from treated grapes than in untreated samples. The fermentations done with treated grapes allowed starter cultures to better implant in the must, although a larger anthocyanin loss (up to 93%) and an increase in hue values (1 unit) towards more yellow hues were observed for treated grapes. The development of biomass was larger in musts from treated grapes. The profile of volatile compounds and oenological parameters reveals that fermentations carried out with untreated grapes are prone to deviations from native microbiota (e.g., production of lactic acid). Finally, no severe damage on the skin was observed with the AFM on treated grapes.
... Torulaspora delbrueckii is also widely used at an industrial level due to its low volatile acidity and contribution to wine fruitiness, structure (Jolly et al., 2013), and improvement in the texture of the wine (González-Royo et al., 2014;Medina-Trujillo et al., 2016). Metschnikowia pulcherrima has a low fermentative power ; however, it produces a large quantity of terpenes, thiols, and esters, such as ethyl octanoate associated with the pear aroma (Clemente-Jimenez et al., 2004;Sadoudi et al., 2012), reduces the alcohol content (Contreras et al., 2014;Ciani et al., 2016), and can produce significant amounts of 2,6-dimethoxyphenol with a smoky aroma (Culleré et al., 2004;González-Royo et al., 2014). All these yeast species are increasingly being commercialized as starter cultures (Roudil et al., 2019). ...
Global warming is causing serious problems, especially, in warm regions, where musts with excess sugars and high pH produce wines with decreased freshness and unstable evolution. This study aimed to determine biocompatibility between yeast species, the capacity for microbiological acidification, and the aromatic profile produced in ternary fermentations in which Lachancea thermotolerans has been co-inoculated with Hanseniaspora vineae, Torulaspora delbrueckii, or Metschnikowia pulcherrima, and the fermentation process is subsequently completed with sequential inoculation of Saccharomyces cerevisiae. For this purpose, different cell culture media and instruments were used such as infrared spectroscopy, enzymatic autoanalyzer, chromatograph coupled with a flame ionization detector, spectrophotometric analysis, among others. The behavior of these yeasts was evaluated alone and in co-inoculation, always finishing the fermentation with sequential inoculation of S. cerevisiae, at a stable temperature of 16°C and with a low level of sulfites (25 mg/L) in white must. Significant results were obtained in terms of biocompatibility using population counts (CFU/ml) in differential plating media that permitted monitoring. Quantification of the five species was studied. Concerning acidification by L. thermotolerans in co-inoculations, we showed some metabolic interactions, such as the inhibition of acidification when H. vineae/L. thermotolerans were used, generating just over 0.13 g/L of lactic acid and, conversely, a synergistic effect when M. pulcherrima/L. thermotolerans were used, achieving 3.2 g/L of lactic acid and a reduction in pH of up to 0.33. A diminution in alcohol content higher than 0.6% v/v was observed in co-inoculation with the L. thermotolerans/M. pulcherrima yeasts, with total sugar consumption and very slow completion of fermentation in the inoculations with H. vineae and T. delbrueckii. The aromatic composition of the wines obtained was analyzed and a sensory evaluation conducted, and it was found that both L. thermotolerans and co-inoculations retained more aromatic esters over time and had a lower evolution toward the yellow tones typical of oxidation and that the best sensory evaluation was that of the Lt + Mp co-inoculation. Lachancea thermotolerans and co-inoculations produced wines with low levels of volatile acidity (<0.4 g/L). This work shows that good consortia strategies with binary and ternary fermentations of yeast strains can be a powerful bio-tool for producing more complex wines.
... Pichia fermentans produced similar amount of ethanol 5.98% and 2.31 g/L glycerol with 39 g/L residual glucose. S. cerevisiae used in the same study, produced 14% ethanol and 0.57 g/L glycerol and consumed almost all amount of glucose, similar to the results obtained in present research [44]. Many strains of T. delbrueckii were able to produce glycerol in the amount 5-6 g/L, but produced significantly higher amount of ethanol [45]. ...
The fermentative potential of native Candida famata isolates from wild and cultivated blackberries was evaluated for potential application in Prokupac grape must fermentation. 5 isolates, out of a total 22 isolated yeasts, were identified as C. famata. After the initial screening of fermentative performances, microfermentation was performed in a sterile grape must. Produced samples were analyzed using the HPLC technique. All isolates showed an ability to grow at lower temperatures, good tolerance to 7% ethanol and 300 ppm of SO2. C. famata isolates WB-1, WB-2 and W-5 had similar fermentation performance, but WB-1 isolate was chosen for validation at a laboratory-scale level according to a pleasant, fruity aroma, highest fermentative vigor and power, good organic acid profile and the highest level of ethanol and glycerol produced in micro-vinification experiments. Good enological performance of selected C. famata WB-1 isolate is confirmed by higher level of glycerol, lower level of ethanol and acetic acid in wine samples produced in pure and sequential fermentation, when compared to the control sample. Throughout the selection of C. famata yeasts with good enological potential, this work gives a contribution in the area of precision enology, aiming to find a perfect match between non-exploited yeasts and “autochthonous” grape cultivar Prokupac.
... In agreement with this result, Merlot wines from SO4 also showed low levels alcohols, especially 2-phenylethanol, vs. other rootstocks [9]. Moreover, 2-phenylethanol has been reported to be a potential contributor to the floral character of wines, and this is attributed to its distinctive rose-like aroma [28,29]. Other alcohols (2-methyl-1-propanol, 3-methyl-1pentanol, 2,3-butanediol and methionol) also reached the highest concentrations for 110R in the 2010 vintage. ...
Background: Rootstock is a viticultural practice used to combat the devastating Phylloxera vitifoliae (Fitch). Additionally, it is well-known that wine aroma composition depends mainly on variety, viticulture management and winemaking; therefore, rootstocks can affect to berry quality. This study evaluated the influence of nine rootstocks (110R, SO4, 196-17C, Riparia G, 161-49C, 420A, Gravesac, 3309C and 41B) on volatile composition of Albariño wine in two consecutive vintages. Material and Methods: Volatile compounds belonging to eight groups (alcohols, C6-compounds, ethyl esters+acetates, terpenes + C13-norisoprenoids, volatile phenols, volatile acids, lactones and carbonyl compounds) were determined in Albariño wines by GC–MS, during 2009 and 2010 vintages. Results: Rootstock 110R had a positive influence on Albariño wines, increasing total volatile concentration, due mainly to 2-phenylethanol, decanoic and hexanoic acids, ethyl esters and acetates, and C13-norisoprenoids. However, the higher contribution of volatile fatty acids to Albariño wine was shown when grapevines were grafted onto SO4. Conclusions: This work provides new information about the impact of rootstocks on Albariño wine volatile composition, where 110R had a positive influence on Albariño wines under the edaphoclimatic conditions of Salnés Valley (Galicia, Spain).
... Similarly to wine colour parameters, the effect of M. pulcherrima on the concentration of total higher alcohols is inconsistent (Vicente et al. 2020). This yeast species, however, generally increases the total concentration of esters (Bisson and Kunkee 1991, Clemente-Jimenez et al. 2004, Rodriguez et al. 2010, Sadoudi et al. 2012) and the total concentration of volatile acids (Benito et al. 2015, Liu et al. 2017, Dutraive et al. 2019) during wine fermentation. Compared to Control ferments, wines produced with M. pulcherrima AWRI3050 had increased concentration of esters in both cultivars and reduced concentration of volatile acids in Cabernet Sauvignon wines. ...
Background and Aims
The use of non‐Saccharomyces yeast strains as starter cultures for wine production has become increasingly popular, particularly due to their positive effect on wine composition, colour, aroma and flavour. Here, we characterise the volatile aroma composition and the sensory profile of Shiraz and Cabernet Sauvignon wines produced with novel active dry yeast preparations of Metschnikowia pulcherrima compared to that of reference strains.
Methods and Results
Winemaking treatments included an uninoculated fermentation, two reference Saccharomyces cerevisiae fermentations, and sequential fermentations inoculated with either M. pulcherrima AWRI1149 or M. pulcherrima AWRI3050 and S. cerevisiae. Amplicon‐based internal transcribed spacer phylotyping was used to determine microbial population dynamics during fermentation. Wines were analysed for volatile composition and subjected to sensory analysis. The M. pulcherrima strains survived and dominated in both grape cultivars, and produced distinctive wine volatile profiles depending on the inoculation treatment. These differences in volatiles resulted in significant differences for several sensory attributes.
Conclusions
Wines made with active dry yeast preparations of M. pulcherrima AWRI1149 and M. pulcherrima AWRI3050 were characterised by increased intensity of desirable sensory attributes and by low scores for negative descriptors.
Significance of the Study
This work provides winemakers with additional yeast preparations that can shape sensory profile and wine style.
... On the 30th day of fermentation, the kimchi was divided into 10 batches of 2 kg each. To prevent bacterial growth, 100 µg/mL of ampicillin [40] was added to five batches of kimchi every three days from 30 to 50 days of fermentation. According to the results of antibiotic susceptibility testing for Lactobacillus and Bifidobacterium [41], the minimum inhibitory concentration of ampicillin was 1 µg/mL or less for all tested strains. ...
The purpose of this study is to investigate the effect of fungi on kimchi metabolites during fermentation. A gas chromatography-mass spectrometry (GC-MS) based metabolite profiling approach in combination with principal component analysis (PCA) is performed to differentiate metabolites produced by fungi or bacteria. To avoid bacterial growth, kimchi is treated with 100 μg/mL of ampicillin every three days from 30 to 50 days of fermentation. The relative content of the major fungi at 50 days of fermentation, between the control group and the ampicillin treatment group, was not significantly different. The administration of ampicillin changed the metabolites in kimchi by affecting the growth of kimchi bacteria. Based on the pattern of change of each metabolite, the changed metabolites are grouped into four categories: (1) metabolites produced or consumed by fungi, (2) metabolites involving both fungi and bacteria, (3) metabolites produced or consumed by bacteria, and (4) metabolites of undetermined origin. Alanine, thymine, galacturonic acid, and malonic acid can be regarded as the metabolites produced by fungi between 30 and 50 days of fermentation. In contrast, malic acid, oxaloacetic acid, galactitol, glucose, and mannitol are presumed to be the metabolites mainly consumed by fungi. This study is meaningful as the first study conducted by inhibiting growth of bacteria to identify the metabolites contributed by fungi or bacteria in the kimchi fermentation process. These results could be used to make customized kimchi that controls the production of desired metabolites by selectively controlling the formation of microbial communities in the kimchi industry.
... In wine production, these organisms are essential for the fermentation process and for the character of each specific wine [2,3]. The most abundant yeasts in a vineyard are generally the yeasts of the genre Candida, Hanseniaspora, Pichia and Metschnikowia [4,5]. The specie S. cerevisiae is present in a smaller proportion. ...
Considering that many Saccharomyces cerevisiae strains exist and that they have different fermentation capacities, the challenge is to select the yeast strain that generates the most interesting wine character and wine flavor for the winemaker. A method based on simple sequence repeats (SSRs) markers, occurring in the yeast genome, was developed to differentiate the collected S.cerevisiae strains. For the amplification of the polymorphic SSR markers performed by polymerase chain reaction (PCR), two primer sets showing different size products for different S. cerevisiae strains were designed. The PCR-method with gel electrophoresis was validated using capillary sequencing and then used as a service for winegrowers combined with a sensory analysis via napping. This approach can be used for the preservation of the yeast diversity associated with given terroirs and as an option for an increased safety of fermentations. The application of S. cerevisiae strains collected in spontaneous fermentations and used for fermentation sustains the initial character of the wine and ensures a secure fermentation at the same time.
... NS species of several yeast genera have been reported as high producers of certain esters in particular 2-phenylethyl acetate (Hanseniaspora), isoamyl acetate (Pichia) or ethyl caprylate (Torulaspora) which may correspond in wine to "flowery", "fruity banana" and "sour apple" descriptors, respectively (Guth et al., 1997;Lambrechts and Pretorius, 2000;Rojas et al., 2001Rojas et al., , 2003Viana et al., 2008). Several species like H. guilliermondii (Romano et al., 1997), H. uvarum (Mateo et al., 1991), M. pulcherrima and Pichia fermentans (Clemente-Jimenez et al., 2004) are known as good producers of esters in general. The associations of S. cerevisiae with H. vinae (Viana et al., 2011), L. thermotolerans (Gobbi et al., 2013) or Williopsis saturnus (Lee et al., 2010) were also reported as resulting on an enhancement of esters composition in wine, when compared with control fermentations by S. cerevisiae. ...
Biogeography is the study of the distribution of species over space and time and aims to
understand where, why, and at what abundance organisms live. Revealing the diversity and
distribution pattern of populations and communities at multiple spatial scales is thus a central issue in ecology. Recent biogeographic studies based on genetic technologies revealed that microorganisms are not randomly distributed over space and time, showing that their distribution is systematically heterogeneous and structured, revealing specific patterns of microbial distribution. In this context, the study of microbial communities associated with vineyards points to the existence of patterns of microbial distribution across viticultural areas, suggesting a microbiological component of the terroir concept. In the light of this knowledge, the composition of the yeast flora present on grapes, besides representing a long-term known factor of wine quality tends to be presently seen as a potential factor of wine typicality. To assess grape yeast diversity and to understand the ecological and geographical factors shaping the yeast communities and populations composition is of great importance for modern oenology. In this chapter we present
on overview of the research developed in this scope.
... Parallèlement, ils produisent peu d'alcools supérieurs (e.g. propyliques, butyliques, et amyliques ) (Romano et Suzzi, 1993;Rojas et al., 2003;Clemente-Jimenez et al., 2004;Moreira et al., 2008), tandis que les esters et les composés soufrés sont principalement produits par Candida, Hansenisapora, T. delbrueckii, et K. gamospora (Rojas et al., 2001;Moreira et al., 2005;Viana et al., 2008). ...
Ce travail de thèse concerne la valorisation des pommes libanaises en évaluant leur potentiel fermentaire en vue de l’élaboration du cidre, spécifiquement l’étape de la fermentation de ce processus. Pour cette fin, l’analyse de la composition chimique des pommes libanaises « Ace spur » a été effectué en comparaison avec une pomme à cidre « Kermerrien » utilisée industriellement en France pour la production du cidre. La variété de pomme libanaise « Ace spur » semble être convenable pour l’élaboration de cidre. De plus, une souche responsable de la fermentation spontanée de cette variété de pommes a été isolée et identifiée. Son potentiel fermentaire dans ces deux variétés de pommes a été par la suite étudié. La levure Hanseniaspora sp. isolée permet la fermentation des jus de pommes jusqu’à 4% (v,v) d’éthanol. Cette étude fournira pour la première fois des données sur la composition chimique de la pomme libanaise, sur une souche présente lors de sa fermentation, ainsi que sur son potentiel fermentaire. Également, les travaux de la thèse consistent à évaluer l’impact des procédés émergents : champs électriques pulsés et ultrasons sur la fermentation. Ces travaux visent à démontrer le potentiel d’implémentation de ces procédés industriellement. Nous proposerons une approche de traitement du jus de pommes en continu qui pourra être transposée de l’échelle laboratoire à l’échelle industrielle. L’application des technologies émergentes (champs électriques pulsés et ultrasons) induit une accélération des cinétiques fermentaires et une réduction du taux d’éthanol.
... Yeasts can affect primary aroma determined by the initial composition of the product and the secondary aromas that are created during the fermentation, as well as the tertiary aromas generated during the maturation of the finished product [86]. Hanseniaspora, Zygosaccharomyces, and Schizosaccharomyces pombe species produce high amounts of volatile fatty acids, such as acetic acid [87][88][89][90][91], and low concentrations of higher alcohols [92][93][94][95]. Esters and sulfur compounds are mainly produced by Candida, Hansenisapora, Torulaspora delbrueckii, and Kazachstania gamospora [93,[96][97][98]. ...
Fermented apple beverages are produced all over the world with diverse characteristics associated with each country. Despite the diversifications, cider producers are confronted with similar issues and risks. The nature of the raw material, also known as the fermentation medium, plays a key role in fermentation. A well-defined composition of apples is, therefore, required to produce cider with good quality. In addition, ferment and its metabolism are important factors in the fermentation process. The producers of cider and other alcoholic beverages are looking in general for novel yeast strains or for the use of native strains to produce "authentic" and diversified beverages that are distinct from each other, and that attract more and more consumers. Research articles on cider production are infrequent compared to wine production, especially on the impact of the chemical composition and microbial diversity of apples on fermentation. Even though the processing of fermented beverages is close in terms of microbial interactions and production, the study of the specific properties of apples and the production challenges of cider production is advantageous and meaningful for cider producers. This review summarizes the current knowledge on apple composition and the impact of the must composition on fermentation and yeast growth. In addition, the microbial diversity of cider, activities, and its influence on fermentation are reviewed.
... To the determinants of the final wine bouquet may be also included acetaldehyde, succinic acid, higher alcohols and esters. Microorganisms characterized by high production of these compounds include Pichia anomala and K. apiculata strains [27]. ...
The international competitiveness of the wine sector and consumer demands for the unique wine styles pose challenges in improving the fermentation process. The basis of proper alcoholic fermentation is knowledge about how individual yeast strains interact with the aroma, taste and color of wine, what results in possibility to select species used as starter cultures. To use the value of non-Saccharomyces yeast strains in wine production and to minimize the possibility of wine deterioration, it is necessary to precisely recognize the yeast cultures present on the fruit of the vine and in grape must, as well as their metabolic properties. The aim of the study was to determine the oenological properties of yeasts isolated from spontaneously fermented grape musts obtained from cool climate grapes. For this purpose, Zweigelt grape must was fermented with yeast monocultures. Alcohol, extract, sugars, glycerol, total acidity and free amine nitrogen were analyzed in the obtained wines. Poor fermentation properties of yeast strains results in obtaining wines with relatively large amounts of residual sugars and low alcohol. A decrease in overall acidity was noted in sets with the participation of M. pulcherrima MG971264, while in other tests the opposite trend was observed. Although some microorganisms have the ability to assimilate organic acids found in wine, they are not able to carry out fermentation or they do it inefficiently. Solution to this problem may, therefore, be use of mixed cultures of noble and non-Saccharomyces yeast, what effectively reduce the concentration of organic acids, while not adversely affecting the organoleptic characteristics of the drink.
... In particular, the highest concentrations of 2-phenylethanol were reached in mixed fermentations including M. pulcherrima (9.2 mg/L) and T. delbrueckii (9.4 mg/L). These results agree with those found by other authors showing that M. pulcherrima and T. delbrueckii produce high level of 2-phenylethanol [3,73,74]. Ethyl acetate was the main ester produced. At high concentrations (>100-150 mg/L) ethyl acetate determines a solvent-like aroma. ...
: The production of wines with peculiar analytical and sensorial profiles, together with the microbiological control of the winemaking process, has always been one of the main objectives of the wine industry. In this perspective, the use of oenological starters containing non-Saccharomyces yeasts can represent a valid tool for achieving these objectives. Here we present the results of seven pilot scale fermentations, each of which was inoculated with a different non-Saccharomyces yeast strain and after three days with a commercial Saccharomyces cerevisiae starter. The fermentations were carried out in double on 70 L of Sangiovese grape must, the most widely planted red grape variety in Italy and particularly in Tuscany, where it is utilized for the production of more than 80% of red wines. Fermentations were monitored by assessing both the development of the microbial population and the consumption of sugars at the different sampling times. The impact of the different starters was assessed after stabilization through the evaluation of the standard analytical composition of the resulting wines, also taking into account polysaccharides and volatile compounds. Moreover, quantitative descriptive sensory analyses were carried out. Compared to the control wines obtained by inoculating the S. cerevisiae starter strain, those inoculated with non-Saccharomyces/Saccharomyces mixed starters presented a significant differentiation in the chemical-analytical composition. Moreover, sensory analysis revealed differences among wines mainly for intensity of color, astringency, and dryness mouthfeel perception.
... For example, inoculation of Metschnikowia pulcherrima was used to increase the production of 2-phenylethanol. 9 Torulaspora delbrueckii has been documented to produce less off-flavor compounds, such as acetaldehyde, 3-hydroxybutan-2-one (acetoin), and acetic acid compared with other non-Saccharomyces strains. 10,11 In comparison with inoculation with S. cerevisiae, fermentations with Hanseniaspora species and Lachancea thermotolerans strain reduced the production of higher alcohols 8,12 and those with Schizosaccharomyces species generated a higher amount of acetaldehyde, 13 whereas fermentation with Issatchenkia orientalis resulted in lower production of acetaldehyde, propan-1-ol, butan-2-ol, and 3-methylbutan-1-ol. ...
The profile of volatile compounds was investigated using HS-SPME-GC-MS during bilberry juice fermentation with nine non-Saccharomyces yeasts, including Pachysolen tannophilus, Metschnikowia pulcherrima, Hanseniaspora uvarum, Torulaspora delbrueckii, Zygosaccharomyces bailii, Schizosaccharomyces pombe, Lachancea thermotolerans, Issatchenkia orientalis, and Saccharomycodes ludwigii. Dynamic changes in volatile compounds were determined simultaneously with the development of ethanol concentration during fermentation. H. uvarum or I. orientalis produced more ethyl acetate than other yeast strains throughout fermentation, while fermentation with M. pulcherrima resulted in a high accumulation of higher alcohols. S. pombe was associated with high productions of pentane-2,3-dione, 3-hydroxybutan-2-one, 2-methylbutanal, and 3-methylbutanal. Among the 59 volatile compounds detected, generally, higher alcohols and monoterpenes accumulated constantly and reached the maximum concentration at the middle or later fermentation stage, whereas aldehydes, ketones, and acetals accumulated first followed by a significant drop. The production and accumulation dynamics of metabolites were highly dependent on the yeast species and the developing ethanol content.
... Coinoculation (all yeast added at once) or sequential inoculation (Saccharomyces yeast added after a period when non-Saccharomyces strain is sole inoculum) can change population dynamics and fermentation outcome (e.g. Clemente-Jimenez et al. 2004). The ratio of S. cerevisiae to non-Saccharomyces strains in the mixed-culture fermentation can also impact the dominance, or otherwise, of a particular yeast and therefore the sensory properties of the beverage (Azzolini et al. 2015;Basso et al. 2016;Curiel et al. 2017). ...
Producers often utilise some of the many available yeast species and strains in the making of fermented alcoholic beverages in order to augment flavours, aromas, acids and textural properties. But still, the demand remains for more yeasts with novel phenotypes that not only impact sensory characteristics but also offer process and engineering advantages. Two strategies for finding such yeasts are (i) bioprospecting for novel strains and species and (ii) genetic modification of known yeasts. The latter enjoys the promise of the emerging field of synthetic biology, which, in principle, would enable scientists to create yeasts with the exact phenotype desired for a given fermentation. In this mini review, we compare and contrast advances in bioprospecting and in synthetic biology as they relate to alcoholic fermentation in brewing and wine making. We explore recent advances in fermentation-relevant recombinant technologies and synthetic biology including the Yeast 2.0 Consortium, use of environmental yeasts, challenges, constraints of law and consumer acceptance.
... These microorganisms are found in fresh must (e.g. Saccharomyces cerevisiae; Pichia spp.; Rhodotorula spp.; Candida stellata; Hanseniaspora guilliermondii; Lachancea thermotolerants; Kluyveromyces marxianus; Torulaspora delbrueckii; Dekkera bruxellensis and Zygosaccharomyces bailii) (Combina et al., 2005;Fleet, 2008;Lonvaud-Funel, 1996;Querol et al., 1990;Torija et al., 2001) and, some of them contribute to the sensorial profile of wine, influencing its organoleptic quality and complexity (Bisson and Kunkee, 1991;Ciani and Ferraro, 1998;Clemente-Jimenez et al., 2004;Fleet, 2003Fleet, , 2008Jolly et al., 2013). However, several non-Saccharomyces species, those as ethanol tolerant as S. cerevisiae (Z. ...
Non-Saccharomyces yeasts have recently garnered significant interest in oenology. When co-inoculated with Saccharomyces cerevisiae, they contribute to the improvement of wine quality from a sensory point of view. In the present study, a group of yeasts previously isolated from manna and honey by-products were subjected to a genotypic identification. The D1/D2 variable domains of the 26-sRNA gene and the ITS region of the 5.8S gene were sequenced. Additionally, a differentiation of strains was carried out by RAPD-PCR. All strains underwent in vitro screening. Subsequently, a micro-vinification experiment was conducted, focusing on strains with favourable technological characteristics: Lachancea thermotolerans, Starmerella lactis-condensi, and Candida oleophila. These strains were sequentially inoculated alongside a control strain of Saccharomyces cerevisiae. Technological screening revealed that some strains exhibited limited H2S production, ethanol tolerance (up to 8% v/v), resistance to potassium metabisulphite (200 mg/L), osmotic stress tolerance (up to 320 g/L of glucose), and copper resistance (on average 5 mM). The findings from this study can guide the selection of new starters and co-starters for regional wine production.
Scotch Whisky, a product of high importance to Scotland, has gained global approval for its distinctive qualities derived from the traditional production process which is defined in law. However, ongoing research continuously enhances Scotch Whisky production and is fostering a diversification of flavour profiles. To be classified as Scotch Whisky, the final spirit needs to retain the aroma and taste of “Scotch”. While each production step contributes significantly to whisky flavour—from malt preparation and mashing to fermentation, distillation, and maturation—the impact of yeast during fermentation is crucially important. Not only does the yeast convert the sugar to alcohol, it also produces important volatile compounds, for example esters and higher alcohols, that contribute to the final flavour profile of whisky. The yeast chosen for whisky fermentations can significantly influence whisky flavour, so the yeast strain employed is of high importance. This review explores the role of yeast in Scotch Whisky production and its influence on flavour diversification. Furthermore, an extensive examination of non-conventional yeasts employed in brewing and winemaking is undertaken to assess their potential suitability for adoption as Scotch Whisky yeast strains, followed by a review of methods for evaluating new yeast strains.
The flavor is one of the sensory attributes that panelists assess and its complexity brings several discussions regarding the definition of its concept. Some authors describe the flavor as a sum of perceptions resulting from the stimulation of the senses by the food/beverage in the digestive and respiratory tracts; other authors define flavor as a combination of taste and mouthfeel. There is a tendency to consider the relevance of the relationship between the flavor and the chemical food/beverage matrix. Acids, alcohols, volatile compounds, and other chemical substances are responsible for distinctive sensations in the mouth promoting different tastes and mouthfeel. In addition, the synthesis path of the chemical substances that respond to flavor compounds is based on relevant biochemical phenomena. In this context, alcoholic fermentation is considered one of the most essential biochemical reactions providing the formation of a set of volatile compounds responsible for influencing the flavor. Wine and mezcal are examples of alcoholic beverages that present several chemical compounds responsible for their complex flavor due to the several biochemical reactions that occur during their production. Esters, higher alcohols, carbonyl compounds, and oak flavors such as lactones, furanic compounds, and methoxyphenols are responsible for providing the complex flavor that differentiates the wines. Other factors such as the intrinsic features of the grape cultivar and the grape vintage also influence the wine flavor. The most relevant biochemical pathway that produces the chemical compounds that respond to wine flavor is the synthesis of different enzymes produced by the different yeasts responsible for alcoholic fermentation. Also, mezcal is produced by different species of maguey and its cooking is the first step that generates caramel and smoky notes related to furans, alcohols, aldehydes, ketones, phenols, and terpenes. The chemical composition of the mezcal depends on the maguey species, age, the chemical composition of the maguey, and the practice of each producer. The use of autochthonous yeasts during alcoholic fermentation and the conditions used for the biochemical reaction respond to the mezcal flavor differences. In summary, biochemical reactions have substantial importance for the flavor complexity of wine and mezcal since the different flavor nuances are explained by the different chemical composition of the grape and maguey, respectively, the conditions used during the process, and the yeasts involved in the alcoholic fermentation. The study of biochemical pathways will be helpful for wineries and mezcal producers to elucidate the mechanism of the reactions involving the synthesis of specific chemical compounds and their respective flavor notes, producing a safe beverage with quality and high acceptance.
Wine production has been performed during most of the recent history of humanity. Yeasts are the primary players involved in the transformation of grape must into wine, and the need for proper control of fermentation requires good microbiological knowledge and appropriate tools to monitor the process. The primary task of microorganisms in nature is to recycle organic matter, including sugars. Among these microorganisms, yeasts have a high capacity to ferment sugars to alcohol whenever fruits are ripe. During the fermentation process, a succession of microorganisms occurs, and the methods available for analyzing microbiological dynamics can be classified as “classical” methods, molecular methods, and next‐generation sequencing methods. Grape must have diverse secondary metabolites that are either volatiles or can be transformed into volatiles and contribute to the aroma. Microbiological control is necessary to produce wines with properties that define the quality and reproducibility of the wines.
Issatchenkia orientalis, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for I. orientalis exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any I. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. The resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host I. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.
Moleculer identification methods beside the conventional identification methods and kit systems using for identification of yeast species have also been using recently. As a part of research, determination of yeast flora on pomegranate surface and raspberry fruits were planned using morphologic features of yeast strains, API ID32C kit system and molecular identification methods and determination of yeast species that can produce industrial importance enzymes were aimed. After genomic DNA isolation from yeast strains, ITS1-5.8S rDNA-ITS2 gene regions were amplified by PCR method using ITS1 and ITS4 universal primer. The groups obtained from the size of PCR products that belongs to yeast strains were cut by using restriction enzymes (HinfI, HaeIII, MspI, AluI ve HhaI). The yeast strains which differ in PCR groups were selected as restriction profile and DNA sequence analysis were carried out and yeast strains were identified using DNA sequences by BLAST program. Extracellular enzyme activities were determined of identified yeast species by API-ZYM test system. Consequence of my study, according to DNA sequence analysis of the pomegranate yeast strains showed high homology with Metschnikowia pulcherrima, Cryptococcus bestiolae, Aureobasidium pullulans, Candida zeylanoides, Hanseniaspora uvarum, Kluyveromyces lactis and Metschnikowia sp. species. Raspberry yeast strains showed high homology M. pulcherrima, A. pullulans, Candida sp. H. uvarum ve Metschnikowia sp. species. According to API-ZYM results, high enzyme activity were determined for pomegranate in alkaline phosphatase, leucine arylamidase, esterase (C 4), esterase lipase (C 8), acid phosphatase, naftol-AS-BI-fosfohidrolase enzymes, were determinated for raspberry yeast strains in leucine arylamydase and β- glukosidase enzymes.
Keywords: Pomegranate, Raspberry, ITS1-rDNA-ITS2, RFLP and API-ZYM
A severe disease occurred in farmed Eriocheir sinensis characterized by milky liquid accumulation in the pectoral cavity, in the province of Liaoning, China, during October 2018–April 2019. Diseased crabs moved sluggishly, exhibited appetite loss and readily lost legs. Under the microscopic analysis, it was observed that the milky liquid contained a large number of yeastlike microorganisms (0.8–1.2 μm × 1.5–1.9 μm), which were also present in the muscle, hepatopancreas and gills. A dominant strain was isolated from the milky liquid and other tissues of diseased crabs. It grew on nutrient agar and formed 1‐ to 3‐mm white opaque colonies, each with a protuberance in the centre. Besides, the results of TEM and SEM also demonstrate a typical multilateral budding model of the yeast clearly. We identified the strain, which we named 2EJM001, as Metschnikowia bicuspidata based on 18S rDNA, ITS and 26S rDNA sequence analyses and on its morphological, physiological and biochemical characteristics. Phylogenetic analysis revealed that 26S rDNA of 2EJM001 was clustered with M. bicuspidata (LNES0119) as reported by Bao et al. In addition, unlike Bao et al., two challenge experiments (injection and immersion) were used in this study. The results of challenge experiments show that 2EJM001 was pathogenic to E. sinensis and caused signs similar to those found in the naturally infected crabs. At the same time, the minimum inhibitory concentrations (MIC80 and MIC90) were determined. This study further confirms that M. bicuspidata 2EJM001 was the pathogen responsible for ‘milky disease’ in E. sinensis from Liaoning Province.
Background
Yeasts are widely spread in nature. Yeasts have a positive role in the fermentation of some products such as wine or beer, although they are also responsible for food spoilage alongside fungi. In addition, some species of yeasts, such as Candida spp., can enter the human body through food and beverages and may cause various types of infections. Therefore, it is necessary to find natural means for inhibition yeast growth in foods where they are undesirable microorganisms.
Scope and approach
This work describes the prevalence of yeasts in food products, the impact of pathogenic yeasts on the human organism, and the possibility to suppress their growth by use of lactic acid bacteria and preservatives of plant origin. It may be applicable in food industries where yeasts are undesirable microorganisms which requires inhibition of their growth.
Key findings
Yeasts have a positive role in food fermentation, but can also cause infections in people, therefore their presence in food should be controlled. Lactic acid bacteria (LAB) are good inhibitors of fungal activity due to the presence of inhibitory compounds, such as lactic, acetic, and ascorbic acids, hydrogen peroxide, bacteriocins, and others. Out of LAB, Lactobacillus plantarum shows the strongest inhibitory effects. Furthermore, plant extracts, such as black walnut, clove, garlic, oregano leaf extracts, as well as anolyte – a natural disinfectant - also display an antifungal effect and could be used for yeast and fungal control.
‘Marselan’ is a promising wine grapevine with unique qualities, studies on its introduction and characteristics have never stopped. However, little is known about the fermentation process of ‘Marselan’ wine. In this study, we compared industrial-scale spontaneous (SMF) and inoculated (IMF) fermentation processes on the succession of microbial community and the change in volatile compounds and predicted their correlations. SMF could ferment ‘Marselan’ to dry red wine with alcohol content of 13.37% ± 0.76% (v/v) in 10 days and enhance the total concentration and odour activity values (OAVs) of volatile compounds. Ethyl caprylate, ethyl caprate, ethyl caproate and citronellol (OAVs > 1.0) were important volatile compounds in SMF and IMF wines. Compared with IMF, SMF was more outstanding in initial non-Saccharomyces diversity, including Hanseniaspora, Metschnikowia and Issatchenkia. In addition, Spearman's correlation (|ρ| > 0.7) and bidirectional orthogonal partial least squares (O2PLS, VIP > 1.0) analysis showed that some indigenous microorganisms in grape were closely related to the formation of volatile compounds, such as lactic acid bacteria, non-Saccharomyces and Saccharomyces cerevisiae. Overall, results showed that SMF could strengthen the flavour of ‘Marselan’ wine, and the possibility of consideration screening indigenous aroma-producing microorganisms as starter to improve the unique characteristics of ‘Marselan’ wine.
Cabernet Sauvignon grape produced in Xinjiang (China) is often overripe, with unusually high sugar content, which impedes utilization. We aimed to establish the optimal combination of indigenous yeast strains to produce a new sweet wine from the overripe grape. Five yeast strains with pronounced enological characteristics were selected from 88 indigenous yeast isolates. Using a series of co-fermentation experiments with different inoculated strategies, we achieved optimal co-fermentation with a combination of strains SC19 and NS68, later identified as Saccharomyces cerevisiae and Pichia kudriavzevii, respectively, simultaneously inoculated in a 1:1 ratio at the early stage of fermentation. The combination was characterized by vigorous fermentation with high resistance to 457.13 g/L sugar and high alcohol yield (16.01% vol). The sweet wine contained 17 aromatic compounds with odor activity value (OAV) ≥ 1 and pronounced sweet fruit, floral, herbaceous, and caramel odors. The co-fermentation has a good potential for utilization of overripe Cabernet Sauvignon grape.
The most mysterious aspect of wine is the endless variety of flavours that stem from a complex, completely non-linear system of interactions among many hundreds of compounds. In its widest sense, wine flavour refers to the overall impression of both aroma and taste components. Aroma is usually associated with odorous, volatile compounds; the bouquet of wine refers to the more complex flavour compounds which evolve as a result of fermentation, elevage and ageing. With the exception of terpenes in the aromatic grape varieties and alkoxypyrazines in the herbaceous cultivars, perceived flavour is the result of absolute amounts and specific ratios of many of these interactive compounds, rather than being attributable to a single "impact" compound. Without underestimating the complexity of these interactive effects or negating the definitive role played by the accumulated secondary grape metabolites in the varietal character of wine, this review will focus mainly on the contribution of yeast fermentation to the sensorial quality of the final product. Yeast and fermentation conditions are claimed to be the most important factors influencing the flavours in wine. Both spontaneous and inoculated wine fermentations are affected by the diversity of yeasts associated with the vineyard and winery. During the primary alcoholic fermentation of sugar, the wine yeast, Saccharomyces cerevisiae, together with other indigenous non-Saccharomyces species, produce ethanol, carbon dioxide and a number of by-products. Of these yeast-derived metabolites, the alcohols, acetates and C4-C8 1tfatty acid ethyl esters are found in the highest concentration in wine. While the volatile metabolites contribute tothe fermentation bouquet ubiquitous to all young wines, the production levels of these by-products are variable and yeast strain specific. Therefore, this article also highlights the importance of untapping the hidden wealth of indigenous yeast species present on grapes, and the selection and genetic development of yeast starter culture strains with improved flavour profiles. In the future, some winemakers may prefer to use mixtures of indigenous yeast species and tailored S. cerevisiae strains as starter cultures to reflect the biodiversity and stylistic distinctiveness of a given region. This will help winemakers to fullfil the consumer's demand for individual wines with intact local character and to ensure the survival of wine's most enthralling aspect - its endless variety.
The aroma formation in the fermentation of two types of natural musts by 12 different yeasts has been analysed. In grape must fermentation Pichia fermentans Coleccin Espaola de Cultivos Tipo (CECT) 11773, Clavispora lusitaniae OJ6 and Pichia anomala OJ5 produced the best balance between concentrations of ethyl acetate and high alcohols. When orange juice was fermented with the 12 yeasts, Pichia fermentans CECT 11773, Rhodotorula mucilaginosa OJ2 and Hanseniaspora uvarum CECT 10885 produced a good beverage with low alcoholic grade. For both types of natural musts Pichia fermentans CECT 11773 increased the presence of higher alcohols and ethyl acetate. After using this strain both alcoholic beverages obtained the highest evaluation in the sensory analysis.
The identification and classification of yeasts have traditionally been based on morphological, physiological and biochemical traits. Various kits have been developed as rapid systems for yeast identification, but mostly for clinical diagnosis. In recent years, different molecular biology techniques have been developed for yeast identification, but there is no available database to identify a large number of species. In the present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts. In many cases, the size of the PCR products and the restriction patterns obtained with endonucleases CfoI, HaeIII and HinfI yielded a unique profile for each species. Accordingly, the use of this molecular approach is proposed as a new rapid and easy method of routine yeast identification.
Molecular and physiological analyses were used to study the evolution of the yeast population, from alcoholic fermentation
to biological aging in the process of “fino” sherry wine making. The four races of “flor” Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, and rouxii) exhibited identical restriction patterns for the region spanning the internal transcribed spacers 1 and 2 (ITS-1 and ITS-2)
and the 5.8S rRNA gene, but this pattern was different, from those exhibited by non-florS. cerevisiae strains. This flor-specific pattern was detected only after wines were fortified, never during alcoholic fermentation, and
all the strains isolated from the velum exhibited the typical flor yeast pattern. By restriction fragment length polymorphism
of mitochondrial DNA and karyotyping, we showed that (i) the native strain is better adapted to fermentation conditions than
commercial strains; (ii) two different populations of S. cerevisiaestrains are involved in the process of elaboration, of fino sherry wine, one of which is responsible for must fermentation
and the other, for wine aging; and (iii) one strain was dominant in the flor population integrating the velum from sherry
wines produced in González Byass wineries, although other authors have described a succession of races of flor S. cerevisiae during wine aging. Analyzing all these results together, we conclude that yeast population dynamics during biological aging
is a complex phenomenon and differences between yeast populations from different wineries can be observed.
Recent ecological surveys indicate that the wine yeast Saccharomyces cerevisiae may be isolated with extreme difficulty from natural substrates, such as vineyard soil or the surface of mature grapes, conventionally believed to be its elected habitat. Conversely, it is amply demonstrated that its preferential location as the only fermenting colonizer is the surfaces of the wineries. Non-conventional methods for the separation, isolation, and enumeration of yeast cells from mature grapes of different varieties produced additional and unequivocal evidence on the numerical inconsistency of S. cerevisiae cells in nature. The taking over of musts by the natural yeast flora was also followed in microfermentations.
The dynamics of the wine yeast strains presented in five spontaneous Malvasia wine fermentations have been studied. Samples were analysed for their microbiological characteristics and chemical substances. All 937 isolates were characterized using electrophoretic karyotyping and tested for their killer activity. The non- Saccharomyces population was identified using a combination of PCR-RFLP analysis of the rDNA spacer region and physiological testing. The total yeast population level in the must after sedimentation was 105cfu ml−1and included the following genera:Candida, Metschnikowia, Hanseniaspora, Rhodotorula, Issatchenkia and Debaryomyces. However, Saccharomyces sp. was not detected in fresh must samples plated on YEPD medium. Based on the chromosome length polymorphism among 649 isolates from the subsequent phases of fermentation, 46 different electrophoretic patterns of Saccharomyces cerevisiae were distinguished. The most abundant karyotypes were L1, L4, L12, P6. A sequential substitution of S. cerevisiae strains occurred during the different phases of fermentation. At the slow fermentation rate, karyotype L4was most abundant in almost all fermenters. At the beginning of the tumultuous fermentation phase, the most frequent karyotype became L1followed by karyotype L4. Finally, during the fermentation process, pattern L4was clearly replaced by karyotype L1followed by pattern L12. Despite the same fermentation source (grape must), differences among five spontaneous fermentations were observed. The population dynamics of S. cerevisiae yeasts, especially the dynamics of the major S. cerevisiae strains (L1, L4, and L12) were quite similar in all five fermenters in opposite to the minor strains of S. cerevisiae.
Recent ecological evidence points to a circulation model for Saccharomyces cerevisiae in nature which is different from that proposed at the end of the last century. The wine yeast ‘par excellence’ is isolated with extreme difficulty from conventional habitats, such as vineyard soil or the surface of ripe grapes, while it is almost the only species colonising the surfaces of the winery equipment.
The source of Saccharomyces cerevisiae responsible for the natural fermentation of grape must and the dominance of added yeast in wine fermentations have been studied using a strain of Saccharomyces cerevisiae selected for wine-making and labelled for not producing hydrogen sulphide (H2S⁻) as fermentation starter.
It was shown that the source of the Saccharomyces flora of fermenting musts was the winery itself and that a selected yeast starter added initially is able to impose itself on the spontaneous grape juice flora naturally.
Commonly used techniques for the identification of industrial yeast strains are usually time-consuming and cumbersome. Moreover, some of these methods may give ambiguous results. A novel strategy has been developed for identifying yeast strain employing polymerase chain reaction technology. Using customised oligonucleotides, some regions of the yeast genome between δ elements are amplified to give an ‘amplified’ sequence polymorphisml (Skolnick and Wallace 1988) characteristic of the strains. With this technique it is possible to identify individual strains of Saccharomyces cerevisiae.
Summary Acetaldehyde is an important aroma compound in wine. This article first reviews the microbial and chemical formation of acetaldehyde, then the effects of sulphur dioxide on acetaldehyde and effects of acetaldehyde on wine colour and physical stability are described briefly. Finally, the microbiological implications of acetaldehyde are emphasized with respect to practical significance in wine fermentation.
A total of 78 strains of non-Saccharomyces yeasts were isolated: 30 strains of Kloeckera apiculata, 20 of Candida stellata, 8 of Candida valida and 20 of Zygosaccharomyces fermentati. The diversity of yeast species and strains was monitored by determining the formation of secondary products of fermentation, such as acetaldehyde, ethyl acetate and higher alcohols. Within each species, the strains were distinguishable in phenotypes through the production of different amounts of by-products. In particular, a great variability was found in C. stellata, where six different phenotypes were identified by means of the production of acetaldehyde, ethyl acetate, isobutanol and isoamyl alcohol. At different stages of the spontaneous fermentation different phenotypes of the non-Saccharomyces yeasts were represented, characterized by consistent differences in some by-products involved in the wine bouquet, such as acetaldehyde.
The capacity to produce 2,3-butanediol by 90 strains of four different species of wine yeasts (Kloeckera apiculata, Saccharomyces cerevisiae, Saccharomycodes ludwigii, Zygosaccharomyces bailii) was tested in grape must by automated multiple development HPTLC. The total amount of 2,3-butanediol produced varied between 23mg l–1 and 857.7mg l–1 according to the yeast species. S. cerevisiae and Z. bailii behaved similarly, producing elevated amounts of 2,3-butanediol. K. apiculata and Sc. ludwigii, in contrast, were low producers. When considerable amounts of 2,3-butanediol were found, little acetoin was present; the amounts of butanediol and acetoin were characteristic of the individual species.
The progress made in the analysis of volatile wine constituents has led to a better understanding of the complexity of wine aroma.Now many factors influencing wine aroma are not only characterized by their sensory impact, but also by chemical data which in many cases allow a more differentiated approach. By correlating sensory and analytical data headway has been made, especially in the field of wine off-odors. Successful analytical cultivar differentiation based on wine volatiles indicates the importance and possibilities of wine aroma for quality control.
Several yeast cultures belonging to five non-Saccharomyces species associated with wine-making were evaluated for their oenological properties. Results showed that Candida stellata and Torulaspora delbrueckii could positively affect the taste and flavour of alcoholic beverages. Apiculate yeasts exhibited large amounts of negative byproducts, particularly ethyl acetate. Nevertheless, Kloeckera apiculata showed a significantly negative correlation between either acetic acid and ethyl acetate formation and ethanol production. Selected non-Saccharomyces yeast cultures could be applied profitably in wine-making for optimization of wine bouquet using new fermentation technologies.
Secondary products in wines obtained by pure, mixed and sequential cultures of Saccharomyces cerevisiae, Hanseniaspora guilliermondii or Kloeckera apiculata were studied. Consistent differences in the composition were determined in wines fermented by sequential cultures. When S. cerevisiae was added to musts partially fermented by apiculate yeasts, its metabolism was significantly affected. In particular it synthesized high amounts of n-propanol and metabolized high quantities of acetoin, produced by apiculate yeasts
A study of the microbiota present during the wine fermentation of five grape varieties from the ‘El Penedès’ area (Spain) was carried out to select autochthonous yeast strains for industrial wine production. In this study we identified members of the genera Candida, Dekkera, Hanseniaspora, Kluyveromyces, Torulaspora, Zygosaccharomyces and Saccharomyces in wine fermentation microbiota. Strains of Saccharomyces cerevisiae, as responsible agents of the alcoholic fermentation, were considered for a selection protocol. In this work we applied different enological criteria for selection, but previously we have characterized and differentiated Saccharomyces isolates by molecular methods to reduce the number of strains to analyse. Three strains were selected to conduct fermentations according to their characteristics. Finally, using mitochondrial DNA restriction analysis we demonstrated that the autochthonous selected strains are important contributors to the wine fermentation.
The effects of sulphur dioxide and a commercial starter inoculum on yeast population dynamics have been analysed by a molecular approach. Yeast identification from fermenting Carinyena grape musts was performed by RFLP's of mtDNA and rRNA-coding DNA. As expected, the use of a commercial inoculum speeded up the start of fermentation, while SO2 addition limited the development of non-Saccharomyces species. However, this effect was also observed with yeast inoculation. Further analysis of population dynamics could lead to a recommendation for the reduction of the dosage of SO2 by the addition of appropriate inoculum of yeasts in the must. Furthermore, the timing of inoculum addition could be modified to allow a proper contribution of non-Saccharomyces species. Molecular biology analysis of population dynamics could provide a tool to efficiently reduce the dosage of SO2 and adjust the timing of inoculum addition.
Production of 2-phenylethanol by Pichia fermentans L-5 as affected by cultivation temperature (20–40°C), medium pH (4.0–11.0) and shaking speed (0–250 rpm) as well as the kinetics of 2-phenylethanol formation during fermentation were investigated in the present study. Results showed the optimum culture temperature, initial medium pH and shaking speed for the 2-phenylethanol production by test organism were 30–35°C, 8.5 and 250 rpm, respectively. Kinetics study revealed that production of 2-phenylethanol during P. fermentans L-5 fermentation increased almost in parallel with the increase in biomass or viable population during the initial stage of fermentation. During a 72-h fermentation period, level of phenylalanine in medium dropped coincidentally with increasing biomass and 2-phenylethyl derivatives, from the start of fermentation. A maximum 2-phenylethanol production of ca 505.5 mg/l with a molar yield of ca 0.74 was obtained after 16 h of fermentation.
Most real-time video recognition systems have historically relied upon region of interest processing based on low level properties such as pixel color to quickly reduce the computational load required for real-time performance. Because these low levelproperties are quite sensitive to lighting variations, they are limited in their application scope. This paper presents an approach which segments objects of interest via higher level relational properties overcoming such limitations. These relations are autoassociatively and incrementally constructed through a processing structure called raster scan video processing in a hierarchical manner. Relational properties of interest are then obtained with local object specific processes. The compactness of this processing structure has allowed it to be implemented on one TMS320C40 digital signal processing chip at a processing rate of three frames per second. The approach is applied to the problem of road sign recognition in realistic outdoor scenes. Video frame recognition results for stop and pedestrian signs are presented along with details of the DSP implementation.
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PCR-RFLP analysis of the rDNA-ITS (internal transcribed spacer) region was applied to 174 yeast strains belonging to 30 species of oenological significance and including 27 type strains in order to define a rapid identification protocol for yeast colonies. DraI-or HaeIII-PCR-RFLP patterns were species-specific with the exception of teleomorphic and anamorphic forms. An improved protocol taking about 30 h was used for the detection and quantification of yeast species occurring in the course of a spontaneous wine fermentation at industrial level. Wine samples were taken and plated daily on an agar medium and the developed colonies were analysed by PCR-RFLP after 24 h of incubation. A representative sample of these colonies was also identified by traditional methods. Both procedures gave identical results. However, PCR-RFLP analysis allowed a more precise enumeration of the yeast populations, proving to be a reliable and simple method for monitoring the development of the yeast community throughout wine fermentation.
The composition of wine yeast populations, present during spontaneous fermentation of musts from two wine-producing areas of Greece (Amyndeon and Santorini) and followed for two consecutive years, were studied using a range of molecular techniques. Internal Transcribed Spacer (ITS) ribotyping was convincingly applied for yeast species identification, proving its usefulness as a reliable tool for the rapid characterization of species composition in yeast population studies. Restriction Fragment Length Polymorphism (RFLP) of mitochondrial DNA (mtDNA) was shown to be a convenient criterion for the detection of intraspecies genetic diversity of both Saccharomyces and non-Saccharomyces isolate populations. Similarly, polymorphism of amplified delta interspersed element sequences provided an additional criterion for S. cerevisiae strain differentiation. Comparative analysis of S. cerevisiae genetic diversity, using mtDNA restriction patterns and delta-amplification profiles, showed a similar discriminative power of the two techniques. However, by combining these approaches it was possible to distinguish/characterize strains of the same species and draw useful conclusions about yeast diversity during alcoholic fermentation. The most significant findings in population dynamics of yeasts in the spontaneous fermentations were (i) almost complete absence of non-S.cerevisiae species from fermentations of must originating from the island Santorini, (ii) a well recorded strain polymorphism in populations of non-Saccharomyces species originating from Amyndeon and (iii) an unexpected polymorphism concerning S. cerevisiae populations, much greater than ever reported before in similar studies with wine yeasts of other geographical regions.
Yeast ecology, biogeography and biodiversity are important and interesting topics of research. The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998). No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations. Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA. This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation. However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains.
Yeast isolates from orange fruit and juice in a spontaneous fermentation were identified and classified by two molecular techniques. The first was analysis of the restriction pattern generated from the polymerase chain reaction (PCR)-amplified 5.8S rRNA gene and the two internal transcribed spacers (ITS) using specific primers. The second technique was sequence analysis of the ITS regions using the same two primers. Nine different restriction profiles were obtained from the size of the PCR products and the restriction analyses with three endonucleases (CfoI, HaeIII and HinfI). These groups were identified as Candida tropicalis, Clavispora lusitaniae, Hanseniaspora uvarum, Pichia anomala, Pichia fermentans, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces unisporus, and Trichosporon asahii. Checking against identification according to morphological, physiological and biochemical traits corroborated this molecular identification. A total concordance was found in the identification with PCR-restriction fragment length polymorphism of the ITS region after analysing certified yeast strains from two different culture collections. Consequently, a rapid and reliable identification of the yeast populations was achieved by using molecular techniques.
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