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Molecular typing techniques as a tool to differentiate non-Saccharomyces wine species
Abstract
A total of 32 yeast strains belonging to four non-Saccharomyces species associated with winemaking was characterized by different molecular techniques. The PCR amplification of 18S rRNA-coding DNA and nontranscribed spacer, followed by restriction analysis with the endonucleases HaeIII and MspI, and PCR fingerprinting with microsatellite primers (GAC)(5) and (GTG)(5) were used. The methods used provided species-specific profiles and proved to be fast and reliable for monitoring the evolution of the four non-Saccharomyces yeast populations throughout wine fermentation.
... Progress in developing molecular techniques with a higher resolving power has led to a more reliable characterization of yeasts, both at the species level and at the strain level (10). Several DNA-based methods such as restriction fragment length polymorphism (RFLP) analysis of 5.8 and 18S rDNA, random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR), and RFLP analysis of mitochondrial DNA have been used to discriminate wine yeast at strain level (7,11). The noncoding internal transcribed spacer regions (ITS1-5.8S ...
... Results found in the literature about suitability of RFLP of the 18S rDNA are contradictory. Thus, this method has been also reported as unsuitable for the differentiation of yeasts while it has been proven as a useful technique for discriminating several yeast species such as Candida stellata, Metschnikowia pulcherrima, Kloeckera apiculata, and Schizosaccharomyces pombe (11). Therefore, this method is not used routinely for the discrimination of yeasts species; however, in our study, it was found to be effective in the discrimination of Y. lipolytica strains. ...
... Obtainment of DNA samples. The extraction of DNA samples for further PCR analysis was performed as described in [7]. Washed and freeze-dried cells of yeast strains (30-50 mg) were incubated in 500 μL of lysing buffer containing DDS-Na, Triton X-100, and proteinase K for 20 min at 65°C. ...
... PCR fingerprinting. To analyze microsatellite loci and interdelta profiling of the strains, we used the primers (GTG)5, (GACA)4 [7] and pair of delta12 (5'-TCAACAATGGAATCCCAAC-3'), delta2 (5'-GTGGATTTTTATTCCAAC-3') as described in [11]. To separate DNA fragments during interdelta profiling, composite agarose polyacrylamide vertical gel electrophoresis of the following composition was performed: 0.6% low-melting agarose, 4% acrylamide (ratio acrylamide/bisacrylamide 30 : 1). ...
Sixteen flor yeast strains from the Magarach Collection of the Microorganisms for Winemaking (Yalta, Crimea), which are used for production of sherry, were analyzed for morphophysiological, cultural, and biochemical properties. Long-term storage did not affect their viability or the preservation of major properties, such as their flor- and aldehyde-forming abilities, and the ability to produce wines with typical sherry properties. Significant variation in the strains was observed mainly in the aldehyde-forming and flor-forming abilities and flor properties. Interdelta typing was shown to be the most informative technique to study the genetic diversity of flor yeast strains. Certain correlations between genetic polymorphisms and the enological properties of the strains were observed. The presence of a 24-bp long deletion in the ITS1 spacer of the ribosomal gene cluster, a typical feature of Spanish flor yeast strains, is correlated with a high level of production of aldehydes and acetales, efficient flor formation, and the ability to produce high quality sherry. The presence of a specific deletion in the promoter of the FLO11 gene appeared to be less informative, since the aldehyde and acetal production and flor formation abilities of such strains were variable. The studies of intraspecies genetic polymorphism by various molecular markers have revealed a high degree of phylogenetic closeness of some yeast flor strains from different geographic regions.
... However these methods are laborious, time-consuming and are influenced by culture conditions thereby leading to uncertain results and thus not suitable for routine analysis. The introduction of DNA based molecular methods [32,33] like RFLP analysis of the 5.8S and 18S rDNA, RAPD-PCR and RFLP analysis of mitochondrial DNA have been used to discriminate yeasts at strain level [19,33]. ...
... However these methods are laborious, time-consuming and are influenced by culture conditions thereby leading to uncertain results and thus not suitable for routine analysis. The introduction of DNA based molecular methods [32,33] like RFLP analysis of the 5.8S and 18S rDNA, RAPD-PCR and RFLP analysis of mitochondrial DNA have been used to discriminate yeasts at strain level [19,33]. ...
In the present investigation, an attempt has been made to screen and identify the isolates of yeast rich in mannan oligosaccharide (MOS) from different food sources collected from local market of Mumbai, India. Out of the forty-eight varied yeast strains obtained using selective and growth media, eighteen isolates were shortlisted on the basis of their MOS yield. The MOS yield obtained from Wickerhamomyces anomalus strain isolated from home-made dahi was even higher (33%) than that obtained from the traditionally used Saccharomyces cerevisiae strain (590.52 ± 8.25 vs 442.85 ± 4.25 mg/L). The reasonably good yield was found in Pichia casonil from grape juice (354.70 ± 1.02 mg/L) and Candida glabrata strain from carrot juice (350.8 ± 2.52 mg/L); however, the lowest yield was of Debaryomyces hansenii SZ10 (73.5 mg/L) grown on yogurt. Identification of the isolates was undertaken using Biomérieux VITEK® 2 system and molecular fingerprinting by polymerase chain reaction-random amplified polymorphism DNA (PCR-RAPD) using microsatellite M13 primer and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of amplified 18S intergenic transcribed spacer region of ribosomal DNA (rDNA) after subjecting it to digest with three restriction endonucleases i.e. HaeIII, MspI and HinfI. Based on the better yield, it was concluded that W. anomalus can be exploited as an alternative of S. cerevisiae yeast stains for commercial mass scale MOS production for human food and animal feed industries in future.
... Several regions have been applied in PCR-RFLP analysis for yeast species identifi cation in food systems including the non-transcribed spacer (NTS) (Baleiras Couto et al. 1996, Pulvirenti et al. 2001, Caruso et al. 2002, Capece et al. 2003, the 18S (Smole Mozina et al. 1997, Redzepovic et al. 2002, Capece et al. 2003 or the 26S rDNA genes (van Keulen et al. 2003, Balerias Couto 2005. The most frequently applied region is the 5.8S-ITS rDNA, usually amplifi ed with the ITS1/ITS4 primer pair (White et al. 1990). ...
... Several regions have been applied in PCR-RFLP analysis for yeast species identifi cation in food systems including the non-transcribed spacer (NTS) (Baleiras Couto et al. 1996, Pulvirenti et al. 2001, Caruso et al. 2002, Capece et al. 2003, the 18S (Smole Mozina et al. 1997, Redzepovic et al. 2002, Capece et al. 2003 or the 26S rDNA genes (van Keulen et al. 2003, Balerias Couto 2005. The most frequently applied region is the 5.8S-ITS rDNA, usually amplifi ed with the ITS1/ITS4 primer pair (White et al. 1990). ...
... p0055 Restriction analysis of other rDNA regions has also been used to identify other yeast species, particularly those belonging to the Saccharomyces sensu stricto complex. This is the case for the NTS region (Baleiras Couto et al., 1996;Capece et al., 2003;Caruso et al., 2002;Nguyen & Gaillardin, 1997;Pulvirenti et al., 2000), the 18S gene (Capece et al., 2003), and various domains of the 26S gene (Baleiras Couto et al., 1996Romancino et al., 2008;Smole-Mozina et al., 1997;van Keulen et al., 2003). However, the absence of a sequence database for these regions means that their use cannot be generalized for the identification of yeasts. ...
... p0055 Restriction analysis of other rDNA regions has also been used to identify other yeast species, particularly those belonging to the Saccharomyces sensu stricto complex. This is the case for the NTS region (Baleiras Couto et al., 1996;Capece et al., 2003;Caruso et al., 2002;Nguyen & Gaillardin, 1997;Pulvirenti et al., 2000), the 18S gene (Capece et al., 2003), and various domains of the 26S gene (Baleiras Couto et al., 1996Romancino et al., 2008;Smole-Mozina et al., 1997;van Keulen et al., 2003). However, the absence of a sequence database for these regions means that their use cannot be generalized for the identification of yeasts. ...
This chapter discusses molecular identification and characterization of wine yeasts. Yeast species can be identified by comparison of nucleotide sequences from rDNA regions. The two most commonly used regions are the D1 and D2 regions at the 5' end of the genes encoding the 26S and 18S ribosomal subunits. The availability of sequences in DNA databases, particularly for the D1/D2 region of the 26S gene, makes this technique particularly useful for assigning unknown yeast to a specific species when the homology of the sequences is greater than 99%. Molecular characterization of commercial yeast strains is necessary for two reasons. First, it is needed for quality-control purposes to confirm that the obtained yeast is the one that was originally selected and not a contaminant, and, second, to detect fraud. Wine is a highly appropriate culture medium for the growth of a large number of microorganisms, in part due to its richness in organic acids, amino acids, residual sugars, growth factors, and mineral salts. Techniques for the detection of spoilage yeasts are essential. These techniques must be very sensitive and allow quantification of the number of microorganisms present. They must also be rapid to allow the application of corrective measures on the production line prior to release of the products onto the market.
... A single primer composed of simple sequence repeats, with or without an anchor made up of a few bases at the 5′ region, can amplify genomic regions between two microsatellite loci with oligonucleotides that anneal to the repeat sequence. ISSR circumvents the requirement for flanking sequence information, and thus has found wide applicability in a variety of plants and fungi (Wolfe et al. 1998;Wolfe 2005;Hantula et al. 1996;Capece et al. 2003). Primers containing tandem repeats (GAC) 5 or (GTG) 5 were used to differentiate non-Saccharomyces species but they were not suitable to discriminate at the subspecies level (Capece et al. 2003). ...
... ISSR circumvents the requirement for flanking sequence information, and thus has found wide applicability in a variety of plants and fungi (Wolfe et al. 1998;Wolfe 2005;Hantula et al. 1996;Capece et al. 2003). Primers containing tandem repeats (GAC) 5 or (GTG) 5 were used to differentiate non-Saccharomyces species but they were not suitable to discriminate at the subspecies level (Capece et al. 2003). Few molecular methods have been reported to differentiate non-Saccharomyces grape yeasts at the strain level. ...
There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomyces cerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleotide based on a conserved tRNA region, specific for ascomycetes. Tandem repeats are randomly dispersed in all eukaryotic genomes and tRNA genes are conserved and present in several copies in different chromosomes. As an example, the method was applied to discriminate native M. pulcherrima strains but it could be extended to differentiate strains from other non-Saccharomyces species. The biodiversity of species and strains found in the grape ecosystem is a potential source of new enzymes, fungicides and/or novel sustainable methods for biological control of phytopathogens.
... In a study conducted by Perez-Brito et al. in 2007, it was demonstrated that by using the primers (GTG) 5 and 6 more it was possible to differentiate between two groups of ascospores forms, so this technique proves to be effective for genotypically distinct strains. The number of bands observed was 5 to 18, in which the species that presented most of the bands was K. marxianus T1.4 whit 18 bands whereas Pichia kluyveri only 5. With this methodology it was possible to group the yeast species using this marker, which agrees with the results obtained by Capece et al. 2003 in non-Saccharomyces native wine strains. The genetic variability of bacteria was high (Figure 4), since is observed the formation of a cluster per strain; therefore, the overall bacterial genetic variability found is greater than 90%. ...
ABSTRACT
Comiteco is an artisanal beverage derived of agave, this is obtained for distillation of aguamiel mixed with sugar
cane and water, and the organoleptic characteristics of beverage depend of proportions to raw material used by
each producer. In this chapter we describe the importance of the beverage for artisan producers of the Meseta
Comiteca, Chiapas, Mexico. For this, we apply semi-structured interviews to primary producers (farmers who get
aguamiel called aguamieleros) and secondary producers (comitequeros) in addition to visits to places where
the beverage is produced and to field where agave plants are; in march and april of 2017 and 2018 to generate
complementary information about the process. The producers of aguamiel and comiteco have knowledges
acquired through different experiences; principally related to their ancestors that are reflected in their environment
social, family, economic and cultural. This is reflected in the value and interest that producers give for the beverage,
that for some, it begins to be one of its main economic activities. In turn, the production has been influenced by
situations that involve political and economic interests. We found some limitations to the realizations of this study: the
current situation of the comiteco influences the distrust of the producers to authorities. This caused many producers
to avoid talking and sharing their story, which explain the low number of interviews achieved. The comiteco has a
promising future for their characteristics, artisan producers are those who keep the tradition alive and those who
face the actual problematic. Work on different aspects that influence in this it is essential.
... In a study conducted by Perez-Brito et al. in 2007, it was demonstrated that by using the primers (GTG) 5 and 6 more it was possible to differentiate between two groups of ascospores forms, so this technique proves to be effective for genotypically distinct strains. The number of bands observed was 5 to 18, in which the species that presented most of the bands was K. marxianus T1.4 whit 18 bands whereas Pichia kluyveri only 5. With this methodology it was possible to group the yeast species using this marker, which agrees with the results obtained by Capece et al. 2003 in non-Saccharomyces native wine strains. The genetic variability of bacteria was high (Figure 4), since is observed the formation of a cluster per strain; therefore, the overall bacterial genetic variability found is greater than 90%. ...
Comiteco is an artisanal beverage derived of agave, this is obtained for distillation of aguamiel mixed with sugar cane and water, and the organoleptic characteristics of beverage depend of proportions to raw material used by each producer. In this chapter we describe the importance of the beverage for artisan producers of the Meseta
Comiteca, Chiapas, Mexico. For this, we apply semi-structured interviews to primary producers (farmers who get aguamiel called aguamieleros) and secondary producers (comitequeros) in addition to visits to places where the beverage is produced and to field where agave plants are; in march and april of 2017 and 2018 to generate
complementary information about the process. The producers of aguamiel and comiteco have knowledges acquired through different experiences; principally related to their ancestors that are reflected in their environment social, family, economic and cultural. This is reflected in the value and interest that producers give for the beverage, that for some, it begins to be one of its main economic activities. In turn, the production has been influenced by
situations that involve political and economic interests. We found some limitations to the realizations of this study: the
current situation of the comiteco influences the distrust of the producers to authorities. This caused many producers
to avoid talking and sharing their story, which explain the low number of interviews achieved. The comiteco has a
promising future for their characteristics, artisan producers are those who keep the tradition alive and those who
face the actual problematic. Work on different aspects that influence in this it is essential.
... These same authors were the first to use these sequences as primers in a PCR reaction, and proved the usefulness of this technique for characterization at the strain level. It has later been used by other authors for typing Saccharomyces (Baleiras Couto et al., 1996;Pérez et al., 2001;Howell et al., 2004;Schuller et al., 2004;Masneuf-Pomarède et al., 2007), non-Saccharomyces (Capece et al., 2003), Brettanomyces (Miot-Sertier and , Hanseniaspora (Caruso et al., 2002), and Zygosaccharomyces (Martorell et al., 2005a) strains. ...
The processes of yeast selection for using as wine fermentation starters have revealed a great phenotypic diversity both at interspecific and intraspecific level, which is explained by a corresponding genetic variation among different yeast isolates. Thus, the mechanisms involved in promoting these genetic changes are the main engine generating yeast biodiversity. Currently, an important task to understand biodiversity, population structure and evolutionary history of wine yeasts is the study of the molecular mechanisms involved in yeast adaptation to wine fermentation, and on remodeling the genomic features of wine yeast, unconsciously selected since the advent of winemaking. Moreover, the availability of rapid and simple molecular techniques that show genetic polymorphisms at species and strain levels have enabled the study of yeast diversity during wine fermentation. This review will summarize the mechanisms involved in generating genetic polymorphisms in yeasts, the molecular methods used to unveil genetic variation, and the utility of these polymorphisms to differentiate strains, populations, and species in order to infer the evolutionary history and the adaptive evolution of wine yeasts, and to identify their influence on their biotechnological and sensorial properties.
... White et al. (1990) used primers ITS1 and ITS4 to amplify regions of the 5.8S gene, which was exclusively used for the identification of yeast strains in wine. The non-transcribed areas, 18S gene, ITS region and 26S gene have been widely used by various authors to identify species in the Saccharomyces sensu stricto group (Baleiras-Couto et al., 1996;Tornai-Lehoczki and Dlauchy, 2000;Caruso et al., 2002Capece et al., 2003. The internal transcribed regions (ITS) has also been targeted by restriction analysis with DraI and HaeIII to identify and characterize yeast populations with enological significance, as well as species in the larger Saccharomyces sensu stricto group (Esteve-Zarzoso et al., 1999;Granchi et al., 1999). ...
“Natural wines”, nowadays, could appear an evocative recall to what wine was like before. During the ages, science allowed winemakers to produce wines with the full control of
vinification condition, obtained mainly by addition of exogenous components, supplements
and microorganisms. Contrarily, “natural wine” come back to basics and simplicity as invasive operations are avoided; but, on the other hand, a deeper knowledge and monitoring of winemaking process is required, in order to obtain high quality products
without exogenous substances. Natural wine has been produced by spontaneous
fermentation of must by yeasts originated from grapes and winery equipment. The wide variety of natural yeasts reflects the biodiversity, which is still under-exploited despite the
large use of commercial Saccharomyces cerevisiae in most grape musts. During
fermentation, several strains compete in the same fermenting must, and the dominance of Saccharomyces cerevisiae takes place when it overcomes all the others. The aim was to investigate Saccharomyces cerevisiae diversity and its technological behavior in two
biodynamic wine farms in Tuscany. Autochthonous S. cerevisiae from Syrah fermentations
were isolated and molecularly characterized in 2009 and 2013 harvests. Saccharomyces cerevisiae strains were isolated starting from Syrah musts with zero or low levels of
sulphite added, according with the tendency of natural wines producers to abait every
invasive operation during wine making. Samples were collected in different steps of winemaking. ITS-PCR method confirmed isolates belong to Saccharomyces sensu stricto complex. The multiplex PCR amplification of microsatellite loci (SC8132X, YOR267C and
SCPTSY7) discriminated S. cerevisiae strains. Genetically diverse S. cerevisiae strains
were subsequently subjected to technological characterization. Micro-fermentations were set up to study fitness and quality traits of biotypes characterized. Weight loss kinetics
were measured and chemical analyses were performed. Collected Saccharomyces
cerevisiae strains were tested also in order to verify the effect of low sulphite additions on biodiversity pursuing information about stress-adaptation mechanisms that allow the
survival in the challenging environment of fermenting must. Sulphite addition may act as a
selective factor to induce the presence and the activity of strains with diverse features about sulphite metabolism. The addition of different dose of sulphite induced a considerable variability among strains. However, some biotypes exhibited tolerance to
certain concentration and only one biotype was classified as resistant to a determinate
concentration. In addition, biotypes were subjected to an other environmental stress factor such as ethanol. The increasing of levels of ethanol limited considerably the activity of selected strains, but some of them highlighted a positive tolerance to various ethanol
concentrations. During the harvest of 2013, the alcoholic fermentation was conducted
inoculating a biotype characterized previously, which expressed good fermentative performance and a multi-strains culture of other S. cerevisiae strains characterized in the
same harvest (2009) in a biodynamic farm from a workgroup of Edmund Mach Foundation
of San Michele all'Adige (TN). The results of 2009 highlighted peculiar behaviors of 13 biotypes (characterized from four hundred isolates) that dominate the spontaneous fermentations. Not surprisingly, some biotypes were also found in fermentations of 2013,
demonstrating yeasts are ubiquitous in that environment. Sensorial analyses of wine,
originated by biotypes fermentation, revealed certain diversity in aroma and flavor traits, but also in the structure. Overall, this work suggests the consolidate relationship existing
between autochthonous yeasts and the terroir, where yeasts are ubiquitous. Furthermore,
autochthonous yeasts surely contribute to the unique identity of wine. In addition, this work suggests that the use of isolated and characterized autochthonous yeast strains could help the winemaking process, where the vintage could be inadequate to develop a particular product such as the wine of a specific wine farm.
... Among them, the Microsatellite/Minisatellite Primed (MSP)-PCR Fingerprinting technique has been widely applied in the literature using primers as (GAC) 5 , (GACA) 4 , (GTG) 5 and M13. For example, the primer (GTG) 5 was frequently used to discriminate species of the genus Saccharomyces [3][4][5][6][7][8], characterize strains of non-Saccharomyces species [9][10][11][12], analyze yeast diversity [13][14][15][16][17][18][19][20], and describe new yeast genus and species [21][22][23][24]. Most of these studies use MSP-PCR fingerprinting as a preliminary clustering step for the choice of representative strains to be sequenced. ...
In microbiology, identification of all isolates by sequencing is still unfeasible in small research laboratories. Therefore, many yeast diversity studies follow a screening procedure consisting of clustering the yeast isolates using MSP-PCR fingerprinting, followed by identification of one or a few selected representatives of each cluster by sequencing. Although this procedure has been widely applied in the literature, it has not been properly validated. We evaluated a standardized protocol using MSP-PCR fingerprinting with the primers (GTG)<sub>5</sub> and M13 for the discrimination of wine associated yeasts in South Brazil. Two datasets were used: yeasts isolated from bottled wines and vineyard environments. We compared the discriminatory power of both primers in a subset of 16 strains, choosing the primer (GTG)<sub>5</sub> for further evaluation. Afterwards, we applied this technique to 245 strains, and compared the results with the identification obtained by partial sequencing of the LSU rRNA ge
... However, there is still a need to develop techniques for improving the characterization of fungal diversity on grape berries without the need for culture, particularly for the minority genera and species present. For other microorganisms, various cultureindependent methods, such as in situ hybridization flow cytometry with fluorescent antibodies and qPCR, have been used, to analyze the yeast ecosystem in grape must, for example (Capece et al., 2003; Xufre et al., 2006; Zott et al., 2010 ). In other environments , such as soil, novel molecular methods have been developed that highly suitable for the microbial communities concerned. ...
... However, there is still a need to develop techniques for improving the characterization of fungal diversity on grape berries without the need for culture, particularly for the minority genera and species present. For other microorganisms, various cultureindependent methods, such as in situ hybridization flow cytometry with fluorescent antibodies and qPCR, have been used, to analyze the yeast ecosystem in grape must, for example (Capece et al., 2003; Xufre et al., 2006; Zott et al., 2010 ). In other environments , such as soil, novel molecular methods have been developed that highly suitable for the microbial communities concerned. ...
a b s t r a c t The grape microflora is complex and includes filamentous fungi, yeasts and bacteria with different physiological characteristics and effects on wine production. Most studies have focused on the wine microbiota, but a few studies have reported the ecology of grape microorganisms. Some of these or-ganisms d such as non-Botrytis bunch rotting fungi, which greatly influence the safety or sensory quality of wine, due to the production of mycotoxins and off-flavors, respectively d are considered to be spoilage agents. We review here the diversity of filamentous fungi on grapes and the factors influencing their development, such as grape ripening stage, environmental factors (climate, rain and cultivation prac-tices), grape variety and grape health status. We also discuss the pathways by which mycotoxins and off-flavors are produced, the control of the population, the metabolites responsible for wine spoilage and the methods for detecting and characterizing the microorganisms involved.
... However, there is still a need to develop techniques for improving the characterization of fungal diversity on grape berries without the need for culture, particularly for the minority genera and species present. For other microorganisms, various cultureindependent methods, such as in situ hybridization flow cytometry with fluorescent antibodies and qPCR, have been used, to analyze the yeast ecosystem in grape must, for example (Capece et al., 2003; Xufre et al., 2006; Zott et al., 2010 ). In other environments , such as soil, novel molecular methods have been developed that highly suitable for the microbial communities concerned. ...
The grape microflora is complex and includes filamentous fungi, yeasts and bacteria with different physiological characteristics and effects on wine production. Most studies have focused on the wine microbiota, but a few studies have reported the ecology of grape microorganisms. Some of these organisms d such as non-Botrytis bunch rotting fungi, which greatly influence the safety or sensory quality of wine, due to the production of mycotoxins and off-flavors, respectively d are considered to be spoilage agents. We review here the diversity of filamentous fungi on grapes and the factors influencing their development, such as grape ripening stage, environmental factors (climate, rain and cultivation practices), grape variety and grape health status. We also discuss the pathways by which mycotoxins and off-flavors are produced, the control of the population, the metabolites responsible for wine spoilage and the methods for detecting and characterizing the microorganisms involved.
... A number of techniques using PCR amplification of ribosomal RNA genes have been suggested for identification of yeast species. Among these methods, those based on the RFLP analysis of the DNA that encodes the ribosomal RNA genes (5, 5.8, 18, and 26S) and the non-coding internal transcribed spacers (ITS) region appear to be useful for the detection of many yeast and fungal species [11][12][13]. Andrade et al. [12], Deak et al. [14], and Polomska et al. [15], who used RFLP analysis of ITS-PCR, have reported that this method is a rapid and easy one for the differentiation of many yeast species. Also, the D1/D2 region of the large subunit (LSU) ribosomal RNA gene (26S rRNA) of all currently recognized ascomycetous yeasts have been sequenced [16,17] and these studies have shown that nucleotide substitutions in the domain do not in general exceed 1% among conspecific strains. ...
In the present study, 120 yeast isolates from different sources (active sludge, soil, and wastewater samples obtained from petroleum refinery and soil contaminated by petroleum) were obtained. The yeast isolates were screened for lipase production and twelve of the isolates (D3, D17, D24, D27, D30, D38, D40, D42, D44, D46, D56, and D57) exhibited lipase activity. Molecular characterization of the yeasts showing the lipase production was performed with RFLP of ITS1-5.8S-ITS2 and 18S rRNA and sequence analysis of D1/D2 domain of 26S rRNA. The 26S rRNA sequencing revealed that four new strains, D38, D40, D44 and D57 identified as Rhodotorula slooffiae, Candida davisiana, Cryptococcus diffluens, and Cryptococcus uzbekistanensis, respectively, are lipase producing yeast species. This study is the first report showed lipase production by these species. The other lipase producing strains identified as Candida parapsilosis (D3), Rhodotorula muciloginosa (D17 and D42), Cryptococcus albidus (D24, D27, D30, and D56), and Wickerhamomyces anomalus (D46).
... This result is in good agreement with previous internal transcribed spacer-PCR data which showed that the procedure could be used to distinguish between yeast species isolated from surface-ripened cheeses (31,32). RAM-PCR was found to be effective in distinguishing between four yeast species, isolated from wine, using primers (GAC) 5 and (GTG) 5 (33). In the present study, the use of the primer (CAC) 5 allowed the differentiation of 11 different yeast species: C. catenulata, C. deformans, C. natalensis, C. musci, D. hansenii, G. candidum, K. lactis, K. marxianus, P. jadinii, T. delbrueckii, and Y. lipolytica (27). ...
Biodiversity of the Surface Microbial Consortia from Limburger, Reblochon, Livarot, Tilsit, and Gubbeen Cheeses, Page 1 of 2
Abstract
Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious “house” flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum, and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum. The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum, Candida catenulata, and Kluyveromyces lactis. B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus, A. arilaitensis, Corynebacterium casei, Corynebacterium variabile, and Microbacterium gubbeenense. S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep-PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.
... Various molecular techniques such as electrophoretic karyotyping, random amplified polymorphic DNA using polymerase chain reaction (RAPD-PCR), PCR-ribotyping have been used by different authors for identifying different wine yeast species. The latter method proved to be useful for the differentiation of wine yeasts at species level (Smole Mozina and Raspor, 1997;Smole Mozina et al., 1998;Guillamón et al., 1998;Esteve-Zarzoso et al., 1999;Granchi et al., 1999;Capece et al., 2003). ...
The number and diversity of yeasts on grape berry surfaces are influenced by several factors, such as grape variety, degree of grape maturity at harvest, climatological conditions, geographic location, physical damage of grapes, the intensity of pest management etc. Cviček is a typical Slovene wine, which has obtained a special protection under the Slovene Wine Law for its geographical origin. This blended red wine is produced from different grape varieties (Vitis vinifera L.), mostly from red grapes of Žametovka and Modra frankinja and from white grapes of Kraljevina. The aim of this study was to evaluate the impact of geographical locations in the Dolenjska vine-growing region and to obtain precise information about the influence of different grape varieties on the composition of yeast community on grape berries. The restriction fragment length polymorphism of PCR-amplified fragments from the rDNA gene cluster (PCR RFLP of rDNA) has been used for the differentiation of yeast species. The standard identification procedure has been performed on representative strains that shared identical RFLP profiles. The number of yeasts and yeast species isolated varied according to different grape varieties, Žametovka, Modra frankinja and Kraljevina (V. vinifera L.) and according to different sampling location. On the surface of grape berries 13 different yeast species have been identified. Saccharomyces cerevisiae has not been found.
... Among these methodologies are included RFLP of genomic or mitochondrial DNA [8,9] chromosome karyotyping [10], AFLP [11] or PCR-fingerprinting [12], to name a few. Of these, PCR-fingerprinting techniques distinguish themselves by being easy, fast, reproducible and relatively inexpensive methods to use having already been described as able to differentiate yeasts in the past [13]. The different fingerprinting patterns obtained by PCR amplification of the extracted DNA with the primer csM13 allowed for a high discrimination of the isolate collection (data not shown). ...
With over 250 different grape varieties distributed throughout 13 officially defined wine regions, for such a small country, Portugal holds within its boundaries an immense trove of potentially great wine. Still, most of it remains untapped.
Wine flavor is the culminating product of a vast number of factors such as grape variety, geographical and viticultural conditions of grape cultivation and the microbial ecology of grapes. Among these, yeast species play a key role in the determination of the quality of the final product.
The metabolic impact of yeasts on wine character is much more complex than the simple fermentation of grape juice sugars. Many studies of wine fermentations in various wine regions of the world have demonstrated the important contribution of indigenous wine yeasts to the sensory properties and overall appeal of the final product. These autochthonous yeasts are presumed to be more competitive than commercial yeasts since they are better adapted to the ecological and technological features of their own wine growing area. Furthermore, a selection of the appropriate local yeasts would ensure the preservation of regional distinctiveness while allowing the production of premium quality wines.
In this study, 768 yeast isolates, belonging to Sogrape Vinhos S.A and originating from four wine regions in Portugal, were studied for their potential use as starter cultures. Rather than the typical pre-selection of any particular genus, a universal approach was applied to all isolates allowing an individual evaluation of each isolate's potential, regardless of genus or species.
The selection protocol consisted of an initial application of molecular fingerprinting methods for determination of variability and genetic characterization, followed by microfermentations in sterile must at different conditions. Throughout the study, by integrative growth analysis and comparison of their performance to that of a commercial wine starter strain, the isolates with the worst scores where sequentially removed until a final group of 33 strains remained. Of these, four were non-Saccharomyces strains.
The final part of the study encompassed a comparative analysis of several molecular fingerprinting methods in search of molecular markers suitable for traceability of these strains at the industrial level.
... Elle est basée sur les différences de Première partie : Etude bibliographique près. L'autre consiste à utiliser la séquence microsatellite en tant qu'amorce unique de la PCR (Lieckfeldt et al., 1993 ;Baleiras Couto et al., 1996 ;Perez et al., 2001 ;Caruso et al., 2002 ;Capece et al., 2003). Elle porte le nom de ISSR (Inter Simple Sequence Repeats)-PCR (Zietkiewicz et al., 1994). ...
La fermentation alcoolique des vins liquoreux français issus de raisin botrytisés est arrêtée brutalement par ajout massif de dioxyde de soufre après qu'un certain équilibre est atteint entre la teneur en alcool formé et la concentration en sucres résiduels. Certaines souches de levures fermentaires survivent et parfois se multiplient provoquant une nouvelle fermentation alcoolique indésirable ; c'est la refermentation. Le suivi microbiologique de nombreux lots de vin a permis de montrer que des levures sont dans un état physiologique similaire à celui décrit chez les bactéries sous l'appellation de viable non cultivable. Cet état explique l'apparente stérilité du vin après le mutage. Au sein de l'espèce Saccharomyces cerevisiae, une sélection naturelle se produit, ne laissant souvent la place qu'à une seule souche de refermentation, tolérante au dioxyde de soufre. Une étude écologique a montré que seules certaines espèces fermentaires et oxydatives survivent. Les plus tolérantes au dioxyde de soufre forment de l'éthanal au cours de l'élevage, malgré un métabolisme ralenti, et augmente la combinaison du dioxyde de soufre libre. Cet éthanal vient progressivement combiner le dioxyde de soufre libre. La sortie de l'état viable non cultivable est probablement la clef des mécanismes engendrant les refermentations. L'utilisation du diméthyldicarbonate au moment du mutage a été étudiée en couplage avec le dioxyde de soufre. Des souches de Saccharomyces cerevisiae de refermentation ont été isolées. Elles exhibent des singularités de séquence de leur ADNr, les apparentant aux souches de voile de certains vins spéciaux. Ces souches surexpriment constitutivement le gène SSU1 et synthétisent rapidement une forte concentration d'éthanal en réponse à la présence de dioxyde de soufre. La présence de fortes concentrations de dioxyde de soufre sélectionne les souches les plus résistantes. La refermentation est donc le résultat d'une adaptation génétique et d'une sélection, fruit d'une multitude de paramètres microbiologiques, physico-chimiques et humains. Botrytis-affected wines microbiology during maturation. Characterization of Saccharomyces cerevisiae strains responsible for refermentation. ABSTRACT : The alcoholic fermentation of Botrytis-affected wines is stopped by addition of sulphur dioxide. Some fermenting yeast species can survive during maturation and grow in spite of high ethanol, sugars and sulphur dioxide concentrations. An undesirable new fermentation, named "refermentation", can sometimes occur. In this study, it was proved that some yeast species were able to survive in a viable but non-culturable-like state. This state explains the apparent sterility of wines during maturation. Within Saccharomyces cerevisiae species, an intraspecific selection was spontaneously operated. After some weeks, only one strain could often survive. An ecological study was realized. Some highly fermentative and oxidative species could survive. In spite of slower metabolism, they synthesized acetaldehyde during maturation. The exit from the VBNC state and the high sulphur dioxide binding power were the keys of refermentations. The use of dimethyldicarbonate to stop alcoholic fermentation was studied. The most efficient action was observed for the mixed sulphur dioxide and DMDC addition. Some Saccharomyces cerevisiae strains responsible for refermentations were isolated. These strains exhibited rDNA sequence singularities, showing that they were close to flor strains, responsible for velum formation in some special wines. Moreover, those strains constitutively over-expressed SSU1 gene and could rapidly synthesize high concentrations of acetaldehyde in response to sulphur dioxide addition. High sulphur dioxide concentrations had probably selected the most resistant strains. Refermentation is the result of genetic adaptation and selection, under the influence of microbiological, physical, chemical and human parameters.
... The isolates were identified at species level by amplification and restriction analysis of 18S rDNA and ITS regions. The 18S rRNA genes were amplified using the primers P108 and M3989, following the protocol reported by Capece et al. (2003). Amplification products were analyzed on 1.2% agarose gel. ...
The composition of yeast microflora in artisanal "Pecorino di Filiano" cheese, a typical product of the Basilicata region of Southern Italy, was studied during ripening. The isolates were identified by restriction analysis of the 18S rDNA amplified region with the combined use of Hinf I and Cfo I enzymes. The majority of the isolates were identified as Debaryomyces hansenii, whereas two yeasts were identified as Kluyveromyces lactis and one as Dekkera anomala. To evaluate natural biodiversity, D. hansenii "Pecorino di Filiano" isolates were submitted to genetic and technological characterization. RAPD-PCR analysis with P80 (5CGCGTGCCCA3) primer revealed significant polymorphism among D. hansenii isolates. About 30% of the isolates showed single molecular profiles, whereas the other D. hansenii yeasts were separated into three main patterns, differing for both the ripening time and the isolation source. Furthermore, the yeasts showed significant variability in their, "proteolytic activity". This work demonstrated the high predominance of D. hansenii among the yeast population of "Pecorino di Filiano" cheese, probably in consequence of the traditional salting process, which was selected for this salt tolerant species. This preliminary study allowed us to isolate autochthonous D. hansenii yeasts potentially useful as starters for the production of this artisanal cheese.
... Currently, discrimination at the strain level becomes a strategic activity for the wine industry because it can link territory, environment, and final products for wine valorization [21]. Moreover, the characterization at the molecular level improves knowledge concerning yeast biodiversity and the dynamics of microbial population during wine production, verifies the starter domination, and allows fast detection of potential spoiling microorganisms [5, 8, 19]. Among the techniques with high discrimination power, multilocus sequence typing (MLST) permits identification at the strain level throughout the analysis of single-nucleotide polymorphisms (SNPs) in loci present in housekeeping genes [11] and characterized by low evolutionary rates [6]. ...
The setting up of new molecular methods for Saccharomyces cerevisiae typing is valuable in enology. Actually, the ability to discriminate different strains in wine making can have a benefit both for the control of the fermentation process and for the preservation of wine typicity. This study focused on the screening of single-nucleotide polymorphisms in genes involved in wine production that could evolve rapidly considering the selective pressure of the isolation environment. Preliminary screening of 30 genes in silico was performed, followed by the selection of 10 loci belonging to 8 genes. The sequence analysis showed a low polymorphism and a degree of heterozygosity. However, a new potential molecular target was recognized in the TPS1 gene coding for the trehalose-6-phosphate synthase enzyme involved in the ethanol resistance mechanism. This gene showed a 1.42% sequence diversity with seven different nucleotide substitutions. Moreover, classic techniques were applied to a collection of 50 S. cerevisiae isolates, mostly with enologic origin. Our results confirmed that the wine making was not carried out only by the inoculated commercial starter because indigenous strains of S. cerevisiae present during fermentation were detected. In addition, a high genetic relationship among some commercial cultures was found, highlighting imprecision or fraudulent practices by starter manufacturers.
... Sequence analysis of the D1/D2 rDNA fragment can discriminate among soaking fluid yeast species, but this method remains relatively laborious. Therefore, researchers have investigated various related molecular techniques such as electrophoretic karyotyping [5], PCR-randomly amplified polymorphic DNA (RAPD) analysis [6][7][8], repeated sequence fingerprinting [9], restriction enzyme fragment length polymorphism (RFLP) 0378 analysis [10,11], heteroduplex mobility assay [12], denaturing gradient gel electrophoresis (DGGE) [13] and temperature-gradient gel electrophoresis (TGGE) [14]. ...
Monitoring the yeast populations within pickle soaking fluid is imperative for ensuring optimum taste, but these analyses have proven time-consuming and expensive, limiting their industrial application. Here, yeasts were identified in the soaking fluid from Japanese radish pickles using fluorescent PCR amplification of the variable D1/D2 region of the 26S rDNA, followed by analysis with microtemperature-gradient gel electrophoresis (micro-TGGE). This smaller version of the normal TGGE apparatus is capable of analyzing samples 10- to 20-fold faster without sacrificing data quality. Each primer set was labeled with a different fluorescent dye, allowing easy isolation of the various PCR products and identification of the bands corresponding to the various yeasts. The results indicate that fluorescent PCR and micro-TGGE may be a useful new method for rapid, easy monitoring of yeast flora in various food industries. This new method can be used on a daily basis to provide overviews of yeast flora during pickle production, allowing producers to quickly grasp pickle readiness at a single glance.
... Among these techniques, the random amplification of polymorphic DNA (RAPD) method is most widely used. RAPD has been exploited for the molecular typing of non-Saccharomyces wine species [14], to characterise yeasts isolated from traditional sausages of southern Italy [15], Italian sourdoughs [16], dairy products [17], brewery strains [18], as well as artisanal Brazilian cachaça [19]. In the wine microbiology field, RAPD-PCR has been used to detect diversity among Saccharomyces sensu stricto strains isolated from Amarone wine [20] and to distinguish between the sibling species Saccharomyces bayanus, S. cerevisiae, Saccharomyces paradoxus and Saccharomyces pastorianus [21]. ...
Saccharomyces cerevisiae strains isolated from two different wineries in central Italy were subjected to enological and molecular characterization to investigate the influence of the winery environment. One of the selected wineries is a modern, working winery, whereas the second one was abandoned since 1914 and was located in an artificial cavern. The results obtained by our analysis of the fermentation traits underline the selectivity of the winery environment (winery effect), since strains isolated from the industrial winery showed higher values for characters typically subjected to selective pressure, such as maximum capability to produce ethanol, fermentation rate and SO(2) resistance. Pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD)-PCR and SAU-PCR were carried out to assesss genetic differences between the two populations studied. Only RAPD-PCR could distinguish between the two populations based on their provenience, whereas PFGE and SAU-PCR gave profiles shared between strains isolated from the industrial and former winery. Moreover, analysis of the karyotypes suggested the presence of chromosomal-length polymorphism; differences in the size and number of chromosomes between the two groups of isolates, as well as within each group, were observed.
... Another disadvantage of these methods is their inability to discriminate among strains within a species. To avoid doubtful identifications or misidentifications, genetic methods, which generate results independent of environmental conditions, have been applied and several studies reported the usefulness of molecular techniques in identifying wine yeast species and in discriminating strains (Baleiras Couto et al. 1996; Cadéz et al. 2002; Caruso et al. 2002; Capece et al. 2003 ). Zarzoso et al. (1999) evaluated the use of restriction fragments length polymorphism (RFLP) of rDNA amplified by polymerase chain reaction (PCR) to generate a database of restriction patterns for the routine identification of yeast species most frequently isolated from food; Guillamón et al. (1998) used the same methods to identify wine yeast species. ...
The characterization by molecular and physiological methods of wild apiculate strains, isolated from 'Aglianico del Vulture' grape must.
The restriction analysis of 18S rDNA allowed the identification of strains at the species level, which were predominantly Hanseniaspora uvarum. The RAPD analysis and the evaluation of technological traits, such as the metabolic and enzymatic activities, were useful to evaluate the polymorphism of this species.
The RAPD analysis clustered the wild H. uvarum strains in four main genetic groups and a very high phenotypic variability confirmed this genetic polymorphism. The technological variables, which determined the strain biodiversity differed significantly, demonstrating that these technological traits are strain dependent. A certain correlation was found between the strain behaviour and its isolation zone, indicating the influence of the environment on the genetic patrimony of the population.
The genetic and technological biodiversity recorded among H. uvarum wild strains represents the basis for organizing a collection of apiculate strains exhibiting oenological characteristics at different levels, such as high/low production of secondary compounds, and, therefore, potentially useful for a selection programme.
Yeast have a fundamental role in winemaking. They carry out alcoholic fermentation and they contribute to the quality of the wine, although they can also cause spoilage during grape must transformation and in the final product. To detect and identify wine yeast and control their activities, a plethora of different methods can be utilized. As reported in the present chapter, these methods have different degrees of complexity and vary in terms of cost, rapidity and sensitivity. Those based on yeast isolation, namely culture-dependent methods, are widely utilized to define the composition of the microflora associated with wine-related environments and for yeast identification at the strain level, besides providing a means for ex-situ preservation of wine yeast biodiversity. Culture-independent methods bypass microorganisms cultivation, thus avoiding any bias introduced by their isolation and uncovering cell populations undetected by culture-dependent methods. These methods can be utilized to evaluate the impact of all of the components of the wine microbiota on the quality of the final product, to implement a quality control system based on real-time detection and quantification of specific targets, such as the inoculated starter (s) or the spoilage yeast, or to provide further insights into the composition of the microbial communities involved in the grape must transformation.
Yeasts are predominant in several fermented foods prepared from ingredients of plant as well as animal origin. The diversity of foods in which, yeasts predominate ranges from alcoholic beverages such as wines (e.g., fruit, palm and rice wines), cereal based leavened products (e.g., sourdough and idli), milk products (e.g., cheese and dahi) and condiments such as soy sauce and papads. In natural food fermentation, yeasts are either dominant alone or mixed with lactic acid bacteria or mycelial fungi. Many yeast strains have been selected from the natural fermentation and successfully utilised as starter culture for industrial food production. They have a significant impact on food quality by improving the taste, flavour, texture, nutritive values, reduction of anti-nutritional factors and improving the functionality (health promoting properties). This chapter focuses on the beneficial role of yeast in fermented foods with special reference in improving the functionality in fermented food products.
Fara të kalibruara të një kultivari komercial u mbollën në kaseta polisteroli. 25 dhe 32 ditë pas
mbjelljes, një pjesë e bimëve u zhvendosën nga kaseta dhe sistemi rrënjor i tyre u shkurtua në 1/3,
ose 2/3 e tyre dhe më pas u trapiantuan në kaseta të tjera me të njëjtat përmasa. Pjesa e mbetur e
bimëve (nps) u la e paprekur, të rritej normalisht në kasetat ku u mbollën fillimisht dhe shërbeu si
kontroll. 40 ditë pas mbjelljes, një numër i barabartë bimësh për secilin nga grupet e mësipërme, të
ndara në tri nëngrupe që ndryshuan midis tyre për nga përqendrimi i kripërave (NaCl) në ujin që u
përdor për ujitjen periodike të bimëve pas trapiantimit (respektivisht 0, 50 dhe 100 mM), u trapiantuan
në vazo individuale. Paralelisht me eksperimentin e mësipërm, sërish 40 ditë pas trapiantimit, një
grup tjetër bimësh iu nënshtruan trajtimeve me ethephton. Për këtë qëllim, një numër i barabartë
fidanësh për secilin nga grupimet e mësipërme, u zhvendosën nga kasetat e tyre dhe u zhytën për pak
sekonda në tretësira me përqendrime të ndryshme ethephtoni (respektivisht, 0, 1, 2 dhe 5 ml L-1), për
t’u trapiantuar më pas në vazo individuale (150 ml) të mbushura me vermikulit. Për secilin nga
variantet e mësipërm u përllogaritën ritmi relativ i rritjes (RGR) dhe ritmi relativ i rritjes së sistemit
rrënjor (RRGR), që u shfrytëzuan si tregues të vlerësimit të ritmeve të lidhjes së bimëve me tokën
pas trapiantimit. Krasitja e sistemit rrënjor ndryshoi në mënyrë thelbësore fluksin e lëvizjes së
lëndëve organike në bimë, duke i dhënë përparësi rikrijimit të rrënjëve të reja anësore. Në këtë
mënyrë, këto bimë realizuan edhe vlera më të larta të ritmeve relative të rritjes së sistemit të tyre
rrënjor (RRGR) dhe mundësuan lidhjen më të shpejtë me tokën pas trapiantimit. Trajtimi i sistemit
rrënjor me produkte që çlirojnë etilen përmirësoi, gjithashtu aftësitë rrënjëzuese të fidanëve të
tejrritur (mplakur) të domates, por përkundrazi, ekspozimi ndaj etilenit i fidanëve në moshë të re i
dëmtoi aftësitë e tyre rrënjëzuese dhe vonoi lidhjen me tokën pas trapiantimit.
Yeast populations used in industrial production of fuel-ethanol may vary according to the plant process conditions and to the environmental stresses imposed on yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as the starter strain instead of less adapted commercial strains. This work reports the use of a PCR-fingerprinting method based on microsatellite primer (GTG)(5) to characterize the yeast population dynamics during the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominated the yeast population and were more commonly present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from the fuel-ethanol producing process.
The conversion of grape sugars to alcohol and other end-products by specific yeast populations may yield wines with distinct organoleptic quality. in order to reduce the risk of undesirable changes of wine flavour, nowadays commercial starter cultures are widespread used in winemaking. in addition to their principal role of transforming grape sugars into alcohol without off-flavours development, starter cultures have to possess technological properties related to the winemaking process, such as useful enzymatic activities and production of secondary compounds related both to wine organoleptic quality and human health. The actual trend is the selection of starter cultures able to complement and optimize grape quality in order to obtain a wine, which could be the result of the optimal interaction yeast/vine. The selection of starter cultures is mainly addressed to the principal actor in wine fermentation, Saccharomyces cerevisiae, characterized by high ethanol and sulphur dioxide tolerance and high fermentation power, which allows to dominate and complete grape must fermentation. Among strains of this species it's demonstrated the existence of a strong polymorphism and it is widely reported that each fermentation seems to have its own population of different S. cerevisiae strains, which contribute to the wine chemical composition and produce wines differing in the expression of technological traits. This paper deals with results of studies performed on numerous wild S. cerevisiae strains, isolated from grapes of different varieties, in order to emphasize the significant biodiversity of this species and the need of a strong selection procedure for the individuation of suitable starter cultures in function of grape must to ferment.
Since the population of indigenous non-Saccharomyces yeasts is displaced from an alcoholic fermentation within a few hours, the use of a selective enrichment broth would be a valid approach for their detection. To accomplish this, yeast carbon base (YCB)–lysine broth without or with different concentrations of cycloheximide was tested. Lysine is an amino acid that cannot serve as the only nitrogen source for Saccharomyces spp., which is in contrast to non-Saccharomyces yeast, while cycloheximide is an antibiotic that is more or less effective against Saccharomyces spp. strains. Metagenomics, the study of genetic material from mixed microbial community as a grape must fermentation has the potential for operating in a culture-independent environment. Direct identification by PCR (dPCR) without previous isolation and a classical approach to analyse the yeast population in a wine inoculated with a mixed population of Saccharomyces cerevisiae and non-Saccharomyces yeasts were compared. Results showed that pre-incubation on YCB–lysine and dPCR combined with classic approaches based on the use of solid media is a suitable strategy for detecting yeast species involved in vinifications.
Among the non-Saccharomyces yeasts which dominate the early fermentation stages, Hanseniaspora uvarum represents the prevalent species, due to its wide diffusion on the grapes and in grape must just pressed. Few years ago strains of this species were considered as spoilage species exhibiting undesirable oenological traits, while recently numerous studies have demonstrated the existence of a significant biodiversity also in H. uvarum population for technological traits. During the last ten years numerous H. uvarum wild strains, isolated and identified at the species level by molecular techniques, have been included in the collection of Wine Microbiology Laboratory of Basilicata University. They have been analyzed to evaluate their genetic and technological variability. For the genetic characterization, the strains were submitted to RAPD-PCR analysis by using the primer P80 (5CGCGTGCCCA3) and M13 and the results obtained emphasized the existence of a significant genetic polymorphism among the strains. The H. uvarum strains were characterized for parameters of technological interest in oenology, such as the evaluation of enzymatic activities influencing wine quality (β-glucosidase and β-xylosidase activities), as well as for the capacity to form by-products, such as higher alcohols, acetic acid, acetaldehyde, acetoin, during grape must fermentation. The evaluation of technological parameters revealed the existence of a wide phenotypic biodiversity, correspondent to the genetic polymorphism.
El uso combinado del análisis de restricción del ADN mitocondrial y del RAPD-PCR permite diferenciar levaduras de jamón ibérico con alta producción de compuestos volátiles implicados en el aroma a curado. En la especie Debaryomyces hansenii se detectan los biotipos más productores, los cuales han sido seleccionados como cultivos iniciadores. Se han encontrado biotipos de levaduras exclusivos de lugar y etapa de maduración.
In order to analyze yeasts that produce industrial compounds, it is essential to identify them accurately. Yarrowia lipolytica is one of the most extensively studied "nonconventional" yeasts, being a strictly aerobic microorganism capable of producing important metabolites and having an intense secretory activity, which justifies efforts to use it in industry (as a biocatalyst), in molecular biology, and in studies of genetics. Therefore, in this study, an accurate identification of Y. lipolytica strains was performed using 3 different molecular biological methods (RFLP analysis of ITS1-5.8S rDNA-ITS2 and 18S rDNA regions and sequencing of the D1/D2 domain of the 26S rDNA region). The 26S rRNA gene sequence of the strains showed sequence homology with various Y. lipolytica strains from the National Center for Biotechnology Information. A number of different lipids (tributyrin, olive oil, and fish oil) were screened in terms of the growth of Y. lipolytica strains and lipase production. It was determined that all lipid-related substrates supported lipase production levels ranging from 4.27 U/mL (tributyrin) to 37.08 U/mL (fish oil). Fish oil (1%) showed maximum specific activity in the supernatant (264.85 U/mg of protein) and TEM TAN 46. The Y. lipolytica strain that was produced in the media containing fish oil was found to be the best lipase producer.
In grapevine (Vitis vinifera L.,), cultivar identification problems have frequently been solved using ampelographic and chemical analysis. However, these methods result in several ambiguities, particularly when different clones of the same cultivar have to be identified. The availability of reliable and reproducible tools to identify genetic differences at clonal level would facilitate the classification of clones and cultivars. At the same time, molecular tools are also well developed in order to classify the autochthonous yeast strains (Saccharomyces cerevisiae) isolated in the area of Aglianico del Vulture cultivation. In this work, six vineyards of the ancient cultivar Aglianico del Vulture and 60 Saccharomyces sensu stricto strains were characterised. Molecular tools such as RAPD-polymerase chain reaction analysis, microsatellites, amplified ribosomal DNA restriction analysis and AFLP analysis were applied in order to study the genetic variability among the vineyards of Aglianico del Vulture and among the S. cerevisiae isolates. The molecular markers revealed different fingerprinting patterns on either grapevine or S. cerevisiae strains. The genetic differences detected in yeast and plant would represent the genetic variability usable for a selection of the best plant–yeast combination in order to preserve the typical Aglianico del Vulture wine features.
In this study, we looked at the yeast population present in four spontaneous alcoholic fermentations in the Rioja appellation
(D.O.Ca. Rioja, Spain). The study was conducted through the identification of the yeasts via the PCR–RFLP technique of the
ITS region of rDNA. In a first harvest, the qualitative diversity of the species present in spontaneous alcoholic fermentation
was studied, and in a second harvest, their quantitative significance. In spontaneous fermentations, 17 different yeast species
were found, although only two of them, Candida stellata and Kloeckera apiculata, as well as Saccharomyces cerevisiae, appeared in major proportions during fermentation. The significance of the non-Saccharomyces yeasts during the spontaneous alcoholic fermentation was conditioned by the presence of Saccharomyces cerevisiae in the medium. Species not cited in literature in connection with winemaking and yeasts associated with wines spoilage have
been detected in all the alcoholic fermentations carried out in the qualitative study.
KeywordsYeasts-Alcoholic fermentation-Non-Saccharomyces
-PCR–RFLP
Our previous work showed that NADP+-dependent glutamate dehydrogenase from K.marxianus behaves similarly to its counterpart in S.cerevisiae. It suggested that the ammonia assimilation pathway might be different between K.marxianus and the genetic closed species K.lactis. In the present work, we analyzed the genetic similarity among the GDH gene family in K.marxianus and closed yeasts. Specific primers for GDH genes were designed based on the K.marxianus sequences deposited in the Génolevures Database. One of them, for the KmGDH2 gene, proved to be specific for K.marxianus DNA samples, which confirmed the molecular identification of environmental yeast isolates, and can be proposed for rapid screening of this yeast from environmental samples. The nucleotide sequence revealed that KmGDH2 belongs to the S.cerevisiae GDH1 gene family together with KlGDH gene.
Spontaneous alcoholic fermentation from grape, agave and others musts into an alcoholic beverage is usually characterized
by the presence of several non-Saccharomyces yeasts. These genera yeasts are dominant in the early stages of the alcoholic fermentation. However the genera Hanseniaspora and Kloeckera may survive at a significant level during fermentation and can influence the chemical composition of the beverage. Several
strains belonging to the species Kloeckera api-culata and Hanseniaspora guilliermondii have been extensively studied in relation to the formation of some metabolic compounds affecting the bouquet of the final
product. Indeed some apiculate yeast showed positive oenological properties and their use in the alcoholic fermentations has
been suggested to enhance the aroma and flavor profiles. The non- Saccharomyces yeasts have the capability to produce and secrete enzymes in the medium, such as β -glucosidases, which release monoterpenes
derived from their glycosylated form. These compounds contribute to the higher fruit-like characteristic of final product.
This chapter reviews metabolic activity of Kloeckera and Hanseniaspora yeasts in several aspects: fermentative capability, aromatic compounds production and transformation of aromatic precursor
present in the must, also covers the molecular methods for identifying of the yeast
The principal agent in winemaking is the yeast Saccharomyces cerevisiae, which is characterized by a significant strain biodiversity. Here we report the characterization of 80 wild S.cerevisiae strains, isolated from grapes of different varieties in southern Italy, for genetic and technological variability. By PCR
amplification with M13 primer a significant polymorphism was recorded and 12 different biotypes were identified among the
strains. The specific strain-pattern could be used to follow the dynamics of different biotypes during the fermentation process.
The analysis of experimental wines obtained by inoculated fermentations with the 80 strains showed significant differences
among the wines. The level of each compound was a function of the strain performing the fermentative process. The main variables
for the strain differentiation were the production of acetaldehyde and acetic acid, which ranged from 53 to 282mg/l and from
0.20 to 1.88g/l, respectively. Selected strains were tested in fermentation with two different grape musts, yielding experimental
wines differing in the levels of secondary compounds and polyphenol content, in function of the interaction “grape must composition/yeast
strain”. This finding has an applicative value for the potentiality of utilizing the resource of strain variability as a tool
to individuate suitable starter cultures, which are able to complement and optimize grape quality.
Traditional winemaking is carried out by spontaneous yeasts from the vineyard that persist throughout the alcoholic fermentation.
This study examined the diversity of yeast species isolated from grape skin and musts of two varieties of Vitis labrusca from a vineyard in the southeast region of Brazil (Jales, São Paulo), which produces artisanal wines. Molecular identification
was achieved by a combination of PCR-RFLP/sequencing of the internal transcribed spacers (ITS) and sequencing of the D1/D2
domain of ribosomal DNA. Eighty yeast samples were isolated from grapes and musts, and seven different species were identified.
The diversity of species varied according to the grape variety. The most frequent species were Hanseniaspora uvarum with 28 isolates, followed by Issatchenkia occientalis with 19 isolates and Issatchenkia orientalis with 16 isolates. Other species with a lower number of isolates were: Issatchenkia terricola, Saccharomyces cerevisiae, Aureobasidium pullulans and Sporidiobolus pararoseus. Our results showed that molecular identification is a very powerful identification method in which natural isolates of ascomycetous
or basidiomycetous yeasts can be rapidly and reliably identified with better reproducibility and higher throughput than conventional
phenotypic methods. This is the first report of the diversity of indigenous yeast species from a vineyard in Brazil.
KeywordsYeasts–Non-Saccharomyces
–Brazil–Wine aroma
The complex microbial ecosystem of grape must and wine harbours a wide diversity of yeast species. Specific oligonucleotide primers for real-time quantitative PCR(QPCR) were designed to analyse several important non-Saccharomyces yeasts (Issatchenkia orientalis, Metschnikowia pulcherrima, Torulaspora delbrueckii, Candida zemplinina and Hanseniaspora spp.) and Saccharomyces spp. in fresh wine must, during fermentation and in the finished wine. The specificity of all primer couples for their target yeast species were validated and the QPCR methods developed were compared with a classic approach of colony identification by RFLP-ITS-PCR on cultured samples. Once the methods had been developed and validated, they were used to study these non-Saccharomyces yeasts in wine samples and to monitor their dynamics throughout the fermentation process. This study confirms the usefulness and the relevance of QPCR for studying non-Saccharomyces yeasts in the complex yeast ecosystem of grape must and wine.
Restriction patterns of amplified regions of ribosomal large subunit RNA encoding genes (26S rDNA) were evaluated as a routine methodology to examine yeast species diversity during red wine fermentation. The results were confirmed by sequencing of D1/D2 region of 26S rDNA. Red wine production was carried out using a yeast starter culture together with different commercial products, namely enzymes, fermentation activators and tannins and their influence on the non-Saccharomyces yeast population was studied. Yeast strains were isolated using lysine agar as a selective medium for non-Saccharomyces yeasts, after morphological characterisation of colonies. Amplification of 26S rDNA followed by digestion with three restriction enzymes applied to the 121 isolates, generated 19 profiles and a very high correlation with sequencing results was achieved. Although a starter yeast culture was added, results showed that several yeast species were present during all stages of fermentation, independent of the conditions tested, emphasizing the diversity of microorganisms associated with winemaking. On the other hand commercial additives did not significantly influence the diversity of yeast population during the fermentation process. For non-Saccharomyces strains, restriction patterns of a PCR amplified 26S rDNA region proved to be an adequate tool for clustering strains at species level and enabled the monitoring of yeast population dynamics during red wine fermentation.
Yeast population used in industrial production of fuel-ethanol may vary according to the plant process condition and to the environmental stresses imposed to yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as starter strain instead of less adapted commercial strains. This work reports the use of PCR-fingerprinting method based on microsatellite primer (GTG)5 to characterize the yeast population dynamics along the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominate the yeast population and were more present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from fuel-ethanol producing process.
Two cation-tolerant yeasts with powdered colonies, K28-3-2T and K26-1-4, were isolated from dry salted shrimp and sewage, respectively, in Siem Reap province, Cambodia. The D1/D2 sequences of the 26S rDNA data showed that the two isolates were conspecific and related to the Pichia burtonii and Candida fennica. Two isolates were examined by a polyphasic taxonomic approach, including molecular phylogenetic analysis, morphological, physiological and biochemical tests, DNA hybridization and MSP-PCR fingerprinting, in comparison with P. burtonii and C. fennica. The two isolates were found to grow by multilateral budding with true and pseudo-mycelium, to not produce ascospores, and to contain ubiquinone Q-8 similar to that of P. burtonii and C. fennica. The two isolates were not differentiated from the two closest species, P. burtonii and C. fennica, by the phenotypic character examined, except for the cation (Li+)-tolerance. From DNA-DNA reassociation studies, however, the two isolates showed low similarities to the closest two species. Based on D1/D2 sequences of 26S rDNA and DNA-DNA reassociation data, they were shown to be a new distinct species from P. burtonii and C. fennica. Therefore, a novel species is proposed, Candida khmerensis sp. nov., represented by strain K28-3-2T (=JCM 13262(T)=CBS 9784T). The novel species, Candida khmerensis sp. nov. can be clearly distinguished from P. burtonii and C. fennica by either the 26S rDNA D1/D2 or ITS region with 5.8S rDNA sequencing, or by the MSP-PCR fingerprinting pattern.
RFLP analysis of the ITS and 18S rDNA, RAPD-PCR using mini- and microsatellite primers and RFLP analysis of mitochondrial DNA were examined to discriminate yeasts related to dry-cured meat products at species and strain level. Seven species and 35 strains of yeasts usually found in dry-cured meat products were tested. RFLP analysis of the ITS1-5.8S rDNA-ITS2 and 18S rDNA did not allow the separation at species level of all of the species tested. RAPD with a M13 primer was found to be useful for differentiation of Rhodotorula mucilaginosa, Candida zeylanoides, Yarrowia lipolytica, Debaryomyces hansenii and Saccharomyces cerevisiae. However, no differences were observed between Debaryomyces polymorphus and Pichia carsonii. RAPD analysis with microsatellite primers (GACA)(4), (GTG)(5) and (GAC)(5) enabled discrimination at species and strain level. However, the degree of discrimination by means of RAPD-PCR depends highly on the primers used. Thus, the PCR fingerprinting with primer (GACA)(4) enabled a higher level of discrimination than primers (GAC)(5) and (GTG)(5). The RFLP analysis of mtDNA allowed the discrimination at the species and strain level except for R. mucilaginosa, where no polymorphisms were observed in the strains tested. RAPD analysis with primer (GACA)(4) and the restriction analysis of mtDNA used in the present work are useful for the differentiation at species and strain level of yeasts related to dry-cured meat products.
1 SUMMARY
The over-all objective for this project is the protection of the consumer’s health by describing measures for decreasing the amount of ochratoxin A in cereals produced in Europe. This has been achieved by identifying the key elements in an effective HACCP programme for ochratoxin A for cereals, and by providing tools for preventive and corrective actions. A summary of the tools provided by this project is presented in Table 1. The project included the whole food chain from primary production to the final processed food product. The objectives and expected achievements were divided into four different tasks, all important steps in a HACCP managing programme for ochratoxin A in cereals: 1. Identification of the critical control points (CCP); 2. Establishment of critical limits for the critical control points; 3. Developing rapid monitoring methods, and 4. Establishment of corrective actions in the event of deviation of a critical limit. The outcome will serve as a pool of knowledge for HACCP-based management programmes, which will increase food safety and support the EU cereal industry.
TASK 1. Investigation of grain samples has revealed that Penicillium verrucosum is the main, if not the only, producer of ochratoxin A in European cereals. It was concluded that P. verrucosum infection was best detected on DYSG media after seven days at 20ºC. Numbers of P. verrucosum found on DYSG and ochratoxin A content in cereals were correlated. . More than 7 % kernels infected with P. verrucosum indicated ochratoxin A contamination. In the action to identify critical control points for infection, the AFLP fingerprinting, which was developed, did not generate additional important information to that gained by the detection of P. verrucosum at species level by “traditional” taxonomic methods. The sources of infection of the grain were the contaminated environments of combines, dryers, and silos. Prompt and effective drying of cereals at harvest is the major CCP for preventing the formation of ochratoxin A. In regions of Europe where the cereal harvest is at greatest risk, measures to avoid mould and toxin problems are often most effective, while areas normally at less risk may not be the best prepared to avoid storage problems when unusual conditions occur. A significant problem arises where conditions at harvest are unpredictable as it may not be economic to have expensive drying machinery idle some years while in others the supply of damp grain may exceed the drying capacity available. Delays in drying may then put the grain at risk. Another problem arises when the infrastructure is such that sufficient funds and expertise are unavailable to advise on and ensure best storage practice.
TASK 2. The studies of the effect of temporal environmental factors on fungal growth, patterns of colonisation and ochratoxin A production revealed interesting characteristics, which may explain why P. verrucosum is the main ochratoxin A producer in cereal grain in Europe. Generally, P. verrucosum was more dominant at lower aw and 15ºC, whereas Aspergillus ochraceus was more dominant at higher aw at 25ºC. Furthermore, results indicated that P. verrucosum was less sensitive to higher concentrations of CO2 than A. ochraceus, which may also be a competitive advantage during storage. A mathematical model for safe storage time before onset of significant growth of P. verrucosum and ochratoxin A production have been developed, which describes the effect of water activity and temperature on the rate of growth of P. verrucosum in cereal grain. The model is valid for aerobic conditions, for instance when drying grain in near-ambient dryers or cooling grain by aeration prior to high-temperature drying.
Table 1. Summary of tools to prevent ochratoxin A in the cereal production chain as provided by the OTA PREV project.
Site Control type Tools provided Comments
(possible % reduction of OTA)
Harvest GAP - Recommendation: Keep machinery and areas, which are in contact with the harvested grain, clean. Remove old grain and dust. (WP1)
(% prevention not possible to estimate, but significant)
Buffer storage before drying and during drying (in near-ambient dryers) CCP -Mathematical model which can predict safe storage time (critical limits). (WP4)
(up to 100 % prevention possible)
- Rapid monitoring methods for OTA and producing fungi. (WP8) Monitoring tolls: LFDs and ELISAs.
- Data on environmental conditions conducive to growth and OTA production. (WP3) (% prevention not possible to estimate, but useful tools in DSS)
Storage GSP/CCP - Recommendations on silo design and maintenance. (WP5) (% prevention not possible to estimate, but significant)
- Critical limits for remoistening. (WP5) (up to 100 % prevention possible)
- Food grade antioxidants and natural control measures to prevent OTA formation in wet grain. (WP 6) (>80 % prevention but not yet economically feasible)
Intake at cereal processing industry
CCP - Rapid monitoring methods for OTA* and OTA producing fungi in grain. (WP8) LFD (with reader for ochratoxin A) and ELISA
- Critical limit: less than 1000 cfu/g P. verrucosum in wheat. (WP4 and WP11) Indicating risk of OTA levels above 5 μg/kg
- Monitoring method for P. verrucosum. (WP1, WP8, and WP9) Monitoring tools: DYSG, LFD, ELISA, and PCR
Milling industry GMP - Reductive measures during milling . (WP10) (cleaning 2-3%, scouring 3-44%, milling up to 60%)
Cereal processing industry GMP - Reductive measures during extrusion and baking. (WP10) (baking up to 5-10%, extrusion up to 40%)
Intake at malting industry CCP - Critical limits: <3% internal infection or <400 cfu/g with P. verrucosum in barley. (WP11) (up to 100 % prevention possible)
Malting industry GMP - Recommendation: effect of temperature on OTA formation during malting. (WP11) (a decrease of temperature from 16-18 to 12-14ºC reduces OTA formation 4 times)
Intake at brewing industry CCP - Rapid monitoring methods for OTA* in malt. (WP8) LFD (with reader) and ELISA
Brewing industry GMP - Fate of OTA during brewing. (WP 11) (up to 80 % reduction)
Official control CCPs - Rapid monitoring methods for OTA*. (WP8) LFD (with reader) and ELISA
* the critical limits at these points are the same as the legislative limits (today 5 and 3 µg/kg for the unprocessed cereals and products, respectively)
Abbreviations used: GAP, Good Agricultural Practice; GSP, Good Storage Practice; GMP, Good Manufacturing Practice; CCP, Critical Control Point; DSS, decision support systems; cfu, colony forming units; OTA, ochratoxin A; LFD, lateral flow device; WP, project workpackage.
The probability, of ochratoxin A levels above the EC maximum limit of 5 µg/kg at different concentration of P. verrucosum in the grain, clearly increased when the levels of P. verrucosum were above 1000 colony forming units/gram. A mathematical model was developed, which describes the risk for condensation in the headspace of a silo during storage of cereal grain. The model has been used to identify the conditions, which cause moistening of the grain, and to develop control strategies to reduce this and the risk for mould growth and ochratoxin A production. Essential oils, resveratrol and lactic acid bacteria (LAB) can control growth and ochratoxin A production by P. verrucosum and A. ochraceus on grain. However, in small-scale storage experiments and experimental maltings, the inhibitory effect of the selected LAB strain could not be clearly shown. Out of twenty-four essential oils tested the most effective were found to be thyme, cinnamon leaf and clove bud.
TASK 3. New diagnostic tools have become available that will provide the means for rapid determination of ochratoxin A in cereals. This will enable the effective implementation of the European legislation and facilitate future internal control and scientific studies. Immunoassays in ELISA format, sensitive enough to meet the EU legislation for ochratoxin A, have been developed where large numbers (100’s) can be analysed in a few hours. In addition a lateral flow device (LFD) taking less than five minutes to perform, which can be used on-site, has also been developed. A number of genes have been cloned, among them a polyketide synthase gene, which is involved in ochratoxin A biosynthesis. PCR primer pairs have been developed which appear to be highly specific for A. ochraceus and P. verrucosum. The primers may find use a in the development of rapid identification protocols for ochratoxigenic fungi. Several advances have been made towards a molecularly imprinted polymer (MIP) specific for ochratoxin A and its integration into a solid phase extraction (SPE) and sensor systems. Several polymers have been designed using a computational method and tested using SPE. The materials demonstrate a high affinity and specificity for the target molecule in aqueous model samples, however integration in real samples with complex biological matrices (grain samples) has proved difficult as interfering compounds affect binding and measurements of ochratoxin A. Attempts to isolate and remove these interfering materials were unsuccessful and consequently the detection limits were not at the level required to meet the legislative requirements
TASK 4. This project has contributed with tools and recommendations for the cereal processing industry. These will will facilitate decisions to be made to enable the dual maximum levels for ochratoxin A described in the Commission Regulation (EC) No 472/2002 of 12 March 2002 setting maximum levels for ochratoxin A in foodstuffs to be followed. Examining the fate of ochratoxin A during milling revealed white flour having the most significant reduction of ochratoxin A of about 50%. An initial cleaning stage and scouring (1-2%) prior to milling, removed small amounts of ochratoxin A. Baking resulted in only a small fall in concentration. However, an overall reduction of about 80% is achievable for white bread with scouring included and up to 35% similarly for wholemeal bread. The increase of ochratoxin A concentration during malting was 2-4-fold in 75 % of the samples studied and process temperature had a pronounced effect. At the higher temperatures of 16-18°C ochratoxin A formation was 20-fold compared to 5-fold at the temperatures of 12-14°C. During the brewing process approximately 20% of the original ochratoxin A from the malt remained in the beer.
Many strategies have been studied for control of mould growth and reduction in mycotoxin production in foods. The most effective strategy for controlling the presence of mycotoxins in foods is prevention of growth of the mycotoxin-producing fungi in foods and field crops in the first place. Mycotoxin contamination may occur prior to harvest of crops and is often the dominant reason for the occurrence of mycotoxins in foods and feeds. However, fungal growth on stored foods and commodities is also a serious and continuing problem. In recent years increased public concern over chemical food additives and fungicides in foods has prompted searches for safe naturally occurring biological agents with antifungal potential. One source of such compounds are the lactic acid bacteria. While only a relatively limited number of studies have reported the inhibitory effects of lactic acid bacteria on fungal growth and mycotoxin production, it is generally believed that it is safe for humans to consume lactic acid bacteria and has been known for many years that lactic acid bacteria may positively influence the gastrointestinal tract
The fermentation of grape juice into wine is a complex microbiological process, in which yeasts play a central role. Traditionally, identification and characterization of yeast species have been based on morphological and physiological characteristics. However, the application of molecular biology techniques represents an alternative to the traditional methods of yeast identification and are becoming an important tool in solving industrial problems. Although Saccharomyces cerevisiae is responsible for the alcoholic fermentation, the presence of non-Saccharomyces species could be important since they produce secondary metabolites, which can contribute to the final taste and flavor of wines.
Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite oligonucleotide primers (GAC)5 and (GTG)5 enabled discrimination between the strains tested. Additionally, restriction enzyme analysis, with TaqI and MseI, of PCR-amplified fragments from the complete internal transcribed spacer and nontranscribed spacer, both present in the rRNA-encoding gene cluster, proved to be suitable for generating intraspecies-specific patterns. Random amplified polymorphic DNA with primers 24 and OPA-11 and PCR fingerprinting with primer (GTG)5 appeared to generate the highest degree of diversity. However, the results indicated that there was no single PCR-mediated typing technique enabling discrimination on the strain level. Discrimination of each individual strain was nevertheless possible by combining the results obtained with all typing techniques.
In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation.
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.
A study was conducted of the dynamics of Saccharomyces and non-Saccharomyces populations during alcoholic fermentation of Albarino musts from two enological subzones located in Galicia (Northwest Spain). Sixteen microvinifications were carried out (8 in each must, M and E) with five indigenous Saccharomyces cerevisiae strains, two commercial active dry strains, and the corresponding spontaneous fermentation. The volatile compounds in the resulting wines were measured using gas chromatography. The study of different physiological and biochemical characteristics allowed us to follow the evolution of the inoculated S. cerevisiae strains. The different cellular concentrations of these strains in the musts produced different growth rates during fermentation. The growth of non-Saccharomyces flora depended on the initial starter culture concentrations and on their growth rate during winemaking. Statistical analysis (factorial and cluster) of data obtained by gas chromatography created statistical relationships between the production of some components of wine aroma and the evolution of yeast flora during alcoholic fermentation.
The spontaneous alcoholic fermentation is characterized by the contribution of different Saccharomyces cerevisiae strains, which grow in succession or in combination throughout the fermentation process and exhibit different metabolic patterns. The formation of secondary compounds is a strain-specific characteristic and the strains are distinguishable in phenotypes through the production of different amounts of by-products. Natural fermentation is a source of indigenous Sacch. cerevisiae strains, which seem more suitable to be used as starter cultures for that particular wine because they were isolated from the original region and, consequently, better adapted to the particular vinification conditions of that enological area. Among the indigenous strains, the cultures for must fermentation should be chosen on the basis of aroma and flavour determinants typical of the wine under study. Successively, the selected cultures should be tested for the genetic segregation of traits under consideration in order to identify strains completely homozygous for the metabolic characteristics. Only a small proportion of natural wine strains is completely homozygous, the majority being heterozygous for one or more traits. In addition, a significant proportion of natural wine strains can sporulate on rich media, such as grape must, and, as a consequence, the progeny of such strains can exhibit differences in the levels of by-products, thereby affecting the organoleptic properties of the final product. Determination of the degree of strain stability overcomes this problem and allows the choice of the most suitable selected culture to be used in inoculated fermentation. The feature, »stability of metabolic phenotype in industrial strains«, represents a selective index, which ensures that the final product is always consistent with the own properties of each wine.
Must and wine from grapes harvested in two vintages (1986 and 1987) were anlyzed during vinification for physicochemical and microbiological characteristics. The 1986 vintage would be considered abnormal or poor vintage because of higher rainfall at harvest, and the 1987 one a normal vintage. Low reducing sugars and high volatile acidity at the beginning of the poor fermentation was observed as compared to normal vinification. The yeast population showed atypical evolution through the process since oxidative yeasts were isolated in the first stages of the poor vinification.
Wine yeasts were isolated from spontaneous alcoholic fermentations performed with white and red grape musts from vintages 1991 and 1992. Yeast cells were analysed by physiological tests and gel electrophoretic karyotyping. It was shown that there is a succession of different strains in the yeast population during the time course of the fermentation process. Furthermore, the composition of the yeast strain population differs from grape must to grape must and from year to year, and may therefore be considered vineyard (terrain)- and vintage-dependent.
Non-Saccharomyces yeast isolates from musts of AC (Appellation Controlee) La Mancha in spontaneous fermentation were comparatively identified by physiological and molecular tests. The criterion of Barnett for the phenotypic identification was followed. Genetic characterization by polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) was made. The region between 18S and 28S rRNA genes was amplified using specific internal transcribed spacers ITS1 and ITS4 primers. The 47 non-Saccharomyces isolates produced nine different phenotypic profiles and 13 different genetic profiles, showing that PCR/RFLP can be more discriminating. The information supplied by the two methodologies was very similar. PCR/RFLP can be used to correct erroneous identifications by phenotype, and in some cases to achieve intra-species differentiation.
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 growth kinetics and fermentation behaviour of five non-Saccharomyces yeast species associated with wine-making were evaluated.The results showed that the Candida stellata and Torulspora delbrueckii species are interesting for biotechnological applications in wine-making, whereas small-size apiculate yeasts could be profitably used in the production of wine for vinegar manufacture.
The phylogenetic interrelationship of species of the genus Zygosaccharomyces was examined by 18S rRNA gene sequencing. Comparative analysis of the sequence data revealed the genus to consist of a number of distinct subdivisions. The most prevalent species associated with food spoilage, Z. bailii, Z. bisporus and Z. rouxii , along with Z. mellis were found to form one subdivision. Zygosaccharomyces cidri and Z. fermentati formed a distinct species pair, as did Z. microellipsoides and Z. mrakii. Zygosaccharomyces florentinus formed a separate line displaying no specific relationship with any of the other Zygosaccharomyces species examined. Comparison with nine published ascosporogenous yeast 18S rRNA gene sequences showed that Z. microellipsoides and Z. mrakii were genealogically very close to Torulaspora delbrueckii (both displaying 99·8% 18S rRNA sequence similarity), raising the possibility that these two Zygosaccharomyces species should be moved to the genus Torulaspora .
The topologies of trees derived from complete 18S rRNA gene sequences and from individual domains within the gene were compared and the implications of using partial sequence data for inferring phylogenetic relationships discussed.
We have used the techniques of DNA fingerprinting and polymerase chain reaction (PCR) with probes specific for hypervariable repetitive DNA sequences (mini‐ and microsatellite DNAs) to analyze 36 yeast strains belonging to 10 species and 2 genera. Using (GTG) 5 , (GACA) 4 , phage M13 DNA and the M13 sequence GAGGGTGGCGGTTCT as probes and primers, respectively, we obtained DNA polymorphisms which allowed us to discriminate 23 biotechnologically important strains of the yeast Saccaromyces cerevisiae and to distinguish them from strains of S. pastorianus, S. bayanus and S. willianus .
Our results demonstrate that both DNA and PCR fingerprinting are suitable tools for an easy, fast and reliable molecular typing of yeasts. The DNA fingerprinting method seems to be more sensitive than PCR fingerprinting with respect to the individualization of strains. Nevertheless, using the PCR fingerprinting technique we were able to unambigously dicriminate between yeast genotypes of different species. Therefore, PCR fingerprinting might become a useful tool in the classification of yeasts on the basis of phylogenetic relatedness.
The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.
The size of the internal transcribed spacer (ITS) region as measured by gel electrophoresis of PCR products, amplified by primers ITS1 and ITS4, was over 800 bp for all Saccharomyces sensu stricto species, but yeasts belonging to other Saccharomyces species had a shorter ITS region, making this characteristic potentially useful in the identification of Saccharomyces isolates. The ITS product length was homogeneous within the species Saccharomyces cerevisiae.
18S rDNA + ITS1 and 25S rDNA PCR products covering more than 95% of the nuclear ribosomal DNA repeat unit of 28 Saccharomyces sensu stricto and Torulaspora yeasts and their anamorph forms were digested with HaeIII, MspI, HinfI and CfoI. Using combinations of two restriction enzymes, specific ribotyping patterns of six species were found. PCR ribotyping offers a convenient tool for quick identification of yeast isolates, but specificity of ribotyping patterns should be checked with a larger number of strains to avoid misidentification because of lack of variation within different taxa or because of strain-specific ribotyping patterns of species type strains.
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.
Kloeckera apiculata and Saccharomyces cerevisiae yeast species are dominant, respectively, at the early and at the following stages of wine fermentation. In the present study, PCR fingerprinting and NTS region amplification and restriction were applied as techniques for monitoring yeast population performing Aglianico of Vulture grape must fermentation.
Thirty S. cerevisiae and 30 K. apiculata strains were typed by PCR fingerprinting with (GAC)5 and (GTG)5 primers and by complete NTS region amplification followed by restriction with HaeIII and MspI enzymes. S. cerevisiae strains generated two patterns with (GAC)5 primer, while (GTG)5 primer yielded a higher genetic polymorphism. Conversely, in K. apiculata Aglianico wine strains (GAC)5 and (GTG)5 primers generated the same profile for all strains. Restriction analysis of the amplified NTS region gave the same profile for all strains within the same species, except for one strain of S. cerevisiae.
The PCR fingerprinting technique was useful in discriminating at strain level S. cerevisiae, particularly with the primer (GTG)5. RFLP patterns generated from the NTS region of the two species can be more easily compared than the patterns resulting from PCR fingerprinting, thus RFLP is more suitable for the rapid monitoring of the species involved in different stages of fermentation.
The molecular techniques used allow discrimination of S. cerevisiae at strain level and monitoring of the ratio of S. cerevisiae/K. apiculata during the fermentation process. Thus, their application can assure technological adjustments in a suitable time.
Yeasts: growth during fermenta-tion
- G H Fleet
- G M Heard
Fleet, G.H., Heard, G.M., 1993. Yeasts: growth during fermenta-tion. In: Fleet, G.H. (Ed.), Wine Microbiology and Biotechnol-ogy. Harwood Academic, Switzerland, pp. 27 – 54.
Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers
- Esteve-Zarzoso