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Identification of spoilage yeasts in a food production chain by microsatellite PCR fingerprinting
Abstract
A survey of yeast strains present in the production chain of mayonnaise and salad dressings was carried out over a period of 14 months. Attempts were made to identify the isolated yeasts with the API system, but identification of all species involved was not possible. In the investigation the performance of the microsatellite polymerase chain reaction (PCR) fingerprinting analysis with the microsatellite oligonucleotide primers (GAC)5and (GTG)5appeared to be superior. Several yeast species were encountered in the production lines but only the speciesZygosaccharomyces bailiiandZygosaccharomyces bisporuswere present in the final products. Only microsatellite PCR fingerprinting analysis allowed discrimination between species of theZygosaccharomycesgenus. In addition, the PCR-based technique allowed the discrimination of different types within theZ. bailiispecies.Z. bailiistrains isolated from spoiled products displayed PCR-fingerprinting types that appeared to be identical to some of those generated by strains isolated from one specific production line. This suggested that microsatellite PCR fingerprinting is useful in tracing back the origin of spoilage outbreaks, and that it can be applied in microbiological quality assurance monitoring systems in industrial environments.
... Several approaches based on nucleic acids polymorphisms have been developed in an attempt to simplify yeast identifi cation, such as electrophoretic karyotyping, temperature gradient gel electrophoresis (TGGE), microsatellite PCR fingerprinting, random amplifi ed polymorphic DNA, ribosomal DNA (rDNA) restriction profi les and partial rDNA sequencing (Török et al.,1993;Baleiras-Couto et al., 1995;Baleiras-Couto et al., 1996;Guillamón et al., 1998;Kurtzman and Robnett, 1998;Esteve-Zarzoso et al., 1999;Hernán-Gómez et al., 2000;Esteve-Zarzoso et al., 2003;Baleiras-Couto et al., 2005;Rodriguez et al., 2010). ...
... White et al. (1990) used this methodology to amplify the ribosomal gene 5.8S and the adjacent intergenic regions ITS1, ITS2 and further to digest with restriction enzymes. Another ribosomal region that is very useful to differentiate at species level is the one that includes 18S gene and the intergenic region ITS1 (Baleiras-Couto et al., 1996;Dlauchy et al., 1999). Since then, this approach has been used for identifying yeast species mainly associated alcoholic beverages and soft drinks (Guillamón et al., 1998;Esteve-Zarzoso et al., 1999;Arias et al., 2002;Ferreira et al., 2009). ...
The complex microbial ecosystem existing in grape, must and wine comprises a wide diversity of yeast species. The knowledge of composition and dynamics of yeast biota occurring along vinification process would provide a better control of wine quality. The sequence of D1/D2 domain of 26S ribosomal DNA (rDNA), reflects ascomycetous yeast phylogenetic relationships and enables their separation at the species level. A region of the 26S rDNA, with around 1100 bp comprising domain D1/D2, was amplified by PCR and then digested with restriction endonucleases (ApaI, HinfI, MseI, HaeIII and CfoI) in order to differentiate yeast species frequently isolated from grape surfaces, wine and cellar equipments. A total of 78 yeast strains (including 36 type strains) belonging to 53 species were used to generate the restriction profiles. Numerical analysis of the profiles generated by the five restriction enzymes enabled to group the strains in 47 different clusters and 42 of them clearly corresponded to different yeast species. The remaining groups comprise closely related species. The enzymes MseI, HaeIII and CfoI revealed a high discrimination power and the restriction profiles generated were sufficient to clearly identify the 42 species mentioned above. Despite one of the clusters included different yeast genera, with different wine characteristics, the common wine spoilage yeasts Zygosaccharomyces bailii and Z. lentus could be separated to one distinctive cluster through the use of ApaI restriction profiles. Since the analysis of restriction profiles of amplified 26S rDNA showed to be a valuable method to identify oenological yeast species, a database comprising the majority of wine yeast biota was created to be applied both at research and industrial environment.
... Mikro-ja minisatelliitti-PCR:n sekä AP-PCR:n erottelukyvyt ovat verrattavissa toisiinsa, ja yleensä satelliittijaksoihin perustuvilla menetelmillä on pystytty erottelemaan hiivoja parhaiten laji-ja alalajitasolla. Menetelmän on todettu soveltuvan erilaisten Candida- (Thanos et al., 1996;Latouche et al., 1997), Zygosaccharomyces- (Baleiras Couto et al., 1996a), Kluyveromyces-ja Saccharomyces-lajien (Lieckfeldt et al., 1993) sekä juustoperäisten hiivojen (Prillinger et al., 1999) tunnistamiseen. Prillinger et al. (1999) pystyivät tunnistamaan IR-PCR:llä useita fenotyyppisiltä ominaisuuksiltaan epätyypillisiä juustoisolaatteja. ...
... Prillinger et al. (1999) pystyivät tunnistamaan IR-PCR:llä useita fenotyyppisiltä ominaisuuksiltaan epätyypillisiä juustoisolaatteja. Baleiras Couto et al. (1996a) onnistuivat jäljittämään mikrosatelliitti-PCR:llä majoneesia ja salaaattikastikkeita pilanneen Z. bailiin alkuperän. ...
... Several approaches based on nucleic acids polymorphisms have been developed in an attempt to simplify yeast identifi cation, such as electrophoretic karyotyping, temperature gradient gel electrophoresis (TGGE), microsatellite PCR fingerprinting, random amplifi ed polymorphic DNA, ribosomal DNA (rDNA) restriction profi les and partial rDNA sequencing (Török et al.,1993;Baleiras-Couto et al., 1995;Baleiras-Couto et al., 1996;Guillamón et al., 1998;Kurtzman and Robnett, 1998;Esteve-Zarzoso et al., 1999;Hernán-Gómez et al., 2000;Esteve-Zarzoso et al., 2003;Baleiras-Couto et al., 2005;Rodriguez et al., 2010). ...
... White et al. (1990) used this methodology to amplify the ribosomal gene 5.8S and the adjacent intergenic regions ITS1, ITS2 and further to digest with restriction enzymes. Another ribosomal region that is very useful to differentiate at species level is the one that includes 18S gene and the intergenic region ITS1 (Baleiras-Couto et al., 1996;Dlauchy et al., 1999). Since then, this approach has been used for identifying yeast species mainly associated alcoholic beverages and soft drinks (Guillamón et al., 1998;Esteve-Zarzoso et al., 1999;Arias et al., 2002;Ferreira et al., 2009). ...
O ecossistema microbiano existente nas uvas, no mosto e no vinho é composto por uma grande diversidade de espécies de leveduras. O conhecimento deste biota de leveduras ao longo do processo de vinificação permite um melhor controlo da qualidade do vinho. Para a identificação de leveduras o ADN ribossómico (ADNr) tem-se revelado muito adequado para estimar relações filogenéticas, consideradas pelas correntes mais actuais da taxonomia como estando na base da classificação taxonómica. No presente trabalho, avaliou-se um método baseado na amplificação do ADNr 26S, compreendendo a região D1/D2, seguido de digestão por enzimas de restrição - Perfis de Restrição - para a identificação de espécies de leveduras envolvidas no processo de produção de vinho. Esta avaliação foi efectuada através do uso de 78 estirpes pertencentes a 53 espécies (incluindo 36 estirpes tipo). Utilizaram-se as enzimas de restrição ApaI, HinfI, MseI, HaeIIIe CfoI e, análise numérica dos perfis de restrição gerados permitiu agrupar as espécies estudadas em 47 grupos, 42 dos quais correspondendo a uma única espécie. As enzimas de restrição MseI, HaeIII e CfoI foram as que apresentaram maior poder discriminante ao nível da espécie, permitindo a identificação das mesmas 42 espécies. Apesar da enzyma ApaIter apresentado o mais baixo grau de polimorfismo, esta enzima poderá ser útil para medidas de controlo uma vez que seu perfil de restrição pôde agrupar em um grupo distinto as leveduras Zygosaccharomyces bailii e Z. Lentus. O método desenvolvido revelou eficácia, rapidez e facilidade de aplicação na identificação de leveduras de interesse enológico. Com o presente trabalho iniciou-se a construção de uma base de dados de perfis de restrição para posterior aplicação em condições industriais e de investigação.
... Microsatellites are short DNA motifs repeated in tandem present in eukaryotic genomes. Although originally designed to study genetic variations in humans due to their high degree of variability, microsatellites have also become a powerful tool to study intraspecific diversity in yeasts, enabling, for example, to discriminate between S. cerevisiae strains from wine and beer [63] or from artisanal versus industrial bread-making processes [64]. Interestingly, a microsatellites analysis of the K. marxianus isolates from henequen and pulque produced four different patterns that corresponded to the four groups already detected in the ITS-5.8S ...
Seven Kluyveromyces marxianus isolates from the elaboration process of pulque and henequen mezcal were characterized. The isolates were identified based on the sequences of the D1/D2 domain of the 26S rRNA gene and the internal transcribed spacer (ITS-5.8S) region. Genetic differences were found between pulque and henequen mezcal isolates and within henequen mezcal isolates, as shown by different branching patterns in the ITS-5.8S phylogenetic tree and (GTG)5 microsatellite profiles, suggesting that the substrate and process selective conditions may give rise to different K. marxianus populations. All the isolates fermented and assimilated inulin and lactose and some henequen isolates could also assimilate xylose and cellobiose. Henequen isolates were more thermotolerant than pulque ones, which, in contrast, presented more tolerance to the cell wall-disturbing agent calcofluor white (CFW), suggesting that they had different cell wall structures. Additionally, depending on their origin, the isolates presented different maximum specific growth rate (µmax) patterns at different temperatures. Concerning tolerance to stress factors relevant for lignocellulosic hydrolysates fermentation, their tolerance limits were lower at 42 than 30 °C, except for glucose and furfural. Pulque isolates were less tolerant to ethanol, NaCl, and Cd. Finally, all the isolates could produce ethanol by simultaneous saccharification and fermentation (SSF) of a corncob hydrolysate under laboratory conditions at 42 °C.
... Microsatellites are short DNA motifs repeated in tandem present in eukaryotic genomes. Although originally designed to study genetic variations in humans due to their high degree of variability, microsatellites have also become a powerful tool to study intraspecific diversity in yeasts, enabling for example to discriminate between S. cerevisiae strains from wine and beer [64] or from artisanal versus industrial bread-making processes [65]. Interestingly, microsatellites analysis of the K. marxianus isolates from henequen and pulque produced four different patterns (Figure 2) that corresponded to the four groups already detected in the ITS-5.8S ...
Seven Kluyveromyces marxianus isolates from the elaboration process of pulque and henequen mezcal were characterized. The isolates were identified based on the sequences of the D1/D2 domain of the 26S rRNA gene and the internal transcribed spacer (ITS-5.8S) region. Genetic differences were found between pulque and henequen mezcal isolates and within henequen mezcal isolates, as shown by different branching patterns in the ITS-5.8S phylogenetic tree and (GTG)5 microsatellite profiles, suggesting that substrate and process selective conditions may originate different K. marxianus populations. All the isolates fermented and assimilated inulin and lactose and some henequen isolates could also assimilate xylose and cellobiose. Henequen isolates were more thermotolerant than pulque isolates which in contrast presented more tolerance to the cell wall-disturbing agent calcofluor white (CFW) suggesting that they had a different cell wall structure. Additionally, depending on their origin, the isolates presented different maximum specific growth rates (umax) patterns at different temperatures. Concerning tolerance to stress factors relevant for lignocellulosic hydrolysates fermentation, tolerance limits were lower at 42 than 30°C except for glucose and furfural. Pulque isolates were less tolerant to ethanol, NaCl, and Cd. Finally, all the isolates could produce ethanol by simultaneous saccharification and fermentation (SSF) of a corncob hydrolysate under laboratory conditions at 42°C.
... For Microsatellite-Primed PCR (MSP-PCR) fingerprinting, genomic DNA was extracted from overnight YEL cultures. The microsatellite oligonucleotide primer (GTG) 5 was used for PCR reaction, as described by Baleiras-Couto et al. (1996). The method developed by Nguyen et al. (2000) was used to extract mtDNA, which was then digested with HaeIII and MboI restriction endonucleases and analysed as described in Csoma et al. (2021). ...
The yeast Starmerella (Candida) lactis-condensi is considered a food contaminant microorganism. The aim of our research was to determine why St. lactis-condensi could become the dominant species of Essences, the top sweet wine speciality of Tokaj wine region in Hungary. We investigated the physiological properties of these yeasts based on parameters that may influence their ability to selectively proliferate and persist during maturation in wines with very high sugar content. These include glucose and fructose, alcohol, and sulphur tolerance. Our studies have shown that St. lactis-condensi is a fructophilic yeast that is able to adapt quickly to very high sugar concentrations (up to 500 g/L) in the Essences. The high glucose concentration inhibits its growth, as well as that of the St. bacillaris (Candida zemplinina) strains tested. The type and amount of sugars in the Essences, together with the sulphur and alcohol content, influence the composition of the dominant yeast biota. Analysis of (GTG)5 microsatellite in the nuclear genome and mtDNA-RFLP studies demonstrate that a diverse population of St. lactis-condensi occurs in the Tokaj wine region, in the Essences. This yeast species is characterised by both physiological and genetic biodiversity. GC-MS analysis of Essences colonised exclusively with these yeasts showed no deterioration in quality.
... The three molecular methods used, RFLPs of mtDNA, MSP-PCR analysis, and electrophoretic karyotyping, have all been applied previously to the comparative study of Zygosaccharomyces. The strain-level resolution power of these techniques was proved for the Zygosaccharomyces species formerly [19,49,50]. Esteve-Zarzoso et al. [19] compared the mtDNA-RFLP patterns (Hinf I) and karyotypes of Z. lentus strains (CBS 3014 and CBS 2900) to those of Z. bailii strains. ...
Tokaj botrytized sweet wines are traditionally aged for several years in wood barrels or bottles. As they have significant residual sugar content, they are exposed to microbial contamination during ageing. Osmotolerant wine-spoilage yeasts are most commonly found in the Tokaj wine-growing region in the species Starmerella spp. and Zygosaccharomyces spp. For the first time, Z. lentus yeasts were isolated from post-fermented botrytized wines. Our physiological studies confirmed that these yeast strains are osmotolerant, with high sulphur tolerance and 8% v/v alcohol tolerance, and that they grow well at cellar temperature in acidic conditions. Low β-glucosidase and sulphite reductase activities were observed, whereas protease, cellulase, and α-arabinofuranosidase extracellular enzyme activities were not detected. Molecular biology analyses carried out by RFLP analysis of mtDNA revealed no remarkable differences between strains, while microsatellite-primed-PCR fingerprinting of the (GTG)5 microsatellite and examination of chromosomal pattern revealed considerable diversity. The fermentative vigour of the tested Z. lentus strains was found to be significantly lower compared to the control Saccharomyces cerevisiae (Lalvin EC1118). It can be concluded that Z. lentus is a potential spoilage yeast in oenology which may be responsible for the initiation of secondary fermentation of wines during ageing.
... For MSP-PCR fingerprinting, genomic DNA was extracted from overnight YEL cultures. The PCR reaction was carried out with the microsatellite oligonucleotide primer (GAC) 5 , as described by Baleiras-Couto et al. [61]. Z. rouxii CBS 732 T was used as the reference strain in both procedures. ...
The conversion of grape juice to wine starts with complex yeast communities consisting of strains that have colonised the harvested grape and/or reside in the winery environment. As the conditions in the fermenting juice gradually become inhibitory for most species, they are rapidly overgrown by the more adaptable Saccharomyces strains, which then complete the fermentation. However, there are environmental factors that even Saccharomyces cannot cope with. We show that when the sugar content is extremely high, osmotolerant yeasts, usually considered as “spoilage yeasts“, ferment the must. The examination of the yeast biota of 22 botrytised Tokaj Essence wines of sugar concentrations ranging from 365 to 752 g∙L−1 identified the osmotolerant Zygosaccharomyces rouxii, Candida (Starmerella) lactis-condensi and Candida zemplinina (Starmerella bacillaris) as the dominating species. Ten additional species, mostly known as osmotolerant spoilage yeasts or biofilm-producing yeasts, were detected as minor components of the populations. The high phenotypical and molecular (karyotype, mtDNA restriction fragment length polymorphism (RFLP) and microsatellite-primed PCR (MSP-PCR)) diversity of the conspecific strains indicated that diverse clones of the species coexisted in the wines. Genetic segregation of certain clones and interactions (antagonism and crossfeeding) of the species also appeared to shape the fermenting yeast biota.
... One of the most popular approaches based on PCR is the PCR fingerprinting technique which uses specific oligonucleotides for simple repetitive DNA sequences. PCR fingerprinting with the oligonucleotide primers (GTG)5, (GAC) 5 , (GACA)4, and the M13 sequence has been widely applied for the identification of yeasts (Lieckfeldt et al., 1993;Couto et al., 1996aCouto et al., , 1996bCaruso et al., 2002;Martorell et al., 2005;Orlić et al., 2010). The DNA sequencing of the genes encoding the D1/D2 domain of the 26S ribosomal RNA gene and the ITS (internal transcribed spacer) region of rDNA has provided highly reliable data for yeast taxonomy, and the characterisation of new genera and species (Kurtzman and Robnett, 1998Robnett, , 2003Groenewald et al., 2011). ...
In the article by Erdem et al. published in Journal of Microbiology 2016; 54, 618–625, the figure 1 should be corrected as below.
... One of the most popular approaches based on PCR is the PCR fingerprinting technique which uses specific oligonucleotides for simple repetitive DNA sequences. PCR fingerprinting with the oligonucleotide primers (GTG)5, (GAC) 5 , (GACA)4, and the M13 sequence has been widely applied for the identification of yeasts (Lieckfeldt et al., 1993;Couto et al., 1996aCouto et al., , 1996bCaruso et al., 2002;Martorell et al., 2005;Orlić et al., 2010). The DNA sequencing of the genes encoding the D1/D2 domain of the 26S ribosomal RNA gene and the ITS (internal transcribed spacer) region of rDNA has provided highly reliable data for yeast taxonomy, and the characterisation of new genera and species (Kurtzman and Robnett, 1998Robnett, , 2003Groenewald et al., 2011). ...
A new method based on high resolution melting (HRM) analysis was developed for the differentiation and classification of the yeast species that cause food spoilage. A total 134 strains belonging to 21 different yeast species were examined to evaluate the discriminative power of HRM analysis. Two different highly variable DNA regions on the 26 rRNA gene were targeted to produce the HRM profiles of each strain. HRM-based grouping was compared and confirmed by (GTG)5 rep-PCR fingerprinting analysis. All of the yeast species belonging to the genera Pichia, Candida, Kazachstania, Kluyveromyces, Debaryomyces, Dekkera, Saccharomyces, Torulaspora, Ustilago, and Yarrowia, which were produced as species-specific HRM profiles, allowed discrimination at species and/or strain level. The HRM analysis of both target regions provided successful discrimination that correlated with rep-PCR fingerprinting analysis. Consequently, the HRM analysis has the potential for use in the rapid and accurate classification and typing of yeast species isolated from different foods to determine their sources and routes as well as to prevent contamination.
... These techniques could also be applied for the early detection of specific spoilage organisms. However, before such techniques could be used for the detection of specific organisms, those microorganisms must be identified for each type of product and their effect on spoilage characteristics must be determined (44,45). ...
This chapter presents an overview on the factors affecting deterioration of foods and approaches used in shelf life testing. It further outlines the need for characterization of the various mechanisms of bio-/chemical and microbial deterioration of each type or group of foods, so that improved methods of testing may be developed, and longer shelf lives can be achieved.
... Out of 60, 18 biochemically identified isolates were confirmed as Kluyveromyces spp. Baleiras et al., (1996) found that the restriction enzyme digests of the ITS region appeared to be relatively discriminative, as this region displayed a higher level of heterogeneity than the NTS region, thereby allowing more detailed information on the subspecies level. The purified PCR products were arranged to be get sequenced from Xcelris Genomic Centre, Ahmedabad, India. ...
K. marxianus and K. lactis happen to be the only lactose
fermenting yeast species found regularly in milk products. These
species are considered to be Generally Regarded As Safe
organisms (GRAS) and have been approved as a food additive.
Since, the information regarding the prevalence of Kluyveromyces
spp. in dairy products is scanty especially under Indian conditions,
hence an attempt has been made in the present study to isolate
and characterize β-galactosidase (β-gal) positive Kluyveromyces
spp. from dairy products. A total number of 110 randomly selected
colonies were isolated from different dairy products. Out of these
60 isolates were identified as Kluveromyces spp. after
morphological and biochemical characterization. However, after
molecular characterization, 18 isolates were confirmed as
Kluyveromyces spp. Out of which 14 isolates were confirmed as
K. marxianus and 4 as K. lactis. The present study has revealed
that indigenous dairy products can be natural and preferred niche
for isolation and growth of native and novel strains of dairy yeasts
such as K. lactis and K. marxianus.
... Occasionally, Saccharomyces cerevisiae and Candida magnolia can be encountered in acidified products (ICMSF, 2005). As from the different Zygosaccharomyces species, Z. bailii is the most recovered from mayonnaise, sauces, and pickles (Couto et al., 1996;James and Stratford, 2003;ICMSF, 2005), this paragraph will mainly focus on this yeast. Species that are most related to Z. bailii are Z. ...
... Salad dressings are among the many foods which are subject to contamination and spoilage by yeasts (Fabian & Wethington, 1950;Kurtzman, Rogers, & Hesseltine, 1971). Zygosaccharomyces bailii has been isolated from spoiled salad dressing and mayonnaise (Couto, Hartog, Veld, Hofstra, & vanderVossen, 1996) Q3 and can grow in products with a pH of 3.6 and water activity of 0.89 (Meyer, Grant, Luedecke, & Leung, 1989). Typically the high acidity (pH < 4.5) of salad dressings is relied on to prevent the growth of spoilage and pathogenic bacteria (Smittle, 2000). ...
... Several approaches based on nucleic acids polymorphisms have been developed in an attempt to simplify yeast identifi cation, such as electrophoretic karyotyping, temperature gradient gel electrophoresis (TGGE), microsatellite PCR fingerprinting, random amplifi ed polymorphic DNA, ribosomal DNA (rDNA) restriction profi les and partial rDNA sequencing (Török et al.,1993;Baleiras-Couto et al., 1995;Baleiras-Couto et al., 1996;Guillamón et al., 1998;Kurtzman and Robnett, 1998;Esteve-Zarzoso et al., 1999;Hernán-Gómez et al., 2000;Esteve-Zarzoso et al., 2003;Baleiras-Couto et al., 2005;Rodriguez et al., 2010). ...
... Molecular biology based methods have been described for the rapid identifi cation of yeast species. Most of these methods such as RAPD, PCR fi ngerprinting and restriction enzyme analysis of rDNA fragments rely on PCR amplifi cation (Baleiras Couto et al., 1995, 1996, 2005. Nevertheless, these methods require previous isolation and cultivation of single cultures which is very laborious and time consuming. ...
A fermentação alcoólica do mosto de uva é um processo dinâmico que envolve diversas populações de leveduras que desempenham um papel importante nas características do vinho. A fim de permitir um controle permanente pelo enólogo urge implementar metodologias rápidas e de baixo custo, para identificação das espécies de leveduras envolvidas na fermentação. O método de Polimorfismo de Conformação do DNA de Cadeia Simples (SSCP) visando os domínios D1 e D2 da região 26S do rDNA foi testado para diferenciar espécies de levedura associadas ao vinho. Foram determinados perfis de SSCP para 17 estirpes de coleção pertencentes a 15 espécies diferentes associadas a ambientes vínicos. A técnica foi depois testada para a identificação de leveduras inoculadas em mosto estéril utilizando uma única espécie ou em misturas de duas espécies diferentes. Foram obtidos perfis idênticos de SSCP a partir de leveduras cultivadas em mosto de uva e em meios de cultura convencionais. A análise de SSCP permitiu a obtenção de bandas específicas de cada espécie, e os perfis de SSPC obtidos a partir de mosto de uva inoculado com misturas de duas estirpes de espécies diferentes revelou a presença de bandas específicas de ambas as espécies. A análise direta por SSCP das leveduras presentes numa fermentação espontânea, realizada após 48 horas e 192 h de fermentação, permitiu comparar a comunidade de leveduras em dois períodos diferentes de fermentação. Os perfis de SSCP revelaram a presença de bandas de espécies associadas com o início e com o fim da fermentação, respetivamente às 48 e às 192 h de fermentação. Em conclusão, a análise SSCP é uma metodologia bastante promissora para monitorizar as populações de leveduras presentes durante a fermentação, que se mostrou ser de fácil aplicação e de baixo custo.
... However, before such techniques could be used for the detection of specific organisms, those microorganisms must be identified for each type of product and their effect on spoilage characteristics must be determined. 55,56 An alternative way is to pinpoint the specific microbial metabolite formed during the metabolism/growth of the specific microorganism causing spoilage or off-flavour in the food product. 57 Formation of such metabolite could be followed upon isolation by sensitive instrumentation, such as high performance liquid chromatography/mass spectrometry (HPLC/MS) or gas chromatography± olfactometry (GCO) and gas chromatography/mass spectrometry (GC/MS). ...
Before attempting to understand shelf-life of foods, it is important to realize that foods are diverse, complex and active systems in which microbiological, enzymatic and physicochemical reactions are simultaneously taking place. These reactions have major consequences in relation to flavor, texture and shelf-life. Food preservation is dependent on the understanding of mechanisms of these reactions and the successful limitation of those most responsible for loss or spoilage of desirable characteristics and sometimes the channeling of other reactions towards beneficial changes. Essentially, the shelf-life of a food can be defined as the period for which it will retain an acceptable level of eating quality, from a safety and sensory point of view. There are four critical factors in this endeavor: · Formulation · Processing · Packaging · Storage conditions. All four factors are critical but their relative importance depends on the food. An understanding of the interplay between these factors is key to shelf-life estimation and testing. For example, a change in a single processing parameter may lead to undesirable chemical or physical changes in a product, or it may require reformulation or a change in packaging in order to attain the required shelf-life. Similarly, the very act of processing may subject the formulated materials and ingredients to conditions that are unfavorable or inhibitory to 9
... Molecular biology based methods have been described for the rapid identifi cation of yeast species. Most of these methods such as RAPD, PCR fi ngerprinting and restriction enzyme analysis of rDNA fragments rely on PCR amplifi cation (Baleiras Couto et al., 1995, 1996, 2005). Nevertheless, these methods require previous isolation and cultivation of single cultures which is very laborious and time consuming. ...
Alcoholic fermentation of grape must is a dynamic process involving diverse yeast populations which play an important role in wine characteristics.
In order to enable a permanent control by the oenologist rapid and low cost methodologies to identify the species of yeasts present during
fermentation should be implemented. Single Strand Conformation Polymorphism technique (SSCP), targeting D1 and D2 domains of 26S rDNA,
was tested to differentiate wine yeast species. SSCP profi les were produced for 17 collection strains belonging to 15 different wine related yeast
species. The technique was further investigated for the identifi cation of yeasts inoculated to sterile grape must as single or in mixtures of two
different species. Identical SSCP profi les were obtained from yeasts grown in grape must and in conventional growth media. SSCP allowed
obtaining species specifi c bands, and SSPC profi les from grape must inoculated with mixtures of two strains revealed the presence of specifi c
bands of both species. Direct SSCP yeast analysis of a spontaneous fermenting must carried out after 48 and 192 h of fermentation, enabled to
compare the yeast community at two different periods of fermentation. SSCP profi les showed the presence of bands from species associated
with the beginning and the end of fermentation, respectively at 48 and 192 h of fermentation. We conclude that SSCP is a quite promising
methodology to monitor yeast populations present during grape must fermentation, which revealed to be easy to implement and low-priced.
... However, this technique is highly time-consuming. Several approaches based on nucleic acids polymorphisms have been developed in an attempt to simplify yeast identifi cation, such as electrophoretic karyotyping, temperature gradient gel electrophoresis (TGGE), microsatellite PCR fingerprinting, random amplifi ed polymorphic DNA, ribosomal DNA (rDNA) restriction profi les and partial rDNA sequencing (Török et al.,1993; Baleiras-Couto et al., 1995; Baleiras-Couto et al., 1996; Guillamón et al., 1998; Kurtzman and Robnett, 1998; Esteve-Zarzoso et al., 1999; Hernán-Gómez et al., 2000; Esteve-Zarzoso et al., 2003; Baleiras-Couto et al., 2005; Rodriguez et al., 2010). Nowadays, innovative wine yeast identifi cation techniques such as DGGE (Denaturing Gradient Gel Electrophoresis) on PCR amplifi ed rRNA genes, FISH (Fluorescence in situ Hybridization), real time quantitative PCR (qPCR) and next-generation DNA sequencing can enable the quantifi cation and/or to monitor yeast dynamics throughout the fermentation process (Hierro et al., 2007; Mardis, 2008; Salinas et al., 2009; Tessonniere et al., 2009; Zott et al., 2010). ...
... Several approaches based on nucleic acids polymorphisms have been developed in an attempt to simplify yeast identifi cation, such as electrophoretic karyotyping, temperature gradient gel electrophoresis (TGGE), microsatellite PCR fingerprinting, random amplifi ed polymorphic DNA, ribosomal DNA (rDNA) restriction profi les and partial rDNA sequencing (Török et al.,1993;Baleiras-Couto et al., 1995;Baleiras-Couto et al., 1996;Guillamón et al., 1998;Kurtzman and Robnett, 1998;Esteve-Zarzoso et al., 1999;Hernán-Gómez et al., 2000;Esteve-Zarzoso et al., 2003;Baleiras-Couto et al., 2005;Rodriguez et al., 2010). ...
The complex microbial ecosystem existing in grape, must and wine comprises a wide diversity of yeast species. The knowledge of composition
and dynamics of yeast biota occurring along vinifi cation process would provide a better control of wine quality. The sequence of D1/D2 domain
of 26S ribosomal DNA (rDNA), refl ects ascomycetous yeast phylogenetic relationships and enables their separation at the species level. A region
of the 26S rDNA, with around 1100 bp comprising domain D1/D2, was amplifi ed by PCR and then digested with restriction endonucleases
(ApaI, HinfI, MseI, HaeIII and CfoI) in order to differentiate yeast species frequently isolated from grape surfaces, wine and cellar equipments.
A total of 78 yeast strains (including 36 type strains) belonging to 53 species were used to generate the restriction profi les. Numerical analysis
of the profi les generated by the fi ve restriction enzymes enabled to group the strains in 47 different clusters and 42 of them clearly corresponded
to different yeast species. The remaining groups comprise closely related species. The enzymes MseI, HaeIII and CfoI revealed a high
discrimination power and the restriction profi les generated were suffi cient to clearly identify the 42 species mentioned above. Despite one of
the clusters included different yeast genera, with different wine characteristics, the common wine spoilage yeasts Zygosaccharomyces bailii
and Z. lentus could be separated to one distinctive cluster through the use of ApaI restriction profi les. Since the analysis of restriction profi les
of amplifi ed 26S rDNA showed to be a valuable method to identify oenological yeast species, a database comprising the majority of wine yeast
biota was created to be applied both at research and industrial environment.
... More anecdotal reports exist on the occurrence of Y. lipolytica in a broad range of food products other than those mentioned so far (for overviews, see Deak & Beuchat, 1987, 1996Fleet, 1992;Heard & Fleet, 2000;Sinigaglia et al., 1994). For instance, Y. lipolytica has been detected in other fermented food products, such as tempeh and sourdough (Romano et al., 2006;Samson et al., 1987), in soft drinks, juices, wine, must and cider (Stratford, 2006), in fruits, fruit concentrates, vegetables and raw plant materials (Sancho et al., 2000), in mayonnaise, salad dressings and salads (Baleiras Couto et al., 1996), as well as in chilled and frozen, processed food, including seafood. All these examples suggest that Y. lipolytica may be a regular, although probably minor, component of food microbiota. ...
Abstract Yarrowia lipolytica has been developed as a production host for a large variety of biotechnological applications. Efficacy and safety studies have demonstrated the safe use of Yarrowia-derived products containing significant proportions of Yarrowia biomass (as for DuPont's eicosapentaenoic acid-rich oil) or with the yeast itself as the final product (as for British Petroleum's single-cell protein product). The natural occurrence of the species in food, particularly cheese, other dairy products and meat, is a further argument supporting its safety. The species causes rare opportunistic infections in severely immunocompromised or otherwise seriously ill people with other underlying diseases or conditions. The infections can be treated effectively by the use of regular antifungal drugs, and in some cases even disappeared spontaneously. Based on our assessment, we conclude that Y. lipolytica is a "safe-to-use" organism.
http://informahealthcare.com/doi/pdf/10.3109/1040841X.2013.770386
... More anecdotal reports exist on the occurrence of Y. lipolytica in a broad range of food products other than those mentioned so far (for overviews, see Deak & Beuchat, 1987, 1996Fleet, 1992;Heard & Fleet, 2000;Sinigaglia et al., 1994). For instance, Y. lipolytica has been detected in other fermented food products, such as tempeh and sourdough (Romano et al., 2006;Samson et al., 1987), in soft drinks, juices, wine, must and cider (Stratford, 2006), in fruits, fruit concentrates, vegetables and raw plant materials (Sancho et al., 2000), in mayonnaise, salad dressings and salads (Baleiras Couto et al., 1996), as well as in chilled and frozen, processed food, including seafood. All these examples suggest that Y. lipolytica may be a regular, although probably minor, component of food microbiota. ...
Yarrowia lipolytica has been developed as a production host for a large variety of biotechnological applications. Efficacy and safety studies have demonstrated the safe use of Yarrowia-derived products containing significant proportions of Yarrowia biomass (as for DuPont's eicosapentaenoic acid-rich oil) or with the yeast itself as the final product (as for British Petroleum's single-cell protein product). The natural occurrence of the species in food, particularly cheese, other dairy products and meat, is a further argument supporting its safety. The species causes rare opportunistic infections in severely immunocompromised or otherwise seriously ill people with other underlying diseases or conditions. The infections can be treated effectively by the use of regular antifungal drugs, and in some cases even disappeared spontaneously. Based on our assessment, we conclude that Y. lipolytica is a "safe-to-use" organism.
... However, bacterial contamination is a problem and improved methods for characterization of contaminations as well as culture independent methods are needed. Various methods using DNA have been developed for the detection of specific microbes in foods (BaleirasCouto, Hartog, Huisin't Veld, Hofstra, & Van der Vossen, 1996;Caggia, Restuccia, Pulvirenti, & Giudici, 2001;Cocolin, Aggio, Manzano, Cantoni, & Comi, 2002;Lieckfeldt, Wieland, & Börner, 1993;López, Querol, Ramón, & Fernández-Espinar, 2001;Manavathu, Vakulenko, Obedeanu, & Lerner, 1996). Among these methods, polymerase chain reaction-based assays (PCR-based methods) are specific, can be extremely sensitive and results are obtained in a few hours. ...
... Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) are fast, reliable methods for molecular characterization of yeasts [10,16]. They are reproducible [7], have possible industrial applications [14] and are useful for characterizing yeasts by species and strains [9]. The region between the 18S rRNA and 28S rRNA genes can be ampli¢ed using speci¢c internal transcribed spacers ITS1 and ITS4 primers. ...
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.
... However, bacterial contamination is a problem and improved methods for characterization of contaminations as well as culture independent methods are needed. Various methods using DNA have been developed for the detection of specific microbes in foods (BaleirasCouto, Hartog, Huisin't Veld, Hofstra, & Van der Vossen, 1996; Caggia, Restuccia, Pulvirenti, & Giudici, 2001; Cocolin, Aggio, Manzano, Cantoni, & Comi, 2002; Lieckfeldt, Wieland, & Börner, 1993; López, Querol, Ramón, & Fernández-Espinar, 2001; Manavathu, Vakulenko, Obedeanu, & Lerner, 1996). Among these methods, polymerase chain reaction-based assays (PCR-based methods) are specific, can be extremely sensitive and results are obtained in a few hours. ...
... Characteristic banding profiles for (GTG) 5 were also found in strains of Fusarium solanum (Brasileiro et al., 2004), allowing the discrimination of all isolates. Similarly, Baleiras Couto et al. (1996) and Meyer et al. (1997) The dendrogram produced using the ISSR data showed a main group containing exclusively A. flavus strains, although divided into two subgroups with a similarity level around 80% (Figure 4). The first subgroup was formed only by A. flavus strains isolated from different substrates in Pernambuco State, which means a high genetic similarity according to the geographical origin. ...
The Aspergillus genus belongs to a filamentous fungal group characterized by wide dispersion in the environment. Some species are associated with diseases, especially in immunocompromised patients, while others are of economical importance due to aflatoxin production or biotechnological applications. Its species identification is nowadays performed by traditional techniques combined with molecular markers, resulting in a higher efficiency of isolate characterization. In the present study, internal transcribed spacer, inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of Aspergillus flavus and strains of other species of the A. flavus group. High genetic diversity was revealed by RAPD and by ISSR, in which the use of the (GACA)4 primer yielded a higher diversity than with the (GTG)5 primer, although the latter showed a characteristic banding profile for each species. These data were used to create a similarity matrix for the construction of dendrograms by means of the UPGMA method. The ISSR and RAPD profiles showed that among the strains previously identificated as A. flavus, one should be A. oryzae, one A. parasiticus and two A. tamarii. On the other hand, a strain previously identified as A. parasiticus should be A. flavus. All these strains were retested by traditional methods and their new species identification was confirmed. These results strongly support the need for using molecular markers as an auxiliary tool in differentiating fungal species and strains.
... PCR amplification with simple repeat primers such as (GAC) 5 (GTG) 5 and M13 core sequence proved to be a useful method for the discrimination at species level of strains of the genus Saccharomyces (Lieckfeldt et al. 1993) or of common food spoilage yeasts (Baleiras Couto et al. 1996). Applicability of M13 primer for the identification of yeast species is confirmed by this work, in which PCR analysis with primers M13 and RF2 allowed the assignment to the species level of 42 of 48 strains isolated from different cheeses. ...
In the present work randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers M13 and RF2 was applied to the identification at species level of yeast strains isolated from cheeses. RAPD-PCR analysis of the type strains of different yeast species gave distinctive band profiles that allowed a clear differentiation of all the considered species. Forty-two of the 48 dairy associated yeasts were clearly assigned to the species Saccharomyces cerevisiae, Kluyveromyces marxianus (anamorph Candida kefyr), Kluyveromyces lactis (anamorph Candida sphaerica), Debaryomyces hansenii (anamorph Candida famata), Yarrowia lipolytica and Torulaspora delbrueckii (anamorph Candida colliculosa). The method, which is rapid and easy to perform, could be a useful tool for the identification of yeasts present in dairy products.
... Nowadays, with the advances in molecular biology, a number of DNA-based analysis methods that are unaffected by culture conditions have emerged and are proving to be extremely useful in taxonomic studies and in distinguishing between strains of the same species. The most commonly used methods of that type are pulsed ¢eld gel electrophoresis of whole chromosomes [4] [5], restriction fragment length polymorphism (RFLP) analysis [6], probe-based detection of speci¢c DNA sequences (`Southern blotting') [7], ampli¢cation of certain rDNA regions followed by RFLP [8], random ampli¢cation of polymorphic DNA [9], and ampli¢cation of microsatellite regions [10]. DNA fragments of the same length but with di¡erent nucleotide sequences are separated on polyacrylamide gels using temperature gradient gel electrophoresis (TGGE) as a result of di¡ering electrophoretic mobilities caused by partial denaturing along a linear temperature gradient [11]. ...
18S rDNA from 74 wine yeast strains was amplified by PCR using specific primers, and the products analyzed by temperature gradient gel electrophoresis (TGGE). TGGE is a useful method in screening the genotypes of the wine yeasts. Intraspecific differentiation was achieved on the basis of TGGE in some cases, whereas in others identical bands for strains classified as separate species were obtained. Heteroduplex analysis was capable of differentiating between similar bands produced by two different species, thereby enhancing the resolution of the TGGE, yielding valuable information in a short time without the need of sequencing or complicated equipment.
The trends toward healthy living, vegetarianism, and busy schedules have increased salad popularity. Salads are usually consumed raw without any thermal treatment, and therefore, without proper care they can become major vehicles for foodborne illness outbreaks. This review examines the microbial quality of 'dressed' salads which contain two or more vegetables/fruits and salad dressings. The possible sources of ingredient contamination, recorded illnesses/outbreaks, and overall microbial quality observed worldwide, besides the antimicrobial treatments available are discussed in detail. Noroviruses were most frequently implicated in outbreaks. Salad dressings usually play a positive role in influencing microbial quality. However, this depends on several factors like the type of contaminating microorganism, storage temperature, dressing pH and ingredients, plus the type of salad vegetable. Very limited literature exists on antimicrobial treatments that can be used successfully with salad dressings and 'dressed' salads. The challenge with antimicrobial treatments is to find ones sufficiently broad in spectrum, compatible with produce flavour which can be applied at competitive cost. It is evident that renewed emphasis on prevention of produce contamination at the producer, processor, wholesale and retail levels plus enhanced hygiene vigilance at foodservice will have a major impact on reducing the risk of foodborne illnesses from salads.
Yeasts are single celled fungi which reproduce vegetatively by budding, or less commonly, divide by fission. This property enables yeasts to increase rapidly in numbers in liquid environments, which favour the dispersal of unicellular microorganisms. Many yeasts grow readily under strictly anaerobic conditions, again favouring their growth in liquids. On the other hand, reproduction as single cells restricts spreading on, or penetration into, solid surfaces, where filamentous fungi have an advantage. Being eukaryotic organisms, yeasts reproduce more slowly than do most bacteria, and hence do not compete in environments which favour bacteria, i.e. at pH values near neutral or at very high temperatures. In common with filamentous fungi, many yeasts are tolerant of acid conditions. In broad terms, then, yeasts are more likely to be active in acidic, liquid environments than elsewhere. However, many yeasts also appear to be highly resistant to sunlight and desiccation and so occur widely in nature on the surfaces of leaves, fruits and vegetables.
Halotolerant yeasts represent a heterogeneous group of unicellular fungi able to survive and thrive under hypersaline conditions. This review examines the biodiversity of halotolerant yeasts in various habitats with high salt content and the potential practical applications of this group of microorganisms in industry and agriculture. Halotolerant yeasts are found in various habitats with elevated salt content, including seawater, hypersaline ponds and salterns, saline soils and wastewaters, salt-containing foods. Habitats with moderate salinity, e.g. seawater, food products, olive fermentation wastewaters can boast a comparatively large biodiversity of yeasts both ascomycetes and basidiomycetes. Hypersaline niches are mostly inhabited by pigmented and melanized yeasts and yeast-like fungi. The adaptability and robustness of halotolerant yeasts could be exploited in several biotechnological fields, mainly the food industry and bioremediation. Yeasts isolated from food products with elevated salt content are studied as potential starter cultures in the corresponding fermenting products due to their enzymatic and antimicrobial activity and probiotic characteristics. Marine yeasts are of an increasing interest due to their production of various hydrolytic enzymes, biofuel production using seawater, bioremediation of saline wastewaters and the probiotic potential in aquaculture. Halotolerant yeasts found in various saline wastewaters could be used in bioremediation of wastewaters with high salinity containing various organic pollutants. However more research is required to achieve practical utilization of this group of microorganisms.
Probabilistic microbial modelling using logistic regression was used to predict the growth/no growth (G/NG) interfaces of Zygosaccharomyces bailii in simulated acid sauces as a function of natamycin, xanthan gum (XG) and sodium chloride concentrations. The growth was assessed colorimetrically by using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride and 2-methoxy-1,4-naphthoquinone as detection reagents. The logistic regression model successfully predicted G/NG probability. The detection reagents used allowed the evaluation of G/NG interfaces in opaque systems with an excellent agreement with the plate count method. Natamycin concentration of 12 mg/L was needed to inhibit Z. bailii growth independently of the presence of XG and/or NaCl. Addition of 3.00 and 6.00% of NaCl exerted an antagonistic effect on natamycin action. Furthermore, addition of 0.25 and 0.50% XG decreased natamycin and/or NaCl action. However, an increased in XG concentration to 1.00% decreased yeast growth. Mentioned results highlighted the importance of the correct selection of stress factors applied to inhibit Z. bailii growth.
The first and second editions of Fungi and Food Spoilage established a reputation as the foremost book on foodborne fungi. This completely revised and updated third edition is an invaluable reference for food microbiologists investigating fungal spoilage and sources of mycotoxin contamination in foods. The introductory chapters of the book deal with the ecology of food spoilage and give an overview of how food processing, packaging and storage affect fungal growth. Subsequent chapters cover the fundamentals of classifying and naming fungi and current methods for isolation and enumeration, including general and special purpose media, incubation conditions, etc. The major part of the book provides keys, descriptions and illustrations of all yeasts and moulds commonly encountered in foods. Characteristics of the species, including their ecology and potential for mycotoxin production, are also included. The broad and practical nature of the coverage will appeal to microbiologists, mycologists and biotechnologists in the food industry, academic, research and public health institutions. Dr John Pitt and Dr Ailsa Hocking are both Honorary Research Fellows at CSIRO Food Science Australia, North Ryde, NSW, Australia. © Springer Science+Business Media, LLC 2009. All rights reserved.
Background and Aims: Microbiological stability of bottled wine is largely dependent on the bottling process as the last technological operation. In this survey, yeast contamination was monitored and characterised during wine bottling.
Methods and Results: Samples of wine, apparatus surfaces and bottles were collected. Yeast counts were recorded and colony morphotypes were differentiated. Isolates were characterised by restriction enzyme digests of polymerase chain reaction amplified regions of 26S ribosomal DNA. For unknown patterns, DNA sequencing was performed. The presence of Zygosaccharomyces bailii and Torulaspora delbrueckii in the filling tubes was also confirmed in the final product. Another important point of contamination was the corker jaws.
Conclusions: A great diversity of yeast species was found, indicating that these habitats are prone to microbial development.
Significance of the Study: Evaluation of the level and diversity of yeast contamination during wine bottling allowed to alert producers for the need to evaluate and control microbial contamination.
Foods and drinks with high solute concentration (fruit juices and concentrates, marzipan, salted and dry-cured meats, olives and cheeses), and foods containing preservatives (wine, beverages and sauces) were selected in order to characterise the contaminant yeast flora by rapid typing techniques. These included the testing of several types of molecular methods (e.g. RFLP, DNA-fingerprinting, PCR-based techniques), analysis of long-chain fatty acids and of isoenzymes. The PCR-based detection methods enabled a faster detection of emerging specific spoilers at earlier stages of processing. Fatty acid characterisation allowed the assessment of the most frequent types of contamination yeasts and supplied the information for the definition of relevant zymological indicators. A selected group of strains was used for further studies of mechanisms underlying the resistance/tolerance of yeasts towards preservatives (weak acids) and other stress factors (temperature, high sugar and salt concentrations). This study enabled the acquisition of data on the basic biology of yeasts used in the development of differential and selective media for Zygosaccharomyces bailii, Yarrowia lipolytica, Kluyveromyces marxianus and Dekkera sp.
This chapter discusses the use of fungal volatiles and molecular techniques, that is, methods based on nucleic-acid sequences or antigens, to detect the presence of toxigenic food-borne fungi. The determination of the number of colony forming units on general or selective agar substrates remains the standard method in food microbiology. Although reasonably well suited to quantify numbers of viable unicellular organism, that is, bacteria and yeast, it is not well adapted for the quantification of filamentous fungi and results of fungal colony-forming units (CFU) determinations are highly influenced by the degree of fungal sporulation. The use of selective substrates for various ecological groups can increase the precision and accuracy of CFU determinations. Fungi produce volatile compounds, both during primary and secondary metabolism. Volatile metabolites from mainly Aspergillus, Fusarium, and Penicillium spp. have been characterized with gas chromatography (GC), mass spectrometry, and sensory analysis and can be used for detection and identification. Common volatiles are 2-methyl-1-propanol, 3-methyl-1-butanol, 1-octen-3-ol, 3-octanone, 3-methylfuran, ethyl acetate, and the malodorous 2-methylisoborneol and geosmin.
Food processing is concerned with the proper conversion of raw materials to value added end products fit for consumption. During the process, microbiological contamination of raw materials has to be inactivated and re-contaminat ion during processing or upon product storage has to be prevented.
In this chapter, we stress the need for active research and surveillance of the ecology and molecular biology of food spoilage yeasts. We briefly highlight the main developments in the area. Subsequently, the research geared to identifying new anti-yeast targets and the understanding of stress resistance using classical approaches is discussed. Most important is the discussion of sorbic acid resistance, the weak-organic acid food preservative used in the majority of cases. We discuss the evidence in favour of an involvement of ATP driven acid and anion extrusion pumps in resistance development, but we also highlight the incompleteness of the current model. Modern approaches using genome-wide screening of stress responses offer new tools for an assessment of the full picture of stress reactions. We indicate examples of this and discuss the bioinformatics methods that go with genomics experiments in order to extract the appropriate knowledge from the data. Yeast stress physiology, genetics and ‘functional genomics’, is put in a perspective of future research and challenges.
Consumers in developed countries have a more critical attitude about what they eat and drink as a consequence of modern life. Food microbiologists are facing a huge challenge regarding food ‘freshness' implicit in the consumer's demand for more natural products. Food with less severe processing, that is additive-free, safer, with satisfactory shelf life and easy to prepare is sought because of higher consciousness about nutrition and health. Besides the development of new methods and preservation concepts, great advances in the fields of chemistry, biochemistry, microbiology, hygiene and food safety have occurred in recent decades. But are such developments properly used to respond to the challenge to food mycology? We will summarize the effects on food of yeast spoilage activity and we will reflect on how this activity can be monitored by food microbiologists.
A rapid assay for detection of yeast species in vacuum packed ham has been developed based on the polymerase chain reaction (PCR) coupled to a 24 h pre-enrichment at 25 °C. DNA was isolated from yeast inoculated ham samples and amplified using primers specific for the 18S rRNA gene sequences of yeasts. A detection limit of 10(2) CFU/cm(2) was achieved following enrichment of samples experimentally inoculated with three yeast species frequently associated with meat products spoilage: Debaryomyces hansenii, Yarrowia lipolytica, and Kluyveromyces marxianus. Likewise, commercial sliced and vacuum packed ham samples were analysed using the PCR-culture technique. The results obtained in this work show that PCR amplification of a conserved region of the 18S rRNA gene in the yeast species could be potentially used as a rapid tool for detection of low levels of viable spoilage yeasts in meat products.
The efficiency of mitochondrial DNA (mtDNA) restriction analysis and random amplification of polymorphic DNA (RAPD)-PCR to characterize yeasts growing on dry-cured Iberian ham was evaluated. Besides, the distribution of the main species and biotypes of yeasts in the different ripening areas of this product was investigated. MtDNA restriction analysis allowed yeast characterization at species and strain level. RAPD-PCR with the primers (GACA)(4) and (GAC)(5) was inappropriate for characterization at species level. Most of the mtDNA restriction patterns detected in dry-cured Iberian ham were consistent with Debaryomyces hansenii. Several yeasts biotypes were associated to specific geographic areas of dry-cured Iberian ham ripening.
The rapid identification of spoilage microorganisms is of eminent importance to the food industry. It provides the food industry with the opportunity to reduce economical losses by designing adequate intervention measures. The use of identification systems based on biochemical and physiological characteristics resulted often in disappointing identification results and misidentifications. This will inevitably lead to inappropriate strategies to prevent spoilage. This review discusses the potential of the DNA based identification technology including the polymerase chain reaction (PCR) for the identification and specific detection of microorganisms. Fingerprinting methods based on the DNA-probe technology enable a clear insight in the identity of microorganisms on different levels, varying from genus to strain level depending on the systems used. Discrimination between subspecies and strain level is shown to be helpful for investigating routes and sources of contamination. Differentiation at the species level is demonstrated to be essential in order to design a highly specific detection system enabling to signalize a microorganism that belongs to a particular species. Also indicated in this review is the necessity and the technical approach to detect microorganisms that display a particular undesirable trait.
Twenty-eight yeast strains presumed to represent Torulaspora delbrueckii were analyzed by randomly amplified polymorphic DNA-PCR analysis. Four strains (HUT 7161, IFO 1138, IFO 1145, and IFO 1956) that were considerably different from the type strain were further investigated. Morphological and physiological characteristics revealed that strains HUT 7161 and IFO 1145 belong to the genus Debaryomyces rather than the genus Torulaspora, and the former strain may represent Debaryomyces hansenii. Strains IFO 1138 and IFO 1956 were classified as either Saccharomyces castellii or Saccharomyces dairensis by identification keys involving physiological tests. On the basis of analysis of the sequences of two rRNA internal spacer regions, strains IFO 1138 and IFO 1956 were closely related to S. castellii and strains HUT 7161 and IFO 1145 were outside members of the genera Torulaspora, Zygosaccharomyces, and Saccharomyces.
The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and
a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous
expression of this toxin inAspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into
the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum
of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone
to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic
spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher
lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence
of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin
HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.
Zygosaccharomyces bailii is an effective spoiler of many foods and beverages, particularly those with low pH and high sugar content. The characteristics that contribute to this virulence are discussed in relation to product composition and process technology. Natural habitats and major sources of infection are identified and the need for suitable quality control methods emphasized.In addition, some desirable characteristics are discussed that may be exploited, in the future, by the food and other industries.
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.
Four problematic areas associated with the identification of foodborne yeasts are discussed. These consist of (1) the inability of conventional identification tests to recognize some common and important foodborne yeasts characterized by genomic differences (e.g., Saccharomyces cerevisiae, S. bayanus and S. pastorianus); (2) the delay in application of non-traditional identification methods such as DNA fingerprinting, chromosome karyotyping, protein electrophoretic patterns and fatty acid profiles for routine identification purposes; (3) the lack of commercially available manual or automated identification systems dedicated to the diagnosis of foodborne yeasts; and (4) the disregard for considering ecological frequency of yeasts in computerized probabilistic identification systems.
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.
A simplified identification key described by Deak and Beuchat (T. Deak and L. R. Beuchat, J. Food Prot. 50:243-264, 1987) and the computer method of Barnett et al. (J. A. Barnett, R. W. Payne, and D. Yarrow, Yeast Identification Program, 1985) were used to identify 12 reference strains and 382 yeasts isolated from cultured milk products. Because the simplified key failed to account for species variability with regard to physiological, morphological, and sexual reproduction characteristics, poor agreement of the identification results was obtained. A reevaluation of the basic theoretical assumptions of the simplified key only confirmed the practical results and indicates that this identification method is unsatisfactory.
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