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1.
SCREENING AND EVALUATION OF ENZYMATIC ACTIVITIES OF WINE RELATED YEAST SPECIES
Adesida Rowland, Melita Sternad Lemut, Lorena Butinar, 2020, published scientific conference contribution abstract

Abstract: A number of indigenous yeast strains isolated from vineyard / cellar-associated samples and non-vineyard sites, from forests with oak trees (a known habitat where Saccharomyces species resides) from existing in-house yeast collection and from ZIM culture collections (Slovenia) were screened for enzymatic activities with enological importance. The yeasts were screened for glycosidase enzymes connected to terpene aroma release, the β-lyase activity responsible for the volatile thiol release, and sulfite reductase activity involved in off-flavours. Yeast strains that showed positive results for qualitative and quick detection of glucoside hydrolase activity on plates were selected for further studies. The glucoside hydrolase activity of 20 selected strains belonging to 15 different species was quantitatively characterized by determinations of the activity on pNPG. All strains demonstrated hydrolase activity, especially strain H. uvarum 116 which showed the highest value of specific activity 6.32 mU/mg for cell-associated activity and T. delbrueckii Sut 94 with highest value of specific activity 1.36 mU/mg for extracellular activity. Increased growth of tested yeast on medium containing substrate S-methyl-L-cysteine indicated β-lyase activity, and by this approach a moderate activity was recorded throughout our tested strains with immense intraspecific inconsistency. Noticeable H2S production was observed in P. manshurica strain whereas, H. uvarum, L. thermotolerance, S. bayanus and S. cerevisiae demonstrated weak/slight H2S-producer strains in our study. Therefore, our screening indicates the importance of strain selection for enological application due to intraspecies differences, as well as the introduction of non-Saccharomyces yeast starters with interesting potential to enhance wine aroma.
Keywords: wine yeasts, non-Saccharomyces, enzymatic activities
Published in RUNG: 08.05.2023; Views: 944; Downloads: 0
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2.
Cider yeasts associated with Hardanger cider during fermentation process
Urban Česnik, Mitja Martelanc, Branka Mozetič Vodopivec, Ingunn Ovsthus, Lorena Butinar, 2022, published scientific conference contribution abstract

Abstract: In the Hardanger area in Western Norway, the production of cider has a long tradition that goes back to the 12th century, when monks introduced apple growing in this area. Nowadays, this is also the main area of fruit production in Norway. Despite the strict regulation of the alcoholic beverage production in Norway, traditional cider is still produced on some farms in this area. Therefore, our aim was to study the ecology and biodiversity of the yeasts associated with the cider production in the Hardanger area during fermentation process; especially of traditional cider, which is produced by a spontaneous fermentation of apple juice, performed by naturally occurring indigenous yeasts that originate from the fruit or the surfaces of the processing equipment. In our study, samples of fermenting juice/cider were taken during fermentation process from 12 producers, located in 12 different locations in Hardanger region. Classical cultivation methods using WL (Wallerstein Laboratories) agar medium with added chloramphenicol enable us to isolate a total of 530 yeast isolates that were stored in in-house yeast collection at the NIBIO and included also at the Wine Research Centre collection. Based on the sequencing of the D1/D2 domain of the 26S rDNA we managed to identify 357 isolates and distinguished 27 different yeast species as follows: Aureobasidium pullulans, Candida californica, C. oleophila, C, sake, Hanseniaspora meyeri, H. uvarum, H. valbyensis. Kregervanrija fluxuum, Kregervanrija sp., Metschnikowia andauensis, M. chrysoperlae, M. fructicola, M. pulcherrima, Metschnikowia sp, Pichia fermentans, P. kluyveri, P. membranifaciens, P. nakasei, Piskurozyma capsuligena, Rhodotorula nothofagi, Saccharomyces bayanus, S. cerevisiae, S. paradoxus, S. pastorianus, Saccharomyces sp., S. uvarum and Torulaspora delbrueckii. Even though we were not able to obtain samples in three different fermentation stages (beginning, middle and at the end of fermentation) from all producers, we could observe yeast succession during fermentation progress. Yeast diversity was higher at the beginning comparing to the middle of fermentation, when mostly different non-Saccharomyces yeast species prevailed, while in the middle of fermentation 11 species were detected (Candida californica, H. uvarum, H. valbyensis, Kregervanrija sp., K. fluxuum, Pichia membranifaciens, Metschnikowia pulcherrima, Saccharomyces sp, S. bayanus, S. uvarum and S. cerevisie). On the other hand, at the end of fermentation mainly Saccharomyces species with high ethanol tolerance were present (Saccharomyces sp., S. cerevisiae, bayanus, S. uvarum and P. fermentans). In samples that were collected from three producers in all three fermentation stages also quality parameters were determined (ethanol, organic acids, sugars, biogenic amines) with in-house developed methods using HPLC-UV/RID. The most important sugars in ciders were fructose and glucose, as expected. Two producers added sugar to increase the level of ethanol in the middle of fermentation, which is a common procedure in the Hardanger area. Ethanol and organic acid analysis indicated that fermentations went in the right direction, since all parameters were within normal limits. Including the acetic acid level, an indicator of low cider quality, was very low (average around 0,06 g/L). The alcohol incised from the beginning to end fermentation in all samples analysed and minimum concentration was 2,71 g/L. In ciders we detected four biogenic amines (putrescin, cadaverine, histamine and tyramine). The average amount was 32 mg/L and the most abundant was tyramine.
Keywords: indigenous yeasts, biodiversity, spontaneous fermentation, cider-making
Published in RUNG: 18.10.2022; Views: 1313; Downloads: 0
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3.
Exploration of yeast biodiversity potential for development of alternative biofungicides in viticulture : dissertation
Rowland Adesida, 2022, doctoral dissertation

Abstract: Botrytis cinerea Pers., the fungal plant pathogen and the causal agent of gray mould diseases in grapevine, is vastly responsible for substantial economic losses in table and wine grapes production worldwide by negatively affecting plant growth and causing the reduction of grape and wine quality. The conventional approach for pathogen control has been up to date based on synthetic fungicides with good effectiveness against pathogens but a negative impact on the environment. The growing level of harmful residues in the environment and some also detected in wines have led the European Union and many winemakers to limit the application of synthetic fungicides to earlier season. However, with a high risk of disease also late in the season, the need for other solutions is clear. Consequently, more and more research is focused on finding potential alternatives in the form of effective biological control agents. Although there are several reports of yeast’s biocontrol activity, they are up to date still poorly commercialized for such purposes. As the yeasts represent an important part of the grape microflora, competing with other microorganisms (including pathogens) for nutrients and space, we decided to examine the potential of autochthonous yeasts as "green" alternatives in fighting against phytopathogens such as B. cinerea in viticulture. With this aim, we tested the biocontrol activity of 119 different indigenous yeasts belonging to 30 different species of 17 genera against filamentous fungus B. cinerea, the causal agent of grey mould or botrytis bunch rot in grape. The yeasts were screened for putative multidimensional modes of action such as antifungal volatiles (VOC), in vitro inhibition of fungal mycelial growth, competition for nutrients, hydrolytic enzyme activities, and yeast tolerance to fungicides like copper, iprodione and cyprodinil/fludioxonil combination. With a qualitative detection of the hydrolysis activity by using screening methods based on solid medium with chitin or βD-glucosides as substrates, we found that many tested yeasts were capable of producing lytic enzymes with the ability to degrade the cell wall of phytopathogenic fungi and are potentially also able to produce VOCs via hydrolysis of grape glycosides as a result of β-glucosidase presence. Furthermore, we observed the capability of tested yeast to inhibit fungal mycelia growth on plate and assimilation of a wide variety of carbon sources; however, no siderophore producers were detected. In general, the yeasts under observation were tolerant to the tested fungicides. Their fungicide resistance can indeed be regarded as a beneficial trait for potential biofungicide agent (PBA) candidates due to open possibilities of applications and combinations within low input pest management strategies in the vineyard. Finally, a field experiment in Pinot noir and Pinot gris vineyards was designed to study different combinations of optimized canopy microclimate manipulation (CMM) techniques and potential biocontrol agent (PBA) application. In experimental conditions, the ability of PBA’s to maintain appropriate population density for disease prevention was observed. In addition, the grape and wine quality parameters were analysed to observe the possible impact of implemented biocontrol yeast on final products. The biocontrol yeast Pichia guilliermondii ZIM 624 was selected and applied in experimental vineyards based on yeast testing results. We were able to detect and confirm PBA yeast’s suitable density on grapes until harvest. In the case of early defoliation for both varieties lower grape compactness was observed together with lower yield/ plant, regardless of PBA yeast/ no yeast application. Among grape basic quality parameters, the optimized techniques showed a positive effect on sugar content. Still, unexpectedly, in the treatments with biocontrol yeasts some trends toward higher acidity were noticed in Pinot gris.
Keywords: sustainable viticulture, Botrytis cinerea, gray mould, yeasts, biocontrol, canopy microclimate manipulation, grapevine metabolite, dissertations
Published in RUNG: 07.07.2022; Views: 1919; Downloads: 137
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4.
Novel analytical approaches in quality and safety control in production of fermented beverages : dissertation
Jelena Topić, 2022, doctoral dissertation

Abstract: The exploitation of microorganisms for fermentation goes back centuries. Two types of fermentation are usually used in the winemaking process – alcoholic fermentation and malolactic fermentation. Nowadays, inoculated fermentations with the use of starter cultures are commonly used in order to produce wine with more consistent quality. However, wines can lack in flavour complexity, so scientists and the industry are constantly looking for new and improved starters that can be adapted to different types of wine. In this work we focused on the development and implementation of novel analytical methods for wine quality control. In the course of method development native yeasts and lactic acid bacteria isolates were characterized for wine starter properties. We focused on the determination of biologically active compounds that determine wine quality and safety. Yeasts can influence wine colour through their adsorption capacity and synthesis of stable colour pigments pyranoanthocyanins and lactic acid bacteria can produce biogenic amines which can have adverse detrimental health effects on sensitive consumers when they are present in wines.
Keywords: Saccharomyces yeasts, non-Saccharomyces yeasts, pyranoanthocyanins, thermal-lens spectrometry, lactic acid bacteria, biogenic amines, thin layer chromatography, dissertations
Published in RUNG: 18.02.2022; Views: 2757; Downloads: 123
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5.
6.
Implementation of high performance liquid chromatography coupled to thermal lens spectrometry (HPLC‑TLS) for quantification of pyranoanthocyanins during fermentation of Pinot Noir grapes
Jelena Topič Božič, Lorena Butinar, Natka Ćurko, Karin Kovačević Ganić, Branka Mozetič Vodopivec, Dorota Korte, Mladen Franko, 2020, original scientific article

Abstract: In this work high performance liquid chromatography coupled to thermal lens spectrometry (HPLC-TLS) was applied for monitoring of vinylphenolic pyranoanthocyanins formation during the fermentation of Pinot Noir wines. Vinylphenolic pyranoanthocyanins are wine pigments, present in low concentrations, but very important for wine colour stability. Fermentation process was conducted with four different yeast strains, used as starters, either in sequential fermentation of non-Saccharomyces with S. cerevisiae yeast or as single fermentation with S. cerevisiae yeasts in order to test the applicability of developed method for monitoring of selected compounds in real wine fermentation experiments. The developed HPLC-TLS method showed higher sensitivity compared to HPLC coupled to diode array detection (DAD) technique for particular wine colour compounds. Obtained limits of detection (LODs), were 6- and 22-times lower in comparison to HPLC–DAD in gradient and isocratic elution mode, respectively, whereas limits of quantification (LOQs) 5 and 18-times lower. Lower LODs enabled earlier observation of vinylphenolic pyranoanthocyanins formation during fermentation (already at day 7) in the case of HPLC-TLS method in gradient mode, while by using HPLC–DAD in gradient elution mode the formation of vinylphenolic pyranoanthocyanins was noticed only after 12 days of fermentation.
Keywords: Thermal lens spectrometry (TLS), High performance liquid chromatography (HPLC), Pyranoanthocyanins, Wine, Yeasts
Published in RUNG: 18.06.2020; Views: 3386; Downloads: 0
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7.
8.
Facilitating green care improvements in viticulture
Melita Sternad Lemut, Lorena Butinar, Adesida Rowland, Urban Česnik, Tjaša Jug, Paolo Sivilotti, Urška Vrhovšek, 2019, published scientific conference contribution

Abstract: The growing demand of consumers for environment-friendly and safe food products already forced many winemakers to restrict the application of synthetic fungicides to earlier season, trying to reduce the risk of residues being detected in wines. Main risk for infection by B. cinerea, however, occurs later in the season. To support an improvement in whole-season B. cinerea green care, the potential of yeasts as biocontrol agent (PBA) was studied. Selected yeasts were tested for their antifungal activities. Furthermore, a field experiment in Pinot noir and Pinot gris vineyards was designed to study different combinations of optimized canopy microclimate manipulation (CMM) techniques and PBA application. In experimental conditions, PBA's ability to maintain appropriate population density for disease prevention was observed. Grape and wine quality parameters were evaluated. Analytical approach of metabolomics was applied to upgrade the understanding of potentially induced changes in plant metabolism. Based on yeasts testing results, biocontrol yeast P. guilliermondii ZIM 624 was selected and after its field application, we were able to detect its suitable density on grapes until harvest. In case of early defoliation for both varieties smaller grape compactness was confirmed together with lower yield. Among grape basic quality parameters, optimized techniques showed positive effect on sugar content, but unexpectedly, in the treatments with biocontrol yeasts the trends toward higher acidity were noticed in Pinot gris. CMM and PBA introduction indicated some effects on grapevine biosynthetic behaviour but the differences in content of these metabolites compared to control were poorly detected in final wines with exception of mainly some flavonols, stilbenes and flavan-3-ols (and consequently total phenolic content).
Keywords: sustainable viticulture, Botrytis cinerea, yeasts, biocontrol, canopy microclimate manipulation, metabolomics, grapevine metabolites
Published in RUNG: 28.02.2020; Views: 3869; Downloads: 0
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9.
Biodiversity of cider yeasts and their cider-making potential
Eivind Vangdal, Melita Sternad Lemut, Branka Mozetič Vodopivec, Lorena Butinar, 2017, published scientific conference contribution abstract

Abstract: In the area of Hardanger, a part of the fjord region in Western Norway, the production of apple wine (cider) has a long tradition that goes back to the 12th century, when monks introduced apple growing in this area. Nowadays, this is also the main area of fruit production in Norway. Despite the strict regulation of the alcoholic beverage production in Norway, traditional cider is still produced on some farms in this area. By tradition cider is produced by a spontaneous fermentation process of apple juice, performed by naturally occurring indigenous yeasts that originate from the fruit or the surfaces of the processing equipment. Therefore, our aim was primarily to study the ecology and biodiversity of the yeasts associated with the production of traditional cider in the Hardanger area. For two consecutive years, we sampled at 11 different locations in the observed region, where we collected cider samples and surface swabs of processing facilities from the cideries, and also soil and various parts of apple trees in orchards owned by the same producers. Thus, by enriching collected samples with the selective medium with high sugar and ethanol concentration, we managed to isolate about 1,300 yeasts. Based on the multiplex PCR results the yeasts were grouped into the Saccharomyces sensu stricto complex and non-Saccharomyces yeasts. The isolates were determined to the species level by performing the restriction analysis of ITS PCR products, and in some cases identifications were confirmed by sequencing of the D1/D2 domain of the 26S rDNA and/ or ITS region. As expected, non-Saccharomyces yeasts from the genus Metschnikowia and Hanseniaspora mainly populated the orchards, while the Saccharomyces yeasts were isolated in the orchards from the soil and fruits. In contrast, in ciders the species S. uvarum was predominantly found, occasionally also S. cerevisiae, Torulaspora delbrueckii and P. membranifacies. Indigenous cider yeasts were further on characterized in micro-plate format for the most important cider-making technological parameters (tolerance to ethanol, SO2, growth at low pH), for the presence of glucoside hydrolase activity, H2S production ability, and assimilation of malic acid. Based on this screenings the micro-scale fermentations of apple juice were performed with 13 different indigenous cider yeasts as monocultures. The most promising indigenous yeasts, T. delbrueckii and S. uvarum, were also tested as mixed cultures in sequential fermentations. Since the tested strain of T. delbrueckii as monoculture was not able to complete the alcoholic fermentation, better results were obtained in sequential fermentation with the mixed culture in combination with S. uvarum.
Keywords: indigenous yeasts, biodiversity, spontaneous fermentation, cider-making
Published in RUNG: 08.11.2017; Views: 6244; Downloads: 0
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10.
Study of yeast biodiversity potential in the development of biological control agents against phytopathogenic fungi in viticulture
Melita Sternad Lemut, Adesida Rowland, Urban Česnik, Lorena Butinar, 2017, published scientific conference contribution abstract

Abstract: Despite public’s growing concerns for human health and environmental pollution, synthetic fungicides are still preferred and massively used for the protection of agricultural crop plants against fungal diseases. However, more and more research is focused on finding potential alternatives in the form of effective biological control agents. Although there are reports of yeast’s biocontrol activity, they are up to date poorly commercialized for such purposes. As the yeasts represent an important part of the grape microflora, competing with other microorganisms (including pathogens) for nutrients and space, we decided to examine the potential of autochthonous yeasts as "green" alternatives in fighting against phytopathogens such as Botrytis cinerea in viticulture. With this aim we tested biocontrol activity of various strains / species of indigenous wine yeasts that were isolated from the vineyard environment in Slovenia and cross-border Italy. The yeasts were tested for the presence of siderophores, hydrolytic enzymes (chitinase, β-glucosidase and β-glucanase) and sensitivity to fungicides (copper, iprodion, cyprodinil + fludioxonil combination). The tests of phytopathogenic fungi growth inhibition in the presence of antagonistic yeast were also performed. The double Petri dish test was used to verify the inhibition of conidia germination by the formation of antifungal volatile organic compounds (VOC), synthetized by the antagonistic yeasts. The presence of hydrolytic enzymes was confirmed in many wine yeasts, particularly in the strains of Hanseniaspora uvarum and Metschnikowia pulcherrima species and in some strains of the Pichia genus. Using a medium, based on natural diluted grape juice in a double Petri dish test, we found that yeasts of the Debaryomyces hansenii, Lachancea thermotolerans, P. kudriavzevii, Saccharomyces kudriavzevii, S. cerevisiae and Torulaspora delbrueckii species were able to inhibit the germination of fungal conidia at the expense of the VOC formation. However, none of the up to date tested yeasts formed siderophores. On the other hand, the yeasts were generally resistant to the tested fungicide concentrations, thus they could be used as potential biofungicides in the combination with chemical fungicides.
Keywords: yeasts, biocontrol, Botrytis cinerea, viticulture
Published in RUNG: 08.11.2017; Views: 4625; Downloads: 0
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