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The role of non-Saccharomyces species in releasing glycosidic bound fraction of grape aroma components - A preliminary study
Abstract
The purpose of the study was to evaluate the effect of beta-glycosidase activity in wine yeasts in releasing terpene glycosides from grape juice.
Glycosidase activity was screened in 160 yeasts by testing their ability to hydrolyse arbutine on agar plates. Only non-Saccharomyces species exhibited beta-glycosidase activity. Enzyme activity, based on hydrolytic activity on p-nitrophenyl-beta-glycoside, was mainly located in the whole cell fraction, with smaller amounts in permeabilized cells being released into the growth medium. The hydrolysis of glycosides was determined by HRGC-MS, confirming the role of yeast in the liberation of monoterpenols, especially linalool and geraniol.
The results indicate the potential of microbial beta-glycosidases for releasing flavour compounds from glycosidically-bound, non-volatile precursors, with significant implications for wines made from less aromatic grapes.
This study confirms the role of non-Saccharomyces species in enhancing wine aroma and flavour, suggesting that the future lies with controlled use of mixed cultures in winemaking.
... 3-Alkyl-2-methoxypyrazines present in grape skin, pulp and bunch stems contribute to wine aroma with their characteristic vegetative, herbaceous, bell pepper or earthy notes, in particular in Cabernet Sauvignon, Sauvignon blanc, Semillon wines [40][41][42][43][44][45]. The level of isobutylmethoxypyrazine in wine can be 10-fold its sensory threshold, secbutylmethoxypyrazine and isopropylmethoxypyrazine are normally present in contents close to their sensory thresholds, 2-methoxy-3-isobutylpyrazine, 2-methoxy-3-secbutylpyrazine and 2-methoxy-3-isopropylpyrazine can impact wine aroma because are characterised by particularly low sensory thresholds (1-2 ng/L in water) [40,46]. ...
... 3-Alkyl-2-methoxypyrazines present in grape skin, pulp and bunch stems contribute to wine aroma with their characteristic vegetative, herbaceous, bell pepper or earthy notes, in particular in Cabernet Sauvignon, Sauvignon blanc, Semillon wines [40][41][42][43][44][45]. The level of isobutylmethoxypyrazine in wine can be 10-fold its sensory threshold, secbutylmethoxypyrazine and isopropylmethoxypyrazine are normally present in contents close to their sensory thresholds, 2-methoxy-3-isobutylpyrazine, 2-methoxy-3-secbutylpyrazine and 2-methoxy-3-isopropylpyrazine can impact wine aroma because are characterised by particularly low sensory thresholds (1-2 ng/L in water) [40,46]. ...
... H. uvarum also showed a higher β-glucosidase activity than S. cerevisiae, explaining the increase of terpenes and norisoprenoids levels in wines in a varietal way [39]. Moreover, Mendes Ferreira et al. [40] showed that in Muscat grape juice, H. uvarum was able to better release linalool, geraniol, nerol, α-terpineol, o-cimenol and citronellol, improving varietal character of the wine. ...
Wine fermentation processes are driven by complex microbial systems, which comprise eukaryotic and prokaryotic microorganisms that participate in several biochemical interactions with the must and wine chemicals and modulate the organoleptic properties of wine. Among these, yeasts play a fundamental role, since they carry out the alcoholic fermentation (AF), converting sugars to ethanol and CO2 together with a wide range of volatile organic compounds. The contribution of Saccharomyces cerevisiae, the reference organism associated with AF, has been extensively studied. However, in the last decade, selected non-Saccharomyces strains received considerable commercial and oenological interest due to their specific pro-technological aptitudes and the positive influence on sensory quality. This review aims to highlight the inter-specific variability within the heterogeneous class of non-Saccharomyces in terms of synthesis and release of volatile organic compounds during controlled AF in wine. In particular, we reported findings on the presence of model non-Saccharomyces organisms, including Torulaspora delbrueckii, Hanseniaspora spp, Lachancea thermotolerans, Metschnikowia pulcherrima, Pichia spp. and Candida zemplinina, in combination with S. cerevisiae. The evidence is discussed from both basic and applicative scientific perspective. In particular, the oenological significance in different kind of wines has been underlined.
... In a previous work, Mendes Ferreira et al. [64] studied the β-glucosidase activity using the pnitrophenyl-β-D-glycoside (pNPG) as substrate in H. uvarum (formerly K. apiculata), Pichia anomala, and Metschnikowia pulcherrima, detecting the highest activity in H. uvarum. Furthermore, these authors demonstrated that H. uvarum was able to release some monoterpenols from an extract of Muscat grape juice, such as linalool, geraniol and in less quantity 3,7-dimethyl-1,7-octadien-3,6-diol and 3,7dimethyl-1,5-octadien-3,7-diol, nerol, trans o-cimenol, α-terpineol, and citronellol [64]. ...
... In a previous work, Mendes Ferreira et al. [64] studied the β-glucosidase activity using the pnitrophenyl-β-D-glycoside (pNPG) as substrate in H. uvarum (formerly K. apiculata), Pichia anomala, and Metschnikowia pulcherrima, detecting the highest activity in H. uvarum. Furthermore, these authors demonstrated that H. uvarum was able to release some monoterpenols from an extract of Muscat grape juice, such as linalool, geraniol and in less quantity 3,7-dimethyl-1,7-octadien-3,6-diol and 3,7dimethyl-1,5-octadien-3,7-diol, nerol, trans o-cimenol, α-terpineol, and citronellol [64]. ...
... In a previous work, Mendes Ferreira et al. [64] studied the β-glucosidase activity using the p-nitrophenyl-β-D-glycoside (pNPG) as substrate in H. uvarum (formerly K. apiculata), Pichia anomala, and Metschnikowia pulcherrima, detecting the highest activity in H. uvarum. Furthermore, these authors demonstrated that H. uvarum was able to release some monoterpenols from an extract of Muscat grape juice, such as linalool, geraniol and in less quantity 3,7-dimethyl-1,7-octadien-3,6-diol and 3,7-dimethyl-1,5-octadien-3,7-diol, nerol, trans o-cimenol, α-terpineol, and citronellol [64]. ...
Apiculate yeasts of the genus Hanseniaspora/Kloeckera are the main species present on mature grapes and play a significant role at the beginning of fermentation, producing enzymes and aroma compounds that expand the diversity of wine color and flavor. Ten species of the genus Hanseniaspora have been recovered from grapes and are associated in two groups: H. valbyensis, H. guilliermondii, H. uvarum, H. opuntiae, H. thailandica, H. meyeri, and H. clermontiae; and H. vineae, H. osmophila, and H. occidentalis. This review focuses on the application of some strains belonging to this genus in co-fermentation with Saccharomyces cerevisiae that demonstrates their positive contribution to winemaking. Some consistent results have shown more intense flavors and complex, full-bodied wines, compared with wines produced by the use of S. cerevisiae alone. Recent genetic and physiologic studies have improved the knowledge of the Hanseniaspora/Kloeckera species. Significant increases in acetyl esters, benzenoids, and sesquiterpene flavor compounds, and relative decreases in alcohols and acids have been reported, due to different fermentation pathways compared to conventional wine yeasts.
... Between veraison (color break) and grape harvest, a substantial increase in nutrients occurs within and at the surface of the berry, which significantly impacts yeast population size and diversity (Coombe and McCarthy 2000;Fleet 2003). Native yeasts can exert a positive or negative effect on any aspect of the vinification process (Fleet 2003;Fugelsang and Edwards 2007), and are responsible for some of the unique flavors, aromas, and textures of premium wines (Clemente-Jimenez et al. 2004;Ferreira et al. 2001; Romano et al. 2003). The mycobiomes of wine grapes or fermentation samples from California, Texas, Brazil, China, Germany, New Zealand and South Africa have been reported (Bokulich et al. 2014;Bougreau et al. 2019;Brysch-Herzberg and Seidel 2015;de Ponzzes-Gomes et al. 2014;Gayevskiy & Goddard 2012;Li et al. 2018;Setati et al. 2015;Vigentini et al. 2016). ...
... Enhanced abundances of A. pullulans, Naganishia uzbekistanensis, the Metschnikowia and Wickerhamomyces anomalus at mid-stage fermentation suggested that these yeasts were tolerant to moderate levels of ethanol, and could J o u r n a l P r e -p r o o f contribute to wine production or quality. For example, species of Hanseniaspora and Metschnikowia have been used as co-starters, introduced sequentially with S. cerevisae (Aplin et al. 2019;Binati et al. 2020;Ferreira et al. 2001;Zhang et al. 2018). ...
Abstract:
To address a knowledge gap about the grape berry mycobiome from Washington State vineyards, next-generation sequencing of the internal transcribed spacer region (ITS1) was used to identify native yeast and fungal species on berries of cultivar ‘Cabernet Sauvignon’ from two vineyards at veraison and harvest in 2015 and 2016. Four hundred fifty-six different yeast amplicon sequence variants (ASV), representing 184 distinct taxa, and 2467 non-yeast fungal ASV (791 distinct taxa) were identified in this study. A set of 50 recurrent yeast taxa, including Phaeococcomyces, Vishniacozyma and Metschnikowia, were found at both locations and sampling years. These yeast species were monitored from the vineyard into laboratory-scale spontaneous fermentations. Taxa assignable to Metschnikowia and Saccharomyces persisted during fermentation, whereas Curvibasidium, which also has possible impact on biocontrol and wine quality, did not. Sulfite generally reduced yeast diversity and richness, but its effect on the abundance of specific yeasts during fermentation was negligible. Among the 106 recurring non-yeast fungal taxa, Alternaria, Cladosporium and Ulocladium were especially abundant in the vineyard. Vineyard location was the primary factor that accounted for the variation among both communities, followed by year and berry developmental stage. The Washington mycobiomes were compared to those from other parts of the world. Sixteen recurrent yeast species appeared to be unique to Washington State vineyards. This subset also contained a higher proportion of species associated with cold and extreme environments, relative to other localities. Certain yeast and non-yeast fungal species known to suppress diseases or modify wine sensory properties were present in Washington vineyards, and likely have consequences to vineyard health and wine quality.
... Fia et al. (2010) confirmed that S. cerevisiae strains possessed low β-glucosidase activities. In addition, Ferreira et al. (2010) analyzed the BGL activity of 160 yeast strains and revealed that most of the BGL was found in non-Saccharomyces yeast strains. In order to study the βglucosidase activity of representative yeast from different wine regions in China, we screened 56 strains of non-Saccharomyces yeast from different wine regions and measured its β-glucosidase activity. ...
... However, many studies mainly focused on the βglycosidase activity secreted by yeast into the supernatant (Ferreira, 2010;Hernández et al., 2003). In order to supplement the current gap in the research of yeast β-glycosidase and find the differentiae in enzyme activity of dissimilar parts, we further investigate the β-glycosidase in different parts of yeast. ...
β‐glucosidase is a pivotal enzyme that hydrolyzes bound volatile aromatic compounds. However, the activity of β‐glucosidase in winemaking and the mechanism by which it affects the flavor and taste of wines have not been fully investigated. In this study, we profiled the characteristics of β‐glucosidase derived from wine‐related yeasts isolated from different wine‐making regions in China, and analyzed the enzyme activity from different parts of the cells under aerobic and anaerobic conditions. A total of 56 strains of wine‐related yeasts producing β‐glucosidases were screened using the YNB‐C medium (YNB 6.7 g L–1, cellobiose 5 g L–1, pH 5.0). We found that strain Clavispora lusitaniae C117 produced the highest enzyme activity (152.39 µmol pNP ml–1 h–1). In most strains, β‐glucosidase were located in whole cells (periplasmic space) and permeabilized cells (intracellular). The non‐Saccharomyces species had the highest enzymatic activity in a strain‐dependent manner. Under aerobic conditions, C. lusitaniae C117, Hanseniaspora guilliermondii A27‐3‐4, Metschnikowia pulcherrima F‐1‐6, and Pichia anomala C84 had the highest β‐glucosidase activity. We further investigated the β‐glucosidase activity during the wine fermentation and the effects of sugar, pH, temperature, and ethanol on the enzyme activities of P. anomala C84 and commercial Saccharomyces yeast strains RC212 and VL1. The presence of fructose, glucose, and sucrose strongly inhibited enzyme activity. Similarly, low pH and low temperature inhibited the activity of β‐glucosidase, whereas ethanol promoted enzyme activity. Our findings provide a theoretical basis on understanding the different yeast characteristics of β‐glucosidase and their potential application for further improving wine aroma complexity.
... Grape-must microbiota is dominated by non-Saccharomyces yeast strains which have for long been considered spoilage agents, not only due to their low fermentative ability but also because of the assumption that they overproduce off-flavor compounds, such as acetic acid, acetaldehyde, acetoin, or ethyl acetate [1,6]. However, the demonstration that these negative traits are species and strain-dependent [7] and that some non-Saccharomyces yeasts even exhibit beneficial traits, not found in S. cerevisiae [8][9][10][11][12][13][14] have led winemakers to take a fresh look at these formerly disregarded species. In this line, over the last years, a massive number of studies searching for autochthonous non-Saccharomyces strains that might impart a unique aroma complexity or mouthfeel to wines, while expressing terroir-associated characteristics have been published [15][16][17][18][19][20][21][22][23]. ...
... Interestingly, S. ludwigii UTAD17 did not present decarboxylase activities responsible for the production of histamine, tyramine and putrescine. On the other hand, S. ludwigii UTAD17 exhibited β-glucosidase and β-lyase activities involved in the liberation of terpenes from glycosylated precursors [12] and volatile thiols from cysteinylated precursors [19]. In line with the results obtained, S. ludwigii UTAD17 presents important features for a wine yeast starter, since it is able to adjust to winemaking stress, can contribute to the improvement of wine aromatic profile and does not compromise consumers' health. ...
Non-Saccharomyces yeasts have received increased attention by researchers and winemakers, due to their particular contributions to the characteristics of wine. In this group, Saccharomycodes ludwigii is one of the less studied species. In the present study, a native S. ludwigii strain, UTAD17 isolated from the Douro wine region was characterized for relevant oenological traits. The genome of UTAD17 was recently sequenced. Its potential use in winemaking was further evaluated by conducting grape-juice fermentations, either in single or in mixed-cultures, with Saccharomyces cerevisiae, following two inoculation strategies (simultaneous and sequential). In a pure culture, S. ludwigii UTAD17 was able to ferment all sugars in a reasonable time without impairing the wine quality, producing low levels of acetic acid and ethyl acetate. The overall effects of S. ludwigii UTAD17 in a mixed-culture fermentation were highly dependent on the inoculation strategy which dictated the dominance of each yeast strain. Wines whose fermentation was governed by S. ludwigii UTAD17 presented low levels of secondary aroma compounds and were chemically distinct from those fermented by S. cerevisiae. Based on these results, a future use of this non-Saccharomyces yeast either in monoculture fermentations or as a co-starter culture with S. cerevisiae for the production of wines with greater expression of the grape varietal character and with flavor diversity could be foreseen.
... Also with the aim of improving the aromatic quality, other selection criterion for non-Saccharomyces yeasts is the high β-glucosidase activity in order to favor the hydrolysis of the nonvolatile aromatic precursors from the grape (Mendes Ferreira et al., 2001). Non-Saccharomyces species usually display greater β-glucosidase activity than Saccharomyces species, which has been defined as intracellular and strain-dependent (Arévalo Villena et al., 2005). ...
... Non-Saccharomyces species usually display greater β-glucosidase activity than Saccharomyces species, which has been defined as intracellular and strain-dependent (Arévalo Villena et al., 2005). K. apiculata and W. anomalus, among others, were found to have a great expression of the β-glucosidase enzyme (Charoenchai et al., 1997;Mendes Ferreira, Climaco, & Mendes Faia, 2001). Pérez et al. (2011) confirmed the effectiveness of an esculin glycerol solid medium (EGA) as semiquantitative measurement technique Ciani and Ferraro (1996), Ferraro et al. (2000), Romano et al. (2003), Canonico et al. (2016) Torulaspora delbrueckii 3-Ethoxy-1-propanol Herraiz et al. (1990), Loira et al. (2014Loira et al. ( , 2015 ...
https://doi.org/10.1016/B978-0-12-816678-9.00015-1
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Abstract
In the last few years, non-Saccharomyces yeasts have been gaining increasing importance in the field of oenology because of its specific contribution to the chemical composition of the wine when they are used alone or in combination with other yeast genera. Moreover, winemakers aim to use indigenous yeasts in order to distinguish their wines in a market currently saturated by supply. Herein, different techniques available for the isolation, selection, and identification of non-Saccharomyces yeasts from the vineyard, such as microscopic observation, physiological tests, selective and chromogenic media, restriction analysis, PCR technique, and mitochondrial DNA analysis, among others, are discussed. In addition, depending on the kind of wine to be elaborated or the physicochemical parameters to be modulated, different yeast selection criteria are considered
... b-glycosidase activity was tested as previously described. 37 Briefly, yeast cells were grown in a medium containing 0.5% of arbutine as the sole carbon source, 0.67% yeast nitrogen base and 2% agar (adjusted at pH 5.0 prior sterilization) and supplemented with 2 ml (per 100 ml of medium) ferric ammonium citrate solution (1% w/v). The plates were incubated at 30 C and b-glycosidase activity was observed after 2, 4, 6 and 8 h by the appearance of a dark brown color in the colonies. ...
... Proteolytic activity was determined assessing growth on skim milk agar plates, after incubation for 3-8 days at 30 C 38 while the potential ability to produce H2S was evaluated by growing yeast cells in Biggy agar. 37 The strains were classified in different categories according with the appearance of a brown-black coloration of the colonies, which is directly correlated with sulfite-reductase activity. Resistance to SO 2 and ethanol were evaluated on YPD agar medium (buffered at pH 3.5), supplemented with increasing concentrations of SO2 (0.25, 0.5, 0.75, 1.0 and 1.5 mM) or ethanol (5.0, 6.0, 7.5, 9.0 and 10% v/v). ...
Hanseanispora species, including H. guilliermondii, are long known to be abundant in wine grape-musts and to play a critical role in vinification by modulating, among other aspects, the wine sensory profile. Despite this, the genetics and physiology of Hanseniaspora species remains poorly understood. The first genomic sequence of a H. guilliermondii strain (UTAD222) and the discussion of its potential significance are presented in this work. Metabolic reconstruction revealed that H. guilliermondii is not equipped with a functional gluconeogenesis or glyoxylate cycle, nor does it harbours key enzymes for glycerol or galactose catabolism or for biosynthesis of biotin and thiamine. Also, no fructose-specific transporter could also be predicted from the analysis of H. guilliermondii genome leaving open the mechanisms underlying the fructophilic character of this yeast. Comparative analysis involving H. guilliermondii, H. uvarum, H. opuntiae and S. cerevisiae revealed 14 H. guilliermondii-specific genes (including five viral proteins and one β-glucosidase). Furthermore, 870 proteins were only found within the Hanseniaspora proteomes including several β-glucosidases and decarboxylases required for catabolism of biogenic amines. The release of H. guilliermondii genomic sequence and the comparative genomics/proteomics analyses performed, is expected to accelerate research focused on Hanseniaspora species and to broaden their application in the wine industry and in other bio-industries in which they could be explored as cell factories.
... Monoterpenes are important compounds determining the flavor of grapes and wine. The yeasts belonging to the genera Brettanomyces, Candida, Debaryomyces, Hanseniaspora/Kloeckera, Kluyveromyces, Issatchenkia, Metschnikowia, Pichia, Rhodotorula, Saccharomycodes, Schizosaccharomyces, Torulaspora, Wickerhamomyces, and Zygosaccharomyces have been shown to produce β-glucosidase in amounts that are species and strain dependent, and with varying degrees of activity and different catalytic activities with respect to grape aroma glycosides (Rosi et al. 1994;Gueguen et al. 1996;Charoenchai et al. 1997;McMahon et al. 1999;Yanai and Sato 1999;Fernández et al. 2000;Manzanares et al. 2000;Mendes Ferreira et al. 2001;Strauss et al. 2001;Spagna et al. 2002;Cordero Otero et al. 2003;Fernández-González et al. 2003;Wallecha and Mishra 2003;Rodríguez et al. 2004Rodríguez et al. , 2007Arévalo Villena et al. 2005González-Pombo et al. 2008Hernández-Orte et al. 2008;Swangkeaw et al. 2009Swangkeaw et al. , 2011Comitini et al. 2011;Domizio et al. 2011;Sadoudi et al. 2012;Cordero-Bueso et al. 2013;López et al. 2014;Sabel et al. 2014;Belda et al. 2015;Hu et al. 2016a;Polizzotto et al. 2016). The activity of these enzymes has been assessed primarily using artificial substrate. ...
... The activity of these enzymes has been assessed primarily using artificial substrate. Some studies have shown the enzymatic activity either hydrolyzes a glycoside extract from grape must, as for the yeasts Debaryomyces hansenii, Hanseniaspora uvarum, Kloeckera apiculata, and Rhodotorula mucilaginosa (Rosi et al. 1994;Yanai and Sato 1999;Mendes Ferreira et al. 2001;Fernández-González et al. 2003;Hu et al. 2016b) or releases terpenols after addition to must or wine, as for the yeasts Hanseniaspora sp., Hanseniaspora uvarum, and Pichia anomala (Swangkeaw et al. 2009;Hu et al. 2016a). ...
Wine is the end product of the fermentative activity of yeast and bacteria. The microbiota of grape juice fermentation can vary significantly as over 40 genera and 100 different species of yeast have been isolated from grapes or wine (Table 3.1). Although the genera listed are commonly identified in surveys of grape mycobiota, some yeast species are more universally found than others, and numerous factors impact the composition of the yeast microbial community of grapes and their persistence during fermentation. Saccharomyces cerevisiae is the primary agent responsible for the conversion of grape sugars into alcohol but other yeast, collectively known as non-Saccharomyces yeast, and bacteria may also contribute to the aroma and flavor profile of the wine. Thus interspecies as well as intraspecies diversity plays an important role in the evolution of wine composition.
... As a result of the vinification process, the aforementioned bonds are hydrolysed by the enzymes present in the fruit, including α-rhamnosidase, α-arabinosidase, or β-xylosidase [47]), although the majority of this hydrolysis is carried out by the glycosidases of yeasts and lactic acid bacteria. It has been shown that yeasts belonging to the Saccharomyces cerevisiae species exhibit a significantly reduced potential to release volatile terpenoids, compared to wild yeasts such as Hanseniaspora uvarum, Debaryomyces hansenii, Metschnikowia pulcherrima, Kloeckera apiculata, Pichia anomala, Meyerozyma guillermondii, and Wickerhamomyces anomalus [48][49][50]. Among lactic acid bacteria, Oenococcus oeni, Lactiplantibacillus (formerly Lactobacillus) plantarum, and Levilactobacillus (formerly Lactobacillus) brevis have also been observed to release free volatile monoterpenoids [9,[51][52][53][54][55][56][57][58]. ...
Volatilomics is a scientific field concerned with the evaluation of volatile compounds in the food matrix and methods for their identification. This review discusses the main groups of compounds that shape the aroma of wines, their origin, precursors, and selected metabolic pathways. The paper classifies fruit wines into several categories, including ciders and apple wines, cherry wines, plum wines, berry wines, citrus wines, and exotic wines. The following article discusses the characteristics of volatiles that shape the aroma of each group of wine and the concentrations at which they occur. It also discusses how the strain and species of yeast and lactic acid bacteria can influence the aroma of fruit wines. The article also covers techniques for evaluating the volatile compound profile of fruit wines, including modern analytical techniques.
... However, the proportion of glycosylated fraction present in all grapes may have a relevant role in the aromatic potential of these grapes, with a concentration ranging from 1.34 to 3.20 mg/L. The bound volatile compounds can be converted into free form by hydrolysis, modifying the aromatic profile of wines and enhancing the varietal character [27]. ...
This chapter synthetizes the main results that our research group has obtained about the specific influence of a commercial Saccharomyces cerevisiae strain on the aromatic profile of fermented musts from four minority grape varieties (Vitis vinifera L.) cultivated in Castilla-La Mancha (Spain), that is, Moribel, Tinto Fragoso, Albillo Dorado and Montonera del Casar. In addition, wines made from the grape cultivars Tempranillo and Airén were evaluated. To determine the main yeast-derived odor relevant in these grape varieties, the aromatic profiles of grape cultivars and the resulting wines were studied by gas chromatography coupled to mass spectrometry and wines were subjected to Napping, a rapid sensory evaluation method. The results revealed wine sensory differences which are consequence of different aromatic profiles of wines produced with these grape cultivars. The combination of quantitative chemical analysis of volatile compounds together with sensory analysis of wines point out different patterns of aroma compound formation and release. Thus, the yeast strain used in the fermentation step is one of the main factors that affect the sensory properties of wines.
... β-Glucosidases can be found in varied organisms, such as plants, fungi, and bacteria [9,10]. The intense aerobic glucosidase activity [3,11] promotes the liberation of varietal volatile compounds from the grape by hydrolyzing bound precursors. However, it is important Microorganisms 2023, 11, 964 2 of 11 to remember that the intensity of activity depends not only on the species but also on the strain [12]. ...
A broad variety of microorganisms with useful characteristics in the field of biotechnology live on the surface of grapes; one of these microorganisms is Metschnikowia pulcherrima. This yeast secretes a β-glucosidase that can be used in fermentative processes to liberate aromatic compounds. In this work, the synthesis of an exocellular β-glucosidase has been demonstrated and the optimal conditions to maximize the enzyme’s effectiveness were determined. There was a maximum enzymatic activity at 28 °C and pH 4.5. Furthermore, the enzyme presents a great glucose and fructose tolerance, and to a lesser extent, ethanol tolerance. In addition, its activity was stimulated by calcium ions and low concentrations of ethanol and methanol. The impact of terpene content in wine was also determined. Because of these characteristics, β-glucosidase is a good candidate for use in enology.
... Higher alcohols play a crucial role in wine aroma and could enhance floral and fruity flavor in wines [35]. It was reported that β-glucosidase from H. uvarum and Kloeckera apiculata increased the higher alcohol content of wine, especially isoamyl alcohol [2,36]. However, according to Zhang et al. (2020) [10], adding SLY-4E, F2-24E and HX-13E decreased the higher alcohol content. ...
The aim of this study was to investigate the effects of crude extracts of β-glucosidase from Issatchenkia terricola SLY-4, Pichia kudriavzevii F2-24 and Metschnikowia pulcherrima HX-13 (termed as SLY-4E, F2-24E and HX-13E) on the flavor complexity and typicality of Cabernet Sauvignon wines. The grape must was fermented using Saccharomyces cerevisiae with single or mixed SLY-4E, F2-24E and HX-13E. The physicochemical characteristics, volatile aroma compounds, total anthocyanins and sensory attributes of the wines were determined. Adding SLY-4E, F2-24E and HX-13E in wines resulted in a decrease in the anthocyanin content, total acids and volatile acids in wines but an increase in the content of terpenes, benzene derivatives, higher alcohols and esters, which may enhance wine sensory qualities and result in loss of wine color. Different adding strategies of β-glucosidase led to a variety of effects on wine aroma. S/H/F-Ew significantly increased the content of benzene derivatives, higher alcohols and long-chain fatty acid esters, which enhanced the fruity and floral flavor of wines. F2-24E significantly increased the content of short- and medium-chain fatty acid esters, acetate esters and carbonyl compounds. The results indicated that the mixed addition of non-Saccharomyces crude extracts and co-fermentation with S. cerevisiae could further improve wine flavor quality.
... Arbutin and esculin were often used in the context of agar plates to which iron compounds like ferric ammonium citrate [20] or ferric chloride [15] are simultaneously added. The complexation of iron by the released aglycons leads to the visible formation of elemental iron which indicates β-glucosidase activity. ...
β-glucosidases are hydrolyzing enzymes which can release many aroma-active compounds from their glycoside form. Several yeasts produce these enzymes and thus are applied during the wine production process. To be able to test specific organisms for the presence of β-glucosidases and to investigate this enzyme activity, four main surrogate substrates have been described. The properties and applicability of these compounds, named arbutin (hydroquinone-β-D-glucopyranoside), esculin (6-O-(-D-glucosyl)aesculetin), 4-nitrophenyl-β-D-glucopyranoside (pNPG) and 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG), are discussed after comparing their advantages and disadvantages. Although all four substrates were found suitable for photometric assays, 4-MUG has proven to be most appropriate due to high sensitivity, high robustness and simple processing. Furthermore, the investigation of β-glucosidase product accumulation is described, which could be used to give indications about β-glucosidase localization.
... Fernández et al. [65] indicated that β-glucosidase activity was mainly related to the species M. pulcherrima. Mendes Ferreira et al. [66] showed the potential of this species for the release of terpenes due to its high β-glucosidase activity, which coincides with the results obtained in the present work. ...
The use of non-Saccharomyces yeasts in sequential fermentations with S. cerevisiae has been proposed to improve the organoleptic characteristics involved in the quality of wine. The present study set out to select a non-Saccharomyces inoculum from the D.O.Ca. Rioja for use in winemaking. Strains included in the study belonged to Torulaspora delbrueckii, Lachancea thermotolerans, Metschnikowia pulcherrima, Zygosaccharomyces bailii, Williopsis pratensis, Debaryomyces hansenii, Pichia kluyveri, Sporidiobolus salmonicolor, Candida spp., Cryptococcus spp. and two mixed inocula of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio. In the first stage of the process, SO2 resistance and presence of enzymatic activities related to wine aroma and wine color and fining (esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, β-glucosidase, pectinase, cellulose, xylanase and glucanase) were studied. In the later stages, selection criteria such as fermentative behavior, aroma compound production or influence on phenolic compounds were studied in laboratory scale vinifications. Taking into account the results obtained in the different stages of the process, a mixed inoculum of Lachancea thermotolerans-Torulaspora delbrueckii in a 30/70 ratio was finally selected. This inoculum stood out for its high implantation capacity, the production of compounds of interest such as glycerol and lactic acid and the consequent modulation of wine acidity. Given these characteristics, the selected inoculum is suitable for the production of quality wines.
... During alcoholic and malolactic fermentation, the content of free terpenes often increases due to the b-glucosidase activity of yeasts (Gil et al. 1996). The ability of non-Saccharomyces yeast to release terpenes from glycosidic precursors has been described by several authors (Maturano et al. 2012;Ferreira et al. 2001) and the amount of the enzymatic hydrolysis is dependent on the yeast strain and the chemical structure of the substrate. In fact the ST1 wine, obtained with M. fructicola have a higher concentration of terpenes (linalol, 4-terpineol, nerolidol and bcitronellol) than ST3 and ST5 wines (Table 2). ...
... Unlike Saccharomyces species, non-Saccharomyces yeasts possess different enzymes: glycosidase, pectinases, proteases β-glucanases, lichenases, β-glucosidases, cellulases, xylanases, amylases, lipases, esterases, etc [9]. These can play an important role both in the technological extractive phase and during the fermentation, in the release or production, of terpenoids, fatty acid esters, higher alcohols, esters, etc [10,11]. The use of these yeasts in fermentation could constitute a valid alternative to the use of enzymes produced by filamentous bacteria and fungi [12]. ...
In this work was evaluated the effect of sequential inoculum of Hanseniaspora uvarum AS27 strain and a commercial Saccharomyces cerevisiae yeast on the physical–chemical and organoleptic features of Aglianico, a traditional red wine of Southern Italy. Four fermentation treatments on a pilot scale were performed. In fermentation treatment A, the alcoholic fermentation was spontaneously conducted by the indigenous yeasts present in grape must. In the fermentation treatments B and C were inoculated respectively S. cerevisiae FE and H. uvarum AS27 strains, as a single starter. The fermentation treatment D was initially inoculated with H. uvarum AS27, and S. cerevisiae strain was added after 72 h (sequential inoculation). Microbiological, physical–chemical parameters and sensory profiles of the wines have been defined. The results showed that the use of H. uvarum AS27, in sequential inoculum with S. cerevisiae FE, influenced the wine composition, enriching it in polyphenolic and volatile compounds. Further, the sensory evaluation showed that the use of H. uvarum AS27 strain, in co-culture with S. cerevisiae, gives the wine more pleasant characteristics. Therefore, the results have highlighted how the use of particular non-Saccharomyces yeasts can represent a biotechnological resource in red wine production.
... The non-Saccharomyces strains of the genera Kloeckera and Hanseniaspora have been reported to be having protein protease activity, which has, in turn, profound impact on the protein profile of finished wines (Charoenchai et al., 1997;Dizy and Bisson, 2000). Non-Saccharomyces yeast have been studied in the context of their ability to produce β-glycosidase enzymes, which are involved in the flavour-releasing processes (McMahon et al., 1999;Yanai and Sato, 1999;Manzanares et al., 2000;Mendes-Ferreira et al., 2001;Cordero-Otero et al., 2003). Recently, the acetate esters formed by enzymatic activities of yeast strains belonging to the genera Hanseniaspora and Pichia have been studied in detail (Rojas et al., 2001;Rojas et al., 2003). ...
... Similarly, β-glucosidase from P. anomala MDD24 has also been reported to be very efficient at releasing aromatic compounds during the final phase of wine fermentation due to its high ethanol tolerance [8,54]. While W. anomalus and M. pulcherrima have been known to produce ethyl acetate and fruity esters (especially ethyl octanoate) during fermentation, respectively [11,56], several researchers have also paid attention to applying the β-glucosidase activities of these strains to improve the volatile terpene compounds in wine [7,8,10,51,54,57]. The formation of volatile aromatic compounds, including monoterpenes, is influenced by many factors, such as grape cultivars, geological differences, soil, climate, water, nitrogen fertilization, and harvest time [58,59]. ...
The Muscat Bailey A (MBA) grape, one of the most prominent grape cultivars in Korea, contains considerable amounts of monoterpene alcohols that have very low odor thresholds and significantly affect the perception of wine aroma. To develop a potential wine starter for Korean MBA wine, nine types of non-Saccharomyces yeasts were isolated from various Korean food materials, including nuruk, Sémillon grapes, persimmons, and Muscat Bailey A grapes, and their physiological, biochemical, and enzymatic properties were investigated and compared to the conventional wine fermentation strain, Saccharomyces cerevisiae W-3. Through API ZYM analysis, Wickerhamomyces anomalus JK04, Hanseniaspora vineae S7, Hanseniaspora uvarum S8, Candida railenensis S18, and Metschnikowia pulcherrima S36 were revealed to have β-glucosidase activity. Their activities were quantified by culturing in growth medium composed of different carbon sources: 2% glucose, 1% glucose + 1% cellobiose, and 2% cellobiose. W. anomalus JK04 and M. pulcherrima S36 showed the highest β-glucosidase activities in all growth media; thus, they were selected and utilized for MBA wine fermentation. MBA wines co-fermented with non-Saccharomyces yeasts (W. anomalus JK04 or M. pulcherrima S36) and S. cerevisiae W-3 showed significantly increased levels of linalool, citronellol, and geraniol compared to MBA wine fermented with S. cerevisiae W-3 (control). In a sensory evaluation, the flavor, taste, and overall preference scores of the co-fermented wines were higher than those for the control wine, suggesting that W. anomalus JK04 and M. pulcherrima S36 are favorable wine starters for improving Korean MBA wine quality.
... The use of only native grape strains in the form of pure cultures as fermentation primers indicates that they exhibit undesirable features, such as production of acetic acid, ethyl acetate, ethanal and acetoin. In addition, most of non-Saccharomyces species have limited fermentation potential and low SO 2 resistance [5][6][7][8]. Despite the unfavorable features, a number of studies have been conducted over the past several years regarding the recognition and activity of non-Saccharomyces yeast in grape must. ...
The international competitiveness of the wine sector and consumer demands for the unique wine styles pose challenges in improving the fermentation process. The basis of proper alcoholic fermentation is knowledge about how individual yeast strains interact with the aroma, taste and color of wine, what results in possibility to select species used as starter cultures. To use the value of non-Saccharomyces yeast strains in wine production and to minimize the possibility of wine deterioration, it is necessary to precisely recognize the yeast cultures present on the fruit of the vine and in grape must, as well as their metabolic properties. The aim of the study was to determine the oenological properties of yeasts isolated from spontaneously fermented grape musts obtained from cool climate grapes. For this purpose, Zweigelt grape must was fermented with yeast monocultures. Alcohol, extract, sugars, glycerol, total acidity and free amine nitrogen were analyzed in the obtained wines. Poor fermentation properties of yeast strains results in obtaining wines with relatively large amounts of residual sugars and low alcohol. A decrease in overall acidity was noted in sets with the participation of M. pulcherrima MG971264, while in other tests the opposite trend was observed. Although some microorganisms have the ability to assimilate organic acids found in wine, they are not able to carry out fermentation or they do it inefficiently. Solution to this problem may, therefore, be use of mixed cultures of noble and non-Saccharomyces yeast, what effectively reduce the concentration of organic acids, while not adversely affecting the organoleptic characteristics of the drink.
... Parmi ces réactions, la libération des terpènes est la plus étudiée. (Fia et al., 2005;Gonzalez-Pombo et al., 2008;King et Richard Dickinson, 2000;Maicas et Mateo, 2005;Mendes Ferreira et al., 2001;Rodríguez et al., 2004;Rosi et al., 1994;Swiegers et al., 2005;Vasserot et al., 1989;Villena et al., 2007). ...
La caractérisation phénotypique de l’espèce Torulaspora delbrueckii en conditions œnologiques, à partir de l’étude d’un grand nombre de souches, a permis de mettre en évidence une grande variabilité au sein de cette espèce. En effet, les souches de T.delbrueckii présentent des différences au niveau des durées de phase de latence et de fermentation, des capacités biotiques mais aussi des productions d’éthanol (maximum 12% vol.). Cette variabilité se retrouve également pour la production d’acidité volatile, de glycérol et de certains arômes. Ce travail confirme les faibles productions d’acidité volatile et de glycérol de cette espèce et met en évidence une réponse au stress osmotique différente de celle de l’espèce Saccharomyces cerevisiae. Au final, l’espèce T. delbrueckii présente une grande « pureté » de fermentation et produit peu de composés indésirables comme le sulfure d’hydrogène, les phénols volatils, l’acetoïne, l’acétaldéhyde et le diacétyle. La réalisation de co-inoculations T. delbrueckii / S. cerevisiae sur moûts liquoreux, permet une réduction systématique de l’acidité volatile des vins, en comparaison à une fermentation pure de l’espèce S. cerevisiae, quelque soit la souche de T. delbrueckii utilisée. De plus, la souche T. delbrueckii OXT1 1//2 a permis de complexifier la composition aromatique d’un moût sec issu du cépage Sauvignon blanc (esters fermentaires +25%, phényl-2-éthanol +51% et thiols volatils +31%). Enfin, la mise au point d’un fermenteur à double compartiment, avec une séparation physique des levures tout en conservant l’homogénéité du milieu de culture, a permis d’aborder l’étude des interactions entre ces 2 espèces. Des inhibitions de type « cell-cell contact » ont ainsi été mises en évidence.
... Also, its high proteolytic activity makes it a candidate to be used in fermentations with S. cerevisiae, releasing amino acids and increasing the available nitrogen sources for the growth of S. cerevisiae [104,105]. It also stands out for its glucosidase-dependent strain activity [106,107], which increases in aerobic conditions [50], promoting the release of varietal aromas by hydrolyzing bound monoterpenes. The expression of β-D-glucosidase favors the release of free terpenes and this activity has been evaluated using the 4-methylumbelliferyl-β-D-glucoside (MUG) and p-nitrophenyl-β-D-glucoside (pNPG) substrates [108]. ...
Wine is a complex matrix that involves compounds of different chemical nature, with volatile compounds being primarily responsible for the aromatic quality of the wine. The formation of these volatile compounds is mainly due to yeasts’ metabolism during alcoholic fermentation. Several studies in the microbiology field have reported that Saccharomyces cerevisiae is responsible for alcoholic fermentation, influencing the sensory quality of the wine and affecting the metabolic activity of other genera and species of yeasts, called non-Saccharomyces, which would positively affect sensory quality. Non-Saccharomyces yeasts, considered until recently as undesirable or spoilage yeasts, can improve the chemical composition and aroma profile of the wine. The activity of these yeasts is considered essential for the final wine aroma profile. Thus, the metabolism of these microorganisms could be a decisive factor that strongly influences the aroma of the wine, impacting on its quality. However, there are few studies that explain the impact of non-Saccharomyces yeasts on the final wine aroma profile. This chapter summarizes relevant aspects and pathways involved in the synthesis of aromatic compounds by non-Saccharomyces yeasts as well as studies at the genetic and transcriptional level associated with their formation.
... The free-form volatiles contribute directly to fruit and associated products flavor, while bound volatiles are usually present as odorless aroma precursors. Bound volatiles are mainly glycosidic and cysteine derivatives and can be transformed into volatile aglycones through hydrolysis [4,5]. ...
Aroma compounds in fruits and related products are known to occur in free and glycosidically bound forms. In this study, free and bound volatiles in juices and wines of two kiwifruit cultivars, Actinidia deliciosa ‘Hayward’ and A. chinensis ‘Hort16A’, were analyzed by solid-phase microextraction-gas chromatography-mass spectrometry. Results showed that 67 free and 79 bound volatiles were found in juices and wines. Alcohols were the most abundant free volatiles, while the most representative bound volatiles were terpenoids. β-Damascenone and 4-methyl-3-penten-2-one released from aroma precursors were found in ‘Hort16A’ juice for the first time. Fruity, floral, and sweet flavors appeared to be the characteristic aroma in kiwifruit juices and wines. Principal component analysis showed that kiwifruit wines had a great aroma potential. Cluster analysis revealed that juices and wines had different profile of free volatiles, while similar bound volatile compositions were found. Moreover, abundant bound volatiles were discovered in these two kiwifruit pomace for the first time, indicating their potential applications in food and cosmetics industry.
... For technological characterization, both extracellular β-glucosidase activity and resistance to sulfur dioxide (SO 2 ) have been determined. Quantitative screening of the extracellular β-glucosidase activity was performed following the protocol described by Mendes Ferreira et al. (2001). Strains were inoculated in 20 mL of YPD liquid medium and, after 48 h of incubation at 26 • C, 1 × 10 6 cells/mL were inoculated in a liquid medium composed by yeast nitrogen base without aminoacids (0.67%), glucose (2%) and 0.4 mL of ferric ammonium citrate solution (1% w/v). ...
A current trend in winemaking has highlighted the beneficial contribution of non-Saccharomyces yeasts to wine quality. Hanseniaspora uvarum is one of the more represented non-Saccharomyces species onto grape berries and plays a critical role in influencing the wine sensory profile, in terms of complexity and organoleptic richness. In this work, we analyzed a group of H. uvarum indigenous wine strains as for genetic as for technological traits, such as resistance to SO2 and β-glucosidase activity. Three strains were selected for genome sequencing, assembly and comparative genomic analyses at species and genus level. Hanseniaspora genomes appeared compact and contained a moderate number of genes, while rarefaction analyses suggested an open accessory genome, reflecting a rather incomplete representation of the Hanseniaspora gene pool in the currently available genomes. The analyses of patterns of functional annotation in the three indigenous H. uvarum strains showed distinct enrichment for several PFAM protein domains. In particular, for certain traits, such as flocculation related protein domains, the genetic prediction correlated well with relative flocculation phenotypes at lab-scale. This feature, together with the enrichment for oligo-peptide transport and lipid and amino acid metabolism domains, reveals a promising potential of these indigenous strains to be applied in fermentation processes and modulation of wine flavor and aroma. This study also contributes to increasing the catalog of publicly available genomes from H. uvarum strains isolated from natural grape samples and provides a good roadmap for unraveling the biodiversity and the biotechnological potential of these non-Saccharomyces yeasts.
... It exhibits tolerance to sugar and oxygen [23]. It is advantageous, because yeast cells synthesize reactive oxygen species (ROS) when the amount of available oxygen is limited [30]. This species shows high physiological variability [23]. ...
... In spite of the fact that it has been known that some non-Saccharomyces yeasts have potentially higher glycosidic activities [30], not many papers deal with the ability of these yeasts to produce aroma compounds from glycosidic precursors. As many aroma glycosides are located in the grape skin, other enzymatic activities, such as pectinase, cellulose, xylanase and glucanase, may contribute to enrich aroma compounds derived from grape glycosidic precursors. ...
The goal of this study is to assess to what extent non-Saccharomyces yeasts can introduce aromatic changes of industrial interest in fermentative, varietal and aged aromas of wine. Aroma precursors from Riesling and Garnacha grapes were extracted and used in two independent sequential experiments. Synthetic musts were inoculated, either with Saccharomyces cerevisiae (Sc) or with Pichia kluyveri (Pk), Torulaspora delbrueckii (Td) or Lachancea thermotolerans (Lt), followed by Sc. The fermented samples were subjected to anoxic aging at 50 °C for 0, 1, 2 or 5 weeks before an aroma analysis. The fermentative aroma profiles were consistently changed by non-Saccharomyces: all strains induced smaller levels of isoamyl alcohol; Pk produced huge levels of aromatic acetates and can induce high levels of fatty acids (FA) and their ethyl esters (EE); Td produced large levels of branched acids and of their EE after aging, and induced smaller levels of FA and their EE; Lt produced reduced levels of FA and their EE. The varietal aroma was also deeply affected: TDN (1,1,6-trimethyl-1,2- dihydronaphthalene) levels in aged wines were reduced by Pk and enhanced by Lt in Garnacha; the levels of vinylphenols can be much reduced, particularly by Lt and Pk. TD and Lt can increase linalool and geraniol in young, but not in aged wines.
... Hanseniaspora uvarum strains showed the capability to produce β-glucosidase enzymes without glucose and low-pH repression (López et al. 2002). M. pulcherrima, Meyerozyma guillermondii, and Wickerhamomyces anomalus also showed high β-D-glucosidase activity (Belda et al. 2016;Mendes-Ferreira et al. 2001). Screening of 370 strains of 20 species of yeasts (Rosi et al. 1994) showed that all of the strains of the species Debaryomyces castelli, Debaryomyces hansenii, Debaryomyces polymorphus, Kloeckera apiculata, and Hanseniaspora anomala exhibited β-D-glucosidase activity. ...
Although there are many chemical compounds present in wines, only a few of these compounds contribute to the sensory perception of wine flavor. This review focuses on the knowledge regarding varietal aroma compounds, which are among the compounds that are the greatest contributors to the overall aroma. These aroma compounds are found in grapes in the form of nonodorant precursors that, due to the metabolic activity of yeasts during fermentation, are transformed to aromas that are of great relevance in the sensory perception of wines. Due to the multiple interactions of varietal aromas with other types of aromas and other nonodorant components of the complex wine matrix, knowledge regarding the varietal aroma composition alone cannot adequately explain the contribution of these compounds to the overall wine flavor. These interactions and the associated effects on aroma volatility are currently being investigated. This review also provides an overview of recent developments in analytical techniques for varietal aroma identification, including methods used to identify the precursor compounds of varietal aromas, which are the greatest contributors to the overall aroma after the aforementioned yeast-mediated odor release.
... During alcoholic and malolactic fermentation, the content of free terpenes often increases due to the b-glucosidase activity of yeasts (Gil et al. 1996). The ability of non-Saccharomyces yeast to release terpenes from glycosidic precursors has been described by several authors (Maturano et al. 2012;Ferreira et al. 2001) and the amount of the enzymatic hydrolysis is dependent on the yeast strain and the chemical structure of the substrate. In fact the ST1 wine, obtained with M. fructicola have a higher concentration of terpenes (linalol, 4-terpineol, nerolidol and bcitronellol) than ST3 and ST5 wines (Table 2). ...
Non-Saccharomyces yeasts are metabolically active during grape must fermentations and can contribute with enzymes and metabolites to enhance the complexity and to define the final wine aroma. Nowadays, the use of non-Saccharomyces yeasts in combination with Saccharomyces cerevisiae is a state-of-the art strategy to improve wine composition and/or wine sensory properties. The present paper deals with the new yeast strains of Metschnikowia fructicola and S. cerevisiae, that were selected as representatives of the yeast microbiota isolated from grapes and grape juice of Aglianico cultivar. S. cerevisiae was utilized both as single strain starter and in combination with M. fructicola in experimental fermentations of Aglianico must. The dynamic of yeast populations was evaluated during the fermentation process analyzing the wine volatile compounds profile. The volatile compounds were identified by SPME-GC/MS. The results, showed that the multiple indigenous yeast starter was able to modulate the volatile compounds profiles and improve the aromatic complexity of wine, with a higher content of esters and terpenes.
... Its high proteolytic activity makes it a very interesting fermentation partner for Sc, since the amino acids released (including those from autolysis) can serve as a source of nutrients for Sc [36]. In addition, its intense glucosidase activity [2], higher under aerobic conditions [37], promotes the release of varietal aromas from the grape by hydrolyzing bound monoterpenes. However, it is important to always remember that the intensity of the enzymatic activity depends not only on the species, but also on the strain [32]. ...
Metschnikowia pulcherrima (Mp) is a ubiquitous yeast that frequently appears in spontaneous fermentations. The current interest in Mp is supported by the expression of many extracellular activities, some of which enhance the release of varietal aromatic compounds. The low fermentative power of Mp makes necessary the sequential or mixed use with Saccharomyces cerevisiae (Sc) to completely ferment grape musts. Mp has a respiratory metabolism that can help to lower ethanol content when used under aerobic conditions. Also, Mp shows good compatibility with Sc in producing a low-to-moderate global volatile acidity and, with suitable strains, a reduced level of H2S. The excretion of pulcherrimin gives Mp some competitive advantages over other non-Saccharomyces yeasts as well as providing some antifungal properties.
... Recently, Bonciani et al. (2018) reported a qualitative and quantitative screening of the β-glucosidase activity in Saccharomyces strains isolated from must. Other investigations have also demonstrated the βglucosidase activity in non-Saccharomyces strains (Rosi et al., 1994;Mendes Ferreira et al., 2001;Barbagallo et al., 2004;Daenen et al., 2004;Pérez et al., 2010). Jolly et al. (2014) also described the potential of non-Saccharomyces yeasts, including those belonging to the genus Pichia, to secrete β-glucosidase. ...
β-glucosidases are glycoside hydrolases that—particularly those from filamentous fungi—have been extensively explored in cellulose fiber saccharification and wine quality improvement. However, these enzymes from yeast have been poorly studied. In this study, an ethanol-glucose tolerant β-glucosidase that is secreted by Pichia guilliermondii (current name Meyerozyma guilliermondii) was purified and characterized. This enzyme exhibited an estimated molecular mass of 97 kDa and the highest activity between pH 3.5–5.5 and 55 °C. The β-glucosidase was also tolerant to acetone, ethanol, isopropanol, and methanol up to 30% and glucose at 1 M. It was also stable up to 55 °C for 80 min, maintaining 70% of its initial activity and in a wide pH range (pH 3–10). The enzyme exhibited 90–100% of its initial activity for 72 h at 20, 25, and 30 °C in presence of 10% ethanol at pH 3.5, which is a similar condition to winemaking. Studies that identify new enzymes and describe their purification are required for oenology applications. The β-glucosidase described herein is a promising candidate for use in the preparation of wine. Additionally, its tolerance to glucose is an important biochemical property that adds value to this enzyme and enables it to be used during the final saccharification process.
... Lors de la croissance de la levure Mp en milieu aéré, la production de l'enzyme βglycosidase peut augmenter jusqu'à 13 fois. Cette activité enzymatique est très importante pour libérer des composés aromatiques à partir de précurseurs non-volatils liés aux glycosides (Mendes Ferreira et al., 2001). Cette particularité a une implication significative lorsque les vins sont élaborés avec des raisins moins aromatiques. ...
The use of yeasts in industry is inseparable from their ability to be produced and dehydrated. This dehydration process causes various dysfunctions in yeast cells that affect their functionality and viability. In order to protect yeasts from dehydration, food additives are often used as emulsifiers and antioxidants. However, yeasts are able to produce naturally protective substances, such as glutathione (GSH) and trehalose (TRE). In this context, three non-Saccharomyces (NS) strains, belonging to the different genera and species Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans, were studied in this thesis. Despite the great interest aroused by their multiple agro-food applications, their dehydration resistance mechanisms associated with the synthesis of GSH and TRE, are currently unknown. This study is ultimately aimed at the formulation of new NS yeast dried strains without any food additives. In a first chapter, the impact of the “reference yeast” Saccharomyces cerevisiae dehydration in a pre-pilot fluidized bed has been correlated with the synthesis of GSH and TRE. It was possible to modulate the culture medium composition in order to optimize cell preservation before, during and after dehydration. In a second chapter, the previously defined conditions were applied to NS yeasts strains in order to understand the effects of dehydration on their microbial functionality. This study demonstrated that GSH plays an important role in NS yeasts protection, depending on the culture and dehydration conditions. In a third chapter, some oxidation resistance phenomena of the three NS strains were studied. It was clearly demonstrated that the susceptibility of cells to oxidative attack was dependent on culture-dehydration conditions and was yeast strain-dependent. Finally, in a fourth chapter, an in-depth biochemical study of the most dehydration-sensitive yeast strain, L. thermotolerans, was performed by synchrotron FTIR micro-spectroscopy. Cells grown in GSM (medium favoring the production of GSH), besides showing a better viability, showed a greater intensity in the spectral bands of lipids CH2 and CH3, associated with the plasma membrane fluidity. In addition, TSM grown cells (TSM is a medium favoring the production of TRE) exhibited a higher protein denaturation, suggested by the intensity of β-sheet peaks, and C=O (lipid oxides) bands correlated with lipid peroxidation. These data can explain the decreased of viability of this strain during production-dehydration process. The fundamental knowledge acquired in this study will be useful to obtain new dehydrated yeast strains without additives and with high performance. It will be useful also to improve the formulation and dehydration methods currently used in industry.
... Nowadays it is widely accepted that the selection of non-Saccharomyces strains through appropriate screening can positively influence the winemaking process (Padilla et al. 2016;Wang et al. 2016). In particular, non-Saccharomyces yeasts have several desired oenological characteristics that are absent in S. cerevisiae, such as the production of high levels of flavor compounds (Ferreira et al. 2001;Mendoza and Farìas 2010;Liu et al. 2016). Different studies have shown that in spontaneous fermentations, Saccharomyces and non-Saccharomyces yeasts do not passively coexist, on the contrary, they appear to interact. ...
In this study, the effect of sequential inoculation with non-Saccharomyces (Hanseniaspora guilliermondii) and Saccharomyces cerevisiae yeast on the distinctive characteristics of the Campanino white wine was investigated. For this purpose, three independent winemaking experiments were carried out on an industrial scale (batches A, B and C). In detail, the first one was carried out using the sequential inoculation technique while the other two, using a S. cerevisiae single-strain starter or no inoculation representing the control batches. Microbiological and chemical parameters and sensorial profiles of the wines were defined. Interestingly, the results showed that when sequential cultures (H. guilliermondii in a sequential mixture with S. cerevisiae) were used, a better wine aroma and quality was observed. More specifically, the wine obtained by sequential inoculation showed lower acetic acid values and enhanced volatile profiles than the wine from the control batches. Finally, sensorial analysis confirmed that the sequential cultures led to an improvement in wine flavour. Therefore, results suggest that the sequential inoculation using non-Saccharomyces and Saccharomyces yeast represents a biotechnological practice that can improve the quality features of traditional white wine. It has been shown for the first time that on an industrial scale H. guilliermondii could be used in sequential inoculum with S. cerevisiae in making white Campanino wine.
Graphical abstract
... The aromatic potential of grapes is influenced by the content of glycosides in wine or in juice. The splitting of glycosides that takes place in the course of processing of grapes causes a release of volatile and aromatic aglycones (Mendes Ferreira et al., 2001). ...
In this study effects of commercial yeast preparations on the aromatic profile of Sauvignon Blanc varietal wine were investigated. Grape juice was divided to 7 experimental variants and fermented spontaneously and using 6 commercial strains of Saccharomyces cerevisiae. In final wine samples, essential analytical parameters and selected aromatic compounds were analysed. The highest content of esters was found out in samples fermented by spontaneous micro‑flora; in this case, concentrations of ethyl hexanoate, ethyl octanoate and ethyl decanoate were 682 µg/L, 735 µg/L and 162 µg/L, respectively. The highest content of acetates was recorded in samples fermented by yeast Vulcaferm Sauvignon; concentrations of isoamyl acetate, 2‑phenylethyl acetate and isobutyl acetate were 7.8 mg/L, 244 µg/L and 137 µg/L, respectively. Yeast strain suitable for cold fermentation (Oenoferm Fredo) produced high amounts of ethyl esters and acetates. As far as the sensory evaluation was concerned, the best rating got the sample fermented by these yeasts; it showed a high degree of smell and flavour cleanness as well as a very good overall harmony.
... b-glucosidase activity has been well characterized in non-Saccharomyces strains belonging to the genera Brettanomyces, Candida, Debaryomyces, Hanseniospora, Hansenula, Kloeckera, Metschnikowia, Pichia, Saccharomycodes, Schizosaccharomyces and Zygosaccharomyces (Rosi et al. 1994;Mendes Ferreira et al. 2001;Barbagallo et al. 2004;Villena et al. 2007). In contrast, the presence of this enzymatic activity has been rarely assayed in the Saccharomyces genus (Mateo and Di Stefano 1997;Spagna et al. 2002;Hern andez et al. 2003;Fia et al. 2005;Daenen et al. 2008). ...
The aim of the present work was to screen a pool of 75 yeasts belonging to the species Saccharomyces cerevisiae and Saccharomyces uvarum in order to select the strains endowed with β‐glucosidase activity. The first screening was a qualitative assay based on chromogenic substrates (arbutin and esculin). The second screening was the quantitative evaluation of the β‐glucosidase activity via a p‐nitrophenyl‐β‐D‐glucopyranoside (p‐NPG) assay. The measurement was performed on three different cell preparations, including the extracellular compartment, the cell lysates and the whole cells. This study pointed out the high frequency of β‐glucosidase activity in S. uvarum strains. In particular, we retrieved three promising S. uvarum strains, CRY14, VA42 and GRAS14, featuring a high enzymatic activity, exploitable for winemaking.
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... All M. pulcherrima isolates, except Mp4 and Mp51, displayed remarkable β-glycosidase activity. Our results are in line with previous reports that verified that M. pulcherrima is one of the non-Saccharomyces species with higher potential to release grape-derived terpenes from their glycosylated precursors [48,71]. Also directly related to varietal aroma enrichment is β-lyase activity, which is involved in the release of volatile thiols from cysteinylated precursors [12]. ...
Some non-Saccharomyces yeasts, including Metschnikowia pulcherrima, have been proposed as selected starters due to their contribution for the overall aroma and chemical profiles of wines. In this work, we aimed to evaluate the genetic and phenotypic diversity of Metschnikowia pulcherrima strains isolated from different locations of Douro Wine Region, and to explore their potential as co-adjuncts of S. cerevisiae in alcoholic fermentation. For that purpose, a set of 64 M. pulcherrima isolates were used. Polymerase chain reaction (PCR) fingerprinting with M13 primers demonstrated to be an efficient tool in intraspecific discrimination of M. pulcherrima strains. No significant associations were found between genotypic profiles and either geographical origin or winery. The isolates were screened for their stress resistance ability (ethanol, SO2, chitosan, copper, H2O2, and Grape Juice Medium), aroma-related activities (resistance to 5, 5′, 5′′-trifluor-d, l-leucine and cerulenin and β-glycosidase, β-lyase and sulfite-reductase activities) as well as other relevant technological proprieties (protease activity and biogenic amines production). M. pulcherrima response to the different enological traits evaluated was greatly strain-dependent. The most discriminant features were the ability of the strains to grow in Grape-Juice Medium (GJM) and sulfite-reductase, and their β-lyase and protease activities. The enological potential of a selected M. pulcherrima strain in mixed-culture with S. cerevisiae was also assessed in natural grape-juice of a local variety, under two nitrogen regimes. M. pulcherrima proved to be promising for future industrial application as a co-starter, lowering ethanol, acetic acid and, reported here for the first time, lowering hydrogen sulfide levels in the wines.
... Saccharomyces cerevisiae shows some enzyme activity in different strains [47] but most studies demonstrate significantly higher enzyme production from non-Saccharomyces species [48][49][50][51]. ...
... Most research on β-glucosidases demonstrated intra-and extracellular activities in non-Saccharomyces yeasts. Selected species of Candida, Hanseniaspora, Pichia, Metschnikowia, Rhodotorula, and Trichosporon with high β-glucosidase could potentially contribute to different aromas in wine (Rosi et al. 1994;Fernández et al. 2000;Ferreira et al. 2001;Rodriguez et al. 2004;Wang et al. 2015;Hu et al. 2016). A Wickerhamomyces anomalus strain AS1 hydrolyzed a number of synthetic and natural glycosides under oenological conditions . ...
Production of wine from grape juice is predominantly the result of complex enzymatic reactions. The primary bioconversion of grape sugar to ethanol and CO2 by the yeast Saccharomyces cerevisiae is catalyzed by cytosolic enzymes. In spontaneous must fermentations, also strains of Saccharomyces bayanus or interspecies hybrids may dominate, probably because of better adaptation to specific environmental conditions. In addition, various enzymes released into the must influence the final composition, color, and sensory properties of wines. These enzymes originate from the grape itself, from epiphytic fungi like Botrytis cinerea, and from yeasts and bacteria associated with vineyards and wine cellars. Especially non-Saccharomyces yeasts, also called “wild” yeasts, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Wickerhamomyces, Zygosaccharomyces, Hanseniaspora, Kluyveromyces, and Metschnikowia, secrete different hydrolytic enzymes (esterases, lipases, glycosidases, glucanases, pectinases, amylases, proteases) which interact with grape compounds. Apart from yeasts, enzymes of lactic acid bacteria have a significant impact on wine quality.
... It is clear that the persistence of non-Saccharomyces yeasts is responsible for increased concentrations of the above-mentioned volatiles because such species can release high amounts of monoterpene alcohols or thiols from their non-volatile precursors (Mendes Ferreira et al. 2001;Zott et al. 2011). The differing ratios between non-Saccharomyces and S. cerevisiae yeasts in each fermentor caused considerable differ- ...
The spontaneous alcoholic fermentations of Moscato Bianco and Welschriesling must were carried out to retrieve indigenous yeasts. We confirmed that those fermentations, conducted with non-Saccharomyces and indigenous Saccharomyces cerevisiae yeasts, can generate high amounts of aroma compounds in wines. Consequently, two of the S. cerevisiae isolates were randomly chosen and further examined in Welschriesling and Sauvignon Blanc must for their ability and efficiency in performing alcoholic fermentation. Alcoholic fermentation with a commercial yeast strain was carried out for comparison. Indigenous isolates showed acceptable fermentation ability and efficiency. Moreover, Sauvignon Blanc produced with indigenous isolates contained significantly higher amounts of 3-mercaptohexyl acetate, linalool, geraniol and 2-phenylethanol and a significantly lower amount of 3-mercaptohexan-1-ol. Differences in Welschriesling wine were less striking but in this case indigenous isolates produced lower amounts of 3-mercaptohexan-1-ol and α-terpineol. Taken together, our results confirm that a suitable aromatic profile of wine can be produced with indigenous S. cerevisiae strains.
... Contrarily, cyclohexenoesculetin-β-glucoside allows the growth of gram-positive bacteria and does not suffer from the diffusion of the 8-hydroxyquinoline-iron complex throughout the plate [40]. The ferric salt may inhibit the growth of microorganisms and misinterpretation of the results may occur [37,41]. ...
Cellulose is the most abundant biomaterial in the biosphere and the major component of plant biomass. Cellulase is an enzymatic system required for conversion of renewable cellulose biomass into free sugar for subsequent use in different applications. Cellulase system mainly consists of three individual enzymes namely: endoglucanase, exoglucanase and β-glucosidases. β-Glucosidases are ubiquitous enzymes found in all living organisms with great biological significance. β-Glucosidases have also tremendous biotechnological applications such as biofuel production, beverage industry, food industry, cassava detoxification and oligosaccharides synthesis. Microbial β-glucosidases are preferred for industrial uses because of robust activity and novel properties exhibited by them. This review aims at describing the various biochemical methods used for screening and evaluating β-glucosidases activity from microbial sources. Subsequently, it generally highlights techniques used for purification of β-glucosidases. It then elaborates various biochemical and molecular properties of this valuable enzyme such as pH and temperature optima, glucose tolerance, substrate specificity, molecular weight, and multiplicity. Furthermore, it describes molecular cloning and expression of bacterial, fungal and metagenomic β-glucosidases. Finally, it highlights the potential biotechnological applications of β-glucosidases.
Climate change has the potential to jeopardize the sustainability of wine production in various geographical areas, primarily by affecting wine quality, and safety. Climate has a considerable influence on wine characteristics, which is based on an intricate interplay between water availability, temperature, plant material, and vineyard management. The primary effects can be summed up as follows: high alcohol content, high pH and low acidity, development of undesirable microorganisms, accumulation of mycotoxins, and biogenic amines (BAs) in wines.
Both yield and quality have been improved by selecting plant material and vineyard management methods based on the climatic conditions. However, because of climate change, several adaptation strategies have been proposed. For instance, to respond to higher temperatures, new varieties have been selected and farming management methods have been modified.
This chapter provides an overview of the main impacts of climate change on grape composition and wine quality. Furthermore, the possibility of utilizing microorganisms to mitigate the negative aspects of climate change is investigated in the final section.
Comprehensive yeast strain characterization is an important issue for the wine industry as market demands require controlled production of distinctive high-quality wines. Glycosides form an important reservoir of varietal grape wine aroma, and their hydrolysis into olfactory-active compounds essentially depends on the fermenting yeast genera and strains. Among the 14 Metschnikowia, Pichia, Torulaspora and 18 Saccharomyces spp., rapid screenings by agar plate and activity assay, including the substrates arbutin, cellobiose and p-nitrophenol-β-D-glucopyranoside, revealed the most glycosidase-active strains. In the novel co-fermentation setups, five selected non-Saccharomyces and a Saccharomyces strain were separated by a 14 kDa cut-off membrane, allowing respective viable cell counts but facilitating metabolite transfer. Chemical analysis focused on aroma glycosides, with extensive quantification by GC-MS with SIDA on the extracted and hydrolyzed compounds. Olfactory profiles obtained for the non-Saccharomyces wines demonstrated a significant impact of these yeasts, albeit mainly correlated with increased hydrolysis of monoterpene glycosides, and surpassed by a technical Aspergillus niger enzyme. While screenings of non-Saccharomyces strains indicated enhanced glucosidase activity under winemaking conditions, their effect was lower than expected and dominated by ester formation. Interestingly, Saccharomyces yeast cell vitality was increased via in co-fermentation, and non-Saccharomyces strains displayed extended viabilities with high ethanol tolerances.
The use of yeast starter cultures consisting of a blend of Saccharomyces cerevisiae and non-Saccharomyces yeasts has increased in recent years as a mean to address consumers’ demands for diversified wines. However, this strategy is currently limited by the lack of a comprehensive knowledge regarding the factors that determine the balance between the yeast-yeast interactions and their responses triggered in complex environments. Our previous studies demonstrated that the strain Hanseniaspora guilliermondii UTAD222 has potential to be used as an adjunct of S. cerevisiae in the wine industry due to its positive impact on the fruity and floral character of wines. To rationalize the use of this yeast consortium, this study aims to understand the influence of production factors such as sugar and nitrogen levels, fermentation temperature, and the level of co-inoculation of H. guilliermondii UTAD222 in shaping fermentation and wine composition. For that purpose, a Central Composite experimental Design was applied to investigate the combined effects of the four factors on fermentation parameters and metabolites produced. The patterns of variation of the response variables were analyzed using machine learning methods, to describe their clustered behavior and model the evolution of each cluster depending on the experimental conditions. The innovative data analysis methodology adopted goes beyond the traditional univariate approach, being able to incorporate the modularity, heterogeneity, and hierarchy inherent to metabolic systems. In this line, this study provides preliminary data and insights, enabling the development of innovative strategies to increase the aromatic and fermentative potential of H. guilliermondii UTAD222 by modulating temperature and the availability of nitrogen and/or sugars in the medium. Furthermore, the strategy followed gathered knowledge to guide the rational development of mixed blends that can be used to obtain a particular wine style, as a function of fermentation conditions.
Most of the glycosidic aroma precursors in grape consist of diglycosides such as rutinoside. Yeast glycosidases have been extensively studied but their activity is limited to monoglucosidic precursors. Yet, there is no study about application of glycosidases active on rutinoside in grape. In this study, we screened Bacillus isolates for their glycosidase activities on rutin and grape aroma precursors. One Bacillus amyloliquefaciens and one Bacillus licheniformis strains produced extracellular glycosidases, active on both rutin and para-nitrophenol-α-l-rhamnoside. The specific rutinosidic activities were 14 and 9.0 μkat/mg respectively, and supernatants were active on Muscat grape glycosidic aroma precursors. Both strains were able to release monoterpenoids and benzenoids. The most released monoterpenoid in grape pomace was neric acid, with 378 μg/kg and the most released benzenoid was 2-phenylethanol, with 189 μg/kg for B. licheniformis. These glycosidases are interesting tools to target rutinosidic glycosides in grape and can be applied as a new way of valorising grape pomace.
Fermentation is one of the most ancient forms of energy metabolism. Wine may involve two forms of alcoholic (yeast and grape cell) and several types of organic acid fermentation in its production. The chapter first begins with a basic outline of winemaking followed by an exploration of prefermentation practices: grape sorting, crushing, destemming, maceration, dejuicing, and pressing as well as clarification and adjustment techniques (sugar and acid contents, enzyme addition, decoloration, blending, sulfiting). Principal alcoholic fermentation is under the control of yeast action, notably but not exclusively by Saccharomyces cerevisiae. Its discussion includes the various types of fermentors that may be employed; the biochemical aspects of yeast fermentation; yeast taxonomy, ecology, inoculation, genetics, and breeding; the influences of grape constituents, additives, pesticide residues, and the physical environment; and slow and stuck fermentation. This is followed by a discussion of malolactic fermentation including the involvement of lactic acid bacteria and their ecology; sensory consequences; chemical, physical, and biologic influences; inoculation; and inhibition.
Organoleptic characteristics of wine, aroma and flavour, are the most important characteristics that define the differences among the vast array of products throughout the world. Yeasts have a prominent role in determining the chemical composition of wine by several mechanisms: by producing enzymes that transform neutral grape compounds into flavour active compounds (pre-fermentative aroma), secondly by producing many hundreds of flavour active, secondary metabolites (fermentative and post-fermentative aroma) and lastly by extracting flavour components from grape solids and by autolytic degradation of dead yeast cells. These reactions vary with the yeast species and strains contributing to the fermentation. This review aims to present an overview on major achievements of yeast role in the formation of wine flavour. Firstly we illustrate the yeast metabolic activities involved in wine aroma production in function of wine styles as well as the main factors affecting flavour quality of wine. Furthermore, the influence of starter cultures (single or mixed) on wine flavour is discussed. Finally, novel methodologies to select wine yeasts in function of their influence on wine aroma are also summarized.
In the past, Saccharomyces spp. yeasts were almost the only option for use in modern winemaking due to their unparalleled ability to metabolize all grape juice sugar into ethanol. For that reason, until some years ago, all commercial dry yeasts were Saccharomyces spp. For several years, non-Saccharomyces were forgotten at industrial level, and even some of them were considered as spoilage microorganisms. Non-Saccharomyces only played a significant role in limited productions that perform spontaneous fermentations following organic polities. However, during the last decade, several researchers have proved numerous non-Saccharomyces to be able to improve wine quality and to solve some modern enology challenges. Some of the factors that can improve are acidity, aromatic complexity, glycerol content, ethanol reduction, mannoproteins, anthocyanins, and polysaccharide concentrations. They can also decrease the concentrations of unwanted compounds that affect food safety, such as ochratoxin A, ethyl carbamate, and biogenic amines. Due to all those scientific advances, the main manufacturers have just started to commercialize dry non-Saccharomyces such as Torulaspora delbrueckii, Schizosaccharomyces pombe, Metschnikowia pulcherrima, Lachancea thermotolerans, and Pichia kluyveri. Other non-Saccharomyces species with special enology abilities such as Candida zemplinina, Kloeckera apiculata, Hanseniaspora vineae, Hanseniaspora uvarum, C. stellata, Kazachstania aerobia, or Schizosaccharomyces japonicus could follow a similar progress. The aim of the chapter is to show which are the main abilities and advantages of these non-Saccharomyces in modern winemaking.
Three hundred and seventeen strains representing 20 species of yeasts were screened for the presence of beta-glucosidase activity. All of the strains of the species Debaryomyces castellii, Deb. hansenii, Deb. polymorphus, Kloeckera apiculata and Hansenula anomala showed beta-glucosidase activity, but only one of 153 strains of Saccharomyces cerevisiae. The other species behaved differently, depending upon the strain. The strains that hydrolysed arbutin were checked to localize the beta-glucosidase activity. A strain of Deb. hansenii exhibited the highest exocellular activity and some wall-bound and intracellular activity. The beta-glucosidase synthesis from this yeast was enhanced by aerobic conditions of growth, was repressed by high glucose concentration (9%) and occurred during exponential growth. The optimum conditions for enzymatic preparations of Deb. hansenii were between pH 4.0 and 5.0 and 40 degrees C. A high concentration of ethanol and glucose did not reduce the enzymatic activity. The enzymatic preparations of Deb. hansenii released monoterpenols and other alcohols from a grape glycoside extract.
This chapter’s topics are diverse. They are grouped here to set the stage. We recommend reading this chapter first as you learn about and consider going into winemaking. An occasional rereading may be helpful to the practicing winemaker.
β-glucosidase activity of differentSaccharomycesstrains has been detected on the basis of its hydrolytic activity onpara-nitrophenyl-β-glucoside (pNPG) and terpene glycosides of Muscat wine. This enzymatic activity is induced by the presence of boundβ-glucose as the only carbon source in the medium and seems to be a characteristic of the yeast strain.β-glucosidase is associated with the cell wall and is found in the insoluble fraction obtained from lysed yeast cells.Saccharomyces β-glucosidase is quite glucose independent, so that its activity is reduced by about 40% in the presence of 200 g/l of glucose, but it is inhibited by about 50% with 5% ethanol in the medium; therefore, its technological use seems to be restricted to the first stages in the wine-making process.
I. ROSI, M. VINELLA AND P. DOMIZIO. 1994. Three hundred and seventeen strains representing 20 species of yeasts were screened for the presence of β-glucosidase activity. All of the strains of the species Debaryomyces castellii, Deb. hansenii, Deb. polymorphus, Kloeckera apiculata and Hansenula anomala showed β-glucosidase activity, but only one of 153 strains of Saccharomyces cerevisiae. The other species behaved differently, depending upon the strain. The strains that hydrolysed arbutin were checked to localize the β-glucosidase activity. A strain of Deb. hansenii exhibited the highest exocellular activity and some wall-bound and intracellular activity. The β-glucosidase synthesis from this yeast was enhanced by aerobic conditions of growth, was repressed by high glucose concentration (9%) and occurred during exponential growth. The optimum conditions for enzymatic preparations of Deb. hansenii were between pH 4.0 and 5.0 and 40d̀C. A high concentration of ethanol and glucose did not reduce the ezymatic activity. The enzymatic preparations of Deb. hansenii released monoterpenols and other alcohols from a grape glycoside extract.
Three enological yeast strains, belonging to the speciesDebaryomyces hansenii, Debaryomyces polymorphus, andSaccharomyces cerevisiae, characterized by an exocellular β-glucosidase activity, were examined for their ability to hydrolize a glycosidic extract from grape juice. The enzymatic preparations (culture supernant fluid) of the different yeasts released different amounts of terpenols such as linalol, α-terpineol, geraniol, nerol, citronellol and benzyl and 2-phenylethyl alcohol. The extent of release of the flavour compounds was related to yeast species. When an enzymatic preparation (concentrate culture supernatant) ofDebaryomyces hansenii was incubated with a wine containing glycosidic precursors, significant production of monoterpenols and benzyl and 2-phenylethyl alcohol was observed.
Synthetic neryl, geranyl, and linalyl β-D-glucopyranosides when hydrolyzed at pH 3.2 and pH 1.0 gave product distributions similar to those from natural monoterpene disaccharides of Vitis vinifera. At pH 3.2 linalool and α-terpineol were major products along with lesser amounts of nerol, 3,7-dimethyloct-1-ene-3,7-diol, 2,6,6-trimethyl-2-vinyltetrahydropyran, and hydrocarbons. At pH 1,1,4- and 1,8-cineoles, isomers of 2,2-dimethyl-5-(1-methylprop-1-enyl)tetrahydrofuran and 2-(5,5-dimethyltetrahydrofuran-2-yl)butan-2-ol, 1- and α-terpineols, and lesser quantities of γ- and 4-terpineols and Z-ocimenol were obtained. Of the model glucosides studied, the α-terpinyl derivative was distinct, giving only cyclic products at each pH. These studies rationalize the pattern of free monoterpenes at the linalool oxidation level, found in muscat juice, wine, and distillates.
The aromatic compounds of must and wine were extracted with a new rapid method using ultrasound. The new technique, showing good recovery, linearity and reproducibility for most of the compounds, together with rapidity and simplicity, has proven itself to be better than resin (C18) extraction. Suitable operating procedures (sample volume, temperature and extraction conditions) gave very good results which, coupled with shorter operating times, allows the method to be applied routinely, particularly for rapid screening in wine yeast selection.
Using a model system, the activities of α-L-arabinofuranosidase, β-glucosidase, and α-L-rhamonopyranosidase were determined
in 32 strains of yeasts belonging to the genera Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Hansenula, Kloeckera, Metschnikowia, Pichia, Saccharomyces, Torulaspora and Brettanomyces (10 strains); and seven strains of the bacterium Leuconostoc oenos. Only one Saccharomyces strain exhibited β-glucosidase activity, but several non-Saccharomyces yeast species showed activity of this enzyme. Aureobasidium pullulans hydrolyzed α-L-arabinofuranoside, β-glucoside, and α-L-rhamnopyranoside. Eight Brettanomyces strains had β-glucosidase activity. Location of enzyme activity was determined for those species with enzymatic activity.
The majority of β-glucosidase activity was located in the whole cell fraction, with smaller amounts found in permeabilized
cells and released into the growth medium. Aureobasidium pullulans hydrolyzed glycosides found in grapes.
Publisher Summary
This chapter focuses on the methods used for the isolation, maintenance, and identification of yeasts. Yeasts have been recovered from widely differing aquatic and terrestrial sources, as well as from the atmosphere. Many types of yeast occur widely, whereas some appear to be confined to restricted habitats. Yeasts seldom occur in the absence of either molds or bacteria. Consequently, selective techniques are often used for recovery of yeasts, employing media which permit the yeast to grow while suppressing molds and bacteria. The composition of such media is determined by the fact that yeasts are, as a rule, capable of developing at pH levels and water activities, which reduce or inhibit the growth of bacteria. Antibiotics may also be used to suppress bacteria. When yeasts are present in low numbers, their isolation may require enrichment using media and conditions which favor the growth of yeasts over other microorganisms. Yeast cultures are best maintained on a medium which contains glucose as the only source of carbon as this reduces the risk of changes in growth and fermentative patterns due to the selection of mutants. Many basidiomycetous yeasts do not survive well during prolonged storage on a glucose-peptone medium, although they grow well on it. Potato-dextrose agar is used when cultures of such yeasts are to be kept for a long time. The majority of yeasts may be stored at temperatures between 4 and 12° C and subcultured at intervals of 6 to 8 months. Yeasts such as Arxiozyma and Malassezia, may have to be subcultured every month.
Les levures et l’expression aromatique des vins blanc
- J.F. Laffort
- H. Romat
- P. Darriet
- J.F. Laffort
- H. Romat
- P. Darriet
Yeast with β-glycosidase activity: proprieties and possible application in winemaking processes
- E. Miklósy
- V. Pölös
- E. Miklósy
- V. Pölös
Etude et exploitation par voie enzymatique des pre Âcurseurs d'aro Ãmes du raisin de nature glycosidique Description of the b-glycosidase activity of wine yeast
- Y Z Gunata
- ±
- J F Laffort
- H Romat
- P Darriet
- ±
- J J Mateo
- Di Stefano
Gunata, Y.Z. (1994) Etude et exploitation par voie enzymatique des pre Âcurseurs d'aro Ãmes du raisin de nature glycosidique. ReÂvue des Oenologues 74, 22±27. Laffort, J.F., Romat, H. and Darriet, P. (1989) Les levures et l'expression aromatique des vins blanc. ReÂvue des Oenologues 53, 9±12. Mateo, J.J. and Di Stefano, R. (1997) Description of the b-glycosidase activity of wine yeast. Journal of Food Microbiology 14, 583±591.
Quanti®cation of glycosidase activities in selected yeasts and lactic acid bacteria Yeast with b-glycosidase activity: proprieties and possible application in winemaking processes Hydrolysis of grape glycosides by enological yeast b-glycosidases
- H Mcmahon
- B W Zoecklein
- K Fugelsang
- Y Jasinski
- E Âsy
- V Po Èlo Ès
McMahon, H., Zoecklein, B.W., Fugelsang, K. and Jasinski, Y. (1999) Quanti®cation of glycosidase activities in selected yeasts and lactic acid bacteria. Journal of Industrial Microbiology and Biotechnology 23, 198±203. Miklo Âsy, E. and Po Èlo Ès, V. (1995) Yeast with b-glycosidase activity: proprieties and possible application in winemaking processes. Acta Alimentaria 24, 167±180. Rosi, I., Domizio, P., Vinella, M. and Salicone, M. (1995) Hydrolysis of grape glycosides by enological yeast b-glycosidases. In Food Flavours: Generation, Analysis and Process In¯uence ed. Charalam-bous, G. pp. 1623±1635. Elsevier Science, Amsterdam, The Netherlands.
Yeast-growth during fermentation
- G H Fleet
- G M Heard
Fleet, G.H. and Heard, G.M. (1993) Yeast±growth during fermentation. In Wine Microbiology and Biotechnology ed. Fleet, G.H.
pp. 27±54. Chur, Switzerland: Harwood Academic Publishers.
Yeast: identi®cation program. Cambridge Micro Software Yeast: Characteristics and Identi®cation 2nd edn
- J A Barnett
- R W Payne
- D Yarrow
- J A Barnett
- R W Payne
- D Yarrow
- R L Baumes
- I Dugeley
- C Grassin
Barnett, J.A., Payne, R.W. and Yarrow, D. (1987) Yeast: identi®cation program. Cambridge Micro Software. Cambridge, UK: Cambridge University Press. Barnett, J.A., Payne, R.W. and Yarrow, D. (1990) Yeast: Characteristics and Identi®cation 2nd edn. Cambridge, UK: Cambridge University Press. Bayonove C., Gunata, Z., Sapis, J.C., Baumes, R.L., Dugeley, I. and Grassin, C. (1992) Augmentation des aro Ãmes dans le vin et utilization d'enzymes. ReÂvue des Oenologues 64, 15±18.
Yeast: Characteristics and Identi®cation 2nd edn
- J A Barnett
- R W Payne
- D Yarrow
Barnett, J.A., Payne, R.W. and Yarrow, D. (1990) Yeast: Characteristics
and Identi®cation 2nd edn. Cambridge, UK: Cambridge University
Press.
Yeast: identification program. Cambridge Micro Software
- J A Barnett
- R W Payne
- D Yarrow
Barnett, J.A., Payne, R.W. and Yarrow, D. (1987) Yeast: identi®cation
program. Cambridge Micro Software. Cambridge, UK: Cambridge
University Press.
Les levures et l'expression aromatique des vins blanc
- J F Laffort
- H Romat
- P Darriet
Laffort, J.F., Romat, H. and Darriet, P. (1989) Les levures et
l'expression aromatique des vins blanc. ReÂvue des Oenologues 53, 9±12.
Description of the b-glycosidase activity of wine yeast
- J J Mateo
- Di Stefano
Mateo, J.J. and Di Stefano, R. (1997) Description of the b-glycosidase
activity of wine yeast. Journal of Food Microbiology 14, 583±591.
Yeast with b-glycosidase activity: proprieties and possible application in winemaking processes
- E Miklo Âsy
- V Po Èlo Ès
Miklo Âsy, E. and Po Èlo Ès, V. (1995) Yeast with b-glycosidase activity:
proprieties and possible application in winemaking processes. Acta
Alimentaria 24, 167±180.