Optimization of a rapid method for studying the cellular location of beta-glucosidase activity in wine yeasts

Abstract

M . A R É V A L O V I L L E N A , J . F . Ú B E D A I R A N Z O , R . R . C O R D E R O O T E R O A N D A . I . B R I O N E S P É R E Z . 2005.Aims: To improve a method for determining b-glucosidase activity and to apply it in yeasts isolated from wine ecosystems from ÔLa ManchaÕ region and to know its cellular location. Methods and Results: A total of 82 wine yeasts were identified (PCR/RFLP) and evaluated for their b-glucosidase activity. First, they were qualitatively evaluated by growth on YNB cellobiose, the activity was quantified using different culture media, under aerobic and anaerobic conditions and cells after 24-72 h of growth. To study the location activity, five fractions were obtained (supernatant, whole cell, cell wall, cytosol and cell membrane). The enzymatic assays were optimized, being: growth in YP cellobiose for 72 h in aeration conditions and, after cell removing, enzyme analysis with 128 g l )1 of cellobiose as substrate, for 30 min at 30°C. The genus that displayed the greatest activity were Pichia, Hanseniaspora and Rhodotorula, and the activity was intracellular. Conclusions:The study showed that b-glucosidase activity was induced by the carbon source and was aerobic dependent. The non-Saccharomyces species displayed the greatest activity, which was intracellular and straindependent.Significance and Impact of the Study: This study developed a reliable method for screening b-glucosidase activity in yeasts isolated from wine ecosystems. This activity is very important in the release of monoterpenols from glycoside precursors for the enhancement of wine aromas.

Spontaneous must fermentation: Identification and biotechnological properties of wine yeasts

“…These were: polygalacturonase (Fernández- González, Úbeda, Vasudevan, Cordero Otero, & Briones, 2004); protease (Fernández- and b-glucosidase (Arévalo-Villena, Úbeda Iranzo, Cordero Otero, & Briones Pérez, 2005), urease, laccase, cinnamate decarboxylase (Briones, Ubeda, Cabezudo, & Martin-Alvarez, 1995) and b-glucanase (Santos, Francisco Rey, Conde, Villanueva, & Nombela, 1979). Strains with reported activity or enzyme crudes were used as positive control, while the non-inoculated substrate was used as negative control.…”

“…These yeasts, which display low fermentative capacity and low ethanol tolerance are also important for the final quality of wine. They are responsible for the oxidative reactions and could enhance wine flavor by increasing concentrations of the volatile compounds responsible for fruity aroma, through hydrolysis of aromatic precursors prompted by b-glucosidase enzyme activity and terpenes liberation (Arévalo-Villena et al, 2005).…”

Modulating Wine Pleasantness Throughout Wine-Yeast Co-Inoculation or Sequential Inoculation

“…Muscat, Gewurztraminer Citrus, floral, geranium [21] Linalool Muscat, Gewurztraminer Floral, lavender [21] Nerol Muscat Floral [21] Tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2,5-cis-2h-pyran (cis-rose oxide) Gewurztraminer Geranium oil [22] 3,6-Dimethyl-3a,4,5,7a-Tetrahydro-3h-1-Benzofuran-2-One Gewurztraminer Coconut, woody, sweet [23] 3-Isobutyl-2-Methoxypyrazines Sauvignon blanc Bell pepper [24,25] 4-Methyl-4-Mercaptopentan-2-One Sauvignon blanc Black currant [24,25] 3-Mercapto-1-Hexanol (R Isomer) Sauvignon blanc Grapefruit, citrus peel [24,25] 1,1,6-Trimethyl-1,2-Dihydronaphthalene Riesling Kerosene [26] 3-Mercapto-1-Hexanol (S Isomer) Semillon Passion fruit [27] Rotundone Shiraz Black pepper [28] During alcoholic fermentation, some yeast, mainly non-Saccharomyces yeasts, can release β-glucosidases that hydrolyze the glycosidic bonds of the odorless non-volatile glycoside linked forms of monoterpenes (geraniol, linalool, nerol, among others), releasing the odor compounds to the wine [29]. Volatile thiols that give Sauvignon blanc wines their characteristic aroma (bell pepper, black currant, grapefruit, and citrus peel) are not present in grape juice but occur in grape must as odorless, non-volatile, cysteine-bound conjugates.…”

Outlining a selection procedure for Saccharomyces cerevisiae isolated from grape marc to improve fermentation process and distillate quality

“…The enzyme activity was tested at two pH values: 5.5 that is the optimum value of yeast glycosidase and 3.5 that is the enological standard. Growth on cellobiose was performed as Villena et al (2005). All the assays were performed in triplicate.…”