Paola Domizio

Selected non-Saccharomyces wine yeasts in controlled multistarter fermentations with Saccharomyces cerevisiae

Non-Saccharomyces yeasts are metabolically active during spontaneous and inoculated must fermentations, and by producing a plethora of by-products, they can contribute to the definition of the wine aroma. Thus, use of Saccharomyces and non-Saccharomyces yeasts as mixed starter cultures for inoculation of wine fermentations is of increasing interest for quality enhancement and improved complexity of wines. We initially characterized 34 non-Saccharomyces yeasts of the genera Candida, Lachancea (Kluyveromyces), Metschnikowia and Torulaspora, and evaluated their enological potential. This confirmed that non-Saccharomyces yeasts from wine-related environments represent a rich sink of unexplored biodiversity for the winemaking industry. From these, we selected four non-Saccharomyces yeasts to combine with starter cultures of Saccharomyces cerevisiae in mixed fermentation trials. The kinetics of growth and fermentation, and the analytical profiles of the wines produced indicate that these non-Saccharomyces strains can be used with S. cerevisiae starter cultures to increase polysaccharide, glycerol and volatile compound production, to reduce volatile acidity, and to increase or reduce the total acidity of the final wines, depending on yeast species and inoculum ratio used. The overall effects of the non-Saccharomyces yeasts on fermentation and wine quality were strictly dependent on the Saccharomyces/non-Saccharomyces inoculum ratio that mimicked the differences of fermentation conditions (natural or simultaneous inoculated fermentation).

Outlining a future for non-Saccharomyces yeasts: selection of putative spoilage wine strains to be used in association with Saccharomyces cerevisiae for grape juice fermentation.

The use of non-Saccharomyces yeasts that are generally considered as spoilage yeasts, in association with Saccharomyces cerevisiae for grape must fermentation was here evaluated. Analysis of the main oenological characteristics of pure cultures of 55 yeasts belonging to the genera Hanseniaspora, Pichia, Saccharomycodes and Zygosaccharomyces revealed wide biodiversity within each genus. Moreover, many of these non-Saccharomyces strains had interesting oenological properties in terms of fermentation purity, and ethanol and secondary metabolite production. The use of four non-Saccharomyces yeasts (one per genus) in mixed cultures with a commercial S. cerevisiae strain at different S. cerevisiae/non-Saccharomyces inoculum ratios was investigated. This revealed that most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces, and which are considered detrimental to wine quality, do not reach threshold taste levels in these mixed fermentations. On the other hand, the analytical profiles of the wines produced by these mixed cultures indicated that depending on the yeast species and the S. cerevisiae/non-Saccharomyces inoculum ratio, these non-Saccharomyces yeasts can be used to increase production of polysaccharides and to modulate the final concentrations of acetic acid and volatile compounds, such as ethyl acetate, phenyl-ethyl acetate, 2-phenyl ethanol, and 2-methyl 1-butanol.

Potential spoilage non-Saccharomyces yeasts in mixed cultures with Saccharomyces cerevisiae

With the aim of exploring the possibility to improve wine quality through the utilization of wine-related yeasts generally considered as spoilage, mixed cultures of Saccharomyces cerevisiae with Hanseniaspora osmophila, Pichia fermentans, Saccharomycodes ludwigii and Zygosaccharomyces bailii were inoculated in grape juice. All the fermentations got to completion and most of the compounds normally produced at high concentrations by pure cultures of non-Saccharomyces yeasts, and considered detrimental for wine quality, did not reach the threshold taste level in mixed fermentations with S. cerevisiae. Interestingly, the association of S. cerevisiae with P. fermentans, S. ludwigii and Z. bailii produced significant increases in the production of polysaccharides as compared to pure cultures of S. cerevisiae. Since polysaccharides improve wine taste and body, and exert positive effects on aroma persistence and protein and tartrate stability, a possible use for these yeasts can be envisaged in mixed starter cultures with S. cerevisiae for the enhancement of the final quality of wine.

Extraction and immobilization in one step of two β-glucosidases released from a yeast strain of Debaryomyces hansenii

Abstract An extracellular, constitutive, and nonglucose repressed β-glucosidase from a yeast strain of Debaryomyces hansenii was purified and immobilized using a one-step procedure on hydroxyapatite (HTP). Analysis of purified enzyme gave two bands both on SDS gel electrophoresis, native gel electrophoresis, and capillary electrophoresis. The two bands on SDS gels were positive for carbohydrate staining. Their apparent molecular mass was estimated to be 122 and 96 kDa with carbohydrates, and 109 and 81 kDa after carbohydrate removal, respectively. Amino acid analysis of electroblotted bands revealed that the n -terminus was blocked in both cases. Gel slices corresponding to the two bands, as obtained after native gel electrophoresis, were found to be reactive when incubated separately with p-nitro-phenyl-β- d -glucopyranoside (pNPG) as substrate. The Km of the two forms coeluted from HTP in the same fractions was 3.68 ± 0.06 m m . The optimum pH was 5. The immobilized enzyme exhibited a lower activity than the purified free enzymes, but both were much more stable than the enzymes in cell-free supernatant. The two enzyme isoforms in the mixture were only active against few glycosides with β-linkage configuration. Since the HTP-bound enzyme was found to be active, stable, easily separable from the substrate, and reusable, it could be potentially used in its immobilized form for the release of specific-bound aroma in wine and fruit juices.

Determination of 2,3-butanediol in high and low acetoin producers of Saccharomyces cerevisiae wine yeasts by automated multiple development (AMD)

P. ROMANO, G. SUZZI, V. BRANDOLINI, E. MENZIANI AND P. DOMIZIO. 1996. High performance thin layer chromatography with automated multiple development was used to determine 2,3‐butanediol levels in wine produced by high and low acetoin‐forming strains of Saccharomyces cerevisiae. An inverse correlation between acetoin and 2,3‐butanediol content was found suggesting a leaky mutation in acetoin reductase of the low 2,3‐butanediol producing strains.

Controlled mixed fermentation at winery scale using Zygotorulaspora florentina and Saccharomyces cerevisiae.

Over the last few years the use of multi-starter inocula has become an attractive biotechnological practice in the search for wine with high flavour complexity or distinctive characters. This has been possible through exploiting the particular oenological features of some non-Saccharomyces yeast strains, and the effects that derive from their specific interactions with Saccharomyces. In the present study, we evaluated the selected strain Zygotorulaspora florentina (formerly Zygosaccharomyces florentinus) in mixed culture fermentations with Saccharomyces cerevisiae, from the laboratory scale to the winery scale. The scale-up fermentation and substrate composition (i.e., white or red musts) influenced the analytical composition of the mixed fermentation. At the laboratory scale, mixed fermentation with Z. florentina exhibited an enhancement of polysaccharides and 2-phenylethanol content and a reduction of volatile acidity. At the winery scale, different fermentation characteristics of Z. florentina were observed. Using Sangiovese red grape juice, sequential fermentation trials showed a significantly higher concentration of glycerol and esters while the sensorial analysis of the resulting wines showed higher floral notes and lower perception of astringency. To our knowledge, this is the first time that this yeasts association has been evaluated at the winery scale indicating the potential use of this mixed culture in red grape varieties.

Cell-recycle batch fermentation using immobilized cells of flocculent Saccharomyces cerevisiae wine strains

Five, highly flocculeng strains of Saccharomyces cerevisiae, isolated from wine, were immobilized in calcium alginate beads to optimize primary must fermentation. Three cell-recycle batch fermentations (CRBF) of grape musts were performed with the biocatalyst and the results compared with those obtained with free cells. During the CRBF process, the entrapped strains showed some variability in the formation of secondary products of fermentation, particularly acetic acid and acetaldehyde. Recycling beads of immobilized flocculent cells is a good approach in the development and application of the CRBF system in the wine industry.

A culture-independent PCR-based method for the detection of Lachancea thermotolerans in wine

When inoculated in association with Saccharomyces cerevisiae, the yeast Lachancea thermotolerans determines a reduction of volatile acidity and an increase in the production of glycerol, 2-phenylethanol, and polysaccharides. Moreover, L. thermotolerans is a natural L-lactic acid producer, thus it contributes to wine acidification and microbiological stabilization. In view of its utilization in winemaking, a culture-independent PCR-based method was developed for the detection of L. thermotolerans during wine fermentations. This method, which utilizes species-specific PCR primer pairs that anneal to intron 2 of the mitochondrial COX1 gene, is rapid and reliable, and detects L. thermotolerans in wine at 104 cells/ml and with a S. cerevisiae/L. termotholerans ratio of 1,000/1.

Impact of mother sediment on yeast growth, biodiversity, and ethanol production during fermentation of Vinsanto wine

The aim of this study was to determine the impact of Vinsanto mother sediment both on the growth and biodiversity of the yeast microflora and on the production of ethanol under natural and inoculated fermentation of Vinsanto wines. To achieve this ten fermentation trials were carried out in 50-L barrels, five without added mother sediment and five with. Moreover, eight of the ten barrels were inoculated with four Saccharomyces cerevisiae wine strains, while the remaining two barrels were not inoculated and were used as controls to study the behaviour of the natural yeast microflora in the absence and presence of mother sediment. The counts of viable yeasts at three different sampling times indicated that the mother sediment had a positive influence on yeast growth and persistence during fermentation. Molecular characterization of the Saccharomyces type colonies isolated after three months of fermentation showed that the addition of mother sediment had no effects on the dominance of the wine starters. In contrast, the mother sediment had a positive influence on the biodiversity of the spontaneous S. cerevisiae yeasts. Moreover, possibly due to its content of fatty acids and sterols and other nutrients, the addition of mother sediment also showed a positive effect on the fermentative activities of wine yeasts as measured by their ethanol production.

An integrated bioprocess to recover bovine milk oligosaccharides from colostrum whey permeate

A major challenge in isolating oligosaccharides from dairy streams is to enrich oligosaccharides while simultaneously reducing the content of simple sugars (mono- and disaccharides) that do not possess the desired prebiotic functions. An integrated approach based on optimized conditions that favor maximum lactose hydrolysis, monosaccharide fermentation and oligosaccharides recovery by nanofiltration was developed. Upon complete lactose hydrolysis and fermentation of the monosaccharides by yeast, nanofiltration of fermented whey permeate from colostrum enabled the recovery of 95% of the oligosaccharides at high purity. While the number of commercially available standards has limited the quantification of only a few sialylated oligosaccharides, the application of both high performance anion-exchange chromatography with pulsed amperometric detection and mass spectrometry provided a complete profile of the final product. Approximately 85% of the oligosaccharides in the final concentrate were sialylated, with the remainder being neutral.