<i>Tetrapisispora phaffii</i> killer toxin is a highly specific β-glucanase that disrupts the integrity of the yeast cell wall

Background. Killer yeasts have been used to combat contaminating wild yeasts in food, to control pathogenic fungi in plants, and in the medical field, to develop novel antimycotics for the treatment of human and animal fungal infections. Among these killer yeasts, Tetrapisispora phaffii (formerly known as Kluyveromyces phaffii) secretes a glycoprotein known as Kpkt that is lethal to spoilage yeasts under winemaking conditions. In the present study, the mode of action of Kpkt, and the specific damage produced by this toxin on sensitive yeasts is investigated. Results. The use of castanospermine, a β-glucanase inhibitor, demonstrated that β-glucanase activity is essential for the Kpkt killer activity in vivo. Accordingly, Kpkt has no killer activity on either sensitive yeast spheroplasts or whole sensitive cells in the presence of isosmothic medium (0.8 molar sorbitol). Kpkt induces ultrastructural modifications in the cell wall of sensitive strains, as shown by confocal microscopy, laser-scanning electron microscopy, and atomic force microscopy. The Kpkt killer action is mediated by the glucidic portion of the toxin. This, in turn, appears to be involved both in the stronger cytocidal activity and in the selectivity for the sensitive strain shown by Kpkt compared to laminarinase. Conclusion. Collectively, these data indicate that the mode of action of Kpkt is directed towards the disruption of cell-wall integrity, and that this is mediated by a highly specific β-glucanase activity. In this, Kpkt differs from other microbial β-glucanases that do not show killer activities

Assessment of Spontaneous Fermentation and Non-Saccharomyces Sequential Fermentation in Verdicchio Wine at Winery Scale

The use of non-Saccharomyces yeasts in sequential fermentation is a suitable biotechnological process to provide specific oenological characteristics and to increase the complexity of wines. In this work, selected strains of Lachancea thermotolerans and Starmerella bombicola were used in sequential fermentations with Saccharomyces cerevisiae and compared with spontaneous and pure S. cerevisiae fermentation trials in Verdicchio grape juice. Torulaspora delbrueckii together with the other two non-Saccharomyces strains (L. thermotolerans, S. bombicola) in multi-sequential fermentations was also evaluated. Wines, obtained under winery vinification conditions, were evaluated for their analytical and sensorial profile. The results indicated that each fermentation gave peculiar analytical and aromatic features of the final wine. L. thermotolerans trials are characterized by an increase of total acidity, higher alcohols and monoterpenes as well as citric and herbal notes. S. bombicola trials showed a general significantly high concentration of phenylethyl acetate and hexyl acetate and a softness sensation while multi-sequential fermentations showed a balanced profile. Spontaneous fermentation was characterized by the production of acetate esters (ethyl acetate and isoamyl acetate), citrus and herbal notes, and tannicity. The overall results indicate that multi-starter fermentations could be a promising tool tailored to the desired features of different Verdicchio wine styles

Starmerella bombicola influences the metabolism of Saccharomyces cerevisiae at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

<p>Abstract</p> <p>Background</p> <p>The use of a multistarter fermentation process with <it>Saccharomyces cerevisiae </it>and non-<it>Saccharomyces </it>wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of <it>S. cerevisiae </it>and immobilized <it>Starmerella bombicola </it>cells (formerly <it>Candida stellata</it>) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of <it>S. bombicola </it>on <it>S. cerevisiae</it>, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied.</p> <p>Results</p> <p>The presence of <it>S. bombicola </it>immobilized cells in a mixed fermentation trial confirmed an increase in fermentation rate, a combined consumption of glucose and fructose, an increase in glycerol and a reduction in the production of ethanol as well as a modification in the fermentation of by products. The alcoholic fermentation of <it>S. cerevisiae </it>was also influenced by <it>S. bombicola </it>immobilized cells. Indeed, Pdc1 activity in mixed fermentation was lower than that exhibited in pure culture while Adh1 activity showed an opposite behavior. The expression of both <it>PDC1 </it>and <it>ADH1 </it>genes was highly induced at the initial phase of fermentation. The expression level of <it>PDC1 </it>at the end of fermentation was much higher in pure culture while <it>ADH1 </it>level was similar in both pure and mixed fermentations.</p> <p>Conclusion</p> <p>In mixed fermentation, <it>S. bombicola </it>immobilized cells greatly affected the fermentation behavior of <it>S. cerevisiae </it>and the analytical composition of wine. The influence of <it>S. bombicola </it>on <it>S. cerevisiae </it>was not limited to a simple additive contribution. Indeed, its presence caused metabolic modifications during <it>S. cerevisiae </it>fermentation causing variation in the gene expression and enzymatic activity of alcohol deydrogenase and pyruvate decarboxilase.</p

Improved Saccharomyces cerevisiae Strain in Pure and Sequential Fermentation with Torulaspora delbrueckii for the Production of Verdicchio Wine with Reduced Sulfites

none4The application of yeast strains that are low producers of sulfur compounds is actually required by winemakers for the production of organic wine. This purpose could be satisfied using a native Saccharomyces cerevisiae strain improved for oenological aptitudes. Moreover, to improve the aromatic complexity of wines, sequential fermentations carried out with S. cerevisiae/non-Saccharomyces yeast is widely used. For these reasons, in the present work an improved native S. cerevisiae low producer of sulfite and sulfide compounds was evaluated in pure and in sequential fermentation with a selected Torulaspora delbrueckii. Additionally, the influence of grape juices coming from three different vintages under winery conditions was evaluated. In pure fermentation, improved native S. cerevisiae strain exhibited a behavior related to vintage, highlighting that the composition of grape juice affects the fermentation process. In particular, an increase in ethyl octanoate (vintage 2017) and phenyl ethyl acetate (vintage 2018) was detected. Moreover, isoamyl acetate was highly consistent and could be a distinctive aroma of the strain. The sequential fermentation T. delbrueckii/S. cerevisiae determined an increase in aroma compounds such as phenyl ethyl acetate and ethyl hexanoate. In this way, it was possible to produce Verdicchio wine with reduced sulfites and characterized by a peculiar aromatic tasteopenAgarbati, Alice; Canonico, Laura; Comitini, Francesca; Ciani, MaurizioAgarbati, Alice; Canonico, Laura; Comitini, Francesca; Ciani, Maurizi

A fast and simple method for wild yeast flora detection in winemaking

Francesca Comitini, Mariza Stringini, Manuela Taccari, Maurizio CianiDipartimento S.A.I.F.E.T. sez. di Microbiologia Alimentare, Industriale e Ambientale, Università Politecnica delle Marche, Ancona, ItalyAbstract: An easy technique for a fast determination of wild yeast population-colonizing grape must before fermentation is described. The mathematical relationship between viable cell number and oxygen consumption rate was determined using a simple pO2 electrode chamber. This relation was determined in pure cultures belonging to six yeast species related to wine environment and in natural samples of grape must collected at the time of the grape delivery in the wineries. Results indicated a significant relationship between oxygen consumption rate and viable cell count of the wine yeast species tested. The evaluation of natural grape must samples indicated that the presence at pre-fermentative of wide contaminant yeast flora at a level commonly believed responsible for uncontrolled microbiological process in winemaking (>106 CFU ml−1), was easily detected. Since the results are available in a short time, this method could be profitable used to detect the presence of contaminant level of wild yeasts reducing the risk of uncontrolled start fermentation that could compromise the quality of final product.Keywords: wild yeasts contamination, oxygen consumption, O2 prob

Saccharomyces and Non-Saccharomyces Starter Yeasts

This chapter describes the importance of yeast in beer fermentation. Initially, the differences between Saccharomyces cerevisiae and Saccharomyces pastorianus in the production of “ale” and “lager” beers are analyzed. Then, the relationships between beer nutrients and yeast growth are discussed, with special emphasis on the production of the flavor compounds. The impact of the wort composition on flocculation is also discussed. Furthermore, conventional approaches to starter yeast selection and the development of genetically modified microorganisms are analyzed. Recent discoveries relating to the use of S. cerevisiae strains isolated from different food matrices (i.e., bread and wine) and the potential for the use of non-Saccharomyces starter strains in beer production are discussed. A detailed review of the selection of starter strains for the production of specialty beers then follows, such as for gluten-free beers and biologically aged beers. Yeast recovery from top-cropping and bottom-cropping systems and the methodologies and issues in yeast propagation in the laboratory and brewery (i.e., re-pitching) are also analyzed. Finally, the available commercial preparations of starter yeast and the methods to evaluate yeast viability prior to inoculation of the must are analyzed

improvement of bioremediation performance for the degradation of petroleum hydrocarbons in contaminated sediments

Microcosm bioremediation strategies were applied to sediments contaminated with hydrocarbons. Experiments were performed in aerobic conditions in a single-step treatment and in a two-step anaerobic-aerobic treatment. In aerobic conditions, either inorganic nutrients or composts were added to the microcosms, while, in the first anaerobic phase of the two-step experiment, acetate and/or allochthonous sulfate-reducing bacteria were used. After the treatment under anaerobic conditions, samples were exposed to aerobic conditions in the presence of compost. In the aerobic treatments, 81% hydrocarbon biodegradation was observed after 43 days in the presence of inorganic nutrients. In aerobic conditions in the presence of mature compost, hydrocarbon biodegradation was 51% after 43 days of treatment, whereas it was 47% after 21 days with fresh compost. The two-step experiment allowed us to obtain a hydrocarbon degradation of 91%, after a first anaerobic step with an inoculum of sulfate-reducing prokaryotes

Multistarter fermentation of high sugar musts

Background and Aims The possibility to decrease wine volatile acidity (VA) is an important aspect in wine production. This applies in particular to wines that are produced from musts with high sugar concentration, where the osmotic pressure promotes an increase in acetic acid production by Saccharomyces cerevisiae. This study aimed to identify suitable yeast strains and fermentation temperature to undertake the alcoholic fermentation of high sugar musts. Methods and Results To lower VA during fermentation of high sugar musts, two non‐Saccharomyces yeast strains, Zygotorulaspora florentina and Starmerella bacillaris, were used in multistarter fermentations with S. cerevisiae at a fermentation temperature of 14 and 20°C. The fermentation temperature influenced the yeast behaviour and the composition of the two mixed fermentations. Conclusions Independent of fermentation temperature, the mixed fermentations with Z. florentina performed best to reduce VA. Significance of the Study Mixed fermentations with the non‐Saccharomyces yeast strains Z. florentina and S. bacillaris may represent a valuable approach for the fermentation of high sugar musts

Lentil Fortification and Non-Conventional Yeasts as Strategy to Enhance Functionality and Aroma Profile of Craft Beer

During the last few years, consumer demand has been increasingly oriented to fermented foods with functional properties. This work proposed to use selected non-conventional yeasts (NCY) Lachancea thermotolerans and Kazachstania unispora in pure and mixed fermentation to produce craft beer fortified with hydrolyzed red lentils (HRL). For this, fermentation trials using pils wort (PW) and pils wort added with HRL (PWL) were carried out. HRL in pils wort improved the fermentation kinetics both in mixed and pure fermentations without negatively affecting the main analytical characters. The addition of HRL determined a generalized increase in amino acids concentration in PW. L. thermotolerans and K. unispora affected the amino acid profile of beers (with and without adding HRL). The analysis of by-products and volatile compounds in PW trials revealed a significant increase of some higher alcohols with L. thermotolerans and ethyl butyrate with K. unispora. In PWL, the two NCY showed a different behavior: an increment of ethyl acetate (K. unispora) and beta-phenyl ethanol (L. thermotolerans). Sensory analysis showed that the presence of HRL characterized all beers, increasing the perception of the fruity aroma in both pure and mixed fermentation

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 winewith high flavour complexity or distinctive characters. This has been possible through exploiting the particular oenological features of somenon-Saccharomyces yeast strains, and the effects that derive fromtheir 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 varietie