Non-Saccharomyces yeast derivatives: Characterization of novel potential bio-adjuvants for the winemaking process.

Winemakers have access to a diverse range of commercially available Inactivated Dry Yeast Based products (IDYB) from various companies and brand names. Among these, thermally inactivated dried yeasts (TIYs) are utilized as yeast nutrients during alcoholic fermentation, aiding in the rehydration of active dry yeasts and reducing ochratoxin A levels during wine maturation and clarification. While IDYB products are generally derived from Saccharomyces spp., this study investigates into the biodiversity of those deriving from non-Saccharomyces for potential applications in winemaking. For that S. cerevisiae and non-Saccharomyces TIYs were produced, characterized for nitrogen and lipid content using FT-NIR spectroscopy, and applied in a wine-like solution (WLS) for analyzing and quantifying released soluble compounds. The impact of TIYs on oxygen consumption was also assessed. Non-Saccharomyces TIYs exhibited significant diversity in terms of cell lipid composition, and amount, composition, and molecular weight of polysaccharides. Compared to that of S. cerevisiae, non-Saccharomyces TIYs released notably higher protein amounts and nHPLC-MS/MS-based shotgun proteomics highlighted the release of cytosolic proteins, as expected due to cell disruption during inactivation, along with the presence of high molecular weight cell wall mannoproteins. Evaluation of antioxidant activity and oxygen consumption demonstrated significant differences among TIYs, as well as variations in GSH and thiol contents. The Principal Component Analysis (PCA) results suggest that oxygen consumption is more closely linked to the lipid fraction rather than the glutathione (GSH) content in the TIYs. Overall, these findings imply that the observed biodiversity of TIYs could have a significant impact on achieving specific oenological objectives.

Addition of Organic Acids to Base Wines: Impacts on the Technological Characteristics and the Foam Quality of Sparkling Wines.

Climate change is causing a significant decrease in the total acidity of grapes and related wines. This represents a serious issue for sparkling wine production. Consequently, before the second fermentation, the acidification of base wines is often necessary. However, the impacts of the most important organic acids on the foam properties of sparkling wines are not yet well known. The impacts of the addition of tartaric, malic, citric, and lactic acid on the quality of Falanghina and Bombino sparkling wines were evaluated. Analyses were performed soon after the second fermentation and one year after aging sur lees. The addition of each different organic acid to the two base wines resulted in significant changes in the sparkling wines not only in terms of pH, titratable acidity, and buffering capacity but also in the content of total amino acids and, in some cases, in the height of the foam and its stability over time. For both grape varieties, acidified wines showed a lower content of total amino acids in comparison with the control wines. The addition of lactic acid determined a higher persistency of the foam even after one year of aging sur lees only in Falanghina wines. The results obtained herein highlight the importance of organic acids and the pH of the base wines for the content of amino acids in sparkling wines. No strict correlation between organic acid addition and the foamability of wines was observed.

Chemical Characterization, Sensory Definition and Prediction Model of the Cider Dryness from New York State Apples.

Cider is a fermented drink obtained from apple juice. As a function of the used apple cultivar, cider can be classified in four different categories (dry, semi-dry, semi-sweet, sweet), distinguished by the attribute of "dryness," which reflects the sweetness and softness perceived. The dryness level is defined by scales (IRF, NYCA scales) based on the residual sugar, titratable acidity and tannin contents. Despite some adjustments, these scales show limitations in the prediction of actual perceived dryness, as they cannot consider the complicated interrelation between combined chemical compounds and sensory perception. After defining the perceived sensory dryness and its sensory description by using the quantitative descriptive analysis (QDA) method, a multivariate approach (PLS) was applied to define a predictive model for the dryness and to identify the chemical compounds with which it was correlated. Three models were developed, based on three different sets of chemical parameters, to provide a method that is easily applicable in the ordinary production process of cider. The comparison between the predicted rating and the relative scales scores showed that the models were able to predict the dryness rating in a more effective way. The multivariate approach was found to be the most suitable to study the relation between chemical and sensory data.

Phormidium ambiguum and Leptolyngbya ohadii Exopolysaccharides under Low Water Availability.

Cyanobacteria can cope with various environmental stressors, due to the excretion of exopolysaccharides (EPS). However, little is known about how the composition of these polymers may change according to water availability. This work aimed at characterizing the EPS of Phormidium ambiguum (Oscillatoriales; Oscillatoriaceae) and Leptolyngbya ohadii (Pseudanabaenales; Leptolyngbyaceae), when grown as biocrusts and biofilms, subject to water deprivation. The following EPS fractions were quantified and characterized: soluble (loosely bound, LB) and condensed (tightly bound, TB) for biocrusts, released (RPS), and sheathed in P. ambiguum and glycocalyx (G-EPS) in L. ohadii for biofilms. For both cyanobacteria upon water deprivation, glucose was the main monosaccharide present and the amount of TB-EPS resulted was significantly higher, confirming its importance in these soil-based formations. Different profiles of monosaccharides composing the EPSs were observed, as for example the higher concentration of deoxysugars observed in biocrusts compared to biofilms, demonstrating the plasticity of the cells to modify EPS composition as a response to different stresses. For both cyanobacteria, both in biofilms and biocrusts, water deprivation induced the production of simpler carbohydrates, with an increased dominance index of the composing monosaccharides. The results obtained are useful in understanding how these very relevant cyanobacterial species are sensitively modifying the EPS secreted when subject to water deprivation and could lead to consider them as suitable inoculants in degraded soils.

Industrial drying for agrifood by-products re-use: Cases studies on pomegranate peel (Punica granatum L.) and stoned olive pomace (pâtè, Olea europaea L.).

The effect of industrial drying processes on phenols and polysaccharides of olive pomace (pâté) and pomegranate peel was studied, with the aim to re-use pomegranate and olive oil by-products. Pomegranate peel (Wonderful and G1 varieties) was oven-dried at different temperatures, taking into account peel thickness and size. Pâté was freeze-dried and oven dried at 50-110 °C, at lab scale; then, an industrial drying system (150 °C) was compared to freeze-drying. All dried samples were analyzed in terms of phenolic and polysaccharides compounds. Drying at room temperature of small pieces of pomegranate peel guaranteed the highest humidity removal and recovery of phenols. Sugar analysis, DLS and 1H NMR confirmed that polysaccharide fractions were not significantly affected by the highest drying temperatures (42 °C for pomegranate, 150 °C for pâté). The two drying procedures at large scale were suitable for avoiding degradation of phenols, maintaining the same profiles of the corresponding freeze-dried samples.

A Methodological Approach to Assess the Effect of Organic, Biodynamic, and Conventional Production Processes on the Intrinsic and Perceived Quality of a Typical Wine: The Case Study of Chianti DOCG.

The aim of this study was to propose a methodological approach to evaluate the impact of the organic, biodynamic, and conventional production processes on the intrinsic and perceived quality of a typical wine. For this purpose, fourteen commercial Chianti DOCG wines from the 2016 harvest were selected based on the type of production management. A survey was set up to get winemaking information from the estate's producer of the wines to estimate the carbon dioxide production under the three types of management. The eligibility, identity, and style properties (the intrinsic quality) of the wines were defined. A group of 45 experts evaluated the differences between wines by the Napping test and rated their typicality (perceived quality). The organic and biodynamic management showed a lower level of estimated values of carbon dioxide production. The overall statistical elaboration of the chemical and sensory data highlighted that the registered differences of the intrinsic, perceived quality, and typicality level of the respective wines, did not depend on the type of management. The comparison of the three kinds of wine by SIMCA modeling, put in evidence that the conventional ones showed a greater homogeneity regarding chemical composition, sensory characteristics, and typicality.

Protection of Wine from Protein Haze Using Schizosaccharomyces japonicus Polysaccharides.

Nowadays commercial preparations of yeast polysaccharides (PSs), in particular mannoproteins, are widely used for wine colloidal and tartrate salt stabilization. In this context, the industry has developed different processes for the isolation and purification of PSs from the cell wall of Saccharomyces cerevisiae. This yeast releases limited amounts of mannoproteins in the growth medium, thus making their direct isolation from the culture broth not economically feasible. On the contrary, Schizosaccharomyces japonicus, a non-Saccharomyces yeast isolated from wine, releases significant amounts of PSs during the alcoholic fermentation. In the present work, PSs released by Sch. japonicus were recovered from the growth medium by ultrafiltration and their impact on the wine colloidal stability was evaluated. Interestingly, these PSs contribute positively to the wine protein stability. The visible haziness of the heat-treated wine decreases as the concentration of added PSs increases. SDS-PAGE Gel electrophoresis results of the haze and of the supernatant after the heat stability test are consistent with the turbidity measurements. Moreover, particle size distributions of the heat-treated wines, as obtained by Dynamic Light Scattering (DLS), show a reduction in the average dimension of the protein aggregates as the concentration of added PSs increases.

Yeast killer toxins: from ecological significance to application.

Killer toxins are proteins that are often glycosylated and bind to specific receptors on the surface of their target microorganism, which is then killed through a target-specific mode of action. The killer phenotype is widespread among yeast and about 100 yeast killer species have been described to date. The spectrum of action of the killer toxins they produce targets spoilage and pathogenic microorganisms. Thus, they have potential as natural antimicrobials in food and for biological control of plant pathogens, as well as therapeutic agents against animal and human infections. In spite of this wide range of possible applications, their exploitation on the industrial level is still in its infancy. Here, we initially briefly report on the biodiversity of killer toxins and the ecological significance of their production. Their actual and possible applications in the agro-food industry are discussed, together with recent advances in their heterologous production and the manipulation for development of peptide-based therapeutic agents.

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.

Purification of caprine oligosaccharides at pilot-scale.

The purification of caprine milk oligosaccharides (COS) by membrane filtration has been hampered by the low concentration of target COS and high concentration of lactose. In addition, their molecular weight proximity hinders the recovery of a COS fraction with high degree of purity and recovery yield. In this work, the recovery of a high purity COS concentrate was obtained by the optimization of an integrated approach including complete lactose hydrolysis, fermentation of the resulting monosaccharides and nanofiltration. All carbohydrates were quantified using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC PAD). Defatted goat whey was ultrafiltered with discontinuous diafiltrations to increase the recovery of COS in the whey permeate which was then subsequently concentrated by nanofiltration. COS recovery yields of 75% with negligible amounts of monosaccharides (0.3% of the initial amount of lactose in the whey permeate) were achieved. A final retentate containing 67.6 and 34.4% of acidic and neutral oligosaccharides respectively was obtained from caprine milk.

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.

Lachancea thermotolerans and Saccharomyces cerevisiae in simultaneous and sequential co-fermentation: a strategy to enhance acidity and improve the overall quality of wine.

In the last few years there is an increasing interest on the use of mixed fermentation of Saccharomyces and non-Saccharomyces wine yeasts for inoculation of wine fermentations to enhance the quality and improve complexity of wines. In the present work Lachancea (Kluyveromyces) thermotolerans and Saccharomyces cerevisiae were evaluated in simultaneous and sequential fermentation with the aim to enhance acidity and improve the quality of wine. In this specific pairing of yeast strains in mixed fermentations (S. cerevisiae EC1118 and L. thermotolerans 101), this non-Saccharomyces yeast showed a high level of competitiveness. Nevertheless the S. cerevisiae strain dominated the fermentation over the spontaneous S. cerevisiae strains also under the industrial fermentation conditions. The different condition tested (modalities of inoculum, temperature of fermentation, different grape juice) influenced the specific interactions and the fermentation behaviour of the co-culture of S. cerevisiae and L. thermotolerans. However, some metabolic behaviours such as pH reduction and enhancement of 2-phenylethanol and glycerol, were shown here under all of the conditions tested. The specific chemical profiles of these wines were confirmed by the sensory analysis test, which expressed these results at the tasting level as significant increases in the spicy notes and in terms of total acidity increases.

Vin Santo.

Vin Santo is the typical dessert wine produced in Tuscany, in particular, and in other areas of central and northern Italy, as well as on the Greek island of Santorini. It remains one of the most important "meditation wines", whose glamour starts with its ancient and mysterious origin. Italy is the country with the World's greatest tradition for sweet wine production. In this contest most of the steps in the Vin Santo making process are still linked to old local traditions. This chapter provides an overview of the different styles of Vin Santo produced in Italy together with its marketing. The main factors affecting grapes drying, alcoholic fermentation, maturation in barrels, are also described. So far there have been few scientific studies that have focused on Vin Santo. However, the increasing demand registered in the last few years for this kind of wine, is now greatly stimulating the research aimed to better manage its peculiar production process.

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.

Controlled mixed culture fermentation: a new perspective on the use of non-Saccharomyces yeasts in winemaking.

Mixed fermentations using controlled inoculation of Saccharomyces cerevisiae starter cultures and non-Saccharomyces yeasts represent a feasible way towards improving the complexity and enhancing the particular and specific characteristics of wines. The profusion of selected starter cultures has allowed the more widespread use of inoculated fermentations, with consequent improvements to the control of the fermentation process, and the use of new biotechnological processes in winemaking. Over the last few years, as a consequence of the re-evaluation of the role of non-Saccharomyces yeasts in winemaking, there have been several studies that have evaluated the use of controlled mixed fermentations using Saccharomyces and different non-Saccharomyces yeast species from the wine environment. The combined use of different species often results in unpredictable compounds and/or different levels of fermentation products being produced, which can affect both the chemical and the aromatic composition of wines. Moreover, possible synergistic interactions between different yeasts might provide a tool for the implementation of new fermentation technologies. Thus, knowledge of the Saccharomyces and non-Saccharomyces wine yeast interactions during wine fermentation needs to be improved. To reach this goal, further investigations into the genetic and physiological background of such non-Saccharomyces wine yeasts are needed, so as to apply '-omics' approaches to mixed culture fermentations.

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.

Staphylococcal enterotoxins G and I, a cause of severe but reversible neonatal enteropathy.

BACKGROUND & AIMS: Staphylococcus aureus is recognized to produce toxins A-E and toxic shock syndrome toxin-1 associated with food poisoning and toxic shock syndrome. Enterotoxins G and I co-exist in the same S aureus strains (staphylococcal enterotoxin G and staphylococcal enterotoxin I) and are implicated in scarlet fever and toxic shock. We report these enterotoxins as causative agents of 2 cases of neonatal intractable diarrhea with enteropathy. METHODS: We used a note review for this study. Stool culture, multiplex polymerase chain reaction for enterotoxin, duodenal biopsy specimens for H&E, periodic acid-Schiff staining, and electron microscopy were used. RESULTS: Infant 1 had diarrhea from age 2 weeks and was referred at age 5 weeks with weight less than the 0.4th percentile. Infant 2 was referred at age 7 weeks with 4 weeks' of diarrhea, weight less than the 0.4th percentile. Both infants were severely malnourished. Elemental feeds were not tolerated and total parenteral nutrition was required. S aureus producing staphylococcal enterotoxin G and staphylococcal enterotoxin I was isolated in stools from both infants. Clinical improvement occurred after intravenous flucloxacillin and parenteral nutrition. Histology showed subtotal villous atrophy (H&E) with abnormal brush border (periodic acid-Schiff). Electron microscopy showed severe microvilli destruction, dilated mitochondria, and lysosomes containing cellular debris. Repeat histology was normal in infant 2, age 3 months, off parenteral nutrition, showed return to normal. Currently, both infants are 2 years of age and are thriving on a normal diet. CONCLUSIONS: Staphylococcal enterotoxin G- and I-induced enteropathy is a life-threatening condition, causing reversible disruption of enterocyte ultrastructure that responds well to supportive treatment with flucloxacillin and parenteral nutrition This condition should be a differential diagnosis of neonatal early onset diarrhea.

Career choices for pathology: national surveys of graduates of 1974-2002 from UK medical schools.

In the past 10 years there has been increasing concern about recruitment of junior doctors into pathology, particularly histopathology, in the UK. In this study, we report on career choices for pathology, derived from postal questionnaire surveys of all qualifiers from all UK medical schools in nine qualification years since 1974. 74% (24,623/33,417) and 73% (20,709/28,468) of doctors responded at 1 and 3 years after qualification. The percentage of doctors choosing pathology 1 year after qualification has fallen sharply in recent years: between 1974 and 1983 it was 4.5% (95% confidence interval 4.1-4.9%) and between 1993 and 2002 it was 2.3% (CI 2.0-2.5%). 57% of doctors who chose pathology 1 year after qualification were still working in pathology at year 10. Hours and conditions of work, the doctor's personal assessment of their aptitudes, and their experience of the subject as a student influenced long-term career choices for pathology. Recruitment of UK graduates into the pathology specialties must increase to meet demand in the new and expanding subspecialties. This depends on developing innovative ways of raising the profile of pathology to medical students and junior hospital doctors.