Wine Yeasts Selection: Laboratory Characterization and Protocol Review.

Wine reflects the specificity of a terroir, including the native microbiota. In contrast to the use of Saccharomyces cerevisiae commercial starters, a way to maintain wines' microbial terroir identities, guaranteeing at the same time the predictability and reproducibility of the wines, is the selection of autochthonous Saccharomyces and non-Saccharomyces strains towards optimal enological characteristics for the chosen area of isolation. This field has been explored but there is a lack of a compendium covering the main methods to use. Autochthonous wine yeasts from different areas of Slovakia were identified and tested, in the form of colonies grown either on nutrient agar plates or in grape must micro-fermentations, for technological and qualitative enological characteristics. Based on the combined results, Saccharomyces cerevisiae PDA W 10, Lachancea thermotolerans 5-1-1 and Metschnikowia pulcherrima 125/14 were selected as potential wine starters. This paper, as a mixture of experimental and review contributions, provides a compendium of methods used to select autochthonous wine yeasts. Thanks to the presence of images, this compendium could guide other researchers in screening their own yeast strains for wine production.

Cultivable microbial ecology and aromatic profile of "mothers" for Vino cotto wine production.

The aim of the present study was to assess the cultivable microbiota of "mothers" of Vino cotto collected from production of different years 1890, 1895, 1920, 1975, 2008. A total of 73 yeasts and 81 bacteria were isolated. Starmerella lactis-condensi, Starmerella bacillaris, Hanseniaspora uvarum, Saccharomyces cerevisiae, Hanseniaspora guillermondi and Metschnikowia pulcherrima were identified. Bacteria isolates belonged to lactic acid bacteria (Lactiplantibacillus plantarum and Pediococcus pentosaceus) and acetic acid bacteria (Gluconobacter oxydans). Remarkable biodiversity was observed for Starm. bacillaris, as well as L. plantarum and G. oxydans. Organic acids and volatile compounds were also determined. Malic and succinic acids were the main ones with values ranging from 8.49 g/L to 11.76 g/L and from 4.15 g/L to 7.73 g/L respectively, while citric acid was present at low concentrations (<0.2 g/L) in all samples. Esters and higher alcohols were the main volatile compounds detected followed by alkanes. This study permits to better understand the microbial communities associated to this product and could be considered a starting point for the definition of tailored starter cultures to improve the quality of Vino cotto preserving its typical traits.

Integral valorization of orange peel waste through optimized ensiling: Lactic acid and bioethanol production.

The management of the huge amount of orange peel waste (OPW) is a complex issue although it has a very high potential in terms of biorefining. One of the main problems in the valorisation of OPW is the seasonality of its production with the ensiling method being largely proposed as a possible solution. During the ensiling process, value added chemicals including lactic acid, acetic acid and ethanol are spontaneously produced together with a significant loss of volatile solids (VS) . In this contribution, the stimulation of lactic acid bacteria by either a biological (inoculation with leachate coming from a previous ensiling process) or chemical (MnCl2 supplementation) methods has been tested with the aim to increase the chemicals production preventing, at the same time, the VS loss. The inoculation with the leachate improves both the VS recovery (+7%) and the concentration of lactic acid (+113%) with respect to the uninoculated one (control). The overall yields of the process are noticeable, up to about 55 g·kgTS-1 of lactic acid, 26 g·kgTS-1 of acetic acid and 120 g g·kgTS-1 of ethanol have been produced. On the other hand, the chemical stimulation enhances the production of liquid products together with a significant VS loss. The proposed preservation method, due to its simplicity, can be easily implemented at full-scale allowing the production of added-value chemicals and the concurrent storage of the OPW that can be further valorised (e.g. animal feed, pectin or biomethane production).

Persistence and Effect of a Multistrain Starter Culture on Antioxidant and Rheological Properties of Novel Wheat Sourdoughs and Bread.

Food consumers make decisions primarily on the basis of a product's nutritional, functional, and sensorial aspects. In this context, this study evaluated the persistence in sourdough of a multistrain starter culture from laboratory to bakery plant production and the effect of the starter on antioxidant and rheological properties of sourdoughs and derived bread. Lactobacillus sanfranciscensis B450, Leuconostoc citreum B435, and Candida milleri L999 were used as a multispecies starter culture to produce a sourdough subsequently used to modify two traditional sourdoughs to make novel bread with improved health and rheological properties. Both these novel bakery sourdoughs showed the persistence of L. sanfranciscensis B450 and C. milleri L999, and showed a significantly different lactic acid bacteria (LAB) concentration from the traditional sourdoughs. The novel sourdough PF7 M had a higher phenolic content (170% increase) and DPPH (8% increase) than the traditional bakery sourdough PF7 F. The novel sourdough PF9 M exhibited an improvement in textural parameters. Further research would be useful on the bioavailability of bio-active compounds to obtain bread with improved characteristics.

Impact of Different Storage Methods on Bioactive Compounds in Arthrospira platensis Biomass.

Arthrospira platensis (spirulina) is considered a source of natural molecules with nutritional and health benefits. As the different storage forms can affect the quantity and quality of bioactive ingredients, the aim of the present work was to evaluate the effects of freezing, oven-drying and freeze-drying on chemical composition of spirulina biomass. Total proteins, photosynthetic pigments and antioxidants, were analyzed and compared to respective quantities in fresh biomass. The frozen sample exhibited the highest content of phycocyanin-C, phenols, and ascorbic acid, also respect to the fresh biomass. The highest total flavonoid amount was in the freeze-dried biomass. HPLC-DAD analysis of phenolic acids revealed the presence of the isoflavone genistein, known for its therapeutic role, in all the spirulina samples. The phosphomolybdenum method (TAC) and DPPH scavenging activity were applied to determine the antioxidant activity of different samples. The highest DPPH scavenging activity was detected in fresh and freeze-dried biomass and it was positively related to carotenoid content. A positive correlation indicated that carotenoids, chlorophyll, ascorbic acid and all phenolic compounds were the major contributors to the TAC activity in spirulina biomass. The results highlighted a different functional value of spirulina biomass, depending on the processing methods used for its storage.

Wild Saccharomyces cerevisiae strains display biofilm-like morphology in contact with polyphenols from grapes and wine.

Polyphenols are a major component of wine grapes, and contribute to color and flavor, but their influence upon yeast growth forms has not been investigated. In this work we have studied the effect of polyphenols on the ability of natural isolates of wine-related Saccharomyces cerevisiae strains to form biofilms attaching to plastic surfaces, to grow as mat colonies, to invade media, and to display filamentous growth. The use of carbon- and nitrogen-rich or deficient media simulated grape juice fermentation conditions. The addition of wine polyphenols to these media affected biofilm formation, and cells exhibited a wide variety of invasiveness and mat formation ability with associated different growth and footprint patterns. Microscopic observation revealed that some strains switched to filamentous phenotypes which were able to invade media. The wide range of phenotypic expression observed could have a role in selection of strains suitable for inoculated wine fermentations and may explain the persistence of yeast strains in vineyard and winery environments.

Depuration in aerated ponds of citrus processing wastewater with a high concentration of essential oils.

Citrus processing wastewater was treated in aerated pilot plants in order to evaluate the following: (a) energy efficiency under different air flow rates and times; and (b) limits of spontaneous microflora in adapting to essential oils. In comparison to permanent air flow, night aeration for 12 hours determined an increase of up to 12% of the monthly removal rate of chemical oxygen demand (COD) and a consequent reduction by 10% of energy consumptions per unit of COD removed from 0.63 to 0.57 kWh/kg(COD). Lowering night aeration from 14 to 7 1/m3/h reduced by only 10% the removal rate of COD; the energy consumption per unit of COD removed (0.32 kWh/kg(COD)) was consequently reduced by more than 40%. Dissolved oxygen was maintained at very low level, rarely exceeding 0.2 ppm, with no bad smell. The consequent high oxygen deficit of 98-99% of saturation induced high oxygen transfer efficiency. The microbial population was characterized mainly by aerobic bacteria; only 5-8% of bacteria were strictly anaerobic. In the deep tank layer under the air diffuser a small amount of sludge settled (0.03-0.04 kg of dry matter per kg of COD removed), containing only 3% of total organic matter detected at the end of the depuration process. The fact that the concentration of essential oils could be progressively increased up to 1400 ppm without noticeably slowing down the biological processes demonstrated the remarkable microbial adaptation.

Ochratoxin A adsorption phenotype: an inheritable yeast trait.

This study aimed to evaluate the inheritance of the trait ochratoxin A adsorption in two wine strains of Saccharomyces cerevisiae and their 46 descendants. Each strain was inoculated in triplicate in test tubes containing 10 ml of must obtained from the Calabrian Zibibbo white grape variety, artificially contaminated with ochratoxin A to reach a total content of 4.10 ng/ml. The microvinification trials were performed at 25°C. After 30 days, ochratoxin A values ranged from 0.74 to 3.18 ng/ml, from 0.01 to 2.69 ng/ml, and from 0.60 to 2.95 ng/ml respectively in wines, in lees after washing, and in the saline solution used to wash the lees. The analysis of OTA in wines was performed to find the residual toxin content after yeast activity, thus obtaining technological evidence of yeast influence on wine detoxification. The analysis of OTA in lees after washing was performed to distinguish the OTA linked to cells. The analysis of OTA in the saline solution used to wash the lees was performed to distinguish the OTA adsorbed on yeast cell walls and removed by washing, thus focusing on the adsorption activity of wine yeast through electrostatic and ionic interactions between parietal mannoproteins and OTA. Ploidy of the two parental strains was controlled by flow cytometry. Results demonstrated that the ochratoxin A adsorption is genetically controlled and is a polygenic inheritable trait of wine yeasts. The majority of the descendants are characterized by a great and significant diversity compared to their parents. Both the parental strains had genome sizes consistent with their being diploid, so validating the observed results. These findings constitute an initial step to demonstrate the mechanisms of inheritance and establish breeding strategies to improve the ochratoxin A adsorption trait in wine yeasts. This will allow a decrease in the ochratoxin A content of contaminated musts during winemaking, by using genetically improved wine yeasts.