Raffaele Guzzon

Immobilization of yeast and bacteria cells in alginate microbeads coated with silica membranes: procedures, physico-chemical features and bioactivity

Salt alginate beads are used to entrap yeast or bacteria cells and exploited as protective environment for deposition of silica gel membranes. This inorganic layer is obtained through different methods: by dipping the beads in a silica sol (method A), by further consolidating them with tetraethoxysilane in an apolar solvent (method B), and by coating the beads with methyltriethoxysilane in the gas phase (method C). Physical–chemical and biological features are investigated. The pyrolysis study elucidates the features of the polymeric organic layer. Elemental analysis and 29Si solid state NMR prove the presence and the condensation degrees of the silica membranes. The bioactivity is studied by evaluating both glucose (Saccharomyces cerevisiae) and L-malic acid (Oenococcus oeni) fermentations. The fermentation performance is discussed considering possible limitations of mass transport across the silica gel layer. Method A produces an inorganic layer made by of 57.3% Si(OSi)4 and 42.7% HO–Si(OSi)3 units). In this case a linear relation is found among different contact times between silica sol and beads and both silica amount and average thickness. Method B appears to be successful in building up the layer, but it is also detrimental to cell viability. Method C is very efficient in terms of mass deposit, cell viability maintenance and leakage reduction. In this case the membrane is constituted by H3C–Si(OSi)2OH and H3C–Si(OSi)3 units in an almost 1 : 1 ratio.

Selection of a new highly resistant strain for malolactic fermentation under difficult conditions.

Malolactic fermentation (MLF) is a biological process that contributes to wine quality, but it is frequently affected by various vinification conditions. Resistance to four wine-limiting factors was studied with respect to 10 Oenococcus oeni strains in order to select a suitable strain for performing reliable MLF in difficult wines. Resistance to low fermentation temperature, high SO2 and/or ethanol concentration, and low pH were assayed in laboratory tests. A pool of the most resistant strains was used in a set of laboratory MLFs. At the end of fermentation, the dominant strains were identified by RAPD-PCR. The PN4 strain was found to be dominant in the majority of cases and under the most detrimental wine conditions, and it was therefore chosen as the single-strain inoculum for the subsequent MLF trials. The effectiveness of the PN4 strain was confirmed in a series of MLFs carried out in three different countries under experimental and industrial conditions. It accomplished MLF in wines with up to 15.8% ethanol, pH as low as 3.0, 60 mg/L of free SO2, and in fermentation temperatures below 17oC. Our findings indicate that the O. oeni PN4 strain could be an effective starter, guaranteeing regular and reliable MLF fermentation.

Evolution of Yeast Populations during Different Biodynamic Winemaking Processes

This work was performed to evaluate the evolution of indigenous yeasts during wine productions carried out following the principles of biodynamic agriculture. Five trials were designed with different technological interventions consisting of the addition of nitrogen (in the form of ammonium salt), thiamine salt, oxygen, and pied de cuvee at varying concentrations. Yeasts were estimated by haemocytometer chamber and plate counts and identified by sequencing of the D1/D2 domain of the 26S rRNA gene. The isolates identified as Saccharomyces cerevisiae were found to dominate must fermentations and were genetically differentiated by interdelta sequence analysis (ISA). Several non-Saccharomyces species, in particular Hanseniaspora spp. and Candida spp., were found at subdominant levels during must fermentation. The trial added with both nitrogen and thiamine (NTV) showed the highest fermentation rate and microbial richness. The internal surfaces of the cellar equipment were characterised by a certain yeast biodiversity and hosted the species found during winemaking; the wooden surfaces represented the primary source of inoculation of a strain of S. cerevisiae found dominant in all winemaking trials.

Use of organo-silica immobilized bacteria produced in a pilot scale plant to induce malolactic fermentation in wines that contain lysozyme

The exploitation of organo-silica immobilized lactic acid bacteria (LAB) to perform malolactic fermentation (MLF) in wine is described. The immobilization of a large amount of Oenococcus oeni cell culture was achieved by a two-step process in an original pilot plant. Cells are entrapped in Ca-alginate microbeads, coated with an organo-silica membrane obtained by two treatments: the first a sol suspension of tetraetoxysilane, the second using methyltriethoxysilane in gas phase. The resulting material improves the physico-chemical features of alginate, avoids cell leakage during fermentation, and protects the cells from antimicrobial compounds. In MLFs carried out at the microvinification scale, the activity of immobilized cells did not differ from that of free cells, and no differences were found in the chemical composition of the wines obtained. The use of immobilized bacteria allowed: (1) simultaneous alcoholic and malolactic fermentations in must inoculated with free yeast and immobilized bacteria; (2) the sequential MLF of three wine lots with the same biomass of immobilized bacteria; (3) the achievement of MLF in a wine with lysozyme added to suppress wild LAB and their potential spoilage.

A new resource from traditional wines: characterisation of the microbiota of “Vino Santo” grapes as a biocontrol agent against Botrytis cinerea

Abstract The microflora of grapes involved in the production of a traditional Italian straw wine, “Vino Santo Trentino”, was evaluated as a biocontrol agent against Botrytis cinerea, one of the main diseases affecting fruit and grapes. The microbiota was described using plate counts and genotypic characterisation (sequencing of 16S rRNA for bacteria and 26s rRNA for yeast), allowing identification of yeasts belonging to the Hanseniaspora, Metschnikowia, Cryptococcus and Issatchenkia genera and bacteria (Bacillus, Microbacterium, Acetobacter and Gluconobacter spp.). The distribution of these species is related to the extent of B. cinerea infection. 7 isolates were able to halt the growth of B. cinerea in antagonistic cultures grown in Petri plates, using both synthetic growth and grape juice media. Technological characterisation of potential biocontrol agents, performed with the help of flow cytometry and HPLC-ECD, demonstrated that these microorganisms did not represent a risk for wine production due to their low resistance to ethanol, low pH and the absence of off-flavours. This ensures that the biocontrol agents disappear during winemaking and excludes a negative impact on the quality of wines. In conclusion, the microflora associated with dried grapes is a precious source of biocontrol agents against B. cinerea, both in terms of preventing disease in the vineyard and in control of the grape drying process for the production of straw wines.

The application of flow cytometry in microbiological monitoring during winemaking: two case studies

In this work, we exploit a general flow cytometry technique involved in the differentiation of live and dead yeast cells for two applications in winemaking. The discrimination of yeast populations is achieved using two fluorescent dyes that measure the metabolic activity and membrane integrity of the yeast. This analytical approach is first applied for quality control of active dry yeast. Results are discussed in comparison with the Codex Oenologique International (International Oenological Codex) of the International Organisation of Vine and Wine (OIV), demonstrating that analysis using flow cytometry is a valuable alternative, given the ease of execution and the high quality of results obtained in terms of reproducibility, repeatability, and confidence interval. In the second case, we apply flow cytometry as a technique for monitoring the production of sparkling wines using the “Champenoise” method, and describe the evolution of yeast through the production process. In this case, results are directly compared with those obtained with the two methods (plate counts and direct microscopic count) listed in the OIV standards, in order to ensure a thorough understanding of the improvements related to the use of flow cytometry.

Simultaneous yeast–bacteria inoculum. A feasible solution for the management of oenological fermentation in red must with low nitrogen content

The simultaneous inoculum of yeasts and bacteria is a feasible solution for improving fermentation in wines with a harsh chemical composition, capable of inhibiting microbial activity. Considering the risk of wine spoilage due to lactic bacteria, co-inoculum is suggested in white wines with a low pH. However, climate change has also caused problems in achieving malolactic fermentation in red wines, due to the high concentration of ethanol and the low nutrient content. In this work, 5 pairs of commercial oenological starters were tested in simultaneous fermentation, using 4 red musts with a low nitrogen content, and compared with a traditional winemaking process. The simultaneous inoculum caused a slowdown in the activity of yeasts, although no problems in the accomplishment of alcoholic fermentations were observed. More reliable malolactic fermentation was performed in the co-inoculum trials, while, in traditional winemaking, some failures in the degradation of malic acid were observed. Microbiological analyses agreed with these observations. No differences were found in yeast density during alcoholic fermentation, demonstrating the absence of negative interaction between the yeast and the bacteria. However, simultaneous fermentation is not without risks; the highest increases of acetic acid were noted in the co-inoculum trials. The addition of yeast and bacteria to must with a serious lack of nutrients would appear to be a promising alternative to traditional fermentation; however, careful control of the chemical composition of must is mandatory to obtain reliable microbiological activity in the first stages of winemaking.

Spoilage potential of brettanomyces bruxellensis strains isolated from Italian wines

Brettanomyces bruxellensis is an important wine spoilage agent. In this study a population of Brettanomyces strains isolated from Italian wines was thoroughly investigated to evaluate adaptability to wine conditions and spoilage potential. The presumptive isolates of Brettanomyces were identified at species level with 26S rRNA gene sequencing and species-specific PCR, and subsequently subjected to analysis of intra-species variability through the study of intron splice sites (ISS-PCR). Although, some strains were tracked in wines from different regions, extensive genetic biodiversity was observed within the B. bruxellensis population investigated. All strains were evaluated for their growth ability in the presence of ethanol, high sugar content, low pH, different temperatures and sulphur dioxide, using optical density and flow cytometry measurement. The ability of yeasts to produce ethyl phenols in red wines with different chemical compositions was evaluated by means of high performance liquid chromatography with electrochemical detection (HPLC-ECD). The results highlighted wide variability in B. bruxellensis in response to wine limiting factors and in terms of the accumulation of ethyl phenols. As regards this last aspect, the differences found among strains were closely related to chemical composition of wine and strain resistance to environmental stress factors, making a priori evaluation of risk of wine alteration quite difficult. These results suggest that strategies for the control of Brettanomyces should be tailored on the basis of strain distribution and wine characteristics.

Silicification of wood adopted for barrel production using pure silicon alkoxides in gas phase to avoid microbial colonisation

The paper presents a new approach, covering wood with silica-based material in order to protect it from spoilage due to microbial colonisation and avoiding the loss of the natural features of the wood. Wood specimens derived from wine barrels were treated with methyltriethoxysilane in gas phase, leading to the deposition of a silica nanofilm on the surface. (29)Si and (13)C solid state Nuclear Magnetic Resonance and Scanning Electron Microscope-Energy Dispersive X-ray analysis observations showed the formation of a silica polymeric film on the wood samples, directly bonding with the wood constituents. Inductively Coupled Plasma-Mass Spectroscopy quantification of Si showed a direct correlation between the treatment time and silica deposition on the surface of the wood. The silica-coated wood counteracted colonisation by the main wine spoilage microorganisms, without altering the migration from wood to wine of 21 simple phenols measured using a HPLC-Electrochemical Coulometric Detection.

Influence of different commercial active dry yeasts on histaminol production during wine alcoholic fermentation

The performance of different selected yeast strains regarding the capacity to convert histidine into histaminol during fermentation has been described and discussed in a model grape must (Chardonnay). First, the activity of 10 commercial active dry yeast strains (ADY) in the native grape must both with and without a nitrogen supplementation has been compared. In the second set of fermentations, after selecting the four best-performing strains, their performances using four different nitrogen sources have been tested. During fermentation process it was not possible to identify a common trend. In some cases, histaminol content appeared related to the nutrient supplementation, however, in other cases a different behaviour emerged. However, as shown by the experimental evidences, among the two variables considered the nature of yeast strain was slightly prevalent.