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

The controversial relationship between chitosan and the microorganisms involved in the production of fermented beverages

Chitosan is a promising antimicrobial agent available in the beverage industry, because it ensures the control of a wide range of spoilage microorganisms. As chitosan does not alter the characteristics of fermented beverages, it is nowadays widely employed in the wine sector. In this work, an exhaustive chemical characterization of 12 commercial chitosans was performed in accordance with the OIV methods. These analyses made it possible to confirm or determine the animal or fungal origin of the 12 samples. Furthermore, ionic chromatography coupled with an amperometric detector (IC-PAD) confirmed peculiar polysaccharide profiles for fungal and animal-derived chitosans. The antimicrobial activity of chitosans was evaluated against the microorganisms involved in beverage fermentation or capable spoil wine, beer and cider. Chitosans were tested in static and stirred conditions, in a synthetic medium that reproduces fermented beverage conditions, to discriminate against the physical settling of cells and their specific antimicrobial activity. Moreover, the activity of the soluble portion of chitosan was checked by inoculating microorganisms in the media after chitosans removal. The results highlighted the different sensitivity of microorganisms to chitosans, allowing selective control of spoilage agents. However, the yeast and bacteria involved in fermentation were damaged by chitosan, and the synthetic media treated with this molecule showed a less fermentative aptitude. These results suggest that chitosan is a promising tool in fermented beverage production, but an in-depth study of the biochemical interaction between chitosan and food microorganisms is necessar

Spatial and seasonal structure of bacterial communities within Alpine vineyards: Trentino as a case study

Bacteria have a fundamental role in determining the fitness of grapevine, the composition of grapes and the features of wines but at present, little information is available. In this work, the bacteria colonizing the different portions of grapevine (bark, leaves and grapes) were explored in the vineyards of the Alpine region of Trentino, considering the impact of different environmental and agronomical variables. The vineyards included in the work were selected based on their different geographical positions (altitude) and grapevine training systems in order to explore the whole variability of the grapevine ecosystem. Moreover, the surface amount of copper was measured on grapes and leaves during the vegetative growth. Bacterial analysis, performed using plate counts and Illumina MiSeq, revealed an increase in the concentration of grape bacteria proportional to the progress of the ripening stage. Conversely, the peak of bacterial concentration onto leaf and bark samples occurred in August, probably due to the more favourable environmental conditions. In bark samples, the bacterial microbiota reached the 7 log CFU/cm2, while 6 log UFC/g were measured in grape samples. A remarkable biodiversity was observed, with 13 phyla, 35 classes, 55 orders, 78 families and 95 genera of bacteria present. The presence of some taxa (Alphaproteobacteria, Desulfovibrionaceae, Clostriadiales, Oscillospira, Lachnospiraceae and Bacteroidales) was ubiquitous in all vineyards, but differences in terms of relative abundance were observed according to the vegetative stage, altitude of the vineyard and training system. Bacteria having oenological implication (Lactobacillus, Pediococcus and Oenococcus) were detected in grape samples collected in August, in low abundance. The data revealed a complex bacterial ecosystem inside the vineyard that, while maintaining common traits, evolves according to environmental and agronomical inputs. This study contributes to define the role of bacteria in the complex balance established in each vineyard between human actions and agricultural environment, known as terroir

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

Selection of lactic acid bacteria species and strains for efficient trapping of Drosophila suzukii

Monitoring of Drosophila suzukii is based on the use of effective traps and baits. The current baits are insufficient to provide efficient monitoring. The use of bacteria as bio-catalyzers to produce bioactive volatiles may improve flies’ attraction. Thus, we conducted this work to improve Droskidrink® bait’s attractiveness using lactic acid bacteria. (2) Different baits that were based on the use of Droskidrink® were assessed for flies’ attraction in a Droso-Trap® in a vineyard. Oenococcus oeni, Pediococcus spp., and Lactobacillus spp. were used. The performance of the most attractive species, O. oeni, inoculated into Droskidrink® was assessed in laboratory tests. The responses of female flies to volatiles produced by Droskidrink® with O. oeni strains were recorded by electroantennography. (3) Preliminary field assessment of baits recorded O. oeni as the most attractive species. Three strain groups showed adaptation to test conditions. Volatiles extracted by the headspace of baits inoculated with O. oeni, elicited electroantennographic responses from fly antennae. (4) Droskidrink® inoculated with O. oeni is a highly attractive bait for monitoring. These findings will be useful for improving the attractiveness of D. suzukii commercial baits based on the utilization of LAB volatiles in a strain-dependent manner

Oenological characterisation of indigenous strains of S. cerevisiae isolated in a biodynamic winery in the Cortona DOC area

Genotypic and technological characterisation of the S. cerevisiae population isolated in a biodynamic winery in the Cortona DOC area was performed to gain better knowledge of the variables that influence winemaking. The oenological performance of 11 S. cerevisiae strains was evaluated with physiological tests; strain typing was performed through analysis of interdelta sequences and 26S rDNA sequencing. The analysis revealed a remarkable variability in terms of S. cerevisiae strains, despite the homogeneity of wine features, underlining the high levels of biodiversity characterising biodynamic agriculture. Some strains were found in wines of different vintages, suggesting the presence of an established microbiota in the winery. Oenological tests demonstrated that while some yeasts provided reliable oenological performance, other strains were not able to accomplish prompt and effective alcoholic fermentation, or were characterised by spoilage characteristics, such as excessive production of volatile phenols or acetic acid. Indigenous strains of S. cerevisiae could be a useful instrument for reliable winemaking without altering the native microbiota of each oenological environment. However, characterisation of their oenological suitability, and the application of practices able to drive the evolution of microbiota, must be employed to reduce the risk of wine spoilage

Characterisation of Diospyros kaki (persimmon) vinegars produced with different microorganisms

The aim of this work was to evaluate the characteristics of nine kaki vinegars produced using different yeasts and bacteria traditionally involved in wine production, and to evaluate their acidity, density, total phenolic content, and antioxidant activity. Furthermore, the study characterized the volatile fingerprinting by headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) and by two-dimensional gas chromatography coupled to mass spectrometry (GCxGC-TOF-MS). Finally, individual carotenoids were characterized using high performance liquid chromatography (HPLC). More than a thousand distinguishing molecules were found. It was discovered that vinegars fermented with S. cerevisiae produced a larger number of volatile chemicals. Among the three vinegars produced with this strain, the one fermented with Acetobacter_vino seemed to have a more elaborate flavour profile than all the other samples. The vinegar produced utilizing the mixture of T. delbruekii and Acetobacter was the only kind to have a high concentration of carotenoids

A novel microbiological approach to impact the aromatic composition of sour loquat beer

The growing interest in novel beer development determined the exploitation of unconventional yeasts isolated from novel ecological niches to generate unexplored sensory profiles. In recent years, there is an increasing interest in generating beers brewed with the addition of fruits. For the first time, Lachancea thermotolerans MNF105 and Saccharomyces cerevisiae MN113 isolated from manna, were tested as starter cultures to process loquat beer to improve the sensory profile. Innovatively, the yeast species L. thermotolerans was investigated for the production of sour fruit beer. Sour fruit beers produced with L. thermotolerans MNF105 were more balanced than the respective control, especially in terms of perceived acidity during sensory analysis. This could be due to the lower lactic acid production (0.49 g/L) compared to the respective control (1.74 g/L). The overall organoleptic investigation showed a preference for S. cerevisiae MN113 (TF1) isolated from manna. Experimental trials conducted with the selected strains demonstrated the absence of off-odour and off-flavour and improved aroma perception. Aldehydes and alcohols were the most abundant compounds emitted from the beers. S. cerevisiae MN113 and L. thermotolerans MNF105, manna related yeasts, showed great technological properties, representing promising starters for the production of fruit beer and sour fruit beer

Lieviti e biofilm: un’interessante strategia evolutiva

Possiamo definire il biofilm come una comunità microbica organizzata all’interno di una struttura polimerica autoprodotta, in grado di aderire solidamente a superfici solide o semisolide, vive o inanimate. La formazione di biofilm è una potente strategia di sopravvivenza attuata da numerosi microrganismi in natur