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 influence of the copper content in grape must on alcoholic fermentation kinetics and wine quality. A survey on the performance of 50 commercial Active Dry Yeasts

The effects of copper on the viability and fermentative activity of 50 active dry yeasts purchased on the northern Italian market were studied, and revealed that Copper excess may cause massive death of yeast cells, leading to a significant delay in the start and progress of alcoholic fermentation. A two-log units reduction in cell viability was observed when copper content of musts was around 20 mg∙L-1. Despite this, the difference noted in the kinetics after 20 days' fermentation was lower than that observed 48 hours after in the grape must. An excess of copper in must affected also the composition of the produced wines. The increase in acetic acid and in the sulphur dioxide concentration, observed in wines made using grape must with a high copper concentration, raises serious doubts both regarding the possibility of obtaining good wines from these raw materials and in relation to the progress of subsequent steps of winemaking, such as malolactic fermentation. While it is an important tool in preventing vine diseases, copper must be used very carefully to avoid serious troubles during wine fermentation, even if some yeasts seem more suited to ferment musts containing up to 20-30 mg∙L-1 copper.

Agenti di biocontrollo contro le contaminazioni microbiche

Il biocontrollo è una strategia per il contenimento dei microbi alterativi che, da un lato, consente una protezione efficace nelle prime fasi della vinificazione e, dall’altro, non altera il successivo processo di affinamento del vin

La posizione del vigneto non cambia il microbiota

I campioni di corteccia, foglie e grappoli isolati in 6 vigneti trentini con altitudini e forme di allevamento diverse non hanno presentato differenze dal punto di vista qualitativo nelle popolazioni microbiche rilevat

Diversity and biomass accumulation in cultured phototrophic biofilms

In the present study, biomass development and changes in community composition of phototrophic biofilms grown under different controlled ambient conditions (light, temperature and flow) were examined. Source communities were taken from a wastewater treatment plant and used to inoculate growth surfaces in a semi-continuous-flow microcosm. We recorded biofilm growth curves in cultures over a period of 30 days across 12 experiments. Biovolume of phototrophs and community composition for taxonomic shifts were also obtained using light and electron microscopy. Species richness in the cultured biofilms was greatly reduced with respect to the natural samples, and diversity decreased even further during biofilm development. Diadesmis confervacea, Phormidium spp., Scenedesmus spp. and Synechocystis spp. were identified as key taxa in the microcosm. While a significant positive effect of irradiance on biofilm growth could be identified, impacts of temperature and flow rate on biofilm development and diversity were less evident. We discuss the hypothesis that biofilm development could have been subject to multistability, i.e. the existence of several possible stable biofilm configurations for the same set of environmental parameters; small variations in the species composition might have been sufficient to switch between these different configurations and thus have contributed to overwriting the original effects of temperature and flow velocity

Evolution of Yeast Populations during Different Biodynamic Winemaking Processes

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

Modulation of wine sensory profile by lactic bacterial activity in a global warming scenario by different malolactic fermentation strategies

Aim. Incoming global warming imposing strategies to adapt wine chemical profile at an unknown scenario. In this sense malolactic fermentation (MLF), the biological deacidification of wines, must become a conscious choice to optimize the content in organic acids and the sensory profile of the wine. The authors present different works to understand how modulate the oenological activity of strains of Oenococcus oeni. The effect of different strategies of bacterial inoculum and correction of acidity were considered. Method. Simultaneous fermentation is an alternative strategy for managing MLF, which involves the inoculation of lactic bacteria into the grape must, instead into the wine at the end of alcoholic fermentation as traditionally practiced in winemaking. This approach has been tested in southern red wines with a high ethanol potential, withe aromatic wines, and sparkling-base wines. Furthermore, on a population of strains of O. oeni was verified the effect of the correction of grape must/wine acidity by different organic acids, in terms of MLF kinetics, cell viability and diacetyl production. Results. In the last years the focus of the selection of bacterial strains for malolactic fermentation was re-oriented from the mere ability to accomplish malic acid degradation, to the adaptability at different winemaking protocols. In this sense, simultaneous inoculum of bacteria and yeast in grape must seems to achieve different goals. In southern, high ethanol and low acidity wines bacteria results more active respect to tests performed by traditional post-alcoholic inoculum, accomplishing MLF without spoilage effects, such as volatile acidity increase. In the case of tests performed in winemaking if aromatic or sparkling base wines the simultaneous inoculum results in a higher varietal character. Tartaric, malic, and citric acid were tested to correct the low acidity of grape must/wine before MLF. Malic acid over the 2.5 g/L affected evolution of MLF, while citric acid stimulates bacterial activity and diacetyl production. The addition of tartaric acid results counterproductive at the higher value. Bacterial activity in grape must is more efficient than in wine in terms of rate and completeness of MLF. Conclusion. The acidic profile of wine is one of the key aspects in a global warming scenario, for longevity and organoleptic quality. This work offers useful information to modulate the activity of lactic acid bacteri

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 affectedby various vinification conditions. Resistance to four wine-limiting factors was studied with respect to 10 Oenococcusoeni strains in order to select a suitable strain for performing reliable MLF in difficult wines. Resistance to lowfermentation temperature, high SO2 and/or ethanol concentration, and low pH were assayed in laboratory tests. Apool of the most resistant strains was used in a set of laboratory MLFs. At the end of fermentation, the dominantstrains were identified by RAPD-PCR. The PN4 strain was found to be dominant in the majority of cases and underthe most detrimental wine conditions, and it was therefore chosen as the single-strain inoculum for the subsequentMLF trials. The effectiveness of the PN4 strain was confirmed in a series of MLFs carried out in three differentcountries 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 theO. oeni PN4 strain could be an effective starter, guaranteeing regular and reliable MLF fermentation

Chitosan in wine industry: identification of origin by a multidisciplinary approach and application in oenology

Aim: Chitosan is a promising antimicrobial agent, capable of providing control of a wide range of spoilage microorganisms. To guarantee the fugal origin of chitosan, the only authorized for oenological practices, a multidisciplinary approach based on the measurement of the stable isotope ratios, Fourier transform infrared spectrometry and thermogravimetric analysis was proposed. In addition, the activity of chitosan against food related microorganisms was evaluated by different experiments aimed to discriminate between chemical and physical action of chitosan vs. the microorganisms. Method: The measurement of the stable isotope ratios (SIR) of carbon δ13C, nitrogen δ15N, oxygen δ18O and hydrogen δ2H of 35 samples of chitosan and the data of maximum degradation temperatures (obtained by TGA) combined with those of the peak areas of amide I and NH2/Amide II (obtained by FTIR) were employed to discriminate chitosan different sources (fungal grown on different substrates vs crustacean). The antimicrobial activity was tested in static and stirred conditions, in a synthetic media, using type strains of most common technological or spoilage microorganism. Viability was evaluated by Petri plate counts. The activity of the soluble portion of chitosan was checked by inoculating microorganisms in the media after chitosan removal. Results: The Kruskal-Wallis test showed that δ13C and δ18O were the most significant parameters able to classified chitosan into three different groups (from fungus grown on C3 photosynthetic cycle plant substrate, from fungus on C4 substrate and from crustacean). HCA and PCA analysis based on TGA, FTIR and SIR data successfully distributed the tested samples into informative clusters. Tests of chitosan antimicrobial activity highlighted the different sensitivity of microorganisms to chitosans, allowing selective control of spoilage agents. However, yeast and bacteria involved in fermentation were damaged by chitosan, and the synthetic media treated with this molecule showed a less fermentative aptitude. Conclusion: A robust analytical strategy for the correct identification of chitosan samples from crustaceans or fungi was presented, based on the observation that diverse biosynthetic pathways during the formation of the chitin influenced the isotopic composition of chitosan. Results of toxicity tests 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 necessary