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

On identification of self-similar characteristics using the Tensor Train decomposition method with application to channel turbulence flow

A study on the application of the Tensor Train decomposition method to 3D direct numerical simulation data of channel turbulence flow is presented. The approach is validated with respect to compression rate and storage requirement. In tests with synthetic data, it is found that grid-aligned self-similar patterns are well captured, and also the application to non grid-aligned self-similarity yields satisfying results. It is observed that the shape of the input Tensor significantly affects the compression rate. Applied to data of channel turbulent flow, the Tensor Train format allows for surprisingly high compression rates whilst ensuring low relative errors

Authentication and geographical characterisation of Italian grape musts through glucose and fructose carbon isotopic ratios determined by LC IRM

The authenticity of grape musts is normally checked through the stable isotopic analysis of carbon (δ13C) after fermentation and distillation by following the official OIV MA AS-312-06 method. Unfortunately, it presents some issues that are difficult to over come. Grape must samples can only be analysed after they have been fermented to obtain ethanol. The process must be carried out under careful control of the fermentation to avoid the presence of unwanted by-products arising from a premature fermentation interruption. Moreover, if the musts have been preserved by the addition of sulphur dioxide (SO2 ), they must undergo an additional step to eliminate the SO2 , which would affect the fermentation. Once the product has been fermented, the ethanol must be separated using specific distillation columns (such as the Cadiot ones) making it possible to obtain ethanol free of isotopic fractionation with a minimum alcohol degree of 95% vol. In this study, the alternative use of a technique based on δ13C isotopic analysis of the major sugars of the grape must by liquid chromatography coupled with isotope ratio mass spectrometry (LC-IRMS) is provided. In LC–IRMS, analytes are separated on an LC system and consecutively oxidized in an online reactor to CO2 , which is required for the determination of compound-specific carbon isotopic ratios. This technique has been already used in the study of matrices such as wine [1], ethanol [1,2], glycerol [2], and honey [3] to detect fraudulent alterations of their natural composition such as the addition of exogenous sugars to the products. The LC-IRMS allows a single separation of the individual components of a sample and makes it possible to determine their δ13C values online, avoiding both the disadvantages of off-line methods and the disadvantages of methods requiring a derivatization step (such as GC-C-IRMS), causing the addition of extra carbons. In order to discriminate between musts from different areas of Italy, a preliminary dataset was considered; the δ13C isotopic ratios of glucose and fructose of around 100 authentic Italian must samples from 16 different sampling regions were analysed. In addition, the δ13C variability in authentic and fake must (added with increasing percentages of exogenous sugars) has been explored and tested to verify their validity as fraud detectors. The two analysed parameters, ranging from −29.8‰ to −21.9‰, are well correlated (R2 = 0.7802) and the northern Italian regions showed significantly more negative δ13C values for both sugars than the rest of the dataset (Figure 1). By using the LC-IRMS technique, the addition of exogenous sugars, such as fructose and glucose from C4 photosynthetic cycle plants, is easily detectable as it modifies the δ13C of the individual sugar

Application of differential pH technique to the determination of urea in Italian wines

A method for the quantification of urea in wine, based on measuring the change in pH when urease is added to the sample, is presented and compared to the conventional dual enzyme (urease/glutamate dehydrogenase) approach. The method is linear in the range 0-30 mg·l-1 in red, white and “raisin” wines, and the detection limit (0.3 mg·l-1) is lower than for the usual enzymatic method. The differential pH technique presented here gives accurate quantification of urea in wine, being unaffected by the presence of ammonium. The amounts of urea in 195 still and sparkling commercially available wines with designation of geographic origin from the most renowned Italian grape growing areas were quantified. 17.4 % of samples were over the 3 mg·l-1 level suggested by the International Organisation of Vine and Wine for urease treatment to limit the potential risk for ethyl carbamate formation during wine ageing. Yeast strains EC1118 and SP665 can minimise urea content in wine.

Status of yeast assimilable nitrogen in Italian grape musts and effects of variety, ripening and vintage

The content of promptly assimilable nitrogen by yeast (PAN) was analysed in 586 juices from technologically ripe grapes collected in several Italian regions. A spectrophotometric method within the reach of each wine laboratory was used, adapting a previous method of the American Society of Brewing Chemists used for wort to grape juices. 58.3 % of the samples were below the classic deficiency threshold of 140 mg·l-1. Among varieties and vintages there were significant differences. An overall trend of PAN to slightly decrease with ripening was observed. The variability of the PAN content of numerous samples harvested in a vineyard to check ripeness seems to be larger than that for sugars and total acidity

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.

Changes in the contents of micro- and trace-elements in wine due to winemaking treatments

Forty-four mineral elements quantified by ICP-OES and ICP-MS were measured in wines, (a) after wine fining, at three pH levels with 10 different bentonites (1 g·l-1), (b) after addition of yeast hulls from 2 suppliers to wine (180 and 360 mg·1-1). Bentonite fining resulted in statistically significant increases of the large majority of elements, but in significant lower levels of Cu, K, Rb and Zn. The addition of yeast hulls caused a statistically significant depletion of the contents of Ce, Cu, Fe, La, Sb, U, V and Y.

I Quaderni di Careggi- Fifth issue- Landscape Observatories

Having regard to Recommendation CM/Rec (2008)3 on the Guidelines for the implementation of the European Landscape Convention, “landscape observatories, centers and institutes” are one of the main instruments for the implementation of landscape policies (II.3.3). They facilitate the collection and exchange of information and study protocols between states and local communities. This issue of the Quaderni di Careggi presents an international overview of the activities of landscape observatories, a reflection on their mission and effectiveness with regard to the ELC objectives, and a reflection on the relationships between the different subjects, thanks to the participation of institutional bodies, public officials as well as researchers and representatives of civil society. It reflects part of the scientific contributions which will be presented during the V Careggi Seminar (Florence, 27-28th June 2013)

Risk of de‐novo formation of chlormequat and mepiquat in industrial cocoa products assessed by ion chromatography coupled with high‐resolution mass spectrometry

Mepiquat (MQ) and chlormequat (CQ) are two main food contaminants that could be naturally present in various animal and plant foods due to heat treatments. Cocoa and its derivative products seem to be a matrix inclined, for both chemical characteristics and production technologies, to the formation of MQ and CQ. In this study, exploiting the selectivity and sensitivity of IC-HRMS, a new reliable analytical method was developed and validated for MQ and CQ quantification. The LOD of the method was set at 1.9 lg kg 1 for both CQ and MQ, and the linearity was defined from 6.25 to 625 lg kg 1 . Average recoveries, at 2 lg L 1 , were 96.5% and 97.5% for CQ and MQ, respectively, while precision as repeatability (RSDr%) was 2.3% and 5.1% respectively. Subsequently, a laboratory-scale experiment was conducted to assess, for the first time, the actual risk of MQ and CQ formation, confirming in cocoa products the presence of MQ at detectable concentration after 55 min at 180 °C, approximately 8 lg kg 1 for cocoa and 7 lg kg 1 for chips. However, the investigation of a wide variety of commercial cocoa and nuts products has fortunately ruled out the presence of such contaminants at detectable concentration