Francesca Borghini and Stefano Ferrari

ISVEA srl, Via Basilicata 1-3-5 
Loc Fosci, Poggibonsi (Siena) 

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Within the framework of the Submeasure 16.2 of the Rural Development Plan of the Region of Tuscany-European Innovation Partnership, the Vintegro project has focused, for over two years, on the study of the integrity and stability of Tuscan wine, in particular on the assessment of macromolecular instability (flavanols, flavonols, pigments, polysaccharides, proteins), which in the setting-up phase was found to be the most in need of innovation according to the opinion of representatives of the Tuscan wine sector.  The macromolecules in wine are in a state of metastable equilibrium, and remain in solution until the equilibrium is broken due to reactions and interactions between them, which are a dependant of environmental conditions and composition. The stability of wine is influenced by a multitude of factors, both intrinsic (variety, properties and concentration of solutes) and extrinsic (temperature, oxygen, humidity and light). If the balance is broken when the wine is already being distributed on the market, obvious precipitates form in the bottle which, although completely harmless, are unwelcome to consumers. The wine is then subject to dispute or rejection, with major economic and commercial consequences. In order to avoid risks, since it is not yet possible to predict whether a wine will give rise to precipitates, the technician may tend to exaggerate with stabilising treatments, which could strip the wine of its typical constituents and which in any case have significant economic and environmental costs. The aim of the Vintegro project was also to develop a predictive test for the instability of a wine, by means of a rapid and inexpensive protocol applicable to a wine at various stages of the production process.
In order to study colloidal and colour stability, the project was developed through different phases (see also Figure 1): wines of different varieties vinified individually and in blends (CBS Cabernet Sauvignon, CBF Cabernet Franc, SG Sangiovese, MRL Merlot and Syrah), at different stages of production (pre-stabilisation, pre-bottling and bottling), were subjected to different tests, both traditional and non-traditional (hot and cold tests, temperature cycles in climatic chambers and hydrogen peroxide additions) and then submitted to a large panel of chemical analyses. Multivariate statistical techniques were then applied to select both the test and the variables to be measured to assess the macromolecular stability of red wines. In addition, during the two years of the project, treatments were carried out in the cellar with different oenological products to verify the stability of the samples.

Figure 1. Articulation of the Vintegro Project for the study of colloidal stability and colour.


The PCA analysis carried out on the non treated samples (different varieties and stages), stressed at different temperatures, in a climate chamber and with hydrogen peroxide shows a clear grouping of the samples according to grape variety rather than treatment (Figure 2). SGs are all in the left-hand quadrants of the score plot and are distinguished from the others by the Cielab colour parameters and a peculiar anthocyanin profile. Syrahs present a more dispersed distribution and, in the quadrants opposite the SG, are characterised by the highest concentrations of anthocyanins and phenols; MRLs form a compact group near the centre of the graph. The latter have high concentrations of flavonols, both free and bound to sugars and glucuronic acid, as well as resveratrol, all compounds with high antioxidant power; the CBS, on the other hand, have higher concentrations of tannins and complexed anthocyanins.

Figure 2. PCA: score plot (top) and loading plot (bottom) of ANT samples. CBF: cabernet franc; CBS: cabernet sauvignon; CBT: blend of cabernet sauvignon and franc; MRL: merlot; SG: sangiovese; SYR: syrah.

A correlation analysis was also carried out in order to identify which of the analysed parameters correlated positively or negatively with the instability of the samples defined by the project partners. The cloudiness measured by the hot laboratory test (equivalent to the test normally used to assess the protein stability of white wines) correlates positively with the theoretical instability; the cloudiness in turn shows a positive and statistically significant correlation with the anthocyanin-tannin complexes and also with the polysaccharides. The latter in turn correlated significantly with tannins (Figures 3-5), while no correlation was found between cloudiness and protein content in red wines.

Figure 3. Correlation between cloudiness from hot test and instability of samples defined by producers.
Figure 4. Correlation between cloudiness from hot test and anthocyan-tannin complexes
Figure 5. Correlation between cloudiness from hot test and polysaccharides

Statistical analysis therefore seems to indicate that in defining the stability of a wine, the ratios between anthocyanins and tannins and their polymerisation are fundamental. Based on these considerations, an instability index was developed, defined as the ratio between anthocyanin-tannin complexes (measured using a spectrophotometric index in Absorbance Units) and total tannins (Atc/T). In particular, when this index is lower than 1, the samples are stable. The evaluation of the ratio between free anthocyanins and tannins was also interesting.
As a function of the hot tests, the samples were stable for ΔNTU values lower than 2. Figure 6 shows the correlation between hot test clouding and index.

Figure 6. Correlation between cloudiness from hot test and polysaccharides

With regard to the tests carried out in the cellar, for the same oenological treatment, the response depends on the variety considered. As an example, we report the results of the heat test of cabernet and sangiovese samples subjected to the addition of skin tannin (B), chestnut (C), alone or in combination with two yeast derivatives (A1 and A2).

Figure 7. Cloudiness after hot testing of cabernet (CBS) and sangiovese (SGV) samples subjected to various oenological treatments in the cellar.

The untreated cabernet is found to be unstable (ΔNTU>3) and its stability seems to improve when treated with tannins, especially when combined with yeast derivatives. If we study in detail how anthocyanins and tannins change in the treated samples, we notice that both anthocyanins, in all their forms, and tannins decrease considerably in the treated samples, as does the Atc/T index (Figure 8), probably due to the precipitation of anthocyanins and tannins by the tannin treatments. This is reflected in a heavy loss of colour intensity in the samples treated with tannins, especially if they are chestnut (Figure 8). The addition of yeast derivatives alone, on the other hand, stabilises the samples without impacting the colour.

Figure 8. Development of Atc/T index and colour intensity in cabernet samples subjected to various oenological treatments in the winery

In Sangiovese, the unaltered samples are stable (Fig. 7) when subjected to a hot test and only destabilise when treated with chestnut tannin. In these samples, however, no phenomena affecting anthocyanins and tannins are noted, so that there are no significant variations in colouring intensity, but there is a decrease in total polysaccharides.
Among the innovative analytical techniques tested during the project, interesting results were obtained from the measurements of the Potential ζ, which is a measure of the repulsive forces between the particles, not so much considering its absolute values as its variations in relation to the oenological treatments applied. In theory, increases in the values of the potential indicate greater repulsion between the colloids in solution, and therefore greater stability. The results of the Potential ζ confirm those of the hot test and the indications of the complexed anthocyanins to tannins ratio on the same samples: the samples treated with tannins, especially if chestnut and in combination with yeast derivatives, are more stable.

Potential ζ shows statistically significant correlations with anthocyanin-tannin complexes. 


Although the hot test seems to be of general validity, the salient quantities found in the description of the colloidal dynamics of red wines are strictly dependent on the matrix and above all on the variety. The phenomena observed are easily traceable to the properties of polyphenols, polysaccharides and macromolecules in general, although many of their aspects still need to be clarified in detail. 

The evaluations carried out are aimed at estimating the intrinsic colloidal stability of wines, but they can also be useful in consideration of colloids of exogenous origin (e.g. protein clarifications, gums, etc.); in such cases, verifications specifically aimed at the products/formulates used must be taken into consideration. In this regard, prior to the use of certain adjuvants, it is specifically prescribed to verify the stability of the wine.


  • Cellar treatments

During the first year of the project, three wines (a Sangiovese, a Merlot and a Cabernet Sauvignon) were treated with either skin tannin or yeast autolysate. In the second year, two types of tannin ( skin and chestnut) and two yeast derivatives were tested on a sangiovese and a cabernet sauvignon, while only the yeast derivatives were tested on a merlot (Table 1).

Table 1. Outline of the oenological treatments to which Sangiovese, Cabernet and Merlot were subjected.
  • Chemical analysis

All samples were subjected to a wide range of chemical analyses by means of diversified analytical approaches, both traditional and innovative. Specifically, within the Isvea srl (SI) laboratories, the following were carried out

  • analyses on the basic chemical profile and on the colorimetric one using traditional analytical techniques of destructive type (OIV Methods);
  • colour analyses were carried out using UV-Vis type techniques;
  • analyses of the polyphenolic and anthocyanin profile using liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) and photodiodarray (PDA);
  • The ζ-potential: The main reason for measuring the ζ-potential is to predict colloidal stability. Interactions between particles play an important role in colloidal stability. Using the ζ-potential in order to predict the stability of the system is an attempt to quantify these interactions. The ζ-potential is a measure of the repulsive forces between particles. And since most aqueous colloidal systems are stabilised by electrostatic repulsion, the likelihood of the repulsive forces between the particles coming together and forming a complex will be quite difficult. Thus a colloid will be more stable. The potential ζ is derived from the measurement of the mobility distribution of a scattering of charged particles when subjected to an electric field. This mobility is defined as the velocity of a particle per unit electric field and is measured by applying an electric field to the dispersion of particles and measuring their average velocity
  • Statistical Analysis

The large number of chemical parameters analysed made it inevitable to process the data using multivariate statistical algorithms. This was done in order to select both the test to be used for the evaluation of the colloidal instability of red wines and the significant variables to be measured. In particular, it was decided to apply principal component analysis (PCA) for the qualitative evaluation of the data. The data was previously normalised. PCA is a data dimensionality reduction technique whose central idea is to reduce a more or less high number of variables (representing as many characteristics of the objects analysed and more or less correlated with each other), into a number of uncorrelated latent variables, which express the largest possible share of the variance present in the data (called “Principal Components”). This is achieved through a linear transformation of the variables, which projects the original ones into a new Cartesian system, in which the new variable with the highest variance is projected on the first axis, the new variable, second in variance size, on the second axis, and so on. The reduction in complexity is achieved by simply analysing the main ones (by share of variance expressed) among the new variables obtained.  

The VINTEGRO project will develop a test to predict wine instability based on several processes and on new technical analysis, the study of suitable technics to remove unstable proteins of the wine from fermentative step. The project aims to increase the knowledge on the role of proteins in red wine stability and the drawing up of guide lines to produce naturally stable wines, without precipitation risks in the bottle and with a longer shelf-life.

The project started at the beginning of February 2019 and will last until April 2022.

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