Use of polyaspartates for the tartaric stabilisation of white and red wines and side effects on wine characteristics

Aim: The stabilising efficacy against tartaric precipitations of polyaspartates-based products (PAs), in particular potassium polyaspartate (KPA), was tested with six different wines (three white and three red). Some side effects on wine characteristics (white wine colour stability, wine turbidity and filterability) were also studied.Results and conclusions: All PAs showed good stabilising efficacy against tartaric precipitations according to the cold test. With the same test, the PAs were stable in wine for 1 year of storage, which was the total duration of the study. The dose of 100 mg/L was sufficient to stabilise the tested wines. No differences in filterability were observed in comparison with MTA (metatartaric acid). The hypothesised protective effect against colour browning in white wines was not observed.Significance and impact of the study: The international wine trade requires stable wines. This paper provides information to support wineries in managing the use of KPA, as little information is available to date in the literature on this stabilising additive.


INTRODUCTION
Potassium bitartrate (KHT) is the main salt present in wine. Its concentration is variable and depends on wine pH and on the content of tartaric acid and potassium. KHT has a low solubility in hydroalcoholic solutions and it can precipitate in wines during bottle ageing, causing the formation of a crystalline sediment (Usseglio-Tomasset et al., 1992). Various oenological practices can be performed to prevent the formation of this deposit, which is not acceptable to most consumers. These practices consist in removing the excess KHT by precipitation at a low temperature (Ribéreau-Gayon et al., 2006), or a part of bitartrate and potassium ions by electrodialysis (Moutounet and Escudier, 1991), or only potassium with cation exchange resins (Mourgue, 1993).
The use of certain additives that can inhibit the growth of KHT crystals or act as protective colloids is also authorised by the EU in order to prevent the appearance of precipitates in the bottle. Metatartaric acid (MTA) was the first additive to be authorised, followed by carboxymethylcellulose (CMC) and mannoproteins (MP), and more recently the use of potassium polyaspartate (KPA) (Commission delegated regulation EU 2017/1961-August 2, 2017. Polyaspartates are homopolypeptides synthesised by thermal polymerisation of L-aspartic acid (ASP) or monoammonium malate, resulting in polysuccinimide (PSI), followed by hydrolysis (Bosso et al., 2015). Polyaspartates are involved in the inhibition of calcium sulphate scale formation (Sarig and Shifrin, 1977) and some of them, such as the sodium salt of polyaspartate, are used to prevent fouling (by changing the crystal structure of calcium salts), as an additive in detergents, for the prevention of corrosion, as an adjuvant in the prevention of dental plaque, and as an excipient in certain drugs. Many works have been published on the stabilising properties of MTA, CMC and MP (Lubbers et al., 1993;Crachereau et al., 2001;Moine-Ledoux and Dubourdieu, 2002;Bosso et al., 2010;Gerbaud et al., 2010;Claus et al., 2014;Guise et al., 2014;Coulter et al., 2015). However, to our knowledge only our previous work (Bosso et al., 2015) has focused on the screening of various types of polyaspartates (PAs), different per producer, type of salt (sodium or potassium) and average molecular weight, added to white and red wines provided by commercial wineries.
The molecular weights of the tested PAs ranged from 2 to 8 KDa. The products were added at a dose of 100 mg/L to the wines before bottling.
All PAs proved to have a good stabilising efficacy against KHT precipitation and a good stability in wine over time, unlike MTA. No differences in stabilising efficacy between the various PAs were observed soon after the addition, whereas some differences were noticed in the duration of their efficacy over time.
The aim of the present work was to verify the stabilising properties of PAs on tartaric precipitations, focusing mainly, but not exclusively, on potassium polyaspartate (KPA), whose use has recently been authorised in oenology. The trials were carried out with some white and red wines provided by commercial wineries, and with two wines (one white and one red) adjusted to two different levels of pH and alcoholic content, in order to modify the degree of tartaric instability by intervening on the dissociation degree (pH variation) and solubility (variation of the alcohol content) of KHT.
As reported above, PAs are obtained by polymerisation of L-aspartic acid. MTA, obtained by polymerisation of tartaric acid, proved to be particularly unstable in wine, where it is rapidly hydrolysed to tartaric acid, thus losing its stabilising efficacy. Storage trials were therefore conducted in order to verify the duration over time of the stabilising efficacy of PAs in wine (a medium with acidic pH). Other possible side effects of KPA were also evaluated, such as the impact on wine turbidity and filterability. Finally, as PAs are used by the industry as de-scaling agents of water pipes due to their chelating properties for the calcium ion (Mocanu et al., 2012), we also wanted to verify whether a chelating effect for other cations, such as iron and copper, could have consequences on the evolution of the colour of white wine. This paper reports the results of the study on a laboratory scale of some oenological properties of KPA that have been investigated during the STABIWINE project (''Use of biopolymers for sustainable stabilisation of quality wines'' -Grant Agreement n.314903 -Seventh Framework Program. Theme: SME-2012-2. Research for SME associations), in parallel with the research on the food safety of the product.
The data collected during the project supported the planning of large-scale trials in commercial wineries whose results, together with those concerning the product safety, led to the EU authorisation of the use of KPA as an additive for the tartaric stabilisation of wines.

Experimental trials
This work was aimed at determining the effect of adding some PAs, in particular KPA, to white and red wines sampled directly from the tank in commercial wineries. The following oenological properties of the additives were studied: • effect on tartaric stability immediately after addition (different wines and different doses) • effect on tartaric stability after 6 or 12 months of bottle ageing • effect on wine filterability immediately after the addition • effect on the colour of white wines Due to the large number of tests, it was impossible to perform all of them simultaneously or delay them over time while still working with the same wines, with the risk of finding important changes in their composition and degree of tartaric instability. The study of the different oenological properties was therefore performed at different times with different wines. At the beginning of each experiment, the wines were in the condition of tartaric instability, but they were ready for bottling according to other parameters, such as protein stability for white wines and limpidity for all wines. Table 1 shows the additives used for the tests, and Table 2 summarises the experiments performed with the different wines.
The effect of KPA on tartaric stability was assessed for all the wines involved in study. All trials were performed in duplicate. For the storage tests the wines were bottled and stored in a thermostatic room at 20 °C.
In addition, for a white and a red wine (W1 and R3, Table 2, Experiment 4) the alcoholic degree and pH were artificially modified to verify whether the stabilising efficacy of KPA could be influenced by these parameters. In particular, a complete factorial design was planned by varying on two levels the factors pH (3.00 and 3.70), alcoholic degree (12 and 15 % v/v) and dose of KPA (100 and 200 mg/L).
As regards the effect on the colour stability of white wines (Table 2, Experiment 2), the chelating effect of PAs for iron and copper was verified using two different products. For the first white wine (W1), the concentration of the two metals (iron and copper) was increased and adjusted to 5.0 and 0.8 mg/L, respectively. The control wine was compared with two treated with KPA (100 and 200 mg/L) and two treated with NaPA-10 (100 and 200 mg/L). For the second white wine (W2), the natural content of 1.5 mg/L iron and 0.075 mg/L copper was not corrected, and the control wine was compared with two treated with KPA at the doses of 100 and 200 mg/L. All trials were oxygenated (4 mg/L oxygen), then bottled in 750 mL bottles and stored in a thermostatic room at 20 °C.

Free aspartic acid (Table 2, Experiment 1)
The free aspartic acid content was determined with HPLC after 12 months of bottle ageing according to the method proposed by Park et al. (2000) and modified by Bosso et al. (2015), based on a pre-column derivatisation reaction between ortho-phthalaldehyde (OPA) and 2-aminoethanol as derivatising agents, and with 2-methylaspartate as internal standard.

Dissolved oxygen (Table 2, Experiment 4)
The concentration of dissolved oxygen in the white wines was measured with a luminescence-based technology (NomaSense™ O 2 Trace, PreSens GmbH, Regensburg, Germany).

Cold test
The tartaric acid content in the wines was determined with HPLC (Cane, 1990) before and after storage at -4 °C for 6 days. After cooling and before the analysis, the wines were filtered (0.45 µm) at a low temperature to avoid the solubilisation of the KHT salts. The concentration of tartaric acid (H 2 T) was measured before and after the period of cold storage, and the difference between the two values due to the precipitation of KHT was calculated: ΔH 2 T represents an index of the stability/instability degree of wines (Motta et al., 2009). The wines are considered stable (no loss of tartaric acid by precipitation) when ΔH 2 T is ≤ 0.1 (Bosso et al., 2016).

Conductivity test (mini-contact test)
The tartaric stability of the wines was assessed by measuring the drop of electric conductivity (Δχ expressed as µS/cm) of 100 mL wine at 0 °C, 4 min after the addition of finely micronised KHT (10 g/L) as a precipitating agent: the higher the difference in conductivity, the higher the tartaric instability (Bosso et al., 2016). The test was performed with Check Stab a-2001 Millennium (Delta Acque, Firenze, Italy). The measurements were taken with a probe made up of two platinum electrodes, which were periodically recalibrated with a 0.01 N KCl solution to ensure a high degree of reliability. The temperature was controlled to a resolution of 0.1 °C. The tartaric stability thresholds, at the described operating conditions, were equal to 150 and 100 µS/cm for white and red wines, respectively.

Physicochemical analyses
Ethanol concentration, total extract, pH, titratable acidity, free and total SO 2 were determined according to EU methods (EEC Regulation 2676/90), and acetaldehyde was measured with a colorimetric method (Di Stefano and Ciolfi, 1982). For white wines, the absorbance at 420 nm (A420) was measured with a quartz cuvette (10 mm o.p.) after filtration with a 0.45 µm polypropylene filter. CIELAB indices (cylindrical coordinates: L* lightness, C* chroma, h* hue) were determined according to Piracci (1994

Experiment 1: Influence of different types of PAs on the tartaric stability of a Dolcetto red wine (R1) after the addition and during aging
After the addition of PAs and MTA all wines were stable to tartaric precipitations (Table 3). After 3 months of bottle aging the MTA trial became unstable, while all PAs trials remained stable.
No statistically significant differences between the PAs trials were observed with the cold test, while according to the conductivity test the NaPA-10 and NaPA-15 trials had significantly lower Δχ values than the NaPA-3 and KPA trials.
After 6 months of bottle aging, the cold test showed that the PAs trials maintained their stability to tartaric precipitations, and the instability degree of the control and MTA trials remained the same. Conversely, the conductivity test showed an important increase in Δχ values for all wines, particularly for the PAs trials that resulted unstable with this test.
The analyses were repeated after 12 months of bottle aging: the Δχ values further increased for all wines, particularly for the PAs trials. Despite this, all PAs trials were still stable with the cold test. An increase in tartaric instability, determined with the cold test, was observed only for the control.
The good stability of PAs to acidic cleavage at room temperature (20 °C) was confirmed by the low increase of free aspartic acid content (monomer released after PAs hydrolysis) in the wines after 12 months of bottle aging: the largest difference in its concentration between the PAs trials and the control was only 3.55 mg/L (3.55 % by weight of the added amount of PAs) (Table 3).
These results were in agreement with a previous work (Bosso et al., 2015): the PAs had a good stabilising efficacy against tartaric precipitations, which was maintained after 12 months of aging; conversely, the MTA trials were unstable 3 months after the additions. The increase of the Δχ parameter over time, which was also observed during further experiments (data not reported), could depend on the fact that during aging KPA interacts permanently with the wine's KHT crystals and loses the ability to react with the excess KHT that is added during the conductivity test. During a recent study on the mechanisms of action of some additives used to prevent the growth of KHT crystals in wines, Lankhorst et al. (2017) hypothesised that the effect of these additives is due to their adsorption on the surface of the crystals. In general, the cold test is considered the reference test to evaluate wine tartaric stability from an objective thermodynamic point of view, while the conductivity test is accepted by the industry due to its rapidity and ease of use, and this discrepancy in the results could lead to negative commercial consequences for wines containing KPA. Therefore, the conductivity test seems to be suitable for verifying the degree of tartaric stability/instability of the wines soon after the addition of KPA, but not always during wine aging. To date, no other works in the literature highlighted this behaviour of the mini-contact test with wines treated with potassium polyaspartate, due to the current lack of research on the use of this additive in oenology. Table 4 shows the results of the cold and conductivity tests performed for the W1 white wine.

Effect on tartaric stability
After storage for 6 days at -4 °C (cold test), the control was significantly unstable compared to the trials with the two PAs added at two different doses (stable). The same result was obtained with the conductivity test (the stability thresholds are reported in the Materials and methods section). No significant differences were observed between the two PAs and the two doses used with the cold test; on the contrary, there were statistically significant differences between the trials with 100 mg/L and the trials with 200 mg/L of PAs with the conductivity test.
After 6 months of bottle aging (   3. Mean values of the stability indexes (∆χ and ∆H 2 T) after the addition of the products to the Dolcetto red wine and after 3, 6 and 12 months of bottle aging, and free aspartic acid content after 12 months of bottle aging. Comparison among different types of PAs and MTA (ANOVA and Tukey's test).
Different letters along the line discriminate the trials significantly different from one another (p < 0.05, Tukey's test). **, *** represent significance at p≤ 0.01 and p≤ 0.001, respectively. test (Δχ = 45.80 and 37.10 µS/cm for the trials with 100 and 200 mg/L of KPA, respectively). The results of the cold test did not change after 12 months, while the Δχ values (conductivity test), especially for the PAs trials, increased (Δχ = 156.6 and 111.7 µS/cm for the trials with 100 and 200 mg/L of KPA, respectively).
The poor reproducibility of the conductivity test during aging, tartaric stability being equal (when measured with the cold test), suggests that the conductivity test is not suitable for measuring over time the tartaric stability of PAs added wines.

Effect on colour stability and on wine oxidation process
Two experiments were carried out to assess the influence of PAs on the colour of white wines and the oxidative evolution, associated with the possible chelating effect for iron and copper. Therefore, all trials were oxygenated before bottling, at the dose of 4 mg/L, corresponding to an amount of oxygen 4-5 times greater than the dose that is normally dissolved in the wines during correct bottling operations.
At the beginning of the first experiment (W1 wine, Table 4), all trials were similar for the content of free and total SO 2 and colour intensity (A420). After 6 months of bottle aging, only small significant differences in acetaldehyde content were present. SO 2 and acetaldehyde concentrations are related to the wine oxidation process: the free and total SO 2 content tends to decrease by oxidation, while the acetaldehyde content increases (Fenton's reaction) (Elias and Waterhouse, 2010). The absence of important differences between the trials for these parameters suggests that PAs have no role in the wine oxidation process. During the first 15 days following the oxygen supplies, the concentration of dissolved molecular oxygen was also monitored (Supplementary material: Figure A). All wines showed a high oxygen consumption rate, similar for all trials. Danilewicz (2011) suggested that the consumption rate of molecular oxygen, soon after the oxygen supplies, is influenced by the presence of metals and in particular iron. In fact, the molecular oxygen reacts directly and rapidly with iron (Fe ++ ), reducing itself to peroxyl radical capable of oxidizing phenols, whereas SO 2 and phenols participate later in the oxidation process by reacting with the peroxyl radical that is formed by iron. Moreover, it seems that copper acts as a catalyser of the reaction between O 2 and Fe ++ (Danilewicz, 2011). When increasing the concentration of iron and copper in wines, an increase was observed in the consumption rate  (Danilewicz, 2007).
According to our results, a chelating effect of PAs for iron and copper should be excluded, because otherwise a decrease of the initial oxygen consumption rate should have been observed in the PAs trials, particularly with the higher dose. The same can be deduced by considering another step of the oxidation process, the Fenton's reaction, during which the hydrogen peroxide that is formed by reduction of the peroxyl radical in the presence of phenols or SO 2 is further reduced to hydroxyl radical in the presence of Fe ++ (Elias and Waterhouse, 2010). A decrease in the rate of this reaction limits the production of acetaldehyde via oxidation of ethanol by the hydroxyl radical. Conversely, in our case the acetaldehyde content did not decrease, but rather it slightly increased in the PAs trials compared to the control.
For this first experiment, iron and copper were added to a wine (W1) in order to accelerate the oxidation reaction and to emphasise the possible differences due to the chelating effect of PAs for metals. The second experiment (W2), without addition of exogenous iron and copper, was performed to verify whether an excess of these metals could have had a masking effect on the chelating action of PAs.
A white wine was used with an original content of 1.5 and 0.075 mg/L of iron and copper, respectively (W2). KPA was added to the wine at two different doses (100 and 200 mg/L). The results (Table 5) confirmed what was observed in the first experiment: no important differences were noticed for free and total SO 2 , acetaldehyde and colour parameters after 1.5, 3, 6 and 12 months of bottle aging. Moreover, the addition of KPA did not slow down the oxygen consumption rate that, on the contrary, resulted increased compared to the control (Supplementary material: Figure B).
The presence of KPA did not influence white wine colour and its oxidation process during bottle aging. Unlike for PAs, some authors (Guise et al., 2014) observed a stabilising effect of CMC on wine colour, variable with the type and dose of CMC, which was probably due to the removal of polyphenols. On the contrary, this effect was not observed during our experiments with PAs.
No differences between the PAs trials and the control were observed soon after the addition of PAs nor after 6 months (first experiment) and 1 year (second experiment) of bottle aging. As an example, soon after the addition of PAs all wines of the first experiment had an average total polyphenols content of 110-111 mg/L, and after 6 months the average concentration dropped to 103 mg/L in the control and 98 and 101 mg/L in the trials with 100 and 200 mg/L of PAs, respectively.

Experiment 3: Effect of increasing doses of KPA on the tartaric stability and turbidity of a red wine
The Syrah red wine (R2 wine) was highly unstable, and after the cold test it had lost, by KHT precipitation, a large amount of tartaric acid  (1.23 g/L) equivalent to 40.6 % of the original content before the cold test (Table 6). The wine became stable with the addition of MTA or KPA at the lower dose (100 mg/L). Turbidity was 9.5 NTU for the control, and it decreased for the wines with 100, 200 and 500 mg/L KPA and 100 mg/L MTA. This experiment was aimed at verifying whether the addition of KPA at doses higher than the maximum authorised level could negatively affect the colloidal equilibrium of the wine, causing an increase in turbidity. The results showed that only doses 10 times higher than the maximum authorised level resulted in a statistically significant increase in turbidity.

White wine (W1)
Table 7 reports the average results of the tartaric stability tests (cold test and conductivity test) for the white wines whose alcoholic degree and pH were artificially modified. All trials had the same content of tartaric acid and potassium.
The wines with pH 3.00 were more stable to KHT precipitations than those with pH 3.70. The pH influences the dissociation degree of tartaric acid: for the same concentration of tartaric acid a decrease of wine pH causes a decrease in the concentration of HTions. Considering the pK values (pK = -logK, where K is the dissociation constant of an acid) of tartaric acid, that is pK 1 = 3.04 and pK 2 = 4.37 as reported by Usseglio-Tomasset (1995), it is possible to calculate the percentages of the three forms of tartaric acid (H 2 T, HTand T 2-) present in a wine at a given pH. In particular, the percentage of HTions that form insoluble salts with K + ions varies from 46.75 % to 69.80 % (50 % variation) of the tartaric acid present in wine when its pH varies from 3.00 to 3.70. After the addition of KPA all wines became stable, but the effect of KPA seemed weaker at low pH values. However, as the wines were originally less unstable at a low pH than at a high pH, a correct evaluation was not possible.
To date, no studies have yet been conducted to define the mechanism of action of KPA in wines. Some authors (Lankhorst et al., 2017) have recently studied the mechanisms of action of CMC, MTA and MP in a model solution and in a deionised white wine, spiked with KHT, with the use of a method based on dynamic light scattering. The method can detect the presence of wine colloids with sizes ranging from 10 -3 to 10 -1 μm, which are already present in wine, and it can distinguish them from the KHT crystals. The results showed that these additives cannot prevent the nucleation of KHT crystals, but rather intervene by slowing down their growth. Crachereau et al. (2001) studied the effect of CMC on the growth of KHT crystals in a supersaturated solution of the salt: CMC caused a slowing down of the KHT crystals growth and a modification of their shape. According to the authors, this effect was due to the interactions between the negatively charged CMC and the faces of the KHT crystals, positively charged by the accumulation of potassium ions, in competition with the bitartrate ions. It could be hypothesised that also the stabilising action of KPA depends on its negative charge; indeed, all additives used for the tartaric stabilisation of wines, including mannoproteins, are negatively charged. Wine pH could therefore influence the stabilising efficacy of KPA by modifying its negative charge, which decreases with decreasing pH.
The increase of the alcoholic degree did not influence the results of the conductivity test (Δχ), but conversely a negative and significant effect on tartaric stability was observed with the cold test (  the formulas proposed by Usseglio-Tomasset et al. (1992), it can be estimated that an increase of 3 % ethanol from 12 to 15 % v/v causes a 25 % reduction of the KHT solubility product in a wine with a saturation temperature ranging from 10 to 15 °C. During the cold test, the appearance and precipitation of KHT crystals in unstable wines are due to the formation of crystallisation nuclei (primary crystallisation) and to their subsequent growth. The formation of the crystallization nuclei depends on the supersaturation state of the wines, that in turn is influenced by the alcohol content. On the contrary, the alcohol content does not influence the conductivity test, during which exogenous KHT crystals are added as crystallisation nuclei, and the measured conductivity drop is only caused by the growth of the added KHT crystals. This is probably the reason for the differences in results observed between the two tests.
After the addition of KPA (independently of the dose) all wines became stable, regardless of their initial level of tartaric instability. Statistically significant differences between wines with different alcoholic degree were observed with the conductivity test, but they were modest and of no practical interest. In all cases, a dose of 100 mg/L was sufficient to stabilise the wines. No differences were observed with the cold test between the trials with different doses of KPA, whereas some significant differences, but without any practical interest, were noticed with the conductivity test.

Red wine (R3)
The experiment was repeated with a Montepulciano red wine (R3) with an initial low degree of tartaric instability. After the adjustment of wine pH, the trials acidified at pH 3.00 became stable, whereas those at pH 3.70 became more unstable than the original wine. As with the white wine, the increase in alcoholic degree caused an increase of tartaric instability, which in this case was significant with both the cold and the conductivity tests. After the addition of KPA, all wines became stable and no dose effect was observed (Table 7).
Finally, wine turbidity was measured before and after the addition of KPA. Statistically significant interactions between pH and alcohol content were observed (Supplementary material: Table A). The initial wine turbidity was on average 5 NTU; after the addition of KPA the turbidity remained virtually unchanged in the trials at pH 3.00, whereas it increased in the trials at pH 3.70, particularly in those with the higher alcoholic degree. The appearance of haze after  the addition of KPA was a rare event throughout the whole research, observed only in this case (R3 red wine) during this work. The appearance of haze observed with an increase of pH could be the consequence of electrostatic interactions between molecules with opposite charges. The destabilising effect of the high alcoholic degree was additive to the pH effect: the trials with the highest alcoholic degree were also the most unstable. However, the pH effect prevailed on the alcohol effect: at low pH no differences in turbidity were observed between the trials with different alcoholic degree.

Experiment 5: Effect of KPA on wine filterability
The filterability of the Cortese white wine (W3) and Barbera red wine (R4) was tested 48 hours after the addition of KPA and MTA. The addition of both additives did not modify wine filterability (Figure 1). For the control, MTA and KPA trials, the filterability index (FI) and the modified filterability index (MIF) were respectively 9, 6, 7 and 7, 6, 6 for the white wine (good filterability), and 17, 19, 18 and 20, 19, 20 for the red wine (values considered as acceptable according to the scale reported in section 2.2.3. above). The addition of KPA caused a similar effect as MTA, that is usually added to wines before filtration at bottling.

CONCLUSION
This paper concerns the study of the tartaric stabilising properties of polyaspartates (PAs), in particular of potassium polyaspartate (KPA). During a screening of different PAs added to a Dolcetto red wine, all products showed a good stabilising efficacy against tartaric precipitations that remained unchanged after 1 year of bottle aging, according to the cold test. The other experiments concerned mainly the study of the stabilising efficacy of KPA when varying the type of wine, its chemical composition (pH, alcoholic degree) and the dose of use.
Regardless of the initial degree of tartaric instability, the dose of 100 mg/L was sufficient to stabilise all tested wines. Moreover, an experiment was carried out with increasing doses of KPA, up to 1000 mg/L, and also in this case the minimum dose (100 mg/L) was sufficient to stabilise the wine, but an increase of wine turbidity was observed with the maximum dose (1000 mg/L).
The modification of wine composition (alcoholic degree and pH) influenced the degree of tartaric instability, which increased with the increase of pH and alcoholic degree. Regardless of the initial degree of instability, the dose of 100 mg/L was sufficient to stabilise all wines.
Moreover, some experiments were performed to verify the influence of KPA on the oxidation process of white wines and on their colour evolution over time. The trend of the oxidation process was studied by monitoring the evolution of free and total SO 2 , acetaldehyde and colour parameters until 6 or 12 months after the addition of PAs. The presence of PAs did not cause statistically significant variations of the studied parameters. The chelating effect of KPA for iron and copper, expected to cause a slowdown of the oxygen consumption rate, was not observed.
The use of additives for wine tartaric stabilisation is generally recommended at bottling, just before the final filtration. Like MTA, the addition of KPA did not modify wine filterability (0.45 µm filter).
Some aspects related to the interactions between PAs, the colouring substance and, in general, the colloidal matter of the red wines, have not been discussed here and will be the object of a future work.