Clarification and Protein Stabilization of White Wines by Ultrafiltration

Clarification and protein stabilization of white wines by ultrafiltration

MARIANA RODRIGUES DE SOUSA1, MARIA NORBERTA DE PINHO1, SOFIA CATARINO1,2,3

* Corresponding author: 1 CeFEMA, Instituto Superior Técnico (IST), Universidade de Lisboa, Av. Rovisco Pais, 1, 1049-001 Lisboa, Portugal 2 LEAF, Instituto Superior de Agronomia (ISA), Universidade de Lisboa, Tapada da Ajuda, 1349-107 Lisboa, Portugal 3 Instituto Nacional de Investigação Agrária e Veterinária, 2651-191 Dois Portos, Portugal

The main objective of this work was to evaluate the influence of ultrafiltration (UF) on the clarification and protein stabilization of white wines. For that, three white wines, protein unstable, form 2016 vintage processed by UF, using a Celfa P-28 instalation with a fluoro polymer UF membrane, FS61PP, from AlfaLaval, with a surface area of 25.25 cm2. The membrane was characterized by hydraulic permeability of 44 kg/(bar h m2) and molecular weight cut-off of 45 kDa. The white wines UF assays were performed at a transmembrane pressure of 1.2 bar, recirculation rate of 0.79 L/min at 24 ºC. UF fractions were evaluated in terms of protein stability, physical-chemical composition, total proteins content, total polysaccharides, total phenols index and turbidity. Total protein content of the wines Viosinho, Lote and Moscatel Graúdo was initially 54.9, 119.2 and 82.3 mg/L, respectively. After UF, the total protein concentration of all the wines was about 5 g/L. In spite of that, protein stability, evaluated in permeate UF stream by applying two thermal stability tests, was not achieved, suggesting that probably proteins of low molecular weight are responsible for protein haze. As expected, significant removal of polysaccharides from the wines was observed. This could justify the heat test result because the polysaccharides that act as protective colloids did not permeate. In respect to total phenols index, decreases close to 10% were verified. Clarification of wines was achieved by UF, resulting in turbidity decreases from 46 (Viosinho), 17 (Lote) and 4.7 NTU (Moscatel Graúdo) to values close to 2 NTU.

KEYWORDS: white wine, protein stabilization, clarification, ultrafiltration.

INTRODUCTION process as a defense mechanism for pathogenesis attacks and infections 12. PR proteins include thaumatin- Protein instability in white wines is a serious problem of like proteins (TLP) and chitinases 13,14 and its physical-chemical instability affecting wine limpidity. concentration in wines could reach several hundred mg/L Despite the numerous studies carried out on this 15. Some factors are known to affect protein stability in phenomenon in the last decades, the mechanisms wines, like protein content, protein nature, the difference involved remain not fully understood, which explains the between wine’s pH and protein’s isoelectric point, wine’s lacking of a satisfactory preventive treatment, specific pH and the presence of non-proteinous compounds 16-18. enough to maintain the wine sensory characteristics. Heat stability tests are used to evaluate wine’s protein Proteins, occurring in concentrations from 15 to stability. The most common ones are: heat test, 300 mg/L, are important components of colloidal stability, trichloroacetic acid test, bentotest, ethanol test and heat as well as polysaccharides. Unfavorable storage and test with tannin addition. Heat stability tests only predict transportation conditions can lead to a slow denaturation protein’s instability phenomena, do not reproduce it. that is thought to originate protein aggregation and Nowadays, protein instability is prevented by removal of flocculation into a hazy suspension, which leads to haze wine proteins with finning agents, namely by using appearance or deposits formation from protein bentonites. Finning agents are substances normally 1–4 precipitation . This unattractive haze does not affect charged that when added to wine adsorb and precipitate olfactory neither gustatory characteristics of the wine, yet, opposite charged particles which are responsible for translucency is a vital visual characteristic to wines quality wine’s haze. Bentonite finning is the most common because this property makes the first impression on the practice used for preventing protein instability in white consumer, who will reject wines containing cloudy wines. However, under some circumstances bentonite precipitates regardless of how the wine tastes. Wine’s finning can affect wine’s quality and change wine protein concentration and proteins fraction depends on sensorial characteristics by removing the components grape variety, grape maturation, climate conditions, and that contribute to color, taste and aroma 13,19. Moreover, 5,6 winemaking process, among others . bentonite finning leads to the production of lees, that Some authors claim that protein instability is related results in wine loss of 3–10 % 20. 7,8 1,2,9,10 with total protein content in wine , but others have Alternatives to bentonite finning have been studied, demonstrated that different protein fractions behave such as alternative finning agents (zirconium oxides, ionic differently, showing different susceptibility to exchange resins, silica gel, hidroxipatite, alumina, quitin denaturation. This leads to the conclusion that protein and natural zeolites), ultrafiltration 2,21, flash 11 instability is related to some specific protein fractions . pasteurization, proteolytic enzymes and protective Proteins responsible for protein casse are originated from colloids such as mannoproteins. grapes and has been identified as pathogenies-related (PR). PR proteins are synthetized during maturation

The aim of this work was to study the effect of the solute concentrations were determined thought total ultrafiltration on a variety of white wines that present carbon concentration measurements. protein instability and visual haze. Membrane compaction and cleaning. Before starting MATERIAL AND METHODS the permeation experiments, the membrane was compacted. Compaction stage aims the minimization of Wines. UF experiments was carried out with white any effects on the membrane’s structure during UF wines, produced in Instituto Superior de Agronomia de assays due to the pressure, insuring that the permeation Lisboa (Tapada da Ajuda, Portugal) by the conventional flow does not decrease. Compaction requires the winemaking technology for white wines Two monovariety permeation of deionized water at a membrane pressure wines (Vitis Vinifera L., Viosinho and Moscatel Graúdo 20% higher than the operation pressure in total grape varieties) and a wine produced from three grape recirculation mode for a period of 2–3 hours. Membrane’s varieties (75% v/v of Viosinho, 12.5% v/v of Alvarinho, cleaning follows every permeation experiment, in total 12.5% v/v of Arinto), from 2016 harvest, were used. The recirculation mode. This operation is performed with general physical-chemical characteristics of the wines deionized water at low pressure (0.5 bar) and maximum are presented in Table 1. recirculation rate for at least 2 hours, up to reach 90% of Table 1 – Physical-chemical composition of white wines. initial flux recovery. White wines Ultrafiltration. Ultrafiltration was carried out using a Analytical parameter Viosinho Lote Moscatel commercial equipment Celfa P-28, represented in Graúdo Figure 1, with the membrane FS61PP with a surface area Alcoholic strenght 12.1(0.3) 12.7(0.2) 13.8(0.1) of 25,25 cm2. (% vol.)

Density 0.9934 0.9920 0.9896 (g/mL at 20 ºC) Total acidity 6.2 6.1(0.1) 5.3 (g tartaric acid/L) Volatile acidity 0.16 0.17(0.01) 0.27(0.02) (g acetic acid/L) Reducing substances 1.5 0.8 (0.03) 0.7(0.03) (g/L) Total dry matter 28.9(0.7) 26.9(0.8) 24.0(0.4) (g/L) Free sulfur dioxide 36 33(1) 26(1) (mg/L) Total sulfur dioxide 65 75(7) 54(2) (mg/L) Absorvance at 420 nm 0.195(0.004) 0.254(0.002) 0.101(0.001) (a.u.) pH 3.27 3.19(0.01) 3.27 Figure 1 - Celfa P-28 unit. Turbidity 602(2) 31 4.07(0.01) (NTU) Ultrafiltration. Reference solute assays. Solutions Total polysaccharides 123(3) 95(4) 93(4) made of reference solutes were permeated at the (mg/L) operation recirculation rate of 0.79 L/min and at a transmembrane pressure of 0.5 bar. Feed tank was filed The total polysaccharides content was determined with 500 mL of solution. Then, before collecting the 22 following the procedure described by Segarra et al., . permeate, the unit runs for 10 minutes in order to stabilize The other enological parameters were determined the feed recirculation rate, system’s pressure and according to the Compendium of International Methods of temperature, and also to stabilize the feed and the Analysis of the International Organization of Wine (OIV, permeate concentrations. For each experiment a volume 23 2011) . of 6 mL of permeate was collected. Protein stability was determined by heat stability test adding tannin to promote proteins’ flocculation and Ultrafiltration. Wine assays. The three white wines precipitation. The wines’ protein instability was confirmed. were treated by ultrafiltration at 1.2 bar with a recirculation rate of 0.79 L/min and controlled temperature at 24 ºC. In Membrane. A commercial membrane composed by each assay, feed tank was filled with 500 mL of wine, then fluoro polymer, FS61PP, from AlfaLaval was used. This the unit ran for a period of 10 minutes to stabilize membrane was characterized in terms of pure water operational conditions and to achieve a stable hydraulic permeability (Lp) and molecular weight cut-off concentration on the permeate. A volume of 160 mL of 2 (MWCO). Lp of 44 kg/(bar h m ) is the slope’s value of the each UF fraction was collected, being the permeate one linear variation of pure water flux vs transmembrane collected several times in smaller volumes (5 times of pressure. MWCO determination involves the permeation 30 mL plus 1 time of 10 mL). Feed’s concentration was of solutions of referent solutes (polyethylenoglycols of 10, maintained during the assays by refilling the same 20 and 35 kDa) with a concentration of 500 mg/L. MWCO volume of wine in the feed tank that was being collected of 45 kDa was obtained by intersection of the curve of in the permeate. UF fractions were evaluated in terms of log(f/(1-f)) vs solute molecular weight with 90,9% protein stability, physical-chemical composition, turbidity, rejection line that corresponds to a value of log(f/(1-f))=1. total protein, total polysaccharides and total phenolic Here, f is the reference solute rejection coefficient and it index. is calculated by f=(Ca-Cp)/Ca, where Ca and Cp are the feed and the permeate concentrations, respectively. The Protein stability tests. Protein stability tests were applied in the initial wine samples and in the UF fractions.

These protein stability tests include the heat test and the value was smaller than 0.05 UF had a significant effect heat test with tannin addition. on the parameter, and whenever p-value is smaller than Heat test. This test was adapted from Moine-Ledoux 0.01 UF has a very significant effect on the parameter. and Dubourdieu 24. A 20 mL of sample centrifuged at 14000 g during 10 minutes was heated for 30 minutes in RESULTS AND DISCUSSION a thermostat-controlled bath at 80 ºC. The difference in Set up of UF operating conditions. The optimization turbidity measured by nephelometry and expressed as of the UF operation means its operation under conditions nephelometric turbidity unit (NTU) before the wine was of minimization of concentration polarization. The heated and after it cooled down again was proportional to maximum feed rate corresponds to the maximum its protein instability. The wine was considered stable if recirculation flow rate allowed by the equipment, the difference in turbidity did not exceed 2 NTU. 0.79 L/min. At this rate, the permeation fluxes of a white Heat test with tannin addition. In two test tubes was wine, Jv, in the broad range of transmembrane pressures, added 20 mL of clear wine (centrifuged at 14000 g for ΔP, from 0 to 5 bar were represented in Figure 2. 10 minutes), 200 mg of ascorbic acid and in one of that 50 tubes 1 mL of a 5 % (w/v) tannin solution in alcohol 10 % 45 was added. Wine samples with and without tannin were 40 maintained for 10 minutes in a water bath at 80 ºC and 35 then cooled under running cold water. The absorbance of 30 each sample was measured at 650 nm in a cuvette of 1 25 cm path length. The difference in absorbance at 650 nm Low pressure 20 measured in absorbance units (a.u.) of the tube with 15 Intermediate pressure tannin and the tube without tannin was proportional to the h¯¹) m¯² (kg Jv 10 High pressure wines protein instability. The wine was considered stable 5 if the difference in absorbance did not exceed 0,1 a.u.. 0 Analytical methods. The total protein content of wines’ 0 1 2 3 4 5 6 UF fractions was determined by the Bradford method 25. ΔP (bar) For an expected protein concentration between 1–25 Figure 2 – Permeate fluxes vs. transmembrane pressure of a μg/mL, in a microplate 150 μL of the Coomassie Reagent white wine. was added to 150 μL of the wine sample and for an Figure 2 displays a low pressures zone (from 0 to expected protein concentration between 100–1500 1.5 bar) where Jv vs. ΔP varies linearly with a slope of μg/mL, 250 μL of the Coomassie Reagent was added to 18.2 kg/(m2 h bar), an intermediate pressures zone (from 5 μL of the wine sample. The microplate was mixed for 30 1.75 to 3.75 bar) and a high pressures zone (from 4 to 5 seconds and then it was incubated for 10 minutes at room bar) where Jv vs. ΔP varies linearly with a slope of temperature. Absorbance at 595 nm was measured for 3.92 kg/(m2 h bar). Transmembrane pressure of 1.2 bar every wine samples and for the blank replicates (with was selected in the zone where Jv varies linearly with a water instead of wine). The average of the absorbance of membrane pressure of 0 to 1.5 bar. the blank replicates was subtracted from the absorbance The recirculation flow rate of 0.79 L/min and the of wine samples and that value was used to determine operating pressure of 1.2 bar ensures a minimization of the total protein concentration by a stand curve of a concentration polarization. known protein (bovine serum albumin, BSA) for low and Influence of UF on protein stability of the wines. high concentrations, between 0–25 μg/mL and 0– Heat tests displayed different results concerning protein 2000 μg/mL, respectively. stability of the three UF fractions, which are the following: The total polysaccharides content was determined initial feed (IF) that corresponds to the initial wine after following the procedure described by Segarra et al., 22 had been dissolved in 19.8 mL of deionized water, where polysaccharides were precipitated with ethanol retentate fraction (R) and permeate fraction (P) and then the precipitate was dried and dissolved in water. The results of both heat tests are shown in Table 2. 26 The phenol-sulphuric method of Dubois et al,. was Table 2 - Protein stability in white wines: heat test with turbidity performed to determine the total polysaccharide content. evaluation, heat test with tannin addiction with absorbance The measurement of total phenolic index (TPI) is based evaluation. IF – initial feed, R – retentate, P – permeate. White UF Heat test with on spectroscopy and consists in evaluating the Heat test 4 absorbance at 280 nm. Previously, wine samples were whine fraction tannin addition 5 centrifuged at 18000 g for 10 minutes and then diluted to IF -0.1 0.50 1:10 with distillate water. Viosinho R 2.1 0.48 Physical-chemical composition of UF fractions of the wines was determined by FTIR (Fourier-transform P 0.1 0.48 infrared spectroscopy) – calibrated methodology for IF 3.6 0.46 wines, including the following parameters: density at 20 Lote R 4.7 0.35 ºC, alcoholic strength at 20 ºC, total dry matter, reducing substances, total and volatile acidity, pH, ashes, tartaric P 0 0.37 acid, malic acid, sulphates and chlorides. Turbidity, total IF 4.3 0.87 protein, total polysaccharides content and total phenolic Moscatel R 2.8 0.44 index were determined by the previous mentioned Graúdo analytical methods. P 0.9 0.72 Statistical Analysis. The effect of UF on each 4 If the result is higher than 2 NTU, it is considered unstable analytical parameter was studied by a variance statistical 5 If the result is higher than 0.1 a.u., it is considered unstable analysis (ANOVA). It was assumed that whenever p-

On one hand, heat test demonstrates that white wines 70 Lote and Moscatel Graúdo that were unstable before UF a IF R P treatment became stable after this treatment. On the 60 other hand, through the same test shows that Viosinho wine was stable before UF treatment and its retentate 50 b fraction became unstable. This could be justified with the 40 concentration of unstable proteins and the retention of polysaccharides which play an important role as 30 a protective colloids. b According to heat test with tannin addition all fractions (NTU) Turbidity 20 of all wines were unstable before and after UF treatment. 10 b a Some authors have studied the influence of intrinsic c c c 27 factors on conventional wine protein stability tests and 0 have concluded that tannin test seems to be influenced Viosinho Lote Moscatel by many factors such as pH, total protein, iron, potassium Graúdo and cooper content, when applied in wines with a total Figure 3 - Development of turbidity in the UF fractions of the protein content above 70 mg/L. These authors suggested white wines. IF – initial feed, R – retentate, P – permeate. For that tannin test should not be recommended to determine each fraction, the value represents the average of two the quantity of finning agent on protein stability process. independent replicates and error bars correspond to the Within a study on heat unstable proteins removal by UF, standard deviation of those determinations. Values followed by the authors concluded that protein stability can be the same letter are not significant at the 0.05 level of significance. achieved with MWCO of 10000–30000, despite small amounts of heat-unstable protein passing through 160 IF R P membranes. It was observed that although stabilization 140 a being not always obtained, reductions of 80–95% in the a a bentonite demand are achieved 28. 120 Effect of UF on whine physical-chemical 100 b characteristics. As regards to the parameters that were 80 determined by FTIR (Table 3) it is verified that Lote wine 60 a was the least influenced by the UF, when compared to a the other samples of wine. For this wine, UF had a 40 significant effect on the reducing substances content and 20 had a very significant effect on the alcohol content, dry (mg/L) content Protein Total b b c extract, total acidity, volatile acidity and malic acid. For 0 the parameters in which a very significant effect was Viosinho Lote Moscatel observed, an evident concentration of the feed after the Graúdo UF assay (C fraction) was verified, with the exception of Figure 4 - Total protein content of the UF fractions of the white the malic acid that had permeated through the wines. IF – initial feed, R – retentate, P – permeate. For each membrane. Concerning Lote wine, it was verified that the fraction, the value represents the average of six independent UF operation had a significant effect on the density and replicates and error bars correspond to the standard deviation of had a very significant effect on the alcohol content. As those determinations. Values followed by the same letter are not regards to Moscatel Graúdo wine, UF had a significant significant at the 0.05 level of significance. effect on density, reducing substances and sulfates, and had a very significant effect on alcohol content, dry 450 extract and total acidity. A similar concentration of feed IF R P after the UF assay was also observed in the parameters 400 a a whose UF effect was significant. 350 In terms of the parameters that are directly related to protein instability, such as turbidity, total protein, total 300 polysaccharides and total phenols index, Figures 3, 4, 5 250 and 6 present the results of these parameters that were 200 obtained for each white wine. (mg/L) a a 150 b b Turbidity is measured by assessing the disturbance b c caused by the diffusion of light by contact with particles in 100 b a liquid, and can be used for the classification of a white 50 wine as shiny or cloudy, with turbidity values below 1.1 content Polysaccharides Total 0 NTU and above 4.4 NTU, respectively. As presented in Viosinho Lote Moscatel Figure 3, and as expected, the turbidity of permeate Graúdo fraction was at maximum of 4.a<4 NTU. A common trend was observed for the three wines Figure 5 - Total Polysaccharides content of the UF fractions of regarding UF effect on turbidity. UF had a very significant the white wines. IF – initial feed, R – retentate, P – permeate. For effect on wine turbidity and all fractions demonstrated to each fraction, the value represents the average of two be different at the 0.05 level of significance, according to independent replicates and error bars correspond to the standard variance analysis. deviation of those determinations. Values followed by the same UF had significant effect on protein content, promoting letter are not significant at the 0.05 level of significance. their removal from wines. Figure 4 shows a significant effect of UF on the protein content of wines and a

Table 3 - Physical-chemical composition of UF fractions. Viosinho Lote Moscatel Graúdo UF UF UF IF R P IF R P IF R P effect effect effect Density at 20 ºC (g/mL) n.s. 0,9922 0,9920 0,9921 * 0,9909 a 0,9905 b 0,9906 b * 0,9887 a 0,9886 b 0,9885 b

Alcoholic strenght (% v/v) ** 10,92(0,01) b 11,23(0,02) a 10,86(0,03) b ** 11,40(0,01) c 11,73(0,02) a 11,56(0,01) b ** 12,65(0,02) c 13,00(0,01) a 12,81(0,01) b

Dry matter (g/L) ** 21,5 b 22,2(0,1) a 21,0 c n.s. 19,6(0,2) 19,8(0,1) 19,3(0,1) ** 17,8(0,1) b 18,6(0,1) a 17,7 b

Reducing substances (g/L) * 2,5(0,1) a 2,6(0,1) a 2,1(0,1) b n.s. 1,77(0,01) 1,8(0,1) 1,59(0,03) * 1,97(0,02) a 2,1(0,1)a 1,80(0,02) b

Total acidity (g tartaric acid/L) ** 5,63(0,02) b 5,7 a 5,65(0,01) b n.s. 5,55(0,03) 5,60(0,01) 5,56(0,04) ** 4,52(0,02) b 4,7 a 4,5 b

Volatile acidity (g acetic acid/L) ** 0,315(0,007) b 0,35 a 0,32 b n.s. 0,24(0,01) 0,25 0,24 n.s. 0,34(0,01) 0,34 0,34(0,01)

pH n.s. 3,20(0,02) 3,217(0,001) 3,22 n.s. 3,11(0,01) 3,110(0,001) 3,12(0,03) n.s. 3,16 3,149(0,001) 3,14(0,01)

Ashes (g/L) n.s. 1,79(0,01) 1,87(0,06) 1,92(0,03) n.s. 1,8(0,2) 1,66(0,04) 1,7(0,1) n.s. 1,67(0,02) 1,74(0,01) 1,72(0,04)

Tartaric acid (g/L) n.s. 2,05(0,08) 2,08(0,01) 2,11(0,08) n.s. 1,7(0,1) 1,6(0,1) 1,7(0,1) n.s. 1,11(0,06) 1,1 1,06(0,02)

Malic acid (g/L) ** 0,88(0,02) b 0,88(0,02) b 1,05(0,03) a n.s. 1,1(0,1) 1,1(0,1) 1,08(0,01) n.s. 0,64(0,05) 0,59(0,04) 0,66(0,01)

Sulphates (mg/L) n.s. 459(3) 471(2) 451(10) n.s. 508(4) a 509(13) a 480(1) b * 530(10) b 558(2) a 532(4) b

Chlorides (mg/L) n.s. 41 41 41 n.s. 40(1) 38 39(1) n.s. 35 36 35 (UF – Ultrafiltration; IF – Initial feed; R – Retentate; P – Permete; * - significant effect for p-value<0,05; ** - very significant effect for p-value<0,01; n.s. – no significant effect for p-value>0,05). For each fraction, the value represents the average of two independent replicates and the standard deviation of those determinations. Values followed by the same letter are not significant at the 0.05 level of significance.

Regarding to total phenolic index (TPI), the effect of UF on this parameter seemed to be very significant for all 8 IF R C wines. In Figure 6 it is shown that all wines had similar a 7 b behaviour in UF assays. However statistical analysis c showed that there are some differences between UF 6 fractions. 5 a TPI indicates the total amount of phenolic compounds b c in wine and as it was observed that UF promotes the 4 decrease of those compounds in wine. The presence of a 3 b c phenolic compounds, namely tannins, contributes to the protein casse, since these compounds bind to proteins 2 Total Phenolic Index Phenolic Total and can lead to their precipitation. 1 Batista et al. 6 subjected a Portuguese white wine 0 (Arinto) to heat stability tests in order to understand the Viosinho Lote Moscatel complex phenomenon of protein precipitation. These Graúdo authors, when evaluating wine after UF with a 3 kDa MWCO membrane, concluded that the permeate fraction Figure 6 - Total Phenolic Index of the UF fractions of the white (<3 kDa) contains low molecular weight components that wines. IF – initial feed, R – retentate, P – permeate. For each play a very important role in protein instability. They fraction, the value represents t the average of two independent replicates and error bars correspond to the standard deviation of namely X factor and defined it as being one or more those determinations. Values followed by the same letter are not constituents of wine of non-protein nature and low significant at the 0.05 level of significance. molecular weight, whose presence promotes the denaturation of the proteins induced by heat, at normal significant difference was observed between feeds and pH wine values. Considering the heat test performed in permeates of all wines, fractions IF and P, respectively. It the present work, the result for the three wines indicates was verified that there were no significant differences the stability of the wines after UF operation. This can be between the IF and R fractions of Viosinho and Lote explained by the difference in the total protein content of wines, although in the case of Moscatel wine there were the permeate fractions of the wines under study significant differences between all UF fractions. (Viosinho, Lote and Moscatel Graúdo) being much lower Considering the heat test in order to evaluate protein (around 5 mg/L) than the protein content of Arinto wine stability of white wines, the permeate fraction, which is added (280 mg/L) to the < 3 kDa UF fraction in the study composed of particles of molecular weight lower than by Batista et al.6. membrane MWCO, resulted in protein stable wines. The obtained results are in agreement with previous studies. Clarification of white wines. The main objective of Peng et al 29. and Esteruelas et al. 10 observed that most wine clarification is the removal of particles in suspension proteins of a Sauvignon Blanc wine had a molecular and colloidal material that are responsible for haze weight range of 12-24 kDa, and thermally unstable formation. proteins have molecular weights of 25-72 kDa. Hsu and Heatherbell 28 identified the protein fractions of 12.6 kDa and 20-30 kDa as being the most important in terms of white wine protein instability. Recently, Bruijn et al. 30 studied the impact of protein composition on the stability of white wines and identified that in the two white wines under study, fractions between 18-60 kDa were related to the protein casse problem. Figure 5 displays the results regarding the effect of UF on polysaccharides content of wines. For all the wines, and as expected, significant effect was observed, with permeate fractions showing significant differences compared with IF fraction, being P fraction lower than IF fraction. Viosinho showed a similar concentration between the IF and C fractions, while Lote and Moscatel Graúdo behave similary to UF treatment. Statistical analysis results shown that IF and R fractions of Viosinho wine are not significant different from each Figure 7 – Colour gradation of UF fractions of Viosinho white wine. Initial feed fraction (in right), retentate fraction (in the other, but are significant different of the P fraction. In the middle) and permeate fraction (on left). case of Lote wine, R and P fractions have no significant differences, but when compared to IF fraction it is In Figure 7 it can be observed a more intense turbidity observed significant differences. All UF fractions of of initial feed and retentate UF fractions when compared Moscatel Graúdo wine are significant different. to the permeate fraction. This optical result was expected Due to membrane nominal MWCO of 45 kDa, it is since the turbidity results from the agglomeration of ensured that the high molecular weight polysaccharides suspended solids and UF operation aims to remove were removed and remained in the R fraction. This could macromolecules with molecular weight higher than have resulted in the removal of mannoproteins (53- membrane MWCO. When appearance of the feed 560 kDa) which act as protective colloids in protein fractions was compared, a higher intensity of haze in the stability of white wines and thus precipitate unstable retentate fraction was observed, which was also expected proteins.

since concentration of macromolecules, namely proteins, The removal of proteins presented similar results for the occurred in the end of UF assay. white wines in study, namely the total protein content Turbidity is an important parameter in a white wine obtained in clarified wine (P fraction) of about 5 mg/L. The clarification. The analysis of Table 4, allowed to conclude removal percentages indicate that the majority of the that UF treatment had a great efficiency, namely in Lote protein content (about 89-95 %) has a molecular weight and Viosinho wines, whose particles that are responsible higher than membrane MWCO. for haze formation were removed by 89.9 and 95.5 %, Regarding the removal of total polysaccharides, a respectively. Moscatel Graúdo wine presented a haze removal of 70.7; 65.8 and 41.1% for Viosinho, Lote and removal of 64.5 %, although this reduction was smaller Moscatel Graudo wines, respectively, could indicate the when compared to the other obtained values, Moscatel mannoprotein composition of these wines, since wine had the lowest initial turbidity value (4.7 NTU), which mannoproteins have molecular weights of 53-560 kDa. was reduced after clarification and reached a value The removal values are lower than those obtained by similar to the one obtained for Viosinho and Lote wines. Escudier et al. 32, which reported a removal of 92% of the Feuillat 31 states that wine turbidity is caused by total polysaccharide content of a clarified UF wine with a suspended material, such as yeast residues and 20 kDa membrane. Other values present in the literature macromolecular compounds with colloidal behavior. The are, for example, related to the study by Gonçalves et al. removal of 95.5% turbidity in the Viosinho wine indicates 33 reported a 16% removal of the total polysaccharide that the majority of the colloidal material responsible for content in the clarification of a white wine per UF with a the turbidity in this wine has molecular weight above 100 kDa membrane. membrane MWCO. The same is true for Lote and Moscatel Graúdo wines.

Table 4 - Decrease of turbidity and total phenolic index, and removal of total protein, total polysaccharides by ultrafiltration. Total Protein content Total Polysaccharides Wines Turbidity (NTU) Total Phenolic Index (mg/L) content (mg/L) Decrease Removal Removal Decrease IF P IF P IF P IF P (%) (%) (%) (%) Viosinho 46,4 2,1 95,5 54,9 5,9 89,3 361 106 70,7 6,4 5,7 11,3

Lote 16,8 1,7 89,9 119,2 5,5 95,4 155 53 65,8 4,0 3,7 6,6 Moscatel 4,7 1,7 64,5 82,3 5,4 93,5 153 90 41,1 2,5 2,4 6,2 Graúdo

CONCLUSIONS characteristics was also observed, according to each wine. The present work focused on the application of the UF Regarding the protein stability of the wines, the results for clarification and protein stabilization of wines as an obtained for the two tests did not always lead to the same alternative to bentonite fining, whose disadvantages are results. In the heat test the precipitate fractions of all the known. For this purpose, experimental tests were carried wines were stable, whereas in the heat test with tannin out with a membrane selected according to the current addition the same fractions were unstable. These results state of knowledge, regarding the characteristics of the can be explained by the presence of potential interferes proteins most commonly involved in protein casse. In 27 and due to the different principle basis of the two tests. parallel with the membrane characterization in terms of The UF operation lead to the clarification of white wines, hydraulic permeability [44 kg / (m2 h bar)] and molecular with a decrease of turbidity in the treated wine compared cut-off (45 kDa), the operating conditions were to the initial feed, above 80%. investigated in terms of flow rate and transmembrane It is concluded that the UF operation in clarification of pressure leading to minimization of the concentration white wines is effective. Regarding the protein stability of polarization, respectively of 0.79 L/min and 1.2 bar. wines, this study constitutes a significant contribution, Subsequently, under the optimized conditions of UF, however it is considered necessary to gather more white wines that were protein unstable were processed. information about the UF effects, since this operation will The protein instability of these three wines was have to ensure that the physical-chemical and sensorial evaluated with a heat test with tannin addition and was characteristics of the wine are not significantly altered and further confirmed by a second test – heat test – based on to that end, it would be necessary to carry out a sensorial the thermal denaturation of the proteins and evaluation of evaluation of the wines after processing by UF. turbidity, of greater applicability. On the basis of the foregoing conclusions the following The UF fractions, especially Permeate that corresponds aspects should be the subject of future work: profile of to the treated wine, were characterized in terms of removed macromolecules, polysaccharides and general physical-chemical composition, total polyphenols, on protein stability; understanding the polysaccharide content, total protein content, total mechanisms of flocculation and precipitation of proteins; phenols index, protein stability and turbidity. if not being possible to achieve the protein stability of As expected, involving macromolecules, a significant wines, development of a treatment process involving UF effect of UF was observed on total polysaccharide and bentonite fining. The development of a process content, total protein content, total phenols index and involving UF for protein stabilization will necessarily turbidity, parameters for which significantly lower values oblige the evaluation of the effect of the treatment on the were found in treated wine. Regarding the other analytical physical-chemical and sensorial characteristics of the parameters, a significant effect of the UF on some wine.

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