Rationale for Haze Formation after Carboxymethyl Cellulose (CMC

Article pubs.acs.org/JAFC

Rationale for Haze Formation after Carboxymethyl Cellulose (CMC) Addition to Red Wine Stephan Sommer,*,† Christian Dickescheid,§ James F. Harbertson,# Ulrich Fischer,§ and Seth D. Cohen† †

Fermentation Sciences, Appalachian State University, 730 Rivers Street, Boone, North Carolina 28608, United States Institute for Viticulture and Enology, Breitenweg 71, 67435 Neustadt/Weinstraße, Germany # Viticulture and Enology Program, Wine Science Center, Washington State University, Richland, Washington 99354, United States §

ABSTRACT: The aim of this study was to identify the source of haze formation in red wine after the addition of carboxymethyl cellulose (CMC) and to characterize the dynamics of precipitation. Ninety commercial wines representing eight grape varieties were collected, tested with two commercial CMC products, and analyzed for susceptibility to haze formation. Seventy-four of these wines showed a precipitation within 14 days independent of the CMC product used. The precipitates of four representative samples were further analyzed for elemental composition (CHNS analysis) and solubility under different conditions to determine the nature of the solids. All of the precipitates were composed of approximately 50% proteins and 50% CMC and polyphenols. It was determined that the interactions between CMC and bovine serum albumin are pH dependent in wine-like model solution. Furthermore, it was found that the color loss associated with CMC additions required the presence of proteins and cannot be observed with CMC and anthocyanins alone. KEYWORDS: carboxymethyl cellulose, red wine, proteins, stabilization, haze

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INTRODUCTION Carboxymethyl cellulose (CMC) is common additive (E 466) to foods, beverages, cosmetics, and various other products. Commercial CMC is a complex mixture of polysaccharides of various molecular sizes and modifications,1 making it a very heterogeneous ingredient. The physicochemical properties of CMC were studied early on2 and applied in various industries over time.3 Today CMC is used as a thickener in toothpaste,4 as a water binder in ice cream and baked goods, as a suspending aid in sauces, cosmetics, and beverages,5 and, among other uses, as a stabilizing agent for tartrate crystals in wine.6,7 CMC inhibits crystal growth of potassium bitartrate and is typically added to wine immediately prior to bottling. If a filtration step is necessary after CMC addition, the selection of liquid preparations is preferable because it shows better solubility and fewer viscosity problems, which might influence filtration characteristics. However, previous research shows that filterability after CMC is not significantly different from a control after 4 days of equilibration.8 A conventional method for preventing tartrate crystallization and precipitation in bottled wine, adding seed crystals and holding temperatures below 0 °C,6 is relatively time-consuming and energy intensive and involves a filtration step to remove the precipitate. However, it is still the most economical stabilization option compared to other technical solutions such as reverse osmosis, electrodialysis, or ion exchange.9 Stabilizing additives used in the industry, like metatartaric acid or mannoproteins, provide only a short-term solution over a few months but no absolute or permanent protection against crystal precipitation.10,11 An additive that remains in solution and provides long-term protection against crystal precipitation without a sensory influence was not available until the approval of CMC by the Organisation Internationale de la Vigne et du Vin (OIV) in 2009.6,7,12 Another additive that was most recently © XXXX American Chemical Society

investigated is polyaspartate, which seems to possess a similar stabilizing effect but a longer stability over time compared to metatartaric acid.13 Although CMC persists in the wine, it presents no risk to human health (U.S. Food and Drug Administration, Code of Federal Regulations 21CFR182.1745). The instability of tartaric salts in wine is caused by several factors that influence the supersaturated solution of potassium bitartrate9 including increasing ethanol levels during fermentation. The growing trend for cold stabilization of red wines is due to higher ethanol concentrations, lower acidity, increased potassium associated with longer ripening periods, and higher pH values.14 This leads to wines that are more likely to show tartrate precipitation and therefore require stabilization. The use of CMC in wine is currently limited to white and sparkling wines,15 because the application to red wines is reported to be less efficient16 and might lead to color loss and haze formation.11 Therefore, manufacturers do not recommend the use in red wine. However, the reasons and mechanisms for the haze formation are not thoroughly understood. A recent study suggests the involvement of proteins in the reaction,17 whereas former experiments tried to explain the color loss by suggesting an interaction between CMC and polyphenols.16 The formation of haze is generally difficult to predict as it may take several days to manifest (manufacturer’s communication, unpublished data). This adds a high risk factor to the application for the winemaker because the precipitation might be visible only after the wine is bottled. The consequences include financial loss for recalling the entire product line and Received: June 5, 2016 Revised: August 14, 2016 Accepted: August 29, 2016

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DOI: 10.1021/acs.jafc.6b02479 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Rentzsch et al.22 The LC instrument used was a Jasco PU 980 lowpressure gradient pump and a three-line degasser Spectra Systems SCM 1000 coupled with a Jasco MD 2010 Plus multiwavelength detector (Jasco, Gross-Umstadt, Germany). Samples were injected by a Spark Basic Marathon autosampler (Spark Holland B.V., Emmen, The Netherlands) on a YMC-Pack ODS-AM separation column with dimensions of 250 × 4.6 mm and a particle size of 5 μm (YMC Europe GmbH, Dinslaken, Germany). The oven temperature was set to 50 °C at a constant flow of 0.5 mL/min. A binary gradient was used with eluent A being water with 5% acetonitrile and a 10 mM KH2PO4/ H3PO4 buffer and eluent B being a 50:50 acetonitrile/water mixture with a 10 mM KH2PO4/H3PO4 buffer. Conditions started at 85% eluent A, decreased to 80% by 15 min, decreased from 80 to 65% by 40 min, and decreased from 65 to 45% by 45 min. After 50 min, conditions were returned to initial (85% A) for 10 min to reach equilibrium (total analysis time of 60 min). Instrument control and data acquisition were performed with Chrompass version 1.8 (Jasco Germany GmbH). Because samples were injected without preparation, no internal standard was used. Anthocyanins were quantified as malvidin-3-glycoside equivalents (standard for external calibration: oenin chloride ≥90%, Sigma-Aldrich). Total phenolics were analyzed as gallic acid equivalents (Folin− Ciocalteu) as described by Singleton and Rossi.23 Bench-Trial Experiments in Wine-like Model Solution. Winelike Model Solution. A wine-like model solution was prepared with 13% (v/v) ethanol and four different pH ranges (3.0, 3.3, 3.7, and 4.0) to evaluate different winemaking conditions. For 1 L of model wine the following components were used: 103 g of ethanol (200 proof, Koptec, King of Prussia, PA, USA), 2.7 g of tartaric acid, 2.0 g of malic acid, 150 mg of potassium metabisulfite (all from VWR International LLC, Radnor, PA, USA), and 878 g of deionized water (Milli-Q, Millipore Corp., Billerica, MA, USA); pH was adjusted using 10 M sodium hydroxide (J. T. Baker). An overview can be found in Table 1.

rebottling the wine as well as also damage to the wineries’ image if the consumer is presented with a hazy product. The objectives of this study with real wines and model systems were to identify the source of haze formation in red wine treated with CMC, explain the mechanisms of the reaction, and identify the putative cause for color loss.

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MATERIALS AND METHODS

Study of Haze Formation in Real Wines. Tartrate Stability Assessment. The potassium bitartrate (KHT) stability of wines was assessed using a CheckStab instrument (Delta Acque, Firenze, Italy) according to the manufacturer’s instructions; the system compares the conductivity of the solution with and without KHT at different temperatures. The resulting change in conductivity due to crystallization of KHT is interpreted by the software (CheckStab Life SW 5.1.7 FW 3.9, Delta Acque, Firenze, Italy). Commercial Wine Selection. Ninety commercial wines representing eight grape varieties (Cabernet Sauvignon (4), Merlot (4), Pinot noir (22), Dunkelfelder (4), Portugieser (14), Dornfelder (30), St. Laurent (5), and Regent (7)) were collected from wineries in the Palatinate region in Germany. The predominant proportion of the wines was not bottled at the time of the experiments but collected as tank samples (vintage 2012); 15 of the 90 wines collected were from the previous vintage (2011) and were also tank samples. Intentional tartrate stabilization had therefore not yet been conducted on the wines. Each sample was tested with two commercial CMC products (VinoStab, Erbslöh Geisenheim AG, Geisenheim, Germany, and Cristab GC, SAS Sofralab, Epernay, France) and analyzed with regard to their susceptibility to haze formation. CMC Treatment and Monitoring. The two commercial CMC preparations were applied to each wine at the highest legal dosage and according to the manufacturer’s instructions. The legal limit of 100 mg/L was chosen to approximate the maximum possible interaction with other wine components. Samples were clarified by centrifugation to remove any previous haze. Stability trials were conducted in duplicate in 100 mL polyethylene sample containers and monitored by nephelometry (Hach Lange turbidimeter 2100N, Hach Lange GmbH, Berlin, Germany) over 15 days. In the case of haze formation at the end of the trial, the sample was centrifuged at 12000 rpm (RCF 13300g) for 15 min to collect the precipitate. After the wine had been decanted, the precipitate was washed twice with approximately 100 mL of sterile deionized water to remove residual wine components and any other water-soluble material, because only the composition of the pure precipitate was of interest. The washed solids were stored in sterile deionized water at 4 °C for further analysis. Elemental Analysis. The precipitates of four samples that showed high levels of precipitation (Dornfelder, Pinot noir, Portugieser, and Regent) were dried in a laboratory oven overnight at 105 °C. CNHS elemental analysis was performed according to DIN EN 1313718 in triplicate. The average values of carbon, nitrogen, and sulfur were used to calculate the percentage of protein in the samples. Because proteins in wine can range from 11 to 65 kDa19 with a range from 13 to 17% nitrogen,20 the average protein factor of 6.2521 was chosen for the calculation from percent nitrogen to percent protein. Solubility Trials. To determine the composition and chemical behavior of the precipitate, several solubility trials were performed with the previously washed solids. The precipitates were suspended in 2 M sodium hydroxide (prepared from pellets, J. T. Baker, Phillipsburg, NJ, USA), 1 M sulfuric acid, and trifluoroacetic acid (both Sigma-Aldrich, St. Louis, MO, USA) to determine solubility under different pH or solvent conditions. The solutions were used at their stated concentration and applied to the centrifuged and decanted precipitate from approximately 100 mL of wine. Because these experiments were qualitative, the exact amounts of precipitate and solvent were not essential. Polyphenol Analysis. To characterize the polyphenol composition of the original wines, the concentrations of anthocyanins and total phenolics were determined by HPLC-DAD and spectrophotometry, respectively. The analysis of anthocyanins was adapted from that of

Table 1. Experimental Design To Evaluate the Interaction between Protein, Tannins, and Carboxymethyl Cellulosea pH range

first component

CMC addition 100 mg/L

3.0 3.3 3.7 4.0

500 mg/L tannin 200 mg/L

100 mg/L 500 mg/L BSA 200 mg/L

a

addition after 24 h 100 400 200 600

mg/L mg/L mg/L mg/L

BSA BSA BSA BSA

100 300 200 500

mg/L mg/L mg/L mg/L

tannin tannin tannin tannin

Each trial was conducted in duplicates (n = 48 × 2).

The CMC used was a food grade sodium salt powder from Modernist Pantry LLC (Portsmouth, NH, USA). Bovine serum albumin (BSA) was purchased as a 7.5% solution (Thermo Fisher Scientific, Waltham, MA, USA), anthocyanins (E163) were added as a spray-dried powder as described in Commission Regulation (EU) No. 231/2012 (prepared in a previous project), and the tannin preparation was the commercial grape skin tannin extract UvaTan Soft (Lallemand via Scott Laboratories, Petaluma, CA, USA). As shown in Table 1, the first component and the CMC were added initially. The second component addition was done 24 h later to assess the specific influence on haze formation and affinity to CMC. In a second set of experiments, the tannin component was replaced with anthocyanins to evaluate differences and monitor color loss. Analysis of Haze Formation. Haze formation was monitored over 32 days at constant room temperature (21 °C) in sterile polypropylene 50 mL centrifuge tubes (VWR International LLC) in duplicates. Turbidity was analyzed via spectrophotometry (Beckman Coulter DU 720 UV−vis spectrophotometer, Beckman Coulter Inc., Brea, CA, USA) at 860 nm after suspension of the precipitate by gentle shaking. B


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Journal of Agricultural and Food Chemistry Absorbance was recorded every 2−4 days to evaluate the change in turbidity over time. In the anthocyanin trials, color was analyzed after centrifugation at 520 nm simultaneously. Data Analysis. Data handling and statistical analysis were performed using XLStat 2014 (AddinSoft, New York, NY, USA) and SigmaPlot 12.5 (Systat Software Inc., San Jose, CA, USA).

the susceptibility to haze formation can be hypothesized. To further investigate this effect, four samples representing the varieties that showed the highest number of precipitation events were chosen for further analysis. These four precipitate samples were analyzed for their elemental composition to determine if proteins are a contributor to wine as haze, as reported by Claus et al.17 The results are shown in Table 3 including the protein content that was calculated from the percentage of nitrogen. The percentage of sulfur was used to evaluate the accuracy of the calculation, because proteins contain between 0.25 and 0.6% sulfur20 and are likely to be the only source of sulfur in a solid precipitate in wine.24 Other forms of sulfur are thiols, sulfides, polysulfides, thioesters, and heterocyclic compounds,25 which are well soluble and therefore less likely to be found in these precipitates. A possible source of error with that procedure was reported by Pocock et al., who stated that sulfate can play a role in protein precipitation.26 However, the level of total sulfur in the precipitates analyzed here suggests only minor effects in red wines. Sulfate in wine is usually formed by oxidation of sulfites and plays a more important role in white wines in which SO2 levels tend to be higher. Elemental analysis represents an indirect method for protein analysis, and although it is not the most precise technique, it can reveal the presence of proteins and the approximate amount. The protein concentration in the precipitate was relatively stable around 50%, which was not to be expected considering the very different wines and varieties that were evaluated. However, the colors of the precipitates were very diverse. Wines with higher concentrations of anthocyanins (Table 4), such as Dornfelder, produced a dark red precipitate, whereas Pinot noir, a lighter colored wine, showed a grayish-brown precipitation. Color loss after CMC addition to red wine has been reported previously;17 however, the percentage of proteins in the reaction product has not been shown before. Another observed difference between the samples was the amount of precipitate found in the wine. The Dornfelder and Portugieser samples showed visually more solid material in the centrifugation tube than Pinot noir and Regent. Possible reasons for that were previously attributed to higher protein instability17 or complex interaction between different macromolecules27 that might be grape cultivar specific. To confirm these results, several solubility trials were conducted with the precipitates previously used in the CNHS analysis and some additional samples. To determine whether the bond between the particles was charge and pH dependent, the decanted precipitate was sequentially exposed to a base and an acid. Most solid samples could be almost completely solubilized in 2 M sodium hydroxide, suggesting that the precipitation reaction was to some extent charge and pH driven. The addition of 1 M sulfuric acid led to reprecipitation of solid material in all cases. This indicates a portion of material either is insoluble at low pH

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RESULTS AND DISCUSSION Study of Haze Formation in Real Red Wines. Of the 90 commercial wines that were collected, 74 showed a haze within 14 days independent of the CMC product used. With >82% forming a precipitate, it could be confirmed that red wines are susceptible to haze formation after being fined with CMC. The comparison between the commercial CMC preparations revealed no obvious differences. Both products led to similar turbidity values. The precipitation is not dependent on a specific product but may be a general phenomenon with CMC in red wine. Table 2 shows a summary of the results including the grape varieties, the percentage of samples showing haze after Table 2. Evaluation of Haze Formation (Average Turbidity in NTU) in the Wine Samples Analyzed after Treatment with Two Different Commercial CMC Productsa haze formation (NTU) cultivar

no. of samples

% haze formation

4 7 5 4 4

75 100 80 50 25

36.2 93.7 31.9 43.1 11.9

30 14 22

90 100 73

64.2 ± 41.1 92.6 ± 48.5 36.3 ± 38.6

Dunkelfelder Regent St. Laurent Merlot Cabernet Sauvignon Dornfelder Portugieser Pinot noir

VinoStab treatment ± ± ± ± ±

23.4 35.2 15.3 39.3 16.8

Cristab GC treatment 45.8 77.2 31.8 48.6 10.3

± ± ± ± ±

35.2 42.1 17.0 47.0 16.3

65.6 ± 36.9 94.7 ± 52.7 36.9 ± 40.2

a

Standard deviation shows the variation among all samples of one cultivar.

treatment, and differences between the two commercial products that were used for treatment. The grape varieties reflect a typical distribution in the Palatinate region of Germany with Dornfelder and Portugieser but also include some types of grapes such as Cabernet Sauvignon and Pinot noir that are of broader relevance. The overview reveals that there seems to be a cultivar effect, based not only on the susceptibility to haze formation but also on the average level of haze formed. Some varieties such as Pinot noir and Merlot display a wide range of turbidity values, whereas others, such as Portugieser and Regent, showed haze in every sample with fairly consistent high values. Even considering the small number of samples for some varieties, a cultivar effect on

Table 3. Elemental Analysis by CNHS Analyzer of Four Representative Precipitation Samples Including the Calculated Protein Content Based on the Percentage of Nitrogena cultivar Dornfelder Pinot noir Portugieser Regent a

nitrogen (%) 8.13 6.91 8.71 7.90

± ± ± ±

0.06 0.04 0.01 0.01

carbon (%) 44.89 46.70 44.27 43.83

± ± ± ±

0.06 0.07 0.14 0.10

sulfur (%) 0.34 0.32 0.29 0.29

± ± ± ±

0.02 0.02 0.01 0.01

protein content (%) 50.8 43.2 54.4 49.4

± ± ± ±

0.40 0.20 0.05 0.05

Standard deviation shows the variation among three analytical repetitions of the same sample. C


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Journal of Agricultural and Food Chemistry Table 4. Total Phenolics (FolinC) and Anthocyanin Concentration of the Grape Varieties Included in This Studya cultivar Dunkelfelder Pinot noir Portugieser Dornfelder Cabernet Sauvignon Merlot St. Laurent Regent

total phenolics (mg/L) 2718.6 1194.4 840.7 1036.6 1182.2 2161.1 1008.0 1610.1

± ± ± ± ± ± ± ±

256.0 252.9 145.9 243.7 368.1 257.2 188.4 623.8

anthocyanins (mg/L) 352.2 105.1 217.9 261.2 127.4 132.8 146.7 157.6

± ± ± ± ± ± ± ±

103.9 39.0 102.1 91.0 27.1 32.5 89.6 47.9

total phenolics/anthocyanin ratio 8.0 13.3 4.4 4.4 9.8 16.5 8.0 10.2

± ± ± ± ± ± ± ±

1.6 6.9 1.5 1.7 5.0 2.1 3.6 2.4

av turbidity (NTU) 58.9 39.3 92.0 74.3 43.2 82.7 38.8 86.3

± ± ± ± ± ± ± ±

6.3 6.6 50.9 38.2 6.9 9.2 7.0 46.5

a

The susceptibility to haze formation is indicated by the average maximum turbidity observed for each cultivar (the two CMC products are also averaged; standard deviation shows the variation among all samples of one cultivar).

Figure 1. Haze formation in red wine samples over time. The four samples were randomly chosen from the data set but are representative of the whole data (two different commercial CMC preparations shown for each sample).

Differences could be due to interactions with polysaccharides that limit the reaction efficiency.27 Interestingly, except for some of the Pinot noir samples, no other cultivar reached a turbidity plateau. Once the haze formation started to be visible, particles grew in size relatively rapidly, making further analysis difficult. The NTU of the sample is therefore actually decreasing after the maximum is reached, indicating an increasing particle size that is harder to relate to average turbidity than small equally distributed particles. To clarify the cultivar dependency, attributes were needed that are more related to the cultivar itself and less to specific production methods. Because the precipitate exhibited various color intensities and the color loss after CMC treatment was mentioned before,17 the anthocyanin concentration was analyzed in the original wine by HPLC. In addition to that, phenolic compounds are reported to influence protein concentration and composition in wine by forming precipitates and therefore removing proteins from the equation.29 Because

or was denatured by the addition of a strong acid. However, the portion that could be precipitated with sulfuric acid could be solubilized in trifluoroacetic acid, indicating the presence of proteins.28 Although these solubility trials might appear very basic, they still provide an important understanding of the solids’ composition and the mechanisms that led to their precipitation. These observations in connection with the benchtrials are discussed in more detail below. Manufacturers describe a delayed precipitation reaction after CMC addition to red wine (personal communication, unpublished data). This observation was confirmed here, whereas the differences in haze formation among the samples suggest that the occurrence may be cultivar dependent. Figure 1 shows haze formation over time for four examples from different grape varieties. In all cases haze formation is delayed by >100 h after CMC addition; however, the actual time to significant haze formation and the extent of haze formation differ among varieties. D


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Figure 2. Spectrophotometric measurements of turbidity in wine-like model solution at different pH conditions over time. Tannins and CMC were added at the beginning of the experiment; the protein (BSA) was added 24 h later. Letters a−c indicate statistically significant differences determined by paired t test.

the level of anthocyanins and color is not well reflected in the total phenolics, and to better display cultivar-specific properties, the proportion of anthocyanins in total phenolics is provided by the total phenol/anthocyanin ratio (TP/A ratio). These numbers including the average turbidity observed for a grape cultivar are listed in Table 4. The relatively broad range among total phenolics and anthocyanins due to different production methods provides interesting information about grape cultivar characteristics in this study. Although it is not a straight correlation, low TP/A ratios lead to a higher risk of haze formation, especially when the level of total phenolics is lower. This observation might be related to the reaction potential of polyphenols with proteins and therefore to a lower general protein level in highly phenolic wines. This, on the other hand, would mean that haze formation in red wine after CMC application is a direct reaction with proteins and that there is a correlation between protein content, CMC addition level, and amount of resulting precipitate. An interesting observation resulted from the analysis of tartrate stability that was conducted with all samples throughout the experiments (data not shown). Some samples that showed a higher degree of precipitation with nephelo-

metric measurements >80 NTU also lost some of the stabilizing effect of CMC during the experiments. This detail led to the conclusion that a portion of the solid precipitate is in fact carboxymethyl cellulose. From an analytical standpoint, proteins account for approximately 50% of the solid material; the other 50% can be assumed to be a larger proportion of CMC with various amounts of anthocyanins or other polyphenols. To confirm and clarify this connection, a number of benchtrials in wine-like model solution were conducted over a typical wine pH range, because protein interactions are mostly charge and therefore pH dependent. Bench-Trial Experiments. A series of bench-trials was designed to evaluate the dynamics of the reaction between CMC, proteins, and polyphenols. To limit the number of variables, the study was conducted on a small scale in a model solution. Because there is no direct evidence in the literature so far of how the precipitation reaction in red wine between CMC and proteins actually happens, the experiment was designed to answer that question as well. All samples, with the exception of controls, contained CMC, proteins, and polyphenols, but the concentration and order of addition were varied. The last addition was done only after 24 h. In the controls, no further E


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Figure 3. Spectrophotometric measurements of turbidity in wine-like model solution at different pH conditions over time. The protein (BSA) and CMC were added at the beginning of the experiment; the tannins were added 24 h later. Letters a−c indicate statistically significant differences determined by paired t test.

protein is not linear. Samples with the lowest additions of both CMC and protein display the lowest haze formation; otherwise, differences among treatments are not as large. Another interesting observation is that the pattern changes in general between pH 3.3 and 3.7. Whereas the graphs at the lower pHsteps are very similar, there is an obvious step toward the higher pH values, which are again fairly similar to one another. Because everything between the experiments was done exactly the same, this could be due to the structural changes in the BSA molecule that might lead to different interactions with the CMC. The slow increase in turbidity could be due to the tannins, which are slowly precipitating proteins that have not already reacted with CMC or are simply making the already existing haze particles bigger. However, the increase is only minor compared to the initial reaction between proteins and CMC. Figure 3 shows the trials in which tannins were added with a 24 h delay to the system that already contained protein and CMC. The results confirm that BSA reacts with CMC, instantly causing a haze, whereas the addition of tannins has only a minor effect on turbidity. Moreover, the influence of protein as well as CMC concentration is clearly visible again. Although the protein level was constant in these trials, increases in CMC led

addition after 24 h was made. This way, a reaction between two components had enough time to happen without being disturbed by another macromolecule. The combined results of the trials are shown in Figures 2 and 3. BSA was chosen as a model protein because it is used in wine-related precipitation assays30 and is of a similar size compared to wine proteins, which can range from 9 to 62 kDa.31 The isoelectric point (pI) of 4.7 is also in the range of wine proteins (pI 4−8).19 However, it was reported before that BSA experiences some structural changes with rising pH32 that might influence its interaction with other macromolecules. The acidic expansion reported to occur between pH 2 and 3.5 should be considered when BSA is used in a wine-like system, because it spans the pH range relevant to wine. Figure 2 shows the results of adding BSA to solutions containing tannins and CMC after a 24 h delay. The controls show that there is no interaction between tannins and CMC independent of pH and CMC concentration. All other trials show a significant haze after protein addition on day 2 with some variation among the pH range. Although the reaction occurs instantaneously, there is a steady increase in turbidity over 30 days. Although more CMC in the sample always leads to more precipitation, the correlation with the amount of F


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The pH dependency of the reaction can be explained by the general structure of proteins and CMC. Whereas the reaction between CMC and protein fraction a1 can occur at any pH within the biologically relevant range, similar to the interaction between proteins and polyphenols (a2 and b), the reaction between CMC and protein fraction a2 is dependent on pH. Only at a medium-low pH when the carboxyl group on the CMC molecule is deprotonated, while the pH is still low enough to allow protonation of the amino group on the protein, can this reaction take place. However, there is the possibility that CMC can interact with proteins at every pH relevant to wine (a1 and c). In addition to that, the color loss connected to CMC application to red wine is likely to occur via a protein-bridged reaction. Carboxymethyl cellulose does not interact with polyphenols alone, thus making a precipitation of anthocyanins unlikely. Only the reaction of anthocyanins with proteins, followed by the precipitation with CMC, can explain this observation. Removing proteins from the wine will therefore lead to fewer precipitation reactions. This hypothesis is consistent with results observed with our initial experiments with wine samples which showed that wines with higher polyphenol levels and consequently lower protein concentrations showed less frequent problems with haze formation. Although the general reaction of proteins and CMC is already reported in the literature,17,33,34 the specific reaction leading to this unpredictable haze in red wines has not been shown before. To prove the resulting hypothesis that CMC is not binding to anthocyanins directly inducing a precipitation, similar model trials using anthocyanins instead of tannins were performed. The results shown in Figure 5 indicate that there is no haze formation and only a minor color change with CMC and anthocyanins alone. At the higher pH of 4.0 the initial color change with only CMC and anthocyanins is more visible than at lower pH and is most likely due to the pH-dependent loss of flavylium cations. With BSA added to the anthocyanins, a color loss in connection with a precipitation reaction is observed. CMC increases the effect resulting in a stronger agglomeration and, by tendency, a more severe color loss. These results confirm that proteins have to be present to cause the haze formation and color loss observed after CMC addition to red wine.

to increased haze, especially at very low and very high pH values. However, the steady increase observed with the later protein addition (Figure 2) does not happen in all cases. The pH differences, on the other hand, are much more pronounced in these trials, with pH 4.0 displaying the most differences. Taking all of these results from the commercial wines and the bench-trials into consideration, a possible reaction can be proposed that helps to explain the interaction between proteins, polyphenols, and CMC in red wine. Figure 4 shows this

Figure 4. Proposed reaction principle between proteins [a], polyphenols [b] (shown as anthocyanin), and carboxymethyl cellulose [c] that could explain the interaction in wine.

reaction involving a high proportion of protein that is reflective of the percentage found in the real wine precipitation samples.

Figure 5. Precipitation trials with anthocyanins, CMC, and BSA [haze formation over time (A) and color loss (B)]. Letters a−d indicate statistically significant differences determined by paired t test. G


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CONCLUSION In conclusion, the haze formation observed in red wine after CMC addition is caused by proteins readily reacting with the additive independent of pH. However, another possible linkage exists that is dependent on pH. This could be shown for BSA as a model protein but is also supported by observations from real wine samples. The color loss of red wines previously reported after CMC addition is most likely the result of protein-bridged precipitation of anthocyanins and not a direct interaction between polyphenols and CMC. The total protein content of any red wine precipitate is around 50%, with only minor variations between cultivars and samples. This supports the strong involvement of proteins in haze formation. The remaining 50% were shown to contain a large proportion of CMC and variable amounts of anthocyanins, colored pigments, or other polyphenols. The results will help to develop strategies to prevent haze formation in red wines and allow the application of CMC. Although the interaction between proteins, polyphenols, and CMC could be shown and explained, the delay of haze formation in some wines remains to be clarified. It is probable that the complex mix of polysaccharides (pectins and glucans) that may be present in wine is able to interfere with the proposed reaction, delaying the aggregation. However, this aspect needs to be addressed in future research.

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AUTHOR INFORMATION

Corresponding Author

*(S.S.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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Rationale for Haze Formation after Carboxymethyl Cellulose (CMC

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