Impact of Bentonite Additions during Vinification on Protein Stability

Article pubs.acs.org/JAFC

Impact of Bentonite Additions during Vinification on Protein Stability and Volatile Compounds of Albariñ o Wines Eugenio Lira,† Juan José Rodríguez-Bencomo,† Fernando N. Salazar,‡ Ignacio Orriols,§ Daniel Fornos,§ and Francisco López*,† †

Departament d’Enginyeria Química, Facultat d’Enologia, Universitat Rovira i Virgili, Av. Països Catalans 26, 43007 Tarragona, Spain Escuela de Alimentos, Facultad de Recursos Naturales, Pontificia Universidad Católica de Valparaíso, Waddington 716, 2360100 Valparaíso, Chile § Estación de Viticultura y Enología de Galicia (EVEGA), Ingacal, Ponte San Clodio, 32427 Leiro, Spain ‡

ABSTRACT: Today, bentonite continues to be one of the most used products to remove proteins in white wines in order to avoid their precipitation in bottles. However, excessive use of bentonite has negative effects on the aroma of final wine, so the optimization of the dose and the time of its application are important for winemakers. This paper analyzes how applying an equal dose of bentonite at different stages (must clarification; beginning, middle, and end of fermentation) affects the macromolecular profile, protein stability, physical−chemical characteristics and aromatic profile of the wine obtained. The results showed the addition during fermentation (especially in the middle and at the end) reduced the total dose required for protein stabilization of Albariño wines and maintained the sensory characteristics of this variety. KEYWORDS: bentonite, protein stability, sensory characteristics, aromas, white wine

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INTRODUCTION Sensory evaluation of white wines by consumers is the final step of a long process that involves different techniques, variables, and treatments that influence the final result. Taste, visual, and aromatic properties, together with the global impression, should be kept the best possible until the wine reaches the consumer’s table. Some of these aspects are closely related to wine proteins, which are present from the grapes to the final product and are one of the main factors affecting the stability of white wine during storage.1−3 These proteins are molecules derived from grapes and yeasts that affect the protein composition of wine both directly (release of mannoproteins with a stabilizing effect)4,5 and indirectly (secretion of additional cell proteases: hydrolysis of grape proteins).6 Grape Vitis vinifera thaumatin-like protein, constituting a large proportion of the total proteins in wine, seems to play a major role in protein haze formation and the turbidity potential is affected by variations in the relative proportions of these macromolecules at the beginning of the vinification.7 Some of the wine proteins as mannoproteins could be associated with aroma compounds that can affect the aromatic profile.8 However, other proteins are unstable and can precipitate hazing the bottled wine, even at low concentrations,9 due to the fact that protein stability does not correlate well with total protein concentration because individual proteins behave differently.1,2 Each wine has a different proportion of proteins, depending on different elements such as the characteristics of the vintage, the pre- and postfermentative treatments, and the variety of grape used.3 Bentonite is an inorganic fining agent that removes wine proteins by electrostatic adsorption.10 At the pH of wine, its surface has a negative charge, which is responsible for the binding of proteins with a net positive charge, thus adsorbing © 2015 American Chemical Society

and removing them from the wine. Bentonite is extensively used because of its efficacy, low cost, and its use being a simple batch process that does not require any specialized equipment or knowledge. However, it has some drawbacks such as significant wine volume losses because of poor settling,3 the cost associated with waste disposal, occupational health and safety issues, interference with common membrane-based winemaking technologies,3,11 and the fact that it is not a specific adsorbent and may reduce both undesirable and desirable compounds such as aroma, flavor, and anthocyanin compounds in the case of rosé wines.12,13 In spite of all the above-mentioned drawbacks, bentonite is still the most effective agent in wine protein stabilization according to recent work published by Chagas et al.14 As mentioned, it is important to consider the effect of bentonite on the aromas of the final wine. Volatile compounds responsible for these aromas could be originated during fermentation, derived from the grape (as free aroma compounds and aroma precursors), or generated during wine aging. Conversely, fining agents can perform unpredictably and may result in over fining, excessive lees production, and loss in wine quality.15 Fining agents have been shown to reduce the concentration of total flavonoids and aroma compounds such as ethyl esters, acetates, and alcohols in various wine typologies.16 Most fermentative aroma compounds are indirectly removed by bentonite via deproteinization, and only a few odor-active molecules are directly removed through adsorption on bentonite.17,18 Received: Revised: Accepted: Published: 3004

July 25, 2014 March 6, 2015 March 9, 2015 March 9, 2015 DOI: 10.1021/acs.jafc.5b00993 J. Agric. Food Chem. 2015, 63, 3004−3011

Article

Journal of Agricultural and Food Chemistry

treatment done in previous years. The bentonite used was natural sodium granular bentonite (Microcol-Alpha, Laffort; Bordeaux, France). The bentonite treatments were performed to grape juice before alcoholic fermentation and removing this bentonite by settling before starting fermentation, at the beginning of alcoholic fermentation, in the middle of alcoholic fermentation, and at the end of alcoholic fermentation. An untreated fermentation without application of bentonite was used as a control. Protein Analysis by Bradford’s Method. Total protein concentration was measured by Bradford’s method using Coomassie brilliant blue reagent. Under acidic conditions, the red form of the dye is converted into its bluer form upon binding to protein. This change was assayed at 595 nm on a spectrophotometer (Cecil CE2021, Cambridge, England) after 5 min of incubation.24 The anionic (bound) form of the dye has a maximum absorption spectrum historically held to be at 595 nm. The cationic (unbound) forms are green or red. Binding of the dye to the protein stabilizes the blue anionic form. The increase of absorbance at 595 nm is proportional to the amount of bound dye and thus to the concentration of protein present in the sample. The protein content was expressed as mg/L of bovine serum albumin (BSA; Sigma, cat. no. A-3803, Madrid, Spain). Unlike other protein assays, the Bradford protein assay is less susceptible to interference by various chemicals that may be present in protein samples. All analyses were done in triplicate. Fast Protein Liquid Chromatography (FPLC). Samples of 200 mL of each must, wine, or sample obtained during fermentation were centrifuged at 4000g for 5 min at 4 °C. Aliquots of 45 mL of the supernatant were immediately dialyzed for 72 h in three dialysis tubes (SIGMA, dialysis tubing−cellulose membrane, D-9652, Madrid, Spain) to remove salts and other low molecular weight compounds. The dialyzed samples were lyophilized and kept at −20 °C. The lyophilized samples were resuspended in freshly obtained pure deionized water (15 × 106 W/cm) until the protein concentration was about 0.5 mg/mL, expressed as BSA. The samples were centrifuged 12000g for 2 min at 4 °C and frozen again. On the day of the analysis, they were lyophilized again and resuspended in 0.6 mL of 0.3 M ammonium acetate solution (pH 6.80) to obtain a protein concentration of 0.25 μg/μL. The samples were centrifuged (5 min at 12000g, 4 °C), and the supernatants were used directly for FPLC analysis.25 Analyses were carried out with a Superdex 75 PC 3.2/30 column on an FPLC system (Smart System, Pharmacia, Uppsala, Sweden). The samples (50 μL) were injected and eluted with a 0.3 M ammonium acetate solution (pH 6.80) at a flow rate of 40 μL/min. The column eluents were continuously monitored at 280 nm using a μPeak Monitor (Pharmacia, Uppsala, Sweden). Determination of Bentonite Dose. The treatment with bentonite included a preliminary test that use a variety of different dose (5−100 g/hL) so that the most appropriate dose could be determined. For every treatment a heat stability test was applied. Heat Stability Test. A wine sample of 20 mL was filtered through a cellulose nitrate membrane with a pore size of 0.45 μm (Whatman, catalogue no. 7184009, England) and heated for 2 h at 80 °C in a bath equipped with a digital control immersion thermostat (Digiterm 100 model). It was then incubated for 2 h at 4 °C. Finally, the turbidity was measured by nephelometry (Turbiquant 1000 IR turbidimeter) and expressed in nephelometric turbidity units (NTU). The difference in turbidity between the initial wine and the wine after the thermal test was proportional to protein instability. The wines were considered stable if this difference did not exceed 2 NTU.4 All analyses were carried out in triplicate. Chemical Analysis of Musts and Wines. Basic parameters of wines (alcohol content, residual sugars, pH, titratable and volatile acidity, tartaric, malic, and lactic acids) were determined by Fourier transform infrared spectrometry (FTIR) using a Bacchus II analyzer (TDI, Barcelona, Spain) calibrated according to the official methods for wine analysis (EC Regulation 479/2008). The spectra were obtained in triplicate and averaged for each sample. In addition, density, total dry matter, and free and total sulfur dioxide were determined using methods of the OIV (June 2012).

In general, studies are focused on the effect of bentonite fining on fermentative and varietal aroma compounds in finished wines.15,18 Moreover, very few works have studied the effects on aroma compounds of the use of bentonite in musts,19,20 and only two previous works have considered bentonite addition during fermentation for a Macabeo wine.20,21 In this way, bentonite fining may affect both the production and the loss of fermentative compounds. The relationship between grape varieties, protein content, and protein stability has been briefly described up to now. The relationship of varieties such as Sauvignon Blanc22 and Macabeo23 and protein stability has been largely described, but for other varieties like Albariño, there is no information available. To avoid the risk of haze in bottled wine, bentonite treatment is often done to remove unstable proteins and protect the wine. However, this is a nonselective treatment, with the result that some of the proteins associated with the good characteristics of the wine are removed as well. This fact makes oenologists especially concerned when applying bentonite for protein stability. The main objective of this work was to determine the best time for treatment with bentonite in a white wine of the Albariño variety both from the standpoint of protein stability and the preservation of aromas, minimizing the bentonite dose.

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MATERIALS AND METHODS

Fermentation Process. The winemaking of Albariño wines was performed on a pilot scale in the “Estación Experimental de Viticultura y Enoloxiá de Ribadumia EGEVAINGACAL” (Pontevedra, SPAIN) between September and October of 2012. Grapes berries used came ́ Baixas, from the same vineyard (Ribadumia, Pontevedra; D.O. Rias Subzone Salnés, Spain), the soil of which has a granitic rock origin, and vines were planted on 2001 on a V trellis conduction system, at a density of 4.000 pl/Ha, over 196−17 Castel rootstock. Albariño grapes were hand harvested in plastic trails of 20 kg, received 8 g/hL of potassium metabisulphite and 6.67 g/hL of ascorbic acid before the pressing of whole clusters in a pneumatic press (Bucher, Vaslin X pro), and then distributed to the different tanks according to each treatment. Each tank contained the free run juice or juice obtained at low pressures (≤1.0 kg/cm2) and treated with 5 g/100 kg of grapes of pectolytic extractive enzymes (Lafazym press, Laffort, Bordeaux, France). Juice clarification was done at low temperatures (18 °C for 48 h), except for the tank clarified also with bentonite as part of the treatments in the study. The volume of the fermentation stainless steel tanks was 30 L, filled up to the recommended height (85% of the tank volume), and the process was carried out in triplicate. Temperature and density controls were carried out on a daily basis according to the center protocols for this variety in the same way as every addition or treatment was usually made. Fermentation was carried out with the commercial yeast IOC18-2007 (Institut Oenologique de Champagne, Reims, France) in a dose of 30 g/hL. In addition, during fermentation, two different nutrients were used: 15 g/hL of Nutriferm (Enartis, Vilafranca del Penedès, Spain) and 10 g/hL Helper (Oenofrance, Bordeaux, France). Fermentation was carried out around 18 °C until dry wines were obtained. Once alcoholic fermentation finished, 6.5 g/hL of potassium metabisulphite was added in order to keep 3.5 g/hL of free sulfur dioxide and the wines were kept at 12 °C the first 2 days and later at 8 °C until tartaric and protein stabilization treatments were applied. Tartaric stabilization treatment was done during 9 days at −6 °C in a cold chamber. Before bottling, wines were filtered with a cellulose plates filter AF-110, checking before that free sulfur dioxide was between 3.0−3.5 g/hL. Bentonite Treatments. A dose of 40 g/hL bentonite was decided together with the oenological center, considering the stabilizing 3005

DOI: 10.1021/acs.jafc.5b00993 J. Agric. Food Chem. 2015, 63, 3004−3011

Article


Table 1. Chemical Analysis of White Wines Obtained after Bentonite Treatment at a Pilot Scale of the Albariño Grape Variety (Average ± Standard Deviation) alcohol content (% v/v)a

wine (dosing time) must clarification beginning of fermentation middle of fermentation end of fermentation control a

14.01 14.22 13.93 13.93 14.03

± ± ± ± ±

0.28 0.28 0.26 0.28 0.28

a a a a a

volatile acidity (g acetic acid/L)a 0.29 0.31 0.31 0.29 0.29

± ± ± ± ±

0.01 0.02 0.02 0.01 0.01

total acidity (g tartaric acid/L)a

a a a a a

6.82 7.27 6.92 7.19 7.34

± ± ± ± ±

0.14 0.15 0.14 0.14 0.15

c a bc ab a

residual sugar (g/L)a

pHa 3.20 3.11 3.11 3.10 3.09

± ± ± ± ±

0.03 0.03 0.03 0.03 0.03

a b b b b

3.15 3.72 3.74 3.39 3.49

± ± ± ± ±

0.03 0.04 0.04 0.03 0.03

c a a b b

Different letters indicate a significant difference (p ≤ 0.05) for different wine treatments.

Table 2. Protein Content, Stability, Bentonite Stabilizing Dose, Bentonite Total Dose, and Protein Profile by FLPC of Musts and Wines Treated with Bentonite at Different Vinification Stages for Albariño Wines dosing time must

beginning of fermentation

middle of fermentation

end of fermentation

control

a

protein content (mg BSA/L)

F1 protein content (mg BSA/L)

F2 protein content (mg BSA/L)a

F3 protein content (mg BSA/L)a

bentonite stabilizing dose (g/hL)

bentonite total treatment (g/hL)

40.8 ± 0.6

0.5 ± 0.0

5.4 ± 0.4

23.3 ± 0.3

40

80

35.1 ± 0.4

0.0 ± 0.0

2.2 ± 0.3

14.2 ± 1.4

32.7 ± 0.1 28.5 ± 0.6

0.0 ± 0.0 0.0 ± 0.0

1.9 ± 0.0 0.4 ± 0.0

11.1 ± 0.3 3.0 ± 0.0

clarified must (with enzyme) pretreatment 24 h post-treatment dry wine stable wine

39.6 ± 0.5

0.6 ± 0.1

5.8 ± 0.1

26.3 ± 0.4

10

50

38.7 ± 0.6 30.1 ± 1.0

0.6 ± 0.1 0.6 ± 0.2

8.0 ± 0.5 4.3 ± 0.4

28.4 ± 2.9 19.7 ± 4.0

31.5 ± 0.2 26.4 ± 0.2

0.0 ± 0.0 0.0 ± 0.0

1.0 ± 0.4 1.1 ± 0.2

4.9 ± 0.9 4.5 ± 0.1


39.6 ± 0.5

0.6 ± 0.1

5.8 ± 0.1

26.3 ± 0.4

0

40

38.8 ± 0.5 26.2 ± 0.4

1.0 ± 0.1 0.2 ± 0.1

9.1 ± 0.2 4.2 ± 0.2

32.8 ± 0.4 23.0 ± 0.1

29.5 ± 0.1 26.9 ± 0.3

0.0 ± 0.0 0.0 ± 0.0

1.8 ± 0.0 0.7 ± 0.1

6.1 ± 0.1 5.7 ± 0.0


39.6 ± 0.5

0.6 ± 0.1

5.8 ± 0.1

26.3 ± 0.4

0

40

36.9 ± 0.4 28.0 ± 0.2

1.1 ± 0.1 0.1 ± 0.0

4.8 ± 0.7 2.4 ± 0.0

25.3 ± 0.4 15.1 ± 2.1

31.0 ± 0.2 26.5 ± 0.3

0.1 ± 0.0 0.0 ± 0.0

1.4 ± 0.1 0.7 ± 0.1

5.1 ± 0.6 4.8 ± 0.0

clarified must (with enzyme) dry wine stable wine

39.6 ± 0.5

0.6 ± 0.1

5.8 ± 0.1

26.3 ± 0.4

70

70

38.7 ± 0.4 26.9 ± 0.4

0.5 ± 0.1 0.0 ± 0.0

2.1 ± 0.6 3.0 ± 0.8

17.0 ± 1.5 5.7 ± 0.7

sample free run juice (with enzyme) clarified must (with bentonite) dry wine stable wine

Protein fractions: F1 (>100 kDa), F2 (60−40 kDa, invertase and β-glucanases), and F3 (20−30 kDa, chitinases and thaumatin like protein TLP).

Wine Sensory Analysis. A test of preference was performed with a panel of 24 untrained tasters (14 male, 10 female). Fourteen tasters had some previous tasting experience against 10 that did not have any, but none of them were professional tasters. They were asked to choose between the five different wines obtained from the four treatments and the control and to sort from the highest to the lowest preference at nose and palate. The objective was to select the best samples to contrast it with their analytical information. Wines were tasted by the panel 5 months after being bottled. The tasting was done in the tasting laboratory of the Enology Faculty of Rovira i Virgili University (Tarragona, Spain), which has the right conditions for the wine service and tasting. Before the sensorial

analysis, wine panelists tasted different wines to make sure that they understood the purpose of the tasting. The results were analyzed using the test of Friedman, performing a global comparison of each series and then a comparison of pairs to determine the differences found by the tasters. Aroma Profile. Because of the wide range of contents that present the volatile compounds of wines, the approach for the aroma compounds analysis was based according to groups of compounds with contents in similar order of magnitude. Thus, three analytical methodologies were used. Major volatile compounds (methanol, higher alcohols, acetaldehyde, ethyl acetate, ethyl lactate, 1-hexanol, γbutyrolactone, and 2-phenylethanol) were determined by gas chromatography−flame ionization detection (GC−FID) using a 3006


Article

Journal of Agricultural and Food Chemistry method of direct injection described in Blanco et al.26 The quantification was carried out by interpolation into calibration curves obtained with model wines spiked at different levels with the pure commercial aroma compounds. For determination of minor alcohols, volatile fatty acids, ethyl esters of fatty acids, and acetates of higher alcohols, a liquid−liquid extraction (with diethyl ether−hexane (1:1 v/ v)) was carried out previous to the injection in a GC−FID system according with the method described in Blanco et al.26 For the quantification, model wines spiked at different levels with the pure commercial aroma compounds were extracted to build the calibration graphs. For the analysis of terpenols, a solid phase extraction (SPE) was carried out according with the method described in López-Vázquez et al. with some modifications.27 A sample of 50 mL of wine diluted with 50 mL of distilled water (spiked with 0.132 mg/L of 4-decanol as internal standard) was percolated through an Isolute ENV+ SPE cartridge (1 g). The free aroma components were eluted with 30 mL of dichloromethane; the eluate was dried with sodium sulfate and concentrated to 1.5 mL on a Vigreux column, stored at −10 °C, and immediately prior to GC analysis, further concentrated to 100 μL under a gentle nitrogen stream. The chromatographic analysis were conducted using Agilent 6890 gas chromatograph with a mass spectrometric detector (MSD) model 5973N (Agilent Technologies). The capillary column was a HP-Innowax (Agilent) (30 m × 0.25 mm i.d. and 0.25 μm of phase thickness) and the column oven program was 35 °C (2 min), at 30 °C/min to 60 °C (0.5 min), at 2 °C/min to 160 °C (10 min), and at 3 °C/min to 230 °C (10 min). Injector temperature was 250 °C, and the injection (2 μL) was in split mode 1:5. The carrier gas was helium at 1 mL/min. The mass spectrometer was operated in the electron ionization mode at a voltage of 70 eV. The temperatures of source, quadrupole, and transfer-line were 230, 150, and 220 °C, respectively. The acquisition mass range was 30−300 amu. The identification was carried out by comparison with the spectra of Nist Mass Library (rev 05) and with those obtained from the injection of the pure commercial compounds (when available). The quantification were carried out by the internal standard method, so the concentrations was expressed in μg/L equivalent of 4-decanol. Quantification ions (m/z) were the following: terpinene (105), linalool (71), hotrienol (71), α-terpineol (59), citronellol (69), nerol (69), geraniol (69), 3,7-dimethyl-1-octen-3,7-diol (71), E-3,7dimethyl-1,5-octadien-3,7-diol (82), E-3,7-dimethyl-1,5-octadien-3,6diol (71), and 4-decanol (55). Linear retention indexes were calculated by injection of the standards of aliphatic alkanes for each analytical method. For theoretical estimation of the sensory importance of each compound, the odor activity values (OAV) were calculated using the formula OAV = (concentration of compound)/(odor threshold) (expressed in units of aroma [ua]) and odor thresholds published in the literature.28−35 Statistical Analysis. All the statistical analyses were performed with the SPSS statistical package (version 17.0, SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was applied to the data for scale and treatment effect and Principal component analysis (PCA) was applied to compounds with odor activity value higher than 1 ua.

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Table 3. Statistical Analysis of Preference Tests (Nose and Palate)a samples compared must/beginning must/middle must/end must/control beginning/middle beginning/end beginning/control middle/end middle/control end/control

nose Ra − Rbb 2.0 35.0 15.0 3.0 33.0 13.0 1.0 20.0 32.0 12.0

ns s ns ns s ns ns ns s ns

palate Ra − Rbb 4.0 4.0 26.0 4.0 8.0 30.0 8.0 22.0 0.0 22.0

ns ns s ns ns s ns s ns s

ns, not significant difference; s,: significant difference. bRa − Rb: difference between ranges. Critical value Ra − Rb = 21.5 (p = 0.05).

a

were due to the time of sampling and the analytical methods used for each case but did not represent a significant difference among the final wines from a practical point of view. Protein Content Evolution and Stability. The protein content, stability, bentonite stabilizing dose, bentonite total dose, and protein profile of samples obtained from the initial grape juice until the finished wine in the whole process is presented in Table 2 for each treatment and the control. Of the five wines obtained, the two treated at the end of fermentation were stable, while the other three were unstable. Unstable wines needed an additional bentonite dose of 40 and 10 g/hL for the wine obtained with the must clarified with bentonite and the wine treated at the beginning of fermentation, respectively. The control wine needed a dose of 70 g/hL for its stabilization. From these data, it can be concluded that wines treated during fermentation need less bentonite for their stabilization. Similar results were obtained by Lira et al.20,21 and Pocock et al.,10 for Macabeo and Sauvignon Blanc wines, respectively. Analysis of the protein profile (Table 2) showed the presence of three fractions: F1 (>100 kDa), F2 (60−40 kDa, invertase and β-glucanases), and F3 (20−30 kDa, chitinases and thaumatin like protein TLP).7,25,38 Fractions F1 and F2 had a lower protein concentration than F3. Fraction F1 showed a high sensitivity to bentonite treatment, as independently of the dosage during fermentation, its diminution was almost complete. The concentration of fraction F2 in free run juice and initial musts clarified with enzymes was in the range of 5.4−5.8 mg BSA/L. Must clarified with bentonite showed a 60% decrease in fraction F2, which remained stable until the end of the fermentation and was further diminished with the additional treatment of bentonite. In wines treated at the beginning and in the middle of fermentation, there was an increase of the concentration of F2 before the treatment, probably due to yeast action, up to values between 8−9 mg BSA/L.39 In the wine treated at the end of fermentation, there was a decrease in F2 compared with the initial content, similar to the control dry wine. The fermentation had a longer action time and followed the normal evolution with an increase of the protein content due to yeast lysis and the later diminution as an effect of the hydrolysis.38 The final effect of bentonite dosage during fermentation led to a decrease of F2 fraction of around 50− 60%, and as bentonite remained in the medium, the F2 fraction continued to decrease, as observed in dry wines. The main fraction was F3, with an initial content in the free run juice of 23.3 ± 0.3 mg BSA/L and a content of 26.3 ± 0.4

RESULTS AND DISCUSSION

Fermentation Kinetics and Chemical Analysis of Wines. The main characteristics of initial must used for all the experiments were: density of 1101 ± 11 kg/m3, total acidity of 8.63 ± 0.09 g tartaric acid/L, potential alcoholic degree of 14.08 ± 0.14% v/v, and pH of 3.30 ± 0.03. Alcoholic fermentation followed a similar tendency in all fermentations, except for the must clarified with bentonite that had a faster evolution in the first 2−5 days, favored by the presence of bentonite not removed by racking.36,37 Fermentations lasted 14 ± 2 days within a temperature range of 17 ± 2 °C. In Table 1 are shown the main analytical parameters for each wine once stabilized and bottled. Differences in analytical values 3007


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Table 4. Volatile Compounds Analyzed in Final Wines (mg/L except Where Indicated Otherwise) and Odor Activity (OAV) Values for Compounds with OAV > 1 (Presented in Brackets) must clarification

compd (odor threshold)

retention index calculated

Alcohols 1-propanol (500c) isobutanol (40g) 1-butanol (150g) (2+3)-methyl-1-butanol (65f)

1036 1100 1153 1220

1-hexanol (8i) t-3-hexen-1-ol (1f) c-3-hexen-1-ol (0.4i) benzyl alcohol (200g) 2-phenylethyl alcohol (14i)

1364 1374 1395 1893 1935

total alcohols Esters ethyl butyrate (0.02g)

1046

ethyl hexanoate (0.014g)

1255

ethyl octanoate (0.005g)

1447

ethyl decanoate (0.2g)

1649

h

ethyl lactate (154 ) isoamyl acetate (0.03g)

1354 1131

hexyl acetate (1.5g) ethyl acetate (12.3c)

1257 885

total esters

average [OAV] 40.1 19.4 1.56 213 [3.28] 2.47 0.013 0.064 2.25 32.4 [2.31] 861 [5.59] 0.456 [22.8] 0.836 [59.7] 1.34 [268] 0.413 [2.06] 14.5 1.01 [33.5] 0.022 50.6 [4.12] 69.2 [390]

SD 2.2 0.7 0.01 2 0.00 0.005 0.002 0.04 1.1 6

0.015 0.002 0.00 0.035 0.6 0.00 0.001 1.3 1.9

beginning of fermentation average [OAV] 50.5 19.1 1.28 220 [3.39] 1.02 0.010 0.082 2.204 27.3 [1.95] 384 [5.34] 0.600 [30.0] 1.21 [86.3] 1.84 [368] 0.523 [2.62] 16.8 1.66 [55.5] 0.056 67.2 [5.47] 89.9 [548]

SD 1.7 1.2 0.02 0 0.00 0.002 0.003 0.03 1.8 5

0.018 0.00 0.00 0.000 1.2 0.02 0.000 1.4 0.2

middle of fermentation average [OAV] 52.9 20.0 1.64 218 [3.36] 2.42 0.013 0.089 4.52 32.8 [2.34] 394 [5.70] 0.597 [29.8] 1.24 [88.4] 1.92 [384] 0.564 [2.82] 17.7 1.72 [57.4] 0.065 64.9 [5.28] 88.7 [568]

SD 0.6 0.5 0.01 1 0.01 0.001 0.007 0.05 1.3 1

0.022 0.00 0.00 0.001 0.5 0.01 0.000 0.5 0.9

end of fermentation average [OAV]

SD

54.0 18.5 1.66 217 [3.34] 1.84 0.018 0.121 2.38 28.0 [2.00] 377 [5.34] 0.694 [34.7] 1.22 [87.5] 1.89 [378] 0.543 [2.72] 15.8 1.80 [60.0] 0.057 67.0 [5.44] 89.0 [568]

0.9 0.2 0.00 1 0.00 0.005 0.002 0.00 1.1 4

0.061 0.00 0.00 0.000 0.4 0.01 0.000 0.9 1.4

Acids isobutyric acid (2.3e) butyric acid (0.173e)

1584 1648

1.33 0.026

0.36 0.002

1.14 0.020

0.05 0.001

1.29 0.080

0.06 0.006

1.68 0.165

0.01 0.191

isovaleric acid (0.033e)

1686

0.05

2070

2.97 [90.1] 7.90 [18.8] 9.04 [18.1] 2.56 [2.56] 24.3 [130]

0.01

octanoic acid (0.5e)

2.04 [61.8] 7.05 [16.8] 9.73 [19.5] 2.65 [2.65] 22.8 [101]

0.03

1863

1.98 [59.9] 6.75 [16.1] 9.41 [18.8] 2.57 [2.57] 21.9 [97.4]

0.02

hexanoic acid (0.42e)

1.64 [49.7] 5.15 [12.3] 7.17 [14.3] 2.19 [2.19] 17.5 [78.5]

e

decanoic acid (1 )

2286

total acids Carbonyl Compounds acetaldehyde (0.5h)

714

γ-butyrolactone (35 )

1642

h

total carbonyl compds Terpenesa terpinenea (1500d) linaloola (25h) hotrienola (110j)

1174 1541 1605

α-terpineola (250h) citronellola (100h) nerola (500f) geraniola (20h) 3,7-dimethyl-1-octen-3,7-diola,b

1691 1760 1793 1847 1967

0.03 0.02 0.05 0.3

36.8 [73.6] 39.7 [1.13] 76.5 [74.7]

1.1

0.141 10.1 181 [1.65] 8.11 1.01 4.36 4.92 6.69

0.005 0.3 9

0.8 0.3

0.06 0.05 0.32 0.37 0.15

0.00 0.00 0.01 0.0

22.2 [44.4] 56.4 [1.61] 78.6 [46.0]

0.8

0.259 8.36 201 [1.83] 8.26 1.35 5.39 10.3 12

0.009 0.24 10

3008

0.3 0.4

0.06 0.07 0.40 0.8 0.3

0.02 0.01 0.01 0.2

22.2 [44.3] 55.2 [1.58] 77.4 [45.9]

0.3

0.316 8.33 130 [1.18] 9.56 1.65 3.46 9.14 13.2

0.011 0.24 6

0.2 0.1

0.07 0.08 0.26 0.68 0.3

0.02 0.00 0.01 0.2

20.9 [41.8] 41.4 [1.47] 72.3 [43.3]

0.5

0.61 8.70 122 [1.11] 8.8 2.57 3.87 11.3 11.5

0.022 0.25 6

0.7 0.2

0.06 0.13 0.29 0.8 0.2

control average [OAV] 50.7 19.5 1.89 226 [3.47] 1.72 0.020 0.154 2.66 29.0 [2.07] 385 [5.54] 0.888 [44.4] 1.11 [78.9] 1.68 [336] 0.560 [2.80] 16.0 1.59 [52.9] 0.047 66.1 [5.37] 88.0 [520]

SD 0.8 0.3 0.02 1 0.01 0.007 0.002 0.25 0.7 3

0.036 0.00 0.00 0.001 0.5 0.01 0.000 0.8 0.3

1.04 0.384 [2.01] 4.34 [131.6] 7.73 [18.4] 7.80 [15.6] 2.29 [2.29] 23.5 [170]

0.03 0.233

19.2 [38.5] 49.2 [1.41] 68.4 [39.9]

1.6

0.043 6.70 149 [1.35] 9.17 1.77 5.27 10.5 9.38

0.002 0.20 7

0.01 0.02 0.02 0.01 0.2

0.4 2.1

0.07 0.09 0.39 0.8 0.20


Article

Journal of Agricultural and Food Chemistry Table 4. continued must clarification retention index calculated

compd (odor threshold) Terpenesa E-3,7-dimethyl-1,5-octadien-3,7-diola,b E-3,7-dimethyl-1,7-octadien-3,6-diola,b total terpenesa

beginning of fermentation

middle of fermentation

end of fermentation

control

average [OAV]

SD

average [OAV]

SD

average [OAV]

SD

average [OAV]

SD

average [OAV]

SD

59.6 2.49 279 [1.65]

1.3 0.05 16

85.3 8.27 340 [1.83]

1.9 0.18 19

122 5.83 303 [1.18]

3 0.13 15

278 6.72 454 [1.11]

6 0.15 20

228 4.36 424 [1.35]

5 0.10 20

1935 2121

Expressed in μg/L equivalent of 4-decanol. bTentative identification (not commercial pure compound available). cOdor thresholds from ref 28. Odor thresholds from ref 29. eOdor thresholds from ref 30. fOdor thresholds from ref 31. gOdor thresholds from ref 32. hOdor thresholds from ref 33. iOdor thresholds from ref 34. jOdor thresholds from ref 35. a

d

Table 5. Principal Components Analysis Results for the Volatile Compound (with Odor Activity Values (OAV) >1) of Musts and Wines Treated with Bentonite at Different Vinification Stages, for Albariño Wines eigenvalue variance explained (%) octanoic acid decanoic acid ethyl octanote γ-butyrolactone ethyl hexanoate isoamyl acetate ethyl acetate ethyl decanoate ethyl butyrate isovaleric acid (2+3)-methyl-1-butanol hexanoic acid ethanal hotrienol 2-phenylethyl alcohol

PC1

PC2

PC3

10.00 66.7 0.991 0.988 0.962 0.951 0.946 0.880 0.803 0.730 0.116 −0.099 0.170 0.581 −0.702 −0.286 −0.336

3.13 20.9 −0.113 −0.121 0.243 0.202 0.319 0.448 0.591 0.617 0.987 0.980 0.897 0.758 −0.709 −0.300 −0.550

1.34 8.9 0.024 0.092 0.121 −0.147 0.061 0.121 −0.750 0.236 0.106 0.168 −0.168 0.236 −0.044 −0.886 0.599

mg BSA/L in musts clarified with enzymes. The diminution of this fraction in the must clarified with bentonite was until 14.2 ± 1.4 mg BSA/L 24 h after applying the treatment. At the end of fermentation, the content of the F3 fraction was 11.1 ± 0.3 mg BSA/L, representing unstable wines. The additional stabilizing treatment with bentonite reduced this concentration to 3.0 ± 0.0 mg BSA/L. The wines treated with bentonite during fermentation showed a diminution of the F3 fraction of 30−40% after 24 h of treatment, similar to the F2 fraction that decreased after the addition of bentonite, by around 50%. In these wines, the content of fraction F3 decreased until the end of fermentation to values in a range of 4.9−6.1 mg BSA/L, representing stable wines, except the wine treated at the beginning of fermentation, which needed an additional dose of 10 g/hL of bentonite. In the control wine, the content of the F3 fraction in dry wine was 17.0 ± 1.5 mg BSA/L, which decreased to 5.7 ± 0.7 mg BSA/L after the stabilizing treatment with bentonite. The results obtained suggest that the F3 fraction would be involved in protein stability.3,7 Sensory Evaluation. The tasting panel evaluated the Albariño wines obtained from the four treatments and the control in nose and palate, ranking them according to their preferences in nose and palate separately. Statistical analysis of results between pairs of wines (for p < 0.05) are presented in Table 3. As can be seen, in the case of nose sensory data, a clear

Figure 1. Principal component analysis of musts and wines treated with bentonite at different vinification stages, for Albariño wines. (○) Must clarification, ( ■ ) beginning fermentation, ( ● ) middle fermentation, (▲) end fermentation, (□) control. Numbers in figure indicate the order in the sensory preference test.

preference of tasters for the wine treated in the middle of fermentation were observed, with a significant difference versus each of the other wines except with the wine treated at the end of fermentation, which was ranked in second place. The other three wines had similar values and, according to the tasters, there was no difference. On palate, the comparative analysis of the wines resulted in a clear preference for the wine treated at the end of fermentation over the other four wines, which did not have significant differences for the panel. Aroma Compounds. The results of the volatile composition analysis of wines are presented in Table 4. In addition, in order to estimate the sensory importance of each compound and the differences between treatments, the odor activity values (OAVs) are also presented in Table 4 (in brackets) for compounds with OAV > 1 ua. As can be seen, treatments with bentonite at different stages of fermentation, in general, affected the production of volatile fermentative compounds. The effect of the treatments on the contents of esters and acids was remarkable and probably affected the wine aroma because most 3009


Article


reduced the total dose required for protein stabilization of Albariño wines and maintained the sensory characteristics of this variety. For practical purposes, the oenological parameters of the fermentations were not affected by any of the treatments. With regard to the sensory quality of wine, the results obtained from aroma analysis allow us to conclude that the wines with higher aroma intensity and quality will be the ones treated with bentonite in the middle or at the end of fermentation. These results were corroborated by the sensory analysis in which the wines treated in the middle and at the end of fermentation were preferred by tasters. In addition, both wines were stable and did not need any extra dose of bentonite, so they were the wines with a less aggressive stabilizing treatment. Nevertheless, further work must be carried out in the future with other varieties and on an industrial winemaking scale to adjust, improve, and adapt this technique.

of them are in contents higher than their odor thresholds. In addition to the possible loss of volatile compounds due to adsorption on bentonite, the effects on the production of fermentative compounds could be related to the variation in nitrogen composition and other nutrients3,40 of musts and wines removed by the bentonite. In the case of varietal compounds, is it remarkable that the total terpenes amounts were highest in control and wines treated at the end of fermentation, while the lowest content was observed for clarified must. These effects could be related to the adsorption on bentonite of bound terpenes (glycoside forms). In addition, the release of free terpenes from the glycoside forms during fermentation could be influenced by the bentonite treatments affecting the interaction of yeast and the specific glycosides.16 Among all terpenes analyzed, it can be observed that studied wines contain high amounts of hotrienol and E-3,7-dimethyl1,5-octadien-3,7-diol, characteristic of this variety, with respect to other Galician varieties41 and that only hotrienol showed contents higher than its odor thresholds, however, some less aromatic terpenes, such as E-3,7-dimethyl-1,5-octadien-3,7-diol, could transform into other more aromatic terpenes during wine aging.42 To obtain more information on the volatile composition analysis and with the aim of focus the discussion on the compounds with sensory importance, a principal component analysis (PCA) was carried out considering only the compounds with odor activity value higher than 1 ua. PCA selected three principal components with eigenvalue higher than 1. Table 5 shows the loading of each variable for the three PC, as well as the eigenvalue for each factor. It was observed that more than 87% of the variation of the values could be explained by the two first principal components. The plot of the different wine samples in the plane defined by the first two PCs is shown in Figure 1. As can be seen, the PC1 (66.7%) showed a clear separation of the treated wines during fermentation (with positive PC1 values) of the control (negative values of PC1 over −0.6) and of the wine obtained from clarified musts (PC1 values over −1.5). On the other hand, the PC2 (20.9%) also allow separation of the samples treated during fermentation (situated between −06 and 0.25 values for PC2) and the control (values of PC2 of 1.6) and musts clarified wines (values of PC2 over −1). As can be seen in Table 5, the PC1 is mainly associated and positively correlated with seven compounds: two acids (octanoic and decanoic acids), five esters (ethyl esters of decanoic, octanoic, and hexanoic acids, and ethyl and isoamyl acetates), and the γbutyrolactone. According with the results of sensory preference test (Wine Sensory Analysis section) and the position of the treated samples during fermentation (preferred by tasters) and the wine worst ranked (must clarified) in the plot (Figure 1), it could be concluded that the sensory quality of these wines is related, at least in part, with the concentration of these seven compounds. On the basis on the bibliography, acids, ethyl esters, and acetates have an important influence on the fruity and freshness character of wine aroma.28 On the other hand, PC2 is mainly positively associate with two acids (isovaleric and hexanoic acids), ethyl butyrate, and isoamyl alcohols and negatively correlated with ethanal. In this case, probably the influence of these compounds on the “preference test” was less important, and it did not show a clear separation of the better and worst wines ranked. In summary, the addition of bentonite during alcoholic fermentation, and especially in the middle or at the end,

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +34 977 558503. E-mail: [email protected]. Funding

J.J.R.B. thanks the fellowship of the program “Beatriu de Pinós” with the support of the Secretariat d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya and the European Union. Notes

The authors declare no competing financial interest.

■

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Impact of Bentonite Additions during Vinification on Protein Stability

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