Influence of Different Fermentation Strategies on the Phenolic Profile

Article pubs.acs.org/JAFC

Influence of Different Fermentation Strategies on the Phenolic Profile of Bilberry Wine (Vaccinium myrtillus L.) Annika Behrends and Fabian Weber* Institute of Nutritional and Food Sciences, Molecular Food Technology, University of Bonn, Römerstrasse 164, D-53117 Bonn, Germany ABSTRACT: Polyphenol rich and especially anthocyanin rich berries like bilberries (Vaccinium myrtillus L.) and derived products such as wine have enjoyed increasing popularity. During winemaking and aging, the phenolic profile undergoes distinct changes, a phenomenon that has been well investigated in grape wine but not in bilberry wine. The present study determined the influence of different fermentation strategies including various pre- and postfermentative heating and cooling concepts on the phenolic profile of bilberry wine. Besides significant differences in total anthocyanin and tannin concentrations, the different fermentation strategies resulted in distinguishable anthocyanin profiles. A very fast aging manifested by a rapid decrease in monomeric anthocyanins of up to 98% during a 12 week storage and a coincident formation of polymeric pigments and pyranoanthocyanins was observed. Several well-known processes associated with production and aging of wine were much more pronounced in bilberry wine compared to grape wine. KEYWORDS: Vaccinium myrtillus, bilberry, phenolics, anthocyanins, wine, fermentation, polymeric pigments

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INTRODUCTION

Today, the use of fruits other than grapes for the production of wine is quite common. Besides apples, elderberries, cherries, peaches, and many other fruits, blueberries and bilberries are also used.11 Especially in the USA and Canada, the production of wine from blueberries has a great economic relevance. Vaccinium corymbosum, Vaccinium ashei, and Vaccinium angustifolium are the species mainly used for winemaking in these countries.14−16 Corresponding wines have been investigated regarding phenolic compounds and antioxidative capacity. The antioxidant capacity of Canadian blueberry wine was shown to be much higher compared to the initial juice.14 According to the authors, this effect is attributed to both an increase in the phenolic content and changes in the phenolic profile. Others highlighted the high antioxidative capacity of American blueberry wine compared to conventional red wine.15,16 To our knowledge, no research has been conducted on the changes of the phenolic profile during production of wine from European bilberry. Accordingly, there is a need to further evaluate the suitability of European bilberry for wine production and the influence of processing. The objective of this study was to investigate the effects of different fermentation strategies commonly applied in grape wine production on the phenolic profile during the production of wine from bilberries and to determine changes in characteristic parameters during storage.

Among many other berries, bilberries (Vaccinium myrtillus L.) and derived products have enjoyed an increasing popularity due to their appealing taste and high amounts of secondary plant metabolites. Regular consumption of berries is associated with numerous health benefits attributed to the presence of a wide range of polyphenols.1−4 Bilberries are especially rich in anthocyanins, which account for contents between 296 mg/ 100 g and 450 mg/100 g fresh weight.5,6 The anthocyanin profile of bilberries is composed of 15 anthocyanins including 3-O-glucosides, 3-O-galactosides, and 3-O-arabinosides of the 5 anthocyanidin aglycones delphinidin (dp), cyanidin (cy), peonidin (pn), petunidin (pt), and malvidin (mn).7,8 Bilberries also contain a relatively large amount of proantho-cyanidins (148 mg/100 g fresh weight),9 which are responsible for the perception of astringency because of their ability to form complexes with salivary proteins.10 Processing of berry fruits into jams, juices, and wine is a common practice to circumvent problems associated with the short shelf life of the fresh crops.11 Fermentation has been shown in numerous studies to increase the release of phenolic compounds and considerably change the polyphenol profile. These changes of the phenolic profile during winemaking and aging are one major focus of research on grape wine. During fermentation, extraction of anthocyanins can be more or less enhanced depending on the fermentation strategy.12 Once extracted, their levels decrease during aging since anthocyanins are involved in distinct reactions with other wine constituents leading to the formation of pyranoanthocyanins and pigmented polymers. Pyranoanthocyanins bear a second pyran ring formed by the addition of compounds with a polarizable double bond to the genuine anthocyanins. Pigmented polymers are formed by incorporation of anthocyanins into tannic material by numerous pathways leading to undefined structures.13 © XXXX American Chemical Society

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MATERIALS AND METHODS

Samples. Frozen bilberries (Vaccinium myrtillus) organically grown and harvested in Romania in 2015 were provided by Haus Rabenhorst Received: Revised: Accepted: Published: A

May 15, 2017 July 26, 2017 July 27, 2017 July 27, 2017 DOI: 10.1021/acs.jafc.7b02268 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

T3 (1.8 μm, 2.1 mm × 150 mm; Waters, Eschborn, Germany), equipped with a security guard cartridge of the same material (1.8 μm, 2.1 mm × 5 mm). Eluent A was water/formic acid (97:3 mL/mL) and eluent B was acetonitrile/formic acid (97:3 mL/mL). A gradient elution program at a flow rate of 0.4 mL/min was used as follows (min/%B): 0/4, 7/8, 13/10, 19/17, 23/30, 26/40, 29/100, 32/100, 33/4, 35/4. Anthocyanins, pyranoanthocyanins, and pigmented polymers (represented by the chromatographic hump) were detected at 520 nm. They were quantified as delphinidin-3-O-glucoside equivalents. For specific retention times, see Figure 3. Although coelution of procyanidins, oligomeric pigments, and the pigmented polymers can be assumed, quantification of pigmented polymers by UHPLC-DAD can be considered as a crude method that yields basic information on the development of the polymeric material in the wines. For LC-MS analysis, a LTQ-XL ion trap mass spectrometer (Thermo Fisher Scientific, Schwerte, Germany) was connected to the UHPLC system via an ESI interface. The analyses were performed as a full scan in positive ionization mode. Helium was used as the collision gas. The following detection parameters were used: sheath gas (N2), 70 arbitrary units; aux gas (N2), 10 units; sweep gas (N2), 1 unit; ion spray voltage, 4 kV; capillary temperature, 325 °C; capillary voltage, 14 V; tube lens, 55 V; collision energy, 35 V. Methyl Cellulose Precipitable (MCP) Tannin Assay. The assay was modified based on Mercurio and Smith.17 Wine was filtered and diluted (dilution factor (df) 2) with water. Aliquots of 25 μL of the diluted wine were transferred to two 1.5 mL microfuge tubes. To the first tube (sample), 300 μL of methyl cellulose solution (0.04% w/v) was added and to the second tube (control), 300 μL water was added. After 3 min, both tubes were vortexed, and 200 μL of saturated ammonium sulfate solution and 475 μL of water were added to both sample and control. After mixing and incubation for 10 min at room temperature, both tubes were centrifuged for 5 min at 11000 g (microcentrifuge Heraeus Pico 17, Thermo Fisher Scientific, Schwerte, Germany). The supernatant was transferred into a UV cuvette and the absorbance was read at 280 nm using a Genesys 6 spectrophotometer (Thermo Fisher Scientific, Schwerte, Germany). The reading resulting from (control − sample) is the amount of tannins precipitable by methyl cellulose. All measurements were performed in triplicate. Tannins were quantified as (+)-catechin equivalents (CE). Adams−Harbertson (AH) Tannin Assay. The assay was performed according to Harbertson et al. with slight modifications.18 The following solutions were used for the analysis: buffer solution (9.86 g/L sodium chloride in 1.2% acetic acid, pH 4.9), bovine serum albumin (BSA) solution (1 g/L BSA in buffer solution), bleaching solution (31.6 g/L sodium metabisulfite in water). Wine was filtered and diluted (df 10) with water. Aliquots of 500 μL of the diluted wine were transferred to two 1.5 mL microfuge tubes. To the first tube, 1 mL buffer solution and 120 μL bleaching solution were added. After mixing and incubation for 10 min at room temperature, the mixture was transferred into a cuvette, and the absorbance was read at 520 nm (reading B). To the second tube, 1 mL BSA solution was added. The mixture was incubated at room temperature for 15 min with occasional upending. The sample was then centrifuged for 5 min at 14300g. Aliquots of 1 mL of the supernatant were transferred into a cuvette, 80 μL of bleaching solution was added, and the absorbance was read at 520 nm after mixing and incubation for 10 min (reading C). The absorbance due to total polymeric pigments (TPP), small polymeric pigments (SPP), and large polymeric pigments (LPP) is given as B, C, and (B − C), respectively. All measurements were performed in triplicate, and results are presented in AU. Statistical Analysis. To determine significant differences, an ANOVA with Bonferroni post hoc test (significance level α = 0.05) was performed using the software XLSTAT (Addinsoft, Paris, France).

O. Lauffs GmbH & Co. KG (Unkel, Germany). The berries were frozen for approximately 10 months. Chemicals. Deionized water was obtained from a Purelab Flex 2 system (Veolia Water Solutions & Technologies, Berlin, Germany). Acetonitrile (HPLC gradient grade; ≥99.9%), water for LC-MS, and sodium chloride were from Th. Geyer (Renningen, Germany). Formic acid (≥99.5%) was purchased from Fisher Scientific (Geel, Belgium). Delphinidin-3-O-glucoside (98%) was from Phytolab GmbH & Co. KG (Vestenbergsgreuth, Germany). Methyl cellulose was obtained from DOW (Schwalbach/Ts., Germany) and ammonium sulfate was from AppliChem (Darmstadt, Germany). (+)-Catechin was purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Acetic acid was obtained from VWR International GmbH (Darmstadt, Germany), sodium hydroxide from Acros Organics (Geel, Belgium), and sodium metabisulfite from Sigma-Aldrich (Steinheim, Germany). Bovine serum albumin (BSA) was from Merck (Darmstadt, Germany). Bilberry Wine Processing. Bilberries (2 kg for each wine trial) were defrosted for 4 h at room temperature and then manually crushed. Further treatment depended on the different fermentation strategies summarized in Table 1. No additional treatment was applied

Table 1. Fermentation Strategies of the Nine Different Elaborated Wines no.

abbreviation

1 2 3

SC NSC SC+OC

4

NSC+OC

5

CSSC

6

CSNSC

7

T55NSC

8

T70NSC

9

SCT45

fermentation strategy fermentation with skin contact fermentation without skin contact fermentation with skin contact and subsequent storage with oak chips for 12 days fermentation without skin contact and subsequent storage with oak chips for 12 days cold soak at 4 °C for 5 days, fermentation with skin contact cold soak at 4 °C for 5 days, fermentation without skin contact thermovinification at 55 °C for 2 h, fermentation without skin contact thermovinification at 70 °C for 3 min, fermentation without skin contact fermentation with skin contact, heating at 45 °C for 24 h after fermentation

to the wines which were processed by fermentation with and without skin contact (SC and NSC). Cold soak entailed maceration during 5 days at 4 °C. These wines were subsequently fermented with (CSSC) or without (CSNSC) skin contact. Thermovinification was conducted on two wines for 2 h at 55 °C or 3 min at 70 °C (T55NSC and T70NSC), respectively. Sample SCT45 underwent a thermal treatment after fermentation for 24 h at 45 °C. Oak chips (6.6 cm × 0.9 cm x 0.9 cm) were added after fermentation to samples SC+OC and NSC +OC and were stored for 12 days. Pressing was done with a Para-Press (Paul Arauner GmbH & Co. KG, Kitzingen, Germany). The wines were fermented at 25 °C with the addition of sucrose (120 g/kg bilberries), nutritional supplement Go-Ferm Protect (1 g/g yeast; Lallemand, Schwarzenbach an der Saale, Germany) and yeast (Saccharomyces cerevisiae; 1 g/kg bilberries; Oenoferm X-treme, Erbslöh Geisenheim AG, Geisenheim, Germany) until they reached a constant alcohol content (between 11.2% and 13.5%, depending on fermentation strategy) Wines were centrifuged twice at 5400g for 15 min (model J2-21 Centrifuge Beckman Coulter GmbH, Krefeld, Germany). Sodium metabisulfite (0.13 g/L) was added. All wines were elaborated in duplicate, yielding a total number of 18 wines. Wines were stored for 12 weeks at 22 °C. Identification and Quantification of Anthocyanins and Anthocyanin-Derived Pigments. For anthocyanin identification and quantification, UHPLC-DAD-ESI-MSn analysis was used. The UHPLC analyses were performed on a Waters Acquity i-Class instrument (Waters, Eschborn, Germany) equipped with a binary pump, a diode-array detector, an autosampler (cooled to 7 °C, injecting 5 μL), and a column oven (at 40 °C). The column was a HSS

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RESULTS AND DISCUSSION Anthocyanins and Anthocyanin-Derived Pigments. Anthocyanins and derived pigments play an important role for the quality of bilberry wine, and thus their evolution was B

DOI: 10.1021/acs.jafc.7b02268 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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their activity results in hydrolysis of anthocyanins leading to their unstable aglycones.24,25 Aglycones of dp and cy were detected in concentrations between 4 and 30 mg/L dp-3-glu equivalents in all wines that were not thermally treated. The decrease in anthocyanins during fermentation of wines without thermal treatment can be explained by enzymatic and chemical processes, while the degradation in thermally treated wines is exclusively caused by chemical processes. Sample SCT45, which was heated after fermentation, however displayed higher anthocyanin contents. The detrimental activity of enzymes during the early stages of fermentation was outbalanced by an enhanced extraction of anthocyanins due to the long heat exposure.26 Anthocyanin concentrations of all wines decreased significantly during 12 weeks storage. The losses ranged from 72.6% to 97.6%. Wines heated before fermentation showed the highest retention. The loss of monomeric anthocyanins during storage cannot be explained by enzymatic degradation because PPO and endogenous glycosidases are inhibited by the increasing amount of ethanol and by the addition of sodium metabisulfite.27 The loss during storage is rather a result of chemical degradation or derivatization caused by condensation reactions and formation of polymeric pigments.28 The concentration of the pigmented polymers quantified as the chromatographic hump, which emerged during fermentation and storage, is shown in Figure 2. This polymeric hump

determined with respect to the different fermentation strategies. Anthocyanins and their derivatives were identified according to their order of elution8 and their specific fragmentation pattern.19−21 Total anthocyanin content is expressed as the sum of individual anthocyanins. The anthocyanin concentration before and immediately after fermentation, as well as after 12 weeks storage time, is shown in Figure 1. The results show that

Figure 1. Anthocyanin content determined by UHPLC-DAD-ESI-MSn before and after fermentation and after 12 weeks (including relative anthocyanin loss), calculated as delphinidin-3-O-glucoside equivalents; different letters indicate significant difference (α = 0.05); n = 2.

different prefermentative treatments caused differences in anthocyanin concentration prior to fermentation. The thermally treated juices (1823.1 mg/L, T55NSC, and 1780.1 mg/L, T70NSC) had highest anthocyanin concentrations. Juices subjected to prefermentative cold soak showed lowest anthocyanin concentrations (866.3 mg/L, CSSC, and 696.0 mg/L, CSNSC). Anthocyanin concentration of the juices with cold soak was expected to be higher due to the prolonged maceration that should have caused an enhanced anthocyanin extraction.12,22 Oxidation or polymerization reactions between anthocyanins and other wine constituents might explain this considerable loss of monomeric anthocyanins. Anthocyanin concentrations of the juices that were neither thermally treated before fermentation nor subjected to cold soak ranged from 1143.9 mg/L (SC and SC+OC) to 1407.9 mg/L (SCT45). The differences observed before fermentation between the samples that were treated in the same way might be explained by the influence of pressing and mashing. All wines had significantly decreased anthocyanin concentration after fermentation compared to the concentration of the juice, with losses ranging from 32.2% to 61.3%. Thermally treated wines generally showed 2-fold to 3-fold higher anthocyanin contents than wines not subjected to thermal treatment. Thermovinification leads to wines with higher amounts of anthocyanins, which might be attributed to inactivation of fruit-borne anthocyanin degrading enzymes like polyphenol oxidase (PPO) and endogenous glycosidases (arabinosidases and galactosidases). Inactivation of PPO prevents the oxidation of other phenolic compounds to reactive quinones, which may subsequently oxidize anthocyanins.23 Inactivation of endogenous arabinosidase and galactosidase enzymes also prevents degradation of anthocyanins because

Figure 2. Concentration of pigmented polymers determined by UHPLC-DAD-ESI-MSn after fermentation and after 12 weeks (including relative changes), calculated as delphinidin-3-O-glucoside equivalents; different letters indicate significant difference (α = 0.05); n = 2.

was not detected before fermentation in any of the different wines. Directly after fermentation, the concentrations ranged from 92.7 mg/L (CSNSC) to 357.3 mg/L (T55NSC). Analogous to anthocyanin concentration, the thermally treated wines had the highest amounts of pigmented polymers. There is a strong correlation (R2 = 0.916) between the concentration of pigmented polymers formed during fermentation and the concentration of anthocyanins before fermentation. The concentration of pigmented polymers increased significantly during storage in the two wines that were thermally treated before fermentation (51.2%, T55NSC, and 74.9%, T70NSC). The wine treated with prefermentative cold soak and fermented without skin contact (CSNSC) also showed a great increase in C


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Figure 3. UHPLC-DAD chromatograms (520 nm) of bilberry wine (SC): (A) before fermentation, (B) right after fermentation, (C) after 3 weeks, (D) after 6 weeks, (E) after 9 weeks, and (F) after 12 weeks, for (1) dp-3-gal, (2) dp-3-glu, (3) cy-3-gal, (4) dp-3-ara, (5) cy-3-glu, (6) pt-3-gal, (7) cy-3-ara, (8) pt-3-glu, (9) pn-3-gal, (10) pt-3-ara, (11) pn-3-glu, (12) mv-3-gal, (13) pn-3-ara, (14) mv-3-glu, (15) mv-3-ara, (I) dp, (II) cy, (i) carboxy-pyrano-del-3-hex, (ii) carboxy-pyrano-pn-3-hex, (iii) carboxy-pyrano-mv-3-hex; (iv) pyrano-mv-3-hex (B-type vitisin), (v) carboxy-pyranopt-3-hex.

bilberry wines was 3.1, whereas commercial red wines commonly show a pH of 3.6.33 Presumably, aging of bilberry wines might be deferred by increasing the pH-value by means of a suitable deacidification. Anthocyanin Profile. Apart from the total amount, the composition of the anthocyanin profile changed during fermentation and storage. Figure 3 shows the chromatograms obtained for bilberry wine (SC) during elaboration and storage. A considerable loss of arabinosides and galactosides was observed during fermentation. After 12 weeks of storage, almost exclusively glucosides were detected. Degradation of anthocyanins by endogenous glycosidases was proven by formation of two aglycones (dp and cy), which diminished later during storage. Further alteration of the anthocyanin profile was manifested in the formation of anthocyanin derivatives like pyranoanthocyanins and increasing proportion of pigmented polymers. Figure 4 shows the relative concentration of glucosides, galactosides, and arabinosides in bilberry wines compared to the initial bilberry juice. The juice was composed of approximately 50% glucosides, 30% galactosides, and 20% arabinosides. The anthocyanin profile was not changed by any prefermentative treatment. The anthocyanin profiles of all bilberry wines were identical at the beginning of fermentation. During fermentation, considerable changes took place. The two wines that underwent prefermentative thermal treatment showed an almost juice-like composition, whereas all other wines displayed losses of arabinosides and galactosides. They were composed of about 7% arabinosides, 8% galactosides, and 85% glucosides at the end of fermentation. As already mentioned, the activity of endogenous glycosidases plays an important role in changes of the anthocyanin profile. The thermally treated wines do not show any effects of

pigmented polymers (44.3%). The other wines displayed inconsistent results ranging from −11.4% (SC+OC) to 23.2% (CSSC). Concentration of pigmented polymers showed a maximum after 6−9 weeks (data not shown). The increase in pigmented polymers in wine is caused by condensation reactions between anthocyanins with other anthocyanins, proanthocyanidins, or flavanols.22,29,30 These condensations may be direct or mediated by acetaldehyde, which is formed during fermentation in the yeasts’ metabolism.31 The decrease in pigmented polymers at the end of storage might be attributed to an insufficient chromatographic separation because the hump of pigmented polymers in UHPLC analysis gets broad and ill-defined during storage, which leads to integration problems of the chromatographic area. A more precise quantification may be achieved by separation via a mixed-mode-phase column.32 Slight formation of a dark-red colored sediment was observed after 12 weeks, which might contribute to the decrease in pigmented polymers. Detailed information on the molecular size and their solubility could confirm this. The observed decrease in monomeric anthocyanins and the corresponding increase in pigmented polymers in bilberry wines can be compared with the changes in conventional red wines.29 However, the time frame is considerably different. Red wine shows a discernible decrease in anthocyanins not before 13 months of storage,29 whereas bilberry wine lost up to 98% of the initial anthocyanins already after 12 weeks. Additionally, very slight color changes of the anthocyanin rich and therefore dark violet colored wines to more brownish hues were observed during 12 weeks storage. Bilberry wine obviously undergoes a very fast aging. The low pH value supposedly influences the chemical reactions during fermentation and storage. The pHvalue of the initial bilberry juice was 2.9 and of the processed D


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pyranoanthocyanins in bilberry wine proceeded quite fast.29,35,36 According to Alcalde-Eon et al., who investigated the pigment composition of red wine during maturity and aging, A-type vitisins were detectable after 8 to 13 months.29 The first A-type vitisins in bilberry wine were already detected in the third week. The elevated storage temperature (22 °C) and the lower pH might be the reason for this fast formation.37 Tannins. Tannin concentration was determined using the MCP tannin assay and quantified as catechin equivalents. Wines that were fermented with skin contact showed higher tannin concentrations than those fermented without skin contact. The former had tannin concentrations between 1147 mg/L (SC) and 2005 mg/L (SCT45), the latter between 592 mg/L (T70NSC) and 1017 mg/L (NSC+OC) (Table 2). The juice contained 381 mg/L tannins. T55NSC and SCT45 showed the highest tannin concentrations. The different amounts of tannins can be reasoned by different extraction kinetics due to the fermentation strategy. Prolonged contact time with the tannin-rich skins and kernels and increasing ethanol content enhances the extraction of tannins. The purpose of thermovinification and the associated aqueousthermal extraction procedure is to extract more anthocyanins than tannins, which leads to more vividly colored and less astringent wines. Heating causes a rapid destruction of cells and, thus, accelerates extraction of water-soluble anthocyanins.12 Accordingly, thermally treated wine fermented without skin contact (T70NSC) showed low tannin concentrations and high anthocyanin concentrations. The wine that was heated at 55 °C (T55NSC), however, displayed very high tannin concentrations. Slow heating up to 55 °C might lead to temporarily elevated enzyme activities, resulting in cell wall decomposition and consequently to higher tannin contents after pressing. Tannin cell wall binding occurs through hydrogen bonding and through hydrophobic interactions between tannins and polysaccharides. This leads to an inclusion of tannins in the complex cell wall network and therefore to a reduced tannin extraction from berry into wine during maceration.12,38 All wines showed increasing tannin amounts within the first 6−9 weeks of storage followed by decreasing concentrations (data not shown). The MCP assay does not distinguish between tannins of different degrees of polymerization. High amounts of oligomeric compounds would lead to similar results as low amounts of high polymeric compounds. Hence, the

Figure 4. Relative anthocyanin concentrations of bilberry wines and juice expressed as the sum of arabinosides, galactosides, and glucosides after fermentation.

enzymatic degradation during fermentation because enzymes were inactivated. The other wines containing active enzymes at the early phase of fermentation show great losses of arabinosides and galactosides, which can be ascribed to a different substrate specificity of the endogenous glycosidases. Buchert et al. showed that enzyme-assisted bilberry juice production with commercially available pectinases results in greater losses of galactosides but not glucosides.34 The fact that the anthocyanin content decreased significantly during storage, whereas the composition of glucosides, arabinosides, and galactosides remained constant (data not shown) can be explained by a consistent anthocyanin decrease caused by nonspecific chemical reactions. These follow first-order kinetics35 resulting in an exponential decrease. Pyranoanthocyanins. The two wines that were thermally treated prior to fermentation showed significantly higher amounts of pyranoanthocyanins (Table 2). Because they also contained much more total anthocyanins (Figure 1), this seems plausible. In comparison to commercial red wine, formation of

Table 2. Concentration of Pyranoanthocyanins Determined by UHPLC-DAD-ESI-MSn, Concentration of Tannins Determined by the Methyl Cellulose Precipitable Assay, and Absorption of Small and Large Polymeric Pigments Determined by the Adams− Harbertson Assay after Fermentationa and after 12 Weeks Storageb,c sample CSSC CSNSC T55NSC T70NSC SCT45 SC SC+OC NSC NSC+OC juice

pyranoanthocyanins (mg/L dp-3-glu eq.) 12.55 15.64 35.31 33.67 15.07 12.26 12.52 15.42 15.32

± ± ± ± ± ± ± ± ±

0.49 0.24 0.12 1.82 0.21 0.07 0.16 0.66 0.66

bc b a a bc c bc bc bc

tannins (mg/L CE) 1405 661 2159 592 2005 1147 1522 767 1017 381

± ± ± ± ± ± ± ± ± ±

68 abc 87 cde 596 a 266 cde 166 ab 300 bcd 108abc 141 cde 12 bcde 126 de

SPP+ (AU) 0.074 0.048 0.184 0.137 0.171 0.088 0.114 0.074 0.084 0.015

± ± ± ± ± ± ± ± ± ±

0.007 0.001 0.004 0.019 0.006 0.006 0.011 0.008 0.012 0.001

SPP* (AU) ef fg a bc ab de cd ef def g

0.163 0.138 0.242 0.377 0.128 0.150 0.147 0.172 0.174

± ± ± ± ± ± ± ± ±

0.004 0.006 0.001 0.010 0.002 0.001 0.008 0.005 0.009

LPP* (AU) cd de b a e cde cde c c

0.007 0.0 c 0.179 0.037 0.123 0.032 0.016 0.019 0.012

± 0.001 c ± ± ± ± ± ± ±

0.012 0.006 0.012 0.010 0.015 0.020 0.011

a c b c c c c

a

Indicated by +; no LPP were detected directly after fermentation. bIndicated by asterisk. cDifferent letters indicate significant difference (α = 0.05); n = 2. E


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inactivation of PPO and of glycosidases naturally occurring in bilberries. Contrary to the expectations, a prefermentative cold soak treatment resulted in lower anthocyanin concentrations compared to traditional grape wine, which might also be attributed to residual enzyme activities or nonenzymatic reactions of the anthocyanins. Wines stored with oak chips after fermentation did not show any significant differences in comparison to the corresponding control. Skin contact during fermentation did not affect polymeric pigment composition in wines not thermally treated, whereas tannin concentration was higher in wines fermented with skin contact due to the prolonged contact time with the tannin-rich skins and kernels. The investigation of the influence of storage on the phenolic profile revealed that bilberry wine undergoes a very fast aging irrespective of the applied fermentation strategy. Thus, the total anthocyanin content was reduced to about 2% of the original amount after 12 weeks, and polymeric pigments as well as pyranoanthocyanins were formed alongside. The low pH-value is assumed to be the main reason for the fast aging. A strategy to increase the pH-value by means of a suitable deacidification might delay early aging. Malolactic fermentation is commonly used in red wine production and has tremendous effects on wine composition. Especially, the consumption of excess acetaldehyde by the applied microorganisms might lead to slower pigment aging. In comparison with grape wine, several well-known processes like specific degradation of anthocyanin glucosides, polymerization reactions, or enzyme inactivation are clearly observable in this study. Further investigation of the process of bilberry wine production and aging may assist to get new insights also in grape wine.

results do not allow conclusions whether storage leads to the formation of larger polymers or to degradation to smaller oligomers. Tannins’ structural diversity renders it nearly impossible to determine the exact concentration. Polymeric Pigments. Polymeric pigments were determined using the AH tannin assay. This assay allows the differentiation between small (SPP) and large polymeric pigments (LPP), whereby SPP have a degree of polymerization between 2 and 4.17,39 The results are given in Table 2. Directly after fermentation, all wines contained only SPP and no LPP, which were formed only during storage. The juice contained lowest amount of SPP, whereas thermal treatment resulted in high amounts. Except for CSNSC, all bilberry wines showed significantly higher amounts of polymeric pigments than pure juice. The formation is based on condensation reactions of anthocyanins with other anthocyanins, flavanols, or proanthocyanidins22,29,30 and via the reaction with acetaldehyde.31 The composition of monomers is also an important factor that influences the formation of polymeric pigments.28 This might explain the different formation kinetics in the wines after cold soak. Apparently, cold soak led to flavanol-to-anthocyanin ratios less favorable for the formation of stable condensation products.40,41 Like the MCP assay, the AH assay does not permit exact conclusions on polymer composition. During storage, a continuous increase in SPP and a slight increase in LPP can be observed after 9 weeks, whereas T55NSC and SCT45 showed a much stronger increase within the first 3−6 weeks (data not shown). A simultaneous decrease in monomeric anthocyanins suggests the incorporation of anthocyanins into stable polymeric pigments. The strong increase in LPP in T55NSC and SCT45 can be explained by enhanced polymerization due to the long heat exposure.42 According to the results obtained by the MCP assay, these two wines exhibited the highest amounts of tannins. This led to an increased formation of LPP due to an increased availability of reactants for anthocyanins to form colored anthocyanin−tannin adducts.43 According to Harbertson et al., the SPP fraction contains vitisins characterized by a low molecular weight.18 T55NSC and T70NSC exhibit the highest amounts of SPP and the highest amounts of vitisin-type pyranoanthocyanins after 12 weeks. Thus, the increase in SPP is not only a consequence of tannin−anthocyanin adduct formation but might also be explained by formation of pyranoanthocyanins. While there was no correlation between SPP and the contents of pyranoanthocyanins (R2 = 0.0003), TPP correlated well with the amount of pigmented polymers quantified by UHPLCDAD (R2 = 0.836 directly after fermentation and R2 = 0.991 after 12 weeks). In conclusion, the phenolic profile of bilberry wine was greatly influenced by the applied fermentation strategy. Different enological techniques resulted in considerably altered concentrations of anthocyanins, tannins, and small and large polymeric pigments. Especially a prefermentative heat treatment affected the characteristics of the wine. These wines were characterized by a significantly higher anthocyanin concentration (approximately 1000 mg/L) than those that were produced using other fermentation strategies. Apart from the differences in the total anthocyanin concentration, the anthocyanin profiles varied significantly. All wines not thermally treated prior to fermentation exhibited a great reduction of galactosides and arabinosides during fermentation, whereas the thermally treated wines showed roughly the same composition as the juice. These observations can be ascribed to an

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +49 228 73 4462. Fax: +49 228 73 4429. ORCID

Fabian Weber: 0000-0003-0830-6461 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully thank Haus Rabenhorst O. Lauffs GmbH & Co. KG for providing the berry material. ABBREVIATIONS USED ara, arabinoside; AU, absorption units; BSA, bovine serum albumin; CE, catechin equivalents; cy, cyanidin; df, dilution factor; dp, delphinidin; gal, galactoside; glu, glucoside; hex, hexoside; LPP, large polymeric pigments; mv, malvidin; pn, peonidin; pt, petunidin; PPO, polyphenol oxidase; SPP, small polymeric pigments; TPP, total polymeric pigments

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REFERENCES

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