Subscriber access provided by UNIVERSITY OF THE SUNSHINE COAST
Article
Influence of Different Fermentation Strategies on the Phenolic Profile of Bilberry Wine (Vaccinium myrtillus L.) Annika Behrends, and Fabian Weber J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02268 • Publication Date (Web): 27 Jul 2017 Downloaded from http://pubs.acs.org on August 1, 2017
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 32
Journal of Agricultural and Food Chemistry
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 *Corresponding author (email:
[email protected], Tel.: +49 228 73 4462; Fax: +49 228 73 4429)
1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 2 of 32
1
ABSTRACT
2
Polyphenol rich and especially anthocyanin rich berries like bilberries (Vaccinium myrtillus L.) and
3
derived products such as wine have enjoyed increasing popularity. During winemaking and aging, the
4
phenolic profile undergoes distinct changes – a phenomenon which has been well investigated in grape
5
wine but not in bilberry wine. The present study determined the influence of different fermentation
6
strategies including various pre- and postfermentative heating and cooling concepts on the phenolic
7
profile of bilberry wine. Besides significant differences in total anthocyanin and tannin concentrations,
8
the different fermentation strategies resulted in distinguishable anthocyanin profiles. A very fast aging
9
manifested by a rapid decrease in monomeric anthocyanins of up to 98 % during a 12 week storage and
10
a coincident formation of polymeric pigments and pyranoanthocyanins was observed. Several well-
11
known processes associated to production and aging of wine were much more pronounced in bilberry
12
wine compared to grape wine.
13
KEYWORDS:
14
Vaccinium myrtillus, bilberry, phenolics, anthocyanins, wine, fermentation, polymeric pigments
2 ACS Paragon Plus Environment
Page 3 of 32
Journal of Agricultural and Food Chemistry
15
INTRODUCTION
16
Among many other berries, bilberries (Vaccinium myrtillus L.) and derived products have enjoyed an
17
increasing popularity due to their appealing taste and high amounts of secondary plant metabolites.
18
Regular consumption of berries is associated with numerous health benefits attributed to the presence
19
of a wide range of polyphenols.1-4 Bilberries are especially rich in anthocyanins, which account for
20
contents between 296 g / 100 g and 450 mg / 100 g fresh weight.5,6 The anthocyanin profile of
21
bilberries is composed of 15 anthocyanins including 3-O-glucosides, 3-O-galactosides and 3-O-
22
arabinosides of the 5 anthocyanidin aglycones delphinidin (dp), cyanidin (cy), peonidin (pn), petunidin
23
(pt) and malvidin (mn).7,8 Bilberries also contain a relatively large amount of proanthocyanidins
24
(148 mg / 100 g fresh weight)9, which are responsible for the perception of astringency because of their
25
ability to form complexes with salivary proteins.10 Processing of berry fruits into jams, juices, and wine
26
is a common practice to circumvent problems associated with the short shelf-life of the fresh crops.11
27
Fermentation has been shown in numerous studies to increase the release of phenolic compounds and
28
considerably change the polyphenol profile. These changes of the phenolic profile during winemaking
29
and aging are one major focus of research on grape wine. During fermentation, extraction of
30
anthocyanins can be more or less enhanced depending on the fermentation strategy12. Once extracted,
31
their levels decrease during aging since anthocyanins are involved in distinct reactions with other wine
32
constituents
33
Pyranoanthocyanins bear a second pyran ring formed by the addition of compounds with a polarizable
34
double bond to the genuine anthocyanins. Pigmented polymers are formed by incorporation of
35
anthocyanins into tannic material by numerous pathways leading to undefined structures.13
leading
to
the
formation
of
pyranoanthocyanins
and
pigmented
polymers.
36
Today, the use of fruits other than grapes for the production of wine is quite common. Besides
37
apples, elderberries, cherries, peaches, and many other fruits, blueberries and bilberries are also used.11
38
Especially in the USA and Canada, the production of wine from blueberries has a great economic 3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 4 of 32
39
relevance. Vaccinium corymbosum, Vaccinium ashei and Vaccinium angustifolium are the species
40
mainly used for winemaking in these countries.14-16 Corresponding wines have been investigated
41
regarding phenolic compounds and antioxidative capacity. The antioxidant capacity of Canadian
42
blueberry wine was shown to be much higher compared to the initial juice.14 According to the authors,
43
this effect is attributed to both an increase in the phenolic content and changes in the phenolic profile.
44
Others highlighted the high antioxidative capacity of American blueberry wine compared to
45
conventional red wine.15,16
46
To our knowledge, no research has been conducted on the changes of the phenolic profile during
47
production of wine from European bilberry. Accordingly, there is a need to further evaluate the
48
suitability of European bilberry for wine production and the influence of processing. The objective of
49
this study was to investigate the effects of different fermentation strategies commonly applied in grape
50
wine production on the phenolic profile during the production of wine from bilberries and to determine
51
changes in characteristic parameters during storage.
52
MATERIAL AND METHODS
53
Samples. Frozen bilberries (Vaccinium myrtillus) organically grown and harvested in Romania in
54
2015 were provided by Haus Rabenhorst O. Lauffs GmbH & Co. KG (Unkel, Germany). The berries
55
were frozen for approx. 10 months.
56
Chemicals. De-ionized water was obtained from a Purelab Flex 2 system (Veolia Water Solutions &
57
Technologies, Berlin, Germany). Acetonitrile (HPLC gradient grade; ≥ 99.9 %), water for LC-MS, and
58
sodium chloride were from Th. Geyer (Renningen, Germany). Formic acid (≥ 99.5 %) was purchased
59
from Fisher Scientific (Geel, Belgium). Delphinidin-3-O-glucoside (98 %) was from Phytolab GmbH
60
& Co. KG (Vestenbergsgreuth, Germany). Methyl cellulose was obtained from DOW (Schwalbach/Ts.,
61
Germany) and ammonium sulfate was from AppliChem (Darmstadt, Germany). (+)-Catechin was
62
purchased from Carl Roth GmbH & Co. KG (Karlsruhe, Germany). Acetic acid was obtained from 4 ACS Paragon Plus Environment
Page 5 of 32
Journal of Agricultural and Food Chemistry
63
VWR International GmbH (Darmstadt, Germany), sodium hydroxide from Acros Organics (Geel,
64
Belgium) and sodium metabisulfite from Sigma-Aldrich (Steinheim, Germany). Bovine serum albumin
65
(BSA) was from Merck (Darmstadt, Germany).
66
Bilberry Wine Processing. Bilberries (2 kg for each wine trial) were defrosted for 4 hours at room
67
temperature and then manually crushed. Further treatment depended on the different fermentation
68
strategies summarized in Table 1. No additional treatment was applied to the wines which were
69
processed by fermentation with and without skin contact (SC and NSC). Cold soak entailed maceration
70
during 5 days at 4 °C. These wines were subsequently fermented with (CSSC) or without (CSNSC)
71
skin contact. Thermovinification was conducted on two wines for 2 h at 55 °C or 3 min at 70 °C
72
(T55NSC and T70NSC), respectively. Sample SCT45 underwent a thermal treatment after
73
fermentation for 24 h at 45 °C. Oak chips (6.6 cm x 0.9 cm x 0.9 cm) were added after fermentation to
74
samples SC+OC and NSC+OC and were stored for 12 days. Pressing was done with a Para-Press (Paul
75
Arauner GmbH & Co. KG, Kitzingen, Germany). The wines were fermented at 25 °C with the addition
76
of sucrose (120 g / kg bilberries), nutritional supplement Go-Ferm Protect (1 g / g yeast; Lallemand,
77
Schwarzenbach an der Saale, Germany) and yeast (Saccharomyces cerevisiae; 1 g / kg bilberries;
78
Oenoferm X-treme, Erbslöh Geisenheim AG, Geisenheim, Germany) until they reached a constant
79
alcohol content (between 11.2 and 13.5 %, depending on fermentation strategy) Wines were
80
centrifuged twice at 5400 g for 15 min (Model J2-21 Centrifuge Beckman Coulter GmbH, Krefeld,
81
Germany). Sodium metabisulfite (0.13 g/L) was added. All wines was elaborated in duplicate, yielding
82
a total number of 18 wines. Wines were stored for 12 weeks at 22 °C.
83
Identification and Quantification of Anthocyanins and Anthocyanin-Derived Pigments. For
84
anthocyanin identification and quantification, UHPLC-DAD-ESI-MSn analysis was used. The UHPLC
85
analyses were performed on a Waters Acquity i-Class instrument (Waters, Eschborn, Germany)
86
equipped with a binary pump, a diode-array detector, an autosampler (cooled to 7 °C, injecting 5 µL) 5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 6 of 32
87
and a column oven (at 40 °C). The column was a HSS T3 (1.8 µm, 2.1 x 150 mm; Waters, Eschborn,
88
Germany), equipped with a security guard cartridge of the same material (1.8 µm, 2.1 x 5 mm). Eluent
89
A was water/formic acid (97:3 mL/mL) and eluent B was acetonitrile/formic acid (97:3 mL/mL). A
90
gradient elution program at a flow rate of 0.4 mL/min was used as follows (min/%B): 0/4, 7/8, 13/10,
91
19/17, 23/30, 26/40, 29/100, 32/100, 33/4, 35/4. Anthocyanins, pyranoanthocyanins and pigmented
92
polymers (represented by the chromatographic hump) were detected at 520 nm. They were quantified
93
as delphinidin-3-O-glucoside equivalents. For specific retention times see Figure 3. Although co-
94
elution of procyanidins, oligomeric pigments, and the pigmented polymers can be assumed,
95
quantification of pigmented polymers by UHPLC-DAD can be considered as a crude method that
96
yields basic information on the development of the polymeric material in the wines.
97
For LC-MS analysis, a LTQ-XL ion trap mass spectrometer (Thermo Fisher Scientific, Schwerte,
98
Germany) was connected to the UHPLC system via an ESI interface. The analyses were performed as a
99
full scan in positive ionization mode. Helium was used as the collision gas. The following detection
100
parameters were used: sheath gas (N2), 70 arbitrary units; aux gas (N2), 10 units; sweep gas (N2),
101
1 unit; ion spray voltage, 4 kV; capillary temperature, 325 °C; capillary voltage, 14 V; tube lens, 55 V;
102
collision energy, 35 V.
103
Methyl Cellulose Precipitable (MCP) Tannin Assay. The assay was modified based on
104
Mercurio and Smith.17 Wine was filtered and diluted (dilution factor (df) 2) with water. Aliquots of
105
25 µL of the diluted wine were transferred to two 1.5 mL microfuge tubes. To the first tube (sample),
106
300 µL methyl cellulose solution (0.04 % w/v) was added and to the second tube (control), 300 µL
107
water was added. After 3 min, both tubes were vortexed and 200 µL saturated ammonium sulfate
108
solution and 475 µL water were added to both sample and control. After mixing and incubation for
109
10 min at room temperature, both tubes were centrifuged for 5 min at 11000 g (microcentrifuge
110
Heraeus Pico 17, Thermo Fisher Scientific, Schwerte, Germany). The supernatant was transferred into 6 ACS Paragon Plus Environment
Page 7 of 32
Journal of Agricultural and Food Chemistry
111
a UV cuvette and the absorbance was read at 280 nm using a Genesys 6 spectrophotometer (Thermo
112
Fisher Scientific, Schwerte, Germany). The reading resulting from (control - sample) is the amount of
113
tannins precipitable by methyl cellulose. All measurements were performed in triplicate. Tannins were
114
quantified as (+)-catechin equivalents (CE).
115
Adams-Harbertson (AH) Tannin Assay. The assay was performed according to Harbertson et al.
116
with slight modifications.18 The following solutions were used for the analysis: buffer solution
117
(9.86 g/L sodium chloride in 1.2 % acetic acid, pH 4.9), bovine serum albumin (BSA) solution (1 g/L
118
BSA in buffer solution), bleaching solution (31.6 g/L sodium metabisulfite in water). Wine was filtered
119
and diluted (df 10) with water. Aliquots of 500 µL of the diluted wine were transferred to two 1.5 mL
120
microfuge tubes. To the first tube, 1 mL buffer solution and 120 µL bleaching solution were added.
121
After mixing and incubation for 10 min at room temperature, the mixture was transferred into a cuvette
122
and the absorbance was read at 520 nm (reading B). To the second tube, 1 mL BSA solution was
123
added. The mixture was incubated at room temperature for 15 min with occasional upending. The
124
sample was then centrifuged for 5 minutes at 14300 g. Aliquots of 1 mL of the supernatant were
125
transferred into a cuvette, 80 µL bleaching solution was added, and the absorbance was read at 520 nm
126
after mixing and incubation for 10 min (reading C). The absorbance due to total polymeric pigments
127
(TPP), small polymeric pigments (SPP) and large polymeric pigments (LPP) is given as B, C and (B-
128
C), respectively. All measurements were performed in triplicate and results are presented in AU.
129
Statistical Analysis. To determine significant differences, an ANOVA with Bonferroni post-hoc
130
test (significance level α = 0.05) was performed using the software XLSTAT (Addinsoft, Paris,
131
France).
132
RESULTS AND DISCUSSION
133
Anthocyanins and Anthocyanin-Derived Pigments. Anthocyanins and derived pigments play an
134
important role for the quality of bilberry wine and thus their evolution was determined with respect to 7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 32
135
the different fermentation strategies. Anthocyanins and their derivatives were identified according to
136
their order of elution8 and their specific fragmentation pattern.19-21 Total anthocyanin content is
137
expressed as the sum of individual anthocyanins. The anthocyanin concentration before and
138
immediately after fermentation as well as after 12 weeks storage time is shown in Figure 1. The results
139
show that different prefermentative treatments caused differences in anthocyanin concentration prior to
140
fermentation. The thermally treated juices (1823.1 mg/L, T55NSC and 1780.1 mg/L, T70NSC) had
141
highest anthocyanin concentrations. Juices subjected to prefermentative cold soak showed lowest
142
anthocyanin concentrations (866.3 mg/L, CSSC and 696.0 mg/L, CSNSC). Anthocyanin concentration
143
of the juices with cold soak was expected to be higher due to the prolonged maceration that should
144
have caused an enhanced anthocyanin extraction.12,22 Oxidation or polymerization reactions between
145
anthocyanins and other wine constituents might explain this considerable loss of monomeric
146
anthocyanins. Anthocyanin concentrations of the juices that were neither thermally treated before
147
fermentation nor subjected to cold soak ranged from 1143.9 mg/L (SC and SC+OC) to 1407. 9 mg/L
148
(SCT45). The differences observed before fermentation between the samples that were treated in the
149
same way might be explained by the influence of pressing and mashing.
150
All wines had significantly decreased anthocyanin concentration after fermentation compared to the
151
concentration of the juice, with losses ranging from 32.2 % to 61.3 %. Thermally treated wines
152
generally showed twofold to threefold higher anthocyanin contents than wines not subjected to thermal
153
treatment. Thermovinification leads to wines with higher amounts of anthocyanins, which might be
154
attributed to inactivation of fruit-borne anthocyanin degrading enzymes like polyphenol oxidase (PPO)
155
and endogenous glycosidases (arabinosidases and galactosidases). Inactivation of PPO prevents the
156
oxidation of other phenolic compounds to reactive quinones, which may subsequently oxidize
157
anthocyanins23. Inactivation of endogenous arabinosidase and galactosidase enzymes also prevents
158
degradation of anthocyanins because their activity results in hydrolysis of anthocyanins leading to their 8 ACS Paragon Plus Environment
Page 9 of 32
Journal of Agricultural and Food Chemistry
159
unstable aglycones.24,25 Aglycones of dp and cy were detected in concentrations between 4 and 30
160
mg/L dp-3-glu equivalents in all wines that were not thermally treated.
161
The decrease in anthocyanins during fermentation of wines without thermal treatment can be
162
explained by enzymatic and chemical processes, while the degradation in thermally treated wines is
163
exclusively caused by chemical processes. Sample SCT45, which was heated after fermentation,
164
however displayed higher anthocyanin contents. The detrimental activity of enzymes during the early
165
stages of fermentation was outbalanced by an enhanced extraction of anthocyanins due to the long heat
166
exposure.26 Anthocyanin concentrations of all wines decreased significantly during 12 weeks storage.
167
The losses ranged from 72.6 % to 97.6 %. Wines heated before fermentation showed the highest
168
retention. The loss of monomeric anthocyanins during storage cannot be explained by enzymatic
169
degradation because PPO and endogenous glycosidases are inhibited by the increasing amount of
170
ethanol and by the addition of sodium metabisulfite.27 The loss during storage is rather a result of
171
chemical degradation or derivatization caused by condensation reactions and formation of polymeric
172
pigments.28
173
The concentration of the pigmented polymers quantified as the chromatographic hump which
174
emerged during fermentation and storage is shown in Figure 2. This polymeric hump was not detected
175
before fermentation in any of the different wines. Directly after fermentation, the concentrations ranged
176
from 92.7 mg/L (CSNSC) to 357.3 mg/L (T55NSC). Analogous to anthocyanin concentration, the
177
thermally treated wines had highest amounts of pigmented polymers. There is a strong correlation
178
(R2 = 0.916) between the concentration of pigmented polymers formed during fermentation and the
179
concentration of anthocyanins before fermentation. The concentration of pigmented polymers increased
180
significantly during storage in the two wines which were thermally treated before fermentation
181
(51.2 %, T55NSC and 74.9 %, T70NSC). The wine treated with prefermentative cold soak and
182
fermented without skin contact (CSNSC) also showed a great increase in pigmented polymers 9 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 10 of 32
183
(44.3 %). The other wines displayed inconsistent results ranging from -11.4 % (SC+OC) to 23.2 %
184
(CSSC). Concentration of pigmented polymers showed a maximum after 6 to 9 weeks (data not
185
shown). The increase in pigmented polymers in wine is caused by condensation reactions between
186
anthocyanins with other anthocyanins, proanthocyanidins, or flavanols.22,29,30 These condensations may
187
be direct or mediated by acetaldehyde, which is formed during fermentation in the yeasts’
188
metabolism.31 The decrease in pigmented polymers at the end of storage might be attributed to an
189
insufficient chromatographic separation because the hump of pigmented polymers in UHPLC analysis
190
gets broad and ill-defined during storage, which leads to integration problems of the chromatographic
191
area. A more precise quantification may be achieved by separation via a mixed-mode-phase column.32
192
Slight formation of a dark-red colored sediment was observed after 12 weeks, which might contribute
193
to the decrease in pigmented polymers. Detailed information on the molecular size and their solubility
194
could confirm this.
195
The observed decrease in monomeric anthocyanins and the corresponding increase in pigmented
196
polymers in bilberry wines can be compared with the changes in conventional red wines.29 However,
197
the time frame is considerably different. Red wine shows a discernible decrease in anthocyanins not
198
before 13 months of storage29, whereas bilberry wine lost up to 98 % of the initial anthocyanins already
199
after 12 weeks. Additionally, very slight color changes of the anthocyanin rich and therefore dark violet
200
colored wines to more brownish hues were observed during 12 weeks storage. Bilberry wine obviously
201
undergoes a very fast aging. The low pH value supposedly influences the chemical reactions during
202
fermentation and storage. The pH-value of the initial bilberry juice was 2.9 and of the processed
203
bilberry wines was 3.1, whereas commercial red wines commonly show a pH of 3.6.33 Presumably,
204
aging of bilberry wines might be deferred by increasing the pH-value by means of a suitable
205
deacidification.
10 ACS Paragon Plus Environment
Page 11 of 32
Journal of Agricultural and Food Chemistry
206
Anthocyanin Profile. Apart from the total amount, the composition of the anthocyanin profile
207
changed during fermentation and storage. Figure 3 shows the chromatograms obtained for bilberry
208
wine (SC) during elaboration and storage. A considerable loss of arabinosides and galactosides was
209
observed during fermentation. After 12 weeks of storage, almost exclusively glucosides were detected.
210
Degradation of anthocyanins by endogenous glycosidases was proven by formation of two aglycones
211
(dp and cy), which diminished later during storage. Further alteration of the anthocyanin profile was
212
manifested in the formation of anthocyanin derivatives like pyranoanthocyanins and increasing
213
proportion of pigmented polymers.
214
Figure 4 shows the relative concentration of glucosides, galactosides, and arabinosides in bilberry
215
wines compared to the initial bilberry juice. The juice was composed of approx. 50 % glucosides, 30 %
216
galactosides and 20 % arabinosides. The anthocyanin profile was not changed by any prefermentative
217
treatment. The anthocyanin profiles of all bilberry wines were identical at the beginning of
218
fermentation. During fermentation considerable changes took place. The two wines which underwent
219
prefermentative thermal treatment showed an almost juice-like composition, whereas all other wines
220
displayed losses of arabinosides and galactosides. They were composed of about 8 % arabinosides,
221
8 % galactosides, and 85 % glucosides at the end of fermentation.
222
As already mentioned, the activity of endogenous glycosidases plays an important role in changes of
223
the anthocyanin profile. The thermally treated wines do not show any effects of enzymatic degradation
224
during fermentation because enzymes were inactivated. The other wines containing active enzymes at
225
the early phase of fermentation show great losses of arabinosides and galactosides, which can be
226
ascribed to a different substrate specificity of the endogenous glycosidases. Buchert et al. showed that
227
enzyme-assisted bilberry juice production with commercially available pectinases results in greater
228
losses of galactosides but not glucosides.34 The fact that the anthocyanin content decreased
229
significantly during storage, whereas the composition of glucosides, arabinosides and galactosides 11 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 12 of 32
230
remained constant (data not shown) can be explained by a consistent anthocyanin decrease, caused by
231
non-specific chemical reactions. These follow first-order kinetics35 resulting in an exponential decrease
232
Pyranoanthocyanins. The two wines which were thermally treated prior to fermentation showed
233
significantly higher amounts of pyranoanthocyanins (Table 2). Because they also contained much more
234
total anthocyanins (Figure 1), this seems plausible. In comparison to commercial red wine, formation
235
of pyranoanthocyanins in bilberry wine proceeded quite fast. 29,35,36 According to Alcalde-Eon et al.
236
who investigated the pigment composition of red wine during maturity and aging, A-type vitisins were
237
detectable after 8 to 13 months.29 The first A-type vitisins in bilberry wine were already detected in the
238
third week. The elevated storage temperature (22 °C) and the lower pH might be the reason for this fast
239
formation.37
240
Tannins. Tannin concentration was determined using the MCP tannin assay and quantified as
241
catechin equivalents. Wines which were fermented with skin contact showed higher tannin
242
concentrations than those fermented without skin contact. The former had tannin concentrations
243
between 1147 mg/L (SC) and 2005 mg/L (SCT45), the latter between 592 mg/L (T70NSC) and
244
1017 mg/L (NSC+OC) (Table 2). The juice contained 381 mg/L tannins. T55NSC and SCT45 showed
245
the highest tannin concentrations. The different amounts of tannins can be reasoned by different
246
extraction kinetics due to the fermentation strategy. Prolonged contact time with the tannin-rich skins
247
and kernels and increasing ethanol content enhances the extraction of tannins. The purpose of
248
thermovinification and the associated aqueous-thermal extraction procedure is to extract more
249
anthocyanins than tannins which leads to more vividly colored and less astringent wines. Heating
250
causes a rapid destruction of cells and, thus, accelerates extraction of water-soluble anthocyanins.12
251
Accordingly, thermally treated wine fermented without skin contact (T70NSC) showed low tannin
252
concentrations and high anthocyanin concentrations. The wine that was heated at 55 °C (T55NSC),
253
however, displayed very high tannin concentrations. Slow heating up to 55 °C might lead to 12 ACS Paragon Plus Environment
Page 13 of 32
Journal of Agricultural and Food Chemistry
254
temporarily elevated enzyme activities, resulting in cell wall decomposition and consequently to higher
255
tannin contents after pressing. Tannin cell wall binding occurs through hydrogen bonding and through
256
hydrophobic interactions between tannins and polysaccharides. This leads to an inclusion of tannins in
257
the complex cell wall network and therefore to a reduced tannin extraction from berry into wine during
258
maceration.12,38
259
All wines showed increasing tannin amounts within the first 6 to 9 weeks of storage followed by
260
decreasing concentrations (data not shown). The MCP assay does not distinguish between tannins of
261
different degrees of polymerization. High amounts of oligomeric compounds would lead to similar
262
results as low amounts of high polymeric compounds. Hence, the results do not allow conclusions
263
whether storage leads to the formation of larger polymers or to degradation to smaller oligomers.
264
Tannins’ structural diversity renders it nearly impossible to determine the exact concentration.
265
Polymeric Pigments. Polymeric pigments were determined using the AH tannin assay. This assay
266
allows the differentiation between small (SPP) and large polymeric pigments (LPP), whereby SPP have
267
a degree of polymerization between 2 and 4.
268
fermentation, all wines contained only SPP and no LPP, which were formed only during storage. The
269
juice contained lowest amount of SPP, whereas thermal treatment resulted in high amounts. Except for
270
CSNSC, all bilberry wines showed significantly higher amounts of polymeric pigments than pure juice.
271
The formation is based on condensation reactions of anthocyanins with other anthocyanins, flavanols,
272
or proanthocyanidins22,29,30 and via the reaction with acetaldehyde.31 The composition of monomers is
273
also an important factor that influences the formation of polymeric pigments.28 This might explain the
274
different formation kinetics in the wines after cold soak. Apparently, cold soak led to flavanol-to-
275
anthocyanin ratios less favorable for the formation of stable condensation products.40,41 Like the MCP
276
assay, the AH assay does not permit exact conclusions on polymer composition.
17,39
The results are given in Table 2. Directly after
13 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 14 of 32
277
During storage, a continuous increase in SPP and a slight increase in LPP can be observed after 9
278
weeks, whereas T55NSC and SCT45 showed a much stronger increase within the first 3 to 6 weeks
279
(data not shown). A simultaneous decrease in monomeric anthocyanins suggests the incorporation of
280
anthocyanins into stable polymeric pigments. The strong increase in LPP in T55NSC and SCT45 can
281
be explained by an enhanced polymerization due to the long heat exposure.42 According to the results
282
obtained by the MCP assay, these two wines exhibited the highest amounts of tannins. This led to an
283
increased formation of LPP due to an increased availability of reactants for anthocyanins to form
284
colored anthocyanin-tannin adducts.43 According to Harbertson et al., the SPP fraction contains vitisins
285
characterized by a low molecular weight.18 T55NSC and T70NSC exhibit the highest amounts of SPP
286
and the highest amounts of vitisin-type pyranoanthocyanins after 12 weeks. Thus, the increase in SPP
287
is not only a consequence of tannin-anthocyanin adduct formation but might also be explained by
288
formation of pyranoanthocyanins. While there was only a weak correlation between SPP and the
289
contents of pyranoanthocyanins (R²=0.397), TPP correlated well with the amount of pigmented
290
polymers quantified by UHPLC-DAD (R² = 0.836 directly after fermentation and R² = 0.991 after 12
291
weeks).
292
In conclusion, the phenolic profile of bilberry wine was greatly influenced by the applied
293
fermentation strategy. Different enological techniques resulted in considerably altered concentrations
294
of anthocyanins, tannins and small and large polymeric pigments. Especially a prefermentative heat
295
treatment affected the characteristics of the wine. These wines were characterized by a significantly
296
higher anthocyanin concentration (approx. 1000 mg/L) than those which were produced using other
297
fermentation strategies. Apart from the differences in the total anthocyanin concentration, the
298
anthocyanin profiles varied significantly. All wines not thermally treated prior to fermentation
299
exhibited a great reduction of galactosides and arabinosides during fermentation, whereas the thermally
300
treated wines showed roughly the same composition like the juice. These observations can be ascribed 14 ACS Paragon Plus Environment
Page 15 of 32
Journal of Agricultural and Food Chemistry
301
to an inactivation of PPO and of glycosidases naturally occurring in bilberries. Contrary to the
302
expectations, a prefermentative cold soak treatment resulted in lower anthocyanin concentrations
303
compared to traditional grape wine, which might also be attributed to residual enzyme activities or non-
304
enzymatic reactions of the anthocyanins. Wines stored with oak chips after fermentation did not show
305
any significant differences in comparison to the corresponding control. Skin contact during
306
fermentation did not affect polymeric pigment composition in wines not thermally treated, whereas
307
tannin concentration was higher in wines fermented with skin contact due to the prolonged contact time
308
with the tannin-rich skins and kernels.
309
The investigation of the influence of storage on the phenolic profile revealed that bilberry wine
310
undergoes a very fast aging irrespective of the applied fermentation strategy. Thus, the total
311
anthocyanin content was reduced to about 2 % of the original amount after 12 weeks and polymeric
312
pigments as well as pyranoanthocyanins were formed alongside. The low pH-value is assumed to be
313
the main reason for the fast aging. A strategy to increase the pH-value by means of a suitable
314
deacidification might delay early aging. Malolactic fermentation is commonly used in red wine
315
production and has tremendous effects on wine composition. Especially, the consumption of excess
316
acetaldehyde by the applied microorganisms might lead to slower pigment aging. In comparison with
317
grape wine, several well-known processes like specific degradation of anthocyanin glucosides,
318
polymerization reactions, or enzyme inactivation are clearly observable in this study. Further
319
investigation of the process of bilberry wine production and aging may assist to get new insights also in
320
grape wine.
321
ACKNOWLEDGMENTS
322
The authors gratefully thank Haus Rabenhorst O. Lauffs GmbH & Co. KG for providing the berry
323
material.
15 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 16 of 32
324
ABBREVIATIONS USED
325
ara, arabinoside; AU, absorption units; BSA, bovine serum albumin; CE, catechin equivalents; cy,
326
cyanidin; df, dilution factor; dp, delphinidin; gal, galactoside; glu, glucoside; hex, hexoside; LPP, large
327
polymeric pigments; mv, malvidin; pn, peonidin; pt, petunidin; PPO, polyphenol oxidase; SPP, small
328
polymeric pigments; TPP, total polymeric pigments
16 ACS Paragon Plus Environment
Page 17 of 32
Journal of Agricultural and Food Chemistry
329
REFERENCES
330
(1) Kalt, W.; McDonald, J. E.; Donner, H. Anthocyanins, phenolics, and antioxidant capacity of
331
processed lowbush blueberry products. J. Food Sci. 2000, 65, 390–393.
332
(2) Katsube, N.; Iwashita. K.; Tsushida, T.; Yamaki, K.; Kobori, M. Induction of apoptosis in cancer
333
cells by bilberry (Vaccinium myrtillus) and the anthocyanins. J. Agric. Food Chem. 2003, 51, 68–
334
75.
335
(3) Faria, A.; Oliveira, J.; Neves, P.; Gameiro, P.; Santos-Buelga, C.; de Freitas, V.; Mateus, N.
336
Antioxidant properties of prepared blueberry (Vaccinium myrtillus) extracts. J. Agric. Food Chem.
337
2005, 53, 6896–6902.
338
(4) Zafra-Stone, S.; Yasmin, T.; Bagchi, M.; Chatterjee, A.; Vinson, J. A.; Bagshi, D. Berry
339
anthocyanins as novel antioxidants in human health and disease prevention. Mol. Nutr. Food Res.
340
2007, 51, 675–683.
341 342
(5) Rimpapa, Z.; Toromanovic, J.; Tahirovic, I.; Šapčanin, A.; Sofic, E. Total content of phenols and anthocyanins in edible fruits from bosnia. Bosn. J. Basic Med. Sci. 2007, 7 (2), 119–122.
343
(6) Aaby, K.; Grimmer, S.; Holtung, L. (2013). Extraction of phenolic compounds from bilberry
344
(Vaccinium myrtillus L.) press residue: Effects on phenolic composition and cell proliferation.
345
LWT Food Sci. Technol. 2013, 55, 257–264.
346 347
(7) Kähkönen, M. P.; Heinämäki, J.; Ollilainen, V.; Heinonen, M. Berry anthocyanins: isolation, identification and antioxidant activities. J. Sci. Food Agric. 2003, 83, 1403–1411.
348
(8) Heffels, P.; Weber, F.; Schieber, A. Influence of accelerated solvent extraction and ultrasound-
349
assisted extraction on the anthocyanin profile of different Vaccinium species in the context of
350
statistical models for authentication. J. Agric. Food Chem. 2015, 63, 7532– 7538.
17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
351 352 353 354 355 356 357 358
Page 18 of 32
(9) Hellström, J. K.; Törrönen, A. R.; Mattila, P. H. Proanthocyanidins in common food products of plant origin. J. Agric. Food Chem. 2009, 57, 7899–7906. (10) Manach, C.; Scalbert, A.; Morand, C.; Rémésy, C.; Jiménez, L. Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 2004, 79, 727–747. (11) Jagtap, U. B.; Bapat, V. A. Wines from fruits other than grapes: Current status and future prospectus. Food Biosci. 2015, 9, 80 – 96. (12) Ribéreau-Gayon, P.; Dubourdieu, D.; Donèche, D.; Lonvaud, A. Handbook of Enology. Volume 1: The Microbiology of Wine and Vinifications, 2nd edition; Wiley & Sons, Chichester, UK, 2006.
359
(13) Dipalmo, T.; Crupi, P.; Pati, S.; Clodoveo, M. L.; Di Luccia, A. Studying the evolution of
360
anthocyanin-derived pigments in a typical red wine of southern Italy to assess its resistance to
361
aging. LWT Food Sci. Technol. 2016, 71, 1–9.
362
(14) Martin, L. J.; Matar, C. Increase of antioxidant capacity of the lowbush blueberry (Vaccinium
363
angustifolium) during fermentation by a novel bacterium from the fruit microflora. J. Sci. Food
364
Agric. 2005, 85, 1477 – 1484.
365
(15) Johnson, M. H. & Gonzalez de Mejia, E. Comparison of chemical composition and antioxidant
366
capacity of commercial available blueberry and blackberry wines in Illinois. J. Food Sci. 2012, 71
367
(1), C141 – C148.
368
(16) Yang, W., Guner, S., Rock, C., Anugu, A., Sims, C. & Gu, L. Prospecting antioxidant capacities
369
and health-enhancing phytonutrient contents of southern highbush blueberry wine compared to
370
grape wines and fruit liquors. Sustainable Agriculture Research 2012, 1 (1), 26 – 35.
371
(17) Mercurio, M. D.; Smith, P. A. Tannin quantification in red grapes and wine: Comparison of
372
polysaccharide- and protein-based tannin precipitation techniques and their ability to model wine
373
astringency. J. Agric. Food Chem. 2008, 56, 5528 – 5537.
18 ACS Paragon Plus Environment
Page 19 of 32
Journal of Agricultural and Food Chemistry
374
(18) Harbertson, J. F.; Picciotto, E. A.; Adams, D. O. Measurement of polymeric pigments in grape
375
berry extracts and wines using a protein precipitation assay combined with bisulfite bleaching.
376
Am. J. Enol. Vitic. 2003, 54, 301 – 306.
377
(19) Wu, X.; Prior, R. L. Systematic identification and characterization of anthocyanins by HPLC-ESI-
378
MS/MS in common foods in the United States: Fruits and berries. J. Agric. Food Chem. 2005, 53,
379
2589 – 2599.
380 381
(20) Zhao, Q.; Duan, C.-Q.; Wang, J. Anthocyanins profile of grape berries of Vitis amurensis, its hybrids and their wines. Int. J. Mol. Sci. 2010, 11, 2212 – 2228.
382
(21) Oliveira, J.; da Silva, M. A.; Teixeira, N.; De Freitas, V.; Salas, E. Screening of anthocyanins and
383
anthocyanin-derived pigments in red wine grape pomace using LC-DAD/MS and MALDI-TOF
384
techniques. J. Agric. Food Chem. 2015, 63, 7636 – 7644.
385
(22) Ribéreau-Gayon, P.; Glories, Y.; Maujean, A.; Dubourdieu, D. Handbook of Enology. Volume 2:
386
The Chemistry of Wine. Stabilization and Treatments, 2nd edition; Wiley & Sons, Chichester, UK,
387
2006.
388
(23) Moreno, J.; Peinado, R. Enological Chemistry, 1st edition; Elsevier, Atlanta, USA, 2012.
389
(24) Arnous, A.; Meyer, A. S. Discriminated release of phenolic substances from red wine grape skins
390
(Vitis vinifera L.) by multicomponent enzymes treatment. Biochem. Eng. J. 2010, 49, 68 – 77.
391
(25) Maier, T.; Göppert, A.; Kammerer, D. R.; Schieber, A; Carle, R. Optimization of a process for
392
enzyme-assisted pigment extraction from grape (Vitis vinifera L.) pomace. Eur. Food Res.
393
Technol. 2008, 227, 267 – 275.
394 395 396 397
(26) Constantin, N. Choisir le bon profil thermique pour mieux gérer la macération en rouge. Guide de la Vinification 2001, 5, 28 – 30. (27) Macheix, J.-J.; Sapis, J.-C.; Fleuriet, A. Phenolic compounds and polyphenoloxidase in relation to browning in grapes and wines. Crit. Rev. Food Sci. Nutr. 1991, 30 (3), 441 – 486. 19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
398 399
Page 20 of 32
(28) Fulcrand, H.; Dueñas, M.; Salas, E.; Cheynier, V. Phenolic reactions during wine-making and aging. Am. J. Enol. Vitic. 2006, 57 (3), 289 – 297.
400
(29) Alcalde-Eon, C.; Escribano-Bailón, M. T.; Santos-Buelga, C.; Rivas-Gonzalo, J. C. Changes in
401
the detailed pigment composition of red wine during maturity and ageing. A comprehensive
402
study. Anal. Chim. Acta 2006, 563, 238 – 254.
403
(30) Calvacanti, R. N.; Santos, D. T.; Meireles, M. A. A. Non-thermal stabilization mechanisms of
404
anthocyanins in model and food systems – An overview. Food Res. Int. 2011, 44, 499 – 509.
405
(31) Weber, F.; Winterhalter, P. Synthesis and structure elucidation of ethyliden-linked anthocyanin-
406
flavan-3-ol oligomers. Food Res. Int. 2014, 65, 69 – 76.
407
(32) Vergara, C.; Mardones. C.; Hermosín-Gutiérrez, I.; von Baer, D. Comparison of high-
408
performance liquid chromatography separation of red wine anthocyanins on a mixed-mode ion-
409
exchange reversed-phase and on a reversed-phase column. J. Chromatogr. A 2010, 1217, 5710 –
410
5717.
411
(33) Avizcuri, J.-M.; Sáenz-Navajas, M.-P.; Echávarri, J.-F., Ferreira, V.; Fernández-Zurbano, P.
412
Evaluation of the impact of initial red wine composition on changes in color and anthocyanin
413
content during bottle storage. Food Chem. 2016, 213, 123 – 134.
414
(34) Buchert, J.; Koponen, J. M.;, Suutarinen, M.; Mustranta, A.; Lille, M.; Törrönen, R.; Poutanen, K.
415
Effect of enzyme-aided pressing on anthocyanin yield and profiles in bilberry and blackcurrant
416
juices. J. Sci. Food Agric. 2005, 85, S. 2548 –2556.
417
(35) Monagas, M.; Gómez-Cordovés, C.; Bartolomé, B. Evolution of polyphenols in red wines from
418
Vitis vinifera L. during aging in the bottle. I. Anthocyanins and pyranoanthocyanins. Eur. Food
419
Res. Technol. 2005, 220, 607 – 614.
20 ACS Paragon Plus Environment
Page 21 of 32
Journal of Agricultural and Food Chemistry
420
(36) Sáenz-Navajas, M.-P.; Echavarri, F.; Ferreira, V.; Fernández-Zurbano, P. Pigment composition
421
and color parameters of commercial Spanish red wine samples: Linkage to quality perception.
422
Eur. Food Res. Technol. 2011, 232, 877 – 887.
423
(37) He, F.; Liang, N.-N.; Mu, L.; Pan, Q.-H.; Wang, J.; Reeves, M. J.; Duan, C.-Q. Anthocyanins and
424
their variation in red wines II. Anthocyanin derived pigments and their color evolution. Molecules
425
2012, 17, 1483 – 1519.
426
(38) Hanlin, R. L.; Hrmova, M.; Harbertson, J. F.; Downey, M. O. Review: Condensed tannin and
427
grape cell wall interactions and their impact on tannin extractability into wine. Aust. J. Grape
428
Wine R. 2010, 16, 173 – 188.
429 430
(39) Landon, J. L.; Weller, K.; Harbertson, J. F.; Ross, C. F. Chemical and sensory evaluation of astringency in Washington State red wines. Am J. Enol. Vitic. 2003, 54, 99 – 104.
431
(40) Durner, D.; Weber, F.; Neddermeyer, J.; Koopmann, K.; Winterhalter, P.; Fischer, U. Sensory and
432
color changes induced by microoxygenation treatments of Pinot noir before and after malolactic
433
fermentation. Am. J. Enol. Vitic. 2010, 61, 474 – 485.
434
(41) Durner, D.; Nickolaus, P.; Weber, F.; Trieu, H.-L.; Fischer, U. Evolution of anthocyanin-derived
435
compounds during micro-oxygenation of red wines with different anthocyanin-flavanol ratios. In
436
Advances in Wine Research; Ebeler, S. B.; Sacks, G.; Vidal, S.; Winterhalter, P., American
437
Chemical Society, 2015, 1203, 253–274.
438
(42) Mansfield, A. K.; Zoecklein B. W. Effect of fermentation, postfermentation, and postbottling heat
439
treatment on Cabernet Sauvignon glycoconjugates. Am. J. Enol. Vitic. 2003, 54 (2), 99 – 104.
440
(43) Villamor, R. R.; Harbertson, J. F.; Ross, C. F. Influence of tannin concentration, storage
441
temperature, and time on chemical and sensory properties of Cabernet Sauvignon and Merlot
442
wines. Am. J. Enol. Vitic. 2009, 60 (4), 442 – 449.
21 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 22 of 32
FIGURE CAPTIONS 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-glucosideequivalents; different letters indicate significant difference (α = 0.05); n = 2 Figure 2. Concentration of pigmented polymers determined by UHPLC-DAD-ESI-MSn after fermentation and after 12 weeks (including realtive changes), calculated as delphinidin-3-O-glucosideequivalents; different letters indicate significant difference (α = 0.05); n = 2 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, (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-3glu, (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-3ara, (I) dp, (II) cy, (i) carboxy-pyrano-del-3-hex, (ii) carboxy-pyrano-pn-3-hex, (iii) carboxy-pyranomv-3-hex; (iv) pyrano-mv-3-hex (B-type vitisin), (v) carboxy-pyrano-pt-3-hex
Figure 4. Relative anthocyanin concentrations of bilberry wines and juice expressed as the sum of arabinosides, galactosides and glucosides after fermentation
22 ACS Paragon Plus Environment
Page 23 of 32
Journal of Agricultural and Food Chemistry
TABLES Table 1. Fermentation strategies of the 9 different elaborated wines
no. abbreviation fermentation strategy 1
SC
fermentation with skin contact
2
NSC
fermentation without skin contact
3
SC+OC
fermentation with skin contact and subsequent storage with oak chips for 12 days
4
NSC+OC
fermentation without skin contact and subsequent storage with oak chips for 12 days
5
CSSC
cold soak at 4 °C for 5 days, fermentation with skin contact
6
CSNSC
cold soak at 4 °C for 5 days, fermentation without skin contact
7
T55NSC
thermovinification at 55 °C for 2 hours, fermentation without skin contact
8
T70NSC
thermovinification at 70 °C for 3 min, fermentation without skin contact
9
SCT45
fermentation with skin contact, heating at 45 °C for 24 hours after fermentation
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 fermentation and after 12 weeks storage (*); different letters indicate significant difference (α = 0.05); n = 2
sample CSSC CSNSC T55NSC T70NSC SCT45 SC SC+OC NSC NSC+OC juice +
pyranoanthocyanins (mg/L dp-3-glu eq.) 12.55 ± 0.49 bc 15.64 ± 0.24 b 35.31 ± 0.12 a 33.67 ± 1.82 a 15.07 ± 0.21 bc 12.26 ± 0.07 c 12.52 ± 0.16 bc 15.42 ± 0.66 bc 15.32 ± 0.66 bc -
tannins (mg/L CE)
SPP+ (AU)
SPP* (AU)
LPP* (AU)
1405 ± 68 abc 661 ± 87 cde 2159 ± 596 a 592 ± 266 cde 2005 ± 166 ab 1147 ± 300 bcd 1522 ± 108abc 767 ± 141 cde 1017 ± 12 bcde 381 ± 126 de
0.074 ± 0.007 ef 0.048 ± 0.001 fg 0.184 ± 0.004 a 0.137 ± 0.019 bc 0.171 ± 0.006 ab 0.088 ± 0.006 de 0.114 ± 0.011 cd 0.074 ± 0.008 ef 0.084 ± 0.012 def 0.015 ± 0.001 g
0.163 ± 0.004 cd 0.138 ± 0.006 de 0.242 ± 0.001 b 0.377 ± 0.010 a 0.128 ± 0.002 e 0.150 ± 0.001 cde 0.147 ± 0.008 cde 0.172 ± 0.005 c 0.174 ± 0.009 c -
0.007 ± 0.001 c 0.0 c 0.179 ± 0.012 a 0.037 ± 0.006 c 0.123 ± 0.012 b 0.032 ± 0.010 c 0.016 ± 0.015 c 0.019 ± 0.020 c 0.012 ± 0.011 c -
no LPP were detected directly after fermentation
* after 12 weeks storage
23 ACS Paragon Plus Environment
ef de - 8.0 %
N SC +O
C
- 58.8 %
- 61.3 %
- 59.5 %
cd c
k
- 94.6 %
- 91.9 %
k
C
bc + 17.3 %
- 97.6 %
k
+O
ef
SC
hi
SC
ef
N
hi
- 11.4 %
- 60.7 %
cd
N
cd - 97.2 %
a
C
b
C
jk
+O
- 42.9 %
- 46.5 %
- 32.2 %
a
NS
bcd
SC
k
- 4.9 %
ef
C
- 87.9 %
- 72.6 %
ij
SC
T4 5
- 74.4 %
fg
SC +O
400 + 13.0 %
500 SC
C
- 60.1 %
de
SC
a + 74.9 %
S
hi
T4 5
T7 0N
ij - 91.6 %
- 39.8 %
bc
SC
600 + 51.2 %
k
T5 5N SC
- 96.9 %
gh
SC
ef + 44.3 %
SC
C
1500
0N
efg
SN
SS
2000
T7
200 C
500
5N SC
300 + 23.2 %
C
0
SC
C ef
SN
SS
anthocyanin concentration (mg/L dp-3-glu eq.) 1000
T5
C
C
concentration of pigmented polymers (mg/L dp-3-glu eq.)
Journal of Agricultural and Food Chemistry
hi
ef de
ACS Paragon Plus Environment
Page 24 of 32
FIGURE GRAPHICS
before fermentation after fermentation after 12 weeks
b c
gh
k
Figure 1
after fermentation after 12 weeks
a
efg
fg ef
100 g
0
Figure 2
24
Page 25 of 32
Journal of Agricultural and Food Chemistry
Figure 3
25 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 26 of 32
100 19.9
7.9
7.9
8.3
10.2
19.9
20.0
31.2
32.1
6.6
7.2
7.1
7.2
8.3
7.8
6.5
6.4
8.3
8.1
85.6
86.3
86.5
84.5
83.6
anthocyanin concentration (%)
80
30.0 60
83.8
81.8
40 48.8 47.9
50.1 20
arabinosides
galactosides
+O C
N
SC
C
NS C
SC
+O SC
SC SC T4 5
T7
0N
SC
SC
5N
SN
T5
CS SC
C
ju
ice
0
glucosides
Figure 4
26 ACS Paragon Plus Environment
Page 27 of 32
Journal of Agricultural and Food Chemistry
GRAPHIC FOR TABLE OF CONTENTS
Figure 5. For Table of Contents Only
27 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
247x212mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 28 of 32
Page 29 of 32
Journal of Agricultural and Food Chemistry
243x206mm (300 x 300 DPI)
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
175x147mm (220 x 220 DPI)
ACS Paragon Plus Environment
Page 30 of 32
Page 31 of 32
Journal of Agricultural and Food Chemistry
288x273mm (300 x 300 DPI)
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
For Table of Contents only 291x157mm (96 x 96 DPI)
ACS Paragon Plus Environment
Page 32 of 32