Influence of Different Fermentation Strategies on the Phenolic Profile

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


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


Page 3 of 32


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


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


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


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


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


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


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


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.


Page 11 of 32


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


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


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



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


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.



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


Page 17 of 32


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.



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.


Page 19 of 32


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


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.


Page 21 of 32


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.



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


Page 23 of 32


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


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.)


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


Figure 3



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


Page 27 of 32


GRAPHIC FOR TABLE OF CONTENTS

Figure 5. For Table of Contents Only



247x212mm (300 x 300 DPI)


Page 28 of 32

Page 29 of 32


243x206mm (300 x 300 DPI)



175x147mm (220 x 220 DPI)


Page 30 of 32

Page 31 of 32


288x273mm (300 x 300 DPI)



For Table of Contents only 291x157mm (96 x 96 DPI)


Page 32 of 32

Influence of Different Fermentation Strategies on the Phenolic Profile

Recommend Documents