Thermal and Photochemical Stability of Anthocyanins from Black

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Food and Beverage Chemistry/Biochemistry

Thermal and Photochemical Stability of Anthocyanins from Black Carrot, Grape Juice and Purple Sweet Potato in Model Beverages in Presence of Ascorbic Acid Violaine Gérard, Emel Ay, Fabrice Morlet-Savary, Bernadette Graff, Christophe Claude Galopin, Thaddao Ogren, William Mutilangi, and Jacques Lalevée J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01672 • Publication Date (Web): 26 Apr 2019 Downloaded from http://pubs.acs.org on April 28, 2019

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Journal of Agricultural and Food Chemistry

Thermal and Photochemical Stability of Anthocyanins from Black Carrot, Grape Juice and Purple Sweet Potato in Model Beverages in Presence of Ascorbic Acid

Violaine Gérard†,‡, Emel Ay†,‡, Fabrice Morlet-Savary†,‡, Bernadette Graff†,‡, Christophe Galopin§, , Thaddao Ogren§, William Mutilangi§, Jacques Lalevée*,†,‡

†

Université de Haute-Alsace, CNRS, IS2M UMR 7361, F-68100 Mulhouse, France

‡

Université de Strasbourg, F-67000, France

§

PepsiCo Global Beverage Research and Development, 100 East Stevens Avenue, Valhalla,

NY 10595, USA Current Address: Givaudan Fragrance, 40 W 57th Street, New York, NY 10019, USA

*Corresponding

author

1 Environment ACS Paragon Plus


1

Abstract

2 3

Anthocyanins are natural dyes widely used in the food industry, but their chemical stability

4

in beverages can be affected by the presence of additives. In the present paper, the

5

interaction between anthocyanins and ascorbic acid (AA) is more particularly investigated.

6

Ascorbic acid is an ubiquitous component in food products. In this study, the thermal

7

stability at 43 °C and the photolysis stability under air and under an inert atmosphere (N2) of

8

anthocyanins extracted from black carrot (BC), grape juice (GJ) and purple sweet potato (SP)

9

have been studied with and without the presence of ascorbic acid (in citrate buffer at pH =

10

3). Discriminating the main environmental factors (i.e. heat and light) affecting the

11

anthocyanins stability is a key point for a better understanding of the degradation pathways.

12

The stability of the anthocyanins has been followed by UV-visible spectrometry. Moreover,

13

to understand the degradation mechanisms both in presence and absence of ascorbic acid,

14

various techniques such as fluorescence quenching, cyclic voltammetry and electron spin

15

resonance (ESR) spectroscopy have also been used to furnish a full coherent picture of the

16

chemical mechanisms associated with the anthocyanins degradation. In addition, molecular

17

orbitals and bond dissociation energies (BDE) have been calculated to extend the

18

investigation. Moreover the effect of some supplementary stabilizers (chlorogenic acid,

19

sinapic acid, tannic acid, fumaric acid, I

20

extract, rosemary extract) and sugars (sucrose, fructose, glucose) on the anthocyanins

21

stability in presence of ascorbic acid has been examined.

isoquercitrin, myrcitrin, green coffee bean

22


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Keywords: anthocyanins, black carrot, grape juice, purple sweet potato, ascorbic acid, color

24

stability, heat, irradiation, atmosphere, degradation mechanism, antioxidants.

25



27

INTRODUCTION

28 29

The visual appearance of food products is an important factor of customer choice.

30

For instance, in beverages, the color constitutes an essential attribute that impacts purchase

31

decision and flavor perception. Synthetic food colorings have been used in industry because

32

of their high stability and low cost. However, public concern about synthetic dyes has

33

increased, leading to a desire to replace them by dyes from natural sources, which constitute

34

a real challenge for the food industry. 1–4

35

Anthocyanins have raised a growing interest as natural colorants in beverages as they

36

are water-soluble, have a wide range of color and promote health benefits. 5–9 Anthocyanins

37

belong to a large group of polyphenolics named flavonoids and can be extracted from many

38

natural sources, e.g. flowers, leaves, fruits, vegetables, roots,...

39

However, anthocyanins are highly sensitive to environmental factors such as pH,

40

light, temperature, oxygen, enzymes, other compounds interaction, co-pigment effect, 6,10,11

41

resulting in a loss of bioactivity and color fading during storage, which are real issues for the

42

industry. Among those degradation factors, the presence of ascorbic acid (AA) is very

43

important in beverages. Indeed, ascorbic acid is commonly used in the food industry for its

44

antioxidant capacity but also for providing an additional source of Vitamin C. However it has

45

been reported to accelerate the thermal degradation, in acidic solution, of anthocyanins

46

from various sources, affecting their color stability

47

16,18,20

48

understand their chemical degradation in acidic solutions that could occur during storage

12–19

proportionally to its concentration.

For this reason, many investigations have been carried out on anthocyanins to


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49

10,21–27

and to improve their stability in beverages containing ascorbic acid, 11,13,28–33 so they

50

can be used more widely in food and beverages.

51

Most of the studies on anthocyanins stability in presence of ascorbic acid focused on

52

thermal stability under heat or at room temperature 12–19 or in combination with light effect

53

30–32

54

the potential reactions involved in the degradation mechanisms, it is necessary to

55

differentiate these major degradation pathways, especially because ascorbic acid has various

56

effects if subjected to thermal or photostress.

57

the effects of heat and light have been discriminated for a better understanding of the

58

interaction processes between the anthocyanins and the ascorbic acid.

whereas only a few investigated both effects separately.

17

17,18

However, to understand

This has been done in the present paper;

59

Thus, this study aims to give an insight into the possible thermal and photolysis

60

degradation mechanisms during storage of anthocyanins from grape juice, black carrot and

61

purple sweet potato with or without ascorbic acid in model beverage systems (pH = 3). Black

62

carrots and purple sweet potatoes have been found to be rich of acylated anthocyanins,

63

which confers them a great color stability during storage. 8 Grapes can also be a source of

64

acylated compounds but this depends on the variety. 34

65

Understanding the chemical mechanisms associated with anthocyanins/ascorbic acid

66

interactions upon different environmental factors can give clues to enhance the stability.

67

The effect of atmosphere, a parameter useful for the conditioning of beverages has also

68

been studied by distinguishing a storage under air and under an inert atmosphere, N2.

69

Moreover, this study explores the effect of some supplementary antioxidants on the stability

70

upon storage of anthocyanins solutions with ascorbic acid.

71

The color stability of anthocyanins was determined by following their absorbance in

72

the visible range. The degradation mechanisms were studied by cyclic voltammetry to



73

determine the potential redox reactions, by fluorescence spectroscopy to assess the

74

quenching potential of ascorbic acid and by electron spin resonance (ESR) spectroscopy for

75

the detection of radicals due to irradiation. Molecular orbitals and bond dissociation

76

energies (BDE) have also been calculated to extend the investigation, by giving an insight

77

into the possible cleavages of anthocyanins molecular bonds due to light irradiation.

78 79 80

MATERIALS AND METHODS

81 82

Chemicals. The following compounds were provided by PepsiCo (Valhalla, NY, USA): mixture

83

of anthocyanins from grape juice (liquid), black carrot (liquid) and purple sweet potato

84

(powder) concentrated extracts, L-ascorbic acid, I

85

(enzymatically modified isoquercitrin, water soluble glycoside of quercetin produced from

86

rutin by enzymatic treatment), myricitrin 30 % Y-AF (blend of glycerol and ethanol with

87

myricitrin), green coffee bean extract, rosemary extract. The other chemicals used in this

88

study were commercially purchased and employed without further purification: pH = 3

89

buffer citric acid / hydrochloric acid / sodium hydroxide Normex (Carlo Erba), hydrochloric

90

acid 37 %, AnalaR NORMAPUR® Reag. Ph. Eur. (VWR chemicals), chlorogenic acid P 95%

91

(Sigma-Aldrich), sinapic acid P 98 % (Aldrich), tannic acid ACS reagent (Sigma-Aldrich), sugar

92

(Cristal Union, Erstein), D-(+)-glucose > 98.0% (TCI), D-(-)-fructose > 99.0 % (TCI), fumaric acid

93

> 99.0 % (TCI), TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) 98 % (Aldrich).

94


isoquercitin R-20 EMIQ 30 %

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Preparation of solutions. All samples were prepared into a citrate buffer solution at pH = 3.0

96

at a fixed concentration of 500 mg L-1 for anthocyanins and 200 mg L-1 for ascorbic acid. The

97

antioxidants and sugars have been added at various concentrations. Then, the solutions

98

were mixed until complete solubilization of the compounds. The concentrations chosen for

99

the study are typical ones used in beverages.

100 101

Accelerated aging study and UV-visible analysis. Thermal treatment of solutions. A volume

102

of 25 mL of sample solution was poured into a glass bottle closed with a cork and wrapped in

103

aluminum. These samples were placed into an oven at 43 °C during several days. Every few

104

days, a part of the solution was removed from the bottle and transferred into a quartz

105

cuvette (1 cm x 1 cm) closed with a cork and the UV-visible spectrum of these solutions was

106

recorded. After each analysis, the samples were placed again in the oven.

107

For the study of thermal stability under air, there was no air exchange during storage.

108

But between each analysis, the bottles were opened, fresh air entering in the bottle. For

109

study under N2, before being placed in the oven, the solutions were degassed with N2 flux.

110

During the storage, there was no exchange of N2 with ambient air. After each analysis and

111

before placing again the samples in the oven, the solutions were degassed with N2.

112

Irradiation of solutions. The solutions to be irradiated were transferred into a quartz cuvette

113

(1 cm x 1 cm, filled with 3 mL of solution) closed with a cork. The solutions were irradiated at

114

room temperature (under air or under N2 flux, see below) each 20 s until 120 s then each 60

115

s until 300 s by an Omni-Cure Series 1000 Lumen Dynamics lamp (100 W, Mercury Arc with

116

intelli-lamp® Technology) at a spectrum range simulating sunlight and artificial light

117

irradiation (Figure S1 in supporting information). After each irradiation, the solutions were

118

analyzed by UV-visible spectrometry.



119

For irradiation under air, there was no exchange of air with the outside during photolysis

120

(the cuvette containing the solution was closed with a cork all along the irradiation and

121

analysis, the exact same cuvette was used for the irradiation and for the analysis). For

122

irradiation under N2, the solution in the cuvette was preliminary degassed by a N2 flux and

123

then kept under continuous N2 flux during the irradiation.

124

Measurement of color stability by UV-visible spectrometry. The UV-visible measurements

125

were performed by a Cary 3 Varian UV-visible spectrophotometer with the spectra

126

processed with the CaryWin UV scan Application software Version 2.0. Before the analysis, a

127

blank for the solvent alone (citrate buffer solution at pH = 3) was recorded and the

128

presented spectra of this study were plotted by subtracting the spectra by the blank

129

spectrum.

130

The UV-visible absorption spectra of the black carrot (BC), purple sweet potato (SP)

131

and grape juice (GJ) extracts in citrate buffer solution at pH = 3 are shown in Figure 1. The

132

spectra are characteristic of anthocyanin pigments, 35 with an absorbance below 400 nm and

133

in the range 450 - 600 nm. The differences of light absorption properties (absorbance and

134

maximum absorption wavelength) indicate that the three extracts are composed by a

135

mixture of different anthocyanin molecules. Indeed it has been shown that natural extracts

136

contain a multitude of anthocyanins, for instance cyaninin-, peonidin-, pelargodinin-,

137

malvidin-, delphinidin-, and petunidin-glucoside derived anthocyanins, with mainly derived

138

molecules of cyanidin for black carrot BC 36–38, cyanidin and peonidin for purple sweet potato

139

SP 33,39–42 and a larger variety of anthocyanins including cyanidin, pelargonidin and peonidin

140

for grape juice GJ. 34,43,44 Furthermore, the extracts seem to be composed of different kinds

141

of anthocyanins with different substitution groups (see the fluorescence quenching part in

142

Results and Discussion) such as glycosylated or acylated groups. Depending on the


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substituent group, the absorption range will differ. For instance, the absorbance below 400

144

nm could be mostly due to acylated phenolic groups. 49

145 146

Note that the anthocyanins composition in the natural extracts depends on the vegetable or fruit variety 36,45,46 but also on the extraction process. 47,48

147

The stability of the anthocyanins in the solutions was determined by measuring the

148

evolution of their absorbance at the maximum wavelength in the visible range (522 nm for

149

black carrot (BC), 527 nm for purple sweet potato (SP) and 529 nm for grape juice (GJ))

150

(Figure 1). These wavelengths were chosen since they correspond to the maximum

151

absorbance of anthocyanins solutions observed in the visible range that corresponds

152

indubitably to the molecule in the extract responsible for the visible pink-purple color.

153

Moreover, at these wavelengths, there was no interference/overlapping with the

154

absorbance of the other compounds used in the study (ascorbic acid, antioxidants and

155

sugars), colorless and absorbing close to the UV range.

156

The remaining proportion “% remaining of anthocyanins” from BC, GJ and SP extracts

157

after thermal treatment or irradiation was calculated from the maximal absorbance of the

158

UV-visible spectra in the range 500 - 550 nm, using the following formula:

159

%remaininganthocyanin = A0 × 100

At

(1)

160

with A0 the absorbance of the solution before irradiation and At the absorbance of the

161

solution after a given time t in the oven at 43 °C for thermal study or after a time t of

162

irradiation for photolysis study.

163

Note that the % remaining proportion of anthocyanins is based on the evolution of

164

the peak in the visible range 500 - 550 nm. However, degradation products may also

165

potentially absorb at the same wavelengths. However, since a decrease in absorbance is

166

observed, the contribution to absorbance of compounds other that initial anthocyanins 9 Environment ACS Paragon Plus


167

would be negligible. Indeed, for high degree of degradation, no products are observed in this

168

spectral range.

169 170

The error bars on the experimental dots in the results figures correspond to the standard deviation calculated from the results of three sets of samples.

171 172

Cyclic voltammetry. For the determination of the oxidation potential of ascorbic acid and

173

the reduction potential of anthocyanins in solution, cyclic voltammetry experiments were

174

performed. A Radiometer analytical PST006 Education VoltaLab was used. It was equipped

175

with a three-electrodes cell (two platinum as working and auxiliary electrodes and one

176

Saturated Calomel Electrode (SCE) as reference). All measurements were performed at room

177

temperature in a sample volume of 60 mL using KCl (at 3.4 10-2 M) as supporting electrolyte

178

in an aqueous solution at pH = 3 (HCl in water) and under N2. For each measurement, the

179

potential was scanned with a 100 mV s-1 scan rate between -2 and 2 V.

180

For each analysis, a blank was done. It corresponded to the cyclic voltammetry plot of

181

the solution (solvent alone with KCl) after degassing under N2. Then, a small amount of the

182

compound of interest, ascorbic acid or anthocyanins was added in the solution. Before each

183

measurement, the solution was stirred and degassed under N2 then the cyclic voltammetry

184

plot was recorded. The blank sample helped to check that the oxidation or reduction peaks

185

were attributed to the anthocyanins and not to the solvent.

186

The error bars on the experimental dots in the results figures correspond to the

187

standard deviation calculated from the results of three sets of samples. An excellent

188

repeatability has always been observed.

189


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Electron Spin Resonance - Spin Trap (ESR-ST) experiment. The Electron Spin Resonance -

191

Spin Trap (ESR-ST) experiment 50,51 was used to prove the presence of radicals formed from

192

anthocyanins

193

tetramethylpiperidine 1-oxyl, (CH2)3(CMe2)2NO•, called TEMPO, as spin trap. 52

194

For this study, a solution of TEMPO at 10-4 M was prepared in aqueous solution at pH = 3

195

(HCl in distilled water). A part of this solution was kept as reference and the other part was

196

added to the BC, GJ and SP extracts to obtain a concentration of 30 g L-1. For the study under

197

N2, the solutions were degassed beforehand during 10 min. For each analyzed solution, a

198

volume of 50 µL was transferred in a glass capillary (Brand disposable Blaubrand

199

micropipette intra Mark 50 µL, inner Ø ~ 0.9 mm) sealed with Crit-O-sealTM and then placed

200

in an ESR-tube with a diameter of 5 mm. The tube containing the solution was then

201

irradiated at room temperature each 20 s until 100 s. After each irradiation, the solution was

202

analyzed by ESR. After the whole irradiation experiment, the ESR signal of the non-irradiated

203

solution (rest of solution, kept in the dark during the time of the experiment, namely Z 30

204

min) was recorded, as a control. The ESR measurements were carried out using a RPE Bruker

205

EMX Plus PremiumX with the software WinEPRAcquisition Version 4.40 rev. 11. The

206

following acquisition parameters were applied: receiver gain 1.0 104; sweep width 80 G

207

(central field: 3500 G); modulation amplitude 1G; time constant 20.48 ms; resolution: 1024

208

pts; power: 2 mW (20 dB); number of scans 3.

209 210

molecules

in

solution

under

irradiation,

using

the

2,2,6,6-

The incertitude on the measurements has been determined from the measurements of the photodegradation of three sets of TEMPO solution.

211 212

Molecular modelling. Molecular orbital calculations, Highest Occupied Molecular Orbital

213

(HOMO) and Lowest Unoccupied Molecular Orbital (LUMO), were carried out with the



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214

Gaussian03 suite of programs. The bond dissociation energies (BDEs) were calculated with

215

the time dependent density functional theory at B3LYP/6-31G* level on the relaxed

216

geometries calculated at UB3LYP/6-31G* level. 53

217

The calculated values from the simulations correspond to the most stable spatial

218

conformation of the molecule. The error bar is determined from the dispersion of the values

219

corresponding to the different realistic molecular conformations of the molecule.

220 221

Fluorescence quenching. To determine if the excited states of anthocyanins are quenched

222

by ascorbic acid (AA), fluorescence quenching experiments were performed. Solutions of

223

anthocyanins with AA in aqueous solution at pH = 3 (HCl in water) were prepared at different

224

AA concentrations and then transferred in a quartz cuvette (1 cm x 1 cm, filled with 3 mL of

225

solution) closed with a cork. The samples were analyzed using a Jasco FP-750 Fluorimeter

226

with the software Spectra Manager (Version 1.53.04) by recording the evolution of the

227

emission spectra at a given excitation wavelength.

228

From the emission fluorescence spectra, Stern-Volmer plots were plotted: 0

229

=1+

0

(2)

230

with I0 the intensity of emission fluorescence (at a given \ex) without quencher (i.e. without

231

AA), I the intensity of emission fluorescence (at a given \ex) with quencher (here with AA), kQ

232

the quencher rate coefficient in L s-1 mol-1, ]0 the lifetime of the excited singlet state of the

233

anthocyanin and [Q] the concentration of the quencher (AA) in mol L-1.

234

The solutions were prepared at a concentration corresponding to an absorbance A

Thermal and Photochemical Stability of Anthocyanins from Black

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