Effect of Pasteurization on Flavonoids and Carotenoids in Citrus

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Effect of pasteurization on flavonoids and carotenoids in Citrus sinensis (L.) Osbeck cv. «Cara Cara» and «Bahia» juices Elisa Brasili, Daniela F. Seixas Chaves, Ana Augusta Odorissi Xavier, Adriana Zerlotti Mercadante, Neuza Mariko Aymoto Hassimotto, and Franco Maria Lajolo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05401 • Publication Date (Web): 01 Feb 2017 Downloaded from http://pubs.acs.org on February 3, 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 30

Journal of Agricultural and Food Chemistry

Effect of pasteurization on flavonoids and carotenoids in Citrus sinensis (L.) Osbeck cv. «Cara Cara» and «Bahia» juices

Elisa Brasili*†1,2, Daniela F. Seixas Chaves†1,2, Ana Augusta O. Xavier3, Adriana Z. Mercadante2,3, Neuza MA. Hassimotto1,2, Franco M. Lajolo1,2

1

Department of Food Science and Experimental Nutrition, School of Pharmaceutical Science,

University of São Paulo, São Paulo, Brazil. 2

Food Research Center (FoRC), CEPID-FAPESP (Research Innovation and Dissemination

Centers São Paulo Research Foundation), São Paulo, Brazil. 3

Department of Food Science, Faculty of Food Engineering, University of Campinas,

Campinas, SP, Brazil

Corresponding author: *(E.B) Phone: (+55) 11-30910128. Fax: (+55) 11-3815 4410. E-mail: [email protected] † These authors contributed equally to this work

1 ACS Paragon Plus Environment


Page 2 of 30

1

Keywords: Citrus sinensis L. Osbeck, Cara Cara orange juice, Bahia orange juice,

2

carotenoids, flavonoids, antioxidant capacity, LC-MS/MS

3 4

ABSTRACT

5

Orange juice is considered an excellent dietary source of several bioactive compounds with

6

beneficial properties for human health. Citrus sinensis Osbeck cv. “Cara Cara” is a bud

7

mutation originated from “Washington” navel orange, also known as “Bahia” navel orange.

8

The ascorbic acid, flavonoid and carotenoid contents in pasteurized and non-pasteurized

9

Bahia and Cara Cara juices using two LC-MS/MS platforms were investigated. Higher

10

ascorbic acid content was observed in Bahia compared to Cara Cara in both pasteurized and

11

non-pasteurized juices. Total flavanones content as well as hesperidin levels were higher in

12

Cara Cara with respect to Bahia pasteurized juice. Cara Cara was also characterized by a

13

significantly higher and diversified carotenoid content compared to Bahia juice with a

14

mixture of (Z)-isomers of lycopene, all- E-β-carotene, phytoene and phytofluene isomers

15

accounting for the highest carotenoid proportion. The exceptional high carotenoid content of

16

Cara Cara may be particularly interesting for nutritional or functional studies of uncommon

17

carotenes in a citrus food matrix.

18 19 20 21 22 23 2 ACS Paragon Plus Environment

Page 3 of 30


24

INTRODUCTION

25

Orange is one of most consumed fruit in the world. Global production in 2012 exceeded 86

26

million metric tons, with an estimated value of $9 billion.1 According to 2015-2016 data from

27

the Food and Agriculture Organization of the United Nations (FAO), Brazil is the world´s

28

largest producer of oranges with 16.8 million tonnes (Mt) per years, accounting for 50% of

29

global production and more than 80% of global exports of the product.2 This production is

30

expected to increase in the coming decade reaching 17.5 Mt in the period of 2023/24. More

31

than 70% of the Brazilian orange fruits are used in the production of juices.

32

Citrus sinensis L. Osbeck orange juice is considered an excellent dietary source of several

33

bioactive compounds with beneficial properties for human health. Numerous epidemiological

34

and intervention studies have provided evidence to support an inverse correlation between the

35

orange juice intake and the occurrence of cardiovascular disease, cancer and aging-related

36

disorders.3,4 In addition to vitamin C, orange juice contains flavonoids, carotenoids, sugars,

37

minerals and fibre. The most abundant flavonoid species that have been identified and

38

quantified in Citrus species, regardless of variety, are hesperidin, narirutin and didymin5 that

39

possess well-known anti-inflammatory, antioxidant, anticarcinogenic and antiproliferative

40

properties.6-8 Orange juice is also rich in carotenoids, a widespread family of natural

41

pigments which provide attractive coloration to flowers and fruits, ranging from yellow,

42

orange to deep red.9,10 Their importance as nutritional components is also well recognized,

43

since specific carotenoids are precursors of vitamin A (e.g. β-carotene, α-carotene and β-

44

cryptoxanthin) and own antioxidant properties11 which could confer to them a protective role

45

in cancer and cardiovascular disease prevention.4

46

It is noteworthy that the distribution and composition of these bioactive compounds differ

47

significantly among cultivars and are also affected by technological processing which

48

impacts on the enzyme activity, potentially changing the chemical composition of orange 3 ACS Paragon Plus Environment


Page 4 of 30

49

juice.12 Despite its low pH, fresh orange juice stability is rather limited, due to microbial

50

growth and enzyme activities. To prolong this shelf life, thermal pasteurization is the most

51

widely applied technique due to its low cost, efficacy in inactivating vegetative

52

microorganisms and enzymes, and for its environmental-friendly nature.13

53

In Brazil, among sweet oranges, it is increasing the interest in Citrus sinensis Osbeck cv.

54

Cara Cara, a bud mutation originated in Venezuela in the 80s from “Washington” navel

55

orange14, also known as “Bahia” navel orange for the Brazilian city from which it was

56

exported into the United States in 1870, according to USDA- ARS National Clonal

57

Germplasm Repository for Citrus (University of California, Riverside).

58

Cara Cara orange pulp is characterized by a bright red coloration which resembles that of

59

pink and red grapefruits. Previous studies have indicated that this deeply coloured flesh is

60

associated with a high content of lycopene accumulated in the juice vesicles.9,10,14-16

61

Nevertheless, the presence of lycopene is an unusual feature in most citrus fruits since has

62

been reported in few orange species,17,18 but is of special interest due to recent nutritional

63

studies that have demonstrated its important role in reduction of cardiovascular disease.19 In

64

Brazil, orange juice cv. Cara Cara is not yet commercially available, although there is an

65

increasingly great interest to introduce this cultivar in orange fruit market. Up to date, there

66

are no data about the bioactive compounds composition and antioxidant capacity of

67

pasteurized and non-pasteurized orange juice from Cara Cara and Bahia cultivars. The main

68

objective of this study was to assess and compare the effect of the thermal pasteurisation on

69

flavonoid and carotenoid contents as well as on the antioxidant capacity of orange juice cv.

70

Cara Cara with respect to orange juice cv. Bahia.

71 72

MATERIAL AND METHODS

73

Samples 4 ACS Paragon Plus Environment

Page 5 of 30


74

Pasteurized and non-pasteurized orange juices obtained from Citrus sinensis L. cv. Cara Cara

75

and Bahia were provided by Fundecitrus company (São Paulo, Brazil). Oranges from both

76

cultivars were collected in Araraquara (São Paulo, Brazil) located in Southeast region of São

77

Paulo at 23°23’19’’ S and 48°43’22’’ W with an elevation of 600 meters above sea level. The

78

fully-mature fruits were hand harvested in July 2015 and then stored at 4 °C for 7 days.

79

Orange juices were obtained by cutting and squeezing, using an industrial extractor (model

80

391, JBT). The pasteurization process was achieved in an industrial pasteurizer (UHT/HTST

81

Lab- 25DH, MicroThermics) at 92-94 °C for 17 s (750 mL/minute). After processing, the

82

juices were ﬁlled into 1L plastic ﬂasks and cooled to 1°C. All juices were immediately stored

83

at -20 °C until further analysis. Subsequently, samples were centrifuged at 8000 g at 4 °C for

84

10 min and the obtained supernatant and pellet were utilized for further chemical analysis.

85 86

Chemicals

87

Fluorescein,

88

diphenylpicrylhydrazyl (DPPH), dithiothreitol, dehydro-ascorbic acid, trifluoroacetic acid,

89

sucrose, glucose, fructose, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

90

Narirutin, hesperidin and didymin were obtained from Extrasynthese (Genay, France). Folin-

91

Ciocalteu reagent, sodium acetate, methanol, metaphosphoric and acetic acid were obtained

92

from Merck Millipore (Darmstadt, Germany). The carotenoid standards (all-E)-lutein, (all-

93

E)-zeaxanthin, (all-E)-β-cryptoxanthin, (all-E)-α-carotene, (all-E)-β-carotene were obtained

94

from DSM Nutritional Products (Basel, Switzerland), and (all-E)-violaxanthin, (all-E)-

95

antheraxanthin and (9ʹZ)-neoxanthin from CaroteNature (Lupsingen, Switzerland).

2,2′-azobis(2-methyl-propanimidamide)dihydrochloride

(AAPH),

2,2-

96 97

Determination of pH and Total Soluble Solids (TSS)



Page 6 of 30

98

The pH of orange juices was measured using an Metrohm pH meter (model 827, Switzerland)

99

at room temperature. The total soluble solids content in the orange juice samples was

100

determined using a digital refractometer (Krüss, Germany). The obtained values were

101

reported as °Brix.

102 103

Analysis of Ascorbic Acid Content

104

Ascorbic acid content was determined according to the method of Rizzolo et al. (1984).20

105

Ascorbic acid was extracted with metaphosphoric acid (0.3% w/v) and analysed by reversed-

106

phase HPLC, in a Hewlett-Packard 1100 system, with an auto-sampler and a quaternary

107

pump, coupled to a diode array detector. The column used was a µ-Bondapack (300 mm x 3.9

108

mm i.d., Waters, Milford, MA, USA) column, and elution (flow rate of 1.5 mL/minute) was

109

carried out in isocratic conditions with 0.2 M sodium acetate/acetic acid buffer (pH 4.2),

110

monitored at 262 nm. Total ascorbic acid was estimated after reduction of dehydro-ascorbic

111

acid (DHA) with 10 mM dithiothreitol, with a calibration curve of ascorbic acid (0−600

112

mg/L).

113 114

Analysis of Total Phenolic Content

115

The total phenolic content of samples was determined using the Folin−Ciocalteu colorimetric

116

method described by Singleton and Rossi (1965)21 with some modifications. A previously

117

shaken sample of orange juice (2 mL) was added to 10 mL of methanol:water (80:20, v/v).

118

The sample was vortexed for 1 min and then placed to an ultrasonic bath (15 min) according

119

to the procedure described by Stella et al. (2011).22 Afterwards, the samples were centrifuged

120

at 10000g during 15 min at 20°C and supernatants were filtered by a Whatman filter and

121

analysed. The absorbance was measured spectrophotometrically at 763 nm. Measurements


Page 7 of 30


122

were recorded on an UV−vis spectrophotometer Helios Epsilon (Thermo Scientific, Madison,

123

WI, USA). The results were expressed as milligrams of gallic acid/100 mL of orange juice.

124 125

Identification and Quantification of Flavonoids

126

Flavonoid identification and quantification was performed according to the method of

127

Hassimotto et al. (2005)23, with slight modifications. Briefly, 5 mL of orange juice was

128

centrifuged for 10 min (7000 g, 4 ºC). An aliquot of the supernatant (1 mL) was filtered by a

129

0.45 µm PVDF Millex filter (Millipore, Tokyo, Japan) and directly injected (10 µl) in the

130

equipment. The pellet was added to 10 mL of DMSO, homogenized for 18 h and then

131

centrifuged and filtered as previously described. Samples were quantified using a 1260

132

Infinity Quaternary LC System (Agilent Technologies, USA) with an autosampler and a

133

quaternary pump, coupled to a diode array detector (DAD). Separation of the analytes was

134

achieved on a column Prodigy 5 µm ODS3 reversed-phase silica (250 mm x 4.60 mm)

135

(Phenomenex Ltd., United Kingdom) with a flow rate of 1 mL/min at 25 °C. The mobile

136

phase consisted of 0.5% formic acid in water (solvent A) and 0.5% formic acid in acetonitrile

137

(solvent B). The solvent gradient was applied as follows: 8% B at the beginning, 10% at 5

138

min, 17% at 10 min, 25% at 15 min, 50% at 25 min, 90% at 30 min, 50% at 32 min, 8% at 35

139

min (run time, 35 min).24,25 The eluates were monitored at 270 and 525 nm. Peak

140

identification was carried out by the combined information provided by comparison of

141

retention times, diode array spectral characteristics and mass spectra, measured by LC-ESI-

142

MS/MS, with the standards and the data available in the literature. The equipment of LC-ESI-

143

MS/MS was a Prominence liquid chromatograph (Shimadzu, Japan) linked to an ion trap

144

mass analyzer (Esquire, Bruker Daltonics, Billerica, MA, USA) with an electrospray

145

ionization (ESI) interface in negative mode for flavonoids. The solvent gradient conditions

146

were the same as those used for the HPLC-DAD process. After passing through the DAD, the



Page 8 of 30

147

flow rate was changed to 0.2 mL/min for application to the mass spectrometer. The mass

148

spectrometer operating conditions were as follows: collision energy was 4000 V and capillary

149

temperature was 275 °C. The analysis was carried out using a full scan from m/z 100 to 1500.

150

Quantification was done by calibration curves of narirutin, hesperidin and didymin.

151 152

Qualitative and Quantitative Analysis of Carotenoids

153

Approximately 2 g of orange juice was exhaustively extracted with acetone according to De

154

Rosso & Mercadante.26 The solid residue retained in the filter was transferred to the mortar

155

and re-extracted until the extract became colorless (4 times). The extract was transferred to

156

petroleum ether/diethyl ether and saponified overnight at room temperature using 10% KOH

157

in methanol. The saponified extract was abundantly washed with deionized water until pH 7,

158

the solvent was evaporated to dryness and the dry extract was stored at -35 °C until analysis.

159

The carotenoids were separated by LC-DAD-MS/MS on a C30 YMC column (5 µm, 250 mm

160

x 4.6 mm), using as mobile phase a gradient of methanol and t-methyl butyl ether from 95:5

161

to 70:30 in 30 min, followed by 50:50 in 20 min, maintaining this condition for 25 min. The

162

flow was 0.9 mL/min, and the column temperature was set as 29 °C. The equipment was a

163

HPLC Shimadzu (Kyoto, Japan) equipped with a quaternary pump (LC-20AD), degasser

164

(DGU- 20A5), Rheodyne injection valve (20 µL loop, Rohnert Park, CA, USA), coupled in

165

series to a diode array detector (DAD) (SPDM20A) and a mass spectrometer with an ion trap

166

analyser with atmospheric pressure chemical ionization (APCI) source (AmaZon Speed ETD,

167

Bruker Daltonics, Bremen, Germany). The MS conditions were: APCI positive mode, corona

168

current of 4000 nA, source temperature of 450 °C, dry gas (N2) temperature of 350 °C at 5

169

L/min, nebulizer at 60 psi. Identification was carried out through analysis of characteristics of

170

UV-Vis and MS/MS spectra and comparison with standards. Quantification was done by


Page 9 of 30


171

calibration curves of (all-E)-lycopene, (all-E)-β-carotene, (all-E)-α-carotene, (all-E)-β-

172

cryptoxanthin, (all-E)-lutein, and (all-E)-zeaxanthin.

173 174

Antioxidant Capacity Assay

175

Antioxidant capacity of hydrophilic and lipophilic fractions was evaluated by Oxygen

176

Radical Absorbance Capacity (ORAC) assay and Radical Scavenging (DPPH) assay. ORAC

177

assay was done according to Prior et al.27 All reagents were prepared in phosphate buffer (75

178

mM, pH 7.0). Briefly, 25 µL of diluted sample (1:300) was added to 150 µL of sodium

179

fluorescein (40 nM) and incubated for 15 min at 37 °C. Subsequently, 0.6 mL of AAPH

180

(4mM) as a peroxyl radical generator and Trolox as a control standard were added, and the

181

fluorescence was read with an excitation wavelength of 485 nm and an emission wavelength

182

of 520 nm, every 5 min for 120 min. Final results were calculated from a standard curve

183

using different concentrations of Trolox (12.5 − 100 µM) and expressed in mmol Trolox

184

equivalent. Measurements were recorded on a SynergyTM HT-multimode microplate reader

185

(Biotek Instrumental Winooki, EUA).

186

The DPPH assay was carried out according to the method described by Brand-Williams et

187

al.28 with modifications. A 0.1 mM solution of DPPH (1,1- diphenyl-2-picrylhydrazyl) in

188

methanol was prepared. An aliquot of 50 µL of deproteinated sample was added to 150 µL of

189

this solution. The decrease in absorbance was determined at 517 nm using a microplate

190

spectrophotometer (Benchmark Plus, Biorad, Hercules, CA) when the reaction reached a

191

plateau (after 20 min). The area under the curve was calculated. The results were expressed

192

as mmol Trolox equivalents.

193 194

Soluble Sugar Content and Total Fibre determination



Page 10 of 30

195

Sugar content was determined according to Shiga et al.29 Samples were extracted three times

196

with 80% ethanol (v/v) at 80 °C for 30 min under stirring. After centrifugation, the

197

supernatants were evaporated under vacuum. The residues were reconstituted with water,

198

filtered through 0.22 µm membrane filters and analysed by LC coupled to a pulse

199

amperometric detector (PAD) using a one-Dionex DX500 system (Dionex, Thermo

200

Scientific, Waltham, MA, USA), equipped with a CarboPac PA1 column (4 mm × 250 mm)

201

(Dionex). The mobile phase consisted of 18 mM NaOH, at flow rate was of 1 mL/min for 25

202

min. The quantification was based on an external calibration using sucrose, glucose, and

203

fructose.

204

The fibre content was measured according to AOAC Official Methods 991.43 and 2009.01.30

205 206

Statistical Analysis

207

All analyses were performed in triplicate. Data were expressed as mean ± standard deviation

208

(SD). The comparisons between pasteurized and non-pasteurized juices from both cultivars

209

were carried out using univariate unpaired Student’s t test and analysis of variance (one-way

210

ANOVA) followed by Holm-Sidak test. The assessment of data normality was performed by

211

the Shapiro-Wilk test. Values p

Effect of Pasteurization on Flavonoids and Carotenoids in Citrus

Recommend Documents