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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

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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


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

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

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ABSTRACT

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

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

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

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

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

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

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

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

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

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

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

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

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carotenes in a citrus food matrix.

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INTRODUCTION

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

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

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

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

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

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

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

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

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bioactive compounds with beneficial properties for human health. Numerous epidemiological

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

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

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

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

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

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

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

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

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

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

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

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in cancer and cardiovascular disease prevention.4

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

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significantly among cultivars and are also affected by technological processing which

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Cara Cara with respect to orange juice cv. Bahia.

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MATERIAL AND METHODS

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Samples 4 ACS Paragon Plus Environment

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

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

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

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

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

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

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

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

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

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

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

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Chemicals

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Fluorescein,

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

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

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

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

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

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

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

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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)



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

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

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

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reported as °Brix.

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Analysis of Ascorbic Acid Content

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

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

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

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

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

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

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

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

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mg/L).

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Analysis of Total Phenolic Content

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

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

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

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

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

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

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


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

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

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Identification and Quantification of Flavonoids

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Flavonoid identification and quantification was performed according to the method of

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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



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

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

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

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

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Qualitative and Quantitative Analysis of Carotenoids

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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Antioxidant Capacity Assay

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Antioxidant capacity of hydrophilic and lipophilic fractions was evaluated by Oxygen

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

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

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

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

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

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

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

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

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

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(Biotek Instrumental Winooki, EUA).

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

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

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

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

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

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

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as mmol Trolox equivalents.

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Soluble Sugar Content and Total Fibre determination



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

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

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

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

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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)

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

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

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fructose.

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

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Statistical Analysis

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

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

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

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

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the Shapiro-Wilk test. Values p

Citrus sinensis (L.) Osbeck cv. - ACS Publications - American

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