Effect of Alcoholic Fermentation on the Carotenoid Composition and

Article pubs.acs.org/JAFC

Effect of Alcoholic Fermentation on the Carotenoid Composition and Provitamin A Content of Orange Juice Isabel Cerrillo,† Blanca Escudero-López,† Dámaso Hornero-Méndez,§ Francisco Martín,†,‡ and María-Soledad Fernández-Pachón*,† Á rea de Nutrición y Bromatologı ́a, Departamento de Biologı ́a Molecular e Ingenierı ́a Bioquı ́mica, Universidad Pablo de Olavide, Carretera de Utrera Km 1, E-41013 Sevilla, Spain § Departamento de Fitoquı ́mica de Alimentos, Instituto de la Grasa−CSIC, Avenida Padre Garcı ́a Tejero 4, E-41012 Sevilla, Spain ‡ CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Universidad Pablo de Olavide, Carretera de Utrera Km 1, E-41013 Sevilla, Spain †

ABSTRACT: Orange juice is considered a rich source of carotenoids, which are thought to have diverse biological functions. In recent years, a fermentation process has been carried out in fruits resulting in products that provide higher concentrations of bioactive compounds than their original substrates. The aim of this study was to evaluate the effect of a controlled alcoholic fermentation process (15 days) on the carotenoid composition of orange juice. Twenty-two carotenoids were identified in samples. The carotenoid profile was not modified as result of the fermentation. Total carotenoid content and provitamin A value significantly increased from day 0 (5.37 mg/L and 75.32 RAEs/L, respectively) until day 15 (6.65 mg/L and 90.57 RAEs/L, respectively), probably due to a better extractability of the carotenoids from the food matrix as a result of processing. Therefore, the novel beverage produced could provide a rich source of carotenoids and exert healthy effects similar to those of orange juice. KEYWORDS: orange juice, alcoholic fermentation, carotenoids, β-cryptoxanthin, provitamin A, bioactive compounds, LC-MS (APCI)

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pulsed electric fields, thermal treatment, high pressure, refrigerated storage, or ultrafrozen on the carotenoid content of different types of orange juice, determining the optimal technological conditions to preserve the potential healthy effect of the resulting product.15 It has been recently found that fermentation might improve the profile of bioactive compounds in fruits and vegetables, so a fermentation process has been carried out in fruits (pomenagrate,16 mulberry,17 apple18) and vegetables (red sorghum,19 onion20), resulting in products that provide higher concentration of bioactive compound content than the substrate. Accordingly, it would be interesting to analyze the effect of a controlled fermentation process on the carotenoid composition of orange juice. On the other hand, this orange fermented product contains a moderate amount of alcohol. Numerous studies have demonstrated that moderate alcoholic consumption produces positive effects on plasma lipid profile, coagulation system, and the atherosclerotic process.21 Therefore, healthy effects of dietary bioactive compounds and moderate alcohol consumption would also act synergistically in a potential beverage based on a controlled alcoholic fermentation of orange juice. Therefore, the aim of this study was to evaluate the effect of the controlled alcoholic fermentation on the carotenoid composition of orange juice with a special emphasis on the provitamin A content. Furthermore, this novel orange juice beverage would suggest a new way to commercialize orange

INTRODUCTION Orange juice is among the most consumed fruit juices worldwide. Orange juice accounts for 60% of all Western Europe consumption of fruit juices and juice-based drinks. In the United States, this juice is the most popular juice per capita, being consumed at 5 times the rate of Americans’ second choice, apple juice.1 Orange juice is considered a rich source of bioactive compounds beneficial for human health, such as ascorbic acid, flavonoids, and carotenoids. Numerous epidemiological and intervention studies have provided evidence to support that orange consumption may reduce the risk of cardiovascular diseases and cancer.2,3 Carotenoids are an important group of natural pigments, responsible for the coloring of most fruits and vegetables such as oranges, red peppers, watermelons, carrots, and tomatoes.4 These compounds are minor constituents in cereal grains.5 Nearly 750 naturally occurring carotenoids have been identified, and this number continues to rise.6 Orange juice has one of the most complex carotenoid profiles reported in any fruit.7 From a nutritional and physiological standpoint, orange juice carotenoids are thought to have diverse biological functions and actions, such as antioxidant capacity,8 provitamin A activity,9 macular protection,10 bone health,11 and anticarcinogenic effect.12 The increasing interest in these pigments has prompted the development of numerous analytical methods for characterizing carotenoids in orange juice7 and other foods13 and has prompted research into adequate production conditions and technological treatments to maintain or enhance carotenoid composition and subsequent application in the food industry.14 Many studies have examined the effect of processing such as © 2014 American Chemical Society

Received: Revised: Accepted: Published: 842

June 28, 2013 January 10, 2014 January 13, 2014 January 13, 2014 dx.doi.org/10.1021/jf404589b | J. Agric. Food Chem. 2014, 62, 842−849

Journal of Agricultural and Food Chemistry

Article

in 3 mL of diethyl ether and 0.5 mL of 20% (w/v) KOH/MeOH was added, followed by a reaction period of 20 min with intermittent agitation. After the addition of 3 mL of distilled water, samples were centrifuged at 5000g and 4 °C for 5 min to separate organic (upper) and aqueous (lower) phases. The supernatant was collected, dried under a nitrogen stream, and kept under the inert atmosphere at −30 °C until chromatographic analysis. Before the analysis, samples were dissolved in 0.5 mL of acetone and centrifuged at 12000g and 4 °C for 5 min. All operations were carried out under dimmed light to prevent isomerization and photodegradation of carotenoids. Pigment Identification. Routine procedures were used to identify the carotenoids, using a combination of different experiments: separation and isolation of pigments by TLC and cochromatography (TLC and HPLC) with pure standards, analysis of the UV−visible and mass spectra, comparison with values in the literature,4,24−28 and chemical test for 5,6-epoxy groups. Samples of β-carotene (β,βcarotene), antheraxanthin (5,6-epoxy-5,6-dihydro-β,β-carotene-3,3′diol), β-cryptoxanthin (β,β-caroten-3-ol), zeaxanthin (β,β-carotene3,3′-diol), lutein (β,ε-carotene-3,3′-diol), violaxanthin (5,6:5′,6′-diepoxy-5,6:5′,6′-tetrahydro-β,β-carotene-3,3′-diol), and neoxanthin (5′,6′-epoxy-6:7-didehydro-5,6:5′,6′-tetrahydro-β,β-carotene-3,5,3′triol) were isolated and purified from natural sources (Capsicum annuum and Mentha arvensis).23 5,8-Epoxy-carotenoids, such as mutatoxanthin (5,8-epoxy-5,8-dihydro-β,β-carotene-3,3′-diol), auroxanthin (5,8:5′,8′-diepoxy-5,8:5′,8′-tetrahydro-β,β-carotene-3,3′-diol), luteoxanthin (5,6:5′,8′-diepoxy-5,6:5′,8′-tetrahydro-β,β-carotene-3,3′diol), and neochrome (5′,8′-epoxy-6,7-didehydro-5,6:5′,8′-tetrahydro-β,β-carotene-3,5,3′-triol) were prepared from the corresponding 5,6-epoxy parent pigments (antheraxanthin, violaxanthin, and neoxanthin) by controlled treatment with diluted HCl. Latochrome (5′,8′epoxy-5,6,5′,8′-tetrahydro-β,β-carotene-3,5,6,3′-tetrol) and karpoxanthin (5,6-dihydro-β,β-carotene-3,3′,5,6-tetrol) were only tentatively identified due to the lack of pure standards. The tentative identification of Z isomers of lutein and mutatoxanthin was based on the presence and relative intensity (%AB/AII) of the cis peak at about 330−340 nm in the UV−visible spectrum; a reduced fine structure and small hypsochromic shift in λmax with respect to the all-E counterpart; and the chromatographic behavior in the C18 HPLC column, showing a slightly greater retention of Z than the all-E isomer.28 Carotenoid Analysis and Quantification by HPLC. Carotenoid pigments were analyzed by reversed-phase HPLC using a method previously developed23 with slight modifications. Briefly, the method uses a C18 reversed-phase column (Mediterranea SEA18, 20 × 0.46 cm i.d., 3 μm particle size; Teknokroma S.C.L., Barcelona, Spain) and a binary gradient elution system of acetone−deionized water at a flow of 1.0 mL/min. The mobile phase started at 75% acetone and rose linearly to 95% within 10 min and continued isocratically for 7 min, then changing to 100% within 3 min and maintaining this composition for 3 min. The injection volume was 10 μL, and detection was carried out simultaneously at 402, 424, and 450 nm (1.2 nm spectral resolution and 10 scan/s scanning speed), to detect and quantify the diverse family of citrus carotenoids characterized as having different chromophores. The column and sample compartment were maintained at 25 and 15 °C, respectively. HPLC analyses were performed with a Waters 2695E Alliance quaternary pump equipped with a Waters 2998 diode array detector and were controlled with Empower2 data acquisition software (Waters Corp., Milford, MA, USA). Pigments were quantified in the saponified extracts by using calibration curves (six to eight concentration levels) prepared with standard stock solutions for each carotenoid in the concentration range of 5−100 μg/mL. Calibration curves were constructed by plotting the peak area at 402 nm (for auroxanthin and ζ-carotene), 424 nm (for neochrome and luteoxanthin), and 450 nm (for mutatoxanthin, zeaxanthin, lutein, β-cryptoxanthin, and β-carotene) versus the pigment concentration. Latochrome and karpoxanthin were quantified by using the calibration curve of neochrome and zeaxanthin, respectively. The Z isomers were quantified by using the calibration curve of the all-E isomer. Concentration values were calculated as milligrams of pigment per liter of juice.

and the possibility of providing the consumer with a new potentially healthful beverage that could extend the supply of other similar drinks with higher alcohol content, such as wine, beer, or cider.

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MATERIALS AND METHODS

Samples. The company Grupo Hespérides Biotech S.L. carried out the controlled alcoholic fermentation by using a novel process (patent WO2012/066176A120120524). A commercial pasteurized orange juice made from Citrus sinensis L. var. Navel late (Huelva, Spain) was used as starting material. The criteria for selection of this orange juice were the compositional homogeneity, microbiological stability, and organoleptic quality. These aspects are necessary for adequate development of the fermentation process and consumer acceptance of the final product. The yeast strain Pichia kluyveri var. kluyveri (Saccharomycetaceae family), isolated by Grupo Hespérides Biotech S.L. from the natural microbiota present in the orange fruit, was used as the inoculum. This yeast was selected for producing a low alcohol percentage (0.8−1.2% v/v) in the final product. The fermentation was carried out in a PVC tank (5 L) at 20 °C for 15 days in repose. Before sample collection, the fermentation liquid was agitated and mixed by the use of magnetic agitator to promote sample homogenization. Samples were aseptically collected on alternate days throughout the fermentation period (days 0, 1, 3, 5, 7, 9, 11, 13, and 15) and immediately stored at −20 °C until analysis. Two simultaneous fermentation processes were performed under identical conditions. Table 1 summarizes the quality parameters in both orange juice and

Table 1. Quality Parameters of Orange Juice before and after Fermentation quality parametera

orange juice

fermented orange juice

pH TAb (g citric acid/L) total glucids (g/L) reducing sugars (g/L) nonreducing sugars (g/L) TSSc (°Brix) % pulp alcohol (% v/v)

3.48 ± 0.20 8.48 ± 0.02 78.20 ± 5.64 48.50 ± 3.63 29.70 ± 2.01 11.00 ± 0.50 12.00 ± 2.00 0.00

3.43 ± 0.20 8.85 ± 0.02 53.70 ± 4.65 24.70 ± 2.13 29.00 ± 2.51 10.00 ± 0.50 8.00 ± 0.50 0.87 ± 0.01

Values are expressed as the mean ± standard deviation. titratable acidity. cTSS, total soluble solids.

a

b

TA,

fermented product: pH, tritatable acidity (TA), total glucids (reducing and nonreducing sugar), total soluble solids (TSS) (°Brix), pulp content (%), and alcohol (% v/v).22 Chemicals and Reagents. HPLC grade methanol and acetone were supplied by BDH Prolabo (VWR International Eurolab, S.L., Barcelona, Spain). Diethyl ether containing 7 ppm of butylated hydroxytoluene (BHT) was purchased from Scharlau S.L. (Barcelona, Spain). HPLC grade deionized water was produced with a Milli-Q 50 system (Millipore Iberica S.A., Madrid, Spain). The remaining reagents were all of analytical grade and purchased from Sigma-Aldrich Quı ́mica S.A. (Madrid, Spain). Carotenoid Extraction. Carotenoid pigments were extracted from the nonfermented and fermented orange juice samples using the method of Mı ́nguez-Mosquera and Hornero-Méndez23 with some modifications. Briefly, an aliquot (10 mL) of a previously shaken sample was centrifuged at 10000g and 4 °C for 10 min to collect the solids in suspension. The pellet containing the carotenoid pigments was extracted with acetone (3 mL) containing 0.5% (w/v) BHT and centrifuged at 5000g and 4 °C for 5 min. The extraction step was repeated once, and both extracts were combined and dried under a nitrogen stream in a round-capped 15 mL polypropylene centrifuge tube. A saponification step was included to simplify the complexity of the HPLC carotenoid profiles by hydrolyzing the carotenoid acyl esters naturally present in orange juice. The dried extract was dissolved 843

dx.doi.org/10.1021/jf404589b | J. Agric. Food Chem. 2014, 62, 842−849


Article

Figure 1. C18 reversed-phase HPLC chromatogram obtained for a saponified carotenoid extract prepared from the orange juice used for fermentation. See text for chromatographic conditions. Peak identification and characterization are given in Table 2.

Table 2. Chromatographic and Spectral Characteristics of the Carotenoid Pigments Present in the Orange Juice Used for Fermentation UV−visible spectrum peak

tR (min)

λmax (nm) in HPLC mobile phase

5.38 5.86 6.05 6.31 6.71 6.87 7.27

401, 424, 401, 423, 399, 422, 420,

424, 450, 424, 448, 421, 445, 442,

450 476 450 476 446 471 469

8 9 10 11 12 13 14 15 16 17 18 19

latochromea karpoxanthina neochrome karpoxanthin isomera neochrome isomer karpoxanthin isomera not identified (lutein-like chromophore) not identified luteoxanthin auroxanthin epimer 1 auroxanthin epimer 2 mutatoxanthin epimer 1 auroxanthin epimer 3 mutatoxanthin epimer 2 (all-E)-zeaxanthin (all-E)-lutein (Z)-mutatoxanthina (Z)-mutatoxanthina (9Z)-lutein

7.37 8.57 8.68 8.79 9.03 9.18 9.31 9.40 9.61 9.82 9.95 10.23

420, 397, 384, 384, 411, 384, 411, 426, 424, 328, 328, 330,

442, 420, 403, 403, 430, 403, 430, 452, 449, 405, 404, 424,

465 446 427 427 457 427 457 480 476 425, 449 426, 448 443, 470

20

(13Z)-lutein

10.45

334, 422, 444, 472

21 22 23 24

β-cryptoxanthin ζ-carotene (all-E)-α-carotene (all-E)-β-carotene

14.15 20.27 20.61 20.72

428, 380, 422, 430,

1 2 3 4 5 6 7

a

carotenoid

454, 401, 446, 454,

λmax (nm) according to the lit.b 398, 419, 398, 419, 400, 419,

422, 448; ethanol 443,472; ethanol 421, 448; acetone 443,472; ethanol 423, 448; acetone 443, 472; ethanol

400, 423, 377, 398, 377, 398, 409, 428, 377, 398, 409, 428, 430, 452, 422, 445, nad na 330, 422, ethanol 332, 422, ethanol 428, 450, 378, 400, 424, 448, 429, 452,

480 425 475 480

% III/II

epoxide test

LC-MS (APCI) m/z [M + H]+

100 94 88 94 87 90 nd

− − − − − − −

619 ndc 601 nd 601 nd nd

nd 105 94 94 87 94 85 20 65

nd 601 601 601 585 601 585 569 569 585 585 569

444, 474;

63

− + − − − − − − − − − −

442, 472;

54

−

569

18 100 55 15

− − − −

553 541 537 537

448; 423; 423; 456; 423; 456; 479; 474;

478; 425; 476; 478;

acetone acetone acetone ethanol acetone ethanol acetone ethanol

ethanol hexane acetone acetone

Tentative identification. bBritton et al.6 cnd, not determined. dna, not available. Carotenoid Analysis and Quantification by Liquid Chromatography−Mass Spectrometry (LC-MS (APCI+)). LC-MS was performed by coupling a chromatographic system with a Micromass ZMD4000 mass spectrometer equipped with a single-quadrupole analyzer (Micromass Ltd., Manchester, UK) and an atmospheric

Provitamin A carotenoid content was expressed as retinol activity equivalents per liter of juice (RAEs/L) following the method of Scott and Rodrı ́guez-Amaya,29 with 1 RAE corresponding to 12 μg of (allE)-β-carotene or 24 μg of Z isomers of β-carotene or any other carotenoid containing one unsubstituted β-ring. 844



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Figure 2. Chemical structures of the carotenoid pigments present in the orange juice used for fermentation.

lutein, β-cryptoxanthin, and several carotenoids containing 5,8epoxy groups in their structure (neochrome, mutatoxanthin, luteoxanthin, auroxanthin, and latochrome), which are respectively derived from neoxanthin, antheraxanthin, violaxanthin, and latoxanthin by the rearrangement of 5,6-epoxy groups under acidic conditions.30 Figure 2 depicts the chemical structures of these pigments. Several geometric isomers corresponding to Z isomers of mutatoxanthin and lutein were also identified. Latochrome was only tentatively identified due to the lack of a pure standard. The [M + H]+ value (m/z 619) was consistent with the formula C40H58O5 (MW = 618.4284) and the presence of at least three to four hydroxy groups, explaining the higher polarity (shorter retention time) with respect to neochrome. The UV−visible absorption maxima at 401, 424, and 450 nm were in agreement with a chromophore of eight conjugated double bonds (as in the case of luteoxanthin) and with the presence of a 5,8-epoxy group, as supported by the negative results of the epoxide test. These data were in accordance with Meléndez-Martı ́nez et al., who reported that numerous authors had erroneously identified latoxanthin (5′,6′-epoxy-5,6,5′,6′-tetrahydro-β,β-carotene3,5,6,3′-tetrol) and its 5,8-epoxy derivative, latochrome, as neoxanthin in citrus fruit.31 The presence of latochrome in orange juice was previously reported by Gross et al.,32 but no further identification work has been carried out in this vegetable product. The UV−visible spectra of latoxanthin (417, 440, 468 nm, %I II/II = 95, in ethanol) and latochrome (398, 422, 448 nm, % III/II = 95, in ethanol) are highly similar to the spectra

pressure chemical ionization (APCI) probe. The system was controlled with MassLynx 3.2 software (Micromass Ltd., Manchester, UK). MS conditions were as follows: positive ion mode (APCI+); source temperature, 150 °C; probe temperature, 400 °C; corona voltage, 3.7 kV; high-voltage lens, 0.5 kV; and cone voltage, 30 V. Nitrogen was used as the desolvation and cone gas at 300 and 50 L/h, respectively. Mass spectra were acquired within the m/z 300−1200 range. The chromatographic conditions were as described above for carotenoid analysis and quantification. Statistical Analysis. The fermentation process was performed in duplicate, and all samples were analyzed in triplicate. Results are expressed as the mean ± standard deviation. Analysis of variance (oneway ANOVA; Duncan’s test) was applied to establish significant differences between the values of carotenoid and provitamin A carotenoid content obtained during the fermentation. A probability value of p < 0.05 was considered as the criteria for significance. These analyses were carried out by SPSS15.0 software (SPSS Inc., Chicago, IL, USA).

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RESULTS AND DISCUSSION Carotenoid Composition of Orange Juice Used in the Fermentation. Figure 1 depicts the chromatogram corresponding to the carotenoid profile present in the orange juice used in the fermentation process, and Table 2 summarizes the chromatographic and spectroscopic data (UV−visible and mass spectra). Twenty-two carotenoids, including some Z geometrical isomers, were identified in the saponified extracts of the orange juice. The carotene fraction was composed of βcarotene, α-carotene, and ζ-carotene, whereas the xanthophyll group was more complex, being composed of zeaxanthin, 845


846

c

4.82 ± 0.31b 71.27 ± 3.86a

0.04b 0.02b 0.01b 0.02b 0.01b 0.02ab 0.02b 0.00b 0.00a 0.00a 0.00a 0.04a 0.01a 0.00ab 0.00ab

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± 0.05ac 0.03a 0.01a 0.03a 0.01a 0.02a 0.01a 0.00a 0.00ab 0.00ab 0.00ab 0.04ab 0.03a 0.00abc 0.00ab

0.00a 0.01ab 0.02a 0.01ab 0.00ab 0.00ab

3 days

5.38 ± 0.32a 74.38 ± 4.40ab

0.15 0.23 0.37 0.24 0.10 0.10 tr 0.67 0.44 0.29 0.31 0.32 0.39 0.30 0.13 0.05 0.02 0.09 0.70 0.36 0.04 0.08 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± 0.03cde 0.01a 0.01a 0.02ab 0.00a 0.02b 0.00ab 0.00a 0.00abc 0.00a 0.00a 0.02ab 0.00a 0.00b 0.00a

0.00a 0.00bc 0.02ac 0.00ab 0.00b 0.00a

5 days

5.41 ± 0.17a 74.46 ± 2.24ab

0.15 0.23 0.41 0.23 0.11 0.10 tr 0.74 0.44 0.28 0.30 0.32 0.33 0.27 0.13 0.05 0.02 0.09 0.72 0.35 0.04 0.07 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± 0.00acd 0.05cd 0.03c 0.03ac 0.03c 0.03cd 0.02cd 0.01c 0.00bcd 0.00bcd 0.00bc 0.05bc 0.02b 0.00cde 0.00bc

0.00ac 0.02cd 0.04c 0.01bc 0.01bc 0.00bcd

7 days

5.94 ± 0.41c 81.86 ± 5.82bc

0.16 0.26 0.42 0.26 0.12 0.11 tr 0.71 0.51 0.32 0.33 0.36 0.43 0.32 0.14 0.05 0.02 0.10 0.77 0.42 0.04 0.08 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± 0.02ef 0.00 cd 0.00cd 0.00ac 0.00c 0.00de 0.00de 0.00cd 0.00cde 0.00cd 0.00c 0.02cd 0.01bc 0.00def 0.00cd

0.00de 0.01de 0.01cd 0.01cd 0.00cd 0.00d

9 days

6.28 ± 0.12 cd 86.07 ± 3.32cd

0.17 0.27 0.45 0.27 0.12 0.11 tr 0.81 0.53 0.33 0.34 0.37 0.44 0.34 0.15 0.05 0.02 0.10 0.80 0.43 0.04 0.09 0.04de 0.01d 0.01cd 0.01ac 0.02cd 0.02e 0.02de 0.00cd 0.00de 0.00d 0.01c 0.03d 0.00c 0.00f 0.00d

0.01cd 0.01de 0.01cd 0.01d 0.00cd 0.00d

6.41 ± 0.23d 90.70 ± 3.97d

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ±

11 days 0.17 0.28 0.44 0.29 0.13 0.12 tr 0.78 0.54 0.34 0.32 0.38 0.47 0.35 0.15 0.06 0.02 0.11 0.85 0.46 0.05 0.09 0.07f 0.03c 0.02d 0.05ac 0.01d 0.01e 0.00e 0.00d 0.00de 0.00d 0.01c 0.07cd 0.04bc 0.00ef 0.00cd

0.01e 0.01e 0.02cd 0.02cd 0.01cd 0.01cd

6.47 ± 0.28d 86.32 ± 7.98cd

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ±

13 days 0.18 0.29 0.47 0.27 0.12 0.11 tr 0.86 0.49 0.36 0.35 0.40 0.47 0.36 0.16 0.06 0.02 0.10 0.81 0.44 0.05 0.09

0.03f 0.02d 0.00d 0.02c 0.00d 0.00e 0.00e 0.00d 0.00e 0.00d 0.00c 0.01d 0.01c 0.00ef 0.00cd

0.00e 0.00e 0.01d 0.00d 0.00d 0.00d

6.65 ± 0.07d 90.57 ± 1.53d

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ±

15 days 0.18 0.29 0.46 0.29 0.13 0.12 tr 0.86 0.55 0.36 0.36 0.40 0.47 0.36 0.16 0.06 0.03 0.11 0.85 0.46 0.05 0.09

The values are the mean of three determinations ± standard deviation. Values with different letters (a−f) in the same row are significantly different (p < 0.05) as determined by Duncan’s test. btr, traces. Provitamin A content calculated according to Scott and Rodrı ́guez-Amaya.29

a

5.37 ± 0.21a 75.32 ± 3.58ab

total carotenoid content provitamin A (RAEs/L)c

0.03a 0.01a 0.01a 0.01a 0.01a 0.02ac 0.01a 0.00a 0.00ab 0.00abc 0.00ab 0.03ab 0.02a 0.00acd 0.00ab

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

± ± ± ± ± ± 0.01b 0.01a 0.02b 0.01a 0.00a 0.00a

1 day

0.00a 0.01ab 0.01a 0.01ab 0.00ab 0.00abc

± ± ± ± ± ± 0.13 0.21 0.32 0.21 0.09 0.10 tr 0.55 0.38 0.25 0.26 0.29 0.36 0.27 0.11 0.04 0.02 0.08 0.66 0.36 0.04 0.07

0 days

0.15 0.22 0.37 0.23 0.10 0.10 trb 0.65 0.43 0.29 0.31 0.32 0.40 0.30 0.13 0.05 0.02 0.09 0.71 0.37 0.04 0.08

carotenoid

latochrome karpoxanthin neochrome karpoxanthin isomer neochrome isomer karpoxanthin isomer luteoxanthin auroxanthin epimer 1 auroxanthin epimer 2 mutatoxanthin epimer 1 auroxanthin epimer 3 mutatoxanthin epimer 2 (all-E)-zeaxanthin (all-E)-lutein (Z)-mutatoxanthin (Z)-mutatoxanthin (9Z)-lutein (13Z)-lutein β-cryptoxanthin ζ-carotene (all-E)-α-carotene (all-E)-β-carotene

concentrationa (mg/L) at a fermentation time of

Table 3. Evolution in the Carotenoid Composition and Provitamin A Content of Orange Juice during the Fermentation Process

Journal of Agricultural and Food Chemistry Article



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ment.39 Fermented chickpeas showed a higher total phenolic content than the unstarted product,40 and fermentation of red sorghum increased the flavonoid content in the final product.19 Our results show that the carotenoid profile at the end of the fermentation process was similar to that of the nonfermented orange juice, with no apparent degradation and/or transformation of carotenoids associated with the processing (Table 3). Carotenoids containing 5,6-epoxy groups were already converted into the corresponding carotenoid 5,8-epoxy derivatives in the nonfermented juice due to the close contact of the pigments with the organic acids naturally present in the starting material (commercial orange juice). In addition, the fact that no increase in Z isomer content was observed during the fermentation suggests that the applied conditions were very smooth and did not affect the integrity of the pigment structures. Hence, the 5,8-epoxy carotenoids and Z isomers in the fermented orange juice would have been formed during the industrial production of the commercial orange juice, including the pasteurization. Auroxanthin (1.77 mg/L; sum of three epimers) and β-cryptoxanthin (0.85 mg/L) also were the most abundant pigments in fermented orange juice (at the end of the process). The sum of the epimers of mutatoxanthin reached a remarkable value of 0.76 mg/L. (all-E)-Zeaxanthin, ζ-carotene, neochrome, and (all-E)-lutein were quantified at similar amounts (0.47, 0.45, 0.46, and 0.36 mg/L, respectively). Karpoxanthin and one of its isomer reached a final content of 0.29 mg/L. The other pigments also were detected in minor concentrations (0.03−0.18 mg/L) in fermented orange juice, as in orange juice (Table 3). As a general trend, the concentration of all individual carotenoids were significantly higher (p < 0.05) after 15 days of the fermentation processing with respect to the original juice. The total carotenoid content, expressed as the sum of individual carotenoid concentrations, showed a significantly higher value (p < 0.05) after the fermentation period (6.65 mg/ L) when compared with the initial content (5.37 mg/L), these data being similar to those reported by Lee and Coates (5.70 mg/L).41 Because de novo biosynthesis of carotenoids is not possible after the processing of orange juice, a feasible explanation for their apparent increase in concentration during fermentation may be due to the occurrence of some changes in the internal structure of the suspended solids, in which the pigments are integrated, enhancing their extractability during the analytical procedure. This aspect is of particular interest because it would indirectly indicate an increased bioaccessibility of the carotenoids in the fermented juice, so that further research should be carried out to confirm this hypothesis. Carotenoids have been implicated in several biological processes including cardiovascular protection,42 immune responses,43 antioxidant activity,44 or bone metabolism.11 Humans need to acquire carotenoids through their diet because they are not able to synthesize them de novo. According to our results, fermentation of orange juice induces an apparent increase of carotenoid content, so the fermented orange juice could be considered as a good source of these bioactive compounds and may have potentially beneficial effects already tested in orange juice. In addition, there is a remarkable presence of carotenoids with provitamin A activity (β-carotene, α-carotene, and β-cryptoxanthin) in the samples, because these compounds have also been assigned with important biological activities (antioxidant capacity, reducing the risk of atherosclerosis process).44,45 The provitamin A value obtained in the present study for the starting orange juice

of neoxanthin (418, 442, 471 nm, % III/II = 85, in ethanol) and neochrome (398, 421, 448 nm, % III/II = 80, in ethanol), respectively. This similarity and their analogous chromatographic behaviors would likely explain the misidentification of these pigments in citrus fruit. For this reason, a complete experimental characterization is essential for their unambiguous identification. In addition, three peaks (eluting at 5.86, 6.31, and 6.87 min) with λmax at 419, 443, and 472 nm (% III/II = 90) were detected, consistent with a chromophore of nine conjugated double bonds plus a β-end ring.30 The negative epoxide test result and the shorter retention time with respect to the dihydroxylated xanthophylls (lutein and zeaxanthin) supported the tentative identification of these pigments as karpoxanthin (5,6-dihydro-β,β-carotene-3,3′,5,6-tetrol) isomers. Unfortunately, no mass spectra could be recorded for these minor chromatographic peaks. Nevertheless, given that latochrome is the 5,8-epoxy derivative of karpoxanthin, it can be conjectured that karpoxanthin was present. Table 3 reports the carotenoid profile and content of the samples. In relation to the quantitative analysis of carotenoids in the orange juice, auroxanthin (1.39 mg/L; sum of three epimers) and β-cryptoxanthin (0.71 mg/L) were the most abundant xanthophylls among the 22 carotenoid pigments identified in this study. As mentioned before, auroxanthin derived from violaxanthin under the acidic conditions of orange juice. These observations were in agreement with previous studies reporting β-cryptoxanthin and violaxanthin as the major carotenoids in orange juice.4,33 Luteoxanthin, the intermediate product between violaxanthin and auroxanthin, was detected in trace amounts. (all-E)-Zeaxanthin, ζ-carotene, and (all-E)lutein were present at 0.40, 0.37, and 0.30 mg/L, respectively. Two epimers of mutatoxanthin (8R and 8S) were detected in orange juice with concentrations of 0.29 and 0.32 mg/L, which derived from antheraxanthin, their 5,6-epoxy precursor, originally present in the fresh juice. Geometric carotenoid isomers were detected in minor concentrations in orange juice, including (9Z)- and (13Z)-lutein and two (Z)-mutatoxanthin isomers, were found at concentrations ranging from 0.02 to 0.13 mg/L. The carotenoid composition of characterized orange juice was similar to other studies,7 but comparisons of the carotenoid profiles of orange juices are notoriously difficult. Their composition depends on the variety of the fruit, climate, stage of maturity, industrial processing, and storage conditions, among other factors. Mouly et al. were able to use the carotenoid profile to identify the geographic origin (cultivation and/or production) of orange juice samples.34 Effect of Alcoholic Fermentation on the Carotenoid Composition of Orange Juice. A limited number of authors have evaluated the effect of fermentation on carotenoid composition in other fruits and vegetables, and the results reported are diverse. Fermentation of carrot slices resulted in carrot chips with higher α-carotene content.35 Fermented sweet potatoes presented higher β-carotene concentration than the starting product.36 Grape must showed increased neoxanthin, violaxanthin, and luteoxanthin contents and decreased αcarotene content during alcoholic fermentation.37 Koh et al. did not find significant variation in lycopene content of tomato juice after fermentation.38 The type of microorganism used and the duration of the fermentation process could explain these differences. In contrast, the influence of fermentation on the content of other bioactive compounds has been widely studied. Fermentation of onion led to an increase in flavononoid metabolites and its consequent biological activity enhance847



Article

Barberán, F. A., Robins, R. J., Eds.; Clarendon Press: Oxford, UK, 1997; pp 11−27. (5) Fratianni, A.; Giuzio, L.; Di Criscio, T.; Zina, F.; Panfili, G. Response of carotenoids and tocols of durum wheat in relation to water stress and sulfur fertilization. J. Agric. Food Chem. 2013, 61, 2583−2590. (6) Britton, G.; Liaanen-Jensen, S.; Pfander, H. Carotenoid Handbook; Birkhäuser Verlag: Basel, Switzerland, 2004. (7) Meléndez-Martínez, A. J.; Vicario, I. M.; Heredia, F. J. Review: analysis of carotenoids in orange juice. J. Food Compos. Anal. 2007, 20, 638−649. (8) Franke, A. A.; Cooney, R. V.; Henning, S. M.; Custer, L. J. Bioavailability and antioxidant effects of orange juice components in humans. J. Agric. Food Chem. 2005, 53, 5170−5178. (9) Meléndez-Martínez, A. J.; Vicario, I. M.; Heredia, F. J. Rapid assessment of vitamin A activity through objective color measurements for the quality control of orange juices with diverse carotenoid profiles. J. Agric. Food Chem. 2007, 55, 2808−2815. (10) Mozaffarieh, M.; Sacu, S.; Wedrich, A. The role of the carotenoids, lutein and zeaxanthin, in protecting against age-related macular degeneration: a review based on controversial evidence. Nutr. J. 2003, 11, 2−20. (11) Sugiura, M.; Nakamura, M.; Ogawa, K.; Ikoma, Y.; Ando, F.; Shimokata, H.; Yano, M. Dietary patterns of antioxidant vitamin and carotenoid intake associated with bone mineral density: findings from post-menopausal Japanese female subjects. Osteoporosis Int. 2011, 22, 143−152. (12) Chatterjee, M.; Roy, K.; Janarthan, M.; Das, S.; Chatterjee, M. Biological activity of carotenoids: its implications in cancer risk and prevention. Curr. Pharm. Biotechnol. 2012, 13, 180−190. (13) Rivera, S. M.; Vilaró, F.; Zhu, C.; Bai, C.; Farré, G.; Christou, P.; Canela-Garayoa, R. Fast quantitative method for the analysis of carotenoids in transgenic maize. J. Agric. Food Chem. 2013, 61, 5279− 285. (14) Alferez, F.; Pozo, L. V.; Rouseff, R. R.; Burns, J. K. Modification of carotenoid levels by abscission agents and expression of carotenoid biosynthetic genes in ‘Valencia’ sweet orange. J. Agric. Food Chem. 2013, 61, 3082−3089. (15) Stinco, C. M.; Fernández-Vázquez, R.; Escudero-Gilete, M. L.; Heredia, F. J.; Meléndez-Martínez, A. J.; Vicario, I. M. Effect of orange juice’s processing on the color, particle size, and bioaccessibility of carotenoids. J. Agric. Food Chem. 2012, 60, 1447−1455. (16) Mena, P.; Ascacio-Valdés, J. A.; Gironés-Vilaplana, A.; Del Rio, D.; Moreno, D. A.; García-Viguera, C. Assessment of pomegranate wine lees as a valuable source for the recovery of (poly)phenolic compounds. Food Chem. 2014, 145, 327−334. (17) Pérez-Gregorio, M. R.; Regueiro, J.; Alonso-González, E.; Pastrana-Castro, L. M.; Simal-Gándara, J. Influence of alcoholic fermentation process on antioxidant activity and phenolic levels from mulberries (Morus nigra L.). LWT−Food Sci.Technol. 2011, 44, 1793− 1801. (18) Ajila, C. M.; Brar, S. K.; Verma, M.; Tyagi, R. D.; Valéro, J. R. Solid-state fermentation of apple pomace using Phanerocheate chrysosporium − liberation and extraction of phenolic antioxidants. Food Chem. 2011, 126, 1071−1108. (19) Svensson, L.; Sekwati-Monang, B.; Lutz, D. L.; Schieber, A.; Ganzle, M. G. Phenolic acids and flavonoids in nonfermented and fermented red sorghum (Sorghum bicolor (L.) Moench). J. Agric. Food Chem. 2012, 58, 9214−9220. (20) Yang, E. J.; Kim, S. I.; Park, S. Y.; Bang, H. Y.; Jeong, J. H.; So, J. H.; Rhee, I. K.; Song, K. S. Fermentation enhances the in vitro antioxidative effect of onion (Allium cepa) via an increase in quercetin content. Food Chem. Toxicol. 2012, 50, 2042−2048. (21) Wang, J. J.; Tung, T. H.; Yin, W. H.; Huang, C. M.; Jen, H. L.; Wei, J.; Young, M. S. Effects of moderate alcohol consumption on inflammatory biomarkers. Acta Cardiol. 2008, 63, 65−72. (22) Office Internationale de la Vigne & du Vin. Recueil des Mèthodes Internationales d’analyse des Vins; OIV: Paris, France, 1990.

(75.32 RAEs/L) was similar to those reported by MeléndezMartı ń ez (9.69−94.8 RAEs/L) for different types of commercial orange juice marketed in Spain.46 As observed for the individual carotenoids, provitamin A content was significantly higher (p < 0.05) in the fermented juice (at day 15 of processing) (90.57 RAEs/L) than in original substrate (75.32 RAEs/L) (Table 3). Some of the most relevant carotenoids with biological activities related with human health (lutein, β-cryptoxanthin, and β-carotene) showed a significantly higher content after fermentation, as indicated above. Consequently, the increase in the carotenoid extractability observed in the fermented orange juice should be considered as an advantage with respect to the original juice. In summary, a potential novel beverage of fermented orange juice may be a rich source of carotenoids and provitamin A compounds, which could exert healthy effects similarly to the original orange juice. Moreover, the controlled alcoholic fermentation would be of great interest to the citrus industry due to the recent interest in the development of innovative practices to meet the demand for natural products rich in bioactive compounds by the consumer, in consonance with their increasing concern about diet and health. In vivo and intervention studies are currently being performed to evaluate the bioavailability of the bioactive compounds of this novel orange drink and its potential effects derived from both the bioactive compounds and the low alcoholic degree. Subsequently, the assessment of its sensory characteristics and consumer acceptance is also necessary.

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AUTHOR INFORMATION

Corresponding Author

*(M.-S.F.-P.) Phone: 34 954977613: Fax: 34 954349813. Email: [email protected]. Funding

This study was supported by funding from the Ministerio de Ciencia e Innovación (Spanish government, Project AGL201014850/ALI) and the Consejerı ́a de Economı ́a, Innovación, Ciencia y Empleo (Junta de Andalucı ́a, Projects P08-AGR03477 and P09-AGR4814, and Grupo PAI BIO311). The Research Project grant of B.E.-L. is supported by the Junta de Andalucı ́a. D.H.-M. is member of the IBERCAROT Network 112RT0445 financed by CYTED. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Grupo Hespérides Biotech S.L. for providing the samples and to Richard Davies for editorial assistance.

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REFERENCES

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Effect of Alcoholic Fermentation on the Carotenoid Composition and

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