Proc. Fla. State Hort. Soc. 123:207–212. 2010.

A Comparison of Processed and Fresh Squeezed ‘Hamlin’ —Nutrients and Phytonutrients

Jinhe Bai, Bryan L. Ford, John A. Manthey, and Elizabeth A. Baldwin* USDA-ARS and Subtropical Products Laboratory, Winter Haven, FL33881

Additional index words. flavonoid, limonoid, alkaloid, carotenoid, ascorbic acid, total phenolic content ‘Hamlin’ orange were prepared using one of following methods: 1) freshly squeezed with a commercial food service squeezer (fresh), 2) freshly squeezed and pasteurized (fresh/pasteurized), and 3) processed with industrial extractor and pasteurized (processed). Samples were taken from the juices directly after extraction and again after 4 d of juice storage at 5 °C for analysis of flavonoids, limonoids, alkaloids, carotenoids, ascorbic acid and total phenolic content. Processed juice had higher levels of insoluble solids, but lower levels of peel oil in comparison to fresh juice regardless of . The majority of flavonoid glycosides including hesperidin, narirutin, narirutin 4´-glucoside, 6,8-di-C- glucosyl apigenin and isosakuranetin rutinoside, which are rich in the albedo and segment membranes, occurred at the highest levels in processed juice. In contrast, the polymethoxylatedflavones associated with peel oil, such as quercetagetin hexamethyl ether, sinensetin, nobiletin, tetramethylscutellarein, heptamethoxyflavone and tangeretin occurred at the highest levels in the fresh juices. Limonoids (limonin glucoside, limonin aglycone, nomilin glucoside, nomilinic acid glucoside, and nomilin aglycone), alkaloids (feruloyl putrescine and an unknown alkaloid) and carotenoids (zeaxanthin, lutein, ß-cryptoxanthin, α-carotene and ß-carotene) occurred at higher levels in the processed juice than in the fresh juice regardless of pasteurization. The processed juice had higher total phenolic content but lower ascorbic acid content than the fresh juices. Thermal pasteurization increased the contents of the polymethoxylated flavones, but decreased the contents of carotenoids. During 4-d storage at 5 °C, 20% to 80% of hesperidin, narirutin, narirutin 4´-glucoside and isosakuranetin rutinoside precipitated in the processed juice but not the fresh juice.

Orange juice is popular worldwide for flavor and nutrition Florida, Jan. 2010, and juiced by three different methods: fresh (Baldwin, 1993). The nutritional value of orange is most squeezed (fresh) using a commercial food service , fresh importantly linked not only to the high levels of vitamins and squeezed then pasteurized (fresh/pasteurized), and processed minerals, but also to the many phytonutrients, including the flavo- using an industrial extractor (set for single-strength juice) and noids (Gattuso et al., 2007; Nogata et al., 2006), limonoids (Maier finisher, and then pasteurized (processed). Pasteurization condi- et al., 1980; Manners et al., 2003), carotenoids (Dhuique-Mayer tions were 90 °C for 10 s with a flow rate of 1.2 L·min–1. Details et al., 2005; Dhuique-Mayer et al., 2007), ascobic acid (O’Neil for the materials and processing conditions can be found in Bai and Nicklas, 2008) and other phenolic compounds (Rapisarda et al. (2010). et al., 1999). After processing, all samples were cooled quickly to 5 °C with There is a perception by some that the nutritional value of orange an ice bath, and stored at 5 °C for 4 d. Three replicate samples per juice decreases during processing and storage, and that freshly treatment were taken at day 0 and day 4 after juicing. Analyses squeezed juice is more nutritious. The latter is also perceived as were carried out immediately or after frozen storage at –20 °C being less adulterated. In this study, the nutritional value of fresh or –80 °C. juice prepared with a commercial juicer – a type typically used in Analysis of insoluble solids, flavonoids, limonoids, and restaurants and institutional food services- was compared to that alkaloids. Juice samples were separated into pellets and super- of a processed juice extracted by an industrial extractor, passed natants by centrifugation at 27,000 gn for 30 min. Pellets were through a finisher, and thermally pasteurized. A portion of the resuspended in deionized water (equivalent volume as original fresh juice was also thermally pasteurized to differentiate the juice) and then centrifuged again at 27,000 gn for 30 min. The effects of extraction and finishing from pasteurization. final pellets were vacuum dried at 55 °C prior to being analyzed. At each step, the amount of juice, supernatant, and pellet were Material and Methods measured by weight to calculate the insoluble solids content. Supernatant samples were passed through a 0.45 μm PTFE Source of fruit and processing methods. ‘Hamlin’ oranges filter and 50 μL of 0.13 mg·mL–1 mangiferin was added to 950 were harvested from a commercial orchard located in central μL filtered supernatant as an internal standard. For pellet sample preparation, 100 mg well-ground dry pellet with 3.0 mL of di- Mention of a trademark or proprietary product is for identification only and does methyl sulfoxide (DMSO) was placed in a Teflon gasket screw-top not imply a guarantee or warranty of the product by the U.S. Department of Ag- test tube and shaken for 18 h with an orbital shaker (VSOS-4P; riculture. The U.S. Department of Agriculture prohibits discrimination in all its Pro Scientific, Oxford, CT) at 120 rpm at room temperature. programs and activities on the basis of race, color, national origin, gender, religion, The extraction was then passed through a 0.45 μm PTFE filter age, disability, political beliefs, sexual orientation, and marital or family status *Corresponding author; phone: (863) 293-4133, ext. 120; email: Liz.Baldwin@ and mangiferin was added as internal standard prior to analysis ars.usda.gov by HPLC-MS.

Proc. Fla. State Hort. Soc. 123: 2010. 207 A Waters 2695 Alliance HPLC (Waters, Medford, MA) con- Peel oil. Peel oil content was analyzed according to Scott- nected in parallel with a Waters 996 PDA detector and a Waters/ Veldhuis bromate titration method (Scott and Veldhuis, 1966). Micromass ZQ single quadrupole mass spectrometer equipped Statistical analysis. SAS Version 9.1 (SAS Institute, Cary, with an electrospray ionization source was used for the analysis. NC) was used for analysis of instrumental analytical data. Each Compound separations were achieved with a Waters XBridge quality attribute with three replicateswas analyzed using analysis C8 column (4.6 × 150 mm), using solvent gradient conditions of variance (ANOVA). The treatment means were separated at as reported previously (Manthey, 2008).Data handling was done the 0.05 significance level by least squares means test (LSD). with MassLynx software version 3.5 (Micromass, Division of Waters Corp., Beverly, MA). Post column split to the PDA and Results and Discussion mass ZQ detector was 10:1. MS parameters were as follows: ionization mode, ES+; capillary voltage 3.0 kV; extractor voltage Insoluble solids and peel oil. Processed juice had 0.52% 5 V; source temperature 100 °C; desolvation temperature 225 °C; (w/w) insoluble solids (precipitated pellet material), 37% higher –1 –1 desolvation N2 flow 465 L·h , cone N2 flow 70 L·h ; scan range than in fresh juice (Table 1). The industrial extractor introduced m/z 150–1600; rate 1 scan/s; cone voltages 20 and 40 V. more albedo and segment membrane tissues into the juice, although Quantification of flavonoids and other secondary metabolites the finisher removed some of the juice vesicle walls. Fresh juice were made using either ESI-MS mass extracted Total Ion Chro- had 0.091% juice oil, over 4-fold higher than the processed juice matograms (TIC) obtained in scanning mode by the ZQ mass (Table 1). The thermal pasteurization did not change insoluble spectrometer, or by single ion response (SIR) mode. To normalize solids and peel oil content in the fresh juice (Table 1). These the mass spectrometer response during sequential runs, an inter- characteristics markedly influenced certain nutritional qualities nal standard, mangiferin, was additionally measured at 423 m/z. of the juices. Analysis of carotenoids. Carotenoids in pellet were analyzed Flavonoids. Eleven flavonoids, including five flavonoid -gly using an HPLC. Juice samples (30 mL) were centrifuged at 10,000 cosides and six polymethoxylated flavones were measured. The gn for 15 min. The pellet extracts were collected by dissolving flavanones, including hesperidin, narirutin, narirutin 4´-glucoside, pellets in acetone. The solution was injected into an HPLC (20 and isosakuranetinrutinoside exhibited more than two times higher µL loop) equipped with an YMC carotenoid column (Waters). concentrations in processed juice compared to fresh-squeezed Elution conditions included a three solvent gradient composed juice in both serum (Fig. 1) and pellets (Fig. 2). The contents initially of water/methanol/methyl tert-butyl ether (4/81/15, v/v/v), of these components in the pellets markedly increased during and changed with linear gradients to 4/6/90 (v/v/v) by 60 min at 4-d storage in the processed juice (Fig. 2), while decreased in a flow rate of 1 mL·min–1, at 30 °C. Compounds were detected the serum (Fig.1). The decreases in the levels of hesperidin and using a photo diode array (PDA) detector scanning 200–700 nm. isosakuranetin rutinoside in the processed juice serum were so Analysis of ascorbic acid (ASA) and dehydroascorbate great that after 4-d storage, the contents of these two compounds (DHA). Juice samples were blended with cold (4 °C) 6% tri- were lower than in the fresh juices (Fig. 1), indicating that the chloroacetic acid (TCA) (v/v, 1:1) at a setting of 4 for 30 s us- composition of the cloud of fresh juice may be different from ing a homogenizer (model PT 10/35, Brinkmann Instruments, that of processed juice, or the high content of oil in the fresh Switzerland). The homogenate was centrifuged for 20 min at juice stabilized turbid juice systems. The adsorption of the oil

12,000 gn at 4 °C, and the supernatant was used for ascorbic globules on the cloud particles enhances their stability in suspen- acid (ASA) and total ASA (which includes dehydroascorbic sion by decreasing their density (Mizrahi and Berk, 1970).The acid (DHA)) analysis using a 96-well microplate reader (Model processed juice contained more insoluble matter, mainly albedo SynergHT, BioTek, Winooski, VT) (Stevens et al., 2006). For and segment membranes, and all particles were broken. Only total ASA assay, supernatant 20 μL was mixed with 20 μL of 5 the particles with sizes smaller than 0.5 mm passed through the mM DTT (in 0.4 M phosphate buffer, pH 7.4) to reduce DHA to screen. In contrast, in the fresh juice, particle sizes were varied ASA, then incubated at 37 °C for 20 min. Then 10 μL of 0.5% with both broken and non-broken juice vesicles. Citrus cloud N-ethyl maleimide (w/v in water) was added and mixed. After 1 particles ranged in size from 0.4 to 5.0 μm, depending on pro- min at room temperature, 80 μL of color reagent was added and cessing method and type of fruit. cloud particles incubated at 37 °C for 40 min, the absorbance was read at 550 smaller than 2 μm are generally stable (Buslig and Carter, 1974; nm. The color reagent was made by mixing solutions A and B Klavons et al., 1994). Crystallization of hesperidin occurs dur- in a ratio of 2.75:1. Solution A consists of 31% orthophosphoric ing different stages of juice processing and storage (Kimball et acid, 4.6% TCA and 0.6% iron chloride (w/v) and solution B al., 2004; Perez-Cacho and Rouseff, 2008). Gil-Izquierdo et al. consists of 4% 2,2-dipyridyl in 70% ethanol (w/v). For ASA, (2001) reported that half of the soluble flavanones precipitate the assay was performed as described for total ASA except equal and integrate into the cloud fraction during storage at 4 °C. They amount of phosphate buffer (0.4 M, pH 7.4) was added instead of dithiothreitol (DTT) and N-ethyl maleimide solutions. DHA Table 1. Peel oil and insoluble solids content in ‘Hamlin’ orange juice.z value was obtained by [total ASA] – [ASA]. Peel oil (%) Insoluble solids (%) Total phenolic content. Total phenolic content (TPC) in supernatants and pellets, prepared as described in Analysis of Processed 0.020 b 0.52 a insoluble solids, flavonoids, limonoids and alkaloids, was as- Fresh squeezed 0.091 a 0.38 b sayed using Folin-Ciocalteu reagent as described by Singleton et Fresh/Pasteurized 0.084 a 0.33 b al.(1999). To eliminate ASA and DHA interference, ASA oxidase zValues followed by the different letters in the same column are signifi- was used to oxidize ASA to DHA and hydrogen peroxide was cantly different at P = 0.05 using Duncan’s multiple range test. y used to oxidize DHA before addition of Folin-Ciocalteu reagent Juice samples were centrifuged at 27,000 gn for 30 min. Pellets were (Ford et al., 2010). Total phenolic content was expressed as resuspended in water and centrifuged again, then vacuum dried at 55 hesperidin equivalence. °C. Values are percentages of dry pellets weight/initial juice weight.

208 Proc. Fla. State Hort. Soc. 123: 2010. Fig. 1. Effect of processing method and storage time on concentration of flavonoids, limonoids and alkaloids (µg·mL–1, excluding *alkaloid, which was expressed with relative peak area) in ‘Hamlin’ orange juice supernatants. Pasteurization condition was 90 °C for 10 s for both processed and

fresh/pasteurized juice. Supernatants were taken after centrifuging at 27,000 gn for 30 min. Vertical bars with the different letters are significantly different at P = 0.05 using Duncan’s multiple range test. reported that the flavanones precipitated in the cloud were not alkaloids (feruloyl putrescine and an unidentified alkaloid) oc- available for digestive absorption, and furthermore, were partly curred at higher concentrations in the serum of processed juice transformed to the corresponding chalcones during pancreatin- compared to the fresh juice, but this was reversed in the pellets bile digestion (Gil-Izquierdo et al., 2001). (Figs. 1–2). Pasteurization decreased the contents of the fol- The flavone glycoside, 6,8-di-C-glucosyl apigenin, in the serum lowing components in the pellets: limonin glucoside, limonin was higher in processed juice than in fresh juices, but this was aglycone, nomilin glucoside, nomilin aglycone and the unknown reversed in the pellets (Figs. 1–2). In contrast, all of the detected alkaloid, but not nomilinic acid glucoside and feruloyl putrescine polymethoxylated flavones occurred in much higher concen- (Figs. 1–2). However, there was no corresponding increase in trations in the fresh juices than in processed juice (Figs. 1–2). the serum for most of the compounds except nomilin aglycone, Pasteurization increased the contents of these compounds in the which completely precipitated in fresh juice (Figs. 1–2). After 4 pellets of the fresh juices by an unknown mechanism (Figs. 1–2). d storage, limonoids and alkaloids decreased in the pellets (Fig. Limonoids and alkaloids. Five limonoids and two alkaloids 2) and decreased or remained the same in the serum (Fig. 1). were measured in the differently prepared orange juice samples. Carotenoids. Five carotenoids were measured, three xantho- The limonoids (limonin glucoside, limonin aglycone, nomilin phylls that contain oxygen: zeaxanthin, lutein and ß-cryptoxanthin, glucoside, nomilinic acid glucoside, and nomilin aglycone) and and two carotenes which only contain carbon and hydrogen in

Proc. Fla. State Hort. Soc. 123: 2010. 209 Fig. 2. Effect of processing method and storage time on concentration of flavonoids, limonoids and alkaloids (µg·mL–1, excluding *alkaloid which was expressed with relative peak area) in ‘Hamlin’ orange juice pellets. Pasteurization condition was 90 °C for 10 s for both processed and

fresh/pasteurized juice. Pellets were taken after centrifuging at 27,000 gn for 30 min, and washed again using water, then extracted by dimethyl sulfoxide. Vertical bars with the different letters are significantly different at P = 0.05 using Duncan’s multiple range test. the molecules: α-carotene and ß-carotene (Fig. 3). ß-carotene, conditions do not damage provitamin A carotenoids.However, α-carotene, and ß-cryptoxanthin have vitamin A activity (meaning Lessin et al. (1997) did find a significant loss of provitamin A they can be converted to retinal), and these and other carotenoids carotenoids (ß-carotene, α-carotene, and ß-cryptoxanthin) due to can also act as antioxidants (Lee and Coates, 2003). Contents thermal processing. Lee and Coates (2003) also found thermal of all five carotenoids in the processed juice were almost twice processing caused carotenoid pigment (violaxanthin and anthe- as much as that in the fresh juice immediately after processing raxanthin) loss in orange juice. (Fig. 3). After 4-d storage at 5 °C, carotenoids decreased in the Ascorbic acid. The ASA content in the fresh juices was processed juice, but generally increased in fresh squeezed juice, higher than in the processed juice, and there was no significant regardless of pasteurization (Fig. 3). Pasteurization decreased decrease during 4-d storage (Fig. 4A). DHA is an oxidized form content of lutein, ß-cryptoxanthin, and α-carotene but not zea- of ASA, and can be reduced back to ASA. The amounts of DHA xanthin and ß-carotene (Fig. 3). Lee and Coates (1999, 2003) were equivalent to about 10% of the ASA (Fig. 4A and 4B). At showed that under the current processing conditions, there were day 0, pasteurization increased DHA content (Fig. 4B). However, no significant losses caused by pasteurization in major after 4 d storage, DHA increased in the fresh juice, but decreased carotenoids: ß-carotene and lycopene, neither in all five carotenoid in the pasteurized fresh juice (Fig. 4B).Although many studies compounds measured in this research in ‘Valencia’ orange juice. showed that the thermal pasteurization caused degradation of ASA Dhuique-Mayer et al. (2007) reported that common pasteurization (Johnson et al., 1995; Lima et al., 1999), Dhuique-Mayer et al.

210 Proc. Fla. State Hort. Soc. 123: 2010. Fig. 3. Effect of processing method and storage time on concentration of carotenoids in ‘Hamlin’ orange juices. Pasteurization condition was 90 °C for 10 s for both processed and fresh/pasteurized juice. Vertical bars with the different letters are significantly Fig. 4. Effect of processing method and storage time different at P = 0.05 using Duncan’s multiple range test. on concentration of ascorbic acid, dehydroascorbate, and total phenolic content in ‘Hamlin’ orange juices. Pasteurization condition was 90 °C for 10 s for both (2007) reported that the classical pasteurization time-temperature processed and fresh/pasteurized juice. Pellets were taken after centrifuging at 27,000 g for 30 min, and combinations did not damage ASA. n washed again using water, then extracted by dimethyl Total phenolic content. The processed juice had higher sulfoxide. Vertical bars with the different letters are TPC in the serum (Fig. 4C) and in pellets at day 4 after storage significantly different at P = 0.05 using Duncan’s (Fig. 4D). Thermal pasteurization decreased the content in the multiple range test. pellets at both day 0 and 4, but only at day 0 in the serum (Fig. 4C and 4D). There was a marked increase of TPC in the processed juice pellets (Fig. 4D). This agrees with the individual phenolic Buslig, B.S. and R.D. Carter. 1974. Particle size distribution in orange juices. Proc. Fla. State Hort. Soc. 87:302–305. compound analysis results by HPLC. Dhuique-Mayer, C., C. Caris-Veyrat, P. Ollitrault, F. Curk, and M.-J. Amiot. 2005. Varietal and interspecific influence on micronutrient Conclusions contents in citrus from the Mediterranean area. J. Agr. Food Chem. 53:2140–2145. The nutritional profiles of the processed juice including the Dhuique-Mayer, C., M. Tbatou, M. Carail, C. Caris-Veyrat, M. Dornier, industrial extraction and finishing was remarkably different from and M.J. Amiot. 2007. Thermal degradation of antioxidant micronu- those of fresh juice prepared with a commercial food service trients in citrus juice: Kinetics and newly formed compounds. J. Agr. juicer. The processed juice had more than two times the amount Food Chem. 55:4209–4216. of flavonoid glycosides, and also had higher levels of limonoids, Ford, B.L., J. Bai, J. Manthey, and E.A. Baldwin. 2010. Toward a facile alkaloids, and carotenoids and total phenolic contents, yet the method to remove ascorbate interference in the Folin-Ciocalteu assay of ‘‘total phenolic content.’’ HortScience 45:498. processed juice contained half or less of the polymethoxylated Gattuso, G., D. Barreca, C. Gargiulli, U. Leuzzi, and C. Caristi. 2007. flavones and ascorbic acid than the fresh juices (pasteurized and Flavonoid composition of citrus juices. Molecules 12:1641–1673. unpasteurized). Thermal pasteurization increased content of Gil-Izquierdo, A., M.I. Gil, F. Ferreres, and F.A. Tomas-Barberan. 2001. flavones, and some limonoids in the serum, but decreased some In vitro availability of flavonoids and other phenolics in orange juice. carotenoids and total phenolic contents. During the 4-d storage J. Agr. Food Chem. 49:1035–1041. at 5 °C, more than 50% hesperidin and isosakuranetin rutinoside Johnson, J., R. Braddock, and C. Chen. 1995. Kinetics of ascorbic acid in the processed juice crystallized and precipitated. The major loss and nonenzymatic browning in orange juice serum: Experimental changes were associated with high content of peel oil and low rate constants. J. Food Sci. 60:502–505. content of insoluble solids in the fresh juice. Kimball, D., M.E. Parish, and R. Braddock. 2004. Oranges and tangerines, p. 617–638. In: D.M. Barrett, L.P. Somogyi, and H.S. Ramaswamy (eds.). Processing : Science and technology. CRC Press, Boca Literature Cited Raton, FL. Klavons, J.A., R.D. Bennett, and S.H. Vannier. ���������������������1994. Physical/chemi- Bai, J., E.A. Baldwin, A. Plotto, R. Cameron, B.L. Ford, G. Luzio, J. cal nature of pectin associated with commercial orange juice cloud. Manthey, J. Narciso, and S. Dea. 2010. A comparison of processed J. Food Sci. 59:399–401. and fresh squeezed ‘Hamlin’ orange juice—Flavor quality. Proc. Fla. Lee, H. and G. Coates. 1999. Thermal pasteurization effects on color of State Hort. Soc. 123 (In press). red grapefruit juices. J. Food Sci. 64:663–666. Baldwin, E.A. 1993. Citrus fruit, p. 107–149. In: G.B. Seymour, J.E. Lee, H.S. and G.A. Coates. 2003. Effect of thermal pasteurization on Taylor, and G.A. Tucker (eds.). Biochemistry of fruit ripening. Chap- Valencia orange juice color and pigments. LWT – Food Sci. Technol. man and Hall, New York. 36:153–156.

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212 Proc. Fla. State Hort. Soc. 123: 2010.