CHEMICAL AND SENSORY CHARACTERIZATION OF ORANGE (CITRUS SINENSIS) PULP, A BY-PRODUCT OF ORANGE JUICE PROCESSING USING GAS-CHROMATOGRAPHY- OLFACTOMETRY

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1 Journal of Food Quality ISSN CHEMICAL AND SENSORY CHARACTERIZATION OF ORANGE (CITRUS SINENSIS) PULP, A BY-PRODUCT OF ORANGE JUICE PROCESSING USING GAS-CHROMATOGRAPHY- OLFACTOMETRY SOPHIE DETERRE, CLOTILDE LECLAIR, JINHE BAI, ELIZABETH A. BALDWIN, JAN A. NARCISO and ANNE PLOTTO 1 U.S. Horticultural Research Laboratory, USDA-ARS, Fort Pierce, FL Corresponding author. TEL: ; FAX: ; anne.plotto@ars.usda.gov Mention of a trademark or proprietary product is for identification only and does not imply a guarantee or warranty of the product by the U.S. Department of Agriculture. The U.S. Department of Agriculture prohibits discrimination in all its programs and activities on the basis of race, color, national origin, gender, religion, age, disability, political beliefs, sexual orientation and marital or family status. Received for Publication September 28, 2015 Accepted for Publication June 2, /jfq ABSTRACT Volatile composition of commercial orange pulp (from Brazil and Florida, U.S.A.) was analyzed by gas chromatography-mass spectrometry (GC-MS) and GC- Olfactometry (GC-O). In both samples 72 volatiles were detected, of which 58 were identified. Odor-active compounds with a high frequency of detection (5 out of 9) or intensity characterizing the aroma of sweet orange pulp were monoterpene hydrocarbons (a-pinene, b-pinene, b-myrcene, a-phellandrene, 3- carene, a-terpinene and limonene), ketones (1-octen-3-one, carvone, (E)-bdamascenone and b-ionone), esters (ethyl-2-methyl butanoate and ethyl hexanoate), aldehydes (methional and octanal), alcohols (linalool and 1-octanol) and 3 unidentified compounds. A few differences in the odor-active volatiles between orange pulp samples were perceived, which might be due to cultivar, growing and processing conditions, but overall, the chemical composition of the two samples was similar. Sensory data described both sweet orange pulp samples with descriptors for orange odor and flavor including orange peel and fruity-noncitrus flavor, sweet and sour taste. PRACTICAL APPLICATIONS Orange pulp is used in the beverage industry to add texture and mouthfeel. It is also added to orange juice for consumer appeal to make it more natural. This study characterized the flavor of orange pulp. Orange pulp consisted of yellow orange floating intact cells. Pulp added to a sugar-acid solution (5% pulp, 10.5% sucrose and 0.25% citric acid) imparted an orange, fruity and fresh flavor. Information from this study on sweet orange pulp flavor will be useful for orange juice processors and beverage manufacturers. INTRODUCTION Orange juice made from sweet oranges (Citrus sinensis [L.] Osbeck) is the most popular juice beverage around the world. Brazil and the U.S.A. (mainly Florida) have long been the two largest producers of orange juice concentrate (658Brix equivalent) with productions of and million Metric Tons, respectively, since These two countries represent 55 and 25% of the world production, respectively (USDA/FAS 2015). During commercial processing, juice is extracted from oranges by squeezing or reaming the fruit by means of mechanical pressure (Ringblom 2004). The resulting pulpy juice (50% of the orange) is then clarified using various types of screens or finishers to obtain a final juice product with the desired consistency. In the early days (in the s), most solids from the orange, including peel, rag (crushed peel), seeds and segment walls, were sent to the feed mill for drying into pellets for animal feed (Ranganna et al. 1983b; Ringblom 2004). 826 Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA.

2 S. DETERRE ET AL. CHEMICAL AND SENSORY EVALUATION OF ORANGE PULP More recently, a diversity of juice types are offered to consumers, with juice having various levels of pulp. Pulp as a by-product of orange juice processing may also be added to other types of drinks and food products to provide a desired texture (Bangert 1976; Loader 1983; Dulebohn et al. 2001; De Moraes Crizel et al.2013). Orange pulp is composed of pieces of membrane materials from the ruptured juice sacs and segment walls, contributing to the texture and mouthfeel of citrus juice. In the orange juice, pulp is characterized as either sinking or floating : the sinking pulp is an integral part of the juice and consists of small particles (<0.5 mm) which settle with time; the floating pulp consists of the larger membrane pieces and broken juice vesicles removed through the finishers (Ringblom 2004). Floating pulp may be added back to clarified juice to provide fiber and consistency, desired by some consumers (AIJN 1993). Another type of pulp is obtained after washing the pulp and solid particles from the clarification or de-oiling process. In both U.S.A. and EU regulations, the washed pulp solids cannot be added back to the orange juice, but are allowed to be used for manufacturing nectar and juice beverages (AIJN 1993; Chen et al. 1993; FAO/WHO 2004). Studies on the effect of pulp on orange juice flavor and texture showed that fresh orange character was increased when pulp was added to low-pulp orange juice, mostly due to increased volatiles, mainly monoterpene and sesquiterpene hydrocarbons (Rega et al. 2004; Rega and Guichard 2004). Moreover, pulp modified the rheological properties of the orange juice matrix, such as texture and viscosity. Nonvolatile components in pulp include proteins, flavonoids, lipids and minerals, as well as solid particles ranging from 0.05 mm to a few hundred micrometers from the tissue fragments (Ranganna et al. 1983a; Klavons et al. 1991). Volatile components detected in the pulp are mainly hydrocarbons, which are nonpolar, whereas polar oxygenated volatiles are largely present in the serum (juice) (Radford et al. 1974; Hernandez et al. 1992). About 100% of myrcene, a-pinene and sabinene, 99% of valencene, and 98% of limonene were detected in the pulp samples, in contrast to compounds mostly found in the serum, including octanal, linalool, ethyl 3-hydroxyhexanoate and ethyl butanoate, which were 12.5, 10, 1.5% and trace amounts in the pulp, respectively (Radford et al. 1974). Quantification of volatile compounds in orange pulp were also reported (on a fresh weight basis): mg/g monoterpene hydrocarbons (of which mg/g is limonene), 99.2 mg/g sesquiterpene hydrocarbons, 25.2 mg/g aldehydes and ketones, 19.5 mg/g esters, 17.2 mg/g aliphatic alcohols, 12.9 mg/g carboxylic acids and 10.3 mg/g monoterpene alcohols (Brat et al. 2003). Except for the report from Hernandez et al. (1992), the source of pulp in the above studies was from hand-squeezed oranges, with pulp and serum separated by centrifugation. As far as we know, there are no studies that report the characteristic aroma volatiles of commercially extracted orange pulp and used in the beverage industry in relation to sensory descriptors. We can only assume which components are involved in the aroma of pulp, with the knowledge of volatile composition and aroma profile of orange juice (Govindarajan et al. 1984; Rega et al. 2004; Berlinet et al. 2007; Sadecka et al. 2014). The aim of this study was to characterize flavor of commercially derived orange pulp cells used as an ingredient in orange juice and other fruit beverages. Two batches of pulp cells were obtained from different commercial sources and analyzed to identify the volatiles by gas chromatography-mass spectrometry (GC-MS) and characterize the odor-active compounds by GC-olfactometry (GC-O). A sensory evaluation was performed, with odor, taste and appearance, to describe both undiluted orange pulp, and orange pulp diluted at 5% in a sucrose-citric acid solution to mimic orange drinks that incorporate this type of pulp by-product. MATERIALS AND METHODS Orange Pulp Samples, Origin and Preparation Sweet orange (C. sinensis (L.) Osbeck) pulp samples comprised of 80% pulp cells and 20% juice were obtained from orange juice processors in Brazil and Florida, U.S.A., using the JBT FoodTech Pulp Recovery System (JBT FoodTech, Lakeland, FL). Samples had been stored frozen for several months. Pulp was observed under a dissecting stereo microscope Olympus SZX12 (Olympus Corporation America) and pulp cells were measured using a Keyence VHX-600E digital microscope equipped with the V-Z20R lens attachment (Keyence Corp., Osaka, Japan) (Fig. 1). For volatile identification by GC-MS, orange pulp samples (3 g straight pulp) were prepared in 20 ml glass vials capped with crimp caps with septa (silicone/ptfe preassembled) (Gerstel Inc., Baltimore, MD). For determination of odoractive compounds by GC-O, initial sample preparation was the same as for GC-MS, but panelists noticed co-elution of several compounds due to a shorter column (30 m) in the GC used for GC-O than for GC-MS (60 m). Pulp was, therefore, diluted with saturated NaCl solution in a 1:4 ratio for a total weight of 6 g in a 20 ml vial. The choice of NaCl as the dilutant was to prevent any bacterial or enzymatic changes, and volatile profiles were similar to dilution with water. For sensory evaluation, undiluted (12.5 g 6 0.5) and diluted (39.5 g 6 0.5) pulp samples were placed in 118-mL plastic souffle cups covered with lids (Solo Cups Co., Urbana, IL). The diluted pulp was a mixture of orange pulp (5%) in a solution of sucrose (10.5% w/w) and citric acid Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA. 827

3 CHEMICAL AND SENSORY EVALUATION OF ORANGE PULP S. DETERRE ET AL. octanoate (99%, FG), 3-(methylthio)-propionaldehyde (methional, 98%, FG), myrcene (90%, FG), neryl acetate (98%, FG), nonanal (95%, FG), (E)-2-nonenal (93%), octanal (92%, FG), 1-octanol (98%, FG), 1-octen-3-one (50 wt % in 1-octen-3-ol, FG), octyl acetate (98%, FG), (S)-(-)-perillaldehyde (92%, FG), a-phellandrene (not pure, FG), (-)-a-pinene (97%, FG), (-)-b-pinene (97%, FG), a-terpinene (89%, FG), g-terpinene (95%, FG), (-)-terpinen-4-ol (95%, sum of enantiomers, GC), a- terpineol (96%, FG), terpinolene (90%, FG) and undecanal (96%, FG), all purchased from Sigma-Aldrich (St. Louis, MO), (-)-alloaromadendrene (98.0%, sum of enantiomers, GC) and (1)23-carene (98.5%, sum of enantiomers) from Fluka (St. Louis, MO), a-cubebene (97%) from International Laboratory USA (San Francisco, CA), b-damascenone (pure) from IFF (Union Beach, NJ), ethyl alcohol (absolute and anhydrous) from Pharmaco-Aaper (Brookfield, CT), sabinene (70%) from Treatt (Lakeland, FL) and valencene (80%) from Bedoukian Research, Inc., (Danbury, CT). Purities were checked by GC-MS. Gas Chromatography Coupled with Mass Spectrometry FIG. 1. (A) ORANGE PULP DILUTED AT 5% IN A SOLUTION OF SUCROSE (10.5% W/W) AND CITRIC ACID (0.25% W/W) AS WOULD BE IN A BEVERAGE. (B). ORANGE PULP CELL OBSERVED UNDER A DISSECTING STEREO MICROSCOPE OLYMPUS SZX12 (OLYMPUS CORPORATION AMERICA). 20X MAGNIFICATION (0.25% w/w), as would be in a typical beverage to which this type of pulp product is added. Chemicals Standards were obtained from the following sources: benzaldehyde (98%, Food Grade [FG]), camphene (95%), (R)-(-)-carvone (98%, optical purity: 98% [GLC]), b-caryophyllene (80%, FG), citral (mixture of cis and trans, 96%, FG), decanal (95%, FG), D-dihydrocarvone (mixture of isomers, 97%, FG), ethyl acetate (99.8%, anhydrous), ethyl butanoate (98%, FG), ethyl hexanoate (98%, FG), ethyl-3-hydroxyhexanoate (99.8%), ethyl-2- methylbutanoate (98%, FG), ethyl octanoate (98%, FG), geraniol (97%, FG), geranyl acetate (98%, FG), guaiacol (98%, FG), hexanal (97%, FG), 2-(E)-hexenal (95%, FG), heptanal (92%, FG), a-humulene (96.0%, GC), b- ionone (predominantly (Z), 97%, FG), (S)-(-)-limonene (95%, FG), linalool (97%, FG), methyl butanoate (98%, FG), methyl hexanoate (99%, FG), methyl Identification of volatile compounds was carried out with the undiluted pulp samples (3 g). Sample headspace was equilibrated for 30 min at 40C, then a 2-cm solid phase microextraction (SPME) fiber (50/30 mm DVB/Carboxen/ PDMS; Supelco, Bellefonte, PA) was exposed to the headspace for 60 min. After exposure, the SPME fiber was inserted into the injector of a gas chromatograph (Agilent Technologies 6890 GC, Santa Clara, CA) to desorb (splitless injection) the fiber for 5 min at 250C. Headspace collection was performed using a Gerstel MPS2 autosampler (Gerstel). All samples were analyzed in triplicate. The GC was configured with a Mass Selective detector (Model 5973 Network MSD, Agilent Technologies) with electron ionization mode generated at 70 ev. Volatile compounds were separated through a DB-5ms capillary column (60 m length, 0.25 mm i.d., 1 mm film thickness; J&W Scientific, Folsom, CA). The flow rate of the carrier gas (He) was 1.75 ml/min. The oven temperature was 40C, increased to 230C at 4C/min, and then held for 12 min. The injector and transfer line temperatures were 250 and 280C, respectively. Mass spectra were recorded in the electron impact mode with an ion source temperature of 230C. Full-scan data acquisition was registered over the range m/z at 3.85 scan/s. The software MSD ChemStation Data Analysis (Rev D) from Agilent was used for control, general operations and data acquisition of the results. Mass spectra matches were made by comparing experimental mass spectra with those of the Adams (Adams 2007) and NIST 02 (National 828 Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA.

4 S. DETERRE ET AL. CHEMICAL AND SENSORY EVALUATION OF ORANGE PULP Institute of Standards and Technology, Gaithersburg, MA) Libraries. Moreover, linear retention indices (I T ) of volatile compounds were calculated using a series of n-alkanes (C-5 to C-15) that were run under the same chromatographic conditions and were then compared to the values given in the literature (Adams 2007; Nist 2011; El-Sayed 2014). To compare the two samples with each other, the following calculations were performed for each compound: average of peak areas over the three injections, and ratio of the average peak areas: Brazil divided by Florida pulp. Gas Chromatography Olfactometry Extraction and desorption of aroma volatiles into the GC injector was performed by the SPME method using the same parameters as described above for the GC-MS analysis, except that the pulp samples were equilibrated in a water bath and injected manually instead of with an autosampler. Further, pulp samples were diluted because pure pulp resulted in poor chromatographic resolution. After exposure, the SPME fiber was manually inserted into the injector of a gas chromatograph (Model 7890A GC, Agilent Technologies) to desorb the extract for 5 min at 250C. The GC was equipped with an HP-5 (Agilent Technologies) capillary column (30 m length, 0.32 mm i.d., 0.25 lm film thickness) and the column effluent was split (1:2) to a flame ionization detector (FID) and an olfactory detection port (Model ODP-2, Gerstel). The olfactory detection port was connected to a humidified air make-up (8.9 ml/min). The oven temperature program was 40C for 2 min, then increased to 180C at 6C/min, then to 250C at 10C/min and then held for 7 min for a total run time of 39.3 min. The choice of sample preparation (diluted pulp samples), column (30 m) and oven program were optimized so that panelists would evaluate samples without odor overlap during the first 30 min of the GC-O runs to avoid fatigue. The carrier gas (He) flow rate was 1.75 ml/min. The chromatographic effluents were evaluated by three female panelists experienced in GC-O of orange products. Prior to evaluating the orange pulp, they practiced to recognize citrus odorant volatiles using chemical standards, as well as using the linear scale. Data were recorded using a computer program written in LabVIEW 8.5 (National Instruments, Austin, TX). Panelists actuated a large slide bar on the computer screen with a mouse to conveniently report aroma intensity on an unstructured linear scale from zero (0 5 no aroma perceived) to ten (10 5 extremely strong). Every 100 ms, the program simultaneously collects graphs and saves voltage data from aroma intensity. This data acquisition rate was sufficiently fast to collect data for the aromagrams. Aroma descriptors were manually written in a notebook. The FID output was simultaneously recorded using EZChrom Elite (v SP2, Agilent Technologies). GC-O analyses of each sample were conducted in triplicate. The samples were run in a random order of presentation to avoid introducing bias into the results. Only those odor-active peaks that were detected in at least two out of three experimental replicates by at least one panelist were retained. The duration(s) and the highest intensities of each odor peak were collected. For identification of odor compounds, I T of volatile compounds and odorous peaks were calculated using a series of n-alkanes (C-5 to C-15) that was run under the same chromatographic conditions. Confirmation of compound identity was then done by comparing I T of the odorous peaks with those obtained by GC-MS analysis, sniffing reference standards by GC-O under the same chromatographic conditions, and comparing with odor data from the literature (Acree and Arn 2004; El-Sayed 2014). Sensory Analysis Odor, flavor, taste and appearance were described for each orange pulp sample, diluted and undiluted, by five panelists trained (>50 h) and evaluating citrus products on a weekly basis. An initial session was performed to choose the best way to present samples and to select the most suitable sensory descriptors for these specific samples. An extensive list of descriptors generated from earlier studies, GC-O and the literature were provided (Perez-Cacho et al. 2008;Plotto et al. 2008). Only the most relevant descriptors were considered: theonesusedbyatleastthreepanelistsoutoffiveforatleast one sample (Table 1). Samples were evaluated in duplicate sessions at several days interval, with only undiluted pulp or diluted pulp served in one sitting, since the ballots were different between the two preparation types. Samples in plastic TABLE 1. SELECTION OF SENSORY DESCRIPTORS USED TO EVALUATE UNDILUTED AND DILUTED SWEET ORANGE PULP Odor Taste/flavor Appearance Undiluted Pulp Orange, orange peel, pine-like, fresh, overripe/ cooked, oxidized Sweet, sour, orange, orange peel, fruity-noncitrus, pine-like, spicy/woody, fresh, overripe/ cooked Yellow, orange, brown, homogeneous (uniform), fibrous Diluted pulp Orange, orange peel, fruity-non-citrus, fresh Sweet, sour, orange, orange peel, fruity-noncitrus, fresh, floral White, yellow, homogeneous (uniform), fibrous Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA. 829

5 CHEMICAL AND SENSORY EVALUATION OF ORANGE PULP S. DETERRE ET AL. souffle cups labeled with three-digit code numbers were served at 25C and were presented in a monadic manner. Panelists evaluated odor, flavor, and taste in isolated booths under red lighting. Appearance was evaluated in a separate room under day-like lighting. Descriptors were rated on a category scale from 0 (none) to 3 (high). Data Analysis Analyses of variance (ANOVA) were performed on the maximum peak intensity and peak duration of the GC-O data. Significance at P < 0.05 was chosen for separation of means. Statistical analyses were performed with XLSTAT v (Addinsoft, Paris, France). Because the samples were rated on a 0-3 scale, sensory descriptive data were transformed into rank and analyzed using the Mann Whitney test, nonparametric equivalent of the t-test (Lawless and Heymann 1998), using XLSTAT. The level of significance was chosen as P < Data are presented as average intensities. RESULTS AND DISCUSSION Identification of Volatile Compounds in Orange Pulp In both orange pulp samples, 72 volatile components were detected and 58 were identified by GC-MS (Table 2). Mono- (73.56 and 69.89% for the Brazil and Florida pulp, respectively) and sesquiterpene (10.90 and 17.17% for Brazil and Florida pulp, respectively) hydrocarbons were the major components of the pulp and among them, limonene was the most abundant ( %). Hydrocarbons are hydrophobic: their octanol-water partition coefficients (such as limonene 4.57, myrcene 4.17 and caryophyllene 6.30) are higher than those of aldehydes (decanal 3.76), esters (ethyl hexanoate 2.83) or alcohols (linalool 2.97) (S.R.C. 2014). They constitute most of the volatiles detected in the pulp, while the hydrophilic compounds remain in the juice during processing (Brat et al. 2003). In these samples, the hydrophilic compounds detected, 5.97% and 4.32% alcohols, 4.24% and 2.93% aldehydes, 1.60% and 1.93% esters, 0.71% and 1.35% ketones and 0.04% and 0.01% furans (2-ethylfuran) in Brazil and Florida pulp, respectively, may come from the 20% fraction of juice (serum) in the pulp. Twenty-three volatiles, representing 2.99 and 2.40% of the detected volatiles (data not shown), remained unidentified in Brazil and Florida pulp, respectively. We found good agreement with previous studies: among the 58 identified components, for example, 28 volatiles were also reported by Brat et al. (2003), 31 by Jordan et al. (2001), and 21 by Radford et al. (1974) (Tables 2 and 3). Differences in volatile profile between the two types of orange pulp in this study were highlighted with the peak area ratios between the Brazil and Florida pulp samples (Table 2). The following compounds were detected in the Brazil pulp in at least twice the amount detected in the Florida pulp (ratio values 2): monoterpene hydrocarbon, linear retention indices on DB-5 (I T ) (ratio value ), sesquiterpene hydrocarbon 1431 (4.39), carvyl acetate (3.69), heptanal (3.60), monoterpene hydrocarbon 995 (2.38), 2- ethylfuran (3.12), sesquiterpene hydrocarbon 1458 (2.25) and decanal (2.13). On the contrary, terpinen-4-ol (0.37), sesquiterpene hydrocarbon 1497 (0.38), ethyl butanoate (0.43) and ethyl-3-hydroxyhexanoate (0.45), were detected in the Florida pulp in amounts at least twice as much as in the Brazil pulp (ratio values 0.5). A few components were only detected in the Brazil or in the Florida pulp (Table 3). Two identified (2-butanone and geraniol) and three unidentified volatiles were only detected in the pulp from Brazil, representing 0.59% of the volatiles detected. Four identified (methyl butanoate, methyl hexanoate, 1-octanol, (E)-dihydrocarvone) and five unidentified volatiles, representing 0.76% of the volatiles detected, were only present in the Florida pulp. Varietal, harvest maturity, seasonal and climatic factors are likely to explain the aroma differences between samples, as well as processing parameters and storage conditions. Differences, such as the larger amount of ethyl butanoate and (E)-2-hexenal in the Florida pulp and the larger amount of linalool, ethyl furan and decanal in the Brazil pulp, could be an indication of higher processing temperatures for the Brazilian pulp (Nisperos-Carriedo and Shaw 1990). But if this were the case, low molecular weight volatiles such as ethanol, ethyl acetate and hexanal would be lower in the Brazilian than in the Florida pulp (Perez-Cacho and Rouseff 2008). Alpha- and b-terpineol, as well as terpinen-4-ol and geraniol are thermally induced volatiles from limonene or linalool (Perez-Cacho and Rouseff 2008). In our study, terpinen-4-ol was higher in the Florida sample, a- and b- terpineol were higher and geraniol was only detected in the Brazil sample, indicating no definite differences in temperature during processing and storage of these samples. Perhaps higher a- and b-terpineol and geraniol in the Brazil sample simply indicate differences in the raw orange fruit material. Overall, the differences in volatiles between the two pulp sources were minimal, as can be seen on a representative chromatogram of each sample (Fig. 2). Identification of Odor-Active Compounds in Orange Pulp from Brazil and Florida Thirty-three odor-active volatiles were detected and characterized in diluted orange pulp samples by one or several odor descriptors (Table 4). Among these 33 volatiles, eight remain unidentified, I T 5 650, 765, 812, 857, 1068, 1147, 1207 and 1229, and nine were not detected by GC-MS but identified by I T and sniffing of pure standards: ethyl Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA.

6 S. DETERRE ET AL. CHEMICAL AND SENSORY EVALUATION OF ORANGE PULP TABLE 2. VOLATILE COMPOUNDS (EXPRESSED AS TOTAL ION CURRENT STANDARD DEVIATION) FROM UNDILUTED SWEET ORANGE PULP FROM BRAZIL AND FLORIDA PRESENTED BY CHEMICAL FAMILY (N 5 3) I T I T Brazil Florida Family DB-5 DB-Wax Compound* Identification Average Std. dev. Average Std. dev. Ratio Alcohols Ethanol c Standard, MS, I T 184, , , , Octanol a,b,c Standard, MS, I T nd - 61, , Linalool a,b,c Standard, MS, I T 613, , , , b-terpineol Standard, MS, I T 19, ,409 12, Terpinen-4-ol a,b,c Standard, MS, I T 59, , , , a-terpineol a,b,c Standard, MS, I T 110, ,094 70, , Geraniol b,c Standard, MS, I T 13, nd - - % Alcohols Aldehydes 793 Hexanal a,b,c Standard, MS, I T 29, ,673 34, , (E)-2-hexenal Standard, MS, I T 8, , , Heptanal Standard, MS, I T 38, ,228 10, , Benzaldehyde b Standard, MS, I T 2, , Octanal a,b,c Standard, MS, I T 223, , , , Nonanal a,b,c Standard, MS, I T 86, ,026 60, , Decanal a,b,c Standard, MS, I T 250, , , , Geranial c Standard, MS, I T 10, , Perillaldehyde c Standard, MS, I T 47, ,933 40, , Undecanal c Standard, MS, I T 10, , % Aldehydes Esters Ethyl acetate b,c Standard, MS, I T 4, , Methyl butanoate a,c Standard, MS, I T nd - 2, Methyl hexanoate Standard, MS, I T nd - 10, Ethyl butanoate a,b,c Standard, MS, I T 24, ,891 57, , Ethyl hexanoate a,b Standard, MS, I T 97, , , , Methyl octanoate Standard, MS, I T 13, , ethyl-3-hydroxyhexanoate a,b Standard, MS, I T 28, ,871 62, , Ethyl octanoate b Standard, MS, I T 27, ,963 20, , n-octyl acetate c Standard, MS, I T 42, ,946 30, , Methyl geraniate MS, I T 5, , (E)-carvyl acetate MS, I T 6, , Neryl acetate Standard, MS, I T 9, , Geranyl acetate Standard, MS, I T 7, , % Esters Ketones Butanone MS, I T 13, ,214 nd (E)-dihydrocarvone Standard, MS, I T nd - 26, , Carvone c Standard, MS, I T 106, , , , % Ketones Furans Ethylfuran MS, I T 6, , % Furans Monoterpene hydrocarbons Standard, MS, I T 1,098, ,486 1,123, , a-thujene MS, I T 4, , a-pinene a,b,c Standard, MS, I T 212, , , , Camphene Standard, MS, I T 3, , n.i 11, , Sabinene a,b,c Standard, MS, I T 8, , /1143 b-myrcene a,b,c 1 (b-myrcene/ b-pinene) b-pinene b,c 1037 n.i 47, ,022 52, , a-phellandrene c Standard, MS, I T 49, ,328 39, , d-3-carene b,c Standard, MS, I T 72, ,503 36, , Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA. 831

7 CHEMICAL AND SENSORY EVALUATION OF ORANGE PULP S. DETERRE ET AL. TABLE 2. CONTINUED I T I T Brazil Florida Family DB-5 DB-Wax Compound* Identification Average Std. dev. Average Std. dev. Ratio a-terpinene b,c Standard, MS, I T 63, , n.i 17, , , n.i 39, ,771 45, , Limonene a,b,c Standard, MS, I T 9,977, ,009 9,780, , n.i 322, , , , c-terpinene a,b,c Standard, MS, I T 120, ,338 91, , Terpinolene b Standard, MS, I T 166, , , , n.i 15, ,221 17, , n.i 26, ,440 31, n.i 8, , n.i 55, ,297 9, n.i 10, , % Monoterpenes Sesquiterpene hydrocarbons 1378 a-cubebene Standard, MS, I T 6, , a-copaene b MS, I T 25, ,399 17, , b-elemene a MS, I T 45, ,271 37, , n.i MS, I T 8, , b-caryophyllene a,b,c Standard, MS, I T 31, ,670 42, , n.i 10, , Alloaromadendrene Standard, MS, I T 31, ,768 61, , a-humulene c Standard, MS, I T 6, , , c-muurolene MS, I T 22, ,184 37, , n.i 18, ,925 49, , n.i 117, , , , Valencene a,b,c Standard, MS, I T 1,176, ,423 1,908, , a-selinene b MS, I T 132, , , , b-selinene MS, I T 57, , , , d-cadinene a MS, I T 35, ,728 45, , (E)-cadina,1,4-diene MS, I T 101, , , , % Sesquiterpenes Ratio of Brazil/Florida (bold italics) showed when compounds were detected twice as much in Brazil than in Florida samples (2), and twice as much in Florida than in Brazil samples (0.5). *Reported in: a Radford et al. (1974), b Brat et al. (2003) and c Jordan et al. (2001). Identification by: Standard: Identification based on co-injection with authentic standards by GC-MS. I T, Identification based on I T matching with DB-5 and DBWax columns; and MS, Identification based on mass spectra matching. Ratio of the averages of peak areas Brazil:Florida. n.i., Non-identified monoterpene or sesquiterpene hydrocarbons (MW or 204, respectively). nd, Not detected. methyl butanoate, methional, 1-octen-3-one, guaiacol, (E)- p-2,8-menthadien-1-ol, (E)-2-nonenal, carvone, (E)-bdamascenone and b-ionone. We can hypothesize that these latter components were not detected by GC-MS (in which undiluted samples were run) but only by smell due to their very low odor thresholds in air: recognition at mg/m 3 for ethyl-2-methyl butanoate (Guth and Grosh 1991), detection and recognition at mg/m 3 for methional (Von Ranson and Belitz 1992), mg/m 3 for 1-octen-3-one (Guth and Grosh 1990), mg/m 3 for guaiacol (Ferreira et al. 1998), detection at mg/m 3 and recognition at mg/m 3 for (E)-2-nonenal (Von Ranson and Belitz 1992), mg/m 3 for carvone (Schieberle and Grosch 1989), mg/m 3 for (E)-b-damascenone (Blank et al. 1989, 1992) and mg/m 3 for b-ionone (Brenna et al. 2002). There are no data reported in the literature for (E)-p-2,8-menthadien-1-ol, and therefore, this compound is only tentatively identified. Guaiacol was reported to be glycosidically bound volatiles in orange pulp (Ren et al. 2015), and 1-octanol and b-damascenone in kiwifruit (Garcia et al. 2013); further, b-damascenone and b- 832 Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA.

8 S. DETERRE ET AL. CHEMICAL AND SENSORY EVALUATION OF ORANGE PULP TABLE 3. VOLATILE COMPOUNDS UNIQUE TO EITHER BRAZIL OR FLORIDA SWEET ORANGE PULP Family I T I T Compound Identification* Brazil Florida DB-5 DB-Wax Total ion current Ketones Butanone MS, I T 14 Alcohols 1282 Geraniol Standard, MS, I T 13 Unknown compounds 651 Unknown Unknown Unknown % Total 0.59 Esters 703 Methyl butanoate Standard, MS, I T Methyl hexanoate Standard, MS, I T 10 Alcohols Octanol Standard, MS, I T 62 Ketones 1240 (E)-dihydrocarvone Standard, MS, I T 26 Unknown compounds 680 Unknown Unknown Unknown Unknown Unknown % Total 0.76 *Identification by: Standard: Identification based on co-injection with authentic standards by GC-MS. MS, Identification based on mass spectra matching. I T, Identification based on I T matching with DB-5 and DBWax columns. FIG. 2. SIDE-BY-SIDE CHROMATOGRAMS OF HEADSPACE VOLATILES OF BRAZIL (TOP) AND FLORIDA (BOTTOM) PULP SAMPLES. IDENTIFIED PEAKS ARE ODOR-ACTIVE BY GC-O (TABLE 4) AND DETECTED BY GC-MS, EXCEPT ETHANOL AND VALENCENE THAT ARE INDICATED AS REFER- ENCE PEAKS Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA. 833

9 CHEMICAL AND SENSORY EVALUATION OF ORANGE PULP S. DETERRE ET AL. TABLE 4. ODOR-ACTIVE COMPOUNDS IDENTIFIED BY GC-O IN DILUTED SWEET ORANGE PULP AND CONFIRMED WITH LITERATURE AND INJEC- TION OF PURE CHEMICALS Occurrence (Out of 9 repetitions)* Maximum intensity (From 0 to 10) Duration (s) I T DB-5 Compounds Odors descriptors Florida Brazil Florida Brazil Florida Brazil 650 Unknown 650 Cheese Unknown 765 Plastic, green Ethyl butanoate1hexanal Fruity, candy, green, cooked Unknown 812 Waxy, solventy, terpeney a 0.7b 0.0a 0.9b 835 Ethyl-2-methyl butanoate Fruity, chemical Unknown 857 Fatty, rubber, cooked b 4.5a 9.9b 5.2a 890 Heptanal Plastic, green, fatty b 0.7a 3.7b 1.2a 898 Methional Mashed potatoes, green, fatty a 14.1b 927 a-pinene Citrus, mint, terpene Octen-3-one Floral, green, mushroom b-pinene 1 Candy, floral, green b-myrcene Fruity, candy, citrus, green Ethyl hexanoate Citrus, lemon, mint a 7.4b Octanal Citrus, lemon, mint a-phellandrene Mint, fresh, floral d-3-carene a-terpinene Limonene 1068 Unknown 1068 Skunk, fatty Octanol Mushroom, metallic, green, fatty Guaiacol Smoke, burnt, vanilla Linalool Floral, cowslip, plant Unknown 1147 Floral, candy, unpleasant (E)-p-2,8-menthadien-1-ol Green, fresh, floral (E)-2-nonenal Cucumber, green, fatty a 3.8b 1.3a 5.8b 1174 b-terpineol Green, garbage, plastic Unknown 1207 Green, floral, mint, plastic Decanal Citrus, plastic, butter a 6.3b 2.4a 8.1b 1229 Unknown 1229 Fatty, skunk Carvone Mint (E)-b-damascenone Cooked apple b-ionone Violet *Occurrence greater than 5 is indicated in bold numbers. Means followed by letter a or b are significantly different from each other for either intensity or duration. Significance indicated in bold highlights. Tentative identification. All other compounds identification was confirmed by sniffing pure standards. ionone are volatiles derived from carotenoid degradation (Perez-Cacho and Rouseff 2008). These volatiles with very low odor threshold and bound to the pulp would contribute to flavor in juice with pulp as they tend to be retained by the nonsoluble fraction of the pulp. Among the odor-active compounds, 20 were detected with an occurrence 5 out of 9 GC-O sessions (Table 4) in both pulp samples, and so can be considered as characteristic of the sweet orange pulp: seven monoterpene hydrocarbons: a-pinene, b-pinene 1b-myrcene (co-eluting compounds), a-phellandrene, 3-carene, a-terpinene, and limonene, all coeluting compounds by GC-O; four ketones: 1-octen-3-one, carvone, (E)-b-damascenone and b-ionone, two esters: ethyl-2-methyl butanoate and ethyl hexanoate; two aldehydes: methional and octanal, two alcohols: linalool and 1- octanol and three unknown components: at I T with fatty, rubber and cooked odors; at I T with green, floral, mint and plastic odors; and at I T with fatty and skunk odors. As we could not find any published report about orange pulp odor components, we compared our findings to orange juice odor volatiles. Some volatiles found in this study were also reported in orange juice with similar odor descriptors such as ethyl butanoate, hexanal, a-pinene, b-pinene, b-myrcene, ethyl hexanoate, octanal, limonene, 834 Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA.

10 S. DETERRE ET AL. CHEMICAL AND SENSORY EVALUATION OF ORANGE PULP 1-octanol, linalool, decanal and b-ionone (Rega et al. 2003, 2004; Perez-Cacho and Rouseff 2008). Regarding the odor differences between the two samples, compounds perceived more frequently in the Brazil pulp than in the Florida pulp were unknown 765 (plastic, green), (E)-2-nonenal (cucumber, green, fatty) and decanal (plastic, butter, citrus). Moreover, unknown 812 (waxy, solventy, terpeney), (E)-2-nonenal and decanal were perceived more intensely and for a longer duration of time in Brazil pulp compared to Florida pulp. In the same way, methional (mashed potato, green, fatty) was perceived for a longer duration time and ethyl hexanoate (citrus, lemon, mint) odor was detected more intensely in the Brazil than in the Florida pulp. On the other hand, co-eluting ethyl butanoate and hexanal (one peak with fruity/candy and green odors), heptanal (plastic, green, fatty), (E)-p22,8-menthadien-1-ol (green, fresh, floral) and b-terpineol (green, garbage, plastic) were perceived with greater frequency in the Florida than Brazil pulp. In the former sample, heptanal and unknown 857 odors were significantly more intense and perceived for a longer duration than in the Brazil pulp. Among all 33 odors compounds, there were also three unknown compounds (at I T 5 650, 1068 and 1147) with unpleasant odors, such as cheese, skunk and fatty, but there were no differences between the two samples for occurrences, intensities and durations. Sensory Attributes of the Orange Pulp Straight undiluted pulp was characterized by orange and overripe/cooked odor, orange, orange peel and fruitynon-citrus flavor, sweet and sour taste as well as a yellow/ orange and homogeneous appearance (rating 1) (Fig. 3). Florida pulp had significantly greater ratings for fresh flavor in comparison with the Brazil pulp, and was more yellow in appearance. The Brazil undiluted pulp had more intense pine-like odor and flavor, and orange and brown appearance in comparison with the Florida pulp. The more intense pine-like odor and flavor intensities in the Brazilian undiluted pulp might be due to the sum of monoterpene hydrocarbons that tended to be either higher or at about the same level in the Brazilian compared to the Florida pulp (Table 2). Differences in appearance and flavor between the two pulp samples might be an indication of more peel components in the pulp from Brazil than in the pulp from Florida, with more carotenoids imparting orange-brown appearance and terpenoids imparting pine-like flavor, or it could be due to varietal differences, processing and/or storage conditions. Slightly higher processing or pasteurization temperatures could induce oxidation and browning, explaining the color and odor differences in the Brazilian pulp (Moshonas and Shaw 1997; Perez-Cacho and Rouseff 2008). But since the FIG. 3. DISTRIBUTION OF THE SENSORY DESCRIPTORS ACCORDING TO THE MEAN OF THE INTENSITIES (FROM 0 TO 3) RATED FOR THE BRAZIL (- - -) AND FLORIDA ( ) UNDILUTED ORANGE PULP Descriptors preceded with the capital letters O, F, T and A refer to ODOR, FLAVOR, TASTE and APPEARANCE, respectively. *Significant difference between the two types of pulp by the Mann Whitney test (P 0.05). raw material and processing conditions were unknown, these explanations remain in the realm of speculations. Sensory tests were also performed after diluting pulp by 5% in a sucrose/citric acid solution, to simulate orange drinks that incorporate this type of pulp by-product. Regardless of the sample batch, diluted pulp was characterized by an orange odor, and orange and fruity-non-citrus flavor, sweet and sour taste, as well as by a yellow and fibrous appearance (rating 1) (Fig. 4). The fibrous appearance was due to the pulp cells floating on the surface of the solution (Fig. 1A). The diluted pulp from Brazil had more intense orange peel odor and flavor than the one from Florida. Pine-like odor and flavor were not included in the ballot describing diluted pulp, but these descriptors were rated higher in the Brazil pure pulp in comparison with the Florida pure pulp, which could explain the contribution to a greater orange peel odor and flavor once diluted. The color differences between the two batches of straight pulp, orange/ brown for Brazil and yellow for Florida (Fig. 3) became yellow and white for the diluted pulp from Brazil and Florida, respectively (Fig. 4). By comparing the two types of pulp preparations (undiluted and diluted), undiluted pulp had more intense orange peel and fruity-non-citrus flavor than the diluted pulp. More descriptors were also chosen for the undiluted pulp, such as pine-like, spicy/woody, overripe/cooked and oxidized, meanwhile floral was only used to describe Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA. 835

11 CHEMICAL AND SENSORY EVALUATION OF ORANGE PULP S. DETERRE ET AL. FIG. 4. DISTRIBUTION OF THE SENSORY DESCRIPTORS ACCORDING TO THE MEAN OF THE INTENSITIES (FROM 0 TO 3) RATED FOR THE BRAZIL (- - -) AND FLORIDA ( ) DILUTED ORANGE PULP Descriptors preceded with the capital letters O, F, T and A refer to ODOR, FLAVOR, TASTE and APPEARANCE, respectively. *Significant difference between the two types of pulp by the Mann Whitney test (P 0.05). diluted pulp. The sweet and sour taste perceived in the straight pulp samples was due to soluble compounds in the 20% juice constituting the pulp sample. Interestingly, bitterness was not perceived in these samples, indicating little to no presence of peel components. Regarding the two geographical origins, it is not clear whether the odoractive compounds that were detected by GC-O with greater frequency in the Florida than in the Brazil pulp including fruity, candy, green, cooked, plastic, fatty and floral odors, are responsible for the fresh flavor perceived more intenselyinthefloridathaninthebrazilundilutedpulp. In the same way, the odor-active compounds detected by GC-O with greater frequency and/or intensity in the Brazil compared to the Florida pulp including plastic, green, cucumber, fatty, butter and citrus odors, may be responsible for the oxidized and pine-like odors perceived more intensely in Brazil than Florida undiluted pulp. CONCLUSIONS Sweet orange pulp is used by the beverage industry to add to juice or drink products for flavor, texture and to add a fresh juice-like quality. To our knowledge, this is the first report on the odor activity of a commercially derived pulp by-product that would be imparted to juice or juice drinks. Odor-active components of two different batches of sweet orange pulp were analyzed by GC-MS, GC-O and a sensory panel. Chemical compositions were similar; however differences in peak area were detected by GC-MS. Among the 33 odor-active volatiles detected, 20 characterized the orange pulp aroma. A few significant odor differences could be highlighted between the two types of pulp by named geographical origin. For example, more intense cucumber, green and fatty notes from (E)-2-nonenal were detected in the Brazil pulp, and more intense plastic, green and fatty notes from heptanal were detected in the Florida pulp. Regardless of the origin, results from sensory analysismadeitpossibletocharacterizeorangepulpcellsas having an orange odor and orange, orange peel and fruity-non-citrus flavor, sweet and sour taste, with yellow to brown homogeneous appearance. Pulp diluted in a drink would contribute to orange odor and flavor, adding pleasant, sweet and sour tastes, with fibrous appearance from the pulp cells. From this study, beverage processors may better target which odor, flavor and color might best complement a particular commercial orange juice or juice drink product by the addition of sweet orange pulp. ACKNOWLEDGMENTS Dr. Jose I. Reyes-De-Corcuera for providing with the GC-O recording software and critical review of the manuscript. REFERENCES ACREE, T.E. and ARN, H Flavornet and Human Odor Space. (accessed September 2014). ADAMS, R.P Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, Allured Publishing, Carol Stream, IL. AIJN Code of practice for evaluation of fruit and vegetable juices. (accessed August ). BANGERT, J.G Nutritious orange drink concentrate, process and drink resultant therefrom. Patent Number US In Google Patents. BERLINET, C., GUICHARD, E., FOURNIER, N. and DUCRUET, V Effect of pulp reduction and pasteurization on the release of aroma compounds in industrial orange juice. J. Food Sci. 72, S535 S543. BLANK, I., FISCHER, K.H. and GROSCH, W Intensive neutral odourants of linden honey. Differences from honeys of other botanical origin. Z. Lebensm.-Untersuch. Forsch. 189, BLANK, I., SEN, A. and GROSCH, W Potent odorants of the roasted powder and brew of Arabica coffee. Z. Lebensm. Untersuch. Forsch. 195, BRAT, P., REGA, B., ALTER, P., REYNES, M. and BRILLOUET, J.M Distribution of volatile compounds in the pulp, cloud, and serum of freshly squeezed orange juice. J. Agric. Food Chem. 51, Journal of Food Quality 39 (2016) VC 2016 This article is a U.S. Government work and is in the public domain in the USA.

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