Differentiation between Flavors of Sweet Orange (Citrus sinensis) and

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Differentiation between the flavors of sweet orange (Citrus sinensis) and mandarin (Citrus reticulata) Shi Feng, Joon Hyuk Suh, Fred G. Gmitter Jr., and Yu Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04968 • Publication Date (Web): 14 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Journal of Agricultural and Food Chemistry

Differentiation between the flavors of sweet orange (Citrus sinensis) and mandarin (Citrus reticulata) Shi Feng §†, Joon Hyuk Suh†, Fred G. Gmitter# and Yu Wang*† §

Department of Food Science and Human Nutrition, University of Florida, 572 Newell Dr., Gainesville, FL 32611, USA † Citrus Research and Education Center, Food Science and Human Nutrition, University of Florida, 700 Experiment Station Rd, Lake Alfred, FL 33850, United States # Citrus Research and Education Center, Horticultural Science, University of Florida, 700 Experiment Station Rd, Lake Alfred, FL 33850, United States

* Corresponding author: Yu Wang, Tel.: 863-956-8673; Fax: (863)-956-4631; Email: [email protected].

1 ACS Paragon Plus Environment


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Abstract Pioneering investigations referring to citrus flavor have been intensively conducted.

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However, the characteristic flavor difference between sweet orange and mandarin has not been

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defined. In this study, sensory analysis illustrated the crucial role of aroma in the differentiation

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between orange flavor and mandarin flavor. To study aroma, Valencia orange and LB8-9

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mandarin were used. Their most aroma-active compounds were preliminarily identified by aroma

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extract dilution analysis (AEDA). Quantitation of key volatiles followed by calculation of odor

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activity values (OAVs) further detected potent components (OAV≥1) impacting the overall

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aromatic profile of orange/mandarin. Follow-up aroma profile analysis revealed ethyl butanoate,

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ethyl 2-methylbutanoate, octanal, decanal and acetaldehyde were essential for orange-like aroma,

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whereas linalool, octanal, α-pinene, limonene and (E,E)-2,4-decadienal were considered key

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components for mandarin-like aroma. Furthermore, an unreleased mandarin hybrid producing

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fruit with orange-like flavor was used to validate the identification of characteristic volatiles in

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orange-like aroma.

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Keywords: sensory analysis, Valencia orange, LB8-9 mandarin, aroma extract dilution analysis,

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aroma profile analysis, orange-like flavor mandarin hybrid


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Introduction

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Sweet orange (Citrus sinensis) and mandarin (Citrus reticulate) fruit are the two most

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widely consumed citrus crops worldwide. The United States is one of the world’s main

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producers of oranges and mandarins, with Florida as a primary contributor. In Florida, the

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majority of orange fruit is processed, whereas mandarins are commonly consumed as fresh fruits.

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The flavors of orange and mandarin have been largely investigated over the last decades.

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As early as 1998, application of the aroma extract dilution analysis (AEDA) helped detect 42

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odor-active compounds in Valencia late orange juice, while application of the static

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headspace/olfactometry (SHO) has been used to reveal the most odor-active compounds in the

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headspace above juice. 1 In a following study, twenty-five odor-active compounds were

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quantitated in the juice of Valencia late and Navel oranges using stable isotope dilution assays to

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evaluate the aroma differences. 2 From these two studies, ethyl 2-methylpropanoate, ethyl

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butanoate, (S)-ethyl 2-methylbutanoate, 3a,4,5,7a-tetrahydro-3,6-dimethyl-2(3H)-benzofuranone

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(wine lactone) and (Z)-hex-3-enal have been proved to be the most potent odorants in orange

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juices based on their high flavor dilution factors (FD factors), as well as OAVs. Moreover, (R)-α-

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pinene, (R)-limonene, ethyl butanoate, (S)-ethyl 2-methylbutanoate and acetaldehyde were also

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demonstrated as the most odor-active compounds in the headspace above the juice. In an aroma

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study of three different Satsuma mandarin varieties, limonene, linalool, γ-terpinene, β-myrcene,

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α-pinene and octanal were demonstrated as key aroma components by headspace solid phase

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microextraction (HS-SPME)-combined with GC-MS. 3 Furthermore, core aroma volatiles in

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mandarin juice were considered to be as linalool, α-terpineol, terpinen-4-ol, nonanal, decanal,

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carvone, limonene, α-pinene and myrcene. 4 According to previous studies, the compositions of

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aroma compounds and even certain key odor-active compounds in orange and mandarin largely



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overlap, which is not surprising since orange is actually a complex hybrid derived from genomic

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introgression of mandarin and pummelo (Citrus maxima). To date, however, it is still not clear

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what makes the characteristic differences between the flavors of orange and mandarin.

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Huanglongbing (HLB), one of the most severe citrus diseases distributed worldwide, is

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presumably caused by the bacterium, Candidatus Liberibacter asiaticus, 5 and currently has no

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established cure. It is now the largest concern for the citrus industry, specifically in the

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production of orange juice. The citrus breeding program in the Citrus Research and Education

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Center (CREC) of the University of Florida (Lake Alfred, FL) has identified some specific

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mandarin hybrid selections that have shown relatively high tolerance to HLB. Moreover, some of

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these HLB-tolerant mandarin hybrid selections, such as the unreleased hybrid 6-2-55, produce

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fruit with a distinctive orange-like flavor, yet they have many of the physical characteristics of

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mandarin fruit and trees. This makes it more interesting to determine the flavor differences

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between orange and mandarin. Furthermore, based on a better understanding of orange versus

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mandarin flavor, it might be possible that some HLB-tolerant mandarin hybrids have the

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potential to be used as supplemental materials in orange juice products.

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Therefore, the aim of the present study was to decode/differentiate the characteristic

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flavor of orange and mandarin by studying Valencia orange and LB8-9 mandarin through aroma

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recombination and omission studies, and to validate the decoded theory using 6-2-55 mandarin

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hybrid as a confirmation.

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Materials and Methods

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Materials

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Fruits of Valencia orange and Hamlin orange were obtained from the local companies. LB8-9 mandarin fruits and 6-2-55 mandarin hybrid fruits were obtained from CREC, UF. Ocean


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Spray Sweet Mandarin fruits were purchased from the local market. For sensory analysis, fresh

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fruit juices were immediately prepared after sample receipt, the remaining fruits were stored at -

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20°C until analytical analysis.

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Chemicals and reference odorants

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Reference compounds (Table 1 and Table 3) were purchased from Sigma-Aldrich (St.

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Louis, MO), Molekula Group, LLC (Santa Ana, CA), Acros Organics (Pittsburgh, PA) and TCI

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America (Portland, OR). 1-decanol and n-alkanes standard were purchased from Sigma-Aldrich.

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Dichloromethane was purchased from Sigma-Aldrich. Methanol, water (LC-MS grade) and

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sodium chloride were purchased from Fisher Scientific (Hampton, NH).

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Sensory analysis

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Sensory differentiation between orange and mandarin. Two sensory tests, including nose-

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pinch and pinch-free tests, were performed simultaneously. A panel consisting of 15 panelists

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(male and female, ages 20-50) was recruited from the CREC. Panelists were trained with fresh

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squeezed orange/mandarin juices from different varieties to become familiar with the flavors of

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orange juice and mandarin juice. For the nose-pinch test, a nose clip was used to block the nasal

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passage of each panelist. For the pinch-free test, panelists conventionally evaluated the samples.

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Orange juice (Hamlin, Brix 7%)) and Mandarin juice (Ocean Spray Sweet Mandarin, Brix 11%)

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were freshly made and presented in covered plastic cups. Both juice samples (15 mL each) were

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randomly presented to panelists. Panelists were asked to consecutively taste the juice without

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being able to see its color, then choose the orange juice for both tests. If no decision could be

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made by the panelist, “I don’t know” was recorded for collected results. Panelists’ comments

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relating to their choices were collected for further interpretation of results.



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Sensory evaluation of 6-2-55 mandarin hybrid. Juice of 6-2-55 mandarin hybrid (Brix

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12%) was freshly made and presented in covered plastic cups. Evaluation of the juice sample

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was performed by 16 panelists (male and female, ages 20-50). Panelists were trained as

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described above. Each panelist was provided 15 mL of juice. Panelists were asked to evaluate

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(drink the juice) and define the juice as “orange juice”, “mandarin juice” or “I don’t know”.

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Comments related to choice were also collected.

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Aroma profile analysis (APA). For APA, a trained panel of seven experienced assessors was asked to evaluate the model mixtures of orange/mandarin juice and their corresponding

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omission samples. 6,7 Panelists were trained to become familiar with the smells of orange juice

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and mandarin juice, as well as to differentiate between them. Each sample (~3 mL) was

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presented in a glass vial (total volume = 20 mL). For model mixtures, aliquots of methanolic

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stock solutions of reference odorants were combined then mixed with corresponding juice bases

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(3 mL). The concentrations of the stock solutions and the volume of the aliquots were adjusted to

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yield final concentrations of each odorant in the model mixture that matched concentrations

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previously determined in the fruit juice. Omission samples were prepared by omitting a group of

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odorants from each mixture. Panelists were asked to define the mixture as “orange-like”,

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“mandarin-like” or “neither”. At completion, the most simplified model mixtures containing

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essential odorants for orange/mandarin aroma profiles were determined. For the model of 6-2-55,

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essential odorants for the aroma profiles of orange determined by the final step were combined

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and mixed with a corresponding juice base as described above. Panelists were asked to define the

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mixture as “orange-like”, “mandarin-like” or “neither”.

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All procedures involving the human participants were in accordance with the ethical standards of the institutional and/or national research committees and with the 1964 Helsinki


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declaration and its later amendments or comparable ethical standards. The study protocol and

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consent procedure received ethical approval from the Institutional Review Board (IRB) of the

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University of Florida. Informed consent was obtained from all individual participants included in

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the study.

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Statistical Analysis. For all the sensory tests mentioned above, three choices for each

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sample/model mixture were provided, which made the chance probability of the test 1/3. We

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took 50% correct performance after adjustment for chance as a working definition. According to

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Abbott’s formula, 8 66.7% of panelists should choose the correct answer to illustrate significant

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differences. Therefore, in the nose-pinch and pinch-free test, at least 10 panelists should choose

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the correct answer to show a significant difference. Similarly, in the evaluation of 6-2-55 juice

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and aroma profile analysis, 11 panelists and five panelists were required to show a significant

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difference, respectively.

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Extraction of volatile compounds HS-SPME analysis was performed following a modified procedure previously reported. 9

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The juice samples were incubated in a water bath at 40°C for 20 min and then extracted by a 2

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cm 50/30 µm DVB/CAR/PDMS fiber (Supelco Inc., Bellefonte, PA) for 30 min (40°C).

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Gas chromatography-mass spectrometry/olfactometry (GC-MS/O) for identification Compound identification was performed following a modified GC-MS/O method

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previously reported. 10 A series of n-alkanes (C7−C30) was used to determine linear retention

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indices (RI) for each compound.

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AEDA

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HS-SPME-AEDA was performed by two experienced panelists to determine the FD factors of aroma-active compounds. The juice sample, extracted by HS-SPME, was stepwise



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diluted using different split ratios, varying from 5:1, 10:1, 20:1, 25:1, 50:1, 100:1, 200:1 to

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500:1. The same GC conditions and column as described above were used. By definition, the

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FD factor obtained for each odorant in the HS-SPME-AEDA is equal to the highest spilt ratio.

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Juice base preparation

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Hand-squeezed fruit juices were used to prepare juice bases. Fruit juice (~300 mL) was

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extracted using an equal volume of dichloromethane and stirring for 1 h at room temperature.

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After solvent extraction, solvent assisted flavor evaporation (SAFE) technique (40°C, 10-3Pa) 11

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was used to remove volatiles from the mixture of juice and solvent while only the juice residue

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(volume less or equal to original juice volume) was collected. The juice residue was then

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extracted with dichloromethane again and the SAFE technique was repeated. The final juice

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residue was collected as the juice base. The juice bases were used as a matrix for the standard

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curve, as well as for aroma recombination.

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Gas chromatography-mass spectrometry (GC-MS) for quantitation

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Quantitation of aroma compounds was performed using identical GC-MS condition with

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compound identification. The preparation and analysis GC program applied to the standard curve

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and juice sample were identical with that mentioned above. Quantitation of volatile compounds

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was conducted by calibration curves that were obtained for each compound from five different

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concentrations in corresponding juice base matrix. Stock solution of the standards and internal

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standard (1-decanol, 100 µg/L) were prepared at appropriate concentrations in methanol and

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stored at -20°C. Standard working solutions were prepared by mixing stock solutions and then

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diluted in methanol to set up the calibration curve. All working solutions were prepared daily

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prior to use.

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Chiral analysis


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The enantiomeric compositions of linalool and limonene were determined by gas

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chromatography-mass spectrometry using an Rt-βDEXsm chiral column (30 m × 0.25 mm, 0.25

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µm film thickness, Restex, PA, USA). Separated by chiral column, two enantiomers of the same

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analyte compound exited at different times due to different affinities to the stationary phase. The

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oven temperature was programmed from 40°C (2 min hold) to 220°C (10 min hold) at 5°C/min.

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Other GC parameters were identical as described above. The juice sample preparation for SPME

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analysis was identical to that mentioned above, while a split ratio of 1:50 was used for sample

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injection. Reference enantiomeric standards were used to fulfill isomer identification. For each

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juice sample, chiral analysis was performed in duplicate.

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Results and Discussion

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Sensory evaluation

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In the nose-pinch test, six panelists selected the correct sample, while one panelist picked

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the incorrect sample. The remaining eight panelists were unable to tell the difference between the

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two samples. From the comments, panelists who chose the correct sample made their decisions

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based on the impression that “orange juice always tastes sourer than mandarin juice” and “orange

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juice has a tart taste/aftertaste”. In the pinch-free test, 12 panelists (more than required number of

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panelists to illustrate a significant difference) selected the correct sample while two panelists

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selected the incorrect sample. Only one panelist could not make a selection. The panelists who

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failed to select the correct sample commented, “I cannot tell which one is orange juice”.

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Flavor perception includes a multitude of sensory input, including smell, taste, tactile

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sensations, visual cues, etc. 12-14 It is mainly based on the responses of olfactory and gustatory

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receptors to chemical stimuli in food. For the nose-pinch test, taste was the major sensory input

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panelists had available to define the juice. In fact, orange juice is not necessarily sourer than



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mandarin, or vice versa; the sourness or sweetness of orange/mandarin varies by cultivars and

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even by individual fruit. Therefore, the different impressions of taste (sourness of juice)

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mentioned by panelists were inappropriately used as evidence for differentiating the orange from

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mandarin samples. Most of the panelists failed to define the juice using the single perception of

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taste, which clearly lessened the importance of taste as an independent factor affecting the

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characteristic flavor of orange/mandarin. On the other hand, once opening olfactory perception

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(pinch-free test), panelists indicated much better perception and interpretation of the flavor of

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both the orange and mandarin samples. This fact indicated that the aroma of both citrus fruits

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played a key role in differentiation of orange and mandarin, although the flavor of

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orange/mandarin is a combined perception of both aroma and taste. From our current

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understanding, the sensations of taste and smell interact with each other resulting in more than

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just the sum of their perceptions. Central neural integration of smell and taste inputs has been

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proven, but only with the precondition of experience with paired taste and odor stimuli. 15-16

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Moreover, it has been reviewed and summarized from both psychophysical and neuroimaging

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findings that if there is a taste-odor association, an odor alone may be sufficient to elicit flavor

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perception (although a taste alone does not seem to do this) due to olfactory-induced synesthesia.

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13, 17-18

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In the sensory evaluation of 6-2-55 mandarin hybrid, eleven out of sixteen panelists

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(equal to required number of panelists to illustrate a significant difference) determined the juice

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samples to be “orange juice”. Four panelists evaluated it as “mandarin juice” mainly due to the

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“thick texture”. Only one panelist selected “I don’t know”. 6-2-55 is an unreleased mandarin

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triploid hybrid, with substantial tolerance of HLB, developed by the UF-CREC citrus breeding

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program; parents on both sides were sexual and somatic hybrids respectively of mandarin with


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sweet orange. According to sensory evaluation, the flavor of 6-2-55 was believed to have

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intensive “orange-like” characteristic, whereas its juice matrix was still recognized as mandarin

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by some panelists. This implied that 6-2-55 mandarin hybrid could be used as a confirmation to

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study the differences of flavor between orange and mandarin. Moreover, the characteristic of 6-

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2-55 indicates the possible future use of it as supplemental material for the orange juice industry,

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although further industry trials, such as juice processing testing, is necessary and required.

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Aroma-active compounds in the juices of Valencia and LB8-9

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AEDA is a useful gas chromatography-olfactometry procedure for the analysis of potent

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odorants in food extracts. 19 Traditional AEDA is usually applied to the solvent extract of food,

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whereas recently, headspace techniques have been widely used to simplify the sample

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preparation procedure before AEDA analysis. Dynamic headspace sampling 20, static headspace

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sampling 21 and HS-SPME 22 have all been combined with the AEDA technique to complete

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identification of key volatiles from various food and beverage samples in recent studies. In this

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study, HS-SPME-AEDA was conducted to determine the aroma-active compounds in juice

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samples.

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To screen for the most potent and aroma-active odorants, HS-SPME-AEDA was

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performed on hand-squeezed juices made from Valencia oranges and LB8-9 mandarins. For both

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samples, in total of 33 odor-active regions in the FD factor range of 1-500 were detected (Table

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1). Overall, the major odor qualities of Valencia and LB8-9 were detected as citrus, fruity, floral,

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grassy and piney, which were mainly raised by esters, aldehydes and terpenes. Moreover,

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Valencia orange and LB8-9 mandarin had largely overlapping aroma-active compounds with

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higher FD factors (FD≥5 at least in one sample).



For Valencia juice, the result was consistent with previous flavor studies of orange fruit, 2,

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23-25

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in the orange aromatic profile. Besides their similarities, different orange cultivars have been

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reported with certain odorants that did not present with high odor intensity in other cultivars,

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such as 2/3 methylbutanol in Valencia Late and nootkatone in blood orange. To date, over 100

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volatiles have been reported in fresh orange juice.26 The natural combination of these volatile

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compounds in a well-balanced system, including sugars, acids and phenolic compounds,

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completes orange flavor.

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in which odorants with high FD factors detected in this study were also shown to be potent

For the juice of LB8-9, odorants identified in this study were previously found with high

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odor intensities in mandarin fruits,4, 27-28 as well as in the peel/peel oil of mandarin. 29-31

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Compared to Valencia orange, LB8-9 mandarin showed a prominent difference in its aroma-

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profile with low, or no, odor activities of esters, including ethyl butanoate, ethyl 2-

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methylbutanoate, ethyl hexanoate and ethyl octanoate, all important odorants with fruity notes in

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orange juice. Similarly, fruit juices of 15 mandarin and mandarin hybrid cultivars were

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investigated in an earlier study while ethyl butanoate and ethyl 2-methylbutanoate were not

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observed in any sample. 32 A more recent study on diversity among 13 mandarin varieties

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revealed ethyl butanoate and ethyl hexanoate were only detected in mandarin hybrids such as

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tangor (C. reticulata × C. sinensis, i.e. mandarin × orange hybrid) and tangelo (C. reticulata× C.

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paradisi, i.e. mandarin × grapefruit hybrid), while ethyl 2-mehtylbutanoate and ethyl octanoate

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were not detected. 33 From AEDA alone, nevertheless, no direct or clear conclusion could be

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drawn to clarify the difference between the aroma profile of orange and mandarin.

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Quantitation of key aroma compounds and calculation of odor activity values (OAVs) in the

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juices of Valencia and LB8-9


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To decode the characteristic difference of the aroma profile between orange and

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mandarin, 15 odorants (FD≥5) in orange juice were quantitated, while 19 aroma compounds and

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4 odorants were quantitated in mandarin and 6-2-55 juice, respectively (Table 2). In addition to

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the aroma-active compounds (FD≥5) determined from AEDA, odorants with low aroma

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activities (myrcene, (E)-2-hexenal, α-terpineol and thymol) were also quantitated in mandarin

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juice due to their abundance and importance in mandarin fruits as reported in previous studies. 33-

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35

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quantitated. Although ethyl butanoate was not detected in the juice made from frozen 6-2-55

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mandarin hybrid fruits, juice from fresh fruit possessed a small amount of ethyl butanoate (50

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µg/L, data not shown). Calibration curves were constructed by plotting the ratio of the analyte

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peak area to the internal standard peak area against the respective ratio of the analyte

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concentration to the internal standard concentration. Good linearity of all compounds was

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observed and demonstrated by the high correlation coefficient (r2) value above 0.99 (data not

265

shown). The precision and accuracy were determined by building three replicates (n=3) of

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standard curves over three consecutive days. Meanwhile, duplicate samples were analyzed on the

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same day as each standard curve and over three consecutive days. Precision was expressed by the

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relative standard deviation (RSD, %), and accuracy was expressed by the percentage of the

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observed value of each standard curve to the average value of all three standard curves. Both the

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accuracy and the precision were within the acceptable range (±15%) (Supplemental Table 1,

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Table 2 and Table 3).

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For 6-2-55, key odorants for orange-like aroma obtained from aroma recombination were

OAV data are required for confirming the contribution of the odorants to the overall

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aroma of orange/mandarin juice. By definition, only odorants with OAV ≥1 are suggested to

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contribute to the overall aroma. Odor thresholds (in water solution) of each aroma compound



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were obtained from literature data. In orange juice, ethyl 2-methylbutanoate showed the highest

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OAV of 2956 followed by ethyl butanoate (436), limonene (106), myrcene (45), decanal (38),

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linalool (24), α-pinene (23), octanal (15), hexanal (13), (E)-2-nonenal (6), nonanal (3) and

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geraniol (1). For mandarin juice, linalool had the highest OAV of 619, followed by octanal (56),

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hexanal (20), α-terpineol (17), limonene (14), decanal (7), myrcene (7), α-pinene (4), (E)-2-

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nonenal (4), (E)-2-hexenal (3), (E)-2-octenal (1), perillaldehyde (1) and geraniol (1). In the juice

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of 6-2-55, OAVs of quantitated compounds were determined: octanal (120), ethyl butanoate (50)

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and decanal (48). These compounds with OAV≥1 implied a key impact to the overall aromatic

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profile of orange juice and mandarin juice, respectively.

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Chiral analysis

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Enantiomeric analysis showed that limonene was present in all three juices as almost pure

286

(R)-(+)-enantiomers (>99%), whereas linalool was predominantly the (S)-(-)-enantiomer (Table

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3 and Figure 1). The results were in good agreement with previous studies on citrus fruits.

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Limonene occurs in nature commonly as (R)-(+)-enantiomer with a strong smell of oranges, 38

289

while the less common (S)-(-)-isomer contributes a piney, turpentine-like odor. Similarly,

290

linalool has been found in many plants existing as two enantiomers. Both of the isomers give

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pleasant scent while different odor qualities and odor thresholds are demonstrated.39-40 The (S)-

292

(+)-linalool is described as a more citrusy, fruity note compared to the (R)-(-)-linalool, which is

293

perceived as a woody, lavender note. However, the odor threshold of (S)-(+)-enantiomer in air is

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3.5-4 times higher than that of (R)-(-)-enantiomer.

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Aroma recombination and omission studies

1, 36-37

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To verify the analytical data, aqueous reconstitution models of Valencia orange and LB8-

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9 mandarin were prepared. Model mixtures containing all odorants with OAVs ≥1 in their actual 14 ACS Paragon Plus Environment

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concentrations were prepared in corresponding juice bases and were presented to the sensory

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panel. A simple recombinate of 6-2-55 mandarin hybrid was also prepared. In this way,

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interactions of a mixture of key odorants at the human odorant receptor level can be addressed.

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Furthermore, to investigate the contributions of these compounds, aroma omission models for

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orange and mandarin were prepared based on aroma descriptions of compounds, as well as their

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OAVs. Each of the reconstitution models was evaluated by the sensory panel and defined as

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“orange-like”, “mandarin-like” or “neither”.

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Orange. Compounds with OAV≥1 (α-pinene, ethyl butanoate, ethyl 2-methyl butanoate,

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hexanal, myrcene, limonene, octanal, nonanal, decanal, (E)-2-nonenal, linalool and geraniol) in

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their actual concentrations were combined and added into orange juice base to make the original

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model mixture. In addition, acetaldehyde (8000 µg/L) was added to the original recombinant to

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include a fresh note, 2 which made model mixture o-1. Different model mixtures for the

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recombination and omission study are listed in Table 4. In model mixture o-1, the odor note,

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floral, was too intense, seeming to cover all the other notes perceived. This might be due to the

312

use of a linalool isomer mixture instead of pure (S)-linalool in model mixture o-1. Therefore,

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model mixture o-2 was prepared, in which linalool was omitted. For model mixture o-2, panelists

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described an orange-like aroma. To further simplify the model, three aldehydes with relatively

315

low OAVs (hexanal, nonanal and (E)-2-nonenal) were omitted from model mixture o-2 to obtain

316

model mixture o-3. Model mixture o-3 was still recognized as orange-like aroma. The final

317

model mixture o-4 was prepared with omission of a group of terpenes (α-pinene, myrcene,

318

limonene, geraniol) based on model mixture o-3. Panelists evaluated model mixture o-4 and

319

defined it as orange-like aroma, which left acetaldehyde, ethyl butanoate, ethyl 2-

320

methylbutanoate, octanal and decanal as the five essential odorants for orange-like aroma. To



321

verify this conclusion, a recombinant of acetaldehyde, ethyl butanoate, ethyl 2-methylbutanoate,

322

octanal and decanal, in their actual concentrations based on 6-2-55 mandarin hybrid, was also

323

prepared. Acetaldehyde (8000 µg/L), ethyl butanoate (50 µg/L), octanal and decanal were added

324

to 6-2-55 juice base to make the recombinant. Panelists described this 6-2-55 recombinant as

325

orange-like aroma. In the juice of 6-2-55 mandarin hybrid, ethyl 2-methylbutanoate was not

326

detected. The omission of ethyl 2-methylbutanoate from 6-2-55 recombinant did not affect the

327

perception of orange-like aroma, possibly suggesting ethyl 2-methylbutanoate was not as

328

important as the other fruity ester, ethyl butanoate, in the aroma profile of orange.

329

Acetaldehyde, a natural aroma component, is present in almost every fruit. The

330

acetaldehyde content along with ethanol content of orange increase during the growing season,

331

offering a measure of maturity in addition to the solids-acid ratio. 41-42 While it seems that the

332

increase in ethanol levels in fruit adversely affected the flavor quality, acetaldehyde has been

333

suggested to improve fruit flavor. 43 It has been proven to contribute a fresh, pungent odor

334

quality to freshly squeezed orange juice, 44 which was also observed in our study. Acetaldehyde

335

contributed freshness to the orange model mixtures and seemed to help the delivery of the

336

overall aroma. Although being very abundant in fresh orange samples, acetaldehyde was

337

observed to decrease during storage of orange juice, 45 which implied its instability and might

338

explain the absence of acetaldehyde in our orange juice samples. Similarly, acetaldehyde has

339

been detected from fresh juice sample prepared by dynamic headspace but not in thermally

340

treated solvent extraction in previous studies. 1, 46 Ethyl butanoate is a fruity ester occurring

341

naturally in many fruits including oranges, 25 pears, 47 pineapples, 48 etc.. It has been considered

342

as the major volatile ester in orange juice and believed to contribute more than all the other esters

343

combined.49 A general decrease in ethyl butanoate in processed orange juice due to thermal


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344

treatment is correlated with decreasing quality of juice product compared to fresh-made juice. 50

345

In the food industry, ethyl butanoate is commonly used as artificial flavoring resembling orange

346

juice. Ethyl 2-methylbutanoate is also an important orange juice ester presenting a pleasant,

347

fruity aroma, although it might not be as important as ethyl butanoate in fresh orange juice.

348

Octanal and decanal, described as citrus-like, soapy notes, are two aldehydes that have been

349

implicated as important contributors to orange flavor. 51 However, contents of octanal and

350

decanal are not necessarily positively correlated to the quality of fresh orange juice since

351

processed juice contains even higher levels mainly from peel oil.

352

Mandarin. Compounds with OAV≥1 (α-pinene, hexanal, myrcene, limonene, (E)-2-

353

hexenal, octanal, (E)-2-octenal, decanal, (E)-2-nonenal, linalool, α-terpineol, perillaldehyde,

354

geraniol) were combined and added into mandarin juice base to make the original model mixture.

355

(E,E)-2,4-decadienal (0.2 µg/L, OAV~1 52) was added to the original recombinant due to its

356

important role in the aroma profile of mandarin 29-30, 32 to introduce an oily note, which made

357

model mixture m-1. Different model mixtures for the recombination and omission study are

358

listed in Table 5. For model mixture m-1, panelists defined it as mandarin-like aroma. To further

359

simplify the model, three odorants (OAVs=1) were omitted from model mixture m-2 compared

360

to model mixture m-1. Model mixture m-2 was still recognized as mandarin-like aroma by

361

panelists. Similarly, three aldehydes ((E)-2-hexenal, (E)-2-nonenal and decanal) with low OAVs

362

were omitted from model mixture m-3 while myrcene, α-terpineol and hexanal were omitted

363

from the last model mixture, mixture m-4. Both model mixture m-3 and model mixture m-4 were

364

perceived as mandarin-like aroma. Therefore, α-pinene, limonene, octanal, linalool as well as

365

(E,E)-2,4-decadienal were determined as the most critical odorants to make the mandarin-like

366

aroma. However, for the mandarin model mixtures, there was a time limitation on the release of



367

aroma. The aroma of fresh prepared mixtures was perceived as mandarin-like, but quickly

368

changed as time passed. Further study is required to clarify this situation.

369

(E,E)-2,4-decadienal, always described as deep fried, oily notes, has been found as a key

370

aroma-active odorant in mandarin juice/oil with high odor activity. The threshold of (E,E)-2,4-

371

decadienal in water was reported ranging from 0.07-0.2 µg/L, which makes it easy to be

372

perceived but hard to be detected. In this study, (E,E)-2,4-decadienal was observed as an

373

indispensable component in mandarin-like aroma. Linalool was added into the mandarin model

374

mixture as the most abundant, as well as the most aroma-active, volatile. From chiral analysis,

375

(S)-(+)-linalool was predominant (95.2%) in mandarin juice, which should contribute a more

376

citrusy fruity note compared to the (R)-(-)-linalool. α-Pinene and limonene are two terpenes

377

contributing citrus-like, pungent aromas. α-Pinene was described as piney, citrus-peel-like while

378

limonene contributed most of the citrus-like, minty notes. Meanwhile, limonene was also one of

379

the most abundant compositions in mandarin, yet with a relatively high sensory threshold. It has

380

been reported that measurements of the stability of limonene and linalool will help with the

381

quality control of mandarin juice. 53-54 Octanal, showed as an important contributor to orange

382

aroma, but also showed great importance to mandarin-like aroma.

383

Comparing essential odorants for orange aroma and mandarin aroma, ethyl butanoate and

384

acetaldehyde stood out as characteristic contributors to fresh orange aroma while linalool and

385

(E,E)-2,4-decadienal seemed to be potent components for mandarin aroma. Aldehydes like

386

octanal could be important for aroma of both orange and mandarin while terpenes were critical

387

for mandarin aroma. Current literature does not define a simple and standardized

388

orange/mandarin aroma. The perception of orange/mandarin aroma seems due to the synergistic

389

effect of several key odorants instead of a characteristic volatile. However, even for the essential


Page 18 of 34

Page 19 of 34


390

odor-active compounds, their contents in citrus fruits vary largely among different varieties and

391

even among fruits from the same variety, but harvested under different conditions (season, year,

392

location, etc.). Meanwhile, orange/mandarin fruits from different varieties may have distinct

393

aromas but still, are perceived and described as orange/mandarin. These facts lead to the

394

hypothesis that the overall perception/impression of orange/mandarin aroma in the human brain

395

remains the same as long as all the key odorants are within a relatively flexible range with

396

respect to each other to maintain a balance. This could be supported by an early study in which

397

the dependence of perceived odor quality on odorant concentration was assessed with the

398

conclusion that variations in olfactory stimulus might be perceived as quality differences.55 The

399

principles underlying the recognition of olfactory stimuli for mammals are complicated and not

400

fully understood.56 The main olfactory system (MOS) is a broadly tuned odor sensor that

401

responds to thousands of volatile chemicals.57 Within MOS, olfactory sensory neurons (OSNs)

402

and main olfactory bulb (MOB) are critical components that are involved in odorant detection.

403

Odors are encoded using a combinatorial strategy since most OSNs can respond to multiple

404

odorants while multiple olfactory receptors recognize the same odorant.57-59 It is believed that

405

odorants initiate distinct spatial activity patterns in the glomerular layer of the olfactory bulb

406

while evoking the perception of distinct odors.60-61 In a study focusing on the mechanism

407

whereby odorants are encoded in the nervous system,62 changes in odorant concentrations for

408

certain compounds, but not others, were illustrated to affect spatial patterns of glomerular

409

activity as well as perceived odor. Due to the complex mechanism of odor perception, the

410

chemical formula for orange/mandarin aroma may fall into a dynamic range with potent odorants,

411

and not necessarily maintained as a single format. Therefore, to decode the differences between

412

orange and mandarin, understanding of the key odorants representing each fruit is the key step.



413

AEDA is a useful tool to screen out the most odor-active compounds in a juice sample, that then

414

require further investigation and quantitation. However, high FD factor does not guarantee the

415

importance of the volatiles’ contribution to the overall aroma. Matrix influence on odorants can

416

significantly change the perception of juice flavor from headspace due to unclear molecular

417

interactions of aroma compounds within the juice sample, e.g., matrix constituents may repress

418

or promote certain aroma notes. On the other hand, synergistic effects, as well as antagonistic

419

effects, among volatile compounds also exist, possibly influencing the release of certain odorants.

420

Therefore, aroma recombination analysis is necessary to verify the analytical data and more

421

importantly, to display the real-time perception of model mixture aroma in its entirety. In

422

addition to the accuracy of quantitation of key odorants, the choice of model matrix is also

423

critical due to its direct influence on the aroma release.

424

This study provided a preliminary understanding of the characteristic flavors of orange

425

and mandarin, as well as their differences. However, there were still limitations in this study and

426

further comprehensive investigation is strongly desired. Future emphasis should be on studying

427

additional citrus varieties, and the aroma compound thresholds should also be measured in

428

corresponding juice matrix. Moreover, dynamic olfactometery could be applied to study the level

429

range of key odorants that characterize orange/mandarin aroma.

430

Acknowledgment

431

We thank Laura Reuss (CREC, UF) for editing the manuscript.

432

Associated Content

433

Supporting information

434

The precisions and accuracies of aroma quantitation in fruit juices (PDF).


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Figure Captions Figure 1. Chromatogram of enantiomeric analysis on limonene and linalool in Valencia juice.



Page 30 of 34

Table 1. The Most Odor-Active Volatiles in the Juices of Valencia Orange and LB8-9 Mandarin. no.a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

odorantb ethyl acetate ethyl propanoate α-pinene ethyl butanoate ethyl 2-methylbutanoate hexanal (Z)-3-hexenal myrcene limonene β-phellandreneg ethyl hexanoate γ-terpinene octanal 1-octen-3-one 6-methyl-5-heptene-2-one 1-hexanol 4-mercapto-4-methylpentan-2-onef nonanal (E)-2-octenal ethyl octanoate 1-octen-3-ol methionalf citronellal α-copaeneg decanal (E)-2-nonenal linalool β-elemene D-carvone perillaldehyde β-damascenonef geraniol perilla acetateg

odor qualityc fruity fruity citrus peel fruity fruity grassy grassy mossy, musty citrus-like, minty terpeny fruity pine tree citrus-like, soapy mushroom sweet, green resin, paper blackcurrant citrus-like floral fruity nutty, earthy cooked potato minty, citrus-like herbal, green green, citrus-like floral floral green sweet, herbal green sweet, syrup floral green

a

RId (FFAP) 873 918 983 1003 1024 1052 1117 1131 1168 1174 1208 1214 1261 1274 1310 1326 1343 1363 1399 1404 1421 1427 1444 1447 1467 1504 1517 1551 1698 1749 1790 1823 1866

FD factore Valencia LB8-9 1

Differentiation between Flavors of Sweet Orange (Citrus sinensis) and

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