Characterization of the Aldehydes and Their Transformations Induced

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Characterization of the aldehydes and their transformations induced by UV irradiation and air exposure of white Guanxi honey pummelo (Citrus grandis (L.) Osbeck) essential oil Li-Jun Li, Peng Hong, Feng Chen, Hao Sun, Yang Yuanfan, Xiang Yu, Gao Ling Huang, Liming Wu, and Hui Ni J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01369 • Publication Date (Web): 26 May 2016 Downloaded from http://pubs.acs.org on May 27, 2016

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Journal of Agricultural and Food Chemistry

Characterization of the Aldehydes and Their Transformations Induced by UV Irradiation and Air Exposure of White Guanxi Honey Pummelo (Citrus Grandis (L.) Osbeck) Essential Oil Li Jun Li1,2,3, Peng Hong1, Feng Chen1,4, Hao Sun1, Yuan Fan Yang1,2,3, Xiang Yu1, Gao Ling Huang1,2,3, Li Ming Wu5, Hui Ni1,2,3 1: College of Food and Biology Engineering, Jimei University, Fujian Province, P.R. China 361021 2: Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen, 361021, China 3: Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China 4: Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC 29634, USA 5: Institute of Apicultural Reaseach, CAAS, Beijing, 100093 China

Professor Hui Ni College of Food and Biology Engineering Jimei University Xiamen, Fujian Province 3616021, China Phone: 13015914929 E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract:

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Aldehydes are key aroma contributors of citrus essential oils. White Guanxi

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honey pummelo essential oil (WPEO) was investigated in its aldehydes constituents

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and their transformations induced by UV irradiation and air exposure by GC-MS,

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GC-O, and sensory evaluation. Nine aldehydes, i.e., octanal, nonanal, citronellal,

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decanal, trans-citral, cis-citral, perilla aldehyde, dodecanal, and dodecenal were

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detected in WPEO. After treatment, the content of citronellal increased, but the

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concentrations of other aldehydes decreased. The aliphatic aldehydes were

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transformed to organic acids. Citral was transformed to neric acid, geranic acid and

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cyclocitral. Aldehyde transformation caused a remarkable decrease in the minty,

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herbaceous and lemon notes of WPEO. In fresh WPEO, β-myrcene, d-limonene,

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octanal, decanal, cis-citral, trans-citral, and dodecenal had highest odour dilution folds.

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After the treatment, the dilution folds of decanal, cis-citral, trans-citral, and dodecenal

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decreased dramatically. This result provides information for the production and

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storage of aldehydes-containing products.

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Keyword: Pummelo essential oil, Aldehyde, Transformation, GC-O, Ultraviolet (UV)

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irradiation, Air exposure

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Introduction

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Citrus essential oils (CEOs) are employed in the food, pharmaceutical and

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perfumery industries for their attractive fragrances and antimicrobial, insect-resistant,

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and antioxidant bioactivities. 1-4

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As reported, CEOs are mainly composed of monoterpenes,5 aldehydes,

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alcohols and esters.6-8 Among these chemicals, d-limonene and β-myrcene account for

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the majority of CEOs in weight.1,

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concentration, they contribute vitally to the aroma of CEOs due to their very low

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threshold values.10, 11 In addition, different CEOs have great variety in the aldehydes

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constituent. Liu et al. reported that decanal was a characteristic aroma compound of

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Mangshanyegan, Kaopan pummelo and Eureka lemon.10 Nguyen et al. found that

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(R)-(+)-citronellal was responsible for the characteristic aroma of Kabosu (Citrus

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sphaerocarpa), rather than the much more abundant constituents limonene and

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β-myrcene, which accounted for 70.5% and 20.2% of the total amount of volatiles,

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respectively.12 Song et al. found that octanal was a characteristic aroma compound of

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daidai (Citrus aurantium L. var. cyathifera Y. Tanaka) peel oil.13 Moreover, the

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concentrations of aldehydes were observed to decrease during production and storage,

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which might be related to the reactions induced by heat, sunlight and oxygen.9, 14

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Previous studies indicated that the decrease in the aldehyde concentration might have

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a great effect on the aroma of a CEO; for instance, Nguyen et al. reported that even a

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0.1% decrease in the citral concentration could significantly change the aroma of

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lemon essential oil.15 Despite all this, our understanding on the aldehyde constituents

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Although aldehydes represent minority in

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and their effects on the aroma of CEOs is still limited.

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Pummelo (Citrus grandis (L.) Osbeck) is a traditional citrus variety of

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Southeast Asia that is now an important and popular citrus species extensively

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cultivated and widely consumed all over the world. Like other citrus peels, pummelo

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peel is rich in fragrant oil;16 which has different aroma from those of other citrus

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essential oils.2,7 Cheong et al. showed that main odorants in pink pomelo peel extract

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were cis,trans-2,6-nonadienal, octanal, citronellal, nonanal, trans-nerolidol, indole,

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6-methyl-5-hepten-2-one,

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nootkatone, while the most intense aromas in white pomelo peel extract were

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terpinolene,

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citronellol, elemol, carvyl acetate, perilla aldehyde and indole,18 indicating the

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different varieties of pummelo have different aroma-active constituents.

terpinolene,

terpinen-4-ol,

trans-2-heptenal,

trans-nerolidol,

trans-linalool

perilla

oxide,

alcohol

and

nootkatone,

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White Guanxi honey pummelo (Citrus grandis (L.) Osbeck) is a traditional

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pummelo variety that has been commercially planted in Fujian province of China with

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an approximate production of 1,200,000 tons, accounting for 40% of the total

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production of China. As the debittering process is becoming more and more effective,

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a few plants have been set up in southeast China to extract the juice. There are now

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about 10,000 tons of pummelo processed in Fujian Province every year, generating

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appropriate 2,000 tons of peels for extracting essential oil. 17

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In recent researches, it has been shown that the essential of white Guanxi

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honey pummelo (WPEO) mainly composed of d-limonene and β-myrcene,

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germacrene D, geranial, neral, β-pinene, linalool, sabinene and α-pinene,2 which are 4


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similar to the major constituents of other CEOs.10,

15, 19

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processes, UV irradiation and oxygen exposure have been shown to have significant

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effects on the constituents and aroma of the WPEO. 2, 13 Moreover, the accumulation

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of linalool and limonene oxides resulted from the degradation of d-limonene is

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illustrated to be the main reason for the strong oily and off-flavor notes.13 However,

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the aroma-active aldehydes and their changes during the production and storage have

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not been sufficiently investigated, yet. In this context, the aim of this study was to

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illustrate the main aldehydes in WPEO and analyze the effects of aldehyde

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transformations on the aroma of WPEO after UV irradiation and air exposure.

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Furthermore, extraction


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Materials and methods

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Chemicals. A standard series of C8−C20 alkanes used for retention index (RI)

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determination, internal standards (i.e., ethyl 2-methylbutyrate and ethyl benzoate)

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were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). Standard

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α-pinene, sabinene, β-pinene, d-limonene, α-phellandrene, β-cis-ocimene, terpinolene,

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perillene, β-elemene, trans-caryophyllene, nerol, geraniol, nerolidol, farnesol,

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γ-elemene, α-humulene, linalool, octanal, nonanal, citronellal, decanal, carvone, and

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citral (mixture of neral and geranial) were purchased from Sigma-Aldrich Co. Ltd. (St.

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Louis, MO, USA); β-myrcene, perilla aldehyde, dodecanal, dodecenal, octanoic acid,

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citral acid (mixture of neric acid and geranic acid), chrysanthemol, carveol (mixture of

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cis- and trans-), neryl acetate, geranyl acetate, linalool oxide (mixture of cis- and

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trans-), and limonene oxide (mixture of cis- and trans-) were purchased from Alfa

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Aesar Co. Ltd. (Heysham, Lancashire, U.K.); α-pinene oxide, caryophyllene oxide,

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and nootkatone were purchased from Ormah Chemical, Inc. Tic Co. (TIC, Hadimkoy,

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Istanbul); indole and octanoic acid were purchased from Penta Manufacturing Co.

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(PMC, Livingston, NJ, USA). HPLC grade n-hexane was purchased from Oceanpak

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Chemical Company (Gothenburg, Sweden). Other chemicals were obtained from

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Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

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Identifying the aldehydes and investigating the concentration changes in

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WPEO after UV irradiation and air exposure. White GuanXi honey pummelo, a

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native pummelo species in southeastern China, was harvested after fully ripe with

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yellow color by Fujian GuoNong Agricultural Development Co. (PingHe County, 6


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Fujian province, China) in October 2014. The fresh flavedo with oil sacs was

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mechanically pressed to squeeze the oil into a tube containing icy brine water. Then,

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the oil-water mixture was centrifuged at 13,500 g for 5 min then dried with Na2SO4 to

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collect fresh WPEO, then stored in -20 ºC.9 Total 10.8 g of WPEO was extracted from

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2,000 g of pummelo peel. To prepare the UV-irradiation and air-exposure co-treated

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sample, 500 µl fresh WPEO was pipetted into small glass tubes (2 mL, 12×32 mm

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bottle diameter 9 mm, borosilicate material) that were then filled with air and sealed

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with a screw cap. The tubes were then irradiated under two ZXC-II×2 UV modulator

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tubes with a peak emission at 305 nm (Yuejin Medical Optical Instruments Factory,

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Shanghai, China) at a distance of 5 cm for 40 h using a power of 40 W and a

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temperature of approximately 25 ˚C. Thereafter, both the fresh and the treated WPEOs

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were subjected to the analysis of their volatile constituents using gas chromatography

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coupled with mass spectrometry (GC-MS) after dilution.

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Identifying the reaction pathways and inducing factors of aldehyde

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transformation. Single standard aldehydes without solvent dilution, i.e., octanal

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(99%), nonanal (96%), citronellal (96%), decanal (95%), citral (97%, mixture of

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trans-citral and cis-citral), perilla aldehyde (92%), dodecanal (95%), and dodecenal

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(93%) were treated with air exposure (Sample 1#), UV irradiation (Sample 2#) or a

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combination of both (Sample 3#), respectively. In short, for the preparation of the

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air-exposed aldehydes (Sample 1#), 500 µL of each aldehyde standards were pipetted

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into small glass tubes that were then filled with air, sealed with a screw cap and stored

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in the dark at 25 ˚C for 40 h. To prepare the UV-irradiated samples (Sample 2#), 500 7



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µL of each aldehyde standards was pipetted into a small glass tube that was filled with

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nitrogen, sealed with a screw cap, and irradiated under UV using the procedure

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described in the section “Investigating the concentration change of aldehydes in

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WPEO after UV irradiation and air exposure”. To prepare the samples undergoing the

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co-treatment of UV irradiation and air exposure (Sample 3#), 500 µL of each

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aldehyde standards was pipetted into a small glass tube that was filled with air, sealed

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using a screw cap, and exposed under UV irradiation using the procedure described

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above. Afterwards, the constituents of the samples were analyzed using GC-MS after

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dilution.

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Calculation of odor activity values (OAVs). Both the fresh and treated

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WPEOs were analyzed for their odor activity values (OAVs) using the equation

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OAV=c/t, where “c” is the concentration of a compound in the WPEO samples and “t”

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is the odor threshold value of a compound in water, as collected from the literature. 10,

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20-22

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Omission Experiment. Flavor models sample (FMS) reported by Sun et. al.

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was used to investigate the contributions of some aroma-impact volatiles to the

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change of the aroma profile.9 In this study, fresh WPEO (FMS 0#) and

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aldehyde-transformed WPEO (FMS 1#) were constructed to illustrate the effects of

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the aldehyde transformation on the aromas using sensory evaluation. In detail, based

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on the GC-O, OAV analysis, sensory evaluation and the study of Sun et al.,9 FMS 0#

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was prepared with a composition of β-myrcene (220,000 µg/mL), octanal (800

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µg/mL), d-limonene (500,000 µg/mL), linalool (2,700 µg/mL), decanal (2,000 µg/mL), 8


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citral (mixtu re of trans- and cis-, 17,000 µg/mL), dodecanal (200 µg/mL) and

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nootkatone (1,000 µg/mL). (Note: because there were some other compounds

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contributing to floral and pummelo note in addition to linalool and nootkatone, FMS

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was formulated with linalool and nootkatone at higher concentrations than their

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counterparts in PEO so that the floral and pummelo note attained the intensity as same

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as those of the fresh WPEO). FMS 1# (only lower concentration of aldehydes

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compared to FMS 0#) contained β-myrcene (220,000 µg/mL), octanal (500 µg/mL),

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d-limonene (500,000 µg/mL), linalool (2,700 µg/mL), decanal (1,000 µg/mL), citral

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(mixture of trans- and cis- citral) (2,000 µg/mL), dodecanal (100 µg/mL), and

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nootkatone (1,000 µg/mL) and was produced by reducing the aldehyde contents of

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FMS 0# according to the decreased concentration in the WPEO after the co-treatment

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of UV irradiation and air exposure. Then, the odors of the fresh WPEO, treated

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WPEO, FMS 0# and FMS 1# were sensory evaluated.

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Gas chromatography-olfactory (GC-O) analysis. An Agilent 7890A was

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equipped with a FID and olfactory detection port Gerstel ODP-2 (Gerstel AG

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Enterprise, Mülheim an der Ruhr, Germany). The GC was fitted with a 60 m×0.32

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mm×0.25 µm Rtx-wax column (Restek Corporation, Bellefonte, PA, USA). The oven

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temperature was programmed as follows: an initial temperature of 40 °C was kept for

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3 min, the temperature was then increased to 130 °C at a rate of 3 °C/min, and then

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increased to 230 ºC at a speed of 5 ºC/min, finally kept at 230 °C for 3 min. Nitrogen

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was used as the carrier gas at a flow rate of 1.8 ml/min. The temperature of the

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injector port was 250 °C, and a 0.2 µL sample was injected into the GC-O system in 9



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splitless mode. The sniffing port was held at 250 ºC to prevent any condensation of

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volatile compounds.

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Aroma extract dilution analysis (AEDA) was used. A series of 4-fold dilutions

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(i.e. 40, 41, 42, 43) of the fresh WPEO and the treated WPEO samples were prepared

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using dichloromethane as a solvent.10 The odor characteristics and flavor dilution (FD)

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factors were evaluated by an expert in fragrance chemicals and flavor technology.

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Operation of gas chromatography coupled with mass spectrometry

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(GC-MS) to analyze the volatile compounds. A QP 2010 Plus gas chromatograph

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coupled with a mass spectrometer was purchased from Shimadzu Corporation (Kyoto,

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Japan). One microliter of sample was injected for the analysis. Helium was used as

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the carrier gas, and the column flow rate was 3 ml/min. The injector temperature was

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set to 250 ºC, and the split ratio was 1:5. The oven temperature was initially

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programmed to 50 ºC for 3 min and then increased to 220 ºC at a speed of 5 ºC/min

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and held at this temperature for 3 minutes. The temperatures of the ion source and the

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interface were both set to 250 ºC. The MS spectrum was operated in positive electron

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ionization mode at 70 eV. The MS spectra were recorded within an m/z range from 29

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to 500 amu.

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Identification of the volatiles was performed using a 60 m×0.32 mm×0.25 µm

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Rtx-5MS column and a 60 m×0.32 mm×0.25 µm Rtx-wax column (Restek

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Corporation, Bellefonte, PA, USA). Most of the volatiles were identified by matching

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their MS spectra and Kovats retention indices (RI) to those of standards on both

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columns. In addition, other volatiles lacking a standard were identified by matching 10


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their MS spectra to those in the Mass Spectral Library (NIST08, NIST08s, FFNSC 1.3)

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and their RIs to those from relevant references or the database book.

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The compounds for which standards were purchased were quantified on the

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Rtx-5MS column using their respective calibration curves, plotted in selective ion

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monitoring (SIM) (Supplemental Table 1) mode according to the signal intensity of a

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series concentration, during which internal references ethyl 2-methylbutyrate and

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ethyl benzoate were used to correct the error. The concentrations of the volatiles that

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lacked standards were estimated by matching the signal intensity to the calibration

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curve of the internal reference ethyl benzoate obtained from Scan mode.

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Sensory evaluation of aroma. The sensory evaluation was conducted by 18

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trained panelists, including 8 women and 7 men in the age range of 20 to 30 years and

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1 woman and 2 men in the age range of 30 to 40 years. Before the sensory evaluation,

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the panelists were instructed to be familiar with the odors of the standards as

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necessary. The panelists were asked to rate the WPEOs in a random order for sweet,

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minty, herbaceous, lemon, floral and oily notes and gave a score within the range 0-9

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according to relevant references and ISO 8589,22-24 in which 0 indicates an

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unperceived attribute intensity and 9 indicates a very strong attribute intensity. The

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odors of limonene oxides, carvone and citral (mixture of trans- and cis-) were used to

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train panelists to score the sweet, minty and herbaceous notes, respectively. A mixture

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of nootkatone and d-limonene was used to train panelists to score the lemon odor, a

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mixture of geraniol and β-myrcene was used to train the panelists to score the floral

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odor, and a mixture of linalool oxides and α-terpineol was used to train panelists to 11



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score the oily note. Among these notes, herbaceous and lemon aromas contributed to

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the fresh odor and sweet and floral aromas contributed to the ripened odor (reduced

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freshness), while the oily aroma was an off-flavor similar to a machinery oil or

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resinous material.25 Mean values were calculated after correcting for the effects of the

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session and the removal of outliers. An interval gap of 20 s was used, which was

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sufficient to separate between the individual odour assessments.

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Statistical analysis. All the experiments were performed in triplicate. The

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means and standard deviations were calculated using the SPSS-IBM 19.0 software

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and Microsoft Excel 2013. Significant difference analysis was performed by ANOVA

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and Duncan’s multiple range tests using SPSS-IBM 19.0 software.

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Results and discussion

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The aldehyde constituents in fresh WPEO and the relative change after

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UV irradiation and air exposure. 49 compounds were detected in the WPEOs (Table

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1), among which 41 volatiles were identified by matching their RI and MS spectra

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with those of standards and the other 8 volatiles were identified by searching the MS

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library and comparing the RIs with those from related references.10, 24, 26-31 Among

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these chemicals, nine were aldehydes (Table 1), including octanal, nonanal, citronellal,

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decanal, trans-citral, cis-citral, perilla aldehyde, dodecanal, and dodecenal. Liu et al.,

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who found 12 aldehydes (including all the 9 aldehydes of the present study) in the

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essential oils of Mangshanyegan (Citrus nobilis Lauriro), Kaopan pummelo (Citrus

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grandis), Eureka lemon (Citrus limon), Huangyanbendizao tangerine (Citrus

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reticulata), and Seike navel orange (Citrus sinensis).10 Furthermore, the five citrus

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essential oils have been detected to have different aldehydes components, e.g., the

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essential oils of Eureka lemon, Huangyanbendizao tangerine, and Seike navel orange

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contain all the 12 aldehydes, while the counterparts of Mangshanyegan and Kaopan

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pummelo have 10 and 5 aldehydes.10 In addition, even the pink and red pomelo

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(Citrus grandis (L.) Osbeck) peel have different aldehyde compositions, the red

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pomelo peel contains 14 aldehydes, including hexanal, octanal, nonanal, citronellal,

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decanal, neral, geranial, dodecanal, tridecanal, trans,trans-2,4-decadienal, perillic

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aldehyde, tetradecanal, α-sinensal and β-sinensal; whereas the white pomelo has 6

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aldehydes, i.e., citronellal, neral, geranial, perillia aldehyde, α-sinensal and

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β-sinensal.32 In comparison, the aldehydes in the present study are different from 13



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those in other citrus essential oils, which is identical to previous studies that the peels

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of citrus fruits have different aldehydes. 10

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Based on the identification, the calibration curves of 41 volatile standards

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(Table 2) were plotted to analyze the concentration changes of the volatile compounds.

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As the result (Table 2), the concentrations of octanal, nonanal, decanal, dodecanal,

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dodecenal, perilla aldehyde, trans-citral, and cis-citral decreased by 13.8, 28.3, 40.5,

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37.8, 85.4, 33.9, 85.6 and 82.1% (p

Characterization of the Aldehydes and Their Transformations Induced

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