Citrus Kinokuni) Compared with Satsuma Mandarin Orange (Citrus Unshiu

90937 (138)

Biosci. Biotechnol. Biochem., 74 (4), 90937-1–8, 2010

Aroma Character Impact Compounds in Kinokuni Mandarin Orange (Citrus kinokuni) Compared with Satsuma Mandarin Orange (Citrus unshiu)

y Norio MIYAZAWA,1;2; Akira FUJITA,1 and Kikue KUBOTA2;3

1Technical Research Institute, R&D Center, T. Hasegawa Co., Ltd., 29-7 Kariyado, Nakahara-ku, Kawasaki, Kanagawa 211-0022, Japan 2Institute of Environmental Science for Human Life, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan 3Graduate School of Humanities and Sciences, Laboratory of Food Chemistry, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan

Received December 16, 2009; Accepted January 9, 2010; Online Publication, April 7, 2010 [doi:10.1271/bbb.90937]

The odor-active volatiles of Kinokuni mandarin tons, while that of Satsuma mandarin has reached about (Citrus kinokuni Hort. ex Tanaka), an original mandarin 800,000 tons.3) Despite these disadvantages, there is still orange in Japan, were characterized by a combination of a demand for Kinokuni mandarin, because the fruit has a instrumental and sensory analyses and compared with pleasant flavor and a distinctive and strong aroma with a thoseAdvance of Satsuma mandarin (Citrus unshiu Marcovitch). View higher sugar content. An aroma extract dilution analysis (AEDA) of the polar Among the citrus fruits, the species mainly cultivated fractions of Kinokuni and Satsuma mandarin peel oils in Japan are Kinokuni mandarin, Satsuma mandarin, identified five odorants in common as the most odor- yuzu (Citrus junos Sieb. ex Tanaka), sudachi (Citrus active volatiles: (Z)-hex-3-enal, decanal, linalool, yuzuol, sudachi Hort. ex Shirai), kabosu (Citrus sphaerocarpa and (2E)-trans-4,5-epoxydec-2-enal. In addition, seven Hort. ex Tanaka), hassaku orange (Citrus hassaku Hort. odorants were identified solely in Kinokuni mandarin as ex Tanaka), and natsudaidai (Citrus natsudaidai significant contributors: octanal, dodecanal, (2E,4E)- Hayata), for example; these are called ‘‘Wa-kankitsu,’’ deca-2,4-dienal, geraniol, yuzunone, (2E,7Z)-trans-4,5- meaning Japanese citrus cultivars, which are distin- epoxydeca-2,7-dienal, and thymol. The odor-active vol- guished from such citrus fruits cultivated throughout the atiles in both the non-polar components of the peel oil world as orange,Proofs grapefruit, lemon, and lime. With yuzu and an extract of the juice prepared from Kinokuni and Satsuma mandarin recently becoming popular all mandarin were also identified. The (S)-isomer of linalool over the world, the other Japanese citrus cultivars are in Kinokuni mandarin peel oil was dominant in the also attracting the attention of the food and beverage enantiomeric distribution (92%), whereas the (R)-isomer industries. Its pleasing olfactometric and organoleptic was dominant in Satsuma mandarin peel oil (90%). flavor features prompted us to reinvestigate the Kinokuni mandarin, which first appeared more than Key words: Kinokuni mandarin orange (Citrus kinokuni 1,000 years ago, for consumption as a rare citrus fruit. Hort. ex Tanaka); Satsuma mandarin orange The aroma of various mandarins and oranges has been (Citrus unshiu Marcovitch); aroma extract well studied by a number of researchers,4–12) and many dilution analysis (AEDA); yuzunone odor-active compounds in mandarin oranges and various ((6Z,8E)-undeca-6,8,10-trien-3-one); yuzuol other citrus species have been identified by gas ((6Z,8E)-undeca-6,8,10-trien-4-ol) chromatography-olfactometry (GC-O) and an aroma extract dilution analysis (AEDA).13–21) Although the Kinokuni mandarin (Citrus kinokuni Hort. ex volatile components of Kinokuni mandarin peel oil have Tanaka), also well-known as Kishu mandarin, used to been studied by Shiota and Ito22,23) in comparison with be the most popular mandarin orange in Japan until those of the other citrus varieties, the odor-active around 1900, but was superseded by a newly introduced compounds have not previously been determined. and more profitable variety, Satsuma mandarin (Citrus Although the odor-active compounds in Satsuma man- unshiu Marcovitch), because Kinokuni mandarin is darin, which is the most popular variety in Japan, have smaller in size (half the size of Satsuma mandarin), been identified by GC-O,24,25) they have not been ranked unattractively rough in appearance, filled with pips, and according to their odor potency. technically difficult to cultivate.1,2) The current annual We analyze in this study the aroma of the peel oil and production of Kinokuni mandarin is only about 1,000 juice of Kinokuni mandarin in order to determine the

y To whom correspondence should be addressed. Tel: +81-44-411-0298; Fax: +81-44-434-5257; E-mail: norio [email protected] Abbreviations: AEDA, aroma extract dilution analysis; ANOVA, analysis of variance; CF, correction factor; df, degree of freedom; EI, electron impact; FD, flavor dilution; FID, flame ionization detector; GC, gas chromatograph; GC-MS, gas chromatography-mass spectrometry; GC-MS-O, gas chromatography-mass spectrometry-olfactometry; GC-O, gas chromatography-olfactometry; IS, internal standard; LSD, least significant differences; MS, mass spectrum; ms, mean square; RI, retention index; SAFE, solvent-assisted flavor evaporation; SEM, standard error measurement; SS, sum of squares 90937-2 N. MIYAZAWA et al. odor-active compounds by using a sensory evaluation program, carrier gas, flow rate and ionization mode were the same with and AEDA. Additionally, to investigate the character- those applied for the GC-MS method just described. The odor-active istics of this original and superior flavored mandarin volatiles were identified by the agreement of their RI, MS, and odor quality data with those of the reference odorant in our laboratory. The orange, the identified odor-active compounds of GC-MS-O analyses were performed twice by two experienced Kinokuni mandarin are compared with those of Satsuma assessors. mandarin which is now the most popular mandarin orange in Japan. Chiral analysis. Chiral analyses were performed with an Agilent 6890 GC combined with a 5973 mass selective detector equipped with Materials and Methods a CHIRAMIX capillary column (0.25 mm i.d. 30 m; GL Sciences Co.). The oven temperature was kept at 40 C for the initial 3 min and Materials. Fresh, orange-colored Kinokuni mandarins (Citrus then increased to 180 C at a rate of 0.7 C/min, with a constant flow kinokuni Hort. ex Tanaka), 50–80 g in weight and 65–70 mm in rate of 0.7 ml/min for the helium carrier gas. The settings for the diameter (68 g mean weight, 6.7 cm mean diameter), grown in sample volume, split rate, injection temperature, and ionization mode Sakurajima, Japan were provided by Shimodozono Co. (Kagoshima, were the same as those applied for the GC-MS method already Japan). Fresh, pale-orange-colored Satsuma mandarins (Citrus unshiu described. The enantiomeric distribution of linalool was calculated by Marcovitch), 130–160 g in weight and 70–80 mm in diameter (146 g integrating the chromatogram obtained by the selected ion. The order mean weight, 7.4 cm mean diameter), grown in Kumamoto, Japan were of the eluates had already been assigned using optically pure reference 36,37) purchased at a local market in Tokyo, Japan. Valencia orange oil samples. prepared by the cold-pressed method from Citrus sinensis (L.) Osbeck was purchased from Polarome International (Jersey City, NJ, USA). Isolation and fractionation of the volatiles. Solvent extraction of peel from Kinokuni mandarin and Satsuma Chemicals. The following compounds were purchased from mandarin. The peel (1.03 kg) of 50 fresh Kinokuni mandarin fruits was commercial sources: hexanal, (Z)-hex-3-enal, 1,8-cineole, octanal, collected, cut into pieces, and extracted with n-pentane (2.15 liter) for (Z)-hex-3-en-1-ol, nonanal, 2-isopropyl-3-methoxypyrazine, acetic 30 min. After removing the fibrous parts of the peel by filtration, the acid, citronellal, decanal, (E)-non-2-enal, linalool, undecanal, (E)- filtrate was dried over Na2SO4, and the volatiles of the filtrate were dec-2-enal, dodecanal, (E)-undec-2-enal, citronellol, perillaldehyde, isolated by the solvent-assisted flavor evaporation (SAFE) method38) at (2AdvanceE,4E)-deca-2,4-dienal, geraniol, (E)-dodec-2-enal, eugenol, View thymol, 40 C. The distillate was dried over Na2SO4 and concentrated to yield isothymol, indole, -pinene, myrcene, limonene, -terpinene, isoamyl 19.5 g of Kinokuni mandarin peel oil. The peel (497 g) of 15 fresh alcohol, oct-1-en-3-one, (E)-hept-2-enal, (E)-oct-2-enal, benzaldehyde, Satsuma mandarin fruits was extracted under the same conditions as (2E,6Z)-nona-2,6-dienal, 2-methylbutanoic acid, 2-acetyl-2-thiazoline, those used for Kinokuni mandarin to yield 3.46 g of Satsuma mandarin 2-phenylethanol, 4-hydroxy-2,5-dimethylfuran-3(2H)-one, methyl N- peel oil. methylanthranilate and vanillin (Sigma-Aldrich Japan, Tokyo, Japan); Silica gel chromatography of the peel oils. Kinokuni mandarin peel -damascenone (Firmenich, Meyrin, Switzerland); a mixture of 2- oil (2.56 g) was pipetted into the top of a water-cooled (10–12 C) glass ethyl-4-hydroxy-5-methylfuran-3(2H)-one and 5-ethyl-4-hydroxy-2- column filled with silica gel slurry (50 g; Wakogel C-100; Wako Pure methylfuran-3(2H)-one (Givaudan, Vernier, Switzerland); and tridec- Chemical Industries, Osaka, Japan) in n-pentane. Chromatography was 1-ene (Tokyo Chemical Industry Co., Tokyo, Japan). The following performed by using n-pentane and then n-pentane/diethyl ether (200:1, compounds were synthesized by the methods reported in the literature: v/v) to give a non-polar fraction, this being followed by n-pentane/ (3E,5Z)-undeca-1,3,5-triene,26) (3E,5Z,8Z)-undeca-1,3,5,8-tetraene,27) diethyl ether (1:1, v/v) and diethyl ether to give a polar fraction. The 6-methylheptanal,28) 6-methyloctanal,28) yuzunone ((6Z,8E)-undeca- solvent in each fractionProofs was evaporated to yield 2.51 g of the non-polar 6,8,10-trien-3-one),29) yuzuol ((6Z,8E)-undeca-6,8,10-trien-4-ol),30) components and 47.6 mg of the polar components. Satsuma mandarin (2E)-cis-4,5-epoxydec-2-enal,31) (2E)-trans-4,5-epoxydec-2-enal,32) peel oil (2.75 g) was fractionated under the same conditions as those (2E,7Z)-trans-4,5-epoxydeca-2,7-dienal33) and wine lactone (3,6- used for Kinokuni mandarin, yielding 2.44 g of the non-polar dimethyl-3a,4,5,7a-tetrahydro-1-benzofuran-2(3H)-one).34,35) All re- components and 25.7 mg of the polar components. agents and solvents were of analytical grade. Solvent extraction of Kinokuni mandarin juice. The pulp (2.35 kg) of 50 fresh Kinokuni mandarin fruits was carefully collected without Gas chromatography-mass spectrometry (GC-MS). The GC-MS mixing with the peel oil, and then hand-squeezed to extract the juice analyses were performed with an Agilent 6890 gas chromatograph (1.61 kg). The volatiles of the juice were isolated by the SAFE 38) (GC) combined with a 5973 mass selective detector and a flame method at 40 C. After adding NaCl (202 g), the distillate was ionization detector (FID, 250 C) equipped with a TC-WAX capillary extracted with diethyl ether (540 ml), dried over Na2SO4 and column (0.25 mm i.d. 60 m; GL Sciences Co., Tokyo, Japan). The concentrated to yield an extract (1.1 mg) of Kinokuni mandarin juice. eluate from the column at the end of the capillary was divided into two branches and respectively routed by deactivated fused silica capillaries AEDA. The flavor dilution (FD) factors of the odor-active to the mass detector and FID. Each 1-ml sample was injected in a split compounds were determined by the AEDA method.39) The polar mode (50:1) at a constant temperature of 250 C. The oven temperature components of the Kinokuni and Satsuma mandarin peel oils and the was kept at 40 C for the initial 3 min and then increased to 230 Cata extract of Kinokuni mandarin juice were each diluted stepwise with rate of 3 C/min, with a constant flow rate of 1.8 ml/min for the helium diethyl ether to obtain dilution ratios of 1:5, 1:25, 1:125, and 1:625. carrier gas. Mass spectra (MS) in the electron impact (EI) mode were Each dilution was analyzed by the GC-MS-O system equipped with the recorded at 70 eV ionization energy. Linear retention indices (RIs) for TC-WAX capillary column. the compounds were calculated from the retention times of n-alkanes. Tridec-1-ene was added to the Kinokuni and Satsuma mandarin peels Sensory evaluation. at a concentration of 1901 ppm as an internal standard (IS). The ratios Samples. The same Kinokuni and Satsuma mandarin peel oil to IS were calculated by using the GC-FID peak area of each samples that had been used for the GC-MS and GC-MS-O analyses compound and that of the IS. were used, while Valencia orange oil was purchased from Polarome International (USA). A 0.5-ml sample of each oil was directly put into Gas chromatography-mass spectrometry-olfactometry (GC-MS-O). a closed sensory vial of 30 ml total volume. The GC-MS-O analyses were performed with an Agilent 6890 GC Evaluation attributes. Seven attributes of fruity, sweet, floral, green, combined with a 5973 mass selective detector and a sniffing port metallic, sour, and balsamic were used. These attributes had been equipped with a TC-WAX capillary column (0.25 mm i.d. 60 m). previously selected by our laboratory to provide a suitable vocabulary The eluate from of the column at the end of the capillary was divided for describing the aroma of citrus fruits30) that is commonly used for into two branches and respectively routed by deactivated fused silica sensory evaluations. capillaries to the mass detector and sniffing port. The settings for the Evaluation conditions. The evaluation panel consisted of seven sample volume, split rate, injection temperature, oven temperature male and four female employees at the Technical Research Institute of Aroma Character Impact Compounds in Kinokuni Mandarin 90937-3 Table 1. Physical Properties of Kinokuni and Satsuma Mandarins Fruity* 5 Mean Mean Yield Nonpolar:Polar weight, diameter, of peel ratio 4 g cm oil, % Balsamic Sweet** 3 Kinokuni mandarin 68 6.7 1.90 98.1:1.9 Satsuma mandarin 146 7.4 0.70 99.0:1.0 2

1 results that the aroma of Kinokuni mandarin was closely similar to that of Satsuma mandarin, with more fruity Sour Floral and less green notes, and partly similar to that of Valencia orange, with more metallic and less sweet odors.

Metallic** Green* Odor-active compounds of Kinokuni and Satsuma mandarin peel oils Isolation and fractionation of the peel oils Fig. 1. Aroma Profile of Kinokuni Mandarin Compared with Satsuma Mandarin and Valencia Orange; ( ) Kinokuni Mandarin Kinokuni and Satsuma mandarin peel oils were Peel Oil, ( ) Satsuma Mandarin Peel Oil, and ( ) Valencia Orange isolated by solvent extraction and subsequent distillation Oil. under high vacuum with SAFE apparatus.38) The yield of Asterisks indicate significant differences between the samples Kinokuni mandarin peel oil (1.90%) was much higher ( p < 0:05, p < 0:01). than that of Satsuma mandarin (0.70%) (Table 1). The odor of the obtained oils was well reproduced and very T. Hasegawa Co., Ltd., Kawasaki, Japan. They had been trained to similar to the odor characteristics of the whole mandarins. recognizeAdvance and quantify the aromas of about 100 odorous Viewchemicals and raw materials. Each panelist was presented with a set of three test Since the preliminary experiments revealed the samples: i) Kinokuni mandarin peel oil, ii) Satsuma mandarin peel oil, excessive presence of monoterpene hydrocarbons in and iii) Valencia orange oil. Each was encoded by a random three-digit the peel oil that prevented the identification and number, and the panelists were instructed to sniff each sample and rate determination of small peaks on GC which might have the intensities of the seven attributes on a five-point linear scale from 1 some contribution to the odor of the samples,30) the (none) to 5 (very strong). The evaluation was conducted in a quiet Kinokuni and Satsuma mandarin peel oils were fractio- room kept at 23 C. Two booths were set up in the room. Each panelist was asked to perform an assessment in a separate booth to eliminate nated by silica gel chromatography into two parts, a non- any influence from each other. polar fraction abundant in hydrocarbons and a polar Statistical analyses. The differences between the average scores for fraction abundant in oxygenated compounds. The the evaluated samples were compared by analyses of variance weight ratio of the fractionated polar components was (ANOVA) and Tukey’s multiple-comparison tests.40–47) The correction almost double inProofs Kinokuni mandarin (1.9) than that in factor (CF), sum of squares (SS), degree of freedom (df), mean square Satsuma mandarin (1.0) (Table 1). The non-polar p < : (ms), and F-value were calculated for each attribute ( 0 05, and fractions of both mandarins had a terpenic and woody 0.01) in ANOVA. The standard error measurement (SEM) and least significant difference (LSD) were calculated for each attribute odor without any strong impact, while the polar fractions (p < 0:05) in Tukey’s multiple-comparison test. The results were of both had a fresh, floral and fruity odor, this being averaged for each attribute and plotted on a spider web diagram. more apparent in Kinokuni mandarin than in Satsuma mandarin. The obtained non-polar and polar fractions Results and Discussions were analyzed by GC-MS and GC-MS-O. Odor-active compounds of the non-polar components Aroma profile of Kinokuni mandarin Non-polar compounds are generally hardly consid- The characteristic aroma of Kinokuni mandarin was ered as main contributors to the characteristic aroma of evaluated according to the seven attributes (fruity, citrus fruits. However, we were able to identify in this sweet, floral, green, metallic, sour, and balsamic) and study six odor-active compounds of -pinene (terpenic compared to two well-known citrus fruits, Satsuma and woody), myrcene (pungent and woody), limonene mandarin which is the most popular mandarin orange in (terpenic and woody), -terpinene (woody and terpenic), Japan at this time, and Valencia orange which is one of (3E,5Z)-undeca-1,3,5-triene (balsamic and fruity), and the most popular oranges throughout the world. The (3E,5Z,8Z)-undeca-1,3,5,8-tetraene (balsamic and average scores for each attribute were plotted on a spider fruity) in the non-polar fractions of both mandarins as web diagram and are shown in Fig. 1. Since there was shown in Table 2. Monoterpene hydrocarbons have been no significant difference in the floral, sour, or balsamic well reported to be the major components and odor- notes among the three orange samples, we considered active compounds in various citrus fruits.3,13–25,48,49) them to represent the basic characteristic aroma of Although present in only small quantities, (3E,5Z)- mandarin oranges. The aroma of Valencia orange was undeca-1,3,5-triene and (3E,5Z,8Z)-undeca-1,3,5,8-tet- significantly sweeter and less metallic than that of raene seemed to be important to some extent in the Kinokuni or Satsuma mandarin. Additionally, the scores aroma of these mandarins, because these undecapo- for the fruity and green notes of Kinokuni mandarin lyenes have only been identified as character impact were not significantly different from those of Satsuma compounds in some citrus fruits, yuzu30) and mandar- mandarin or Valencia orange (p < 0:05), while those of in.50,51) The relative amount by IS ratio of the non-polar Satsuma mandarin significantly differed from those of components of Kinokuni mandarin peel oil was almost 3 Valencia orange (p < 0:05). We found from these times more than that of Satsuma mandarin, although the 90937-4 N. MIYAZAWA et al. Table 2. Odor-Active Volatiles in the Nonpolar Components of Kinokuni and Satsuma Mandarin Peel Oils

Kinokuni mandarin Satsuma mandarin RI on Identification Odoranta Odor qualityb TC-WAX GC-FID area, Ratio to IS GC-FID area, Ratio to IS modee % (103)c %d (103)c,d -Pinene terpenic, woody 1033 1.027 125.94 0.918 37.46 M, R, O Myrcene pungent, woody 1168 1.815 222.57 1.773 72.35 M, R, O Limonene terpenic, woody 1209 89.623 10990.46 89.828 3665.58 M, R, O -Tterpinene woody, terpenic 1251 4.238 519.71 4.047 165.15 M, R, O (3E,5Z)-Undeca-1,3,5-triene balsamic, fruity 1387 0.002 0.25 trace trace M, R, O (3E,5Z,8Z)-Undeca-1,3,5,8-tetraene balsamic, fruity 1449 0.002 0.25 trace trace M, R, O

aEach odorant was identified by the agreement of its mass spectrum, retention index, and odor quality with the reference odorant. bOdor quality perceived at the sniffing port cIS was added at 1901 ppm to the peel. dtrace, trace amount (<0:001 for GC-FID area %, <0:1 for ratio to IS) eM, reference mass spectrum; R, reference retention index; O, gas chromatography coupled with olfactometry composition ratios in the GC-FID area percentages of identified in Satsuma mandarin. Yuzunone (balsamic the compounds in both mandarins were approximately and fruity) was also tentatively identified in Satsuma the same. These findings explain well that there was no mandarin, although the FD factor was not high enough significant difference between the aroma qualities of the to be certain (FD 5). non-polar components of the Kinokuni and Satsuma Linalool, which has been reported as an odor-active mandarin peel oils, making it difficult to characterize the compound of various citrus fruits,13–21,24,25,30) was also odor of each mandarin by the non-polar components. found to contribute strongly to the aroma of both AdvanceOdor-active compounds of the polar components ViewKinokuni and Satsuma mandarins (both FD 625). We observed by GC-MS-O that there were more Octanal, which has previously been reported as the odor-active compounds in the polar fraction than in the most odor-active compound in Valencia orange,13,14) non-polar fraction, so the aroma of these components was found to contribute strongly to the aroma of was investigated in detail. AEDA was carried out to rank Kinokuni mandarin (FD 625), but not to that of Satsuma the odorants in the polar components of both the mandarin (FD 25). The relative amounts by IS ratio of Kinokuni and Satsuma mandarin peel oils according to the saturated aliphatic aldehydes (except for hexanal) their odor potency. were 7–400 times more in Kinokuni mandarin than in The polar components of Kinokuni mandarin peel oil Satsuma mandarin. Previous studies21,24) have found yielded 19 odor-active compounds within the FD factor thymol and methyl N-methylanthranilate to be the range of 25–625 (Table 3). Ten odorants of the 13 odor- characteristic volatiles in mandarins. On the other hand, active compounds within the FD factor range of 125– only thymol wasProofs detected in Kinokuni and Satsuma 625 were positively identified by the agreement of their mandarins, with not even a trace of methyl N-methyl- RI, MS, and odor quality data with those of the standard anthranilate being apparent either sensorially or instru- odorants in our laboratory. Octanal (citrus and fatty) and mentally. Thymol was one of the contributors to the linalool (floral) had the highest FD factor (FD 625) as Kinokuni mandarin aroma (FD 125), while isothymol the most characteristic odor-active compounds. The was one of the contributors to the Satsuma mandarin other odor-active compounds with FD 125 were (Z)-hex- aroma (FD 125). Wine lactone, which has also pre- 3-enal (green and herbal), decanal (citrus and fatty), viously been reported to be the most odor-active dodecanal (fatty), (2E,4E)-deca-2,4-dienal (oily), gera- compound in Valencia orange,13,14) was found to con- niol (citrus), yuzuol (balsamic and fruity), (2E,7Z)- tribute strongly to the aroma of Satsuma mandarin (FD trans-4,5-epoxydeca-2,7-dienal (fruity and metallic), 625), but not to that of Kinokuni mandarin (FD 25). The and thymol (medicinal). In addition, yuzunone (balsamic relatively higher FD factor, IS ratio, and GC-FID area and fruity) and (2E)-trans-4,5-epoxydec-2-enal (metallic percentage of each of the C6 compounds (hexanal, and fruity) were tentatively identified (both FD 125) by (Z)-hex-3-enal, and (Z)-hex-3-enol) in the Satsuma the agreement of their RI and odor quality data with mandarin components indicated that these odorants those of the reference odorants. contributed to the green note considered as one of the The polar components of Satsuma mandarin peel oil characteristic aroma features of Satsuma mandarin in the yielded 21 odor-active compounds within the FD factor sensory evaluation. The FD factors of the major odor- range of 25–625 (Table 3). Seven odorants of the 11 active compounds in the polar components were odor-active compounds within the FD factor range of significantly different between Kinokuni and Satsuma 125–625 were positively identified by their RI, MS, and mandarins. odor quality data. (Z)-Hex-3-enal (green and herbal) and Kinokuni mandarin was sensorially evaluated as more linalool (floral) had the highest FD factor (FD 625) as fruity than Satsuma mandarin, and this fruity evaluation the most characteristic odor-active compounds, followed could be attributed to the higher FD factor and IS ratio by 6-methyloctanal (citrus and fruity), decanal (citrus of octanal. Additionally, the high FD factor and IS ratios and fatty), yuzuol (balsamic and fruity), eugenol (spicy of linalool and decanal in Kinokuni mandarin also and medicinal), and isothymol (medicinal and woody), significantly affected the stronger fruity note. all with FD 125. Furthermore, wine lactone (medicinal The identification of the novel key odor-active and buttery, FD 625) and (2E)-trans-4,5-epoxydec-2- compounds, yuzunone and yuzuol, in yuzu elsewhere,30) enal (metallic and fruity, FD 125) were tentatively and in Kinokuni and Satsuma mandarins in this study, Aroma Character Impact Compounds in Kinokuni Mandarin 90937-5 Table 3. Most Odor-Active (FD 5) Volatiles in the Polar Components of Kinokuni and Satsuma Mandarin Peel Oils

Kinokuni mandarin Satsuma mandarin

a b RI on GC-FID Ratio Identifi- GC-FID Ratio Identifi- Odorant Odor quality FD FD TC-WAX area, to IS cation area, to IS cation factor factor %c (103)c,d modee %c (103)c,d modee Hydrocarbons (3E,5Z)-Undeca-1,3,5-triene fruity, balsamic 1387 25 trace trace M, R, O 5 trace trace M, R, O (3E,5Z,8Z)-Undeca-1,3,5,8-tetraene balsamic, fruity 1449 5 trace trace M, R, O trace trace M, R Aldehydes Hexanal green, herbal 1083 5 0.061 0.08 M, R, O 25 0.586 0.15 M, R, O Octanal citrus, fatty 1297 625 5.927 7.94 M, R, O 25 0.069 0.02 M, R, O Nonanal citrus, fatty 1395 5 3.767 5.05 M, R, O 5 0.712 0.18 M, R, O Decanal citrus, fatty 1502 125 22.819 30.57 M, R, O 125 10.070 2.50 M, R, O Undecanal citrus, fatty 1611 25 1.532 2.05 M, R, O 5 1.130 0.28 M, R, O Dodecanal fatty 1713 125 3.121 4.18 M, R, O 25 2.075 0.51 M, R, O (Z)-Hex-3-enal green, herbal 1139 125 0.531 0.71 M, R, O 625 0.480 0.12 M, R, O (E)-Non-2-enal fatty, 1539 0.032 0.04 M, R 5 trace trace M, R, O watermelon-like (E)-Dec-2-enal oily 1654 25 0.599 0.80 M, R, O 5 0.320 0.08 M, R, O (E)-Undec-2-enal fatty, cosmetic 1753 0.024 0.03 M, R 5 trace trace M, R, O Deca-2,4-dienalf,g oily 1771 5 0.199 0.27 M, O 5 0.128 0.03 M, O (2E,4E)-Deca-2,4-dienal oily 1816 125 0.299 0.40 M, R, O 25 0.046 0.01 M, R, O (E)-Dodec-2-enal fatty 1859 1 0.026 0.04 M, R, O 5 0.028 0.01 M, R, O (2E)-cis-4,5-Epoxydec-2-enalf metallic, fruity 1988 5 nd nd R, O 1 nd nd R, O (2E)-trans-4,5-Epoxydec-2-enalf metallic, fruity 2017 125 nd nd R, O 125 nd nd R, O (2E,7Z)-trans-4,5-Epoxydeca-2,7-dienal fruity, metallic 2074 125 trace trace M, R, O 25 trace trace M, R, O 6-MethylheptanalAdvance citrus, fruity View 1258 5 0.024 0.03 M, R, O trace trace M, R 6-Methyloctanal citrus, fruity 1361 0.008 0.01 M, R 125 trace trace M, R, O Ketones and lactones Yuzunonef balsamic, fruity 1899 125 nd nd R, O 5 nd nd R, O 3-Methyldecano-4-lactonef,h peach-like, creamy 2134 25 0.057 0.08 M, O 5 0.068 0.02 M, O Wine lactonef medicinal, buttery 2271 25 nd nd R, O 625 nd nd R, O Alcohols (Z)-Hex-3-en-1-ol green, herbal 1393 5 0.239 0.32 M, R, O 5 1.346 0.33 M, R, O Yuzuol balsamic, fruity 1986 125 trace trace M, R, O 125 trace trace M, R, O Terpenes 1,8-Cineole medicinal 1207 0.068 0.09 M, R 5 0.525 0.13 M, R, O Citronellal cosmetic, citrus 1483 5 1.136 1.52Proofs M, R, O 25 1.721 0.43 M, R, O Linalool floral 1550 625 32.013 42.88 M, R, O 625 45.269 11.21 M, R, O Citronellol cosmetic, citrus 1763 5 1.444 1.93 M, R, O 5 1.825 0.45 M, R, O Perillaldehyde sour, fruity 1797 5 0.930 1.25 M, R, O 5 0.410 0.10 M, R, O Geraniol citrus 1842 125 0.035 0.05 M, R, O 0.022 0.01 M, R Aromatics Eugenol spicy, medicinal 2173 125 0.064 0.02 M, R, O Thymol medicinal 2191 125 3.828 5.13 M, R, O 1 0.056 0.01 M, R, O Isothymol medicinal, woody 2222 1 0.041 0.06 M, R, O 125 0.049 0.01 M, R, O Others 2-Isopropyl-3-methoxypyrazine earthy, medicinal 1446 5 trace trace M, R, O 25 trace trace M, R, O Acetic acid acidic, sour 1455 25 trace trace M, R, O 1 trace trace M, R, O Indole medicinal 2454 trace trace M, R 25 0.534 0.13 M, R, O Unknown fatty 1420 5 nd nd O 5 nd nd O Unknown fatty 1527 1 nd nd O 125 nd nd O Unknown green, 1559 5 nd nd O 125 nd nd O watermelon-like Unknown cucumber-like, 1705 5 nd nd O green Unknown oily, green 1710 5 nd nd O Unknown medicinal, fruity 1888 5 nd nd O 1 nd nd O Unknown medicinal, plastic 1996 1 nd nd O 25 nd nd O Unknown fruity, metallic 2028 5 nd nd O Unknown fruity, metallic 2063 5 nd nd O Unknown fatty, phenolic 2287 1 nd nd O 25 nd nd O Unknown powdery, cosmetic 2290 125 nd nd O 1 nd nd O

aEach odorant was identified by the agreement of its mass spectrum, retention index, and odor quality with the reference odorant. bOdor quality perceived at the sniffing port cnd, not detected; trace, trace amount (<0:001 for GC-FID area %, <0:01 for ratio to IS) dIS was added at 1901 ppm to the peel. eM, reference mass spectrum; R, reference retention index; O, gas chromatography coupled with olfactometry f Tentatively identified odorant gThe geometric isomer was not determined. hThe MS data were obtained from a commercial database. 90937-6 N. MIYAZAWA et al. Table 4. Most Odor-Active (FD 5) Volatiles in the Extract of Kinokuni Mandarin Juice

RI on FD GC-FID Identification Odoranta Odor qualityb TC-WAX factor area, %c moded Hydrocarbons (3E,5Z)-Undeca-1,3,5-trienee balsamic, fruity 1387 5 nd R, O (3E,5Z,8Z)-Undeca-1,3,5,8-tetraenee balsamic, fruity 1449 25 nd R, O Aldehydes Dodecanal oily, fatty 1713 25 trace M, R, O (E)-Hept-2-enal metallic, fruity 1346 25 0.183 M, R, O (E)-Oct-2-enal medicinal, plastic 1435 25 0.369 M, R, O (2E,6Z)-Nona-2,6-dienal green, cucumber-like 1596 25 trace M, R, O Deca-2,4-dienale,f green, plastic 1771 25 0.240 M, O (2E,4E)-Deca-2,4-dienal oily, plastic 1816 625 0.042 M, R, O (2E)-trans-4,5-Epoxydec-2-enal metallic, fruity 2017 625 0.321 M, R, O (2E,7Z)-trans-4,5-Epoxydeca-2,7-dienale metallic 2074 25 nd R, O Ketones, lactones and furanones Oct-1-en-3-one mushroom-like 1320 25 0.017 M, R, O -Damascenone honey 1826 5 trace M, R, O Wine lactonee medicinal 2271 625 nd R, O 4-Hydroxy-2,5-dimethylfuran-3(2H)-onee sweet 2030 125 nd R, O 2-Ethyl-4-hydroxy-5-methylfuran-3(2H)-onee sweet 2086 5 nd R, O 5-Ethyl-4-hydroxy-2-methylfuran-3(2H)-onee Alcohols Isoamyl alcohol oily, inky 1200 25 8.096 M, R, O 2-Phenylethanol sweaty 1929 5 0.542 M, R, O Terpenes AdvanceLinalool View cosmetic, floral 1550 625 1.693 M, R, O Geraniol cosmetic 1842 25 trace M, R, O Aromatics Benzaldehyde woody, fatty 1532 25 0.089 M, R, O Eugenol spicy 2169 125 0.021 M, R, O Thymol inky, medicinal 2191 125 2.277 M, R, O Vanillin vanilla-like 2597 5 0.020 M, R, O Others 2-Methylbutanoic acid buttery 1691 25 0.593 M, R, O 2-Acetyl-2-thiazolinee oily, popcorn-like 1778 25 nd R, O Unknown metallic, fruity 1510 25 nd O Unknown oily, green 1727 25 nd O Unknown green, fatty 1762Proofs 5 nd O Unknown metallic 1893 5 nd O Unknown sweaty 2049 625 nd O Unknown medicinal 2196 5 nd O Unknown phenolic 2262 25 nd O Unknown cosmetic 2290 25 nd O

aEach odorant was identified by the agreement of its mass spectrum, retention index, and odor quality with the reference odorant. bOdor quality perceived at the sniffing port cnd, not detected; trace, trace amount (<0:001) dM, reference mass spectrum; R, reference retention index; O, gas chromatography coupled with olfactometry eTentatively identified odorant f The geometric isomer was not determined. both of which are Japanese citrus cultivars, may make it of the juice differed from that of the peel oil, so AEDA possible to distinguish Japanese citrus fruits from citrus was conducted to identify the potent odorants in the fruits produced in other countries, although this assump- juice. A total of 25 odor-active compounds were tion needs further studies. detected within the FD factor range of 25–625 (Table 4). The following five odorants of the eight odor-active Odor-active compounds in hand-squeezed Kinokuni compounds within the FD factor range of 125–625 were mandarin juice identified as very important: linalool (cosmetic and Intrigued by the outstanding total flavor of this edible floral, FD 625), (2E,4E)-deca-2,4-dienal (oily and fruit,1,2) the aroma of the juice of Kinokuni mandarin plastic, FD 625), (2E)-trans-4,5-epoxydec-2-enal (me- was also investigated. An earlier study has reported that tallic and fruity, FD 125), eugenol (spicy, FD 125), and citrus juice contained an oil with minor compositional thymol (inky and medicinal, FD 125). Two more differences from that of the peel (flavedo).4) Kinokuni odorants, wine lactone (medicinal, FD 625) and 4- mandarin juice was prepared by careful hand-squeezing, hydroxy-2,5-dimethylfuran-3(2H)-one (sweet, FD 125), taking precautions to prevent any mixing of the peel oil were tentatively identified by the agreement of their RI with the juice. An aroma extract from the juice was and odor quality data with those of the reference prepared by the SAFE method.38) The yield of the odorants. extract of the juice (0.47 ppm) was 40,000 times lower Thymol, wine lactone, and such unsaturated aliphatic than that of the peel oil (1.90%). We felt that the aroma aldehydes as (2E,4E)-deca-2,4-dienal and (2E)-trans- Aroma Character Impact Compounds in Kinokuni Mandarin 90937-7 Table 5. Biological Classification of Mandarin Species and Enantiomeric Distribution of Linalool Identified in Mandarins

Enantiomeric Classificationa Mandarin distribution, % Enantiomeric excess, %ee Genus Subgenus (R)(S) Satsuma peel oil Citrus Metacitrus Acrumen Euacrumen 89.9 10.1 79.8 (R) Kinokuni peel oil Citrus Metacitrus Acrumen Microacrumen 7.8 92.2 84.4 (S) Kinokuni juice Citrus Metacitrus Acrumen Microacrumen 4.0 96.0 92.0 (S)

aThe classification is based on Tanaka Systematics.58–61)

4,5-epoxydec-2-enal were found to contribute strongly Biological classification of mandarin species to the Kinokuni mandarin aroma of both the juice and It is interesting that the enantiomeric distribution of the peel oil, while such saturated aliphatic aldehydes as linalool was different in the mandarin species. Although octanal and decanal, compounds which contributed the enantiomeric distribution of linalool in Kinokuni strongly to the aroma of the peel oil, were not detected mandarin was similar to that in most of the other in the juice by AEDA. Monoterpene hydrocarbons, mandarins such as Mediterranean, Nova and ponkan, it which were the major and odor-active components of was different in Satsuma. From the point of view of the peel oil, were not considered to contribute to the biological classification, Kinokuni mandarin is similarly aroma of the juice. Eugenol and 4-hydroxy-2,5-dime- grouped with Satsuma mandarin as Citrus-Metacitrus- thylfuran-3(2H)-one were identified as odor-active com- Acrumen in the classification table of citrus fruits by pounds solely in the extract of the juice, but not in the Tanaka.58–61) However, in the more detailed classifica- peel oil. Yuzunone and yuzuol, substances which have tion under Acrumen, Kinokuni mandarin belongs to beenAdvance identified as the character impact aroma View com- Microacrumen which is also the group with Mediterra- pounds in the peel oil, were not identified in the extract nean, Nova and ponkan mandarins, and not the Euacru- of the juice. It was found from these results that the men group with Satsuma mandarin (Table 5). Thus, aroma of the juice was significantly different from that there was a correlation between the enantiomeric of the peel oil both sensorially and instrumentally. distribution of linalool and the biological classifica- tion of mandarins. Earlier studies have discussed Chiral analysis and biological classification of the the correlation between the biological classification Mandarin species of citrus fruits and such non-volatiles as auraptene Chiral analysis of linalool and carotenoids.60,61) Our study has revealed that the The enantiomeric distribution of linalool, which was volatile compound, linalool, also played a significant the most contributive compound in the aroma compo- role as an indicator of the biological classification of nents of Kinokuni peel and juice and of Satsuma peel citrus fruits. Proofs was analyzed by GC-MS with a CHIRAMIX36,37) chiral The character impact compounds in the peel oils of capillary column. The enantiomeric distribution of Kinokuni and Satsuma mandarin oranges and in the linalool is well known to vary greatly among the citrus juice of Kinokuni mandarin orange were identified in species.52,53) In bergamot (Citrus bergamia Risso et this study for the first time. Three of the findings here are Poit.)53,54) and yuzu,54) for example, the distribution is especially significant and very interesting: first, the predominantly (R)-isomer, while in Valencia orange,52) characteristic aroma of Kinokuni mandarin was attrib- it is predominantly (S)-isomer. It has been reported that utable to the polar fraction, and to the different odor- (R)-linalool (88%) was dominant in Satsuma mandar- active compounds with Satsuma mandarin; second, in,10,54) while (S)-linalool was dominant in Mediterra- yuzunone and yuzuol, two compounds only previously nean mandarin (Citrus deliciosa Tenore), Nova man- found in yuzu, were also identified in other Japanese darin (Citrus reticulata Blanco cv. Nova), and ponkan citrus cultivars, Kinokuni and Satsuma mandarins; third, mandarin (Citrus reticulata Blanco) as 84%, 96%, and the enantiomeric distribution of linalool in Kinokuni 94%.9,10,53) Our chiral analysis of Kinokuni mandarin mandarin was dominated by the (S)-isomer, whereas that peel oil showed (S)-linalool (92%) to be dominant, while in Satsuma mandarin was dominated by the (R)-isomer (R)-linalool (90%) was dominant in Satsuma mandarin. which correlates with the biological classification of Additionally, in the extract of Kinokuni mandarin juice, citrus fruits. (S)-linalool (96%) was found to be dominant, similarly to the peel oil. The enantiomeric distribution of linalool Acknowledgments in mandarins is summarized in Table 5. Previous studies55–57) have judged the odor quality and threshold We thank Shimodozono Co., Ltd., for graciously value for each enantiomer of linalool to be different. providing us with fresh Kinokuni mandarins. 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