_ Food Science and Technology Research, 23 (1), 79 89, 2017 Copyright © 2017, Japanese Society for Food Science and Technology doi: 10.3136/fstr.23.79

http://www.jsfst.or.jp

Original paper

Comparison of Volatile Compounds in ‘Fuji’ Apples in the Different Regions in China

1 2* 3 2 2 2 Ling Qin , Qin-Ping Wei , Wen-Huai Kang , Qiang Zhang , Jian Sun and Song-Zhong Liu

1Department of Life Science, Hebei Normal University of Science and Technology, Qinhuangdao 066004 P. R. China 2Institute of Forestry & Pomology, Beijing Academy of Agriculture & Forestry Sciences,Beijing 100093 P.R. China 3College of Bioscience and Bioengineering, Hebei University of Science and Technology, Shijiazhuang 050018 P.R. China

Received May 25, 2015 ; Accepted September 28, 2016

The characteristics of the volatiles from 43 ‘Fuji’ apples representing 14 different apple production regions in China were investigated using headspace-solid phase micro-extraction (HS-SPME) combined with gas chromatography–mass spectrometry (GC-MS). The results obtained from this experiment showed that sixty- four volatile compounds were identified in ‘Fuji’ apples collected from 43 counties in China. The major volatile compounds were identified as 2-methyl butyl acetate and hexyl acetate. The composition of volatiles and their contents in ‘Fuji’ apples varied in different regions. All of the ‘Fuji’ apple samples could be classified into the following groups using a principal component analysis of the volatiles: (1) apples with high concentrations of hexyl acetate and (Z)-3-hexenyl acetate, which were collected in Shandong (Qixia, Wendeng, Penglai, Zhaoyuan, Jiaonan and Yishui), Shanxi (Wanrong, Ruicheng and Linyi), and Gansu Ninglang, (2) apples with high contents of 2-methyl butyl acetate and 1-hexanol, which mainly came from North Shaanxi, Henan Sanmenxia, Liaoning Wafangdian and Liaoning Suizhong, (3) apples with high contents of hexyl butanoate, butyl acetate and hexyl 2-methyl butyrate, which were mainly collected in Gansu (excluding Ninglang), and (4) apples without any characteristic volatile composition. In addition, it was found that mean annual temperature was significant correlated with 2-methyl butyl acetate,butyl 2-methyl butanoate, hexyl acetate, and (E)-2-hexenyl acetate. Longitude was significantly correlated with butyl acetate, (Z)-3-hexenyl acetate, and ethyl hexanoate.

Keywords: Apple (Malus domestica), aroma compounds, GC-MS (gas chromatography-mass spectrometry), HS-SPME (headspace - solid phase microextraction)

Introduction chains including combinations of acetic, butanoic, and hexanoic Aroma is one of the essential components of fruit quality. The acids with ethyl, butyl, and hexyl alcohols, have been reported to relative contributions of specific aroma volatile compounds to the be the major contributors to the aroma of apples (López, Lavilla, flavor of apples have been examined by many investigators, and Riba & Vendrell, 1998; Matich, Rowan & Banks, 1996; Song & more than 300 compounds have been identified (Dixon & Hewett, Bangerth, 1993). 2000; Elss, Preston, Appel, Heckel & Schreier, 2006; Fallik, Investigations have focused on the characteristic volatiles Archbold, Hamilton-Kemp, Loughrin & Collins, 1997; Mehinagic, produced by ripening apples and the evolution of apple aromas Royer, Symoneaux, Jourjon & Prost, 2006). The volatile post-harvest (Bangerth, Song & Streif, 2012; Echeverrıa, Graell, compounds of apples (Malus domestica Borkh) include alcohols, López & Lara, 2004; Song et al., 1993). Bangerth and colleagues aldehydes, carboxylic esters, ketones, and ethers (Dixon et al., (Bangerth et al., 2012) reviewed physiological impacts of fruit 2000). The esters, particularly those with even numbered carbon ripening and storage conditions on the formation of aroma volatiles

*To whom correspondence should be addressed. E-mail: [email protected] 80 L. Qin et al. in apples. Apple volatile production had been categorized from the above locations when the apples were fully ripe (based on according to: type and quantity of esters or alcohols (Dirinck & the days after full bloom). Forty fruits per orchard were sampled Schamp, 1988; Paillard, 1990), skin color (Paillard, 1979), or according to an equatorial pattern (East–West–North–South) from aroma production patterns (Dirinck et al., 1988). Several studies the periphery of the canopy of each tree individually, avoiding (Defilippi, Kader & Dandekar, 2005; Elss et al., 2006; Matich et fruits situated at the top, bottom or deep inside the foliage. The al., 1996; Schaffer et al., 2007; Schumacher, Asche, Heil, sampled fruits were healthy and without any symptoms of pest Mittelstädt, Dietrich & Mosandl, 1998; Song & Bangerth, 2003; infestation or disease infection. The sampled fruits were put into Xiaobo & Jiewen, 2008) have discussed the biochemical origin of plastic bags and transferred to the laboratory in an insulated box aroma volatiles and improvements in methods for the separation filled with ice packs. In the laboratory the apple samples were and identification of volatile compounds. Several studies washed in deionised water and surface-dried with gauze. (Mpelasoka & Hossein Behboudian, 2002; Nielsen, Jägerstad, Öste Analysis of aroma volatile compounds Two kilograms of & Wesslén, 1992; Plotto, McDaniel & Mattheis, 1999; Song et al., sampled fruits from each location were ground to a powder and 1993) have also investigated the effect of culture techniques and homogenized using a blender. Juice from the collected apples was management on the composition and content of volatiles. ‘Fuji’ centrifuged at 4000 rpm for 10 min. The supernatant was stored at apple cultivar (Malus domestica Borkh CV. Red Fuji) is becoming -80℃ until analysis. Volatiles from the apple juice samples were one of the major apple cultivars in China, and it is grown both in extracted using HS-SPME. A 50/30 µm DVB / CAR / PDMS fiber northern and southern China. In the two dominant apples (Tupelo, Inc., Bellefonte, PA) was used for aroma extraction. production zones in China, the Loess Plateau regions and Apple juice (8 g) was weighed in a 20 mL headspace vial and Surrounding Bohai Gulf regions, the cultivated area of ‘Fuji’ capped with a septum. The juice was saturated with sodium apples has exceeded 80%. Until now, there have been no reports on chloride (2.4 g) and a 2-octanol internal standard solution was aroma volatile characteristic and quality parameters in different added to each vial to give a final 2-octanol concentration of 80 µg _ apple production regions. L 1. In this study, headspace-solid phase micro-extraction (HS- Each sample was equilibrated at 45℃ in a thermostatic bath for SPME) combined with gas chromatography – mass spectrometry 45 min, and then extracted for 30 min at the same temperature (GC-MS) was applied to study the volatiles compounds in ‘Fuji’ while stirring. After extraction, the fiber was inserted into the apples in the different production regions in China. The aim of this injection port of the GC (250℃) to adsorb the analysis. GC-MS work focused on comparing the aroma composition and content, was carried out using an Agilent GC 6890-5975 Mass Selective which was used to assess aroma volatile characteristic and quality Detector (MSD). Samples were analyzed on a DB-Wax column parameters in ‘Fuji’ apples, particularly among the different apple (30 m × 0.32 mm I.D., 0.25 µm film thickness; J&W Scientific, production regions in China that have not been extensively studied. Folsom, CA, and USA). Helium was used as the carrier gas at a constant flow rate of 2 mL/min. The oven temperature was Materials and Methods maintained at 50℃ for 2 min then the temperature was increased to Plant materials ‘Fuji’ apple (Malus domestica Borkh CV. 230℃ and maintained for 25 min. The MS detector was operated Red Fuji) samples were collected from October 30 through in the scan mode (mass range 50-200) and the transfer line to the November 6, 2011 in 43 well-managed orchards located in 14 MS system was maintained at 250℃ apple production regions in China. Twenty-one ‘Fuji’ fruits Identification of volatile compounds was based on the samples came from the Loess Plateau, which included the comparison of their mass spectra and retention indices (RI) with following locations: three East Gansu areas, four Gansu Tianshui standards and published data, as well as standard mass spectra in areas, three North Shaanxi areas, four Shaanxi Guanzhong areas, the NIST05a. L database (Agilent Technologies Inc.). Retention one collection from Lingwu Ningxia, five middle and south Shanxi indices were calculated using a mixture of n-paraffin C6-C30 as locations, and one collection from Sanmenxia Henan. Twenty-two standards. The volatile compound content was calculated from the ‘Fuji’ apple samples came from areas surrounding the Bohai Gulf, GC-peak areas that relate to the GC-peak area of the internal including six locations in southern and western Liaoning, five in standard. the Hebei Yanshan mountainous area, five in the Shandong Statistical analysis Data for each apple sample were averaged Peninsula, three in the Shandong TaiYi mountainous areas, and one among the three replications. A principal component analysis collection from Changping, Beijing. Two apple samples came from (PCA) was done to detect clustering formations and establish other regions, one from Malong Yunnan and one from Fengxian relationships between samples and volatile compounds using SPSS Jiangsu. Table 1 gives the geographical location and main climatic for Windows Version 17.0 (SPSS Inc.). The correlation among factors of samples in 43 counties. major aroma volatile compounds, geographic location and main The ‘Fuji’ apples were collected from five trees at each orchard climatic factors was also calculated using SPSS. Comparison of ‘Fuji’ Apples in China 81 ℃ 9.8 9.4 8.2 9.5 8.0 8.6 8.9 12 / 10.3 12.2 10.6 11.5 11.5 11.9 11.4 11.9 11.8 11.6 12.1 12.8 12.3 MAT AP 645 675 550 550 578 650 741 480 743 800 800 735 /mm 652.5 636.3 613.2 659.1 762.2 606.2 798.3 690.9 550.3 40°07′ 39°29′ 40°31′ 37°33′ 37°23′ 37°32′ 27°39′ 40°37′ 41°06′ 41°26′ 39°07′ 39°34′ 40°45′ 40°36′ 38°42′ 36°08′ 36°02′ 36°13′ 36°23' 37°50′ ~ ~ ~ ~ ~ ~ ~ 40°23′ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 122°16′/39°20′ 121°41′/39°21′ 122°33′/39°55′ 120°38′/37°05′ 122°19′/36°52′ 121°15′/37°05′ 103°56′/27°07′ 120°31′/39°59′ 120°18′/40°24′ 119°37′/40°35′ 115°17′/38°45′ 119°18′/39°05′ 119°10′/40°17′ 119°36′/40°04′ 114°41′/38°20′ 120°11′/35°35′ 118°15′/35°27′ 118°31′/35°55' 119°03′/35°36′ 116°29′/40°2′ 121°09′/37°25′ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Longitude / Latitude 121°13′ 119°34′ 121°34′ 119°13′ 121°57′ 118°50′ 114°50′ 118°40′ 118°10′ 118°33′ 114°09′ 120°08′ 121°43′ l20°35′ 120°33′ 119°30′ 117°45′ 117°54′ 118°13′ 115°50′ 103°08′ Qixia Leting Yishui Yishui Yiyuan Yiyuan County Penglai Jiaonan Malong Gaizhou Mengyin Xingtang Wendeng Wendeng Suizhong Qinglong Lingyuan Shunping Jianchang Zhaoyuan Changping Kuancheng Sanshilibao Wafangdian Wafangdian area area regions Liaoning peninsula Shandong Mountainous Mountainous Hebei Yanshan Hebei Yanshan South and West South and West Shandong TaiYi Hebei Yunan Beijing Province Liaoning Shandong Apple Samples LN-WFD LN-SZ LN-SSLB LN-JC LN-GZ LN-LY HB-SP HB-LT HB-KC HB-QL HB-XT SD-ZY SD-WD SD-PL SD-QX SD-JN SD-MY SD-YY SD-YS BJ-CP YN-ML 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 NO ℃ 11 10 10 14 / 7.1 9.9 9.4 9.6 9.2 8.8 8.8 9.9 11.4 11.4 11.9 12.7 10.4 12.9 12.4 13.5 13.8 MAT AP /mm 625 546 592 450.8 488.2 537.5 555 920 580 507.3 577.8 622 505.3 521.1 308 600 441.8 500 550 508.7 630 34°45′ 34°50′ 34°37′ 36°23′ 38°01′ 34°31′ 36°17′ 27°56′ 34°56′ 35°11′ 35°27′ 36°04′ 37°19′ 35°31′ 34°48′ 35°31′ 35°18′ 35°05′ 35°52′ 35°45' ~ ~ ~ ~ 37°28' ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ 106°.37′/37°.60 105°36′/33°35′ 108°05′/35°42′ 107°45′/35°11′ 101°16′/26°35′ 106°30′/34°32′ 106°02′/34°44′ 109°45′/35°04′ 109°45′/35°26′ 109°26′/36°30′ 110°32′ / 35°42′ / 110°32′ 107°38′ / 34°20′ 108°41′ / 34°20′ 108°03′ / 34°12′ 110°42′/34°36′ 110°59′/35°13′ 110°54′/34°58′ 112°01′/33°31′ 112°03′/35°23′ 112°39'/37°15′ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ Longitude / Latitude Geographic location and main climatic factors of ‘Fuji’ apple samples collected from 43 counties in China Geographic location and main climatic factors of ‘Fuji’ 105°20'~106°05'/35°01' 104°37′ 107°16′ 107°15′ 100°21′ 105°45′ 105°20′ 109°16′ 109°13′ 108°05′ 109°41′ 107°10′ 108°17′ 107°45′ 105°.59 110°36′ 112°12' 110°25′ 111°34′ 110°17′ 110°21′ Table 1.Table County Jingning Lixian Qingcheng Jingchuan Ninglang Qingshui Qin’an Baishui Luochuan An’sai Yichuan Fengxiang Liqua, Fufeng Lingwu Ruicheng Qixian Wanrong Yicheng Linyi Sanmenxia area area North Gansu Henan regions Shaanxi Sanmenxia East Gansu Middle and Guanzhong Shaanxi area Tianshui area Tianshui South Shanxi Gansu Henan Shanxi Shaanxi Ningxia Province b Apple Samples GS-JN GS-LX GS-QC GS-JC GS-LN GS-QS GS-QA SAX-BS SAX-LC SAX-AS SAX-YC SAX-FX SAX-LQ SAX-FF NX-LW SX-RC SX-QX SX-WR SX-YC SX-LY HN-SMX a : Province - County : NO showed the number of apple samples, 1-21 collected from Loess Plateau regions, 22-41 in areas surrounding Bohai Gulf 42-43 other regions 1 2 3 4 5 6 7 8 9 a b Mean annual temperature Annual precipitation; MAT: AP: 11 10 12 13 14 15 16 17 18 19 20 21 NO 82 L. Qin et al.

Results and Discussion butyrate (C4), hexanoate (C6), and 2-methyl butyrate (2M) esters Identification of volatiles Using HS-SPME-GC-MS, 64 (Dixon et al., 2000). volatile compounds in ‘Fuji’ apples were identified and the Eleven types of acetate ester (EC2) compounds, including ethyl amounts of volatile compounds were measured. We identified 43 acetate, pentyl acetate, hexyl acetate, butyl acetate, n-propyl esters, nine alcohols, two aldehydes, one terpenoid, two acids, and acetate, 2-ethyl hexyl acetate, 2-methyl butyl acetate, 2-methyl seven alkanes. Table 2 gives the average content of all volatiles propyl acetate, (Z)-4-hexen-1-ol acetate, (Z)-3- hexenyl acetate, based on the location of the different ‘Fuji’ apple samples. Of all and (E)-2- hexenyl acetate, were detected. Acetate ester compounds the aroma compounds identified, the following 10 were the most of all of the apple samples contributed 76.9% of the total volatile abundant: 2-methyl butyl acetate, hexyl acetate, (E)-2-hexen-1-ol content (Table 2). There was significant (F=3.318, p=0.002) acetate, hexyl-2-methyl butyrate, butyl acetate, hexyl butanoate, difference in the content of EC2 in 14 apple production regions. In (Z)-3- hexenyl acetate, (Z)-ethyl acetate, 1-hexanol, and ethyl all regions, the content of acetate ester compounds produced in hexanoate. ‘Fuji’ apples was the highest in mid- and southern Shanxi (80.3%). Although a large number of volatile compounds were identified The lowest acetate ester content was found in southern and western in ‘Fuji’ apples collected from 43 counties in China, esters and Liaoning (66.5%; Figure 1). Of the 11 types of acetate ester alcohols were responsible for almost all of the total compounds found, 2-methyl butyl acetate and hexyl acetate were chromatographic area, 89.9% and 6.0%, respectively. The most the major acetate ester compounds. As the major components, abundant esters compounds in ‘Fuji’ apples were six to ten 2-methyl butyl acetate contributed 32.8% to the total volatile numbered carbon chains esters, which included combinations of content (Table 2). Five Shaanxi ‘Fuji’ apples (including Baishui acetic, butanoic, methyl butyrate, and hexanoic acids with ethyl, Shaanxi (SAX-BS), Luochuan Shaanxi (SAX-LC), An’sai Shaanxi butyl, methyl butyrate, and hexyl alcohols. Aldehydes and (SAX-AS), Liquan Shaanxi (SAX-LQ), and Yichuan Shaanxi terpenoids were only responsible for 1.7% and 0.9% of the total (SAX-YC)) and two Liaoning ‘Fuji’ apples (Wafangdian Liaoning chromatographic area, respectively. (LN-WFD) and Suizhong Liaoning (LN-SZ)) were characterized Some studies have identified over 300 volatile compounds in with high 2-methyl butyl acetate content (Table 3). The Baishui the aroma profile of apples (Dixon et al., 2000). These compounds Shaanxi apple samples had the highest 2-methyl butyl acetate included alcohols, aldehydes, carboxylic esters, ketones, and ethers content among all of the apple samples. The second major acetate (Bangerth et al., 2012). About 20 of these chemicals were ester compound was hexyl acetate (25.3%; Table 2), and apple ‘character impact’ compounds. For example, the senses of 2-methyl samples collected in Ruicheng Shanxi (SX-RC) had the highest butyl acetate, hexyl acetate, hexanal, ethyl-2-methyl butanoate, content, followed by Ninglang Gansu (GS-LN), Penglai Shandong acetaldehyde, pentyl acetate, and et al, were described as the (SD-PL), Jiaonan Shandong (SD-JN), and Yiyuan Shandong (SD- ‘characteristic’ apple tastes. In this work, 2-methyl butyl acetate YY) apples. All of these apples had more than 40% of the total and hexyl acetate were found to be the main ‘character impact’ volatile. Butyl acetate was also common in the 43 ‘Fuji’ apple volatile compounds in ‘Fuji’ apples. 2-Methyl butyl acetate has samples (Table 3). Butyl acetate contributed 2.3% _ 7.8% of the been described as having a fruity, floral, sweet, berry aroma. Hexyl total volatiles (Table 3). The content of (Z)-3- hexenyl acetate was acetate has been described as the complex aromas of sweet, fruity, different in all 43 locations that the ‘Fuji’ apples were collected flowery and pear-scented (Klesk, Qian & Martin, 2004). from. In Qixia Shandong (SD-QX) the apple sample had the Composition of volatiles highest content of (Z)-3- hexenyl acetate (21.03%; Table 3), but Esters In this work, the ester family composed the major (Z)-3- hexenyl acetate was not detected in Gansu and Liaoning ‘Fuji’ apple aroma volatiles and accounted for 89.9% of the total apples. The other six types of acetate ester compounds had a low volatiles. The non-substituted aliphatic chain esters with a high content of less than 2%. content in ‘Fuji’ apples were: hexyl acetate (25.3%), butyl acetate Butanoate ester (EC4) volatiles identified in ‘Fuji’ apple fruits (5.0%), (Z)-3-hexenyl acetate (3.5%), (E)-2-hexenyl acetate were the second most abundant esters aroma components (Figure (6.5%), hexyl butanoate (4.4%), and ethyl hexanoate (2.0%; Table 1), and included ethyl butanoate, pentyl butanoate, hexyl butanoate, 2). Substituted aliphatic chains esters with a high content were: butyl butanoate, propyl butanoate, 2-methylbutyl butanoate, (Z)-3- 2-methyl butyl acetate (32.8%), butyl 2-methyl butanoate (1.9%), hexenyl butanoate, and (E)-2-hexenyl butanoate. EC4 volatiles and hexyl 2-methyl butyrate (5.9%; Table 2). contributed 8.9% of the total volatiles in ‘Fuji’ apples (Table 2). Although 43 esters in total were found in 43 ‘Fuji’ apple The content of EC4 was significantly (F=4.860,p < 0.001) different samples in 14 apple production regions, 2-methyl butyl acetate, in 14 apple production regions. In all apple regions, the content of hexyl acetate, (Z)-2-hexen-1-ol acetate, hexyl 2-methyl butyrate butanoic acid ester compounds produced in ‘Fuji’ apple samples accounted for more than 90% of total esters (Table 2). Because the were high in Gansu Tianshui and Shandong TaiYi mountainous ester family was abundant in ‘Fuji’ apple fruits, we separated them areas, especially in Gansu Tianshui area. The lowest butanoic acid into the following groups for further discussion: acetate (C2), ester content was found in southern and eastern Shanxi (4.17%; Comparison of ‘Fuji’ Apples in China 83

Table 2. Volatiles detected in fruits of ‘Fuji’ apple samples in 43 counties in China

Codes Compoundsa Formula Contentb % Compounds Codes Formula Content %

Esters Ethyl acetate EC2-1 C4H8O2 1.58 ± 0.69 Alcohols 2-Methyl-1-Butanol A-1 C5H12O 0.55 ± 0.28

Pentyl acetate EC2-2 C7H14O2 0.70 ± 0.19 Ethanol A-2 C2H6O 1.40 ± 1.00

Hexyl acetate EC2-3 C8H16O2 25.3 ± 10.1 1-Pentadecanol A-3 C15H32O 0.01 ± 0.01

Butyl acetate EC2-4 C6H12O2 4.97 ± 1.43 1-Heptadecanol A-4 C17H36O 0.15 ± 0.10

n-Propyl acetate EC2-5 C5H10O2 0.84 ± 0.98 1-Nonadecanol A-5 C19H40O 0.43 ± 0.25

2-Ethyl hexyl acetate EC2-6 C10H20O2 0.26 ± 0.10 1-Hexanol A-6 C6H14O 2.11 ± 0.87

2-Methyl butyl acetate EC2-7 C7H14O2 32.8 ± 10.9 1-Butanol A-7 C4H10O 1.12 ± 1.78

2-Methyl propyl acetate EC2-8 C6H12O2 0.15 ± 0.05 (E)-3-Hexen-1-ol A-8 C6H12O 0.18 ± 0.08

(Z) -4-Hexen-1-ol acetate EC2-9 C8H14O2 0.33 ± 0.25 (E)-2-Hexen-1-ol A-9 C6H12O 0.23 ± 0.14

(Z)-3- Hexenyl acetate EC2-10 C8H14O2 3.45 ± 4.28 Aldehydes Hexanal A’-1 C6H12O 1.15 ± 0.84

(E)-2- Hexenyl acetate EC2-11 C8H14O2 6.50 ± 3.60 (E)-2-Hexenal A’-2 C6H10O 0.56 ± 0.48

Hexyl propanoate EC3-1 C9H18O2 0.29 ± 0.19 Terpenoids α-Farnesene T-1 C15H24 0.90 ± 0.79

Butyl propanoate EC3-2 C7H14O2 0.34 ± 0.21 Other Nonane, 3,7-dimethyl- O-1 C11H24 0.13 ± 0.08

Propyl propanoate EC3-3 C6H12O2 0.21 ± 0.17 compounds Undecane, 3,8-dimethyl- O-2 C13H28 0.15 ± 0.07

Pentyl propanoate EC3-4 C8H16O2 0.14 ± 0.05 Tetradecane O-3 C14H30 0.36 ± 0.29

2-Methyl butyl propanoate EC3-5 C8H16O2 0.14 ± 0.06 Dodecane, 2,6,11-trimethyl- O-4 C15H32 0.12 ± 0.05

Ethyl butanoate EC4-1 C6H12O2 0.89 ± 0.43 Pentadecane O-5 C15H32 0.20 ± 0.12

Pentyl butanoate EC4-2 C9H18O2 0.23 ± 0.09 Heptadecane O-6 C17H36 0.21 ± 0.12

Hexyl butanoate EC4-3 C10H20O2 4.40 ± 2.06 Hexadecane O-7 C16H34 0.14 ± 0.08

Butyl butanoate EC4-4 C8H16O2 1.05 ± 0.62 Tetradecanoic acid O-8 C14H28O2 0.18 ± 0.09

Propyl butanoate EC4-5 C7H14O2 0.65 ± 0.48 n-Hexadecanoic acid O-9 C16H32O2 0.29 ± 0.17

2-Methyl butyl butanoate EC4-6 C9H18O2 0.19 ± 0.08

(Z)-3-Hexenyl butanoate EC4-7 C10H18O2 0.39 ± 0.37

(E)-2-Hexenyl butanoate EC4-8 C10H18O2 1.12 ± 0.64

Isopentyl hexanoate EC6-1 C11H22O2 0.10 ± 0.04

Ethyl hexanoate EC6-2 C8H16O2 2.00 ± 1.70

Pentyl hexanoate EC6-3 C11H22O2 0.16 ± 0.10

Hexanoate EC6-4 C7H14O2 0.17 ± 0.08

Hexyl hexanoate EC6-5 C12H24O2 1.00 ± 0.44

Propyl hexanoate EC6-6 C9H18O2 0.50 ± 0.34

(Z)-3-hexenyl hexanoate EC6-7 C12H22O2 0.39 ± 0.39

(E)-2-hexenyl hexanoate EC6-8 C12H22O2 0.38 ± 0.22

Ethyl -2-methyl butanoate E2M-1 C9H18O2 0.44 ± 0.29

Hexenyl 2-methyl butyrate E2M-2 C11H20O2 0.37 ± 0.21

Pentyl -2-methyl butyrate E2M-3 C10H20O2 0.18 ± 0.08

Hexyl 2-methyl butyrate E2M-4 C11H22O2 5.91 ± 2.48

Butyl 2-methyl butanoate E2M-5 C9H18O2 1.86 ± 0.75

Propyl 2-methyl butyrate E2M-6 C8H16O2 0.64 ± 0.48

3-Methylbutyl-2-methyl butyrate E2M-7 C10H20O2 0.20 ± 0.11

2-Methylbutyl-2-methyl butyrate E2M-8 C10H20O2 0.32 ± 0.10

Hexyl 2-methyl propanoate E2M-9 C10H20O2 0.13 ± 0.05

Hexyl n-valerate EO-2 C11H22O2 0.10 ± 0.10

2-Ethylhexyl salicylate EO-3 C15H22O3 0.12 ± 0.06 a: Identities confirmed by comparing mass spectra and retention time with standards. b: Content calculated using the average of the relative amount in 43 ‘Fuji’ apple samples.

Figure 1). Hexyl butanoate was the main volatile in all butanoic Nine types of 2-methyl butyrate ester (E2M) volatiles were acid ester compounds. The hexyl butanoate content of apples in found in ‘Fuji’ apples in different regions, including ethyl 2-methyl Qingshui Gansu (GS-QS), Qin’an Gansu (GS-QA), Qingcheng butanoate, hexenyl 2-methyl butyrate, pentyl 2-methyl butyrate, Gansu (GS-QC), and Jingning Gansu (GS-JN) was high (Table 3), hexyl 2-methyl butyrate, butyl 2-methyl butanoate, butyl 2-methyl while in apples of mid- and southern Shanxi and the butanoate, propyl 2-methyl butyrate, 3-methyl butyl 2-methyl Shandong Peninsula was low. butyrate, and 2-methyl butyl 2-methyl butyrate. 2-Methyl butyrate 84 L. Qin et al.

Table 3. Contents of the major volatiles (%) in ‘Fuji’ apples from 43 counties in China

b EC2-3 EC2-4 EC2-7 EC2-10 EC2-11 EC4-3 EC6-2 E2M-4 E2M-5 A-6 BJ-CPa 26.93 5.32 28.89 0.22 10.54 3.13 1.42 9.92 1.91 1.41 GS-JC 21.87 7.45 41.29 - c 2.92 5.22 0.56 6.66 2.24 1.54 GS-JN 16.34 4.48 34.26 - 3.48 8.12 0.44 11.53 2.87 3.73 GS-LX 25.06 6.78 30.89 - 8.71 4.44 0.95 7.3 2.34 1.71 GS-NL 41.61 2.45 26.48 0.76 11.16 1.75 2.26 4.05 0.87 0.74 GS-QA 23.27 4.11 30.43 0.29 7.04 8.94 1.73 7.62 2.08 2.34 GS-QC 18.19 6.31 31.98 - 5.29 8.64 1.19 7.04 2.28 1.94 GS-QS 18.06 7.07 39.28 - 2.38 8.55 0.44 5.96 2.18 1.92 HB-KC 20.24 4.95 41.32 0.53 6.21 4.61 0.62 4.4 2.47 2.81 HB-LT 19.09 7.76 39.83 - - 7.14 1.25 4.87 3.28 2.46 HB-QL 25.1 5.01 32.12 1.87 11.18 4.06 1.68 6.24 2.08 2.02 HB-SP 21.51 7.56 36.09 - 2.48 5.26 0.26 5.77 1.98 2.57 HB-XT 33.11 4.81 25.89 0.62 6.20 4.82 1.01 10.01 2.01 1.71 HN-SMX 19.79 6.46 32.19 - 6.52 6.73 0.49 4.81 1.51 3.42 JS-FX 27.30 4.8 36.11 - 8.50 2.6 1.02 6.2 1.4 2.22 LN-GZ 14.75 6.04 41.18 - 1.59 6.82 1.35 8.90 2.52 2.25 LN-JC 12.54 5.23 37.78 - 2.23 6.71 1.51 11.16 3.03 2.21 LN-LY 25.71 5.49 32.08 - 5.54 4.25 4.28 8.98 2.36 1.05 LN-SSL 16.81 4.79 29.42 - 5.77 4.89 1.94 9.83 2.57 1.72 LN-SZ 12.51 5.08 45.81 0.34 3.32 4.31 1.87 3.84 2.41 2.15 LN-WFD 10.52 5.21 52.18 - 2.8 4.25 2.76 6.18 2.45 2.39 NX-LW 33.02 5.82 22.40 1.83 12.9 3.44 2.11 6.59 1.22 1.22 SAX-AS 15.12 3.47 48.88 1.99 5.98 2.69 0.53 3.81 1.74 3.14 SAX-BS 7.74 6.58 57.63 - 0.89 4.01 0.22 4.52 3.04 1.83 SAX-FF 32.88 4.21 27.61 - 8.41 3.53 2.63 7.73 1.48 1.32 SAX-FX 27.74 4.41 29.31 - - 2.87 3.29 9.97 1.54 1.31 SAX-LC 11.42 5.56 50.37 0.62 3.21 2.58 - 4.48 1.88 2.81 SAX-LQ 16.02 5.25 44.42 - 5.82 4.03 1.04 4.86 2.97 3.11 SAX-YC 13.81 3.56 54.65 - 3.80 3.03 0.47 6.01 3.23 2.02 SD-JN 40.11 2.27 25.73 2.87 6.44 1.84 3.74 5.29 1.54 3.52 SD-MY 26.61 5.47 24.16 3.31 11.21 6.63 1.08 4.35 1.41 4.51 SD-PL 44.84 3.29 15.91 5.66 9.65 1.75 6.94 3.38 0.54 0.97 SD-QX 35.22 2.77 14.12 21.03 5.53 1.55 7.28 1.68 0.36 1.35 SD-WD 35.87 2.94 11.70 15.71 7.81 4.49 4.42 3.23 0.69 1.88 SD-YS 31.82 4.01 24.12 2.11 6.66 6.37 2.94 2.87 1.64 3.51 SD-YY 39.65 3.86 24.55 0.62 9.49 4.07 2.36 3.54 0.91 1.85 SD-ZY 20.41 4.85 34.10 9.92 8.93 2.64 1.03 4.11 1.34 2.19 SX-LY 31.12 3.32 21.89 3.91 10.4 2.64 1.32 4.42 1.33 1.61 SX-QX 43.91 4.36 24.4 1.32 7.29 0.71 2.31 4.26 0.80 0.69 SX-RC 45.55 3.04 21.53 2.25 14.23 1.12 2.66 2.69 0.53 1.06 SX-WR 31.21 4.01 33.94 1.32 10.62 2.19 0.65 4.17 1.39 1.84 SX-YC 31.22 3.22 29.93 0.36 9.49 3.19 1.48 5.84 1.66 1.47 YN-ML 37.88 7.82 18.71 0.72 10.18 4.91 2.68 2.92 1.01 1.61

a: Capital letter represent the apple samples corresponding to the accession number in Table 1. b: The letter plus the number represents compounds corresponding to the code in Table 2. c: Not detected in sample. ester volatiles contributed to 8.9% of the total volatiles (Table 2). 2-methyl butyrate and butyl 2-methyl butanoate were common,and The content of E2M in ‘Fuji’ apples was extremely significantly contributed 5.9% and 1.9% of the total volatiles,respectively (Table (F=3.873, p=0.002) higher in the Shaanxi Guanzhong area, 2). southwest Liaoning, and the Yanshan and Taihang mountain areas Five types of propanoate ester volatiles in ‘Fuji’ apples fruits than in other regions. The lowest content was found in apples were identified. The sum of propanoate esters accounted for less collected on the Shandong Jiaodong Peninsula (Figure 1). Hexyl than 1% of the total volatiles (Figure 1). Other types of esters, such Comparison of ‘Fuji’ Apples in China 85

Fig. 1. Average percent content of the groups of volatiles in apple samples collected from different regions in China. (A) Average percent ** ** content of alcohols, aldehydes, and terpenoids.[F-value: Falcohols= 4.643 , Faldehydes=0.679, Fterpenoids=3.604 ]. (B) Average percent content ** of acetate (EC2), propanoate (EC3), butyrate (EC4), hexanoate (EC6), and 2-methyl butyrate (E2M) esters. [ F-value: FEC2=3.318 , ** * ** FEC3=1.049, FEC4=4.860 , FEC6=2.566 , FE2M=3.873 ]. The values are means ± standard error (SE). as hexyl 2-methyl propanoate, hexyl n-valerate, and 2-ethylhexyl occurred in ‘Fuji’ apples. salicylate, had a very low concentration. These esters was not Alcohols Nine types of alcohols were found in ‘Fuji’ apples discussed here in detail because of their low levels. (Table 2), and they accounted for 0.01% _ 2.1% of the total In apples, the majority of volatiles were esters (Defilippi et al., volatiles (Figure 1). There was extremely significant (F= 4.643, 2005; Knee & Hatfield, 1981; Paillard, 1979), the formation of p < 0.001) difference in 14 apple production regions. In ‘Fuji’ which was dependent on the availability of C2-C8 acids and apples from northern Shaanxi and Sanmenxia Henan the alcohols alcohols (Bangerth et al., 2012; Elss et al., 2006; Mehinagic et al., were more abundant than in other regions. Apples collected from 2006). Ester production in fruit tissue is the result of esterification Sanmenxia Henan had the highest alcohol content in apples from of alcohols, carboxylic acids, and acyl CoAs in an oxygen the 43 counties. In all of the ‘Fuji’ apple samples, 1-hexanol and dependent reaction (Berger, Drawert & Nitz, 1983; Echeverrıa et 2-methyl-1-butanol were major components and accounted for al., 2004). In this study, the branched chain esters were high in more than 70% of total alcohol compounds. 1-hexanol was ‘Fuji’ apples, 2-methyl butyl acetate and hexyl 2-methyl butyrate dominant with contents ranging from 0.7% to 4.5% of total were found to be the major ester compounds in ‘Fuji’ apples, and volatiles, and was the highest in Mengyin Shandong (Table 3). The they accounted for 32.6% and 5.9% of all volatiles in ‘Fuji’ apples, second most common alcohol was 2-methyl-1-butanol, which had respectively. Heath and Reineccius (Heath & Reineccius, 1986) a content of less than 1.0%. reported that branched chain alcohols, carbonyls, and esters are Aldehydes Two aldehydes, hexanal and (E)-2-hexenal, were produced by the metabolism of amino acids. Iso-leucine was detected in ‘Fuji’ apples in 14 apple production province in China, considered to be the biosynthetic precursor of 2-methyl butanoic and accounted for 1.8% of the total volatiles. The content of acid and its esters in apples (Paillard, 1990). Our results indicated aldehydes was not significantly (F=0.679, p=0.785) different in 14 that the high ratios of amino acid conversion to volatiles, in apple production regions. The content of hexanal was higher than particular the differential rates of metabolism of iso-leucine, (E)-2-hexenal. The sum of the aldehydes in the apples collected 86 L. Qin et al.

Fig. 2. Positions of PC scores of 43 ‘Fuji’ apple samples according to PC1 and PC2 obtained using content of volatile compositions. (A) The scores scatter plot of PCA, and (B) the loadings plot of PCA. The capital letter in Figure 2 (A) represent the accession number, which corresponds to the same number as in Table 1. The letter plus the number in Figure 2 (B) is the code of volatiles, which correspond to the same code as in Table 2. from Shaanxi Guanzhong had the highest aldehyde content of all importance of the variables is shown in Figure 2B. The scores tested apple production regions (Figure 1). Aldehydes were not scatter plot (Figure 2A) shows that the first principal component detected in apples collected from southern and mid- Shanxi, the (PC1-axis; explaining 49.0% of the variability in the data) is Shandong Peninsula, Changping Beijing, Malong Yunnan, and influenced by most of compounds. The second principal component Fengxian Jiangsu. (PC2-axis) only explains 12.2% of the variability in the data. The Terpenoids α-Farnesene was the only terpenoid compound in apple samples from different regions can be divided into four the aroma profile of apples. The total content of α-farnesene groups (Figure 2A) based on their position in the scores scatter accounted for 0.9% of the total volatiles. The content of plot. α-farnesene was extremely significantly (F=3.604, p=0.002) Group I: The cluster in the far lower left of the scatter plot is different in 14 apple production regions. α-Farnesene in Liaoning composed of apples from the six Shandong locations (SD-QX, SD- was the highest out of all apple regions (Figure 1). There was no WD, SD-PL, SD-YS, SD-JN and SD-ZY), the three Shanxi α-farnesene detected in apples collected from Shunping Hebei, locations (SX-RC, SX-LY and SX-WR), and one Gansu location Leting Hebei, Kuancheng Hebei, or Qinglong Hebei. (GS-NL). This clustering was likely due to high concentrations of

Other compounds We also detected two acids and seven other hexyl acetate (EC2-3, described as the complex aromas of sweet, carbonyl compounds. The sum of the acids and other carbonyl fruity, flowery and pear), ethyl hexanoate (EC6-2), and (Z)-3- _ _ compounds accounted, respectively, for 0.2% 0.3% and 0.1% hexenyl acetate (EC2-10). These compounds make apples in this 0.4%, of the total volatiles (Table 2). Acids and other carbonyl cluster the sweet tasting. compounds are not discussed here in detail because of their low Group II: On the right of the PC1 axis and below the PC2 axis levels. there is a cluster composed of apples from the five Shaanxi Principal component analysis The aroma composition and locations (SAX-BS, SAX-LC, SAX-AS, SAX-YC, and SAX-LQ), content in ‘Fuji’ apples varied in different regions. In order to study one Henan location (HN-SMX), two Liaoning locations (LN-WFD the principal sources of variation among our results and to and LN-SZ), and three Hebei locations (HB-KC, HB-LT and HB- understand the relationships between samples in each apple SP). The apples in this cluster were characterized by high contents production region and the compound identified, the principal of 2-methyl butyl acetate (EC2-7, described as having fruity, sweet, component analysis (PCA) was applied. The PCA scores scatter flowery, and over all aroma,) and 1-hexanol (A-6, described as plot of the different apple growing regions is shown in Figure 2A, having the aromas of green apple and grass-liking). These and the corresponding loading plot establishing the relative compounds make apples in this cluster the best smelling. Comparison of ‘Fuji’ Apples in China 87

Table 4. Correlation among major aroma volatile compounds, geographic location and main climatic factors

a b AP MAT Longitude Latitude EC2-7 EC2-3 EC2-11 E2M-4 EC2-4 EC4-3 EC2-10 A-6 EC6-2 MAT 0.380* Longitude 0.357* -0.016 Latitude -0.250 -0.447** 0.730**

EC2-7 -0.210 -0.380* -0.073 0.246

EC2-3 -0.018 0.428** -0.136 -0.354* -0.849**

EC2-11 0.130 0.387* 0.011 -0.204 -0.611** 0.678** E2M-4 -0.132 -0.205 0.098 0.267 0.174 -0.356* -0.325*

EC2-4 -0.044 -0.269 -0.369* -0.222 0.332* -0.436** -0.415** 0.216

EC4-3 0.141 -0.152 0.016 0.037 0.166 -0.481** -0.409** 0.388* 0.529**

EC2-10 0.266 0.138 0.346* 0.124 -0.478** 0.295 0.186 -0.450** -0.418** -0.276 A-6 0.214 0.136 0.153 0.104 0.297 -0.434** -0.230 -0.036 0.188 0.451** -0.126

EC6-2 0.161 0.156 0.328* 0.067 -0.627** 0.581** 0.190 -0.251 -0.450** -0.352* 0.603** -0.371* E2M-5 -0.068 -0.409** 0.042 0.281 0.759** -0.831** -0.655 0.532** 0.486** 0.535** -0.534** 0.336* -0.557**

AP: Annual precipitation MAT: Mean annual temperature * and **: significant at 0.05 and 0.1 levels

Group III: On the right side of the PC1 axis and the top of the Gansu and other six Gansu locations were not identified as the PC2 axis there is a cluster composed of apples from six Gansu same group, which might be due to the difference of rainfall or locations (GS-TS, GS-QA, GS-JN, GS-LX, GS-QC and GS-QA) temperature. These results indicated that geographic changes could and three Liaoning locations (LN-SSL, LN-JC and LN-GZ). This affect the concentration of chemical compounds in ‘Fuji’ apple due cluster was characterized by high contents of hexyl butanoate (EC4- to the difference of temperatures, rainfall or geographic location.

3), butyl acetate (EC2-4, described as the aromas of having fruity, Correlation among major aroma volatile compounds, sweet, and grassy), hexyl 2-methyl butyrate (E2M-4, described as geographic location and main climatic factors It was found ten having apple, grape fruit taste ), and butyl 2-methyl butanoate (E2M- major volatile compounds in ‘Fuji’ apples in the experiment,

5, described as having fruity). These compounds make apples in including EC2-7, EC2-3, EC2-11, E2M-4, EC2-4, EC4-3, EC2-10, this cluster the best fruity aroma. A-6, EC6-2 and E2M-5. Moreover, correlation analysis of ten major Group IV: The last cluster is composed of apples from Yunnan aroma volatile compounds, geographic location and main climatic (YN-ML), Jiangsu (JS-FX), Beijing (BJ-CP), Ningxia (NX-LW), factors was done and shown in the table 4. It was found that mean three Sanxi locations (SX-YC, SX-WR, SX-LY), and one Liaoning annual temperature (MAT) was significant and extremely location (LN-LY). In this group it was difficult to identify the significant negatively correlated with EC2-7 ( p < 0.05) and E2M-5 dominant volatile and there was not characteristic composition for ( p < 0.01) respectively, and significant and extremely significant this group. positively correlated with EC2-3 ( p < 0.01) and EC2-11 ( p < 0.05) The composition and content of volatile compounds in ‘Fuji’ respectively. Annual precipitation was not correlated with ten apples varied in different regions. This difference may be due to major volatile compounds. Longitude was significantly ( p < 0.05) the climate, elevation, or geographic location, which may interfere correlated with concentration of EC2-4, EC2-10, and EC6-2. It was with the activity of some flavoring enzymes. Some studies has found that latitude showed significantly ( p < 0.05) negative reported that grape composition is related to many environmental correlation with EC2-3 concentration, and no correlation with other factors, such as macro, meso and microclimate, soil, altitude, and major volatile compounds. At the same time, it was found that topography (Styger, Prior & Bauer, 2011; Webb, Whetton & there were some significant correlation between the major Barlow, 2008). In addition, it has been reported that altitude compounds. changes affect the chemical composition of black tea, with changes in temperatures and rainfall altering the taste, aroma, and potential Conclusions health benefits of the beverage (Owuor, Obaga & Othieno, 1990). The volatile composition and content in ‘Fuji’ apples varied in In this study, the rainfall, temperature and altitudes of the locations different regions in China. Esters were the major volatile collected apple samples were different (table 1), the result showed compounds in ‘Fuji’ apple aroma volatiles. 2-methyl butyl acetate that the composition and content of main volatile compounds in and hexyl acetate contributed 32.5% and 24.5% to total volatile ‘Fuji’ apples in different locations were varied (table 3). Otherwise, content, respectively. All of the ‘Fuji’ apple samples from 43 annual precipitation and mean annual temperature in Ninlang different counties could be classified into following groups: (1) Gansu were higher than those in six other Gansu locations. Ninlang apples with high concentrations of hexyl acetate and (Z)-3-hexenyl 88 L. Qin et al. acetate, came from Shandong (Qixia, Wendeng, Penglai, AVI Publishing Company. Zhaoyuan, Jiaonan, and Yishui), Shanxi (Wanrong, Ruicheng, and Klesk, K., Qian, M., and Martin, R. R. (2004). Aroma extract dilution Linyi), and Gansu Ninglang; (2) apples characterized by high analysis of cv. Meeker (Rubus idaeus L.) red raspberries from Oregon contents of 2-methyl butyl acetate and 1-hexanol came from and Washington. J. Agr. Food Chem., 52, 5155-5161. northern Shaanxi, Henan Sanmenxia, Liaoning Wafangdian, and Knee, M. and Hatfield, S. G. (1981). The metabolism of alcohols by apple Suizhong; (3) apples characterized by high contents of hexyl fruit tissue.J. Sci. Food Agr., 32, 593-600. butanoate, butyl acetate, and hexyl 2-methyl butyrate came from López, M., Lavilla, M., Riba, M., and Vendrell, M. (1998). Comparison of Gansu (except Ninglang), and (4) a group that lacked any volatile compounds in two seasons in apples: Golden Delicious and characteristic volatile composition. Moreover, correlation analysis Granny Smith. J. Food Quality, 21, 155-166. of ten major aroma volatile compounds, geographic location and Matich, A. J., Rowan, D. D., and Banks, N. H. (1996). Solid phase main climatic factors showed that mean annual temperature (MAT) microextraction for quantitative headspace sampling of apple volatiles. was significant correlated with 2-methyl butyl acetate (EC2-7) and Anal. Chem., 68, 4114-4118. butyl 2-methyl butanoate (E2M-5), hexyl acetate (EC2-3) and (E)- Mehinagic, E., Royer, G., Symoneaux, R., Jourjon, F., and Prost, C. (2006).

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