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

Ling Qin; Qin-Ping Wei; Wen-Huai Kang; Qiang Zhang; Jian Sun; Song-Zhong Liu

doi:10.3136/fstr.23.79

Abstract

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.

Introduction

Aroma is one of the essential components of fruit quality. The relative contributions of specific aroma volatile compounds to the flavor of apples have been examined by many investigators, and more than 300 compounds have been identified (Dixon & Hewett, 2000; Elss, Preston, Appel, Heckel & Schreier, 2006; Fallik, Archbold, Hamilton-Kemp, Loughrin & Collins, 1997; Mehinagic, Royer, Symoneaux, Jourjon & Prost, 2006). The volatile compounds of apples (Malus domestica Borkh) include alcohols, aldehydes, carboxylic esters, ketones, and ethers (Dixon et al., 2000). The esters, particularly those with even numbered carbon chains including combinations of acetic, butanoic, and hexanoic acids with ethyl, butyl, and hexyl alcohols, have been reported to be the major contributors to the aroma of apples (López, Lavilla, Riba & Vendrell, 1998; Matich, Rowan & Banks, 1996; Song & Bangerth, 1993).

Investigations have focused on the characteristic volatiles produced by ripening apples and the evolution of apple aromas post-harvest (Bangerth, Song & Streif, 2012; Echeverrıa, Graell, López & Lara, 2004; Song et al., 1993). Bangerth and colleagues (Bangerth et al., 2012) reviewed physiological impacts of fruit ripening and storage conditions on the formation of aroma volatiles in apples. Apple volatile production had been categorized according to: type and quantity of esters or alcohols (Dirinck & Schamp, 1988; Paillard, 1990), skin color (Paillard, 1979), or aroma production patterns (Dirinck et al., 1988). Several studies (Defilippi, Kader & Dandekar, 2005; Elss et al., 2006; Matich et al., 1996; Schaffer et al., 2007; Schumacher, Asche, Heil, Mittelstädt, Dietrich & Mosandl, 1998; Song & Bangerth, 2003; Xiaobo & Jiewen, 2008) have discussed the biochemical origin of aroma volatiles and improvements in methods for the separation and identification of volatile compounds. Several studies (Mpelasoka & Hossein Behboudian, 2002; Nielsen, Jägerstad, Öste & Wesslén, 1992; Plotto, McDaniel & Mattheis, 1999; Song et al., 1993) have also investigated the effect of culture techniques and management on the composition and content of volatiles. ‘Fuji’ apple cultivar (Malus domestica Borkh CV. Red Fuji) is becoming one of the major apple cultivars in China, and it is grown both in northern and southern China. In the two dominant apples production zones in China, the Loess Plateau regions and Surrounding Bohai Gulf regions, the cultivated area of ‘Fuji’ apples has exceeded 80%. Until now, there have been no reports on aroma volatile characteristic and quality parameters in different apple production regions.

In this study, headspace-solid phase micro-extraction (HS-SPME) combined with gas chromatography — mass spectrometry (GC-MS) was applied to study the volatiles compounds in ‘Fuji’ apples in the different production regions in China. The aim of this work focused on comparing the aroma composition and content, which was used to assess aroma volatile characteristic and quality parameters in ‘Fuji’ apples, particularly among the different apple production regions in China that have not been extensively studied.

Materials and Methods

Plant materials ‘Fuji’ apple (Malus domestica Borkh CV. Red Fuji) samples were collected from October 30 through November 6, 2011 in 43 well-managed orchards located in 14 apple production regions in China. Twenty-one ‘Fuji’ fruits samples came from the Loess Plateau, which included the following locations: three East Gansu areas, four Gansu Tianshui areas, three North Shaanxi areas, four Shaanxi Guanzhong areas, one collection from Lingwu Ningxia, five middle and south Shanxi locations, and one collection from Sanmenxia Henan. Twenty-two ‘Fuji’ apple samples came from areas surrounding the Bohai Gulf, including six locations in southern and western Liaoning, five in the Hebei Yanshan mountainous area, five in the Shandong Peninsula, three in the Shandong TaiYi mountainous areas, and one collection from Changping, Beijing. Two apple samples came from other regions, one from Malong Yunnan and one from Fengxian Jiangsu. Table 1 gives the geographical location and main climatic factors of samples in 43 counties.

Table 1. Geographic location and main climatic factors of ‘Fuji’ apple samples collected from 43 counties in China

NO^a	Apple Samples	Province	regions	County	Longitude / Latitude	AP/mm	MAT/°C
1	GS-JN ^b	Gansu	East Gansu area	Jingning	105°20′∼106°05′/35°01′∼35°45′	450.8	7.1
2	GS-LX		East Gansu area	Lixian	104°37′∼105°36′/33°35′∼34°31′	488.2	9.9
3	GS-QC			Qingcheng	107°16′∼108°05′/35°42′∼36°17′	537.5	9.4
4	GS-JC			Jingchuan	107°15′∼107°45′/35°11′∼35°31′	555	10
5	GS-LN			Ninglang	100°21′∼101°16′/26°35′∼27°56′	920	12.7
6	GS-QS		Gansu Tianshui area	Qingshui	105°45′∼106°30′/34°32′∼34°56′	580	9.6
7	GS-QA		Gansu Tianshui area	Qin'an	105°20′∼106°02′/34°44′∼35°11′	507.3	10.4
8	SAX-BS	Shaanxi	North Shaanxi area	Baishui	109°16′∼109°45′/35°04′∼35°27′	577.8	11.4
9	SAX-LC		North Shaanxi area	Luochuan	109°13′∼109°45′/35°26′∼36°04′	622	9.2
10	SAX-AS			An'sai	108°05′∼109°26′/36°30′∼37°19′	505.3	8.8
11	SAX-YC			Yichuan	109°41′∼110°32′ / 35°42′∼36°23′	521.1	10
12	SAX-FX		Shaanxi Guanzhong area	Fengxiang	107°10′∼107°38′ / 34°20′∼34°45′	625	11.4
13	SAX-LQ			Liqua, Fufeng	108°17′∼108°41′ / 34°20′∼34°50′	546	12.9
14	SAX-FF			Liqua, Fufeng	107°45′∼108°03′ / 34°12′∼34°37′	592	12.4
15	NX-LW	Ningxia		Lingwu	105°.59∼106°.37′/37°.60∼38°01′	308	8.8
16	SX-RC	Shanxi	Middle and South Shanxi Sanmenxia Henan	Ruicheng	110°36′∼110°42′/34°36′∼34°48′	600	14
17	SX-QX			Qixian	112°12′∼112°39′/37°15′∼37°28′	441.8	9.9
18	SX-WR			Wanrong	110°25′∼110°59′/35°13′∼35°31′	500	11.9
19	SX-YC			Yicheng	111°34′∼112°03′/35°23′∼35°52′	550	11
20	SX-LY			Linyi	110°17′∼110°54′/34°58′∼35°18′	508.7	13.5
21	HN-SMX	Henan		Sanmenxia	110°21′∼112°01′/33°31′∼35°05′	630	13.8
22	LN-WFD	Liaoning	South and West Liaoning	Wafangdian	121°13′∼122°16′/39°20′∼40°07′	645	10.3
23	LN-SZ		South and West Liaoning	Suizhong	119°34′∼120°31′/39°59′∼40°37′	652.5	9.8
24	LN-SSLB			Sanshilibao	121°34′∼121°41′/39°21′∼39°29′	675	9.4
25	LN-JC			Jianchang	119°13′∼120°18′/40°24′∼41°06′	550	8.2
26	LN-GZ			Gaizhou	121°57′∼122°33′/39°55′∼40°31′	636.3	9.5
27	LN-LY			Lingyuan	118°50′∼119°37′/40°35′∼41°26′	550	8.0
28	HB-SP	Hebei	Hebei Yanshan	Shunping	114°50′∼115°17′/38°45′∼39°07′	578	12.2
29	HB-LT		Mountainous area	Leting	118°40′∼119°18′/39°05′∼39°34′	613.2	10.6
30	HB-KC		Mountainous area	Kuancheng	118°10′∼119°10′/40°17′∼40°45′	650	8.6
31	HB-QL			Qinglong	118°33′∼119°36′/40°04′∼40°36′	741	8.9
32	HB-XT			Xingtang	114°09′∼114°41′/38°20′∼38°42′	480	12
33	SD-ZY	Shandong	Shandong peninsula	Zhaoyuan	120°08′∼120°38′/37°05′∼37°33′	659.1	11.5
34	SD-WD		Shandong peninsula	Wendeng	121°43′∼122°19′/36°52′∼37°23′	762.2	11.5
35	SD-PL			Penglai	120°35′∼121°09′/37°25′∼37°50′	606.2	11.9
36	SD-QX			Qixia	120°33′∼121°15′/37°05′∼37°32′	743	11.4
37	SD-JN			Jiaonan	119°30′∼120°11′/35°35′∼36°08′	798.3	12.1
38	SD-MY		Shandong TaiYi	Mengyin	117°45′∼118°15′/35°27′∼36°02′	800	12.8
39	SD-YY		Mountainous area	Yiyuan	117°54′∼118°31′/35°55′∼36°23′	690.9	11.9
40	SD-YS		Mountainous area	Yishui	118°13′∼119°03′/35°36′∼36°13′	800	12.3
41	BJ-CP	Beijing		Changping	115°50′∼116°29′/40°2′∼40°23′	550.3	11.8
42	YN-ML	Yunan		Malong	103°08′∼103°56′/27°07′∼27°39′	735	11.6

^a: NO showed the number of apple samples, 1–21 collected from the the Loess Plateau regions, 22–41 collected in areas surrounding Bohai Gulf regions, 42–43 collected in other regions

^b: Province - County

AP: Annual precipitation; MAT: Mean annual temperature

The ‘Fuji’ apples were collected from five trees at each orchard from the above locations when the apples were fully ripe (based on the days after full bloom). Forty fruits per orchard were sampled according to an equatorial pattern (East-West-North-South) from the periphery of the canopy of each tree individually, avoiding fruits situated at the top, bottom or deep inside the foliage. The sampled fruits were healthy and without any symptoms of pest infestation or disease infection. The sampled fruits were put into plastic bags and transferred to the laboratory in an insulated box filled with ice packs. In the laboratory the apple samples were washed in deionised water and surface-dried with gauze.

Analysis of aroma volatile compounds Two kilograms of sampled fruits from each location were ground to a powder and homogenized using a blender. Juice from the collected apples was centrifuged at 4000 rpm for 10 min. The supernatant was stored at −80°C until analysis. Volatiles from the apple juice samples were extracted using HS-SPME. A 50/30 µm DVB / CAR / PDMS fiber (Tupelo, Inc., Bellefonte, PA) was used for aroma extraction. Apple juice (8 g) was weighed in a 20 mL headspace vial and capped with a septum. The juice was saturated with sodium chloride (2.4 g) and a 2-octanol internal standard solution was added to each vial to give a final 2-octanol concentration of 80 µg L⁻¹.

Each sample was equilibrated at 45°C in a thermostatic bath for 45 min, and then extracted for 30 min at the same temperature while stirring. After extraction, the fiber was inserted into the injection port of the GC (250°C) to adsorb the analysis. GC-MS was carried out using an Agilent GC 6890-5975 Mass Selective Detector (MSD). Samples were analyzed on a DB-Wax column (30 m × 0.32 mm I.D., 0.25 µm film thickness; J&W Scientific, Folsom, CA, and USA). Helium was used as the carrier gas at a constant flow rate of 2 mL/min. The oven temperature was maintained at 50°C for 2 min then the temperature was increased to 230°C and maintained for 25 min. The MS detector was operated in the scan mode (mass range 50–200) and the transfer line to the MS system was maintained at 250°C

Identification of volatile compounds was based on the comparison of their mass spectra and retention indices (RI) with standards and published data, as well as standard mass spectra in the NIST05a. L database (Agilent Technologies Inc.). Retention indices were calculated using a mixture of n-paraffin C6-C30 as standards. The volatile compound content was calculated from the GC-peak areas that relate to the GC-peak area of the internal standard.

Statistical analysis Data for each apple sample were averaged among the three replications. A principal component analysis (PCA) was done to detect clustering formations and establish relationships between samples and volatile compounds using SPSS for Windows Version 17.0 (SPSS Inc.). The correlation among major aroma volatile compounds, geographic location and main climatic factors was also calculated using SPSS.

Results and Discussion

Identification of volatiles Using HS-SPME-GC-MS, 64 volatile compounds in ‘Fuji’ apples were identified and the amounts of volatile compounds were measured. We identified 43 esters, nine alcohols, two aldehydes, one terpenoid, two acids, and seven alkanes. Table 2 gives the average content of all volatiles based on the location of the different ‘Fuji’ apple samples. Of all the aroma compounds identified, the following 10 were the most abundant: 2-methyl butyl acetate, hexyl acetate, (E)-2-hexen-1-ol acetate, hexyl-2-methyl butyrate, butyl acetate, hexyl butanoate, (Z)-3- hexenyl acetate, (Z)-ethyl acetate, 1-hexanol, and ethyl hexanoate.

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

	Compounds^a	Codes %	Formula	Content^b
Esters	Ethyl acetate	EC₂-1	C₄H₈O₂	1.58 ± 0.69
	Pentyl acetate	EC₂-2	C₇H₁₄O₂	0.70 ± 0.19
	Hexyl acetate	EC₂-3	C₈H₁₆O₂	25.3 ± 10.1
	Butyl acetate	EC₂-4	C₆H₁₂O₂	4.97 ± 1.43
	n-Propyl acetate	EC₂-5	C₅H₁₀O₂	0.84 ± 0.98
	2-Ethyl hexyl acetate	EC₂-6	C₁₀H₂₀O₂	0.26 ± 0.10
	2-Methyl butyl acetate	EC₂-7	C₇H₁₄O₂	32.8 ± 10.9
	2-Methyl propyl acetate	EC₂-8	C₆H₁₂O₂	0.15 ± 0.05
	(Z) -4-Hexen-1-ol acetate	EC₂-9	C₈H₁₄O₂	0.33 ± 0.25
	(Z)-3- Hexenyl acetate	EC₂-10	C₈H₁₄O₂	3.45 ± 4.28
	(E)-2- Hexenyl acetate	EC₂-11	C₈H₁₄O₂	6.50 ± 3.60
	Hexyl propanoate	EC₃-1	C₉H₁₈O₂	0.29 ± 0.19
	Butyl propanoate	EC₃-2	C₇H₁₄O₂	0.34 ± 0.21
	Propyl propanoate	EC₃-3	C₆H₁₂O₂	0.21 ± 0.17
	Pentyl propanoate	EC₃-4	C₈H₁₆O₂	0.14 ± 0.05
	2-Methyl butyl propanoate	EC₃-5	C₈H₁₆O₂	0.14 ± 0.06
	Ethyl butanoate	EC₄-1	C₆H₁₂O₂	0.89 ± 0.43
	Pentyl butanoate	EC₄-2	C₉H₁₈O₂	0.23 ± 0.09
	Hexyl butanoate	EC₄-3	C₁₀H₂₀O₂	4.40 ± 2.06
	Butyl butanoate	EC₄-4	C₈H₁₆O₂	1.05 ± 0.62
	Propyl butanoate	EC₄-5	C₇H₁₄O₂	0.65 ± 0.48
	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
Alcohols	2-Methyl-1-Butanol	A-1	C₅H₁₂O	0.55 ± 0.28
	Ethanol	A-2	C₂H₆O	1.40 ± 1.00
	1-Pentadecanol	A-3	C₁₅H₃₂O	0.01 ± 0.01
	1-Heptadecanol	A-4	C₁₇H₃₆O	0.15 ± 0.10
	1-Nonadecanol	A-5	C₁₉H₄₀O	0.43 ± 0.25
	1-Hexanol	A-6	C₆H₁₄O	2.11 ± 0.87
	1-Butanol	A-7	C₄H₁₀O	1.12 ± 1.78
	(E)-3-Hexen-1-ol	A-8	C₆H₁₂O	0.18 ± 0.08
	(E)-2-Hexen-1-ol	A-9	C₆H₁₂O	0.23 ± 0.14
Aldehydes	Hexanal	A'-1	C₆H₁₂O	1.15 ± 0.84
	(E)-2-Hexenal	A'-2	C₆H₁₀O	0.56 ± 0.48
Terpenoids	α-Farnesene	T-1	C₁₅H₂₄	0.90 ± 0.79
Other compounds	Nonane, 3,7-dimethyl-	O-1	C₁₁H₂₄	0.13 ± 0.08
Other compounds	Undecane, 3,8-dimethyl-	O-2	C₁₃H₂₈	0.15 ± 0.07
	Tetradecane	O-3	C₁₄H₃₀	0.36 ± 0.29
	Dodecane, 2,6,11-trimethyl-	O-4	C₁₅H₃₂	0.12 ± 0.05
	Pentadecane	O-5	C₁₅H₃₂	0.20 ± 0.12
	Heptadecane	O-6	C₁₇H₃₆	0.21 ± 0.12
	Hexadecane	O-7	C₁₆H₃₄	0.14 ± 0.08
	Tetradecanoic acid	O-8	C₁₄H₂₈O₂	0.18 ± 0.09
	n-Hexadecanoic acid	O-9	C₁₆H₃₂O₂	0.29 ± 0.17

^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.

Although a large number of volatile compounds were identified in ‘Fuji’ apples collected from 43 counties in China, esters and alcohols were responsible for almost all of the total chromatographic area, 89.9% and 6.0%, respectively. The most abundant esters compounds in ‘Fuji’ apples were six to ten numbered carbon chains esters, which included combinations of acetic, butanoic, methyl butyrate, and hexanoic acids with ethyl, butyl, methyl butyrate, and hexyl alcohols. Aldehydes and terpenoids were only responsible for 1.7% and 0.9% of the total chromatographic area, respectively.

Some studies have identified over 300 volatile compounds in the aroma profile of apples (Dixon et al., 2000). These compounds included alcohols, aldehydes, carboxylic esters, ketones, and ethers (Bangerth et al., 2012). About 20 of these chemicals were ‘character impact’ compounds. For example, the senses of 2-methyl butyl acetate, hexyl acetate, hexanal, ethyl-2-methyl butanoate, acetaldehyde, pentyl acetate, and et al, were described as the ‘characteristic’ apple tastes. In this work, 2-methyl butyl acetate and hexyl acetate were found to be the main ‘character impact’ volatile compounds in ‘Fuji’ apples. 2-Methyl butyl acetate has been described as having a fruity, floral, sweet, berry aroma. Hexyl acetate has been described as the complex aromas of sweet, fruity, flowery and pear-scented (Klesk, Qian & Martin, 2004).

Composition of volatiles

Esters In this work, the ester family composed the major ‘Fuji’ apple aroma volatiles and accounted for 89.9% of the total volatiles. The non-substituted aliphatic chain esters with a high content in ‘Fuji’ apples were: hexyl acetate (25.3%), butyl acetate (5.0%), (Z)-3-hexenyl acetate (3.5%), (E)-2-hexenyl acetate (6.5%), hexyl butanoate (4.4%), and ethyl hexanoate (2.0%; Table 2). Substituted aliphatic chains esters with a high content were: 2-methyl butyl acetate (32.8%), butyl 2-methyl butanoate (1.9%), and hexyl 2-methyl butyrate (5.9%; Table 2).

Although 43 esters in total were found in 43 ‘Fuji’ apple samples in 14 apple production regions, 2-methyl butyl acetate, hexyl acetate, (Z)-2-hexen-1-ol acetate, hexyl 2-methyl butyrate accounted for more than 90% of total esters (Table 2). Because the ester family was abundant in ‘Fuji’ apple fruits, we separated them into the following groups for further discussion: acetate (C2), butyrate (C4), hexanoate (C6), and 2-methyl butyrate (2M) esters (Dixon et al., 2000).

Eleven types of acetate ester (EC2) compounds, including ethyl acetate, pentyl acetate, hexyl acetate, butyl acetate, n-propyl acetate, 2-ethyl hexyl acetate, 2-methyl butyl acetate, 2-methyl propyl acetate, (Z)-4-hexen-1-ol acetate, (Z)-3- hexenyl acetate, and (E)-2- hexenyl acetate, were detected. Acetate ester compounds of all of the apple samples contributed 76.9% of the total volatile content (Table 2). There was significant (F=3.318, p=0.002) difference in the content of EC2 in 14 apple production regions. In all regions, the content of acetate ester compounds produced in ‘Fuji’ apples was the highest in mid- and southern Shanxi (80.3%). The lowest acetate ester content was found in southern and western Liaoning (66.5%; Figure 1). Of the 11 types of acetate ester compounds found, 2-methyl butyl acetate and hexyl acetate were the major acetate ester compounds. As the major components, 2-methyl butyl acetate contributed 32.8% to the total volatile content (Table 2). Five Shaanxi ‘Fuji’ apples (including Baishui Shaanxi (SAX-BS), Luochuan Shaanxi (SAX-LC), An'sai Shaanxi (SAX-AS), Liquan Shaanxi (SAX-LQ), and Yichuan Shaanxi (SAX-YC)) and two Liaoning ‘Fuji’ apples (Wafangdian Liaoning (LN-WFD) and Suizhong Liaoning (LN-SZ)) were characterized with high 2-methyl butyl acetate content (Table 3). The Baishui Shaanxi apple samples had the highest 2-methyl butyl acetate content among all of the apple samples. The second major acetate ester compound was hexyl acetate (25.3%; Table 2), and apple samples collected in Ruicheng Shanxi (SX-RC) had the highest content, followed by Ninglang Gansu (GS-LN), Penglai Shandong (SD-PL), Jiaonan Shandong (SD-JN), and Yiyuan Shandong (SD-YY) apples. All of these apples had more than 40% of the total volatile. Butyl acetate was also common in the 43 ‘Fuji’ apple samples (Table 3). Butyl acetate contributed 2.3% −7.8% of the total volatiles (Table 3). The content of (Z)-3- hexenyl acetate was different in all 43 locations that the ‘Fuji’ apples were collected from. In Qixia Shandong (SD-QX) the apple sample had the highest content of (Z)-3- hexenyl acetate (21.03%; Table 3), but (Z)-3- hexenyl acetate was not detected in Gansu and Liaoning apples. The other six types of acetate ester compounds had a low content of less than 2%.

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: F_alcohols= 4.643^**, F_aldehydes=0.679, F_terpenoids=3.604^**]. (B) Average percent content of acetate (EC2), propanoate (EC3), butyrate (EC4), hexanoate (EC6), and 2-methyl butyrate (E2M) esters. [ F-value: F_EC2=3.318^**, F_EC3=1.049, F_EC4=4.860^**, F_EC6=2.566^*, F_E2M=3.873^**]. The values are means ± standard error (SE).

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

	EC₂-3^b	EC₂-4	EC₂-7	EC₂-10	EC₂-11	EC₄-3	EC₆-2	E2M-4	E2M-5	A-6
BJ-CP^a	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.

Butanoate ester (EC4) volatiles identified in ‘Fuji’ apple fruits were the second most abundant esters aroma components (Figure 1), and included ethyl butanoate, pentyl butanoate, hexyl butanoate, butyl butanoate, propyl butanoate, 2-methylbutyl butanoate, (Z)-3-hexenyl butanoate, and (E)-2-hexenyl butanoate. EC4 volatiles contributed 8.9% of the total volatiles in ‘Fuji’ apples (Table 2). The content of EC4 was significantly (F=4.860, p < 0.001) different in 14 apple production regions. In all apple regions, the content of butanoic acid ester compounds produced in ‘Fuji’ apple samples were high in Gansu Tianshui and Shandong TaiYi mountainous areas, especially in Gansu Tianshui area. The lowest butanoic acid ester content was found in southern and eastern Shanxi (4.17%; Figure 1). Hexyl butanoate was the main volatile in all butanoic acid ester compounds. The hexyl butanoate content of apples in Qingshui Gansu (GS-QS), Qin'an Gansu (GS-QA), Qingcheng Gansu (GS-QC), and Jingning Gansu (GS-JN) was high (Table 3), while in apples of mid- and southern Shanxi and the Shandong Peninsula was low.

Nine types of 2-methyl butyrate ester (E2M) volatiles were found in ‘Fuji’ apples in different regions, including ethyl 2-methyl butanoate, hexenyl 2-methyl butyrate, pentyl 2-methyl butyrate, hexyl 2-methyl butyrate, butyl 2-methyl butanoate, butyl 2-methyl butanoate, propyl 2-methyl butyrate, 3-methyl butyl 2-methyl butyrate, and 2-methyl butyl 2-methyl butyrate. 2-Methyl butyrate ester volatiles contributed to 8.9% of the total volatiles (Table 2). The content of E2M in ‘Fuji’ apples was extremely significantly (F=3.873, p=0.002) higher in the Shaanxi Guanzhong area, southwest Liaoning, and the Yanshan and Taihang mountain areas than in other regions. The lowest content was found in apples collected on the Shandong Jiaodong Peninsula (Figure 1). Hexyl 2-methyl butyrate and butyl 2-methyl butanoate were common,and contributed 5.9% and 1.9% of the total volatiles, respectively (Table 2).

Five types of propanoate ester volatiles in ‘Fuji’ apples fruits were identified. The sum of propanoate esters accounted for less than 1% of the total volatiles (Figure 1). Other types of esters, such as hexyl 2-methyl propanoate, hexyl n-valerate, and 2-ethylhexyl salicylate, had a very low concentration. These esters was not discussed here in detail because of their low levels.

In apples, the majority of volatiles were esters (Defilippi et al., 2005; Knee & Hatfield, 1981; Paillard, 1979), the formation of which was dependent on the availability of C2–C8 acids and alcohols (Bangerth et al., 2012; Elss et al., 2006; Mehinagic et al., 2006). Ester production in fruit tissue is the result of esterification of alcohols, carboxylic acids, and acyl CoAs in an oxygen dependent reaction (Berger, Drawert & Nitz, 1983; Echeverrıa et al., 2004). In this study, the branched chain esters were high in ‘Fuji’ apples, 2-methyl butyl acetate and hexyl 2-methyl butyrate were found to be the major ester compounds in ‘Fuji’ apples, and they accounted for 32.6% and 5.9% of all volatiles in ‘Fuji’ apples, respectively. Heath and Reineccius (Heath & Reineccius, 1986) reported that branched chain alcohols, carbonyls, and esters are produced by the metabolism of amino acids. Iso-leucine was considered to be the biosynthetic precursor of 2-methyl butanoic acid and its esters in apples (Paillard, 1990). Our results indicated that the high ratios of amino acid conversion to volatiles, in particular the differential rates of metabolism of iso-leucine, occurred in ‘Fuji’ apples.

Alcohols Nine types of alcohols were found in ‘Fuji’ apples (Table 2), and they accounted for 0.01% – 2.1% of the total volatiles (Figure 1). There was extremely significant (F= 4.643, p < 0.001) difference in 14 apple production regions. In ‘Fuji’ apples from northern Shaanxi and Sanmenxia Henan the alcohols were more abundant than in other regions. Apples collected from Sanmenxia Henan had the highest alcohol content in apples from the 43 counties. In all of the ‘Fuji’ apple samples, 1-hexanol and 2-methyl-1-butanol were major components and accounted for more than 70% of total alcohol compounds. 1-hexanol was dominant with contents ranging from 0.7% to 4.5% of total volatiles, and was the highest in Mengyin Shandong (Table 3). The second most common alcohol was 2-methyl-1-butanol, which had a content of less than 1.0%.

Aldehydes Two aldehydes, hexanal and (E)-2-hexenal, were detected in ‘Fuji’ apples in 14 apple production province in China, and accounted for 1.8% of the total volatiles. The content of aldehydes was not significantly (F=0.679, p=0.785) different in 14 apple production regions. The content of hexanal was higher than (E)-2-hexenal. The sum of the aldehydes in the apples collected from Shaanxi Guanzhong had the highest aldehyde content of all tested apple production regions (Figure 1). Aldehydes were not detected in apples collected from southern and mid-Shanxi, the Shandong Peninsula, Changping Beijing, Malong Yunnan, and Fengxian Jiangsu.

Terpenoids α-Farnesene was the only terpenoid compound in the aroma profile of apples. The total content of α-farnesene accounted for 0.9% of the total volatiles. The content of α-farnesene was extremely significantly (F=3.604, p=0.002) different in 14 apple production regions. α-Farnesene in Liaoning was the highest out of all apple regions (Figure 1). There was no α-farnesene detected in apples collected from Shunping Hebei, Leting Hebei, Kuancheng Hebei, or Qinglong Hebei.

Other compounds We also detected two acids and seven other carbonyl compounds. The sum of the acids and other carbonyl compounds accounted, respectively, for 0.2% – 0.3% and 0.1% – 0.4%, of the total volatiles (Table 2). Acids and other carbonyl compounds are not discussed here in detail because of their low levels.

Principal component analysis The aroma composition and content in ‘Fuji’ apples varied in different regions. In order to study the principal sources of variation among our results and to understand the relationships between samples in each apple production region and the compound identified, the principal component analysis (PCA) was applied. The PCA scores scatter plot of the different apple growing regions is shown in Figure 2A, and the corresponding loading plot establishing the relative importance of the variables is shown in Figure 2B. The scores scatter plot (Figure 2A) shows that the first principal component (PC1-axis; explaining 49.0% of the variability in the data) is influenced by most of compounds. The second principal component (PC2-axis) only explains 12.2% of the variability in the data. The apple samples from different regions can be divided into four groups (Figure 2A) based on their position in the scores scatter plot.

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.

Group I: The cluster in the far lower left of the scatter plot is composed of apples from the six Shandong locations (SD-QX, SD-WD, SD-PL, SD-YS, SD-JN and SD-ZY), the three Shanxi locations (SX-RC, SX-LY and SX-WR), and one Gansu location (GS-NL). This clustering was likely due to high concentrations of hexyl acetate (EC₂-3, described as the complex aromas of sweet, fruity, flowery and pear), ethyl hexanoate (EC₆-2), and (Z)-3- hexenyl acetate (EC₂-10). These compounds make apples in this cluster the sweet tasting.

Group II: On the right of the PC1 axis and below the PC2 axis there is a cluster composed of apples from the five Shaanxi locations (SAX-BS, SAX-LC, SAX-AS, SAX-YC, and SAX-LQ), one Henan location (HN-SMX), two Liaoning locations (LN-WFD and LN-SZ), and three Hebei locations (HB-KC, HB-LT and HB-SP). The apples in this cluster were characterized by high contents of 2-methyl butyl acetate (EC₂-7, described as having fruity, sweet, flowery, and over all aroma,) and 1-hexanol (A-6, described as having the aromas of green apple and grass-liking). These compounds make apples in this cluster the best smelling.

Group III: On the right side of the PC1 axis and the top of the PC2 axis there is a cluster composed of apples from six Gansu locations (GS-TS, GS-QA, GS-JN, GS-LX, GS-QC and GS-QA) and three Liaoning locations (LN-SSL, LN-JC and LN-GZ). This cluster was characterized by high contents of hexyl butanoate (EC₄-3), butyl acetate (EC₂-4, described as the aromas of having fruity, sweet, and grassy), hexyl 2-methyl butyrate (E2M-4, described as having apple, grape fruit taste ), and butyl 2-methyl butanoate (E_2M-5, described as having fruity). These compounds make apples in this cluster the best fruity aroma.

Group IV: The last cluster is composed of apples from Yunnan (YN-ML), Jiangsu (JS-FX), Beijing (BJ-CP), Ningxia (NX-LW), three Sanxi locations (SX-YC, SX-WR, SX-LY), and one Liaoning location (LN-LY). In this group it was difficult to identify the dominant volatile and there was not characteristic composition for this group.

The composition and content of volatile compounds in ‘Fuji’ apples varied in different regions. This difference may be due to the climate, elevation, or geographic location, which may interfere with the activity of some flavoring enzymes. Some studies has reported that grape composition is related to many environmental factors, such as macro, meso and microclimate, soil, altitude, and topography (Styger, Prior & Bauer, 2011; Webb, Whetton & Barlow, 2008). In addition, it has been reported that altitude changes affect the chemical composition of black tea, with changes in temperatures and rainfall altering the taste, aroma, and potential health benefits of the beverage (Owuor, Obaga & Othieno, 1990). In this study, the rainfall, temperature and altitudes of the locations collected apple samples were different (table 1), the result showed that the composition and content of main volatile compounds in ‘Fuji’ apples in different locations were varied (table 3). Otherwise, annual precipitation and mean annual temperature in Ninlang Gansu were higher than those in six other Gansu locations. Ninlang Gansu and other six Gansu locations were not identified as the same group, which might be due to the difference of rainfall or temperature. These results indicated that geographic changes could affect the concentration of chemical compounds in ‘Fuji’ apple due to the difference of temperatures, rainfall or geographic location.

Correlation among major aroma volatile compounds, geographic location and main climatic factors It was found ten major volatile compounds in ‘Fuji’ apples in the experiment, including EC₂-7, EC₂-3, EC₂-11, E2M-4, EC₂-4, EC₄-3, EC₂-10, A-6, EC₆-2 and E2M-5. Moreover, correlation analysis of ten major aroma volatile compounds, geographic location and main climatic factors was done and shown in the table 4. It was found that mean annual temperature (MAT) was significant and extremely significant negatively correlated with EC₂-7 (p < 0.05) and E2M-5 (p < 0.01) respectively, and significant and extremely significant positively correlated with EC₂-3 (p < 0.01) and EC₂-11 (p < 0.05) respectively. Annual precipitation was not correlated with ten major volatile compounds. Longitude was significantly (p < 0.05) correlated with concentration of EC₂-4, EC₂-10, and EC₆-2. It was found that latitude showed significantly ( p < 0.05) negative correlation with EC₂-3 concentration, and no correlation with other major volatile compounds. At the same time, it was found that there were some significant correlation between the major compounds.

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

	AP^a	MAT^b	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

Conclusions

The volatile composition and content in ‘Fuji’ apples varied in different regions in China. Esters were the major volatile compounds in ‘Fuji’ apple aroma volatiles. 2-methyl butyl acetate and hexyl acetate contributed 32.5% and 24.5% to total volatile content, respectively. All of the ‘Fuji’ apple samples from 43 different counties could be classified into following groups: (1) apples with high concentrations of hexyl acetate and (Z)-3-hexenyl acetate, came from Shandong (Qixia, Wendeng, Penglai, Zhaoyuan, Jiaonan, and Yishui), Shanxi (Wanrong, Ruicheng, and Linyi), and Gansu Ninglang; (2) apples characterized by high contents of 2-methyl butyl acetate and 1-hexanol came from northern Shaanxi, Henan Sanmenxia, Liaoning Wafangdian, and Suizhong; (3) apples characterized by high contents of hexyl butanoate, butyl acetate, and hexyl 2-methyl butyrate came from Gansu (except Ninglang), and (4) a group that lacked any characteristic volatile composition. Moreover, correlation analysis of ten major aroma volatile compounds, geographic location and main climatic factors showed that mean annual temperature (MAT) was significant correlated with 2-methyl butyl acetate (EC2-7) and butyl 2-methyl butanoate (E2M-5), hexyl acetate (EC₂-3) and (E)-2-hexenyl acetate (EC₂-11). Longitude was significantly correlated with Butyl acetate (EC₂-4), (Z)-3-hexenyl acetate (EC₂-10), and ethyl hexanoate (EC₆-2). Latitude was correlation with hexyl acetate (EC₂-3). Annual precipitation was not correlated with ten major volatile compounds.

Acknowledgements This study was funded by the China Agriculture Research System (CARS-28).

References

Bangerth, F. K., Song, J., and Streif, J. (2012). Physiological impacts of fruit ripening and storage conditions on aroma volatile formation in apple and strawberry fruit: a review. HortScience, 47, 4-10.
Berger, R. G., Drawert, F., and Nitz, S. (1983). Sesquiterpene hydrocarbons in pineapple fruit. J. Agric. Food Chem., 31, 1237-1239.
Defilippi, B. G., Kader, A. A., and Dandekar, A. M. (2005). Apple aroma: alcohol acyltransferase, a rate limiting step for ester biosynthesis, is regulated by ethylene. Plant Sci., 168, 1199-1210.
Dirinck, P. and Schamp, N. (1988). Instrumental aroma analysis for objective evaluation of the parameters influencing aroma formation in apples and for prediction of the optimum picking date. In: International Symposium on Postharvest Handling of Fruit and Vegetables, 258, pp. 421-428.
Dixon, J. and Hewett, E. W. (2000). Factors affecting apple aroma/flavour volatile concentration: a review. New Zeal. J. Crop. Hort., 28, 155-173.
Echeverrıa, G., Graell, J., López, M., and Lara, I. (2004). Volatile production, quality and aroma-related enzyme activities during maturation of ‘Fuji'apples. Postharvest Biol. Tec., 31, 217-227.
Elss, S., Preston, C., Appel, M., Heckel, F., and Schreier, P. (2006). Influence of technological processing on apple aroma analysed by high resolution gas chromatography-mass spectrometry and on-line gas chromatography-combustion/pyrolysis-isotope ratio mass spectrometry. Food Chem., 98, 269-276.
Fallik, E., Archbold, D. D., Hamilton-Kemp, T. R., Loughrin, J. H., and Collins, R. W. (1997). Heat treatment temporarily inhibits aroma volatile compound emission from Golden Delicious apples. J. Agr. Food Chem., 45, 4038-4041.
Heath, H. B. and Reineccius, G. (1986). Flavor chemistry and technology. AVI Publishing Company.
Klesk, K., Qian, M., and Martin, R. R. (2004). Aroma extract dilution analysis of cv. Meeker (Rubus idaeus L.) red raspberries from Oregon and Washington. J. Agr. Food Chem., 52, 5155-5161.
Knee, M. and Hatfield, S. G. (1981). The metabolism of alcohols by apple fruit tissue. J. Sci. Food Agr., 32, 593-600.
López, M., Lavilla, M., Riba, M., and Vendrell, M. (1998). Comparison of volatile compounds in two seasons in apples: Golden Delicious and Granny Smith. J. Food Quality, 21, 155-166.
Matich, A. J., Rowan, D. D., and Banks, N. H. (1996). Solid phase microextraction for quantitative headspace sampling of apple volatiles. Anal. Chem., 68, 4114-4118.
Mehinagic, E., Royer, G., Symoneaux, R., Jourjon, F., and Prost, C. (2006). Characterization of odor-active volatiles in apples: influence of cultivars and maturity stage. J. Agr. Food Chem., 54, 2678-2687.
Mpelasoka, B. S. and Hossein Behboudian, M. (2002). Production of aroma volatiles in response to deficit irrigation and to crop load in relation to fruit maturity for ‘Braeburn'apple. Postharvest Biol. Tec., 24, 1-11.
Nielsen, T. J., Jägerstad, I., Öste, R. E., and Wesslén, B. O. (1992). Comparative absorption of low molecular aroma compounds into commonly used food packaging polymer films. J. Food Sci., 57, 490-492.
Owuor, P. O., Obaga, S. O., and Othieno, C. O. (1990). The effects of altitude on the chemical composition of black tea. J. Sci. Food Agr., 50, 9-17.
Paillard, N. (1979). Biosynthesis of organic volatile products from apple (pirus-malus l)-ketones synthesis and metabolic differences of varieties. Comptes Rendus Hebdomadaires Des Seances De L Academie Des Sciences Serie D, 288, 77-80.
Paillard, N. M. (1990). flavour of apples, pears and quinces. Developments in food science.
Plotto, A., McDaniel, M. R., and Mattheis, J. P. (1999). Characterization of Gala'Apple aroma and flavor: differences between controlled atmosphere and air storage. J. Am. Soc. Hortic. Sci., 124, 416-423.
Schaffer, R. J., Friel, E. N., Souleyre, E. J., Bolitho, K., Thodey, K., Ledger, S., Bowen, J. H., Ma, J.-H., Nain, B., and Cohen, D. (2007). A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosynthetic pathway. Plant Physiol., 144, 1899-1912.
Schumacher, K., Asche, S., Heil, M., Mittelstädt, F., Dietrich, H., and Mosandl, A. (1998). Methyl-branched flavor compounds in fresh and processed apples. J. Agr. Food Chem., 46, 4496-4500.
Song, J. and Bangerth, F. (1993). Production and development of volatile aroma compounds of apple fruits at different times of maturity. In: International Symposium on Postharvest Treatment of Horticultural Crops 368, pp. 150-159.
Song, J. and Bangerth, F. (2003). Fatty acids as precursors for aroma volatile biosynthesis in pre-climacteric and climacteric apple fruit. Postharvest Biol. Tec., 30, 113-121.
Styger, G., Prior, B., and Bauer, F. F. (2011). Wine flavor and aroma. J. Ind. Microbiol. Biot., 38, 1145-1159.
Webb, L., Whetton, P., and Barlow, E. (2008). Climate change and winegrape quality in Australia. Clim. Res., 36, 99.
Xiaobo, Z. and Jiewen, Z. (2008). Comparative analyses of apple aroma by a tin-oxide gas sensor array device and GC/MS. Food Chem., 107, 120-128.

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