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Article

Characterization of Changes in Ripening Process of Volatile Apple Compounds Based on HS-SPME-GC-MS Analysis

by
Jun Ma
1,
Xiaolong Li
1,
Yannan Chu
1,
Haiying Yue
1,
Zehua Xu
1,
Baiyun Li
1,
Xianyi Wu
2,
Jun Gan
3 and
Yonghua Jia
1,*
1
Horticultural Research Institute of Ningxia Academy of Agriculture and Forestry 1, Yinchuan 750002, China
2
People’s Government of Touzha Township, Shizuishan 753499, China
3
Ningxia Zhongwei Tailong Agricultural Science and Technology Co., Zhongwei 755000, China
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(10), 1787; https://doi.org/10.3390/agriculture14101787
Submission received: 23 July 2024 / Revised: 15 September 2024 / Accepted: 17 September 2024 / Published: 11 October 2024
(This article belongs to the Section Agricultural Product Quality and Safety)
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Abstract

:
The aim of this study was to identify the aromatic compounds present in different apple varieties and to gain insights into the changes in the aromatic compounds during ripening. Three apple varieties (“Red Astrachan”, “Ning Qiu”, and “Golden Delicious”) at different stages of ripening were selected for this study; their peel and pulp were analyzed via headspace solid-phase microextraction (HS-SPME)–gas chromatography–mass spectrometry (HS-SPME-GC-MS), and 30 volatile compounds were identified. The samples’ differences were analyzed using heat map cluster analysis, principal component analysis (PCA), and an independent samples t-test. The results showed that the content of aromatic compounds in the peel was higher than that in the pulp. The relative content of esters in the aromatic compounds of the three apple varieties followed the order of pulp > peel and “Ning Qiu” > “Golden Delicious” > “Red Astrachan”. This suggests that “Ning Qiu” combines the advantages of its parents in terms of its aroma content. The highest concentrations of aroma compounds in “Red Astrachan” and “Ning Qiu” accumulate before the ripening stage, and care should be taken to choose an appropriate harvesting time according to the different needs during production. The main compounds of “Red Astrachan” are aldehydes and C8 esters, while those of “Ning Qiu” and “Golden Delicious” are alkenes and esters. After analyzing the relative odor activity values (rOAVs) of key volatile compounds and their aroma descriptors during the harvest period, acetic acid pentyl ester, butanoic acid hexyl ester, hexanoic acid hexyl ester, and 2-ethyl-1-Hexanol were found to contribute the most to the overall flavor of the peel and pulp. “Ning Qiu” combines the parental advantages of the concentrated peel of “Red Astrachan” and the astringent pulp of “Golden Delicious”, with compounds in its composition that give a pleasing aroma. Mature “Ning Qiu” fruits have a more intense aroma and fruity flavor. The development of flavor-specific varieties has provided the theoretical basis for future research in molecular hybridization, molecular-assisted breeding, and the molecular biology of apple flavor synthesis and metabolism.

1. Introduction

With high nutritional value, strong ecological adaptability, high storage resistance, and a long supply cycle, apple is one of the world’s four major fruits and is favored by the majority of consumers. The apple aroma consists of a complex mixture of multiple volatile substances and is a key component in the composition of apples, mainly influenced by volatile compounds, concentrations, and odor thresholds. To date, more than 300 volatile compounds have been identified in apples [1]. Most of the volatiles are esters (70–90% of total volatiles), alcohols (5–15% of total volatiles), aldehydes, thetaenoids, and ketones [2]. However, the composition and concentrations of volatiles are influenced by many factors, including the variety [3], processing [4], the maturity stage [5], the climate [6], and terroir effects [7].
Golden Crown (Malus × domestica Borkh., “Golden Delicious”) is one of the world’s major apple varieties, known for its adaptability, abundance, stability, and high quality, and is well loved due to its golden yellow color and its sweet and sour taste when ripe [8]. Ningxia is a high-quality production area for Golden Crown in China, and the Golden Crown apples produced here are famous for their smooth fruit surface, bright golden color, lack of fruit rust, and fine, crispy, and juicy pulp [9]. “Ning Qiu” (for which Golden Crown is the mother and Red Kui is the father) has a strong floral aroma and sweet flavor. However, due to its tendency to fall off, its soft pulp, its intolerance to storage and transportation, and the tendency of mature fruits to rot, this variety has not yet been widely planted. Nonetheless, with the in-depth study of the synthesis and metabolism of the apple aroma, the research value of “Ning Qiu”, with its strong aroma, will become apparent. The development of selective breeding of apples as a horticultural crop began with the use of and improvement in local varieties by the local people. For example, the Volga variety Red Astrachan (first described in 1780) [10], known as “Hong Kui” since its introduction in the Ningxia region, usually ripens in mid- to late July and belongs to a family of early-ripening varieties. When fruit production shifted to specialized agriculture and the planting strategy shifted to staple plants, such as rice, vegetables, etc., it began to be neglected. At the same time, it was displaced by marketable mainstream apple varieties; thus, only a small amount of Red Astrachan exists and is cultivated by the population.
Aromatic compounds are present in the peel and pulp in both free and bound forms. The bound form is odorless but can be converted into odor-active compounds (free form) via hydrolysis during apple development [11]. Most of the volatile flavor components are produced after color change and prior to harvest [12]. “Ning Qiu” apples have a unique and strong flavor that is very useful for research and needs to be further explored. Due to the direct consumption of the fruit, either pre-prepared or processed into fermented beverages and canned foods, most studies have focused on the concentration of flavor compounds in apples during ripening [13,14], but few studies have investigated the uptake of flavor compounds in apples. Furthermore, the study of the synthesis and metabolism of flavor compounds during apple growth and development may help to elucidate the pathways and genes involved in the metabolism of flavor compounds in apples.
Headspace solid-phase microextraction (HS-SPME) is a technique that integrates extraction, sampling, concentration, and injection, and it is characterized by good yields, simple operation, and high efficiency [15]. Gas chromatography–mass spectrometry (GC-MS) is an effective method for the identification and quantification of volatile compounds and is usually used in combination with HS-SPME as an effective pretreatment process for the extraction of volatile organic compounds. Thus far, HS-SPME-GC-MS has been widely used for species identification and quality analysis of rice, fermented beverages, pulp, edible fungi, tobacco, and other fermented foods and agricultural products [16]. Combined with heat map cluster analysis and principal component analysis (PCA), the large amount of information generated by GC-MS has also been effectively utilized [17]. In addition, aroma thresholds and relative odor activity values (rOAVs) play a key role in assessing the contributions of aroma substances to the overall aroma [18].
In this experiment, the cultivars “Golden Delicious”, “Ning Qiu”, and “Hong Kui” were selected for the qualitative and quantitative analysis of the aroma components in apple growth and development. This provides a theoretical basis for a better understanding of the differences in quality and characteristics among apple varieties and has practical relevance for the selection of the best time for fruit harvest and the breeding of new varieties.

2. Materials and Methods

2.1. Materials

Samples of “Hong Kui”, “Ning Qiu”, and “Golden Delicious” apples were collected from the National Comprehensive Experimental Base for Apples (38°38′ N, 106°09′ E) of the Institute of Horticulture, Ningxia Academy of Agriculture and Forestry, Ningxia Province, China. This area has a temperate continental climate, with rainfall and average annual temperatures ranging from 200 mm to 220 mm and from 5 °C to 18 °C, respectively. The average temperature difference between day and night can reach 12–15 °C. Thirty apple trees with uniform growing conditions were selected. Randomly selected undamaged, disease- and pest-free apples were tested for the right-angle amyloid-like iodine index using the Blampied and Silsbee methods on a small portion (5 kg) of 15 kg of apples of each variety to identify apples at the same stage of maturity [15] and determine the ripening stage (green fruit stage S1, pre-transition S2, late transition S3, and ripening S4). After screening and sampling, the pulp and rind were separated and immediately frozen in liquid nitrogen and stored in a refrigerator at −80 °C before being set aside.

2.2. Fruit Quality

The fruits’ soluble solid content (TSS) was determined using a digital refractometer (model PAL-1, ATAGO, Tokyo, Japan). The single-fruit weight (METTLER TOLEDO, Columbus, OH, USA) was determined using a precision analytical balance (accuracy 0.001 g). The fruit longitudinal and transverse diameters were measured using electronic vernier calipers (accuracy of 0.001 mm) (Mitutoyo, Kawasaki, Japan). The ratio of fruit length to fruit width was calculated as follows: fruit shape index = fruit length/fruit width. Every 5 fruits were taken as 1 replication, 3 replications were created, and the average value was taken.

2.3. HS-SPME

Headspace solid-phase microextraction (HS-SPME) was used to extract aromatic compounds from the apples [16]. The apple peel or pulp stored at −80 °C was ground to a powder in liquid nitrogen using a mortar and pestle. We weighed 15 g of apple pulp or peel powder into a 50 mL centrifuge tube and added 0.5 g of D-(+)-glucono-1,5-lactone and 1 g of polyvinyl pyrrolidone (PVPP) to the tube. The tubes were left to macerate for 2 h and the clarified juice was obtained at 4 °C, after 10,000 r for 20 min. We then took a sample of the above clarified juice (100 mL), and a saturated sodium chloride solution (2 mL) and n-alkanes (C7–C21, Sigma-Aldrich, St. Louis, MO, USA) (10 μL) were mixed in 15 mL headspace vials. Meanwhile, 5 μL 2-octanol (diluted 200-fold in anhydrous ethanol) was used as the internal standard. Then, the headspace vial was sealed, and an activated 75 μm polydimethylsiloxane/carbon sieve/divinylbenzene (PDMS/CAR/DVB) extraction head (57328-u, Supelco, Stockbridge, GA, USA) was inserted into the headspace vial 1 cm above the liquid surface. The sample was placed in a magnetic stirring and heating station at 60 °C for 30 min, so as to allow the volatility of the volatile substances in the headspace vial to reach equilibrium. The extraction head was then removed from the vial and inserted into the GC injection port at a resolution temperature of 250 °C for a resolution duration of 5 min.

2.4. GC-MS

The carrier gas was high-purity helium (purity > 99.9%), the column was an Agilent HP-5MS 30 m × 250 μm (25 μm), and the flow rate was 1 mL/min. The heating program consisted of 55 °C as the initial temperature, which was held for 1 min, and then, the temperature was increased to 145 °C at a rate of 4 °C/min and then to 260 °C at a rate of 15 °C/min. The total running time of the GC process was 36 min. The auxiliary heater temperature was 250 °C. An electron bombardment ion source (EI) was used. The collision energy was 70 eV. The temperature of the ion source was 230 °C. The temperature of the MS quadrupole was 150 °C, and the scanning mass range was 22~500 amu.

2.5. Qualitative and Quantitative Analysis of Aromatic Compounds

Mass spectra of unknown compounds were identified using qualitative and semi-quantitative methods. For the qualitative analysis, the NIST Chemistry WebBook database was used to check the reference values of the retention indices (RIs), and the retention indices (RIs) of the compounds were calculated using C7~C27 n-alkane standards to accurately characterize the volatile compounds.
Retention index (RI) = 100n + 100 × (Tx − Tn)/(Tn + 1 − Tn), where n is the number of carbon atoms of n-alkanes; Tx, Tn, and Tn + 1 are the retention times/min of n-alkanes with carbon atoms of substance x, n, and n + 1, respectively.
The method of quantification was semi-quantitative, in which the peak area of each volatile component was divided by the peak areas of all volatile components measured and compared with the mass concentration of the internal standard. The formula was as follows: relative content of each compound (μg/kg) = peak area of volatile component to be measured/peak area of internal standard × mass concentration of internal standard (μg/L) × volume of internal standard (L)/volume of sample (kg).

2.6. Calculation of rOAVs

The rOAV was calculated using the following expression: rOAV = 100 × (Ci/Cstan) × (Tstan/Ti). Here, Ci and Ti are the relative content and odor threshold of the volatile compounds, respectively; Cstan and Tstan are the relative content of the compounds contributing the most to the flavor of the samples and the odor thresholds of the compounds contributing the most to the flavor of the samples, respectively. If rOAV > 1, it means that the substance is a key flavor compound and contributes more to the overall flavor; if 0.1 < rOAV ≤ 1, it means that the substance has a modifying effect on the overall flavor. In general, the larger the rOAV, the greater the contribution to the flavor [19,20].

2.7. Data Analysis

Statistical analyses were performed using IBM SPSS Statistics 27 (Armonk, NY, USA), and the data were verified for homogeneity using the Kolmogorov–Smirnov method prior to analysis. To determine significant differences between varieties and maturity stages, comparative analyses at the 5% level of significance (p < 0.05) were performed using the independent samples t-test. Then, principal component analysis and cluster analysis were used to determine the relationship between the aroma compound content and variety in order to identify the differences in the interaction between the variety and maturity stage. Then, using the radar chart area visualization function, the comprehensive rOAV evaluation value was used to assess the apple flavor. Finally, based on the above information, the results of the analysis were plotted using Origin 2021 (OriginLab Corp., Northampton, MA, USA).

3. Results

3.1. Fruit Quality

As shown in Figure 1, the fruit mass was in the order “Ning Qiu” (260 g) > “Hong Kui” > “Golden Delicious” (Figure 1A). The three varieties reached 12–14% total soluble solids at maturity (Figure 1B). This indicates that all three varieties are in the mature stage (TSS > 10%). The fruit length and fruit width values of “Ning Qiu” and “Hong Kui” were significantly larger than those of “Golden Delicious”. The indices of the fruit shape at harvest were similar for the three varieties (0.81–0.86) (Figure 1E).

3.2. Volatile Compound Content of Apple Variety

Changes in the volatile compounds in “Hong Kui”, “Ning Qiu”, and “Golden Delicious” during fruit development were found, as shown in Figure 2. The total aroma content of the peel of “Ning Qiu” peaked at S3 (late color change) (160,650 μg/kg) and decreased thereafter (Figure 2A). The aroma content of the “Golden Delicious” peel peaked at S2 (pre-transition stage) (125,938 μg/kg) and decreased thereafter, but exhibited a small peak at S4 (maturity stage) (118,230 μg/kg). The volatile content of the “Hong Kui” peel increased and then decreased with the fruit’s growth and development, reaching a peak (89,153 μg/kg) at S3 (late color change stage), and then decreased. The total aromatic content of the “Ning Qiu” pulp was significantly higher than that of “Hong Kui” and “Golden Delicious”, reaching a peak at S3 (123,024 μg/kg) and decreasing thereafter (Figure 2B). The total aroma content of the pulp of “Hong Kui” peaked at S3 (52,715 μg/kg) and decreased thereafter. The total aroma content of the “Golden Delicious” pulp reached a small peak at S2 (71,415 μg/kg) but peaked at S4 (85,544 μg/kg).

3.3. Percentage Content of Aroma Types in Apple Variety

The proportion of esters increased with fruit maturity (Figure S1), whereas the proportion of aldehydes tended to decrease gradually. (Figure S1A–C). The proportions of alcohols, ketones, and terpenes fluctuated throughout development. The proportions of ester aromas in the peel of the early-ripening red variety “Hong Kui” (21.55–43.41%) and the early–medium-ripening red variety “Ning Qiu” (18.38–52.26%), as well as in the peel of the medium–late-ripening yellow variety “Golden Delicious” (14.13–30.8%), are shown in Figure S1A–C. Among the esters, the compounds in the pulp of “Hong Kui” (19.81–58.81%), “Golden Delicious” (17.46–39.29%), and “Ning Qiu” (9.31–65.75%) are shown in Figure S1D,E. The proportion of esters in the pulp of “Ning Qiu” reached the maximum value (65.75%) in the S3 period and decreased thereafter.

3.4. Changes in Content of Volatile Compounds in Apple Variety

There were 30 volatile compounds identified in the fruits of “Hong Kui”, “Ning Qiu”, and “Golden Delicious”, including 5 C6-type (alcohol, ester, aldehyde) compounds, 12 esters (except C6 type), 2 alcohols (except C6 type), 6 aldehydes, 1 acid, 3 thetenes, and 1 ketone.
Some volatile compounds in “Ning Qiu” gradually accumulated in the pericarp as the ripening stage increases (Figure 3), such as esters (2-methyl-1-butanol acetate, (E,E)-2,4-hexadienal hexanoic acid hexyl ester), aldehydes (benzaldehyde, octanal, (E)-3,7-dimethyl-2,6-octadienal), and postene (α-farnesene). In addition, other aromatic compounds showed a general trend of increasing and then decreasing in anabolism, especially from the S2 period onwards. The anabolism of the aromatic compounds during the maturation of “Golden Delicious” showed a general “N”-shaped trend, where 6-methyl-5-hepten-2-one, octanal, propanoic acid hexyl ester, naphthalene, isopentyl hexanoate, (E)-3,7-dimethyl-2,6-octadienal, and hexanoic acid hexyl ester were found at high levels during the fruit’s growth and development. In “Hong Kui”, aldehydes ((E)-2-hexenal, (E)-2-octenal) and esters (acetic acid pentyl ester, butanoic acid butyl ester, acetic acid hexyl ester) peaked at S3, and butanoic acid and 2-methyl-, heptyl ester also peaked during the S3 period. In addition, most of the other 24 compounds varied at low levels throughout the growth and development stages.
The trend of the aromatic compounds in the pulp of “Golden Delicious” was similar to that in its peel; most of them showed an “N” trend and increased with ripening, especially the aldehydes (Figure 4). Some of the volatiles in the peel of “Ning Qiu” reached their peaks at the late stage of color transfer (S3), such as heptanal, acetic acid pentyl ester, benzaldehyde, 6-methyl-5-hepten-2-one, butanoic acid butyl ester, benzeneacetaldehyde, propanoic acid hexyl ester, heptyl ester 2-methyl-butanoic acid, and hexanoic acid hexyl ester. The majority of the remaining volatiles peaked at fruit ripening (S4). As the “Hong Kui” matured, the concentration of esters increased, but the concentration of alcohols and aldehydes decreased, which occurred mainly prior to color change (S2) and included compounds such as aldehydes (hexanal, (E)-2-hexenal, benzaldehyde, octanal, benzeneacetaldehyde) and alcohols ((s)-2,5-dimethyl-2-hexanol).

3.5. Principal Component Analysis (PCA) of Aromas of Three Varieties

Volatile compounds in the skin and flesh of three apple varieties at different stages of ripening were analyzed using principal component analysis (PCA), as shown in Figure 5. The results showed that there were significant differences in the characteristic volatile compounds in the peel and pulp of different apple varieties at different stages of ripening. The representative compounds of different apple varieties at different maturity stages were also different. Among the pericarp, the representative aromatic compounds of the “Hong Kui” variety were (E)-2-hexenal and (E)-3,7-dimethyl-2,6-octadienal; terpenes and esters were the most important aromatic compounds in the “Ning Qiu” variety. Terpenes and esters are representative aromatic compounds of the “Ning Qiu” variety, including α-farnesene, butanoic acid butyl ester, and acetic acid pentyl ester; thetenes, aldehydes, and esters are the main representative aromatic compounds of the “Golden Delicious” variety. The main representative aromatic compounds of the “Golden Delicious” variety also include hexanal, (E)-2-hexenal, and naphthalene. The aromatic characteristics of the pulp of these three varieties were similar to those of the peel before color change, and the main representative aromatic compounds of the “Ning Qiu” variety are 1-hexanal, (E)-2-hexenal, naphthalene, etc. After color change, the main aromatic compounds in “Ning Qiu” were 1-hexanol, acetic acid butyl ester, butanoic acid butyl ester, α-farnesene, and 6-methyl-5-hepten-2-one; esters and aldehydes were the main compounds in the “Golden Delicious” variety.

3.6. Cluster Analysis of Aromatic Components of Different Varieties and Maturation Processes

The aromatic compounds and their relative content in the different varieties at different stages of growth and development were analyzed using cluster analysis, as shown in Figure 6. The aromatic compounds of the “Ning Qiu” peel after color transfer were similar during S3 and S4 (Figure 6A), mainly consisting of esters and theta-ene aromatic compounds, especially α-farnesene, acetic acid butyl ester, and butanoic acid hexyl ester. The aromatic compounds in the “Hong Kui” peel were also similar during the S3 and S4 stages, mainly consisting of aldehydes and esters. They were similar to the compounds of the “Golden Delicious” peel at S3, especially (E)-2-octenal and butanoic acid butyl ester. The compounds in the rind of “Golden Delicious” were also similar during the S2 and S4 phases, with benzeneacetic acid ethyl ester, (Z)-butanoic acid 3-hexenyl ester, (E)-2-hexenal, naphthalene, and acetic acid butyl ester being the major differential compounds. In terms of changes in the content of the aromatic compounds, the three aldehydes, hexanal, (E)-2-hexenal, and benzeneacetaldehyde, were similar, and the esters and alcohols were close to each other, with 1-hexanol and butanoic acid 2-methylbutyl ester being the main ones. The variations in terpenes and ketones, including naphthalene, 6-methyl-5-hepten-2-one, and α-farnesene, were consistent.
In the pulp, the “Ning Qiu” aroma compounds at the S3 and S4 stages clustered together (Figure 6B). “Hong Kui” is similar to the “Golden Delicious” apple at the S3 stage, they are clustered together at S1 (green fruit stage), with similar aromatic compounds. At S2 and S4, the pulp aromas of the three varieties varied considerably. As with the peel, the variations in hexanal and (E)-2-hexenal were similar in the pulp.

3.7. rOAV Analysis of Key Volatile Compounds in Fruits of Different Varieties at Maturity Stage

Figure 7 shows the rOAV data of 16 volatile aroma (rOAV > 0.1) compounds screened from 30 major volatile compounds, where acetic acid pentyl ester, butanoic acid hexyl ester, hexanoic acid hexyl ester, and 2-ethyl-1-Hexanol were found to contribute the most to the overall flavor of peel and pulp. Except for acetic acid pentyl ester, butanoic acid hexyl ester, hexanoic acid hexyl ester, and hexanal, the rOAVs of all of the compounds in the peel and pulp of “Hong Kui” were basically lower than those of “Ning Qiu” and “Golden Delicious”, and this difference might be the reason why the aroma of “Hongkui” was not obvious.
The compounds with an rOAV > 1 were the major flavor compounds that contributed more to the overall flavor, as shown in Table 1; the major flavor compounds in the peels of “Hong Kui”, “Ning Qiu”, and “Golden Delicious” had an rOAV of 5, 4, and 5 in order, and the number of major flavor compounds in the pulp was 2, 4, and 5 in order, as can be seen from the number of major flavor compounds. The decrease in the number of major flavor compounds in the pulp of “Hong Kui” indicated that the compounds contributing more to the overall flavor of “Hong Kui” were concentrated in the peel.
In addition, focusing on the four main flavor compounds, acetic acid pentyl ester, butanoic acid hexyl ester, hexanoic acid hexyl ester, and 2-ethyl-1-Hexanol (Figure 7), it is noteworthy that the “Hong Kui” and “Ning Qiu” pericarp had much higher contents of compounds with pleasant aromas, such as acetic acid pentyl ester (Fruity) and butanoic acid hexyl ester (Fruity), than those of “Golden Delicious”. However, the rOAVs of butanoic acid hexyl ester and hexanoic acid hexyl ester (fatty wax flavor) compounds in the pulp of “Ning Qiu” were higher, whereas the rOAVs of “Hong Kui” and “Golden Delicious” had lower rOAVs in the fruit pulp. The rOAVs of 2-ethyl-1-Hexano (floral aroma) compounds in the pulp of “Ning Qiu” remained high, while the rOAVs in the pulp of “Golden Delicious” were also high. In addition, butanoic acid hexyl ester, hexanoic acid hexyl ester, and 2-ethyl-1-Hexanol compounds in “Hong Kui ‘’ all had very low levels of odor activity in the fruit pulp and low contributions. This indicates that “Ning Qiu” combines the advantages of its parents in the composition of compounds with a pleasant aroma. As noted above, “Ning Qiu” has the advantage of containing compounds that embody the pleasing aromas of the pulp and peel of “Hong Kui” and “Golden Delicious”.

4. Discussion

The aroma is an important indicator of an apple’s flavor and quality, and it is a characteristic substance for quality diversification. The cultivation of new varieties with high quality and a strong aroma will become one of the major directions of apple breeding in the future [21], and screening and breeding apple varieties with different aromas and flavors are of great significance for the development of the apple industry. Research on the genetic patterns of the apple aroma will provide a theoretical basis for apple breeding to efficiently select and breed new apple varieties. Meanwhile, among the various fruit parts, the peel is where the aroma is more easily detected [22,23]. Thus, the study and analysis of the aroma components of different parts of the apple (peel and pulp) are of guiding significance for the understanding of the apple aroma system and the comprehensive development and utilization of waste resources.

4.1. Aroma Characterization

The aroma of apples is characterized as aromatic, fruity, herbaceous, floral, and sweet [24,25]. Fruits can produce compounds that give them a distinctive aroma as they grow [26], and the various types of volatile compounds and their amounts in different varieties of apples produce different aromas. However, during apple ripening, the concentration of aldehydes decreases, while esters and alcohols become the main volatiles and their concentrations increase [27]. In addition, the apple variety is another key factor that has a strong influence on the volatiles, as studies on different varieties have shown many differences in the volatile composition and concentrations [28]. In this study, the aromatic compounds were more abundant in the peel than in the pulp of the three apple varieties (Figure 2), and the proportion of higher esters increased with fruit development (Figure 3), while the proportion of aldehydes gradually decreased, e.g., aldehydes dominated in immature apples. After the late color change (S3), “Ning Qiu” had a higher content of esters and thalloids than “Hong Kui” and “Golden Delicious”. The next representative aromatic compounds of the “Hong Kui” variety are E-2-hexenal and E-3,7-dimethyl-2,6-octadienal. The “Ning Qiu” variety carries epitaxial aromatic compounds such as α-farnesene, butanoic acid butyl ester, and acetic acid pentyl ester. The “Golden Delicious” variety mainly contains representative aromatic compounds such as hexanal, E-2-hexenal, and naphthalene. Esters are important contributors to the fruit aroma and are usually synthesized through fatty acid metabolism; they have a low threshold and usually produce pleasant odors [29]. Aldehydes gradually become less abundant during ripening [30]. C6 compounds and aldehydes are produced primarily through fatty acid metabolism, and key precursors include linolenic and linoleic acids, which contribute primarily to green aromas [31]. They are also key green leaf volatiles (GLVs) [32]. For example, the precursor substance of hexanal and 1-hexanol is linoleic acid. Hexanal is initially formed via the lipoxygenase (LOX) pathway, followed by the production of hexanol by alcohol dehydrogenase (ADH) [33]. Moreover, hexanal can be generated from linolenic acid to E-2-hexenal [34]. Since the concentrations of 1-hexanol, hexanal, and E-2-hexenal were high in this study, the precursor content of linoleic and linolenic acids in these three varieties may also exhibit high levels for the same reason. Ethyl 2-methylbutanoate and 2-methyl-1-butanol can be formed via the transamination and decarboxylation of leucine and isoleucine, respectively [35]. It has also been reported that the ascorbic acid content of apples is one of the factors that may lead to an increase in the concentrations of hexanal, E-2-hexenal, and E-2-hexen-1-ol [36]. Meanwhile, the aldehyde content is higher in fruits with more acidic and yellow peels during ripening [37], which may explain the higher aldehyde content in the early-ripening type “Hong Kui” and the yellow peel type “Golden Delicious” [38]. In addition, as an acyclic sesquiterpene, farnesene is an intermediate in the synthesis of vitamins E and K1 and is the most abundant sesquiterpene [38]. α-Farnesene further characterizes the “Ning Qiu” variety.
Naphthalene has been reported to be present in apples, cider, mangoes, and pears [39,40,41,42]. Coincidentally, Zhu et al. [43] found naphthalene in apples, and there may be several reasons for its presence. Naphthalene-derived compounds, such as 1-naphthalene acetic acid, are commonly used as plant growth regulators to improve the yields of fruits (e.g., apples, pears, and grapes) [39]. Moreover, some authors have suggested that naphthalene may be produced via the thermal degradation of β-carotene [44], which could be related to the unique high light and diurnal temperature conditions in the area of origin where the present experiment was conducted [45].

4.2. Changes in Volatile Compounds during Apple Ripening and Choice of Harvest Time

The green fruit stage (S1 ripening stage) of “Ning Qiu” has a low content of volatile compounds, mainly aldehydes. From the color change stage to the ripening stage, aromatic compounds gradually accumulated, mainly including esters and terpenoids. The pattern of aromatic compounds in the pulp of “Golden Delicious” is similar to that in its peel; most of them showed an “N” trend with increasing ripeness, especially the aldehydes. Among the three apple varieties, the content of aromatic compounds in the peel was higher than that in the pulp, and studies have shown that volatile metabolizing enzymes with high activity and content in the peel have a greater ability to synthesize aroma volatiles [46].
In this experiment, the aroma content of “Hong Kui” and “Ning Qiu” reached the maximum before harvest (Figure 2), and the “Ning Qiu” aroma compounds in stages S3 and S4 were clustered together (Figure 6). The aromatic compounds of “Hong Kui” at S1 (green fruit stage) were similar to those of “Golden Delicious” at S3 (late color change stage). The aromatic compounds of “Golden Delicious” apples showed an “N” trend with increasing maturity (Figure 4 and Figure 5). In the production process, it is possible to choose the appropriate harvesting period for different uses.

4.3. Evaluation of Aromatic Compounds’ rOAVs in Different Apple Varieties at Harvest

Humans perceive fruit odor through the interaction between the volatile compounds released by the fruit and the G-protein-bound odor receptors (ORs) in the olfactory epithelium of the nose. However, not all volatile compounds produce an odor, although the concentrations of volatile compounds may reflect their contributions [47,48]. Olfactory thresholds must also be taken into account. The rOAV is the ratio of the concentration of volatile compounds in a fruit to the olfactory threshold of the volatile compound and is used to assess the contribution of the compound to the overall flavor of the fruit. A higher rOAV indicates that the compound contributes more to the flavor of the fruit; this parameter is widely used for fruits of the Rosaceae family [49]. The rOAV of the volatile aroma compounds was calculated to determine which compounds play a crucial role in modulating the overall flavor of the fruit at different stages of ripening in apple peel and pulp. In the present study, the rOAVs for different compounds in the pulp and peel were obtained in mature fruits from different varieties. Hexanoic acid hexyl ester, benzaldehyde, and butanoic acid hexyl ester were identified as the key compounds affecting the overall aromas of the three varieties. Among the compounds with a pleasant aroma, those in “Hong Kui” were concentrated in the peel, those in “Golden Delicious” were concentrated in the pulp, and “Ning Qiu” combined the advantages of both parents (“Hong Kui” and “Golden Delicious”). The reason for this is the high or low contribution of 2-ethyl-1-Hexanol (aromatic), butanoic acid hexyl ester (fruity), acetic acid pentyl ester (fruity), and hexanoic acid hexyl ester (fatty wax aroma) in the pulp and peel to the odor. In addition to the four key volatile aroma compounds mentioned above, the 2-methyl-1-Butanol acetate, 2,4-di-tert-butylphenol key compound (rOAV > 1) was also present, a finding that suggests that certain compounds can only affect the flavor of “Ning Qiu”, and they are essential compounds in “Ning Qiu”, which may be the reason for its more pronounced odor.

4.4. Research Value of Aroma of “Ning Qiu”

Since the early days of breeding, the cost and complexity of olfactory phenotyping research has been a major concern [50]. The process of breeding requires the improvement in existing germplasm resources through the collection and conservation of abundant landraces, wild species, and wild relatives. “Ning Qiu” has a sweet taste, a distinct strawberry aroma, and excellent quality. Compared with its sister variety, “Ning Guan”, “Ning Qiu” is more resistant to drought and salt and has a richer flavor [28].
Currently, there are few studies on the synthesis of aromas in “Ning Qiu” apples. In this study, the total aroma content in the peel and pulp of “Ning Qiu” was higher than that of “Hong Kui” and “Golden Delicious”. The relative contents of esters in volatile compounds of different apple varieties were in the order of pulp > peel and “Ning Qiu” > “Golden Delicious” > “Hong Kui”. “Ning Qiu” has high value for flavor research, and the fruit aroma serves an important function in horticulture, being appealing to humans. In fact, horticultural breeding has adopted the selection of fruit aromas to improve the eating quality and economic value of fruits, and this should be further studied in the future for apple production and the cultivation of new varieties [51].
The composition of apple flavor is therefore very complex and many factors influence the formation and accumulation of apple flavorings. Among these factors, genotypic differences are the main causes of the variations in the types and amounts of flavor substances. In addition, there are also differences in flavor metabolism in apples with different flavors, which need further research. Advances in genomics, proteomics, and metabolomics have made it possible to study in detail the specific metabolic pathways and key genes involved in apple flavor metabolism.

5. Conclusions

In this study, 30 volatile compounds were identified and quantified in the peel and pulp of three apple varieties at four stages of maturity. The volatiles showed different results in the four periods. The PCA results revealed similarities and differences between the different maturity stages of the apple varieties and in the peel and pulp, but the extent of differentiation was low. The clustering results showed better differentiation based on the volatile characteristics of the apple varieties at different stages of ripening. Aldehydes such as hexanal and benzeneacetaldehyde, esters and alcohols such as 1-hexanol and butanoic acid 2-methylbutyl ester, and terpenes and ketones such as naphthalene, 6-methyl-5-hepten-2-one, and α-farnesene were important compounds. Considering the odor thresholds, the most critical odor compounds characterized in the apples were acetic acid pentyl ester, butanoic acid hexyl ester, hexanoic acid hexyl ester, and 2-ethyl-1-Hexanol. In addition, there were differences in the volatile concentrations and rOAVs among the apple varieties at the four stages of maturity, which implies that geoclimatic factors have a significant influence on the volatility formation of certain varieties.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14101787/s1, Table S1: Concentration of apple volatiles in peel (μg/kg); Table S2: Concentration of apple volatiles in pulp (μg/kg); Figure S1: Proportion of various volatile aromatic compounds.

Author Contributions

Conceptualization, J.M., X.L. and Y.J.; methodology, Y.C. and J.M.; validation, H.Y. and Z.X.; formal analysis, B.L.; investigation, X.W. and J.G.; resources, Z.X.; writing—preparation for first draft, J.M.; writing—comments and revisions, Y.J., X.L. and Z.X.; funding acquisition, X.L. and Y.J. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Yinchuan Apple Comprehensive Experimental Station of Modern Apple Industry Technology System of the Ministry of Agriculture of China (CARS-27), the Central Finance Forestry Science and Technology Promotion and Demonstration Funding Program (2023ZY06), and the Ningxia Academy of Agriculture and Forestry’s “Fourteenth Five-Year Plan” Demonstration Project of Science and Technology Innovation for High-Quality Agricultural Development and Ecological Protection (NGSB-2021-1-01) funds.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data supporting the results of this study are included in this manuscript and its Supplementary Information File. Also, the datasets used and/or analyzed in this study are available upon request from the corresponding author.

Conflicts of Interest

Jun Gan was employed by the Company Ningxia Zhongwei Tailong Agricultural Science and Technology Co. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Agriculture 14 01787 g001
Figure 1. Fruit quality of “Hong Kui”, “Ning Qiu”, and “Golden Delicious”. (A) Single-fruit weight, (B) soluble solids, (C) fruit length, (D) fruit width, and (E) fruit shape index. Based on t-test between “Hong Kui”, “Ning Qiu”, and “Golden Delicious” at maturity. Different lowercase letters at the same maturity stage indicate significant differences between varieties (p < 0.05).
Figure 1. Fruit quality of “Hong Kui”, “Ning Qiu”, and “Golden Delicious”. (A) Single-fruit weight, (B) soluble solids, (C) fruit length, (D) fruit width, and (E) fruit shape index. Based on t-test between “Hong Kui”, “Ning Qiu”, and “Golden Delicious” at maturity. Different lowercase letters at the same maturity stage indicate significant differences between varieties (p < 0.05).
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Figure 2. Total content of volatile compounds during ripening of “Hong Kui’”, “Ning Qiu”, and “Golden Delicious”. Total content: (A) peel; (B) pulp. n = 3. Based on t-test between “Hong Kui”, “Ning Qiu”, and “Golden Delicious” at maturity (S4). Letters at harvest represent time of fertility of three varieties.
Figure 2. Total content of volatile compounds during ripening of “Hong Kui’”, “Ning Qiu”, and “Golden Delicious”. Total content: (A) peel; (B) pulp. n = 3. Based on t-test between “Hong Kui”, “Ning Qiu”, and “Golden Delicious” at maturity (S4). Letters at harvest represent time of fertility of three varieties.
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Figure 3. Concentrations of volatile compounds in the peel of “Hong Kui”, “Ning Qiu”, and “Golden Delicious” at different stages of ripening. n = 3. Different lowercase letters at the same maturity stage indicate significant differences between varieties (p < 0.05).
Figure 3. Concentrations of volatile compounds in the peel of “Hong Kui”, “Ning Qiu”, and “Golden Delicious” at different stages of ripening. n = 3. Different lowercase letters at the same maturity stage indicate significant differences between varieties (p < 0.05).
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Figure 4. Concentrations of volatile compounds in the pulp of “Hong Kui”, “Ning Qiu”, and “Golden Delicious” at different stages of ripening. n = 3. Different lowercase letters at the same maturity stage indicate significant differences between varieties (p < 0.05).
Figure 4. Concentrations of volatile compounds in the pulp of “Hong Kui”, “Ning Qiu”, and “Golden Delicious” at different stages of ripening. n = 3. Different lowercase letters at the same maturity stage indicate significant differences between varieties (p < 0.05).
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Figure 5. PCA of various volatile aromatic compounds in peel and pulp of “Hong Kui”, “Ning Qiu”, and “Golden Delicious” during ripening and maturation. (A) Peel; (B) pulp.
Figure 5. PCA of various volatile aromatic compounds in peel and pulp of “Hong Kui”, “Ning Qiu”, and “Golden Delicious” during ripening and maturation. (A) Peel; (B) pulp.
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Figure 6. Cluster analysis of aromatic compounds at different stages of maturity in three varieties. (A) Peel; (B) pulp. Normalization of relative content data.
Figure 6. Cluster analysis of aromatic compounds at different stages of maturity in three varieties. (A) Peel; (B) pulp. Normalization of relative content data.
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Figure 7. rOAV radar plots of the volatile aroma compounds at the ripening stage in the three varieties. (A) Peel; (B) pulp.
Figure 7. rOAV radar plots of the volatile aroma compounds at the ripening stage in the three varieties. (A) Peel; (B) pulp.
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Table 1. rOAV used for volatile aroma compounds in fruits of different varieties at harvest time.
Table 1. rOAV used for volatile aroma compounds in fruits of different varieties at harvest time.
Structure TypeCompound NameTa (mg/kg) ArOAVOdor Description B
PeelPulp
Hong KuiNing QiuGolden
Delicious		Hong KuiNing QiuGolden
Delicious
C6Hexanal0.230.125 0.268 0.231 3.594 0.774 0.778 Herbal, green, grass, fruity
C6Acetic acid butyl ester0.461.611 0.184 0.547 0.693 0.926 0.619 Fruity, fruity, banana
C61-Hexanol0.034<0.1 <0.1 <0.1 0.129 0.260 0.111 Green; herbaceous; woody; sweet
C72-methyl-1-Butanol acetate0.140.709 1.084 0.136 0.768 1.681 0.547 Estery, fruity, banana, pear, sweet
C7Heptanal0.260.250 <0.1 0.237 <0.1<0.1 <0.1 Aromatic, nutty, almond flavor
C7Acetic acid pentyl ester2.27.555 15.338 1.476 1.061 0.497 0.856 Ethereal, fresh–fruity, reminiscent of pear, banana, and apple
C7Benzaldehyde0.0850.309 0.143 0.125 <0.1<0.1 0.871 Aromatic, nutty, almond flavor
C86-methyl-5-Hepten-2-one0.50.312 1.271 1.679 0.259 0.479 2.946 Phenolic aroma, moldy aroma, ketone aroma
C8Butanoic acid butyl ester0.0280.147 <0.1 <0.1 <0.1 <0.1 0.102 Fruity, banana, pineapple
C8Octanal0.17<0.1 <0.1 <0.1<0.1 <0.10.301 Fruity, green, herbal, diffused
C82-ethyl-1-Hexanol0.8<0.10.205 2.817 <0.1 0.237 7.855 Citrus, fresh, floral, oily, sweet
C10Naphthalene0.450.322 0.302 0.602 0.065 <0.1 0.355 Pungent, dry, tarry
C10Butanoic acid hexyl ester528.817 41.480 5.527 1.509 34.717 3.715 Fruity, pineapple, strawberry
C11Isopentyl hexanoate0.321.220 0.108 <0.1 0.996 0.928 1.893 Fruity, sweet, pineapple, a slightly pungent sour cheesy note
C12Hexanoic acid hexyl ester6.423.116 11.872 13.729 0.562 47.005 7.842 Fatty wax flavor, soybean flavor, fruit flavor
C142,4-Di-tert-butylphenol0.50.144 0.329 0.320 <0.1 1.501 0.865 Pungent, dry resin scent
A: Ta, odor thresholds—Compilation of Data Representing Odor Thresholds for Air, Water, and Other Media (Compendium of Olfactory Thresholds for Compounds, Expansion and Revision of the Original Book, Second Edition). Beijing: Science Press. 2018. B: Odor descriptions adapted from http://www.thegoodscentscompany.com (accessed on 13 September 2024).
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Ma, J.; Li, X.; Chu, Y.; Yue, H.; Xu, Z.; Li, B.; Wu, X.; Gan, J.; Jia, Y. Characterization of Changes in Ripening Process of Volatile Apple Compounds Based on HS-SPME-GC-MS Analysis. Agriculture 2024, 14, 1787. https://doi.org/10.3390/agriculture14101787

AMA Style

Ma J, Li X, Chu Y, Yue H, Xu Z, Li B, Wu X, Gan J, Jia Y. Characterization of Changes in Ripening Process of Volatile Apple Compounds Based on HS-SPME-GC-MS Analysis. Agriculture. 2024; 14(10):1787. https://doi.org/10.3390/agriculture14101787

Chicago/Turabian Style

Ma, Jun, Xiaolong Li, Yannan Chu, Haiying Yue, Zehua Xu, Baiyun Li, Xianyi Wu, Jun Gan, and Yonghua Jia. 2024. "Characterization of Changes in Ripening Process of Volatile Apple Compounds Based on HS-SPME-GC-MS Analysis" Agriculture 14, no. 10: 1787. https://doi.org/10.3390/agriculture14101787

APA Style

Ma, J., Li, X., Chu, Y., Yue, H., Xu, Z., Li, B., Wu, X., Gan, J., & Jia, Y. (2024). Characterization of Changes in Ripening Process of Volatile Apple Compounds Based on HS-SPME-GC-MS Analysis. Agriculture, 14(10), 1787. https://doi.org/10.3390/agriculture14101787

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