Analyzing Volatile Compounds of Young and Mature Docynia delavayi Fruit by HS-SPME-GC-MS and rOAV

This study focused on the examination of the volatile compounds and fragrance properties of the young and mature fruit of Docynia delavayi. Headspace solid-phase microextraction combined with gas chromatography–mass spectrometry (HS-SPME-GC-MS) was applied for identifying 42 volatile compounds, with young and mature fruit containing 36 and 42 compounds, respectively. Heat map cluster analysis, principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA), and independent sample t-testing were used to analyze sample differences. Based on a variable importance in projection (VIP) > 1 and p < 0.05, 23 key volatile compounds such as octanal, geranylacetone, butyl acetate, and dihydro-β-ionone were screened. β-Ionone and phenethyl acetate made the largest contribution to the aroma of D. delavayi after analyzing the relative odor activity value (rOAV) of the key volatile compounds and their aroma descriptors. Young D. delavayi fruit exhibited a prominent woody scent, while mature D. delavayi fruit had more intense floral and rosy aromas. The findings may lay a foundation for comprehensively developing and utilizing D. delavayi fruit.


Introduction
Docynia delavayi is a plant of the Docynia genus in the Rosaceae family and a plant resource with medicinal food value that is highly distributed in southwest China [1]. D. delavayi fruit is rich in a variety of nutrients and polyphenolic compounds that improve appetite, prevent oxidation, and lower blood glucose [2]. The fruit can be directly eaten, preserved, or processed into fruit wine and jam, among others; it is a wild fruit with great development potential [3]. While local and international studies into D. delavayi have mainly focused on breeding and genetic diversity [4], functional analyses of secondary metabolite bioactivities [5], coloring mechanisms [6], processing, and applications [7], studies on aroma have been scarce thus far.
Fruit aroma critically indicates fruit quality and plant breeding, and vitally impacts fruit production and processing applications [8,9]. Aldehydes, esters, terpenoids, and other volatile compounds released by fruits contribute to the production of fruit aromas. The types and quantities of these compounds depend on the fruit variety, maturity, and environmental conditions [10][11][12]. Therefore, analyzing the volatile compounds and aroma characteristics of D. delavayi fruit at various stages of maturity is expected to provide an understanding of the volatile compounds in D. delavayi fruit and their aromas.
Headspace solid-phase microextraction (HS-SPME) is a common extraction method that adsorbs volatile compounds using silica salt fibers coated with sorbents, which not only reduces sample loss but also reduces analysis time and cost [13]. HS-SPME combined with gas chromatography-mass spectrometry (GC-MS) is a convenient, highly sensitive, solventfree, and non-toxic method commonly used for the analysis of volatile compounds in fruits of the Rosaceae family [14,15]. GC-MS can produce a large amount of data. Therefore, the information in a large data set can effectively be exploited by combining heat map cluster analysis with principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) [16,17]. In addition, aroma thresholds and the relative odor activity value (rOAV) play key roles in assessing the contribution of the aroma substance to the overall aroma [18].
Herein, HS-SPME-GC-MS in combination with OPLS-DA was employed for the investigation of the key volatile compounds that differentiate young and mature D. delavayi fruit. Moreover, key volatile flavor compounds were identified using odor-threshold and rOAV methods, and the differences in the aromas of young and mature fruit were compared to offer a research basis for comprehensively evaluating D. delavayi fruit quality and utilization.

D. delavayi Samples
D. delavayi fruit was collected from Lancang County, Yunnan Province, China (22 • 29 55 N, 100 • 14 59 E). Three disease-and insect-pest-free fruit trees of the same age and with similar growth were selected for sample collection. Ten pieces of D. delavayi fruit of similar size, shape, and maturity were collected from each tree (total of 30 samples). Voucher samples were deposited in the Collaborative Innovation Center of Forest Resource Breeding and Utilization in Yunnan, Southwest Forestry University, under accession number DY2019053. Plant species were identified by Assoc. Prof. Jianghua Liu (College of Forestry, Southwest Forestry University). The collected young and mature fruit were mixed separately, 10 pieces of fruit were randomly selected and used as biological replicates, and three biological replicates were used for each maturity level. The chopped small pieces of D. delavayi fruit were frozen in liquid nitrogen, and were maintained at −80 • C for use in subsequent experiments.

HS-SPME Conditions
Samples were taken out of the −80 • C freezer and crushed using zirconia beads in a mixing mill (MM 400, Verder Shanghai Instruments and Equipment Co., Ltd., Shanghai, China) at 30 Hz for 1.5 min before any experiment. D. delavayi volatile compounds were extracted using 50/30 µm DVB/CAR/PDMS fibers (Supelco, Bellefonte, PA, USA). An automatic injection method was used. (PAL RTC 120, Agilent Technologies, Santa Clara, CA, USA).
The sample (100 mg), saturated sodium chloride solution (2 mL), and n-alkanes (C 7 -C 27 , Sigma-Aldrich, St. Louis, MO, USA) (10 µL) were mixed in a 20 mL headspace vial and heated at 60 • C for 10 min, followed by exposure to SPME fibers (aged at 250 • C for 30 min before use). The volatile compounds were finally volatilized at the inlet for 5 min.

GC-MS Conditions
An Agilent 7890B GC system coupled with an Agilent 7000D mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) was used to identify the volatile compounds in the various samples using an Agilent DB-5MS capillary column (30 m × 0.25 mm × 0.25 µm) and helium (purity > 99.999%) as the carrier gas at a flow rate of 1 mL/min. The front injection temperature and inlet mode were set to 250 • C and splitless. The GC oven temperature was set to 40 • C for 1 min, and then increased to 280 • C at 5 • C/min. EI energy of 70 eV was used during MS, the ion source and quad temperatures were at 230 • C and 150 • C, respectively. Full-scan acquisition mode (from 30 to 350 aum) was used with a solvent delay time of 3.0 min.

Qualitative and Quantitative Analysis of GC-MS
Acquired mass spectra were compared with spectra in the National Institute of Standards and Technology Library (NIST 14. L). Retention index (RI) values were calculated for each compound and compared with the RI values on the website. Matching volatile compounds with RI > 80 were retained, and the relative contents of the identified compounds were confirmed by dividing individual peak areas by the total peak area. The calculation of RI was performed by the expression: RI = 100 × [n + (Rt (x) − Rt (n) )/(Rt (n+1) − Rt (n) )], where Rt (x) denotes the corrected retention time (RT) of the tested compound; n and n + 1 denote the number of carbon atoms in the previous and next n-alkane, respectively; Rt (n) and Rt (n+1) represent the RT of n-alkanes with n and n + 1 carbon atoms, respectively; R (n) < R (x) < R (n+1) .

Calculating rOAV
rOAV often serves for the assessment of the effect on the overall aroma of individual volatile compounds [19]. The rOAV of the volatile flavor compound that most obviously affected the overall flavor of the sample was set to 100. Compounds with rOAV ≥ 1 are considered critical volatile aroma compounds of the sample, and compounds with 0.1 < rOAV < 1 are considered to have a modifying effect on the flavor of the sample [20]. rOAV values were calculated using the expression: rOAV = 100 × (C i /C max ) × (T max /T i ), where C i represents the peak area of the target volatile compound, and C max represents that of the compound with the highest odor activity value; T i is the odor threshold in air and T max is the largest odor threshold in air.

Statistical Analyses
Experimental data are in the form of means ± standard deviations (SDs). Excel software (Excel 2016, Addinsoft, New York, NY, USA) and OriginPro 2022 (Origin Lab Corporation, Northampton, Massachusetts, USA) were used to collect data. IBM SPSS Statistics 26 (SPSS, Chicago, IL, USA) was used to Z-score-process all peak areas and for independent sample t-testing. TBtools (version 1.0986) was used to create heat maps. SIMCA 14.1 (Umetrics, Malmö, Sweden) was used for the PCA and OPLS-DA analyses.
The DY and DM groups displayed an obviously different number of volatile compounds ( Figure 2a). Among the 42 volatile compounds, DM contained unique volatile compounds that included butyl butanoate, butyl hexanoate, hexyl hexanoate, benzylcarbinyl caproate, cis-α-bergamotene, and cis-bisabolene. The relative contents of the volatile compounds in DY and DM are shown in Figure 2B. Alkanes mainly originate from the homogeneous decomposition of fatty acid alkoxy radicals [14]. The alkane contents of DY and DM were 0.83% and 0.46%, respectively; however, the alkanes were observed to have a high threshold, which indicated that they hardly affected the flavor of D. delavayi fruit. In addition, the alkene contents in DY and DM were also relatively low (0.04% and 0.15%, respectively), as observed in other Rosaceae fruits [21]. Interestingly, small amounts of 1-octanol and 2-ethylfuran, with green aromas, were detected in both DY and DM. The volatile compound content is in the form of mean value ± standard deviation (mean ± SD), "-" information was not found in the literature.

Key Volatile Compounds in D. delavayi Fruit of Varying Maturities
A cluster heat map was produced and colored according to the relative expression change (Z-score) following the normalization of the volatile compounds in D. delavayi fruit samples (Figure 3a). Overall, the volatile compounds in DY were mainly upregulated, while those in DM were mainly downregulated. Twenty two volatile compounds in DY (mainly ketones and esters) were revised upwards, while 14 volatile compounds (mainly aldehydes) were revised downwards. Meanwhile, 16 volatile compounds in DM (mainly aldehydes) were revised upwards, while 20 volatile compounds (mainly alkanes) were revised downwards. These 36 volatile compounds form the basis for the different aroma characteristics of DY and DM. The key volatile compounds in DY and DM were also shown to significantly differ when column clustering analysis was used. The normalized peak areas of the 36 volatile components in the cluster heat map were subjected to PCA (Figure 3b). Principal components 1 (PC1) and 2 (PC2) accounted for 76.3% and Aldehydes are oxidation products of unsaturated fatty acids and a category of volatile compounds commonly found in fruits, such as tomatoes, oranges, and apples [22,23]. In this study, the number of aldehydes in both DY and DM was 8, while the contents of aldehydes were 1.07% and 2.38%, respectively, among which both DY and DM exhibited the highest (E)-2-hexenal contents (0.39% and 1.49%, respectively). The average ketone contents accounted for 11.19% and 6.43% of the volatile compounds identified in DY and DM, respectively. Among them, the levels of 2,6-dimethyl-4-heptanone, 2,3-butanedione and 2-heptanone were highest in both DY and DM, with each exhibiting a green aroma and a sweet fragrance.
Esters are critical fruit-aroma compounds and are mainly formed by esterifying acids with alcohols [24]. As shown in Figure 2, the number of esters in DY was fewer than that in DM (5 and 9, respectively); however, the total content of esters in DY was higher than that in DM (71.22% and 40.80%, respectively), with relatively high phenethyl acetate and ethyl caprylate contents. Phenethyl acetate is aromatic; its low threshold and high content significantly affect the overall flavor of DY and DM. Terpenes, which are considered to form through the oxidative cleavage of carotenoids, are among the most important groups of volatile compounds and play irreplaceable roles in determining the characteristic aromas of berries as well as essential roles in the phytopharmacological properties (e.g., antiinflammatory and anticancer) of the fruit [25,26]. In the present study, terpenes accounted for 13.68% and 47.96% (on average) of the identified volatiles in DY and DM, including theaspirane, dihydro-β-ionone, geranylacetone, β-ionone, cis-α-bergamotene, farnesene, and cis-bisabolene; both sets of fruit contained theaspirane as the dominant terpene. Apart from β-ionone, DY exhibited lower terpene content than DM. Moreover, theaspirane, with a mixture of fruity and woody aromas, was the most abundant terpenoid in both DY and DM (12.19% and 34.9%, respectively).
Taken together, the volatile components of the two fruit samples differed in type and content; hence, the data were further analyzed to identify the key aromas that differentiate them.

Key Volatile Compounds in D. delavayi Fruit of Varying Maturities
A cluster heat map was produced and colored according to the relative expression change (Z-score) following the normalization of the volatile compounds in D. delavayi fruit samples (Figure 3a). Overall, the volatile compounds in DY were mainly upregulated, while those in DM were mainly downregulated. Twenty two volatile compounds in DY (mainly ketones and esters) were revised upwards, while 14 volatile compounds (mainly aldehydes) were revised downwards. Meanwhile, 16 volatile compounds in DM (mainly aldehydes) were revised upwards, while 20 volatile compounds (mainly alkanes) were revised downwards. These 36 volatile compounds form the basis for the different aroma characteristics of DY and DM. The key volatile compounds in DY and DM were also shown to significantly differ when column clustering analysis was used. The normalized peak areas of the 36 volatile components in the cluster heat map were subjected to PCA (Figure 3b). Principal components 1 (PC1) and 2 (PC2) accounted for 76.3% and 14.6% of the total variation (90.9%), respectively. In addition, the volatile compounds were mainly distributed in two regions. Most alkanes, ketones, and esters were located on the left side of the vertical scale, in similar positions to DY, whereas most terpenoids and aldehydes were located on the right side of the vertical scale, in similar positions to DM. Moreover, no overlap was observed between DY and DM, which indicated that their volatile components were clearly distinguishable.
OPLS-DA serves as a new multivariate statistical method to conduct regression modeling on multiple independent variables and is capable of accurately determining the key variables impacting a group [27], and was adopted for further screening of the key volatile compounds affecting the aroma characteristics of young and mature D. delavayi fruit. As shown in Figure 3c, DY and DM have entirely separated score plots, with excellent fit parameters (R 2 X = 0.908, R 2 Y = 0.997, Q 2 = 0.99) that confirmed the model's accuracy. The model was further verified using 200 cross-validations. According to the test outcome [R 2 = (0.0, 0.636), Q 2 = (0.0, −0.878)], the OPLS-DA model did not undergo overfitting (Figure 3d). Variable importance in projection (VIP) can be used to quantify the contribution of each OPLS-DA variable to the classification; variables with VIP > 1 played vital classification roles [28]. In this study, the OPLS-DA model was developed for the identification of 25 volatile compounds with VIP > 1 (Figure 3e), with 23 key volatile compounds that satisfied VIP > 1 and p < 0.05 finally identified from among these 25 volatile compounds ( Figure 4 and Table 2).

rOAV Analysis of Key Volatile Compounds in D. delavayi Fruit of Varying Maturities
rOAV data for the 14 volatile aroma compounds identified among the 23 key volatile compounds are shown in Figure 5, from which β-ionone and phenethyl acetate were found to mostly contribute to the overall flavor of DY and DM, respectively. Except for β-ionone and (E)-2-octenal, all compounds in DM exhibited higher rOAV values than those in DY; this discrepancy might be the reason why DY lacks a pronounced aroma. To more intuitively analyze the differences in the aroma characteristics of DY and DM, the critical volatile aroma compounds were classified and summarized according to aroma type. Figure 6 shows that the aroma characteristics of DY and DM can be described as being woody (more prominent in DY) followed by floral and fruity. DM exhibited a more prominent floral aroma. Moreover, woody, green, cedar, fruity, and sweet aromas contributed to the more intense aroma of DM.   β-Ionone is a 9,10/9 ,10 cleavage product of β-carotene, and can be extensively found in fruits, flowers, and vegetables [29]. β-Ionone has a low threshold; hence, the compound presents a strong woody and floral aroma at low levels. β-Ionone is receiving increasing levels of attention in the biomedicine field owing to its anti-inflammatory, antibacterial, and anticancer properties [30]. Phenethyl acetate is a high-value natural volatile ester with an intense floral aroma; it is the primary aromatic ester in roses, jasmine, and other plants, and has been widely added to cosmetics, food, and beverages to enhance aroma and flavor [31,32]. As shown in Table 3, volatile aroma compounds that exhibit green odors, such as (E)-2-hexenal (green), 1-octanol (green, citrus), and hexanal (green, grass), showed stronger rOAV in DM than in DY, which indicated that DM has a greener aroma. It is worth noting that compounds with pleasant aromas, such as benzeneacetaldehyde (sweet, honey), ethyl heptanoate (fruity, sweet), and ethyl caprylate (pineapple, fruity), were more abundant in DM than DY. Dihydro-β-ionone has a mellow, sweet, and fresh cedar aroma; it is an attractive compound for commercial flavor and fragrance applications, and is used in the cosmetics, perfumery, food, and beverage industries [33]. While dihydro-β-acetone was found to be the critical volatile aroma component in DM, it only modified the fragrance of DY. This difference is possibly one of the reasons for the different fragrances of the two samples, as was also observed for hexanal and benzeneacetaldehyde. To more intuitively analyze the differences in the aroma characteristics of DY and DM, the critical volatile aroma compounds were classified and summarized according to aroma type. Figure 6 shows that the aroma characteristics of DY and DM can be described as being woody (more prominent in DY) followed by floral and fruity. DM exhibited a more prominent floral aroma. Moreover, woody, green, cedar, fruity, and sweet aromas contributed to the more intense aroma of DM.

Discussion
Fruit aroma is an important indicator for plant breeding and influences consumer acceptance. Fruits can produce compounds that give them a characteristic aroma during the growth process; therefore, various types of volatile compounds and their quantities result in different aromas [34]. In the present study, the number of esters and terpenes in DM was more than that of DY. Esters are essential contributors to fruit aromas; they have

Discussion
Fruit aroma is an important indicator for plant breeding and influences consumer acceptance. Fruits can produce compounds that give them a characteristic aroma during the growth process; therefore, various types of volatile compounds and their quantities result in different aromas [34]. In the present study, the number of esters and terpenes in DM was more than that of DY. Esters are essential contributors to fruit aromas; they have low thresholds and generally produce pleasant odors [35]. Esters are generally synthesized through fatty acid metabolism, with critical precursors including linolenic and linoleic acids [36]; these acids are catalyzed by lipoxygenase (LOX) enzymes to generate 9hydroperoxide (9-HPO) and 13-hydroperoxide (13-HPO) that form C6 and C9 aldehydes by the action of hydroperoxide lyase (HPL), with alcohol dehydrogenase (ADH) and alcohol acyltransferase (AAT) catalyzing their conversion to the corresponding linear esters [35].
In addition, metabolic pathways involving amino acids, such as alanine, valine, and aspartic acid, in fruit also participate in the ester synthesis [10]. Amino transferase (ATF) catalyzes the conversions of amino acids into α-ketoacids, which undergo decarboxylation, reduction, and esterification reactions to generate the corresponding aldehydes, esters, and other volatile compounds. Furthermore, α-ketoacid acids can combine with coenzyme A (CoA) to form acyl-CoA, which generates branched-chain esters by AAT catalysis [37]. Studies have shown that changes in the enzyme activities of the various synthetic pathways and sharp increases in free amino acid content during fruit ripening are possible reasons for the higher number of esters and the obvious aroma associated with fruit during ripening [21,38].
Terpenoids are the most diverse secondary metabolites and tend to have low aroma thresholds and characteristic aromas. Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), with C5 structures, act as the main synthetic precursors of terpenoids, with the main synthetic pathways proceeding through methylerythritol phosphate (MEP) and mevalonic acid (MVA), among which monoterpenes (C10), diterpenes (C20), and tetraterpene (C40) are mainly formed through the MEP pathway, while sesquiterpenes and triterpenes are mainly associated with the MVA pathway [26,39]. In this study, three monoterpenes (β-ionone, dihydro-β-ionone, and geranyl acetone), three sesquiterpenes (cis-α-bergamotene, farnesene, and cis-bisabolene), and one cyclovanilene derivative (theaspirane) were detected, with DY containing more monoterpenes and DM containing more sesquiterpenes and theaspirane. β-Ionone is a monocyclic monoterpene and an end-loop analog of β-carotenoids extensively found in fruits and vegetables, and it forms the basic structures of retinoic acid, retinol, beta-carotene, and vitamin A [40]. According to previous studies, β-ionone not only exhibits outstanding woody incense properties but also possesses significant pharmacological properties, including anti-inflammatory, antibacterial, and anticancer activities [30,41]. Dihydro-β-ionone is a monocyclic monoterpene compound with a strong cedar aroma that is often detected in Osmanthus fragrans flowers and tea [42,43]. Geranyl acetone is an acyclic monoterpene characterized by a rose and green fragrance; it has also been detected in blueberries and grapes [25,44,45]. Cis-α-bergamotene and cis-bisabolene were detected only in DM; the latter is a monocyclic sesquiterpene that exhibits antitumor, antifungal, and antimicrobial activities [46]. As an acyclic sesquiterpene, farnesene, which is an intermediate in the synthesis of vitamins E and K1, is the most abundant sesquiterpene (among cis-α-bergamotene, farnesene, and cis-bisabolene) [47]. C6 aldehydes, which are most common in DY and DM, are a class of compounds produced through fatty acid metabolism and mainly contribute to the green aroma; they are also critical green leaf volatiles (GLVs) [48]. Esters are the essential compounds in most ripe fruits, and aldehydes gradually become less abundant during ripening. Aldehydes are reportedly present in higher levels in fruits with more acidic and yellow peels during ripening [49], which may account for the higher aldehyde content in DM.
Human perception of fruit aroma occurs through interactions between the volatile compounds released from fruit and G protein-coupled odorant receptors (OR) in the olfactory epithelial cells of the nasal cavity. However, not all volatile compounds contribute to the aroma of the fruit, and only when the compound reaches a certain threshold will fruit aroma be affected [34]. rOAV is the ratio of the concentration of a volatile compound in the fruit to the odor threshold of the volatile compound, which assesses the promoting effect of the component on the overall fruit flavor. A higher rOAV indicates that the compound contributes more to the fruit aroma profile; this method is widely used for Rosaceae fruits [50]. The rOAV of the volatile aroma compounds was calculated to elucidate which compounds play crucial roles in regulating the overall flavor of D. delavayi fruit. In the present study, different rOAV scores for the same compounds in the young and mature fruit were obtained. (E)-2-Hexenal, β-ionone, 1-octanol, ethyl caprylate, and phenethyl acetate were determined to be key compounds that contribute to the overall aroma of DY, while DM also contains dihydro-β-ionone, hexanal, and benzeneacetaldehyde in addition to the five abovementioned key volatile aroma compounds. These findings showed that certain compounds could only decorate flavor in DY, whereas they were essential and critical compounds in DM; the overall flavor of DM was affected by more volatile aroma compounds, which may be the reason for its more pronounced odor.
D. delavayi is mainly produced in the Xishuangbanna and Shangri-La regions of Yunnan Province, and its fruit is commonly consumed by local ethnic minorities. D. delavayi has not been well promoted, and its fruit has not been well researched owing to the geography of the region. In addition, product research and development continue to focus on simple processed products, such as beverages, preserved fruits, and jams. Therefore, this study explored the differences between DY and DM in terms of the volatile components and aromas, which may effectively contribute to the comprehensive utilization of D. delavayi fruit.

Conclusions
The volatile components in young and mature D. delavayi fruit were comparatively analyzed by HS-SPME-GC-MS. DY and DM exhibited different volatile-compound profiles and aroma characteristics. Twenty three key volatile compounds, including octanal, geranylacetone, and butyl acetate, were identified using two indicators: VIP > 1 and p < 0.05. The rOAV of the volatile aromatic compounds revealed that DY had fewer key volatile aromatic compounds than DM, with the latter mainly exhibiting a floral aroma. Moreover, differences in young and mature D. delavayi fruit were analyzed from the perspective of volatile compounds and aroma characteristics. This study provides reference data for the comprehensive development, utilization, and evaluation of D. delavayi fruit. Furthermore, volatile compounds need to be sensory-assessed and analyzed throughout the ripening process to comprehensively understand the aroma changes exhibited by D. delavayi.

Conflicts of Interest:
The authors declare no conflict of interest.