Abstract
Knowing the content, compound, and key odor of volatile oil in the fruit of prickly ash during the development is important for improving product quality and determining a suitable harvesting period. Samples of Zanthoxylum bungeanum ‘Hanyuan’ fruit were collected at 31−115 day after flowering (AF) used for volatile oil extraction by the steam distillation method, and the volatile compounds were determined by gas chromatography-mass spectrometry (GC-MS), and the cluster and principal component analysis (PCA) were used for odor evaluation. The volatile oil content increased gradually with the fruit development, ranging from 0.4% to 8.5%, and no significant (P > 0.05) difference was observed between 100 d AF and 115 d AF. A total of 65 compounds were detected in volatile oils at the different stages; these compounds mainly included hydrocarbons, alcohols, esters, ethers, aldehydes, and ketones, which accounted for 96.36%−99.52% of the volatile oils. The main volatile compounds were alkenes, alcohols, and esters according to the number and relative content of the compounds in volatile oils. A total of 25 common compounds were detected in volatile oils at the different stages, which accounted for 93.99%−98.36% of the volatile oils, and (+)-limonene, linalool, myrcene, linalyl acetate, and 1,8-cineole had higher relative content than other compounds. The main compounds of volatile oils at the different stages were similar, but the relative content varied greatly. The relative content of (+)-limonene was observed to be the highest at all stages, myrcene and linalyl acetate showed an increasing trend, and linalool showed a decreasing trend with fruit development. The key odor compounds of Zanthoxylum bungeanum ‘Hanyuan’ fruit at the different stages were varied. Linalool, (+)-limonene, myrcene, 1,8-cineole, (+)-delta-cadinene, β-ocimene, (E)-β-ocimene, and geranyl acetate were key flavor compounds during the entire fruit period of Zanthoxylum bungeanum ‘Hanyuan’. The comprehensive score of odor evaluation at the different stages ranked as 115 d AF > 100 d AF > 86 d AF > 74 d AF > 39 d AF > 64 d AF > 47 d AF > 56 d AF > 31 d AF. The optimal harvest period for Zanthoxylum bungeanum ‘Hanyuan’ fruit for obtaining a higher content of volatile oil and a comprehensive score of odor evaluation was 100−115 d AF.
Keywords: Zanthoxylum bungeanum ‘Hanyuan’, Fruit development, Volatile oil, Volatile compounds, Odor evaluation
Introduction
Zanthoxylum genus, commonly known as prickly ash, is a kind of shrub, small arbor, or woody vine, and belongs to the Rutaceae family. There are approximately 250 species worldwide and widely planted in tropical and subtropical regions of Asia, America, Africa, and Oceania, of which China has 45 species and 13 varieties [1]. The mature fresh fruit of prickly ash is red or purple-red in color, with scattered raised oil sacs on the fruit surface. Its fruit is a traditional Chinese medicinal herb and was listed in “Pharmacopoeia of the People’s Republic of China”. It has the function of warming to relieve pain, and insecticidal to relieve itching. As a Chinese medicinal material, it is widely used in the medical field and is also one of the “Eight Traditional Condiments”. Therefore, it has been recognized as a dual-use plant for medicine and food [2]. Plant volatile oil is a type of secondary metabolite derived from plants, with a small molecular weight that can be evaporated with water vapor. They are mainly oily liquid substances at usual temperature, with highly volatile, strong, and irritating odor. They have been called essential oils in botany, aromatic oils in commerce, and volatile oils in chemistry and medicine [3]. Volatile oil of prickly ash is a kind of oily liquid extracted from the fruit pericarp, which can be dissolved with organic solvents. The substance structure in the volatile oil of prickly ash contains a large number of double bonds and hydroxyl, ester, carboxyl, and thiol groups, and most of them contain a nearly planar cyclic structure [4]. Volatile oil is the main aroma substance of prickly ash; it mainly contains volatile compounds such as alkenes, alcohols, esters, aldehydes, and ketones, which are important compounds affecting the quality of prickly ash [5]. The aroma of prickly ash comes from the volatile oil contained in the fruit, which has the activity of lowering blood sugar [6] and antioxidation [7]. The extraction of volatile oil from prickly ash usually uses the steam distillation method, and the gas chromatography-mass spectrometry (GC-MS) technology was used to analyze [5].
Previous researchers had conducted extensive studies on the compounds in the volatile oil of prickly ash, and they varied with geographical location and species. Kazuaki et al. [8] found that limonene, β-phellandrene, citronellal, and geranyl acetate as the main volatile compounds of Zanthoxylum piperitum by GC and GC/MS analysis, and the compounds changed with the producing area. Similarly, Wu et al. [9] found that temperature, precipitation, and wind speed effectively impacted the compounds accumulation in the volatile oil of prickly ash by comparing these compounds from 10 different regions. However, Farouil et al. [10] found that the metabolic compounds of Zanthoxylum caribaeum in Guadeloupe were similar to those of the same plant species in Brazil, Costa Rica, and Paraguay. The preservation and processing technology of prickly ash after harvest has an important impact on volatile oil compounds. The compound and content of odor in the fruit of prickly ash may vary depending on storage conditions, drying methods, and development stages. Liu et al. [11] measured the changes in volatile oil compounds of prickly ash under different drying methods and found that the peak area of 33 compounds was significantly (P < 0.05) decreased under hot air drying at 60 ℃, 30 compounds were decreased under radio frequency combined with 60 ℃ hot air drying, and 27 compounds were reduced under radio frequency combined with 50 ℃ hot air drying, while radio frequency treatment did not affect the content of limonene and linalool as compared to fresh fruit. Zhu et al. [12] identified 38 compounds from volatile oils in leaves and pericarps of Z anthoxylum bungeanum at different development stages using headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) technology and found that linalyl acetate, limonene, linalool, germacrene-D, β-myrcene, cineole, caryophyllene, and β-phellandrene were the main compounds, which accounted for more than 81% of the total odor compounds, and the relative content of linalyl acetate (23.53%−44.95%) at different maturity levels were generally higher than that of limonene (14.40%−27.25%).
There were many reports on the compounds and changes of odor during the fruit development of plants such as grapes [13], peppers [14], and loquat [15]. Previous researchers on the odor of prickly ash mainly focused on the comparison of odor compounds between different species [9, 16, 17], and the study of odor compounds in the fruit of prickly ash mainly focused on the mature harvesting stage. However, there are fewer reports on odor compounds and changes during fruit development. The planting area of prickly ash in China was 1.67 million hectares; both the planting area (0.39 million hectares) and production (190000 T) were the highest in Sichuan Province, and the quality was generally better than in other regions [1]. Hanyuan County is a famous prickly ash production area in Sichuan Province. It has a cultivation history of more than 2100 years and is known as the “Hometown of Prickly Ash in China”. Zanthoxylum bungeanum ‘Hanyuan’ is the main landrace species in Hanyuan County and was a tribute to the court since the Tang Dynasty, and is known as “Gongjiao” [18].
The contribution of volatile compounds to overall flavor depends on the concentration and threshold of odor [19]. The relative odor activity value (ROAV) is a useful indicator to quantitatively evaluate the contribution of different compounds to the flavor of food. It calculates key flavor compounds by combining their sensory threshold and relative content. The scientific and objective nature of the ROAV method makes it widely used in food flavor research [20]. However, there is no literature on the ROAV dynamics of key volatile compounds of prickly ash fruit during the different development periods. Therefore, the objectives of this study were to (1) determine the content of volatile oil; (2) and identify the key volatile compounds and the ROAV in fruit during the development period. This study used the fruit of Zanthoxylum bungeanum ‘Hanyuan’ at different development stages as material to extract volatile oil by the steam distillation method; both GC-MS and ROAV methods were used to analyze volatile oil compounds to reveal the key odor compounds dynamics; and provide a theoretical basis for suitable harvesting time determination and comprehensive utilization of Zanthoxylum bungeanum ‘Hanyuan’.
Materials and methods
Experiment material
The experimental material is Zanthoxylum bungeanum ‘Hanyuan’ in the demonstration base of Qingxi Town, Hanyuan County, Sichuan Province, China (102°7′E, 29°39′N). The area is a typical subtropical monsoon humid climate, with an annual average air temperature of 17.9 ℃, a frost-free period of 300 d, sunshine hours of 1475.8 h, active accumulated temperature of 5844.7 ℃, and an average annual rainfall of 741.8 mm. Soil type is yellow loam with the following initial properties: pH 6.0, organic matter 30.2 g kg−1, total N 1.8 g kg−1, total P 0.8 g kg−1, and total K 6.6 g kg−1[21]. Zanthoxylum bungeanum ‘Hanyuan’ (Fig. 1) was verified as an improved variety of Zanthoxylum bungeanum Maxim. by the authorized committee of forest improved varieties in Sichuan Province, China, in 2013, and planted conventionally, with row spacing of 2 × 3 m and a tree age of 10 years. The cultivation and management measures were consistent, and conventional pest control measures were adopted. The trees of Zanthoxylum bungeanum ‘Hanyuan’ with healthy and similar growth were selected for testing and sample collection.
Fig. 1.
Fruit of Z. bungeanum ‘Hanyuan’
Experiment design
The experiment was conducted from April to August 2022. Three repeated experimental plots were selected, each with an area of 10 m × 10 m, the same soil type, similar elevation (1740 m), aspect (SW 223), and slope (7°). Five trees of Zanthoxylum bungeanum ‘Hanyuan’ with similar growth were selected from each plot. April 12 was designated as the first day after flowering (AF) based on the observation and investigation, and May 12 (31 d AF) was the first fruit sampling time. Afterward, samples of fruit were collected every 1–2 weeks from 9:00 to 10:00 in the morning. The specific sampling times were 31 d AF, 39 d AF, 47 d AF, 56 d AF, 64 d AF, 74 d AF, 86 d AF, 100 d AF, and 115 d AF.
Measurement parameters
Fruit traits
The fruit size, shape index, weight of a thousand fresh fruits (TFF), dry fruits (TDF), dry pericarps (TDP), and dry seeds (TDS) were measured, and the pericarp rate was calculated by separating the pericarp and seed from dried fruit. The fruits were dried for 1 week indoors at room temperature, and the pericarp and seed were separated after drying. The 50 representative fruits in every plot were selected to be measured at each stage and to calculate the fruit shape index. The longitudinal and transverse diameters of the fruit were measured from fresh fruit by using a stereomicroscope, taking the widest transverse diameter as the transverse diameter and the widest longitudinal diameter as the longitudinal diameter.
Fruit shape index = longitudinal diameter/transverse diameter.
Pericarp rate (%) = dry fruit pericarp weight (g)/fresh fruit weight (g) × 100%.
Extraction of volatile oil
25 g of Zanthoxylum bungeanum ‘Hanyuan’ pericarp powder was used for volatile oil, which was extracted by the steam distillation method for 2 h. Volatile oil was dried with anhydrous sodium sulfate overnight and stored in a −20 ℃ refrigerator for later use [11, 22]. Volatile oil content was calculated by the weight of the pericarp and the volume of volatile oil distilled from the pericarp.
Volatile oil (%) = volume of volatile oil collected (mL)/weight of pericarp (g) × 100%.
Determination of compounds in volatile oil
The dried volatile oil had been dissolved in n-hexane (chromatographically pure) at a volume fraction of 5%. In brief, 50 µL of volatile oil was dissolved in 950 µL of n-hexane solution, mixed well, and passed through a 0.22 μm filter membrane. The volatile oil components were determined by the GC-MS method [23] using a gas chromatography-mass spectrometer (Agilent 8890-5977B). The chromatographic column was a HP-5MS (60 m × 250 μm × 0.1 μm) capillary gas chromatography column. The carrier gas was high-purity helium gas with a flow rate of 1.0 mL/min. The inlet temperature was 250 ℃, and the interface temperature was 280 ℃. The initial temperature was 60 ℃, held for 2 min, heated to 135 ℃ at a rate of 2 ℃/min, held for 10 min, and then heated to 240 ℃ at a rate of 20 ℃/min and held for 4 min. Quality full scan mode, scanning range was 33–650 amu. Electron bombardment ionization source with an electron energy of 70 eV. Ion source temperature was 230℃; transmission line temperature was 250 ℃; solvent delay was 3 min.
Attribute query
The chemical compounds in volatile oils were determined by searching for similarity-matching values as compared to the mass spectrum corresponding to each chromatographic peak with the standard spectral library (NIST17.0). The compounds with similarity higher than 80% (maximum value of 100%) in the spectral library were identified, and the area normalization method was used for quantification to obtain the relative content of compounds [9, 19].
The key flavor compounds in fruit were determined based on the relative content of every compound in volatile oils, and their odor thresholds and contributions to volatile flavor. The odor threshold of each volatile compound referred to by Qian et al. [20], which could not be queried, was not analyzed. The ROAV method was used to evaluate the contribution of volatile flavor. The compound that contributed the maximum to the overall flavor in fruit was defined as 100 (ROAVmax = 100), and the ROAV calculation formula for other compounds was as follows:
 |
1 |
Where Ci is the relative content of the compound in volatile oil (%), T is the sensory threshold of the volatile compound (µg/kg), and Cmax is the relative content of the volatile compound that had a maximum contribution to the overall flavor in fruit (%), Tmax is the sensory threshold of the volatile compound that had a maximum contribution to the overall flavor in fruit (µg/kg).
All compounds with ROAV ≤ 100.00 and higher ROAV contributed more to the overall flavor in fruit. In this study, the compounds with ROAV ≥ 1.00 were identified as key flavor compounds in fruit, while the compounds with 0.10 ≤ ROAV < 1.00 were identified as having important modifying effects on the overall flavor in fruit [20].
Comprehensive evaluation
TBtools software was used for heatmap clustering analysis to make the differences in the relative content of volatile oil compounds intuitively and clearly understood. The heat map of the relative content of volatile oil compounds at the different stages was obtained by using the relative content of 65 identified volatile compounds as variables, and each variable has been standardized. The standard for color intensity in a heatmap is from a maximum value of 2.0 (red) to a minimum value of −2.0 (blue), representing the abundance of volatile compounds from high to low [24].
The odor compounds in volatile oils were used for odor evaluation and key characteristic flavors determination by principal component analysis (PCA), in which the ROAV of volatile compounds should be 1−100 d AF, and characteristic values should be greater than 1. Standardized the raw data into dimensionless data with a mean of 0 and a standard deviation of 1. The function and score of the comprehensive evaluation were obtained by linearly weighting the principal component scores and corresponding weights based on the variance contribution rates of the principal components’ rotated loads as weights [9].
Data collection and statistical analyses
The weight of fresh fruit and dry fruit (including pericarp and seed) was expressed as the average of a thousand fruit samples. Excel 2010 and SPSS 25.0 were used for data statistics and analyses. Indices at the different stages of fruit traits and volatile oil content were analyzed by one-way ANOVA, followed by a post-hoc test that employed Duncan’s multiple range test to compare the differences among the different stages. The figures were drawn using Origin 2018 software. The heatmap clustering analysis was drawn using TBtools software.
Results
Fruit traits at the different stages
The growth of fruit transverse and longitudinal diameters was similar, both showed an increased trend with the fruit development and increased by 17.0% and 21.8%, respectively, from 31 d AF to 115 d AF (Fig. 2A). There were two growth peaks both for transverse and longitudinal diameters during the fruit development period, which were observed at 31−56 d AF and 74−100 d AF, respectively. There was no significant (P > 0.05) difference in transverse and longitudinal diameters at 115 d AF as compared to 100 d AF, but those at the two stages (100 d AF and 115 d AF) were significantly (P < 0.05) higher than those at the other stages. The fruit shape index was the lowest (1.08) and highest (1.14) at 31 d AF and 39 d AF, respectively, and the fruit was nearly spherical (Fig. 2B). The longitudinal diameter was higher than the transverse diameter at every stage, and the fruit shape index was always greater than 1.
Fig. 2.
Dynamic changes of the fruit shape index and weight of Z. bungeanum ‘Hanyuan’. A Fruit diameter. B Shape index of fruit. C Weight of a thousand fresh fruits. D Weight of a thousand dry fruits. Different letters (little letters for the longitudinal diameter, shape index of fruit, weight of a thousand fresh fruits, and weight of a thousand seeds; little letters in brackets for the weight of a thousand pericarps; capital letters for the weight of a thousand dry fruits) represent significant differences at the P < 0.05 level at the different stages
The weight of a thousand fresh fruits (TFF) had two growth peaks, which were observed at 39−47 d AF and 56−74 d AF with an average daily growth rate of 1.39% and 0.64%, respectively, and TFF reached the highest value (63.37 g) at 115 d AF (Fig. 2C). The weight of a thousand dry fruits (TDF) gradually increased from 10.49 g at 31 d AF to 30.15 g at 115 d AF, and significant (P < 0.05) differences were observed among the different stages (Fig. 2D). The weight of a thousand dry pericarps (TDP) rapidly increased from 8.39 g at 31 d AF to 12.71 g at 56 d AF with an increase of 51.5%, and significant (P < 0.05) differences were observed among the different stages. The TDP reached the highest value (13.00 g) at 86 d AF, while no significant (P > 0.05) difference was observed among the stages from 56 d AF to 115 d AF. The TDS increased from 2.09 g to 17.41 g during the whole period. It increased slowly by 1.17 times from 31 d AF to 56 d AF, and increased rapidly by 2.84 times from 56 d AF to 115 d AF. The increase in pericarps was dominant at 31−56 d AF, while the growth of seeds was dominant at 56−115 d AF.
Volatile oil content at the different stages
The volatile oil content increased with the development, and obvious differences were observed among the different stages (Fig. 3). The fruit growth belonged to the early stage at 31 d AF, and the fruit surface had few oil sacs; meanwhile, the volatile oil content was the lowest (0.4%). Oil sacs on the fruit surface began to gradually protrude at 39−56 d AF. The volume increased from small to large, the formation and accumulation of volatile oil were slow during this period, and no significant (P > 0.05) difference was observed in volatile oil content among the different stages. The fruit development accelerated after 56 d AF, the oil sacs on the fruit surface continued to develop and expand, and the volatile oil continued to form and accumulate. The period from 64 d AF to 86 d AF belonged to the stage of oil sac enlargement. The volatile oil contents at 64 d AF, 74 d AF, and 86 d AF were significantly (P < 0.05) different, and the latter stage was always significantly (P < 0.05) higher than the former stage. The volatile oil content reached the maximum value of 8.5% at 115 d AF. There was no significant (P > 0.05) difference in volatile oil content between 100 d AF and 115 d AF. The fruit entered the mature stage, and the accumulation of volatile oil was almost completed at 100 d AF.
Fig. 3.
Dynamic changes of the volatile oil content in the fruit of Z. bungeanum ‘Hanyuan’. Different little letters represent significant differences at the P < 0.05 level at the different stages
Volatile oil compounds at the different stages
Based on the obtained volatile oils in fruit at the different stages, GC-MS was further used to qualitatively analyze the compounds of volatile oils, and the GC-MS total ion chromatogram of volatile oils was obtained (Fig. 4). The chromatographic peak separation of volatile oil at each stage was good under the established GC-MS analysis condition, the horizontal axis showed similar peak time and concentrated in 5–25 min, while the vertical axis showed obvious differences in abundance. Most of the compounds contained in volatile oils had low boiling points, and the compounds at the different stages were similar, but the differences in relative content were obvious.
Fig. 4.
Total ion chromatogram of volatile oil at the different stages
A total of 65 compounds were identified in volatile oils during the development period, which accounted for 96.36%−99.52% of the total volatile substances, and included 26 hydrocarbons, 18 alcohols, 14 esters, 5 aldehydes, 1 ether, and 1 ketone (Table 1; Fig. 5). A total of 25 common and 40 non-common compounds were identified during the development period, and the common compounds included 14 alkenes, 5 alcohols, 4 esters, 1 ether, and 1 ketone (Fig. 6). The 25 identified common compounds accounted for 93.99%−98.36% of the total volatile substances, indicating that the volatile oil compounds were extremely similar at the different stages, but the content of common compounds varied greatly. The main common compounds were terpenes, alcohols, and esters, which accounted for 52.02%−66.81%, 17.78%−35.17%, and 3.65%−10.28%, respectively. The main common compounds included (+)-limonene, linalool, myrcene, β-ocimene, 1,8-cineole, (E)-β-ocimene, α-terpineol, and linalyl acetate, totaling 8 compounds, and their relative contents were higher than 1% during the development period.
Table 1.
Dynamic changes of the compounds and their relative contents in volatile oils of Z. bungeanum ‘Hanyuan’ fruit at different days after flowering
| No. | Compound name | Relative content (%) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| 31 d | 39 d | 47 d | 56 d | 64 d | 74 d | 86 d | 100 d | 115 d | ||
| 1 | Isobutyl propionate | — | — | — | — | — | — | 0.01 | — | 0.01 |
| 2 | Isoamyl acetate | — | — | — | — | — | 0.01 | 0.01 | 0.01 | 0.01 |
| 3 | Methyl 4-methylpentanoate | — | — | — | 0.01 | 0.02 | 0.02 | 0.02 | 0.01 | 0.01 |
| 4 | α-phellandrene | — | — | — | — | — | — | 0.01 | 0.01 | 0.01 |
| 5 | α-Pinene | 0.06 | 0.10 | 0.11 | 0.17 | 0.19 | 0.25 | 0.24 | 0.18 | 0.17 |
| 6 | Isobutyl isobutyrate | — | — | 0.01 | — | 0.01 | — | — | — | — |
| 7 | Isobutyl butyrate | — | — | — | 0.01 | — | 0.01 | 0.01 | 0.01 | 0.02 |
| 8 | 2-Methylvaleraldehyde | — | — | — | 0.01 | — | — | — | — | — |
| 9 | 2-Methylheptanal | — | — | — | — | — | 0.02 | 0.01 | 0.01 | 0.02 |
| 10 | 2-Methylbutanal | — | — | — | — | 0.01 | — | — | — | — |
| 11 | Sabenene | 0.10 | 0.20 | 0.21 | 0.23 | 0.29 | 0.40 | 0.43 | 0.47 | 0.49 |
| 12 | (1S)-(1)-β-Pinene | 0.04 | 0.04 | 0.04 | 0.04 | 0.05 | 0.07 | 0.08 | 0.08 | 0.09 |
| 13 | Myrcene | 5.27 | 6.80 | 7.76 | 8.79 | 9.50 | 12.06 | 13.73 | 14.44 | 15.05 |
| 14 | β-Pinene | — | — | — | 0.02 | 0.02 | 0.02 | 0.02 | 0.03 | — |
| 15 | β-Terpinene | — | 0.02 | 0.02 | — | — | — | — | — | 0.03 |
| 16 | Isoterpinolene | — | — | — | 0.01 | — | 0.01 | — | 0.02 | 0.02 |
| 17 | o-Cymene | — | 0.05 | 0.06 | 0.02 | 0.01 | 0.03 | — | — | — |
| 18 | (+)-Limonene | 38.33 | 43.66 | 44.17 | 40.21 | 38.89 | 42.05 | 43.39 | 41.25 | 40.30 |
| 19 | 1,8-Cineole | 3.10 | 3.84 | 3.91 | 4.34 | 5.63 | 5.44 | 3.35 | 3.42 | 3.15 |
| 20 | (E)-β-Ocimene | 2.05 | 1.98 | 1.80 | 1.82 | 2.19 | 2.23 | 3.10 | 3.39 | 3.41 |
| 21 | β-Ocimene | 3.34 | 2.33 | 2.01 | 2.38 | 2.53 | 2.48 | 3.66 | 3.87 | 3.88 |
| 22 | γ-Terpinene | 0.07 | 0.02 | 0.01 | 0.02 | 0.01 | 0.02 | 0.03 | 0.02 | 0.02 |
| 23 | (Z)-4-Thujanol | — | 0.03 | 0.03 | — | 0.03 | — | — | — | — |
| 24 | Terpinolene | — | — | 0.08 | — | — | — | 0.16 | 0.17 | 0.17 |
| 25 | α-Terpinene | 0.20 | 0.10 | — | 0.13 | 0.11 | 0.12 | — | — | — |
| 26 | Linalool | 31.44 | 30.19 | 29.64 | 28.57 | 28.42 | 19.40 | 18.10 | 16.48 | 16.23 |
| 27 | Cis-4-(isopropyl)-1-methylcyclohex-2-en-1-ol | 0.08 | 0.11 | 0.11 | 0.09 | 0.05 | 0.06 | 0.03 | 0.03 | 0.02 |
| 28 | neo-Alloocimene | — | — | — | — | — | — | — | 0.01 | — |
| 29 | (E)-1,2-Limonene oxide | — | 0.02 | 0.02 | — | — | — | — | — | — |
| 30 | (+)-Citronellal | — | — | — | — | — | 0.04 | — | 0.07 | 0.09 |
| 31 | Decanal | — | — | — | 0.07 | 0.06 | 0.06 | 0.05 | — | — |
| 32 | (-)-Terpinen-4-ol | — | — | 0.20 | — | — | — | 0.09 | 0.09 | 0.08 |
| 33 | 4-Carvomenthenol | 0.35 | 0.19 | — | 0.22 | 0.13 | 0.15 | — | — | — |
| 34 | α-Terpineol | 1.93 | 1.57 | 1.62 | 2.42 | 1.80 | 1.88 | 1.37 | 1.37 | 1.23 |
| 35 | (Z)-Geraniol | 0.14 | 0.17 | 0.12 | 0.18 | 0.11 | 0.18 | 0.10 | 0.13 | 0.12 |
| 36 | Piperitone | 0.05 | 0.04 | 0.04 | 0.05 | 0.03 | 0.04 | 0.02 | 0.02 | 0.02 |
| 37 | Linalyl acetate | 1.58 | 1.64 | 1.99 | 3.67 | 4.75 | 6.98 | 7.28 | 8.42 | 9.30 |
| 38 | Isobornyl formate | — | 0.01 | — | — | — | — | — | — | — |
| 39 | (Z)-p-Menth-2,8-diene-1-ol | — | 0.09 | 0.09 | 0.03 | — | — | — | — | — |
| 40 | δ-Terpineol acetate | — | 0.02 | — | 0.02 | — | — | — | — | — |
| 41 | [(1R,4S,6S)-1,3,3-trimethyl-2-oxabicyclo[2.2.2]octan-6-yl] acetate | — | 0.04 | — | 0.06 | 0.04 | 0.09 | — | — | — |
| 42 | Terpinyl acetate | 1.08 | 0.78 | 0.76 | 0.82 | 0.62 | 0.57 | 0.41 | 0.44 | 0.42 |
| 43 | Citronellyl acetate | — | — | — | — | — | — | — | — | 0.02 |
| 44 | Nerol acetate | 0.33 | 0.28 | 0.29 | 0.42 | 0.30 | 0.33 | 0.19 | 0.22 | 0.18 |
| 45 | (3E,5E)-2,6-Dimethylocta-3,5,7-trien-2-ol | — | 0.10 | 0.12 | — | — | — | — | — | — |
| 46 | Geranyl acetate | 0.67 | 1.01 | 1.07 | 0.97 | 0.80 | 0.81 | 0.42 | 0.41 | 0.38 |
| 47 | β-Caryophyllene | 0.30 | 0.19 | 0.18 | 0.15 | 0.13 | 0.15 | 0.15 | 0.18 | 0.15 |
| 48 | Perilla alcohol | — | — | — | — | — | 0.02 | — | — | — |
| 49 | Perilla acetate | 0.14 | 0.07 | 0.07 | 0.05 | — | — | — | — | — |
| 50 | Humulene | 0.12 | 0.07 | 0.06 | 0.07 | 0.07 | 0.07 | 0.07 | 0.09 | 0.08 |
| 51 | β-Copaene | 1.02 | 0.45 | 0.41 | 0.86 | 0.84 | 1.11 | 1.26 | 1.81 | 1.64 |
| 52 | (+)-Bicyclogermacrene | 0.28 | 0.13 | 0.11 | 0.22 | 0.20 | 0.27 | 0.30 | 0.44 | 0.39 |
| 53 | (-)-α-muurolene | — | — | — | 0.08 | — | — | — | 0.08 | — |
| 54 | α-Muurolene | 0.12 | 0.05 | 0.04 | — | — | 0.07 | 0.05 | — | 0.06 |
| 55 | γ-Muurolene | — | — | — | — | — | 0.06 | 0.06 | 0.11 | 0.10 |
| 56 | (+)-delta-Cadinene | 0.77 | 0.26 | 0.24 | 0.46 | 0.20 | 0.27 | 0.28 | 0.31 | 0.25 |
| 57 | Hedycaryol | 0.16 | 0.10 | 0.09 | 0.08 | 0.05 | 0.03 | — | 0.03 | — |
| 58 | Germacrene B | 0.29 | 0.17 | 0.16 | 0.12 | 0.08 | 0.08 | 0.10 | 0.11 | 0.09 |
| 59 | Cubebol | — | — | 0.24 | 0.24 | 0.51 | — | 0.42 | — | — |
| 60 | 1H-Cyclopenta[1,3]cyclopropa[1,2]benzen-3-ol, octahydro-3,7-dimethyl-4-(1-methylethyl)-, (3R,3aR,3bR,4S,7R,7aR)- | 0.25 | 0.27 | — | — | — | 0.37 | — | 0.56 | 0.48 |
| 61 | T-Muurolol | 0.74 | 0.35 | 0.31 | 0.37 | 0.15 | 0.19 | — | — | 0.18 |
| 62 | T-Cadinol | — | — | — | — | — | — | — | 0.14 | 0.10 |
| 63 | α-muurolol | 0.13 | — | — | 0.08 | — | — | — | — | — |
| 64 | α-Cadinol | 1.58 | 0.67 | 0.62 | 0.79 | 0.32 | 0.39 | 0.29 | 0.24 | 0.17 |
| 65 | Shyobunol | 0.29 | 0.25 | 0.20 | 0.17 | 0.15 | 0.11 | — | 0.09 | 0.07 |
| Total | 96.36 | 98.45 | 99.00 | 99.52 | 99.34 | 99.00 | 99.27 | 99.25 | 98.69 | |
| The number of compounds | 34 | 42 | 40 | 44 | 39 | 44 | 38 | 42 | 43 | |
Fig. 5.
Number of types in compounds. A and their relative content. B in volatile oils of Z. bungeanum ‘Hanyuan’ fruit at the different stages
Fig. 6.
Number of compounds and common compounds in volatile oils of Z. bungeanum ‘Hanyuan’ fruit at the different stages after flowering (AF)
There were 34, 42, 40, 44, 39, 44, 38, 42, and 43 volatile compounds in volatile oils were detected at 31 d AF, 39 d AF, 47 d AF, 56 d AF, 64 d AF, 74 d AF, 86 d AF, 100 d AF, and 115 d AF, respectively (Table 1; Fig. 5, and Fig. 6). The minimal (only 34) compounds in volatile oil were detected at 31 d AF, while the maximal (44) compounds were detected at 56 d AF and 74 d AF, and the main compounds were terpenes, alcohols, and esters at the different stages. There were 25 common compounds in volatile oils at the different stages. Except for these stages of 31 d AF, 47 d AF, and 86 d AF had non-unique compounds in volatile oils, all other stages had one unique compound in volatile oils. The unique compounds were isobornyl formate, 2-methylvaleraldehyde, 2-methylbutanal, perilla alcohol, neo-alloocimene, and citronellyl acetate at 39 d AF, 56 d AF, 64 d AF, 74 d AF, 100 d AF, and 115 d AF, respectively.
Compounds and their relative content in volatile oils at the different stages
A total of 26 alkenes, included 25 hydrocarbons and 1 aromatic hydrocarbon, were detected during the development period, which included 14 common compounds, and the lowest (52.34%) and highest (67.10%) relative content of those volatile substances was observed at 31 d AF and 86 d AF, respectively (Table 1). The hydrocarbons had the most compounds and highest relative content in volatile oils. The identified monoterpene alkenes mainly included (+)-limonene, myrcene, (E)-β-ocimene, and β-ocimene. The highest relative content of compound in volatile oils at the different stages was (+)-limonene, which accounted for 38.33%−44.17%. The relative content of sabinene and myrcene was 0.10%−0.49% and 5.27%−15.05%, respectively, both increased continuously with fruit development. The relative content of α-pinene showed a trend of first increasing and then decreasing with the fruit development. On the contrary, the relative content of (1S)-(1)-β-pinene and (E)-β-Ocimene showed a trend of first decreasing and then increasing. The sesquiterpene hydrocarbons mainly included β-copaene, (+)-delta-cadinene, β-caryophyllene, and (+)-bicyclogermacrene, and the relative content of sesquiterpene hydrocarbons was much lower than that of monoterpenes. O-cymene was not detected during the early and mature stages of the fruit development of Zanthoxylum bungeanum ‘Hanyuan’; it only appeared at 39 d AF, 47 d AF, 56 d AF, 64 d AF, and 74 d AF.
A total of 18 alcohols were detected during the fruit development, accounting for 18.68%−37.09% of the volatile oils. The number and their relative content of alcohols were second only to hydrocarbons in the volatile oils. Linalool, cis−4-(isopropyl)−1-methylcyclohex-2-en-1-ol, α-terpineol, α-cadinol, and (Z)-geraniol were common compounds, in which the relative content of linalool changed more obviously than other compounds and decreased from 31.44% at 31 d AF to 16.23% at 115 d AF, and showed a trend of continuous decrease with the fruit maturity. The relative content of alcohols varied greatly at the different stages, generally showing a trend of decreasing with the fruit maturity and reaching their maximum and minimum at 31 d AF and 115 d AF, respectively.
A total of 14 esters were detected during the fruit development; the relative content of those compounds accounted for 3.79%−10.34% of the volatile oils, and the relative contents of esters were lower than those of alkenes and alcohols. Linalyl acetate, nerol acetate, geranyl acetate, and terpinyl acetate were common compounds during fruit development. The relative content of linalyl acetate changed obviously at the different stages, showed a trend of increasing with the fruit maturity, and increased from 1.58% at 31 d AF to 9.30% at 115 d AF with an increase of 4.9 times. The relative content of non-common compounds was low (less than 1%), and the variation pattern was not obvious.
A total of 5 aldehydes were detected in volatile oils, including 2-methylvaleraldehyde, 2-methylheptanal, 2-methylbutanal, (+)-citronellal, and decanal, which appeared at 56−115 d AF with low relative content. Only 1 ketone compound, piperitone, was detected, which was a common compound during the development period. The relative content of piperitone was the lowest at all development stages and had no obvious change pattern. In addition, only 1 ether compound, 1,8-cineole, was detected, which was a common compound during the development period. The highest relative content of 1,8-cineole was 5.63% at 64 d AF, which was higher than that of aldehydes and ketones at every development stage.
Except the above groups of classification, these volatile oil compounds also can be classified monoterpene hydrocarbons, oxygenated monoterpenoids, sesquiterpene, oxygenate sesquiterpene, and other compounds, which relative content was 49.46%−64.85% (average 57.87%), 20.94%−37.09% (average 30.50%), 1.20%−3.12% (average 2.13%), 0.71%−3.15% (average 1.45%), and 3.80%−10.37% (average 6.84%) from 31 d AF to 115 d AF, respectively.
Hierarchical cluster analysis of volatile oil compounds
The compounds in volatile oils at the different stages could be classified into four categories, in which 31 d AF and 86 d AF were separately classified into two categories, 100 d AF and 115 d AF were classified into one category, and the rest of the stages of 39 d AF, 47 d AF, 56 d AF, 64 d AF, and 74 d AF were classified into one category (Fig. 7). The number of compounds in volatile oils was minimum (only 34) at 31 d AF, the compounds and relative content of alkenes and esters were the lowest, while the relative content of alcohols was the highest (37.09%), other compounds (e.g., linalool, α-terpineol, and α-cadinol) were observed with higher relative content. The volatile oil at 86 d AF contained the least number of alcohols and the highest relative content of alkenes. The volatile oils at 100 d AF and 115 d AF contained many alkenes, esters, and aldehydes, which showed higher relative content, while the relative content of alcohols was low at the two stages. The relative content of piperitone and 1,8-cineole in volatile oils at 39 d AF, 47 d AF, 56 d AF, 64 d AF, and 74 d AF was high, while the relative content of alkenes was low. Cluster analysis could distinguish and classify compounds at the different stages, and development time has a significant impact on the formation of flavor compounds in the fruit of Zanthoxylum bungeanum ‘Hanyuan’.
Fig. 7.
Heat map and hierarchical cluster analysis of compounds in volatile oils of Z. bungeanum ‘Hanyuan’ fruit at the different stages after flowering (AF)
Key flavor compounds in fruit at the different stages
The key flavor compounds contained in Zanthoxylum bungeanum ‘Hanyuan’ fruit at the different stages were the same, while the ROAVs were different (Table 2). The 8 key flavor compounds (ROAV ≥ 1) included linalool, (+)-limonene, myrcene, 1,8-cineole, (+)-delta-cadinene, β-ocimene, (E)-β-ocimene, and geranyl acetate. The highest contribution to fruit flavor at the different stages was linalool (ROAV = 100), which has an odor similar to bergamot oil and lavender. The key flavor compounds (ROAV ≥ 10) at 31 d AF were (+)-limonene and 1,8-cineole; the ROAVs of myrcene and (+)-delta-cadinene were also very high, and the fruit at this stage generally has an odor of sweet, balsamic, lemon, camphor, and woody. The ROAVs of key flavor compounds ranked as (+)-limonene > 1,8-cineole > myrcene at 39 d AF, 47 d AF, 56 d AF, 64 d AF, and 74 d AF, and (+)-limonene > myrcene > 1,8-cineole at 86 d AF, 100 d AF, and 115 d AF. The 1,8-cineole was increased in compounds of key flavor at the other stages as compared to 31 d AF.
Table 2.
Dynamic changes of the key volatile compounds and their relative odor activity values of Z. bungeanum ‘Hanyuan’ fruit at different days after flowering
| No. | Compound name | Odor characteristic | Relative odor activity value | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 31 d | 39 d | 47 d | 56 d | 64 d | 74 d | 86 d | 100 d | 115 d | |||
| 1 | α-phellandrene | Pepper, citrus | — | — | — | — | — | — | < 0.1 | < 0.1 | < 0.1 |
| 2 | α-Pinene | Pine, turpentine | < 0.10 | 0.14 | 0.16 | 0.25 | 0.28 | 0.55 | 0.57 | 0.46 | 0.45 |
| 3 | Sabenene | Turpentine | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 |
| 4 | (1S)-(1)-β-Pinene | Pine | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 |
| 5 | Myrcene | Sweet | 7.73 | 10.40 | 12.08 | 14.19 | 15.42 | 28.70 | 35.01 | 40.43 | 42.79 |
| 6 | β-Pinene | Pine | — | — | — | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | — |
| 7 | Isoterpinolene | Turpentine, woody | — | — | — | < 0.1 | — | < 0.1 | — | < 0.1 | < 0.1 |
| 8 | (+)-Limonene | Citrus | 21.51 | 25.52 | 26.30 | 24.84 | 24.14 | 38.25 | 42.31 | 44.17 | 43.81 |
| 9 | (E)-β-Ocimene | Herbal | 1.15 | 1.16 | 1.07 | 1.13 | 1.36 | 2.03 | 3.02 | 3.63 | 3.71 |
| 10 | β-Ocimene | Herbal | 1.87 | 1.36 | 1.19 | 1.47 | 1.57 | 2.25 | 3.57 | 4.14 | 4.22 |
| 11 | γ-Terpinene | Citrus | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 |
| 12 | Terpinolene | Pine | — | — | < 0.1 | — | — | — | < 0.1 | < 0.1 | < 0.1 |
| 13 | α-Terpinene | Herbal, lemon | < 0.1 | < 0.1 | — | < 0.1 | < 0.1 | < 0.1 | — | — | — |
| 14 | neo-Alloocimene | Floral, fatty | — | — | — | — | — | — | — | < 0.1 | — |
| 15 | (E)−1,2-Limonene oxide | Fresh, citrus | — | < 0.1 | < 0.1 | — | — | — | — | — | — |
| 16 | β-Caryophyllene | Cloves, turpentine | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | 0.1 | < 0.1 |
| 17 | Humulene | Spicy, woody | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 |
| 18 | (+)-delta-Cadinene | Herbal, woody | 7.33 | 2.57 | 2.40 | 4.81 | 2.08 | 4.21 | 4.57 | 5.58 | 4.55 |
| 19 | Linalool | Bergamot, lavender | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 | 100.00 |
| 20 | (-)-Terpinen-4-ol | Pepper, woody | — | — | < 0.1 | — | — | — | < 0.1 | < 0.1 | < 0.1 |
| 21 | 4-Carvomenthenol | Pine | < 0.1 | < 0.1 | — | < 0.1 | < 0.1 | < 0.1 | — | — | — |
| 22 | α-Terpineol | Sweet | 0.11 | < 0.10 | < 0.10 | 0.15 | 0.12 | 0.18 | 0.14 | 0.15 | 0.14 |
| 23 | (Z)-Geraniol | Fresh, sweet, rose | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 |
| 24 | Perilla alcohol | Linalool, terpineol | — | — | — | — | — | < 0.1 | — | — | — |
| 25 | Isoamyl acetate | Bergamot odor | — | — | — | — | — | < 0.1 | < 0.1 | < 0.1 | < 0.1 |
| 26 | Isobutyl isobutyrate | Pineapple | — | — | < 0.1 | — | < 0.1 | — | — | — | — |
| 27 | Linalyl acetate | Bergamot | < 0.10 | < 0.10 | < 0.10 | < 0.10 | 0.10 | 0.22 | 0.24 | 0.31 | 0.34 |
| 28 | Terpinyl acetate | Fresh bergamot-lavender | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 |
| 29 | Citronellyl acetate | Rose | — | — | — | — | — | — | — | — | < 0.1 |
| 30 | Nerol acetate | Sweet, floral | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 |
| 31 | Geranyl acetate | Ethereal fruity | 1.41 | 2.23 | 2.41 | 2.26 | 1.88 | 2.79 | 1.55 | 1.66 | 1.58 |
| 32 | 2-Methylbutanal | Slightly fruity, chocolate | — | — | — | — | 0.26 | — | — | — | — |
| 33 | (+)-Citronellal | Lemon | — | — | — | — | — | < 0.10 | — | < 0.10 | 0.11 |
| 34 | Decanal | Sweet | — | — | — | 0.49 | 0.46 | 0.66 | 0.53 | — | — |
| 35 | Piperitone | Minty, fresh | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 | < 0.1 |
| 36 | 1,8-Cineole | Camphor | 12.85 | 16.61 | 17.18 | 19.81 | 25.86 | 36.57 | 24.16 | 27.10 | 25.30 |
The volatile oil at each fruit development stage contained 1−5 flavor compounds that had a modifying effect on the overall flavor (0.1 ≤ ROAV ≤ 1). The volatile substances, α-terpineol at 31 d AF, o-cymene and α-pinene at 39 d AF and 47 d AF, o-cymene, α-pinene, 2-methylvaleraldehyde, decanal, and α-terpineol at 56 d AF, α-pinene, linalyl acetate, 2-methylbutanal, decanal, and α-terpineol at 64 d AF, o-cymene, α-pinene, linalyl acetate, decanal, and α-terpineol at 74 d AF, α-pinene, linalyl acetate, decanal, and α-terpineol at 86 d AF, α-pinene, β-caryophyllene, α-terpineol, and linalyl acetate at 100 d AF, α-pinene, α-terpineol, linalyl acetate, and (+)-citronellal at 115 d AF, had modifying effects on the overall flavor of volatile oils. Therefore, the key flavor compounds in Zanthoxylum bungeanum ‘Hanyuan’ fruit at the different stages were the same, but the ROAVs of each key flavor and the modifying flavor at the different stages were different, resulting in the overall flavor in fruit varying to some extent at the different stages.
PCA of key flavor compounds and odor evaluation in fruit at the different stages
The cumulative contribution of the two principal component factors PC1 (57.6%) and PC2 (25.5%) reached 83.1% (Fig. 8). The comprehensive scores of key odor compounds of volatile oils in fruit at the different stages could be divided into three groups based on the distance on the coordinate axis, in which the first group was 31 d AF, the second group was 39 d AF, 47 d AF, 56 d AF, 64 d AF, and 74 d AF, and the third group was 86 d AF, 100 d AF, and 115 d AF. The compounds in PC1 and PC2 were (E)-β-ocimene and (+)-limonene with the highest positive load, and geranyl acetate and (+)-delta-cadinene with the highest negative load, respectively. (E)-β-ocimene, geranyl acetate, (+)-limonene, and (+)-delta-cadinene contributed the most to the overall odor in Zanthoxylum bungeanum ‘Hanyuan’ fruit; these were the main odors in fruit and could be used to effectively distinguish Zanthoxylum bungeanum ‘Hanyuan’ fruit at the different stages.
Fig. 8.
Scores and loadings of key odor compounds in Z. bungeanum ‘Hanyuan’ fruit at the different stages after flowering (AF)
The comprehensive scores of key odor compounds in fruit at the different stages ranked as 115 d AF > 100 d AF > 86 d AF > 74 d AF > 39 d AF > 64 d AF > 47 d AF > 56 d AF > 31 d AF (Fig. 9).
Fig. 9.
Comprehensive score of odor in Z. bungeanum ‘Hanyuan’ fruit at the different stages
Discussion
Traits and volatile oil content of Zanthoxylum bungeanum ‘Hanyuan’ fruit at the different stages
The fruit longitudinal and transverse diameters, weights of a thousand dry fruits (TDF), pericarps (TDP), and seeds (TDS) were observed to be gradually increasing with maturity, and the fruit traits of Zanthoxylum bungeanum ‘Hanyuan’ at the different stages were significantly (P < 0.05) different in the present study. This was similar to the findings of Ahmet et al. [25]. In addition, there was no significant (P >0.05) difference in the weight of a thousand fresh fruits (TFF) at 74−115 d AF, while the TDF continued to increase during this period and showed significant (P < 0.05) differences among the different stages, indicating that the physiological activity of Zanthoxylum bungeanum ‘Hanyuan’ gradually shifted to solid matter storage and a decrease in water content during the late maturity stage. Meanwhile, the present study also found that the TDP remained relatively stable at 56−115 d AF, and the main reason for the continuous increase in the TDF during this stage was the accumulation of inclusions in the seed. The decrease in water content and the increase in dry matter in the seed not only provide optimal conditions for seed storage and dissemination [26], but also increase the volatile oil content in Zanthoxylum bungeanum ‘Hanyuan’ fruit, which contributes to the improvement of fruit quality in production.
Volatile oil has many flavor compounds in Zanthoxylum bungeanum fruit and is also an important indicator for evaluating the quality. It is mainly located in oil sacs on the fruit surface [23], and the accumulation rate varies depending on the physiological activities in the fruit at different stages. This study found that the TDP significantly (P < 0.05) increased at 31−56 d AF, but there was no significant (P >0.05) change in volatile oil content among these stages. This is possibly due to fruit during this period was young, and the absorbed nutrients were mainly used for fruit enlargement and morphology construction. The content of volatile oils significantly (P < 0.05) increased at 56 −100 d AF and remained stable at 100−115 d AF, indicating that the secondary metabolism in Zanthoxylum bungeanum fruit was dominant during this period, and secondary metabolites continued to accumulate [27]. This was consistent with the research results of Huang et al. [28], who found that the volatile oil content of Zanthoxylum armatum differed significantly (P < 0.05) at the different stages, and increased continuously with the fruit development. This implies that maturity affects the volatile oil content in the fruit of Zanthoxylum bungeanum ‘Hanyuan’; the fruit was almost mature, and the accumulation of volatile oil was also basically completed at 100 d AF. Harvesting the Zanthoxylum bungeanum ‘Hanyuan’ fruit at 100−115 d AF could maximize the volatile oil content, resulting in the best fruit quality, and avoiding the loss of volatile oil caused by excessive ripening. This study provides a scientific basis for determining the suitable harvesting period of Zanthoxylum bungeanum ‘Hanyuan’, which could be beneficial to increase and improve the fruit’s key flavor compounds and economic value, and further increase the market competitiveness.
Odor compounds of Zanthoxylum bungeanum ‘Hanyuan’ fruit at the different stages
The composition and distribution of volatile compounds could provide a theoretical basis for the construction of a sensory quality evaluation system. The flavor quality of prickly ash is influenced by the relative content and proportion of volatile compounds, and the odor is more easily affected by changes in compounds [29]. In the present study, GC-MS was used to determine the total ion chromatogram of volatile oil in Zanthoxylum bungeanum ‘Hanyuan’ fruit at different stages. A total of 65 volatile compounds were detected during the entire development period, mainly including alkenes, alcohols, and esters. The number of alkenes in Zanthoxylum bungeanum ‘Hanyuan’ fruit at every growth stage was dominant in volatile compounds, and most of them were monoterpenes (C10Hn) and sesquiterpenes (C15Hn) with a relative content of over 49% at each stage. The relative content of sesquiterpenes (C15Hn) was only 1.20%−3.12%, indicating that monoterpenes were the main terpenoids in Zanthoxylum bungeanum ‘Hanyuan’ fruit in the present study. This is probably due to the content of monoterpenes in leaf decreasing and the content of monoterpenes in fruit increasing with fruit development, resulting in the monoterpenes in leaf being sustainably transferred to fruit with fruit development [12]. Similarly, it has been found that the relative content of monoterpene hydrocarbons in green fruit pepper accounted for 76% of the volatile oil and was the key flavor compound [30]. Previous research results had shown that the volatile oil of prickly ash was mainly composed of sesquiterpenes, and the immature and mature fruits were rich in monoterpenes and sesquiterpenes, respectively [31]. This further demonstrates the importance of monoterpenes in the fruit flavor of prickly ash. Therefore, the flavor characteristic of prickly ash is closely related to the composition, relative content, and growth stage of the volatile compounds, in which monoterpenes dominate the volatile oil. The results of this study revealed the basic compounds in Zanthoxylum bungeanum ‘Hanyuan’ fruit, and provided a certain reference for improving and optimizing the flavor quality.
The main common compounds in volatile oils include (+)-limonene, linalool, myrcene, β-ocimene, 1,8-cineole, (E)-β-ocimene, α-terpineol, and linalyl acetate, based on the composition and their relative contents. Previous studies on GC-MS analysis of volatile oils found that (+)-limonene, linalool, and linalyl acetate were the main compounds in the volatile oil of Zanthoxylum bungeanum ‘Hanyuan’ fruit [12, 23]. The main compounds in volatile oils of other prickly ash species, such as ‘Dahongpao’ from different producing areas and ‘Shizhitou’ from Hancheng, included β-myrcene, (E)/(Z)-β-ocimene, sylvestrene, eucalyptol, and terpinen-4-ol [16]. This was not entirely consistent with the results obtained in the present study; the difference in species may be one of the important factors affecting the compounds in volatile oils, and the key odor of different prickly ash species is often directly related to the contained volatile substances [24]. In addition, environmental factors such as climate conditions [32], soil fertility, and cultivation management [10, 33] may also cause changes in volatile substances of prickly ash from different producing areas and result in a unique odor [34].
The relative content of compounds in volatile oils of Zanthoxylum bungeanum ‘Hanyuan’ fruit varied greatly at the different stages in this study, and the relative contents of alkenes dominated by (+)-limonene and myrcene were always the highest, the represented substance of esters (e.g., linalyl acetate) and alcohols (e.g., linalool) increased and decreased continuously, respectively, with the fruit development. Zhu et al. [34] studied the volatile substances in young, semi-mature, and mature fruits of Zanthoxylum bungeanum and found that the relative content of myrcene and linalyl acetate increased with fruit maturity, which was consistent with the results of the present study. However, Devi et al. [35] studied the changes of volatile substances in Zanthoxylum armatum fruit at different stages and found that the represented alcohols (e.g., linalool) gradually accumulated with the fruit maturity. This may be due to the differences caused by different species and growing regions of prickly ash. In addition, linalool has the functions of anti-bacteria, anti-tumor, and analgesic [36], and is a raw material for the synthesis of linalyl acetate [37]. Therefore, the possible reason for the decrease in the relative content of linalool was that the linalool in Zanthoxylum bungeanum ‘Hanyuan’ fruit continuously converted to linalyl acetate, resulting in the relative content of linalool decreasing.
Key flavor and odor evaluation of Zanthoxylum bungeanum ‘Hanyuan’ fruit at the different stages
Alkene compounds often have the odor of woody, citrus, camphor, and lemon [35], and prickly ash usually has a unique odor due to a large number of alkene compounds working together. Linalool and linalyl acetate were the main compounds in the essential oil of lavender, both with similar flavors and the same odor of spicy, citrus, floral, and woody [38]. A previous study found that not every identified compound in volatile oil contributed to the odor of prickly ash; only the compounds that can be smelled during gas chromatography-olfaction (GC-O) analysis and whose content is higher than the threshold are the ones that truly contribute to the odor of prickly ash [20]. In the present study, the key flavor compounds in Zanthoxylum bungeanum ‘Hanyuan’ fruit at the different stages determined by the ROAV method were linalool, (+)-limonene, myrcene, 1,8-cineole, (+)-delta-cadinene, β-ocimene, (E)-β-ocimene, and geranyl acetate. Therefore, those compounds in volatile oils had an important impact on the key flavor and odor in Zanthoxylum bungeanum ‘Hanyuan’ fruit.
Extraction and analysis methods for volatile oils may have certain impacts on their compounds. Feng et al. [39] found that linalool, linalyl acetate, d-limonene, and myrcene were the key odors of prickly ash by using HS-SPME-GC-MS combined with partial least squares discriminant analysis (PLS-DA), while acetone, 1,8-cineole, isoamyl acetate, isobutyl acetate, α-terpineol, linalool, caryophyllene, and geraniol were the key odor compounds of prickly ash by using GC-IMS combined with a PLS-DA model. Zhao et al. [40] found that myrcene, (+)-limonene, (E)-β-ocimene, β-cubebene, germacrene D, cineole, linalool, and linalyl acetate were the main odor compounds in Zanthoxylum bungeanum ‘Hanyuan’ fruit by using solid-phase microextraction (SPME) combined with gas chromatography-olfactory mass spectrometry (GC-O-MS) and full two-dimensional gas chromatography-olfactory mass spectrometry (GC×GC-O-MS). In the present study, linalool, (+)-limonene, and myrcene were observed as the main odor compounds in Zanthoxylum bungeanum ‘Hanyuan’ fruit by using steam distillation and GC-MS analysis. This was not entirely consistent with previous research results [39, 40]. The reason for this difference may be due to the influence of different extraction and analysis methods on the retention and detection accuracy of odor in volatile oils of prickly ash [41].
Terpenes are key factors determining the aromatic quality of prickly ash, and fruits at the different stages have different aromatic characteristics, volatile compounds, and odor intensities [31]. In the present study, the cluster analysis on compounds detected at every stage was conducted, combined with principal component analysis (PCA) on key flavor compounds to better classify and evaluate the odor in Zanthoxylum bungeanum ‘Hanyuan’ fruit; both analysis results were inconsistent. This indicates that the key odor compounds could accurately characterize the flavor attribute of the samples and are the key aromatic active substances that lead to differences in flavor of prickly ash at different stages [20]. The PCA results showed that (E)-β-ocimene, geranyl acetate, (+)-limonene, and (+)-delta-cadinene were key aromatic active substances with the most obvious changes in relative content at every stage. The highest comprehensive score of odor evaluation in Zanthoxylum bungeanum ‘Hanyuan’ fruit during the development period was 115 d AF, which had the strongest key flavor, followed by 100 d AF. Both cluster analysis and PCA grouped the odor compounds at 100 d AF and 115 d AF into a group, indicating that there was no obvious difference in volatile substances, and the flavor attributes were similar. The odor in Zanthoxylum bungeanum ‘Hanyuan’ fruit at the different stages varied due to differences in the relative content of key substances in volatile oils, resulting in differences in their flavor attributes, and the highest flavor intensity in fruit was observed at 100−115 d AF. Therefore, the fruit of Zanthoxylum bungeanum ‘Hanyuan’ could be considered to start to harvest from 100 d AF to obtain the products with higher flavor intensity. The ROAV can be used to estimate the key odor compounds. In addition, the highest total ROAV of all odor compounds was observed at 115 d AF, the 100 d AF ranked second, and it increased with the fruit development time until it reached the peak (Table 2). This further indicated 100−115 d AF was the suitable harvest time, and harvest the fruit of Zanthoxylum bungeanum ‘Hanyuan’ at this period can obtain optimal quality. Thus, the ROAV can also be used to estimate the quality of Zanthoxylum bungeanum ‘Hanyuan’ fruit. Both GC-MS [42] and GC-FID [43, 44] methods can be used to determine volatile oil compounds; the former and the latter can identify and quantify the components, respectively. The GC-MS and GC-FID methods can be combined for future research. This study provided the scientific basis for the determination of a suitable harvest period by analyzing the volatile compounds changes at the different stages, which has important meaning for improving the flavor quality and industrial value of Zanthoxylum bungeanum ‘Hanyuan’ fruit.
Conclusion
All fruit traits of Zanthoxylum bungeanum ‘Hanyuan’ showed obvious changes with maturity, and these were significantly (P < 0.05) impacted by the fruit development stages. The volatile oil content in the fruit increased with maturity and varied greatly at the different stages. The accumulation of volatile oil was almost completed at 100 d AF. A total of 65 compounds were identified in the volatile oils of Zanthoxylum bungeanum ‘Hanyuan’ fruit during the development, including 25 common compounds and 40 non-common compounds. The main compounds in volatile oils were alkenes, esters, and alcohols. The compounds and the relative content in fruit at the different stages were varied, and linalool, (+)-limonene, myrcene, 1,8-cineole, (+)-delta-cadinene, β-ocimene, (E)-β-ocimene, and geranyl acetate were key flavor compounds in Zanthoxylum bungeanum ‘Hanyuan’ fruit. The changes in the relative content of key flavor compounds resulted in different flavors in Zanthoxylum bungeanum ‘Hanyuan’ fruit during the development period, and the comprehensive score of odor evaluation was the highest at 115 d AF. This research plays an important role in determining the optimal harvest time to obtain a superior quality of Zanthoxylum bungeanum ‘Hanyuan’ fruit.
Acknowledgements
This study was conducted at the College of Forestry, Sichuan Agricultural University. The authors would like to extend their gratitude to the teachers for their valuable support in this research, and the manager for taking care of the research base to sample the plant material.
Authors’ contributions
Wei Gong, Jingyan Wang, and Wenkai Hui have supervised related experiments and revised the manuscript. Jing Qiu, Shuaijie Lu, Yafang Zhai, and Jing Xv performed the experiments. Jing Qiu and Jiayuan Gong have compiled the data and written the manuscript. All authors have read and approved the manuscript.
Funding
This work was supported financially by the Projects of Science and Technology Department of Sichuan Province (Grant No. 2021YFYZ0032, 2016NYZ0035), the National Key Research and Development Program of China (Program No. 2020YFD1000700, 2018YFD1000605), the Sichuan Chinese Prickly Ash Innovation Team Project of National Modern Agricultural Technology Systems (SCCXTD-2024-23), and the Creating National Modern Agricultural Industry Science and Technology Innovation Center in Ya'an City of China (kczx2023-2025-03).
Data availability
All datasets generated for this study are included in the article.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Jing Qiu and Yuanjia Gong contributed equally to this work.
Contributor Information
Wei Gong, Email: gongwei@sicau.edu.cn.
Wenkai Hui, Email: wkxi@sicau.edu.cn.
Jingyan Wang, Email: wangjingyan@sicau.edu.cn.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All datasets generated for this study are included in the article.