Characterization of the Key Aroma Compounds in the Fruit of Litsea pungens Hemsl. (LPH) by GC-MS/O, OAV, and Sensory Techniques

,e key aroma compounds in the fruit of Litsea pungens Hemsl. (LPH) were concentrated through solvent-assisted flavor evaporation (SAFE) and characterized by gas chromatography-mass spectrometry-olfactometry (GC-MS/O), quantitative descriptive analysis (QDA), odor activity values (OAVs), and addition test. ,e results showed that LPH contained 31 aroma-active compounds (flavor dilution, FD� 9). Among them, 30 odorants were quantified by the standard curve method.,e OAV analysis results showed that 25 odorants had OAVs≥ 1, which could be considered as the potent odorants. D-Limonene and 3,7-dimethyl2,6-octadienal had the highest OAVs (OAV� 9803 and 8399), followed by (Z)-3,7-dimethylocta-2,6-dienal (OAV� 1893), β-myrcene (OAV� 1798), (E)-3-phenyl-2-propenoic acid ethyl (OAV� 1603), and β-caryophyllene (OAV� 1129). Addition experiments further confirmed that 3,7-dimethyl-2,6-octadienal, (Z)-3,7-dimethylocta-2,6-dienal, and D-limonene contributed to lemon attribute, β-myrcene contributed to green attribute, citronellal contributed to mint and fresh note, and eucalyptol contributed to eucalyptus-like note were the key odorants.


Introduction
Litsea pungens, as a genus belongs to Lauraceae's family, is an evergreen or deciduous tree or shrub with about 200 species distributed worldwide (mainly distributed in tropical and subtropical regions of Asia and America) [1]. In China, there are 72 species of Litsea pungens Hemsl. (LPH) distributed in 20 provinces (Figure 1). Among them, Yunnan Province has the most species (37), followed by Guangdong (24), Sichuan (18), Guizhou (15), and Hunan Province (12). e fruit, root, branch, and leaves of LPH have a wide range of applications in traditional Chinese medicine, fragrance industry [2,3], cosmetics industry, and food industry due to the functional compounds existing inside, such as the aromatic compounds (essential oil), flavonoids, terpenoids, butanolides, and butenolactones steroids, lignans, amides, and alkaloids. e LPH, rich in citral, is an important raw material for ionone and damascene [4,5]. Due to the large and broad market demand, several decades ago, LPH species had been industrially cultivated, especially in Yunnan, Hubei, Hunan, Sichuan, Chongqing, and Guizhou provinces of China. e therapeutic effects of LPH include removing dampness, regulating spleen deficiency, helping digestion, dysmenorrhea, expelling cold, and analgesia. All these benefits have been widely recorded in ancient Chinese medicine books such as "Guizhou folk medicine," "Chongqing Herbs," and "Hunan yaowuzhi." Modern molecular biology technologies have also elucidated that the functional compounds in the fruits, roots, branches, and leaves of LPH have anti-inflammatory activity, antimicrobial activity, hepatoprotection, antidiabetic, antiasthma activity, anticholelithiasis activity, immunomodulation, and miscellaneous bioactivities [3]. is information confirmed the abundance of pharmacological properties of LPH. According to the Chinese natural spices classification standard (GB/T 21725-2017), LPH with a strong aroma is characterized as one of the 20 pungent type spices [20,21]. Based on its unique characteristics of citrus, sweet lemon, camphor wood, and green aroma, LPH is also a vital fragrance applied to many southwestern Chinese dishes, such as cooking beef or lamb, chili sauce, and pickled vegetables, especially the traditional Zhijiang Blood Duck (Huaihua, Hunan province) [6][7][8].
e LPH is an important pharmaceutical/food resource; therefore, elucidating the flavor chemistry of LPH is meaningful to the application of standardized LPH.
e objectives of this work are to identify the key aroma compounds in LPH's fruit by (1) isolating the volatile compounds from LPH's fruit by SAFE; (2) characterizing the aroma-active compounds by gas chromatography-mass spectrometry-olfactometry (GC-MS/O); and (3) quantifying the aroma-active compounds and calculating their OAVs. (4) Confirming the key odorants by addition experiment.

Sensory Evaluation.
Quantitative descriptive analysis (QDA) was used to evaluate the aroma profiles of LPH's fruit. Twelve panelists with no rhinitis and no smoking (6 females and 6 males, age of 22-30) were recruited from our laboratory. e sensory evaluation room temperature was 23∼25°C, the humidity was 50∼55%, and filament lamp (36 W) was used. All panelists were informed of the aim, detailed experimental steps, and requirements of sensory evaluation before participating in this experiment. ey were trained for 3 weeks before the QDA analysis: (1) all panelists were requested to sniff and describe the aroma characteristics of 54-aroma kit (Le Nez du Vin ® , France) with 3 times a week (each training lasted for 30 min); (2) then, panelists were requested to analyze the aroma profiles of LPH's fruit descriptively. e final 6 aroma attributes (lemon, floral and sweet, mint and fresh, green, eucalyptuslike, and sour) were determined according to the frequency of descriptors, and their corresponding referenced standards were D-limonene, nerol, menthol, 1-hexanol, 1,8-cineole, and propionic acid, respectively; (3) finally, 12 panelists were qualified to score the intensity of 6 aroma attributes on a scale from 1 to 9 (1-3, weak; 4-6, medium; 7-9, strong). e LPH's fruit sample (4.00 g) loaded in 200 mL transparent glasses was presented to the panelist.

Isolation of the Volatile Compounds by SAFE.
Dried LPH's fruit (20.00 ± 0.20 g) and dichloromethane solvent (80 mL) were loaded in a conical flask (250 mL) and extracted for 15 min by ultrasonication (KH-500 DE, Jiangsu, China) in 500 W at 10°C. en, the organic phase was collected after filtration. After 3 extractions, the collected solvents were combined and submitted to the SAFE apparatus for volatile isolation. Isolation of the volatile compounds from the solvents was reference from our previous work with some modifications [25,26]. e recycled water in SAFE apparatus was (40 ± 1)°C; the distillation flask was bath at (40 ± 1)°C; the collection flask was immersed in liquid nitrogen; the extraction system was operated under vacuum (10 −5 ∼10 −6 Pa) via molecular turbine pump (Edwards, England), and the filtrate was added dropwise to the distillation flask. en, the extract was concentrated to 1∼2 mL with rotary evaporation instrument (EYELA N-1100, Tokyo Physical and Chemical Equipment Co., Ltd, Japan) after drying with anhydrous sodium sulfate. Finally, the concentrate was reduced to 1.00 mL by nitrogen (99.99%) before GC-MS and GC-MS/O analysis. All analyses were repeated in triplicate.

GC-MS and GC-MS/O Analysis.
e identification and quantification of the aroma compounds were conducted by a single quadrupole gas chromatograph-mass spectrometer (GC-MS) ( ermo Fisher Trace 1310, ermo Fisher Technology Co., Ltd, USA) in a split ratio of 50 : 1 (optimized in our lab). e aroma-active compounds were screened by GC-MS equipped with a sniffing port (ODP3, Gerstel, Germany) (GC-MS/O). e temperature of sniffing port was 220°C, and the humidifier with flow rate of 10 mL/min (nitrogen, 99.999%) was used to humidify the air at sniffing port. e GC effluent was split at a ratio of 1 : 1 between the MS and sniffing port for the GC-MS/O's special structure in splitless injection. Separation of the aroma compounds in LPH's fruit extract was achieved on TG-5MS and TG-WAX columns (both 30 m × 0.25 mm i.d. × 0.25 μm, ermo Fisher). Helium (99.999%) was the carrier gas, and the carrier gas flow rate was constant at 1.200 mL/min and 2.000 mL/min in GC-MS and GC-MS/O, respectively. e oven temperature of TG-WAX column analyzer was initially held at 40°C for 2 min, increased to 100°C (temperature rise rate, 4°C/min) and held for 1 min, and then increased to 175°C (temperature rise rate, 2°C/min) and held for 1 min, finally increased to 230°C (temperature rise rate, 5°C/min). e oven temperature of TG-5MS column analysis was initially held at 40°C, then increased to 100°C (temperature rise rate, 3°C/min), increased to 170°C (temperature rise rate, 1°C/min) and held for 1 min, and finally increased to 230°C (temperature rise rate, 5°C/min). e temperature of the sniffing port was kept at 230°C. e injector temperature was 250°C, and the ion source temperature was 280°C. e electronic-impact mass spectra ionization mode with ionization energy of 70 eV was used. e full scan mode (m/z range from 40 to 350 amu) was used.

Gas Chromatography-Olfactometric (GC-O) Analysis.
e aroma frequency, combined with the aroma dilution method, was used in the GC-O study. Firstly, the concentrated organic extract was diluted to 1 : 9 with dichloromethane solvent. e diluted sample was then submitted to the GC-MS/O with the TG-WAX column to screen the aroma-active compounds with flavor dilution (FD) factor over 9. e diluted sample was repeated 3 times by 3 trained panelists. Only the aroma compounds detected over 5 of 9 were recorded. Panelists underwent GC-MS/O training by sniffing 31 standards aroma compounds in dichloromethane solvent (1,000 μg/L) three times before this experiment.

Identification and Quantification.
e identification of the aroma compounds was based on comparing the mass spectra (MS) database NIST 2020, with retention indexes (RIs, on nonpolar and polar GC columns), pure standards (S), and the odor characteristics (O). All quantifications of key odorants were performed by constructing standard curves. e abscissa was referred to the ratio of the peak area of each compound to the three internal standards (1,2-dichlorobenzene, 2,500 µg/mL; 2-octanol, 2,900 µg/mL; 3methylacetophenone, 3,000 µg/mL) that are obtained by GC-MS and the ordinate was the concentration ratio of aroma compounds to the three internal standards [29]. Each quantified aroma compound referenced from the specifically internal standard was labeled in Table 1. Each of the internal standards (100 µL) was added when LPH's fruit was extracted by dichloromethane solvent.

Calculation of the Odor Activity Value (OAV).
e OAVs of aroma-active compounds were measured by dividing their concentration detected in the LPH's fruit sample by their odor threshold detected in water. Each threshold value was referenced from the corresponding literature studies and book, which labeled in Table 1 [30-33, 36, 37]. e aroma-active compounds with OAV ≥ 1 are considered to be the potent key odorants of LPH fruit.

Addition Experiment.
e addition tests were conducted to validate the potent odorants and elucidate their specifically contributions with high OAV to LPH's fruit sample by adding the aroma compounds to the LPH's fruit sample (5.00 g) based on the detected concentration. Two original LPH's fruit samples (5.00 g) and one aroma added sample were subjected to the panelists. Panelists were requested to evaluate the difference by triangle tests and quantitative descriptive analysis (Section 2.3) [38] as shown in Table 2.

Sensory Evaluation.
e QDA result data of LPH's fruit were plotted on a spider diagram shown in Figure 2, suggesting that lemon note was the strongest, followed by floral and sweet, mint and fresh, eucalyptus-like, and green characteristics. e sour note of LPH's fruit had the lowest intensity. is result also elucidated the LPH's fruit had a potent flavor enhancer or improving ability.

Conclusions
e aroma compounds in LPH's fruit were isolated by SAFE. By application of frequency combined with aroma dilution analysis, 31 aroma-active compounds were detected, and 30 of them were further quantified by the external standard method. OAVs ≥ 1 were obtained for 25 odorants among which D-limonene (OAV � 9803) and 3,7-dimethyl-2,6-octadienal (OAV � 8399) had the highest OAV value.
ese results elucidated that they played an important role to the overall aroma profiles if LPH's fruit. Based on the addition tests, 3,7-dimethyl-2,6-octadienal and (Z)-3,7-dimethylocta-2,6-dienal and D-limonene contributing to lemon attribute, β-myrcene contributing to green attribute, citronellal contributing to mint and fresh note, and eucalyptol contributing to eucalyptus-like note were confirmed as the key odorants in LPH's fruit.

Data Availability
All the date used in this work could be found in the manuscript and the supplemental materials.

Conflicts of Interest
e authors declare that they have no conflicts of interest.