Aromatic Characterization of Mangoes (Mangifera indica L.) Using Solid Phase Extraction Coupled with Gas Chromatography–Mass Spectrometry and Olfactometry and Sensory Analyses

Mangoes (Mangifera indica L.) are wildly cultivated in China with different commercial varieties; however, characterization of their aromatic profiles is limited. To better understand the aromatic compounds in different mango fruits, the characteristic aromatic components of five Chinese mango varieties were investigated using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry-gas chromatography-olfactometry (GC-MS-O) techniques. Five major types of substances, including alcohols, terpenes, esters, aldehydes, and ketones were detected. GC-O (frequency detection (FD)/order-specific magnitude estimation (OSME)) analysis identified 23, 20, 20, 24, and 24 kinds of aromatic components in Jinmang, Qingmang, Guifei, Hongyu, and Tainong, respectively. Moreover, 11, 9, 9, 8, and 17 substances with odor activity values (OAVs) ≥1 were observed in Jinmang, Qingmang, Guifei, Hongyu, and Tainong, respectively. Further sensory analysis revealed that the OAV and GC-O (FD/OSME) methods were coincided with the main sensory aromatic profiles (fruit, sweet, flower, and rosin aromas) of the five mango pulps. Approximately 29 (FD ≥ 6, OSME ≥ 2, OAV ≥ 1) aroma-active compounds were identified in the pulps of five mango varieties, namely, γ-terpinene, 1-hexanol, hexanal, terpinolene trans-2-heptenal, and p-cymene, which were responsible for their special flavor. Aldehydes and terpenes play a vital role in the special flavor of mango, and those in Tainong were significantly higher than in the other four varieties.


Introduction
Mango (Mangifera indica L.), a native crop of South Asia, is a member of the Anacardiaceae family [1] and has been historically grown for more than 4000 years [2], thereby earning the title "king of fruits". It is recognized as one of the most popular fruits around the world, with the highest rates of production, marketing, and consumption [3,4]. Among tropical fruits, mango is the second most common crop involved in international trade, following banana. Global mango production has been estimated at 50.65 million tons, with China being the second largest mango-growing country, 2017 production reaching close to 4.94 million tons [5]. Common mango cultivars in China featuring specific regional characteristics include Guifei, Hongyu Jinmang, Qingmang, and Tainong.
Aroma is a major factor that influences the quality and consumer acceptance of mango products. Investigating various aromatic components would improve our understanding and facilitate controlling critical quality parameters that could influence mango processing. Hundreds of compounds have been characterized in various mango cultivars, which mainly include aldehydes, alcohols, esters, ketones, and terpenes [6]. However, previous studies have mainly focused on the volatiles in various mangoes in China [7,8], and a few volatiles were detected because of their odor threshold. Thus, scientific information relating to aromatic constituents as well as sensory characteristics of various mango cultivars is limited. Therefore, an in-depth investigation is required to identify the volatile or aromatic components of various mango cultivars in China.
To determine the aromatic components of mango, simultaneous solvent-assisted flavor evaporation (SAFE), solid phase microextraction (SPME), and distillation and extraction (SDE) have been employed in food aroma extraction [9][10][11][12]. The procedures of SDE and SAFE isolate aromatic compounds from food matrices using organic solvents [13]; however, these methods are highly laborious, time-consuming, and entail preconcentration of extracts. Unlike well-established protocols, SPME has been extensively used in the preparation of volatile and semi-volatile compounds from various types of samples [14]. This technique was developed more than two decades ago and is a rapid, simple, sensitive, and solvent-free technique for the analysis of volatile organic compounds (VOCs) [15]. Coupling of the methods of aroma extraction with the GC-MS/O technique, in particular, headspace solid-phase microextraction (HS-SPME) for extraction, together with detection frequencies (FD) and order-specific magnitude estimation (OSME) for GC-MS/O, creates a highly reliable method of identifying potent odorants.
Thus, to fully understand the aromatic compounds present in typical mango varieties, this study conducted the following studies: (1) identification and quantification of volatiles of different types of mangoes by HS-SPME-GC-MS; (2) discrimination of the major aroma-active compounds in various types of mangoes using combined GC-O detection (FD, OSME) and odor activity value (OAV); and (3) validation of the sensory differences using quantitative descriptive analysis (QDA). In summary, objectives of this investigation were to reveal the major aromatic compounds of mangoes in China.

Samples Preparation
This study used seven mature samples of the cultivars Jinmang (JM), Qingmang (QM), Hongyu (HY), Guifei (GF), and Tainong (TN), which were purchased from the regional market in Guangzhou (133.35 • N, 23.12 • E), Guangdong Province (April 2019). The samples were shipped to the laboratory and kept at 25 • C until complete maturity (2 days). Full maturity and maturity of the mangoes were based on fruit color (green to yellow-orange or red, except for the green lawn, Table S1), odor (sweet scent), and hardness (pulp hardness index changed from 5.28 to 4.32 N). Finally, the mango samples with the same maturity were washed, and the peeled pulp was immediately frozen in liquid nitrogen, and then stored at −80 • C for further studies.

HS-SPME-GC-MS
Based on previous studies, the optimized SPME experimental conditions were established [16][17][18]. Approximately 5.0 g of the juice with 1.5 g of NaCl were blended in a 15 mL vial tightly capped with a PTFE-silicon septum at a stirring speed of 80 rpm. The flavor compounds in mango pulp are formed during equilibrium and during the extraction process. Therefore, the extraction temperature was set at 40 • C. After the vial containing the sample was equilibrated at 40 • C for 10 min on a heating platform agitation, the pretreated (conditioned at 270 • C for 30 min) SPME fiber (50/30 µm DVB/Carboxen/PDMS, Supelco, Bellefonte, PA, USA) was then inserted into the headspace, and extraction was performed for 30 min with continued heating and agitation. Afterward, the fiber was withdrawn and instantly introduced to the GC for desorption and analysis.
GC-MS analysis was performed on an Agilent 7890 (Agilent Technologies, Palo Alto, CA, USA) gas chromatography and Agilent 5977 mass selective detector. Samples were separated using both HP-5 and DB-WAX (both 30 m × 0.25 mm i.d., 0.25 mm film thickness, Agilent Technologies, Palo Alto, CA, USA). Helium was used as carrier gas at a flow rate of 1.7 mL/min, and the GC inlet was set in the split-less mode. The injector temperature was 250 • C. The temperature program was from 40 • C (2 min hold) to 160 • C at 4 • C/min and finally raised to 280 • C at 50 • C/min. Then, electron ionization mode (EI) was used with a 70 eV ionization energy. The ion source temperature was 230 • C, and the mass range was from m/z 35 to 450. The volatile compounds were determined by authentic standards, retention indices (RI), and NIST 14.0 library. The retention indices (RIs) of compounds were determined via sample injection with a homologous series of straight-chain alkanes (C6-C30) (Sigma Aldrich, St. Louis, MO, USA).

Identification of Aroma-Active Compounds by GC-O
The odorant compounds were analyzed using a sniffing port (ODP3, Gerstel, Germany) coupled with a GC-MS (7890B-5977B, Agilent Technologies, Inc.). Upon exiting the capillary column, the effluents were divided to a ratio of 3:1 (by volume) into a sniffing port as well as an MS detector using an Agilent capillary flow technique. The transfer line directed to the GC-O sniffing port was set at a temperature of 270 • C. The GC-MS settings were similar to those described earlier. Aroma extraction was conducted by four highly skilled personnel (in an alternate order of 50 min intervals) using reference compounds. All personnel were extensively trained on the GC-O technique for at least 90 h. Frequency analysis was conducted by four trained sensory panelists (i.e., two males and two females). Retention time and odor quality, together with substance detection, were recorded. Frequency analysis was performed in duplicate by every panelist. Odorants with an FD ≥2 (determined by at least two analysts) were considered to have potential aroma activity [19].
The OSME reflected the aromatic intensity of the stimulus that was based on a five-point scale that ranged from 0 to 5, where 0 = none, 3 = moderate, and 5 = extreme. Every sample was sniffed thrice by each panelist, and then the average aromatic intensity values were calculated. If the panelists did not utilize a similar attribute for an aroma that was eluted by GC, the analysis was repeated, and only the descriptors used for the same aroma were included in the analysis [20].

Quantitative Analysis of Aromatic Compounds
Quantitative data on the identified compounds were gathered by calculating their relative quantitative correction factors (RQCFs) with the "single-point correction method", which is similar to the standard addition method. Similar GC conditions as described above were used in GC-MS analysis, with solvent delay time set at 3 min to prevent the solvent in the standard solutions to reach the filament. The method of obtaining RQCFs consisted of the following: 5.0 g of mango pulp was analyzed using SPME-GC-MS, resulting in an ion peak area for each identified compound. A similar volume of mango pulp with defined amounts of various authentic and internal standards (to avoid run-to-run variations) was then analyzed to generate a new quantifying ion peak for each detected compound and quantitative correction factor for the internal standard. The RQCF of each volatile was generated using the following equation: where f i was the RQCF of a detected compound (i); m s and m i were the respective known contents of authentic (i) and internal standard (s); A s was the peak area of the quantifying ions of (s); and A i was the peak area of the quantifying ions of (i) before and after the addition of the standard solution to the juice sample.
To determine the amounts of the identified volatiles in mango pulp, approximately 5.0 g of juice per volume containing a similar amount of internal standard as that of the calculated RQCF was prepared and used in GC-MS analysis. The concentration was computed using the equation: where m i was the amount of compound (i); m s was the known amount of (s), A i and A s were the respective peak areas of the quantifying ions of detected compound (i) and internal (s) standards; and f i was the RQCF of (i). The peak area of the quantifying ion of every component in selected ion chromatograms was assessed in triplicate, and the average value was computed. Then, the concentration of every identified volatile in mango pulp is described in nanograms per milliliter of juice [19].

Odor Activity Value (OAV)
OAV pertains to the concentration of the odor divided by its threshold in water. Compounds with an OAV ≥1 were considered as major contributors to the aromatic profile of each sample [21].

Sensory Panel and Aroma Profile Analysis
Aromatic profiling was performed using descriptive sensory analysis, as earlier described [22]. The mango pulps were analyzed by a highly skilled panel of 10 members consisting of five males and five females. Prior to quantitative descriptive analysis, 50 mL of mango pulp was placed in a 100 mL cubage of a plastic cup with a Teflon lid, which was handed over to a panelist in the laboratory without peculiar smell at a temperature of 25 • C. Then, the panelists assessed the aromatic profile of the mango pulp using three preliminary sessions (each spent approximately 2 h), until all of them attained a consensus as to the degree of aromatic flavor. Then, the organoleptic characteristic descriptors were assessed using eight sensory features (i.e., "overall aroma", "tropical fruit", "citrus", "floral", "fresh", "rosin", "honey/sweet", and "green") to evaluate aroma negative and positive mango pulp features. The descriptors were described as the following odors: linalool for the "floral" descriptor, β-phellandrene for "citrus", butyl acetate for the "fruity" descriptor, β-damascenone for the "honey/sweet" descriptor, phenylacetaldehyde for "fresh", (E)-2-hexenal for "green and grassy", and terpinolene for "rosin". The complete profile of each sample was randomly assessed in triplicate for each treatment. The assessors then rated the odor intensities a seven-point scale ranging from 0 to 3, with 0 as not perceivable, 1 as weak, 2 as significant, and 3 as strong. The results were then averaged for every odor note and plotted using a spider web diagram. Figure 1 shows that the five mango pulps had similar aromatic intensities, and these can be divided into six aroma attributes, including tropical fruit, flower aroma, sweet aroma, green grass aroma, green melon aroma, wood aroma, and rosin aroma, although to different extents. SPSS was used to distinguish differences between the mango samples through sensory evaluation scores (Table S2). In most cases, the five mango pulps exhibited significant differences in intensity of fruit aroma (p < 0.05), with some exceptions for "tropical fruit" and sweetness between JM and TN, tropical fruit between GF and QM, green between JM and HY, melon between GF and TN, and wood between JM and GF, where no significant difference was observed (p > 0.05). In general, innate differences among the mango varieties significantly influenced the intensities of most of the key sensory attributes. The overall (decreasing) order of flavor scores was: TN > GF > HY > JM > QM. The intensity of fruit aroma was highest in both GF and QM, followed by TN and JM. The intensity of flower aroma was highest in both GF and HY, followed by QM and JM. TN had the lowest. GF and HY had the highest intensity of sweet aroma, followed by TN, QM, and JM. TN had the highest intensity of green grass aroma, followed by GF and QM. The intensity of green grass aroma in JM and HY was almost identical. The intensities of green melon aroma in QM, GF, and TN were markedly higher than those of JM and HY. TN had the highest intensity of green melon aroma. In addition, the intensity of wood aroma in GF and TN was higher than in JM, QM, and HY. TN had the highest intensity of wood aroma.

Sensory Analysis of Five Mango Cultivars
In general, GF had the strongest flower and sweet aromas, HY had the strongest fruit aroma, and TN had the strongest green grass, green melon, wood, and Rosin aromas. According to Bonneau [9], different mango varieties significantly influenced the intensities of key sensory attributes of mango, which was due to the different amounts of aroma-active components in mango.

Comparasion of the GC-MS Results of Five Mango Cultivars
The MS and RI results preliminarily identified 47 volatile compounds ( Figure 2). There were 25, 24, 24, 29, and 23 volatile compounds in JM, QM, GF, HY, and TN, respectively. These were generally composed of alcohols, alkenes, aldehydes, esters, ketones, and ethers. Most of the volatiles found in this study were similar to those in the findings of previous studies [1,6,23]. Six compounds, namely, pcymen-8-ol, 2-(vinyloxy)ethanol, 1-methyl-4-(1-methylethyl)-1,3-cyclohexadiene, 3-methyl butanal, 2cyclohepten-1-one, and tetraethyl orthosilicate, were first observed in the volatile composition of mango, although those were not the major contributors to the mango aroma. This difference might be The overall (decreasing) order of flavor scores was: TN > GF > HY > JM > QM. The intensity of fruit aroma was highest in both GF and QM, followed by TN and JM. The intensity of flower aroma was highest in both GF and HY, followed by QM and JM. TN had the lowest. GF and HY had the highest intensity of sweet aroma, followed by TN, QM, and JM. TN had the highest intensity of green grass aroma, followed by GF and QM. The intensity of green grass aroma in JM and HY was almost identical. The intensities of green melon aroma in QM, GF, and TN were markedly higher than those of JM and HY. TN had the highest intensity of green melon aroma. In addition, the intensity of wood aroma in GF and TN was higher than in JM, QM, and HY. TN had the highest intensity of wood aroma.
In general, GF had the strongest flower and sweet aromas, HY had the strongest fruit aroma, and TN had the strongest green grass, green melon, wood, and Rosin aromas. According to Bonneau [9], different mango varieties significantly influenced the intensities of key sensory attributes of mango, which was due to the different amounts of aroma-active components in mango.

Identification of Key Aromatic Compounds in the Pulps of Five Mango Cultivars
Not all volatile substances in mango contribute to its aroma, and thus aromatic intensity identification was conducted to determine whether the high content or unique volatile components in mango contribute to its overall aroma. The frequency-of-detection (FD) method requires the evaluator to smell the same aromatic substance and record the peak time and aromatic properties of the aromatic substance. The more times the aromatic substance was detected, the greater the contribution to the

Identification of Key Aromatic Compounds in the Pulps of Five Mango Cultivars
Not all volatile substances in mango contribute to its aroma, and thus aromatic intensity identification was conducted to determine whether the high content or unique volatile components in mango contribute to its overall aroma. The frequency-of-detection (FD) method requires the evaluator to smell the same aromatic substance and record the peak time and aromatic properties of the aromatic substance. The more times the aromatic substance was detected, the greater the contribution to the overall aroma. The intensity (OSME) method was a GC-O detection method that was used to evaluate flavor contribution based on the odor intensity of the aroma substance. After the aromatic substances are separated by GC-MS, the method directly describes the change in odor intensity (measured) and the frequency of aroma attributes. It is considered to be the simplest and most effective GC-O analysis method because it is less time-consuming and less demanding on assessors. Thirty-three characteristic aromatic components were identified in the five mango pulps by GC-O combined with FD and OSME methods, including three alcohols, 10 aldehydes, 11 terpenes, three esters, and four ketones. The differences in aromatic components in the pulp of five mango cultivars were not significant.
Further analysis, the FD analysis identified 23 aromatic substances (FD ≥ 2) in JM. According to the FD statistical data of each substance in the Table 2, one of the substances (FD = 8) was identified in all of the tests: γ-terpinene with citrus aroma. The substances with FD = 7 were phellandrene (citrus-like aroma) and γ-octanoic (floral, violet aroma). The substances with FD = 6 were ethyl cyclopropanecar-boxylate (fruit aroma) and terpinolene (rosin aroma). In QM, 18 aromatic substances with FD ≥2 were identified. According to the FD statistical data of each substance in the table, three kinds of substances (FD = 8) were identified in all of the tests, including 1-penten-3-one, 3-hexenal, and terpinolene. 1-penten-3-one had mushroom-like aroma, 3-hexenal had a grassy aroma, and terpinolene had a rosin-like aroma. The substance with FD = 7 was β-pinene, which has a green-grass-type aroma. The substances with FD = 6 were p-cymene (citrus and green aroma) and γ-terpinene (citrus and lemon-like aroma). In GF, 18 aromatic substances with FD ≥2 were identified. According to the FD statistical data of each substance in Table, 2 substances with FD = 8 were identified in all of the tests: ethyl cyclopropanecarboxylate (fruit aroma) and γ-terpinene (citrus and lemon aroma). The substances with FD = 7 were 3-hexenal (green grass aroma) and terpinolene (rosin-like aroma). The substance with FD = 6 was p-cymene (citrus and green aroma). In HY, 23 aromatic substances with FD ≥2 were identified. The substances with FD = 8 were ethyl cyclopropanecarboxylate (fruit aroma). The substances with FD = 7 were cis-2-penten-1-ol (grass and tea aroma), 3-hexenal (green grass aroma), and terpinolene (rosin-like). The substance with FD = 6 was p-cymene (citrus and green aroma). In TN, there were 24 aromatic substances with FD ≥ 2. The substances with FD = 8 were terpinolene and γ-terpinene, which mainly had a rosin citrus-like aroma. The substances with FD = 6 were ethyl cyclopropanecarboxylate, β-myrcene, 2-carene, phellandrene, and p-cymene. They had a fruity rosin and sweet aroma.
The OSME analysis showed that the aromatic intensities of the five mango pulps had significant differences ( Figure 5). There were 23 kinds of aromatic components in JM, and the substances with the aromatic intensity greater than 2 were phellandrene (4.1), ocimene (2.33), γ-terpinene (2.3), and terpinolene (2.3). This suggests an overall aromatic profile of citrus, green grass, and flowers. There were 19 kinds of aromatic components in QM, and the substances with an aromatic intensity greater than 2 were 1-hexanol (2.12), methyl-3-methylcyclohex-3-en-1-one (2.67), β-phellandrene (2.1), and decanal (3) showing an overall aromatic profile of lemon, sweet, and green grass. Seventeen aromatic components were identified in GF, and the substances with aroma intensities greater than 2 were 1-hexanol (2.13), phellandrene (2.5), terpinolene (2.33), and p-menthol (2.25) showing an overall aroma profile of citrus and lemon, fresh green leaves, and green grass. There were 22 aromatic components in HY, and the substances with aromatic intensity greater than 2 were phellandrene (3)  There were no significant differences in the numbers of overall aromatic substances identified using the two methods (FD and OSME). The number of substances with an FD ≥6 identified in TN by the FD method was the highest, followed by QM, JM, HY, and GF. The highest number of aromatic components with intensity values >2 was also found in TN, followed by HY and JM. QM and GF had the same number of aromatic substances. Therefore, the identification results obtained using FD and OSME were similar. These were highly consistent in identifying key aromatic substances. 2-Cyclohepten-1-one, ethyl cyclopropanecarboxylate, 1,3,8-p-menthatriene, and citral were identified for the first time and regarded as a characteristic aroma substance in mangoes, regardless of analytical method used (FD or OSME), although 1,3,8-p-menthatriene and citral were previously detected in lychee fruits [16]. In contrast to previous findings of aromatic substances in mangoes, some potent odorants, such as linalool and isoamyl acetate, were not detected, which might be due to differences in varieties, storage conditions, and extraction techniques. According to sensory analysis, the main aromatic profiles (fruit, sweet, flower, and rosin aromas) of pulp from the five mango cultivars were similar to those identified using the FD and intensity methods, indicating that the intensity method combined with the FD method can accurately illustrate the characteristic aromatic components with high or low intensity.

Odor Activity Values (OAVs) for the Pulps of Five Mango Cultivars
Aromatic analysis techniques such as the FD and intensity methods can effectively analyze the major aromatic compounds in mango pulp, but these cannot accurately reflect the contributions of individual components to the overall aroma characteristics [24]. Therefore, OAVs may be a more accurate scale to evaluate the contribution of volatile substances to fully consider the interactions between the food matrix and aromatic substances. The main component of mango pulp is water, and the calculation of the OAVs of each substance was based on the results of accurate quantitative analysis performed in this study, and the aroma threshold of each compound in water was previously reported [25,26].
According to the literature [19], substances with OAVs >1 contribute to the overall aroma of the sample. Table 3 and Figure 6 show the results of OAV analysis of pulp from five mango cultivars. There were 25 characteristic aromatic components with an OAV ≥ 1 in pulps of five mango cultivars, including two alcohols, seven aldehydes, three esters, 11 terpenes, and two ketones. Terpenes (44%) and aldehydes (28%) were the main aromatic components of mango, of which γ-terpinene had the highest OAV (3.04-10.04), followed by β-phellandrene (2.41-3.41), hexanal (1.10-16.97), and 1-nonanal (5.37-56.2), which were also considered as major aroma-active compounds in Australian mango There were no significant differences in the numbers of overall aromatic substances identified using the two methods (FD and OSME). The number of substances with an FD ≥6 identified in TN by the FD method was the highest, followed by QM, JM, HY, and GF. The highest number of aromatic components with intensity values >2 was also found in TN, followed by HY and JM. QM and GF had the same number of aromatic substances. Therefore, the identification results obtained using FD and OSME were similar. These were highly consistent in identifying key aromatic substances. 2-Cyclohepten-1-one, ethyl cyclopropanecarboxylate, 1,3,8-p-menthatriene, and citral were identified for the first time and regarded as a characteristic aroma substance in mangoes, regardless of analytical method used (FD or OSME), although 1,3,8-p-menthatriene and citral were previously detected in lychee fruits [16]. In contrast to previous findings of aromatic substances in mangoes, some potent odorants, such as linalool and isoamyl acetate, were not detected, which might be due to differences in varieties, storage conditions, and extraction techniques. According to sensory analysis, the main aromatic profiles (fruit, sweet, flower, and rosin aromas) of pulp from the five mango cultivars were similar to those identified using the FD and intensity methods, indicating that the intensity method combined with the FD method can accurately illustrate the characteristic aromatic components with high or low intensity.

Odor Activity Values (OAVs) for the Pulps of Five Mango Cultivars
Aromatic analysis techniques such as the FD and intensity methods can effectively analyze the major aromatic compounds in mango pulp, but these cannot accurately reflect the contributions of individual components to the overall aroma characteristics [24]. Therefore, OAVs may be a more accurate scale to evaluate the contribution of volatile substances to fully consider the interactions between the food matrix and aromatic substances. The main component of mango pulp is water, and the calculation of the OAVs of each substance was based on the results of accurate quantitative analysis performed in this study, and the aroma threshold of each compound in water was previously reported [25,26].