Bei Wng, Qing Meng,*, Lin Xio, Ruili Li, Chunhi Peng, Xueli Lio, Jingn Yn,Honglin Liu, Gunhu Xie, Chi-Tng Ho, Hurong Tong,*

a College of Food Science, Southwest University, Chongqing 400715, China

b Guizhou Aerospace Vocational and Technical College, Zunyi 563000, China

c Department of Food Science, Rutgers University, New Brunswick, NJ 08901, USA

Keywords:

Pu-erh ripen tea

SAFE

GC-MS

GC-O

Methoxyphenyl compounds

A B S T R A C T

Pu-erh ripen tea becomes popular worldwide owing to its unique taste and aroma.In the present study,volatile compounds from Pu-erh ripen tea were extracted using a solvent assisted flavor evaporation(SAFE) and analyzed with a gas chromatography-mass spectrometry (GC-MS) and a gas chromatography olfactometry (GC-O).Results demonstrated that 58 major volatile compounds and 24 aroma active substances of Pu-erh ripen tea were identified by GC-MS and GC-O analysis.Among volatile compounds identified,methoxyphenyl compounds were considered as the dominate compounds in the aroma of Pu-erh ripen tea.Further investigation showed that 6 methoxybenzenes were responsible for the aging aroma in Pu-erh ripen tea; α-ionone, 3,4-dimethoxytoluene, 1,3-dimethoxybenzene, etc.were responsible for the wood-like aroma.Linalool, α-terpineol, α-ionone, β-ionone, and benzeneacetaldehyde were responsible for the floral odor.Compared the odor detection threshold (ODT) among the 8 methoxyphenyl compounds in water,1,2,3-trimethoxybenzene showed the highest ODT, followed by 3,4-dimethoxyphenol.1,3-dimethoxybenzene(105 μg/L) and 1,2,3-trimethoxy-5-methylbenzene (100 μg/L) were present at relatively low odor threshold values.The result indicated that methoxyphenyl compounds play a vital role in the unique flavor of Pu-erh ripen tea.

1.Introduction

Pu-erh tea is a native kind of tea (Camellia sinensis) of Yunnan Province, China.It becomes popular in Asia owing to its unique flavor and health benefits [1-4].Pu-erh tea is made by the leaves of the tea plant cultivar (Camellia sinensis(L.) O.Kuntze var.assamicaKitamura) [5].According to the processing of post-fermentation, Pu-erh tea is generally classified into Pu-erh raw tea and Pu-erh ripen tea.The former is more like green tea, the latter belongs to dark tea due to its rapid fermentation under high temperature and humidity conditions.It makes Pu-erh ripen tea possess unique aroma and taste,which distinguishes it from Pu-erh raw tea [1,5].

Recently, several studies have shown that Pu-erh ripen tea not only contained common aroma substances in other teas such as alcohols, ketones, and aldehydes, but also possessed a large number of volatile methoxyphenyl compounds, such as 1,2,3-trimethoxybenzene,1,2,4-trimethoxybenzene, and 1,2-dimethoxybenzen [6,7].The contents of methoxyphenyl compounds could account for more than 30% of the total but rarely detected in green tea or black tea [8].Thus, methoxyphenyl compounds could be the dominate ingredients for the characteristics of Pu-erh ripen tea.Besides, how a kind of volatile compound contributing tea aroma is not only influenced by its high contents but also by its level of the odor detection threshold(ODT).Up to now, there have been minor kinds of literature focused on the active aroma components of Pu-erh ripen tea or the ODT of methoxyphenyl compounds.

To date, gas chromatography-mass spectrometry (GC-MS)and gas chromatography olfactometry (GC-O) are commonly used to characterize volatile compounds in different kinds of samples and identify their aroma-active components [9-12].Published studies on Pu-erh ripen active tea aroma was conducted using either aroma extract dilution analysis and time-intensity analysis.In comparison, time-intensity analysis is more efficient and effective, identifying the intensity and duration of the active compounds simultaneously.The higher the intensity of the compounds sniffed, the more significant the odor contribution.To further exploring tea flavor, an appropriate extraction method should be selected as a prerequisite, to ensure that the volatile compounds were extracted at utmost, and their structures are not being distorted [13].Previously, simultaneous distillation extraction and headspace solid-phase microextraction are mainly developed methods for extracting volatile compounds in Pu-erh ripen tea [14,15].Simultaneous distillation extraction can separate and concentrate volatile compounds simultaneously, but this method may result in selectivity bias or the formation of artifacts due to high extracting temperatures.Headspace solid-phase microextraction is relatively simple to operate, however, limited adsorption capacity may lead to deviation.Thus, solvent assisted flavor evaporation (SAFE) is an optimal extraction technique allowing the efficient and effective isolation of volatile compounds from the tea matrix [16].On the one hand, liquid nitrogen is used to provide a low-temperature environment, which could maintain the relative stability of volatile compounds.On the other hand, high recovery can be achieved even for high-boiling compounds [17].Therefore, in this study, the aroma-active compounds of Pu-erh ripen tea were extracted by SAFE.And the ODTs of 8 methoxyphenyl compounds in Pu-erh ripen tea were measured.Furthermore, the key aroma compounds in Pu-erh ripen tea were identified through GC-O and odor activity values (OAV), respectively.The OAV and GC-O result is promising to provide markers to explore the relation between aroma formation and the manufactory process of Pu-erh ripen tea, which offers a theoretical basis for improving the quality of Pu-erh ripen tea.

2.Materials and methods

2.1 Tea samples

Three kinds of Pu-erh ripen teas numbered D1, D2, and D3 were obtained from Douji Tea Co., Ltd.(Kunming, China).D1 and D3 made in 2014 and D2 made in 2015.The manufactory process of samples is similar, whereas the production batches and raw materials are different.All samples were collected and sealed in aluminum foil compound bags and stored in a refrigerator (at –80 °C).

2.2 Chemicals

A mixture of hydrocarbons ranging from octane (C8) to triacontane(C30) and ethyl caprate (99%) was purchased from Sigma-Aldrich(Shanghai, China).GC-grade ethanol and methylene dichloride, and analytical-grade anhydrous sodium sulfate were purchased from Chengdu Kelong Chemical Reagent Co., Ltd.(Chengdu, China).Ultra-pure water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA).Purity and purchase channel of standard aroma compounds for identity confirmation were listed in Table S1.

2.3 Sensory evaluation of Pu-erh ripen tea

An analysis of aroma characteristics and quality scores for the tea samples were employed based on the national standards in“Methodology of sensory evaluation of tea” (GB/T 23776, 2018) [18].To be specific, 150 mL boiled water poured into a cup with 5 g Pu-erh ripen tea and immersed for 4.0 min.Promptly, the tea infusion was transferred to a tea bowl and ready to be evaluated.A panel consisting of 5 well-trained members was asked to evaluate aroma characteristics and give descriptions.Before the sensory evaluation, all the members were previously trained using Pu-erh ripen tea samples and the standard aromatic compounds to obtain the ability to recognize, describe, and discriminate of different aroma qualities.Five sensory attributes were selected to represent the aroma profile which defined as the following aromas: 1,2,3-trimethoxy-5-methylbenzene for “aging aroma” attribute, 1,3-dimethoxybenzene for“wood-like” note, linalool for “ floral” attribute, 2,5-dimethylpyrazine for “roasted”, geraniol for “sweet” note.The odor intensities of the five sensory attributes was evaluated by a 7-point intensity scale from 1 to 7 (1, weak; 4, moderate; 7, strong).The final sensory scores were determined by calculating the averages of the panel and descriptors.

2.4 Aroma extraction by SAFE

Tea samples were ground to pass through 40 mesh to obtain tea leaves of homogeneous particle size.5 g Pu-erh ripen tea powder was extracted by methylene dichloride for 3 times, each duration time for 3, 13, 1 h, and each volume for 40, 30 and 30 mL, respectively [19].The extracts were combined after filtration.Volatiles from the methylene dichloride phase was isolated using the SAFE apparatus(Kiriyama Glass Works Co.Japan) according to the reported method [13], and the distillate was concentrated to 4 mL using a rotary evaporator (HB10 IKA Germany).The evaporation conditions were: vacuum pressure of 500 mbar, water bath temperature of 40 °C,chiller temperature of 5 °C, and rotation speed of 150 r/min.Then, the concentrate was dried using anhydrous sodium sulfate overnight, and then it was filtered and concentrated again to 0.2 mL under a gentle stream of N2(purity > 99.999%), finally stored in 2 mL glass vials with a lid at –40 °C for further analysis.

2.5 GC-MS analysis

GC-MS (GCMS-QP2010 Plus, Shimadzu, Japan) coupled with DB-5MS capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) was employed.Highly pure helium (> 99.99 %) used as the carrier gas, and the flow rate was 1 mL/min.The injector temperature was 250 °C, and the injection mode was splitless.The oven temperature programming was as follows: 40 °C raised to 130 °C at a rate of 5 °C/min, to 150 °C at a rate of 10 °C/min, to 180 °C at a rate of 3 °C/min held for 1 min, last to 260 °C at a rate of 10 °C/min, held for 3 min.The mass ion source temperature was 250 °C, and the ionization energy was 70 eV.The transfer temperature was 250 °C.The scan range wasm/z40-400.The solvent delay time was 4 min.The injection volume was 1 μL.

The volatile compounds were tentatively identified by using the mass spectra library NIST014s and further confirmed by the retention index (RI) calculated based on literature [20].The RI of each compound was calculated using ann-alkane mixture (C8-C30)which was run under the identical conditions.The values were then compared with previously published reports to verify their reliability.The standard reference compounds were used to confirm the positive identification by comparing their mass spectral data and RI.The concentrations of compounds detected in samples were calculated by their corresponding standard curve, as shown in Table S1.

2.6 ODT of methoxyphenyl compounds

The measurement of odor threshold was based on ASTM (E679-04)(2011) [18].Panelists included 30 healthy participators (15 males,15 females) with ages ranged 20-25 years old.The three-alternative forced-choice (3-AFC) method was used in the determination of the odor threshold of methoxyphenyl compounds.The test samples were prepared with varying concentrations of methoxyphenyl compounds in water.Methoxyphenyl compounds dissolved in ethanol(100 μL) at first, then diluted by Milli-Q water.And the concentration of ethanol in mixture is below its threshold value.For each test, three samples consisting of two control samples, and one test sample were coded with random three digits to blind the sample identity.Then,each sample (50 mL) was prepared in odor-free flasks (100 mL) with ground glass stopper lids and were presented to the panelists.The panelists had to identify the odd sample of each test.A choice that must be made despite no difference was sensed to all statistics that can be used.Besides, all samples for each methoxyphenyl compounds were arranged in descending order of concentration.The tests of each concentration repeated three times, and if the panelists made two or three correct judgments, we believed that the concentration was above the threshold values (x).Through the results of the pretest,the threshold values (x) could be roughly estimated.Then, another 6 test samples with concentrations around the threshold values(x), namely 8x, 4x, 2x,x, 1/2x, and 1/4xwere again prepared and presented to the panelists for further verification of threshold values(Table 1).Individual best estimate threshold (BET) was calculated as the geometric mean concentration of the highest “incorrectly”identified concentration and the adjacent higher “correctly”.If all concentrations were identified correctly (or incorrectly), the BET was the geometric mean of the lowest (or highest) concentration and the next hypothetical concentration in the series [21].The final results of odor thresholds were the panel’s thresholds which calculated using the geometric mean of all individual threshold estimates.

Table 1The designed concentrations of methoxyphenyl compounds for the 3-AFC threshold evaluation.

2.7 Calculation of OAVs

OAV was calculated by the ratio of the concentration of each compound to its threshold value in water which obtained from references.Compounds which with OAV ≥ 1 were considered as potential contributors to tea aroma profile.OAVs were calculated according to the Equ (1).

WhereCiis the concentration of the odorant (i) calculated from D1, D2, and D3, andODTiis its corresponding odor threshold in water found in the literature.

2.8 GC-O time-intensity analysis

GC-O analysis was performed on a gas chromatograph (GC 2010ATF, Shimadzu, Japan) equipped with a flame ionization detector (FID), a sniffing port (Sniffer 9000, Brechbühler,Switzerland) and RTX-5 capillary column (30 m × 0.25 mm i.d., 0.25 μm film thickness) [22].High purity nitrogen (＞ 99.99%) was used as the carrier gas at a flow rate of 1 mL/min.The temperature of injector and FID was 230 and 250 °C, respectively.The oven temperature and injection volume were programmed as same as that of GC-MS.Post column flow was split at a ratio of 1:1 to the FID and the sniffing port.The transfer line to the GC-O sniffing port was held at 250 °C.Humidified air was added in the sniffing port at 20 mL/min to help maintain the olfactory sensitivity.

The sniffing panel consisted of 3 assessors who have already trained for 10 h over a period of 2 weeks to obtain relatively objective results.The training procedure was based on previous research with partial modification [12].According to previous studies [14,23-25],volatile compounds (Table 1) were selected to train the panelist by GC-O procedure mentioned above.Each assessor was asked to note the perceived odor characteristic and intensity.The aroma intensity was evaluated using a 7-point scale, where 1, 4, and 7 represented for weak,moderate, and extremely strong aroma intensity, respectively [26].Each sample was sniffed 3 times by each assessor, and the aroma intensity values were averaged for all 3 analyses.Only if at least 5 times of the total 9 trials found a similar odor quality at the same retention time, the corresponding ef fluent was considered as aromaactive compounds [19].The RI of each aroma-active compound was calculated using then-alkane mixture (C8-C30), which was run under the GC-O conditions.Then we compared these RI values with those in the related literature to verify the exact aroma compounds.

2.9 Statistical analysis

All quantitative analysis was carried out in triplicate, and the experimental results were expressed as means and standard deviations.The statistical data were analyzed by one-way analysis of variance(ANOVA) to determine the statistical difference (P＜ 0.05) using IBM SPSS Statistics 25 (Chicago, IL, USA).Average concentration expressed in microgram per kilo of the tea infusion with RSD (%)below 15% (n= 3).

3.Results and discussion

3.1 Analysis of volatile compounds in Pu-erh ripen tea

ThePu-erh ripen tea was described by the sensory panel.All tea samples possessed typical odors of Pu-erh ripen tea i.e.woody,and aging aroma (Fig.1).The content of compounds category differed in 3 samples, while small difference of the proportion accounting for the total content.Specifically, the concentration of methoxyphenyl compounds in D1, D2 and, D3 was 84 301.64,121 503.4 and 27 196.42 μg/kg, respectively (Fig.2a), whilst the proportion in D1, D2 and, D3 was similar, at nearly 60%(Fig.2b).The proportion of other category of aroma substances were also showed the same trend.Combined with the sensory evaluation results, it could be concluded that the similar aroma profiles of different Pu-erh ripen teas were due to the similar proportion of compounds category.Meanwhile, the differences of contents might be the reason for the intensity of aroma attributes.

Fig.1 Aroma profiles of different Pu-erh tea infusions.

Fig.2 Constitution of volatile compounds in Pu-erh ripen tea infusion.(a) Compounds category in D1, D2, and D3 obtained with headspace SAFEGC-MS: (1) phenol, (2) alcohol, (3) methoxyphenyl compound, (4) lactone,(5) aldehyde, (6) ketone, (7) heterocyclic compound, and (8) ester.The mean ±standard error, n = 3.(b) Rate of volatiles compounds category in D1, D2, and D3.

As shown in Table 2, 58 volatiles were identified and quantified in 3 different Pu-erh ripen tea extracts.Which including 18 alcohols,12 ketones, 9 aldehydes, 9 methoxyphenyl compounds, 3 lactones, 2 phenol compounds, 2 esters, and 3 heterocyclic compounds.Among these substances, the content of methoxyphenyl compounds was highest, followed by that of ketones.Most of these components are well-known odorants in Pu-erh ripen tea [14,25,27,28].Interestingly,several volatiles like methylpyrazine, dihydrocitronellol and benzothiazole have not been reported in Pu-erh ripen tea before.While several compounds such as 2-pentylfuran, 2-ethyl-1-hexanol and 2-methylnaphthalene were not detected in this research [25,27].

Table 2Concentrations, ODT and OAV for selected odorants in Pu-erh ripen tea infusions.

3.1.1 Alcohols and aldehydes

Alcohols play a key role in the formation of tea aroma [29].Nerolidol, phenylethyl alcohol, geraniol, and linalool were generally believed to have floral and sweet notes [30-32], and were adduced as being impact compounds of tea aroma [31,33].As shown in Table 3, nerolidol, 1-octen-3-ol, phenylethyl alcohol, and linalool oxide showed comparatively high contents in the tea matrices and all of those were higher than 250 μg/kg.The development of aldehydes was mainly from two pathways, i.e.Strecker degradation occurred under heat condition and the degradation of unsaturated fatty acid under thermal or lipoxygenase-mediated conditions [34].Benzeneacetaldehyde was derived from Strecker degradation of phenylalanine and showed comparatively high contents ranged from 179.10 μg/kg to 301.59 μg/kg.

3.1.2 Ketones and lactones

Ketones were mainly derived from carotenoid precursors in tea and were considered significant contributors to tea flavor [35,36].α-Ionone was considered to be the potential maker explaining the differences between raw and ripened Pu-erh tea [14], and it showed high contents ranging from 559.95 μg/kg to 1 667.87 μg/kg.Besides,other ketone volatiles also demonstrated high concentration in all samples such as geranylacetone (1 070.42 μg/kg to 5 726.94 μg/kg)and 6-methyl-5-hepten-2-one (334.52 μg/kg to 520.83 μg/kg).Furthermore, several lactone compounds which rarely reported in Pu-erh ripen tea previously such as pantolactone, dehydromevalonic lactone were detected in this study.In addition, dihydroactinidiolide,which is derived from carotenoid and considered as characteristic flavor compound of black tea [37]were also detected.

3.1.3 Methoxyphenyl compounds

Methoxyphenyl compounds are thought to be the significant aroma components of in Pu-erh ripen tea.Previous studies have suggested that methoxyphenyl compounds and their derivate had important effects on the unique aging aroma of Pu-erh ripen tea [8,23].As reported in the literature, the vast majority of methoxyphenyl compounds showed comparatively higher concentration in this study such as 1,2-dimethoxybenzene,1,2,3-trimethoxybenzene and 1,2,3-trimethoxy-5-methylbenzene.In addition, 1,3-dimethoxybenzene, 1,2,3-trimethoxy-5-methylbenzene,1,2,4-trimethoxybenzene, 4-ethyl-1,2-dimethoxybenzene, and 3,4-dimethoxytoluene also had relatively high level in all samples.

In a recent paper, Pu-erh raw tea and Pu-erh ripen tea were employed to investigate the role of post-fermentation in Pu-erh tea aroma formation.The study used orthogonal projection to latent structure discriminant analysis to distinguish the aroma profile differences based on acquired OAVs of Pu-erh raw tea and Pu-erh ripen tea.Its results showed the formation of methoxyphenyl compounds in Pu-erh ripen tea, lead to the characterized stale or aging flavor.Similarly, it was reported in another research that the characteristic volatiles in ripen tea were 1,2,3-trimethoxybenzene,1,2,4-trimethoxybenzene, and 1,2,3-trimethoxy-5-methyl-benzene,etc.[24].Thus, a conclusion could be drawn that the unique process of Pu-erh ripen tea, post-fermentation processing, could be the key impact factor to help in the formation of methoxyphenyl compounds.

Microbe was generally considered to promote a series of complex biochemical changes during the post-fermentation processing and be responsible for the methoxyphenyl compounds, which showed aging aroma and mellow flavor in Pu-erh ripen tea [5,23].The methylation of gallic acid by microbial enzymes and thermal degradation to product these compounds [5].Yeast,Penicillium,Rhizopus, andAspergillus nigerhave been considered to related to methoxyphenyl compounds formation during the post-fermentation process [6,28].Haas et al.[38]identified fungi in Pu-erh ripen tea,and the results showed thatA.nigerwas identified in 29 out of 36 samples.This result is consistent with another study: the formation of methoxyphenyl compounds may be due to the activity ofA.nigerin Pu-erh during the fermentation process [28].

Storage time is also a key factor affecting the flavor formation of Pu-erh ripen tea.The Pu-erh ripen tea aged 1 year and Pu-erh raw tea aged 6 years show similar waveforms and absorption peaks in the infrared spectrum, indicating that they have similar characteristic chemical components [5].Gao et al.[39]conducted sensory evaluation on Pu-erh ripen tea with different storage time, and found that the content of amino acids and caffeine in tea increased significantly with the increase of storage time.Besides, the score of heavy and thick attribute of Pu-erh ripen tea stored for 10 years is significantly better than that of tea stored for one year.Interestingly,1,3-dimethoxybenzene was only identified in D1 (19 528.18 μg/kg).However, the manufacturing process of samples is similar.Thus, we speculate that different raw materials could significantly differentiate volatile compounds for Pu-erh ripen tea aroma formation.

3.2 Analysis of ODT and OAVs calculation

Table 2 listed the odor threshold values of 8 methoxyphenyl compounds in water.1,2,3-trimethoxybenzene (58 349 μg/L) showed the highest ODT value, compared to 1,2,4-trimethoxybenzene(2 004 μg/L), 1,3-dimethoxybenzene (105 μg/L) and 1,2,3-trimethoxy-5-methylbenzene (100 μg/L) presented relatively low ODT values(< 200 μg/L).

As shown in Table 2, there were a total of 17 volatiles’OAVs > 1.The odorants whose OAVs > 100 were considered to greatly contribute to the characteristic aroma of Pu-erh ripen tea.It mainly included ketones, methoxyphenyl compounds, and terpenes.1,2,3,4-Tetramethoxybenzene presented the highest OAVs ranging 582-2 634, followed by 1,2-dimethoxybenzene (67-295),1,2,3-trimethoxy-5-methylbenzene (33-180); besides, the OAVs of 1,2,4-trimethoxybenzene (1-3) and 4-ethyl-1,2-dimethoxybenzene(1.5) were also above 1.It meant that methoxyphenyl compounds might be the dominant components in aroma of Pu-erh ripen tea.These findings were in accord with the literature but the OAV of 1,2,3-trimethoxybenzene was quite different (OAV ＜ 1) [23,25].The ODT of 1,2,3-trimethoxybenzeneis was also far exceed that in other research (0.75 μg/kg) [14].The reason why the ODT of a certain compound varies in similar researches maybe attributed to the scale and make-up of the panel, the ways of sniff, the matrix, the test procedure, and other factors.Normally, the panel constituted by selected or trained panelists usually shows lower ODT than that of the unselected or untrained [40].In addition, ODTs obtained by a smallscale panel are more decisive, however, ODTs acquired by a relatively large-scale panel is more representative of the general levels [40].For instance, the ODT of (E)-2-pentenal reported by Tandon et al.[41](55 × 10-9) with an 11 members panel, and Plotto et al.[42](2 770 × 10-9)with a 33-58 members panel differed significantly.Moreover, it was reported that panelist, who was very insensitive for one compound,could be very sensitive for another [43].

Generally, a certain of terpenoid compounds such as geranylacetone (107-573) and nerolidol (106-411) presented relatively higher OAVs than other compounds.Moreover,α-ionone showed an OAV of 1 400 to 4 170 whileβ-ionone with woody notes presented OAV of 4 723 and 7 426 in D1 and D2, respectively.Thus,these compounds were considered as the most important compounds impacting the aroma profile of Pu-erh ripen tea.Besides, 1-octen-3-ol,linalool, hexanal, heptanal, linalool oxide II, and linalool oxide I with their OAVs > 10 have also been reported as the active-compounds in Pu-erh ripen tea [6,14,24,25].

It is noteworthy that heptanal (80-105), benzeneacetaldehyde(45-75), butyl butanoate (10) were not known as the active compounds of Pu-erh ripen tea in previous studies [7,14].Nevertheless, these compounds possessed high OAVs in this study.It may be contributed to the characteristic aroma of Pu-erh ripen tea.

3.3 Odor-active components identified by GC-O

A total of 24 aroma active substances with their odor descriptions and intensities were detected in 3 samples (Table 3).These aromaactive substances included 7 alcohols, 7 methoxyphenyl compounds, 4 ketones, 6 aldehydes, and they may be divided into 3 main categories based on their primary aroma characteristics: aging aroma, floral, and wood-like.

According to Table 3, the aging aroma mainly consist of 3,4-dimethoxytoluene (2.75-4.33), 4-ethyl-1,2-dimethoxybenzene(2.45-4.25), 1,2,3-trimethoxy-5-methylbenzene (3.00-4.00),1,2,3,4-tetramethoxybenzene (2.00-4.00), 1,2,3-trimethoxybenzene(2.40-3.75), and 1,2-dimethoxybenzene (2.30-2.50), which agreed with reports in the literature [7,25].The former 3 compounds presented higher aroma intensity and were considered as the primary contributors to the aging aroma of Pu-erh ripen tea.According to previous findings, the wood-like aroma was related to linalool oxide [15].It could be seen from Table 3 thatα-ionone (3.00-5.00),3,4-dimethoxytoluene (2.75-4.33), 1,3-dimethoxybenzene(1.50-4.00) were also the dominate constitutes of the wood-like aroma beside linalool oxide I (3.00-3.50), and linalool oxide II(3.60).Especially,α-ionone showed the highest intensity in D1 which possessed typical wood-like aroma.It meant thatα-ionone would be the greatest contributor to the characteristic.Based on the literature,the floral aroma was mainly made up of terpenes and ketones [15,47].As shown in Table 3, the floral aroma was highly related to linalool,α-terpineol,α-ionone,β-ionone, and benzeneacetaldehyde.In contrast, linalool is one of the most common ingredients in green teas but presented a relatively low intensity (2.75-3.00) compared withβ-ionone (3.50-4.00) in this study.Besides, benzeneacetaldehyde presented a relatively high aroma intensity ranging from 2.50 to 4.00.

Combined the results with OAV and GC-O.It concluded thatα-ionone, benzaldehyde, benzeneacetaldehyde,1,2-dimethoxybenzene, 1,2,3-trimethoxy-5-methylbenzene,1-octen-3-ol, and 1,2,3,4-tetramethoxybenzene were detected as the aroma active components of Pu-erh ripen tea.However, there were still differences between the results of OAV and intensity.For instance, the OAV of 1,2,3-trimethoxybenzene (2.40-3.75)and 3,4-dimethoxytoluene (2.75-4.33) ＜ 1 but showed moderately high intensity by GC-O.Heptanal (80-126) and linalool (220-307)were identified as the aroma-active compounds by OAV but failed to be detected in GC-O analysis.It is supposed that there were still limitations in the calculation of OAV.On the one hand, the ODTs of compounds in water may be difficult to present all of their characteristics in the tea infusion matrix.On the other hand, it is difficult to exclude the interaction among compounds when the OAV calculating.For example, a binary model aqueous solutions contained the authentic odorants, and the concentration of odorants was below the odor threshold.However, the odor intensity of the model solution was significantly enhanced when only 5% of each odorant replaced by 4-hexanolide [47].

4.Conclusion

In summary, 58 major volatile compounds were identified in Pu-erh ripen tea by SAFE/GC-MS analysis, and 24 potent odorants have been identified by GC-O analysis.ODT was detected and OAVs was also calculated.Alcohols, aldehydes, methoxyphenyl compounds,ketones, lactones were abundant components of aroma in Pu-erh ripen tea.In addition, methoxyphenyl compounds are potentially responsible for the special characteristic of aging aroma which discriminate Pu-erh ripen tea from other teas.1,2,3-Trimethoxybenzene showed the highest threshold value in 8 methoxyphenyl compounds in water.Ketones, methoxyphenyl compounds, and terpenes which OAVs value higher than 100 were considered to greatly contribute to the characteristic aroma of Pu-erh ripen tea.Our findings contribute new insight to quantitative description in sensory investigation of the quality characteristics of Pu-erh ripen tea in different kinds and regions.This work has the potential to provide a foundation for continuing efforts to improve quality of Pu-erh ripen tea via the optimization of processing and storage methods.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

This research was supported by grants from the Chongqing Municipal Agriculture Committee (2020-7).

Appendix A.Supplementary data

Supplementary data associated with this article can be found in the online version, at http://doi.org/10.1016/j.fshw.2021.12.018.