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2022 Volume 2
Article Contents
Abstract
Introduction
Materials and methods
Materials and preparation of heat-induced black lycium barbarum
Preparation of melanoidins
Isolation and evaluation of melanoidin fractions
Orthogonal experimental design of resin purification
Determination of the chemical constituents of melanoidin fractions
Antioxidant activity determination
Hydroxyl radical scavenging activity
Ferric-reducing antioxidant power
Dpph radical scavenging activity
Fourier transform infrared spectroscopy (ftir)
Stability assay
Antioxidant activity in vitro simulated digestion
Statistic analysis
Results and discussion
Isolation and preliminary evaluation of melanoidin fractions
Content of isolated melanoidin fractions
Chemical compositions of isolated melanoidin fractions
Antioxidant activities of isolated melanoidin fractions
Optimization of uf3 purification conditions
Ftir analysis of purified uf3
Stability of purified uf3
Changes in antioxidant activities of purified uf3 in in vitro digestion
Conclusion
Author contributions
Data availability
Acknowledgments
Conflict of interest
Rights and permissions
References
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ARTICLE   Open Access    

Essential aroma substances and release pattern of Xinhui Chenpi

  • 1.

    College of Food Science and Biological Engineering, Tianjin Agricultural University, No. 22, Jinjing Road, Tianjin 300384, PR China

  • 2.

    Key Laboratory of Tea Science of Ministry of Education, Hunan Agricultural University, Changsha, Hunan 410128, PR China

More Information
  • This study focuses on analyzing aqueous solutions of aroma-active compounds in Xinhui Chenpi distilled after being heated with headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC/MS). Feasibility of this method was also tested by comparison with crushed samples. The study also analyzed the aqueous solutions of the aroma-active compounds employing gas chromatography-olfactometry (GC-O) defense, aroma extraction dilution analysis (AEDA) and odor activity value (OAV) as well as their emission patterns. According to the study, there are 24 major aroma-active detected in the aqueous solution. Linalool, d-limonene, 2-methoxy-4-vinylphenol, and α-terpineol with sweet, spicy, and woody aroma contributed the most to the aroma-active compounds and were considered to be the essential aroma substances of Chenpi. When heated, the aroma of aromatic-active compounds rich in Chenpi volatilized rapidly and the release dropped dramatically in a short time. The substantial aroma-active compounds can be collected from the first ten released segments.

  • Introduction

    The Maillard reaction (MR) is known as non-enzymatic browning, which occurs widely during the heating process of food. Melanoidins, the product of the MR, are formed by the condensation of reducing sugar carbonyl and amino group[1]. Melanoidins have been shown to improve food quality, such as color, flavor, and biological activity[2]. For example, melanoidins extracted from bread and biscuits exhibit antimicrobial effects on certain molds and yeasts, which could improve the shelf life and safety of food as a natural antimicrobial agent; melanoidins extracted from bread could serve as a carbon source for Bifidobacterium. Melanoidins from black vinegar significantly suppressed adipogenesis of 3T3-L1[3−5]. Currently, there are many reports on the antioxidant capacity of melanoidins. It is generally recognized that melanoidins have excellent antioxidant activities[6−9]. These excellent properties demonstrate melanoidins have considerable application value in the food industry, which can be used as novel food ingredients.

    Lycium barbarum L. has been used as a traditional food and herbal medicine in China, as described in the Compendium of Materia Medica. A large number of bioactive substances, including polysaccharides, phenols, proteins, alkaloids, and vitamins, have been found in Lycium barbarum L.[10,11]. Importantly, the bioactivity of Lycium barbarum L. can be significantly enhanced by the concoction process. It has been reported that this increase in activity can be attributed to the production of melanoidin[12]. Black garlic and black apple, which have been extensively studied, also belong to this category of food products and the presence of melanoidins in the system also increases the functional properties of the products[13−15]. However, the development of novel products with melanoidin from Lycium barbarum L. as a raw material by heat-high humidity or low-temperature-induced processing has not been intensively investigated; in particular, it was carried out with the Ningxia wolfberry as the selective target. Therefore, melanin was extracted and purified from Lycium barbarum L. and a preliminary study was conducted on its physicochemical properties, digestive properties, and functional activities. This study will provide theoretical support for further research on the application of melanoidins extracted from Lycium barbarum L. as functional foods or dietary additives, and provide a new avenue for the utilisation of Lycium barbarum L.

    Materials and methods

    Materials and preparation of heat-induced black Lycium barbarum

    Dried Lycium barbarum L. with 20% water content was provided by Zaokang Wolfberry Incorporated Company (Ningxia Province, China). Dried Lycium barbarum L. was placed in a constant temperature and humidity chamber, then treated for 48 h at 60 °C and 50% humidity to obtain black Lycium barbarum L. (BLB). Pepsin (10,000 U/g) was purchased from Hefei Qiansheng Biotechnology Co., Ltd. ( Anhui Province, China). Trypsin (250 U/mg) and α-amylase (10,000 U/g) were purchased from Jiangsu Ruiyang Biotechnology Co., Ltd. (Jiangsu Province, China).

    Preparation of melanoidins

    Melanoidins were extracted according to the method described by Liu et al.[16] with some modifications, as described in the Supplemental File 1. BLB powder (20 g) was extracted with 200 mL of 30% ethanol solution (v/v) in a water bath at 45 °C for 90 min and then filtered to collect the supernatant. The collected supernatant was added to an anhydrous alcohol solution to achieve an ethanol concentration of 70% and then allowed to stand for 12 h at 4 °C to precipitate. Then the supernatant was filtered with filter paper. The filtrate was dialyzed for 48 h to remove compounds with a molecular weight (MW) below 1 kDa and then freeze-dried. Finally, the crude melanoidins were extracted and packed in vacuum-sealed bags for further use.

    Isolation and evaluation of melanoidin fractions

    The isolation of crude melanoidins was conducted using a method previously reported by Tores de la Cruz et al.[17] with some modifications. The prepared crude melanoidin solution was sequentially ultrafiltered three times using ultrafiltration membranes with molecular weight cut-off (MWC) of 50, 10, and 3 kDa to separate the fractions UF1 (ultrafiltration fraction 1, MWC > 50 kDa)，UF2 (10 kDa < MWC < 50 kDa)，UF3 (3 kDa < MWC < 10 kDa) and UF4 (< 3 kDa) respectively. The ultrafiltration procedure was repeated three times to collect the four melanoidin fractions. The fractions were freeze-dried, weighed and then stored at −20 °C. The chemical composition and antioxidant activity were analyzed to preliminarily evaluate the properties of the four fractions.

    Orthogonal experimental design of resin purification

    According to the preliminary analysis of four fractions, the target fraction was confirmed. The target fraction was further purified according to the method proposed by Zhang et al.[18] with some modifications. Briefly, dynamic adsorption and desorption tests were performed with the pretreated AB-8 resin. Twenty grams of the pretreated AB-8 resin was loaded into the glass column and a sample of the target fraction solution was loaded, and then eluted with 40% ethanol. First, three variables (loading concentration, elution volume, and elution flow rate) that could influence the yield of melanoidin purification were evaluated in single factor experiment. The conditions for the single factor experiment were set as follows: Loading concentrations (2, 4, 6, 8, and 10 mg·mL−1), elution volume (4, 5, 6, 7, and 8 BV) and elution flow rate (1, 2, 3, 4, and 5 mL·min−1). Based on the results of the single factor experiments, an orthogonal L9 (33) test (as shown in Table 1) was performed. The purified melanoidin obtained under the optimal conditions was then analyzed for structure, stability, and antioxidant activity.

    Table 1.  Analysis and results of orthogonal L9 (33) experimental design.
    Number Factors Results
    a: Loading concentration (mg·mL−1) b: Elution volume (BV) c: Elution flow rate (mL·min−1) Yield (%)
    1 6 6 1 0.487
    2 6 7 2 0.332
    3 6 8 3 0.243
    4 8 6 2 0.398
    5 8 7 3 0.265
    6 8 8 1 0.255
    7 10 6 3 0.253
    8 10 7 1 0.348
    9 10 8 2 0.273
    k1 0.351 0.376 0.360
    k2 0.306 0.315 0.334
    k3 0.291 0.257 0.253
    R 0.0594 0.119 0.107
    Order of importance b > c > a
    Optimal level a1b1c1
     | Show Table
    DownLoad: CSV

    Determination of the chemical constituents of melanoidin fractions

    The contents of reducing sugar and total sugar were determined by the DNS method and phenol sulfate colorimetry method, respectively. The total phenolic content was determined using the Folin-Ciocalteu method[19]. The protein content was carried out using the Bradford method using bovine serum albumen as standard[20]. Using rutin as the standard sample, the total flavonoid content was determined. A 0.5 mL sample was mixed with 0.4 mL of 5% NaNO2 and then incubated for 6 min. Afterward, 0.4 mL of 10% Al(NO3)3 was added and reacted for 6 min. Finally, 4 mL of 4% NaOH and 4.7 mL of 70% ethanol were added to the reacted solution. Then the sample solution was incubated for 10 min and the absorbance was determined at 505 nm.

    Antioxidant activity determination

    Hydroxyl radical scavenging activity

    The hydroxyl radical scavenging activity was determined using the method described by Wu et al.[21] with some modifcations. Briefly, 1 mL of FeSO4 solution (5 mmol·L−1), 4 mL of salicylic acid solution (5 mol·L−1) and 3 mL of distilled water were mixed with 1 mL sample. Then, 1 mL of H2O2 (5 mmol·L−1) was added and reacted at 37 °C for 10 min. The absorbance of the reacted solution was measured at 510 nm, and the hydroxyl radical scavenging activity was calculated using the following equation:

    Hydroxylradicalscavengingability(%)=A0−(A1−A2)A0×100% (1)

    where, A0: the absorbance of the blank; A1: the absorbance of the control; A2: the absorbance of the sample.

    Ferric-reducing antioxidant power

    The antioxidant power was measured according to the method described by Oracz & Nebesny[22]. One mL of sample (0.5 mg·mL−1) was mixed with 2.5 mL of sodium phosphate buffer (pH = 6.6, 0.2 mol·L−1) and 2.5 mL of potassium ferricyanide (1%, v/v). The mixed solution was incubated in a water bath at 50 °C for 20 min. 2.5 mL of 10% trichloroacetic acid was added when the solution was cooled to room temperature. One mL of ferric chloride (1%, v/v) and 2.5 mL of distilled water were added into 2.5 mL of this mixture. Finally, the absorbance of the mixture was determined at 700 nm after incubation for 10 min.

    DPPH radical scavenging activity

    The DPPH radical scavenging activity of melanoidin was evaluated following previous reports by Kim[23] with slight modifications. Four mL of 95% ethanol solution and DPPH-ethanol mixed solution (2 mL, 0.05 mg·mL−1) were added to 1 mL of sample solution and then allowed to react for 30 min under light protection. The absorbance value of the solution was determined at 517 nm. The DPPH radical scavenging activity (%) was calculated as follows:

    DPPHradicalscavengingactivity(%)=A0−(A1−A2)A0×100% (2)

    where, A0: the absorbance of the blank; A1: the absorbance of the control; A2: the absorbance of the sample.

    Fourier transform infrared spectroscopy (FTIR)

    The sample obtained above (1 mg) was mixed with dried KBr (100 mg) and pressed into a thin sheet with a tablet press after being ground. Then the sample was analyzed using a spectrometer in the 400−4,000 cm−1 range.

    Stability assay

    The stability of the purified melanoidins was examined according to a previous study[24]. Briefly, the stability was estimated by exposing the samples to different heating temperatures (40, 50, 60, 70, and 80 °C) and different light conditions (sunlight, incandescent light, darkness). The absorbance of the samples exposed to different heating temperatures was determined every 1 h at 420 nm. And the absorbance of the samples exposed to different light conditions were determined every 1 d. The retention rate (R) was calculated using the following equation:

    R=A1−A0A0×100% (3)

    where, R: Retention rate; A0: The absorbance value of the initial sample; A1: The absorbance value of the sample after treatment under different conditions.

    Antioxidant activity in vitro simulated digestion

    The purified melanoidins were digested according to the method described by Peña-Correa et al.[25]. Briefly, in the oral digestion phase, 1.0 g of sample and 20 mL of simulated salivary fluid (pH adjusted to 7.0) were mixed and incubated at 37 °C for 2 min. In the gastric digestion phase, the sample after oral digestion was mixed with 10 mL of simulated gastric fluid (pH adjusted to 1.2) and incubated at 37 °C for 2 h. In the intestinal digestion phase, 10 mL of simulated intestinal fluid (pH adjusted to 7.0) was added to the above gastric digestion mixture and incubated at 37 °C for 2 h. The samples before and after each digestion step were collected and freeze-dried for further analysis of antioxidant activity. Antioxidant activity was analyzed according to the method described above.

    Statistic analysis

    All experimental results were determined in triplicate and the data were expressed as mean ± standard deviation (Mean ± SD). ANOVA accompanied and Duncan’s multiple range tests were performed to examine statistical differences across groups using SPSS software (version 19.0 for Windows, SPSS Inc., Chicago, IL, USA).

    Results and discussion

    Isolation and preliminary evaluation of melanoidin fractions

    Content of isolated melanoidin fractions

    Four melanoidin fractions with different MW were obtained by ultrafiltration. As shown in Fig 1a, the melanoidin fraction with HMW presented a deeper brown color than in the LMW fractions. The results indicate that the HMW melanoidins were stronger colorants that contribute more to the color of thermally treated foods[21,26]. As shown in Fig. 1b, UF3 showed the highest content (59.37% ± 3.66%), followed by UF4 (24.21% ± 3.01%), UF2 (12.90% ± 1.25%) and UF1 (3.52% ± 0.98%), indicating that melanoidin fractions with LMW were dominant fractions. This result could be due to the low temperature of the reaction system, which leads to the formation of a large amount of LMW melanoidin in the early phase of MR. Melanoidin is the product of the Maillard reaction between proteins and sugars. It has been shown that the reaction time and temperature are related to the size of the molecular weight. When the reaction temperature is low, a large amount of LMW melanoidin is formed in the early phase of MR. LMW melanoidin can be used as a reaction intermediate with other Maillard reaction products to polymerize or crosslink to form HMW melanoidin in the later stage of the reaction with the extension of heating time[27,28]. This finding is consistent with the fact that the molecular weight of melanoidin in ale beers and lager beers increased with the roasting time of the ingredients and the intensity of the heating[28].

    Figure 1.  The physicochemical properties of melanoidin fractions with different MW extracted from induced black Lycium bararum L., including (a) color, (b) proportion of ultrafiltration fraction, and (c) chemical composition. UF1 (Ultrafiltration fraction 1, MWC > 50 kDa), UF2 (10 kDa < MWC < 50 kDa), UF3 (3 kDa < MWC < 10 kDa) and UF4 (< 3 kDa).
    DownLoad: Full-Size Img PowerPoint
    Chemical compositions of isolated melanoidin fractions

    Chemical compositions commonly retained in melanoidins by ultrafiltration membranes include total sugar, reducing sugar, total phenol, and protein[21]. The content of chemical compositions in four melanoidin fractions are shown in Fig. 1c. The total flavonoid, which is abundant in Lycium barbarum L., was not detected in the four melanoidin fractions. The total phenol exhibited a low content of less than 0.5% in four melanoidin fractions. The total phenol content increased with the increase in MW. The findings were similar to those of Naibaho & Korzeniowska[29], who observed that melanoidins in Brewers' spent grain with the higher molecular weight contained more total polyphenol. The reducing sugar and protein also presented low content indicating that the reducing sugar was fully reacted with protein in the Maillard reaction. The total sugar in four melanoidin fractions showed the highest content in the range of 5.91% to 7.14%. And a significant difference in the total sugar content was observed between UF4 and other melanoidin fractions. This is probably the result of the ultrafiltration membrane with MWC < 3 kDa efficiently removing the unreacted high molecular weight sugars.

    Antioxidant activities of isolated melanoidin fractions

    As shown in Fig. 2a, the reducing power of all melanoidin fractions increased with the increase of sample concentration. The reducing power of melanoidin fractions with LMW was found to be significantly higher than those of melanoidin fractions with HMW. The reducing power of UF4 in concentration of 2.0% was about 0.46 times higher than that of UF1. The scavenging activities of four melanoidin fractions with different concentrations against the hydroxyl radical are shown in Fig. 2b. The scavenging activity against hydroxyl radicals of the four melanoidin fractions was positively associated with the concentration. The scavenging activity was enhanced significantly with the decreasing of MW. The scavenging activity against hydroxyl radicals of UF4 at a concentration of 2.0% was 18% higher than that of UF1. The results consistent with a previous report[28] indicated that melanoidin with LMW prepared from beers showed stronger antioxidant activity. It can be seen from Fig. 2c, a positive correlation between the concentrations and radicals scavenging activities of all melanoidin fractions was also observed. The DPPH radicals scavenging activity of UF2 and UF3 was significantly stronger than that of UF1 and UF4. The reason for this result may be that the ability of melanin to scavenge DPPH free radicals is expressed by the donation of hydrogen atoms and UF2 and UF3 may contain more hydrogen to form stable DPPH-H molecules[13,30].

    Figure 2.  The antioxidant activities of melanoidin fractions with different MW, including (a) reducing power, (b) hydroxyl radical scavenging activity, and (c) DPPH radical scavenging activity.
    DownLoad: Full-Size Img PowerPoint

    Optimization of UF3 purification conditions

    According to the above analysis of four fractions, UF3 was confirmed as the target fraction for further research and application. Seen from the results of single-factor experiments presented in Fig. 3a−c, the highest yield of melanoidins were 33.5%, 38.5%, and 43.5% at the loading concentration of 8 mg·mL−1, the elution volume of 7 BV and the elution flow rate of 2 mL·min−1 respectively. Based on the single factor experiments, orthogonal experiments with these three variables were performed. Table 1 presents the optimization results. Among these factors that influenced the yield of melanoidins, the elution volume (b) was the most significant influencing factor, followed by the elution flow rate (c), and the loading concentration (a). The optimum parameters for UF3 purification were a1b1c1. The highest yield of UF3 was obtained when the loading concentration, the elution volume, and the elution flow rate were 4 mg·mL−1, 6 BV, and 1 mL·min−1 , respectively.

    Figure 3.  Impact of (a) loading concentration, (b) elution volume, and (c) elution flow rate on the purification yields.
    DownLoad: Full-Size Img PowerPoint

    FTIR analysis of purified UF3

    The FTIR spectra of the purified UF3 are shown in Fig. 4. An absorption band at 3,394.98 cm−1 was observed. The absorption bands at 3,600−3,200 cm−1 is assigned to stretching vibration of the hydroxyl group (O-H) or amide group (N-H)[29]. The signal peak presented at 2,923.63 cm−1 could be attributed to the stretching vibration of C-H. The C-H (CH3 and CH2) band are found around 2,943 cm−1[31]. In previous studies, the melanoidin skeleton is rich in O-H, N-H, and -CH functional groups[31−33]. The absorption bands of amide were observed at 1,639.25, 1,404.61, and 1,242.00 cm−1, which was attributed to C=O stretching and C=N stretching modes (amide I, 1,700−1,600 cm−1), N-H bending and C-N stretching modes (amide III, 1,450−1,240 cm−1), respectively[34]. Two absorption peaks at 1,075.28 and 1,038.23 cm−1 can be assigned to C-O-C bending. In addition, a band was observed at 581.29 cm−1 ascribed to the skeletal modes of pyranose rings[35]. In summary, the structure of purified UF3 was close to the typical melanoidins structure, indicating that the UF3 was effectively prepared.

    Figure 4.  FT-IR spectra of purified UF3.
    DownLoad: Full-Size Img PowerPoint

    Stability of purified UF3

    The stability of purified UF3 under different light conditions and temperatures was investigated. Under different light conditions, the retention rate of all melanoidin samples showed a downward trend with increasing exposure time (Fig. 5a). The downward trend of melanoidin samples exposed to sunlight showed a greater decrease than the other two groups. The retention rate of the melanoidin sample was reduced by about 17.23% after 10 d of sunlight exposure. It could be that the ultraviolet rays contained in sunlight accelerated the oxidative degradation of melanoidin. Similarly, melanoidin from black garlic also showed a decreasing trend under ultraviolet conditions[24]. The retention rate of melanoidin treated with incandescent light was similar to that of dark conditions with a retention rate of about 90%, suggesting that the purified UF3 can be stored and used under incandescent light. As shown in Fig. 5b, the retention rate of the melanoidin samples treated with different heating temperatures showed a fluctuating trend, indicating that the heat treatment causes heat polymerization and heat degradation of the purified UF3[28]. Therefore, heat treatment could aggravate the thermal instability of the melanoidin.

    Figure 5.  Stability results of purified UF3 under (a) different light conditions, and (b) different temperatures.
    DownLoad: Full-Size Img PowerPoint

    Changes in antioxidant activities of purified UF3 in in vitro digestion

    To assess the effect of in vitro enzymatic digestion on the antioxidant activity of purified UF3, the change of hydroxyl radical scavenging activity, DPPH scavenging activity, and reducing power of purified UF3 during the in vitro digestion process were investigated. As presented in Fig. 6a−c, the in vitro enzymatic digestion weakened the antioxidant activity of purified UF3. The antioxidant activities of melanoidins may be directly related to its structure, and in vitro digestion could result in a change in the structure of purified UF3. There are two reasons for this: firstly, the generation of new non-covalently bound melanoidin compounds in the presence of digestive enzymes, and secondly, the free radical scavenging activity of food melanoidins attributed to the presence of phenolic groups in their structure[32,36]. Although the antioxidant activity of digested samples was lower than that of undigested samples, all digested samples still exhibited a good antioxidant activity. After in vitro intestinal digestion, the hydroxyl radical scavenging activity, DPPH scavenging activity, and reducing power of digested UF3 was 65%, 50%, and 0.33, respectively. It could be seen that the purified UF3 extracted from BLB has potential antioxidant application value in vivo.

    Figure 6.  The changes on antioxidant activities of purified UF3 in vitro digestion. (a) Hydroxyl radical scavenging activity, (b) DPPH scavenging activity, and (c) reducing power. Different letters indicate significant difference at p < 0.05.
    DownLoad: Full-Size Img PowerPoint

    Conclusion

    This study demonstrated that melanoidin fractions with LMW (3 kDa < MWC < 10 kDa) were dominant fractions in low thermal induced BLB, and the dominant fractions exhibited excellent antioxidant activities. The dominant melanoidin fractions (UF3) were purified, preliminarily characterized, and its antioxidant activities checked by in vitro digestion. The results of FTIR spectroscopy revealed that purified UF3 showed a typical structure of melanoidins. The optimum purification conditions proved to be efficient for the purification of UF3 from BLB. The purified UF3 was stable in the dark and under light conditions without ultraviolet rays. However, the purified UF3 with LMW was unstable under heat treatment. The antioxidant activities of purified UF3 decreased after in vitro intestinal digestion, yet it exhibited a certain level of antioxidant activity. These results suggest that the UF3 extracted from low thermally induced BLB could be considered as a potential antioxidant ingredient and as a potential antioxidant component for the development of orally consumable functional foods.

    Author contributions

    The authors confirm contribution to the paper as follows: study conception and design: Chen J; experimental processing: Wang J, Liu Y; data collection and validation: Wang J, Li H; analysis and interpretation of results: Li H, Wang W; draft manuscript preparation: Chen J, Pan Y; language editing, manuscript revision: Hu Y. All authors reviewed the results and approved the final version of the manuscript.

    Data availability

    Data are available from the corresponding author on reasonable request.

    Acknowledgments

    This study was partly supported by the Innovative Team of Modern Agricultural Industry Technology System in Tianjin (ITTHRS2021000) and Key R & D Project of Xinjiang Uygur Autonomous Region (2022B02030).

  • The authors declare that they have no conflict of interest.

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  • Cite this article

    Yang D, Wu X, Shi H, Zhang J, Wang C. 2022. Essential aroma substances and release pattern of Xinhui Chenpi. Beverage Plant Research 2:22 doi: 10.48130/BPR-2022-0022
    Yang D, Wu X, Shi H, Zhang J, Wang C. 2022. Essential aroma substances and release pattern of Xinhui Chenpi. Beverage Plant Research 2:22 doi: 10.48130/BPR-2022-0022
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Essential aroma substances and release pattern of Xinhui Chenpi

  • 1.

    College of Food Science and Biological Engineering, Tianjin Agricultural University, No. 22, Jinjing Road, Tianjin 300384, PR China

  • 2.

    Key Laboratory of Tea Science of Ministry of Education, Hunan Agricultural University, Changsha, Hunan 410128, PR China

  • Received: 12 September 2022
  • Accepted: 16 November 2022
  • Published online: 09 December 2022
Beverage Plant Research  2,  Article number: 22  (2022)  |  Cite this article

Abstract: This study focuses on analyzing aqueous solutions of aroma-active compounds in Xinhui Chenpi distilled after being heated with headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC/MS). Feasibility of this method was also tested by comparison with crushed samples. The study also analyzed the aqueous solutions of the aroma-active compounds employing gas chromatography-olfactometry (GC-O) defense, aroma extraction dilution analysis (AEDA) and odor activity value (OAV) as well as their emission patterns. According to the study, there are 24 major aroma-active detected in the aqueous solution. Linalool, d-limonene, 2-methoxy-4-vinylphenol, and α-terpineol with sweet, spicy, and woody aroma contributed the most to the aroma-active compounds and were considered to be the essential aroma substances of Chenpi. When heated, the aroma of aromatic-active compounds rich in Chenpi volatilized rapidly and the release dropped dramatically in a short time. The substantial aroma-active compounds can be collected from the first ten released segments.

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    INTRODUCTION

    • Chenpi (Tangerine peel) is made from ripe peels of citrus (Citrus reticulata Blanco), dried in the sun or low temperatures[1]. The production of dried Chenpi mainly includes fruit picking and washing, peeling, drying in the shade, turning, drying in the sun, storage, turning and drying, and flesh sweeping, as shown in Fig. 1. Chenpi is known for its 'chen' (in Chinese it means a long time), so the best Chenpi comes from being stored for a very long period of time. It should be stored at least for three years. Citrus are usually produced from Guangdong, Fujian, Sichuan, Guangxi and Zhejiang, etc, in China. Multiple kinds of citrus from different places can be made into Chenpi of various qualities. And Xinhui Chenpi is top in its family, Chenpi is usually hard and brittle with a fragrant-pungent, and bitter taste. It plays an important part in promoting digestion and curing-vomiting, coughs, and respiratory diseases[2−8].

      Figure 1. 

      Xinhui Chenpi production process flow chart.

      The unique aroma of Chenpi is among the many criteria used to evaluate its quality. According to available literature, the aroma of food comes mainly from its volatile compounds, also called aroma-active compounds[9]. Much progress has been made in studying aroma-active compounds of Chenpi with the method of GC-MS. Duan et al.[10] used the GC-MS metabolomics approach combined with principal component analysis and orthogonal partial least squares discriminant analysis to show that the method effectively differentiated samples. Finally, 15 compounds such as methyl methanthranilate, α-sinensal, geranyl acetate, thymol were identified as chemical markers of Chenpi samples. With the method of GC-MS, Luo et al.[2] detected 98 compounds in Chenpi volatile oil and their main components were d-limonene, γ-pinene, α-pinene, linalool and myrcene.

      The extraction of volatile compounds of Chenpi is a key technology for its quality analysis. At present, most studies on aroma-active compounds of Chenpi are based on shearing treatment[11] or extraction of volatile compounds with the help of solvent apparatus, and the main extraction methods involved are supercritical fluid extraction (SFE)[12], hydro-distillation (HD)[9], vacuum steam fraction (VSF)[13], solvent extraction (SE)[6,14] and simultaneous distillation extraction (SDE)[15], each of which has its own advantages and disadvantages. The main disadvantages of these methods including the solvent being contaminated and the time consuming nature of the complicated steps, as well as the variety of equipment needed. In this study, water vapor condensation reflux method was used to extract the aroma-active compounds, which is simple and easy to operate. This method resulted in an aqueous solution of aroma-active compounds (hereafter referred to as aroma water), which fully enriched the aroma-active compounds of Chenpi without solvent contamination.

      In addition, GC-O was more often used to screen aroma-active compounds. Analyzing Chenpi oil by GC-O and AEDA, Dharmawan et al.[16] concluded that β-pinene, α-pinene, linalool, and 2-methoxy-3-(2-methylpropyl) pyrazine were characteristic aroma substances. Xiao et al.[17] used GC-O to analyze five citruses and concluded that substances such as nonanal, hexanal, linalool and limonene (OAVs ≥ 1) were the characteristic aroma compounds of orange juice samples.

      This experiment collects Chenpi aroma water from Chenpi volatiles obtained by heating and hydro distillation. Then the aroma water was analyzed by HS-SPME-GC-MS, and its essential aroma substances were selected using the method of GC-O, AEDA results were expressed as flavor dilution factors (FD), and OAV was used to identify the unique contribution of each compound to the characteristic aroma. In addition, the essential aroma-active compounds of Chenpi varied with heating time during the aroma collecting process. Therefore, the release pattern of essential aroma substances of Chenpi was identified.

    MATERIAL AND METHODS

      Materials

    • Xinhui Chenpi sample (WG20210023, WG20210045, and WG20210066) were obtained from Yunnan Tasly Deepure Biological Tea Group Co., Ltd. (Pu’er, Yunnan, China).

      Reagents and chemicals

    • Furfural (PubChem CID: 7362; ≥ 99.5%), octanal (PubChem CID: 454; 99%), myrcene (PubChem CID: 31253; ≥ 90%), γ-terpinene (PubChem CID: 7461; 95%), thymol (PubChem CID: 6989; > 99%), (−)-carvone (PubChem CID: 379; ≥ 95%), cymenol (PubChem CID: 10364; 99%), 2-methoxy-4-vinylphenol (PubChem CID: 332; ≥ 98%), citronellyl acetate (PubChem CID: 9017; ≥ 96%), methyl methanthranilate (PubChem CID: 6826; 98%), geranyl acetate (PubChem CID: 7780; 96%), α-terpinene (PubChem CID: 7462; 95%), octanoic acid (PubChem CID: 379; ≥ 99.5%) and n-decanol (PubChem CID: 8174; 98%) were purchased from McLean Biochemical Technology Co., Ltd (Shanghai, China). Geraniol (PubChem CID: 637566; 99%), 1-nonanol (PubChem CID: 8175; 99.5%), 4-terpineol (PubChem CID: 11230; 98%), α-terpineol (PubChem CID: 17100; > 95%), β-ionone (PubChem CID: 638014; 97%), p-cymene (PubChem CID: 7463; ≥ 99.5%), 1-octanol (PubChem CID: 957; > 99.5%), n-decanoic acid (PubChem CID: 2969; > 99%) and β-ionone (PubChem CID: 638014; 97%) were purchased from Aladdin (Shanghai, China). nonanal (PubChem CID: 31289; ≥ 95%), linalool (PubChem CID: 6549; 98%) and d-limonene (PubChem CID: 440917; > 95%) were purchased from TCI (Shanghai, China). NaCl (analytical purity 99.5%) were purchased from Tianjin Zhiyuan Chemical Reagant Co. n-Alkanes solution (C8-C32) was employed to calculate the retention index (RI) of the detected components.

      Extraction of the volatile compounds

    • Samples were treated using two different methods. Samples were crushed with a universal crusher (Tianjin Teste Instruments Co., Ltd, Tianjin, China) and passed through 30 mesh sieves before the experiment. The second method is as follows: 100 g of sample was weighed in a 2000-mL round bottom flask, 1,500 mL of hot water was added, soaked for 40 min (in order to shorten the extraction time, and the aroma substances are concentrated), then the samples were distilled by heating. The hydro-distillation method was used to recover condensed water (rich in volatile aroma-active components) and saved for HS-SPME pretreatment. The experiments were repeated in triplicate.

      HS-SPME procedure

    • The analytical conditions of HS-SPME, GC-MS, and GC-O were adapted based on the proven method of Liu et al.[11]. The temperature rise procedure, shunt ratio, sample volume, and NaCl addition were optimized to establish the detection method, as shown in Fig. 2. The results showed good peak patterns and were suitable for this study.

      Figure 2. 

      Total ion chromatogram (TIC) of volatile compounds of Chenpi.

      New extraction heads were activated, as follows: the 50/30 μm DVB/CAR/PDMS extraction head was placed on the GC-MS inlet for about 30 min under the inlet temperature of 250 °C.

      TriPlus RSH autosampler (Thermo Fisher Scientific, USA) coupled with 50/30 μm divinylbenzene/carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) (purchased from Supelco) was used for HS-SPME analysis. Respectively, 1.0 g of sample crushing (add 5 mL of ultra-pure water), 5 mL aroma water sample and 1.5 g of NaCl were placed into a 20-mL headspace bottle. Extraction temperature 65 °C, extraction time 30 min, equilibrium temperature 65 °C, equilibrium time 10 min, desorption time 5 min.

      GC-MS analyses and quantitation of aroma-active compounds

    • The equipment used was Rtx-5MS column (30 m × 0.25 mm internal diameter, 0.25 μm film thickness (Restek, Bellefonte, PA, USA), TRACE1300-ISQ gas chromatograph-mass spectrometer (Thermo Fisher Scientific, USA), Thermo Scientific Barnstead water purification system (Thermo Fisher Scientific, USA). The procedures of temperature increase are as follows: initial temperature 40 °C, ramp-up to 70 °C at 10 °C/min, ramp-up to 190 °C at 3 °C/min, ramp-up to 250 °C at 10 °C/min, hold for 3 min; flow rate: 1.0 mL/min; inlet temperature: 250 °C; injection volume: 1.0 μL; split ratio: 70:1; carrier gas: 99.999% high purity helium. EI ion source; ion source temperature: 250 °C; transfer line temperature: 250 °C; ionization energy: 70 eV; scan mode: full scan; mass scan range 40−500 m/z.

      Quantitative analysis of volatile compounds was conducted using a standard curve that obtained for each compound. The stock solution composed of 24 aroma-active compounds was configured and diluted with ethanol to six gradients of 2, 2.5, 10/3, 5, 10 and 20 to produce the standard curve. Meanwhile, the retention indices and aroma characteristics of each aroma-active compound were also used to verify the qualitative results.

      GC-O analyses

    • GC-O analysis was performed using a Thermo Trace 1300 gas chromatograph (Thermo Fisher Scientific Inc., USA) equipped with a flame ionization detector and a sniffing port (ODP2, Gerstel, Inc., Germany), Rtx-5MS column (30 m × 0.25 mm internal diameter, 0.25 µm film thickness; Restek, Bellefonte, PA, USA) was used for the separation, other equipment used was SGH-300 high-purity hydrogen generator, SGK-2L low-noise air pump (Beijing Zhongke Jirui Technology Co. Ltd., Beijing, China).

      Experienced sensory evaluators completed GC-O sniffing experiments, three panelists were selected and trained based on GB/T 16291.1-2012 (Sensory analysis-general guidance for the selection, training, and monitoring of assessors)[18]. The time of odor appearance, end time, and odor description were recorded by the selected panelists.

      Odor activity value (OAV)

    • OAV is often used to evaluate and screen the contribution of aroma-active compounds to the aroma formation of samples[19]. According to the calculation formula, OAV is the ratio of concentration of aroma-active compounds relative to their respective threshold in water, the compound concentration is the absolute concentration corrected by the GC-MS standard curve, and the odor threshold in water is obtained from previous literature[16,20−24].

      Aroma extraction dilution analysis (AEDA)

    • Aroma water was diluted in gradient with water at 2n to obtain dilution multiples of 1:2, 1:4, 1:8, 1:16, etc. GC-O detection, based on sniffing until the aroma-active compound odor disappears, was determined as the FD factor of the compound.

      Aroma-active compound release pattern

    • In order to study the content changes of aroma-active compounds of samples with heating time during the aroma enrichment process, segmented interception of aroma extracts was performed during the extraction of aroma substances, with each 50 mL segment being collected 20 times. The relative quantification was performed by adding 0.01 mL of n-decanol (0.13 mg/kg) to analyze the release pattern of aromatic compounds. The process was repeated three times.

    RESULTS AND DISCUSSION

      Optimization of sample processing method

    • The study analyzed crushed samples, as well as aroma water samples. The number of aroma-active compounds in the samples and their relative content was examined to select the best treatment method. As shown in Fig. 3, 24 aroma-active compounds were identified in the detected values of the two forms of samples, such as d-limonene, linalool, 2-methoxy-4-vinylphenol, furfural, and α-terpinene, etc.; aroma-active compounds specific to the heated enriched aroma-active water such as p-cymene, 1-octanol, 4-terpineol, etc. There were 19 kinds of aroma-active compounds particular to the directly crushed samples such as undecanal, citronellol, neral, etc. There were eight kinds of aromatic compounds.

      Figure 3. 

      Comparison of aroma-active compounds in two forms, (a) concentration and number; (b) the number of compounds.

      The relative content of aroma-active compounds was collected using the normalization method. The sum of the relative content of aroma-active compounds in the water sample was greater than that of the crushed sample, and the aroma-active compounds were also more in the aroma water sample. In conclusion, the experimental feasibility of studying the aroma-active compounds of aroma water met expectations, and the aroma-active compounds of the Xinhui Chenpi samples could be characterized in this way.

      Quantitative analysis of aroma-active compounds

    • The GC-MS qualitative analysis of the volatile compounds contained in the aroma water detected a total of 24 volatile aroma-active compounds with a matching index greater than 80% compared with the database, including six alcohols, four olefins, three aldehydes, two ketones, one acid, three phenols and three esters, as shown in Table 1. The corresponding standard curves were established for each compound with good linearity (equations of standard curves, where y is the area of the peak of an authentic standard, and x is the concentration of the authentic standard). Among them, d-limonene (3,291.64 mg/kg) showed highest in all compounds, followed by linalool (561.39 mg/kg), 4-terpineol (370.81 mg/kg), γ-terpinene (354.97 mg/kg), furfural (274.80 mg/kg), 2-methoxy-4-vinylphenol (253.67 mg/kg) and α-terpineol (250.54 mg/kg). According to the available literature, compounds such as d-limonene, γ-terpinene, linalool, and myrcene are essential components of the aroma composition of Chenpi[25−30], and substances such as 4-terpineol, furfural, 2-methoxy-4-vinylphenol, and p-cymene in the Chenpi aroma-active substances have been rarely reported previously. In the present study, octanal (78.71 mg/kg), thymol (38.18 mg/kg), cymenol (29.62 mg/kg), and myrcene (21.19 mg/kg) were the compounds that contributed significantly to the aroma of Xinhui Chenpi.

      Table 1.  The concentration of volatile compounds detected in aroma water samples.

      NoTime
      (min)      		RICompoundMolecular formulaCalibration curvesR2Linear range
      (mg/kg)      		Content
      (mg/kg)a      		RSD (%)b
      14.12840FurfuralC5H4O2y = 2.08E+09x + 1.76E+06R2 = 0.996592.34-4.62274.8017.64
      27.42983MyrceneC10H16y = 3.55E+11x – 1.25E+07R2 = 0.99391.37-0.0721.1924.52
      37.741002OctanalC8H16Oy = 3.05E+10x – 4.37E+06R2 = 0.9922.34-0.1278.7131.41
      48.191009α-TerpineneC10H16y = 2.73E+11x – 6.85E+06R2 = 0.99190.51-0.033.4727.55
      58.441017p-CymeneC10H14y = 4.27E+11x – 5.02+05R2 = 0.99590.41-0.0212.8513.06
      68.601023d-LimoneneC10H16y = 1.06E+11x + 2.55E+08R2 = 0.9916127.98-6.403291.6428.02
      79.531054γ-TerpineneC10H16y = 1.50E+11x – 3.95E+07R2 = 0.994112.92-0.65354.9726.94
      89.8710711-OctanolC8H18Oy = 1.62E+11x + 6.65E+06R2 = 0.99230.40-0.043.3220.64
      910.911101LinaloolC10H18Oy = 1.37E+11x + 9.32E+08R2 = 0.990233.41-3.34561.3917.00
      1011.081105NonanalC9H18Oy = 1.61E+11x + 4.96E+06R2 = 0.99150.38-0.046.4518.43
      1113.5811681-NonanolC9H20Oy = 1.05E+11x + 3.83E+07R2 = 0.99421.38-0.1410.1021.04
      1213.9011924-TerpineolC10H18Oy = 9.17E+10x + 3.02E+08R2 = 0.990913.14-0.66370.8125.28
      1314.401202α-TerpineolC10H18Oy = 1.08E+11x + 1.66E+08R2 = 0.990819.11-1.91250.5430.68
      1414.701219Octanoic acidC8H16O2y = 2.00E+09x + 1.41E+07R2 = 0.9946--
      1516.521291(-)-CarvoneC10H14Oy = 2.07E+11x + 4.62E+07R2 = 0.9909092-0.096.1824.79
      1616.921304GeraniolC10H18Oy = 2.00E+11x + 4.34E+07R2 = 0.99011.39-0.1416.9928.71
      1718.511358ThymolC10H14Oy = 6.54E+11x + 6.06E+08R2 = 0.99227.88-0.7938.1826.40
      1818.891371CymenolC10H14Oy = 6.14E+11x + 7.10E+07R2 = 0.99160.77-0.0829.6221.95
      1919.4013402-Methoxy-4-vinylphenolC9H10O2y = 3.41E+10x + 2.15E+08R2 = 0.991131.33-3.92253.6730.06
      2021.001360Citronellyl acetateC12H22O2y = 8.53E+11x + 1.20E+07R2 = 0.99070.14-0.011.6826.58
      2121.561380n-Decanoic acidC10H20O2y = 3.55E+11x – 1.42E+07R2 = 0.99240.48-0.057.1731.25
      2222.221385Geranyl acetateC12H20O2y = 1.01E+12x + 6.69E+06R2 = 0.99060.08-0.0080.5430.38
      2323.221453Methyl methanthranilateC9H11NO2y = 1.16E+11x + 7.55E+06R2 = 0.99140.53-0.0515.2010.21
      2426.401486β-IononeC13H20Oy = 5.98E+11x + 3.47E+06R2 = 0.99940.06-0.0071.9423.68
      a: The data of concentration is mean (n = 3 for aroma water sample). b: The RSD is standard deviation (n = 3 for aroma water sample). −: Indicates no detection results.

      GC-O verifies the aroma-active compounds in samples

    • The odor activity of 24 aroma-active compounds detected by GC-MS was characterized by GC-O combined with AEDA and expressed as FD dilution factor, which was used in combination with the OAV value of each aroma-active compounds to verify its contribution to the aroma-active compounds of Xinhui Chenpi[24,31,32]. As shown in Table 2, the FD dilution factors ranged from 2 to 8,192, the higher FD factors, the stronger the odor of the compounds, and the greater the contribution to the aroma of the sample. The compounds with the highest FD factors were linalool (8,192) with sweet, cymenol (8,192) with pungent and refreshing odors, 2-methoxy-4-vinylphenol (8,192) with pungent and flower odors, followed by β-ionone (4,096) with woody odor, 4-terpineol (2,048) with woody and loamy incense odors, α-terpineol (2,048) with woody and flower odors, (−)-carvone (2,048) with mint and spicy odors, geranyl acetate (2,048) with medicinal odor, n-decanoic acid (2,048) with flower odor. The aroma-active compounds of Xinhui Chenpi are mainly alcohols, olefins, esters and aldehydes, and other compounds such as ketones and phenols. Among them, alcohols and olefins accounted for the highest proportion, such as d-limonene and linalool, which mainly show the typical orange and sweet odor of Chenpi, are the most abundant compounds in Xinhui Chenpi, with FD factors of 32 and 8,192, respectively. Aldehydes also contributed to the aroma-active formation of Chenpi, such as octanal, which has a typical orange flavor with an FD factor of 512. Furfural has a nut odor, which has rarely been reported in previous studies of aroma-active compounds of Chenpi.

      Table 2.  The FD factor of aroma-active compounds.

      NoCompoundOdor description*FD**
      1FurfuralNut8
      2MyrcenePungent32
      3OctanalOrange flavor512
      4α-TerpineneWax, orange16
      5p-CymeneRefreshing8
      6d-LimoneneCitrus32
      7γ-TerpineneWoody8
      81-OctanolOily, fruity128
      9LinaloolFlowers, sweet8192
      10NonanalOily, sweet, orange8
      111-NonanolOrange scent2
      124-TerpineolWoody, loamy incense2048
      13α-TerpineolFlowers, woody2048
      14Octanoic acidFruity32
      15(−)-CarvoneMint, spicy2048
      16GeraniolRose512
      17ThymolMedicine1024
      18CymenolPungent, refreshing8192
      192-Methoxy-4-vinylphenolPungent, flowers8192
      20Citronellyl acetateFlowers16
      21n-Decanoic acidFlowers2048
      22Geranyl acetateMedicine2048
      23Methyl methanthranilateOrange, flowers1024
      24β-IononeWoody4096
      * Description of the sniffing results by the sensory evaluator (n = 3 for sensory evaluator).
      ** Maximum dilution of the aroma-active compound.

      To identify the contribution of aroma-active compounds to the aroma of Xinhui Chenpi, the OAV was calculated to verify that each compound's odor threshold was known from the literature. The literature has reported that an aroma-active compound OAV ≥ 1 indicates that the compound contributes to aroma formation[24,33,34]. As shown in Table 3, the compounds have been ranked in order of OAV from largest to smallest. Octanoic acid was not detected in the content; other than that, all 22 aroma-active compounds revealed had OAV > 1 in aroma water. The results showed that d-limonene had the highest OAV (24,027) in all compounds, followed by linalool (20,050), 2-methoxy-4-vinylphenol (13,351), geraniol (1,699), thymol (382), octanal (342), α-terpineol (291), β-ionone (231). The above results show that the most important aroma-active compounds were d-limonene and linalool in Chenpi. Notably, although the contents of geraniol (16.99 mg/kg) and β-ionone (1.94 mg/kg) were not very high, their OAVs were the highest, because the thresholds were low (0.01 and 0.0084 mg/kg).

      Table 3.  The results of OAVs calculation of aroma-active compounds.

      NoCompoundConcentration
      (mg/kg)      		Odor threshold in
      water (mg/kg)      		OAV
      6d-Limonene3291.640.14b24026.58
      9Linalool561.390.03a20049.61
      192-Methoxy-4-vinylphenol253.670.02b13351.10
      16Geraniol16.990.01a1699.27
      17Thymol38.180.10b381.80
      3Octanal78.710.23b342.23
      13α-Terpineol250.540.86b291.32
      24β-Ionone1.940.01c231.49
      18Cymenol29.620.18b164.56
      81-Octanol3.320.02b144.30
      15(-)-Carvone6.180.07a92.26
      1Furfural274.803.00c91.60
      10Nonanal6.450.10a64.50
      124-Terpineol370.816.40a57.94
      21n-Decanoic acid7.170.13b55.19
      23Methyl methanthranilate15.200.35b43.54
      2Myrcene21.190.67a31.63
      111-Nonanol10.101.00a10.10
      7γ-Terpinene354.9755.00c6.45
      22Geranyl acetate0.540.15a3.60
      5p-Cymene12.857.20b1.78
      20Citronellyl acetate1.681.00b1.68
      4α-Terpinene3.472.40b1.45
      14Octanoic acid−0.86b−
      Odor thresholds in water found in the literature. a: Indicates reference[18]. b: Indicates reference[35]. c: Indicates reference[21]. −: Indicates no detection.

      Release pattern of aroma-active compounds

    • The changes of 24 aroma-active compounds with heating time when samples were heated to enrich the aroma-active compounds were averaged over three replicate values. As shown in Table 4 and Fig. 4, a decreasing pattern was seen, with a sharp decrease after the 1st time, and a slight increase from the 2nd to the 3rd time, followed by a slight decrease.

      Table 4.  Changes in the concentration of aroma-active compounds of Chenpi with extraction time.

      Time (min)CompoundConcentration (mg/Kg)
      1234567891011121314151617181920
      4.12Furfural4.072.462.492.482.592.692.712.702.532.412.242.322.632.582.542.662.702.612.512.21
      7.42Myrcene1053.510.370.250.220.310.400.190.510.200.370.350.391.090.440.850.691.200.770.770.98
      7.74Octanal69.184.196.485.876.805.306.975.645.255.565.535.086.294.985.104.965.897.615.994.75
      8.19α-Terpinene114.060.560.590.360.520.510.440.560.500.520.560.420.750.590.780.721.100.970.881.11
      8.44p-Cymene53.161.181.180.891.241.180.881.380.821.161.171.171.341.340.721.711.581.411.331.97
      8.60d-Limonene36762.6620.3020.3321.2420.2921.3218.2344.9220.0032.1830.1533.8045.8236.9142.0830.5020.7921.7929.8426.29
      9.53γ-Terpinene3591.331.631.521.721.811.611.593.731.782.802.763.124.003.053.774.133.674.073.653.33
      9.871-Octanol20.452.862.861.721.871.811.421.081.171.020.881.110.750.580.470.390.450.550.42-
      10.91Linalool1080.19396.41408.52350.55346.02352.47270.33240.68232.30211.12194.25173.38180.08161.11140.28138.62121.05112.0861.1259.41
      11.08Nonanal58.693.083.303.003.723.613.492.962.913.433.593.293.563.283.503.563.543.343.043.39
      13.581-Nonanol16.307.727.627.316.966.806.625.514.713.903.233.293.283.233.293.362.983.002.972.79
      13.904-Terpineol498.61147.36144.59136.17133.69123.46117.43108.56112.45110.63104.22104.54103.89103.29102.11106.1089.2784.6550.5679.04
      14.40α-Terpineol394.39134.60127.01114.39113.81100.9979.6168.7970.1967.8461.1756.7056.0356.1255.6255.0043.9041.6241.0140.69
      14.70Octanoic acid−4.284.11−−−−−−−−−−−−−−−−−
      16.52(−)-Carvone30.1115.9014.4113.3211.479.656.815.314.924.143.342.862.872.101.841.551.040.980.910.78
      16.92Geraniol24.1618.2617.6715.0914.8513.2410.377.848.167.786.635.705.605.284.824.953.853.432.842.66
      18.51Thymol207.17187.75184.75180.91168.43134.25103.7586.0483.9574.1057.6454.9951.6047.0840.0638.6728.1923.1318.9317.40
      18.89Cymenol72.98152.32175.42189.58198.40198.78116.7182.0820.8720.1520.0120.9321.0327.2430.9424.918.897.4914.3213.62
      19.402-Methoxy-4-vinylphenol22.9723.2131.7062.0368.2271.4954.0653.2151.4550.4650.3450.1049.7248.8548.5349.5449.7044.4344.7239.54
      21.00Citronellyl acetate21.935.325.305.205.555.263.653.401.731.521.481.411.731.671.481.441.561.461.862.09
      21.56n-Decanoic acid−12.0514.5214.5215.4516.5812.0510.008.037.427.317.737.307.335.275.735.615.115.725.50
      22.22Geranyl acetate7.131.121.161.291.161.291.201.181.181.161.161.151.161.041.161.171.091.091.181.13
      23.22Methyl methanthranilate−5.126.935.655.024.894.844.823.102.822.522.161.981.681.541.491.431.050.890.73
      26.40β-Ionone7.726.955.234.884.883.823.213.022.852.622.362.232.061.841.721.661.461.090.990.99
      Total concentration44839.781155.541187.931138.381132.861081.40826.57743.90641.10615.12562.88537.87554.55521.93498.49483.52400.94373.74326.45310.42
      - indicates no detection.

      Figure 4. 

      Changes of total concentration of aroma-active components in Chenpi with heating time.

      As shown in Table 4, the nine compounds including furfural, myrcene, octanal, α-terpinene, p-cymene, d-limonene, γ-terpinene, nonanal, and geranyl acetate showed a pattern of rapidly decreasing to a minimum and then being continuously maintained; the 10 compounds including 1-octanol, linalool, 1-nonanol, 4-terpineol, α-terpineol, (−)-carvone, geraniol, thymol, citronellyl acetate, β-ionone showed a gradually decreasing pattern; cymenol, 2-methoxy-4-vinylphenol, n-decanoic acid, and methyl methanthranilate showed a pattern of increasing first, reaching a maximum value, and then gradually decreasing; octanoic acid was detected only in the 2nd and 3rd times.

      The alcohols and ketones in the aroma-active compounds of Xinhui Chenpi mainly showed a gradually decreasing pattern. For example, among the alcohols, linalool (1,080.19 mg/kg), 4-terpineol (498.61 mg/kg), α-terpineol (394.39 mg/kg) are of the highest proportion, linalool decreased from 1,080.19 mg/kg to 59.41 mg/kg; 4-terpineol decreased from 498.61 mg/kg to 79.04 mg/kg; α-terpineol decreased from 394.39 mg/kg to 40.69 mg/kg. This indicates that the alcoholic compounds of Xinhui Chenpi are persistent in aroma and slow in release.

      The olefins and aldehydes mainly showed a pattern of rapidly decreasing at the lowest level and then being continuously maintained. Among the olefins, d-limonene (36,762.66 mg/kg), γ-terpinene (3,591.33 mg/kg), myrcene (1,053.51 mg/kg) and α-terpinene (114.06 mg/kg) are of the highest proportion, and d-limonene decreased from 36,762.66 mg/kg to 20.30 mg/kg in the 2nd and maintained since then; The γ-terpinene decreased from 3,591.33 mg/kg to 1.63 mg/kg in the 2nd and maintained since then; Myrcene decreased from 1,053.51 mg/kg to 0.37 mg/kg in the 2nd and maintained since then; α-terpinene decreased from 114.06 mg/kg to 0.56 mg/kg in the 2nd and maintained at a consequent level after that. It indicates that the olefins of Xinhui Chenpi are easily soluble and released extremely fast.

      Other compounds, such as thymol gradually decreased from 207.17 mg/kg to 17.40 mg/kg; cymenol gradually increased from the initial 72.98 mg/kg, reaching a maximum value of 198.78 mg/kg at the 6th pass, and then started to decrease to 13.62 mg/kg; 2-methoxy-4-vinylphenol gradually increased from the initial 22.97 mg/kg to a maximum of 71.49 mg/kg at the 6th time and then started to decline to 39.54 mg/kg. This indicates that the aroma was persistent and remained in the release phase during the heating extraction.

      Furfural differed from the other substances, with a small amount of 4.07 mg/kg initially, which decreased to 2.46 mg/kg in the 2nd and then continued to be maintained, indicating that Furfural is more difficult to volatilize than the other substances.

    CONCLUSIONS

    • Aroma water obtained by heating and enrichment has obvious advantages. The aroma water of Xinhui Chenpi was comprehensively analyzed using the methods of GC-MS, GC-O and AEDA. It was concluded that linalool, d-limonene, 2-methoxy-4-vinylphenol, and α-terpineol were the four aroma-active compounds with high content and high contribution to the aroma formation, which were identified as the essential aroma substances of Xinhui Chenpi, and mainly presenting orange, sweet, spicy, woody and floral aromas. The heat release pattern was studied, and the results showed that the content of the aroma-active compounds generally showed a decreasing pattern with the heating time in the process of heating enrichment. In particular, the overall content decreased sharply after the 1st time, and it was speculated that this occurred because the Chenpi samples were soaked in heated water before being enriched by heating and distillation. However, this conclusion needs to be further verified. Although the overall content changed significantly after the 1st time, and combined with the respective emission pattern of 24 aroma-active compounds, it is difficult to volatilize all the aroma-active compounds in a short time, so this experiment recommends that the first 10 times of aroma water can be collected to achieve a shorter time and enrichment of the most aroma-active compounds.

      Acknowledgments

      • This work was supported by Youth Top Talents Project in Tianjin Special Support Program, the Science and technology Project of Tianjin (Grant No. 21ZYCGSN00410), and the Science and technology Project of Tianjin (Grant No. 21YDTPJC00910). This work was supported by the National Natural Science Foundation of China [32102004]; the Project funded by China Postdoctoral Science Foundation [2021M701169, 2022T150206], the China Agriculture Research System of MOF and MARA; the Construction of World Big-leaf Tea Technology Innovation Center and Industrialization of Achievements [202102AE090038]; and the Scientific and Technological Talents and Platform Plan (Academician Expert Workstation) [202104AC100001-B01].

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      Rights and permissions
      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press, Fayetteville, GA. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (4)  Table (4) References (35)
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    Cite this article
    Yang D, Wu X, Shi H, Zhang J, Wang C. 2022. Essential aroma substances and release pattern of Xinhui Chenpi. Beverage Plant Research 2:22 doi: 10.48130/BPR-2022-0022
    Yang D, Wu X, Shi H, Zhang J, Wang C. 2022. Essential aroma substances and release pattern of Xinhui Chenpi. Beverage Plant Research 2:22 doi: 10.48130/BPR-2022-0022
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  • Introduction
  • Material and methods

  • Materials

  • Reagents and chemicals

  • Extraction of the volatile compounds

  • Hs-spme procedure

  • Gc-ms analyses and quantitation of aroma-active compounds

  • Gc-o analyses

  • Odor activity value (oav)

  • Aroma extraction dilution analysis (aeda)

  • Aroma-active compound release pattern

  • Results and discussion

  • Optimization of sample processing method

  • Quantitative analysis of aroma-active compounds

  • Gc-o verifies the aroma-active compounds in samples

  • Release pattern of aroma-active compounds

  • Conclusions
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