Characterization of volatile flavor compounds of cigar with different aging conditions by headspace–gas chromatography–ion mobility spectrometry

1 Introduction

Cigars, distinctive tobacco products composed entirely of cigar leaves, are cherished by consumers for their unique flavor profiles. The manufacturing process of cigars primarily encompasses cultivation, harvesting, fermentation, rolling, and aging [1]. A cigar consists of three parts, including wrapper, binder, and filler, each made from different varieties, grades, and parts of fermented cigar tobacco leaves [2]. After rolling, the tobacco leaves are independent of each other, resulting in cigars with greater stimulation, poor harmony, and softness. To enhance the flavor of cigars, they must undergo an aging process before being sold [3]. The slang goes “the quality of cigars is 30% determined by tobacco leaves and 70% by aging.” The aging process is the final stage of cigar production and plays an important role in the cigar production.

The aging process of cigars is usually stored in a cabinet or room with controlled temperature and humidity. The aging conditions are usually determined by the characteristics of cigars. The aging time of cigars usually ranges from several months to several years. With the change in aging time, the coordination of full-bodied cigars continuously improved, resulting in a more mellow flavor, However, light cigars should not be too long, as this can lead to a dull and tasteless [4]. The aging temperature of cigars is usually controlled at 16–24°C. When the aging temperature is excessively high, the cigar is hard and easy to burst. Additionally, the wrapper is wrinkled with obvious veins, and the smoke of the cigar is dry and irritating. Moreover, high temperatures can accelerate the maturation of cigars, leading to the loss of their mellow taste and flavor [5]. The aging humidity of cigars is usually controlled at 60–70%, which can prevent cigars from becoming too dry or overly moist, retain the flavor of cigars, and avoid mold and pests. In general, the humidity at the bottom of the aging cabinet or room is higher, but not absolute, so it is necessary to regularly flip the cigars during the aging process. Suitable aging time, temperature, humidity, and flipping frequency are conducive to improving the quality of cigars.

Recently, more attention has been paid to the aging media of cigars, hoping to improve the quality and stability of cigars. Hu et al. [3] demonstrated that adding cocoa and coffee media during the aging process can improve the smoke and aroma characteristics of cigars. Another study suggested that the aging treatment of Cunninghamia lanceolata and Sabina pingii var wilsonii can enrich the variety of volatile components in cigars, mainly including olefins and nitrogen-containing compounds [6]. Additionally, Xue et al. [5] investigated the biodegradation of biomass and alkaloids of cigars under different aging conditions through metagenomics; however, they did not conduct a comprehensive and continuous analysis of volatile flavor compounds (VFCs) of cigar samples under different aging conditions.

Flavor is an important index of cigar quality, significantly influencing consumer preferences. It is also an essential index for the cigar production process. The type and quantity of VFCs determine the flavor of the cigar. Zheng et al. [7] studied the differences in cigar leaves from different production areas with flavor substances as key indicators. Zong et al. [8] evaluated the VFCs of cigar tobacco leaves of the adding cacao medium agricultural fermentation. Hu et al. [9,10] also evaluated the VFCs of cigar tobacco leaves treated with exogenous additives during industrial fermentation. Headspace–gas chromatography–ion mobility spectrometry (HS–GC–IMS) combines the high separation ability of gas chromatography with the fast response ability of ion migration spectrometry. In recent years, a novel analytical technique has emerged, known for its simplicity, speed, and sensitivity in detecting VFCs. This technique facilitates the processing of numerous samples promptly, without extensive pretreatment, and is capable of real-time detection of VFCs at ppb concentrations [11,12]. As a result, the HS–GC–IMS technology has been widely applied in detecting VFCs in various tobacco products, including electronic cigarettes and cigarette paper [13,14,15,16,17,18].

The purpose of this research was to identify the differences and potential changing pathways of the VFCs of cigar samples under different aging conditions by HS–GC–IMS. The study characterized the aging cigar samples based on the VFCs data through the application of Orthogonal projection to latent structures discriminant analysis (OPLS-DA). This research offers a theoretical foundation and practical guidance for quality control and cigar aging technology during the aging cigar process.

2 Methods

3 Results

3.1 Analysis of the topographic plots of VFCs of cigar via GC–IMS

GC–IMS was used to analyze the VFCs of cigar samples under different aging conditions. Figure 1 shows the VCFs of cigars under different aging conditions, with each point symbolizing a VFC. One sample’s spectral diagram was elected as the reference, with the spectral diagrams of the other samples undergoing subtraction by this reference. Instances, where the concentration of two VFCs was found to be congruent, resulted in a white backdrop post-subtraction. The appearance of red spots signified that a substance’s concentration exceeded that of the reference, whereas blue spots indicated a lower concentration relative to the reference. The analysis revealed notable differences in the molecular structure and concentrations of VFCs of cigars under varying conditions of aging time, temperature, humidity, and flipping frequency. This suggests that the aging conditions significantly influence the VFC profiles of cigar samples. With the increase in aging time, humidity, and flipping frequency, the concentration of the VFCs of cigar samples significantly increases, while with the aging temperature increases, the concentration of the VFCs of cigar samples initially increases before subsequently decreasing.

Figure 1

Comparison of topographic plots of GC–IMS analysis of cigar under different aging conditions: (a) time, (b) temperature, (c) humidity, and (d) flipping frequency.

3.3 Fingerprints of VFCs of cigar

Fingerprints visually represent the results of flavor measurement of samples. The visually distinct VFCs were selected and collectively presented in a gallery plot for intuitive comparison (Figure 2). In this representation, individual rows correspond to samples, and columns correspond to VFCs. Color intensity reflects VFC content, with deeper hues indicating higher concentrations. Accordingly, the differences in the VFCs of cigar samples under different aging conditions were observed. Figure 3 shows the differences in the total content of the VFCs across various functional groups in cigars under different aging conditions. The significance of these differences was assessed through one-way ANOVA.

Figure 2

Fingerprints of the VFCs of cigar samples under different aging conditions: (a) time, (b) temperature, (c) humidity, and (d) flipping frequency.

Figure 3

Response intensity of the VFCs of cigar samples under different aging conditions: (a) time, (b) temperature, (c) humidity, and (d) flipping frequency. The same letter indicates that the proportion of VFCs in cigars aged under varying conditions was not statistically significant (P > 0.05), whereas different letters denote statistical significance (P ≤ 0.05).

The results illustrated in Figures 2 and 3 reveal that the VFC content in cigar samples undergoes significant variations with the escalation of aging time, temperature, humidity, and flipping frequency. As the aging time increased, the content of the VFCs including N-heterocycles, ketones, O-heterocycles, alcohols, and olefins of cigars increased significantly (Figure 3a). Notably, the concentration of N-heterocycles such as 2-ethyl-5-methylpyrazine, ketones like 1-octen-3-one and 2-pentanone, O-heterocycles such as 2-ethylfuran and 2-heptylfuran, alcohols including 1-penten-3-ol, and olefins such as β-pinene, myrcene, and α-pinene either increase or newly generated with aging time (Figure 2a), whereas the content of esters initially significantly increase and then decrease with aging time, peaking at 60 days of aging (Figure 3a). The concentration of hexyl acetate and ethyl 2-methylbutyrate is either added or newly generated with increasing aging time (Figure 2a). In contrast, aldehydes such as 3-methylbutanal, heptanal, and 2-methyl-2-pentenal decrease or disappearance with increasing aging time, while the concentration hexanal first slightly decreases and then increases to the initial value with increasing aging time (Figure 2a). Furthermore, cigar samples aging at 90 days display a greater diversity and concentration of VFCs, suggesting a richer flavor profile at this aging day. Therefore, the changes in the VFCs of cigar samples under different aging temperatures were investigated at age 90 days.

Figure 3b shows that the total contents of aldehydes, ketones, and O-heterocycles of cigars were significantly higher at 16 and 20°C than at 24°C. When the aging temperature is 20°C, N-heterocycles such as 2-methylpyrazine and 2-ethyl-5-methylpyrazine, as well as aldehydes including benzaldehyde, 2-methyl-2-pentenal, and hexanal, ketones such as 1-octen-3-one, 4-methyl-2-pentanone, 2-pentanone, and 6-methyl-5-hepten-2-one, O-heterocycles like 2-ethylfuran, 2-n-butylfuran, alcohols including (Z)-3-hexen-1-ol, 1-penten-3-ol, hex-2-ol, 1-penten-3-ol, and linalool are either added or newly generated (Figures 2b and 3b). The content of esters and acids significantly increases with the increase in aging temperature, peaking at 24°C, especially 2-methylbutyrate ethyl ester, lactate ethyl ester, and butyrate methyl ester. The VFCs of cigar samples are abundant and diverse under the aging temperature of 20°C. Therefore, the changes in the VFCs of cigar samples under different aging humidities were investigated after 90 days of aging at 20°C.

Figures 2c and 3c show that when the aging humidity is 60%, the content of aldehydes in cigars is significantly higher, particularly 3-methylbutyraldehyde. The contents of alcohols, esters, and aromatic compounds in cigars were significantly higher at 65 and 70% aging humidity than at 60% aging humidity. When the aging humidity is 65%, the N-heterocycles 2-methylpyrazine, O-heterocycles such as 2-heptyl furan, 2-ethyl furan, 2-furanmethol, and 2-n-butyl furan were significantly higher. When the aging humidity was 70%, the ketones and alkenes were significantly higher, with notable increases in the ketone 2-pentanone, butane-2-one, hexyl-2-one, and alkene myrcene. The VFCs of cigar samples are abundant under aging humidity of 65 and 70%; however, higher humidity levels increase the risk of mildew. Therefore, the aging humidity of 65% was selected for further investigation into the influence of flipping frequency on the VFCs of cigar samples.

Figures 2d and 3d show that when the flipping frequency is 90 d/f, it is more conducive to the generation of the VFCs of cigar samples. With the decrease in flipping frequency, the N-heterocycles, ketones, O-heterocycles, esters, and olefins significantly increased, especially 2-ethyl pyrazine, 2,5-dimethyl pyrazine, 2-ethyl-5-methylpyrazine, 4-methyl-2-pentanone, 2,3-pentanedione, butyl-2-one, 1-octen-3-one, 2-heptylfuran, 2-n-butyl furan, 2-methylmercaptan furan, ethyl 2-methylbutyrate, hexyl acetate, ethyl lactate, isobutyl acetate, dibutyl sulfide, (E)-ethyl 2-hexanoate, myrcene, α-pinene, and 1-methyl-4-(1-methylvinyl) cyclohexene. The content of alcohols and acids was significantly higher under 30 d/f, particularly propanol-2-ol. When the flipping frequency was 45 d/f, the aldehydes, particularly methacrolein and 3-methylbutyraldehyde, were significantly differences.

3.4 OPLS-DA analysis of cigar characteristics under different aging conditions

OPLS-DA is a regression modeling technique that analyzes multiple dependent variables to multiple independent variables. It is a supervised statistical method of discriminant analysis. To assess the similarities and differences among cigars subjected to various aging conditions, OPLS-DA was performed according to the intensity of the VFCs of cigar samples under different aging conditions. As shown in Figure 4, the cigar samples are distinctly separated and clustered in a relatively independent space according to the intensity of the VFCs. The OPLS-DA model R ² X ≥ 0.577, R ² ≥ 0.93, Q ² ≥ 0.808, indicating that the model has good interpretation ability. Figure 5 shows R ² intercept values greater than 0, and Q ² intercept values less than 0, indicating the robustness, reliability, and absence of overfitting in the OPLS-DA model.

Figure 4

OPLS-DA analysis of VFCs of cigars under different aging conditions: (a) time, (b) temperature, (c) humidity, and (d) flipping frequency.

Figure 5

Corresponding OPLS-DA validation plots of cigars under different aging conditions: (a) time, (b) temperature, (c) humidity, and (d) flipping frequency.

4 Discussion

Since the processing conditions for cigars are relatively mild without high temperatures, the aging process of cigars is a continuation of the fermentation process of cigar tobacco leaves. A large number of enzymes and microorganisms in cigars are retained and active under appropriate aging temperature and humidity conditions [5]. These biological agents can degrade macromolecular substances such as proteins, starches, and cellulose of cigar tobacco leaves, resulting in the production of amino acids, sugars, and terpenoids. These compounds can further generate small-molecule flavor substances through the Maillard reaction, including furans, aldehydes, ketones, phenolic compounds, and alkenes, or continue to degrade [21,22]. There are also some macromolecular substances, such as cembranoids and labdanoids, that can be degraded into small molecules, including ketones, furans, and esters. These small molecules of VFCs play a crucial role in determining the characteristics of cigars [7]. The Maillard reaction, along with the degradation of terpenoids, cembranoids, and labdanoids, is a complex process influenced by factors such as reaction time, temperature, and humidity [23,24].

The content of aldehydes of cigar leaves is high, which plays an important role in the formation of cigar flavor. The findings of our study indicate that the total content of aldehydes is significantly affected by aging time, temperature, and humidity, especially 3-methylbutanal, heptanal, benzaldehyde, 2-methyl-2-pentenal, hexanal, and 3-methylbutyraldehyde. Previous research indicates that aldehydes such as benzaldehyde, 3-methylbutyral, and hexanal have pleasant fruity and nutty aromas [25] and that heptanal is positively correlated with the burnt sweet of cigar smoke [26]. Aldehydes like benzaldehyde, 3-methylbutyraldehyde, and hexanal are primarily generated through the degradation of amino acids and microbial transformation [27]. In the Maillard reaction, amino acids are deaminated and decarboxylated, leading to the formation of aldehydes [28]. Amino acid degradation, microbial transformation, and Maillard reaction are affected by reaction time, temperature, and humidity [27,28].

Additionally, our study revealed that the total content of esters and ketones is also significantly affected by aging time, temperature, and humidity, particularly for compounds such as ethyl acetate, ethyl 2-methylbutyrate, methyl butyrate, 2-pentanone, 6-methyl-5-heptene-2-one, and 4-methyl-2-pentanone. Esters make an important contribution to the flavor of tobacco; monomer esters have sweet, fruity, or wine-like aromas that harmonize with tobacco aromas, while mixing esters can endow cigars with a specific odor, reduce irritation, and make smoke aromas soft [29]. Ketones, including 2-pentanone, 6-methyl-5-heptene-2-one, and 4-methyl-2-pentanonecan, can endow cigars with sweet and fruity aromas [30].

Furthermore, our research indicated that the content of linalool initially increased and then decreased with the increase in aging temperature. Previous research has indicated that linalool is a degradation product of carotenoids, which gradually increases during the fermentation of cigar tobacco leaves [31]. The degradation of carotenoids is affected by the environment, and the degradation rate is faster and earlier at low temperature in the yellowing stage [32,33].

The heterocyclic compounds of the VFCs of cigar samples include N-heterocycles, O-heterocycles, and S-heterocycles, especially N-heterocycles and O-heterocycles, which have a higher content, consistent with the research of He et al. [34,35]. It may be related to the high nitrogen content of cigar tobacco leaves [36]. During the aging process of cigars, N-heterocycles and O-heterocycles are significantly affected by aging time, temperature, and humidity, especially 2-methylpyrazine, 2-ethylpyrazine, and 2-ethylfuran, 2-butylfuran [37]. The aroma threshold of these heterocyclic compounds is relatively low, which can endow cigars with roasted, nutty, fruity, and caramel aromas, thereby significantly enhancing their flavor [28]. N-heterocycles and O-heterocycles are mainly important products of the Maillard reaction of tobacco [38]. Key factors such as temperature, humidity, and reaction time play crucial roles in determining both the products and the rate of the Maillard reaction. The higher the temperature, the faster the reaction rate. The reaction time mainly affects the completeness of the reaction. The longer the reaction time, the more intermediate substances are produced, and the stronger the aroma. However, excessive reaction time can lead to adverse effects [39,40].

5 Conclusion

In this article, we utilized HS–GC–IMS rapid qualitative technology in conjunction with OPLS-DA to analyze the variations in VFCs of cigar samples under different aging conditions, establishing a distinctive flavor fingerprint. A total of 82 VFCs were identified by HS–GC–IMS, including 8 N-heterocycles, 11 aldehydes, 13 ketones, 8 O-heterocycles, 13 alcohols, 15 esters, 6 olefins, 3 aromatic compounds, 1 acid, and 4 sulfides. Topographic plots and fingerprints revealed that the changes in the VFCs of cigar samples under different aging conditions were mainly based on amino acid degradation, microbial transformation, the Maillard reaction, and the degradation products of carotenoids. These degradation products include benzaldehyde, 3-methylbutanal, hexanal, 2-methylpyrazine, 2-ethylpyrazine, 2-ethylfuran, 2-n-butylfuran, and linalool. Additionally, other compounds such as ketones and esters, including 2-pentanone, 6-methyl-5-hepten-2-one, ethyl acetate, 2-methylbutyrate ethyl ester, and butyrate methyl ester, play important roles in regulating and forming the flavor of cigar. The results of OPLS-DA demonstrated that cigar samples aged for different days exhibited greater dispersion compared to those subjected to varying temperature, humidity, and flipping frequency. Additionally, the impact of aging temperature, humidity, and flipping frequency on VFC alterations surpassed that of aging time. This study indicates that HS–GC–IMS combined with OPLS-DA can be used as reliable techniques to identify and classify the VFCs of cigar samples under different aging conditions quickly and sensitively. This research provides a theoretical basis for quality control and flavor change mechanism of the cigar aging process.