Ingredients used in this study were purchased from TIANGEN Technology Co., Ltd. (Beijing, China), unless noted otherwise. PBS solution (pH 7.2), Potato Dextrose Broth (PDB), agar powder, Ampicillin sodium, were purchased from Solarbio Technology Co., Ltd. (Beijing, China). Fungal genomic DNA extraction kit was purchased from Biospin Co., Ltd. (Hangzhou, China). QuEChERS extraction kit, QuEChERS SPE kit and other GC–MS required materials were purchased from Agilent (Santa Clara, United States). The cigar tobacco leaves for fermentation were collected and provided by China Tobacco Sichuan Industrial Co., Ltd. (Sichuan, China).

To isolate Cyberlindnera strains from cigar tobacco leaves, 5 g CTLs sourced from Yunnan, Hubei, and Sichuan. were separately added into 200 mL PBS solution (pH 7.2) and under shaking conditions with rpm of 180, 30°C for 3 h. The mixture was filtered to remove CTL fragments, and the suspension was then spread on PDB plates (with ampicillin sodium). After incubation for 2.5 days at 30°C. The microorganisms with yeast colony morphology were isolated and cultured individually. The genomic DNA was extracted by fungal DNA kit and amplified with primer pairs ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′)/ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and NL1 (5′-GCATATCAATAAGCGGAGGAAAAG-3′)/NL4 (5′-GGTCCGTGTTTCAAGACGG-3′) (Brysch-Herzberg et al., 2021). The PCR products of ITS and D1/D2 region was sequenced in Sangon Biotech (Shanghai, China), and the sequences were compared with the identified species using BLAST (Basic local alignment search tool). After identification, the isolated Cyberlindnera fabianii strain F4 was deposited in the China General Microbiological Culture Collection Center (CGMCC), Beijing, China, under accession number CGMCC 30447.

C. fabianii strains were cultured individually in PDB medium for 2 days at 28°C with 200 rpm shaking. The yeast cells were inoculated into 5 kg CTLs. The initial cell density of CTL fermentation was 1 × 10⁶ CFU/g C. fabianii, the initial water content was 30%. CTLs with same water content but without inoculation were used as control. Subsequently, all samples were fermented at 30°C (70% humidity) for 28 days and prepared for further testing.

Flavor components of CTLs were analyzed with untargeted metabolomics techniques by gas chromatography–mass spectrometry (GC–MS), which has been reported previously (Hu et al., 2022; Jia et al., 2023). In brief, 2 g CTL powder were added into 10 mL water, after the powder was completely infiltrated by water through shaking. Subsequently, 10 mL acetonitrile and 50 μL phenylethyl acetate (10.477 mg/mL, internal standard) were added, the mixture was shaken for 2 h at 2,000 rpm. After frozen at −20°C for 10 min, QuEChERS extraction kit was used for metabolites extraction. A pre-formulated salt packet, containing 4 g MgSO₄, 1 g NaCl, 1 g NaCitrate, and 0.5 g disodium citrate sesquihydrate was added and immediately shaken for dehydration. Subsequently, 1 mL supernatant was mixed with 0.15 g MgSO₄ and shaken at 2000 rpm for 2 min. The supernatants were subsequently analyzed by GC–MS (DB-5MS column 60 m × 1.0 μm × 0.25 mm, 26–400 atomic mass units mass scan range). Helium served as the carrier gas with a flow rate of 1.2 mL/min. The GC oven started at 60°C, increased to 250°C at a rate of 2°C/min, then to 290°C at 5°C/min, and was held at the final temperature for 20 min. The MS operated in electron impact mode with a 230°C ion source temperature and a 70 eV ionization voltage. The detected compounds were identified by the NIST17 database.

The major chemical components of CTLs, including total alkaloids, total sugar, reducing sugar and total nitrogen were analyzed by continuous flow analytical system. The contents of total alkaloids were determined according to the Tobacco Industry Standard YC/T468-2013. The contents of total sugar and reducing sugar were determined according to the Tobacco Industry Standard YC/T159-2019. The contents of total nitrogen were determined according to the Tobacco Industry Standard YC/T161-2002.

The fermented CTLs were rolled into 110 mm length, 14 mm diameter cigars. The cigars were balanced water content under a temperature of 20°C and relative humidity of 60%. Subsequently, the sensory quality was evaluated according to the Standard Evaluation Form provided by Great Wall Cigar Factory (Jia et al., 2023). Ten well-trained assessors who specialized in cigar production and evaluation were invited to conduct sensory quality evaluation. The sensory quality evaluation was conducted through two aspects: quality characteristics and flavor characteristics. For quality characteristics, 12 parameters (e.g., richness, matureness, irritation,) were rated on a 0–9 scale, where higher scores indicated better performance. As for the Flavor characteristics, such as bean, baking, nutty, etc., were evaluated using a 1–5 scale, with higher scores representing stronger flavor intensities. All panelists reached agreement on the evaluation scores for each sample.

Previous study has demonstrated that Cyberlindnera, as an aroma-producing yeast strain, has the capability to produce ester compounds (van Rijswijck et al., 2017). This result indicates that Cyberlindnera had the potential to increase flavor components content through CTL fermentation. In this study, five Cyberlindnera strains with identical colony morphology (Figure 1A shows a representative strain) were isolated from domestic CTLs originating from Yunnan, Hubei, and Sichuan. The strains exhibited highest sequence similarities with Cyberlindnera fabianii, showing >99.26% identity in the ITS region and >99.64% identity in the D1/D2 region. These high sequence similarities strongly suggest that the isolated strains belong to C. fabianii. Phylogenetic tree of these isolated strains was constructed based on ITS sequence by MEGA11 (Figure 1B). The analysis revealed that five isolated C. fabianii strains (F1-F5) and C. fabianii CBS 5640 were in the same cluster, confirming their close genetic relationship. Interestingly, several Candida species were found to be interspersed within the Cyberlindnera clade, which suggests a close evolutionary relationship between certain members of the Candida genus and Cyberlindnera.

The flavor components determine the aroma profile of CTLs, which is an essential impactor of cigar quality (Hu et al., 2022; Zhu et al., 2024). To assess whether isolated C. fabianii strains can increase flavor component content in CTLs during fermentation, we analyzed the fermented CTLs using GC–MS. The metabolites were classified by precursors (Figure 2B) and chemical classes (Figure 2C). Based on the total flavor component content in CTLs after fermentation by C. fabianii strains (Figure 2A), the fermented groups were ranked from highest to lowest as follows: F4 > F3 > F1 > F5 > CK > F2. Notably, only the F2 group exhibited a lower total flavor component content after fermentation. The F4 group, which had the highest total flavor component content, showed a 14.4% increase compared with the water fermentation control group. The result also indicated that most C. fabianii strains (e.g., F1, F3, F4, F5) could increase the content of chlorophyll degradation products (from 1.65 mg/g to 1.85 ~ 2.06 mg/g) and esters (1.18 mg/g to 1.46 ~ 1.66 mg/g) during fermentation. This result is consistent with previous studies, which have shown that C. fabianii can produce ester compounds (van Rijswijck et al., 2017; Liang et al., 2024).

Figure 3 illustrates the changes of flavor components detected in different fermentation groups. The CK group exhibits higher levels in several carotenoid degradation products, whereas, the C. fabianii groups possess higher content in other flavor components. The F4 group, which exhibited the highest total flavor content, contained 15 flavor components with higher content than the control group, including phytyl acetate, sandalrome, sclareolide, D-Limonene, (E)-beta-bergamotene, 3,5-di-tert-butyl phenol, and 7,11-Epoxymegastigma-5(6)-en-9-one. Among these compounds, phytyl acetate is particularly noteworthy. It serves as both a chlorophyll degradation product and an ester compound. Compared to the control group, the concentration of phytyl acetate was higher in most C. fabianii fermentation groups, except for strain F2. Particularly, the F4 fermentation group showed a 41% increase in phytyl acetate over the control group. The above results suggest that the aroma and ester-producing properties of C. fabianii contribute to enhancing the flavor component content in CTLs through fermentation. Strain F4, in particular, produces more flavor components and has the potential to improve CTL quality.

The intrinsic quality of CTLs is influenced by major chemical compositions such as total nitrogen, total sugar, total alkaloids, and reducing sugar. To determine whether C. fabianii strains can enhance the intrinsic quality of CTLs during fermentation, we performed continuous flow analysis to monitor changes in these major chemical compositions. Results (Figure 4) showed that strain F3 and F4 significantly reduced four major chemical compositions in CTLs after fermentation, including total alkaloids, total sugar, reducing sugar and total nitrogen. Furthermore, compared with the water fermentation control group, all five isolated C. fabianii fermentation groups showed a significant decrease in both total sugar (Figure 4A) and reducing sugar content (Figure 4B). Since total sugar and reducing sugar are crucial precursors of neutral aroma-enhancing compounds (Huang et al., 2024), their reduction in C. fabianii fermentation groups suggests an acceleration of biochemical processes. These processes may involve the conversion of sugars, like sucrose and glucose, into flavor compounds through degradation, oxidation, and esterification (Forney and Song, 2017).

High protein content in CTLs can lead to bitterness, irritation, and excessively strong smoke intensity during combustion (Zhang W. et al., 2023), which negatively impacts CTL quality. Since total nitrogen content is a reliable indicator of protein level (Boulos et al., 2020), the total nitrogen content of C. fabianii fermented CTLs was detected (Figure 4C). The F3, F4, and F5 groups showed a significant decrease in total nitrogen content compared to the control group, with reductions of 7.11, 10.11, and 8.58%, respectively. This result shows that certain C. fabianii strains possess the capacity for utilizing nitrogen-containing substances such as proteins. Furthermore, the amino acids produced by protein degradation can act as precursors of the Maillard reaction, and enhance aromatic compound generation during cigar combustion (Liu et al., 2022).

Excessive alkaloids in cigars can not only cause bitterness and irritation during smoking but also are associated with numerous health risks (Jia et al., 2023; Xue et al., 2023). Figure 4D shows that, strain F1, F3, and F4 significantly reduce the total alkaloids content in CTLs by 4.8, 16.5, and 14.9%, respectively, compared with the control group. The degradation of alkaloids is commonly believed to diminish the harshness of tobacco leaves, resulting in a milder and smoother taste, which contributes to the CTL quality improvement (Xue et al., 2023).

Taken together, post-fermentation, strain F4 exhibited the most significant reduction in total nitrogen (Figure 4C) and a near-maximal decrease in total alkaloids (Figure 4D). Considering the results of flavor components analysis, strain F4 also showed the highest total flavor content (Figure 2A). The above results indicate that strain F4 could be a promising microbial fermentation agent capable of reducing irritation and increasing aroma. Therefore, we chose the strain F4 fermented group for subsequent sensory evaluation.

Sensory evaluation is the most effective and definitive method to assess CTL fermentation effect and overall cigar quality. To verify that the isolated C. fabianii strain F4 can enhance CTL quality through fermentation, we conducted sensory evaluations on CTLs fermented by strain F4, using water-fermented CTLs as a control. The results, illustrated in Figure 5, indicate that the total sensory score of the CTLs fermented with C. fabianii strain F4 was higher than that of the control. Specifically, Figure 5A showed that compared with the control group, in terms of irritation and off-flavor, strain F4 could improve cleanliness, matureness and reduce irritation. In terms of aroma quantity, strain F4 could enhance mellowness, richness, and sweetness. As for the aroma profile (Figure 5B), strain F4 could enhance caramel, baking, hay, and resinous flavor of CTLs. The sensory evaluation result was consistent with chemical properties analysis, confirming that C. fabianii strain F4 can improve CTL quality through fermentation.