The fermented tobacco leaves from the Dominican Republic, Brazil, Indonesia, and China were collected and provided by China Tobacco Sichuan Industrial Co., Ltd. (Sichuan, China). Potato dextrose agar (PDA), potato dextrose broth (PDB), and yeast extract peptone dextrose agar (YPD) medium were purchased from Solarbio Technology Co., Ltd. (Beijing, China). Ampicillin sodium was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The fungal DNA kit was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). High-Performance Liquid Chromatography (HPLC) grade reagents used for chromatography were obtained from Thermo Fisher Scientific (Waltham, MA, United States). Phenylethyl acetate was purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, United States). The QuEChERS extraction kit (Kit contents: 4 g MgSO₄, 1 g NaCl, 1 g NaCitrate, 0.5 g disodium citrate sesquihydrate, 50 mL tubes with ceramic homogenizers) was purchased from Agilent (Santa Clara, CA, United States).

To isolate Candida strains, the fermented tobacco leaves were mixed with a sterile saline solution and homogenized. The suspension was then gradient diluted and spread on the surface of YPD and PDA plates added with ampicillin sodium (inhibiting bacterial growth) and propionate sodium (inhibiting fungal growth), and the plates were incubated at 30°C for 3 days. The genomic DNA of isolated strains was extracted using a fungal DNA kit. The internal transcribed spacer region was amplified with primers ITS1 (5′-TCC​GTA​GGT​GAA​CCT​GCG​G-3′) and ITS4 (5′-TCC​TCC​GCT​TAT​TGA​TAT​GC-3′) (White et al., 1990). The DNA sequencing of the polymerase chain reaction (PCR) products was performed by Sangon Biotech Co., Ltd. (Shanghai, China). The isolates were identified by Basic Local Alignment Search Tool (BLAST) alignment (http://www.ncbi.nlm.nih.gov/BLAST/).

Then, the isolated Candida strains were separately inoculated into PDB medium and grown at 30°C with shaking for 2 days, and then the cultures were centrifuged at 12000 rpm for 30 min to collect the cells. Next, the Candida strains were individually inoculated into 5 kg tobacco leaves with an initial cell density of 1 × 10⁶ CFU/g. Then, the tobacco leaves were evenly stacked in a constant temperature and humidity incubator at 30°C and 70% humidity for 21 days of fermentation. The non-inoculated sample was used as a control to evaluate the fermentation ability of different Candida strains by detecting the chemical indexes and flavor substances of the fermented tobacco leaves.

As described by F. Liu et al., the contents of total sugar (TS), reducing sugar (RS), total alkaloids (NIC), and total nitrogen (TN) were detected by an active pharmaceutical ingredient (API) continuous flow instrument (Liu et al., 2021). The metabolite components were extracted by QuEChERS extraction kit, and then the contents of metabolites were examined by gas chromatography-mass spectrometry (GC-MS).

Extraction step: first, 2 g of sample and 10 mL water were added to a 50 mL tube with ceramic homogenizers, and shaken until the sample was sufficiently infiltrated by water. Then, the metabolites were extracted by adding 10 mL acetonitrile and 50 μL phenylethyl acetate internal standard (9.06 mg/mL) and shaking at 2000 rpm for 2 h. After the mixture was frozen at −20°C for 10 min, a salt packet (4 g MgSO₄, 1 g NaCl, 1 g NaCitrate, 0.5 g disodium citrate sesquihydrate) was added and immediately shaken to remove water. Finally, 0.15 g MgSO₄ was added to 1 mL of the supernatant and shaken at 2000 rpm for 2 min. After centrifuging at 6,000 rpm for 2 min, the supernatant was collected for further analysis.

The GC-MS conditions were described by (Jia et al., 2023). Separations of the metabolites compounds were performed on a DB-5MS column (60 m × 1.0 μm × 0.25 mm). The initial temperature of the GC oven was 60°C, and ramped to 250°C at 2 C/min, then ramped to 290°C at 5°C/min, finally held for 20 min at final temperature. Helium was used as the carrier gas at the flow rate of 1.2 mL/min. MS was operated in the electron impact mode with an ion source temperature of 230°C and an ionization voltage of 70 eV. Mass scan range was 26–400 atomic mass units. Qualitative analysis of compounds was performed by matching the mass spectra with the National Institute of Standards and Technology and Wiley Library. Compounds quantification was calculated according to the ratio between the peak area of a particular compound and the internal standard.

After fermentation, the tobacco leaves were rolled into cigarettes with a length of 110 mm and a diameter of 14 mm. Then, the cigarette was conserved for 1 month in a constant temperature and humidity incubator with a temperature of 20°C and relative humidity of 60% for smoking evaluation. The sensory quality evaluation was performed following the Standard Evaluation Form provided by Great Wall Cigar Factory (Sichuan, China) (Hu et al., 2022). The quality characteristics consisted of mellowness, richness, matureness, irritation, smoothness, fluentness, sweetness, cleanliness, aftertaste, combustibility, ash color, and ash coagulation. The flavor characteristics mainly included caramel, beany, baking, nutty, cocoa, honey, resinous, hay, leather, and pepper flavor. A well-trained sensory panel consisting of 10 assessors majoring in cigar production was invited for the sensory quality assessment.

The Spearman’s pairwise correlations and the significance of the correlations were calculated using corr. test function and the psych package in R (Version R-3.5.1). Significance difference analysis and Z-score normalization were performed using SPSS (Version 22.0, SPSS Inc., Chicago, IL, United States). Further statistical analysis and graphics were performed in EXCEL 2017 software (Microsoft Office, United States) and GraphPad Prism Software (Version 8.0, GraphPad Software, San Diego, California, United States).

In a previous study, correlations between physicochemical metabolites and microbial communities during cigar fermentation have been analyzed (Jia et al., 2023). Correlation results indicated that the Candida genus was significantly negatively correlated with total nitrogen and alkaloids contents, while positively correlated with the total contents of flavor components, which implied that the Candida genus had the functional potential to degrade nitrogen-containing substances and synthesize flavor substances during the cigar fermentation (Supplementary Table S1). Therefore, this study conducted directional isolation of Candida strains from cigar tobacco leaves collected from the Dominican Republic, Brazil, Indonesia, and China. As shown in Table 1, a total of 4 strains of C. metapsilosis and 5 strains of C. parapsilosis were isolated. Next, 9 strains of Candida were separately inoculated into cigar tobacco leaves with an initial cell density of 1 × 10⁶ CFU/g, and the physicochemical indexes of the fermented tobacco leaves were detected and analyzed.

As shown in Figures 1A, B, the contents of total sugar and reducing sugar in the bioaugmentation fermentation groups (P1∼P5, M1, M3, M4) were significantly reduced by 38.8%–89.2% compared with the control group. However, compared with the control group, the total sugar content in the M2 group was decreased by 7.1%, and the content of reducing sugar was increased by 6.2%, which reflected the differences in the utilization ability of sugar compounds among different Candida strains (Rivera and Tyx, 2021).

The protein in cigar tobacco leaves would produce a burnt feather flavor when smoked, and its content is one of the important indicators affecting the irritancy and cleanliness of cigars. Compared with the control group, the total nitrogen contents in the M2, M4, M1, P4, and P5 groups were significantly decreased by 8.3%, 6.7%, 5.5%, 3.4%, and 2.8%, respectively (Figure 1C). In contrast to the high sugar content in the M2 group, its total nitrogen content was the lowest among all the bioaugmentation groups, indicating that the M2 strain might have a higher utilization capacity for nitrogen-containing substances such as proteins.

In addition, higher alkaloids content could not only cause bitterness and irritancy of tobacco leaves but also have potential addictive and carcinogenic effects on humans (Kaminski et al., 2020; Rehder Silinski et al., 2020). As shown in Figure 1D, the alkaloids contents in all bioaugmentation groups showed a significant decrease compared to the control group. The order of alkaloids content in these 10 groups from low to high was: M4 < P4 < P2 < M2 < P1 < P5 < P3 < M3 < M1 < CK. Compared with control group, the alkaloids contents of M4, P4, P2, M2, and P1 groups decreased by 32.6%, 32.5%, 30.9%, 27.5%, and 25.3%, respectively.

Furthermore, qualitative and quantitative analysis of different alkaloids in tobacco leaves were conducted by GC-MS, and a total of seven alkaloids were detected, including cotinine, nornicotine, nicotine, N-acetyl-DL-nornicotine, isonicoteine, (1′s, 2′s)-nicotine-N′-oxide (Figure 2). Among them, nicotine accounted for 94.2–98.8% of the alkaloids content and was the most dominant alkaloids. The nicotine contents of these 10 groups were ranked from low to high as: P2 < M4 < P1 < P4 < P3 < M1 < P5 < M2 < M3. Compared with the control group, the nicotine content of the P2, M4, and P1 groups decreased by 56.3%, 51.8%, and 47.6%, respectively. In general, the alkaloids contents were significantly decreased after bioaugmentation with the P1, P2, P4, and M4 strains, and the contents of seven alkaloids in the M4 group were lower than those in other bioaugmentation groups. This might be due to the conversion of alkaloids such as nicotine to amino acids, organic acids, and volatile ammonia by Candida strains during fermentation.

Previous studies have shown that the total content of flavor components is an important indicator of the quality of cigar tobacco leaves (Banozic et al., 2021; Hu et al., 2022; Li et al., 2020). According to the classification of precursors, flavor components were mainly classified into chlorophyll degradation products, carotenoid degradation products, Maillard reaction products, cembranoids degradation products, and other flavor components (Figure 3). The total contents of flavor components of cigar leaves after bioaugmentation fermentation by different Candida strains were ranked from high to low as follows: P1 > M4 > P2 > P5 > P3 > CK > P4 > P5 > M3 > M2 > M1. Compared with the control group, the total content of flavor components in the P1, M4, P2, and P5 groups significantly increased by 25.2%, 18.9%, 11.1%, and 6.0%, respectively, while those in M1, M2, M3, and P4 groups significantly decreased by 19.5%, 17.7%, 13.5%, and 6.6%, respectively. The content of flavor components in tobacco leaves after bioaugmentation with different strains suggested that P1 and M4 strains might have higher abilities to synthesize flavor substances.

Figure 4 showed the changes in the content of 34 flavor substances in different fermentation groups, with the majority of flavor components in the P1 and M4 groups being higher than in the other bioaugmentation groups. Compared with the control group, 25 and 21 flavor components were found to be higher in the P1 and M4 groups, respectively. In group P1, chlorophyll degradation products (e.g., phytol, neopphytodiene) and carotenoid degradation products (e.g., 1- (2,6,6-trimethyl-1-cyclohexen-1-yl)-1-Penten-3-one, phytone, α-farnesene, megastigmatrienone) were more abundant than those in the control group (Table 2). In the group M4, the contents of chlorophyll degradation products (e.g., neophytodiene), carotenoid degradation products (e.g., 6,10-dimethyl-5,9-undecadien-2-one, trans-geranylgeraniol, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-2 (4 h)-benzofuranone, megastigmatrienone, 1-methyl-4- (1-methylethenyl)-7-oxabicyclo [4.1.0] heptane, cis-beta-bergamotene), cembranoids degradation products (e.g., solanone), and Maillard reaction products (e.g., 3-ethyl-3,4-dihydro-2 (1H)-quinoxalinone, tetrahydro-2,4-dimethyl-furan, tetrahydro-2,2,5,5-tetramethyl-furan) were higher than those in the control group. Overall, the total contents of chlorophyll degradation products, carotenoid degradation products, and Maillard reaction products were significantly higher in the P1 group than those in the control group, while the total contents of chlorophyll degradation products, cembranoids degradation products, and Maillard reaction products were significantly higher in M4 group (Table 2).

By comparing the metabolic characteristics of nine Candida strains, it was found that C. parapsilosis P1 and C. metapsilosis M4 strains could not only increase the total content of flavor components but also reduce the alkaloids content in cigar tobacco leaves, which have great potential as starter. Therefore, the bioaugmentation effects of the P1 and M4 strains were verified by sensory evaluation. It was found that both P1 and M4 strains could improve the irritation and cleanliness, enhance the matureness, smoothness, fluentness, sweetness, and combustibility, and improve the coagulation and color of ash (Figure 5A). Among them, P1 could improve the beany and nutty flavor of cigar tobacco leaves, while M4 could enhance the baking, nutty, cocoa, honey, resinous, and hay flavor (Figure 5B).