Three traditional Chinese starters were collected from Yuanhui District, Luohe City (LH), Hongqi District, Xinxiang City (XX) in Henan Province, and Donggang District, Rizhao City (RZ) in Shandong Province. Wheat flour was purchased from Henan Jinyuan Grain and Oil Co., Ltd., Zhengzhou, China, and conformed to the national standard GB/T 1355-2021 for first-grade wheat flour. Highly active dry yeast (AQ) was purchased from Angel Yeast Co., Ltd., Yichang, China.

Glucose, yeast extract powder, agar powder, tryptone, magnesium sulfate, manganese sulfate, peptone, beef extract, calcium carbonate, triammonium citrate, sodium acetate, yeast extract, Tween 80, tris, hydrochloric acid, isoamyl alcohol, isopropanol, glacial acetic acid, bromocresol green, ferric chloride, calcium chloride, potassium dihydrogen phosphate, 1,2-dichlorobenzene, potassium chloride, and anhydrous ethanol were supplied by Tianjin Kemiou Chemical Reagent Co., Ltd. The 50 × TAE Buffer, agarose, DNA Marker, 2 × Power Taq PCR Master Mix, and Taq PCR Master Mix were obtained from Sangon Biotech (Shanghai) Co., Ltd. The C₇ - C₃₀ n-alkane mixture standard was acquired from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

Fungal and bacterial DNA were extracted from the starters using the E. Z. N. A™ Mag-Bind Soil DNA Kit (Omega Bio-tek, Norcross, GA, United States, Cat. No. D3370-01), and the concentration and purity of the extracted DNA were analyzed. For fungal DNA, the internal transcribed spacer (ITS) region was amplified with the extracted fungal DNA as the template using primers SSU 0817F (5’-TTAGCATGGAATAATRRAATAGGA-3′) and SSU 1196R (5’-TCTGGACCTGGTGAGTTTCC-3′). For bacterial DNA, the V3-V4 hypervariable region was amplified with the extracted bacterial DNA as the amplification template using primers 338F (5’-ACTCCTACGGGAGGCAGCAG-3′) and 806R9 (5’-GGACTACHVGGGTWTCTAAT-3′). The purified amplicons were quantified, and then sequenced on the Illumina MiSeq PE300 platform. The sequencing results were uploaded to the National Center for Biotechnology Information (NCBI) database. Usearch was used to conduct operational taxonomic unit (OTU) clustering analysis on sequences with > 97% similarity (excluding singletons), and chimeras were removed to obtain representative OTU sequences. The optimal alignment results for the sequences were selected using an alignment threshold of 90%.

Yeast genomic DNA was extracted using the E. Z. N. A.® Yeast DNA Kit (Omega Bio-tek, D3370-01) following the manufacturer’s protocol: enzymatic pre-lysis with lyticase (30 min, 37 °C), bead-beating with 0.5 mm glass beads (FastPrep-24, 6.0 m·s⁻¹, 30 s × 3), followed by silica-column binding and elution in 50 μL elution buffer.

Bacterial DNA was obtained using the DNeasy UltraClean Microbial DNA Kit (Qiagen, 12,224–50) according to the manufacturer’s instructions: samples were resuspended in MicroBead solution, mechanically lysed in a bead-beater, treated with MD1/MD2 lysis buffers (65 °C, 10 min), and finally purified on silica spin filters with MD4 wash buffer and eluted in 100 μL MD5 buffer.

Using the extracted yeast and bacterial DNA as templates, the yeast ITS region was amplified with primers ITS4 (5’-TCCTCCGCTGACTAATATGC-3′) and ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3′), and the bacterial 16S rRNA gene was amplified with primers P0 (5’-GAGAGTTTGATCCTGGCTCAG-3′) and P6 (5’-CTACGGCTACCTT-3′).

PCR was conducted using a PTC Genesy 96 T thermal cycler (Xi’an Tianlong Technology Co., Ltd.). All amplifications were performed in 25.5 μL reactions with four replicates. Each reaction volume contained 1 μL of DNA template, 1 μL of Primer ITS4/P0, 1 μL of Primer ITS5/P6, 12.5 μL of 2 × Power Taq PCR Master Mix (Thermo Scientific, United States) and 10 μL of Double-distilled water.

The following cycle parameters were used for yeast: initial denaturation for 5 min at 94 °C; 30 cycles of 30 s at 94 °C (denaturation), 45 s at 52 °C (annealing), and 45 s at 72 °C (extension); and final extension for 10 min at 72 °C.

The following cycle parameters were used for bacteria: initial denaturation for 5 min at 94 °C; 30 cycles of 60 s at 94 °C (denaturation), 60 s at 52 °C (annealing), and 90 s at 72 °C (extension); and final extension for 10 min at 72 °C.

Electrophoresis analysis was performed using a 0.8% agarose gel with a DNA Marker as reference, under conditions of 100 V voltage, 100 mA current, and 25 min duration using an electrophoresis apparatus (Model YJ300C, Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd., Beijing, China). After staining with ethidium bromide solution for 40 min, the PCR amplification fragments were observed under an ultraviolet analyzer (UVP LLC, Upland, CA, United States) to determine their clarity, and the results were photographed and recorded.

The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequence determination. Homology search was then performed in the NCBI BLAST database to identify the species of the tested strains.

Yeast and LAB were streaked onto YPD medium and MRS medium, separately, and incubated at 28 °C and 36 °C for 48 h. Single colonies were then picked up and inoculated into YPD liquid medium and MRS liquid medium, respectively. After 20 h of cultivation, the supernatant was discarded to obtain fresh wet cells of yeast and LAB.

The fermentation capacity of yeast was determined using the Durham tube fermentation method. Activated yeast was inoculated into YPD liquid medium containing an inverted Durham tube, followed by incubation at 28 °C for 48 h. The volume of gas in the Durham tube was observed every hour, and the gas production initiation time of the strain and the gas production status at 48 h were recorded.

Yeast seed culture (100 μL) was inoculated into YPD liquid media with different acid and ethanol concentrations, followed by shaking cultivation at 28 °C and 180 r/min for 20 h. The absorbance was then measured at a wavelength of 600 nm. The absorbance value was used to represent the growth density of yeast under different acid and ethanol concentrations, thus reflecting the acid tolerance and ethanol tolerance of the yeast. The volume ratios (v/v) of lactic acid and acetic acid added were as follows: (0 + 0) %, (0.4 + 0.1) %, (0.8 + 0.2) %, (1.2 + 0.3) %, (1.6 + 0.4) %, and (2 + 0.5) %. The addition amounts of ethanol were 0, 5, 10, and 12%, respectively.

Fresh wet yeast cells were inoculated into flour at a concentration of 10⁸ CFU/g for dough mixing, and the gas production properties of the dough were determined using a Chopin F3 rheological fermentation instrument (Chopin Technologies, Villeneuve-la-Garenne, France). The test conditions were set as follows: temperature 35 °C; determination duration 3 h; weight of the counterbalance 1.5 kg.

Fresh wet yeast cells were added to 200 g of wheat flour at an inoculum size of 10⁷ CFU/g, with 48% water addition for dough mixing. After forming a dough with a smooth and elastic surface, it was placed in a proofing box for the first fermentation (temperature: 30 °C; relative humidity: 85%; duration: 6 h). After proofing, wheat flour (100 g) and water (45 mL) were added for remixing into dough, which was then repeatedly rolled out to fully expel air. The dough was subsequently divided into 100 g portions, which were shaped into round dough blanks using a round dough press, and then placed in the proofing box for the second fermentation (temperature: 30 °C; relative humidity: 85%; duration: 40 min). The proofed dough blanks were then transferred to an oven for baking with the top heat set to 180 °C and the bottom heat to 200 °C, for 16 min. After baking, the products were naturally cooled at room temperature for 30 min, and subsequently stored in a sealed container for later use.

The sensory evaluation was carried out by 20 trained panelists (10 males and 10 females, 20–40 years old) in an isolated sensory booth (room temperature 20–22 °C). The panelists were trained according to ISO standards (ISO 8586: 2012) for sensory analysis and subjected to performance monitoring (ISO 11132: 2012). Compusense software (version 5.2, Compusense Business Avionics B. V., Canada) was used for sample distribution. Panelists finished the sensory evaluation within 10 min and rinsed their mouths with spring water between samples. The appearance, color, palatability, toughness, viscosity, smoothness, and flavor of the CBF was scored by panelists on the basis of Supplementary Table S1.

Volatiles were extracted from CBF samples using headspace solid-phase microextraction (HS-SPME) and analyzed by Gas chromatography–mass spectrometry (GC–MS) instrument (Model 8,890-5977B, Agilent Technologies (China) Co., Ltd., China) with a DB-5 capillary column (60 m × 0.25 mm × 0.25 μm) (7).

Solid-phase microextraction conditions: CBF sample (2 g) was placed into a 20 mL headspace vial and internal standard solution (1 μL, 1,2-dichlorobenzene, 100 mg/L) was immediately added. Sampling was performed using an automatic headspace injection. The extraction head was aged for 30 min, the incubator was preheated at 60 °C for 10 min, the extraction time was 40 min, and the sample was desorbed at 250 °C for 300 s at the injection port.

In Formula, C_X is the relative mass concentration of compound X in the CBF sample, μg/kg; S_X is the peak area of compound X in the CBF sample; S(internal) is the peak area of the internal standard 1,2-dichlorobenzene; C(internal) is the concentration of the internal standard 1,2-dichlorobenzene; V(internal) is the volume of the internal standard added; M(sample) is the mass of the sample, g.

All experiments were performed in triplicate, and the results were expressed as “mean ± standard deviation.” For significant difference analysis, one-way analysis of variance (ANOVA) and Duncan’s multiple range test (p < 0.05) were used, with statistical analysis conducted using IBM SPSS Statistics 25 (IBM Corporation, Armonk, NY, United States). Graphs were plotted using Origin Pro 2025 (OriginLab Corporation, Northampton, MA, United States).

Alpha diversity analysis, a crucial method for evaluating microbial community complexity, reflects the species richness and evenness of samples (8). As shown in Table 1, for both fungi and bacteria in the traditional starter samples of RZ, XX, and LH, the Coverage indices were all greater than 99%, indicating that the sequencing results were reliable. The LH starter sample exhibited significantly higher species richness in both fungi (Chao1 = 271.86; ACE = 274.36) and bacteria (Chao1 = 341.76; ACE = 351.64) than the other samples. This value range is consistent with the species richness characteristics of traditional dough starters reported in existing studies which detected 490 fungal OTUs and 234 bacterial OTUs in Chinese northwest traditional jiaozi (9). The high species richness of LH may be linked to the combination of its unique raw material composition or fermentation processes and potential cross-kingdom synergistic effects (8).

In contrast, although the fungal richness in the XX starter was moderate, its Shannon index (2.29) and Simpson index (0.22) indicated that the fungal community possessed higher evenness and niche differentiation capacity. This aligns with the findings of which reported that traditional sourdoughs with balanced microbial evenness often establish stable metabolic interaction networks (10). Such high evenness might arise from a more complex metabolic interaction network in its fermentation environment, which is also supported by the findings that high niche differentiation in traditional fermented foods contributes to functional stability (8, 11). Giving that the Coverage indices of all samples exceeding 99%, the Sobs index (271) of the LH fungal community was almost consistent with the Chao1 estimate (271.86). This consistency not only confirms the accuracy of the sequencing data but also suggests that rare microbial groups (abundance < 0.1%) in LH were fully captured. Rare taxa, despite their low abundance, often harbor unique functional genes that may play crucial roles (9). This effective detection provides a valuable dataset to explore uncharacterized microbial functions in traditional starters.

Analysis of the relative abundance of fungal community composition is recognized as a critical method for uncovering the proportional distribution of distinct fungal species within a community (12). The relative abundance of fungal communities in XX, LH, and RZ starters are shown in Figures 1a,b. At the phylum level, three identifiable fungal phyla were detected: Ascomycota, Mucoromycota, and Basidiomycota. Among these, Ascomycota had the highest relative abundance (RA: > 1%), in XX (RA: 91.11%), LH (RA: 96.18%), and RZ (RA: 91.71%) starters, respectively. At the genus level, 15 fungal genera with relative abundances greater than 1% were detected.

Saccharomyces, as the dominant genus within Ascomycota, exhibited extremely high relative abundances in samples XX (46.98%), LH (56.83%), and RZ (59.86%). Saccharomyces is the primary microbial group involved in dough fermentation, exerting significant influence on dough expansion height and gluten network structure (13). Furthermore, Saccharomycopsis has an excellent ability to produce β-glucosidase, acid protease, and amylase, which helps promote microbial community succession enriching the flavor of malt (14). Previous studies have shown that bread fermented with exhibits superior flavor properties and lower hardness, as the genus Wickerhamomyces anomalus can convert precursor substances into aromatic substances such as esters, acids, alcohols, and aldehydes via synthetases (15).

Moreover, Saccharomyces exhibited the highest relative abundance in RZ sample, indicating that RZ starter possessed superior fermentation capacity. In addition, the main dominant fungal genera in XX sample also include Saccharomycopsis, Pichia, Rhizomucor, and Thermomyces. In LH sample, the main dominant fungal genera were Hyphopichia (RA: 12.12%) and Wickerhamomyces, (RA: 10.30%). In RZ sample, the main dominant fungal genera were Rhizomucor and Pichia.

Notably, Aspergillus, Monascus Rhizopus, and Rhizomucor were detected in XX, LH, and RZ samples. These are important fermentation strains in mold culture, possessing strong saccharification enzyme activity, which can provide the fermentation agent with multiple enzyme systems, supplying a large amount of small-molecule monosaccharides and free amino acids in the fermentation agent for metabolic utilization by lactic acid bacteria and yeast (16).

Analysis of the relative abundance of bacterial community composition is recognized as a critical method for uncovering the proportional distribution of distinct bacterial species within a community, and the structural characteristics of dominant and rare taxa can be reflected (12). The relative abundances of bacterial communities in XX, LH, and RZ starters are shown in Figures 1c,d. At the phylum level, three bacterial phyla were detected: Firmicutes, Cyanobacteria, and Proteobacteria, and they are commonly found in traditional Chinese starters (17). At the genus level, 12 dominant bacterial genera were detected. The dominant bacterial genera in XX sample were Companilactobacillus (RA: 12.55%) and Lactiplantibacillus (RA: 6.00%). The main dominant bacterial genera in RZ sample were Latilactobacillus (RA: 19.35%), Lactiplantibacillus (RA: 12.97%), and Acetobacter (RA: 8.10%). In LH sample, the main dominant bacterial genera were Companilactobacillus (RA: 18.71%), Latilactobacillus (RA: 19.98%), and Lactiplantibacillus (RA: 8.47%). Companilactobacillus, Latilactobacillus, and Lactiplantibacillus produce lactic acid and other organic acids during dough fermentation, which enrich the flavor of fermented bread products (18). Acetobacter was the only dominant bacterial genus in RZ sample, and it oxidizes ethanol to produce acetic acid during dough fermentation and improves the texture and taste of bread products (14).

Based on morphological differences such as colony shape, color, size, and protrusion degree, a total of 94 yeast strains were selected from XX, LH, and RZ starters, with their colony morphologies shown in Supplementary Table S2, exhibiting seven distinct colony morphologies. Through isolation, purification, analysis, and identification, species composition within microbial communities can be recognized, relative abundances and distribution characteristics of different bacteria in the community determined, and interactions among microorganisms explored (19).

The ITS sequence is extensively applied in species identification and widely recognized as the “barcode” region for fungal classification (20). The gel electrophoresis detection results of partial yeast strains are presented in Supplementary Figure S1a. Target fragments were successfully amplified from all samples. The fragment size was consistent with the expected ITS region length (500–1,000 bp), indicating that the primer design and amplification conditions exhibited favorable specificity and effectiveness.

The molecular identification results of the yeast strains are shown in Table 2. Three yeast species were identified from the three starters (XX, LH, and RZ): Saccharomyces cerevisiae, Wickerhamomyces anomalus, and Hyphopichia burtonni. Strains Y1, Y2, Y3, Y4, and Y5 were all identified as Saccharomyces cerevisiae through molecular identification, and their colony morphology characteristics differed. This is because the colony morphology exhibited by Saccharomyces cerevisiae is related to intraspecific genetic diversity (21).

During dough fermentation, Saccharomyces cerevisiae primarily metabolizes various sugars to produce CO₂, causing the dough to rise, and can also undergo esterification reactions through metabolic processes, enriching the flavor of fermented foods (13). Moreover, Saccharomyces cerevisiae can impart good flavor to steamed breads and produce substances that inhibit fungal growth during dough fermentation and bread storage (15). Wickerhamomyces anomalus possesses excellent capabilities for producing various glycosidases, including β-D-glucosidase and α-L-arabinofuranosidase, and is widely present in traditional sourdough and mold culture (22).

The results of gel electrophoresis detection for partial bacterial strains are presented in Supplementary Figure S1b. Fragment sizes were mainly concentrated in the range of 1,000–2000 bp. The sizes of the fragments were consistent with the typical length of bacterial 16S rRNA genes, verifying the reliability of the amplification products.

The molecular identification results for the three species of bacteria from XX, LH and RZ are shown in Table 2. A total of four LABs and one acetic acid bacterium (Acetobacter malorum) were identified. The four LABs were Furfurilactobacillus rossiae, Latilactobacillus curvatus, Lactiplantibacillus plantarum, and Companilactobacillus crustorum. Lactiplantibacillus plantarum is a common LAB in traditional Chinese starters, capable of producing large amounts of organic acids during dough fermentation, which positively improves the quality of fermented dough products (18). Acetobacter malorum is an obligate aerobic bacterium that metabolizes ethanol produced by yeast fermentation to generate acetic acid and water, positively influencing the flavor of fermented dough products made with traditional Chinese starters (23).

Sensory evaluations of CBFs made with seven yeast strains (Y1–Y7) were conducted, and AQ yeast was as the control. As shown in Table 3 and Supplementary Table S3, CBF fermented individually by the seven yeast strains showed no significant differences in surface color and surface morphology that the sensory scores of these CBFs ranged 79.20–82.80. CBFs fermented with the five Saccharomyces cerevisiae exhibited larger specific volume, better springiness, fine and uniform internal pores, soft texture, crisp taste, and no stickiness to the teeth. Saccharomyces cerevisiae Y4 exhibited remarkable advantages, and the CBF fermented by this strain showed higher volumetric ratios, indicating stronger gas-producing and dough expansion capacities. Moreover, it achieved the highest overall sensory score (82.80 ± 0.5) and performed well in multiple dimensions. In contrast, Y6 and Y7 had lower sensory scores (81.00 ± 0.8 and 79.20 ± 0.7). The sensory score (81.90 ± 0.8) of control AQ performed on par with most Saccharomyces cerevisiae strains lower than Y4.

The composition of volatiles in baked food serves as the fundamental material basis for reflecting the aroma characteristics of baked food (7). As shown in Table 4, a total of 40 volatiles were detected in the CBF (Y4) sample, including alcohols (12), esters (5), aldehydes (10), acids (6), ketones (2), furan (1), and other compounds (4). CBF (Y4) and CBF (AQ) samples had 38 and 36 volatiles, respectively.

During dough fermentation, yeast metabolizes amino acids via the Ehrlich pathway to generate various alcoholic metabolites, which exert a positive impact on the herbal and wine-like aromas of fermented products (27). CBF (Y4) contained high relative contents of phenethyl alcohol, 2-methylbutanol, and isopentanol, consistent with previous research findings. Phenethyl alcohol has a rose-like aroma and is positively correlated with bread aroma, while 2-methylbutanol can add rich, fruity, and floral aromas to fermented dough products (28). Isoamyl alcohol, derived from leucine metabolism, is positively correlated with aroma and exhibits “fragrant, mellow, and malt-like” flavors (29). Compared to CBF (AQ), CBF (Y4) uniquely contained 1-octen-3-ol and trans-5-decenoic alcohol, and the former contributed mushroom and grassy aromas to add a fresh layer of complexity, while the latter further enriched the stereochemical structure of alcohol compounds (7). Moreover, relative content of isobutyl alcohol in CBF (Y4) was significantly lower than in CBF (AQ), avoiding a harsh alcoholic taste (28).

Furthermore, aldehydes are produced via the oxidation of lipids and amino acids. Among the detected aldehyde volatiles, benzaldehyde, nonanal, phenylacetaldehyde, n-hexanal, and trans-2-nonenal are the main ones, characterized by flavor profiles of nutty, floral, and fruity aromas (7). Meanwhile, acidic compounds not only produce acidity during dough fermentation but also serve as important precursors for other aromatic compounds, positively contributing to flavor formation (30). The acidic volatiles detected in CBF (Y4) primarily included caproic acid, benzoic acid, and caprylic acid. An appropriate amount of caproic acid can contribute a cheesy flavor with an oily aroma (27). Caprylic acid exhibits a mild cheesy taste and can react with ethanol to form caprylic acid ethyl ester, imparting a fruity aroma to CBF (Y4).

Esters, as important aromatic compounds, are formed via the esterase-catalyzed reaction between unsaturated fatty acids and alcohols during fermentation (31). Among ester volatiles, those with relatively high contents include ethyl caprylate, phenethyl acetate, γ-nonalactone, and ethyl decanoate. The content of ethyl phenylacetate in CBF (Y4) is 2.46 times that of CBF (AQ), contributing a fruity aroma to CBF (29). In addition, both 3-hydroxy-2-butanone and geranyl propionate were detected in two CBF samples. Ketones are mainly produced through amino acid degradation and β-oxidation of saturated fatty acids (18). 3-Hydroxy-2-butanone contributes a buttery aroma, while geranyl propionate provides a fruity aroma. 2-Pentyl furan imparts a floral aroma to CBF (Y4). The unique indole in CBF (Y4) further enhance the uniqueness of the flavor. The results showed that CBF (Y4) had abundant unique volatiles, synergistically enhancing flavor complexity and uniqueness.